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Objectives: To investigate a possible relationship between hypertriglyceridemia and the coagulation system, a Cardiovascular Risk Factor Two-township Study was conducted in Taiwan. Design: A case-control study. This longitudinal, prospective study focused on the evolution of cardiovascular disease risk factors with emphasis on haemostatic factors. Subjects: Hypertriglyceridemic subjects (triglyceride <2.26 mmoll+1, n = 327) and age-matched normal controls from a population screening program. Main outcome measures: Haemostatic parameters measured in this study included prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, factors VIIc and VIIIc, and antithrombin-III and plasminogen levels. Results: In our male hypertriglyceridemic subjects, aPTT was not significantly reduced, while significant elevations of factor VIIIc, factor VIIc, and plasminogen and antithrombin-III levels were noted. In the female hypertriglyceridemic subjects, the elevation of factor VIIc, factor VIIIc, and plasminogen and antithrombin-III levels was highly-significant, whereas aPTT was not significantly reduced. Unexpectedly, the levels of the established coronary risk factor, fibrinogen, did not show a statistically different change. Similar to previous data, our hypertriglyceridemic subjects also presented with hyperinsulinemia, glucose intolerance, upper body obesity, and elevated blood pressure. Conclusions: Despite the fact that in population studies, triglycerides do not consistently appear to be an independent risk factor for coronary heart disease, our data suggest that a pronounced increase in triglycerides warrants aggressive therapy, because this increase may be associated with a hypercoagulable state. This phenomenon contributes another perspective to the study of higher cardiovascular mortality in hypertriglyceridemic subjects.

The gene responsible for hereditary haemochromatosis (HH) has recently been identified. One mutation in this gene, termed HFE, has been found in all Australian HH patients. We previously identified a predominant HH ancestral haplotype covering 4.5Mb at 6p21.3, and showed that patients with two copies of this haplotype express a more severe form of the disorder. One key question to now be resolved is why haplotype related variation in phenotypic expression of HH is present if all patients tested have the same HFE mutation. A cosmid resource covering the 4.5Mb HH ancestral haplo type region was obtained. These cosmids provide the material for the completion of a transcript map of this region, and will assist the identification of candidate modifiers of HFE expression.

Antibodies to phospholipids (APL) were studied in 97 patients which had ischemic stroke in young age (up to 46 years). These antibodies (Abs) were found in 25 patients (26%): Abs to cardiolipin--in 15 patients (60%), Abs to lupoid anticoagulant in 18 from 24 patients (75%), Abs to phosphatidylethanolamine--in 4 from 13 patients (31%). Disorders of cerebral circulation (DCC) usually began at the age of 14-45 years and were characterized by ischemic strokes and by transitory DCC. There was no correlation between the occlusion of extracranial arteries and their hemodynamic significant stenosis. There was occlusion of intracranial arteries in 7 from 12 patients (58%). Other risk factors of the stroke development were also found in 19 patients (76%) together with Abs to phopholipids. Other manifestations of antiphospholipid syndrome (APLS) were observed in 68% of the patients: miscarriage of pregnancy (63%), thrombosis of peripheric veins (16%), thrombocytopenia (32%), ischemic heart disease (28%). Comparison of APL+ and APL- patients revealed that transitory DCC, occlusion of intracranial arteries, intact extracranial arteries, widening or condensation of the cardial valves on echo-ECG, abortions, increase of ESR were significantly more frequently observed in the former group. For confirmation of APLS diagnosis it was necessary to study simultaneously Abs to cardiolipin and lupoid anticoagulant. Prophylaxis of repeated DCC in APLS included administration of both aspirin and anticoagulants of indirect action.

Platelet aggregation was undertaken in platelet rich plasma in 34 heat-stroke patients during the Muslim pilgrimage (Haj) to Makkah; 18 were males and 16 were females; their ages ranged from 36 to 80 years (mean SD = 58 10). Platelet aggregability, on arrival at the Heatstroke Centres, was markedly inhibited in response to adrenaline, collagen, arachidonic acid and ristocetin but not to ADP. Responses to decreasing ADP doses (20.0, 2.0, 1.0 and 0.5 micromol/l) showed hyperaggregability in 12 patients, inhibited responses in 16 and normal responses in 6 patients. Aggregation responses were not significantly different when comparing patients with bleeding manifestations ( n = 10), with those without bleeding ( n = 24). Haemostatic parameters including plasma fibrinogen, ATIII and platelet count, were markedly reduced in the two patient groups who showed hyperaggrebable and depressed aggregation responses, but not in those with normal responses. These results lead us to conclude that: (1) platelet activation is a frequent feature of heatstroke; (2) in heatstroke altered aggregation responses, whether hyperaggregable or depressed, occur simultaneously with a consumption coagulopathy.

Phe(12) of deacetyl-thymosin beta(4) is one of the structural essentials for restorative effect on the impaired blastogenic response of uraemic T-lymphocytes. In order to evaluate the functional roles of this phenyl group in the restorative effect on impaired T-lymphocytes, two analogues, [1- Nal(12)]deacetyl-thymosin beta(4) and [Cha(12)]deacetyl4 thymosin beta(4), were synthesized by a solid-phase method and evaluated for restorative effect on the impaired blastogenic response of uraemic T-lymphocytes. The results indicated that [1-Nal(12)]deacetyl-thymosin beta(4) which had a bulky naphthyl ring showed a stronger restorative effect than that of deacetyl-thymosin beta(4), but it was slightly weaker than that of [Phe(4F)(12)]deacetyl-thymosin beta(4). However, [Cha(12)]deacetyl-thymosin beta(4) showed no restorative effect on the impaired blastogenic response of uraemic T-lymphocytes.

Purpose. Amplification of genetic sequences on chromosome 12q13 is frequently found in soft tissue tumours. However, for the MDM2 gene, over-expression of the MDM2 protein has not always been shown to accompany gene amplification, raising the possibility that amplification of genetic sequences targets alternative genes on chromosome 12q13 for over-expression. To investigate this discrepancy, we have examined 129 soft tissue tumours for amplification of the MDM2 gene using Southern analysis, and 39 of these tumours were also examined by immunohistochemical staining for MDM2 over-expression.Results. Gene amplification was identified in 14/114 (12.3%) of the malignant tumours, but was not identified in any of the benign tumours; 21/39 (54%) of the malignant tumours also demonstrated MDM2 over-expression. Within this group the MDM2 gene was over-expressed in every tumour in which the gene amplification was found, and over-expression in the absence of gene amplification was also found in an additional 10 tumours.Discussion. These data demonstrate a clear correlation between the presence of MDM2 amplification and MDM2 over-expression, and provide persuasive evidence therefore that the amplification of genetic sequences on chromosome 12q13 in soft tissue sarcomas targets the MDM2 gene for over-expression. These data also indicate that alternative mechanisms may contribute to MDM2 over-expression within some tumours.

Purpose. The prognosis, treatment principles and prediction of clinical outcome of patients with chondrosarcoma currently rest on histologic grading which is somewhat ambiguous due to difficulty in pathologic interpretation of this neoplasm. Immunohistochemistry, flow cytometry and oncogene/tumor suppressor gene expression have been examined as alternative indices to predict the biologic behavior of these tumors. Because of partial successes obtained with flow cytometry and because of the improvement in predicting recurrence offered by examining the S-phase fraction, we undertook the current study to determine if expression of specific regulators of the cell cycle would act as prognostic indicators for these patients.Subjects/methods. We examined archival pathologic specimens from 39 patients with at least 2 years' clinical follow-up for the presence of p53, Rb, src and MIB-1 by immunohistochemistry and correlated this with clinical histories and incidence of recurrence.Results. While Rb, p53 and src gene products were identified to a variable extent in these specimens, there was no prognostic significance to their expression. In contrast, MIB-1, an epitope expressed only during semiconservative replication and an accepted marker of cell proliferation, served as a significant prognostic indicator. MIB-1 staining was present in 14.5% of tumor cells in all specimens (range 0-59%). When MIB-1 staining was examined with respect to disease recurrence, there was a statistically significant association between staining and histologic grade (p < 0.05) as well as event-free survival (p < 0.02). Comparing survival curves stratified by MIB-1 expression, there was a significant decrease in event-free survival associated with increasing MIB-1 indices (p < 0.003). Covariates that were associated with event-free survival include histologic grade (p = 0.025) and stage (Musculoskeletal Tumor Society) (p = 0.014). There was no statistical association with patient age (p = 0.15), tumor size (p = 0.47), tumor histology (p = 0.62) or anatomic location (p = 0.316).Discussion. These results indicate that determination of the proliferation index by MIB-1 immunostaining may serve as a useful adjunct to current histopathologic classification. Patients with a high proliferation index may benefit from established adjuvant therapies or experimental approaches including immunotherapy or biologic modulation.

No abstract available.

An impairment in serotonergic neurotransmission may be associated with alcoholism. We recently identified a high-affinity serotonin transporter (5-HTT) in human peripheral blood lymphocytes (PBLs). Moreover, molecular analysis of RNA samples of human lymphocytes using reverse transcription, coupled with polymerase chain reaction, enabled us to confirm the expression of a 5-HTT identical to the one reported in neuronal tissues, as evidenced by hybridization and sequence analysis. In this investigation, we measured the serotonin (5-HT) uptake in PBLs of recovering alcoholics (N = 10) with long-term abstinence (2-10 years) and non-alcoholic controls (N = 10). 5-HT uptake was measured by incubating 1 x 10(7) cells of PBLs with [3H]5-HT (3-1000 nM; sp. act. 23 Ci/mmol) for 10 min at 37 degrees. The results of this preliminary study revealed that abstinent alcoholics had significantly (P < 0.01) increased uptake of 5-HT (43.6 +/- 5.70 pmol/10(7) cells) as compared with controls (23.33 +/- 2.50 pmol/10(7) cells). An enhanced uptake of 5-HT in PBLs of abstinent alcoholics agrees with previously reported observations of increased 5-HT uptake in brain and platelets of former alcoholics and their descendants. This suggested that a serotonergic mechanism may be linked to the heredity of alcoholism.

The human homologue of the Drosophila segment polarity gene patched is implicated in the development of nevoid basal cell carcinoma syndrome (NBCCS) and in the genesis of sporadic basal cell carcinomas. In order to examine the phenotypic variability in NBCCS and to highlight functionally important domains of the PTCH protein, we have now screened 71 unrelated NBCCS individuals for mutations in the PTCH exons. We identified 28 mutations that are distributed throughout the entire gene, and most (86%) cause protein truncation. As part of this analysis, we demonstrate that failure of one NBCCS family to show clear linkage to chromosome 9q22.3-31 is most likely due to germinal mosaicism. We have identified three families bearing identical mutations with variable phenotypes, suggesting phenotypic variability in NBCCS is a complex genetic event. No phenotype genotype correlation between the position of truncation mutations and major clinical features was evident. Two missense mutations have been identified, and their location within transmembrane domains supports the notion that PTCH may have a transport function. The preponderance of truncation mutants in the germ line of NBCCS patients suggests that the developmental defects associated with the disorder are most likely due to haploinsufficiency.

The anti-factor Xa activity of the synthetic pentasaccharide SR 90107A/ORG 31540 was assayed by a chromogenic method at pH 8.4 and pH 7.35, comparatively to the 4th International Heparin Standard (IHS) or to the Ist International Low Molecular Weight Heparin Standard (LMWHS). At pH 8.4, SR 90107A/ORG 31540 was found to have a specific anti-factor Xa activity of 639 +/- 14 and 659 +/- 19 IU/mg (mean +/- sem, n = 6) when assayed in comparison with the 4th ISH and the Ist LMWHS respectively. At pH 7.35, the corresponding figures were 864 +/- 6 and 1160 +/- 51 IU/mg (mean +/- sem, n = 6) respectively. The dissociation constants of the ATIII-pentasaccharide complex formed by SR 90107A/ORG 31540 and by two close analogues: SR 80327A and SR 80027A in the presence of purified human ATIII were found to be 41 +/- 8, 96 +/- 1 and 3 +/- 1.4 nM (mean +/- sem, n = 3) respectively. For the three compounds, the pseudo-first order molar catalytic constants for factor Xa inactivation by the ATIII-pentasaccharide complex were shown to be statistically comparable, in the range of 7-8 x 10(7) min-1 per mole. It is concluded that the differences in specific anti-factor Xa activities between SR 90107A/ORG 31540 and its synthetic chemical analogues can be attributed to variations of the dissociation constants whereas the catalytic constants for factor Xa inactivation remain unchanged.

The cause of the hepatic siderosis and iron overload that is common in porphyria cutanea tarda (PCT) is uncertain. Heterozygosity for genetic hemochromatosis has been supported by some studies of the association between the HLA-A3 antigen and porphyria cutanea tarda but not by others. The hemochromatosis gene is now believed to be located telomeric to HLA-A3 and close to the DNA microsatellite marker D6S1260. We have used this and other microsatellite markers, which together define an ancestral haplotype that is strongly linked to hemochromatosis, to reinvestigate the relationship between these disorders in 41 British patients with sporadic PCT. Fifteen patients carried the hemochromatosis-associated alleles D6S265-1 and D6S105-8. Four of these were homozygous for the ancestral haplotype D6S265-1 : D6S105-8: D6S1260-4. We estimate that approximately 37% of British patients with sporadic PCT carry at least one hemochromatosis gene compared with 10% of the general population.

Antithrombin-III (AT-III) activity was determined in the plasma of 32 healthy cows. The range of activities was between 105 and 160 per cent of a human reference plasma; the median activity was 132.5 per cent, and the 5 per cent and the 95 per cent quartiles were 111.1 per cent and 153.9 per cent of the standard. The activity of AT-III was also determined in 82 cows with a variety of internal diseases. In cows with hepatopathy, acute enteritis, generalised peritonitis, blood loss, idiopathic cardiomyopathy, glomerulonephritis and disseminated coagulopathy, the activity of AT-III was lower than in healthy animals. The lower values were due to decreased synthesis, increased consumption, or the loss of AT-III. An acquired deficiency of AT-III appears to favour the development of thrombophlebitis.

Thirty-four uterine serous carcinomas, a type of endometrial carcinoma with aggressive behavior and a high frequency (90%) of p53 gene mutations, were analyzed for microsatellite instability (MI). Genomic DNA isolated from paired normal and tumor tissue was analyzed at eight microsatellite loci (D2S119, D2S123, D2S147, D10S197, D13S175, D18S58, D18S69, and ATn) located on four different chromosomes. All 34 tumors failed to meet the criteria for MI, defined as an alteration in the size of at least two of the microsatellite loci in tumor DNA when compared with normal DNA. Only three tumors demonstrated a shift in the size of a single microsatellite locus. Previously we reported MI in 20% of uterine endometrioid carcinomas, the most common type of endometrial carcinoma. The observed difference in the MI frequency between endometrioid and serous carcinoma is statistically significant (P = 0.003). Our data demonstrate that MI is uncommon in uterine serous carcinoma and support that different pathogenetic mechanisms are involved in the development of the two most common types of endometrial carcinoma.

Nin1p, a component of the 26S proteasome of Saccharomyces cerevisiae, is required for activation of Cdc28p kinase at the G1-S-phase and G2-M boundaries. By exploiting the temperature-sensitive phenotype of the nin1-1 mutant, we have screened for genes encoding proteins with related functions to Nin1p and have cloned and characterized two new multicopy suppressors, SUN1 and SUN2, of the nin1-1 mutation. SUN1 can suppress a null nin1 mutation, whereas SUN2, an essential gene, does not. Sun1p is a 268-amino acid protein which shows strong similarity to MBP1 of Arabidopsis thaliana, a homologue of the S5a subunit of the human 26S proteasome. Sun1p binds ubiquitin-lysozyme conjugates as do S5a and MBP1. Sun2p (523 amino acids) was found to be homologous to the p58 subunit of the human 26S proteasome. cDNA encoding the p58 component was cloned. Furthermore, expression of a derivative of p58 from which the N-terminal 150 amino acids had been removed restored the function of a null allele of SUN2. During glycerol density gradient centrifugation, both Sun1p and Sun2p comigrated with the known proteasome components. These results, as well as other structural and functional studies, indicate that both Sun1p and Sun2p are components of the regulatory module of the yeast 26S proteasome.

Recently, a novel human major histocompatibility complex (MHC) class I-like gene (HLA-H) was reported as a candidate gene for human hereditary haemochromatosis, a recessive disease of iron metabolism with a remarkably high incidence in northern Europeans. Independently we have isolated this gene in the course of a search for new human MHC class I-related genes and named it MR2. Here we report a mouse homolog of this human gene. The mouse MR2 gene is similar to the human counterpart with an overall predicted amino acid sequence similarity of approximately 66% and it is expressed in various tissues as in human. The extra eight amino acid residues between the alpha1 and the alpha2 domains in the mouse molecule compared to the human counterpart can be explained by the creation of the coding sequence from the intron. While the human gene is located at the site telomeric to the MHC region on human chromosome 6, our study indicated the translocation of the mouse homolog from the site telomeric to the MHC on mouse chromosome 17 to chromosome 13 along with other genes. This mouse gene should be important in clarifying a possible role in iron metabolism.

Immunosuppression has been sporadically discontinued by noncompliant liver allograft recipients for whom an additional 4 1/2 years of follow-up is provided. These anecdotal observations prompted a previously reported prospective drug withdrawal program in 59 liver recipients. This prospective series has been increased to 95 patients whose weaning was begun between June 1992 and March 1996, 8.4+/-4.4 (SD) years after liver replacement. A further 4 1/2 years follow-up was obtained of the 5 self-weaned patients. The prospectively weaned recipients (93 livers; 2 liver/kidney) had undergone transplantation under immunosuppression based on azathioprine (AZA, through 1979), cyclosporine (CsA, 1980-1989), or tacrolimus (TAC, 1989-1994). In patients on CsA or TAC based cocktails, the adjunct drugs were weaned first in the early part of the trial. Since 1994, the T cell-directed drugs were weaned first. Three of the 5 original self-weaned recipients remain well after drug-free intervals of 14 to 17 years. A fourth patient died in a vehicular accident after 11 years off immunosuppression, and the fifth patient underwent retransplantation because of hepatitis C infection after 9 drug-free years; their allografts had no histopathologic evidence of rejection. Eighteen (19%) of the 95 patients in the prospective series have been drug free for from 10 months to 4.8 years. In the total group, 18 (19%) have had biopsy proved acute rejection; 7 (7%) had a presumed acute rejection without biopsy; 37 (39%) are still weaning; and 12 (13%, all well) were withdrawn from the protocol at reduced immunosuppression because of noncompliance (n=8), recurrent PBC (n=2), pregnancy (n=1), and renal failure necessitating kidney transplantation (n=1). No patients were formally diagnosed with chronic rejection, but 3 (3%) were placed back on preexisting immunosuppression or switched from cyclosporine (CsA) to tacrolimus (TAC) because of histopathologic evidence of duct injury. Two patients with normal liver function died during the trial, both from complications of prior chronic immunosuppression. No grafts suffered permanent functional impairment and only one patient developed temporary jaundice. Long surviving liver transplant recipients are systematically overimmunosuppressed. Consequently, drug weaning, whether incomplete or complete, is an important management strategy providing it is done slowly under careful physician surveillance. Complete weaning from CsA-based regimens has been difficult. Disease recurrence during drug withdrawal was documented in 2 of 13 patients with PBC and could be a risk with other autoimmune disorders.

To elucidate the role of ras gene mutations during the early stage of colorectal tumour progression, K-ras gene mutations were analysed in 32 benign adenomas and 36 adenomas with focal carcinoma in the colorectum by microscraping of histologically pure regions from tissue sections, polymerase chain reaction-restriction fragment length polymorphism and in part by direct sequencing. Several regions were scraped out and analysed when an adenoma contained areas with different grades of dysplasia. The frequencies of K-ras gene mutation in mild dysplasia, moderate dysplasia and focal carcinoma were 19% (7/36), 51% (25/49) and 39% (14/36) respectively. The K-ras gene status was heterogeneous in 4 of the 11 benign adenomas from which multiple samples were obtained, and mutations were always found in the regions with more advanced dysplasia in these adenomas. Thirteen of the 36 adenomas with focal carcinoma showed heterogeneity of mutations between the adenoma region and the focal carcinoma. Seven of which had mutations only in the adenoma region. These findings indicated that the K-ras gene mutations occur during the late stage of adenoma progression and may confer a more advanced morphological phenotype of adenoma, but these mutations are not mainly involved in malignant transformation from adenoma to carcinoma.

DCC (Deleted in Colorectal Carcinoma) is a candidate tumor suppressor gene located on the long arm of chromosome 18. DCC was initially identified and cloned during a search for the target gene located in a region of 18q that demonstrated loss of heterozygosity (LOH) in 70 to 80% of colorectal cancers. More recently, the region of 18q harboring the DCC gene has been shown to undergo LOH in approximately 14 to 30% of endometrial carcinomas. These findings suggest that DCC may be a target of LOH in at least some endometrial carcinomas and, therefore, may have a role in the pathogenesis of this common malignancy of the female genital tract. To address this possibility, we analyzed 26 cases of endometrioid endometrial carcinoma for DCC LOH and alterations in an AT microsatellite repeat located in an intron of the DCC gene. LOH was detected in one case (4%). Allelic shifts at the DCC AT repeat were detected in five (19%) additional cases. We also evaluated DCC protein expression by immunohistochemical analysis in normal, hyperplastic, and neoplastic endometrial tissues. Three proliferative and five secretory endometria and one simple endometrial hyperplasia demonstrated staining for DCC. Four of the 26 endometrioid endometrial carcinomas for which frozen tissue was available, including at least one from each histologic grade, and a case of endometrioid carcinoma confined to the endometrium completely lacked detectable staining for DCC. Although DCC LOH was infrequent in endometrial carcinomas, alterations of the gene (LOH or AT repeat alterations) were not uncommon (23% of our cases). In addition, DCC was expressed in normal endometrial tissue, whereas expression was lost in all of the five endometrial carcinomas. The combination of the genetic alterations and loss of DCC protein expression suggests that inactivation of the DCC gene may play a role in the pathogenesis of endometrial carcinoma.

Two thrombosis models in rats are described in which mixed type thrombi are formed at arterial and venous flow rates. The models, containing a silk thread in the aorta and vena cava, respectively, were characterised for the activity of three platelet inhibitors, three thrombin active site inhibitors and five glycosaminoglycans (GAGs). In the two models a similar highly platelet-dependent thrombus developed both in size and composition during the first 10 min after insertion of the silk thread. The thrombotic processes were self-limiting, thus maintaining blood flow, but persisted twice as long in the vena cava model. In both models the thrombus consisted for more than 65% of platelets. Thrombus development under arterial as well as under venous flow conditions was inhibited dose dependently by all tested compounds including aspirin and the synthetic alpha-methyl glycoside copy of the ATIII binding pentasaccharide within heparin, Org31540/SR90107A. Simultaneous fibrin deposition and platelet activation, which represents an essential element of arterial thrombosis, initially dominated-in both models. The gradual thrombus outgrowth, in the cava model, was more sensitive to factor Xa selective anti-coagulants, as is venous thrombosis.

Hereditary haemochromatosis (HFE) is a recessive genetic disease of iron overload which has been shown by linkage analysis to reside on the short arm of chromosome 6, close to the major histocompatibility complex (MHC). Positional cloning of the putative HFE locus has been hampered, in part, by the lack of a structural alteration on 6p. In this report, we describe a pedigree with HFE which carries a balanced paracentric inversion of chromosome 6, inv(6)(p21.1p23), a rarely reported chromosomal rearrangement in this region. We have determined the inheritance of the chromosome harbouring the inversion, which segregates as an HFE chromosome. Because the HFE locus has been mapped distal to the HLA-F class I locus at 6p21.3, the breakpoints associated with this chromosomal rearrangement may provide a significant genomic landmark for positional cloning of the HFE gene.

Hereditary haemochromatosis (HFE) is a common inherited disorder, affecting approximately five per thousand white people of northern European descent. Genetic linkage and linkage disequilibrium studies indicate that the disease locus is tightly linked to HLA-A and D6S105. Recombination between HFE and HLA class I loci is known to be rare. We report here two pedigrees in which recombinations telomeric of HLA-A occurred. These recombinant events define new centromeric and telomeric borders for the HFE locus.

A candidate gene for hemochromatosis has recently been localized on the short arm of chromosome 6, about 4 megabases telomeric to the major histocompatibility complex. It encodes a protein that exhibits significant similarity to the HLA class I molecules and can be provisionally designated HLA-hc. Genotype analysis of 94 hemochromatosis patients living in France and a similar number of controls confirms that the disease is strongly associated with homozygosity at nucleotide 845 (72% of the patients and none of the controls carry two copies of the 845A variant). The data are consistent with hemochromatosis being a heterogeneous disease: about 79% of the cases in this sample would be caused by a defect in HLA-hc and 21% by an unrelated mechanism. A second variant (187 G) enriched on patient chromosomes that do not carry the 845A mutation might influence the affinity of a ligand for HLA-hc; the exact nature of this ligand remains to be discovered. The 845A variant is the best genetic marker for the disease identified to date, and the detection of 845A homozygosity should now permit diagnosis of a readily curable disease and the prevention of sometimes deadly complications in at least 72% of the patients.

Oestrogen receptor (ER) and progesterone receptor (PR) contents were determined by biochemical (dextran charcoal-coated (DCC) assay) and immunohistochemical (ICA) methods in biopsies from 145 primary endometrial adenocarcinomas. Correlations between receptor contents and cancer specific survival were examined in a multivariate analysis including histopathological characteristics. Median patient follow-up time was 67 months with 18 cancer deaths. "High" PR levels correlated significantly (p = 0.004) with survival, independently of stage risk group (stages Ia-Ib vs Ic-IV). Patient age and histological grade were prognostic factors in a univariate setting but these parameters were eliminated in the multivariate model. The association between PR contents and cancer survival suggests that determination of PR can be of importance in the evaluation of endometrial cancer.

The three known mechanisms of cellular transformation and oncogenesis include mutations in proto-oncogenes, inactivation of both copies of a tumor suppressor gene, and defects in DNA mismatch repair genes. Examples of each are included to substantiate the importance of understanding these mechanisms. RET is a proto-oncogene that is fundamental to the pathogenesis, and in the current era, molecular diagnosis of MEN 2 syndromes. TP53 is a tumor suppressor gene that is mutated in individuals with Li-Fraumeni syndrome. CDKN2 is a tumor suppressor gene that is mutated in pancreatic cancers and is associated with a poorer prognosis and the development of melanoma. MSH2 is a mismatch repair gene that is important in the pathogenesis of HNPCC and Muir-Torre syndrome. Altered gene function such as loss of DCC in colon cancers may affect cell adhesion properties and promote metastases. As we begin to better define and understand the mechanisms of neoplasia, we will be able to improve current diagnosis and treatment.

<h4>Objectives</h4>To study thrombin and plasmin activation during elective abdominal aortic aneurysm surgery.<h4>Design</h4>Prospective study.<h4>Setting</h4>University Hospital.<h4>Materials</h4>Nine consecutive patients undergoing elective surgery were included. The mean age was 72 years (range 60-79). Blood samples were drawn: (1) before induction of anaesthesia; (2) after induction and Swan Ganz catherisation; (3) just before cross-clamping; (4) before declamping; (5) 8 h postoperatively; (6) 18 h postoperatively.<h4>Chief outcome measures</h4>Assays included: prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen, prothrombin fragments (F 1 + 2), anti-thrombin III (ATIII), plasminogen, alpha 2-antiplasmin, haematocrit, platelet and serum protein for correction of haemodilution. Data were expressed as mean (S.D.). Differences between mean values were tested by means of the ANOVA for repeated measures and the Wilcoxon signed rank test.<h4>Main results</h4>The APTT and TT did not change until heparinisation. The F 1 + 2 were already elevated preoperatively. After correction for haemodilution the AT III and alpha 2-antiplasmin decreased in time (p = 0.009 and 0.0023, respectively) and the F1 + 2 increased (p < 0.0001). Postoperatively (t5 and 6) the values normalised again.<h4>Conclusions</h4>The coagulation and fibrinolytic systems are activated during and after elective aortic replacement. Standard tests, like the prothrombin and partial thromboplastin time, are unreliable when assessing the coagulation status of the patient.

Telomerase is a ribonucleoprotein capable of replacing telomeric DNA sequences that are lost at each cell division. Under normal circumstances, it is active in rapidly dividing embryonic cells and in stem cell populations but not in terminally differentiated somatic cells. Much attention has recently focused on the hypothesis that activity of this enzyme is necessary for cells to become immortal. This predicts that telomerase activity should be detectable in malignant cells and tissues but not in their normal counterparts, which slowly senesce and die. In accordance with this notion, telomerase activity has been reported in a wide range of malignancies, including those of the gastrointestinal tract, breast and lung. In the present study, we used a polymerase chain reaction (PCR)-based assay for telomerase activity, designated the "telomeric repeat amplification protocol (TRAP)', to examine initially 35 colonic carcinomas, their corresponding normal tissues and 12 inflammatory bowel disease (IBD) lesions. We detected strong enzyme activity in 32 (92%) of the 35 colon carcinomas while there was no activity in 30 (86%) of 35 matched normal colonic tissue specimens and only very weak activity in the remainder. Four of seven specimens of ulcerative colitis and two of five Crohn's disease lesions were negative, and the rest were only weakly positive. These results led us to examine whether telomerase could be detected in carcinoma cells exfoliated into the colonic lumen. We assayed lysates of exfoliated cells in luminal washings from colectomy specimens of 15 patients with colon carcinoma and nine with IBD. Telomerase activity was detected in washings from 9 (60%) of the 15 colon carcinoma cases but not in any from cases with IBD, suggesting that it can be a good marker for the detection of colon carcinoma, possibly even in non-invasively obtained samples.

We have recently reported that about 30-40% of female breast tumours produce prostate-specific antigen (PSA) and that PSA production is associated with the presence of oestrogen (ER) and progesterone (PR) receptors. We have now developed a tissue culture system to study the regulation of the PSA gene in breast cancer. The breast carcinoma cell line T-47D produces PSA when stimulated by androgens, progestins and glucocorticoids/mineralocorticoids but not oestrogens. PSA mRNA appears approximately 2 h after stimulation; PSA protein appears after 4-8 h. Among 38 compounds tested, only androgens and progestins were able to stimulate PSA production at concentrations below 10(-9) M. Evidence that the progesterone and androgen receptors can regulate the PSA gene independently was provided as follows: (a) the progestin norgestimate, which does not bind to the androgen receptor, up-regulates the PSA gene at concentrations as low as 10(-10) M; (b) triamicinolone acetonide, which does not bind to the androgen receptor (AR) but binds to the PR, acts similarly to norgestimate; (c) the antiandrogen cyproterone acetate, which blocks the androgen receptor but has progestational activity, up-regulates the PSA gene at concentrations as low as 10(-10) M; (d) the antiprogestine mifepristone completely blocks the stimulation of the specific progestin norgestimate. Our tissue culture system identified androgen-progestin agonist activities of 17 alpha-ethinyloestradiol, the antioestrogen RU56, 187 and the antiprogestin mifepristone. Our data suggest that the expression of the PSA gene in the female breast is under the control of androgens and progestins. Our tissue culture system is a highly sensitive in vitro method for evaluating the biological activity of candidate compounds having agonist and antagonist steroid hormone activity.

Today we known that the coagulation system of the neonate differs in many ways from that of the adult system. We see a general reduction of the coagulation factors II-XIII (except VIII), the fibrinogen and of the coagulation inhibitors ATIII, protein C and heparin cofactor II at the same time. In addition the newborns show different levels of the coagulation factors for premature and full-term infants. This situation necessitates the generation of not one, but several, reference ranges for the various components of the hemostatic system. While the above-mentioned factors of the coagulation and fibrinolytic system of the newborn are well studied, our knowledge of others is still lagging behind that of the adult. That applies to the vWF and PAI and even more to D-Dimer. Our aim was to collect some data for vWF, PAI and D-Dimer to complete the understanding of the physiology of the hemostatic system in the newborn. Therefore we used the blood samples from 59 newborns and their mothers to obtain a number of different components of the coagulation and fibrinolytic system. Besides some rheological parameters (erythrocyte aggregation, plasma viscosity) we measured the aPTT, PT and the plasma levels of ATIII, fibrinogen and protein C (PC). But we paid special attention to the plasma levels of von Willebrand factor (vWF), plasma activator inhibitor (PAI) and D-Dimer. The blood samples were collected from the umbilical cord and the cubital vein of the mother immediately after delivery, anti-coagulated, centrifuged and stored until measurement at -70 degrees C. ATIII, aPTT, PT and fibrinogen were measured by the clotting technique test on the Chromotimer (Behring, Marburg, Germany). D-Dimer, vWF and protein C were determined with a commercially available ELISA obtained from Boehringer Mannheim (Mannheim, Germany). PAI activity was measured by a two-stage amiolytic method (Behring, Marburg). Besides the known differences of the neonatal hemostatic components to their mothers, such as a prolonged aPTT (52.19 s +/- 13.4 newborn, 35.26 s +/- 4.2 mother), reduced PT (64.34 s +/- 20.15 vs. 91.03 s +/- 1.25), ATIII (83.36% +/- 24.42 vs. 92.58% +/- 11.43) and fibrinogen (156.05 mg/dl +/- 91.68 vs. 344 mg/dl +/- 55.25), we found some peculiarities. The newborns show a clear reduction in the vWF (97.79 ng/ml +/- 36.33 vs. 183.43 ng/ml +/- 16.03) as well as the PAI (0.846 U/ml +/- 1.44 vs. 7.652 U/ml +/- 0.764). D-Dimer levels in the umbilical cord samples (1514.02 ng/ml +/- 953.56) are clearly prolonged in contrast to the maternal levels (740.2 ng/ml +/- 139.68). We were also able to find a difference of these factors related to gestational age. Premature infants showed a clearly higher level of PAI (2.2 U/ml +/- 2.86 vs. 0.69 +/- 1.13; p = 0.0136) compared with full-term infants, as well as significantly lower levels of ATIII (48.8% +/- 23.19 vs. 86.62 +/- 22.04; p = 0.0006) and protein C (25.33% +/- 9.37 vs. 35.73 +/- 7.53; p = 0.0027). D-Dimer, vWF, fibrinogen and the rheological parameters are similar. This seems to show an increased tendency for bleeding with the premature infant. Newborns show a balance of the coagulation system solely on a lower level. This is due to the general reduction of the coagulation factors with a reduction of the coagulation inhibitors at the same time. The physiologically low level of the vitamin K dependent and other coagulation factors and ATIII leads to a prolonged aPTT and reduced PT of the newborn. As well as these differences, known from literature, we found the following specialities: lower vWF and PAI and higher D-Dimer levels of the newborn compared with their mothers.

A method is described to sulfate PSP toxins at various positions in the molecule and to prepare 35S labelled compounds using H2(35)SO4 in the presence of dicyclohexylcarbodiimide (DCC). The 11-sulfates of saxitoxin and neosaxitoxin, known as gonyautoxins, are often the most abundant of the PSP toxins in algae and contaminated shellfish. Receptor site binding and antibody assays based on these analogues should, therefore, better reflect toxicity than those in which saxitoxin is used. Although the specific activity of 35S-gonyautoxins is lower than that of commercially available 3H-saxitoxin, the label is strongly bound and is not lost through proton exchange with water as occurs with tritiated saxitoxin. The labelling procedure is rapid, inexpensive and can be done on a small scale. Sulfate can be removed from the 11-position of GTX's in methanolic-HCl and from the 21-position by mild acid hydrolysis and H2(35)SO4 added in 5-10-fold excess. Addition or exchange occurs rapidly on mixing DCC in dimethylformamide with dry toxin and sulfate. Reaction conditions were optimized and reaction products identified by capillary electrophoresis, autoradiography and ionspray mass spectrometry. Together with methods for selective removal of sulfate, the sulfation reaction provides an additional way to prepare some of the naturally occurring derivatives of saxitoxin, many of which are sulfates.

The high-affinity folate-binding protein (FBP) is primarily involved in the uptake of the 5-methyltetrahydrofolate, and its expression may be physiologically regulated by the intracellular folate content. The overexpression of FBP on the cell surface of ovarian carcinoma cells may be responsible for an increased folate uptake. We tested the hypothesis of the existence of a defect in the 5, 10-methylenetetrahydrofolate reductase (MTHFR) in ovarian tumours that could cause reduced intracellular regeneration of the 5-methyltetrahydrofolate and induce increased FBP expression. No sequence mutations were found in the MTHFR gene, but allelic deletions of this gene were frequently detected in ovarian tumours (59%). Chromosomal losses appeared to be confined to the 1p36.3 region to which the MTHFR gene maps. Although it cannot be stated that MTHFR is the target gene of the chromosomal loss involving the 1p36.3 region, a correlation between loss of heterozygosity at this locus and decrease in MTHFR activity was shown, suggesting a role of these allelic deletions in generating a biochemical defect in folate metabolism. Further studies are needed to assess further the relationship between MTHFR and FBP overexpression, but the demonstration of the alteration of a key metabolic enzyme of the folate cycle in a subset of human ovarian tumours is in accordance with the hypothesis of an altered folate metabolism in these neoplasias and might be exploited for therapeutic purposes.

<h4>Background and objective</h4>Hereditary hemochromatosis (HC) is an inborn error of iron metabolism leading to increased intestinal iron absorption and progressive iron overload. There have been definite advances in our knowledge of the pathogenesis and management of idiopathic hemochromatosis in recent years, which prompted us to review this subject.<h4>Information sources</h4>The material examined in the present review includes articles and abstracts published in the journals covered by the Science Citation Index and Medline. In addition, both authors have been working in this field for several years and have contributed twelve of the papers cited in the references.<h4>State of art and perspectives</h4>The disease is a late onset autosomic recessive condition, especially frequent in Caucasians. If unrecognized, severe clinical symptoms develop in mid-life related to organ failure. Early diagnosis prevents complications, since an intensive phlebotomy course removes excess iron and offers patients a normal life expectancy. Transferrin saturation is the first examination step, but liver biopsy is still essential for diagnosis and prognosis of HC. The biochemical defect is unknown. Positional cloning of the HC gene has led to the isolation of all the candidate region on the short arm of chromosome 6, telomeric to HLA-A. Recently a putative HC gene has been cloned from this region and found to be mutated in a large proportion of patients. The gene, known as HLA-H, is an atypical MHC class I gene. Although its biological function remains unknown, HLA-H is the first strong HC candidate gene. Molecular screening of patients and carriers is now possible in a significant portion of cases, thereby permitting better control of the disease. If it is unequivocally confirmed that the HLA-H gene is responsible for the disease, understanding of its biological function will provide information on the type and activity of the involved protein, revealing new insights into iron uptake and metabolism in humans.

1. Hydrocortisone (HC; 80 mg/kg body weight, intraperitoneally for 4 days), both alone and in combination with acetylsalicylic acid (ASA; 160 mg/kg body weight, orally, for 4 days), decreased ASA general and specific toxicity via metabolic modulation of drug-metabolizing enzyme systems (intestinal ASA-esterase and hepatic UDP-glucuronyltransferase) and did not change the ASA analgesic effect. 2. ASA alone, given 4 for days, did not change the specific ASA toxicity, but increased its general acute toxicity, which was probably due to alterations in some intestinal and hepatic metabolizing enzyme systems.

Dystrophic cardiac calcification (DCC) is a post-mortem finding in mice of various strains frequently used in biomedical research. The major aim of this study was to see whether DCC severity can be assessed by chemical analysis of calcium in the heart. Histological examination was used as the method of reference. Hearts of mice of four strains (BALB/c, C3H, C57BL/6 and DBA/2) were halved and the two resulting parts were subsequently subjected to histology or chemical analysis. Within hearts, the halves generally yielded similar results. The DCC scores and calcium contents were directly correlated within hearts. Thus, calcium analysis could serve as an alternative to histological examination in the assessment of DCC severity in mice. DBA/2 and C3H mice were found to be affected by DCC. Plasma magnesium concentrations were lower in these strains than in the DCC-free C57BL/6 and BALB/c strains. The tongue, lungs and diaphragm were also found to be calcified in DCC positive animals. Possibly, DCC is just one component of a generalized soft tissue calcification.

CDKN2A, the gene encoding the cell-cycle inhibitor p16CDKN2A, was first identified in 1994. Since then, somatic mutations have been observed in many cancers and germline alterations have been found in kindreds with familial atypical multiple mole/melanoma (FAMMM), also known as atypical mole syndrome. In this review we tabulate the known mutations in this gene and discuss specific aspects, particularly with respect to germline mutations and cancer predisposition.

The netrins are laminin-like axon guidance molecules that are conserved among Caenorhabditis elegans, Drosophila, and vertebrates and that have chemoattractive and chemorepellant properties. To study the possible actions of this gene family in the developing and adult mammalian nervous systems, we have cloned a partial cDNA which corresponds to a region conserved among chick netrin-1, netrin-2, and unc-6 and studied its expression and that of a netrin receptor, dcc, the deleted in colorectal cancer gene, in the developing and adult rat CNS. The localization of cells expressing netrin or dcc suggests that these genes, in addition to their actions in defining the ventral midline, may act in controlling retinal ganglion cell axon guidance in the optic nerve, cell migration in the developing cerebellum and olfactory epithelium, and development and maintenance of connections to the substantia nigra and corpus striatum.

The 'deleted in colon carcinoma' (DCC) gene has been considered a candidate tumour-suppressor gene that encodes for a transmembrane protein with strong structural similarity to members of the superfamily of neural cell adhesion molecules. It has been mapped to the chromosomal region 18q21.1 and it is implicated in cellular differentiation and developmental processes. In human osteosarcoma allelic loss frequently occurs on the long arm of chromosome 18, suggesting a possible involvement of the DCC gene in the pathogenesis of this tumour entity. In the present study the mRNA and protein expression and rearrangements at the DNA level of the DCC gene were addressed in 25 osteosarcomas and several tumour cell lines, including osteosarcoma- and colon carcinoma-derived cell lines. Using an reverse transcriptase polymerase chain reach in (RT-PCR)-based approach DCC expression was found to be lost or substantially reduced in 14 of 19 high-grade osteosarcomas, in three of six lower grade osteosarcomas and most of the tumour cell lines, in contrast to normally differentiated osteoblasts. Immunohistochemical studies on DCC protein expression of 14 selected tumours correlated well with the RT-PCR-based results. In view of the putative tumour-suppressor characteristics of the DCC gene its loss or reduction of expression could be a specific event in the development or progression of many high-grade osteosarcomas.

<h4>Background</h4>Colitis associated dysplasia and cancer often have deletions involving the long arm of chromosome 18q, suggesting the location of a tumour suppressor gene critical for their tumorigenesis. The DPC4 gene, which is genetically inactivated in pancreatic and other cancers, has recently been described.<h4>Aim</h4>Because DPC4 is located at 18q21.1, the hypothesis that it could be a mutated tumour suppressor gene in colitis associated neoplasms was tested.<h4>Patients</h4>Advanced neoplastic lesions from six patients having chronic colitis were analysed for DPC4.<h4>Methods</h4>Individual exons of DPC4 were amplified by the polymerase chain reaction (PCR) and sequenced from genomic DNA of tissue specimens dissected by cryostat.<h4>Results</h4>DPC4 was found to have biallelic inactivation in one of three neoplasms shown to have allelic loss of 18q. The mutation had been acquired somatically in a plaque of high grade dysplasia. The mutation created a non-sense codon, which would cause premature termination of protein translation.<h4>Conclusion</h4>The DPC4 gene is a target of 18q LOH events in colitis associated neoplasia.

MIB1 proliferation rate (MIB1-PR) and total S-phase fraction (SPF) were retrospectively determined in formalin-fixed, paraffin-embedded sections of 90 primary node-negative breast carcinomas. None of the patients had received adjuvant systemic therapy. Median follow-up in patients still alive at the time of analysis was 37.5 months (16-72 months). Immunostaining of Ki-67 antigen was performed using the monoclonal antibody MIB1 and the APAAP technique. An adjacent 50-microm paraffin section was used for flow cytometric S-phase determination. Results were compared to established clinicopathological prognostic factors. MIB1-PR was significantly correlated to grading (P = 0.018); SPF was significantly correlated with tumour size (P = 0.041) and inversely with steroid hormone receptor status (P = 0.03). A significant correlation between MIB1-PR and SPF was found in aneuploid (P = 0.025) but not in diploid tumours (P = 0.164). In univariate analysis, both MIB1-PR (optimized cut-off of 25%) and SPF (optimized cut-off of 8%) were significant prognostic factors for disease-free survival (DFS) (MIB1-PR, P = 0.0224; SPF, P = 0.0028). In multivariate analysis, however, only SPF remained significant; it was the strongest prognostic factor for DFS (P = 0.0073), stronger than MIB1-PR or established clinicopathological prognostic factors. We thus conclude that MIB1-PR and SPF provide additional prognostic information in node-negative breast cancer. However, in our study, flow cytometrically determined SPF had the greater prognostic impact.

A Celtic origin for hemochromatosis, a common genetic iron metabolism disorder, has been postulated for a long time. To check whether the two mutations recently identified in the HLA-class I candidate gene for this disease were found only in Caucasians, we examined their frequencies in individuals originating from Algeria, Ethiopia, and Senegal. The presumably disease-causing mutation, responsible for the Cys282Tyr substitution, was not found in any member of these ethnic groups, although it was shown to be highly prevalent in populations of European ancestry. This geographic distribution supports the previously suggested Celtic origin for the disease. In contrast, the mutation responsible for the His63Asp substitution is not restricted to European populations. Although absent in the Senegalese, it was found on about 9% of the chromosomes of the Central Ethiopians and Algerians (Mzab) genotyped for this study. This second mutation, which probably represents a common variant unrelated to hemochromatosis, thus appears to have occurred earlier than that responsible for the Cys282Tyr substitution. More detailed population studies are needed to provide information on the age of these two mutations and eventually show how the hemochromatosis-causing mutation chronologically spread throughout Europe.

Hereditary hemochromatosis is a common disorder in people of European origin. The HLA-H gene has been found to have two mutations that apparently cause hemochromatosis. The principal mutation, 845G-->A (C282Y), is believed to have arisen relatively recently in the Celtic population. To determine the incidence of this mutation and the other hemochromatosis-associated mutation, 187C-->G (H63D), among Ashkenazi Jews, a people who are believed to have arrived in Europe in about the 8th Century A.D., we have examined the DNA from 381 unrelated Jewish subjects and 206 non-Jewish white controls. The gene frequency for the 845G-->A mutation among Jewish subjects was only 0.013 compared with a frequency of 0.070 among controls, a difference that is significant at the 0.00001 level. The phenotypically milder nt 187C-->G mutation had a frequency of 0.155 in the non-Jewish population and 0.097 in the Jewish population, a difference that was also statistically significant at the <0.01 level.

We evaluated Alabama hemochromatosis probands (n = 74) and normal control subjects (n = 142) for expression of the hemochromatosis-associated mutations nt 845G-->A (845A; Cys282Tyr) and nt 187C-->G (His63Asp) in a gene linked to the major histocompatibility complex (MHC). We also tabulated parameters of iron metabolism and iron overload in probands and in obligate heterozygote family members of homozygous Cys282Tyr probands. Among probands, 59.4% were Cys282Tyr homozygotes and 20.3% were heterozygotes; 20.3% did not express this mutation. In normal control subjects, 14.7% were heterozygous for the Cys282Tyr mutation; one normal control subject was homozygous for the Cys282Tyr mutation. None (0 of 44) of our Cys282Tyr-homozygous hemochromatosis probands had the His63Asp mutation. Of the Cys282Tyr-heterozygous and -negative probands, the His63Asp mutation occurred in 26.7% (4/15) and 53.3% (8/15), respectively. In normal control subjects, 23.2% were heterozygous for the His63Asp mutation; 2.8% were homozygous. Induction phlebotomy requirements and other manifestations of iron overload were significantly greater in Cys282Tyr homozygotes than among other probands. Cys282Tyr-heterozygous probands had significantly higher values of serum iron parameters than did obligate Cys282Tyr heterozygotes whose values were, on the average, normal. Co-expression of HLA-A3, HLA-B7, and D6S105(8) was significantly more frequent in all subgroups of probands stratified by Cys282Tyr expression than in normal control subjects. These results demonstrate that the severity of iron overload in hemochromatosis is affected significantly by genetic factors. Further, our findings support the hypothesis that one or more MHC-linked genes other than that corresponding to the Cys282Tyr and His63Asp mutations contributes to increased iron absorption and iron overload in hemochromatosis probands.

Cellular protooncogenes, tumor suppressor genes (antioncogenes), and DNA mismatch repair mutators are generally the key molecular genetic biomarkers undergoing alterations during carcinogenesis, i.e., activation of oncogenes, inactivation of tumor suppressors, and DNA mismatch repair gene defects are essential events in cancer causation. In pancreas cancer, high incidence of oncogene K-ras point mutations at the codon 12th is associated with premalignant and malignant transformation. Mutation in p53 tumor suppressor is also detected in pancreas adenocarcinoma. Concurrent loss of p53 and K-ras function may contribute to the clinical aggressiveness of pancreas cancer. Microsatellite instability and DNA mismatch repair defects may represent new mutator phenotype for pancreas carcinogenesis. Mutation of cell cycle regulators, such as inhibitor of CDK4 or p16 tumor suppressor gene, is a new molecular event in pancreas cancer. Mutation of cyclin-dependent kinases also may be involved in pancreas carcinogenesis. Loss or mutation of a new candidate tumor suppressor, DPC4 (deleted in pancreas carcinoma locus 4), is reported in pancreas cancer. The protein products of these gene mutations are potential tumor antigens, thus genotype expression can be detected by phenotype. Most of these emerging molecular genetic biomarkers are associated with regulation of cell growth and recognition, as well as gene expression, and may offer new insight into the cellular precursors to and genesis of pancreas cancer.

A total of 169 colorectal adenocarcinomas, obtained from patients with a median follow-up of 6.5 years, were studied with immunohistochemical staining on cryosections using a monoclonal anti-tenascin antibody to evaluate the possible association between the staining patterns and tumour stage, tumour differentiation and survival. We found two different staining patterns in the tumour stroma--a diffuse stromal fibrillar staining in 92 out of 169 (54%) tumours and a subglandular staining in the remaining 77 tumours. When the entire group of patients (P < 0.01) and the group of potentially cured patients (P < 0.03) were analysed univariately, it was found that diffuse stromal fibrillar staining was associated with a shorter survival time than subglandular staining. In a multivariate analysis, the Dukes' stage and age were independent prognostic factors, whereas the tenascin expression did not retain a clear independent relationship to survival (P = 0.06). Hence, it appears that the tumour expression of tenascin may be a potential prognostic marker in colorectal cancer, in so far as a diffuse stromal fibrillar staining pattern seems to indicate an increased risk of poor outcome. However, after adjustment for age and Dukes' stage, the additional prognostic value of tenascin remains to be established in further analyses.

The distribution of lymphocyte subsets in blood, thymus, spleen, mesenteric lymph nodes, Peyer's patches and bone marrow was evaluated in an experimental model of secondary hemochromatosis in rats. The values of CD2, CD4, CD8, B and NK cells in these different lymphoid compartments did not differ between the control group and the experimental group. These results suggest that the abnormalities of lymphocyte subsets previously reported in patients with secondary hemochromatosis may be due to factors other than iron overload.

Tumor behavior is the result of specific genetic changes that alter gene expression. From our cytogenetic studies chromosome 18 loss emerged as a common genetic change in squamous carcinoma cell lines. In this report we summarize data that link loss of 18 to tumor progression and reduced survival, indicating that one or more tumor suppressor gene(s) are located on this chromosome. Tumors grown in vitro were karyotyped either as short-term or permanent cultures. Loss of chromosome 18 was measured by karyotype, decreased frequency of heterozygosity at the DCC locus, and loss of heterozygosity (LOH) for microsatellite repeat polymorphisms (MSRP). Loss of any part of chromosome 18 was observed in approximately 63% of cultured tumors. Primary and secondary tumors from the same individuals sometimes differed in loss of 18 indicating that this genetic change is associated with tumor progression. Heterozygosity for DCC was present in only 3/19 cultured SCC (16%), compared with 68% (11/16) of blood samples from unrelated donors, which is consistent with LOH in roughly one half of the cases. Of 4 informative cases with normal and tumor tissue, LOH was observed in 2. Microsatellite analysis also shows loss of 18q in 55% of fresh tumors. Analysis of tumor tissue and cell lines from the same patient gave identical results. There was an excess of deaths from cancer in the group with 18 loss (20/25) when compared with the group without (5/15). Loss of chromosome 18 appears to be a marker of tumor progression in SCC. It is likely that mutation affecting DCC or another gene on 18 affects tumor growth or spread, leading to more rapid progression and reduced survival.

<h4>Objectives</h4>The aim of this study was to determine the epidemiological characteristics of hepatocellular carcinoma in a non-selected population.<h4>Methods</h4>Between 1984 and 1990, all cases of hepatocellular carcinoma were registered at the Registry of Digestive Tumors of Calvados. Standardized incidence rates were calculated for males and females. Prognostic factors were determined by the Cox multivariate method.<h4>Results</h4>213 patients with hepatocellular carcinoma were registered. Standardized incidence rates were 7.5/100,000 in men and 0.4/100,000 in women. Sex-ratio was 18.3. Mean age was 66.4 years; hepatocellular carcinoma was uncommon (3%) before the age of 50. Cirrhosis was associated in 85.9% of patients. The cause of cirrhosis was known in 150 cases: alcoholic: 73.3%, cryptogenetic: 8.7%, viral B or C: 7.3%, alcoholic and viral B or C: 5.3%, and genetic hemochromatosis: 4.7%. The overall survival rate in 203 patients was 21%, 8% and 3% at 1 year, 3 years and 5 years, respectively. The multivariate study identified 4 prognostic factors: number of tumors < or = 2, lack of ascites, serum alpha-fetoprotein < or = 10 mg/mL, and hepatocellular carcinoma revealing a well-compensated liver disease until the time of diagnosis.<h4>Conclusion</h4>The occurrence of hepatocellular carcinoma seems to be linked to cirrhosis, male sex, and age > 50, which could be used as the main selection criterias for the screening of hepatocellular carcinoma.

A human colon cancer cell line, OCUC-LM1(LM), was established from a liver metastasis in our laboratory. Intrasplenic injection of LM into nude mice was repeated three and five times, and the daughter cell lines were designated as LM-H3 and LM-H5 respectively. The level of sialyl Lewis A (SLA) in the supernatant of LM-H3 and LM-H5 was 3 and 4.5 times higher than that of LM respectively. Flow cytometric analysis of SLA expression showed that the peak channel for LM was 113; for LM-H3, 126; and for LM-H5, 146. The mean fluorescence intensity of LM was 102.3 +/- 43.5; for LM-H3, 126.2 +/- 28.4; and for LM-H5, 144.8 +/- 23.4. In endothelial cell adhesion assays, the percentages of adherent LM-H3 and LM-H5 cells were significantly higher than for LM. The activity of alpha1-->4 fucosyltransferase was higher in LM-H3 and LM-H5 than in LM, but there was no difference in alpha2-->3 sialyltransferase activities for type 1 chain among the cell lines. Our results suggest that SLA expression is associated with acquisition of a high capacity for liver metastasis of colon cancer; increased SLA expression is due mainly to increased fucosyltransferase activity.

Sclerotherapy combined with ligation has become a widely accepted treatment for varicose veins; however, it is associated with some risk of the serious complications of deep vein thrombosis (DVT). We investigated the incidence of thrombophilia in 164 consecutive patients undergoing treatment for varicose veins and determined the activities of antithrombin-III, protein C, and plasminogen. Of the 164 patients, 10 were diagnosed as having dysplasminogenemia (DPG), showing an incidence of 6.1%, in accordance with previous reports. DVT was not found to be caused by DPG in any patient, and no difference was found between patients with and those without DPG, suggesting that DPG is not a risk factor for varicose veins. We also investigated the activation of coagulation by measuring the thrombin-antithrombin III complex (TAT). The activation of coagulation after sclerotherapy was inhibited when ligation was performed 1 month prior to sclerotherapy, whereas it was increased when sclerotherapy and ligation were performed simultaneously. Of the 10 patients with DPG, 5 were treated uneventfully, and their TAT level increased to 4.0 micrograms/l, which was comparable to the level after sclerotherapy and ligation. These findings indicate that sclerotherapy can be performed safely in the majority of patients with DPG, and that the temporal separation of sclerotherapy and surgery is an alternative for these patients to prevent the activation of coagulation.

Nasopharyngeal carcinoma is a subset of head and neck squamous cell cancers with unique endemic distribution and aetiological co-factors. Epstein-Barr virus has been revealed to be an important aetiological factor for most nasopharyngeal carcinomas. Nevertheless, additional genetic alterations may be involved in their development and progression. The aim of this study was to determine the likely chromosomal locations of tumour-suppressor genes related to Epstein-Barr virus-associated nasopharyngeal carcinoma. Fifty-six microsatellite polymorphic markers located on every autosomal arm were used to estimate the incidence of loss of heterozygosity in 27 Epstein-Barr virus-associated nasopharyngeal carcinomas. High frequencies of allelic loss were observed on chromosome 3p (75.0%) and 9p (87.0%). Chromosome 9q, 11q, 13q and 14q displayed loss in over 50%, while chromosome 3q, 6p, 16q, 19q and 22q exhibited loss in 35-50%. Furthermore, several other chromosomal arms demonstrated allelic loss in 20-35%. Additionally, 1 of the 27 cases showed microsatellite instability at multiple loci. These findings provide evidence of multiple genetic alterations during cancer development and clues for further studies of tumour-suppressor genes in Epstein-Barr virus-associated nasopharyngeal carcinoma.

The incidence of infection by Actinobacillus actinomycetemcomitans, one of the important pathogens in human periodontal diseases, has been reported to be associated with racial background and genetic factors. We attempted to determine the genetic regulation of immune responses to A. actinomycetemcomitans fimbriae, an attachment factor, using various inbred strains of mice. For this purpose, we synthesized an oligopeptide antigen using the amino acid sequence of the fimbriae and conjugated this antigen to branched lysine polymer resin beads. After immunization with the synthetic A. actinomycetemcomitans fimbrial antigen, serum antibody levels and the delayed-type hypersensitivity (DTH) reaction to the antigen were measured by enzyme-linked immunosorbent assay (ELISA) and footpad swelling responses, respectively. The strains of mice found to be high-IgG responders to the antigen were B10.HTT, B10.RIII, B10.A (5R) and B10.S (9R). These results indicate that mice with E(beta s):E(alpha k), E(beta r):E(alpha r) and E(beta b):E(alpha k) respond strongly to the synthetic peptide. All of the high-IgG responders showed a high DTH response. A cell transfer experiment confirmed that CD4 T cells mediated with DTH response to the synthetic peptide. Thus, the results of this study demonstrate that the immune responses to A. actinomycetemcomitans fimbriae are genetically controlled.

We performed a detailed and comprehensive study of the involvement of tumor suppressor genes in human prostate cancer. We utilized primers flanking either the restriction fragment length polymorphism (RFLP) or variable number of tandem repeat [VNTR; microsatellite or simple repeat site (SRS)] polymorphic sites to polymerase chain reaction (PCR) amplify the genomic DNA and detect loss of heterozygosity of the target genes. Quantitative reverse transcription (RT)-PCR was performed to measure the mRNA expression levels and PCR/single strand conformational polymorphism (SSCP) and DNA sequencing carried out to detect mutation of the tumor suppressor genes. We found that multiple tumor suppressor genes (e.g., p53, DCC, APC, MCC, BRCA1, and WAF1/CIP1) were inactivated at different frequencies via various mechanisms [e.g., loss of heterozygosity (LOH), loss of expression (LOE), mutation, and inactivation by cellular binding protein]. Several important and novel findings are as following: LOH and LOE of the DCC gene, LOH, LOE, and possible mutation of the APC/MCC genes, LOH of the BRCA1 locus, and mutation of the WAF1/CIP1 gene. For p53 tumor suppressor gene alone, multiple inactivation mechanisms (i.e., LOH, LOE, mutation, and amplification of the cellular inactivating protein MDM2) were identified. A possible involvement of genomic instability or mutator phenotype in human prostate cancer was investigated by microsatellite typing using PCR. A high frequency of microsatellite instability was detected and the microsatellite instability found to correlate with advanced stage and poor differentiation of prostate cancer, suggesting that genes functioning in DNA mismatch repair or general stabilization of the genome may be involved in prostate cancer. The results obtained in this study suggested that multiple tumor suppressor genes (both known and unknown genes) may share the role in prostate cancer; a pattern which has been found in a number of human malignancies such as cancers of the esophagus, colon and breast. In fact, we performed deletion studies aimed at localizing potential tumor suppressor loci on various chromosomal regions. A number of chromosomal regions (i.e., 6p12-24 and 17q21) were found to potentially harbor unidentified tumor suppressor genes. Detailed deletion mapping has localized the potential tumor suppressor loci to a < 2 Mb region centromeric to the BRCA1 gene on chromosome 17q. In addition, we identified a number of novel mechanisms of tumor suppressor gene inactivation, in prostate cancer such as loss of mRNA expression of the DCC, APC, MCC and p53 gene, and mutator phenotype. And for the very first time, we identified somatic mutations of the WAF1/CIP1 gene in primary human malignancy-human prostate cancer. This finding provides the first evidence in primary tumor that the WAF1/CIP1 gene may be a tumor suppressor gene and may be involved in prostate cancer. We identified 12-lipoxygenase (12-LOX) as a potential prognostic marker for human prostate cancer. mRNA expression levels of the 12-LOX gene was measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and semi-quantitative in situ hybridization (ISH) in 122 pairs of matched normal and tumor tissues from prostate cancer patients. We found that 12-LOX expression levels were elevated in approximately half of the patients analyzed and the 12-LOX elevation correlates with advanced stage, poor differentiation, and surgical margin positivity. Our data suggest that 12-LOX may serve as a correlative marker for a more aggressive phenotype of prostate cancer and therefore for poor prognosis. We are currently refining our assays for possible clinical applicability. Since not all patients with loss of expression of the DCC gene showed LOH of the DCC locus, there must be other mechanism(s) responsible for loss of expression of the DCC gene. When we analyzed the relationship between DCC loss of expression and 12-LOX elevation in prostate cancer pati

We have used human mammary cells of the MCF-7 strain, which constitutively express high levels of the small heat shock protein HSP27 and we have compared the changes in the phosphorylation status of this protein together with changes in cell growth and/or morphology induced by the action of one of the following agents: (1) TPA (12-O-tetradecanoylphorbol-13-acetate), known as a differentiation inducer in MCF-7 cells; (2) OH-TAM (hydroxytamoxifen), which exerts a cytostatic and cytotoxic action; or (3) TNF alpha (tumour necrosis factor), which induces apoptotic cell death in this cell line. Our data show that TPA and TNF stimulate an immediate and massive phosphorylation of HSP27, whereas OH-TAM affect the phosphorylation status of the protein only after a 3 day delay. In the case of TPA, high levels of HSP27 phosphorylation were maintained for at least 4 days, along with growth inhibition and acquisition by the cells of a secretory phenotype. TPA and OH-TAM exerted similar immediated effects on cell growth, despite the different time course of their action on HSP27 phosphorylation. This excludes the possibility that the latter is a necessary consequence of, or an absolute requisite to, growth inhibition. With OH-TAM and TNF the increase in HSP27 phosphorylation was concomitant with the appearance of apoptosis, not observed with TPA. This indicates that increased phosphorylation of HSP27 is not specifically associated with the triggering or the execution of apoptosis in these cells. Altogether, our data support the concept that phosphorylated HSP27 is involved (and might then be rate limiting in some instances) in the execution of vital cell programmes (including resistance to stress, proliferation and differentiation), as well as in that of cell death. This is consistent with its role in actin polymerization and its position downstream of the p38/RK-type MAPkinase, itself a point of convergence for diverse signal transduction pathways.

Recent knowledge about biological role of tumor suppressor genes and their products: RB1, p53, WT1, DCC, APC/FAP, NF1, NF2, VHL, MCC and MTS1 is presented. The main approaches of these agents as physiological regulators of cell growth and proliferation are discussed. Views on the tumor suppressor genes involvement in the development of inherited and sporadic forms of cancer have been reviewed.

Most changes of tumor suppressor p53 and its pathway involve a protein with prolonged half-life that permits immunohistochemical detection. The goal of this study was to compare the prognostic relevance of five different p53 antibodies in primary soft-tissue sarcomas (STS) with known p53 mutation status, using a multivariate Cox regression model (adjusted to tumor grading, staging, localization, tumor type, and therapy). A group of 198 primary STS of six types were investigated for p53 overexpression, using p53 antibodies DO-1, DO-7, Pab1801, Pab240, and CM-1. A positive marker frequency between 36.2% and 62.6% was detected. Out of 65 patients whose primary tumor reacted positively to all five antibodies, 52 (80%) died within the study period. Only the N-terminal-binding monoclonal antibodies DO-1, DO-7 and Pab1801 showed a multivariate correlation with survival (P = 0.0014, 0.0048 and 0.02). CM-1 and Pab240 had a univariate, but not a multivariate correlation, with a confounding effect of grading. The prognostic relevance for the five p53 antibodies was: DO-1 > Pab1801 > DO-7 > CM-1 > Pab240. This is the first study that investigates multivariately the prognostic relevance of p53 immunostaining in STS. If monoclonal antibodies with an epitope in the N-terminal region of the p53 protein (DO-1, Pab1801, DO-7) are applied, p53 immunohistochemistry provides an independent prognostic marker in STS.

Pancreatic cancer is the fifth leading cause of cancer death in the United States, and despite improvements in the results of surgical treatment for this disease, little impact has been made upon overall mortality. New advances in treatment will depend upon improved adjuvant therapy, early diagnosis, and a better understanding of tumor biology. This article summarizes the results of molecular genetic studies in pancreatic cancer and their potential clinical significance. Familial predisposition to pancreatic cancer, cytogenic studies, DNA ploidy analysis, and examination of specific oncogenes and tumor suppressor genes are reviewed. The most frequent mutations detected have been in the K-ras oncogene, which occur in 80% of pancreatic cancers. These mutations do not correlate with tumor stage or survival, but can be useful in differentiating pancreatic exocrine from endocrine tumors and chronic pancreatitis. Mutations in the p53 gene occur in approximately 50% of tumors, and appear to be an independent prognostic factor for patient survival. Mutations in the CDKN2 gene are frequently seen in sporadic pancreatic cancers, and have been implicated in cases of familial pancreatic cancer. The significance of mutations in APC, MCC, DCC, c-erbB-2, RB-1, and mismatch repair genes in the genesis of pancreatic cancer is less clear.

The short arm of the chromosome 11, known to harbour a number of putative and established tumour-suppressor genes, is frequently hypermethylated in various human neoplasms. We subjected the promoter regions of two genes residing at 11p, namely the tumour-suppressor gene WT1 (Wilms' tumour gene) (11p13) and the calcitonin gene (11p15.5), to methylation analysis in human sporadic colorectal cancer using genomic sequencing. Both genes showed significant hypermethylation of CpG sites within their promoter regions in adenomas and carcinomas compared with normal colonic mucosa. Although the WT1 promoter region was significantly hypermethylated, two CpG sites located in Sp1 motifs were unmethylated in the majority of cases (68-74% of carcinomas). The expression of WT1 gene, as revealed by in situ hybridization, showed no differences between normal colonic mucosa and malignant carcinoma. Together with earlier observations, our present results support the view that the short arm of human chromosome 11 is subjected to widespread regional hypermethylation in various human malignancies.

Medulloblastoma is the most frequent paediatric brain tumour. Because of the uniform histology, a common genetic mechanism has been postulated. Loss of heterozygosity (LOH) studies support evidence that a candidate gene, which functions as a tumour-suppressor gene, is located in 17p13. Eighteen tumours were examined for loss of heterozygosity at 15 different loci at chromosome 17p. Nine of 18 (50%) tumours had allelic loss in 17p 13.3-13.2. The smallest region of overlap, which harbours the disease gene, includes markers from UT222 (D17S675) to UT49 (D17S731) and spans a region of less than 6 cM. Candidate genes within this region are HIC-1, a potential tumour-suppressor gene, and DPH2L, a gene that has been cloned from the ovarian critical region. The putative region excludes the p53 gene and the ABR gene, which have been favoured by others. LOH of chromosome 17p may be used as a new prognostic biological marker. Children with an allelic loss had a poorer prognosis than those patients without loss of heterozygosity (P<0.05).

Between 1984 and 1990, 94 women presenting to the Edinburgh Breast Unit with operable breast cancer of 4 cm or greater in diameter (T2, T3, N0, N1, M0) were given preoperative systemic therapy. Initially, all women received hormone therapy, with CHOP (cyclophosphamide 1 g m(-2), doxorubicin 50 mg m(-2), vincristine 1.4 mg m(-2) to a maximum of 2 mg and prednisolone 40 mg per day orally for 5 days) chemotherapy being administered to those who failed to respond by 3 months. After April 1987, first-line hormone therapy was only offered to women with oestrogen receptor (ER)-moderate/-rich (> 20 fmol mg(-1) protein) tumours, and CHOP was reserved for those women whose tumours failed to respond to hormone therapy and for those with ER-negative/-poor tumours. Response data have been published previously (Anderson et al, 1991). After a median follow-up of 7.5 years, there is no difference in survival between those women given initial hormone therapy and those given chemotherapy, with neither group having yet reached its median survival. The two key factors that predicted for a poor survival were the number of involved axillary nodes after preoperative systemic therapy (P < 0.00001) and a lack of response to preoperative therapy (P < 0.05). These data suggest that many women with ER-moderate/-rich tumours will have a good prognosis after preoperative hormone therapy alone. However, it is possible to identify, by their post-systemic therapy axillary node status, a group of women who still have an appalling prognosis after preoperative chemotherapy or hormone therapy.

Between 1977 and 1993, 384 breast cancer patients were followed up post-operatively every 4 or 6 months with a serum tumour marker panel (CEA-TPA-CA15-3) and the usual imaging techniques. Twenty-eight patients were treated 13.5 +/- 10 months (mean +/- s.d.) before the clinical and/or radiological occurrence of distant metastases that were suspected because of an increase in the tumour markers (patients treated 'early'). Their outcome was compared with that of 22 similar patients who were treated only after a definite radiological diagnosis was achieved (patients treated 'not early'). The median survivals from mastectomy and salvage treatment were also compared for the two groups. The groups were similar for all the major prognostic factors (menopause, staging, hormone dependency). In the group treated 'early', the lead time from the tumour marker increase to the clinical and radiological signs of metastases was significantly longer than that of the group not treated 'early' (13.5 +/- 10 vs 3.4 +/- 2.8 months respectively; P < 0.001 by unpaired t-test). For patients treated 'early', the survival curves up to 30 months after salvage treatment and up to 72 months after mastectomy showed greater survival than those for the patients treated later (42.9% vs 13.6% and 42.9% vs 22.7% respectively; P = 0.04 in both instances). These data suggest that treatment triggered by rising tumour markers before clinical and/or radiological appearance of distant metastases can be useful in prolonging both the asymptomatic interval and the duration of response of some relapsed patients. Randomized prospective trials must be encouraged to confirm these data and to better evaluate the effect on the disease-free survival (DFS) and overall survival (OS) of 'early' salvage treatment protocols.

Urokinase-type plasminogen activator (u-PA), which cleaves plasminogen to yield plasmin, is a serine protease of fibrinolysis and is presumed to play a key role in extracellular proteolysis and facilitate the migration of cancer cells. This study was conducted prospectively to evaluate the prognostic significance of u-PA antigen level in breast cancer tissues. u-PA concentrations in the cytosol of 226 breast cancer tissues were determined prospectively by enzyme-linked immunosorbent assay using cytosol fractions prepared for steroid hormone assay. The median follow-up period of the patients was 60 months. Various prognostic factors were evaluated by univariate analysis or multivariate analysis using the Cox proportional-hazards method. Patients with primary breast cancer containing high levels of u-PA had a significantly shorter disease-free survival than patients with low levels of u-PA antigens. In multivariate analysis, a high level of u-PA was an independent risk factor for disease-free survival, being independent of age, axillary node status, and estrogen receptor status. Among the major prognostic factors, a high u-PA antigen level, lymph node involvement, and a positive estrogen receptor status were the most important for predicting relapse-free survival (P = 0.044, P < 0.0001, P = 0.0039). This first prospective study confirmed the prognostic significance of the u-PA antigen level in association with other major prognostic factors. The results of our present study suggest that u-PA in breast cancer tissue might be involved in breast cancer invasion and metastasis.

Several lines of evidence implicate a role for the serotonergic system in body weight regulation and eating disorders. The magnitude and duration of postsynaptic responses to serotonin (5-HT) is directed by the transport into and release from the presynaptic neuron. Recently, a common polymorphism of a repetitive element in the region of the serotonin transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) was identified that results in a system of two common alleles. The activity of the 5-HTT, as measured in in vitro assays and in human lymphoblastoid cell lines, is dependent on the respective genotype. We thus hypothesized that this polymorphism is relevant for weight regulation in general and is possibly involved in the etiology of anorexia nervosa (AN). Allele frequencies and genotypes were determined in a total of 385 unrelated obese children, adolescents and adults, 112 underweight subjects and 96 patients with AN. Furthermore, both parents of 98 obese children and adolescents and of 55 patients with AN, respectively, were genotyped, thus allowing to test for both association and linkage. The comparison of allele frequencies between obese and underweight probands provided no evidence for a major role of the 5-HTTLPR in weight regulation. Patients with AN had allele frequencies not significantly different to those observed for obese and underweight individuals.

A candidate gene for Hereditary Hemochromatosis (HFE) has been recently cloned from a region 4 cM telomeric to HLA-A on the short arm of chromosome 6. This gene, defined HFE, is mutated in a high proportion of HFE patients. Positional cloning of HFE has been difficult, because of the extended region of linkage disequilibrium observed around the gene and of the rarity of recombination events in this DNA area. Here we describe a crossover in an Italian HFE patient which occurred close to the HFE gene in a restricted interval between D6S2221 and D6S2240-D6S2238 markers. The molecular analysis of this event and the segregation of the HFE mutations in the family are consistent with the position of the HFE gene telomeric to D6S2221.

The purpose of this study was to determine estrogen receptor (ER) and progesterone receptor (PR) expression assessed by dextran-coated charcoal method (DCC) and standardized immunohistochemical method (IHC) in a prospective series of 557 primary breast carcinomas, to assess the concordance between the two assays, and to evaluate the association between hormone receptor expression and various clinicopathological parameters. For ER, results of both methods were in agreement in 73.6% of the cases (277 positive, 133 negative), 74 tumors (13.3%) where IHC+/DCC- and 73 (13.1%) were IHC-/DCC+. For PR, concordant results were observed in 72.7% of the cases (201 positive and 204 negative), 127 tumors (22.8%) were IHC+/DCC- and 25 (4.5%) were IHC-/DCC+. Irrespective of the method used, ER and PR positivity showed a strong negative association with tumor grade. ER+ tumors were significantly more common among older patients. With IHC, PR+ cases were more common among tumors of lobular and mucinous type and among node positive tumors. The only parameter that was related to the concordance rate of ER determination by the DCC and IHC method was the age of the patients, with agreement being significantly lower in the group of patients younger than 50 years. On the other hand, discordant PR determination was more often observed in tumors of lower grade, node positive tumors and in tumors of lobular and mucinous type.

Loss of heterozygosity (LOH) at several chromosomal loci is a common event in human malignancies. Frequent LOH on the long arm of chromosome 7 has been reported in various human malignancies, and investigators have identified the most common site of LOH as 7q31.1. We have identified ten chromosomal loci, including chromosome 7q, that have been shown by previous allelotype study to be sites of frequent LOH in differentiated adenocarcinoma of the stomach. In the present study, we performed a polymerase chain reaction (PCR) microsatellite analysis to define the common deleted region on 7q, using 14 polymorphic microsatellite markers in matched tumour and non-tumour DNAs from 53 patients with primary gastric carcinoma of the differentiated type. LOH at any locus on 7q occurred in 34% (18 out of 53) of the tumours. Although many tumours exhibited total or large interstitial deletions, we determined the smallest common deleted region to be at D7S480 (7q31.1). This is identical to the region identified for other human malignancies. These observations indicate that a putative tumour suppressor gene at 7q31.1 may be involved in the pathogenesis of differentiated adenocarcinoma of the stomach.

The incidence of colorectal carcinoma is rising at an alarming pace in Asian urban societies such as Hong Kong. Detailed examination of the epidemiological pattern and genetic mutation of colorectal cancer in the Hong Kong Chinese population is overdue. We compared the reported age incidence of colorectal carcinoma in Hong Kong with that of Scotland and other countries. Hong Kong showed a much higher incidence of colorectal carcinoma among the young age groups. By comparison with other countries, this raised incidence among the young appeared to be related to southern Chinese societies. The recent dramatic rise in colorectal cancer in Hong Kong was largely attributable to an increase in the over 50 years age group, while the young incidence remained unchanged. We also defined the mutation spectrum of p53 and Ki-ras in 67 unselected cases by direct DNA sequencing. Interestingly, insertion/deletion mutations in p53 from colorectal carcinoma in Hong Kong showed a significantly higher frequency (17.2%) than the Scottish data (0%) and the world database (6.6%), although the overall frequency of p53 mutation (43%) in Hong Kong was similar to others. The high incidence of colorectal carcinoma in young people and the raised proportion of frameshift mutations in p53 encourage further search for a genetic basis for susceptibility to this disease in the Hong Kong Chinese population.

NCAM, L1, and DCC--immunoglobulin cell adhesion molecules (Ig CAMs)--are widely expressed during development. Many workers have dismissed a role for such molecules in the control of axonal growth and guidance because they do not show highly restricted expression patterns. Yet evidence from a number of model systems suggests all three CAMs play a role in the development of specific projections in the nervous system. For example, there is a reduction in mossy fiber tracts in the hippocampus of mice that lack NCAM, a requirement for DCC in the response of commissural neurons to a floor plate-derived chemoattractant, and a loss of corticospinal tracts in humans who carry mutations in the L1 gene. The above paradox might be explained by the observation that differential post-translational processing can modulate CAMs function and that alternative splicing can generate functionally distinct isoforms of a CAM. Activation of the FGF tyrosine kinase receptor is required for the responses stimulated by NCAM and L1, and the importance of regulated tyrosine phosphorylation for growth and guidance is underscored by the involvement of receptor tyrosine phosphatases in this process.

The work was aimed to estimate the interrelation and indexes which define the main alterations of hemostatic deviations in the patients with abdominal bleeding. For this reason the authors have analysed interrelations between 31 hemostasis indexes in 30 patients after operations. Fourteen groups of interrelated indications have been revealed. They are as follows: 1. Prothrombin index, activated partial thromboplastin index; 2. Prothrombin's inactive forms (prethrombin-1, decarboxylated prethrombin etc.), ecamulin index; 3. Velocity bend of distraction of fibrin clot, velocity bend of forming of fibrin clot, height of clot using turbidimetric method; 4. Half-lysis time, lysis time, time of fibrin clot using turbidimetric method; 5. Velocity, Intensity, remainder of platelet aggregation induced by ADP 10 mcg/ml, 2.5 mcg/ml, 1.9 mcg/ml; 6. Thrombin time; 7. Protein C; 8. Fibrinogen; 9. Platelet count; 10. Soluble fibrin; 11. Ancystron time; 12. X factor; 13. Antithrombin-III; 14. Fibrin(ogen) degradation products. It was shown how different groups affect hemostasis. The authors have suggested to use the data of mathematical analysis and laboratory tests for the estimation of hemostatic deviations.

Mood, emotion, and cognition are modulated by the serotonergic midbrain raphe system, which seems to be involved in the pathogenesis of psychiatric disorders like those of the affective spectrum. Since a dysregulation of serotonin transporter expression might be important in the course of those disorders, we isolated and cloned the 5'-regulatory region of the serotonin transporter gene, which is inducible by cAMP- and PKC-depended signal transduction pathways. Systematic screening for length variations and functional promoter analyses revealed a genetic polymorphism with allelic variation in transcriptional activity and protein expression. This 5-HTT gene polymorphism comprises a tandemly repeated sequence in the 5'-regulatory promoter region of the serotonin transporter gene. Recent population association studies demonstrated the 5-HTT gene promoter polymorphism accounting for 4-5% of population variation of anxiety-related behavioral traits.

There is vast evidence in support of the idea that accumulated genetic changes (mutations) are the underlying cause of neoplasia development. This multi-step process is aptly illustrated by colorectal carcinoma (CRC), usually developing in the course of decades, and presumably requiring at least seven genetic events to complete its development. In CRC the oncogenes most frequently undergoing mutation are c-k-ras and c-myc, and among tumor suppressant genes--APC, MCC, DCC, p53. An updated model of the molecular bases for adenoma occurrence and its evolution into carcinoma is presented. Inheritance of a single gene only which has undergone mutation augments substantially the predisposition to CRC. This is noted in a clearcut manner in the hereditary syndromes familial adenomatous polyposis (FAP) and hereditary non-polypous colorectal carcinoma (HNPCC). Recent studies along these lines suggest that the genetic defect in FAP increases the incidence of tumor initiation through functional impairment of the APC gene which is a gene regulator of the enhanced colorectal mucosa proliferation. Contrarily, the defect in HNPCC involves mainly the tumor progression through mutation of the DNA repair genes (MMRs), which are regulators of the genome stability. The study of hereditary syndromes give rise to a new concept for the occurrence and development of sporadic and inherited cancer in humans.

This chapter lists the various terms discussed in the book Methods in Cell Biology, Volume 54, which includes Abercrombie correction factor, acetone, acid hydrolases, acid loading, acousto-optic modulator, and amino acids.