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The identification of homozygous deletions in sporadic neoplasms has been pivotal in the positional cloning of several tumor suppressor genes. Chromosomal arm 18q harbors the DPC4, SMAD2, and DCC genes and is suspected on the basis of high frequencies of allelic loss to harbor additional tumor suppressor genes. We applied high-resolution sequence-tagged site (STS) marker scanning to a panel of 106 pancreatic adenocarcinomas to identify novel regions of homozygous deletions on 18q. Three homozygous deletions were identified. Physical mapping of these deletions showed them to be nonoverlapping, but clustered in an approximately 7- to 10-Mb region of chromosome band 18q22. Each deletion spanned physical distances of nearly 1.3 to 3 Mb. A number of transcribed genes map within these deletions. The identification of these homozygous deletions might aid in the identification of novel tumor suppressor genes on chromosomal arm 18q. Genes Chromosomes Cancer 25:370-375, 1999.

Mutation analysis was performed on DNA samples of 965 individuals from four different ethnic groups in South Africa, in an attempt to determine the spectrum of sequence variants in the haemochromatosis ( HFE ) gene. This population screening approach, utilizing a combined heteroduplex and single-strand conformation polymorphism (HEX-SSCP) method, revealed three previously described and four novel missense mutations. Novel variants V53M and V59M were identified in exon 2, Q127H in exon 3 and R330M in exon 5. The exon 5 variant was identified in one of 13 patients referred for a molecular diagnosis of hereditary haemochromatosis (HH), who tested negative for the known C282Y and H63D mutations. Mutation Q127H was detected in exon 3 of the HFE gene together with mutation H63D in an apparently severely affected patient previously shown to carry the protoporphyrinogen oxidase ( PPOX ) gene mutation R59W, which accounts for dominantly inherited variegate porphyria (VP) in >80% of affected South Africans. The mutant allele frequency of the C282Y mutation was found to be significantly lower in 73 apparently unrelated VP patients with the R59W mutation than in 102 controls drawn from the same population ( P = 0.005). The population screening approach used in this study revealed considerable genotypic variation in the HFE gene and supports previous data on the involvement of this gene in the porphyria phenotype.

<h4>Background</h4>Following orthotopic liver transplantation (OLT) histological examination of explant livers from patients with alcoholic liver disease (ALD) sometimes shows extensive iron deposits in a distribution suggestive of homozygous haemochromatosis.<h4>Aims</h4>To use haemochromatosis gene (HFE) assays to distinguish between ALD with notable siderosis and hereditary haemochromatosis. To evaluate the possible influence of spur cell haemolytic anaemia on hepatic iron loading.<h4>Patients</h4>Thirty seven patients with ALD were abstinent for at least six months prior to OLT. Twenty three patients had transferrin saturations greater than 55%, 16 also had increased serum ferritin (>350 micrograms/l). Eight of 37 (22%) explant livers had grade 3 or 4 hepatic iron deposition, predominantly in hepatocytes. Of these, four had a hepatic iron index greater than 1. 9 and most seemed to have spur cell haemolytic anaemia.<h4>Methods</h4>Mutation analysis for C282Y and H63D mutations was performed on DNA extracts from peripheral blood or explant liver. Spur cell haemolytic anaemia was diagnosed when the haemoglobin was 105 g/l in the presence of notable acanthocytosis.<h4>Results</h4>None of the eight patients with grade 3 or 4 hepatic iron had evidence of the C282Y mutation. Two of the eight were heterozygous for H63D. None of the remaining 28 patients tested showed homozygous HFE mutations. Spur cell anaemia was present in six of the eight patients with heavy iron deposition and only one of the remaining patients.<h4>Conclusions</h4>The HFE mutation was not present in these patients with advanced ALD and heavy iron loading. Spur cell haemolytic anaemia provides an alternative potential mechanism for the heavy iron loading.

Our aim was to reveal the significance of tumor-suppressor genes and genomic instability in 99 Hong Kong Chinese colorectal carcinoma (CRC) patients by PCR-LOH analysis and PCR-PTT assay. The frequencies of allelic loss of Apc, Mcc and Dcc and of APC truncation were 31.3% (15/48), 11.6% (5/43), 44.4% (20/45) and 46/93 (49.5%), respectively. The frequency of Apc LOH was similar to, the Mcc LOH was lower than, and the Dcc LOH was higher than that reported for Caucasians and Japanese. In Hong Kong CRC patients, the replication error-positive (RER(+)) phenotype occurred with a frequency of 10% (10/99), which was similar to other results using microsatellite markers where RER(+) frequencies ranged from 11% to 28%. The rates of genetic alteration in RER(+) tumors were lower in tumors harboring p53, Mcc and Dcc alterations; similar in Apc; and higher in Ki-ras tumors compared with RER(-) tumors, though these differences did not achieve statistical significance. None of the biomarkers examined were predictive of survival independently, but strong trends confirming earlier observations of associations between RER(+) phenotypes with proximal tumor location and poorly differentiated tumor status were noted. The RER(+) phenotype was correlated significantly to the less aggressive Duke's stage B and improved prognosis. Additionally, tumors with RER(+) phenotypes were positively correlated with young age and sex. Our results support the observation that a subset of younger male CRC patients in Hong Kong may develop CRC via the RER pathway and show differences in RER status and sex. A significantly higher percentage of older Hong Kong Chinese CRC patients had APC truncations. Int. J. Cancer (Pred. Oncol.) 84:404-409, 1999.

Over half of the neurons in Caenorhabditis elegans send axons to the nerve ring, a large neuropil in the head of the animal. Genetic screens in animals that express the green fluorescent protein in a subset of sensory neurons identified eight new sax genes that affect the morphology of nerve ring axons. sax-3/robo mutations disrupt axon guidance in the nerve ring, while sax-5, sax-9 and unc-44 disrupt both axon guidance and axon extension. Axon extension and guidance proceed normally in sax-1, sax-2, sax-6, sax-7 and sax-8 mutants, but these animals exhibit later defects in the maintenance of nerve ring structure. The functions of existing guidance genes in nerve ring development were also examined, revealing that SAX-3/Robo acts in parallel to the VAB-1/Eph receptor and the UNC-6/netrin, UNC-40/DCC guidance systems for ventral guidance of axons in the amphid commissure, a major route of axon entry into the nerve ring. In addition, SAX-3/Robo and the VAB-1/Eph receptor both function to prevent aberrant axon crossing at the ventral midline. Together, these genes define pathways required for axon growth, guidance and maintenance during nervous system development.

Haemophilus ducreyi, the causative agent of chancroid, produces a hemolysin, whose role in virulence is not well defined. To assess the possible role of hemolysin in pathogenesis, we evaluated its target cell range by using wild-type H. ducreyi 35000, nonhemolytic mutants with the hemolysin structural gene deleted, and isogenic strains expressing different amounts of hemolytic activity. The cytotoxicity of the various cell types was assessed by quantitating the release of lactate dehydrogenase into culture supernatants as a measure of cell lysis. In these experiments, human foreskin fibroblasts, human foreskin epithelial cells, and, to a lesser extent, HEp-2 cells were lysed by H. ducreyi hemolysin. Hemolysin also lysed human blood mononuclear cells and immune system cell lines including U937 macrophage-like cells, T lymphocytes, and B lymphocytes. In contrast, human polymorphonuclear leukocytes were not sensitive to hemolysin under the conditions tested. We also analyzed the effect of hemolysin on invasion of human epithelial cells and found that H. ducreyi strains expressing cloned hemolysin genes showed a 10-fold increase in invasion compared to the control strain. These data support the hypothesis that the H. ducreyi hemolysin is important in the pathogenesis of chancroid and may contribute to ulcer formation, invasion of epithelial cells, and evasion of the immune response.

The androgen and glucocorticoid hormones evoke specific in vivo responses by activating different sets of responsive genes. Although the consensus sequences of the glucocorticoid and androgen response elements are very similar, this in vivo specificity can in some cases be explained by differences in DNA recognition between both receptors. This has clearly been demonstrated for the androgen response element PB-ARE-2 described in the promoter of the rat probasin gene. Swapping of different fragments between the androgen- and glucocorticoid-receptor DNA-binding domains demonstrates that (i) the first Zn-finger module is not involved in this sequence selectivity and (ii) that residues in the second Zn-finger as well as a C-terminal extension of the DNA-binding domain from the androgen receptor are required. For specific and high-affinity binding to response elements, the DNA-binding domains of the androgen and glucocorticoid receptors need a different C-terminal extension. The glucocorticoid receptor requires 12 C-terminal amino acids for high affinity DNA binding, while the androgen receptor only involves four residues. However, for specific recognition of the PB-ARE-2, the androgen receptor also requires 12 C-terminal residues. Our data demonstrate that the mechanism by which the androgen receptor binds selectively to the PB-ARE-2 is different from that used by the glucocorticoid receptor to bind a consensus response element. We would like to suggest that the androgen receptor recognizes response elements as a direct repeat rather than the classical inverted repeat.

Ceramic green bodies can be produced by enzyme-catalyzed reactions from suspensions with high solids loading. There are two ways of destabilizing an electrostatically stabilized suspension: shifting the pH to the isoelectric point (IEP) or increasing the ionic strength. Both can be done via the urease-catalyzed decomposition of urea. The properties of wet green bodies produced by these two methods were compared using compressive strength measurements. Ionic-strength samples needed 24 h or more to achieve sufficient strength because the high urea concentrations required for these slurries inhibit the enzyme. Due to the long coagulation time and to the weak attractive forces formed, the particles can rearrange. This causes the wet green strength and the Young's modulus of these bodies to be considerably higher than those of the pH shift bodies. For the latter, coagulation needs only about one hour, and the interparticle attractive forces are strong. pH shift bodies are more or less insensitive to vibration, whereas ionic-strength bodies tend to flow under oscillatory stress. Copyright 1999 Academic Press.

We have investigated the molecular determinants that mediate the differences in voltage-dependent inactivation properties between rapidly inactivating (R-type) alpha(1E) and noninactivating (L-type) alpha(1C) calcium channels. When coexpressed in human embryonic kidney cells with ancillary beta(1b) and alpha(2)-delta subunits, the wild type channels exhibit dramatically different inactivation properties; the half-inactivation potential of alpha(1E) is 45 mV more negative than that observed with alpha(1C), and during a 150-ms test depolarization, alpha(1E) undergoes 65% inactivation compared with only about 15% for alpha(1C). To define the structural determinants that govern these intrinsic differences, we have created a series of chimeric calcium channel alpha(1) subunits that combine the major structural domains of the two wild type channels, and we investigated their voltage-dependent inactivation properties. Each of the four transmembrane domains significantly affected the half-inactivation potential, with domains II and III being most critical. In particular, substitution of alpha(1C) sequence in domains II or III with that of alpha(1E) resulted in 25-mV negative shifts in half-inactivation potential. Similarly, the differences in inactivation rate were predominantly governed by transmembrane domains II and III and to some extent by domain IV. Thus, voltage-dependent inactivation of alpha(1E) channels is a complex process that involves multiple structural domains and possibly a global conformational change in the channel protein.

Motor patterns are selected from multifunctional networks by selective activation of different projection neurons, many of which contain multiple transmitters. Little is known about how any individual projection neuron uses its cotransmitters to select a motor pattern. We address this issue by using the stomatogastric ganglion (STG) of the crab Cancer borealis, which contains a neuronal network that generates multiple versions of the pyloric and gastric mill motor patterns. The functional flexibility of this network results mainly from modulatory inputs it receives from projection neurons that originate in neighboring ganglia. We demonstrated previously that the STG motor pattern selected by activation of the modulatory proctolin neuron (MPN) results from direct MPN modulation of the pyloric rhythm and indirect MPN inhibition of the gastric mill rhythm. The latter action results from MPN inhibition of projection neurons that excite the gastric mill rhythm. These projection neurons are modulatory commissural neuron 1 (MCN1) and commissural projection neuron 2 (CPN2). MPN excitation of the pyloric rhythm is mimicked by bath application of proctolin, its peptide transmitter. Here, we show that MPN uses only its small molecule transmitter, GABA, to inhibit MCN1 and CPN2 within their ganglion of origin. We also demonstrate that MPN has no proctolin-mediated influence on MCN1 or CPN2, although exogenously applied proctolin directly excites these neurons. Thus, motor pattern selection occurs during MPN activation via proctolin actions on the STG network and GABA-mediated actions on projection neurons in the commissural ganglia, demonstrating a spatial and functional segregation of cotransmitter actions.

In the Caenorhabditis elegans embryo, some ventral midline motoneurons extend a process circumferentially to the dorsal midline and a process longitudinally along ventral nerve cord interneurons. Circumferential migrations are guided by netrin UNC-6, which repels motoneuron axons dorsally. Although the motoneuron cell bodies and the longitudinal axons are positioned along UNC-6-expressing interneurons in the ventral nerve cord, the circumferential processes extend only from the motoneuron cell bodies and from defined locations along some longitudinal axons. This implies a mechanism regulates motoneuron branching of UNC-6-responsive processes. We show that expression of unc-6DeltaC, which encodes UNC-6 without domain C, partially rescues circumferential migration defects in unc-6 null animals. This activity depends on the netrin receptors UNC-5 and UNC-40. These results indicate that UNC-6DeltaC can provide the circumferential guidance functions of UNC-6. Furthermore, we show that expression of unc-6DeltaC causes motoneuron branching and the extension of processes from abnormal positions along the ventral nerve cord. This activity is also UNC-5- and UNC-40-dependent. We propose that local interactions mediated by domain C regulate motoneuron branching and responsiveness to the UNC-6 cue.

T-cell-mediated myocardial damage has been shown to be involved in acute myocarditis and dilated cardiomyopathy. It is necessary for T-cells to receive a co-stimulatory signal as well as the main signal through the T-cell receptor for antigen-specific T-cell activation to occur. To investigate the roles of the co-stimulatory molecules CD40/CD40L, CD30/CD30L, CD27/CD27L, and OX40/OX40L, which belong to the tumour necrosis factor (TNF) receptor/ligand superfamily, in the development of chronic ongoing myocarditis, the expression of CD40, CD30L, CD27L, and OX40L was analysed in the hearts of A/J mice with myocarditis induced by Coxsackie virus B3 (CVB3). The expression of CD40L, CD30, CD27, and OX40 was also examined on the infiltrating cells. Furthermore, the induction of CD40, CD30L, CD27L, and OX40L was evaluated on cultured cardiac myocytes treated with interferon (IFN)-gamma. CVB3-induced myocarditis resulted in the induction of CD40 and CD30L on the surface of cardiac myocytes. Induction of CD40 and CD30L on cardiac myocytes was confirmed by treatment with IFN-gamma in vitro. CD27L and OX40L were expressed on cardiac myocytes in vivo and in vitro. The expression of CD27L and OX40L on cardiac myocytes was increased, at least partly, by CVB3-induced myocarditis in vivo. Many infiltrating cells expressed CD27 and OX40, whereas much smaller numbers expressed CD40L and CD30. The induction of these molecules, especially CD40 and CD30L, on cardiac myocytes strongly suggests that cardiac myocytes may co-stimulate T-cells and induce cytokine production by T-cells and humoral immune responses. This may play an important role in the pathogenesis of the resulting myocardial damage.

Huntington disease (HD) belongs to the group of neurodegenerative disorders characterized by unstable expanded trinucleotide repeats. In the case of HD, the expansion of a CAG repeat occurs in the IT15 gene. The detection of the expanded CAG repeats has usually involved the electrophoretic separation of polymerase chain reaction (PCR) amplification products using conventional agarose and acrylamide gel electrophoresis. We have undertaken the comparative analysis of sizing CAG repeats of the IT15 gene using radioactive and fluorescent PCR amplification, and the subsequent separation of these products by slab gel and capillary electrophoresis. The assays have been performed on both cloned and sequenced CAG repeats, as well as genomic DNA from HD patients with a wide range of repeat lengths. The mobility of the CAG repeat amplification products of the IT15 gene is greater using capillary electrophoresis compared to slab gel electrophoresis. The analysis of 40 DNA samples from HD patients indicates that the mobility difference increases with the length of the repeat. However, we have devised an allele ladder for sizing the CAG repeats. This ladder provides a mandatory internal calibration system for diagnostic purposes and enables the confident use of either capillary or slab gel electrophoresis for sizing HD alleles.

The Authors point out the basis for a better characterisation of colo-rectal cancer and precursory lesions. In fact the etiology of familial adenomatous polyposis (FAP), aberrant crypt foci (ACF), hereditary non polyposis colon cancer syndrome (HNPCC) seems to be correlated to molecular pathology. Therefore the Authors review colo-rectal cancer natural history which frequently appears to be not related to clinical evolution.

<h4>Objective</h4>Hepatocellular carcinoma (HCC) is one of the dreaded complications of cirrhosis. Although there are no randomized controlled studies showing improved survival with screening, patients with cirrhosis are screened for HCC. Little is known about the practice of HCC screening in the United States. Our aim was to describe the practice of HCC screening in patients with cirrhosis in the United States.<h4>Methods</h4>In March 1998, we mailed a standard questionnaire to 1021 physician members of the American Association of Study for Liver Diseases and the same questionnaire was re-sent to nonrespondents 4 weeks later.<h4>Results</h4>We received a response from 554 members (54%). After excluding those not involved in active adult patient care, 473 responses were eligible for analysis. Eighty-four percent of the respondents routinely screened patients with cirrhosis for HCC (screening respondents). Nearly half of the screening respondents limited the HCC screening to patients with high-risk etiologies such as hepatitis B or C or hemochromatosis. Although alpha-fetoprotein (99.7%) and ultrasound (93%) were the two most frequently used screening methods, a sizable proportion of the screening respondents (25%) used abdominal computed tomography for routine screening. On multivariate analysis, the following variables predicted screening for HCC by the respondents: seeing more than one new cirrhotic per week (odds ratio [OR]: 5.4, 95% confidence interval [CI]: 2.5-11.7); practicing for < 10 yr (OR: 4.0, 95% CI: 1.2-13.4); an opinion that screening is cost-effective (OR: 6.4, 95% CI: 1.6-25); an opinion that screening prolongs survival (OR: 5.7, 95% CI: 1.8-17.9); and an opinion that not screening poses malpractice liability (OR: 9.3, 95% CI: 4.2-20.8).<h4>Conclusions</h4>The majority of respondents routinely screen patients with cirrhosis for HCC. Approximately half of the screening respondents limit their screening to only patients with high-risk etiologies. On multivariate analysis, several variables predicted screening for HCC by the respondents.

Transition metals are essential for many metabolic processes and their homeostasis is crucial for life. Aberrations in the cellular metal ion concentrations may lead to cell death and severe diseases. Metal ion transporters play a major role in maintaining the correct concentrations of the various metal ions in the different cellular compartments. Recent studies of yeast mutants revealed key elements in metal ion homeostasis, including novel transport systems. Several of the proteins discovered in yeast are highly conserved, and defects in some of the yeast mutants could be complemented by their human homologs. The studies of yeast metal ion transporters helped to unravel the molecular mechanism of macrophage defense against bacterial infection and hereditary diseases.

Loss of tumor suppressors that restrain important oncoproteins such as c-Myc may contribute to malignant progression. Bin1 is an adapter protein with features of a tumor suppressor that was identified through its interaction with and inhibition of the oncogenic properties of c-Myc. In this study, we analyzed the patterns of Bin1 expression in normal melanocytes and melanoma cells at different stages of tumor progression. Evidence is provided that Bin1 function is abrogated in melanoma cells by a mechanism based on aberrant splicing of a tissue-specific exon. Specifically, most melanoma cells inappropriately expressed exon 12A, which is spliced alternately into Bin1 isoforms found in brain but not into isoforms found in melanocytes and many other nonneuronal cells. Exon 12A sequences abolished the ability of Bin1 to inhibit malignant transformation by c-Myc or adenovirus E1A. Similarly, these sequences abolished the ability of Bin1 to induce programmed cell death in melanoma cells that endogenously expressed exon 12A. Our findings suggest that aberrant splicing of Bin1 may contribute to melanoma progression, and they define a mechanism by which the activity of a tumor suppressor can be eliminated in cells.

The HFE gene and its mutations C282Y and H63D cause hereditary haemochromatosis (HH). Among 54 affected individuals from North-East Scotland, 91% were homozygous for C282Y and 5.5% were compound heterozygotes for C282Y and H63D. The general population allele frequencies were high (8% and 15.7% for C282Y and H63D respectively). Although it is likely that HH is under diagnosed, these figures suggest that disease expression is variable, and many of those with the genetic predisposition HH will never develop the clinical consequences of iron overload. This has implications for diagnosis and predictive testing.

The homeobox gene Msx-2 is expressed specifically in sites of skin appendage formation. To explore its part in skin morphogenesis, we produced transgenic mice expressing Msx-2 under the control of the cytomegalovirus promoter. The skin of these transgenic mice was flaky, exhibiting desquamation and shorter hairs. Histologic analysis showed thickened epidermis with hyperproliferation, which was restricted to the basal layer. Hyperkeratosis was also evident. A wide zone of suprabasal cells were misaligned and coexpressed keratins 14 and 10. There was reduced expression of integrin beta 1 and DCC in the basal layer. Hair follicles were misaligned with a shrunken matrix region. The dermis showed increased cellularity and empty vacuoles. We suggest that Msx-2 is involved in the growth control of skin and skin appendages.

Huntington's disease is a neurodegenerative disorder resulting from expansion of the polyglutamine region in huntingtin. Although huntingtin is normally cytoplasmic, in affected brain regions proteolytic fragments of mutant huntingtin containing the polyglutamine repeat form intranuclear inclusions. Here, we examine the contribution of nuclear localization to toxicity by transiently transfecting neuro-2a cells with an N-terminal huntingtin fragment similar in size to that believed to be present in patients. The huntingtin fragment, HD-N63, was targeted either to the cytoplasm with a nuclear export signal (NES) or to the nucleus with a nuclear localization signal (NLS). The NES decreased the number of cells with aggregates in the nucleus while an NLS had the opposite effect. By cotransfecting HD-N63 with GFP as a marker, we observed direct cell loss with constructs containing expanded polyglutamine repeats. Compared to unmodified HD-N63-75Q, adding an NES reduced cell loss by 57% while an NLS increased cell loss by 111%. These results indicate that nuclear localization of mutant huntingtin fragments plays an important role in cell toxicity.

Loss of heterozygosity (LOH) of loci on chromosome 18q occurs in a majority of colorectal cancers. The DPC4/SMAD4 gene, lying in close proximity to the DCC gene at 18q21.1, was recently identified as a candidate tumor suppressor for the genesis of pancreatic cancer as well as a predisposing gene for Juvenile Polyposis Syndrome (JPS). The gene product functions as a cytoplasmic mediator in the signaling pathway of transforming growth factor beta (TGF-beta). To investigate the potential role of DPC4/SMAD4 gene in colorectal cancers, we examined 73 tumors of clinical stages II or III from Japanese patients, for LOH at 18q21 and also for subtle mutations anywhere within the coding region of DPC4/SMAD4. LOH was identified in 50 (78%) of the 64 tumors that were informative for polymorphic markers in the region. Somatic mutations were identified in seven of those tumors: two frameshift mutations, a 1-bp deletion (326 del T) in exon 8 and a 1-bp insertion (50-51 ins A) in exon 1; two nonsense mutations, Arg445Ter in exon 10 and Glu538Ter in exon 11; and three missense mutations, Asn129Lys in exon 2, Tyr95Asn in exon 2, and Asp355Glu in exon 8. Three of the seven mutations were observed in the mad homology 1 (MH1) domain encoded by exons 1 and 2. In all of the tumors carrying intragenic mutations of one allele, LOH analysis had shown that the other allele was missing. The results demonstrated that inactivation of both alleles of the DPC4/SMAD4 gene occurs in a substantial proportion of advanced colorectal cancers, and that the DPC4/SMAD4 gene probably exerts a tumor-suppressor effect for colorectal carcinogenesis that fulfills the criterion of the two-hit concept proposed by Knudson [A.G. Knudson, Hereditary cancer, oncogenes, and anti-oncogenes, Cancer Res. 45 (1985) 1437-1443.].

Telomerase is a ribonucleoprotein complex which, by de novo synthesized telomeric TTAGGG repeats, prevents telomere erosion. While telomerase is active in most cancers, conflicting results exist for normal tissues and premalignant lesions. To establish the telomerase status of normal gastrointestinal mucosa and to elucidate whether telomerase up-regulation is an early or late event in carcinogenesis, we determined the telomerase activity of 88 biopsies of normal mucosa from esophagus, stomach, and intestine and compared it with that of 21 samples of premalignant lesions and 6 adenocarcinomas using the telomere-repeat amplification protocol assay. Telomerase was found in all normal tissues, revealing most activity in esophagus (11 samples), followed by intestine (45 samples), and stomach (32 samples). In 53% of the stomach samples, enzyme activity could only be demonstrated when telomerase inhibitors were eliminated by a modified telomerase assay. In the 21 precancerous lesions (5 Barrett's esophagus, 3 stomach intestinal metaplasias, and 13 colorectal adenomas of type I/II dysplasia) a similar or even reduced telomerase activity was seen, while the adenocarcinomas showed high activity. These data demonstrate that telomerase activity is expressed in all epithelia along the gastrointestinal tract, thus confirming our previous hypothesis that telomerase is constitutively expressed in permanently renewing epithelia. Furthermore, activity was not increased in preneoplastic lesions, suggesting that telomerase up-regulation is a late event during carcinogenesis of the esophagus, stomach, and intestine.

One hundred and seventeen elderly residents of seven geriatric health facilities located in Tokyo participated in this study. The Mini-Mental State, Metamemory in Adulthood, Geriatric Depression Health Scale and demographic questionnaires were used in face-to-face interviews. The subjects were 32 males and 85 females, with a mean age of 83.08 years. Depression was found to be a key factor for explaining metamemory. Depression accounted for 17% of the variance in capacity and 23% in change. There were no differences between the mild cognitive impairment group and the cognitively intact group on achievement, capacity, change, locus and strategy subscales. When depression was considered as a moderating factor, a difference arose between two cognitive levels. History of stroke was not related to metamemory in this study; however, future studies should emphasize memory awareness in brain injury residents since 41% of the sample had a history of CVA.

Serum iron studies have high values in 30% of patients with virus C (HCV) chronic hepatitis even in hemochromatosis range. These indices do not represent hepatic iron storage which has a low to moderate intensity. Heterozygosity for hereditary hemochromatosis could be responsible of the iron liver storage in some patients. Nowadays the relationship between hepatic iron loading and HCV infection is unknown. It has been suggested that iron storage could allow HCV infection or, by other way, it may be caused by the infection. Iron, per se, has hepatolesive effect and acts synergistically with HCV worsening liver injury. Also, iron storage reduces the interferon response rate, and iron depletion with phlebotomies improves transaminases level. However, it is discussed if the association: iron removal-interferon treatment improves interferon alone treatment response. In this work all these points are reviewed.

A 28-year-old man presented in 1992 with decreased libido and erectile dysfunction. He was found to have central hypogonadism with low serum levels of testosterone, LH and FSH. Computer tomography of the cella turcica was normal. Apart from hypogonadism, the pituitary function was normal. He was successfully treated with testosterone injections. In June 1995, elevated levels of serum ferritin (4,094 micrograms/l) and transferrin saturation (94%) raised suspicion of hemochromatosis. The diagnosis was confirmed by a percutaneous liver biopsy, and treatment started with regular phlebotomies. After three years of treatment the serum levels of LH and testosterone were normalized. Hypogonadism is, except for diabetes mellitus, the most frequent endocrine disturbance in hemochromatosis. It is found almost exclusively in male patients. It is important to exclude hypogonadism in male patients with hemochromatosis, but it is also important to exclude hemochromatosis in male patients with hypogonadism.

From March 1988 to December 1990, we detected estrogen receptor (ER) from breast cancer tissues with the dextran-coated charcoal (DCC) method. From September 1990 to March 1998, we repeated the above with the frozen section method (cloneH222; DAINABOT). With the paraffin-embedded section method using two antibodies of anti-ER (clone6F11; NOVOCASTRA and clone 1D5; MBL), we examined the ER of the same 185 primary breast cancer tissues. We had already detected these tissues with the frozen section method, and we also applied the same procedure for the 43 primary breast cancer tissues which had already been detected with the DCC method. We compared these data. The positive rates of ER with DCC, H222, 6F11 and 1D5 were 49%, 53%, 53% and 52% respectively, which were within the reported range. The accuracy between H222 and 6F11 which was calculated as the percentage that were in positive and negative concordance by the two methods, was 88%. The accuracies between H222 and 1D5, between 6F11 and 1D5, between DCC and 6F11, between DCC and 1D5 were respectively 89%, 96%, 79%, and 77%. Although the accuracy between DCC and the paraffin-embedded section was not necessarily high, we obtained a higher concordance between the frozen and paraffin-embedded sections. The highest concordance existed between 6F11 and 1D5. The 30% of negative cases with DCC were positive with paraffin-embedded section. Among these methods of ER detection from breast cancer tissues, the paraffin-embedded section method seemed to be the most useful.

Estrogen receptors (ERs) in the prostate and prostatic urethra were examined in 33 men with benign prostatic hyperplasia (BPH) and in 11 with prostate cancer (PC). The Abbot monoclonal ER-ICA assay was used for immunohistochemical investigation. In the BPH group, ERs were revealed in the prostatic stroma in eight cases and in the glandular epithelium in one. In four cases ERs were seen in the prostatic stroma and in the glandular epithelium. In the prostatic urethra, ERs were found in 19 cases located in the urothelium, lamina propria and/or periurethral glands. In the PC group, ERs were demonstrated in the prostatic stroma and/or prostatic urethra in 6 out of 11 cases. In both BPH and PC patients, immunoreactivity was weak and confined to few cells, indicating low ER content in the prostate as well as in the prostatic urethra. Dextran-coated charcoal (DCC) analysis was used for detection and quanticization of cytosolic and nuclear ERs. In the BPH group, ERs were detected once in the prostate and prostatic urethra in the nuclear and cytosol, and additionally in the prostatic urethra in the cytosol fraction in three cases. In all cases, ER content was low, ranging from 10-15 fmol/mg protein. In the PC group, ERs were detected in the prostatic urethra and/or prostate in the cytosol fraction from two patients. The contents were low, ranging from 10-13 fmol/mg protein. We conclude that in human BPH and PC, ERs can be present in the prostate and prostatic urethra. In the prostate, ERs are mainly located in the stroma, but in BPH specimens they can also be found in the glandular epithelium. Biochemically, the use of the DCC analysis is of limited value, since ER content in the human prostate and prostatic urethra is at the limit of detection with this method.

Dimethylarsinic acid (DMA), a main metabolite of arsenicals which are carcinogenic in man, exerts tumor-promoting activity on rat urinary bladder carcinogenesis initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Sodium L-ascorbate (Na-AsA) is also a strong tumor promoter in this animal model. In this study, we used (LewisxF344)F, rats to compare molecular alterations in urinary bladder tumors caused by BBN followed by DMA or Na-AsA. Male, 6-week-old rats were given 0.05% BBN in their drinking water for 4 weeks, and then the rats in group 1 were maintained with no further treatment for 40 weeks. The animals of groups 2 and 3 were administered 0.01% DMA in their drinking water (group 2) or 5% Na-AsA in the powder diet (group 3) after the BBN treatment. Group 4 rats were given 0.05% BBN continuously for 36 weeks. At weeks 12, 20, 36 and 44, subgroups of rats were killed. Histopathological examination revealed promoting activity for DMA and, to a greater extent, Na-AsA on urinary bladder carcinogenesis. Loss of heterozygosity (LOH), detected with the polymerase chain reaction using 36 microsatellite markers, was found to be present in 2 of 9 (22%) urinary bladder tumors after treatment with DMA and 3 of 22 (14%) induced by continuous administration with BBN. No LOH was, however, detected in urinary bladder tumors after treatment with Na-AsA. The results thus suggest that the mechanisms of action of these two promoters, DMA and Na-AsA, may differ in rat urinary bladder carcinogenesis.

Cerebral venous and sinus thrombosis (CVST) is still considered a severe clinical problem that is difficult to diagnose and manage and is linked to a poor prognosis. Nonetheless, conventional cerebral angiography and magnetic resonance imaging (MRI), or more recently, MR angiography allow a more rapid and precise diagnosis, and prognosis has improved with the use of anticoagulant treatment. We report 23 cases of CVST consecutively admitted to the Institute of Neurology of the University of Parma during the period 1990-1997. In all cases diagnosis was confirmed by means of MRI or conventional angiography of brain vessels. Among the patients, 22 were female and 1 was male. In all patients, plasma levels of protein C, protein S, antithrombin III (ATIII) and antiphospholipid antibodies (APA) were evaluated. In 15 of 23 patients, the presence of factor V Leiden mutation was also determined, and found positive in 3 patients (20%). Of the 22 female patients, 15 (68%) were on low-oestrogen (containing less than 50 microg oestrogen) oral contraceptive (OC) treatment. This percentage of OC use by patients with CVST is much higher than that of the rest of the female Italian population. OC use was associated with the presence of factor V Leiden mutation in two cases, with a deficiency of protein C in 1 case and a deficiency of protein S in another.Whether low-oestrogen Ocs may induce cerebral thromboembolic events is an open matter. According to our data, it may be argued that Ocs, even if at low oestrogen content, represent a major risk factor for CVST. The use of Ocs, as is the case for systemic venous thromboembolic events, may further increase the risk of CVST in women carrying the factor V Leiden mutation or other inherited hyperthrombotic conditions.

<h4>Background</h4>Hereditary haemochromatosis (HH) is a multi-organ disease characterized by increased deposition of iron in some tissues. When discovered early the disease is curable. The Caucasian population is burdened with a high incidence of carriers and homozygotes. The recent discovery of the gene for haemochromatosis (HFE) and of the major mutation C282Y makes direct diagnosis possible.<h4>Methods and results</h4>Exploiting a newly-designed polymerase chain reaction system, we determined the frequency of the C282Y mutation of the HFE gene in the Czech population by analysis of 278 chromosomes from anonymous clients of an in vitro fertilization centre (128 male and 150 female karyotypes respectively). There were 14 heterozygotes of the C282Y mutation (6 males and 8 females) and no asymptomatic homozygotes. All 12 patients with classically diagnosed haemochromatosis were homozygous for the C282Y mutation. Twelve Guthrie cards ten years old were tested as a source of the PCR signal for C282Y with a positive result in all cases (100% efficiency).<h4>Conclusions</h4>Our data suggest that the approximate frequency of heterozygotes (carriers) of hereditary haemochromatosis in the Czech population is 1:10 which is comparable with data from the Central European region. The frequency of the major mutation among patients with HH was 100% (12/12), however, the difference with data from neighbouring countries (Austria, Germany) cannot be considered significant due to the small number of patients tested. We also showed the possibility of a retro-diagnosis of the presence/absence of C282Y mutation from Guthrie cards.

During and following significant surgical intervention, deep venous thrombosis prophylaxis by application of anticoagulants is routinely used. However, patients with malignant disorders are subject to an especially high risk of deep venous thrombosis progressing in severe cases to subsequent pulmonary embolism. The present study focuses on appraising modern markers of deep vein thrombosis in 34 patients undergoing major maxillofacial surgery, with some malignant disorders. No significant differences between the two patient groups were noted using the markers of the kallikrein-kinin-system. From the first postoperative day plasma levels of the coagulation indicator thrombin-antithrombin-III complexes were significantly higher in the group of tumour patients. Markers of fibrinolysis indicated corresponding results: on the first postoperative day tissue-plasminogen activator values rose to 18.9 +/- 3.2 micrograms/l in the group of malignant patients, but only to 7.4 +/- 1.1 micrograms/l (P < 0.05) in the control group. Also postoperative D-dimer concentrations in the malignancy group were significantly above those of the control group. In the present study it could be demonstrated that patients with malignant neoplasia undergoing major maxillofacial surgery are exposed postoperatively to a particularly high risk of developing thromboembolic complications. All in all, the status of anti-thrombotic therapy requires reappraisal with respect to the current treatment approach adopted in tumour patients.

<h4>Background</h4>It has been shown previously in ulcerative colitis tissue that E-cadherin can occasionally be mutated in the extracellular domain early in neoplastic progression. E-cadherin is known to maintain differentiation and inhibits invasion in vivo.<h4>Aims</h4>To assess the mechanisms by which such dysfunction occurs.<h4>Methods</h4>Four human colorectal cancer cell lines, HCA-7 colonies 1, 3, 6, and 30, derived from a single heterogeneous colorectal cancer were studied. The HCA-7 cell line has p53 mutations and a random errors of replication "positive" phenotype, as is seen in early colitis associated cancers or hereditary nonpolyposis coli cancer (HNPCC).<h4>Results</h4>Cell lines 6 and 30 expressed E-cadherin abundantly and this correlated positively with their degree of differentiation and organisation; however, both cell lines had loss of heterozygosity of E-cadherin. Interestingly, E-cadherin production was downregulated in the poorly differentiated cell line 1, and this was associated with major chromosomal rearrangements of 16q. This cell line also had a mutation in the homophilic binding domain of exon 4, which was associated with disaggregation by low titres of a function blocking antibody, and an invasive phenotype.<h4>Conclusions</h4>These multiple biological alterations further characterise the complex association that E-cadherin has with tumour heterogeneity and suggest that this series of cell lines may be a useful model of colitis associated or HNPCC associated tumorigenesis.

The serotonin transporter gene (SERT) plays an important role in the serotonin uptake into neurons. Recently, several polymorphisms including a variable-number-tandem-repeat (VNTR) in the second intron and an insertion/deletion polymorphism (5-HTT linked polymorphic region, 5-HTTLPR) were identified and reported to be associated with a variety of mental illnesses, including major depression, bipolar disorder, anxiety-related traits, and autism. In our study, we performed an association study between the SERT VNTR polymorphism and schizophrenia (n = 260), bipolar disorder (n = 137), and unipolar depression (n = 33) in the Han Chinese. A large group of ethnically matched control individuals (n = 362) were also genotyped. Allele 12 of the VNTR polymorphism was associated with schizophrenia (P = 0.007) and unipolar depression (P = 0.011). Bipolar disorder was not associated with the VNTR (P = 0.93). Thus, we conclude that the SERT VNTR polymorphism may be a risk factor for both schizophrenia and unipolar depression, but not for bipolar disorder, in the Han Chinese.

Although significant technical advances in surgical and radiation treatment for brain tumors have emerged in recent years, their impact on clinical outcome for patients has been disappointing. A fundamental source of the management challenge presented by glioma patients is the insidious propensity of the malignant cells to invade into adjacent normal brain. Invasive tumor cells escape surgical removal and geographically dodge lethal radiation exposure. Recent improved understanding of the biochemistry and molecular determinants of glioma cell invasion provide valuable insight to the underlying biological features of the disease, as well as illuminating possible new therapeutic targets. Heightened commitment to migrate and invade is accompanied by a glioma cell's reduced proliferative activity. The microenvironmental manipulations coincident to invasion and migration may also impact the glioma cell's response to cytotoxic treatments. These collateral aspects of the glioma cell invasive phenotype should be further explored and exploited as novel antiglioma therapies.

<h4>Objective</h4>To investigate the role of multiple tumor suppressor gene abnormality in gastrointestinal metaplasia (IM).<h4>Methods</h4>The loss of heterozygosity (LOH) of YNZ22, APC, MCC and DCC genes was examined by PCR-RFLP technique in 60 IM cases.<h4>Results</h4>LOH of YNZ22, APC, MCC and DCC genes were found in 19.4% (6/31), 12.0% (3/25), 13.0% (3/23) and 4.3% (2/47) respectively. IM was classified into types I, II and III. It was found that the LOH rates of each tumor suppressor gene of type III IM were higher than those of types I & II IM, but the difference was not significant (P > 0.05). Two cases of type III IM were found to also have 2 gene alterations (APC, MCC or YNZ22, MCC) respectively. All LOH were found in IM of adjacent carcinoma. None were found in IM of cancer-free patients.<h4>Conclusion</h4>The alteration of tumor suppressor gene may play a role in the development and carcinogenesis of IM.

No abstract available.