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The protease domain of tissue plasminogen activator (tPA), a key fibrinolytic enzyme, was expressed in Escherichia coli with a yield of 1 mg per liter of media. The recombinant protein was titrated with the Erythrina caraffa trypsin inhibitor (ETI) and characterized in its interaction with plasminogen and the natural inhibitor plasminogen activator inhibitor-1 (PAI-1). Analysis of the catalytic properties of tPA using a library of chromogenic substrates carrying substitutions at P1, P2, and P3 reveals a strong preference for Arg over Lys at P1, unmatched by other serine proteases like thrombin or trypsin. In contrast to these proteases and plasmin, tPA shows little or no preference for Pro over Gly at P2. A specific inhibition of tPA by Cu2+ was discovered. The divalent cation presumably binds to H188 near D189 in the primary specificity pocket and inhibits substrate binding in a competitive manner with a Kd = 19 microM. In an attempt to engineer Na+ binding and enhanced catalytic activity in tPA, P225 was replaced with Tyr, the residue present in Na+-dependent allosteric serine proteases. The P225Y mutation did not result in cation binding, but caused a significant loss of specificity (up to 100-fold) toward chromogenic substrates and plasminogen and considerably reduced the inhibition by PAI-1 and ETI. Interestingly, the P225Y substitution enhanced the ability of Cu2+ to inhibit the enzyme. Elimination of the C136-C201 disulfide bond, that is absent in all Na+-dependent allosteric serine proteases, significantly enhanced the yield (5 mg per liter of media) of expression in E. coli, but caused no changes in the properties of the enzyme whether residue 225 was Pro or Tyr. These findings point out an unanticipated crucial role for residue 225 in controlling the catalytic activity of tPA, and suggest that engineering of a Na+-dependent allosteric enhancement of catalytic activity in this enzyme, must involve substantial changes in the region homologous to the Na+ binding site of allosteric serine proteases.

<h4>Background</h4>Iron overload is common in the livers of alcoholics and may play a role in disease pathogenesis. An MHC like gene, HFE, has recently been identified that is mutated in most patients with hereditary haemochromatosis (C282Y in 90% and H63D in 45% of the remainder).<h4>Aim</h4>To examine the hypothesis that these mutations determine hepatic iron status in alcoholics and play a role in pre-disposition to advanced alcoholic liver disease.<h4>Methods</h4>The HFE gene was genotyped in 257 patients with alcoholic liver disease and 117 locally matched healthy volunteers. In addition, iron staining was scored (0-4) on biopsy specimens from fibrotic/cirrhotic patients with and without HFE mutations matched for age and sex.<h4>Results</h4>Some 15.7% of fibrotic/cirrhotic patients were C282Y heterozygotes compared with 13.7% of controls (p = 0.77). One control and three patients were C282Y homozygotes. Of chromosomes without the C282Y mutation, 68/442 (15.4%) of patients' chromosomes carried the H63D mutation compared with 36/216 (16.6%) of control chromosomes (p = 0.91). Significant (> grade 1) hepatocyte iron staining was seen in 6/23 C282Y heterozygotes and 4/26 H63D heterozygotes compared with 4/23 controls.<h4>Conclusions</h4>Possession of a single copy of either of the two HFE mutations influences neither liver iron content nor the risk of fibrotic disease in alcoholics.

Eight patients with pancreatic abscesses secondary to acute necrotizing pancreatitis underwent drainage of their abscesses under laparotomy. Two of them died of acute pulmonary thromboembolism (PTE) within 1 week. Autopsy revealed a large thrombus at the main trunk of the pulmonary artery and in the left common iliac vein. Femoral catheter insertion/indwelling, immobilization, surgery, increased trypsin/kinin/kallikrein, increased endotoxin, and decreased antithrombin-III (AT-III) were present following drainage of the pancreatic abscesses. With respect to the bedside diagnosis of acute PTE, alveolar-arterial oxygen gradients obtained by blood gas analysis and mean pulmonary artery pressure estimated by pulsed Doppler echocardiography are very useful. In terms of the treatment, attention should be paid to the following to prevent deep venous thrombosis: prophylactic administration of low molecular weight heparin and administration of AT-III (AT-III > or = 80%), use of the subclavian vein whenever possible as blood access for apheresis therapy, as short a compression time as possible after removing the blood access catheter (< or =6 h), and application of intermittent pneumatic compression devices or elastic compression stockings on the lower extremities.

<h4>Objective</h4>To explore the relationship between the genetic instability of microsatellite and the evolution of chronic myelogenous leukemia(CML).<h4>Methods</h4>The loss of heterozygosity(LOH) and the microsatellite instability(MSI) of two polymorphic microsatellite markers, DCC and Mfd41, which located on chromosome 18q and 17p respectively, were assayed by standard PCR-silver staining analysis in bone marrow cells from 17 CML patients progressing from chronic phase to accelerated phase or blast crisis.<h4>Results</h4>LOH or MSI of the two microsatellites were demonstrated in 8/17 (47.5%) patients in accelerated/blastic phases. For DCC, genetic instability was revealed in 2 of 9 patients in accelerated phase and in 4 of 8 in blast crisis. For Mfd41, genetic instability was revealed in 1 of 9 patients in accelerated and in 1 of 8 in blast crisis.<h4>Conclusion</h4>Genetic instability of DCC and Mfd41 may play a role in the evolution of CML.

AIM:To clone novel gastric cancer-associated genes and investigate their roles in gastric cancer occurrence.METHODS:A method called differential display was used which allows the identification of differentially expressed genes by using PAGE to display PCR-amplified cDNA fragments between gastric cancer cells and normal gastric mucosa cells. These fragments were cloned into plasmid vector pUC18. Homology analysis was made after sequencing these fragments.RESULTS:Two novel genes were identified compared with sequences from GenBank. One was registered with the AD number AF 051783.In situ hybridization showed that these two novel genes expressed specifically in gastric cancer tissues.CONCLUSION:The two novel genes obtained by differential display were confirmed to be gastric cancer-associated genes using in situ hybridization.

An alternatively spliced form of c-myb exists that encodes an additional 120 amino acids in chicken and 121 amino acids in human and mouse. These amino acids are encoded by an additional exon, termed exon 9A. This exon is not present in v-myb, and proteins containing these amino acids have never been tested for oncogenic transformation. A series of myb constructs was therefore created in order to compare the functions of Myb proteins on the basis of their inclusion or exclusion of the amino acids encoded by exon 9A (E9A). We found that the presence of E9A resulted in a robust increase in transactivation for full-length c-Myb (CCC), as well as the singly truncated derivatives dCC and CCd, while doubly truncated Myb proteins v-Myb (dVd) and dCd did not exhibit any differences in transactivation. The increase in transactivation requires the Myb DNA-binding domain. When the leukemic transformation by the Myb proteins was tested, it was found that cells transformed by dVd resembled monoblasts, while cells transformed by CCC and its derivatives, dCd, dCC, and CCd, resembled myelomonoblasts. Despite differences in the morphology of the hematopoietic cells, the cell surface phenotypes and cell cycle profiles of transformed cells did not change for each pair of Myb proteins in the presence or absence of E9A. Thus, there was no direct correlation between the level of transcriptional activation and the strength of leukemic transformation.

Pathfinding mechanisms underlying retinal ganglion cell (RGC) axon growth from the optic chiasm into the optic tract are unknown. Previous work has shown that mouse embryos deficient in GAP-43 have an enlarged optic chiasm within which RGC axons were reportedly stalled. Here we have found that the enlarged chiasm of GAP-43 null mouse embryos appears subsequent to a failure of the earliest RGC axons to progress laterally through the chiasm-tract transition zone to form the optic tract. Previous work has shown that ventral diencephalon CD44/stage-specific embryonic antigen (SSEA) neurons provide guidance information for RGC axons during chiasm formation. Here we found that in the chiasm-tract transition zone, axons of CD44/SSEA neurons precede RGC axons into the lateral diencephalic wall and like RGC axons also express GAP-43. However unlike RGC axons, CD44/SSEA axon trajectories are unaffected in GAP-43 null embryos, indicating that GAP-43-dependent guidance at this site is RGC axon specific or occurs only at specific developmental times. To determine whether the phenotype results from loss of GAP-43 in RGCs or in diencephalon components such as CD44/SSEA axons, wild-type, heterozygous, or homozygous GAP-43 null donor retinal tissues were grafted onto host diencephalons of all three genotypes, and graft axon growth into the optic tract region was assessed. Results show that optic tract development requires cell autonomous GAP-43 function in RGC axons and not in cellular elements of the ventral diencephalon or transition zone.

The dioxin dioxygenase of Sphingomonas sp. strain RW1 activates dibenzo-p-dioxin and dibenzofuran for further metabolism by introducing two atoms of oxygen at a pair of vicinal carbon atoms, one of which is involved in one of the bridges between the two aromatic rings, i.e., an angular dioxygenation. The dxnA1 and dxnA2 cistrons encoding this dioxygenase have been cloned and shown to be located just upstream of a hydrolase gene which specifies an enzyme involved in the subsequent step of the dibenzofuran biodegradative pathway. Genes encoding the electron supply system of the dioxygenase are not clustered with the dioxygenase gene but rather are located on two other distinct and separate genome segments. Moreover, whereas expression of dxnA1A2 is modulated according to the available carbon source, expression of the dbfB gene encoding the ring cleavage enzyme of the dibenzofuran pathway, which is located in the neighborhood of dxnA1A2 but oriented in the opposite direction, is constitutive. The scattering of genes for the component proteins of dioxin dioxygenase system around the genome of Sphingomonas sp. strain RW1, and the differential expression of dioxin pathway genes, is unusual and contrasts with the typical genetic organization of catabolic pathways where component cistrons tend to be clustered in multicistronic transcriptional units. The sequences of the alpha and beta subunits of the dioxin dioxygenase exhibit only weak similarity to other three component dioxygenases, but some motifs such as the Fe(II) binding site and the [2Fe-2S] cluster ligands are conserved. Dioxin dioxygenase activity in Escherichia coli cells containing the cloned dxnA1A2 gene was achieved only through coexpression of the cognate electron supply system from RW1. Under these conditions, exclusively angular dioxygenation of dibenzofuran and dibenzo-p-dioxin was obtained. The dioxin dioxygenase was not active in E. coli cells coexpressing a class IIB electron supply system. In the course of the isolation of the dxnA1 and dxnA2 cistrons, a number of other catabolic genes dispersed over different genome segments were identified, which may indicate greater catabolic potential than was previously suspected. This finding is consistent with the catabolic versatility of members of the genus Sphingomonas, which is becoming increasingly evident, and may indicate a less well evolved and regulated but more dynamic genetic organization in this organism than is the case for better-studied pathways in organisms such as Pseudomonas species.

The three-dimensional structure of the human Rap30 DNA-binding domain has been solved by multinuclear NMR spectroscopy. The structure of the globular domain is strikingly similar to that of linker histone H5 and its fold places Rap30 into the "winged" helix-turn-helix family of eukaryotic transcription factors. Although the domain interacts weakly with DNA, the binding surface was identified and shown to be consistent with the structure of the HNF-3/fork head-DNA complex. The architecture of the Rap30 DNA-binding domain has important implications for the function of Rap30 in the assembly of the preinitiation complex. In analogy to the function of linker histones in chromatin formation, the fold of the Rap30 DNA-binding domain suggests that its role in transcription initiation may be that of a condensation factor for preinitiation complex assembly. Functional similarity to linker histones may explain the dependence of Rap30 binding on the bent DNA environment induced by the TATA box-binding protein. Cryptic sequence identity and functional homology between the Rap30 DNA-binding domain and region 4 of Escherichia coli sigma70 may indicate that the sigma factors also possess a linker histone-like activity in the formation of a prokaryotic closed complex.

Serotonin (5-HT) is an important regulator of morphogenetic activities during early central nervous system development, including cell proliferation, migration, and differentiation. The 5-HT transporter (5-HTT) plays a pivotal role in brain 5-HT homeostasis. It is also the initial target for both antidepressant drugs and drugs of abuse, some of which are potent neurotoxins. A polymorphism in the 5'-flanking regulatory region of the 5-HTT gene that results in allelic variation of 5-HTT expression is associated with anxiety-related personality traits and may influence the risk of developing affective disorders. Progress in 5-HTT gene inactivation studies are also changing views of the relevance of adaptive 5-HT uptake function in brain development and plasticity as well as processes underlying drug dependence and neurodegeneration. Despite evidence for a potential role of the 5-HTT in the integration of synaptic connections in the mammalian brain during development, adult life, and old age, detailed knowledge of the molecular mechanisms involved in these fine-tuning processes is just beginning to emerge. Integration of various strategies, including molecular genetic, transgenic, and gene transfer techniques, will allow elucidation of the 5-HTT's role in brain development, plasticity, and degeneration as well as in affective illness, drug abuse, and dementia.

In preliminary studies, the generation of thrombin in vivo was found to induce a 92% loss of functional activity of factor IX (F.IX) despite the detection by Western blotting of a product resembling activated F.IX (F.IXa) and a 25% increase in F.IX antigen levels (Hoogendoorn et al, Thromb Haemost 69:1127, 1993 [abstr]). These changes were associated with evidence of increased elastase availability. To study the possibility that these two observations were related, a detailed physical and functional characterization of the hydrolysis of purified human F.IX by human neutrophil elastase (HNE) was performed in vitro. An activated partial thromboplastin time (aPTT) clotting assay demonstrated that, although HNE eliminated the potential of F.IX to be activated, it only marginally reduced the F.IXa activity. Reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that HNE treatment of F.IX generated cleavage products of 30 and 20 kD that could not be distinguished from the respective heavy and light chain peptides that were identified in parallel studies when F.IX was activated by activated bovine F.XI (F.XIa), one of its physiological activators. In addition, nonreducing SDS-PAGE demonstrated that HNE-treated F.IX formed no complexes with antithrombin III (ATIII) in the presence of heparin. Furthermore, HNE-treated F.IX was unable to (1) bind the active site probe p-aminobenzamidine; (2) hydrolyze the synthetic peptide substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide; and (3) activate human factor X (F.X). In contrast to dansyl-Glu-Gly-Arg-chloromethyl ketone (dEGR)-inactivated F.IXa, HNE-treated F.IX (0.01 to 10,000 pmol/L) failed to inhibit the clotting activity of F.IXa (10 pmol/L) in the aPTT. NH2-terminal sequencing indicated that HNE cleaved human F.IX at Thr140, Thr144, Ile164, Thr172, and Val181. The cleavages at Thr140/Thr144 and at Thr172/Val181 are both very close to the normal F.XIa alpha-(Arg145) and beta-(Arg180) cleavage sites, respectively. In summary, the results suggest that the activatability of F.IX is eliminated after cleavage by HNE and that the inability of HNE-treated F.IX to support F.IXa-like coagulant function is a consequence of improper active site formation. These in vitro observations support the possibility that increased HNE cleavage of F.IX in vivo may contribute to the disregulation of hemostasis that occurs in conditions such as disseminated intravascular coagulation (DIC).

Colorectal carcinogenesis is widely thought to follow the adenoma-adenocarcinoma sequence. However, there are two morphologically distinct subtypes of colorectal cancer (CRC), polypoid and ulcerative. We conducted a comparative study to clarify whether different combinations of some commonly involved genetic alterations (including mutations in K-ras, p53, DCC, APC, and Rb genes) may exist between polypoid- and ulcerative-type CRCs, the two morphologically distinct types of CRC. By using PCR-based RFLP, single-strand conformational polymorphism, and loss of heterozygosity analysis, we found that K-ras codon 12 mutation was preferentially involved in polypoid tumor (P < 0.0001). There were no other significant correlations with p53 point mutation or loss of heterozygosity in chromosomes 5q, 17p, and 18q and Rb gene, which have been suggested to be involved in the progression of CRC of both morphological types. Therefore, different combinations of molecular genetic alterations may be involved in morphologically distinct types of colorectal carcinogenesis, and the K-ras codon 12 mutations may play an important role in polypoid growth of CRC. These results shed light on the function of K-ras oncogenes involved in colorectal carcinogenesis and may be important in the future design of genetic screening programs, determination of prognosis, and treatment for patients with CRC.

<h4>Objective</h4>Pathogenesis of diabetes-related microvascular complications involving oxidative damage by free radicals has been demonstrated. Free radical generation has been shown to derive largely from iron. Our objectives, therefore, were to determine if there is an increased incidence and/or an accelerated course of nephropathy in patients with diabetes, secondary to transfusional hemochromatosis, and to examine whether free radical activity contributes to the development of this complication.<h4>Research design and methods</h4>We evaluated nine patients with homozygous beta-thalassemia, complicated by clinically overt diabetes, for diabetic nephropathy over a 7-year period. Lipid peroxidation was quantified by measuring the presence of 20 saturated and unsaturated aldehydes, and results were compared with five normotensive type 1 diabetic patients without iron overload.<h4>Results</h4>Nephropathy developed in five of nine patients (55%) after a mean duration of overt diabetes of 3.6 +/- 2.0 years. Three patients showed evidence of progressive microalbuminuria over a 7-year period (24.7-46.2, 52.2-430.1, and 17.7-54.3 micrograms/min, respectively). Two patients with borderline microalbuminuria (19.9 and 14.5 micrograms/min, respectively) demonstrated stable albumin excretion rates over the follow-up period. Total aldehyde concentration was significantly higher in beta-thalassemia diabetic patients, compared with nonthalassemic diabetic control subjects (8,106 +/- 1,280 vs. 4,594 +/- 247 nmol/l; P < 0.0001). The three patients with progressive microalbuminuria demonstrated significantly higher total aldehyde concentration, compared with the other beta-thalassemia diabetic patients with stable albumin excretion (9,428 +/- 337 vs. 7,445 +/- 1,003 nmol/l; P < 0.01). Serum vitamin E concentrations were significantly lower in beta-thalassemia patients with diabetes, compared with diabetic patients without iron overload (12.1 +/- 6.0 vs. 25.9 +/- 11.4 mumol/l; P = 0.02). Serum vitamin C concentrations did not differ between the two groups. Multiple regression analysis demonstrated total aldehyde concentration to be the most significant predictor for the development of microalbuminuria (P = 0.01), followed by the duration of diabetes (P = 0.02) and glycemic control (P = 0.02).<h4>Conclusions</h4>Early development and an accelerated course of diabetic nephropathy in iron-loaded patients with beta-thalassemia are observed. These findings may be attributed to high oxidative stress in these patients, which is secondary to iron-derived free radicals and to the patients' diminished antioxidant reserves.

The mechanism by which a novel major histocompatibility complex class I protein, HFE, regulates iron uptake into the body is not known. HFE is the product of the gene that is mutated in >80% of hereditary hemochromatosis patients. It was recently found to coprecipitate with the transferrin receptor (Feder, J. N., Penny, D. M., Irrinki, A., Lee, V. K., Lebron, J. A., Watson, N., Tsuchihashi, Z., Sigal, E., Bjorkman, P. J., and Schatzman, R. C. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1472-1477; Parkkila, S., Waheed, A., Britton, R. S., Bacon, B. R., Zhou, X. Y., Tomatsu, S., Fleming, R.E. , and Sly, W. S. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 13198-13202) and to decrease the affinity of transferrin for the transferrin receptor (Feder et al.). In this study, HeLa cells were transfected with HFE under the control of the tetracycline-repressible promoter. We demonstrate that HFE and the transferrin receptor are capable of associating with each other within 30 min of their synthesis with pulse-chase experiments. HFE and the transferrin receptor co-immunoprecipitate throughout the biosynthetic pathway. Excess HFE is rapidly degraded, whereas the HFE-transferrin receptor complex is stable. Immunofluorescence experiments indicate that they also endocytose into transferrin-positive compartments. Combined, these results suggest a role for the transferrin receptor in HFE trafficking. Cells expressing HFE have modestly increased levels of transferrin receptor and drastically reduced levels of ferritin. These results implicate HFE further in the modulation of iron levels in the cell.

The aim of this study was to add to existing information on the effects of certain tumour markers expressed by breast cancers on tumour malignancy as evidenced by size of primary and occurrence of lymph node invasion. One hundred freshly resected breast cancers were examined by immunocytochemical staining of imprint smears for Cathepsin D and pS2. Oestrogen receptor (ER) and progesterone receptor (PR) were tested for by dextrose-coated charcoal (DCC) assay and the results correlated with tumour size, histology and presence or absence of lymph node metastases at the time of surgery using chi(2) analysis. A significant positive correlation was demonstrated between Cathepsin D positivity and ER, PR and pS2 positivity. In tumours < 2 cm in diameter at surgery a positive correlation was observed between Cathepsin D positivity and the presence of lymph node metastases. The findings support the hypothesis that Cathepsin D may promote early metastasis, possibly by its proteolytic activity.

No abstract available.

The high incidence of loss of heterozygosity (LOH) on chromosome 18q in advanced non-small cell lung carcinomas indicates the presence of tumor suppressor gene(s) on this chromosome arm, which plays an important role in the acquisition of malignant phenotypes in lung cancers. In the present study, we examined 62 lung cancer specimens and 54 lung cancer cell lines for allelic imbalance at 11 microsatellite loci to define common regions of 18q deletions. Allelic imbalance of 18q was detected in 24 (55.8%) non-small cell lung carcinoma specimens and in 6 (31.6%) small cell lung carcinoma specimens, whereas a similar frequency of LOH was statistically inferred to occur in cell lines by analyzing marker homozygosity as an indirect measure of LOH. Five specimens and 11 cell lines showed partial or interstitial deletions of chromosome 18q, and 2 of them had homozygous deletions at the 18q21.1 region. A commonly deleted region was assigned between the D18S46 and y953G12R loci. The size of this region is less than 1 Mb, and the coding exons of three candidate tumor suppressor genes, Smad2, Smad4, and DCC, were mapped outside the region. This result suggests that the common region harbors a novel tumor suppressor gene involved in the progression of lung cancer.

1. In order to improve the in vivo stability of the opioid peptide dermorphin we synthesized O-betaglucosylated analogs ([Ser7-O-betaGlc]dermorphin and [Ser7-O-betaGlc(Ac)4]-dermorphin) and C-alphagalactosylated analogs ([Ala7-C-alphaGal]dermorphin and [Ala7-C-alphaGal(Ac)4]-dermorphin). 2. O- and C-glycosylation of dermorphin halved the peptide affinity for brain mu-opioid receptors and the biological potency in guinea-pig ileum assay (GPI). Despite their lower opioid receptor affinity, when administered intracerebroventricularly (i.c.v., 8-40 pmol) and subcutaneously (s.c., 0.5-3 micromol kg(-1)) in rats, glycosylated analogs were two times more potent than dermorphin in reducing the nociceptive response to radiant heat. Acetylation of sugar hydroxyl groups reduces 5-10 times both biological activity on GPI and mu-receptor affinity, whereas the antinociceptive potency was equal to (i.c.v.) or only two-three times lower (s.c.) than dermorphin potency. 3. Blood-Brain Barrier Permeability Index (BBB-PI) of the glycodermorphins was significantly higher than that of dermorphin, indicating a facilitated entry into the brain: O-beta-linked glucoconiugates are expected to enter CNS by the glucose transporter GLUT-1 of the endothelial barrier. However the calculated BBB-PI for the C-alphagalactoside was about two times higher than that of the O-betaglucoside, excluding the implication of GLUT-1 that is known to be selective for O-beta-links and preferring for the exose glucose. 4. The enhanced brain permeability with the subsequent decrease in peripheral dosage of these opioid peptides did not result in lowering constipation.

The anticoagulant activity of antithrombin III (ATIII), as observed in a plasma-free system consisting of thrombin and fibrinogen, is readily reduced by acetaldehyde (AcH) at concentrations of 447, 89.4, and 17.9 mM. Whereas control thrombin-fibrinogen mixtures clotted in 17.7+/-0.75 sec, ATIII prolonged clotting time to 55.0+/-1.75 sec on preincubation with thrombin for 30 min at room temperature. On subsequent preincubation of ATIII with the AcH for 30 min at room temperature and passage of the mixture through Sephadex G-25 minicolumns to remove excess AcH, the eluates were tested for anticoagulant activity. Clotting times of 20.9+/-1.0, 32.3+/-1.0, and 45.3+/-1.6 sec were obtained with 447, 89.4, and 17.9 mM AcH-ATIII mixtures, respectively. These data suggest that functional groups on ATIII, such as guanidiniums, aminos, and others are susceptible to adduct formation with AcH, thereby altering the shape and charge of the anticoagulant. As a consequence of this type of reaction, an altered molecule of reduced biological activity may be produced. These experimental results may explain, in part, the reduction in ATIII levels reported by others in patients with alcoholic liver disease.

Genetic haemochromatosis (GH) is one of the most common hereditary diseases, with a prevalence of 1-5/1000 in the Western world. In 90 per cent of cases a mutation is found in an MHC-class-like gene designated HFE, involving a substitution at position 282 of the HFE protein and resulting in defective binding of beta(2)-microglobulin. Animals with beta(2)-microglobulin deficiency develop iron overload, indicating this protein to be involved in the regulation of iron metabolism. Hepatic iron overload results in increased production of oxygen free radicals and peroxidation of membrane lipids, thus causing damage to lysosomes, mitochondria and the endoplasmic reticulum. These cellular events may progress to cell death, fibrogenesis, and the development of liver cirrhosis which is associated with a 200-fold increase in risk of hepatocellular carcinoma. In addition to the risk of diabetes, arthralgia, cardiac arrhythmia, pituitary insufficiency and hypogonadism, iron excess is also associated with aggravation of the cytotoxic effects exerted on hepatocytes by other agents such as alcohol or hepatotrophic viruses. The treatment of iron overload in GH consists of weekly venesection until the serum ferritin level is normalized, followed by maintenance therapy. Survival rates are normal if the disease is detected and treated before complications have developed.

A population association between a regulatory variation in the promoter of the serotonin transporter gene (HTT) and severe alcohol dependence was recently reported. We analyzed this potential association in a large number of systematically ascertained families in the United States; these families had at least three first-degree relatives who were alcohol-dependent. Analyses focused on individuals defined as alcohol-dependent by criteria from ICD-10 and on subsets of these individuals reporting withdrawal-related symptoms. Application of the transmission disequilibrium test did not provide support for either linkage or association between this functional polymorphism and alcohol dependence; there was no significant bias in the transmission of either allele to the alcohol-dependent offspring. We also report that African Americans differ from Caucasians in allele frequencies for this polymorphism.

Surface-bound thrombin which contributes to vessel wall hyperplasia, is resistant to inhibition by heparin/antithrombin III (/ATIII) but not to inhibition by dermatan sulphate/heparin cofactor II (/HCII). To determine the effects of heparin and dermatan sulphate on vessel wall hyperplasia after a first or second injury, rabbit carotid arteries first were injured, using a standard procedure (first injury). Half of the first-injury rabbits were given heparin, dermatan sulphate, or saline, 5 minutes before and at 30-minute intervals over 2 hours post-injury, and then allowed to recover. Four weeks later, the first-injury treated animals were killed and their injured carotid arteries were processed histologically. The remaining untreated first-injury rabbits were also allowed to recover. At 4 weeks, those rabbits were re-anesthetized and their first-injury arteries (which were occluded >75%), were isolated, and vessel wall lumen patency was re-established by endarterectomy (second injury). During this second injury, the animals were treated with heparin, dermatan sulphate, or saline as described above. Four weeks after this second injury, these rabbits were killed and their second injury arteries were processed histologically. Intimal hyperplasia determined histologically, was expressed as an x-fold increase in vessel wall cross-sectional area (i.e., [(media+intima area) media area]). Vessel wall lumen occlusion was expressed as [1-(lumen area/internal elastic lamina area) x 100; % occlusion]. Vessel wall area in the saline-treated animals, increased 2.6+/-1.2 and 2.4+/-1.0 fold respectively, means+/-SD, n = 12, within 4 weeks of the first and second injuries. These increases were due to intimal hyperplasia and associated with 75+/-19% and 79+/-21% occlusion of the vessel wall lumen, respectively. Heparin had little effect, whereas dermatan sulphate (1) decreased hyperplasia by 45% after the first injury and by 47% after the second injury, p<0.008 and <0.03, respectively, and (2) decreased vessel wall occlusion 47+/-12% and 33+/-5% after the first and second injury, respectively. We conclude that (1) dermatan sulphate/HCII may be a useful inhibitor of vessel wall hyperplasia following vessel wall injury, and (2) this effect can be achieved by an acute anticoagulant treatment at the time of injury, unlike heparin/ATIII.

L-Type voltage operated calcium channel plays an important role in the contraction-relaxation of muscle cells. The alpha-1 subunit of this pentameric protein performs catalytic functions. Multiple mRNA isoforms of this subunit are generated by alternative splicing. For example, an exon encoding 11 amino acids (aa) between the third and fourth transmembrane domains produces two mRNA isoforms in gastrointestinal (GI) tract. The corresponding exon in brain encodes 15 aa. Whether the alpha-1 mRNA isoforms are translated into the corresponding isozymes remain unknown. To address this issue and to characterize the exon in brain, isozymes specific anti-peptide polyclonal antibodies were raised. Both the antibodies reacted with a protein of Mr 180 kDa in the heart and GI-tract, while no reaction was obtained in the kidney or liver. Brain expressed the isoform containing the same exon encoding 11 amino acids present in GI-tract, but the corresponding isozymes were of Mr 145-150 kDa. These findings suggest a tissue-specific translation of the alpha-1 isozymes.

We evaluated the effect of antithrombin III (ATIII), a serine protease inhibitor (SERPIN), on induction of nitric oxide (NO) synthesis in murine peritoneal macrophages. Incubation of macrophages with ATIII plus interferon-gamma (IFN-gamma) but not ATIII alone induced nitrite accumulation (a metabolite of NO) in a dose-dependent manner. Expression of the inducible nitric oxide synthase isoform was confirmed by Western blot. NO synthesis was inhibited by NG-monomethyl-l-arginine, by complexing ATIII with thrombin or by rabbit anti-human ATIII antiserum. Addition of polymyxin B to macrophage cultures failed to inhibit ATIII/IFN-gamma-induced NO synthesis, excluding lipopolysaccharide contamination. 125I-ATIII bound to macrophages in a dose-dependent, specific, and saturable manner, with a Km of approximately 7.1 nM. Our results demonstrate that ATIII, but not ATIII/thrombin complex, acts to costimulate macrophage activation and NO synthesis via a novel receptor mediated mechanism, which may indicate a role for SERPINs in macrophage activation.

The functional levels of AT-III were determined to the following groups: A. Eleven healthy non pregnant women B. Thirteen healthy pregnant women (third trimester). C. Six preeclamptic patients. D. Five patients with eclampsia and/or HELP syndrome. The results were as follows: [table: see text] A different grade of DIC may explain the low activity of AT-III in preeclampsia and a more severe coagulation disorder in eclampsia and HELP syndrome. Our preliminary results encourage other prospective studies including larger populations to determine its usefulness as early diagnostic test and severity marker of the disease.

Although viral variants which are not recognized by epitope-specific cytotoxic T lymphocytes (CTL) have been shown to arise during a number of persistent virus infections, in many cases their significance remains controversial: it has been argued that the immune response is sufficiently plastic to contain their replication. In this review, we describe the mechanisms by which amino acid changes in viral proteins may affect epitope recognition by virus-specific CTL, and discuss the viral and immunological basis for the emergence of viral variants bearing such amino acid changes during infection. We then consider the impact that viral variation may have on the host CTL response and its ability to contain virus replication. We argue that the emergence of a viral variant demonstrates that it must have an in vivo replicative advantage, and that as such, the variant must tip the balance between virus replication and immune control somewhat in favor of the virus. Further, we suggest that although the immune response can evolve to recognize new viral epitopes, the CTL generated following such evolution frequently have a reduced ability to contain virus replication. We conclude that this escape mechanism likely does make a significant contribution to persistence/pathogenesis during a number of different virus infections.

We prospectively evaluated the antenatal changes in antithrombin-III (AT-III) activity and liver enzymes in a woman with a triplet pregnancy. A gradual decline in AT-III activity occurred in the absence of clinical signs of preeclampsia and preceded the onset of the typical HELLP syndrome in this patient. Monitoring of AT-III activity might help to avoid the development of severe HELLP syndrome.

<h4>Aim</h4>To investigate the role of DCC gene inactivation in the development and progression of gastric cancer.<h4>Methods</h4>Loss of heterozygosity and loss of expression of the DCC gene was studied in 51 surgical specimens of gastric cancer using detection based on polymerase chain reaction.<h4>Results</h4>Loss of heterozygosity was found in 35.3% (18 of 51) of specimens and was detected more often in stage III and IV (50%) than in stage I and II cancers (14.3%) (p < 0.05). Occurrence of loss of heterozygosity was not correlated with histological type, tumour size, depth of invasion, or lymph node metastasis. Loss of expression was found in 49% of cases (25 of 51). Loss of expression was not significantly correlated with any clinicopathological variable.<h4>Conclusions</h4>Loss of heterozygosity and loss of expression of the DCC gene are often encountered in gastric cancer. Loss of heterozygosity of the DCC gene is a late event and associated with malignant progression.

<h4>Introduction</h4>Low doses of androgen are used in women for the symptomatic treatment of sexual dysfunction and premenstrual syndrome (PMS). However, little is known about the long-term safety of androgen use in women. This study investigated the effects of low dose exogenous testosterone (T) on lipid metabolism, markers of activation of the coagulation system and ultrasonographic ovarian morphology in women.<h4>Patients</h4>Twenty-two patients with severe PMS (age 39.6 +/- 3.1 years, mean +/- SD) treated with subcutaneous T implants (100 mg six monthly) for at least two years (mean duration 3.3 (+/- 0.9 years) were compared with 22 age-matched (age 37.7 +/- 2.9 years) control patients with severe PMS who had not previously received T treatment. All women continued to have regular menses.<h4>Measurements</h4>Fasting blood samples were obtained for measurement of lipids and clotting factors and ovarian ultrasound examination carried out between days 1-4 of the menstrual cycle (2.3 +/- 1.2 months after the T implant in T-treated group).<h4>Results</h4>Mean plasma T was 4.5 +/- 2.2 nmol/l, and 1.9 +/- 0.6 nmol/l in the treated and control groups, respectively. In the T-treated group apolipoprotein-A1 (Apo-A1) (treated 99.2 +/- 12 vs controls 116.2 +/- 27.7 g/l, P < 0.01) and high density lipoprotein cholesterol (HDL-C) (treated 1.3 +/- 0.3 vs controls 1.5 +/- 0.4 nmol/l, P < 0.01) were significantly decreased. In addition very low density lipoprotein cholesterol (VLDL-C) (treated 0.4 +/- 0.3 vs controls 0.2 +/- 0.1 nmol/l, P < 0.05) was increased in T-treated patients. There were no differences in total serum cholesterol and triglyceride or low density lipoprotein cholesterol (LDL-C), apolipoprotein B (Apo-B), lipoprotein(a), lecithin:cholesterol acyltransferase and cholesteryl ester transfer protein activity. There was no difference in clotting factors between the two groups which included prothrombin time, fibrinogen, antithrombin-III, protein-C, protein-S (total and free), tissue plasminogen activator, plasminogen activator inhibitor, beta-thromboglobulin and prothrombin fragments 1.2. Ultrasound showed normal ovarian architecture with no evidence of polycystic ovarian changes in any patients in the T-treated group. No patient experienced adverse symptoms while on T treatment, in particular, there were no complaints of hirsutism or acne and no one requested termination of treatment.<h4>Conclusion</h4>Low-dose testosterome administration to women for over two years did not induce changes in ovarian architecture but had small, potentially atherogenic effects on some parameters of lipid and lipoprotein metabolism. However, no differences were detected in markers of activation of the clotting system to indicate an actual increase in the risk of thrombosis. Overall, this study provides largely reassuring data about the safety of low-dose androgen treatment in women. However, caution should be exercised in women with existing or a familial predisposition to lipid abnormalities, because of the small but significant changes found in HDL-C, apo-A1 and VLDL-C.

The cell adhesion molecules are ubiquitous recognition molecules that allow cells to communicate with one another and their environment. Through these molecules, complex alterations in the cytoplasmic messenger pathways and the microfilamentous cytoskeleton can lead to profound alterations in cell division, differentiation, behaviour, and function (fig 9). It is difficult to conceive of a group of molecules that could be more important to pathologists and to their understanding of disease processes.

<h4>Aim</h4>To provide a more efficient method for isolating DNA from peripheral blood for use in diagnostic DNA mutation analysis.<h4>Methods</h4>The use of blood impregnated filter paper and Chelex-100 in DNA isolation was evaluated and compared with standard DNA isolation techniques.<h4>Results</h4>In polymerase chain reaction (PCR) based assays of five point mutations, identical results were obtained with DNA isolated routinely from peripheral blood and isolated using the filter paper and Chelex-100 method.<h4>Conclusion</h4>In the clinical setting, this method provides a useful alternative to conventional DNA isolation. It is easily implemented and inexpensive, and provides sufficient, stable DNA for multiple assays. The potential for specimen contamination is reduced because most of the steps are performed in a single microcentrifuge tube. In addition, this method provides for easy storage and transport of samples from the point of acquisition.

Hereditary haemochromatosis is an autosomal recessive disease in which there is defective regulation of iron absorption, causing gradual accumulation of excessive amounts of iron in certain organs. Recently, a candidate gene for hereditary haemochromatosis has been identified, located on the short arm of chromosome 6, telomeric to the major histocompatibility complex (MHC) and showing sequence homology to the human leucocyte antigen (HLA) class I genes. Two mutations have been found in this gene that are potential markers for haemochromatosis. The first, a cysteine to tyrosine substitution (Cys282Tyr) is strongly associated with the disease, whereas the second mutation, a histidine to aspartic acid substitution (His63Asp) shows a less obvious relation. To examine the importance of this second mutation in hereditary haemochromatosis it is important to study the links between this genotype and abnormalities of iron metabolism. A polymerase chain reaction method using sequence specific primers is described which might be useful for identifying those individuals carrying the mutation that encodes the His63Asp substitution, who might be at risk from a milder form of haemochromatosis.

<h4>Objectives</h4>To compare the results of magnetic resonance imaging (MRI) and thallium-201 (201Tl) SPET scanning with laboratory analyses including CSF DNA detection, brain biopsy, and necropsy in the discrimination of cerebral lymphoma and toxoplasmosis in patients with AIDS.<h4>Methods</h4>A retrospective study of 32 patients infected with HIV who had focal CNS lesions on MRI as a result of either lymphoma or toxoplasmosis.<h4>Results</h4>18 patients had lymphoma, 12 had toxoplasmosis, and two had both. Toxoplasma IgG antibodies were detected in only seven patients--four with toxoplasmosis, two with lymphoma, and one with both diagnoses. Epstein-Barr virus DNA was detected in CSF of all six patients with lymphoma and none of two with toxoplasmosis. MRI showed multiple lesions in 23 patients, appearances did not discriminate between lymphoma and toxoplasmosis; nine patients had single lesions, of these eight had lymphoma (p = 0.044, two tailed Fisher's exact test) 201Tl SPET showed accumulation in 17 with lymphoma and six with toxoplasmosis (p = 0.034, two tailed Fisher's exact test). Of nine patients with single lesions on MRI and 201Tl SPET with focal accumulation eight had lymphoma. 201Tl SPET uptake ratios of > or = 2.9 were only seen with lymphoma.<h4>Conclusion</h4>Knowledge of patients' toxoplasma serostatus does not aid discrimination between lymphoma and toxoplasmosis. Single lesions on MRI with focal accumulation of 201Tl strongly suggest lymphoma. Multiple lesions on MRI with 201Tl SPET uptake ratios > or = 2.9 also suggest lymphoma; uptake ratios less than 2.1 do not aid discrimination. Detection of Epstein-Barr virus DNA in CSF is highly sensitive and specific for cerebral lymphoma.