Gene Literature Dashboard

This study dealt with the effect of citrate phosphate dextrose adenine (CPDA) whole blood stored at 4 degrees C for 5 weeks and fresh frozen plasma at -30 degrees C for 12 months on coagulation, fibrinolytic and kallikrein system activation. Stored whole blood showed a significant decrease in ATIII activity by the second week with a significant decrease in thrombin-antithrombin complex by the fourth week. alpha 2-antiplasmin and plasminogen decreased significantly by the first and second week, respectively, accompanied by a significant increase in D-dimer level by the fourth week. A significant decrease in C1-inhibitor activity occurred by the first week associated with a significant increase in kallikrein activity by the third week. However, all measured parameters were minimally affected in fresh frozen plasma. Therefore, fresh frozen plasma supplemented with packed RBCs are preferred to whole blood stored over 3 weeks especially in patients with proteolytic enzyme system activation.

Synthesis of 1-methyl-2-fluoro-4,5-dicyanoimidazole was done by halogen exchange between 1-methyl-2-bromo-4,5-dicyanoimidazole and potassium fluoride. Halogen exchange between 1-methyl-2-bromo-4,5-dicyanoimidazole and lithium chloride in N-methylpyrrolidinone at 150 degrees C yielded 1-methyl-2-chloro-4,5-dicyanoimidazole, and additional heating to 210 degrees C resulted in the demethylation to yield 2-chloro-4,5-dicyanoimidazole. Thermolyses of the 2-halo-4,5-dicyanoimidazole derivatives (F, Cl) and 1-iodo-2-halo-4,5-dicyanoimidazole derivatives (Cl, Br, I) between 100 and 290 degrees C were found to yield Tris(imidazo)[1,2-a:1,2-c:1,2-e]-1,3,5-triazine-2,3,5,6,8,9-hexacarbonitrile, or HTT, with (C(5)N(4))(3) composition. HTT has been characterized and purified and the crystal structure obtained. Thermolysis of HTT at 490-500 degrees C gives a material with C/N = 1.020. The thermal properties of HTT and its decomposition products show thermal stability to 350 degrees C.

The diagnosis of many hemostatic defects in infancy and childhood depends on the establishment of normal levels of various hemostatic factors. In this study, measurements of the natural anticoagulants (proteins C, S, and antithrombin III), as well as the fibrinolytic factors (tPA and PAI) were undertaken in healthy neonates (cord blood; n = 56), as well as in healthy children, up to 12 years of age (n = 103). The results were compared to normal adult values obtained from blood donors (n = 49). Neonatal values were found to be 50% of those obtained in adults and their mean concentrations were as follows: ATIII antigen = 48.4%, ATIII activity = 61.6%, protein C antigen = 47.7%, protein C activity = 57.2%, total protein S = 41.8% and tPA = 1.9 ng/mL. PAI level (25.7 ng/mL) was similar to adult values. In the first three years of life, almost all the hemostatic factors, other than PAI, gained adults levels. The diminished concentrations of the natural anticoagulants, in addition to the hypofibrinolysis in neonates, shifts the hemostatic balance towards fibrin formation and safeguards effective hemostasis. The values obtained in this study may serve as local reference values.

Organophosphorus pesticides are one of the most commonly used insecticide classes. They act through a potent inhibition of acetylcholinesterase (AChE). Many of them must undergo transformation into the corresponding oxon analogs to inhibit AChE. This study showed that a brain tissue subfraction transformed methyl parathion (O,O-dimethyl O-p-nitrophenyl phosphorothioate) in vitro. Methyl parathion activation was assayed by solvent extraction of the products followed by HPLC and GC-MS analyses and, indirectly, by the inhibition of AChE present in the incubation mixture. The lack of impairment of AChE after 2 h of incubation of the brain subfraction with methyl parathion and, alternatively, with NADPH, CO, SKF 525-A, piperonyl butoxide or nitrogen indicated that this brain subfraction transformed methyl parathion without the involvement of a mixed-function oxidative pathway. The results from HPLC analysis did not show a peak corresponding to methyl paraoxon (O,O-dimethyl O-p-nitrophenylphosphate), but showed the production of an unidentified peak which eluted nearby standard methyl parathion (retention times of 10.65 and 8.86 min, respectively). GC-MS analysis suggested that the unidentified product could be a methyl parathion isomer.

We report on the use of spectral map analysis of the inter- and intraclade neutralization data of 14 sera of human immunodeficiency virus type 1 (HIV-1)-infected individuals and 16 primary isolates, representing genetic clades A to H in group M and group O. This multivariate analysis has been used previously to study the interaction between drugs and receptors and between viruses and antiviral compounds. The analysis reveals the existence of neutralization clusters, not correlated with the known genetic clades. The structural factors that have been identified may correlate with the most important neutralization epitopes. Three key primary HIV-1 isolates, which allow discrimination of sera that are likely or unlikely to neutralize primary isolates from most of the genetic clades, were identified. Our method of analysis will facilitate the evaluation as well as the design of suitable HIV-1 vaccines, which induce high-titer interclade cross-neutralizing antibodies.

Huntington's disease (HD) is caused by a genetic mutation that results in a polyglutamine expansion in huntingtin. The time course of neuronal loss in the HD striatum and other affected brain regions before the onset of symptoms is unknown. To determine the potential influence of huntingtin on brain development, we examined its expression in the developing mouse and in human control and HD brain. By Western blot, huntingtin was detected throughout the adult mouse brain and at all stages of embryonic and postnatal brain development. The protein increased significantly between postnatal day 7 (P7) and P15, which marks a period of active neuronal differentiation and enhanced sensitivity to excitotoxic injury in the rodent striatum. Immunoreactivity was found in neurons throughout the brain and localized mostly to the somatodendritic cytoplasm and to axons in fiber bundles. Staining was variable in different groups of neurons and within the same cell population. In developing brain, huntingtin was limited primarily to neuronal perikarya. Increased immunoreactivity in large neurons followed the gradient of neurogenesis and appeared in the basal forebrain and brainstem by embryonic days 15-17, in regions of cortex by P0-P1, and in the striatum by P7. In human brain at midgestation (19-21 weeks), huntingtin was detected in all regions. The brain of a 10-week-old infant with the expanded HD allele expressed a higher molecular weight mutant form of huntingtin at levels comparable to those of the wild-type protein. Thus, mutant huntingtin is expressed before neuronal maturation is complete. Results suggest that huntingtin has an important constitutive role in neurons during brain development, that heterogeneity in neuronal expression of the protein is developmentally regulated, and that the intraneuronal distribution of huntingtin increases in parallel with neuronal maturation. The presence of mutant huntingtin in the immature HD brain raises the possibility that neurons may be affected during brain development and possibly in the postnatal period when vulnerability to excitotoxic injury is at its peak.

Using the shell vial assay and sequence analysis of a variable region of the glycoprotein B (gB) gene, cytomegalovirus (CMV) excretion rates in urine and virus transmission were studied among 93 children from six day care centers (DCCs). During a 6-month period, excretion rates differed significantly between DCCs (P < .001). The 6 gB gene sequences, obtained from 24 CMV-infected children, were classified in four previously defined groups. In five DCCs, 2 or 3 strains cocirculated, and none was dominant. Infection could have been acquired outside the DCC for 2 children and inside it for 9. Two children from the same DCC had mixed infections. No differences in hygiene, child care practices, or experience and level of qualification of the staff could explain this wide variety of excretion rates between DCCs. The distribution of gB gene patterns observed does not suggest that 1 type was dominant or more efficiently transmitted.

HHC is the most common inherited metabolic disease among the white population worldwide, with a gene frequency of about 10% and a frequency of homozygosity of about 1 of 250. Many patients harbor a common haplotype of informative markers on chromosome 6p2l.23, suggesting a strong founder effect exerted by a common Celtic ancestor. With the advent of screening tests (serum Tf saturation, fe), many subjects with HHC are being identified before development of cirrhosis or diabetes mellitus, and early detection is important because prompt and vigorous iron reduction prevents development of such complications and assures normal life expectancy. The HIC can be estimated as accurately by specialized magnetic resonance imaging or susceptometric measurements as by chemical measurements on liver biopsy specimens. However, biopsy specimens retain value for showing fibrosis/cirrhosis and dysplastic hepatocytes, both of which increase risks of HCC development. There is growing evidence that iron in the liver plays an important role in non-HHC diseases, such as alcoholic liver disease, chronic viral hepatitis, and porphyria cutanea tarda. The complicated, manifold roles of iron in pathogenesis of the latter disorder include enhancement of production and irreversible oxidation of uroporphyrinogen, as well as formation of an inhibitor targeted specifically at hepatic uroporphyrinogen decarboxylase. The nature of the gene and gene product that are abnormal in HHC remain elusive, despite the intense efforts of several investigative groups. The search has been hampered by a dearth of informative markers in HHC patients in the relevant region of chromosome 6p. Note added in proof: The cloning of a candidate gene, the mutation of which may perhaps cause HLA-linked hemochromatosis, has just been reported (Feder et al: A novel MHC class I-like gene is mutated in patients with hereditary haemochromatosis. Nature (Genetics) 1996;399-408). These workers identified a 250-kb region move than three megabases telomeric of the MHC that was identical in 85% of chromosomes of HHC patients. Within this region, they identified a gene related to the MHC class I family, termed HLA-H, containing two missense alterations one of which is predicted to inactivate this class of proteins. 83% of 178 patients were homozygous for this mutation (Cys 282Tyr). This variant was also found on 3.2% of control chromosomes, as would be expected for such a common disorder. Functional studies are awaited with great interest.

The prostacyclin synthase gene isolated from human genomic libraries (PTGIS) consists of 10 exons spanning approximately 60 kb. All the splice donor and acceptor sites conform to the GT/AG rule. Genomic Southern blot and fluorescence in situ hybridization analyses revealed that the human prostacyclin synthase gene is present as a single copy per haploid genome and is localized on chromosome 20q13. 11-q13.13. The 1.5-kb sequence of the 5'-upstream of the translational initiation site contained both GC-rich and pyrimidine-rich regions and consensus sequences of the transcription factor recognition sites such as Sp1, AP-2, the interferon-gamma response element, GATA, NF-kappaB, the CACCC box, and the glucocorticoid response element. The core binding sequence (GAGACC) of the shear stress responsive element was also found in the 5'-flanking region of the gene. The major product of the primer extension analysis suggested that the transcription of the gene started from the positions around 49 bp upstream of the translational initiation codon. Transient transfection experiments using human aortic and bovine arterial endothelial cells demonstrated that the GC-rich region (positions -145 to -10) possessed a significant promoter activity. The 6-kb downstream sequence of the translational termination codon contained multiple polyadenylation signals, Alu repeat sequences, and the consensus sequence of the primate-repetitive DNA element, MER1. Two sizes of the prostacyclin synthase mRNAs (approximately 6 and 3.3 kb) were detected with the human aorta and lung. RNA blot hybridization analysis using the 3'-untranslated region as probe indicated that the sizes of the 3'-flanking regions were different in the major 6-kb and minor 3.3-kb mRNAs.

Determination of breast cancer estrogen receptor (ER) status as a predictor of tumor response to adjuvant endocrine therapy remains a mainstay of breast cancer management. Recent second generation anti-ER antibodies and new epitope retrieval methods have produced paraffin-based immunohistochemical results that correlate closely with the dextran-coated charcoal (DCC) assay and appear to represent a superior method of ER assay. The authors determined the ER status of 103 invasive breast cancers by paraffin-based, automated immunohistochemistry on the Ventana ES using a new monoclonal antibody, CC4-5, and compared the results to those of parallel DCC biochemical analysis and manual immunohistochemical analysis using anti-ER monoclonal antibody ER1D5. The specificity of the CC4-5 antibody for ER protein was confirmed by Western blot analysis. Sixty of 103 cases were positive for ER by CC4-5 automated immunohistochemistry. With a ligand binding assay threshold value of 20 fmol/mg protein, there were 50 positive cases by biochemical assay. The biochemical results corresponded to an 88% rate of agreement with automated CC4-5 staining. Analysis of discordant cases revealed that the majority of CC4-5 immunopositive only cases (8 of 11) were strongly positive, stroma rich tumors, suggesting that corresponding biochemical measurements were diluted by non representative stromal tissue. There was only one immunonegative, biochemically positive case (27 fmol/mg protein). Semiquantitation of CC4-5 staining using percent positive tumor cells or weighted average staining intensity (HSCORE) showed moderate to good correlation with quantitative DCC results (r = 0.64 and 0.62, P < .0001). ER1D5 was not suitable for use on the Ventana ES, most likely due to temperature constraints of the instrument. By manual ER1D5 staining, 40 of 79 examined cases were positive corresponding to a 99% rate of agreement with automated CC4-5 staining. Semiquantitation of ER1D5 staining by percent positive tumor cells and weighted average staining intensity (HSCORE) showed excellent correlation with semiquantitation of automated CC4-5 results (r = 0.90 and 0.88, P < .0001). Automated immunohistochemistry using the Ventana ES and monoclonal antibody CC4-5 is a reliable method for determining breast cancer ER status. As with other immunohistochemical methods, direct correlation with morphology precludes errors due to tissue sampling, allowing for accurate analysis of stroma-rich or partially necrotic tumors and small neoplasms that otherwise would yield insufficient tumor tissue for biochemical analysis.

Twenty-four cases of histologically defined follicle centre cell (FCC) lymphoma have been examined for allele imbalance at 19 microsatellite loci spanning the length of chromosome 6, including six markers within the major histocompatibility complex (MHC), using fluorescent polymerase chain reaction (PCR) to amplify microsatellites. Nineteen cases were observed in which imbalance of one or more markers on chromosome 6 had occurred (79%). The frequency of allele imbalance was significantly higher on 6p than 6q, and two regions of deletions, 6p24-25 and 6p21.3-23, were identified in which the loci showed a significantly high allele imbalance frequency.

In spite of the fact that planar lipid bilayers are still the best-suited artificial membrane system for the study of reconstituted ion channels and receptors, data dealing with their physical characterization, especially as regards dynamics, are scanty. A combined electrical and optical chamber was designed and allowed fluorescence recovery after photobleaching recovery curves to be recorded from stable virtually solvent-free bilayers. D, the lateral diffusion coefficient of N-(7-nitrobenzoyl-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn- glycero-3-phosphoethanolamine, was found to be relatively insensitive to the phospholipid composition (headgroup, chain unsaturation, etc.), whereas inclusion of 33-50% cholesterol in the membrane reduced D by a factor of 2. Divalent cations significantly reduced D of negatively charged bilayers. These results compare well with data gathered on other model and natural systems. In addition, the incorporation of the voltage-dependent pore-former alamethicin did slightly reduce lipid lateral mobility. This study demonstrates the feasibility of such experiments with planar bilayers, which are amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.

<h4>Objective</h4>To investigate the clotting and fibrinolytic activities in synovial fluid (SF) from patients with rheumatoid arthritis (RA) and to examine the role of thrombin in synovial hyperplasia.<h4>Methods</h4>We measured the amounts of thrombin-antithrombin-III complex (TAT), antithrombin-III (AT-III), thrombin, plasminogen, alpha 2-plasmin inhibitor (alpha 2-PI), and plasmin-alpha 2-antiplasmin complex (PAP) in SF of 20 patients with RA and 16 patients with osteoarthritis (OA). The proliferative response of synovial fibroblast-like cells to thrombin was measured using [3H] thymidine incorporation. Expression of platelet derived growth factors (PDGF) in conditioned medium was analyzed using a Western blot method, and expression of the mRNA of PDGF and their receptors was analyzed by reverse transcription polymerase chain reaction.<h4>Results</h4>The amounts of clotting factors (TAT, AT-III) and fibrinolytic factors (plasminogen, alpha 2-PI, and PAP) were significantly higher in the patients with RA than in patients with OA (p < 0.01). Moreover, SF thrombin concentrations of patients with RA correlated significantly with erythrocyte sedimentation rates (rs = 0.751, p < 0.01) and serum C-reactive protein concentrations (rs = 0.531, p < 0.05). Thrombin exhibits mitogenic activity toward synovial fibroblast-like cells in vitro, and this mitogenic activity is associated with an increase in the expression of mRNA of both PDGF-alpha receptor and PDGF-beta-receptor.<h4>Conclusion</h4>The high levels of thrombin activity in the SF of patients with RA and strong mitogenic activity of thrombin toward the synovial fibroblast-like cells suggest that thrombin plays an important role in the pathogenesis of RA.

Dexfenfluramine (DF), given in high doses, can produce long-lasting decreases in brain levels of serotonin (5-HT) and 5-HT transporter (5-HTT) protein. The purpose of this study was to determine if DF-induced decreases in 5-HT and 5-HTT in rat forebrain are correlated with compensatory changes in the expression of the genes for tryptophan hydroxylase (TPH) and 5-HTT in the dorsal raphe nucleus. Gene transcripts were measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR). Rats were treated with either one or eight injections of DF at either high (10 mg/kg) or low (2 mg/kg) doses. A positive control group for 5-HT cell loss received a single cerebroventricular injection of 5,7-dihydroxytryptamine (DHT). Rats were killed either 5, 15 or 30 days after their last treatment. Paroxetine binding to the 5-HTT protein in frontal cortex was, as expected, reduced in all of the treated groups relative to vehicle controls. TPH mRNA levels in the dorsal raphe of animals that received DHT were significantly higher than those measured in all other treatment groups 15 days following treatment. By 30 days, the amount of TPH mRNA in DHT-treated rats had fallen to well below control levels. None of the DF regimens significantly affected TPH mRNA levels. Unlike the TPH mRNA changes in DHT-treated rats, the 5-HTT mRNA levels in the dorsal raphe declined progressively throughout the 30 day survival period. None of the DF regimens significantly affected 5-HTT mRNA levels. The significance of these data are discussed in terms of whether loss of forebrain markers for 5-HT reflects either the loss of fine caliber 5-HT axon terminals or a decrease in the expression of these markers in the somata of these cells which are located in the dorsal raphe.

The study assessed the effects of supplementary feeding over 180 consecutive days on iron status of infants and toddlers at six tea plantation in West Java, Indonesia. The design used was a clinical trial: two eohorts (i.e., 12 and 18 months old children) and three treatment groups (i.e., energy + micronutrient, micronutrient alone, and placebo) per cohort. Every day except Sunday, the infants attended day-care centers. Twenty four centers and 136 infants were selected. The infants were screened for weight and length and those meeting the criteria (i.e., < -1 SD of length-for-age, and between -1 and -2 SD of weight-for-length of the NCHS reference) were included. The experimental unit was the day-care centers (DCC), where each DCC was randomly assigned to one of the three treatment. As expected, groups of energy + micronutrient and micronutrient alone of the 12 months cohort experienced a significant upward shift in hemoglobin, ferritin and TS and a downward change in FEP, while the values for the group of placebo remain about the same as at base line. In the first 6 month of treatments, the ANOVA for each iron indicator yielded significant main effects of treatment (P < 0.01) and for Hb with (P = 0.059) on 12 months cohort. On the other hand, the main effects of treatment on hemoglobin, TS, ferritin and FEP were not significant for the 18 months cohort. In the second 6 months of treatments, the only significant of the treatment effect (P < 0.01) was in serum ferritin on 18-month cohort. Under these circumstances, energy has a positive role in improving iron stores. It is likely that the equilibrium of hemoglobin and each iron indicators were reached in 6 months of treatment except ferritin still continued to increase up to 12 month. The effects of treatment on the improvement of iron status was stronger in 12 months than in 18 months.

Plasma antithrombin-III (AT-III), protein S, and protein C were measured during myocardial stunning (MS) and acute myocardial infarction (AMI). The effects of magnesium (Mg), diltiazem, and a Mac-1 inhibitor on their plasma levels were elucidated. Forty-nine open-chest swine underwent brief (8 min) or prolonged (50 min) coronary artery occlusion followed by reperfusion. During MS an increase in the plasma AT-III (from 98.5 +/- 3.38% to 138.1 +/- 3.6%) during the early occlusion phase, without any further changes was observed. The profile of total protein S was not changed during MS. Protein C increased at the end of occlusion (from 45.3 +/- 1.8% to 55.7 +/- 1.4%) reaching a peak (64.5 +/- 1.4%) at the beginning of reperfusion. When compared with controls, no significant differences were found in the antithrombotics profile during MS after pretreatment with Mac-1 inhibitor. For the AMI, the AT-III decreased during occlusion (from 98.5 +/- 3.4% to 61.0 +/- 3.6%). The protein S decreased during occlusion with the lowest level at 1 h of reperfusion (from 71.8 +/- 2.2% to 46.7 +/- 1.0%), followed by an increase during late reperfusion (59.2 +/- 1.5%). Contrarily, protein C increased during occlusion and early reperfusion (from 44.7 +/- 2.6% to 79.4 +/- 2.4%), but declined to 49.6 +/- 2.5% thereafter. In both Mg and diltiazem-treated swine, protein C was higher at the end of occlusion and during the entire reperfusion period compared with controls. Mg and diltiazem therapy was associated with the slight elevation of plasma AT-III. The patterns for protein S level during ischemia-reperfusion were similar with the controls. Protein S was higher at the end of occlusion and through the entire reperfusion in the NPC 15669-treated animals when compared with the controls. Mac-1 inhibition was associated with the elevated protein C during late reperfusion. Ability of Mg, diltiazem, and Mac-1 inhibitor to favorably modulate the plasma level of antithrombotics have direct clinical implications for the use of these agents in patients with acute coronary artery syndromes.

Genetic hemochromatosis is an autosomal recessive disease characterized by increased intestinal iron absorption and consequent tissue iron overload. The hemochromatosis gene has been localized on the short arm of chromosome 6, in close proximity to the HLA locus, but has yet to be identified. Neither the gene product nor the pathogenetic defect have been characterized. Clinical manifestations vary according to the degree of iron overload, ranging from the asymptomatic state to the features of cirrhosis and hepatocellular carcinoma. Early diagnosis remains essential, since the survival of patients without established cirrhosis is comparable to that of the general population. Transferrin saturation and ferritin levels are suggestive of the diagnosis, but measurement of the hepatic iron concentration still remains the gold standard, despite the utilization of computerized tomography and magnetic resonance imaging. Routine phlebotomies constitute the principal therapeutic option, despite the recent preliminary data on oral iron chelators.

The interobserver reproducibility in the immunocyto-chemical assessment of estrogen- and progesterone receptor status was investigated in a series of 102 cases of primary breast carcinomas. Immunostaining was performed on 4 microns cryostat sections using estrogen- and progesterone immunocytochemical assays (ER-ICA and PR-ICA) with methylgreen counterstaining. The slides were evaluated independently by two observers. The proportion and staining intensity of ER- or PR-positive tumor cells in areas of invasive tumor growth was subjectively assessed based on the examination of the entire slide. Scoring was performed according to the proposals of Remmele and Reiner. Additionally, the cytosol estrogen- and progesterone receptor content was determined by a standard dextran-coated charcoal (DCC-) assay. Observer 1 interpreted 60 (59%) of the ER-stained specimens and 50 (49%) of the PR-stained specimens as receptor-positive; for observer 2 the respective values were 59 (58%) for ER-stained specimens and 51 (50%) for PR-stained specimens. Positive biochemical receptor status was found in 68 cases with the ER-DCC (67%) and in 47 with the PR-DCC (46%). The interobserver agreement between the two observers on the immunocytochemical receptor status was 89% for the estrogen receptor and 93% for the progesterone receptor 8.8% of the specimens were interpreted differently. Using the Remmele score, the concordance within the group of cases, which had been interpreted as receptor-positive by both observers, was 54% for the estrogen receptor and 57% for the progesterone receptor. The Reiner score showed concordant scorings of 72% for the estrogen receptor and 79% for the progesterone receptor. The present study indicates that complete agreement between scorings of different observers may not be expected, mainly due to differences in the interpretation of the specificity of staining and of the histological structures after immunostaining. The concordance of positive results using the three-graded Reiner score is comparable to that of the three-graded Bloom and Richardson grading system of breast cancer and reflects the limitation of subjective evaluation of morphology in general.

Estimation of oestrogen receptors (ER) in breast carcinoma has important therapeutic and prognostic implications. The dextran-coated charcoal (DCC) biochemical technique is being replaced in many institutions by immunohistochemical techniques. The aim of this study was to assess the level of agreement between ER counts in frozen and paraffin sections. Ninety-seven infiltrating breast carcinomas were studied immunohistochemically, for ER in frozen and paraffin sections. Cases were classified as ER-positive if > or = 10% of the tumor cells were positive. Both techniques gave a high level of correlation and agreement. There was complete agreement in classifying cases as ER-positive or ER-negative. ER estimation on only one case was outside 2SD of complete agreement. Oestrogen-receptor content can be assayed satisfactorily in paraffin sections and offers many advantages over the frozen section technique.

2-Methoxyethanol (2-ME) produces adverse reproductive effects in humans at an exposure level that is about 60-fold lower (2.6 mg/m3) than the concentration toxic to rat testes (167 mg/m3), suggesting that humans are much more sensitive to the testicular toxicity of 2-ME than rats. Previous studies found that 2-ME-induced germ cell death seen in vivo could be faithfully mimicked in vitro only in cultured seminiferous tubules, using the active metabolite methoxyacetic acid (MAA). To test whether human testis per se is more sensitive than rat testis to MAA, we compared the responses of cultured rat seminiferous tubules (RSTs) and human testicular tissues (HTT) in vitro. Degeneration in spermatocytes was observed in RSTs 19 hours after a 5-hour exposure to MAA at and above 1 mM. The dying germ cells showed necrotic-like morphology, as seen in vivo. Germ cells in HTT were also killed by doses > or = 1 mM, although the dying germ cells appeared apoptotic, rather than necrotic. For both species, doses lower than 1 mM were without visible effect. Interestingly, agarose gel electrophoresis of DNA from tubules of both species showed internucleosomal DNA fragmentation after MAA treatment, indicating that MAA induced apoptosis in both human and rat germ cells, though the dying cells showed different morphology in the two species. Furthermore, MAA-induced germ cell apoptosis in both species could be significantly attenuated by calcium channel blockers such as nifedipine or verapamil, which inhibit calcium movement through plasma membranes. In conclusion, the results suggest that: 1) human testis is equally sensitive to MAA compared to rat testis; and 2) MAA induces germ cell apoptosis both in human and rat, probably through similar, calcium-dependent mechanism(s). The precise steps in this germ cell apoptosis are under investigation.

The identity of the protein responsible for hemochromatosis, the iron overload disease, has eluded scientists for years. However, a recent report identifies the gene where the hemochromatosis defect lies. It is a gene that encodes a major histocompatibility complex (MHC) class-1-like protein called HLA-H. The mechanism by which an HLA-H defect alters iron metabolism is still unidentified. However, this new discovery will certainly ignite a new wave of study into the physiology of iron metabolism and its regulation.

To evaluate the role of DCC gene alteration in the development and progression of gastric cancer, loss of heterozygosity (LOH) and mRNA expression of DCC gene was examined in surgically resected gastric cancer specimens using PCR-RFLP and RT-PCR analyses. LOH was detected in 18 (35.3%) of 51 informative cancers specimens. The frequency of LOH in stages III and IV (50.0%) was significantly higher than that in stages I and II (14.3%, P < 0.05%), but LOH was not correlated to histological types, tumor size, invasion depth and lymph node metastases. Expression of DCC mRNA in 26/51 cases was examined and the DCC expression was lost in 8/26 (30.7%) cancer, and no significant correlation was found between loss of DCC expression or LOH and clinico-pathologic parameters. LOH and loss of expression at DCC gene occur frequently in gastric cancers, LOH at DCC locus is a late event.