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No abstract available.

1. Effects of two volatile anesthetics [halothane (Hal) and enflurane (Enf)] and a volatile convulsant [hexafluorodiethyl ether (HFE)] on amino acid-induced membrane currents in neurons dissociated from the nucleus tractus solitarius of the rat were examined. The dissociated neurons were voltage clamped in the whole-cell mode of the patch-clamp technique. All drugs were applied with a microperfusion system, termed the "Y-tube" method. 2. The glutamate (Glu)-induced excitatory response was slightly reduced by both the anesthetics. The responses to three agonists at Glu receptor were depressed by Hal (10(-3) M) in the rank order of quisqualate greater than N-methyl-D-aspartate greater than kainate. HFE slightly increased the Glu response at a high concentration of 2 x 10(-3) M. 3. The gamma-aminobutyric acid (GABA)-induced chloride current (ICl) was enhanced by both anesthetics. The dissociation constant (Kd) for the enhancement was 2.3 x 10(-4) M for Hal and 2.1 x 10(-4) M for Enf, and the Hill coefficient was 1.6 for Hal and 1.5 for Enf. HFE depressed the GABA response with a Kd of 8.7 x 10(-5) M and a Hill coefficient of 0.84. 4. Hal (10(-3) M) and Enf (10(-3) M) decreased the Kd of the GABA concentration-response curve from 3.5 x 10(-6) to 10(-6) and 1.9 x 10(-6) M, respectively, without changing the maximum response or the Hill coefficient (1.5). In the presence of HFE (10(-4) M), the Kd was increased to 1.4 x 10(-5) M and the Hill coefficient was slightly changed to 1.2.(ABSTRACT TRUNCATED AT 250 WORDS)

No abstract available.

Suppressor gene loci involved in the development of hepatocellular carcinoma (HCC) have not been fully identified. The aim of this study was to look for consistent allele loss, or loss of heterozygosity (LOH), in HCC which might represent such gene loci. We have prepared DNA from tumour and non-tumour material from 16 patients with HCC (nine with and seven without liver cirrhosis). Tumour DNA was compared with non-tumour DNA by Southern analysis performed with a panel of 22 probes recognising restriction fragment length polymorphisms assigned to chromosomes 1, 4, 5, 7, 9, 11, 12, 13, 14, 16, 17, 18 and 20. Non-tumour DNA from five of the seven patients with HCC without cirrhosis was heterozygous with the probe Lambda MS8 (5q35-qter), and in all five there was LOH in tumour DNA. Probes for other regions of chromosome 5 have as yet shown no LOH in this group of patients. Cirrhotic HCC patients exhibited LOH on chromosomes 1q and 5p but not in the region 5q35-qter. Both groups of HCC showed LOH on chromosome 17p13. Screening with other probes has not shown any consistent LOH in either group as yet. A comparison of LOH on chromosome 5 in seven patients with colorectal metastasis in the liver showed a different pattern, which suggests that the proposed tumour suppressor gene locus for HCC without cirrhosis on chromosome 5 appears to be distinct from the familial adenomatous polyposis coli gene.

To determine the efficiency of image analysis in immunohistochemical progesterone receptor (PgR) measurement, 94 primary breast carcinoma tissue samples were evaluated for PgR by biochemical dextran-coated charcoal assay (DCC) and an immunohistochemical method. Frozen sections immunostained for PgR with a monoclonal antibody (Abbott PgR-ICA, Chicago, IL) and the peroxidase-antiperoxidase technique were scored semiquantitatively histologic score by microscopy and quantitatively (percentage nuclear area immunopositivity [PNA] using the CAS 200 image analyzer (Cell Analysis Systems, Elmhurst, IL). There was a positive correlation between dextran-coated charcoal assay and both histologic score (r = 0.82) and PNA (r = 0.69). Selected cutoff points of 60 histologic score and 6.5% PNA based on sensitivity/specificity calculations yielded a predictive value of a negative test of 73% and 80%, respectively, and a positive predictive value of 100% for both; ranges of fmol/mg protein PgR correspond to ranges of histologic score and PNA. The use of an image analyzer to measure PNA in PgR-immunostained sections is a viable alternative to dextran-coated charcoal assay, especially when insufficient fresh tissue is available.

Ovarian hyperstimulation syndrome (OHSS) is occasionally seen following hMG-hCG treatment in combination with a GnRH agonist. Increased coagulability and decreased renal perfusion may be life threatening in some severe cases. In order to evaluate coagulo-fibrinolytic activity, several related factors in the general circulation were examined for approximately 2 weeks after admission in 11 patients with severe OHSS. The results are as follows. 1. Fibrinopeptide A (FPA) was increased during the initial stage of OHSS followed by a gradual decrease. However, the level remained slightly higher than normal for 2 weeks after the onset of severe OHSS. 2. Fibrinopeptide B beta 15-42 (FPB beta 15-42) showed grossly similar patterns to those of FPA. 3. D-dimer levels were constantly higher than normal from the initial to the late stages of OHSS. 4. Thrombin-Antithrombin III complex (TAT) was markedly increased on the days of admission followed by a gradual decrease during the following week. 5. Antithrombin III (ATIII), plasminogen and alpha 2 plasmin inhibitor (alpha 2PI) showed only minimal decreasing patterns throughout blood samplings. 6. Increases in FPA, FPB beta 15-42 and D-dimer were greater in the cases with severe hemoconcentrations. Our present data suggest that severe OHSS brings on hypercoagulability resulting in microthrombosis. In order to avoid development of coagulopathy, prophylactic treatment should be considered for patients with OHSS.

Parameters of the kallikrein-kinin, fibrinolytic, and coagulation systems before (control), during, and after occlusion of the hepatic artery and the portal vein were studied in anesthetized pigs. Hepatic synthesis was observed for kallikrein, plasmin, and plasminogen. The other factors, prekallikrein, kallikrein inhibitor, alpha 2-antiplasmin, prothrombin, and antithrombin-III were all released by the liver and underwent partial hepatic inactivation. The splanchnic area showed a significant synthesis of antithrombin-III. After 90 min of hepatic arterial occlusion, the hepatic release of all factors declined, and synthesis of kallikrein, plasmin, and plasminogen fell an average of 54%. The inactivation of prekallikrein and alpha 2-antiplasmin was increased and the splanchnic synthesis of antithrombin-III fell. Portal vein blood flow fell by 38%. After 15 min of portal vein occlusion (combined occlusion of the hepatic artery and portal vein) the splanchnic area started to synthetize kallikrein and the hepatic inflow of kallikrein increased by more than 50%. Splanchnic metabolism of other factors was also altered, but after revascularization of the hepatic artery changes were mostly insignificant. Hepatic synthesis of kallikrein and plasmin remained depressed after reperfusion, and that of plasminogen fell to zero. After this short period of occlusion of the hepatic artery and portal vein, plasma concentrations of prekallikrein, plasmin, and alpha 2-antiplasmin were decreased, causing a state of fibrinolysis. A role for kallikrein/bradykinin in hepatic blood flow regulation is proposed and implications of these observations for liver transplantation are discussed.

As an initial step towards the characterization of replicative DNA polymerases of trypanosomes, we have cloned, sequenced and examined the expression of the Trypanosoma (Trypanozoon) brucei brucei gene that encodes the DNA polymerase alpha catalytic core (pol alpha). The protein sequence contains the six conserved regions that have been recognized previously in eukaryotic and viral replicative DNA polymerases. In addition, we have identified a seventh region which appears to be conserved primarily in alpha-type DNA polymerases. The T.brucei DNA pol alpha core N-terminus is 123 and 129 amino acids smaller than that of the human and yeast homologue, respectively. The gene is separated by 386 bp from an upstream open reading frame (ORF) of 442 codons. Stable transcripts of the upstream sequence are detected in both dividing and non-dividing forms, while pol alpha transcripts are detected principally in dividing forms. Allelic copies of the T.brucei pol alpha region exhibit restriction site polymorphisms; one such sequence polymorphism affects the amino acid sequence of the T.brucei DNA pol alpha core. The T.brucei pol alpha region cross-hybridizes weakly with that of T.(Nannomonas) congolense and T.(Duttonella) vivax.

Specific radioligand [3H] methyltestosterone incubation and dextran-coated charcoal absorption technique (DCC) were used to test cytosol androgen receptors (AR) of laryngeal carcinoma in 33 patients. In all specimens, high level AR ranged from 3 to 16.9 fmol/mg of cytosol protein (mean 10 +/- 3.9 fmol/mg), and dissociation constant was 1.7 x 10(-9) mole. Laryngeal carcinoma is considered a sex hormone-dependent tumor. High level of AR in laryngeal carcinoma may indicate a super-demand on androgen; when the content of AR increases the tumor proliferates perniciously. Antiandrogen endocrine therapy may be helpful in treating laryngeal carcinoma.

Platelet aggregation and release, platelet number, mean platelet volume, antithrombin-III activity, and fibrinogen concentration were evaluated in heartworm-negative and heartworm-infected dogs at baseline and on days 3, 10, and 21 after treatment with thiacetarsamide. Platelet reactivity was enhanced in a group of dogs naturally infected with Dirofilaria immitis, compared with 2 groups of heartworm-negative dogs, but platelet reactivity was not further enhanced after treatment with thiacetarsamide. A significant decrease in antithrombin-III activity was detected 21 days after treatment. The platelets from a group of laboratory Beagles implanted with 50 adult D immitis displayed enhanced reactivity 6 months after implantation, but by 18 months, platelet reactivity had returned to near, or less than, baseline. Platelet reactivity was enhanced after thiacetarsamide treatment in this group. Thiacetarsamide-associated changes were not observed in platelet number or size; antithrombin-III activity decreased, but the change was not significant. Fibrinogen concentration was increased significantly (P less than 0.05) on day 10. Enhanced adenosine diphosphate (ADP)-induced platelet aggregation was observed on days 3, 10, and 21 after treatment in heartworm-negative dogs. This change was not observed in 6 control Beagles not treated with thiacetarsamide. Although antithrombin-III activity was decreased on day 3 and fibrinogen concentration was increased on day 10, paralleling changes observed in the heartworm-infected dogs, the changes were not statistically significant. In this study, thiacetarsamide was procagulatory in heartworm-negative dogs and may be an important contributing factor to the thromboembolism observed with adulticidal therapy.

The mechanism of the antiproliferative effects of progestins on human breast cancer cells is not known. In view of the ability of estrogen to stimulate human breast cancer cell production of peptide growth factors, and since previous studies have suggested that the inhibitory action of progestins is dependent on estrogen-stimulated growth, the present study examined the interaction of growth factors and the synthetic progestin R5020 on the proliferation of T47D human breast cancer cells. In this study, the concentrations of estradiol, insulin, and EGF for optimal stimulation of T47D cell growth in 3% dextran-charcoal treated fetal bovine serum (DCC-FBS) were determined to be 1 nM, 100 nM, and 1 nM, respectively. Furthermore, incubation with these optimal concentrations of estradiol, insulin, and EGF in various combinations produced additive effects on T47D cell proliferation, suggesting that these agents act, at least in part, by different mechanisms. In contrast, in a chemically defined medium (DM), both estradiol and EGF were unable to stimulate T47D cell proliferation. In the case of estradiol, the inability to demonstrate stimulation of T47D cell growth in DM was not due to down-regulation of the estrogen receptor. R5020 inhibited the growth of T47D cells, although its effect was more marked in the presence of 3% DCC-FBS than in DM. Stimulation of T47D cell growth by either estradiol or insulin in 3% DCC-FBS was effectively inhibited by R5020. In contrast, growth of T47D cells stimulated by EGF in the absence of estradiol was not markedly inhibited by R5020, the growth being comparable to that of untreated control cells. These findings suggest that the inhibitory effect of R5020 on T47D cell proliferation is dominant over the action of some, but not all, breast cancer mitogens.

Based on the high incidence of loss of heterozygosity for loci on chromosome 17p in the vicinity of the p53 locus in human breast tumors, we investigated the frequency and effects of mutations in the p53 tumor suppressor gene in mammary neoplasia. We examined the p53 gene in 20 breast cancer cell lines and 59 primary breast tumors. Northern blot analysis, immunoprecipitation, and nucleotide sequencing analysis revealed aberrant mRNA expression, over-expression of protein, and point mutations in the p53 gene in 50% of the cell lines tested. A multiplex PCR assay was developed to search for deletions in the p53 genomic locus. Multiplex PCR of genomic DNA showed that up to 36% of primary tumors contained aberrations in the p53 locus. Mutations in exons 5-9 of the p53 gene were found in 10 out of 59 (17%) of the primary tumors studies by single-stranded conformation polymorphism analysis. We conclude that, compared to amplification of HER2/NEU, MYC, or INT2 oncogene loci, p53 gene mutations and deletions are the most frequently observed genetic change in breast cancer related to a single gene. Correlated to disease status, p53 gene mutations could prove to be a valuable marker for diagnosis and/or prognosis of breast neoplasia.