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Previous studies have shown that the E7 gene of human papillomavirus (HPV) type 16 or 18 alone was sufficient for immortalization of human foreskin epithelial cells (HFE) and that the efficiency was increased in cooperation with the respective E6 gene, whereas the HPV6 E6 or E7 gene was not active in HFE. To detect weak immortalizing activities of the HPV6 genes, cells were infected with recombinant retroviruses containing HPV genes, alone and in homologous and heterologous combinations. The HPV6 genes, alone or together (HPV6 E6 plus HPV6 E7), were not able to immortalize cells. However the HPV6 E6 gene, in concert with HPV16 E7, increased the frequency of immortalization threefold over that obtained with HPV16 E7 alone. Interestingly, 6 of 20 clones containing the HPV16 E6 gene and the HPV6 E7 gene were immortalized, whereas neither gene alone was sufficient. Thus, the HPV6 E6 and E7 genes have weak immortalizing activities which can be detected in cooperation with the more active transforming genes of HPV16. Acute expression of the HPV6 and HPV16 E6 and E7 genes revealed that only HPV16 E7 was able to stimulate the proliferation of cells in organotypic culture, resulting in increased expression of the proliferative cell nuclear antigen and the formation of a disorganized epithelial layer. Additionally, combinations of genes that immortalized HFE cells (HPV16 E6 plus HPV16 E7, HPV16 E6 plus HPV6 E7, and HPV6 E6 plus HPV16 E7) also stimulated proliferation.

A strategy involving polymerase chain reaction amplification of cDNAs was designed to study the expression of the hamster polyomavirus (HaPV) early region in HaPV-transformed rat fibroblasts, productively HaPV-infected cells, and HaPV-induced lymphoma. We identified three mRNAs resulting from alternative splicing of open reading frames leading to coding capacities for three polypeptides with molecular weights similar to those of the murine polyomavirus large T, middle T (MT), and small T (ST) antigens. The corresponding intronless cDNAs direct the in vitro synthesis of polypeptides with the expected electrophoretic mobilities. The biological activities carried by the HaPV early genes were assayed by transfection of appropriate cell systems. The fragment of genomic viral DNA that encodes the three early antigens contains all of the genetic information necessary for immortalization of primary rat embryo fibroblasts and transformation of F111 rat cells. The large T antigen is sufficient for immortalization, although the MT and ST antigens stimulate the growth and modify the phenotype of immortal cell lines. A stringent cooperative effect was observed in the transformation of F111 cells, which requires the simultaneous presence of the MT and ST antigens, as opposed to the transformation by murine polyomavirus, which can be carried out by the MT antigen alone.

Two hundred twelve Italian patients with genetic hemochromatosis (181 men, mean age 50 +/- 11 yr; and 31 women, mean age 49 +/- 10 yr) were followed for a median period of 44 mo (range = 3 to 218 mo). Alcohol abuse was present in 31 subjects (15%), and chronic HBV and HCV infection were seen in 19 (9%) and 35 (24%) of 145 cases tested, respectively. Twenty-four patients (11%) had concomitant beta-thalassemia trait. Liver biopsy revealed cirrhosis in 146 and a noncirrhotic pattern in the other 66. Perls' stain was degree III in 37 patients and IV in 171 patients. One hundred eighty-five patients underwent weekly venesection, and iron depletion was achieved in 122 cases after total iron removal of 3 to 41 gm. Death occurred in 44 patients after 3 to 198 mo and was due to hepatocellular carcinoma in 20 cases, liver failure in 10, extrahepatic cancer in six, heart failure in three and hemochromatosis unrelated causes in five. Cancer has developed in seven other patients still alive (hepatocellular in five and extrahepatic in two). No deaths were observed among noncirrhotic patients; cumulative survival rates in cirrhotic patients were 85%, 75%, 60% and 47% at 3, 5, 8 and 10 yr, respectively. Univariate analysis in the 146 cirrhotic patients showed that age greater than 60 yr, alcohol abuse, cardiomyopathy, skin pigmentation, portal hypertension, hypoalbuminemia, hypergammaglobulinemia and Child class B or C had significant negative prognostic value. At multivariate analysis, only alcohol abuse, gamma-globulins greater than 2.0 gm/dl and Child class B or C maintained their negative prognostic values (p less than 0.01, hazard ratio 2.7; p less than 0.001, hazard ratio 2.8; and p less than 0.001, hazard ratio 4.3, respectively).

The thromboresistance of endothelium is maintained as long as natural anticoagulant pathways are functionally present on endothelial plasma membranes. The principal anticoagulant pathways in human hearts and kidneys are thrombomodulin (TM) and heparan sulfate proteoglycan-antithrombin III (HSPG-ATIII). The downregulation of TM or the loss of ATIII is associated with fibrin deposition. This sequence of events occurs when stable allografts of hearts or kidneys become unstable or rejected. Human placentae do not contain the HSPG-ATIII natural anticoagulant pathway, but the TM system is uniformly represented on endothelium of normal chorionic villi. However, many villi in placentae from preeclamptic pregnancies contain thrombomodulin-negative endothelium, and these vessels contain fibrin thrombi. These thrombi compromise blood flow through the placental microcirculation and are associated with ischemic changes either with or without the presence of cellular infiltrates.

A simple method was developed to establish serum-independent Chinese hamster ovary (CHO) cells that grew and secreted high level of human antithrombin-III (AT-III). First, human AT-III and mouse dihydrofolate reductase (DHFR) cDNAs were transfected into DHFR-deficient CHO cells. Transfected cells were treated with increasing concentrations of methotrexate (MTX) and clones secreting high levels of AT-III (10-20 micrograms/ml/3 day) in a serum-containing medium were obtained. Serum-independent clones were derived from the serum-dependent clones by simply culturing the cells for a few weeks in a serum-free medium. In a serum-free medium the established serum-independent clones grew at normal rate and produced almost equivalent amount of AT-III to that of the serum-dependent, parent clones. In addition, AT-III from the serum-independent clones has specific activity similar to that of plasma-derived AT-III.

The mode of action of gliotoxin against poliovirus has been analyzed in detail. This fungal metabolite inhibits the appearance of poliovirus proteins when present from the beginning of infection but has no effect on viral translation when added at late times. In agreement with previous findings, this toxin potently inhibited the incorporation of [3H]uridine into poliovirus RNA soon after its addition to the culture medium. Analysis of the synthesis of poliovirus plus- or minus-stranded RNA in the presence of gliotoxin suggests that this compound effectively hampered both processes. This result contrasts with the mode of action of other inhibitors of poliovirus RNA synthesis, such as guanidine or flavones, that selectively block plus-stranded RNA synthesis and suggests that the target of gliotoxin differs from the target of guanidine, i.e., poliovirus protein 2C. Indeed, gliotoxin was found to be a potent inhibitor of poliovirus RNA synthesis in cell-free systems, using membranous crude replication complexes, a reaction that is not blocked by guanidine or Ro 09-0179. Moreover, in vitro activity of the purified poliovirus polymerase 3Dpol was efficiently inhibited by gliotoxin. These results indicate that this toxin acts on the poliovirus polymerase 3Dpol, providing the first description of an inhibitor of this viral enzyme.

The BTG1 gene locus has been shown to be involved in a t(8;12)(q24;q22) chromosomal translocation in a case of B-cell chronic lymphocytic leukemia. We report here the cloning and sequencing of the human BTG1 cDNA and establish the genomic organization of this gene. The full-length cDNA isolated from a lymphoblastoid cell line contains an open reading frame of 171 amino acids. BTG1 expression is maximal in the G0/G1 phases of the cell cycle and is down-regulated when cells progress throughout G1. Furthermore, transfection experiments of NIH3T3 cells indicate that BTG1 negatively regulates cell proliferation. The BTG1 open reading frame is 60% homologous to PC3, an immediate early gene induced by nerve growth factor in rat PC12 cells. Sequence and Northern blot analyses indicate that BTG1 and PC3 are not cognate genes. We then postulate that these two genes are the first members of a new family of antiproliferative genes.

Three methods for evaluation of serum iron-binding capacity have been described: biochemical, immunological based on transferrin assay, and biophysical based on electron-paramagnetic resonance (EPR) spectroscopy of transferrin. Interrelation has been shown between transferrin and general serum iron-binding capacity. Basing on the data presented it is suggested that in secondary hemochromatosis plasma contains an iron pool that is not specifically bound with transferrin, while in primary hemochromatosis such pool was not detected.

A negative regulator gene for synthesis of arylsulfatase in Klebsiella aerogenes was cloned. Deletion analysis showed that the regulator gene was located within a 1.6-kb cloned segment. Transfer of the plasmid, which contains the cloned fragment, into constitutive atsR mutant strains of K. aerogenes resulted in complementation of atsR; the synthesis of arylsulfatase was repressed in the presence of inorganic sulfate or cysteine, and this repression was relieved, in each case, by the addition of tyramine. The nucleotide sequence of the 1.6-kb fragment was determined. From the amino acid sequence deduced from the DNA sequence, we found two open reading frames. One of them lacked the N-terminal region but was highly homologous to the gene which codes for diadenosine tetraphosphatase (apaH) in Escherichia coli. The other open reading frame was located counterclockwise to the apaH-like gene. This gene was highly homologous to the gene which codes for dihydrofolate reductase (folA) in E. coli. We detected 30 times more activity of dihydrofolate reductase in the K. aerogenes strains carrying the plasmid, which contains the arylsulfatase regulator gene, than in the strains without plasmid. Further deletion analysis showed that the K. aerogenes folA gene is consistent with the essential region required for the repression of arylsulfatase synthesis. Transfer of a plasmid containing the E. coli folA gene into atsR mutant cells of K. aerogenes resulted in repression of the arylsulfatase synthesis. Thus, we conclude that the folA gene codes a negative regulator for the ats operon.

The interaction of heparin with glia-derived nexin (GDN) has been characterized and compared to that observed between heparin and antithrombin III (ATIII). Heparin was fractionated according to its affinity for immobilized GDN, and the ability of various fractions to accelerate the inhibition rate of thrombin by either GDN or ATIII was examined. Fractions with different affinities for GDN accelerated the thrombin-GDN reaction to a similar extent; heparin with a high affinity for immobilized GDN stimulated the reaction only about 30% more than the fraction that did not bind to immobilized GDN. Slightly greater differences were observed for the effect of these fractions on the thrombin-ATIII reaction; heparin that did not bind to the GDN affinity column was about 60% more effective than heparin with a high affinity for GDN in accelerating the inhibition of thrombin by ATIII. The CNBr fragment of GDN between residues 63 and 144 was able to reduce the heparin-accelerated rate of inhibition of thrombin by GDN indicating that this region of GDN was able to bind the heparin molecules responsible for the acceleration. Shorter synthetic peptides within this sequence did not significantly reduce the rate, suggesting that the heparin-binding activity of fragment 63-144 depends on a specific conformation of the polypeptide chain. Fragment 63-144 was less effective in decreasing the heparin-accelerated rate of inhibition of thrombin by ATIII. The results are discussed in terms of the heparin species that are responsible for the acceleration of the GDN- and ATIII-thrombin reactions and the heparin-binding sites of GDN and ATIII.

To determine whether release of tumor necrosis factor-alpha (TNF-alpha), a cytokine that affects iron homeostasis, may be selectively altered in hereditary hemochromatosis, we measured concentrations of TNF-alpha and interleukin-1 beta (IL-1 beta) in supernatants of cultured peripheral blood monocytes from 11 homozygotes for hereditary hemochromatosis, 11 healthy individuals, and five patients with iron-loading anemia. The gene for hereditary hemochromatosis is tightly linked to the HLA locus on chromosome 6, but its exact site and product are not known. The gene for TNF-alpha also is located within the HLA region. Monocytes were incubated from 4 to 36 hours in medium alone or with added lipopolysaccharide. Mean concentrations of immunoreactive TNF-alpha in supernatants were significantly lower for subjects with hereditary hemochromatosis as compared to healthy controls (P less than .037) and patients with iron-loading anemia (P less than .005); differences between homozygotes for hemochromatosis and healthy controls were up to 4.5-fold at 4 hours (P = .008), 1.9-fold at 12 hours (P = .036), and 7.0-fold at 36 hours (P = .001). Importantly, concentrations of IL-1 beta in supernatants were not significantly different among the three groups. We conclude that release of TNF-alpha by monocytes may be selectively impaired in hereditary hemochromatosis. Deficient activity of TNF-alpha may contribute to the disordered iron metabolism of this disease.

The effects of the major human serum bile acid, glycochenodeoxycholic acid (GCDC), as well as unconjugated chenodeoxycholic acid (CDC), on the MCF-7 human breast cancer cell line have been studied in vitro under oestrogen and bile acid deprived culture conditions. GCDC increased the growth of the breast cancer cells over the range 10-300 microM. At concentrations in excess of the bile acid binding capacity of the medium cell growth was prevented. In contrast 10 microM CDC tended to reduce cell growth. Oestrogen (ER) and progesterone (PgR) receptors, pS2 and total cathepsin D were quantified by monoclonal antibody based immunoassays. Ten to 100 microM GCDC and 10 microM CDC down-regulated ER protein and this was accompanied by induction of the oestrogen-regulated proteins PgR, pS2 and possibly cathepsin D, including increased secretion of the latter two proteins into the culture medium. All these changes were quantitatively similar to those observed with 10 nM oestradiol. The bile acid effects on ER and PgR were not due to interference with the assay procedures. Cells incubated with 50 microM GCDC or 10 microM CDC had higher pmolar concentrations of the bile acids than controls. This study suggests that naturally occurring bile acids influence the growth and steroid receptor function of human breast cancer cells.

Forty-two endometrial carcinomas of various stages of progression were analyzed to search for loss of chromosomal regions and for point mutations of ras genes and amplification of Int-2 gene. This approach is particularly favorable for observation of genetic events and their significance in the process of neoplastic conversion by considering the clinico-pathological characteristics of each tumor. At least 3 genetic events, including 18q, 17p deletions, and point mutations at codon 12 of the K-ras gene, are implicated in the development of endometrial carcinomas. Likely targets for allelic losses on chromosomes 18q and 17p are the DCC gene and the p53 gene sequences, respectively. Overall numbers of allelic losses in individual tumors appeared to increase in case of advanced stage tumors, thereby indicating the association of allelic loss accumulation with tumor progression. The genetic features seen in 2 juvenile-type adenocarcinomas and 2 clear-cell carcinomas suggested the possibility that etiological factors providing selective pressure for particular mutation sub-sets during carcinogenesis are probably heterogeneous.

Four mouse POU domain genomic DNA clones--Brain-1, Brain-2, Brain-4, and Scip--and Brain-2 cDNA, which are expressed in adult brain, were cloned and the coding and noncoding regions of the genes were sequenced. The amino acid sequences of the four POU domains are highly conserved; sequences in other regions of the proteins also are conserved but to a lesser extent. The absence of introns from the coding regions of the four POU domain genes and the similarity of amino acid sequences of the corresponding proteins suggest that the coding region of the ancestral class III POU domain gene lacked introns and therefore may have originated by reverse transcription of a molecule of POU domain mRNA followed by insertion of the cDNA into germ cell genomic DNA. Additional duplications of the ancestral class III POU domain gene (or mRNA) would create the Brain-1, Brain-2, Brain-4, and Scip genes.

This is the first study of the antithrombin III-heparan sulfate natural anticoagulant pathway in human kidneys. Immunocytochemical experiments were done to demonstrate the pathway on normal renal endothelial cells. Enzymatic studies were done to show that the antithrombin III was anchored to endothelium by molecules of heparan sulfate. Displacement studies were done with glycosaminoglycans to show that the antithrombin III was bound to its glycosaminoglycan anchor via a heparinlike binding site, and replacement studies showed that antithrombin III could be returned to the same endothelial cells from which it was displaced. Immunocytochemical studies of biopsies showed that normally functioning renal allografts manifested the endothelial antithrombin III-heparan sulfate anticoagulant pathway. The pathway was compromised or absent from the microcirculation of biopsies from rejecting or rejected renal allografts, and the diminishment of endothelial ATIII was associated with the presence of fibrin deposition. It is concluded that compromise of the antithrombin III-heparan sulfate natural anticoagulant pathway results in compromised renal function in transplanted kidneys.

Treatment of T lymphocytes with mitogenic antibodies against the T-cell receptor/CD3 complex induces within seconds a rise in the concentration of intracellular free Ca2+. We recently reported that free myristic acid, but not its methyl ester, inhibits both the anti-CD3-induced Ca2+ influx across the cell membrane and the Ca2+ release from intracellular stores in Jurkat T cells. Here we show that myristic acid induced a rapid hyperpolarization of the cell membrane potential and a decrease in intracellular pH in Jurkat cells. Lauric acid and palmitic acid caused minor hyperpolarization, whereas other saturated non-esterified fatty acids tested were without effect. Hyperpolarization of the membrane potential in Jurkat cells with valinomycin did not, however, inhibit the anti-CD3-induced Ca2+ signal, and the blocking effect on the Ca2+ signal in myristic acid-treated Jurkat cells was not reversed after normalization of the cell membrane potential by treatment with gramicidin. The inhibitory effect of myristic acid on the Ca2+ fluxes thus cannot be explained by changes in membrane potential. We also present evidence that the blocking effect of myristic acid on the receptor-operated Ca2+ flux is not due to the myristic acid-induced decrease in intracellular pH. Moreover, we demonstrate that myristic acid does not prevent the release of Ca2+ triggered by inositol 1,4,5-trisphosphate from intracellular pools in permeabilized cells. Our findings indicate that myristic acid blocks anti-CD3-induced Ca2+ traffic in Jurkat cells by interfering with the regulation of Ca2+ mobilization, apparently by blocking an early step in signal transduction from the T-cell-antigen receptor/CD3 complex.

Ten healthy dogs and 10 dogs with multicentric lymphoma were given a single dose of L-asparaginase at a rate of 10,000 IU/m2 of body surface. Assessment of concentrations of contributors to the coagulation process and of the ability to coagulate including antithrombin III, one-stage prothrombin time, prothrombin-proconvertin time, activated partial thromboplastin time, plasminogen, fibrinogen, and platelet number were performed prior to drug administration (day 0). These tests were repeated 24 hours (day 1), 48 hours (day 2), and 7 days after treatment with L-asparaginase. Antithrombin-III concentrations were significantly lower in the dogs with lymphoma than in healthy dogs on days 0, 1, 2, and 7; however, with the exception of day 1, mean values remained within normal limits. There was also a difference between the 2 groups in prothrombin/proconvertin values on day 7 and in platelet number on day 2, with the lymphoma group having significantly shorter prothrombin/proconvertin time than healthy dogs, and the difference in platelet numbers being associated with increased counts in the healthy dogs. Data obtained from the healthy dogs and dogs with lymphoma for each coagulation test were pooled for each treatment day (0, 1, 2, and 7), and day-0 values for each coagulation test were compared with data obtained on days 1, 2, and 7. Antithrombin-III concentration on day 7 was significantly lower than on day 0, prothrombin/proconvertin time on day 1 was significantly longer than on day 0, and fibrinogen concentrations on days 1 and 2 were significantly lower than on day 0.(ABSTRACT TRUNCATED AT 250 WORDS)

A 23-year-old woman developed thrombosis of the superior mesenteric vein and underwent an extensive enterectomy. She was diagnosed to have ATIII deficiency with extrahepatic portal vein thrombosis and esophagogastric varices. She was admitted to our department and underwent esophageal mucosal transection and splenectomy. Her activities of ATIII were 46%, but ATIII activities of her family were over 90%. ATIII activities during perioperative period were kept more than 70% following administration of ATIII drug. After splenectomy thrombocythemia which was over 300 x 10(4)/mm3 appeared with severe headache and slight pain of hands. She was discharged on 76th postoperative day with no complications and collapse of esophageal varices.

No abstract available.