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PubMed2006

Characterization of a Cys329Gly mutation causing hereditary factor VII deficiency.

We have previously reported a homozygous Cys329Gly mutation in a Chinese patient with factor VII (FVII) deficiency. Others have found a heterozygous Cys329Gly mutation in the F7 gene from patients of three different pedigrees. However, none of the reports included the expression and characterization of the mutant FVII in vitro. To investigate the effect of Cys329Gly on FVII function, we carried out transient transfections of baby hamster kidney cells (BHK-21) with a mutant FVII construct and compared the results to those obtained using a wild-type FVII construct and vector control. The results demonstrate that the level of FVII:Ag secreted into the medium by transfected BHK-21 cells with mutant construct was not affected, but the coagulation activity of the mutant FVII was undetectable. We conclude that Cys329 is critical to FVII coagulation, and the replacement of cysteine 329 by glycine leads to the loss of coagulation activity in the patients, possibly the molecular basis for FVII deficiency in the patients.

Amino Acid Substitution↗
PubMed1998

Isolated superfused juxtaglomerular cells from rat kidney: a model for study of renin secretion.

Freshly isolated rat juxtaglomerular cells (JGC) were superfused to study renin secretion rate (RSR) at the cellular level. Effluates from the superfusion chamber collected in 20-min intervals showed a time-dependent decline in RSR from 85.5 +/- 32 to 4.0 +/- 2.4 ng ANG I. ml-1. h-1. mg protein-1. min-1 within 100 min of collection (mean +/- SE, n = no. of JGC preparations/superfusion chambers = 9/18). Addition of adenosine deaminase type II (ADA II, 3 U/1.4 mg protein) to the superfusion medium increased RSR more than fourfold to 402 +/- 100 ng in the first collection period, which dropped to 237.5 +/- 67 ng ANG I. ml-1. h-1. mg protein-1. min-1 (n = 9/18) within 100 min. This ADA II effect was rapid in onset and fully reversible. When the purified ADA type VII, with a 40-fold higher specific activity, was added to the superfusate, RSR was increased only by 96 +/- 17.8% compared with controls. This ADA VII (5 U/30 microgram) effect could be mimicked by the selective adenosine A1-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 10(-6) mol/l). Since albumin stimulated RSR in a concentration-dependent fashion, to an extent similar to that of ADA II, we assume that the ADA II effect was largely unspecific in nature. We conclude that 1) superfusion of isolated JGC from rats is suitable for investigations of renin secretion at the cellular level, 2) the increase in RSR by ADA II appears to be only in part due to deamination of endogenously generated adenosine, and 3) albumin in the superfusate induces a similar stimulatory effect as ADA II.

Adenosine↗
PubMed1997

The hemopexin-like domain (C domain) of human gelatinase A (matrix metalloproteinase-2) requires Ca2+ for fibronectin and heparin binding. Binding properties of recombinant gelatinase A C domain to extracellular matrix and basement membrane components.

The binding properties of the COOH-terminal hemopexin-like domain (C domain) of human gelatinase A (matrix metalloproteinase-2, 72-kDa gelatinase) were investigated to determine whether the C domain has binding affinity for extracellular matrix and basement membrane components. Recombinant C domain (rC domain) (Gly417-Cys631) was expressed in Escherichia coli, and the purified protein, identified using two antipeptide antibodies, was determined by electrospray mass spectrometry to have a mass of 25,925 Da, within 0.1 Da of that predicted. As assessed by microwell substrate binding assays and by column affinity chromatography, the matrix proteins laminin, denatured type I collagen, elastin, SPARC (secreted protein that is acidic and rich in cysteine), tenascin, and MatrigelTM were not bound by the rC domain. Unlike the hemopexin-like domains of collagenase and stromelysin, the rC domain also did not bind native type I collagen. Nor were native or denatured types II, IV, V, and X collagen, or the NC1 domain of type VII collagen bound. However, binding to heparin and fibronectin (Kd, 1.1 x 10(-6) M) could be disrupted by 0.58-0.76 and 0.3 M NaCl, respectively. Using nonoverlapping chymotrypsin-generated fragments of fibronectin, binding sites for the rC domain were found on both the 40-kDa heparin binding and the 120-kDa cell binding fibronectin domains (Kd values, approximately 4-6 x 10(-7) M). The Ca2+ ion, but not the potential structural Zn2+ ion, were found to be essential for maintaining the binding properties of the protein. The apo-form of the rC domain did not bind heparin, and both ethylenediaminetetraacetic acid and the specific Ca2+ ion chelator 1, 2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid, but not the Zn2+ ion chelator 1,10-phenanthroline, eluted the holo form of the rC domain from both heparin-Sepharose and fibronectin. Inductive coupled plasma mass spectrometry also did not detect a Zn2+ ion in the rC domain. In contrast, reduction with 65 mM dithiothreitol did not interfere with heparin binding, further emphasizing the crucial structural role played by the Ca2+ ion. Together, these data demonstrate for the first time that the hemopexin-like domain of gelatinase A has a binding site for fibronectin and heparin, and that Ca2+ ions are important in maintaining the structure and function of the domain.

Binding Sites↗
PubMed1999

Targeting tumor vasculature endothelial cells and tumor cells for immunotherapy of human melanoma in a mouse xenograft model.

An immunotherapy treatment for cancer that targets both the tumor vasculature and tumor cells has shown promising results in a severe combined immunodeficient mouse xenograft model of human melanoma. The treatment involves systemic delivery of an immunoconjugate molecule composed of a tumor-targeting domain conjugated to the Fc effector domain of human IgG1. The effector domain induces a cytolytic immune response against the targeted cells by natural killer cells and complement. Two types of targeting domains were used. One targeting domain is a human single-chain Fv molecule that binds to a chondroitin sulfate proteoglycan expressed on the surface of most human melanoma cells. Another targeting domain is factor VII (fVII), a zymogen that binds with high specificity and affinity to the transmembrane receptor tissue factor (TF) to initiate the blood coagulation cascade. TF is expressed by endothelial cells lining the tumor vasculature but not the normal vasculature, and also by many types of tumor cells including melanoma. Because the binding of a fVII immunoconjugate to TF might cause disseminated intravascular coagulation, the active site of fVII was mutated to inhibit coagulation without affecting the affinity for TF. The immunoconjugates were encoded as secreted molecules in a replication-defective adenovirus vector, which was injected into the tail vein of severe combined immunodeficient mice. The results demonstrate that a mutated fVII immunoconjugate, administered separately or together with a single-chain Fv immunoconjugate that binds to the tumor cells, can inhibit the growth or cause regression of an established human tumor xenograft. This procedure could be effective in treating a broad spectrum of human solid tumors that express TF on vascular endothelial cells and tumor cells.

Adenoviridae↗
PubMed1991

Effects of interleukin-6, interleukin-2, and tumor necrosis factor alpha on transferrin release from Sertoli cells in culture.

Recent studies demonstrate that several cytokines are potent modulators of steroid release from the testis. In an attempt to determine whether these agents may influence other types of secreted substances, we used plaque assays to measure the effect of interleukin-6 (IL-6), interleukin-2 (IL-2), and tumor necrosis factor alpha on transferrin (TF) release from Sertoli cells in culture. Because Sertoli cells from different parts of the tubule respond differently to modulatory factors, we used cultures obtained by microdissection from stages III-V, VII, IX-XI, and XIII of the cycle of the seminiferous epithelium. Our results revealed that each agent increased the rate of TF plaque formation from cultures of IX-XI, and XIII staged segments but not from those staged III-V and VII. Moreover, IL-6, but not the other cytokines, modified the response of Sertoli cells to another regulator, FSH. This was evidenced by our findings that pretreatment with IL-6 for 1 h resulted in FSH-induced increases in the rate of plaque formation for cells from IX-XI segments, in addition to those segments which are normally responsive without pretreatment (III-V and VII segments). Further experiments revealed that IL-6 also had a chronic influence on the proportion of TF secretors present in certain staged cultures. Treatment for 24 h with IL-6 markedly reduced the percentage of TF secretors in cultures from stage XIII segments and resulted in a slight increase in TF cells for stage VII cultures. However, no chronic influences in TF secretors were detected with either IL-2 or tumor necrosis factor alpha treatment. Our results demonstrate very clearly that certain cytokines acting in a stage specific manner have acute and/or chronic influences on the release of TF from Sertoli cells. These findings, when viewed in light of reports of the presence of these factors in the testis, suggest strongly that cytokines or cytokine-like substances, by modulating the release of Sertoli cell substances, may play an important role in testis function.

Animals↗
PubMed1997

Apoptosis pattern elicited by oestradiol treatment of the seminiferous epithelium of the adult rat.

It is widely assumed that oestrogen administration in the male mimics hypophysectomy by suppressing gonadotrophin secretion. Nevertheless, oestradiol treatment can increase germ-cell apoptosis mainly at stages IV-X of the spermatogenic cycle, rather than at stage VII when apoptotic germ-cell death is mainly triggered by gonadotrophin withdrawal caused by hypophysectomy. Since the roles of testicular oestrogens in spermatogenic regulation, if any, are unknown, we re-evaluated the germ-cell types that undergo apoptosis after oestradiol treatment. Adult male rats were injected daily with 50 micrograms oestradiol, oestradiol plus testosterone propionate (25 mg every 3 days) or oestradiol plus human menopausal gonadotrophin (equivalent to 25 iu FSH plus 25 iu LH) for 15 days. Apoptosis was assessed by in situ 3'-end labelling of internucleosomal DNA fragments in plastic semithin sections; the germ-cell types involved were identified by high-resolution light microscopy. The quantitative analysis of our results shows that the apoptosis pattern elicited by oestradiol treatment of the seminiferous epithelium differs from that reported to be caused by gonadotrophin or testosterone withdrawal, suggesting a possible role for oestradiol in the modulation of germ-cell death in the adult testis of the rat.

Animals↗
PubMed2005

Of four mutations in the factor VII gene in Tunisian patients, one novel mutation (Ser339Phe) in three unrelated families abrogates factor X activation.

Hereditary factor VII (FVII) deficiency is a rare bleeding disorder. Dysfunctional FVII variants characterized by normal or reduced levels of FVII antigen and discordantly low FVII activity have been described. In this study, seven unrelated Tunisian patients with FVII deficiency were examined. Molecular analysis revealed that three probands harbored a novel Ser339Phe mutation, one proband was inferred to have a novel splice site mutation in intron 2, c.226-2 A>G and three probands had two previously described mutations, Arg304Gln and Cys310Phe. Expression of Ser339Phe in baby hamster kidney cells yielded secretion of FVII antigen at a concentration of 225+/-50 ng/ml, compared with 181+/-47 ng/ml in cells transfected with wild-type FVII but with no demonstrable FVII activity. FVII Ser339Phe bound to tissue factor similarly to the binding of commercial recombinant activated FVII or recombinant wild-type FVII and was normally activated by activated factor X. The major defect of FVII Ser339Phe was its inability to activate factor X in the presence of tissue factor. Modeling predicted that the substitution of Ser339 by Phe abrogated substrate docking.

DNA Mutational Analysis↗
PubMed1989

Regulation of urokinase- and tissue-type plasminogen activator gene expression in the rat seminiferous epithelium.

The secretion of plasminogen activator by seminiferous tubules at defined stages of the epithelial cycle is influenced both by neighboring spermatogenic cells and by hormones. We have used cRNA probes for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators to analyze their mRNA levels in different stages of the epithelial cycle. Urokinase-type PA mRNA was most abundant in stages VII-VIII, while tPA mRNA levels showed smaller variations between the different stages. Both FSH and (Bu)2cAMP increased the steady-state level of tPA mRNA and tPA production without affecting those of uPA in stages VII-IX in vitro, whereas retinoic acid treatment selectively increased the concentration uPA mRNA and uPA production in stages II-VI. The results show that the expression of the uPA and tPA genes is differentially regulated in specific stages of the rat seminiferous epithelium.

Animals↗
PubMed2000

Synaptotagmin VII regulates Ca(2+)-dependent exocytosis of lysosomes in fibroblasts.

Synaptotagmins (Syts) are transmembrane proteins with two Ca(2+)-binding C(2) domains in their cytosolic region. Syt I, the most widely studied isoform, has been proposed to function as a Ca(2+) sensor in synaptic vesicle exocytosis. Several of the twelve known Syts are expressed primarily in brain, while a few are ubiquitous (Sudhof, T.C., and J. Rizo. 1996. Neuron. 17: 379-388; Butz, S., R. Fernandez-Chacon, F. Schmitz, R. Jahn, and T.C. Sudhof. 1999. J. Biol. Chem. 274:18290-18296). The ubiquitously expressed Syt VII binds syntaxin at free Ca(2+) concentrations ([Ca(2+)]) below 10 microM, whereas other isoforms require 200-500 microM [Ca(2+)] or show no Ca(2+)-dependent syntaxin binding (Li, C., B. Ullrich, Z. Zhang, R.G.W. Anderson, N. Brose, and T.C. Sudhof. 1995. Nature. 375:594-599). We investigated the involvement of Syt VII in the exocytosis of lysosomes, which is triggered in several cell types at 1-5 microM [Ca(2+)] (Rodríguez, A., P. Webster, J. Ortego, and N.W. Andrews. 1997. J. Cell Biol. 137:93-104). Here, we show that Syt VII is localized on dense lysosomes in normal rat kidney (NRK) fibroblasts, and that GFP-tagged Syt VII is targeted to lysosomes after transfection. Recombinant fragments containing the C(2)A domain of Syt VII inhibit Ca(2+)-triggered secretion of beta-hexosaminidase and surface translocation of Lgp120, whereas the C(2)A domain of the neuronal- specific isoform, Syt I, has no effect. Antibodies against the Syt VII C(2)A domain are also inhibitory in both assays, indicating that Syt VII plays a key role in the regulation of Ca(2+)-dependent lysosome exocytosis.

Animals↗
PubMed2004

A patient homozygous for a Gly354Cys mutation in factor VII that results in severely impaired secretion of the molecule, but not complete deficiency.

We investigated the molecular basis of factor VII (FVII) deficiency in a Japanese woman who suffered occasional epistaxis. The patient had low levels of both FVII coagulant activity (FVII:C) and antigen (FVII:Ag) (5.0% and 7.3% of normal controls respectively). DNA sequence analysis of the FVII gene showed that the patient was homozygous for a mutation that resulted in a Cys for Gly354 substitution, a novel missense mutation in the catalytic domain. Haplotype analysis showed that this missense mutation was inherited from her consanguineous parents. Transient expression experiments showed that secreted FVII Cys354, FVII:C and FVII:Ag levels in conditioned media were reduced to 4% and 5%, respectively, of levels secreted from wild-type FVII. However, the intracellular FVII Cys354 was 67% that of normal recombinant protein. Immunohistochemical analysis showed that intracellular FVII:Ag from FVII 354Cys was present diffusely throughout the cytoplasm. Substitution of FVII 354Gly with amino acids other than Cys (Arg, Asp, Ser and Phe), did not produce a phenotype similar to FVII Cys354Gly. Molecular modelling indicated that FVII Gly354 was located outside the FVII heavy chain, and that Cys135 in the EGF2 domain, Cys262 in the catalytic domain and Cys127 all exist within 10 A of Gly354. Therefore, we propose that the introduction of an additional free cysteine residue in the FVII molecule results in the formation of illegitimate disulphide bonds and a mis-folded domain, leading to defective secretion.

Adult↗
PubMed2002

Human factor VII deficiency caused by S339C mutation located adjacent to the specificity pocket of the catalytic domain.

This report documents our identification of a novel factor VII (FVII) gene mutation in a Japanese boy with FVII deficiency. The proband's FVII activity was 34% and his FVII antigen level was 40% of normal controls. DNA sequence analysis of the proband's FVII gene identified a C to G point mutation at nucleotide position 10 933 in exon 8, which results in the substitution of Cys (TGC) for Ser339 (TCC). Hinf I digestion results indicate the proband and his mother were heterozygous for the mutation. Both wild-type and mutant FVIIs were transiently expressed in COS-1 cells. FVII levels measured in the culture medium of FVII Ser339Cys mutants were markedly reduced as compared to those of cells with FVII wild-type. The amount of intracellular FVII in FVII Ser339Cys mutants was 80% of that in wild-type. In the wild-type FVII, Ser339 is juxtaposed to Asp338, which is positioned at the bottom of the substrate-binding pocket in the protease domain and located adjacent to FVII Cys340, that forms a disulphide bond with Cys368. We suspect that the creation of a novel unpaired cysteine through this mutation leads to abnormal disulphide bonding during protein folding, thereby reducing the secretion of FVII.

Catalytic Domain↗
PubMed1998

Factor VII deficiency caused by a structural variant N57D of the first epidermal growth factor domain.

We have previously described a kindred with factor VII (FVII) deficiency whose members exhibited reduced procoagulant activity relative to FVII antigen concentration. In this report, the molecular genetic basis of the FVII defect has been determined to be a heterozygous substitution of Asp for Asn at position 57 in the first epidermal growth factor (EGF) domain. Recombinant FVII (N57D) cDNA was created by site-directed mutagenesis and transiently expressed in human 293 cells. The transfected cells synthesized an immunoprecipitable protein with an apparent molecular weight of 50 kD. Quantitation of expression by FVII enzyme-linked immunosorbent assay indicated that mutant protein yields were consistently low, typically 10% to 30% of wild-type FVII. FVII (N57D) protein did not accumulate intracellularly, and Northern blot analysis indicated equivalent FVII mRNA levels in 293 cells expressing either wild-type FVII or FVII (N57D). Secreted FVII (N57D) protein did not bind tissue factor, exhibited no procoagulant activity, and failed to bind a conformation-dependent monoclonal antibody specific for the first EGF domain of FVII. Molecular modeling of the first EGF domain of FVII predicted that the N57D amino acid substitution would disrupt tertiary bonding structure. We conclude that the N57D mutation affects folding of the first EGF domain of FVII resulting in decreased cellular secretion of a mutant FVII molecule, which is unable to bind tissue factor and is therefore biologically inactive.

Animals↗
PubMed1997

Amphotropic and ecotropic retroviral vector viruses transduce midgestational murine fetal liver cells in a dual-chambered cocultivation system.

A beta-glucuronidase cDNA was transferred into fetal liver cells (FLC) from mice affected with mucopolysaccharidosis (MPS) type VII and normal littermates using a retrovirus vector. The cells were transduced by direct cocultivation or by culturing the FLC and vector packaging cells separated by a 0.45 micron filter in a dual-chambered cocultivation system. Gene transduction occurred using an ecotropic or amphotropic vector in FLC obtained from 13.5- and 15.5-day-old murine fetuses. Histochemical staining assays and measurement of enzyme activity demonstrated gene expression in the midgestational FLC. Enzyme secreted into the supernatant from transduced FLC obtained from 13.5-day-old affected fetuses surpassed secreted enzyme from normal, age-matched untransduced FLC. The 13.5 day FLC, transduced by direct cocultivation or by using the dual-chambered system, were transplanted in utero into 13.5-day-old murine fetuses. Proviral sequences were detected in various organs shortly after birth. The results indicate that midgestational FLC can be transduced with an amphotropic vector virus in a dual-chambered cocultivation system without contaminating virus-producing packaging cells and that the transduced cells survive in utero transplantation.

Animals↗
PubMed1999

Characterization of two naturally occurring mutations in the second epidermal growth factor-like domain of factor VII.

We investigated the mechanisms responsible for severe factor VII (FVII) deficiency in homozygous Italian patients with either Gly97Cys or Gln100Arg mutations in the second epidermal growth factor domain of FVII. Transient expression of complementary DNA coding for the mutations in COS-1 cells showed impaired secretion of the mutant molecules. Using stably transfected Chinese hamster ovary (CHO) cells, we performed pulse-chase labeling studies, immunohistochemistry, and experiments with inhibitors of protein degradation, showing that FVII-Cys97 did not accumulate intracellularly but was degraded in a pre-Golgi, nonlysosomal compartment by a cysteine protease. In stably transfected CHO cells expressing FVII-Arg100, the level of intracellular FVII was not increased by several inhibitors of protein degradation, but FVII-Arg100 was retained in the endoplasmic reticulum for a longer period of time than wild-type FVII. FVII-Arg100 had a lower apparent molecular weight than did wild-type FVII under nondenaturing conditions, which is attributable to misfolding due to abnormal disulfide bond formation.

Animals↗
PubMed1976

Two chromosomal genes required for killing expression in killer strains of Saccharomyces cerevisiae.

The killer character of yeast is determined by a 1.4 X 10(6) molecular weight double-stranded RNA plasmid and at least 12 chromosomal genes. Wild-type strains of yeast that carry this plasmid (killers) secret a toxin which is lethal only to strains not carrying this plasmid (sensitives).--We have isolated 28 independent recessive chromosomal mutants of a killer strain that have lost the ability to secrete an active toxin but remain resistant to the effects of the toxin and continue to carry the complete cytoplasmic killer genome. These mutants define two complementation groups, kex1 and kex2. Kex1 is located on chromosome VII between ade5 and lys5. Kex2 is located on chromosome XIV, but it does not show meiotic linkage to any gene previously located on this chromosome.--When the killer plasmid of kex1 or kex2 strains is eliminated by curing with heat or cycloheximide, the strains become sensitive to killing. The mutant phenotype reappears among the meiotic segregants in a cross with a normal killer. Thus, the kex phenotype does not require an alteration of the killer plasmid.--Kex1 and kex2 strains each contain near-normal levels of the 1.4 x 10(6) molecular weight double-stranded RNA, whose presence is correlated with the presence of the killer genome.

Chromosome Mapping↗
PubMed1982

Stage-dependent secretion of ABP by rat seminiferous tubules.

The secretion rate of the Sertoli-cell-specific androgen-binding protein (ABP), has been studied in isolated rate seminiferous tubules, where the stages of the spermatogenic cycle have been identified by a transillumination method. The secretion of ABP was highest in stages VII-XII, as determined by steady-state polyacrylamide-gel electrophoresis as well as by radioimmunoassay. More specifically, when the sensitive RIA technique permitted the assay of ABP secretion from a total amount of 10-30 mm of isolated tubules, it was found that maximal ABP secretion occurred at stages VIII-XI and minimal at stages IV-V. The present results show that the secretory activity of the Sertoli cells (ABP) is influenced by the type of germ cell at each cell association. It is postulated that the variation is Sertoli-cell secretory activity is of importance for the normal maintenance of spermatogenesis.

Androgen-Binding Protein↗
PubMed2004

[Role of the hemostatic system in pathogenesis of atherosclerosis as the main etiology of coronary ischemia] [corrected].

Coronary artery disease is a leading cause of mortality in highly developed societies. This occurs in spite of growing therapeutic opportunities. Atherosclerosis begins as a functional or/and structural damage of endothelium, which in turn causes its discontinuation and impairs humoral and secreting function. Haemostasis plays an important role in the progression of atherosclerosis and development of cardiac complications--acute coronary syndromes. Research still continues to determine precisely role of each of haemostasis disorders in increased risk of coronary artery disease and its complications. The aim of this paper is to review the literature data concerning haemostatic risk factors and their role of development of coronary artery disease. Fibrinogen, thrombocytes, factor VII, factor VIII, von Willebrand factor (vWF), thrombomodulin (TM), plasminogen activator inhibitor--type 1 (PAI-1), tissue-plasminogen activator (t-PA) and other haemostatic factors, were described as more or less helpful in estimation of risk of occurrence of coronary artery disease and its cardiovascular complications. Only some of the described hematologic factors were verified so far in large prospective studies, and were recognized as independent risk factors of cardiovascular diseases.

Arteriosclerosis↗