Zonal necrosis prevented by transduction of the artificial anti-death FNK protein.

Protection of cells from necrosis would be important for many medical applications. Here, we show protein transduction domain (PTD)-FNK therapeutics based on protein transduction to prevent necrosis and acute hepatic injury with zonal death induced by carbon tetrachloride (CCl4). PTD-FNK is a fusion protein comprising the HIV/Tat PTD and FNK, a gain-of-function mutant of anti-apoptotic Bcl-x(L). PTD-FNK protected hepatoma HepG2 from necrotic death induced by CCl4, and additionally, increased the apoptotic population among cells treated with CCl4. A concomitant treatment with a pan-caspase inhibitor Z-VAD-FMK (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone), which alone could not prevent the necrosis, protected these cells from the apoptosis. When pre-injected intraperitoneally, PTD-FNK markedly reduced zonal liver necrosis caused by CCl4. Moreover, injection of PTD-FNK accompanied by Z-VAD-FMK suppressed necrotic injury even after CCl4 administration. These results suggest that PTD-FNK has great potential for clinical applications to prevent cell death, whether from apoptosis or necrosis, and organ failure.

A case report: verrucous carcinoma of the endometrium--the difficulty of diagnosis, and a review of the literature.

A verrucous carcinoma is a subtype of well-differentiated squamous-cell carcinomas, arising in the vagina, vulva, and uterine cervix. But a verrucous carcinoma very rarely arises in the uterine endometrium. The present paper presents a case of a verrucous carcinoma of the endometrium that is described in association with the tumor marker SCC; this paper also includes a review of the relevant literature.

Suppression of calphobindin I (CPB I) production in carcinoma of uterine cervix and endometrium.

Calphobindin I (CPB I) is a member of the family of Ca(2+)-dependent phospholipid binding proteins collectively termed as annexins. CPB I (Annexin V) has recently been shown to be an endogenous inhibitor of protein kinase C, a key enzyme in the cellular signal transduction and its inhibition by CPB I is presumed to be related ultimately to carcinogenesis. We therefore examined the level of production of CPB I in uterine cancer cells. Immunohistochemical analysis, northern blot, and in situ hybridization showed that the production of CPB I was markedly suppressed at the level of transcription in both cervical and endometrial carcinoma cells when compared to their normal counterparts. Decrease in production of CPB I may lead to dysregulated activation of protein kinase C and, accordingly, may be involved in a disorder of cell differentiation, proliferation, and carcinogenesis.

Antibody to annexin V has anti-phospholipid and lupus anticoagulant properties.

Anti-annexin V (anti-ANXV) IgG and lupus anticoagulant (LAC) were both shown to be capable of inducing apoptosis in umbilical vein endothelial cells [Nakamura et al.: Biochem Biophys Res Commun 205:1488-1493, 1994]. In the present study, we have demonstrated that anti-ANXV IgG prolongs the activated partial thromboplastin time and has an affinity for phospholipids in enzyme-linked immunosorbent assay. This indicates overlapping of anti-ANXV and LAC activities, suggesting that ANXV may be involved in the autoimmune mechanism for LAC production.

Lupus anticoagulant autoantibody induces apoptosis in umbilical vein endothelial cells: involvement of annexin V.

The effects of lupus anticoagulant (LAC) on cultured human umbilical vein endothelial cells were studied. All five monoclonal antibodies from a patient with systemic lupus erythematosus (SLE), as well as plasma samples with LAC activity from six SLE patients, induced apoptosis. Anti-annexin V IgG also induced apoptosis. Since monoclonal antibodies and plasma from SLE patients had an affinity for annexin V, an endothelial apoptosis pathway mediated by annexin V was suggested as the molecular pathogenesis of the hemostatic derangement associated with LAC.

Effects of danazol at the immunologic level in patients with adenomyosis, with special reference to autoantibodies: a multi-center cooperative study.

OBJECTIVE: We attempted to evaluate the effects of danazol on autoantibodies, in particular, to phospholipids, and on the immune system in patients with adenomyosis. STUDY DESIGN: Forty-two patients with adenomyosis who had high titers of autoantibodies were randomly chosen, and they received 400 mg/day of danazol for 4 months (n = 22) or underwent hysterectomy (n = 20). RESULTS: Among the six autoantibodies we investigated, the incidence of antiphosphatidylinositol immunoglobulin G was the highest (42.9%), followed by antiphosphatidylglycerol immunoglobulin G (38.1%). The autoantibody titers decreased with time and were comparable to the control values 16 weeks after treatment in both groups. Total serum levels of immunoglobulin G and M were high before treatment, but immunoglobulin M levels decreased significantly in week 8 during treatment with danazol, whereas C4 levels increased and C3 levels decreased with danazol. CONCLUSION: Danazol has an inhibitory effect on the autoimmunologic response associated with adenomyosis.

Phospholipid-binding properties of calphobindin-II(annexin VI), anticoagulant protein from human placenta.

Calphobindin-II (CPB-II, annexin VI), is a calcium dependent phospholipid binding protein that can be classified as a member of the annexin family. The phospholipid-binding properties of CPB-II were investigated by measuring the binding constants of [125I]-CPB-II using phospholipid vesicles consisting of 80% phosphatidylcholine and 20% phosphatidylserine. A dissociation constant (Kd) of CPB-II with the phospholipid vesicles was determined to be 0.2 to 0.3 nM in the presence of Ca2+ ranging from 0.3 to 30 mM. The number of CPB-II capable of binding to the phospholipid vesicles at 0.3 mM Ca2+ decreased to about 1/2 in the presence of Ca2+ of more than 1 mM. Prothrombin and factor X were effective in competing with the binding of CPB-II to the phospholipid vesicles, although their affinities were lower by two or three orders of magnitude than that of unlabeled CPB-II at 30 nM Ca2+. Competitive effects of CPB-II, calphobindin-I (CPB-I, annexin V) and calphobindin-III (CPB-III, annexin III) on binding of [125I]-CPB-II to phospholipid vesicles, were similarly observed.

An enzyme-linked immunosorbent assay system for quantitative determination of calphobindin I, a new placental anticoagulant protein, and its application to various specimens.

We developed a sandwich enzyme-linked immunosorbent assay (ELISA) system for calphobindin I (CPB-I), a new placental coagulation inhibitor, using two monoclonal antibodies. This ELISA system can detect CPB-I at concentrations of between 0.4 and 25 ng/ml in buffer and allow almost quantitative determination of it in human plasma. Using this ELISA system, CPB-I levels in many kinds of specimens were measured. Levels in the plasma and urine of women were as low as 10 ng/ml, and no significant differences were observed throughout the trimesters of pregnancy and during different stages of the menstrual cycle. Toxemic patients were slightly higher in CPB-I levels than normal pregnant women, and levels in body fluids such as the amniotic fluid, saliva, milk, ascites, and semen were higher than those in the plasma. The high levels of CPB-I were found, being in the order of micrograms/ml, in the ascites of carcinomatous peritonitis as well as seminal plasma. Measurements of the levels in ovarian follicular fluid samples at different stages of the menstrual cycle showed that those in the immature and atretic stages were higher than those in mature stages. CPB-I levels in many types of cultured human cells ranged from 0.023 to 10.30 micrograms/mg protein, and levels in cultured human lymphocytes were less than those in other types of cells measured. Little of this inhibitor was secreted into media from cultured human lymphocytes, and it was found in all measured tissues of Macacus irus at levels ranging from 0.232 to 1.557 micrograms/mg protein. From these results, it was suggested that CPB-I might be a ubiquitous protein in the body that has an important physiological role.

Structure and properties of calphobindin II, an anticoagulant protein from human placenta.

The structure of human placental calphobindin-II (CPB-II) was investigated by amino acid composition and amino acid sequence analyses of peptides generated by protease digestion of the protein. The 45 peptides obtained from the lysyl endopeptidase digest of CPB-II, and the amino-terminal peptide prepared from its tryptic digest, were analyzed, and they accounted for over 98% of total amino acids of CPB-II. The structure of CPB-II determined by protein sequencing was identical to that previously predicted from its cDNA sequence (Iwasaki, A. et al. (1989) J. Biochem. 106, 43-49), except for the amino terminus. Since the amino terminus of CPB-II was blocked to Edman degradation, fast-atom-bombardment mass spectrometric analysis was used to demonstrate that the amino-terminal residue was acetyl-alanine. The carboxyl-terminal residue of CPB-II was identified as aspartic acid by the hydrazinolytic procedure. Calcium-binding studies indicated that 1 mol of CPB II binds 1 mol of calcium in the absence of phospholipid and 8 mol of calcium in the presence of phospholipid.

Structure and expression of cDNA for calphobindin II, a human placental coagulation inhibitor.

Calphobindin II, with Mr 73,000, is one of the human placental anticoagulant proteins. The cDNA encoding calphobindin II was obtained by screening a human placental lambda gt11 cDNA library using a specific antibody as a probe. The longest cDNA insert consisted of 2,361 nucleotides and a 64-nucleotide-long poly(A) tract. An open reading frame encoding 673 amino acids was predicted. The deduced sequence includes an 8-fold repeat of a conserved 70-amino-acid-long segment that has a high degree of sequence identity with the repeated segments in members of the Ca2+-dependent phospholipid binding protein family. The cDNA fragment including the open reading frame was introduced into the expression vector pKK223-3 and subsequently expressed in Escherichia coli JM105 cells. The resulting recombinant protein reacted with the specific monoclonal antibodies to calphobindin II and prolonged the blood coagulation time as did placental calphobindin II.

Isolation and characterization of an anticoagulant protein from human placenta.

An anticoagulant protein was purified from the EDTA extract of human placental tissue. The purified protein had a molecular weight of 73,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis under both reducing and non-reducing conditions. Because this protein had the ability to bind phospholipids such as phosphatidylserine, phosphatidylinositol, and cardiolipin in the presence of Ca2+, this protein was designated as calphobindin II (CPB-II). CPB-II prolonged the clotting time of normal plasma when coagulation was induced by tissue factor, cephalin and ellagic acid or recalcification, but did not affect thrombin-initiated fibrin formation. CPB-II also inhibited the activation of prothrombin by the complete prothrombinase complex or factor Xa-phospholipid-Ca2+ but not that by phospholipid-free factor Xa. In addition, CPB-II had an inhibitory activity against phospholipase A2.

[Fundamental study of imipenem/cilastatin sodium in obstetrics and gynecology].

Following results were obtained from drip intravenous administration of imipenem/cilastatin sodium (MK-0787/MK-0791) (500 mg/500 mg) by measuring concentration of MK-0787 in uterine arterial plasma, cubital venous plasma, oviduct, ovary and several sites in uterine tissue in cases of simple hysterectomy, and pelvic cavity fluid in cases of radical operation. Cervix uteri, portio vaginalis, myometrium showed higher concentration among various uterine tissues in any time after the end of administration. In cases of radical operation, the pelvic cavity fluid showed 6.6 approximately 7.8 micrograms/ml at 5 hours after the end of administration. In the field of obstetrics and gynecology, it was considered that MK-0787/MK-0791 has good efficacy in infections especially caused by Gram-positive aerobic bacteria.

[Acting point of placental coagulation inhibitor].

In the previous paper, the methods of purification of placental coagulation inhibitor (PCI) were reported. This paper deals with the acting point of the PCI on the coagulation process. Prothrombin time, activated partial thromboplastin time, Russell's viper venom time, and recalcification time were all prolonged by PCI. Neither thrombin time nor Echis carinatus venom time was affected by PCI. Factors XII, XI, X, IX, VIII, VII and V were not inactivated by the incubation with PCI. PCI had no activities of fibrinolysis, antifibrinolysis and protein Ca. In the intrinsic pathways, activity of phospholipid seemed to be inhibited by PCI. The tissue thromboplastin activity was completely inactivated by incubation with PCI.

[Isolation and purification of placental coagulation inhibitor].

The placenta contains such thrombotic factors as tissue thromboplastin, placental factor XIII, and placental urokinase inhibitor. On the other hand, there are some antithrombotic factors, for example, placental plasminogen activator and platelet aggregation inhibitor. This paper deals with another antithrombotic factor isolated from the human placenta. The results obtained are as follows: The placental coagulation inhibitor (PCI) was isolated from the human placental extract, by delipidation and chromatographic procedures with Con-A Sepharose, DEAE-Sephacel and gel filtration with Sephacryl S-300 and Sephadex G-100. The PCI was a protein, having a molecular weight of approximately 45,000 daltons. Immunological examination revealed that the PCI was different from such well-known anticoagulants as AT-III, alpha 1-AT, alpha 2-M, C1-INA, and the PCI had no heparin like characteristics. The PCI had neither fibrinolytic nor antifibrinolytic activity. Platelet aggregation was not inhibited by the PCI. The PCI had anticoagulant activity which prolongs both intrinsic and extrinsic coagulation systems.