Endogenous basic fibroblast growth factor-dependent induction of collagenase and interleukin-6 in tumor necrosis factor-treated human microvascular endothelial cells.

Tumor necrosis factor-alpha (TNF-alpha) has been shown to enhance the synthesis of interleukin-6 (IL-6) and collagenase in human omental microvascular endothelial (HOME) cell (Mawatari, M., Kohno, K., Mizoguchi, H., Matsuda, T., Asoh, K., Van Damme, J. V., Welgus, H. G., and Kuwano, M. (1989) J. Immunol. 143, 1619-1627). In the present study, we have examined whether the TNF-alpha-induced synthesis of IL-6 or collagenase in HOME cells is mediated by an inducible growth factor. Among several growth factors examined, we found that the expression of basic fibroblast growth factor (bFGF) mRNA was the one most prominently enhanced when HOME cells were treated with TNF-alpha. The increase of bFGF mRNA by TNF-alpha in HOME cells was observed in both a dose- and time-dependent manner when assayed by Northern blot analysis. The induction of bFGF mRNA was observed by 3 h after incubation with TNF-alpha, and the maximal increase of 5-fold was obtained after 12 h of incubation with 100 units/ml TNF-alpha. Western blot analysis confirmed the enhanced synthesis of bFGF by TNF-alpha. Metabolic labeling and immunoprecipitation assays of bFGF showed that exposure to TNF-alpha enhanced secretion of bFGF into culture medium and also that TNF-alpha stimulated the production of bFGF molecules with molecular masses of 18, 21, and 22.5 kDa in HOME cells. TNF-alpha induced the expression of collagenase mRNA and IL-6 mRNA in HOME cells as well, and the coadministration of neutralizing anti-bFGF antibody almost completely blocked the effects of TNF-alpha. The treatment of HOME cells with exogenous bFGF significantly stimulated the expression of collagenase mRNA and IL-6 mRNA in HOME cells. Therefore, the biological effects of TNF-alpha on HOME cells may be mediated, at least in part, by TNF-alpha-induced bFGF.     

Enzymatic preparation of organometallic compounds with second-order optical nonlinearities

Twelve species of optically active metallocene derivatives having a 4-nitrophenyl group were prepared with horse liver alcohol dehydrogenase- or lipase-catalyzed optical resolution as the key step. The second harmonic generation (SHG) efficiently of these products was measured by the power method using the fundamental light from the Nd:YAG laser. (-)-1- (4-Nitrophenylthio)ethylferrocene (9), (+)-1-(4-nitrophenylthio)ethylruthenocene (16), (+)-1-(4-nitrophenylthio)ethylosmocene (19), (+) -1-(5-nitro-2-pyridylthio)ethylruthenocene (21), and (+) -1-[(4-nitrophenylhydrazono)methyl] -2-methylferrocene (12) showed SHG signals. The highest SHG efficiency was found with (+) -16, being 27 times more intense than the commonly used urea standard.     

2-Chlorodeoxyadenosine inhibits the repair of DNA double-strand breaks and does not inhibit the repair of DNA single-strand breaks in X-irradiated Chinese hamster V79 cells.

The exposure of log-phase Chinese hamster V79 cells to 2-chlorodeoxyadenosine (CdA) for 3 h after X irradiation enhanced the lethal effects of X-rays in a concentration-dependent manner. The enhancement of the killing efficiency of X-rays by CdA was mainly observed in the reduction of quasi-threshold doses (Dq) of the dose-response curves. When the ability of CdA to inhibit the repair of X-ray-induced double- and single-strand breaks (dsb and ssb) of DNA was investigated by neutral- and alkaline-filter elution techniques, respectively, it was observed that 90% of dsb were rejoined in the absence of CdA within 30 min after X irradiation and 15-40% of dsb rejoining was suppressed by co-incubation of the cells with 5-10 microM of CdA for 3 h after X irradiation, whereas almost 100% of ssb were rejoined within 15 min regardless of the presence or absence of CdA. From these results it was concluded that CdA interfered exclusively with the repair of DNA dsb in X-irradiated Chinese hamster V79 cells and thereby increased the lethality of X-rays.     

Anti-pituitary antibodies in patients with hypopituitarism and their families: longitudinal observation.

In an attempt to investigate the clinical significance of anti-pituitary antibodies in patients with hypopituitarism, anti-pituitary antibody in plasma was examined in 10 such patients (7 cases of isolated ACTH deficiency, 1 of partial hypopituitarism, and 2 of Sheehan's syndrome), on two or three occasions with an interval of more than 6 months (longitudinal study). In a total of 16 relatives of these 4 patients (2 cases of Sheehan's syndrome, one in each of partial hypopituitarism and isolated ACTH deficiency) and one patient not involved in the longitudinal study, anti-pituitary antibodies were also examined (family study). Anti-pituitary antibodies reacting with rat pituitary cytoplasmic antigens (pituitary cell antibodies: PCA) and pituitary cell surface antibodies (PCSA) reacting with GH3 cells and/or AtT-20 cells were measured with indirect immunofluorescence. The longitudinal study revealed the disappearance of antibodies in 3 patients, 2 PCA positive and one both PCA and PCSA positive. In 3 patients, altered antibody titers or a newly appearing antibody were found during the follow-up period. In 4 patients, the pituitary antibodies were negative during the follow-up periods. Of 16 family members studied, positive PCA was found in 3 members (2 in the families of patients with PCA positive Sheehan's syndrome, and 1 in the family of the patients with PCA positive partial hypopituitarism). Positive PCSA was found in 4 members (one in each of families of patients with partial hypopituitarism and isolated ACTH deficiency and of two cases of Sheehan's syndrome), and weakly positive PCSA was found in one family member of a patients with PCA positive Sheehan's syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide sequence and characterization of the sfs1 gene: sfs1 is involved in CRP*-dependent mal gene expression in Escherichia coli.

We have cloned at least 12 different Escherichia coli genes which enable strain MK2001 to use maltose. The genes were designated sfs1 through sfs12 (sugar fermentation stimulation). Previously, one (sfs7) of them was mapped at 65 min on the E. coli chromosome and identified as nlp, which has high homology to repressor protein (Ner) of Mu phage, which contains a putative DNA binding region (Y.-L. Choi, T. Nishida, M. Kawamukai, R. Utsumi, H. Sakai, and T. Komano, J. Bacteriol. 171:5222-5225, 1989). In this study, another gene (sfs1) located at 3.5 min was newly found and analyzed. The nucleotide sequence of sfs1 encoded a protein of 234 amino acids (molecular mass, 26,227 Da) which also has a putative DNA binding domain. Overexpression of the sfs1 gene in MK2001 resulted in a 10-fold increase of amylomaltase, which was still dependent on MalT. These results suggest that Sfs1 could be a new regulatory factor involved in maltose metabolism.     

Species-specific differences in heterogonic development of serially transferred free-living generations of Strongyloides planiceps and Strongyloides stercoralis

The direction of free-living development (homogonic vs. heterogonic) in Strongyloides stercoralis and Strongyloides planiceps was examined by successive transplantation of the uterine eggs of free-living females into a test tube culture system containing fresh feces. The eggs from the first-generation free-living females of S. stercoralis did not develop into second-generation free-living adult worms, but all developed into filariform larvae. However, the majority of S. planiceps eggs from the first-generation free-living females developed into second-generation free-living adults. By successive transfer of uterine eggs of each generation, 9 generations developed, and in every cycle more adult worms developed than filariform larvae. However, the number of free-living generations was not infinite; in experiments repeated twice, the number of worms developing from the eggs of eighth or ninth generations was too small to continue further culture. These findings indicate that the pattern of free-living development is different between the 2 species.     

cis-4-Hydroxy-L-proline and ethyl-3,4-dihydroxybenzoate prevent myogenesis of C2C12 muscle cells and block MyoD1 and myogenin expression.

cis-4-Hydroxy-L-proline (cis-OH-Pro) and ethyl-3,4-dihydroxybenzoate (EDHB), two distinct inhibitors of collagen synthesis, prevented myogenesis in C2C12 mouse skeletal muscle cells. Both inhibitors blocked myotube formation and the expression of sarcomeric myosin heavy chain. Northern blot analysis showed that cis-OH-Pro- and EDHB-treated C2C12 muscle cells did not express the myogenic regulatory genes, MyoD1 and myogenin, but continued to express non-muscle isoforms of actin (beta and gamma) and alpha-tropomyosin. 10TFL2-3B cells, a C3H10T1/2 cell line permanently transfected with myogenin cDNA, constitutively expressed exogenous myogenin in the presence of cis-OH-Pro but failed to activate endogenous myogenin and to undergo myogenesis. These results demonstrate that commitment to terminal differentiation and activation of myogenic regulatory genes requires active synthesis of the extracellular matrix component collagen.     

Regulation of the superoxide-generating NADPH oxidase by a small GTP-binding protein and its stimulatory and inhibitory GDP/GTP exchange proteins.

The superoxide-generating NADPH oxidase system in phagocytes consists of at least membrane-associated cytochrome b558 and three cytosolic components named SOCI/NCF-3/sigma 1/C1, SOCII/NCF-1/p47-phox, and SO-CIII/NCF-2/p67-phox. p47-phox and p67-phox were isolated, and their primary structures were determined, but SOCI has not been well characterized. In the present study, we first purified SOCI to homogeneity from the cytosol fraction of the differentiated HL-60 cells. The purified SOCI was a small GTP-binding protein (G protein) with a M(r) of about 22,000. The guanosine 5'-(3-O-thio)triphosphate-bound form, but not the GDP-bound form, of this small G protein showed the SOCI activity. The partial amino acid sequence of SOCI thus far determined was identical to the amino acid sequence deduced from the cDNA encoding rac2 p21. None of the purified small G proteins, including Ki-ras p21, smg p21B/rap1B p21, rhoA p21, and rac1 p21, showed the SOCI activity. These results indicate that SOCI is a small G protein very similar, if not identical, to rac2 p21. The GDP/GTP exchange reaction of SOCI was stimulated and inhibited by stimulatory and inhibitory GDP/GTP exchange proteins for small G proteins, named smg GDS and rho GDI, respectively. The NADPH oxidase activity was also stimulated and inhibited by smg GDS and rho GDI, respectively. These results indicate that the superoxide-generating NADPH oxidase system is regulated by both smg GDS and rho GDI through rac2 p21 or the rac2-related small G protein in phagocytes.     

Deficiency of X and Y chromosomal pairing at meiotic prophase in spermatocytes of sterile interspecific hybrids between laboratory mice (Mus domesticus) and Mus spretus

The normal association between the X and Y chromosomes at metaphase I of meiosis, as seen in air-dried light microscope preparations of mouse spermatocytes, is frequently lacking in the spermatocytes of the sterile interspecific hybrid between the laboratory mouse strains C57BL/6 and Mus spretus. The purpose of this work is to determine whether the separate X and Y chromosomes in the hybrid are asynaptic, caused by failure to pair, or desynaptic, caused by precocious dissociation. Unpaired X-Y chromosomes were observed in air-dried preparations at diakinesis, just prior to metaphase I. Furthermore, immunocytology and electron microscopy studies of surface-spread pachytene spermatocytes indicate that the X and Y chromosomes frequently fail to initiate synapsis as judged by the failure to form a synaptonemal complex between the pairing regions of the X and Y Chromosomes. Several additional chromosomal abnormalities were observed in the hybrid. These include fold-backs of the unpaired X or Y cores, associations between the autosome and sex chromosome cores, and autosomal univalents. The occurrence of abnormal autosomal and XY-autosomal associations was also correlated with cell degeneration during meiotic prophase. The primary breakdown in hybrid spermatogenesis occurs at metaphase I (MI), with the appearance of degenerated cells at late MI. In those cells, the X and Y are decondensed rather than condensed as they are in normal mouse MI spermatocytes. These results, in combination with the previous genetic analysis of spermatogenesis in hybrids and backcrosses with fertile female hybrids, suggest that the spermatogenic breakdown in the interspecific hybrid is primarily correlated with the failure of XY pairing at meiotic prophase, asynapsis, followed by the degeneration of spermatocytes at metaphase I. Secondarily, the failure of XY pairing can be accompanied by failure of autosomal pairing, which appears to involve an abnormal sex vesicle and degeneration at pachytene or diplotene.by C. Heyting     

Characterization of an abnormal fibrinogen Osaka V with the replacement of gamma-arginine 375 by glycine. The lack of high affinity calcium binding to D-domains and the lack of protective effect of calcium on fibrinolysis.

Prolonged thrombin time was completely corrected by the addition of millimolar concentrations of calcium in a new abnormal fibrinogen, Osaka V. Analysis of lysyl endopeptidase digests of A alpha-, B beta-, or gamma-chains by high performance liquid chromatography, and the following amino acid sequence analysis of relevant peptides revealed that about 50% of the gamma-chain has a replacement of gamma-arginine 375 by glycine. When fibrinogen was digested with plasmin in the presence of millimolar concentration of calcium, the amount of fragment D1 was about 50% of the normal control, and the rest was further cleaved to fragment D2, D3, or D62 with an apparent Mr of 62,000. Plasmic digestion of cross-linked fibrin in the presence of calcium resulted in the appearance of an abnormal fragment with an apparent Mr of 123,000 as well as fragments D2, D3, and D62, concomitant with the decrease of D dimer. The gamma-remnant of the abnormal fragment proved to be a cross-linked complex of the normal D1 gamma-remnant and residues 374-406/411 of the abnormal gamma-chain. The number of high affinity Ca(2+)-binding sites for the normal fibrinogen and fibrinogen Osaka V obtained by equilibrium dialysis was 2.88 (about 3) and 1.85, respectively, and that for the abnormal molecules was calculated as 0.9 (about 1) from their relative amounts in the samples, suggesting the lack of two Ca(2+)-binding sites in the D-domains. These data suggest that the normal structure of the COOH-terminal portion of the gamma-chain including residue 375 is required for the full expression of high affinity calcium binding to D-domains, the ability to be protected by calcium against plasmic digestion, and fibrin polymerization. During these studies, we found that the NH2-terminal amino acid of the gamma-remnant in fragments D or D dimer which were obtained after prolonged digestion with plasmin is gamma-Met89.