Dissociation of fibrinogen and fibronectin binding from transglutaminase-mediated cross-linking at the hepatocyte surface

The interaction of fibrinogen and fibronectin with hepatocytes has been dissociated into distinct binding and cross-linking steps. Binding and cross-linking of 125I-labeled ligands were both decreased by transglutaminase inhibitors, but not by heparin or hirudin. Transglutaminase activity was manifest by Ca2+-dependent incorporation of [14C]putrescine into cells. Preferential cross-linking of fibrinogen A alpha over gamma chains, and lack of inhibition by heparin or hirudin indicates the involvement of tissue transglutaminase, and not Factor XIIIa. Hepatic transglutaminase activity, as well as binding and cross-linking of fibrinogen and fibronectin, were maximally supported by Ca2+, partially supported by Mn2+ and Sr2+, and markedly decreased by Mg2+ and Ba2+. In contrast, Co2+ supported binding but not cross-linking or transglutaminase activity, indicating that binding and cross-linking are dissociable events. This conclusion was corroborated by the finding that fibrinogen fragments D95 and D78 both inhibited Ca2+-dependent fibrinogen binding without being cross-linked themselves. Ligand binding in the presence of either cation was localized to the cell surface as evidenced by its trypsin sensitivity. Thus, fibrinogen and fibronectin binding to hepatocytes is independent of transglutaminase activity, whereas cross-linking of these adhesive macromolecules requires an enzymatically active cellular transglutaminase. In addition, fibrinogen binding appears to be mediated by molecular determinants present in fragment D78.     

Differential effects on active avoidance performance and locomotor activity of two major enkephalin metabolites, tyr-gly-gly and des-tyr-[leu]enkephalin

We examined the effects of two enkephalin metabolites, des-tyr-[leu]enkephalin and tyr-gly-gly, on one-way active avoidance conditioning in mice. These metabolites are products of the two major enkephalin hydrolyzing enzymes in plasma, aminopeptidase and angiotensin converting enzyme. Like [leu]enkephalin from which it may be formed, tyr-gly-gly impaired avoidance acquisition, and its dose-response function for this effect was U-shaped. Also like [leu]enkephalin, tyr-gly-gly did not alter locomotor activity. On the other hand, des-tyr-[leu]enkephalin, at the doses tested, was without effect on avoidance conditioning but produced decreased locomotion. These data suggest that the tyrosine end of the enkephalin molecule may be important for its effects on conditioning. Because of their low opioid potencies, it is unlikely that the behavioral actions of tyr-gly-gly and des-tyr-[leu]enkephalin are mediated through opioid receptors.     

Culture conditions for arresting and stimulating the proliferation of a rainbow trout fibroblast cell line,RTG-2

Summary Conditions for arresting and stimulating the proliferation of the rainbow trout fibroblast cell line RTG-2 have been examined and the time course of events after stimulation determined. Quiescent populations were achieved in two ways. Cultures grown to confluency without a medium change for at least 7 d had fewer than 5% of the cells in S phase and few mitotic figures. Cultures deprived of serum, which could be done for up to 3 d without a loss in cell number, also achieved quiescence. After 3 d without serum, less than 1% of cells were in S phase and mitotic figures were infrequent. Addition to these cultures of fresh serum-containing medium brought about the synchronous entry of cells into S phase and mitosis. For cultures in which either the medium had been changed after 7 d without a change or serum-containing medium had been added after 3 d of serum deprivation, DNA synthesis increased after a lag period of 20 to 24 h, was pronounced between 30 and 45 h, and then declined. This was followed by a peak in the mitotic index. These protocols for arresting and subsequently stimulating RTG-2 proliferation should allow the G₁-S transition to be studied in a representative of teleosts. This research was supported by Natural Sciences and Engineering Research Council of Canada grant to N. C. B.     

Biological effects of human gastrin I and II chemically modified at the C-terminal tetrapeptide amide.

Binding to gastrin receptors and gastric acid secretion experiments were performed with gastrin derivatives modified at the C-terminal tetrapeptide amide from HG-13 sequence. 1. When the ultimate phenylalanine amide was replaced by a phenethylester or a phenetylamide moiety, the resulting compound bound to gastrin receptors (Kd approximately 10 nM) and exhibited antagonist activity on gastrin-induced acid secretion in the anesthetized rat. 2. Changing the peptide bond between Trp and Leu residues to a -omega(CH2-NH)- bond resulted in a compound which also bound to gastrin receptors (Kd approximately 10 nM) but presented agonist activity on acid secretion in the rat. In contrast, when the peptide bond between Leu and Asp residues was replaced by a -omega(CH2-NH)- bond, the resulting compound was devoid of any affinity for gastrin receptor (Kd greater than 10(-6) M) and of any biological activity. 3. The HG-13 derivatives were synthesized in sulfated and unsulfated forms: O-sulfation of the HG-13 tyrosine residue did not change its intrinsic in vivo activity but enhanced its affinity for gastrin receptors (Kd approximately 0.3 nM). On the contrary, O-sulfation of the various chemically modified HG-13 had no significant effect in either in vitro or in vivo experiments. 4. Finally, no significant difference between binding on parietal (F3) and nonparietal (F1) cells was observed, in agreement with the presence of a gastrin-type receptor in these two cell populations.     

Respiratory electron transport activity in plankton of the Weddell and Scotia Seas during late spring — early summer: relationships with other biological parameters

Summary As a means to estimate potential oxygen consumption, profiles of elctron transport system (ETS) activity were made along three transects across the Weddell-Scotia Confluence zone (WSC) and the marginal ice zone (which overlapped in part) during the EPOS leg 2 cruise of the RV Polarstern. The integrated ETS activity between 0 and 100 m depth (referred to in situ temperatures) ranged from 261 meq (mili-electron equivalents) m^–2 day^–1 in the WSC to 45 meq m^–2 day^–1 in the southernmost stations at 62° S. The temporal changes in the overall distribution of ETS activity were small compared with the spatial variations. The main feature of the ETS activity distribution was the presence of maxima located in the WSC, coinciding with peaks of phytoplankton biomass. Different relationships between ETS and chlorophyll a concentration in these maxima appeared to be related to diatom or flagellate dominance. Vertically integrated ETS activities were significantly correlated with chlorophyll a and paniculate organic carbon concentrations, primary production and bacterial thymidine uptake.Data presented here were collected during the European Polarstern Study (EPOS) sponsored by the European Science Foundation     

Organisation and functions of the actV A region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor

Summary Sequence analysis of the actVA region of the actinorhodin biosynthetic gene cluster of Streptomyces coelicolor revealed a succession of six open reading frames (ORFs), all running in the same direction and extending over 5.32 kb. The protein product of actVA-ORF1 strongly resembles that of another gene, elsewhere in the act cluster (actII-ORF2), which codes for a trans-membrane protein previously implicated in actinorhodin export from the mycelium. This suggests that the two gene products may co-operate in actinorhodin export, perhaps being sufficient for self-protection of the organism against suicide. At least four of the other five ORFs are implicated in the control of the C-6 and C-8 ring-hydroxylation reactions, lacking in actVA mutants, that occur at middle to late stages in the actinorhodin biosynthetic pathway. This conclusion was reached by genetic mapping of actVA mutants to actVA-ORF3 and-ORF5 (and perhaps -ORF4), and by the finding of strong resemblances between the protein products of actVA-ORF2 and -ORF6 and the products of genes of the oxytetracycline or tetracenomycin gene clusters that have been implicated in ring-hydroxylation reactions in the biosynthesis of these other aromatic polyketide antibiotics.     

Further characterization of the in vitro hydrolysis of [Leu]- and [Met]enkephalin in rat plasma: HPLC-ECD measurement of substrate and metabolite concentrations.

Hydrolysis of [Leu]- and [Met]enkephalin was determined in whole rat plasma in vitro by using HPLC-ECD to measure Tyr, Tyr-Gly and Tyr-Gly-Gly formation. Although [Leu]- and [Met]enkephalin did not differ in Tyr or Tyr-Gly accumulation, the amount of Tyr-Gly-Gly resulting from [Met]enkephalin hydrolysis was greater than that resulting from [Leu]enkephalin hydrolysis, and [Met]enkephalin's half-life in plasma was slightly shorter than that of [Leu]enkephalin. By comparing metabolite formation in the presence and absence of peptidase inhibitors with high selectivity for their respective enzymes, these studies demonstrated that aminopeptidase M and angiotensin converting enzyme are the major peptidases that hydrolyze enkephalins in rat plasma.     

Modulation of xanthine dehydrogenase and oxidase activities during the hormonal induction of vaginal epithelial differentiation in ovariectomized mice

In situ hybridization and immunocytochemical techniques have been used to examine the distribution of vitamin-D-induced calbindin mRNA and calbindin protein in enterocytes lining the crypts and villi of chicken small intestine. Basal villus enterocytes contained approximately twice as much calbindin but over three times as much calbindin mRNA compared to values found in basal crypt and upper villus enterocytes, all values being measured 2 days after vitamin D injection into D-deficient chickens. Virtually no calbindin mRNA was detected in tissues taken from control D-deficient birds. Direct proportionality found between calbindin mRNA and calbindin content in enterocytes of basal crypt, mid and upper villus suggests pre-translational control over calbindin synthesis. The implications of possible inefficient translation of calbindin mRNA in basal villus enterocytes are discussed. Present methods of analysis provide a novel way to study mechanisms controlling gene expression throughout the whole process of enterocyte differentiation.