Physiological adaptations of an under-ice population of Pseudocalanus in Barrow Strait (N.W.T) to increasing food supply in spring

Summary Zooplankton and water samples were collected at weekly intervals between April 25 and May 30, 1986 in Barrow Strait, N.W.T. (Canadian Arctic Archipelago). In tows from 0–30 m, the zooplankton community (>202 m) was dominated by Pseudocalanus. The population was apparently growing and developing as shown by an increase in the proportion of adults (stage VI) and decreases in the proportion of stages III, IV, and V as the season progressed. Respiration and excretion rates of the Pseudocalanus populations were probably linked, there being an immediate increase in excretion rate, accompanying an increase in feeding rate when chlorophyll concentrations increased, which was followed by a smaller increase in respiration rate after a time lag. Hence, there was a large decrease in the ON ratio. Increased metabolism coincided with changes in the population structure, as did protease and bodily protein, but could not be clearly linked to dietary acclimation. Only laminarinase activity could be statistically related to an identifiable fraction of potential nutritional value in the water, particulate soluble carbohydrate, but neither showed overall seasonal change.     

The precursor of a metalloendopeptidase from human rheumatoid synovial fibroblasts. Purification and mechanisms of activation by endopeptidases and 4-aminophenylmercuric acetate.

Two active forms (Mr 45,000 and 28,000) of a metalloendopeptidase that digest proteoglycans and other extracellular matrix components of connective tissues have previously been purified from rheumatoid synovial cells and characterized [Okada, Nagase & Harris (1986) J. Biol. Chem. 261, 14245-14255]. To study the mechanisms of activation the precursor of this metalloendopeptidase has now been purified. The final products are homogeneous on SDS/polyacrylamide-gel electrophoresis and identified as a set of zymogens of Mr 57,000 and 59,000, in which the latter form is probably the product of post-translational glycosylation of the Mr 57,000 zymogen, as it binds to concanavalin A. The zymogen can be activated by trypsin, chymotrypsin, plasma kallikrein, plasmin and thermolysin, but not by thrombin. Although the activated metalloendopeptidase is further degraded by trypsin, plasma kallikrein and thermolysin during a prolonged incubation, it is relatively stable against plasmin and chymotrypsin. Activation with 4-aminophenylmercuric acetate is dependent on its concentration. It requires the reaction with the zymogen, possibly through thiol groups, and the continued presence of the agent. During this treatment the zymogen undergoes a sequential processing; first it becomes active without changing its apparent molecular mass, and then it is processed to low-Mr species of Mr 46,000, 45,000 (HMM) and 28,000 (LMM). The rate of conversion of the precursor into an initial intermediate of Mr 46,000 follows first-order kinetics (t1/2 2.0 h with 1.5 mM-4-amino-phenylmercuric acetate at 37 degrees C) and is independent of the initial concentration of the zymogen or the presence of up to a 676-fold molar excess of substrate, whereas the generation of HMM and LMM species is affected by these parameters. These results indicate that activation of the prometalloendopeptidase by an organomercurial compound is initiated by the molecular perturbation of the zymogen that results in conversion into the 46,000-Mr intermediate by an intramolecular action; the subsequent processing of this intermediate in HMM and LMM species is a bimolecular reaction. In vivo it is probable that the precursor of this metalloendopeptidase is activated either by direct limited proteolysis by tissue or plasma endopeptidases, or, alternatively, by factors that cause certain conformational changes in the zymogen molecule.     

Sequence identity between an inverted repeat family of transposable elements in Drosophila and Caenorhabditis.

The Tc1-like transposable elements, originally described in Caenorhabditis elegans, have a much wider phylogenetic distribution than previously thought. In this paper, we demonstrate that Tc1 shares sequence identity in its open reading frame and terminal repeats with a new transposable element Barney (also known as TCb1-Transposon Caenorhabditis briggsae 1). Barney was detected and isolated by Tc1 hybridization from the closely related nematode species, Caenorhabditis briggsae. The conserved open reading frames of Tc1 and Barney share identity with a structurally similar family of elements named HB found in Drosophila melanogaster, after the introduction of 3 small centrally located deletions in HB1. These reading frames would code for proteins with 30% amino acid identity (42% when conservative changes are included). Tc1, Barney and HB1 contain highly conserved blocks of amino acids which are likely to be in the functional domains of the putative transposase.     

Cystic fibrosis typing with DNA probes: experience of a screening laboratory

Summary A sample of 125 individuals from 37 British cystic fibrosis (CF) families with at least one living affected child were typed with probes for restriction fragment length polymorphisms (RFLPs) known to be linked to the CF gene. These probes were MetD, MetH, pJ3.11 and 7C22. Using this combination of probes, 30 out of the 37 families were sufficiently informative to enable prenatal diagnosis of the disease. Linkage analysis has also proved to be useful in excluding CF in two cases where diagnosis of the disease was equivocal in the sibling of an affected child.     

Effect of culture media and growth phase on the morphology of lactobacilli and on their ability to adhere to epithelial cells

Previous studies have demonstrated that the ability of lactobacilli to attach to and colonize uroepithelial surfaces is an important characteristic that enhances interference against uropathogenic bacteria. This adherence capacity was found to vary amongst lactobacillus strains and with the type of growth medium used to culture the organisms. The present study was undertaken to examine further the effect of culture media and growth phase on lactobacillus adherence to uroepithelial cells in vitro. In addition, a freeze substitution technique was developed to examine the morphology of strainsLactobacillus casei ssrhamnosus RC-17,L. casei GR-1, andL. acidophilus T-13 in relation to growth conditions and adhesion. A growth curve was plotted for strain GR-1, and adherence was found to be lowest for bacteria in early log phase (39 bacteria per uroepithelial cell) and highest in stationary phase (59 bacteria per uroepithelial cell). Strains RC-17 and GR-1 attached in high numbers to uroepithelial cells, whereas T-13 was poorly adherent. The latter formed a long, relatively dense, fibrous capsule after growth in brain heart infusion yeast extract agar, unlike strains GR-1 and RC-17, which formed a short, tightly bound, electron-dense capsule which surrounded the cells in a radial fashion. Growth of RC-17 in batch cultures of human urine, with and without addition of carbohydrates, resulted in formation of an irregular, fibrous extracellular matrix. These experiments illustrate that growth phase and culture conditions affect the extracellular structure of lactobacilli and also affect the adherence capacity of these bacteria. Structural changes mediated by availability of nutrients may partly explain why lactobacilli vary between species and between hosts in their colonization of the urogenital tract.     

Synthesis of eicosanoids by gamma-interferon-differentiated U937 cells

Interferons induce morphological, biochemical and functional alterations in monocyte macrophage and myeloid cell lines. We studied the effect of 3 days incubation with gamma-interferon from human buffy coats on the global synthesis of arachidonic acid metabolites by U937 cells. Interferon-induced morphologic changes including cytoplasmic and nuclear changes and the appearance of multiple lysosomal-like granules consistent with cellular differentiation were observed by electron microscopy. The labeling of phosphatidylserine, phosphatidylcholine and phosphatidylethanolamine was increased and that of phosphatidylinositol, free fatty acids as 3H-arachidonic acid and neutral lipids reduced, when interferon-treated cells were incubated with 3H-arachidonic acid. Interferon caused qualitative and quantitative changes in the synthesis of cyclooxygenase and lipoxygenase products. A23187, a calcium ionophore, and the tumor promotor, phorbol myristate acetate, greatly increased the synthesis by interferon-differentiated cells of 2 cyclooxygenase products; synthesis of lipoxygenase products was reduced. In the presence of indomethacin, 'shunting' into putative lipoxygenase products occurred. The relationship between interferon-induced morphologic and functional changes, the development of altered phospholipid and eicosanoid metabolism and the identity of these metabolites are yet to be established.     

Poliovirus proteinase 3C: large-scale expression, purification, and specific cleavage activity on natural and synthetic substrates in vitro.

Proteinase 3C of poliovirus type 2 (Sabin) was expressed at 4% total protein in Escherichia coli. The protein was soluble and could be purified by a simple scheme. It was weakly active on the capsid precursor P1 (expressed in vitro), which contains two cleavage sites. The products of processing P1 were 1ABC and 1D (VP1). The activity was insensitive to Triton X-100. Crude extracts of cells infected with poliovirus type 1 (Mahoney) gave strong processing and yielded 1AB (VP0), 1C (VP3), and 1D in the same assay system but were sensitive to detergent. 3C from cell extracts that was separated from its precursors resembled the recombinant proteinase in its activity. Recombinant 3C cleaved the peptide dansyl-Glu-Glu-Glu-Ala-Met-Glu-Gln-Gly-Ile-Thr-Asn-Lys-NH2 at the Gln-Gly bond. We conclude that 3C is merely the core of the Gln-Gly-cleaving activity which processes P1 in vivo and that there is probably a hydrophobic contact between a larger 3C precursor and its P1 substrate which allows the second processing reaction: 1ABC, 1D----1AB, 1C, 1D.     

Epitope mapping of the human immunodeficiency virus type 1 gp120 with monoclonal antibodies.

A soluble form of recombinant gp120 of human immunodeficiency virus type 1 was used as an immunogen for production of murine monoclonal antibodies. These monoclonal antibodies were characterized for their ability to block the interaction between gp120 and the acquired immunodeficiency syndrome virus receptor, CD4. Three of the monoclonal antibodies were found to inhibit this interaction, whereas the other antibodies were found to be ineffective at blocking binding. The gp120 epitopes which are recognized by these monoclonal antibodies were mapped by using a combination of Western blot (immunoblot) analysis of gp120 proteolytic fragments, immunoaffinity purification of fragments of gp120, and antibody screening of a random gp120 gene fragment expression library produced in the lambda gt11 expression system. Two monoclonal antibodies which blocked gp120-CD4 interaction were found to map to adjacent sites in the carboxy-terminal region of the glycoprotein, suggesting that this area is important in the interaction between gp120 and CD4. One nonblocking antibody was found to map to a position that was C terminal to this CD4 blocking region. Interestingly, the other nonblocking monoclonal antibodies were found to map either to a highly conserved region in the central part of the gp120 polypeptide or to a highly conserved region near the N terminus of the glycoprotein. N-terminal deletion mutants of the soluble envelope glycoprotein which lack these highly conserved domains but maintain the C-terminal CD4 interaction sites were unable to bind tightly to the CD4 receptor. These results suggest that although the N-terminal and central conserved domains of intact gp120 do not appear to be directly required for CD4 binding, they may contain information that allows other parts of the molecule to form the appropriate structure for CD4 interaction.     

Bleomycin and X-ray-hypersensitive Chinese hamster ovary cell mutants: genetic analysis and cross-resistance to neocarzinostatin

We have previously reported the isolation of 3 mutants of Chinese hamster ovary cells which exhibit hypersensitivity to bleomycin. 2 mutants were isolated on the basis of bleomycin-sensitivity [designated BLM-1 and BLM-2, Robson et al., Cancer Res., 45 (1985) 5304-5309] and 1 as adriamycin-sensitive [ADR-1, Robson et al., Cancer Res., 47 (1987) 1560-1565]. Because bleomycin generates DNA-strand breaks via a free-radical mechanism, we have studied the survival response of these mutants to a range of drugs which also generate free radicals and consequently DNA-strand breaks. The mutants are all hypersensitive to phleomycin, which differs from bleomycin in being unable to intercalate due to a modified bithiazole moiety. However, BLM-2 cells alone are hypersensitive to pepleomycin, a semi-synthetic bleomycin analogue. In contrast, BLM-1 cells are more sensitive than BLM-2 to streptonigrin (which operates via a hydroquinone intermediate). ADR-1 cells show wild-type resistance to streptonigrin. The results obtained with neocarzinostatin, an antibiotic requiring thiol activation, are unusual in that both BLM-1 and BLM-2 are approximately 3-fold more resistant than parental cells. However, the steady-state intracellular level of the major non-protein thiol, glutathione, is not altered in BLM-1 or BLM-2 cells. ADR-1 cells show essentially wild-type resistance to neocarzinostatin. Analysis of cell hybrids shows that BLM-1 and BLM-2 cells are phenotypically recessive in combination with parental CHO-K1 cells and represent different genetic complementation groups not only from one another, but also from the bleomycin-sensitive mutant xrs-6, isolated on the basis of X-ray sensitivity by Jeggo and Kemp [Mutation Res., 112 (1983) 313-319]. These results indicate that at least 3 gene products are involved in cellular protection against bleomycin toxicity in mammalian cells.