Enzymatic characterization of oral and non-oral black-pigmented Bacteroides species

The purpose of this study was to investigate the production of various enzymes by oral and non-oral black-pigmented Bacteroides species using chromogenic substrates. The 19 substrates present in the API ZYM system did not differentiate between B. melaninogenicus, B. denticola, B. loescheii and B. levii. The asaccharolytic black-pigmented Bacteroides species showed each species specific enzyme activity, however, differences were based on one enzyme only as far as B. asaccharolyticus and B. endodontalis are concerned. An extended number of 40 chromogenic substrates were tested in order to find more species specific enzyme. With a set of 20 substrates it appeared to be possible to discriminate between all species tested. The possibility to use enzymes for the identification of black-pigmented Bacteroides is discussed.     

Degradation of 4-hydroxyphenylacetate by Xanthobacter 124X

Xanthobacter 124X when grom on 4-hydroxyphenylacetate was able to hydroxylate this compound yielding homogenisate. Ring fission of this latter compound gave maleylacetoacetate which was isomerized to fumarylacetoacetate. The isomerase involved resembled maleylacetoacetate isomerases in Gram-negative bacteria in that glutathione was required for activity. Fumarate and acetoacetate were both detected as products of the hydrolysis of fumarylacetoacetate.     

Functional constituents of the active site of human neutrophil collagenase

A series of chemical modification reactions has been carried out to identify functional constituents of the active site of human neutrophil collagenase. The enzyme is reversibly inhibited by the transition metal chelating agent 1,10-phenanthroline, and inhibition is fully reversed by zinc. Removal of weakly bound metal ions by gel filtration inactivates collagenase, and activity is fully restored on immediate readdition of calcium. The enzyme is unaffected by reagents that modify serine, cysteine, and arginine residues. However, reaction with the carboxyl reagents cyclohexylmorpholinocarbodiimide and Woodward's Reagent K lowers the activity of the enzyme substantially. Acetylimidazole inactivates the enzyme, but activity is completely restored on addition of hydroxylamine. The enzyme is also inactivated by tetranitromethane, indicating that it contains an essential tyrosine residue. Acylation of collagenase with diethyl pyrocarbonate, diketene, acetic anhydride, or trinitrobenzenesulfonate inactivates the enzyme, and activity is not restored on addition of hydroxylamine, indicating the presence of an essential lysine residue.     

Phorbol ester stimulates the synthesis and phosphorylation of a 48 kDa-intracellular protein in plasminogen activator secreting melanoma cells

Phorbol ester (12-O-tetradecanoyl-phorbol 13-acetate) stimulates the secretion of tissue-type plasminogen activator by the melanoma cell line, Bowes. This effect is associated with increased levels of mRNAs for both tissue-type plasminogen activator and a 48 kDa-protein. Labelling of melanoma cells with L-[35S]methionine allowed to identify an intracellular protein which, by 3 criteria, was identical with the in vitro translation product of the 48kDa-protein mRNA: a Mr of 48,000 on electrophoresis in the presence of sodium dodecyl sulphate; inducibility by phorbol ester and failure of reducing agents to affect electrophoretic mobility. As detectable by L-[35S]methionine labelling, the protein was mainly localized in the cytosol. In vitro phosphorylation reactions, carried out on subcellular fractions revealed a membrane-associated protein which also had the three characteristics of the aforementioned 48 kDa-protein. Phosphorylation did not require Ca2+-ions. Addition of phorbol ester to the reaction mixtures increased the phosphorylation. Reconstitution experiments between membrane and cytosol fractions of phorbol ester-treated and untreated cells showed that the 48kDa protein occurs in a cytosolic, unphosphorylated and a membrane-bound, phosphorylated form and that the former is converted to the latter by a phorbol ester activated, membrane-associated protein kinase.     

Predicted membrane topology of the coronavirus protein E1

The structure of the envelope protein E1 of two coronaviruses, mouse hepatitis virus strain A59 and infectious bronchitis virus, was analyzed by applying several theoretical methods to their amino acid sequence. The results of these analyses combined with earlier data on the orientation and protease sensitivity of E1 assembled in microsomal membranes lead to a topological model. According to this model, the protein is anchored in the lipid bilayer by three successive membrane-spanning helices present in its N-terminal half whereas the C-terminal part is thought to be associated with the membrane surface; these interactions with the membrane protect almost the complete polypeptide against protease digestion. In addition, it is predicted that the insertion of E1 into the membrane occurs by the recognition of the internal transmembrane region(s) as a signal sequence.     

Calmodulin and troponin C: a comparative study of the interaction of mastoparan and troponin I inhibitory peptide [104-115]

Recent studies using bee and wasp venom peptides have led to the hypothesis that proper complex formation with calmodulin (CaM) requires the presence of a basic amphiphilic helix on the surface of the target protein [Cox, J. A. (1984) Fed. Proc., Fed. Am. Soc. Exp. Biol. 43, 3000]. We have tested this hypothesis by examining CaM and troponin C (TnC) complex formation with two basic peptides, the wasp venom tetradecapeptide mastoparan and the physiologically relevant synthetic troponin I (TnI) inhibitory peptide [104-115], using far-ultraviolet circular dichroism as a secondary structure probe. Complex formation between mastoparan and either CaM or TnC results in an increase in helical content, whereas the helical content of TnI inhibitory peptide does not increase when bound to either protein. Significantly, mastoparan is 78% alpha-helical in a 50% solution of the helix-inducing solvent trifluoroethanol and has a high helix-forming potential according to the Chou-Fasman rules while TnI inhibitory peptide contains none and is not predicted to have any. We interpret these data as indicating that these peptides exhibit substantially different secondary structures upon binding to CaM or TnC. The ability of mastoparan to regulate the acto-subfragment 1-tropomyosin ATPase has also been examined. Mastoparan and TnI inhibitory peptide inhibited 31% and 45% of the activity, respectively. TnC and CaM promote differing degrees of Ca2+-sensitive release of inhibition by both peptides. Sequence comparison suggests that the basic residues present in both peptides are important for binding. However, we conclude that an alpha-helical structure is not a prerequisite for the binding of target proteins to CaM and TnC.     

Opioid peptides modulate production of interferon gamma by human mononuclear cells

The opioid neuropeptides have previously been shown to bind to and affect leukocyte function including lymphocyte proliferation, NK-cell activity, mononuclear cell chemotaxis, immunoglobulin synthesis, and lymphokine production. The effect of the opioid peptides beta-endorphin and Met-enkephalin on interferon gamma (IFN) production by concanavalin A-stimulated human mononuclear cells was examined. Both beta-endorphin and Met-enkephalin enhanced IFN production by the majority of donor mononuclear cells tested and did so at concentrations between 10(-14) and 10(-10) M. When 10(-12) M beta-endorphin or Met-enkephalin were included in concanavalin A-stimulated mononuclear cell cultures, IFN concentrations were significantly enhanced to 205 +/- 45 and 252 +/- 67% of control, respectively. Although the majority of cell preparations tested exhibited an enhanced production of IFN in response to these opioid peptides, some did not. When beta-endorphin or Met-enkephalin were utilized at 10(-11) M, 10 of 15 and 7 of 11 responded with IFN production greater than 20% above the control (untreated) level. There was not an absolute correlation between an enhanced response to beta-endorphin and Met-enkephalin, suggesting the presence of multiple receptor types on these cells for opioids. The opioid receptor antagonist, naloxone, did not significantly prevent the opiate effect. When 10(-8) M naloxone was included in cultures containing 10(-12) M beta-endorphin or Met-enkephalin no significant inhibition of the effect of either opioid on IFN production was observed.     

Selective breeding for variations in patterns of mystacial vibrissae of mice. Bilaterally symmetrical strains derived from ICR stock

The establishment of certain patterns of mystacial vibrissae in mice has been the aim of an extensive breeding program carried on in this laboratory since 1977. In a companion paper we have reported on variations in this pattern in an outbred population of ICR mice. Starting with 21 ICR animals we bred, mostly by brother-sister mating, for 13 bilaterally symmetric patterns of mystacial vibrissae characterized by the presence (or absence) of supernumerary whiskers (SWs). The strains are classified as follows: I, a mouse strain with the standard pattern; II, eight strains bred for the occurrence of SWs at a given site or sites; and III, four mouse strains bred for a maximal number of SWs in different regions of the whiskerpad. Commonly, SWs occur in regions that coincide with the zones of mergence between the three facial processes except for two class II strains in which we bred for SWs in the "straddler" row of vibrissae, and for one class III strain, in which we cultivated the tendency (that appeared late in our program) to have SWs at the crest of a facial process. For classes I and II we analyzed the results for about 18 generations in terms of "improvement," meaning an increase in the percentages of animals with the desired phenotype together with a decreased frequency of undesired SWs. For class III, success in breeding meant the increase of the mean number of the desired SWs. All results led to the same conclusion: there is a genetic basis for the occurrence of SWs. The side preference of a particular SW is not strain dependent. It disappears in those class I and II strains in which almost 100% of animals obtained the desired phenotype. The increase in number of SWs in one zone of mergence does not depend on the presence of SWs in the other. Where tested, we almost always found a representation of an SW in a topologically equivalent location within the "barrelfield" area of the somatosensory cerebral cortex. Except for some diseases early in the breeding program, and some side effects of inbreeding that were eliminated, the population was without obvious defects. Where tested, there was no correlation between the occurrence of SWs and sex. The observed variations in pattern of mystacial vibrissae and their genetic background led us to propose a morphogenetic model for the formation of the pattern of mystacial vibrissae.     

Rheumatoid factor production in 129/Sv mice: involvement of an intestinal infectious agent

High titers of IgG2a-specific rheumatoid factors (RF) are frequently observed in colonies of 129/Sv mice. The involvement of a transmissible agent in this phenomenon was shown by the following findings: (i) cesarean-derived and isolator-reared offsprings of RF-positive dams were free of RF, ii) a single intragastric inoculation with intestinal fluid from RF-positive donors elicited chronic RF production in RF-negative recipients, and iii) intestinal fluid collected from these primary recipients induced a comparable RF response in a second set of animals. The nature of this RF-inducing agent, however, remained elusive. Although its ability to pass through filters that efficiently retained bacteria could be unequivocally established, systematic serological analysis failed to detect any significant correlation between RF production and antibody responses to common mouse viruses or Mycoplasma. Moreover, all attempts to identify the RF-inducing agent by electron microscopy or to grow it in nude or newborn mice, as well as in cell cultures, remained unsuccessful.     

Effect of T3 modulation on pokeweed mitogen-induced T cell activation: evidence for an alternative pathway of T cell activation

Modulation of the T3 molecule on human T cells with monoclonal anti-T3 antibodies has been shown to result in the disappearance of the T3-Ti complex from the membrane and to preclude subsequent T cell activation by various mitogenic and antigenic stimuli. We have examined the effect of T3 modulation on pokeweed mitogen (PWM)-induced T cell activation. T3 modulation was accomplished by incubating peripheral blood mononuclear cells (PBMC) or mixtures of T cells and non-T cells at 37 degrees C for 18 hr in the presence of UCHT-1, a mouse IgG1 anti-T3 monoclonal antibody. Only donors whose PBMC were unresponsive to the mitogenic activity of this antibody were selected. Although T3 modulation resulted in complete to substantial inhibition of T cell proliferation induced by low PWM concentrations of 5 or 50 ng/ml, it had no effect on T cell proliferation when PWM was added at a concentration of 0.5 and 5 micrograms/ml. The results demonstrate that the higher doses of PWM can induce T cell proliferation via an alternative pathway that does not involve participation of the T3-Ti complex. In contrast, irrespective of the PWM dose added, T3 modulation almost totally inhibited PWM-induced interleukin 2 (IL 2) production. The differential effect of T3 modulation on IL 2 production and on T cell proliferation induced by high doses of PWM suggests that this alternative pathway of T cell proliferation is IL 2 independent. This suggestion was additionally substantiated by the lack of effect of anti-Tac, and anti-IL 2 receptor antibody, on PWM-induced proliferation of T3-modulated T cells. In conclusion our data demonstrate that high doses of PWM can induce T cells to proliferate via an alternative pathway that does not involve perturbation of the T3-Ti complex.