Consequences of terbium (111) binding on the conformation and enzymatic activity of Guinea pig liver transglutaminase

Calcium ions are crucial for expression of transglutaminase activity. Although lanthanides have been reported to substitute for calcium in a variety of protein functions, they did not replace the calcium requirement during transglutaminase activity measurements. Furthermore, lanthanides strongly inhibited purified liver transglutaminase activity using either casein or fibrinogen as substrates. Terbium (III) inhibition of transglutaminase-catalyzed putrescine incorporation into casein was not reversed by the presence of 10–200 fold molar excess of calcium ions (Ki for Tb(III)=60 µM). Conformational changes in purified liver transglutaminase upon Tb(III) binding were evident from a biphasic effect of Tb(III) on transglutaminase binding to fibrin. Low concentrations of Tb(III) (1 µM to 10 µM inhibited the binding of transglutaminase to fibrin, whereas higher concentrations (20 µM to 100 µM promoted binding. Conformational changes in purified liver transglutaminase consequent to Tb(III) binding were also demonstrated by fluorescence spectroscopy due to Forster energy transfer. Fluorescence emission was stable to the presence of 200 mM NaCl and 100 mM CaCl₂ only partially quenched emission. Purified liver transglutaminase strongly bound to Tb(III)-Chelating Sepharose beads and binding could not be disrupted by 100 mM CaCl₂ solution. Our data suggest that Tb(III)-induced conformational changes in transglutaminase are responsible for the observed effects on enzyme structure and function. The potential applications of Tb(III)-transglutaminase interactions in elucidating the structure-function relationships of liver transglutaminase are discussed.     

Growth dynamics of a ctenophore (Mnemiopsis) in relation to variable food supply. I. Carbon biomass, feeding, egg production, growth and assimilation efficiency

This paper contains new experimental data on the growth dynamicsof a lobate coastal ctenophore, Mnenmiopsis mccradyi, whichadd significantly to our understanding of the nutritional ecologyof ctenophores and their role as opportunistic predators. Theseexperimental observations were necessary to refine the dynamiccarbon budget presented as a simulation model in another report.The ratio of carbon biomass to dry weight may vary several-folddepending on the nutritional state and size from >12% inwell-fed larvae to <1% in starved adults. Feeding effort(clearance rate) is higher for previously starved animals, fallingsharply within a few hours after re-exposure to food. Directvisual observations of feeding activity of animals confirmedthis. Assimilation efficiency can be high (72%) in these animalsbut if they continue to feed at high food concentrations, incomingfood displaces material which is only partially digested andassimilation efficiency decreases substantially. Except at verylow food concentrations, growth efficiency ranges between 20and 45%. Mnemiopsis, begins to produce eggs at a size much lessthan its maximum. Egg production is very sensitive to food supply,and somatic growth is favored over egg production at low fooddensities. Even though several thousand eggs may be producedover a few days, they represent <2% day^–1 of the carbonbiomass of the ctenophore and several-fold less than respiratorycarbon.     

Production of alkaline phosphatase by immobilized growing cells of Escherichia coli excretory mutants

Summary In continuous cultures, alkaline phosphatase was synthesised and excreted for more than 250 h by immobilized growing cells in contrast to free cells for which the excretion decreased after 150 h of culture. This observed increase in alkaline phosphatase synthesis and excretion by immobilized cells may have resulted from growing conditions within the gel beads.Offprint requests to: C. Manin     

Evaluation of strains of Geotrichum candidum for lipase production and fatty acid specificity

Summary Three strains of Geotrichum candidum (ATCC 34614, NRRL Y-552 and NRRL Y-553) were examined for lipase production and activity. Variables including medium, pH, temperature, agitation rate and incubation time were examined to define the optimal culture conditions. Growth on oil in complex medium at 30°C, 300 rpm, and pH 7 produced maximal lipase activity. Fatty acid specificity of these strains and of two crude G. candidum enzyme preparations (lipase 26557 RP, Rhône Poulenc and lipase GC-4, Amano) was measured using equimolar mixtures of methyl or butyl esters of palmitic and oleic acids. The lipase from NRRL Y-553 and lipase 26557 RP displayed preferential specificity for hydrolyzing oleic acid esters, while the lipases from ATCC 34614, NRRL Y-552 and lipase GC-4 failed to discriminate between plamitic and oleic acids.     

Cloning, sequence analysis and expression of a cDNA encoding a novel insulin-like growth factor binding protein (IGFBP-2).

Insulin-like growth factors bind with high affinity to specific binding proteins in extracellular fluids. To identify structural characteristics of IGF-binding proteins that might define their physiological roles, we determined the complete primary structure of a novel human IGF-binding protein (IGFBP-2) from a cloned cDNA. The cDNA encodes a 328 amino acid IGF-binding protein precursor which contains a 39-residue signal peptide. The mature 289 amino acid IGFBP-2 has a predicted Mr of 31,325. Chinese hamster ovary (CHO) cells stably transformed with the IGFBP-2 cDNA secreted a 36 kd protein which bound, with different affinities, IGFII and IGFI, but did not bind insulin. The predicted protein sequence of this IGF-binding protein shares extensive amino acid homology (greater than 85%) with the IGF-binding protein secreted by rat BRL-3A cells, but less than 40% homology with human IGFBP-1. Therefore IGFBP-2, and not IGFBP-1 as previously suggested, represents the human homologue of the rat BRL-BP (alpha IGFBP-2). Moreover, from alignment of the predicted protein sequences of IGFBP-1 and IGFBP-2, extensive conservation of the distribution of cysteine residues is observed. Although the overall amino acid homology shared by these proteins is not high, we suggest that they represent a family of structurally related human IGFBPs. Southern blot analysis of human DNA demonstrates that IGFBP-2 is encoded by a single-copy gene, different from that of IGFBP-1.     

Expression of a mouse metallothionein-Escherichia coli β-galactosidase fusion gene (MT-βgal) in early mouse embryos

We have microinjected DNA containing the inducible mouse metallothionein-I (MT-I) promoter, coupled to the structural gene for Escherichia coli β-galactosidase (lacZ), into the pronuclei of one-cell mouse embryos. A qualitative histochemical assay, with 5-bromo-4-chloro-3-indolylβ- -galactopyranoside (X-Gal) as a substrate, was used to detect expression of lacZ at several preimplantation stages. We observed staining indicative of exogenous β-galactosidase activity in 5–17% of DNA-injected embryos assayed at preimplantation stages after 16–24 h treatment with ZnSO₄. Thus, lacZ can be used as an indicator gene for promoter function during early mouse embryogenesis, and the incorporation of the MT-I promoter into fusion genes can be a useful means of controlling the expression of exogenous genes in preimplantation mouse embryos.     

Molecular approaches to problems in biogeochemical cycling

Summary By using molecular probe techniques in combination with activity and expression measurements, it is possible to estimate bacterial populations in nature. This information can be expooited to study a number of important environmental problems. For instance, it will be possible to study ecosystem perturbation and microbial competition, by altering an ecosystem or a laboratory model of an ecosystem, and assessing corresponding changes in key activities and populations. In addition, regulation of activities in the laboratory can be compared to the response of activities and populations in situ, to develop an understanding of the key parameters that control these processes in nature. These types of approaches are important steps for determining the role of microorganisms in geochemical cycling, in both specific habitats and on a global basis.     

Field Study Comparing Growth and Viability of a Population of Phototrophic Bacteria

The growth and viability of an anoxygenic, phototrophic bacterial community in the hypolimnion of Zaca Lake, Calif., were compared throughout the summer. The community is dominated by a single species, “Thiopedia rosea,” that inhabits the entire hypolimnion (6 to 8 m) for approximately 11 months. Suboptimal conditions in the hypolimnion (extremely low light intensity, high or low H₂S levels) result in zero or extremely low growth rates (doubling times > 1 month) for most of the population, most of the time, yet cells remain viable and capable of high specific growth rates (doubling times of 1 to 10 days) when placed under favorable conditions (higher light intensities and temperatures). We first conclude that phototrophic bacterial populations in situ may frequently exist in a viable yet nongrowing state. Second, the viability of cells is likely to be reduced with depth owing to higher concentrations of potentially toxic chemicals and to changes in the physiological state associated with the prolonged periods of darkness commonly found at the bottom of bacterial plates.     

Adenosine, a cytoprotective autocoid: effects of adenosine on neutrophil plasma membrane viscosity and chemoattractant receptor display

At inflammatory sites neutrophils are stimulated to produce a variety of toxic agents, yet rarely harm the endothelium across which they migrate. We have recently found that endothelium releases adenosine which, acting via receptors on the surface of human neutrophils, inhibits generation of toxic metabolites by stimulated neutrophils but, paradoxically, promotes chemotaxis. Agents which diminish plasma membrane viscosity affect neutrophil function similarly, possibly by modulating chemoattractant receptor number or affinity. We therefore determined whether adenosine receptor agonists modulate neutrophil function by decreasing membrane viscosity and/or chaning the affinity of chemoattractant (N-fMet-Leu-Phe, FMLP) receptors. Surprisingly, 5′-(N-ethylcar☐amido)adenosine (NECA, 10 μM), the most potent agonist at neutrophil adenosine receptors, increased plasma membrane viscosity, as measured by fluorescence anisotropy of the plasma membrane specific probe 1-(4-trimethylaminophenyl)-6-diphenyl-1,3,5-hexatriene (TMA-DPH), in unstimulated neutrophils from a mean microviscosity of 1.67 ± 0.02 (S.E.) to 1.80 ± 0.02 (p < 0.001) while inosine (10 μM), a poor adenosine receptor agonist, had no effect (1.73 ± 0.04, p =n.s. vs. control, p < 0.01 vs. NECA). Adenosine receptor agonists increased plasma membrane viscosity in neutrophils with the same order of potency previously seen for inhibition of superoxide anion generation and enhancement of chemotaxis (NECA > adenosine = N⁶-phenylisopropyladenosine). The adenosine receptor antagonist 8-(p-sulfophenyl)theophylline reversed the effect of NECA on plasma membrane viscosity. Unlike other agents which modulate plasma membrane viscosity, NECA (10 μM) did not significantly change the number or affinity of [³H]FMLP binding sites on neutrophils. In contrast to the hypothesis of Yuli et al. these results indicate that occupancy of adenosine receptors on neutrophils increases plasma membrane viscosity without affecting chemoattractant receptor display.     

Cloning and expression of the toxin B gene ofClostridium difficile

Results from our cloning studies on toxin A indicated that the gene for toxin B resided approximately 1 kb upstream of the toxin A gene. Clone pCD19, which contains the 5-end of the toxin A gene and a small open reading frame, was found to contain 1.2 kb of DNA which, when subcloned, expressed a nontoxic peptide that reacted with toxin B antibodies. The rest of the toxin B gene was located on the 6.8 kb cloned fragment of plasmid pCD19L. The two fragments overlapped 0.8 kb. Lysates containing protein expressed by the 6.8 fragment were cytotoxic and lethal, and were neutralized by toxin B antibody. The two fragments were ligated to give the complete toxin B gene. The protein expressed by the complete gene was cytotoxic and lethal, and showed complete immunological identity with toxin B. Further analysis of the expressed protein and the toxin B gene confirmed our earlier findings showing that toxin B has a molecular weight of 240,000 or greater.