Chaperone-mediated activation in vivo of a Pseudomonas cepacia lipase

An extracellular Pseudomonas cepacia lipase, LipA, is inactive when expressed in the absence of the product of the limA gene. Evidence has been presented that LimA is a molecular chaperone. The lipA and limA genes have been cloned in separate and independently inducible expression systems in Escherichia coli. These systems were used to test the molecular chaperone hypothesis by investigating whether LimA could activate presynthesized prelipase and whether presynthesized LimA could activate newly synthesized prelipase. The results show that LimA cannot activate presynthesized prelipase and that presynthesized LimA can activate only a limited number of de novo synthesized prelipase molecules. Co-immunoprecipitation of prelipase/lipase with LimA generated a 1:1 complex of prelipase/lipase and LimA. The results suggest that a 1:1 complex of LipA and LimA is required for prelipase processing and secretion of active lipase.     

EPR characterization of oxide supported transition metal ions: Relevance to catalysis

EPR spectroscopy is a powerful tool to identify at a molecular level, the different steps of catalyst preparation, and of catalytic reactions:

1. Deposition of paramagnetic transition metal ions onto a support is monitored, and the coordination sphere of the metallic center is characterized by EPR.
2. The catalyst is also characterized after activation (thermal oxidation or reduction):

- the distribution among the different sites in zeolites can be determined;

- the dispersion of the active phase may be appreciated;

- the unsaturation degree of the active site may be evaluated using probe molecules such as water or¹³C enriched carbon monoxide.

1. The catalytic mechanisms can be investigated by studying the elementary steps of the catalytic reaction, as illustrated for methanol oxidation over Mo/SiO₂ catalysts whose EPR results have extended the reaction mechanism proposed on the basis of kinetic data. In addition, reaction intermediates may be isolated inquasi-in situ conditions as in the case of olefin oligomerization catalyzed by Ni/SiO₂ systems.

     

An enzyme-immobilized microplate for determination of linamarin for large number of samples

Summary An enzyme-immobilized microplate for determination of linamarin was prepared by covalently linking cassava leaf linamarase to the microplate. For linamarin determination, cassava roots were homogenised in 0.1 Mo-phosphoric acid and the filtrate adjusted to pH 6 with NaOH prior to adding into the wells. The cyanide released was then determined spectrophotometrically. One nmol linamarin can be detected. The microplate method is suitable for analysis of large number of samples and is useful for screening purposes.     

Correlation ofLegionella pneumophila virulence with the presence of a plasmid

Legionella pneumophila is the causative agent of Legionellosis in man and considered an opportunistic intracellular Gram-negative bacterium that preferentially infects macrophages. The presence of a plasmid in these organisms was determined in cultures of the bacteria grown in vitro. A correlation was observed between the growth of virulent strains of theLegionella in murine macrophages and growth on standard buffered charcoal yeast extract agar supplemented with 0.1% -ketoglutarate (BCYE) agar medium rich in cysteine and widely used for growth of the bacteria in vitro. In contrast, the avirulent isolates of these strains grew well on supplemented Mueller-Hinton (SMH) agar utilized for differentiating virulent from avirulentLegionella. However, one virulent strain ofLegionella (the Iowa strain) was found to grow moderately well on the SMH agar. In addition, test strains ofLegionella that infect in vitro human monocytes were found to grow moderately well on the BCYE- agar, but did not grow on the SMH agar. Examination of these strains for plasmid DNA expression showed that extra chromosomal DNA-containing molecules were present in theL. pneumophila strains characterized as virulent for in vitro growth in macrophages. However, the avirulent strains that replicated in the human monocytes readily but only poorly in the permissive murine macrophages did not show evidence of similar plasmid DNA expression.     

Ethylbenzene degradation by Pseudomonas fluorescens strain CA-4

Abstract Pseudomonas fluorescens strain CA-4 is a bioreactor isolate capable of ethylbenzene degradation. Transposon mutagenesis and enzyme assays have been performed which allow us to propose the ethylbenzene degradative pathway in operation in this strain. Ethylbenzene is initially converted to 2-phenylethanol. This is degraded to phenylacetaldehyde and then to phenylacetic acid. The major inducer of the pathway is ethylbenzene itself. The pathway is regulated by the presence of non-aromatic carbon sources. Oxidation of ethylbenzene is repressed by glutamate, but not by citrate or glucose. A clone from a chromosomal library has been found to complement a mutant deficient in the ability to convert ethylbenzene to 2-phenylethanol.     

The expression of cecropin peptide in transgenic tobacco does not confer resistance to Pseudomonas syringae pv tabaci

Summary We used in vitro growth inhibition assays to demonstrate that synthetic cecropin protein has potent activity against a range of plant pathogenic bacteria. We then prepared transgenic tobacco plants which express cecropin mRNA and protein. We have used Pseudomonas syringae pv tabaci infection of these transgenic tobacco as a model system to evaluate whether the plants which express cecropin protein also have increased tolerance to infection. We found no dramatic difference in disease response between plants which are expressing cecropin protein and control plants which were derived from the transformation with a binary vector which did not carry the gene encoding cecropin protein.     

Effects of omnivorous shrimp in a montane tropical stream: sediment removal,disturbance of sessile invertebrates and enhancement of understory algal biomass

Freshwater shrimp dominate the faunal biomass of many headwater tropical streams: however, their role in community organization is unclear. Enclosure/exclosure experiments in a montane Puerto Rican stream examined direct and indirect effects of two dominant taxa of atyid (Atyidae) shrimp, Atya lanipes Holthuis and Xiphocaris elongata Guerin-Meneville. Both shrimp taxa caused significant reductions in sediment cover on rock substrata, reducing sedimentation and enhancing algal biovolume on clay tiles in cages. When tiles incubated in shrimp exclosures for 2 wks were placed outside of cages, atyid shrimp removed 100% of the sediment cover within a 30 min observation period. Atyid shrimp appear to play an important role in stream recovery after high discharge events by rapidly removing sediments and detritus deposited on benthic substrata in pools. We evaluated the mechanism by which A. lanipes influences algae and benthic insects by comparing patterns of algal biomass, taxonomic composition, and insect abundance between shrimp-exclusion and shrimp-presence treatments both with and without manual sediment removal. The shrimp exclusion treatment without manual sediment removal bad significantly lower algal biomass and greater sedimentation than all other treatments. The treatment in which shrimp were excluded but sediment was manually removed, however, accrued almost the same algal biovolume as the shrimp enclosure treatment, supporting the hypothesis that sediment removal enhances the biovolume of understory algal taxa. Algal community composition was similar between stream bottom bedrock exposed to natural densities of shrimp and all experimental treatments for both Atya and Xiphocaris: a diatom community strongly dominated (78–95%) by the adnate taxon, Achnanthes lanceolata Breb ex. Kutz. Atyid shrimp are important in determining the distribution and abundance of benthic insects through both direct and indirect effects. Sessile, retreat-building chironomid larvae (Chironomidae: Diptera) are negatively affected by both A. lanipes and X. elongata, through direct removal by foraging activities and/or indirectly through depression of sediment resources available to larvae for the construction of retreats. In constrast, the mobile grazer, Cloeodes maculipes (Baetidae: Ephemeroptera) was not adversely affected and atyid shrimp have the potential to exert positive indirect effects on this taxon by facilitating its exploitation of algal resources and/or through enhancement of understory algal food resources through sediment removal.     

Characterization of the oligosaccharide moiety of VIP receptor from the human pancreatic cell line BxPC-3

The human pancreatic cell line BxPC-3 displays two classes of binding sites with high and low affinity for VIP. The order of potency of VIP-related peptides in inhibiting either [¹²⁵I]VIP or [¹²⁵I]N-AcPACAP27 binding and in stimulating cAMP production was typical of the human VIP receptor. By combining affinity labeling with glycosidase treatments, we have characterized the VIP receptor as a M_r = 68,200 glycoprotein, consisting of a M_r = 39,300 polypeptide core with at least three N-linked oligosaccharide chains. In addition, our results revealed the presence of a low amount of sialic acid residues in the carbohydrate moiety of receptor.     

Citrate metabolism in 16 Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum strains

Citrate metabolism was studied in non-growing cells of Leuconostoc mesenteroides subsp. mesenteroides and subsp. dextranicum with respect to energetics, formation of degradation products and stoichiometry. The use of selective ionophores and uncoupler showed that citrate utilization was coupled to the proton motive force generated by ATP hydrolysis. Differences in citrate metabolism observed in 20 Leuconostoc strains were related to strains but not to the species or subspecies studied. Citrate metabolism was stimulated by glucose up to a concentration of 25 mmol 1^-1 and decreased at higher concentrations. The main degradation products resulting from the co-metabolism of citrate (10 mmol 1^-1) and glucose (2 mmol 1^-1) were acetate, lactate and pyruvate. Only four Leuconostoc strains produced low levels of acetoin and diacetyl. No strains produced ethanol or acetaldehyde. Citrate degradation ability was stable for at least 130 generations in 81% of the Leuconostoc strains.     

Stimulating effect of pyroglutamylglutamylprolineamide,a prostatic,TRH-related tripeptide,on mouse sperm capacitation and fertilizing ability in vitro

Pyroglutamylglutamylprolineamide, a prostatic tripeptide with structural similarities to thyrotrophin-releasing hormone (TRH), has been found in the seminal plasma of several mammalian species, suggestive of a biological function relating to spermatozoa. Using chlortetracycline (CTC) fluorescence analysis and in vitro fertilization, we have obtained evidence that the tripeptide stimulates mouse sperm capacitation and fertilizing ability in vitro. The tripeptide at concentrations from 5–500 nM was added to sperm suspensions and cells were assessed with CTC after 40 min, insufficient time for complete capacitation by a majority of spermatozoa under standard conditions of incubation. Concentrations of 25 nM and higher significantly promoted capacitation, as evidenced by a decrease in the proportion of acrosome-intact F pattern spermatozoa, characteristic of uncapacitated cells, and an increase in the proportion of acrosome-intact B pattern spermatozoa, characteristic of capacitated cells. However, there was no significant stimulation of acrosomal exocytosis. These results suggested that peptide-treated cells would be more fertile than their untreated counterparts. This was confirmed using in vitro fertilization, where the presence of 100 nM peptide during sperm preincubation and gamete coincubation significantly stimulated fertilizing ability (peptide, 56.5% of oocytes fertilized; controls, 26.5%). Comparison of the prostatic tripeptide and TRH effects on capacitation revealed that TRH at a concentration of 250 nM was as effective as the prostatic tripeptide in promoting the F & B transition but was less effective or ineffective at lower concentrations. In vitro fertilization assessment of the two peptides, at 100 nM, revealed that only the prostatic tripeptide significantly stimulated fertility. Again, this was consistent with the CTC analyses. Because the prostatic tripeptide can stimulate sperm function in vitro, it is possible that it plays a similar role in vivo and promotes fertilizing ability of ejaculated spermatozoa. We therefore propose that this tripeptide be referred to as fertilization promoting peptide (FPP). © 1994 Wiley-Liss, Inc.