Genetic engineering of Escherichia coli inorganic pyrophosphatase. Tyr55 and Tyr141 are important for the structural integrity.

Two tyrosines are supposed to be essential for the activity and to participate in the stabilization of Escherichia coli inorganic pyrophosphatase (PPiase) against heat denaturation [Samejima, T., Tamagawa, Y., Kondo, Y., Hachimori, A., Kaji, H., Takeda, A. and Shiroya, Y. (1988) J. Biochem. (Tokyo) 103, 766-772]. To locate these two tyrosines in the amino acid sequence, we substituted all the eight tyrosines of E. coli PPiase with phenylalanine and studied the properties of these YF mutant PPiases. Interestingly, substitution of the tyrosines (Tyr51, Tyr55 and Tyr141) conserved with the amino acid sequence of yeast PPiase [Lahti, R., Kolakowski, L. F., Heinonen, J., Vihinen, M., Pohjanoksa, K. and Cooperman, B. (1990) Biochim. Biophys. Acta 1038, 338-345] exerted the most drastic effects on the structure and activity of E. coli PPiase. PPiase variants YF51, YF55 and YF141 had 64%, 7% and 22% of the wild-type PPiase activity, respectively. Furthermore, PPiase variant YF141 had an increased sensitivity to heat denaturation, whereas mutant PPiase YF55 displayed a profound conformational change, as demonstrated by the binding of the fluorescent dye 9-(diethylamino)-5H-benzo(alpha) phenoxazine-5-one (Nile red) that monitors the hydrophobicity of protein surfaces. None of the tyrosines of E. coli PPiase seem to be essential for catalysis, but Tyr55 and Tyr141 are important for the structural integrity of E. coli PPiase.     

Intracellular concentration of inorganic pyrophosphate in the cells of Escherichia coli: A method for its determination

A new method was developed for the separation of labelled inorganic pyrophosphate (PP₁) from the extracts of bacterial cells grown in the presence of labelled phosphate of high specific activity. First PP₁ is precipitated as manganous salt. Further purification is achieved in two chromatographic runs performed in the opposite directions on the same sheet of paper. The first solvent system consists of ethyl methyl ketone-methanol-water-36% HCl (20:40:10:1, by vol) and the second of ethyl methyl ketone-methanol-water-trichloroacetic acid-25% NH₄-EDTA (40 ml: 10 ml: 15 ml: 5 g: 0.8 ml: 50 mg). Each run takes only about 40 min of time. Thus it is possible to treat tens of samples in one working day.Using this procedure the intracellular concentration of PP₁ in the exponential phase cells of Escherichia coli growing aerobically in minimal medium at 37°C was determined to be about 1.3 mm or 5.5 nmoles/mg of dry weight.     

On the annual variation of phytoplankton biomass in Finnish inland waters

Annual variations in phytoplankton biomass in 63 lakes in Southern and Central Finland are discussed. Biomass is rather small during winter (January–April), usually <0.05 mg l^–1 (fresh weight) and there are no differences between oligotrophic and eutrophic lakes. In early spring and in autumn biomass varies widely, depending mainly on water temperature. Phytoplankton biomass is smaller in July than in June and August in oligotrophic lakes (biomass <0.20 mg l^–1 fresh weight) and mesotrophic (biomass 1.0–2.5 mg l^–1) lakes, but greater in eutrophic (biomass 2.5–10.0 mg l^–1) and hypereutrophic (biomass >10.0 mg l^–1) lakes. The standard deviation of phytoplankton biomass in Finnish inland waters is usually smallest in July, which facilitates the comparison of phytoplankton between different kinds of lakes.     

Glutathione depletion in isolated rat hepatocytes caused by styrene and the thermal degradation products of polystyrene

Depletion of reduced glutathione (GSH) was induced in isolated rat hepatocytes incubated with styrene or exposed for 120 min to products from oxidative thermal degradation of polystyrene. The depletion depended on the concentrations of styrene and on the degradation temperature. Styrene (1 mM) or products from degradation of polystyrene at 200°C (concentration of styrene in exposure atmosphere 0.7 ppm) had no detectable effect on glutathione levels in isolated hepatocytes. At higher degradation temperatures (250°C and 300°C, with styrene concentrations of 2.5 and 25 ppm, respectively) a rapid depletion was detected as well as with 3 mM styrene in incubation mixture. The latency of lactate dehydrogenase was affected neither by the polystyrene degradation products nor by the styrene added to the incubation mixture.     

Thiaminase activity of gastrointestinal contents of salmon and herring from the Baltic Sea

Potential thiaminase activity of Baltic herring Clupea harengus ranged from 0 to c. 55 nmol g-1 min-1 while potential thiaminase activity in Baltic salmon Salmo salar gastrointestinal (GI) contents ranged from 7 to c. 60 nmol g-1 min-1. About 30% of the Baltic herring analysed had a potential thiaminase activity equivalent to Baltic salmon GI contents. The results are consistent with the hypothesis that thiaminase in the forage fish of Baltic salmon may be an important link in the aetiology of the thiamine deficiency syndrome, M74, in Baltic salmon and indicate that Baltic salmon might feed selectively on Baltic herring with high thiaminase activity.     

Characterization of AmphiF-spondin reveals the modular evolution of chordate F-spondin genes

The F-spondin genes are a family of extracellular matrix molecules united by two conserved domains, FS1 and FS2, at the amino terminus plus a variable number of thrombospondin repeats at the carboxy terminus. Currently, characterized members include a single gene in Drosophila and multiple genes in vertebrates. The vertebrate genes are expressed in the midline of the developing embryo, primarily in the floor plate of the neural tube. To investigate the evolution of chordate F-spondin genes, I have used the basal position in chordate phylogeny of the acraniate amphioxus. A single F-spondin-related gene, named AmphiF-spondin, was isolated from amphioxus. Based on molecular phylogenetics, AmphiF-spondin is closely related to a particular subgroup of vertebrate F-spondin genes that encode six thrombospondin repeats. However, unlike these genes, expression of AmphiF-spondin is not confined to the midline but is found through most of the central nervous system. Additionally, AmphiF-spondin has lost three thrombospondin repeats and gained two fibronectin type III repeats, one of which has strong identity to a fibronectin type III repeat from Deleted in Colorectal Cancer (DCC). Taken together, these results suggest a complex evolutionary history for chordate F-spondin genes that includes (1) domain loss, (2) domain gain by tandem duplication and divergence of existing domains, and (3) gain of heterologous domains by exon shuffling.      

Transmission potential,skin inflammatory response,and parasitism of symptomatic and asymptomatic dogs with visceral leishmaniasis

Background

Visceral leishmaniasis in Brazil is caused by the protozoan Leishmania (Leishmania) chagasi and it is transmitted by sandfly of the genus Lutzomyia. Dogs are an important domestic reservoir, and control of the transmission of visceral leishmaniasis (VL) to humans includes the elimination of infected dogs. However, though dogs are considered to be an important element in the transmission cycle of Leishmania, the identification of infected dogs representing an immediate risk for transmission has not been properly evaluated. Since it is not possible to treat infected dogs, they are sacrificed when a diagnosis of VL is established, a measure that is difficult to accomplish in highly endemic areas. In such areas, parameters that allow for easy identification of reservoirs that represents an immediate risk for transmission is of great importance for the control of VL transmission. In this study we aimed to identify clinical parameters, reinforced by pathological parameters that characterize dogs with potential to transmit the parasite to the vector.

Results

The major clinical manifestations of visceral leishmaniasis in dogs from an endemic area were onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. The transmission potential of these dogs was assessed by xenodiagnosis using Lutzomyia longipalpis. Six of nine symptomatic dogs were infective to Lutzomyia longipalpis while none of the five asymptomatic dogs were infective to the sandfly. Leishmania amastigotes were present in the skin of all clinically symptomatic dogs, but absent in asymptomatic dogs. Higher parasite loads were observed in the ear and ungueal region, and lower in abdomen. The inflammatory infiltrate was more intense in the ears and ungueal regions of both symptomatic and asymptomatic dogs. In clinically affected dogs in which few or none Leishmania amastigotes were observed, the inflammatory infiltrate was constituted mainly of lymphocytes and macrophages. When many parasites were present, the infiltrate was also comprised of lymphocytes and macrophages, as well as a larger quantity of polymorphonuclear neutrophils (PMNs).

Conclusion

Dogs that represent an immediate risk for transmission of Leishmania in endemic areas present clinical manifestations that include onicogriphosis, skin lesions, conjunctivitis, lymphadenopathy, and weight loss. Lymphadenopathy in particular was a positive clinical hallmark since it was closely related to the positive xenodiagnosis.