Exogenously applied 20-hydroxyecdysone increases the net photosynthetic rate but does not affect the photosynthetic electron transport or the content of photosynthetic pigments in Tetragonia tetragonioides L.

Phytoecdysteroids are steroid compounds present in many plant species (sometimes in rather large amounts), but their biological role is still far from being clear. We have found that the exogenous application of 20-hydroxyecdysone (20E) to leaves of Tetragonia tetragonioides L. causes stimulation of its net photosynthetic rate (P _N) but does not positively affect the photosynthetic electron transport or the content of photosynthetic pigments. The increase in P _N was observed shortly after 20E treatment and was statistically significant during the 4th and 6th hours after treatment but not later, which could be perhaps caused by a strictly short-term window of opportunity for ecdysteroids to significantly affect photosynthetic processes. To our knowledge, these results are the first to suggest a new potential biological function of phytoecdysteroids—regulation of photosynthesis.     

Hexamers of the Type II Secretion ATPase GspE from Vibrio cholerae with Increased ATPase Activity

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Phenotypic and genotypic analysis of<Emphasis Type="Italic">Borrelia</Emphasis> spp. isolated from<Emphasis Type="Italic">Ixodes ricinus</Emphasis> ticks by using electrophoretic chips and real-time polymerase chain reaction

The genotype of Borrelia burgdorferi sensu lato was detected in 371 out of 1244 ticks. Borrelia determination was based on partial sequencing of the 16S rRNA gene and real-time polymerase chain reactions for identification and quantitation of ospA and recA genes. Different Borrelia spp. were identified; B. garinii in 40% ticks followed by B. afzelii (36.3%), B. burgdorferi sensu stricto (12.9%), B. valaisiana (3.5%), B. lusitaniae (0.8%), B. bissettii (0.5%) and B. miyamotoi-like (0.5%). Cultivation of 30 borrelia strains in BSK-H medium, among them B. valaisiana, B. bissettii-like and B. miyamotoi-like strains was unique in Czechia. Calibrated microfluidic-based quantification showed differences in the concentration of the nucleic acids and molar mass of the outer surface proteins of different Borrelia spp. with standard sensitivity and specificity and was helpful for their identification. The outer surface protein OspA was absent in B. miyamotoi-like and the OspB protein in B. valaisiana, B. lusitaniae and in three subtypes of B. garinii.     

Radiochromatographic assay of metabolites of the oostatic peptide labeled in different positions of the peptide chain

Reversed-phase high-performance liquid radio-chromatography (radio-HPLC) was set up to detect the time course of labeled degradation product formation of the pentapeptide H-Tyr-Asp-Pro-Ala-Pro-OH (5P), which has oostatic effects in different insect species. The detection limit of the system was in the range of 80-150 Bq. To follow formation of the degradation products, three amino acid residues in 5P were independently tritiated: Tyr1, Pro3 and Pro5. Each of the three tritiated peptides was analyzed after incubation with fresh hemolymph or ovaries of Neobellieria bullata. In the incubation mixture, free terminal amino acids and shortened sequences of 5P were identified. A metabolite of tyrosine represented the only exception; it was finally identified as water using degradation of [3H]Tyr by tyrosinase. Metabolic degradation of [3H]Tyr-5P was found to be considerably quicker than that of H-[3H]Tyr-Asp-Pro-Ala-OH (4P). The degradation of 5P was considerably slower in ovaries in comparison to hemolymph.     

Using a galactose library for exploration of a novel hydrophobic pocket in the receptor binding site of the Escherichia coli heat-labile enterotoxin

The binding of the B subunits of Escherichia coli heat-labile enterotoxin (LT) to epithelial cells lining the intestines is a critical step for the toxin to invade the host. This mechanism suggests that molecules which possess high affinity to the receptor binding site of the toxin would be good leads for the development of therapeutics against LT. The natural receptor for LT is the complex ganglioside GM1, which has galactose as its terminal sugar. A chemical library targeting a novel hydrophobic pocket in the receptor binding site of LT was constructed based on galactose derivatives and screened for high affinity to the receptor binding site of LT. This screening identified compounds that have 2-3 orders of magnitude higher affinity toward the receptor binding site of LT than the parent compound, galactose. The present findings will pave the way for developing simple and easily synthesizable molecules, instead of complex oligosaccharides, as drugs and/or prophylactics against LT-caused disease.     

Pretreatment with granulocyte colony-stimulating factor reduces myelopoiesis in irradiated mice

The purpose of this study was to investigate effects of the treatment prior to irradiation with granulocyte colony-stimulating factor (G-CSF) on hematopoiesis in B10CBAF1 mice exposed to a sublethal dose of 6.5 Gy of 60Co gamma radiation. G-CSF was administered in a 4-day regimen (3 microg/day); irradiation followed 3 h after the last injection of G-CSF. Such a treatment was found to stimulate granulopoiesis, as shown by increased counts of granulocyte-macrophage progenitor cells (GM-CFC) and of granulocytic cells in the femoral marrow and spleen at the time of irradiation. However, postirradiation counts of GM-CFC and granulocytic cells in the marrow of mice pretreated with G-CSF were reduced up to day 18 after irradiation. Interestingly, the D0 values for marrow GM-CFC determined 1 h after in vivo irradiation were 1.98 Gy for controls and 2.47 Gy for mice pretreated with G-CSF, indicating a decreased radiosensitivity of these cells after drug treatment. The inhibitory effects of the pretreatment with G-CSF on the postirradiation granulopoiesis could be attributed to the phenomenon of "rebound quiescence" which can occur after cessation of the treatment with growth factors. Postirradiation recovery of erythropoiesis in the spleen of mice pretreated with G-CSF exhibited a dramatic increase and compensated for the decreased erythropoiesis in the marrow at the time of irradiation. This complexity of the hematopoietic response should be taken into account when administering G-CSF in preirradiation regimens.     

The Development of Chloroplast Ultrastructure and Hill Reaction Activity During Leaf Ontogeny in Different Maize (Zea Mays L.) Genotypes

Changes in Hill reaction activity (HRA) and ultrastructure of mesophyll cell (MC) chloroplasts were studied during the ontogeny of third leaf of maize plants using polarographic oxygen evolution measurement, transmission electron microscopy, and stereology. The chloroplast ultrastructure was compared in young (actively growing), mature, and senescing leaves of two different inbreds and their reciprocal F1 hybrids. Statistically significant differences in both HRA and MC chloroplast ultrastructure were observed between different stages of leaf ontogeny. Growth of plastoglobuli was the most striking characteristic of chloroplast maturation and senescence. The chloroplasts in mature and senescing leaves had a more developed system of thylakoids compared to the young leaves. Higher HRA was usually connected with higher thylakoid volume density of MC chloroplasts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Leishmania mexicana glycerol-3-phosphate dehydrogenase showed conformational changes upon binding a bi-substrate adduct

Certain pathogenic trypanosomatids are highly dependent on glycolysis for ATP production, and hence their glycolytic enzymes, including glycerol-3-phosphate dehydrogenase (GPDH), are considered attractive drug targets. The ternary complex structure of Leishmania mexicana GPDH (LmGPDH) with dihydroxyacetone phosphate (DHAP) and NAD(+) was determined to 1.9A resolution as a further step towards understanding this enzyme's mode of action. When compared with the apo and binary complex structures, the ternary complex structure shows an 11 degrees hinge-bending motion of the C-terminal domain with respect to the N-terminal domain. In addition, residues in the C-terminal domain involved in catalysis or substrates binding show significant movements and a previously invisible five-residue loop region becomes well ordered and participates in NAD(+) binding. Unexpectedly, DHAP and NAD(+) appear to form a covalent bond, producing an adduct in the active site of LmGPDH. Modeling a ternary complex glycerol 3-phosphate (G3P) and NAD(+) with LmGPDH identified ten active site residues that are highly conserved among all GPDHs. Two lysine residues, Lys125 and Lys210, that are presumed to be critical in catalysis, were mutated resulting in greatly reduced catalytic activity. Comparison with other structurally related enzymes found by the program DALI suggested Lys210 as a key catalytic residue, which is located on a structurally conserved alpha-helix. From the results of site-directed mutagenesis, molecular modeling and comparison with related dehydrogenases, a catalytic mechanism of LmGPDH and a possible evolutionary scenario of this group of dehydrogenases are proposed.