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.     

Starch degradation by glucoamylase Glm from Saccharomycopsis fibuligera IFO 0111 in the presence and absence of a commercial pullulanase

This study describes the course of enzymatic hydrolysis of the native corn starches Maritena 100 and Maritena 300. Hydrolyses were carried out with glucoamylase Glm produced by Saccharomycopsis fibuligera IFO 0111, which degrades also native starch, with the purpose to substitute a two-step hydrolysis (amylase followed by glucoamylase) by a one-step process (glucoamylase only). Hydrolysis generally became more effective by adding the pullulanase Promozyme D, which cleaves alpha-1,6-glycosidic bonds more effectively than glucoamylase Glm does. The time course (kinetics) of hydrolysis was followed by determination of the glucose concentration and calculation of dextrose equivalents.     

Structural protein analysis of the polyvalent staphylococcal bacteriophage 812

Phage 812 is a polyvalent phage with a very broad host range in the genus Staphylococcus, which makes it a suitable candidate for phage therapy of staphylococcal infections. This proteomic study, combining the results of both 1-DE and 2-DE followed by PMF, led to the identification of 24 virion proteins. Twenty new proteins, not yet identified by proteome analysis of closely related staphylococcal phages K and G1 were identified using this approach. Fifteen proteins were assigned unambiguously to the head-tail genome module; the remaining nine proteins are encoded by genes of the left or right arms of the phage genome. As expected, the most abundant proteins in the electrophoretic patterns are the major capsid protein, the major tail sheath protein and proteins identical to ORF 50 and ORF 95 of phage K, although their function is only putative. Identification of these 20 new proteins contributes substantially to a detailed characterization of phage virions, knowledge of which is necessary for rational phage therapy.     

Caspases in yeast apoptosis-like death: facts and artefacts

Various findings suggest that programmed cell death (PCD) is induced in yeast as a response to the impact of a deleterious environment and/or an intracellular defect. Moreover, the specifically localized PCD within multicellular colonies seems to be important for the safe degradation of cell subpopulations to simple compounds that can be used as nutrients by healthy survivors occurring in propitious colony areas, being thus important for proper development and survival of the yeast population. In spite of this, the question remains whether yeast dies by real apoptosis, i.e. death involving caspases, or by other kinds of PCD. A large group of mammalian caspases includes those that are responsible for monitoring of the stimulus and initiating the dying process, as well as those involved in the execution of death. Additionally, paracaspases and metacaspases, that share some homology with real caspases, but possibly differ in substrate specificity, have been identified in plants, fungi, Dictyostelium and metazoa. In yeast, one homologue of caspases, metacaspase Mca1p/Yca1p, has been identified so far, although there are several indications of the presence of other caspase-like activities in yeast. In this minireview, we summarize various data on the possible involvement of Mca1p and other caspase-like activities in yeast PCD.     

Rice Resistance to Brown Spot Mediated by Nitrogen and Potassium

This study was undertaken to investigate the effects of both nitrogen (N) and potassium (K) rates on rice resistance to brown spot, caused by the fungus Bipolaris oryzae. Rice plants (cultivar ‘Metica 1’) were grown in soil corrected with 0, 25, 50, 75 and 100 mg of N / kg (as NH₄NO₃) of soil as well as with 25, 50, 75, 125 and 150 mg of K / kg (as KCl) of soil. Thirty‐three‐day‐old plants were inoculated with a suspension of Bipolaris oryzae conidia and the incubation period (IP), number of lesions (NL) per cm² of leaf area and disease severity was evaluated. Disease severity was scored at 24, 48, 72, 96, 120 and 144 h after inoculation and data were used to obtain the area under brown spot progress curve (AUBSPC). Soil plant analysis development (SPAD) index, plant dry weight and concentration of N and K in leaf tissues were also determined for both non‐inoculated (NI) and inoculated (IN) plants. Concentration of N in leaf tissue increased as the N rates in the soil increased. Concentration of K in leaf tissue increased sharply as the K rates in the soil increased for both NI and IN plants. Concentration of K in leaf tissue was not affected by N rates. The IP increased as the N rates increased, but was somewhat less impacted by increasing K rates. The NL decreased as the N rates increased. The NL dramatically declined at the highest K rates. The AUBSPC dramatically declined as the N and K rates in the soil increased. SPAD index values increased as the N and K rates in the soil increased for both NI and IN plants. Plant dry weight increased as the N and K rates in the soil increased for both NI and IN plants. Results from this study suggest that combining high N and K rates may contribute to reducing the intensity of brown spot in rice while improving plant development.     

Molecular dynamics of DNA quadruplex molecules containing inosine, 6-thioguanine and 6-thiopurine

The ability of the four-stranded guanine (G)-DNA motif to incorporate nonstandard guanine analogue bases 6-oxopurine (inosine, I), 6-thioguanine (tG), and 6-thiopurine (tI) has been investigated using large-scale molecular dynamics simulations. The simulations suggest that a G-DNA stem can incorporate inosines without any marked effect on its structure and dynamics. The all-inosine quadruplex stem d(IIII)(4) shows identical dynamical properties as d(GGGG)(4) on the nanosecond time scale, with both molecular assemblies being stabilized by monovalent cations residing in the channel of the stem. However, simulations carried out in the absence of these cations show dramatic differences in the behavior of d(GGGG)(4) and d(IIII)(4). Whereas vacant d(GGGG)(4) shows large fluctuations but does not disintegrate, vacant d(IIII)(4) is completely disrupted within the first nanosecond. This is a consequence of the lack of the H-bonds involving the N2 amino group that is not present in inosine. This indicates that formation of the inosine quadruplex could involve entirely different intermediate structures than formation of the guanosine quadruplex, and early association of cations in this process appears to be inevitable. In the simulations, the incorporation of 6-thioguanine and 6-thiopurine sharply destabilizes four-stranded G-DNA structures, in close agreement with experimental data. The main reason is the size of the thiogroup leading to considerable steric conflicts and expelling the cations out of the channel of the quadruplex stem. The G-DNA stem can accommodate a single thioguanine base with minor perturbations. Incorporation of a thioguanine quartet layer is associated with a large destabilization of the G-DNA stem whereas the all-thioguanine quadruplex immediately collapses.     

Enzymatic glycosylation using 6-O-acylated sugar donors and acceptors: beta-N-acetylhexosaminidase-catalysed synthesis of 6-O,N,N'-triacetylchitobiose and 6'-O,N,N'-triacetylchitobiose

p-Nitrophenyl 6-O-acetyl-2-acetamido-2-deoxy-beta-D-glucopyranoside (5a) was used as the glycosyl donor in a beta-N-acetylhexosaminidase-catalysed (from Penicillium brasilianum) glycosylation of GlcNAc yielding 6'-O,N,N'-triacetylchitobiose (6), while 6-O-acetyl-2-acetamido-2-deoxy-beta-D-glucopyranose (3a) served as a selectively protected acceptor in a transglycosylation reaction catalysed by the same enzyme to yield 6-O,N,N'-triacetylchitobiose (4).     

Molecular shape of the cationic lipid controls the structure of cationic lipid/dioleylphosphatidylethanolamine-DNA complexes and the efficiency of gene delivery

Pyridinium amphiphiles, abbreviated as SAINT, are highly efficient vectors for delivery of DNA into cells. Within a group of structurally related compounds that differ in transfection capacity, we have investigated the role of the shape and structure of the pyridinium molecule on the stability of bilayers formed from a given SAINT and dioleoylphosphatidylethanolamine (DOPE) and on the polymorphism of SAINT/DOPE-DNA complexes. Using electron microscopy and small angle x-ray scattering, a relationship was established between the structure, stability, and morphology of the lipoplexes and their transfection efficiency. The structure with the lowest ratio of the cross-sectional area occupied by polar over hydrophobic domains (SAINT-2) formed the most unstable bilayers when mixed with DOPE and tended to convert into the hexagonal structure. In SAINT-2-containing lipoplexes, a hexagonal topology was apparent, provided that DOPE was present and complex assembly occurred in 150 mm NaCl. If not, a lamellar phase was obtained, as for lipoplexes prepared from geometrically more balanced SAINT structures. The hexagonal topology strongly promotes transfection efficiency, whereas a strongly reduced activity is seen for complexes displaying the lamellar topology. We conclude that in the DOPE-containing complexes the molecular shape and the nonbilayer preferences of the cationic lipid control the topology of the lipoplex and thereby the transfection efficiency.     

Changes of E-cadherin and beta-catenin in human and mouse intestinal tumours

An immunohistochemical analysis of E-cadherin and -catenin was performed in human colorectal cancer as well as in surrounding normal intestinal tissue. We also analysed the expression of these two cell adhesion proteins in transgenic Apc1638N mice as a model of human familial adenometous polyposis syndrome. In the normal intestinal mucosa of both species, E-cadherin and -catenin were localized along the lateral plasma membrances of epithelial cells. In intestinal tumour cells, however, they were also present in the cytoplasm. The expression of both proteins was reduced in human and mouse tumours. The pattern of their distribution was frequently heterogenous with strongly positive cells in a mosaic of negative ones. Further, E-cadherin and -catenin expression did not correlate to the Duke's staging of tumours and therefore neither can be used as prognostic criteria.     

Phylogenetic analysis of the rplA genes encoding ribosomal protein L1

Previously we have identified therplA gene encoding ribosomal protein L1 inStreptomyces aureofaciens. Sequence comparison of ribosomal protein L1 among several bacterial genera revealed a high level of conservation. Based on this conservation, these proteins were used as a phylogenetic tool to compare evolutionary relationships among eubacteria and archaebacteria. This phylogenetic analysis of L1 ribosomal proteins including theS. aureofaciens rplA gene product revealed, except similar bacterial groupings, some new evolutionary relationships.