Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

l-Glutamic acid was formed from d-, l-, and dl-PCA with cell-free extract of Pseudomonas alcaligenes ATCC-12815 grown in the medium containing dl-PCA as a sole source of carbon and nitrogen. The enzyme(s) involved in this conversion reaction was distributed in the soluble fraction within the cell and in 0.5 saturated fraction at the fractionation procedure with the saturation of ammonium sulfate. Optimum pH of this enzyme(s) lied at pH 8.5 and optimum temperature was 30°C. Cu (5 × 10^?3 m) inhibited the reaction considerably while Ca or Fe accelerated it. PALP (1×10^?3 m) also gave an enhanced activity to some extent. The enzyme preparation converted dextro-rotatory enan-thiomorph of PCA to its laevo-rotatory one which in turn was not converted to the opposite rotation direction by this enzyme. Furthermore, the preparation did not, if any, show d-glutamic acid racemase activity. Isotopic experiments with using dl-PCA-1-¹⁴C revealed that l-glutamic acid-1-¹⁴C was formed by the cleavage of –CO–NH– bond of pyrrolidone ring of PCA. It was concluded that dl-PCA when assimilated by the present bacterium is at first transformed to l-PCA by the optically isomerizing enzyme and subsequently is cleaved to l-glutamic acid probably by the PCA hydrolysing enzyme.     

Ab initio molecular orbital study of the mispairing ability of a nucleotide base analogue, N4-aminocytosine

The intrinsic properties of N4-aminocytosine, a base analogue of cytosine, are analyzed by an ab initio molecular orbital method. Relative stabilities of four possible isomeric structures of N4-aminocytosine are shown. The more stable isomer has the smaller dipole moment, so the relative stabilities of the isomers in solutions are subject to solvent polarity. The mutagenicity of this base analogue must arise because it can behave like either cytosine or thymine. It can form a guanine-cytosine-like base pair more easily than cytosine, and an adenine-thymine-like base pair less easily than thymine.     

Overexpression of the Escherichia coli catalase gene, katE, enhances tolerance to salinity stress in the transgenic indica rice cultivar, BR5

Salinity stress is a major limiting factor in cereal productivity. Many studies report improvements in salt tolerance using model plants, such as Arabidopsis thaliana or standard varieties of rice, e.g., the japonica rice cultivar Nipponbare. However, there are few reports on the enhancement of salt tolerance in local rice cultivars. In this work, we used the indica rice (Oryza sativa) cultivar BR5, which is a local cultivar in Bangladesh. To improve salt tolerance in BR5, we introduced the Escherichia coli catalase gene, katE. We integrated the katE gene into BR5 plants using an Agrobacterium tumefaciens-mediated method. The introduced katE gene was actively expressed in the transgenic BR5 rice plants, and catalase activity in T₁ and T₂ transgenic rice was approximately 150% higher than in nontransgenic plants. Under NaCl stress conditions, the transgenic rice plants exhibited high tolerance compared with nontransgenic rice plants. T₂ transgenic plants survived in a 200 mM NaCl solution for 2 weeks, whereas nontransgenic plants were scorched after 4 days soaking in the same NaCl solution. Our results indicate that the katE gene can confer salt tolerance to BR5 rice plants. Enhancement of salt tolerance in a local rice cultivar, such as BR5, will provide a powerful and useful tool for overcoming food shortage problems.     

Impaired spermatogenesis associated with changes in spatial arrangement of Sertoli and spermatogonial cells following induced diabetes

The current study was conducted to assess the relationship between testicular cells in spermatogenesis, through which the production of healthy and mature sperm is essential. However, it seems necessary to obtain more information about the three-dimensional pattern of the testis cells arrangement, which is directly related to the function of the testis after induction of diabetes. Twelve adult mice (28-30 g) were assigned into two experimental groups: (1) control and (2) diabetic (40 mg/kg STZ). The epididymal sperm collected from the tail of the epididymis and testes samples were taken for stereology, immunocytochemistry and RNA extraction. Our data showed that diabetes could notably decrease the number of testicular cells, together with a reduction of total sperm count. In addition, the results from the second-order stereology indicated the significant changes in the spatial arrangement of Sertoli cells and spermatogonial cells in the diabetic groups, in comparison with the control (P < .05). Moreover, the immunohistochemistry results showed a significant reduction in Sex-determining Region Y (SRY) box 9 gene (SOX9), vimentin, occludin, and connexin-43 positive cells in the diabetic groups compared with the control (P < .05). Furthermore, our data showed that the expression of steroidogenic acute regulatory protein steroidogenic acute regulatory protein (StAR) and peripheral benzodiazepine receptor peripheral benzodiazepine receptor (PBR) was significantly reduced in the diabetic groups, in comparison with the control (P < .05). These findings suggest that structural and functional changes of testis cells after induction of diabetes cause the alterations in the spatial arrangement of Sertoli and spermatogonial cells, ultimately influencing the normal spermatogenesis in mice.     

Sequential fractionation and two-dimensional gel analysis unravels the complexity of the dimorphic fungus Candida albicans cell wall proteome

The cell wall proteins of Candida albicans play a key role in morphogenesis and pathogenesis and might be potential target sites for new specific antifungal drugs. However, these proteins are difficult to analyze because of their high heterogeneity, interconnections with wall polysaccharides (mannan, glucan, and chitin), low abundance, low solubility, and hydrophobic nature. Here we report a subproteomic approach for the study of the cell wall proteins (CWPs) from C. albicans yeast and hyphal forms. Most of the mannoproteins present in this compartment were extracted by cell wall fractionation according to the type of interactions that they establish with other structural components. CWPs were solubilized from isolated cell walls by hot SDS and dithiothreitol treatment followed by extraction either by mild alkali conditions or by enzymatic treatment with glucanases and chitinases. These highly enriched cell wall fractions were analyzed by two-dimensional PAGE, showing that a large number of proteins are involved in cell wall construction and that the wall remodeling that occurs during germ tube formation is related to changes in the composition of CWPs. We suggest that the CWP-chitin linkage is an important retention mechanism of CWPs in C. albicans mycelial forms. This article also highlights the usefulness of the combination of sequential fractionation and two-dimensional PAGE followed by Western blotting using specific antibodies against known CWPs in the characterization of incorporation mechanisms of such CWPs into the cell wall and of their interactions with other wall components. Mass spectrometry analyses have allowed the identification of several cell surface proteins classically associated with both the cell wall and other compartments. The physiological significance of the dual location of these moonlighting proteins is also discussed. This approach is therefore a powerful tool for obtaining a comprehensive and integrated view of the cell wall proteome.     

Expression and Functional Characterization of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Recombinant <Emphasis Type="SmallCaps">l</Emphasis>.Asparaginase

Recombinant _l.asparaginase, L.ASNase, from Pseudomonas aeruginosa was purified using nickel affinity chromatography. The affinity purified L.ASNase exhibited a protein band with a molecular weight of 72.4 kDa on a native polyacrylamide gel and 36.276 kDa using SDS–PAGE. The activity of the purified L.ASNase was enhanced by Mg²⁺ and inhibited by Zn²⁺ at a concentration of 5 mM. The specificity of the recombinant L.ASNase towards different substrates was examined, and it was found that the enzyme showed the highest activity towards l.asparagine. Moreover, the enzyme showed lower activity towards other substrates such as _L.glutamine, urea and acrylamide. The in vitro hemolysis assay revealed that the purified L.ASNase did not show hemolysis effect on blood erythrocytes. Serum and trypsin half-life of L.ASNase suggested that the recombinant L.ASNase retained 50% of its initial activity after 90 and 60 min incubation period in serum and trypsin separately.     

Retinoids control anterior and dorsal properties in the developing forebrain

We have previously shown that retinoic acid (RA) synthesized by the retinaldehyde dehydrogenase 2 (RALDH2) is required in forebrain development. Deficiency in RA due to inactivation of the mouse Raldh2 gene or to complete absence of retinoids in vitamin-A-deficient (VAD) quails, leads to abnormal morphogenesis of various forebrain derivatives. In this study we show that double Raldh2/Raldh3 mouse mutants have a more severe phenotype in the craniofacial region than single null mutants. In particular, the nasal processes are truncated and the eye abnormalities are exacerbated. It has been previously shown that retinoids act mainly on cell proliferation and survival in the ventral forebrain by regulating SHH and FGF8 signaling. Using the VAD quail model, which survives longer than the Raldh-deficient mouse embryos, we found that retinoids act in maintaining the correct position of anterior and dorsal boundaries in the forebrain by modulating FGF8 anteriorly and WNT signaling dorsally. Furthermore, BMP4 and FGF8 signaling are affected in the nasal region and BMP4 is ventrally expanded in the optic vesicle. At the optic cup stage, Pax6, Tbx5 and Bmp4 are ectopically expressed in the presumptive retinal pigmented epithelium (RPE), while Otx2 and Mitf are not induced, leading to a dorsal transdifferentiation of RPE to neural retina. Therefore, besides being required for survival of ventral structures, retinoids are involved in restricting anterior identity in the telencephalon and dorsal identity in the diencephalon and the retina.     

Ethylene and in vitro rooting of hazelnut (Corylus avellana) cotyledons

Ethylene may be one of the many factors that play a role in rooting. However, in some studies ethylene promoted rooting, while in others it was inhibitory or had no effect. Using cotyledons of hazelnut ( Corylus avellana L. cv. Casina) observations were made of the effect of ethylene precursors on adventitious root formation. l-methionine (Met) or 1-aminocyclopropane-1-carboxylic acid (ACC) added to a standard indole-3-butyric acid (IBA)-kinetin-containing medium did not enhance rooting, while 2-chloroethylphosphonic acid (CEPA) did. The ethylene inhibitor, aminoethoxyvinylglycine (AVG), inhibited root formation, but its effect was reversed by ACC when cotyledonary segments were transferred to rhizogenic medium plus ACC at day 10. Ethylene production by cotyledons cultured on rhizogenic medium or rhizogenic medium plus CEPA was high at the beginning of rooting. Thus, the wound-induced ethylene is a key stimulatory factor in the formation of root primordia. The data support the hypothesis that ethylene plays a positive role in root formation.