Development of sporeless/low sporing strains of Pleurotus through mutation

Summary The sporophores of Pleurotus are gymnocarpous and continuously release spores in the atmosphere causing respiratory allergies like hay fever and farmer’s lung disease among workers. The allergy is caused by the antigens present on the walls of the spores. Apart from this, during commercial production, these spores settle on the fruit bodies, germinate and form a velvety film which gives an unpleasant appearance to the mushrooms. The spores emitted may include new genotypes likely to attack wood or trees. Spore allergy is one of the most important limiting factors for the large scale cultivation of this species. Different approaches are being adopted at IIHR for the production of commercial sporeless/low-sporing strains of Pleurotus to alleviate the spore allergy problem. Attempts were made during the present investigation to produce sporeless or low-sporing mutants through u.v. mutation. Mutation of the mycelium did not yield the desired results. Mutation of the spores of Pleurotus sajor-caju yielded an extremely low-sporing mutant after 75 min exposure. The character has been found to be stable for more than 10 generations of subculturing.     

The Phylogenetic Relationships of the Members of the DROSOPHILA ROBUSTA Group

The phylogenetic relationships among the species of the D. robusta group were investigated by the analysis of chromosomal differences. Six of the ten known members of the D. robusta group were available for the study: D. colorata and D. robusta from the United States, and D. sordidula, D. pseudosordidula, D. lacertosa, and D. moriwakii from Japan. Analysis of the metaphase chromosomes from larval ganglion cells suggests that D. moriwakii and D. colorata, with rod-shaped X-chromosomes, are the more ancestral species, while D. sordidula, D. pseudosordidula, D. robusta, and D. lacertosa, with V-shaped X-chromosomes, are derived. The ancestral position of D. colorata and D. moriwakii is further strengthened by the fact that these are the two species in the D. robusta group that are cytologically closest to D. nigromelanica of the related D. melanica group. Of the four derived species, D. sordidula was found to be the closest to the ancestral species. The phylogeny based on the analysis of the gene sequences in the homologous chromosomes agreed with that indicated by the metaphase chromosomes. Since all attempts to obtain hybrids were unsuccessful except for the cross involving D. moriwakii females and D. colorata males, photographic maps of the salivary chromosomes were used to determine homology between the chromosomes of the different species. Evidence is presented to indicate that the D. robusta group originated in Asia (Japan), and that there were two migrations to the New World, the first leading to D. robusta, and the second to D. colorata. It is suggested that the route of migrations was across the Bering Land Bridge, and further, that the migrations occurred during the period from late Oligocene to middle Miocene, 20-25 million years ago.     

Effects of starvation,chilling, and injury on endocrine gland function in Galleria mellonella

Starvation, chilling, and injury of last instar Galleria mellonella larvae typically elicit extra larval molts or a delay in pupation. The primary sites of action and the nature of the signals by which these treatments affect development are not known. However, since the connections of the brain to the nerve cord are crucial for the effects of starvation and chilling, these signals apparently affect the brain-centered program of developmental regulation via the nerve cord. Chilling, and occasionally starvation, cause extra larval molts in last instar larvae treated prior to the nervous inhibition of their corpora allata; release of a cerebral allatotropin, which stimulates the production of juvenile hormone, appears to be involved in this effect. After this time, a delay in pupation is the principal effect of starvation and chilling, and is apparently due to a temporal inhibition of the release of the prothoracicotropic hormone. Chilling also appears to inhibit unstimulated ecdysteroid production by the prothoracic glands. The effect of injury is not mediated by the nerve cord, but appears to involve an inhibitory humoral factor that affects either the brain or the prothoracic glands themselves. Injury also stimulates juvenile hormone production, an effect which is enhanced when the brain is separated from the nerve cord and which is evidenced by a delay of ecdysis and the occasional retention of some larval features in the ecdysed insects. None of the effects of these various treatments on the brain and the endocrine glands persist when the brains or glands are implanted into untreated hosts.     

Synthesis of 3-O-β-d-xylopyranosyl-l-serine (xylosylserine) andO-β-d-galactopyranosyl-(1-4)-O-β-d-xylopyranosyl-l-serine (galactosylxylosylserine) and use of the synthetic products for detection of galactosyltransferase I activity in rat liver

3-O-β-d-Xylopyranosyl-l-serine (xylosylserine) was synthesized by the following three-step procedure: 1) 2,3,4-tri-O-benzoyl-α-d-xylopyranosyl bromide (benzobromoxylose) was condensed withN-carbobenzoxy-l-serine benzyl ester using the silver triflate-collidine complex as promoter; 2) theN-carbobenzoxy and benzyl ester groups in the resultant glycoside were cleaved by transfer hydrogenation with palladium black as catalyst and ammonium formate as hydrogen donor; and 3) the benzoyl groups were removed with methanolic ammonia. Xylosylserine was obtained in an overall yield of 70%. O-β-d-Galactopyranosyl-(1-4)-O-β-d-xylopyranosyl-(1-3)-l-serine (galactosylxylosylserine) was also synthesized by this methodology and was characterized by 2-dimensional (2D) NMR spectroscopy techniques. The two serine glycosides (xylosylserine and galactosylxylosylserine) were used in detection and partial purification of galactosyltransferase I (UDP-d-galactose:d-xylose galactosyltransferase) from adult rat liver.     

Use of the cytokinesis-block method for the analysis of micronuclei in V79 Chinese hamster lung cells: results with mitomycin C and cyclophosphamide

The cytochalasin B (CYB)-blocked binucleated cell assay has been explored to analyze micronuclei and cell cycle kinetics using 2 known mutagenic carcinogens in V79 Chinese hamster lung cells. To determine the optimum time to obtain the maximum number of binucleated cells for micronucleus analysis, duplicate cultures of exponentially growing cells were treated with 3 micrograms/ml CYB for varying durations (8-48 h). A peak appearance of binucleated cells at 16 h in the presence of CYB suggested this as an optimum time for micronucleus analysis in binucleated V79 cells. To evaluate the capacity for induction of micronuclei in V79 cells, 2 mutagenic carcinogens, mitomycin C (0.125-1.0 micrograms/ml) and cyclophosphamide (2-12 micrograms/ml) were tested in duplicate cultures. Mitomycin C, a direct-acting alkylating agent, caused approximately an 18-fold increase in micronucleus frequency over controls at the highest concentration tested (1.0 micrograms/ml), and this increase occurred in a dose-related manner (r = 0.92). The concentrations of mitomycin C tested also caused a significant dose-related cell cycle delay, thus suggesting cytotoxicity to V79 cells. Cyclophosphamide, an indirect-acting alkylating agent, requiring the presence of S9 mix, caused approximately a 17-fold increase in micronucleus frequency over controls at the highest tested concentration (12 micrograms/ml), with a clear dose response (r = 0.99). The various concentrations of cyclophosphamide also caused cytotoxicity in a dose-related fashion. Thus, this study demonstrates the usefulness of the cytokinesis-block method in V79 cells as a possible screen to analyze micronucleus induction and cytotoxicity. Because this approach is much less labor intensive than conducting a structural chromosomal analysis, this assay has great potential both as an initial screen for clastogenic activity and as a tool for investigating the underlying mechanisms for clastogenicity.     

A baiting technique for the isolation of bacteria producing starch-hydrolysing enzymes

By baiting litter soils with raw potatoes, species of bacilli producing thermostable amylases and raw starch-degrading amylase were selectively isolated.     

Effects of endogenous calcium transport inhibitor from heart muscle on the active calcium uptake and passive calcium release properties of sarcoplasmic reticulum

In the present study, the effects of the cytosolic Ca2+ transport inhibitor on ATP-dependent Ca2+ uptake by, and unidirectional passive Ca2+ release from, sarcoplasmic reticulum enriched membrane vesicles were examined in parallel experiments to determine whether inhibitor-mediated enhancement in Ca2+ efflux contributes to inhibition of net Ca2+ uptake. When assays were performed at pH 6.8 in the presence of oxalate, low concentrations (less than 100 micrograms/mL) of the inhibitor caused substantial inhibition of Ca2+ uptake by SR (28-50%). At this pH, low concentrations of the inhibitor did not cause enhancement of passive Ca2+ release from actively Ca2+-loaded sarcoplasmic reticulum. Under these conditions, high concentrations (greater than 100 micrograms/mL) of the inhibitor caused stimulation of passive Ca2+ release but to a much lesser extent when compared with the extent of inhibition of active Ca2+ uptake (i.e., twofold greater inhibition of Ca2+ uptake than stimulation of Ca2+ release). When Ca2+ uptake and release assays were carried out at pH 7.4, the Ca2+ release promoting action of the inhibitor became more pronounced, such that the magnitude of enhancement in Ca2+ release at varying concentrations of the inhibitor (20-200 micrograms/mL) was not markedly different from the magnitude of inhibition of Ca2+ uptake. In the absence of oxalate in the assay medium, inhibition of Ca2+ uptake was observed at alkaline but not acidic pH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proton nuclear magnetic resonance characterization of the aromatic residues in the variant-3 neurotoxin from Centruroides sculpturatus Ewing

The amino acid sequence for the variant-3 (CsE-v3) toxin from the venom of the scorpion Centruroides sculpturatus Ewing contains eight aromatic residues. By use of 2D NMR spectroscopic methods, the resonances from the individual protons (NH, C alpha H, C beta H',H", and the ring) for each of the individual aromatic residues have been completely assigned. The spatial arrangement of the aromatic ring systems with respect to each other has been qualitatively analyzed by 2D-NOESY techniques. The results show that Trp-47, Tyr-4, and Tyr-42 are in close spatial proximity to each other. The NOESY contacts and the ring current induced shifts in the resonances of the individual protons of Tyr-4 and Trp-47 suggest that the aromatic ring planes of these residues are in an orthogonal arrangement. In addition, the spatial proximity of the rings in the pairs Tyr-4, Tyr-58; Tyr-42, Tyr-40; and Tyr-40, Tyr-38 has also been established. A comparison with the published crystal structure suggests that there is a minor rearrangement of the aromatic rings in the solution phase. No 2D-NOESY contacts involving Phe-44 and Tyr-14 to any other aromatic ring protons have been observed. The pH dependence of the aromatic ring proton chemical shifts has also been studied. These results suggest that the Tyr-58 phenolic group is experiencing a hydrogen-bonding interaction with a positively charged group, while Tyr-4, -14, -38, and -40 are experiencing through-space interactions with proximal negatively charged groups. The Trp-47 indole NH is interacting with the carboxylate groups of two proximal acidic residues. These studies define the microenvironment of the aromatic residues in the variant-3 neurotoxin in aqueous solution.