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.     

Selection of Lactobacillus acidophilus strains for use in “acidophilus products”

Recent studies by DNA-DNA hybridization revealed that strains now designated as L. acidophilus, can be divided into several groups and only one group should be classified as L. acidophilus. We studied several phenotypic characteristics in representative strains from the six DNA-homology groups of L. acidophilus. No group specific pattern was observed among the strains for fermentation of eight carbohydrates, growth at 15 and 45°C, resistance to 0.2% oxgall, lysis by lysozyme or sensitivity to 17 antibiotics. However, some differences among groups were observed in u564151146742518/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-galactosidase (u564151146742518/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-gal) activity and surface layer (s-layer) protein. Strains in B1 do not have a s-layer or u564151146742518/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-gal while B2 strains also lack a s-layer but do possess u564151146742518/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-gal. All strains in groups A1, A2, A3 and A4, capable of growing in lactose, have u564151146742518/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-gal activity and also have a s-layer composed of protein subunits of different molecular weights (MW). Strains in A1 homology group have a s-layer with 46 Kd protein subunits while strains in other A groups have s-layer protein subunits that varied in MW within each group. On the basis of these two traits several isolates of unknown homology groups have been tentatively placed in A1, B1 or B2 groups. L. acidophilus from A1 group showed strain variation in u564151146742518/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-gal specific activity and rate of acid production and growth. For use in dietary adjuncts, L. acidophilus strains should be selected for these three and other desirable traits. They should be maintained and grown in media containing lactose.     

Abnormal subcellular distribution of β-glucuronidase in mice with a genetic alteration in enzyme structure

Liver u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase is structurally altered in inbred strain PAC so that a peptide subunit with a more basic isoelectric point, GUS-SN, is produced. This allele of u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase was transferred to strain C57BL/6J by 12 backcross matings to form the congenic line B6 · PAC-Gus up>nup>. Liver u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase activity was halved in males of the congenic strain compared to normal males. The lowered activity was specifically accounted for by a decrease in the lysosomal component. There was no alteration in the concentration of microsomal activity. This alteration in the subcellular distribution of u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase in Gus up>nup>/Gus up>nup> mice was confirmed by two independent gel electrophoretic systems which separate microsomal and lysosomal components. u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-Glucuronidase activity was likewise approximately halved in mutant spleen, lung, and brain, organs which contain exclusively or predominantly lysosomal u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase. The loss of liver lysosomal u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase activity was shown by immunotitration to be due to a decrease in the number of u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase molecules in lysosomes of the congenic strain. The Gus up>nup> structural alteration likely causes the lowered lysosomal u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase activity since the two traits remain in congenic animals. Heterozygous Gus up>nup>/Gus up>bup> animals had intermediate levels of liver u484606n/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-glucuronidase. Also, the effect was specific, in that three other lysosomal enzymes were not reproducibly lower in Gus up>nup>/Gus up>nup> mice. Gus up>nup> is, therefore, an unusual example of a mutation which causes a change in the subcellular distribution of a two-site enzyme.This work was supported by National Institutes of Health Grants GM-33559 and GM-33160 and National Science Foundation Grant PCM-8215808.     

Structural analysis of the carbohydrate moieties of α-l-fucosidase from human liver

Acid u7/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-l-fucosidase (EC 3.2.1.51) was obtained from human liver and purified to homogeneity. The enzyme consists of four subunits; each of these has a molecular mass of 50 kDub>aub> and bears oneN-linked carbohydrate chain. The structures of these chains were studied at the glycopeptide level by methylation analysis and 500-MHzup>1up>H-NMR spectroscopy. Oligomannoside-type chains andN-acetyllactosamine-type chains are present in an approximate ratio of 3u7/xxlarge8758.gif" alt="ratio" align="BASELINE" BORDER="0">1. While the oligomannoside-type chains show some heterogeneity in size (Manub>5–8ub>GlcNAcub>2ub>), theN-acetyllactosaminetype chains are exclusively bi-u7/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">(2–6)-sialyl, bi-antennary in their structure.These observations on the carbohydrate moieties of u7/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-l-fucosidase substantiate our hypothesis [Overdijket al. (1986) Glycoconjugate J 3:339–50] with respect to the relationship between the oligosaccharide structure of lysosomal enzymes and their residual intracellular activity in I-cell disease. For the series of enzymes examined so far, namely, u7/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-N-acetylhexosaminidase, u7/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-l-fucosidase and u7/xxlarge946.gif" alt="beta" align="MIDDLE" BORDER="0">-galactosidase, the relative amount ofN-acetyllactosamine-type carbohydrate increases, while the residual intracellular activity in I-cell disease tissue decreases in this order. The system which is responsible for preferentially retaining hydrolases with (non-phosphorylated) oligomannoside-type chains both in I-cells and in normal cells has yet to be identified.     

Immunocytochemical markers revealing retinal and pineal but not hypothalamic photoreceptor systems in the Japanese quail

Summary The retinal proteins opsin,u753424n56x5201p/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-transducin, S-antigen and interstitial retinol-binding protein (IRBP) are essential for the processes of vision. By use of immunocyto-chemistry we have employed antibodies directed against these u753424n56x5201p/xxlarge8220.gif" alt="ldquo" align="MIDDLE" BORDER="0">photoreceptor proteinsu753424n56x5201p/xxlarge8221.gif" alt="rdquo" align="MIDDLE" BORDER="0"> in an attempt to identify the photoreceptor systems (retina, pineal and deep brain) of the Japanese quail. Opsin immunostaining was identified within many outer (basal portion) and inner segments of retinal photoreceptor cells and limited numbers of photoreceptor perikarya. Opsin immunostaining was also demonstrated in limited numbers of pinealocytes with all parts of these cells being immunoreactive. These results differ from previous observations. In contrast to the results obtained with the antibody against opsin, S-antigen andu753424n56x5201p/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-transducin immunostaining was seen throughout the entire outer segments and many photoreceptor perikarya of the retina. In the pineal organ immunostaining was seen in numerous pinealocytes in all follicles. These results conform to previous findings in birds. In addition, IRBP has been demonstrated for the first time in the avian retina and pineal organ. These findings underline the structural and functional similarities between the retina and pineal organ and provide additional support for a photoreceptive role of the avian pineal. No specific staining was detected in any other region of the brain in the Japanese quail; the hypothalamic photoreceptors of birds remain unidentified.     

Abnormal expression ofα-L-fucosidase in lymphoid cell lines of fucosidosis patients

Fucosidosis is an autosomal recessive lysosomal storage disease due to a deficiency ofu0u667345w567836/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-L-fucosidase activity in tissues and body fluids. Exponentially growing lymphoid cell cultures from four fucosidosis patients had 2.7-fold to 15.6-fold less extracellularu0u667345w567836/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-L-fucosidase protein and 28.8-fold to 144.0-fold less intracellularu0u667345w567836/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-L-fucosidase protein with negligible catalytic activity, compared to the mean of 19 control cultures. The percentage of totalu0u667345w567836/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-L-fucosidase protein released extracellularly by cultures from the four patients was 64 to 85%, compared to 35±9% for control cultures. Intracellular and extracellular enzyme forms in fucosidosis and control cell lines were glycoproteins containing polypeptide chains ofM ub>rub>=52,000. During a 1.5-hr pulse-label withup>35up>S-methionine,u0u667345w567836/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-L-fucosidase was synthesized by control cells and two fucosidosis cell lines as an intracellular form withM ub>rub>=58,000. During a subsequent 21-hr chase with unlabeled methionine, mutant enzyme was almost entirely processed to an extracellular form withM ub>rub>=62,000. In contrast, only 25–30% of control enzyme was processed to an extracellular form (M ub>rub>=62,000), with the remainder retained intracellularly (M ub>rub>=60,000). In the other two fucosidosis cell lines,u0u667345w567836/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-L-fucosidase was synthesized as an intracellular form withM ub>rub>=56,000 that was processed to an extracellular form withM ub>rub>=60,000. In summary, the fucosidosis mutation(s) affected the catalytic activity, quantity, and extracellular release ofu0u667345w567836/xxlarge945.gif" alt="agr" align="BASELINE" BORDER="0">-L-fucosidase as expressed by lymphoid cells.This work was funded by NIH Grants DK 32161 to R. A. DiCioccio and GM 28428 to J. K. Darby.     

Selective labeling of α-bungarotoxin with fluorescein isothiocyanate and its use for the study of toxin-acetylcholine receptor interactions

The main product of the reaction of fluorescein isothiocyanate (FITC) and bungarotoxin (Bgt) under near stoichiometric conditions is a monofluorescein derivative preferentially labeled at Lys 26, a highly conserved residue known to be involved in the binding (McDaniel, C. S., Manshouri, T., and Atassi, M. Z. (1987)J. Prot. Chem. 6, 455–461; Garcia-Borron, J. C., Bieber, A. L., and Martinez-Carrion, M. (1987)Biochemistry 26, 4295–4303) of postsynaptic neurotoxins specific for the nicotinic acetylcholine receptor (AcChR). The fluorescently labeled toxin retains a high affinity for the AcChR, and an unaltered specificity. Binding of FITC-Bgt to AcChR results in a significant decrease in the fluorescence intensity of the probe. This AcChR-mediated quenching of FITC-Bgt fluorescence allows for a continuous monitoring of the binding process. The quenching of free and bound FITC-Bgt by charged and neutral quenchers shows few fluorophore accessibility changes as induced by the toxin-bound state. The results are consistent with a model in which the positively charged concave surface of the toxin interacts with a negatively charged complementary surface in the receptor molecule.     

Pollination biology of two species ofKielmeyera (Guttiferae) from Brazilian cerrado vegetation

The pollination biology and breeding systems ofKielmeyera coriacea andK. speciosa, two sympatric woody species common in the cerrado vegetation of C. Brazil, were studied. Both species have similar nectarless, polystemonous u6347w4n894/xxlarge8220.gif" alt="ldquo" align="MIDDLE" BORDER="0">Papaver-typeu6347w4n894/xxlarge8221.gif" alt="rdquo" align="MIDDLE" BORDER="0"> flowers which are visited by a similar spectrum of insects, though they bloom in different seasons and are thus phenologically isolated. Large carpenter bees seem to be the most important pollinators and these and other bees effect u6347w4n894/xxlarge8220.gif" alt="ldquo" align="MIDDLE" BORDER="0">buzz pollenu6347w4n894/xxlarge8221.gif" alt="rdquo" align="MIDDLE" BORDER="0"> retrieval despite the fact that anthers are not poricidal. Both species ofKielmeyera possess strong xenogamous breeding systems. The presence of staminate flowers and andromonoecy inK. coriacea, as well as the longevity ofK. speciosa flowers are discussed as alternative strategies to improve pollination success and reproductive efficacy.