The Isolation of Soluble Proteins,Glycoproteins, and Proteoglycans from Bone

Procedures are described for the isolation from bone of fractions containing proteins, glycoproteins and proteoglycans. Extraction of powdered bone with solutions of the sodium salts of ethylendiaminetetra-acetic acid (EDTA) at pH 7.5 solubilised about 7% of the organic material. These extracts contained about 1.8% of the total collagen and at least 60% of the total non-collagenous protein of bone. The extracts were dialysed against water to remove EDTA and then against a pH 5 buffer. At this stage a precipitate (Cl) formed which was removed by centrifugation. The supernatant was applied to a column of the carboxylio ion-exchange resin, Amberlite CG-50. The effluent at pH 5 contained the proteoglycans and more-acidic glycoproteins and was therefore named the Acidic Fraction (AF). The material adsorbed to the resin (Fraction G2) was eluted by equilibration to pH 8. AF was further fractionated by cetylpyridinium chloride (CPC) precipitation into three relatively pure components:(i) CP-S, a glycoprotein soluble in CPC, (ii) bone sialoprotein (BSP) which formed a CPC precipitate soluble in 0.2M-MgCl₂; and (iii) a proteoglycan fraction which formed a CPC precipitate insoluble in 0.2M-MgCl₂. The G2 fraction contained most of the soluble collagen together with glycoproteins and other non-collagenous proteins. These were fractionated by chromatography on DEAE-cellulose at pH 7.2 using stepwise elution with increasing concentrations of NaCl. Some resolution of the mixture was obtained, though most of the fractions contained more than one component. These procedures have been used on an analytical scale to assess the yields and recoveries of total protein, hydroxyproline and sialic acid in the fractions described above. This has been compared with the large scale procedure for the preparation of the fractions, which have been studied in previous work.     

Toxic polyketides produced by Fusarium sp., an endophytic fungus isolated from Melia azedarach

A new isocoumarin derivative named fusariumin (1), together with two known related resorcylic acid lactones aigialomycin D (2) and pochonin N (3), has been isolated from the cultures of Fusarium sp. LN-10, an endophytic fungus originated from the leaves of Melia azedarach. Their structures were established on the basis of extensive spectroscopic analyzes including 1D- and 2D- NMR (¹H-¹H COSY, HSQC, HMBC, and NOESY) experiments. Compounds 1-3 displayed significant growth inhibitory activity against the brine shrimp (Artemia salina).     

Aspergilones A and B, two benzylazaphilones with an unprecedented carbon skeleton from the gorgonian-derived fungus Aspergillus sp

Two novel benzylazaphilone derivatives with an unprecedented carbon skeleton, aspergilone A (1), and its symmetrical dimer with a unique methylene bridge, aspergilone B (2), have been isolated from the culture broth of a marine-derived fungus Aspergillus sp. from a gorgonian Dichotella gemmacea. Their structures and relative stereochemistries of 1 and 2 were elucidated using a combination of NMR spectroscopy and X-ray crystallography. Compound 1 not only exhibited in vitro selective cytotoxicity but also showed potent antifouling activity.     

Effect of inoculation with Penicillium expansum on the microbial community and maturity of compost

Compost prepared from wheat straw and cattle/chicken mature was inoculated with the lignocellulolytic fungus, Penicillium expansum. Compared to uninoculated compost, the inoculated compost exhibited a 150% higher germination index, more than 1.2 g kg(-1)-dw of changes in NH(4)(+)-N concentrations, a ca. 12.0% higher humus content and a lignocellulose degradation that proceeded 57.5% faster. Culture-based determinations of microbial populations demonstrated that aerobic heterotrophic bacteria and fungi were about 1-2 orders of magnitude higher in inoculated than in uninoculated compost. The number of ammonifying, ammonium-oxidizing, nitrite-oxidizing, denitrifying bacteria and cellulose-decomposing bacteria was 6.1-9.0 log(10) CFU g(-1)-dw, 1.2-4.3 log(10) MPN g(-1)-dw, 3.5-6.8 log(10) MPN g(-1)-dw, 3.58-4.34 log(10) MPN g(-1)-dw, 1.4-3.8 log(10)MPN g(-1)-dw, and 4.2-8.8 log(10) CFU g(-1)-dw higher in the compost inoculated with P. expansum.     

Rice RING protein OsBBI1 with E3 ligase activity confers broad-spectrum resistance against Magnaporthe oryzae by modifying the cell wall defence

Emerging evidence suggests that E3 ligases play critical roles in diverse biological processes, including innate immune responses in plants. However, the mechanism of the E3 ligase involvement in plant innate immunity is unclear. We report that a rice gene, OsBBI1, encoding a RING finger protein with E3 ligase activity, mediates broad-spectrum disease resistance. The expression of OsBBI1 was induced by rice blast fungus Magnaporthe oryzae, as well as chemical inducers, benzothiadiazole and salicylic acid. Biochemical analysis revealed that OsBBI1 protein possesses E3 ubiquitin ligase activity in vitro. Genetic analysis revealed that the loss of OsBBI1 function in a Tos17-insertion line increased susceptibility, while the overexpression of OsBBI1 in transgenic plants conferred enhanced resistance to multiple races of M. oryzae. This indicates that OsBBI1 modulates broad-spectrum resistance against the blast fungus. The OsBBI1-overexpressing plants showed higher levels of H(2)O(2) accumulation in cells and higher levels of phenolic compounds and cross-linking of proteins in cell walls at infection sites by M. oryzae compared with wild-type (WT) plants. The cell walls were thicker in the OsBBI1-overexpressing plants and thinner in the mutant plants than in the WT plants. Our results suggest that OsBBI1 modulates broad-spectrum resistance to blast fungus by modifying cell wall defence responses. The functional characterization of OsBBI1 provides insight into the E3 ligase-mediated innate immunity, and a practical tool for constructing broad-spectrum resistance against the most destructive disease in rice.     

Freezing and freeze-drying of the bacterium Rahnella aquatilis BNM 0523: study of protecting agents, rehydration media and freezing temperatures

Aims: The aim of the present study was to evaluate and compare freezing and freeze‐drying treatments for conserving Rahnella aquatilis (BNM 0523) with the goal to achieve an adequate commercial formulation of this biocontrol agent. Methods and Results: The effect of several protective agents, rehydration media and freezing temperatures on the viability and functional activity of the R. aquatilis was investigated. The storage stability at 3 months and 4 years was determined by checking the viability of the cells and their biocontrol capability against Botrytis cinerea by measuring the percentage of reduction of disease severity on apple. The best results were obtained by the freeze‐drying of the cells using a mixture of skimmed nonfat milk 10%, yeast extract 0·5% and glucose 1% as the protecting and rehydrating medium, and a quickly freezing (?70°C) before the freeze‐drying. In this case, the viability of the cells after 4 years was 98%, and their antagonistic ability showed a little decrease with respect fresh cells. Conclusions: The studies showed that R. aquatilis was resistant to freezing and freeze‐drying when it was used a mixture of cryoprotectants and that it was possible to obtain inoculums with high viability and good effectiveness for reduction of decay caused by B. cinerea. Significance and Impact of the study: This study is probably the first report about the resistance of R. aquatilis to freezing and freeze‐drying treatments and shows that these operations could be useful for obtaining a commercial formulation of this biocontrol agent.     

白木香内生真菌Fimetariella rabenhorstii的甾体类代谢产物

从白木香(Aquilaria sinensis(Lour.)Gilg)内生真菌Fimetariella rabenhorstii A20的发酵菌丝体中分离得到4个甾体类化合物.通过波谱分析,分别鉴定为5α,8α-桥氧-(22E,24R)-麦角甾-6,22-二烯-3β-醇(1)、3β,6β,7α-三羟基-(24R)麦角甾...     

Genetic population structure and distribution of a fungal polypore, Datronia caperata (Polyporaceae), in mangrove forests of Central America

Aim We examine the genetic structure of a fungal polypore, Datronia caperata (Berk.) Ryvarden (Polyporaceae), colonizing white mangrove, Laguncularia racemosa (L.) Gaertn. f. (Combretaceae), of Central America. Location Mangrove forests of Costa Rica and Panama. Methods Sequences of elongation factor alpha (EFA), beta tubulin (BTUB) and nuclear ribosomal internal transcribed spacer (ITS) regions were obtained from 54 collections of D. caperata collected from Caribbean and Pacific L. racemosa forests in Central America. Measures of haplotype and nucleotide diversity, nested clade analyses and coalescent analyses were used to estimate the direction and extent of migration of the fungus, and the factors promoting population divergence. We also conducted phylogenetic analyses using Bayesian estimation to test whether putative D. caperata collected from L. racemosa was conspecific with D. caperata colonizing other hosts from diverse Neotropical forests. Results Our results demonstrate that there is genetic isolation between D. caperata populations from Caribbean mangroves and those from Pacific mangroves. Our data suggest that the best explanation for the observed haplotype distribution is a recent range expansion from the Caribbean to the Pacific coasts, with subsequent isolation. This is supported by the infrequent overlap of haplotypes, unidirectional migration estimates from the Caribbean to the Pacific and the older estimated age of mutations in the Caribbean low‐copy BTUB and EFA loci. In addition, our data suggest that D. caperata from mangroves are not conspecific with collections from other hosts found in diverse Neotropical forests. Main conclusions The low frequency of shared haplotypes between coasts, coupled with the incomplete lineage sorting after cessation of gene flow, is consistent with isolation during the last Pleistocene glaciation. We hypothesize that the greater haplotype and nucleotide diversity in the Pacific occurs either because larger effective population sizes of D. caperata are maintained in Pacific mangroves or because D. caperata populations underwent a significant bottleneck as a result of local extinction followed by recolonization. In addition, we found that D. caperata found on L. racemosa was not conspecific with D. caperata from non‐mangrove hosts and suggest that D. caperata found on L. racemosa may be a host specialist.