Intraspecific aflatoxin inhibition in Aspergillus flavus is thigmoregulated, independent of vegetative compatibility group and is strain dependent

Biological control of preharvest aflatoxin contamination by atoxigenic stains of Aspergillus flavus has been demonstrated in several crops. The assumption is that some form of competition suppresses the fungus's ability to infect or produce aflatoxin when challenged. Intraspecific aflatoxin inhibition was demonstrated by others. This work investigates the mechanistic basis of that phenomenon. A toxigenic and atoxigenic isolate of A. flavus which exhibited intraspecific aflatoxin inhibition when grown together in suspended disc culture were not inhibited when grown in a filter insert-plate well system separated by a .4 or 3 μm membrane. Toxigenic and atoxigenic conidial mixtures (50∶50) placed on both sides of these filters restored inhibition. There was ～50% inhibition when a 12 μm pore size filter was used. Conidial and mycelial diameters were in the 3.5-7.0 μm range and could pass through the 12 μm filter. Larger pore sizes in the initially separated system restored aflatoxin inhibition. This suggests isolates must come into physical contact with one another. This negates a role for nutrient competition or for soluble diffusible signals or antibiotics in aflatoxin inhibition. The toxigenic isolate was maximally sensitive to inhibition during the first 24 hrs of growth while the atoxigenic isolate was always inhibition competent. The atoxigenic isolate when grown with a green fluorescent protein (GFP) toxigenic isolate failed to inhibit aflatoxin indicating that there is specificity in the touch inhibiton. Several atoxigenic isolates were found which inhibited the GFP isolate. These results suggest that an unknown signaling pathway is initiated in the toxigenic isolate by physical interaction with an appropriate atoxigenic isolate in the first 24 hrs which prevents or down-regulates normal expression of aflatoxin after 3-5 days growth. We suspect thigmo-downregulation of aflatoxin synthesis is the mechanistic basis of intraspecific aflatoxin inhibition and the major contributor to biological control of aflatoxin contamination.     

Psychosine,the cytotoxic sphingolipid that accumulates in globoid cell leukodystrophy,alters membrane architecture

Globoid cell leukodystrophy (GLD) is a neurological disease caused by deficiency of the lysosomal enzyme galactosylceramidase (GALC). In the absence of GALC, the cytotoxic glycosphingolipid, psychosine (psy), accumulates in the nervous system. Psychosine accumulation preferentially affects oligodendrocytes, leading to progressive demyelination and infiltration of activated monocytes/macrophages into the CNS. GLD is characterized by motor defects, cognitive deficits, seizures, and death by 2–5 years of age. It has been hypothesized that psychosine accumulation, primarily within lipid rafts, results in the pathogenic cascade in GLD. However, the mechanism of psychosine toxicity has yet to be elucidated. Therefore, we synthesized the enantiomer of psychosine (ent-psy) to use as a probe to distinguish between protein-psy (stereo-specific enantioselective) or membrane-psy (stereo-insensitive nonenantioselective) interactions. The enantiomer of psychosine has equal or greater toxicity compared with psy, suggesting that psy exerts its toxicity through a nonenantioselective mechanism. Finally, in this study we demonstrate that psy and ent-psy localize to lipid rafts, perturb natural and artificial membrane integrity, and inhibit protein Kinase C translocation to the plasma membrane. Although other mechanisms may play a role in disease, these data strongly suggest that psy exerts its effects primarily through membrane perturbation rather than through specific protein-psy interactions.     

Enhancement of <Emphasis Type="Italic">Trichoderma</Emphasis> endochitinase secretion in tobacco cell cultures using an <Emphasis Type="Italic">α-</Emphasis>amylase signal peptide

An α-amylase signal peptide from rice was synthesized and fused with endochitinase (ech42) gene cloned from Trichoderma virens. The chimeric gene was designated as PSPα-amyech42, and this was transferred to a plant transformation vector, referred to as pMASGK. Leaf explants of tobacco cv White Burley were co-cultivated with Agrobacterium tumefaciens strain LBA4404 carrying ech42 with its own signal peptide(ech42SP) and PSPα-amyech42(pMASGK) separately. Putative transformants were selected on Murashige and Skoog (MS) medium, supplemented with 1 mg/l bezyladenine (BA), 0.5 mg/l naphthalene acetic acid(NAA), and containing 200 mg/l kanamycin and 200 mg/l cefotaxime. Transformation was further confirmed by PCR with specific primers and Southern blot hybridization. Endochitinase secretion was quantified in 1-week-old cell suspension cultures obtained from 3-week-old callus cultures of transformants carrying PSPα-amyech42, transformants with ech42 and of control (untransformed) plant. Callus cultures of PSPα-amyech42 showed higher endochitinase activity (9–12 times) than those carrying ech42SP (7–8 times) in both medium and cell extracts. Media collected (200 μg of total protein) from PSPα-amyech42 suspension cultures in Potato Dextrose Agar plates showed growth inhibition of 73 and 53% against Sclerotium rolfsii and Rhizoctonia bataticola, respectively, whereas media collected (200 μg of total protein) from ech42SP suspension culture showed inhibition of 14 and 24% against Sclerotium rolfsii and Rhizoctonia bataticola, respectively.     

Synthesis and characterization of diethyltin-based three dimensional self-assemblies derived from sulfonate-phosphonate ligands

Diethyltin(methoxy)methanesulfonate reacts readily with equimolar amount of t-butylphosphonic acid (RT, 8-10 h) in moist dichloromethane and methanol to afford [(Et₂Sn)₆(O₃PBu^t)₄(OSO₂Me)₄·CH₂Cl₂]_n (1) and [(Et₂Sn)₆(O₃PBu^t)₄(OSO₂Me)₄·2H₂O]_n (2), respectively. The identity of these compounds has been established by IR, multinuclear (¹H, ¹³C, ³¹P and ¹¹⁹Sn) NMR as well as X-ray crystallographic studies. Despite apparent similarity in the composition, a marked difference in the structural motifs between 1 and 2 is evident in the solid state. For 1, the formation of three-dimensional self- assembly results from μ₃-phosphonate and μ₂, μ₃-sulfonate binding modes and exhibits continuous channel of voids which are occupied by disordered CH₂Cl₂ molecules. In contrast, the sulfonate ligands in 2 act exclusively in μ₂-fashion and hydrogen bonding interactions (O-H?O) between coordinated water molecules and methanesulfonate groups is significant in the construction of 3D supramolecular assembly.     

Rice straw fermentation by Schizophyllum commune ARC-11 to produce high level of xylanase for its application in pre-bleaching

Rice straw is valuable resource that has been used as substrate for cost effective production of xylanase under solid-state fermentation by a newly isolated white rot fungi, S. commune ARC-11. Out of eleven carbon sources tested, rice straw was found most effective for the induction of xylanase that produced 4288.3?IU/gds of xylanase by S. commune ARC-11. Maximum xylanase production (6721.9?IU/gds) was observed on 8^th day of incubation at temperature (30?°C), initial pH (7.0) and initial moisture content (70.0%). The supplementation of ammonium sulphate (0.08% N, as available nitrogen) enhanced the xylanase production up to 8591.4?IU/gds. The xylanase production by S. commune ARC-11 was further improved by the addition of 0.10%, (w/v) of Tween-20 as surfactant. The maximum xylanase activities were found at pH 5.0 and temperature 55?°C with a longer stability (180?min) at temperature 45, 50 and 55?°C. This xylanase preparation was also evaluated for the pre-bleaching of ethanol-soda pulp from Eulaliopsis binata. An enzyme dosage of 10?IU/g of xylanase resulted maximum decrease in kappa number (14.51%) with a maximum improvement 2.9% in ISO brightness compared to control.     

Towards automation of glycomic profiling of complex biological materials

Glycosylation is considered one of the most complex and structurally diverse post-translational modifications of proteins. Glycans play important roles in many biological processes such as protein folding, regulation of protein stability, solubility and serum half-life. One of the ways to study glycosylation is systematic structural characterizations of protein glycosylation utilizing glycomics methodology based around mass spectrometry (MS). The most prevalent bottleneck stages for glycomic analyses is laborious sample preparation steps. Therefore, in this study, we aim to improve sample preparations by automation. We recently demonstrated the successful application of an automated high-throughput (HT), glycan permethylation protocol based on 96-well microplates, in the analysis of purified glycoproteins. Therefore, we wanted to test if these developed HT methodologies could be applied to more complex biological starting materials. Our automated 96-well-plate based permethylation method showed very comparable results with established glycomic methodology. Very similar glycomic profiles were obtained for complex glycoprotein/protein mixtures derived from heterogeneous mouse tissues. Automated N-glycan release, enrichment and automated permethylation of samples proved to be convenient, robust and reliable. Therefore we conclude that these automated procedures are a step forward towards the development of a fully automated, fast and reliable glycomic profiling system for analysis of complex biological materials.     

Increased Nitric Oxide Production in Patients with Systemic Sclerosis

Nitric oxide (NO, nitrogen monoxide) is a messenger molecule whose synthesis can be induced by proinflammatory cytokines. Increased production of NO has been reported in various inflammatory and autoimmune diseases. We studied serum nitrite and citrulline as surrogate markers for NO production in patients with systemic sclerosis (SSc) and looked for correlation with extent of disease, disease duration, age, and systemic involvement. Thirty-four patients were studied against 20 controls. The nitrite levels were significantly higher in the disease group (1588.4 +/- 998.2 nmol/ml compared to 327.8 +/- 137.7 nmol/ml; P < 0.001). The citrulline levels of the disease group were also significantly higher (5490.1 +/- 2518.3 nmol/ml compared to 3264.5 +/- 2509.7 nmol/ml in the controls; P = 0.005). There was no significant difference among limited and diffuse subgroups. There was no significant difference in patients with or without arthritis or interstitial lung disease or with other systemic involvement. On multivariate analysis there was a trend toward a rising level of nitrite with worsening lung functions (P = 0.07). Hence, there is evidence of increased NO production in patients with SSc. There is no difference between NO levels in disease subgroups or those with systemic involvement.