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.     

Enhancement of water status by calcium pretreatment in groundnut and cowpea plants subjected to moisture stress

Summary The effect of calcium in the water relations and tolerance to moisture deficits was tested in groundnut and cowpea. In both species, enrichment of tissue with calcium resulted in maintenance of a higher water status under stress associated with low proline accumulation. The extent of membrane damage (as reflected by the absorbance at 273 nm) was lesser in leaves of plants fed with higher levels of Ca⁺⁺ when subjected to simulated stress. The rate of water loss from the leaves of Ca⁺⁺-enriched plants was also lower. The possible role of Ca⁺⁺ in inducing membrane stability and maintenance of higher water status is discussed.     

A general multistate model for the analysis of hydrogen-exchange kinetics

In proton nmr, the chemical exchange rates of slowly exchanging labile hydrogens (with lifetimes in the range ～ 10 msec – ～ 1 sec) of peptides, proteins, and nucleic acids can be measured in H₂O by a combination of two separate experiments: (1) the transfer of solvent saturation and (2) saturation-recovery experiments. When these molecules exist in a dynamic equilibrium among different conformations, the experiments cannot be analyzed in a straightforward manner to derive the intrinsic exchange rates. In the present study we have derived analytical expressions for the above two experiments on a biomolecule under certain limiting conditions: (1) the extreme low-motility limit, where each of the conformational transitions is much slower than the corresponding hydrogen exchange rate with the solvent; (2) the high-motility limit (EX₂ mechanism), which is the opposite extreme of the previous limit; and (3) the low-motility limit (EX₁ mechanism), which is a mixture of limits (1) and (2), i.e., for some of the conformations, the exchange rate with the solvent is much faster than their conformational transition rates, while for the remaining conformations the reverse situation is realized. The results may be considered as a generalization to an arbitrary number of states of the two-state model treated by Hvidt. Equations have also been derived that are applicable to the iostope exchange method of measuring very slow exchange rates (with life-times of the order of minutes and longer) in biomolecules. The saturation recovery experiments performed in H₂O on the active pentapeptide fragment of thymopoietin serve to illustrate the high-motility limit. The theoretical formulation presented in this study can be easily adapted to other double-resonance techniques and also to situations where the kinetics of an arbitrary system existing in a multistate equilibrium are of interest.     

Synthesis of lysine-containing fragments of the Proteus mirabilis O27 O-specific polysaccharide and neoglycoconjugates therefrom.

Amide-linked lysine mono- and di-uronic acid fragments of the O-specific polysaccharide from P. mirabilis O27 have been synthesised. N epsilon-Boc-L-lysine tert-butyl ester was condensed with 2-azidoethyl glycosides of glucuronic acid and beta-D-GlcpNAc-(1----3)-beta-D-GlcpA. Transformation of the products into 2-acrylamidoethyl glycosides, followed by deprotection using trifluoroacetic acid, gave the target monomers that were converted into high-molecular-weight copolymer-type neoglycoconjugates.     

Pentacyclic steroids,part XVI,studies on the total syntheses of racemic 1,6-dithiabenz[3,4]estra-3, 5(10),8,14-tetraen-17-one and its D-homo analogue

The total syntheses of racemic 1,6-dithiabenz[3,4]-estra-3,5(10), 8,14-tetraen-17-one [VII]and 1,6-dithiabenz-[3,4]-D-homoestra-3,5(10),8,14-tetraen-17a-one [IX]starting fron isothiochroman-4-one [I]are described.     

Association of RAGE gene polymorphism with circulating AGEs level and paraoxonase activity in relation to macro-vascular complications in Indian type 2 diabetes mellitus patients

Background and aims

Sustained interaction of advanced glycation end products (AGEs) with their receptor RAGE and subsequent signaling plays an important role in the development of diabetic complications. Genetic variation of RAGE gene may be associated with the development of vascular complications in type 2 diabetes mellitus (T2DM).

Objectives

The present study aimed to explore the possible association of RAGE gene polymorphisms namely − 374T/A, − 429T/C and G82S with serum level of AGEs, paraoxonase (PON1) activity and macro-vascular complications (MVC) in Indian type 2 diabetes mellitus patients (T2DM).

Methods

A total of 265 diabetic patients, including DM without any complications (n = 135), DM-MVC (n = 130) and 171 healthy individuals were enrolled. Genotyping of RAGE variants were assessed by polymerase chain reaction-restriction fragment length polymorphism. Serum AGEs were estimated by ELISA and fluorometrically. and PON1 activity was assessed spectrophotometrically.

Results

Of the three examined SNPs, association of − 429T/C polymorphism with MVC in T2DM was observed (OR = 3.001, p = 0.001) in the dominant model. Allele ‘A’ of − 374T/A polymorphism seems to confer better cardiac outcome in T2DM. Patients carrying C allele (− 429T/C) and S allele (G82S) had significantly higher AGEs levels. − 429T/C polymorphism was also found to be associated with low PON1 activity. Interaction analysis revealed that the risk of development of MVC was higher in T2DM patients carrying both a CC genotype of − 429T/C polymorphism and a higher level of AGEs (OR = 1.343, p = 0.040).

Conclusion

RAGE gene polymorphism has a significant effect on AGEs level and PON1 activity in diabetic subjects compared to healthy individuals. Diabetic patients with a CC genotype of − 429T/C are prone to develop MVC, more so if AGEs levels are high and PON1 activity is low.     

Analysis of differentiation antigens on normal and carcinogen-altered rat epithelial cells in vivo and in culture

Two murine monoclonal antibodies, 3BG8 and 9BG8, which were raised against a rat tracheal squamous-cell-carcinoma cell line, recognize cell-surface antigens on normal rat squamous epithelium (skin, esophagus, vagina, and cornea) as well as on carcinogen-exposed, immortalized, rat tracheal epithelial cells. Monoclonal antibody 3BG8 binds to a 115-kilodalton cell-surface protein on undifferentiated basal cells of the epithelium, while the binding of the other antibody, 9BG8, occurs in both differentiated and undifferentiated populations of normal squamous epithelium and squamous cell carcinomas. Undifferentiated tracheal carcinomas bound only the 3BG8 antibody. No binding of either antibody was detected on normal tracheal mucociliary epithelium. Only under conditions that induce squamous differentiation of rat tracheal epithelium was binding of 3BG8 and 9BG8 detected. For reasons which are not clear at present, 9BG8 dramatically inhibits the growth of normal tracheal and esophageal cells in primary culture, whereas only 3BG8 affects the growth of carcinogen-altered tracheal cell lines. Based on antigen characterization and distribution, it is concluded that the 3BG8 and 9BG8 epitopes are localized on differentiation antigens which differ from others that have been previously described.     

A Novel Folate-Targeted Nanoliposomal System of Doxorubicin for Cancer Targeting

Targeted drug delivery systems for cancer improves anti-tumor efficacy and reduces systemic toxicity by restricting availability of cytotoxic drugs within tumors. Targeting moieties, such as natural ligands (folic acid, transferrin, and biotin) which are overexpressed on tumors, have been used to enhance liposome-encapsulated drug accumulation within tumors and resulted in better control. In this report, we explored the scope of targeting ligand folic acid, which is incorporated in liposome systems using folic acid-modified cholesterol (CPF), enabled highly selective tumor-targeted delivery of liposome-encapsulated doxorubicin and resulted in increased cytotoxicity within tumors. Folate-tagged poloxamer-coated liposomes (FDL) were found to have significantly higher cellular uptake than conventional poloxamer-coated liposomes (DL), as confirmed by fluorometric analysis in B16F10 melanoma cells. Biodistribution study of the radiolabeled liposomal system indicated the significantly higher tumor uptake of FDL as compared to DL. Anti-tumor activity of FDL against murine B16F10 melanoma tumor-bearing mice revealed that FDL inhibited tumor growth more efficiently than the DL. Taken together, the results demonstrated the significant potential of the folate-conjugated nanoliposomal system for drug delivery to tumors.     

Using homology modeling to interrogate binding affinity in neutralization of ricin toxin by a family of single domain antibodies

In this report we investigated, within a group of closely related single domain camelid antibodies (V_HHs), the relationship between binding affinity and neutralizing activity as it pertains to ricin, a fast‐acting toxin and biothreat agent. The V1C7‐like V_HHs (V1C7, V2B9, V2E8, and V5C1) are similar in amino acid sequence, but differ in their binding affinities and toxin‐neutralizing activities. Using the X‐ray crystal structure of V1C7 in complex with ricin's enzymatic subunit (RTA) as a template, Rosetta‐based homology modeling coupled with energetic decomposition led us to predict that a single pairwise interaction between Arg29 on V5C1 and Glu67 on RTA was responsible for the difference in ricin toxin binding affinity between V1C7, a weak neutralizer, and V5C1, a moderate neutralizer. This prediction was borne out experimentally: substitution of Arg for Gly at position 29 enhanced V1C7's binding affinity for ricin, whereas the reverse (ie, Gly for Arg at position 29) diminished V5C1's binding affinity by >10 fold. As expected, the V5C1_R29G mutant was largely devoid of toxin‐neutralizing activity (TNA). However, the TNA of the V1C7_G29R mutant was not correspondingly improved, indicating that in the V1C7 family binding affinity alone does not account for differences in antibody function. V1C7 and V5C1, as well as their respective point mutants, recognized indistinguishable epitopes on RTA, at least at the level of sensitivity afforded by hydrogen‐deuterium mass spectrometry. The results of this study have implications for engineering therapeutic antibodies because they demonstrate that even subtle differences in epitope specificity can account for important differences in antibody function.