Mechanistic studies on carboxypeptidase a from goat pancreas — part II: Evidence for carboxyl group

Studies with substrate analogues and the pH optimum indicated the involvement of carboxyl group in the active site of goat carboxypeptidase A. Chemical modification of the enzyme with 1-cyclohexyl-3-(2-morpholinoethyl) carbodiimide methoI -p-toluene sulphonate, a carboxyl specific reagent, led to loss of both esterase and peptidase activities. Protection studies showed that this carboxyl group was in the active site and was protected by Βp-phenylpropionic acid and glycyl-L-tyrosine. Kinetic studies also confirmed the involvement of carboxylic group because the enzyme modification with water soluble carbodiimide was a two step reaction which excluded the possibility of tyrosine or lysine which are known to give a one step reaction with this reagent     

Function in the Immune System of Antigenic Peptides from S-Antigen (Arrestin)

A significant amount of information concerning immunologic domains of an antigenic molecule can be obtained by studying its peptides. We describe a method for identifying and characterizing immunologically relevant T-cell and B-cell epitopes in S-antigen, a well-characterized, highly pathogenic retinal autoantigen for the induction of experimental autoimmune uveitis. The method involves the generation of peptide fragments by enzymatic treatment of native S-antigen and by the simultaneous synthesis of large numbers of peptides in small quantities for screening and testing. Peptides demonstrating T- or B-cell activity are then synthesized in large quantity for additional studies. Although useful information was obtained by the use of enzymatically generated peptides, synthetic peptides provided the greatest flexibility and specificity, allowing the precise localization of amino acid sequences of S-antigen required for a particular immunological function such as antibody binding, T-cell proliferative responses, pathogenicity, and the induction of tolerance. These studies have wide applicability to the study of other antigenic molecules and have led to a better understanding of the immune mechanisms involved in the pathogenesis of experimental autoimmune uveitis. This, in turn, provides a basis for the processes that may be occurring in certain forms of human uveitis.     

Conformations of blood group H-active oligosaccharides of ovarian cyst mucins

Spectroscopic data and conformational energy calculations are reported for eight oligosaccharides from ovarian cyst mucins and from human milk, the nonreducing terminals of which have fucose (α1 → 2)galactose linked either (β1 → 3) (type I) or (β1 → 4)(type II) to N-acetylglucosamine or in (β1 → 3) linkage to galactosaminitol. The fully assigned proton nmr spectra are reported along with nuclear Overhauser enhancement (NOE) data. Amide proton coupling constants and vacuum-uv CD spectra provide information on the amide plane orientation and amide environment. Our results imply that the fucosidic dihedral angles are similar for all three cases and that the substantial differences in the chemical shifts of the fucosyl protons of type I, type II, and 3-ol chains result from different perturbations by the amide group of the residue to which the β-galactose is linked. Stereopair diagrams of conformational models for both type I and II H chains are presented that are consistent with NOE, coupling constants, conformational energy calculations, and the CD data. While the temperature dependence of the observed NOE of penta- and hexasaccharides indicates that their rotational correlation times are strongly temperature dependent, we conclude that the conformations are essentially independent of temperature.     

Fungal spores are an important component of library air

The airborne fungal spore types were studied in different libraries in Delhi, using an Andersen sampler and a Burkard personal sampler, for culturable and non-culturable fungi respectively. The concentration inside the libraries, before and after the agitation of books, were compared with outside air. The major contributors to the library air areCladosporium, aspergilli/penicillia, smuts andAlternaria, varying from 50 to 14%. Some fungi (Cladosporium, Alternaria, smut,Penicillium chrysogenum andnigricans) showed seasonal occurrence, corresponding to their occurrence in the extramural environment. Aspergilli/penicillia,Drechslera, Curvularia andAspergillus flavus had a significantly higher concentration (P<0.01) inside the library, and recorded a significant increase in concentration after agitation of books. Air-conditioned libraries have low fungal spore concentrations, as compared to naturally ventilated libraries.     

An active-site carboxyl group in liquefying α-amylase: specific chemical modification

Bacillus amyloliquefaciens -amylase activity is pH-dependent and the plot log (Vmax/Km) versus pH implicated a carboxyl group of aspartic acid/glutamic acid at the active site. Chemical modification of -amylase with EDC confirmed this view. Further, analysis of inactivation kinetics showed that modification of a single carboxyl group led to complete loss of the enzymic activity.     

Association genetics of the parameters related to nitrogen use efficiency in Brassica juncea L.

Key message

Genome wide association studies allowed prediction of 17 candidate genes for association with nitrogen use efficiency. Novel information obtained may provide better understanding of genomic controls underlying germplasm variations for this trait in Indian mustard.

Abstract

Nitrogen use efficiency (NUE) of Indian mustard (Brassica juncea (L.) Czern & Coss.) is low and most breeding efforts to combine NUE with crop performance have not succeeded. Underlying genetics also remain unexplored. We tested 92 SNP-genotyped inbred lines for yield component traits, N uptake efficiency (NUPEFF), nitrogen utilization efficiency (NUTEFF), nitrogen harvest index (NHI) and NUE for two years at two nitrogen doses (N_o without added N and N₁₀₀ added @100 kg/ha). Genotypes IC-2489-88, M-633, MCP-632, HUJM 1080, GR-325 and DJ-65 recorded high NUE at low N. These also showed improved crop performance under high N. One determinate mustard genotype DJ-113 DT-3 revealed maximum NUTEFF. Genome wide association studies (GWAS) facilitated recognition of 17 quantitative trait loci (QTLs). Environment specificity was high. B-genome chromosomes (B02, B03, B05, B07 and B08) harbored many useful loci. We also used regional association mapping (RAM) to supplement results from GWAS. Annotation of the genomic regions around peak SNPs helped to predict several gene candidates for root architecture, N uptake, assimilation and remobilization. CAT9 (At1g05940) was consistently envisaged for both NUE and NUPEFF. Major N transporter genes, NRT1.8 and NRT3.1 were predicted for explaining variation for NUTEFF and NUPEFF, respectively. Most significant amino acid transporter gene, AAP1 appeared associated with NUE under limited N conditions. All these candidates were predicted in the regions of high linkage disequilibrium. Sequence information of the predicted candidate genes will permit development of molecular markers to aid breeding for high NUE.

     

Augmented Dried versus Cryopreserved Amniotic Membrane as an Ocular Surface Dressing

Purpose

Dried amniotic membrane (AM) can be a useful therapeutic adjunct in ophthalmic surgery and possesses logistical advantages over cryopreserved AM. Differences in preservation techniques can significantly influence the biochemical composition and physical properties of AM, potentially affecting clinical efficacy. This study was established to investigate the biochemical and structural effects of drying AM in the absence and presence of saccharide lyoprotectants and its biocompatibility compared to cryopreserved material.

Methods

AM was cryopreserved or dried with and without pre-treatment with trehalose or raffinose and the antioxidant epigallocatechin (EGCG). Structural and visual comparisons were assessed using electron microscopy. Localisation, expression and release of AM biological factors were determined using immunoassays and immunofluorescence. The biocompatibility of the AM preparations co-cultured with corneal epithelial cell (CEC) or keratocyte monolayers were assessed using cell proliferation, cytotoxicity, apoptosis and migration assays.

Results

Drying devitalised AM epithelium, but less than cryopreservation and cellular damage was reduced in dried AM pre-treated with trehalose or raffinose. Dried AM alone, and with trehalose or raffinose showed greater factor retention efficiencies and bioavailability compared to cryopreserved AM and demonstrated a more sustained biochemical factor time release in vitro. Cellular health assays showed that dried AM with trehalose or raffinose are compatible and superior substrates compared to cryopreserved AM for primary CEC expansion, with increased proliferation and reduced LDH and caspase-3 levels. This concept was supported by improved wound healing in an immortalised human CEC line (hiCEC) co-cultured with dried and trehalose or raffinose membranes, compared to cryopreserved and fresh AM.

Conclusions

Our modified preservation process and our resultant optimised dried AM has enhanced structural properties and biochemical stability and is a superior substrate to conventional cryopreserved AM. In addition this product is stable and easily transportable allowing it to be globally wide reaching for use in clinical and military sectors.