Molecular characterization of intergeneric hybrid between Aspergillus oryzae and Trichoderma harzianum by protoplast fusion.

AIMS: Protoplast fusion between Aspergillus oryzae and Trichoderma harzianum and application of fusant in degradation of shellfish waste. METHODS AND RESULTS: The filamentous chitinolytic fungal strains A. oryzae NCIM 1272 and T. harzianum NCIM 1185 were selected as parents for protoplast fusion. Viable protoplasts were released from fungal mycelium using enzyme cocktail containing 5 mg ml(-1) lysing enzymes from T. harzianum, 0.06 mg ml(-1) ß-glucuronidase from Helix pomatia and 1 mg ml(-1) purified Penicillium ochrochloron chitinase in 0.8 mol l(-1) sorbitol as an osmotic stabilizer. Intergeneric protoplast fusion was carried out using 60% polyethylene glycol as a fusogen. At optimum conditions, the regeneration frequency of the fused protoplasts on colloidal chitin medium and fusion frequency were calculated. Fusant showed higher rate of growth pattern, chitinase activity and protein content than parents. Fusant formation was confirmed by morphological markers, viz. colony morphology and spore size and denaturation gradient gel electrophoresis (DGGE). CONCLUSIONS: This study revealed protoplast fusion between A. oryzae and T. harzianum significantly enhanced chitinase activity which ultimately provides potential strain for degradation of shellfish waste. Consistency in the molecular characterization results using DGGE is the major outcome of this study which can be emerged as a fundamental step in fusant identification. SIGNIFICANCE AND IMPACT OF THE STUDY: Now it is need to provide attention over effective chitin degradation to manage shrimp processing issues. In this aspect, ability of fusant to degrade shellfish waste efficiently in short incubation time revealed discovery of potential strain in the reclamation of seafood processing crustacean bio-waste.

Fatal rabies despite post-exposure prophylaxis.

Only sporadic reports of failure of post-exposure prophylaxis for rabies exist in the published literature. We are reporting such a case in a 3-year-old boy. The child had Category III dog bite on his right thigh. He presented with progressive ascending paralysis, finally developing quadriplegia and respiratory paralysis. Typical hydrophobia and aerophobia were absent. He received four doses of antirabies cell culture vaccine. He did not receive antirabies immunoglobulin. The boy succumbed on the 23 rd day of the dog bite. Diagnosis of rabies was confirmed in the laboratory by demonstration of Negri bodies, direct fluorescent antibody test and reverse transcriptase-polymerase chain reaction either on impression smear of brain or a piece of brain taken during autopsy.

Rheumatoid arthritis (RA)-associated HLA-DR alleles form less stable complexes with class II-associated invariant chain peptide than non-RA-associated HLA-DR alleles.

Certain HLA-DR alleles confer strong susceptibility to the autoimmune disease rheumatoid arthritis (RA). We compared RA-associated alleles, HLA-DR*0401, HLA-DR*0404, and HLA-DR*0405, with closely related, non-RA-associated alleles, HLA-DR*0402 and HLA-DR*0403, to determine whether they differ in their interactions with the class II chaperone, invariant chain (Ii). Ii binds to class II molecules in the endoplasmic reticulum, inhibits binding of other ligands, and directs class II-Ii complexes to endosomes, where Ii is degraded to class II-associated Ii peptide (CLIP). To evaluate the interaction of Ii and CLIP with these DR4 alleles, we introduced HLA-DR*0401, *0402, and *0404 alleles into a human B cell line that lacked endogenous HLA-DR or HLA-DM molecules. In a similar experiment, we introduced HLA-DR*0403 and *0405 into an HLA-DM-expressing B cell line, 8.1.6, and its DM-negative derivative, 9.5.3. Surface abundance of DR4-CLIP peptide complexes and their susceptibility to SDS-induced denaturation suggested that the different DR4-CLIP complexes had different stabilities. Pulse-chase experiments showed CLIP dissociated more rapidly from RA-associated DR molecules in B cell lines. In vitro assays using soluble rDR4 molecules showed that DR-CLIP complexes of DR*0401 and DR*0404 were less stable than complexes of DR*0402. Using CLIP peptide variants, we mapped the reduced CLIP interaction of RA-associated alleles to the shared epitope region. The reduced interaction of RA-associated HLA-DR4 molecules with CLIP may contribute to the pathophysiology of autoimmunity in RA.

Autoantigenic HCgp39 epitopes are presented by the HLA-DM-dependent presentation pathway in human B cells.

It is hypothesized that autoimmune diseases manifest when tolerance to self-Ags fails. One possible mechanism to break tolerance is presentation of self-Ag in an altered form. Most Ags are presented by APCs via the traditional presentation pathway that includes "epitope editing" by intracellular HLA-DM, a molecule that selects for stable MHC-peptide complexes. We were interested in testing the hypothesis that autoreactive MHC-peptide complexes may reach the cell surface by an alternate pathway without being edited by HLA-DM. We selected a cartilage autoantigen human cartilage glycoprotein 39 to which T cell responses are observed in rheumatoid arthritis (RA) patients and some DR(*)04 healthy subjects. RA is genetically associated with certain DRB1 alleles, including DRB1(*)0401 but closely related allele DRB1(*)0402 is either neutral or mildly protective with respect to RA. We generated human B lymphoblastoid cell line cells expressing DR(*)0401 or DR(*)0402 in the presence or absence of intracellular HLA-DM and assessed their ability to present a candidate autoantigen, human cartilage glycoprotein 39. Our results show that the presence of intracellular HLA-DM is critical for presentation of this autoantigen to CD4(+) T cell hybridomas generated from DR(*)04-transgenic mice. Presentation of an autoantigen by the traditional HLA-DM-dependent pathway has implications for Ag presentation events in RA.

Accessory molecules for MHC class II peptide loading.

Accessory molecules, such as HLA-DM and invariant chain, modulate the ligands bound to MHC class II molecules in antigen-presenting cells. Recent investigations, including gene targeting experiments, have shed light on the functions of these molecules, their mechanisms of action, interactions with class II molecules, and the relationships with associated molecules such as tetraspanins and HLA-DO.

A developmentally regulated deletion element with long terminal repeats has cis-acting sequences in the flanking DNA.

Approximately 6000 specific DNA deletion events occur during development of the somatic macro-nucleus of the ciliate Tetrahymena. The eliminated Tlr1 element is 13 kb or more in length and has an 825 bp inverted repeat near the rearrangement junctions. A functional analysis of the cis -acting sequences required for Tlr1 rearrangement was performed. A construct consisting of the entire inverted repeat and several hundred base pairs of flanking DNA on each side was rearranged accurately in vivo and displayed junctional variability similar to the chromosomal Tlr1 rearrangement. Thus, 11 kb or more of internal element DNA is not required in cis for DNA rearrangement. A second construct with only 51 bp of Tetra-hymena DNA flanking the right junction underwent aberrant rearrangement. Thus, a signal for determination of the Tlr1 junction is located in the flanking DNA, 51 bp or more from the right junction. Within the Tlr1 inverted repeat are 19 bp tandem repeats. A construct with the 19mer repeat region deleted from the right half of the inverted repeat utilized normal rearrangement junctions. Thus, despite its transposon-like structure, Tlr1 is similar to other DNA rearrangements in Tetrahymena in possessing cis -acting sequences outside the deleted DNA.

Alternate junctions and microheterogeneity of Tlr1, a developmentally regulated DNA rearrangement in Tetrahymena thermophila.

A large number of developmentally regulated DNA rearrangements occur during the development of the macronucleus in Tetrahymena thermophila. Tlr1 is a deletion element which has large inverted repeats near the rearrangement junctions and deletes more than 13 kbp of internal DNA. Previous analysis of caryonidal lines revealed alternate left junctions for the Tlr1 rearrangement in B strain cells. We show here that C2 strain Tetrahymena also use alternate rearrangement junctions. We have mapped and sequenced two additional rearrangement variants and find that both the left and right junctions can vary over a range of approximately 200 bp. We also demonstrate the presence of sequence microheterogeneity in the most commonly found Tlr1 rearrangement product.

Macroporous poly(sucrose acrylate) hydrogel for controlled release of macromolecules.

We have studied the controlled release of proteins from poly(sucrose acrylate) hydrogels. The hydrogels were prepared by a two-step procedure in which sucrose was first acylated to sucrose-1'-acrylate followed by free radical polymerization. By adjusting the cross-link ratio and initial monomer concentration, the swelling ratio of the hydrogel was varied from five to 28. The mechanical strength of these hydrogels was comparable to that of the hydrogels with approximately the same swelling ratio. Scanning electron micrographs and mesh size calculations indicate that the hydrogel is macroporous, suggesting it may be suitable for a variety of biomedical applications. The release kinetics of beta-lactoglobulin, bovine serum albumin and gamma-globulin were studied as a function of initial monomer concentrations for the sucrose-based hydrogel. All of the release profiles were characterized by an initial burst of protein in the first 25 h followed by a long period of sustained release (> 500 h). The magnitude of the initial burst was reduced by increasing the initial monomer concentration and by increasing the molecular weight of the protein. A quantitative model based on the heterogeneous nature of hydrogel was developed to explain the observed release kinetics.

Adaptive larval thermotolerance and induced cross-tolerance to propoxur insecticide in mosquitoes Anopheles stephensi and Aedes aegypti.

Fourth-instar larvae of mosquitoes Anopheles stephensi and Aedes aegypti normally died within 90 min at 43 degrees C. Pre-exposure to high but sublethal temperatures conferred adaptive thermotolerance, dependent on the temperature and the duration of pre-exposure. Adaptive cross-tolerance to propoxur (a carbamate insecticide) was also induced in larvae by pre-exposing them to sublethal temperatures. Pre-exposure to sublethal concentrations of propoxur was found to confer cross-thermotolerance to a lower extent. These results suggest that the shock proteins (e.g. heat shock proteins) induced by unrelated stress factors play an important role in the development of adaptive cross-protection (stress response) to other stress conditions.