Physicochemical characterization of β-crystallins from bovine lenses: Hydrodynamic and aggregation properties

A detailed investigation of the hydrodynamic and aggregation behaviors has been made on the -crystallins of bovine lens. Results from this study indicated that H (high-molecular-weight -crystallin) and L (low-molecular-weight -crystallin) exhibited considerable heterogeneity in their native structures and subunit polypeptides. Low-speed sedimentation equilibrium showed a heterogeneous paucidisperse system in each -crystallin fraction. Viscosity and circular dichroism studies pointed to a compact and globular shape and the presence of -sheet and -turns in these crystallins. Dissociation of H by urea and guanidinium HCl followed by reassociation during gel-filtration chromatography produced an elution pattern with two fractions corresponding to L crystallin and high-molecular-weight aggregates without the formation of native H. By contrast, under similar treatment, about 60% L reassociated into the correct native structure and the rest into high-molecular-weight fractions. Amino acid analyses of H and L and their corresponding subunit polypeptides demonstrated the close similarity of these crystallins. Trace element analyses indicated that both Ca and Mg are present in H and L crystallins and may be involved in maintaining the native quarternary structures of these proteins.     

Thermal stabilization of fungal β-glucosidase through glutaraldehyde crosslinking

Summary The thermal stability of a purifiedAspergillus niger -glucosidase (EC 3.2.1.21) has been dramatically increased by intramolecular crosslinking with glutaraldehyde at pH 9.0, followed by borohydride reduction. The stabilization has been shown to be due to the formation of a distinct subpopulation of enzyme molecules, the activity of which has a half-life at 65°C of approximately 40 hours, which is some 80 times as long as that of the native enzyme at this temperature.     

Characterization and molecular genetic complementation of mutants affecting dimorphism in the fungus ustilago maydis

Ustilago maydis, the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a unicellular, nonpathogenic yeast-like form and a dikaryotic, pathogenic filamentous form. Previously, a constitutively filamentous haploid mutant was obtained. Complementation of this mutant led to the isolation of the gene encoding adenylate cyclase, uac1. Secondary mutagenesis of a uac1 disruption strain allowed the isolation of a large number of suppressor mutants, termed ubc, for Ustilago bypass of cyclase, lacking the filamentous phenotype. Analysis of one of these suppressor mutants previously led to the identification of the ubc1 gene, encoding the regulatory subunit of cAMP-dependent protein kinase. In this report we describe the isolation of cosmids containing three new ubc genes, termed ubc2, ubc3, and ubc4. We also describe the morphology of the ubc2, ubc3, and ubc4 mutants in a uac1- background as well as in a background with a functional uac1 gene. In addition, we describe several mutant strains not complemented with any of the genes currently in hand and that are thus presumed to possess mutations in additional ubc genes. Copyright 1998 Academic Press.     

The taste of polycose in hamsters

Hamsters show a preference for Polycose, a mixture of starch-derived glucose polymers, that is as strong as their preference for sucrose. However, in the hamster, taste aversions to Polycose may be less easily acquired than taste aversions to sucrose and the qualitative aspects of Polycose are unknown in this species. In order to examine the taste of Polycose in the hamster, we utilized a taste-aversion protocol with two conditioning trials. Animals were trained to avoid one of three different conditioning stimuli: 50 mM sucrose, 100 mM Polycose and a mixture of 50 mM sucrose with 100 mM Polycose. Control animals were conditioned with deionized water. After the second conditioning trial, generalization testing began for the three conditioning stimuli plus 3 mM citric acid, 300 mM KCI and 30 mM NaCl. The results showed that aversions to Polycose, sucrose or the Polycose/sucrose mixture cross- generalized, demonstrating that Polycose and sucrose share a common taste percept in the hamster. None of the aversions generalized to NaCl, citric acid or KCI. In addition, comparisons among the patterns of taste generalizations indicated that the tastes of Polycose and sucrose also had distinct qualitative components. Finally, although the taste of 100 mM Polycose was more salient than the taste of 50 mM sucrose, the taste of sucrose could still be detected in a mixture with Polycose.      

The 67-kDa laminin receptor originated from a ribosomal protein that acquired a dual function during evolution

The 67-kDa laminin receptor (67LR) is a nonintegrin cell surface receptor that mediates high-affinity interactions between cells and laminin. Overexpression of this protein in tumor cells has been related to tumor invasion and metastasis. Thus far, only a full-length gene encoding a 37-kDa precursor protein (37LRP) has been isolated. The finding that the cDNA for the 37LRP is virtually identical to a cDNA encoding the ribosomal protein p40 has suggested that 37LRP is actually a component of the translational machinery, with no laminin-binding activity. On the other hand, a peptide of 20 amino acids deduced from the sequence of 37LR/p40 was shown to exhibit high laminin-binding activity. The evolutionary relationship between 23 sequences of 37LRP/p40 proteins was analyzed. This phylogenetic analysis indicated that all of the protein sequences derive from orthologous genes and that the 37LRP is indeed a ribosomal protein that acquired the novel function of laminin receptor during evolution. The evolutionary analysis of the sequence identified as the laminin-binding site in the human protein suggested that the acquisition of the laminin-binding capability is linked to the palindromic sequence LMWWML, which appeared during evolution concomitantly with laminin.      

Visualization of Trichoderma reesei Cellobiohydrolase I and Endoglucanase I on Aspen Cellulose by Using Monoclonal Antibody-Colloidal Gold Conjugates

Monoclonal antibodies (MAbs) specific for cellobiohydrolase I (CBH I) and endoglucanase I (EG I) were conjugated to 10- and 15-nm colloidal gold particles, respectively. The binding of CBH I and EG I was visualized by utilizing the MAb-colloidal gold probes. The visualization procedure involved immobilization of cellulose microfibrils on copper electron microscopy grids, incubation of the cellulose-coated grids with cellulase(s), binding of MAb-colloidal gold conjugates to cellulase(s), and visualization via transmission electron microscopy. CBH I was seen bound to apparent crystalline cellulose as well as apparent amorphous cellulose. EG I was seen bound extensively to apparent amorphous cellulose with minimal binding to crystalline cellulose.     

Cross-reactive and specific monoclonal antibodies against cellobiohydrolases I and II and endoglucanases I and II of Trichoderma reesei.

Splenocytes derived from mice inoculated with a commercial cellulase preparation or purified cellulases were fused with a stable myeloma cell line (SP2/0). Specific monoclonal antibodies to cellobiohydrolases I and II and endoglucanases I and II were established. In addition to specific monoclonal antibodies, we were also able to establish stable hybridoma cell lines which produced monoclonal antibodies that recognized similar epitopes possessed by two or more of the above cellulases. By obtaining monospecific antibodies for all four individual cellulases, the role and function of the individual cellulases can thus be studied in greater detail.     

Investigation of the protective effects of crocin on acrylamide induced small and large intestine damage in rats

We investigated repair of acrylamide (AA) induced damage in intestines by administration of crocin. We used 40 male Wistar rats in four groups of 10 animals: control, AA, crocin, and AA + crocin groups. We investigated biochemical and histological changes to small and large intestine. AA ingestion decreased glutathione (GSH) levels and total antioxidant status (TAS) in the intestine compared to the control group, while superoxide dismutase (SOD) and catalase (CAT) activities, and total oxidant status (TOS) and malondialdehyde (MDA) levels were increased. Villi were shortened and villus degeneration was observed in ileum of the AA group. Degeneration of surface epithelium and Liberkühn crypts were observed in colon sections. GSH and TAS levels increased after administration of AA together with crocin, while SOD and CAT levels and TOS and MDA levels decreased; significant recovery of histological damage also was observed. We found that crocin exhibits protective effects on AA induced small and large intestine damage by inhibiting oxidative stress.     

Decreased brain dopamine synthesis rate and increased [3H]spiroperidol binding in streptozotocin-diabetic rats

The rate of accumulation of 3,4-dihydroxy-phenylalanine following decarboxylase inhibition and of homovanillic acid following probenecid treatment were significantly decreased in streptozotocin-diabetic rats. These changes were observed in both the striatum and limbic forebrain. The Bmax for [3H]spiroperidol receptor binding was significantly increased in both brain regions. All of these neurochemical changes were reversed by insulin replacement therapy. Whether these neurochemical changes are attributable to chronic hyperglycemia or some other aspect of the diabetic state is not known.