Inhibitory Effect of Glycerin on Vibrio parahaemolyticus and Salmonella

In a study of the effect of glycerin in transport media on Vibrio parahaemolyticus and Salmonella, it was found that a concentration of 30% glycerin was highly inhibitory for V. parahaemolyticus and to a lesser degree for Salmonella. The incorporation of peptone or human feces in media did not reduce the inhibitory effect of glycerin. In media with 15% glycerin, viable counts of V. parahaemolyticus and Salmonella increased after 24 hr of incubation both in the presence and absence of feces. Due to the concurrent increase in the total bacterial count in the media containing feces, no enrichment effect was noted.     

Expression of the p80 region of bovine viral diarrhea virus and identification of specific antibodies to this recombinant protein in bovine sera.

A 917-base pair segment of the bovine viral diarrhea virus (BVDV) genome encoding part of the p80 region was cloned into plasmid Gex-2T expression vector for expression as a fusion protein with glutathione-S-transferase (GST). When the p80 and GST sequences were in the same reading frames, the resulting GST-p80 fusion protein had a molecular mass of 58 kilodaltons (kDa) in SDS-PAGE. Extracts of control E. coli carrying only the vector plasmid (Gex-2T) did not contain this new 58-kDa protein band. Mouse monoclonal antibody specific to BVDV-p80 recognized this recombinant protein. Seventy cattle sera that had an SN titer (to TGAC isolate of cytopathic BVDV) greater than 1:8 reacted with this recombinant protein in Western blots. Of 28 cattle sera that had SN titers less than or equal to 1:8, only one serum tested positive on Western blots.     

UHRF2, a Ubiquitin E3 Ligase,Acts as a Small Ubiquitin-like Modifier E3 Ligase for Zinc Finger Protein 131

Small ubiquitin-like modifier (SUMO), a member of the ubiquitin-related protein family, is covalently conjugated to lysine residues of its substrates in a process referred to as SUMOylation. SUMOylation occurs through a series of enzymatic reactions analogous to that of the ubiquitination pathway, resulting in modification of the biochemical and functional properties of substrates. To date, four mammalian SUMO isoforms, a single heterodimeric SUMO-activating E1 enzyme SAE1/SAE2, a single SUMO-conjugating E2 enzyme ubiquitin-conjugating enzyme E2I (UBC9), and a few subgroups of SUMO E3 ligases have been identified. Several SUMO E3 ligases such as topoisomerase I binding, arginine/serine-rich (TOPORS), TNF receptor-associated factor 7 (TRAF7), and tripartite motif containing 27 (TRIM27) have dual functions as ubiquitin E3 ligases. Here, we demonstrate that the ubiquitin E3 ligase UHRF2 also acts as a SUMO E3 ligase. UHRF2 effectively enhances zinc finger protein 131 (ZNF131) SUMOylation but does not enhance ZNF131 ubiquitination. In addition, the SUMO E3 activity of UHRF2 on ZNF131 depends on the presence of SET and RING finger-associated and nuclear localization signal-containing region domains, whereas the critical ubiquitin E3 activity RING domain is dispensable. Our findings suggest that UHRF2 has independent functional domains and regulatory mechanisms for these two distinct enzymatic activities.     

Continuous culture of a recombinantEscherichia coli and plasmid maintenance for tryptophan production

Summary Production of tryptophan by a temperature sensitive recombinant microorganism (Escherichia coli W3110 trpLDtrpR ^ts tna ^– (pCRT185)) was investigated. In a single-stage continous culture, at an elevated temperature, 42°C (derepressed condition), tryptophan concentration increased in an early phase of the fermentation, and then gradually decreased with time. The reduction in the production rate was mostly due to the segregation of the plasmid and subsequent increase of plasmid-free cells. However, the plasmid could be maintained stable at 37°C, with repressed condition oftrp-operon, over 200 generations. A two-stage continuous culture system, i.e. cell growth was maintained in the first stage at 37°C and gene expression was induced in the second stage at 42°C, was therefore tested to improve the performance of the fermentation system. Operation of the two-stage system showed that the plasmid stability was significantly improved, and the specific rate of tryptophan production was maintained almost constant for more than 500 hours in the second stage.     

Simian retrovirus-D serotype 1 (SRV-1) envelope glycoproteins gp70 and gp20: expression in yeast cells and identification of specific antibodies in sera from monkeys that recovered from SRV-1 infection.

The gp70 and transmembrane gp20 envelope proteins of simian retrovirus-D serotype 1 (SRV-1) were expressed in Saccharomyces cerevisiae as fusion proteins with human superoxide dismutase (SOD). Expression of the SOD-gp70 and SOD-gp20 sequences yielded fusion proteins of 52 and 29 kilodaltons, respectively. The yeast-expressed SRV-1 envelope proteins were used in an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in the sera of rhesus macaques that recovered from SRV-1. Sera from 47 of 49 such monkeys tested positive for antibodies to the SOD-gp70 fusion protein, while 45 of 49 reacted positively to SOD-gp20. None of 26 SRV-1-nonexposed monkeys tested positive in either ELISA. Monkeys immunized with the recombinant SRV-1 gp20 and gp70 proteins made good ELISA and Western blot (immunoblot) antibodies to whole SRV-1. This antibody was not neutralizing in vitro, however.     

Islet-cell Carcinoma (Zollinger—Ellison Syndrome) with Fulminating Adrenocortical Hyperfunction and Hypokalemia

The production of ACTH-like material by tumours arising in non-endocrine tissue may initiate severe adrenocortical hyperfunction. The pathogenesis and clinical and laboratory features of Cushing''s syndrome associated with such tumours are characteristic. The autonomous production by the tumour of ACTH-like material cannot be suppressed by exogenous corticoids. The onset of clinical symptoms is rapid; muscle wasting, general weakness, thirst and peripheral edema predominate, and the classical signs of Cushing''s syndrome may be absent. High levels of plasma 17-hydroxycorticosteroids and urinary 17-hydroxycorticosteroids and 17-ketosteroids, usually with normal levels of urinary aldosterone, commonly occur. Hypokalemic alkalosis unresponsive to replacement therapy may cause death. In the case reported herein, the intriguing possibility exists that two hormone-like substances were produced by the primary growth and its metastases: one, ACTH-like, to account for the adrenal hyperplasia and Cushing''s syndrome; and another, gastrin-like, giving rise to the ulcerogenic diathesis.     

Crystallization,molecular replacement solution,and refinement of tetrameric β-amylase from sweet potato

Sweet potato β-amylase is a tetramer of identical subunits, which are arranged to exhibit 222 molecular symmetry. Its subunit consists of 498 amino acid residues (Mr 55,880). It has been crystallized at room temperature using polyethylene glycol 1500 as precipitant. The crystals, growing to dimensions of 0.4 mm × 0.4 mm × 1.0 mm within 2 weeks, belong to the tetragonal space group P4₂2₁2 with unit cell dimensions of a = b = 129.63 Å and c = 68.42 Å. The asymmetric unit contains 1 subunit of β-amylase, with a crystal volume per protein mass (V_M) of 2.57 ų/Da and a solvent content of 52% by volume. The three-dimensional structure of the tetrameric β-amylase from sweet potato has been determined by molecular replacement methods using the monomeric structure of soybean enzyme as the starting model. The refined subunit model contains 3,863 nonhydrogen protein atoms (488 amino acid residues) and 319 water oxygen atoms. The current R-value is 20.3% for data in the resolution range of 8–2.3 Å (with 2 σ cut-off) with good stereochemistry. The subunit structure of sweet potato β-amylase (crystallized in the absence of α-cyclodextrin) is very similar to that of soybean β-amylase (complexed with α-cyclodextrin). The root-mean-square (RMS) difference for 487 equivalent Cα atoms of the two β-amylases is 0.96 Å. Each subunit of sweet potato β-amylase is composed of a large (α/β)₈ core domain, a small one made up of three long loops [L3 (residues 91–150), LA (residues 183–258), and L5 (residues 300–327)], and a long C-terminal loop formed by residues 445–493. Conserved Glu 187, believed to play an important role in catalysis, is located at the cleft between the (α/β)₈ barrel core and a small domain made up of three long loops (L3, L4, and L5). Conserved Cys 96, important in the inactivation of enzyme activity by sulfhydryl reagents, is located at the entrance of the (α/β)₈ barrel. © 1995 Wiley-Liss, Inc.