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.     

Heat generation in laser irradiated tissue

Many medical applications involving lasers rely upon the generation of heat within the tissue for the desired therapeutic effect. Determination of the absorbed light energy in tissue is difficult in many cases. Although UV wavelengths of the excimer laser and 10.6 microns wavelength of the CO2 laser are absorbed within the first 20 microns of soft tissue, visible and near infrared wavelengths are scattered as well as absorbed. Typically, multiple scattering is a significant factor in the distribution of light in tissue and the resulting heat source term. An improved model is presented for estimating heat generation due to the absorption of a collimated (axisymmetric) laser beam and scattered light at each point r and z in tissue. Heat generated within tissue is a function of the laser power, the shape and size of the incident beam and the optical properties of the tissue at the irradiation wavelength. Key to the calculation of heat source strength is accurate estimation of the light distribution. Methods for experimentally determining the optical parameters of tissue are discussed in the context of the improved model.     

Solution structure of an unusually stable RNA tetraplex containing G- and U-quartet structures.

A model for the solution structure of an RNA tetraplex, (rUGGGGU)4, has been obtained by two-dimensional NMR spectroscopy and molecular dynamics. The molecule is parallel stranded and Hoogsteen base-paired in 50 mM KCl, and it is so stable that three of its six imino protons have exchange half-lives measured in days at 40 degrees C. The tetraplex is stabilized by base stacking and by the hydrogen bonds in four G quartets and at least one U quartet. This is the first indication of the existence of U-quartet structures of which we are aware.     

Lamellar stacking in three-dimensional crystals of Ca(2+)-ATPase from sarcoplasmic reticulum.

Electron microscopy of multilamellar crystals of CA(2+)-ATPase currently offers the best opportunity for obtaining a high-resolution structure of this ATP-driven ion pump. Under certain conditions small, wormlike crystals are formed and provide views parallel to the lamellar plane, from which parameters of lamellar stacking can be directly measured. Assuming that molecular packing is the same, data from these views could supplement those obtained by tilting large, thin platelike crystals. However, we were surprised to discover that the lamellar spacing was variable and depended on the amount of glycerol present during crystallization (20% versus 5%). Projection maps (h,0,l) from these womklike crystals suggest different molecular contacts that give rise to the different lamellar spacings. Based on an orthogonal projection map (h,k,0) from collapsed, wormlike crystals and on x-ray powder patterns, we conclude that molecular packing within the lamellar plane is the same as that in thin, platelike crystals and is unaffected by glycerol. Finally, the orientation of molecules in the lamellar plane was characterized from freeze-dried, shadowed crystals. Comparing the profile of molecules in these multilamellar crystals with that previously observed in helical tubes induced by vanadate gives structural evidence of the conformational change that accompanies binding of calcium of Ca(2+)-ATPase.     

Assessment of cytoplasmic effects on the development of mouse embryonic nuclei transferred to enucleated zygotes

In this study, cytoplasmic effects on the development of nuclear transplant embryos were examined. In addition, the production of offspring from nuclear transplant embryos was attempted. Nuclei from cleavage-stage embryos were transplanted to enucleated zygotes at different cell cycle stages and with different cytoplasmic volumes. A greater developmental rate to the blastocyst stage was observed in reconstituted late stage zygotes that received nuclei from late 2-cell stage embryos than in early stage zygotes (46.3% vs. 16.9%). A further increase in developmental rate to the blastocyst stage (85.5%) and in cell number was obtained in reconstituted late stage zygotes with reduced cytoplasmic volume. However, developmental potential of nuclei from 4- and 8-cell stage embryos was very limited, although they were transferred to enucleated late stage zygotes with reduced cytoplasm. After the transfer of blastocysts derived from nuclear transplant embryos to recipient females, live young were obtained from reconstituted embryos that received nuclei from late 2-cell stage embryos (28.6%). These results confirm that the development of nuclear transplant embryos can be affected by recipient cell cycle stage and cytoplasmic volume. Furthermore, the nuclei from late 2-cell stage embryos in which activation of the embryonic genome had occurred can be reprogrammed to a certain extent when transplanted into enucleated zygotes, especially late stage zygotes with reduced cytoplasmic content.     

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.     

Genetics of glucosephosphate isomerase in Aedes togoi (Diptera: Culicidae)

The genetics of glucosephosphate isomerase (E.C. 5.3.1.9) in two strains (Malaysian and Taiwan) of Aedes togoi is reported. Three electrophoretic phenotypes were present in both sexes. The zymogram patterns were identical in both strains of A. togoi. The phenotypes were governed by a pair of codominant alleles. The allele frequency of the slow-moving band was 0.63 in the Malaysian strain and was 0.86 and 0.82 in F161 and F169 generations, respectively, of the Taiwan strain. The sample studied was in good accord with Hardy-Weinberg expectations.     

Isolation and characterization of a reducing polyketide synthase gene from the lichen-forming fungus Usnea longissima

The reducing polyketide synthases found in filamentous fungi are involved in the biosynthesis of many drugs and toxins. Lichens produce bioactive polyketides, but the roles of reducing polyketide synthases in lichens remain to be clearly elucidated. In this study, a reducing polyketide synthase gene (U1PKS3) was isolated and characterized from a cultured mycobiont of Usnea longissima. Complete sequence information regarding U1PKS3 (6,519 bp) was obtained by screening a fosmid genomic library. A U1PKS3 sequence analysis suggested that it contains features of a reducing fungal type I polyketide synthase with β-ketoacyl synthase (KS), acyltransferase (AT), dehydratase (DH), enoyl reductase (ER), ketoacyl reducatse (KR), and acyl carrier protein (ACP) domains. This domain structure was similar to the structure of ccRadsl, which is known to be involved in resorcylic acid lactone biosynthesis in Chaetomium chiversii. The results of phylogenetic analysis located U1PKS3 in the clade of reducing polyketide synthases. RT-PCR analysis results demonstrated that UIPKS3 had six intervening introns and that UIPKS3 expression was upregulated by glucose, sorbitol, inositol, and mannitol.     

Expression of the IL-2 gene in the potato (Solanum tuberosum cv. Superior)

We intended to examine the expression of the T-cell growth factor (human interleukin-2) so that a binary vector, pSSK-1, carrying the IL-2 gene was constructed and transferred intoA. tumefaciens for the purpose of the transformation of the potato (Solanum tuberosum cv. Superior). All of theAgrobacterium-infected potato explants were regenerated to shoots in modified MS medium and 81% of them rooted on the medium containing kanamycin (200 mg/L). Southern and Northern analysis were performed to verify the transformation events. EL-ISA test indicated that IL-2 protein was produced from IL-2-transformed potatoes. These results suggested expression and production of the IL-2 protein from the transgenic potato.