Molecular substructure of a viral receptor-recognition protein. The gp17 tail-fiber of bacteriophage T7

The bacteriophage T7 tail complex consists of a conical tail-tube surrounded by six kinked tail-fibers, which are oligomers of the viral protein gp17 (Mr 61,400). We have derived a molecular model for the tail-fiber by integrating secondary structure predictions with ultrastructural information obtained by correlation averaging of electron micrographs of negatively stained tail complexes. This model has been further refined by high-resolution scanning transmission electron microscopy of purified fibers, both negatively stained and unstained. Mass measurements made from the latter images establish that the fiber is a trimer of gp17. The proximal half-fiber is a uniform rod, about 2.0 nm in diameter and 16.4 nm long, which we infer to be a triple-stranded coiled-coil, containing three copies of an alpha-helical domain of about 117 residues, starting at Phe151. The distal half-fiber is 15.5 nm long, and is made up of four globules, 3.1 to 4.8 nm in diameter, in rigid linear array: it contains the carboxy-terminal halves (residues approximately 268 to 553) of the constituent gp17 chains, arranged with 3-fold symmetry around its long axis. The amino-terminal domains (residues 1 to 149) link the fiber to the tail-tube. We conclude that the three gp17 chains are quasi-equivalent in the proximal half-fiber, equivalent in the distal half-fiber, and non-equivalent in the kink region that separates the two half-fibers: such localized non-equivalence may represent a general mechanism for the formation of kinked joints in segmented homo-oligomeric proteins.     

Lipopolysaccharide-induced NF-kappa B activation in mouse 70Z/3 pre-B lymphocytes is inhibited by mevinolin and 5''-methylthioadenosine: roles of protein isoprenylation and carboxyl methylation reactions.

We show that both the lipopolysaccharide (LPS)-induced activation of NF-kappa DNA binding and kappa gene expression are blocked by treating murine pre-B lymphocyte 70Z/3 cells with 5'-methylthioadenosine (MTA), an inhibitor of several S-adenosylmethionine-dependent methylation reactions. We further show that the LPS-induced incorporation of radioactivity from [methyl-3H]methionine into methyl ester-like linkages on a group of membrane polypeptides is also inhibited by MTA treatment, suggesting the involvement of protein methylation reactions in the LPS signal transduction pathway. We also find that NF-kappa B and kappa gene activation in LPS-treated 70Z/3 cells is blocked by mevinolin, an inhibitor that prevents protein isoprenylation. Interestingly, mevinolin-treated cells also exhibited a marked reduction in the methylation of membrane proteins. Neither MTA nor mevinolin significantly inhibited NF-kappa B activation by phorbol myristate acetate, suggesting that these agents act early in signal transduction. These results provide the first evidence that carboxyl methylated and/or isoprenylated proteins play an essential role in the LPS-signaling pathway.     

四川省西部鱼类寄生粘孢子虫——1.粘体虫属六新种(粘孢子纲:双壳目)

本文报导四川省西部鱼类寄生粘孢子虫粘体虫属六新种,即异型粘体虫,新种Myxosoma disparis sp.nov.,四川粘体虫,新种Myxosoma sichuanensis sp.nov.,光唇粘体虫,新种Myxosoma acrossochilusi sp.nov.鳅粘体虫,新种Myxosoma nemachilusi sp.nov.斜囊粘体虫,新种Myxosoma obliqua sp.nov.,雅安粘体虫,新种Myxosoma yaanensis sp.nov.。     

Conformational analysis of 16 S ribosomal RNA from Escherichia coli by scanning transmission electron microscopy

Digitized images of molecules of 16 S rRNA from Escherichia coli, obtained by scanning transmission electron microscopy (STEM), provide quantitative structural information that is lacking in conventional electron micrographs. We have determined the morphology, total molecular mass, mass distribution within individual rRNA molecules and apparent radii of gyration. From the linear density (M/L) we have assessed the number of strands in the structural backbone of rRNA and studied the pattern of branching and folding related to the secondary and tertiary structure of rRNAs under various buffer conditions. Even in reconstitution buffer 16 S RNA did not show any resemblance to the native 30 S subunit.     

Developmental competence of domestic cat follicular oocytes after fertilization in vitro

Empirical evaluation of variables affecting oocyte collection, in vitro fertilization, and embryo transfer resulted in establishing a successful procedure for the artificial production of offspring in the domestic cat. Female cats were treated with pregnant mare's serum gonadotropin (PMSG, 150 IU) followed 72 or 80 h later with 100 or 200 IU human chorionic gonadotropin (hCG). After laparoscopic collection, follicular oocytes were inseminated in vitro with ejaculated, processed spermatozoa, cultured (37 degrees C, 5% CO2), and then examined for evidence of fertilization. Two- to 4-cell stage embryos were transferred to the oviducts of oocyte donors. Oocyte donor cats and naturally mated controls also were subjected to sequential laparoscopic examinations and blood sampling to assess corpora lutea (CL) function. At 24-30 h of culture, fewer (p less than 0.001) degenerate oocytes were observed in cats receiving 100 IU hCG (8.2%) compared to those receiving 200 IU (20.6%), regardless of the PMSG-hCG interval. Overall fertilization (48.1%) and cleavage (45.2%, at 30 h post-insemination) rates were greatest following an 80-h PMSG-hCG interval combined with the 100 IU hCG dose. Five of the 6 cats receiving 6 to 18 embryos became pregnant and produced from 1 to 4 kittens/litter. Gonadotropin-treated females subjected to follicular aspiration produced morphologically normal CL and circulating progesterone patterns that were qualitatively similar (p greater than 0.05) to control cats. These data indicate that domestic cat follicular oocytes are capable of fertilization in vitro, but success is dependent on both the timing and dose of the hCG stimulus. Follicles subjected to aspiration appear capable of forming normal, functional CL and the birth of live young after embryo transfer unequivocally demonstrates, for the first time, the developmental competence of in vitro-fertilized carnivore oocytes.     

Intersubunit transfer of fatty acyl groups during fatty acid reduction

Fatty acid reduction in Photobacterium phosphoreum is catalyzed in a coupled reaction by two enzymes: acyl-protein synthetase, which activates fatty acids (+ATP), and a reductase, which reduces activated fatty acids (+NADPH) to aldehyde. Although the synthetase and reductase can be acylated with fatty acid (+ATP) and acyl-CoA, respectively, evidence for acyl transfer between these proteins has not yet been obtained. Experimental conditions have now been developed to increase significantly (5-30-fold) the level of protein acylation so that 0.4-0.8 mol of fatty acyl groups are incorporated per mole of the synthetase or reductase subunit. The acylated reductase polypeptide migrated faster on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than the unlabeled polypeptide, with a direct 1 to 1 correspondence between the moles of acyl group incorporated and the moles of polypeptide migrating at this new position. The presence of 2-mercaptoethanol or NADPH, but not NADP, substantially decreased labeling of the reductase enzyme, and kinetic studies demonstrated that the rate of covalent incorporation of the acyl group was 3-5 times slower than its subsequent reduction with NADPH to aldehyde. When mixtures of the synthetase and reductase polypeptides were incubated with [3H] tetradecanoic acid (+ATP) or [3H]tetradecanoyl-CoA, both polypeptides were acylated to high levels, with the labeling again being decreased by 2-mercaptoethanol or NADPH. These results have demonstrated that acylation of the reductase represents an intermediate and rate-limiting step in fatty acid reduction. Moreover, the activated acyl groups are transferred in a reversible reaction between the synthetase and reductase proteins in the enzyme mechanism.     

Ammonium uptake in Rhodopseudomonas capsulata

Investigations of the uptake of ammonium (NH ₄ ⁺ ) by Rhodopseudomonas capsulata B100 supported the presence of an NH ₄ ⁺ transport system. Experimentally NH ₄ ⁺ was determined by electrode or indophenol assay and saturation kinetics were observed with two apparent K _m's of 1.7 M and 11.1 M (pH 6.8, 30°) and a V _max at saturation of 50–60 nmol/min·mg protein. The optimum pH and temperature were 7.0 and 33° C, respectively. The Q₁₀ quotient was calculated to be 1.9 at 100 M NH ₄ ⁺ , indicating enzymatic involvement. In contrast to the wild type, B100, excretion of NH ₄ ⁺ , not uptake, was observed in a glutamine auxotroph, R. capsulata G29, which is derepressed for nitrogenase and lacks glutamine synthetase activity. G29R1, a revertant of G29, also took up NH ₄ ⁺ at the same rate as wild type and had fully restored glutamine synthetase activity. Partially restored derivatives, G29R5 and G29R6, grew more slowly than wild type on NH ₄ ⁺ as the nitrogen source, remained derepressed for nitrogenase in the presence of NH ₄ ⁺ , and displayed rates of NH ₄ ⁺ uptake in proportion to their glutamine synthetase activity. Ammonium uptake and glutamine synthetase activity were also restored in R. capsulata G29 exconjugants which had received the plasmid pPS25, containing the R. capsulata glutamine synthetase structural gene. These data suggest that NH ₄ ⁺ transport is tightly coupled to assimilation.Abbreviations used CHES cyclohexylaminoethanesulfonic acid - GS glutamine synthetase - SDS sodium dodecylsulfate     

Effect of computer use in the consultation on the delivery of care.

The effects of the use of a computer on the delivery of care in consultations in general practice were examined. In this trial a computer system provided for the review and update of patients'' medical histories, notes on doctor-patient contacts, and information on repeat prescribing. Thirty consultations in which the computer system was used and 30 consultations in which no computer was used were matched individually for the doctor consulted, the sex and age of the patient, and the presenting problem(s). Six independent general practitioners blind rated each consultation for the standard of care attained. A minor negative effect of computer use on the doctors'' clinical performance was found. We suggest, however, that this small effect would disappear if a computer system was used routinely.