Purification and properties of a methionine formylmethionyl-tRNA-binding initiation factor from pig liver

(i) A factor, EIF-2, that binds methionyl-tRNA_f^Met in the presence of GTP has been isolated from pig liver. (ii) Dodecylsulfate-gel electrophoresis and sedimentation equilibrium centrifugation indicate that the factor has a molecular weight of 122,000 and that it consists of three unequal subunits. (iii) The apparent K_D for binding of methionyl-tRNA_f^Met varies with factor concentration. GTP participates in the binding with a K_D of 0.5 μm. β,γ-Methylene-guanosine triphosphate supports 40% of the binding observed with GTP. GDP is a competitive inhibitor with a K_i of 0.2 μm. The optimal, free Mg²⁺ concentration is approximately 50 μm. GTP and Mg²⁺ stabilize the factor against thermal inactivation and inactivation by N-ethyl maleimide. (iv) The factor is required for the formation of a sucrose gradient-stable complex between methionyl-tRNA_f^Met and the 40S ribosomal subunit. The presence of template is not necessary, but poly(A,U,G) increases the binding observed 1.5-fold. (v) The factor markedly stimulates synthesis in a reconstituted protein-synthesizing system with globin messenger RNA as template.     

Rapid two-step procedure for large-scale purification of pediocin-like bacteriocins and other cationic antimicrobial peptides from complex culture medium

A rapid and simple two-step procedure suitable for both small- and large-scale purification of pediocin-like bacteriocins and other cationic peptides has been developed. In the first step, the bacterial culture was applied directly on a cation-exchange column (1-ml cation exchanger per 100-ml cell culture). Bacteria and anionic compounds passed through the column, and cationic bacteriocins were subsequently eluted with 1 M NaCl. In the second step, the bacteriocin fraction was applied on a low-pressure, reverse-phase column and the bacteriocins were detected as major optical density peaks upon elution with propanol. More than 80% of the activity that was initially in the culture supernatant was recovered in both purification steps, and the final bacteriocin preparation was more than 90% pure as judged by analytical reverse-phase chromatography and capillary electrophoresis.     

A PATO-compliant zebrafish screening database (MODB): management of morpholino knockdown screen information

Background  

The zebrafish is a powerful model vertebrate amenable to high throughput in vivo genetic analyses. Examples include reverse genetic screens using morpholino knockdown, expression-based screening using enhancer trapping and forward genetic screening using transposon insertional mutagenesis. We have created a database to facilitate web-based distribution of data from such genetic studies.     

Characterization of the FAM110 gene family

We have previously characterized the centrosome/spindle pole-associated protein (CSPP) involved in cell cycle progression. The open reading frame C20orf55 was identified in a yeast two-hybrid screen in a search for CSPP-interacting proteins. A homology search revealed that C20orf55 belongs to a gene family consisting of three members that have not yet been described. The HUGO Nomenclature Committee has assigned these genes the names FAM110A-FAM110C. Studies of transfectants showed that the FAM110 proteins localized to centrosomes and accumulated at the microtubule organization center in interphase and at spindle poles in mitosis. In addition, overexpression of FAM110C induced microtubule aberrancies. Our data also indicated a cell cycle-regulated expression of FAM110A. Moreover, ectopic expression of FAM110B and FAM110C proteins impaired cell cycle progression in G1 phase. To summarize, we have characterized a novel family of genes encoding proteins with distinct conserved motifs, of which all members localize to centrosomes and spindle poles.     

Mutational effects on conformational changes of the dephospho- and phospho-forms of the Na+,K+-ATPase

Gly263 of the rat kidney Na(+),K(+)-ATPase is highly conserved within the family of P-type ATPases. Mutants in which Gly263 or the juxtaposed Arg264 had been replaced by alanine were expressed at high levels in COS-1 cells and characterized functionally. Titrations of Na(+),K(+), ATP, and vanadate dependencies of Na(+),K(+)-ATPase activity showed changes in the apparent affinities relative to wild-type compatible with a displacement of the E(1)-E(2) conformational equilibrium in favor of E(1). The level of the K(+)-occluded form was reduced in the Gly263-->Ala and Arg264-->Ala mutants, and the rate constant characterizing deocclusion of K(+) or Rb(+) was increased as much as 20-fold in the Gly263-->Ala mutant. Studies of the sensitivity of the phosphoenzyme to K(+) and ADP showed a displacement of the E(1)P-E(2)P equilibrium of the phosphoenzyme in favor of E(1)P, and dephosphorylation experiments carried out at 25 degrees C on a millisecond time scale using a quenched-flow technique demonstrated a reduction of the E(1)P to E(2)P conversion rate in the mutants. Hence, the mutations displaced the conformational equilibria of dephosphoenzyme and phosphoenzyme in parallel in favor of the E(1) and E(1)P forms. The observed effects were more pronounced in the Gly263-->Ala mutant compared with the Arg264-->Ala mutant. Leu332 mutations that likewise displaced the conformational equilibria in favor of E(1) and E(1)P were also studied. Unlike the Gly263-->Ala mutant the Leu332 mutants displayed a wild-type like rate of K(+) deocclusion. Thus, the effect of the Gly263 mutation on the E(1)-E(2) conformational equilibrium seems to be caused mainly by an acceleration of the K(+)-deoccluding step, whereas in the Leu332 mutants the rate of the reverse reaction seems to be reduced.     

Interrelations between pulmonary liquid volumes and lung compliance.

We have investigated the relative effects of lung edema and of increases in pulmonary blood volume (PBV) on lung compliance (CL), and also the effects of selective elevations of pulmonary arterial (Ppa) and left atrial (Pla) pressures on PBV and on CL, using an isolated, perfused, and ventilated rabbit lung preparation. Lung weight was continuously recorded. A step rise in Pla at constant flow caused a rapid rise in PBV accompanied by an immediate fall in CL. With maintained high vascular pressures interstitial edema accumulated with no further fall in CL. Not until 3 times the normal amount of extra-vascular fluid had accumulated did a further, secondary reduction in CL occur. When Ppa was elevated to the same level by 1) a rise in flow and 2) a rise in Pla, the latter type of experiment gave 3-5 times larger increases in PBV. Pla elevations with or without rise in Ppa (flow adjusted) gave almost the same rises in PBV. The fall in CL was related to rises in PBV regardless of how such rises were obtained. Our conclusion is that increases in PBV, but not accumulation of interstitial edema, reduced CL in this preparation.