Membrane protein synthesis in Micrococcus lysodeikticus and selective effect of chloramphenicol.

Micrococcus lysodeikticus cytoplasmic membranes labeled with ]-14C]arginine plus [-14C]-threonine were prepared and subjected to mild washing treatments to fractionate membrane proteins. Polyacrylamide gel electrophoresis of total membranes, in the presence of sodium dodecyl sulfate, results in the separation of 28-30 bands of labeled protein. Three peaks of protein show higher specific radioactivity than the others. Chloramphenicol at 100 mug/ml inhibits the incorporation of labeled precursors into membrane proteins by 45-70 percent, some of them being more affected by the antibiotic. From all available results, we suggest that the partial inhibitory effect shown by this antibiotic could be due to the existence of specific biosynthetic sites for some membrane proteins, which are differently affected by chloramphenicol.     

Post-thawing survival of motile ram sperm after isolation by layering on protein columns

Post-thawing survival of ram sperm was examined after semen which had been layered on top of isolation columns containing solutions of bovine serum albumin (BSA) in Tris diluent was processed for freezing by the pellet method. Sperm isolated from the bottom of the BSA column had better post-thawing survival than sperm from the top of the column. The efficiency of sperm isolation was affected by the concentration of BSA in the column, the holding time of semen on the column and the concentration of sperm in the layered semen. The best post-thawing survival of sperm occurred when semen diluted to a concentration of 200 x 10(6) sperm/ml was layered on a column of 6% BSA in Tris diluent and the bottom layer of the column was isolated for freezing after two hours' holding time.     

Effect of monensin on osmotic water flow across the toad bladder and its stimulation by vasopressin and cyclic AMP

Summary The effects of the sodium ionophore monensin on osmotic water flow across the urinary bladder of the toadBufo marinus were studied. Monensin alone did not alter osmotic water flow; however, the ionophore inhibited the hydrosmotic response to vasopressin and cyclic AMP in a dose-dependent manner. The inhibitory effects of monensin were apparent when the ionophore was added to the serosal bathing solution but not when it was added to the mucosal bathing solution. The inhibitory effect of serosal monensin required the presence of sodium in the serosal bathing solution but not the presence of calcium in the bathing solutions. Thus, it appears that intracellular sodium concentration is a regulator of the magnitude of the hydrosmotic response to vasopressin and cyclic AMP.     

Effect of ACE inhibition on the fetal kidney: decreased renal blood flow.

The kidneys of nine fetuses whose mothers were chronically hypertensive were examined microscopically. Three of these mothers used antihypertensive agents throughout pregnancy including one who used an angiotensin-converting enzyme (ACE) inhibitor. The tubular defects found in these kidneys were compared to the kidneys of 20 normal controls, 13 fetuses with various multiple malformation syndromes and six cases of the twin to twin transfusion syndrome. Evidence from these cases as well as the literature suggest that the primary mechanism by which ACE inhibitors affect development of the fetal kidney is through decreased renal blood flow.     

Histidine ligand variants of a flavo-diiron protein: effects on structure and activities

Flavo-diiron proteins (FDPs) contain non-heme diiron and proximal flavin mononucleotide (FMN) active sites and function as terminal components of a nitric oxide reductase (NOR) and/or a four-electron dioxygen reductase (O₂R). While most FDPs show similar structural, spectroscopic, and redox properties, O₂R and NOR activities vary significantly among FDPs. A potential source of this variability is the iron ligation status of a conserved His residue that provides an iron ligand in all known FDP structures but one, where this His residue is rotated away from iron and replaced by a solvent ligand. In order to test the effect of this His ligation status, we changed this ligating His residue (H90) in Thermotoga maritima (Tm) FDP to either Asn or Ala. The wild-type Tm FDP shows significantly higher O₂R than NOR activity. Single crystal X-ray crystallography revealed a remarkably conserved diiron site structure in the H90N and ?A variants, differing mainly by either Asn or solvent coordination, respectively, in place of H90. The steady-state activities were minimally affected by the H90 substitutions, remaining significantly higher for O₂R versus NOR. The pre-steady-state kinetics of the fully reduced FDP with O₂ were also minimally affected by the H90 substitutions. The results indicate that the coordination status of this His ligand does not significantly modulate the O₂R or NOR activities, and that FDPs can retain these activities when the individual iron centers are differentiated by His ligand substitution. This differentiation may have implications for the O₂R and NOR mechanisms of FDPs.     

The new higher level classification of eukaryotes with emphasis on the taxonomy of protists

This revision of the classification of unicellular eukaryotes updates that of Levine et al. (1980) for the protozoa and expands it to include other protists. Whereas the previous revision was primarily to incorporate the results of ultrastructural studies, this revision incorporates results from both ultrastructural research since 1980 and molecular phylogenetic studies. We propose a scheme that is based on nameless ranked systematics. The vocabulary of the taxonomy is updated, particularly to clarify the naming of groups that have been repositioned. We recognize six clusters of eukaryotes that may represent the basic groupings similar to traditional "kingdoms." The multicellular lineages emerged from within monophyletic protist lineages: animals and fungi from Opisthokonta, plants from Archaeplastida, and brown algae from Stramenopiles.     

Derived amino acid sequence and identification of active site residues of Escherichia coli beta-hydroxydecanoyl thioester dehydrase

The nucleotide sequence of the fabA gene encoding beta-hydroxydecanoyl thioester dehydrase, a key enzyme of the unsaturated fatty acid synthesis pathway of Escherichia coli, has been determined by the dideoxynucleotide sequencing technique. Most of the sequence was obtained by sequencing intragenic insertions of the transposon, Tn1000, isolated in vivo. A synthetic primer complementary to a portion of the inverted repeat sequences at the ends of the transposon was used to prime DNA synthesis into the flanking fabA sequences. The gene is composed of 516 nucleotides (171 amino acid residues) encoding a protein with a molecular weight of 18,800. Approximately half of the derived amino acid sequence was confirmed by automated Edman sequencing of peptides obtained by cyanogen bromide cleavage. The active site histidine residue (His-70) has been identified by analysis of the peptides labeled by reaction with 14C-labeled 3-decynoyl-N-acetylcysteamine, a specific mechanism-activated inhibitor. A cysteine residue (Cys-69) adjacent to the active site histidine may play the role in catalysis previously assigned to a tyrosine residue. We also report a simplified purification process for the dehydrase beginning with extracts of a brain which greatly overproduces the enzyme.     

Quantifying apoprotein synthesis in rodents: coupling LC-MS/MS analyses with the administration of labeled water

Stable isotope tracer studies of apoprotein flux in rodent models present difficulties as they require working with small volumes of plasma. We demonstrate the ability to measure apoprotein flux by administering either (2)H- or (18)O-labeled water to mice and then subjecting samples to LC-MS/MS analyses; we were able to simultaneously determine the labeling of several proteolytic peptides representing multiple apoproteins. Consistent with relative differences reported in the literature regarding apoprotein flux in humans, we found that the fractional synthetic rate of apoB is greater than apoA1 in mice. In addition, the method is suitable for quantifying acute changes in protein flux: we observed a stimulation of apoB production in mice following an intravenous injection of Intralipid and a decrease in apoB production in mice treated with an inhibitor of microsomal triglyceride transfer protein. In summary, we demonstrate a high-throughput method for studying apoprotein kinetics in rodent models. Although notable differences exist between lipoprotein profiles that are observed in rodents and humans, we expect that the method reported here has merit in studies of dyslipidemia as i) rodent models can be used to probe target engagement in cases where one aims to modulate apoprotein production and ii) the approach should be adaptable to studies in humans.