Reproducible high yield sequencing of proteins electrophoretically separated and transferred to an inert support

A method allowing initial sequencing yields of 60-85% to be consistently obtained from samples prepared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer is described in detail. Conducting electrophoresis at a pH near neutrality is the single most important of the modifications made to earlier procedures, but pre-electrophoresis in the presence of glutathione or sodium thioglycolate and use of Immobilon polyvinylidene difluoride membranes all contribute to the success of the technique. When tryptophan was the NH2 terminus of a protein, the phenylthiohydantoin (PTH)-derivative recovered appeared to be an irreversible oxidation product if pre-electrophoresis was not performed. Following pre-electrophoresis, the PTH-derivative recovered co-migrated with that of unmodified tryptophan, and the recovery was higher. Recovery of methionine as its PTH-derivative was not affected by pre-electrophoresis suggesting that thioglycolate in the electrophoresis buffer during sample separation prevented or reversed oxidation of methionine sulfur but did not protect tryptophan.     

Structure of two human beta-actin-related processed genes one of which is located next to a simple repetitive sequence.

From a human gene library we have isolated and sequenced a beta-actin-like pseudogene, H beta Ac-psi 2, which lacks intervening sequences and contains several mutations resulting in frame-shifts, stop codons and in a departure from the known beta-actin protein sequence. We have also extended our sequence work on the intronless human beta-actin-related pseudogene H beta Ac-psi 1 described previously and we find that both genes are processed genes ending in a poly(dA) tract and flanked by direct repeats. The gene H beta Ac-psi 2 is preceded by a 230-bp region in which the simple sequence 5'-GAAA-3' is repeated greater than 40 times. This satellite-like sequence is highly repetitive in the human genome.     

Blood–Brain Barrier Permeability to Sodium. Modification by Glucose or Insulin?

In order to explore the pathogenetic mechanism underlying the changes in blood-brain barrier sodium transport in experimental diabetes, the effects of hyperglycemia and of hypoinsulinemia were studied in nondiabetic rats. In untreated diabetes, the neocortical blood-brain barrier permeability for sodium decreased by 20% (5.6 +/- 0.7 versus 7.0 +/- 0.8 X 10(5) ml/g/s) as compared to controls. Intravenous infusion of 50% glucose for 2 h was associated with a decrease in the blood-brain barrier permeability to sodium (5.4 +/- 1.2 X 10(5) ml/g/s), whereas rats treated with an inhibitor of insulin-secretion (SMS 201-995, a somatostatin-analogue) had normal sodium permeability (7.3 +/- 2.0 X 10(5) ml/g/s). Acute insulin treatment of diabetic rats normalized the sodium permeability within a few hours as compared to a separate control group (7.7 +/- 1.1 versus 6.9 +/- 1.4 X 10(5) ml/g/s). To elucidate whether the abnormal blood-brain barrier passage is caused by a metabolic effect of glucose or by the concomitant hyperosmolality, rats were made hyperosmolar by intravenous injection of 50% mannitol. Although not statistically significant, blood-brain barrier sodium permeability increased in hyperosmolar rats as compared to the control rats (8.3 +/- 1.0 and 7.0 +/- 1.9 X 10(5) ml/g/s, respectively). It is concluded that either hyperglycemia per se or a glucose metabolite is responsible for the blood-brain barrier abnormality which occurs in diabetes. Further, we suggest that the specific decrease of sodium permeability could be the result of glucose-mediated inhibition of the Na+K+-ATPase localized at the blood-brain barrier.     

Cell Proliferation Inhibition in Reduced Gravity

Extended durations of spaceflight have been shown to be deleterious on an organismic level; however, mechanisms underlying cellular sensitivity to the gravitational environment remain to be elucidated. The majority of the gravitational studies to date indicates that cell regulatory pathways may be influenced by their gravitational environment. Still, few cell biology experiments have been performed in space flight and even fewer experiments have been repeated on subsequent flights. With flight opportunities on STS-50, 542 and 57, Sf9 cells were flown in the BioServe Fluids Processing Apparatus and cell proliferation was measured with and without exposure to a cell regulatory sialogycopeptide (CeReS) inhibitor. Results from these flights indicate that the Sf9 cells grew comparable to ground controls, that the CeReS inhibitor bound to its specific receptor, and that its signal transduction cascade was not gravity sensitive.     

The effect of acetic acid and specific growth rate on acetic acid tolerance and trehalose content of Saccharomyces cerevisiae

Summary The effects of acetic acid and specific growth rate on acetic acid tolerance and trehalose content of Saccharomyces cerevisiae CBS 2806 were studied using anaerobic chemostat cultures. Cells grown in the presence of acetic acid at a defined specific growth rate showed a higher acetic acid tolerance and a slightly lower trehalose content. Cells grown at a low specific growth rate showed a lower energy demand, a higher acetic acid tolerance, and a higher trehalose content. These results indicate that trehalose plays a growth rate dependent role in the tolerance of S. cerevisiae to acetic acid.     

Separation of 5'-ribonucleoside monophosphates by ion-pair reverse-phase high-performance liquid chromatography

We have developed computer programs for characterization of ligand-binding systems in terms of continuous affinity distributions of arbitrary shape based on a numerical finite difference method. This method provides an excellent initial estimate of the affinity distribution, which can be further refined by means of nonlinear least-squares curve fitting. The method has been extensively tested for several cases including receptor heterogeneity, cooperativity, and for several examples of experimental design (e.g., ligand concentrations), and various levels of random and systematic experimental errors. The results provide a guide to experimental design, and indicate limits to the resolution obtained by ligand-binding studies, irrespective of the method of analysis.     

Purification and partial characterization of the 17 kDa sperm coating protein from boar seminal plasma.

Sperm coating proteins of 16, 17, and 19 kDa have been purified from boar seminal plasma. The 17 kDa protein has been identified as an antigen recognized by monoclonal antibody ACR.3 and is thus identical to low molecular mass zona pellucida binding protein from boar spermatozoa (Moos et al., 1990). The 17 and 19 kDa proteins are glycosylated and tend to form hetero-complexes. The 17 kDa ACR.3 antigen is sequentially released from the sperm cell surface during capacitation and, after induction of the acrosome reaction, the 16 kDa form was also observed. Immunocytochemical studies on boar reproductive tissues have suggested that the seminal vesicle epithelium may be the source of these proteins.     

The role of a cell surface inhibitor in early signal transduction associated with the regulation of cell division and differentiation

Serum stimulation of quiescent human fibroblast cultures resulted in a hyperphosphorylation of the nuclear retinoblastoma gene susceptibility product (RB). However, serum stimulation in the presence of 9 x 10(-8) M of a purified bovine sialoglycopeptide (SGP) cell surface inhibitor abrogated the hyperphosphorylation of the RB protein and the subsequent progression of cells through the mitotic cycle. The experimental results suggest that the SGP mediated its cell cycle arrest at a site in the cell cycle that was at the time of RB phosphorylation or somewhat upstream of the modification of this regulatory protein of cell division. Both cells serum-deprived and serum stimulated in the presence of the SGP displayed only a hypophosphorylated RB protein, consistent with the SGP-mediated cell cycle arrest point being near the G1/S interface.     

Pyrophosphate contamination in crystalline phosphocreatine: a source of error in kinetic studies using coupled enzymes.

Commercial crystalline phosphocreatine generally contains a contaminant, probably pyrophosphate, that can inhibit nucleoside triphosphatases and may therefore cause errors in steady-state kinetic studies of creatine kinase-coupled r reactions. Ion-exchange chromatography reveals the presence of pyrophosphate in some batches of phosphocreatine sufficient to account for the observed inhibition. Chromatographically purified phosphocreatine shows no inhibitory effect.