Structure of the platelet membrane glycoprotein IIb. Homology to the alpha subunits of the vitronectin and fibronectin membrane receptors

The platelet membrane glycoprotein IIb X IIIa heterodimer complex (GPIIb X IIIa) is the platelet receptor for adhesive proteins, containing binding sites for fibrinogen, von Willebrand factor, and fibronectin on activated platelets. GPIIb X IIIa also appears to be a member of a family of membrane adhesive protein receptors that plays a major role in cell-cell and cell-matrix interactions. GPIb is the larger component of this platelet receptor and is composed of two disulfide-linked subunits. In this report we describe the analysis of cDNA clones for human GPIIb that were isolated from a lambda gt11 expression library prepared using RNA from HEL cells. A total of 3.3 kilobases of cDNA was sequence, revealing a continuous open reading frame encoding both GPIIb subunits. The cDNA encodes 1039 amino acids: 137 constituting the smaller subunit, 871 constituting the larger subunit, and 30 constituting an NH2-terminal signal peptide. No homology was found between the larger and smaller subunits. The smaller subunit contains a 26-residue hydrophobic sequence near its COOH terminus that represents a potential transmembrane domain. Four stretches of 12 amino acids present in the larger subunit are homologous to the calcium binding sites of calmodulin and troponin C. Northern blot analysis using HEL cell RNA indicated that the mature mRNA coding for GPIIb is 4.1 kilobases in size. A comparison of the GPIIb coding region with available cDNA sequences of the alpha-chains of the vitronectin and fibronectin receptors revealed 41% DNA homology and 74% and 63% amino acid homology, respectively. Our data establish the amino acid sequence for the human platelet glycoprotein IIb and provide additional evidence for the existence of a family of cellular adhesion protein receptors.     

Influence of oxygen and substrate supply on the metabolism of Candida maltosa during cultivation on n-alkanes

Summary The influence of different states of oxygen and alkane substrate supply on the metabolism of Candida maltosa during cultivation on n-alkanes has been investigated. At sufficient oxygen and substrate supply a nearly equimolar ratio between the formation of biomass and alkane oxidation was observed. About 45% of the carbon source utilized was incorporated into the biomass. Strong oxygen limitation decreased protein formation and carbon incorporation into the biomass with a simultaneous increase in CO₂ formation, whereas periodic changes of oxygen supply only caused a decrease in carbon incorporation into the biomass and an increase in CO₂ formation. During cultivation in the presence of an inert hydrocarbon (pristane) it was found that carbon limitation and oxygen saturation diminished the formation of total and nitrogen-containing biomass, whereas carbon and oxygen limitation reduced the formation of total biomass.Offprint requests to: P. Riege     

The structure and evolution of the spider monkey delta-globin gene

We have isolated the delta-globin gene of the New-World spider monkey, Ateles geoffroyi, and compared its nucleotide sequence with those of other primate delta- and beta-globin genes. Among primate delta-globin genes, the rate of nonsynonymous substitutions is much less than the rate of synonymous substitutions. This suggests that primate delta- globin genes may remain under evolutionary conservation, perhaps because hemoglobin A2 has an as yet unknown physiological importance.      

Stoichiometric translocation of adipocyte insulin receptors from the cell-surface to the cell-interior. Studies using a novel method to rapidly remove detergent and concentrate soluble receptors

A rapid one-step method was developed for harvesting and concentrating insulin receptors from solubilized adipocytes, which entails precipitating soluble receptors with polyethylene glycol and resuspending the receptor-containing pellet in a reduced volume of binding buffer. With this procedure 90-100% of receptors were recovered, while 80% of cellular protein was removed, thus resulting in a marked reduction of both ligand and receptor proteases and about a 5-fold purification of the receptor. More importantly, greater than 98% of the Triton X-100 detergent was removed during this procedure so that the reduced receptor affinity observed in solubilized extracts (due to detergent) was restored to normal. Reconstituted receptors exhibited normal binding characteristics similar to those observed for plasma membrane receptors. The general utility of our receptor precipitation-reconstitution method is highlighted by studies on insulin-induced translocation of receptors from the cell-surface to the cell-interior of adipocytes and studies on the assessment of the binding affinity of nascent intracellular receptors. The results of these studies are consistent with the following. 1) Insulin initiates endocytotic uptake of insulin receptors, which then recycle back to the cell-surface. 2) Chloroquine impairs the recycling of internalized receptors while preventing receptor degradation, resulting in the progressive trapping and accumulation of receptors within cells during insulin treatment. 3) Receptor translocation during acute insulin-induced down-regulation is stoichiometric in that receptors lost from the cell-surface can be quantitatively recovered within the cell-interior. 4) In the absence of ligand, these receptors within adipocytes are mainly newly synthesized receptors enroute to the cell-surface, and they possess an affinity similar, if not identical, to mature receptors on the plasma membrane.     

Biochemical pathways in prokaryotes can be traced backward through evolutionary time

For the first time, a credible prokaryotic phylogenetic tree is being assembled by Woese and others using quantitative sequence analysis of oligonucleotides in the highly conservative rRNA. This provides an evolutionary scale against which the evolutionary steps that led to the arrangement and regulation of contemporary biochemical pathways can be measured. This paper presents an emerging evolutionary picture of aromatic amino acid biosynthesis within a large superfamily assemblage of prokaryotes that is sufficiently developed to illustrate a new perspective that will be applicable to many other biochemical pathways.      

Pertussis toxin catalyzed ADP-ribosylation of a 41 kDa G-protein impairs insulin-stimulated glucose metabolism in BC3H-1 myocytes

In this study, we examined the effects of pertussis toxin (PT) on the ADP-ribosylation of guanine nucleotide binding proteins (G-proteins) and various insulin-stimulated processes in cultured BC3H-1 myocytes. Treatment of intact myocytes with 0.1 microgram/ml PT for 24 hours resulted in the complete ribosylation of a 41 kDa protein. The 41 kDa PT substrate was immunoprecipitated with antibodies directed against a synthetic peptide corresponding to a unique sequence in the alpha subunit of Gi-proteins. PT treatment of intact cells had no effect on insulin receptor binding or internalization. However, PT inhibited insulin-stimulated glucose transport at all insulin-concentrations tested (1-100 ng/ml). Maximally stimulated glucose transport was reduced by 50% +/- 15%. Insulin-stimulated glucose oxidation was also decreased by 31% +/- 8%. The toxin had no significant effect on the basal rates of glucose transport and glucose oxidation. The time course of PT-induced inhibition on glucose transport correlated with the time course of the "in vivo" ADP-ribosylation of the 41 kDa protein. The results suggest that a 41 kDa PT-sensitive G-protein, identical or very similar to Gi, is involved in the regulation of glucose metabolism by insulin in BC3H-1 cells.     

Increased inactivation of Saccharomyces cerevisiae by protraction of UV irradiation.

The principle of equi-effectivity of the product of intensity and exposure time (principle of Bunsen-Roscoe) of UV irradiation has been assumed to be valid for the inactivation of microorganisms in general. Earlier studies claimed higher survival of Escherichia coli B/r with fractionated irradiation compared with single-exposure survival. However, data on the inactivation effect of protraction of UV irradiation are not available. By means of a specially designed UV irradiation apparatus which secured absolute UV dose measurements throughout the experiments, the effects of variation of UV irradiation intensities (253.7 nm) and exposure times were tested on the inactivation of a bacterial virus (Staphylococcus aureus phage A994), a vegetative bacterial strain (E. coli ATCC 25922), and bacterial spores (Bacillus subtilis ATCC 6633) as well as three haploid laboratory strains (RC43a, YNN281, and YNN282) and two diploid strains (commercial bakery yeast strain and laboratory strain YNN281 x YNN282) or yeast (Saccharomyces cerevisiae) and spores of the latter diploid yeast strain. Each test organism was exposed to three UV intensities (0.02, 0.2, and 2 W/m2), with corresponding exposure times resulting in three dose levels for each intensity. Differences in inactivation rates were tested by analyses of variance and Newman-Keuls tests. Virus and bacteria showed no differences in inactivation rates by variation of intensities and exposure times within selected UV doses; hence, the principle of Bunsen-Roscoe could not be rejected for these strains. However, in the eukaryotic test strains of S. cerevisiae longer exposure times with lower intensities led to enhanced inactivation in both haploid and diploid strains, with a more pronounced effect in the diploid yeast strains, whereas in yeast spores in this dose rate effect could not be observed.     

The evolutionary history of the amylase multigene family in Drosophila pseudoobscura

In Drosophila pseudoobscura, the amylase (Amy) multigene family is contained within a series of inversions, or gene arrangements, on the third chromosome. The Standard (ST), Santa Cruz (SC), and Tree Line (TL) inversions are central to the phylogeny of arrangements, and have clusters of other arrangements derived from them. The gene arrangements belonging to each of these three clusters have a characteristic number of Amy genes, ranging from three in ST to two in SC to one in TL. This distribution pattern can reflect a history of either duplications or deletions, although the data available in the past did not permit a decision between these alternatives. We provide unambiguous evidence that three Amy genes were present before the divergence of the ST, SC, and TL arrangements. Thus, the current status of the Amy multigene family is the result of deletions in the TL and SC arrangements, which created three new pseudogenes: TL Amy2-psi, TL Amy3-psi, and SC Amy3- psi. Analysis of pseudogene sequences revealed that, in the SC and ST arrangements, pseudogene evolution has been retarded, most likely due to the homogenization effect of gene conversion. Finally, by determining the original copy number, we have reconstructed the evolutionary history of the Amy multigene family and linked it with the evolution of the central gene arrangements.      

USE OF SENSORY EVALUATION FOR MEASURING DEODORIZING EFFECTS OF AN AIR CONDITIONER

The application of sensory methodology for measuring deodorizing effect of an air conditioner equipped with electric plasma was introduced. Deodorizing effect was measured using chemical and sensory methods at different time (0, 30 and 60 min) and mode (control, blowing and cooling) of an air conditioner. Smoke from a roll of cigarette in a closed room was used as a source of odor and the concentrations of acetic acid and ammonia were measured as odorous chemical components. As one of the sensory methods triangle test was used and as a first step to obtain deodorizing effects by triangle test, the threshold of each panelist was obtained as the log dilution ratio of odor concentration at which the difference from odorless air was detected. The odor concentration at each time and mode was calculated using the threshold of the panel and the deodorizing effect was obtained on the basis of the odor concentration. In addition to a triangle test, scaling methods such as category scaling or magnitude estimation were used to measure deodorizing effect of an air conditioner. Deodorizing effects by scaling methods were calculated based on odor intensity with time at each mode. The regression analysis was done between the efficacy of deodorizing effect by sensory test and those by acetic acid and ammonia, the R² values of the regression equations for triangle test, category scale, and magnitude estimation were 0.84, 0.72 and 0.69, respectively. Deodorizing effect by triangle test explained the decrease of acetic acid and ammonia better than those by category scaling or magnitude estimation while high cost and time consuming labor involved in triangle tests reduced the merit. The results of this study demonstrated that various sensory methods could be used to measure deodorizing effect of air conditioners and further researches on fast and reliable methods are needed to establish the official procedures.