The effects of general anaesthetics on glucose and phosphate transport across the membrane of the human erythrocyte

A study has been carried out into the effects of clinically important general anaesthetics, althesin, thiopentone and propanidid, on the transport of glucose and phosphate across the membrane of the human erythrocyte. In general these three substances all inhibit both transport processes but with characteristic inhibition profiles and varying degrees of efficacy. Glucose transport was more sensitive to the hydrophobic steroids and phosphate transport to propanidid. Some hydrophobic agents, e.g., iodobenzene and its azide, were not inhibitory. Removal of cholesterol to some extent augmented the inhibitory effects of most of these compounds (not propanidid). It is argued that these effects are due to the penetration of the anaesthetics into the lipid bilayer and either subsequent disruption of the lipid annuli surrounding the integral membrane proteins and/or direct anaesthetic-protein interaction.     

The identification of O-glycosylated precursors of insulin-like growth factor II.

A procedure that combined ion exchange, gel permeation, and insulin-like growth factor-binding protein 3 (IGF-BP-3) affinity chromatography with chromatofocusing and reversed-phase high pressure liquid chromatography was used to isolate high molecular weight precursors of human insulin-like growth factor II (IGF-II) from acetic acid extracts of Cohn fraction IV1. Two precursors had isoelectric points (pI) of 5.1 and 5.4 and apparent Mr values of 15,000 and 11,500, respectively. An apparent Mr = 16,000 RLPG/Ser29 variant of IGF-II was also identified in the acetic acid extracts. Amino-terminal amino acid sequencing of the major E domain-containing peptide that had been isolated from apparent Mr = 15,000 IGF-II (pI 5.1), following its digestion with the endoprotease Lys-C, indicated the carboxyl terminus of this precursor was near or at Lys88. During the sequencing of this peptide, a sharply reduced yield of derivatized amino acid occurred at cycle 10, indicating that Thr75 had been posttranslationally modified, possibly by O-glycosylation. To evaluate this possibility, the 125I-labeled high molecular weight IGF-IIs and their endoprotease-generated peptides were treated with glycosidases, and their effects were determined from the change in relative mobilities of the polypeptide and peptides during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Neuraminidase treatment of apparent Mr = 15,000 and 11,500 IGF-II reduced their Mr values to a common value of 10,500. When the desialylated precursors of IGF-II were treated with O-glycosidase, but not when treated with N-glycosidase, the Mr values were reduced further to about 10,000. This was the Mr value that would be predicted for an unglycosylated form of precursor IGF-II that had a carboxyl-terminal end at or near Lys88. When the Ser66-Lys88 endoprotease-generated E domain peptides from pI5.1 and 5.4 high Mr IGF-II were treated with the glycosidases, they had relative mobility changes during sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were similar to those of the intact precursors. Finally, the association of O-linked oligosaccharide with the E domain peptide of IGF-II was confirmed by demonstrating the specificity of binding of the Ser66-Lys88 asialoglycopeptide to jackfruit lectin.     

Human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation mediated by vascular cell adhesion molecule-1: evidence for involvement of cell adhesion molecules in HTLV-1 biology.

While studying the potential role of vascular cell adhesion molecule-1 (VCAM-1) in infection of endothelial cells by human immunodeficiency virus (HIV), we found that VCAM-1 can mediate human T-cell lymphotropic virus type 1 (HTLV-1)-induced syncytium formation. Both expression-vector-encoded and endogenously expressed VCAM-1 supported fusion of uninfected cells with HTLV-1-infected cells. Fusion was obtained with cell lines carrying the HTLV-1 genome and expressing viral proteins but not with an HTLV-1-transformed cell line that does not express viral proteins. In clones of VCAM-1-transfected cells, the degree of syncytium formation observed directly reflected the level of VCAM-1 expression. Syncytium formation between HTLV-1-expressing cells and VCAM-1+ cells could be blocked with antiserum against HTLV-1 gp46 and with a monoclonal antibody (MAb) against VCAM-1. Fusion was not blocked by antiserum against HIV or a MAb against VLA-4, the physiological counter-receptor for VCAM-1. The results indicate that VCAM-1 can serve as an accessory molecule or potential coreceptor for HTLV-1-induced cell fusion and provide direct evidence of a role for cell adhesion molecules in the biology of HTLV-1.     

Isolation of Aspergillus nidulans Mutants That Overcome brlA-Induced Growth Arrest

The Aspergillus nidulans brlA gene is a primary regulator of development-specific gene expression during conidiation. Forced activation of brlA in vegetative cells leads to inappropriate induction of conidiophore formation and causes growth to stop. In fact, when conidia containing a nutritionally inducible brlA gene fusion are placed on inducing medium, they fail to germinate. We used this phenotype to select 174 mutants that continue growing following such forced brlA activation. Forty-six of these mutants also produced abnormal developmental structures during air-induced conidiation as expected if the mutations resulted in an altered response to BrlA (designated sbr mutants for suppressors of brlA response). The predominant mutant class identified was defective in a known developmental regulatory gene, abaA. We also identified mutants with defects in the previously characterized early acting developmental regulatory genes flbB and flbD and in four previously undescribed loci designated sbrA-D. sbrA mutants represent the second largest group and are characterized by production of conidiophore stalks that lack a normal vesicle and form branching sterigmata that rarely make spores. Because abaA expression could not be detected in sbrA mutants following brlA activation we propose that sbrA functions as a developmental modifier, participating in brlA-dependent activation of other developmental regulators.     

Role of 26S proteasome and HRD genes in the degradation of 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein.

3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R), a key enzyme of sterol synthesis, is an integral membrane protein of the endoplasmic reticulum (ER). In both humans and yeast, HMG-R is degraded at or in the ER. The degradation of HMG-R is regulated as part of feedback control of the mevalonate pathway. Neither the mechanism of degradation nor the nature of the signals that couple the degradation of HMG-R to the mevalonate pathway is known. We have launched a genetic analysis of the degradation of HMG-R in Saccharomyces cerevisiae using a selection for mutants that are deficient in the degradation of Hmg2p, an HMG-R isozyme. The underlying genes are called HRD (pronounced "herd"), for HMG-CoA reductase degradation. So far we have discovered mutants in three genes: HRD1, HRD2, and HRD3. The sequence of the HRD2 gene is homologous to the p97 activator of the 26S proteasome. This p97 protein, also called TRAP-2, has been proposed to be a component of the mature 26S proteasome. The hrd2-1 mutant had numerous pleiotropic phenotypes expected for cells with a compromised proteasome, and these phenotypes were complemented by the human TRAP-2/p97 coding region. In contrast, HRD1 and HRD3 genes encoded previously unknown proteins predicted to be membrane bound. The Hrd3p protein was homologous to the Caenorhabditis elegans sel-1 protein, a negative regulator of at least two different membrane proteins, and contained an HRD3 motif shared with several other proteins. Hrd1p had no full-length homologues, but contained an H2 ring finger motif. These data suggested a model of ER protein degradation in which the Hrd1p and Hrd3p proteins conspire to deliver HMG-R to the 26S proteasome. Moreover, our results lend in vivo support to the proposed role of the p97/TRAP-2/Hrd2p protein as a functionally important component of the 26S proteasome. Because the HRD genes were required for the degradation of both regulated and unregulated substrates of ER degradation, the HRD genes are the agents of HMG-R degradation but not the regulators of that degradation.     

Analysis of Lung Microbiota in Bronchoalveolar Lavage,Protected Brush and Sputum Samples from Subjects with Mild-To-Moderate Cystic Fibrosis Lung Disease

Individuals with cystic fibrosis (CF) often acquire chronic lung infections that lead to irreversible damage. We sought to examine regional variation in the microbial communities in the lungs of individuals with mild-to-moderate CF lung disease, to examine the relationship between the local microbiota and local damage, and to determine the relationships between microbiota in samples taken directly from the lung and the microbiota in spontaneously expectorated sputum. In this initial study, nine stable, adult CF patients with an FEV₁>50% underwent regional sampling of different lobes of the right lung by bronchoalveolar lavage (BAL) and protected brush (PB) sampling of mucus plugs. Sputum samples were obtained from six of the nine subjects immediately prior to the procedure. Microbial community analysis was performed on DNA extracted from these samples and the extent of damage in each lobe was quantified from a recent CT scan. The extent of damage observed in regions of the right lung did not correlate with specific microbial genera, levels of community diversity or composition, or bacterial genome copies per ml of BAL fluid. In all subjects, BAL fluid from different regions of the lung contained similar microbial communities. In eight out of nine subjects, PB samples from different regions of the lung were also similar in microbial community composition, and were similar to microbial communities in BAL fluid from the same lobe. Microbial communities in PB samples were more diverse than those in BAL samples, suggesting enrichment of some taxa in mucus plugs. To our knowledge, this study is the first to examine the microbiota in different regions of the CF lung in clinically stable individuals with mild-to-moderate CF-related lung disease.     

Krill aggregation characteristics: Spatial distribution patterns from hydroacoustic observations

Summary The distributional features and physical characteristics of some 1500 krill (Euphausia superba Dana) aggregations detected and sized acoustically in the south-west Indian Ocean are described. The aggregations were on average considerably smaller than elsewhere in the Antarctic. Aggregation biomass was log-normally distributed with a mean of 0.18 t. Only 3% of the aggregations were heavier than 1 t. The aggregations were on average 4.2 km apart along-track, and on 10 of the 15 survey days were randomly distributed, judged by a Chisquare test based on expected Poisson statistics. The distribution of aggregations heavier than 1 t, and of aggregations 20 km on either side of such aggregations was similarly judged to be random. These observations suggest widespread randomness throughout the survey area, which is consistent with the absence of pronounced hydrographic or topographic concentrating mechanisms in the region. On the 5 non-random days, aggregations were smaller than average and were clustered on a scale of 1 km, suggesting that they could have been in the process of fragmenting or amalgamating. Estimates of the mean nearest-neighbour distance were derived for the random distributions. It is suggested that these could be useful in the development of fisheries-dependent indices of krill abundance.