Induction of autocrine epidermal growth factor receptor ligands in human keratinocytes by insulin/insulin-like growth factor-1

Autocrine activation of the epidermal growth factor (EGF) receptor on keratinocytes has been recognized as an important growth regulatory mechanism involved in epithelial homeostasis, and, possibly, hyperproliferative diseases. Insulin-like growth factor (IGF)-1 and insulin have been shown to be paracrine keratinocyte mitogens that bind to the type I IGF receptor which is expressed on actively proliferating keratinocytes in situ. In this report, we demonstrate that IGF-1/insulin induced production of keratinocyte-derived autocrine growth factors that bind to the EGF receptor. Increased steady-state mRNA levels for transforming growth factor alpha (TGF-α) and for amphiregulin (AR) were observed upon incubation of keratinocytes with mitogenic concentrations of IGF-1. IGF-1 also induced production and secretion of TGF-α and AR proteins as detected by immunoassays. An EGF receptor antagonistic monoclonal antibody abolished the mitogenic effect of IGF-1 on cultured keratinocytes. These results suggest that stimulation of keratinocyte growth by IGF-1 requires activation of an EGF receptor-mediated autocrine loop. © 1995 Wiley-Liss, Inc.     

Ultrastructure of Nitrosospira sp. X101 with crystalline outer membrane protein (COMP)

Abstract The outer membrane (OM) structure of Nitrosospira sp. X101 was studied by different electron microscopic techniques and SDS-PAGE. A crystalline outer membrane protein was visible in freeze-etched cells, occasionally seen also in the thin sectioned cells, but was difficult to see in a negatively-stained preparation. The lattice probably consists of large globular protein subunits with a hexagonal arrangement. The molecular weights of the major proteins in the cell envelope are 35 kDa, 40 kDa and 42 kDa.     

Analysis of clozapine and norclozapine by high-performance liquid chromatography

A simple and reliable method for analyzing the concentrations of clozapine and its biologically active metabolite, norclozapine, in human serum or plasma has been developed. This method is based on reversed-phase high-performance liquid chromatography (HPLC) with automated solid-phase extraction (SPE). For HPLC analysis, samples and standards are prepared with an ASPEC automatic sample preparator using 100 mg Bond-Elut C₁₈ SPE columns. The HPLC assay is an isocratic method with a mobile phase of acetonitrile-methanol-10 mM dipotassium hydrogenphosphate, pH 3.7 (30:2:100, v/v/v) at a flow-rate of 1.5 ml/min with a C₁₈ reversed-phase column. Detection is performed with a diode array detector set at 220 nm and with peak purity analyses at 210–365 nm. The absolute recovery varied from 85 and 95%. The intra-assay coefficients of variation (C.V.s) were from 4.2 and 8.0% and the inter-assay C.V.s were from 1.1. to 9.3% at therapeutic drug concentrations. The detection limit is 15 nmol/l. The method has been developed for use in a clinical laboratory for therapeutic drug monitoring.     

Relationship between the seed rain and the establishment of vegetation in two areas abandoned after peat harvesting

In this study the number and species composition of diaspores dispersing into two newly abandoned peat harvesting areas in Finland were investigated. In an area abandoned six years earlier a total of 2978 living seeds m⁻², representing 18 species, was captured by using water-filled traps during two summers. In a one-year-old succession area the total number of living seeds m⁻² was 2241, representing 16 plant species. The soil seed banks were found to be empty of viable seeds. In neither area there appeared to be any relation between number of dispersing seeds and of plant individuals of the same species in the pioneer vegetation. It is suggested that unfavourable conditions on the soil surface for seed germination and seedling growth of most dispersed species are majors factor in determining the structure of the established vegetation.     

Human immune responses to major human cytomegalovirus glycoprotein complexes.

Sera from both human cytomegalovirus (HCMV)-seropositive adults and infants with congenital HCMV infection recognized two major HCMV glycoprotein complexes. However, proliferative responses of peripheral blood mononuclear cells to these complexes varied among seropositive adults and were not detected in any of the infants. Thus, these glycoproteins alone may not be sufficient to develop a subviral HCMV vaccine.     

A multigene family encodes the human cytomegalovirus glycoprotein complex gcII (gp47-52 complex).

The HXLF (HindIII-X left reading frame) gene family is a group of five genes that share one or two regions of homology and are arranged in tandem within the short unique component of the human cytomegalovirus genome (K. Weston and B.G. Barrell, J. Mol. Biol. 192:177-208, 1986). These genes were cloned into an SP6 expression vector in both the sense and antisense orientations. An abundant 1.62-kilobase (kb) bicistronic mRNA, predicted to originate from HXLF1 and HXLF2, was detected in the cytoplasm of infected human fibroblast cells by Northern (RNA) blot analysis. Less abundant RNAs of 1.0 and 0.8 kb, predicted to originate from the HXLF5 and HXLF2 genes, respectively, were also detected. Monocistronic, bicistronic, and polycistronic RNAs synthesized in vitro by using SP6 polymerase were translated in rabbit reticulocyte lysates with or without canine pancreatic microsomal membranes. The HXLF1 or the HXLF1 and HXLF2 translation products were detected when the above mRNAs were used. The HXLF3, HXLF4, and HXLF5 gene products were not detected by in vitro translation of the SP6-derived polycistronic mRNA. Nonglycosylated or glycosylated HXLF1 and HXLF2 gene products were immunoprecipitated by monoclonal antibody 9E10, which is specific for a virion envelope glycoprotein complex designated gcII (gp47-52 complex). In addition, the monoclonal antibody 9E10 immunoprecipitated a diffuse glycoprotein band, designated gp47-52, from HCMV-infected cell lysates. The amino acid composition of gp47-52 purified from viron envelopes has the highest similarity to the predicted amino acid composition of the HXLF1 plus HXLF2 open reading frames, but it is more similar to HXLF2 than to HXLF1. The Northern blot results imply that gp47-52 is synthesized predominantly from the abundant 1.62-kb bicistronic mRNA encoded by the HXLF1 and HXLF2 genes. However, the glycoprotein could also be synthesized by the monocistronic 0.8-kb mRNA encoded by the HXLF2 gene as well as by the mRNAs predicted from the other HXLF genes.     

Identification and characterization of three distinct families of glycoprotein complexes in the envelopes of human cytomegalovirus

Several disulfide-linked glycoprotein complexes were identified in the envelope of human cytomegalovirus (HCMV). These glycoprotein complexes were fractionated by rate-zonal centrifugation in sucrose density gradients in the presence of detergents. Fractionated glycoproteins and complexes were immunoprecipitated with three different monoclonal antibodies specific for HCMV glycoproteins and a rabbit polyclonal antiserum prepared against detergent-extracted virion and dense-body envelope glycoproteins. Three distinct families of disulfide-linked glycoprotein complexes were observed and designated glycoprotein complex gcI, gcII, and gcIII. The gcI family, recognized by monoclonal antibody 41C2 under nonreducing conditions, consisted of three complexes with approximate molecular masses of 250 to 300, 190, and 160 kilodaltons (kDa). These complexes consistently sediment more rapidly than other HCMV glycoproteins or complexes in sucrose density gradients. Upon reduction of the gcI family, two size classes of glycoproteins with average molecular masses of 93 to 130 and 55 kDa were observed. The gcII family was recognized by monoclonal antibody 9E10. Under nonreducing conditions, as many as six electrophoretic forms were observed for gcII. When reduced, the major component of the gcII family was a heterogeneous glycoprotein designated gp47-52. The gcIII family was recognized by monoclonal antibody 1G6. It consisted of a complex of approximately 240 kDa without reduction of disulfide bonds. When reduced, two glycoprotein size classes with average molecular masses of 145 and 86 kDa were observed. Polyclonal antiserum R-7 reacted strongly with the gcI and gcIII families, but weakly with the gcII family.     

Variation in the carbon content of two Asplanchna species

The rotifers of the genus Asplanchna were sampled four times during the summer from eight lakes of different types. The mean individual carbon content in the population varied between 0.15–0.66 µg C ind.^-1 (n = 21) for A. priodonta and 1.0–1.6 µg C ind.^-1 (n = 3) for A. herricki. The carbon content and the size of A. priodonta varied considerably between the populations of both different lakes and dates.The carbon level of both Asplanchna species (sample mean 0.2–1% of wet weight) was considerably lower than is generally found for rotifers. Much of the variation of carbon level could be explained by an inverse relationship with wet weight. The high variation in the carbon content of individuals suggests that Asplanch population may adapt their mean body size to fit prevailing environmental conditions.