Studies on proinsulin and proglucagon biosynthesis and conversion at the subcellular level: II. Distribution of radioactive peptide hormones and hormone precursors in subcellular fractions after pulse and pulse- chase incubation of islet tissue

Anglerfish proinsulin and insulin were selectively labeled with [(14)C]isoleucine, while proglucagon, conversion intermediate(s), and glucagon were selectively labeled with[(3)H]tryptophan. After various periods of continuous or pulse-chase incubation, islet tissue was subjected to subcellular fractionation. Fraction extracts were analyzed by gel filtration for their content of precursor, conversion intermediate(s), and product peptides. Of the seven subcellular fractions prepared after each incubation, only the microsome and secretory granule fractions yielded significant amounts of labeled insulin-related and glucagon-related peptides. After short-pulse incubations, levels of both [(14)C]proinsulin and [(3)H]proglucagon (mol wt approximately 12,000) were highest in the microsome fraction. This fraction is therefore identified as the site of synthesis. With increasing duration of continuous incubation or during chase incubation in the absence of isotopes, proinsulin, proglucagon, and conversion intermediate(s) are transported to secretory granules. Conversion of proinsulin to insulin and proglucagon to a approximately 4,900 mol wt conversion intermediate and 3,500 mol wt glucagon occurs in the secretory granules. Converting activity also was observed in the microsome fraction. The recovery of most of the incorporated radioactivity in microsome and secretory granule fractions indicates that the newly synthesized islet peptides are relegated to a membrane-bound state soon after synthesis at the RER is completed. This finding supports the concept of intracisternal sequestration and intragranular maintenance of peptides synthesized for export from the cell of origin.     

Characterization and Expression of the cDNA Coding for the Human Myelin/Oligodendrocyte Glycoprotein

Abstract: We report here the characterization of a full-length cDNA encoding the human myelin/oligodendrocyte glycoprotein (MOG). The sequence of the coding region of the human MOG cDNA is highly homologous to that of other previously cloned mouse, rat, and bovine MOG cDNAs, but the 3' untranslated region differs by an insertion of an Alu sequence between nucleotides 1,590 and 1,924. Accordingly, northern blot analyzes with cDNA probes corresponding to the coding region or the 3' untranslated Alu-containing sequence revealed a single band of 2 kb, rather than the 1.6 kb of bovine, rat, or mouse MOG cDNA(s). Immunocytochemical analysis of HeLa cells transfected with human MOG cDNA, which was performed using a specific antibody raised against whole MOG, clearly indicated that MOG is expressed at the cell surface as an intrinsic protein. These data are in accordance with the predicted amino acid sequence, which contains a signal peptide and two putative transmembrane domains. The knowledge of the human MOG sequence should facilitate further investigations on its potential as a target antigen in autoimmune demyelinating diseases like multiple sclerosis.     

The Deuterator: software for the determination of backbone amide deuterium levels from H/D exchange MS data

Background  

The combination of mass spectrometry and solution phase amide hydrogen/deuterium exchange (H/D exchange) experiments is an effective method for characterizing protein dynamics, and protein-protein or protein-ligand interactions. Despite methodological advancements and improvements in instrumentation and automation, data analysis and display remains a tedious process. The factors that contribute to this bottleneck are the large number of data points produced in a typical experiment, each requiring manual curation and validation, and then calculation of the level of backbone amide exchange. Tools have become available that address some of these issues, but lack sufficient integration, functionality, and accessibility required to address the needs of the H/D exchange community. To date there is no software for the analysis of H/D exchange data that comprehensively addresses these issues.     

Sperm surface galactosyltransferase activities during in vitro capacitation

Studies using genetic and biochemical probes have suggested that mouse sperm surface galactosyltransferases may participate during fertilization by binding N- acetylglucosamine (GlcNAc) residues in the egg zona pellucida. In light of these results, we examined sperm surface galactosyltransferase activity during in vitro capacitation to determine whether changes in enzymatic activity correlated with fertilizing ability. Results show that surface galactosyltransferases on uncapacitated sperm was preferentially loaded with poly N-acetyllactosamine substrates. As a consequence of capacitation in Ca(++)-containing medium, these polylactosaminyl substrates are spontaneously released from the sperm surface, thereby exposing the sperm galactosyltransferase for binding to the zona pellucida. Sperm capacitation can be mimicked, in the absence of Ca(++), either by washing sperm in Ca(++)-free medium, or by pretreating sperm with antiserum that reacts with the galactosyltransferase substrate. In both instances, sperm galgactosylation of endogenous polylactosaminyl substrates is reduced, coincident with increased galactosylation of exogenous GlcNAc, and increased binding to the zona pellucida. Binding of capacitated sperm to the egg can be inhibited by pronase-digested high molecular weight polyactosaminyl glycoside extracted from epidymal fluids or from undifferentiated F9 embryonal carninoma cells. Thus, these glycosides function as “decapacitation factors” when added back to in vitro fertilization assays. These glycoside “decapacitation factors” inhibit sperm-egg binding by competeing for the sperm surface galactosyltransferase, since (a) they are galactosylated by sperm in the presence of UDP[(3)H]galactose, and (b) enzymatic removal of terminal GlcNAc residues reduces “decapacitation factio” competition. On the other hand “conventional” low molecular weight glycosides, isolated from either epididymal fluid or differentiated F9 cells, fail to inhibit capacitated sperm binding to the zona pellucida. These results define a molecular mechanism for one aspect of sperm capacitation, and help explain why removal of “decapacitation factos” is a necessary prerequisite for sperm binding to the zona pellucida.     

Annexins in rat enterocyte and hepatocyte: an immunogold electron-microscope study

In the present study, immunogold labeling of ultrathin sections of rat small intestine and liver has been used to obtain insights into the ultrastructural localization and possible functions of annexins. In enterocytes, annexins II, IV, and VI are found at the periphery of the core of each microvillus and of the rootlets, but are absent from the interrootlet space. Annexins II, IV, and VI are also observed close to the interdigitated plasma membrane. In hepatocytes, only annexin VI is found to be concentrated within the microvilli in the bile canaliculi, on the inner face of the sinusoidal cell surface, particularly in the space of Disse, and all along the plasma membrane. Annexin VI is also detected in mitochondria of enterocytes and hepatocytes. These localizations are in agreement with the concept of a close calcium-dependent association of annexins with membranes and cytoskeletal proteins, particularly with actin. Moreover, they support the hypothesis of an involvement of annexins in exocytotic and endocytotic processes, which take place in epithelial cells.     

Developmental regulation of calmodulin gene expression in rat brain and skeletal muscle.

Three different calmodulin genes that encode the identical protein have been identified in the rat (Nojima, 1989); however, calmodulin gene expression at the various stages of tissue differentiation and maturation has not been previously determined. We have quantitated the content of mRNAs encoding calmodulin in the developing brain and skeletal muscle using RNA blot analysis with three specific cDNA probes. Our results show that five species of calmodulin mRNAs: 4.0 and 1.7 kb for CaM I, 1.4 kb for CaM II, and 2.3 and 0.8 kb for CaM III are detectable at all ages in the brain as well as in skeletal muscle but exhibit a tissue-specific developmental pattern of expression. The comparison of the temporal pattern of calmodulin gene expression with both mitotic activity, as demonstrated by cyclin A mRNA levels, and differentiation and maturation of specific brain or muscle regions is consistent with calmodulin involvement in development.     

Exercise-induced modulation of calcineurin activity parallels the time course of myofibre transitions

This study establishes a causal link between the limitation of myofibre transitions and modulation of calcineurin activity, during different exercise paradigms. We have designed a new swimming-based training protocol in order to draw a comparison between a high frequency and amplitude exercise (swimming) and low frequency and amplitude exercise (running). We initially analysed the time course of muscle adaptations to a 6- or 12-week swimming- or running-based training exercise program, on two muscles of the mouse calf, the slow-twitch soleus and the fast-twitch plantaris. The magnitude of exercise-induced muscle plasticity proved to be dependent on both the muscle type and the exercise paradigm. In contrast to the running-based training which generated a continuous increase of the slow phenotype throughout a 12-week training program, swimming induced transitions to a slower phenotype which ended after 6 weeks of training. We then compared the time course of the exercise-induced changes in calcineurin activity during muscle adaptation to training. Both exercises induced an initial activation followed by the inhibition of calcineurin. In the muscles of animals submitted to a 12-week swimming-based training, this inhibition was concomitant with the end of myofibre transition. Calcineurin inhibition was a consequence of the inhibition of its catalytic subunit gene expression on one hand, and of the expression increase of the modulatory calcineurin interacting proteins 1 gene (MCIP1), on the other. The present study provides the first experimental cues for an interpretation of muscle phenotypic variation control.     

Dynamics of mutated GFAP aggregates revealed by real-time imaging of an astrocyte model of Alexander disease

Alexander disease (AxD) is a rare neurodegenerative disorder characterized by large cytoplasmic aggregates in astrocytes and myelin abnormalities and caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), the main intermediate filament protein in astrocytes. We tested the effects of three mutations (R236H, R76H and L232P) associated with AxD in cells transiently expressing mutated GFAP fused to green fluorescent protein (GFP). Mutated GFAP-GFP expressed in astrocytes formed networks or aggregates similar to those found in the brains of patients with the disease. Time-lapse recordings of living astrocytes showed that aggregates of mutated GFAP-GFP may either disappear, associated with cell survival, or coalesce in a huge juxtanuclear structure associated with cell death. Immunolabeling of fixed cells suggested that this gathering of aggregates forms an aggresome-like structure. Proteasome inhibition and immunoprecipitation assays revealed mutated GFAP-GFP ubiquitination, suggesting a role of the ubiquitin-proteasome system in the disaggregation process. In astrocytes from wild-type-, GFAP-, and vimentin-deficient mice, mutated GFAP-GFP aggregated or formed a network, depending on qualitative and quantitative interactions with normal intermediate filament partners. Particularly, vimentin displayed an anti-aggregation effect on mutated GFAP. Our data indicate a dynamic and reversible aggregation of mutated GFAP, suggesting that therapeutic approaches may be possible.