A new member of a secretory protein gene family in the dipteranChironomus tentans has a variant repeat structure

Summary We describe the structure of a gene expressed in the salivary gland cells of the dipteranChironomus tentans and show that it encodes 1 of the approximately 15 secretory proteins exported by the gland cells. This sp115,140 gene consists of approximately 65 copies of a 42-bp sequence in a central uninterrupted core block, surrounded by short nonrepetitive regions. The repeats within the gene are highly similar to each other, but divergent repeats are present in a pattern which suggests that the repeat structure has been remodeled during evolution. The 42-bp repeat in the gene is a simple variant of the more complex repeat unit present in the Balbiani ring genes, encoding four of the other secretory proteins. The structure of the sp115,140 gene suggests that related repeat structures have evolved from a common origin and resulted in the set of genes whose secretory proteins interact in the assembly of the secreted protein fibers.     

70-Kilodalton Heat Shock Protein Induction in Cerebellar Astrocytes and Cerebellar Granule Cells In Vitro: Comparison with Immunocytochemical Localization After Hyperthermia In Vivo

Induction of the 70-kDa heat shock protein, hsp70, was evaluated in cultured cerebellar astrocytes and granule cell neurons subjected to a hyperthermic stress, using a monoclonal antibody and an oligonucleotide probe that selectively recognize stress-inducible species of hsp70-related proteins and RNAs, respectively. Immunoblots of cultures enriched in either granule cells or astrocytes, and immunocytochemical localization studies in cocultures of these cell types, demonstrated that hsp70 induction was restricted to the astrocyte population. Amino acid incorporation experiments showed little difference in the loss and recovery of overall protein synthesis activity in these two cell types following transient hyperthermic stress. RNA blot hybridizations confirmed the preferential glial induction of hsp70. In vivo immunocytochemical studies in brains of adult rats following hyperthermia were consistent with earlier observations that suggested a primarily glial and vascular localization of the heat shock response in most brain regions, although the intense immunoreactivity in the cerebellar granule cell layer suggests that there is induction of hsp70 in these neurons under in vivo conditions. These results suggest the potential value of such defined cell cultures in identifying mechanisms responsible for differences in the heat shock response of various cell types in vitro, and in revealing factors that may account for the apparent absence of the stress response in cultured cerebellar granule cell neurons.     

Intraspecific evolution of a gene family coding for urinary proteins

Summary The genome of the laboratory mouse contains about 35 major urinary protein (MUP) genes, many of which are clustered on chromosome 4. We have used distance and parsimony methods to estimate phylogenetic relationships between MUP genes from nucleotide sequence and restriction maps. By analyzing coding sequences we show that the genes fall into four main groups of related sequences (groups 1–4). Comparisons of restriction maps and the nucleotide sequences of hypervariable regions that lie 50 nucleotides 5 to the cap sites show that the group 1 genes and probably also the group 2 pseudogenes fall into subgroups. The most parsimonious trees are consistent with the evolution of the array of group 1 and 2 genes by mutation accompanied by a process tending toward homogenization such as unequal crossing-over or gene conversion. The phylogenetic grouping correlates with grouping according to aspects of function. The genomes of the inbred strains BALB/c and C57BL contain different MUP gene arrays that we take to be samples from the wild population of arrays.     

Most Synaptic Vesicles Isolated from Rat Brain Carry Three Membrane Proteins, SV2, Synaptophysin, and p65

We have prepared highly purified synaptic vesicles from rat brain by subjecting vesicles purified by our previous method to a further fractionation step, i.e., equilibrium centrifugation on a Ficoll gradient. Monoclonal antibodies to three membrane proteins enriched in synaptic vesicles--SV2, synaptophysin, and p65--each were able to immunoprecipitate specifically approximately 90% of the total membrane protein from Ficoll-purified synaptic vesicle preparations. Anti-SV2 precipitated 96% of protein, anti-synaptophysin 92%, and anti-p65 83%. These results demonstrate two points: (1) Ficoll-purified synaptic vesicles appear to be greater than 90% pure, i.e., less than 10% of membranes in the preparation do not carry synaptic vesicle-associated proteins. These very pure synaptic vesicles may be useful for direct biochemical analyses of mammalian synaptic vesicle composition and function. (2) SV2, synaptophysin, and p65 coexist on most rat brain synaptic vesicles. This result suggests that the functions of these proteins are common to most brain synaptic vesicles. However, if SV2, synaptophysin, or p65 is involved in synaptic vesicle dynamics, e.g., in vesicle trafficking or exocytosis, separate cellular systems are very likely required to modulate the activity of such proteins in a temporally or spatially specific manner.     

Modification of Gs-Stimulated Adenylate Cyclase in Brain Membranes by Low pH Pretreatment: Correlation with Altered Guanine Nucleotide Exchange

Pretreatment of rat brain membranes at pH 4.5 before assay at pH 7.4 modifies the function of GTP-binding proteins (G-proteins) by eliminating Gs-stimulated adenylate cyclase activity while increasing opiate-inhibited adenylate cyclase activity. To help characterize the molecular nature of the low pH effect, we labeled Gs and Gi alpha-subunits in both control and low pH-pretreated membranes with the GTP photoaffinity analog [32P]P3 (4-azidoanilido)-P1-5'-GTP ([32P]AAGTP). When membranes were preincubated with unlabeled AAGTP, a persistent inhibitory state of adenylate cyclase was produced, which was overcome in untreated membranes with high (greater than 1 microM) concentrations of guanylyl-5'-imidodiphosphate [Gpp(NH)p]. In low pH-pretreated membranes, this inhibition could not be overcome, and stimulation by Gpp(NH)p was eliminated. Maximal inhibition of adenylate cyclase achieved by incubation with AAGTP was not altered by low pH pretreatment. Incorporation of [32P]AAGTP into Gs (42 kilodaltons) or Gi/o (40 kilodaltons) was unaffected by low pH pretreatment; however, transfer of 32P from Gi/o to Gs, which occurs with low (10 nM) concentrations of Gpp(NH)p in untreated membranes, was severely retarded in low pH-pretreated membranes. Both the potency and efficacy of Gpp(NH)p in producing exchange of [32P]AAGTP from Gi/o to Gs were markedly reduced by low pH pretreatment. These results correlate the loss of Gs-stimulated adenylate cyclase with a loss of transfer of nucleotide from Gi/o to Gs alpha-subunits and suggest that the nucleotide exchange participates in the modulation of neuronal adenylate cyclase.     

Age-dependent modifications of mitochondrial proteins in cerebral cortex and striatum of rat brain

The protein composition of free mitochondria purified from cerebral cortex and striatum during aging was analyzed by gel electrophoresis. Mitochondria were isolated from cerebral cortex and striatum of 4-, 12-, and 24-month-old rat brain. The percent amount of mitochondrial proteins after gel-electrophoretic separation was determined densitometrically. A significant decrease in the amount of two polypeptides (with molecular weights of 20 and 16 kDa, respectively) in both brain regions during aging was found. The decrease was higher in the striatum indicating a greater vulnerability of this brain area to the aging process. The age-dependent modifications of mitochondrial proteins observed may play an important role in several mitochondrial functions, such as energy transduction and transport processes as well as in structural changes occurring with age, causing altered membrane permeability and fluidity.     

Outer membrane proteins of gentamicin induced small colony variants of Pseudomonas aeruginosa

Small colony variants (SCVs) of Pseudomonas aeruginosa NCTC 6750 (WT) were repeatedly isolated in an in vitro kinetic model after exposure to gentamicin (GM). There were minor differences biochemically and in phage and serotyping between the wild type (WT) strain and SCVs. Changes in outer membrane protein profiles were found. SCVs were more resistant to polymixin and to a range of aminoglycosides (except kanamycin), but were more susceptible to a range of other antibiotics (hydrophilic and hydrophobic) with differing modes of action.     

Response of seeds ofLupinus termis andVicia faba to the interactive effect of salinity and ascorbic acid or pyridoxine

The interactive effect of salinity and presoaking in ascorbic acid or phyridoxine on germination, seedling growth, and some relevant metabolic changes ofLupinus termis andVicia faba seeds were studied. Germination studies indicated that broad bean tolerated NaCl salinity up to 240mM NaCl and lupin to 200mM NaCl. The lengths of roots and shoots and their water content, as well as dry matter yield, remained more or less unchanged up to the level of 80mM NaCl. Salinity induced marked progressive increases of carbohydrates and proline in broad bean and soluble protein in lupin seedlings, irrespective of the salinity level used. The other organic solutes (soluble protein in broad bean and carbohydrates in lupin seedlings) remained more or less unchanged at low and moderate levels of NaCl. However, under the higher salinity levels, in lupin the losses in carbohydrates were accompanied by increases in soluble protein, whereas in broad bean an opposite effect was obtained. The level of 40mM NaCl had a pronounced stimulatory effect on the all the variables studied. Presoaking seeds in either ascorbic acid or pyridoxine counteracted the adverse effects of salinity on germination and seedling growth as well as on some metabolic mechanisms of lupin and broad bean plants. The importance of these processes to the salinity tolerance of broad bean and lupin have been discussed.