High Ammonia Levels in Brain Induce Tubulin in Cerebrum but Not in Cerebellum

Ingestion of large amounts of ammonium increases markedly the content of tubulin in brain. The effect on tubulin induction of ammonium ingestion for up to 100 days was investigated. Brain tubulin content showed a rapid initial increase (28%) at 2 days and reached 50% after 100 days on the diet. To discern if ammonia, the increase in urea synthesis, or both was responsible for tubulin induction, rats were maintained at several levels of uremia (by administering diets containing 0 to 80% protein) or in hyperammonemia (by urease treatment). Only ammonium administration in the diet and urease injection induced tubulin in brain. Tubulin was quantified in three different brain regions. There was a regional selectivity of tubulin induction by ammonia in rat brain. Whereas the cerebellum remained unaltered, the paleencephalon showed the highest increase, and the cerebral cortex exhibited only a modest increase.     

Localization of Nitrogen-Assimilating Enzymes in the Chloroplast of Chlamydomonas reinhardtii

The specific activities of nitrate reductase, nitrite reductase, glutamine synthetase, glutamate synthase, and glutamate dehydrogenase were determined in intact protoplasts and intact chloroplasts from Chlamydomonas reinhardtii. After correction for contamination, the data were used to calculate the portion of each enzyme in the algal chloroplast. The chloroplast of C. reinhardtii contained all enzyme activities for nitrogen assimilation, except nitrate reductase, which could not be detected in this organelle. Glutamate synthase (NADH- and ferredoxin-dependent) and glutamate dehydrogenase were located exclusively in the chloroplast, while for nitrite reductase and glutamine synthetase an extraplastidic activity of about 20 and 60%, respectively, was measured. Cells grown on ammonium, instead of nitrate as nitrogen source, had a higher total cellular activity of the NADH-dependent glutamate synthase (+95%) and glutamate dehydrogenase (+33%) but less activity of glutamine synthetase (−10%). No activity of nitrate reductase could be detected in ammonium-grown cells. The distribution of nitrogen-assimilating enzymes among the chloroplast and the rest of the cell did not differ significantly between nitrate-grown and ammonium-grown cells. Only the plastidic portion of the glutamine synthetase increased to about 80% in cells grown on ammonium (compared to about 40% in cells grown on nitrate).     

Rhizosphere microorganism effects on soluble amino acids,sugars and organic acids in the root zone ofAgropyron cristatum,A. smithii andBouteloua gracilis

Three axenic and rhizosphere microorganism-inoculated shortgrass steppe plant species were evaluated for possible differences in residual organic carbon and nitrogen present as sugars, organic acids and amino acids. IntroducedAgropyron cristatum was compared toA. smithii andBouteloua gracilis, which are dominant species in the native shortgrass steppe. These plants, grown for 90 days in root growth chambers, showed differences in residual organic carbon and nitrogen per gram of root, and rhizosphere microbe presence resulted in additional changes in these compounds. The root biomass ofB. gracilis was significantly increased with microbes present. TheAgropyron species had significantly lower amino acid levels with microbes present, while under the same conditions, theB. gracilis showed significant decreases in residual sugars. Based on the amino acids, sugars and organic acids, the C/N ratio of the sterileA. cristatum was higher than forB. gracilis. Rhizosphere microbe presence did not result in changes in these C/N ratios. These results suggest thatA. cristatum, with microbes present, will have lower levels of amino acids present, whileB. gracilis, with a lower C/N ratio, will have sugars used to a greater extent by the rhizosphere microbes. This resulted in the higher levels of residual soluble organic C and N in the rhizosphere ofB. gracilis, in comparison with the introducedA. cristatum. These differences may be critical in influencing the course of nutrient accumulation and plant competition in short-grass steppe communities, and in understanding basic aspects of plant-rhizosphere microorganism interactions.     

Glomerular basement membrane proteoglycans are derived from a large precursor

The basement membrane heparan sulfate proteoglycan produced by the Englebreth-Holm-Swarm (EHS) tumor and by glomeruli were compared by immunological methods. Antibodies to the EHS proteoglycan immunoprecipitated a single precursor protein (Mr = 400,000) from [35S]methionine-pulsed glomeruli, the same size produced by EHS cells. These antibodies detected both heparan sulfate proteoglycans and glycoproteins in extracts of unlabeled glomeruli and glomerular basement membrane. The proteoglycans contained core proteins of varying size (Mr = 150,000 to 400,000) with a Mr = 250,000 species being predominant. The glycoproteins are fragments of the core protein which lack heparan sulfate side chains. Antibodies to glomerular basement membrane proteoglycan immunoprecipitated the precursor protein (Mr = 400,000) synthesized by EHS cells and also reacted with most of the proteolytic fragments of the EHS proteoglycan. This antibody did not, however, react with the P44 fragment, a peptide situated at one end of the EHS proteoglycan core protein. These data suggest that the glomerular basement membrane proteoglycan is synthesized from a large precursor protein which undergoes specific proteolytic processing.     

Molecular basis of growth cone adhesion: anchoring of adheron- containing filaments at adhesive loci

Adhesive contacts made by filopodia of neuronal growth cones are essential for proper neurite elongation and may have a role in the formation of synaptic junctions. Previously we described the appearance of filamentous materials extending from growth cone surfaces that seem to be associated with the strongly adhesive behavior of filopodia (Tsui, H.-C., K. L. Lankford, and W. L. Klein. 1985. Proc. Natl. Acad. Sci. USA. 82:8256-8260). Here, we have used immunogold labeling to determine whether known adhesive molecules might be localized at points of adhesion and possibly be constituents of the filamentous material. Antibodies to an adhesive molecule (neural cell adhesion molecule [N-CAM]) and to an adhesive macromolecular complex of proteins and proteoglycans (adheron) were localized at the EM level in whole mounts of cultured avian retina cells. Labeling of fixed cells showed that N-CAM and adheron molecules were both present on growth cones and on filopodia. However, filamentous materials extending from the cell surface were labeled with anti-adheron but not with anti-N-CAM. If cells were labeled before fixation, patches of anti-N-CAM labeling occurred in random areas over the growth cones, but adheron antibodies concentrated at points of apparent adhesion. Particularly dense clustering of anti-adheron occurred at individual filopodial tips and at points of contact between pairs of filopodia. The different patterns of labeling imply that N-CAMS do not associate with the main antigenic components of adheron on the membrane surface. Most importantly, the data indicate the N-CAMs were mobile in the membrane but that constituents of adherons were anchored at adhesive loci. An appealing hypothesis is that molecules found in adheron preparations have an important role in establishing the adhesive junctions formed by growth cone filopodia.     

Tandem duplication and divergence of a sea urchin protein belonging to the troponin C superfamily

The Spec1 and Spec2 proteins of the sea urchin Strongylocentrotus purpuratus are related to calmodulin, troponin C, and myosin light chains by sequence similarity in their four calcium binding domains. These domains, the EF-hands, are distinct helix-loop-helix structures of about 40 amino acids. The Spec1 and Spec2 genes are expressed specifically in aboral ectoderm cells of the developing embryo; however, the function of the Spec proteins in these cells is unknown. To find conserved regions of the proteins that might be important for structure and function, Spec homologues from Lytechinus pictus, a distantly related sea urchin, were sought. L. pictus embryos do not synthesize detectable amounts of the 14,000-17,000-Da Spec proteins as determined by two-dimensional gel electro-phoresis, but do synthesize three 34,000-Da proteins that cross-react with Spec1 antibodies and display a similar ontogenetic pattern of expression. cDNA clones were isolated by hybridization to a synthetic oligonucleotide corresponding to the EF-hand. One clone, LpS1, encodes an mRNA with developmental properties like those of the S. purpuratus Spec mRNAs. However, LpS1 contains an open reading frame for a protein of 34,000 Da rather than 17,000 Da, and antibodies raised against part of the LpS1 reading frame demonstrate that LpS1 encodes a 34,000-Da protein in L. pictus embryos. The sequence of LpS1 reveals the presence of eight EF-hand domains, which share structural homology with the Spec1 or Spec2 EF-hands; however, little else in the protein sequence is conserved. The results support the hypothesis that the LpS1 gene arose from a duplication of an ancestral Spec gene and that the overall structural features of the Spec family of proteins are more conserved than the amino acid sequences.     

Altered amount and activity of superoxide dismutase in sickle cell anemia

The amount and activity of superoxide dismutase (SOD) (EC 1.15.1.1) were measured in red cells collected from 50 white controls, 101 black controls, 50 patients with sickle hemoglobin (SS Hb), 12 with sickle trait, and 11 with other sickling hemoglobinopathies. Red cells from normal black subjects had more SOD amount and activity than normal whites (1.77 U/mg Hb and 2.96 micrograms/mg Hb vs. 1.47 U/mg Hb and 2.64 micrograms/mg Hb, respectively) or blacks with SS Hb or other sickling hemoglobinopathies. Patients with more severe manifestations of SS Hb had lower levels of SOD activity than those with milder symptoms but had the same amount of enzyme protein. Individuals with sickle trait had amounts and activities of SOD comparable to black controls. An alteration in defense to free radical oxygen may play a role in the severity of symptoms experienced by patients with homozygous sickle cell disease.     

Differentiation of the melanotrophic cells of rat pituitary primordium in organotypic culture in defined medium

Summary Organotypic cultures, in defined medium, of pituitary primordia obtained from 15-day-old rat fetuses were performed in order to study the in vitro differentiation of melanotrophic cells. The morphological and ultrastructural features of the transplants resembled those of the gland developing in vivo. In situ hybridization on semi-thin sections, using a ³⁵S-labelled oligonucleotide probe, revealed pro-opiomelanocortin-mRNA-containing cells on the first day of culture in the anterior lobe and after 2–3 days in the intermediate lobe. Immunoperoxidase labelling of adjacent sections showed that the same cells reacted with antibodies against -melanocyte-stimulating hormone (MSH), ₃ and adrenocorticotropic hormone in both lobes. The pro-opiomelanocortin-mRNA-containing cells formed progressively conspicuous areas in the intermediate lobe, which was almost uniformly labelled after 6 days. In the anterior lobe, these cells remained scattered in small cell groups, and colloidal gold immunolabelling showed the progressive disappearance of MSH labelling from the secretory vesicles in cells exhibiting morphological features of adult corticotrophic cells. Both the MSH content of the explants and MSH release into the culture medium increased with time. Treatment with the dopamine agonist bromocriptine induced a strong dose-dependent decrease in MSH secretion, which was significant after 3 days in culture, indicating that dopamine D₂ receptors are able to regulate hormonal release of melanotrophic cells at early stages. This system constitutes a suitable model for further studies of factors controlling cell differentiation and cellular interactions involved in histogenesis.     

Rhabdomeric membrane and smooth endoplasmic reticulum in photoreceptors of Manduca sexta: modulations associated with the diurnal light/dark cycle and effects of chromophore deprivation

Summary Aberrations of photoreceptor ultrastructure resulting from carotenoid/retinoid (vitamin A) deprivation were studied in the retina of Manduca sexta. The syndrome of chromophore deficiency included hypertrophy of smooth endoplasmic reticulum, variable dilation of rhabdomeric microvilli, the insertion of endomembrane fingers into such enlarged microvilli, and the formation of rhabdomeric vacuoles, intracellular compartments containing microvilli similar to those of the rhabdomere. Retinas were processed either with conventional procedures employing preliminary aldehyde fixation followed by heavy metal postfixation, or by fixation and incubation in unbuffered OsO₄. The latter method deposits osmium throughout the endomembrane system, within the rhabdomeric vacuoles, and in the extracellular space of the rhabdom. However, the intravillous fingers were rarely impregnated with osmium, despite their continuity with densely stained cisternae of the smooth endoplasmic reticulum. We suggest that the insertion of endomembrane fingers into dilated microvilli results from a cytoskeleton-mediated link between cisternae of the smooth endoplasmic reticulum and the rhabdomeric membrane, an association that may be important in the turnover of photoreceptor membrane. We interpret endomembrane hypertrophy and development of rhabdomeric vacuoles as symptoms of disturbance in the pathway leading to the assembly of the rhabdomere resulting from reduced synthesis of visual pigment.     

TGA cysteine codons and intron sequences in conserved and nonconserved positions are found in macronuclear RNA polymerase genes of Euplotes octocarinatus.

The gene sequences of the second largest subunits of RNA polymerases I and II of Euplotes octocarinatus, RPA2 and RPB2, were determined and compared to the respective known sequences of Saccharomyces cerevisiae. The similarity of the derived polypeptide sequences permitted their assignment to the respective polymerases and allowed the comparison of the zinc binding regions. In frame TGA codons were detected, which are likely to encode conserved cysteinyl residues in the putative zinc-finger region of the RPA2 gene. They were also found in other positions in both the RPA2 and RPB2 genes. The RPB2 gene contains a 30 bp intron close to the 5'-end of its coding region. The 5'-ends of the coding regions of all three genes encoding the largest subunits of the three different polymerases were also analyzed. The zinc finger structures again show the use of TGA codons for conserved cysteinyl residues in two of the genes. An N-terminal intron is located in the RPB1 gene at a conserved position as compared to the respective genes of several other eucarya.