Prediction of the packing arrangement of strands in β-sheets of globular proteins

A method is proposed for predicting the adjacency order in which strands pack in a -sheet in a protein, on the basis of its amino acid sequence alone. The method is based on the construction of a predicted contact map for the protein, in which the probability that various residue pairs are close to each other is computed from statistically determined average distances of residue pairs in globular proteins of known structure. Compact regions, i.e., portions of the sequence with many interresidue contacts, are determined on the map by using an objective search procedure. The proximity of strands in a -sheet is predicted from the density of contacts in compact regions associated with each pair of strands. The most probable -sheet structures are those with the highest density of contacts. The method has been tested by computing the probable strand arrangements in a five-strand -sheet in five proteins or protein domains, containing 62–138 residues. Of the theoretically possible 60 strand arrangements, the method selects two to eight arrangements as most probable; i.e., it leads to a large reduction in the number of possibilities. The native strand arrangement is among those predicted for three of the five proteins. For the other two, it would be included in the prediction by a slight relaxation of the cutoff criteria used to analyze the density of contacts.     

Cryptococcus neoformans: A central nervous system isolate from an AIDS patient that is rhinotropic in a normal mouse model

A strain of Cryptococcus neoformans that was isolated from the cerebrospinal fluid of a human diagnosed as having acquired immunodeficiency syndrome (AIDS), and that produced cutaneous lesions in experimentally infected, normal mice is described. Although no unusual cutaneous manifestations were noted in the patient's records, this isolate of C. neoformans proved to be dermotropic when injected intravenously into CD-1 mice. The LD₅₀ at 28 days post infection ranged from 3.6–7.5×10⁵ cells per mouse, and in vitro growth rate studies demonstrated that this isolate grew well at 35 °C and at 37 °C, but did not grow at 40 °C and higher. This isolate was rhinotropic producing large granulomatous lesions in the nasal tissues. Other cutaneous tissues affected were the periocular tissues, ears, feet and tail, although the granulomas were nodular in structure and less necrotic than the nasal lesions. The brain, lungs, liver, kidneys and spleen also were culture positive for C. neoformans. Histopathologically, each affected tissue examined had large densities of yeast cells and a chronic, granulomatous host response. Animals surviving the infection appeared to develop a commensal-type relationship with the infective yeast. This is the first report of an isolate of C. neoformans from an AIDS patient that has caused cutaneous manifestations in an animal model. The model described in this report may be useful for elucidating pathogenic mechanisms of cryptococcosis, particularly cutaneous manifestations of the disease.     

Aerial dispersal of the two-spotted spider mite (Tetranychus urticae) from field corn

Field data from 48 plots monitored during diverse weather conditions in two separate years were subjected to multiple regression analysis to determine which factors were related to spider-mite aerial dispersal. With the number of aerially dispersing mites as the dependent variable, partial regression coefficients (b) for mite population density and percent corn-leaf area infested with mites were positive, while those for percentNeozygites-infected mites and hours per week 90% r.h. were negative. When an aerial dispersal index (number of aerially dispersing mites/mites per plant) was used as the dependent variable, the partial regression coefficient for percent leaf area infested was positive, while coefficients for hours per week 90% r.h. and percentNeozygites-infected mites were negative. Mite aerial dispersal was greatest in predator-suppressed field plots under dry weather conditions. Mite aerial dispersal was substantially reduced in plots where moist weather conditions induced epizootics ofNeozygites floridana before corn plants became entirely infested with mites.     

Structure and expression of the cAMP cell-surface receptor

Using antibodies specific for the 3',5'-cyclic AMP (cAMP) cell surface receptor of Dictyostelium discoideum, we have screened lambda gtll expression libraries and isolated a series of cDNAs derived from cAMP receptor mRNA during early development. The identity of the cDNA clones was verified by multiple criteria: 1) beta-galactosidase fusion proteins synthesized by isolated cDNA clones stain intensely with cAMP receptor directed antiserum, 2) these fusion proteins affinity purify antibodies specific for the cAMP receptor, 3) the cDNA probes hybridize to a 2 kb mRNA whose change in relative level of abundance during development parallels that of receptor mRNA as assayed by in vitro translation, 4) the 2 kb mRNA size equals that of receptor mRNA as determined by in vitro translation of size fractionated poly (A)+ RNA, and 5) RNA transcribed in vitro from cDNAs containing the entire protein-coding region produces a polypeptide by in vitro translation with an apparent molecular weight in close agreement with that of nascent cAMP receptor protein produced by in vitro translation of cellular RNA. The DNA sequence predicts an open reading frame of 392 amino acids. The deduced amino acid sequence contains seven domains enriched in hydrophobic residues. A model is proposed in which the cAMP cell-surface receptor traverses the lipid bilayer seven times in a pattern similar to that of other receptors, such as rhodopsin, which interact with G-proteins. The structural similarities suggest a gene family of related surface receptors from such evolutionarily diverse species as Dictyostelium, yeast, and mammals.     

Transformation with a mutant Arabidopsis acetolactate synthase gene renders tobacco resistant to sulfonylurea herbicides

Summary A gene encoding acetolactate synthase was cloned from a chlorsulfuron-resistant mutant of Arabidopsis. The DNA sequence of the mutant gene differed from that of the wild type by a single base pair substitution. When introduced into tobacco by Ti plasmid-mediated transformation the gene conferred a high level of herbicide resistance. These results suggest that the cloned gene may confer agronomically useful levels of herbicide resistnace in other crop species, and that it may be useful as a selectable marker for plant transformation experiments.     

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.