A conserved proline-rich region of the Saccharomyces cerevisiae cyclase-associated protein binds SH3 domains and modulates cytoskeletal localization.

Saccharomyces cerevisiae cyclase-associated protein (CAP or Srv2p) is multifunctional. The N-terminal third of CAP binds to adenylyl cyclase and has been implicated in adenylyl cyclase activation in vivo. The widely conserved C-terminal domain of CAP binds to monomeric actin and serves an important cytoskeletal regulatory function in vivo. In addition, all CAP homologs contain a centrally located proline-rich region which has no previously identified function. Recently, SH3 (Src homology 3) domains were shown to bind to proline-rich regions of proteins. Here we report that the proline-rich region of CAP is recognized by the SH3 domains of several proteins, including the yeast actin-associated protein Abp1p. Immunolocalization experiments demonstrate that CAP colocalizes with cortical actin-containing structures in vivo and that a region of CAP containing the SH3 domain binding site is required for this localization. We also demonstrate that the SH3 domain of yeast Abp1p and that of the yeast RAS protein guanine nucleotide exchange factor Cdc25p complex with adenylyl cyclase in vitro. Interestingly, the binding of the Cdc25p SH3 domain is not mediated by CAP and therefore may involve direct binding to adenylyl cyclase or to an unidentified protein which complexes with adenylyl cyclase. We also found that CAP homologous from Schizosaccharomyces pombe and humans bind SH3 domains. The human protein binds most strongly to the SH3 domain from the abl proto-oncogene. These observations identify CAP as an SH3 domain-binding protein and suggest that CAP mediates interactions between SH3 domain proteins and monomeric actin.     

Isolation of the MurineNbr1Gene Adjacent to the MurineBrca1Gene

The humanNBR1cDNA has previously been identified using polyclonal sera to CA125, an ovarian tumor antigen used in monitoring ovarian cancer. The gene was mapped to theBRCA1region on chromosome 17q21 and subsequently found to lie in close proximity to the recently identifiedBRCA1gene. The NBR1 protein has a B-box motif but the function of the protein is as yet unknown. To investigate the function and importance of this gene, we have studied the conservation of this gene in other species and in particular in the mouse. We have isolated murineNbr1cDNA and genomic clones. Translation of the cDNA sequence indicates that the protein is highly conserved, being 89% similar and 84% identical to the human. Analysis of the murineNbr1genomic clones indicates that it maps less than 1 kb from theBrca1gene and that, unlike that in human, this region is not duplicated.     

Phosphorylation of glucose by a guanosine-5′-triphosphate (GTP)-dependent glucokinase in Fibrobacter succinogenes subsp. succinogenes S85

Cell extracts of Fibrobacter succinogenes subsp. succinogenes S85 phosphorylated glucose with a GTP-dependent glucokinase. The enzyme showed little activity with ATP (12% of that with GTP). Of other phosphate donors tested, only dGTP and ITP gave high glucokinase activities. Dialyzed extracts required Mg⁺² and K⁺ for maximal activity. In potassium phosphate buffer, glucokinase showed maximum activity at pH 7.5 with glucose-6-phosphate dehydrogenase as the coupling enzyme. In this assay, glucokinase was active with glucose (100%), 2-deoxy-d-glucose (40%), and mannose (20%). Partially purified glucokinase had a molecular weight of 82,000 and a pl of 4.82. Double-reciprocal plots of substrate concentration versus velocity were linear and the enzyme had apparent K_m values of 55 M for glucose and 72 M for GTP. Dialyzed cell extracts of Fibrobacter intestinalis C1A also contained a GTP-dependent glucokinase that showed little activity with ATP. Potassium also stimulated the activity of this enzyme. These results suggest that this unusual glucokinase may be characteristic of the genus Fibrobacter.Abbreviations CHES cyclohexylaminoethanesulfonic acid - GK glucokinase - PEP phosphoenolpyruvate Published with the approval of the Director of the North Dakota Agricultural Experiment Station as journal article no. 2186     

Influence of Plumbing Materials on Biofilm Formation and Growth of Legionella pneumophila in Potable Water Systems

A two-stage chemostat model of a plumbing system was developed, with tap water as the sole nutrient source. The model system was populated with a naturally occurring inoculum derived from an outbreak of Legionnaires' disease and containing Legionella pneumophila along with associated bacteria and protozoa. The model system was used to develop biofilms on the surfaces of a range of eight plumbing materials under controlled, reproducible conditions. The materials varied in their abilities to support biofilm development and the growth of L. pneumophila. Elastomeric surfaces had the most abundant biofilms supporting the highest numbers of L. pneumophila CFU; this was attributed to the leaching of nutrients for bacterial growth from the materials. No direct relationship existed between total biofouling and the numbers of L. pneumophila CFU.     

EVOLUTIONARY INTERACTIONS BETWEEN SEXUAL AND ALL-FEMALE TAXA OF CYPRINOTUS (OSTRACODA: CYPRIDIDAE)

Freshwater ostracodes show both an exceptionally high incidence of transitions to unisexuality and, in some cases, an extraordinary level of clonal diversity. There is no understanding of the agents promoting these transitions to thelytoky, although it has been suggested that their frequency may set the stage for sexual taxa to infuse clonal diversity into unisexuals. This study examines the nature and origins of clonal diversity in the unisexual ostracode Cyprinotus incongruens. A combination of allozyme and cytogenetic studies revealed the presence of two diploid clones of this species at three temperate sites and ten clones at one arctic site including three diploids, five triploids, and two tetraploids. The low heterozygosity (0%–20%) of its diploid clones suggests that parthenogenesis has arisen spontaneously in C. incongruens rather than through hybridization, as in vertebrate asexuals. Polyploid clones appear to owe their origin to genome additions from sexual taxa, although subsequent mutational divergence has played a role in further enhancing diversity. Two triploid clones have apparently originated from the incorporation of a haploid genome from the sexually reproducing C. glaucus, as evidenced by their high heterozygosity and possession of alleles otherwise found only in that species. Other polyploid clones have likely arisen as a result of interbreeding between bisexual and unisexual C. incongruens. These results suggest that both the incidence of spontaneous transitions to clonality and the frequency of interbreeding with relatives may be the key processes that govern clonal diversity in unisexual ostracodes.     

Feasibility of expanded granular sludge bed reactors for the anaerobic treatment of low-strength soluble wastewaters

The application of the expanded granular sludge bed (EGSB) reactor for the anaerobic treatment of low-strength soluble wastewaters using ethanol as a model substrate was investigated in laboratory-scale reactors at 30oC. Chemical oxygen demand (COD) removal efficiency was above 80% at organic loading rates up to12 g COD/L . d with influent concentrations as low as 100 to 200 mg COD/L. These results demonstrate the suitability of the EGBS reactor for the anaerobic treatment of low-strength wastewaters. The high treatment performance can be attributed to the intense mixing regime obtained by high hydraulic and organic loads. Good mixing of the bulk liquid phase for the substrate-biomass contact and adequate expansion of the substrate-biomass contact and adequate expansion of the sludge bed for the degassing were obtained when the liquid upflow velocity (V(up)) was greater than 2.5 m/h. Under such conditions, an extremely low apparent K(s) value for acetoclastic methanogenesis of 9.8 mg COD/L was observed. The presence of dissolved oxygen in the wastewater had no detrimental effect on the treatment performance. Sludge piston flotation from pockets of biogas accumulating under the sludge bed occurred at V(up) lower than 2.5 m/h due to poor bed expansion. This problem is expected only in small diameter laboratory-scale reactors. A. more important restriction of the EGSB reactor was the sludge washout occurring at V(up) higher than 5.5 m/h and which was intensified at organic loads higher than 7 g COD/L. d due to buoyancy forces from the gas production. To achieve an equilibrium between the mixing intensity and the sludge hold-up, the operation should be limited to an organic loading rate of 7 g COD/L d. and to a liquid up-flow velocity between 2.5 and 5.5 m/h (c) 1994 John Wiley & Sons, Inc.     

Bioluminescent Salmonella typhimurium provides a rapid assay for measuring the efficacy of freeze-drying suspension media

A strain of Salmonella typhimurium , transformed to a bioluminescent phenotype, was used to compare three freeze-drying suspension media: inositol serum broth with and without added gelatin and sterile skimmed milk. Recovery and growth studies performed by measuring changes in bioluminescence demonstrated that of the three media tested, the routinely used inositol serum broth was the most effective freeze-drying suspension medium.     

Distribution of the enantiomers of hydroxychloroquine and its metabolites in ocular tissues of the rabbit after oral administration of racemic-hydroxychloroquine

The disposition of the enantiomers of hydroxychloroquine (HCQ) and its major metabolites in ocular tissues of rabbits has been studied. Both albino, New Zealand White (NZW), and pigmented animals were administered daily oral doses of rac-HCQ, (S)-HCQ or (R)-HCQ (20 mg/kg) over 1, 6, or 8 day periods or for 8 days followed by a 7-day washout period. At the end of the study periods, plasma and whole blood samples were collected and the rabbits were sacrificed. The eyes were collected, the aqueous humor removed with a syringe, and the eyes separated into the cornea, lens, vitreous body, iris, choroid-retina, sclera, and conjunctiva. The concentrations of (R)-HCQ, (S)-HCQ, and their respective metabolites were determined using a validated enantioselective liquid chromatographic assay. The data from these studies indicate that HCQ accumulated in both pigmented and nonpigmented ocular tissues. In the pigmented tissues, HCQ and its metabolites were bound to melanin and the binding was not enantiospecific. In the nonpigmented tissues and in the iris and retina-choroid of the NZW rabbits, the accumulation appeared to be the result of a reversible and enantioselective binding of HCQ and its metabolites to an unidentified biopolymer present in these ocular tissues. © 1994 Wiley-liss, Inc.