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Biomedical subjects

G B Cox

Publications and source records attributed to G B Cox.

At least 55 records · Page 3Linked to original sources
PubMed1992

A combination of human alpha 1 and beta 1 subunits is required for formation of detectable GABA-activated chloride channels in Sf9 cells.

The baculovirus expression system was used to produce alpha 1 and beta 1 subunits of the human GABAA receptor in Sf9 cells. In cells infected with both alpha 1 and beta 1 recombinant viruses, GABA elicited an outwardly rectifying chloride current that was blocked by bicuculline and potentiated by pentobarbitone. GABA did not produce detectable currents in cells infected with either alpha 1 or beta 1 recombinant viruses alone. In these cells, and in control (non-infected) Sf9 cells, pentobarbitone depressed the leakage current (Ki = 55 microM). Fluorescently labelled monoclonal antibodies to the alpha 1 subunit showed greater amounts of the alpha 1 subunit in cells infected with only the alpha 1 recombinant virus than in cells co-infected with the alpha 1 and beta 1 recombinant viruses. Fluorescence of the plasma membrane was seen in cells co-infected with the alpha 1 and beta 1 recombinant viruses, but was absent in cells infected with only the alpha 1 recombinant virus. It was concluded that the alpha 1 subunit normally interacts with the beta 1 subunit to be transported to the plasma membrane in Sf9 cells.

Animals↗
PubMed1992

Mutational analysis of the Escherichia coli phosphate-specific transport system, a member of the traffic ATPase (or ABC) family of membrane transporters. A role for proline residues in transmembrane helices.

The Escherichia coli Pst system is a periplasmic phosphate permease. A mutational analysis of the requirement for function of specific charged residues or proline residues in the two hydrophobic subunits (PstC and PstA) has been carried out. No residues, among 19 charged residues altered, were found to be essential for phosphate uptake, although some alterations resulted in partial effects. Evidence was obtained that the 3 residues, R220 in the PstA protein and R237 and E241 in the PstC protein, previously shown to be required for phosphate transport (Cox, G. B., Webb, D., Godovac-Zimmermann, J., and Rosenberg, H. (1988) J. Bacteriol. 170, 2283-2286; Cox, G. B., Webb, D., and Rosenberg, H. (1989) J. Bacteriol. 171, 1531-1534), interact with each other. A feature of the proposed structures of the PstA and PstC proteins was 2 pairs of proline residues in putative transmembrane helices 3 and 4. While individual substitutions of these proline residues by leucine resulted in loss of phosphate transport activity substitution by alanine only had partial effects. However, if the proline to alanine changes were paired then, depending on the particular subunit, markedly different effects were obtained. The double mutation in the PstA protein resulted in a permanently "closed" system, whereas the double mutation in the PstC protein resulted in a permanently "open" transport system.

ATP-Binding Cassette Transporters↗
PubMed1992

The expression, purification and crystallization of the epsilon subunit of the F1 portion of the ATPase of Escherichia coli.

The epsilon subunit of the F0F1-ATPase from Escherichia coli has been expressed in E. coli as a fusion protein with glutathione S-transferase from the parasitic helminth Schistosoma japonicum. The epsilon subunit released by thrombin treatment of the purified fusion protein carried two amino acid changes, A1G and M2S, and was obtained in a yield of about five milligrams per litre of cultured cells. The two amino acid changes were shown not to affect function. The protein has been crystallized in a form suitable for X-ray diffraction structure analysis. The crystals are hexagonal, space group P6(1)22 (or P6(5)22), with a = b = 94.9 A, c = 57.1 A and gamma = 120 degrees. The diffraction from small crystals extends to at least 2.9 A resolution.

Crystallization↗
PubMed1992

Second-site revertants of an arginine-210 to lysine mutation in the a subunit of the F0F1-ATPase from Escherichia coli: implications for structure.

Arg-210 of the a subunit of the Escherichia coli F0F1-ATPase has been proposed previously as a component of the proton pore. A mutant in which lysine was substituted for Arg-210 was generated and was found to be unable to translocate protons. A plasmid carrying this mutation, along with wild-type genes encoding the c and b subunits, was unusual in that it failed to complement a chromosomal c-subunit mutation on succinate minimal medium. Three revertants on succinate minimal medium contained plasmids that showed complementation with chromosomal c-subunit but not with a-subunit mutations. One of these had a deletion in the a subunit. The other two were point mutations, resulting in the substitution of aspartic acid by Gly-53 and of arginine for Leu-211. The Gly-53 to aspartic acid change implied that Gly-53 and Arg-210 are normally in close proximity. To test this idea further, a series of mutants in which aspartic acid was placed in helix I at positions ranging from 42 to 57 was generated. Full complementation was regained only when the aspartic acid residue was present on the same side of a putative helix as Gly-53 over a span of three turns of the alpha-helix. These results and others suggest modifications of a previously proposed model for the transmembrane helices of the F0 portion of the F0F1-ATPase. The implications of these modifications for the mechanism of proton translocation are discussed.

Arginine↗
PubMed1992

Influence of operating parameters on the preparative gradient elution chromatography of insulins.

The mass-overloaded separation of bovine and porcine insulins has been studied in the reversed-phase gradient elution mode. Strong solute-solute displacement effects have been found, which are related to the efficiency of the column used. Low flow-rates and small particle diameters maximise the displacements, as well as improve the resolution between the parent insulin and its desamido contaminant. The gradient slope did not substantially affect the separation between the parent insulins, but, due to the relative "S" values of the solutes, an increase in gradient slope improved the separation of the parent insulin from its desamido compound. An optimum pore size of 150 A was found for the insulins. Experiments to optimise the loadability were not carried out, but a recovery of 90% at a purity of 99.5% was obtained with a loading of 12 mg/g of porcine insulin.

Animals↗
PubMed1991

The chloroplast beta-subunit allows assembly of the Escherichia coli F0 portion of the energy transducing adenosine triphosphatase.

The effect of the expression of the chloroplast F1-ATPase beta-subunit in two Escherichia coli beta-subunit mutant strains was investigated. The amount of chloroplast beta-subunit formed in E. coli was increased by introducing a 'Shine-Dalgarno' sequence upstream from the translation start site. The chloroplast beta-subunit was membrane bound but was unable to functionally replace the mutant beta-subunit in a strain carrying the uncD409 allele [corrected]. However, in an E. coli mutant strain unable to form the beta- and epsilon-subunits the presence of the chloroplast beta-subunit enabled the assembly of a functional proton pore [corrected]

Base Sequence↗
PubMed1991

Separation and purification of oligonucleotides using a new bonded-phase packing material.

We describe a new bonded-phase packing material, based upon surface-stabilised microparticulate silica, suitable for the rapid separation and purification of oligonucleotides. Columns packed with this material were demonstrated to give rapid separations of individual oligonucleotide species of up to 44 base units with high purity; agarose gel electrophoresis showed that the products were essentially single bands, with only trace quantities of the (n-1)-mer present. Baseline resolution of the desired oligomer from (n +/- 1)-mer was achieved under preparative loading conditions, where up to 200-300 micrograms of oligonucleotide could be separated. The separation was essentially independent of structure or sequence of the oligonucleotides. The retention mechanism of the oligonucleotides was investigated, and the results used to determine the optimum column configuration and separation conditions.

Base Sequence↗
PubMed1991

Morning or evening bright light treatment of winter depression? The significance of hypersomnia.

In a randomized crossover design 19 patients with winter depression were treated with 7 days of bright morning light (6:00 to 8:00 AM) and 7 days of evening light (7:00 to 9:00 PM). Bright light in the morning reduced the Hamilton Depression Rating Scale score from 22.3 to 5.5; bright light in the evening decreased the Hamilton score from 21.0 to 12.2. Improvement in the depression as measured by the Hamilton Depression Rating scores was greater with morning light compared with evening lights. Hypersomnia was associated (p less than 0.05) with a superior response to morning light.

Adult↗
PubMed1990

The chloroplast CF0I subunit can replace the b-subunit of the F0F1-ATPase in a mutant strain of Escherichia coli K12.

The amino acid sequence of the CF0I subunit from the chloroplast F0F1-ATPase has only a low similarity to the amino acid sequence of the b-subunit of the E. coli F0F1-ATPase. However, secondary and tertiary structure predictions plus the distribution of hydrophobic and hydrophilic amino acids have indicated that these two subunits serve a similar function. This proposition was investigated directly. A cDNA clone for the chloroplast atpF gene, encoding the CF0I subunit, was altered by site-directed mutagensis such that the translation start site corresponded to the N-terminus of the mature protein. An E. coli mutant strain carrying a chain-terminating mutation in the uncF gene, encoding the b-subunit, was transformed with the plasmid carrying the altered atpF gene. The resultant transformant was able to grow on succinate and gave a growth yield similar to that of a wild-type control. Assays on membrane preparations from the transformant also clearly indicated that the mature CF0I subunit from spinach chloroplasts was able to replace the E. coli b-subunit in the E. coli F0F1-ATPase.

Amino Acid Sequence↗
PubMed1990

Mutational analysis of the function of the a-subunit of the F0F1-APPase of Escherichia coli.

In a model proposed for the structure of the a-subunit of the Escherichia coli F0F1-ATPase (Howitt, S.M., Gibson, F. and Cox, G.B. (1988) Biochim. Biophys. Acta 936, 74-80), a cluster of charged residues, including one arginine and four aspartic acid residues, lie on the periplasmic side of the membrane. On the cytoplasmic side, three pairs of lysine residues and an arginine residue are present. Site-directed mutagenesis was used to investigate the roles of these residues. It was found that none was directly involved in the proton pore. However, the substitutions of Asp-124 or Asp-44 by asparagine or Arg-140 by glutamine had similar effects in that the membranes from such mutants from which the F1-ATPase was removed were proton-impermeable. A combination of the Asp-44 mutation with either the Asp-124 or Arg-140 mutations in the same strain resulted in complete loss of oxidative phosphorylation. It was tentatively concluded that Asp-124 and Arg-140 form a salt bridge, as did Asp-44 with an unknown residue, and these salt bridges were concerned with the maintenance of correct a-subunit structure. Further support for this conclusion was obtained when second site revertants of a Glu-219 to histidine mutant were found to have either histidine or leucine replacing Arg-140. Thus, the lack of the Asp-124/Arg-140 salt bridge might enable repositioning of the helices of the a-subunit such that His-219 becomes a functional component of the proton pore.

Base Sequence↗
PubMed1990

Bright light treatment of winter depression: morning versus evening light.

In a randomized crossover design, 7 patients with winter depression were treated with 7 d of bright morning light (0600 to 0800) and 7 d of evening light (2000 to 2200). Bright lights in the morning significantly reduced the Hamilton Rating Scale for Depression (HRSD) score (18.4 to 5.0); the bright light in the evening moderately decreased the HRSD score (19.4 to 15.1). The improvement in the HRSD score was significantly greater with morning light than with evening light.

Adult↗
PubMed1989

Ethnic populations: community mental health services ten years later.

In a replication of a series of studies conducted by Sue and colleagues in the mid-1970s, demographic and service data were retrieved for the Seattle-King County area from the Washington Mental Health Information System. Caucasian clients were compared against Asian, black, Hispanic, and Native American client groups, and, where possible, against the findings reported earlier by Sue. These clients were compared in terms of basic demographic characteristics, characteristics of staff providing the services, dropout rates, and average number of services received. The most notable findings are (a) that failure-to-return rates are dramatically lower for the current sample than for Sue's and not greatly different for minorities than for Caucasians, (b) that variability in failure-to-return rates is most strongly related to level of functioning and not related to minority status, and (c) that although Asian Americans still average fewer services than Caucasians (other minorities do not differ significantly), the mean number of services had increased substantially for all groups but more for minorities than for Caucasians.

Adult↗
PubMed1989

Specific amino acid residues in both the PstB and PstC proteins are required for phosphate transport by the Escherichia coli Pst system.

Three mutant alleles of the pstC gene and one mutant allele of the pstB gene were produced by site-directed mutagenesis. The pstC gene encodes an integral membrane protein of the phosphate-specific transport (Pst) system of Escherichia coli. The amino acid substitutions resulting from the pstC gene mutations, Arg-237----Gln, Glu-240----Gln, or a combination of both, caused the loss of phosphate transport through the Pst system, but the alkaline phosphatase activity remained repressed. The pstB gene encodes a peripheral membrane protein of the Pst system which carries a putative nucleotide-binding site. The amino acid substitutions Gly-48----Ile and Lys-49----Gln, resulting from the pstB mutations, caused the loss of phosphate transport through the Pst system and the derepression of alkaline phosphatase activity. The residues Gly-48 and Lys-49 are key residues in the putative nucleotide-binding site.

Alkaline Phosphatase↗
PubMed1988

The proton pore of the F0F1-ATPase of Escherichia coli: Ser-206 is not required for proton translocation.

A series of experiments was carried out to investigate the role of some polar amino acids in the a-subunit of the ATP synthase of Escherichia coli. Site-directed mutagenesis resulted in the amino acid substitutions Ser-199----Ala, Ser-202----Ala, Ser-206----Ala, Arg-61----Gln or Asp-44----Asn. None of these amino acid substitutions affected the ability of the cells to carry out oxidative phosphorylation. It was concluded therefore that the effect of the substitution of leucine for Ser-206 reported previously (Cain, B.D. and Simoni, R.D. (1986) J. Biol. Chem. 261, 10043-10050) was due to the presence of the leucine rather than the absence of serine. Even though cells carrying the Asp-44----Asn substitution were able to carry out oxidative phosphorylation, membranes from such cells remained proton-impermeable after removal of the F1-ATPase. It appears likely that the proton pore of the F0 of the ATP synthase of E. coli consists of four amino acids, namely Arg-219, Glu-210 and His-245 of the a-subunit and Asp-61 of the c-subunit.

Alanine↗
PubMed1988

Preparative separation of peptide and protein samples by high-performance liquid chromatography with gradient elution. I. The Craig model as a basis for computer simulations.

The Craig model (assuming a Langmuir isotherm) has been used by us previously to successfully simulate isocratic high-performance liquid chromatographic (HPLC) separation in a mass-overload mode. Here we have extended this approach to the case of gradient elution for large samples. These simulations support our earlier conclusion that so-called "corresponding" isocratic and gradient separations provide similar sample resolution when the sample size is the same. "Corresponding" separations refer to the case where isocratic retention k' is equal to average gradient retention k, and where other conditions (column, flow-rate, etc.) are the same. Craig simulations reported here also provide further insight into the factors that affect preparative HPLC separations under mass-overload conditions.

Chromatography, High Pressure Liquid↗
PubMed1988

Preparative separation of peptide and protein samples by high-performance liquid chromatography with gradient elution. II. Experimental examples compared with theory.

Craig simulations of mass-overloaded gradient elution reported in the preceding paper have been extended to the case of non-Langmuir isotherms. Isotherms were selected that appear to be characteristic of peptide and protein samples in reversed-phase high-performance liquid chromatography. The dependence of bandwidth on sample size and gradient conditions was examined by Craig simulation and compared with experimental data for 13 different experimental systems involving four different proteins. There is a good correspondence between simulations and experimental data, and it seems possible to quantitatively predict bandwidth and resolution as a function of small-sample retention data, experimental conditions, and sample size. A systematic approach for designing the preparative or process-scale separation of protein mixtures by reversed-phase gradient elution is proposed.

Chemical Phenomena↗
PubMed1988

Community mental health staff utilization in Washington State: characteristics and target groups.

This study addresses human resource development issues for the State of Washington by examining relationships among clinical staff characteristics such as sex, educational level, discipline of academic study, race-ethnic group, and years of mental health treatment experience, and relationships between these staff characteristics and characteristics of staff caseloads, primarily percent of caseload falling into various state priority populations: chronically mentally ill, children, elderly, and minority groups. Results are interpreted in terms of past and future state goals for the mental health service system.

Chronic Disease↗