Search PubMed⌕ Search

Biomedical subjects

K D Young

Publications and source records attributed to K D Young.

At least 37 records · Page 2Linked to original sources
PubMed1995

Identification and cloning of the gene encoding penicillin-binding protein 7 of Escherichia coli.

Penicillin-binding protein (PBP) 7 of Escherichia coli is a poorly characterized member of the family of enzymes that synthesize and modify the bacterial cell wall. The approximate chromosomal position of the gene encoding this protein was determined by measuring the expression of PBPs during lytic infection of E. coli by each of the 476 miniset members of the Kohara lambda phage genomic library. Phages lambda 363 and lambda 364, encompassing the region from 47.7 to 48 min of the chromosome, overproduced PBP 7. One open reading frame, yohB, was present on both these phages and directed the expression of PBPs 7 and 8. The predicted amino acid sequence of PBP 7 contains the consensus motifs associated with other PBPs and has a potential site near the carboxyl terminus where proteolysis by the OmpT protein could occur, creating an appropriately sized PBP 8. The PBP 7 gene (renamed pbpG) was interrupted by insertion of a kanamycin resistance gene cassette and was moved to the chromosome of E. coli. No obvious growth defects were observed, suggesting that PBP 7 is not essential for growth under normal laboratory conditions.

Amino Acid Sequence↗
PubMed1994

slyD, a host gene required for phi X174 lysis, is related to the FK506-binding protein family of peptidyl-prolyl cis-trans-isomerases.

Recessive mutations in the slyD gene were isolated by selecting for survival after induction of the cloned lysis gene E of bacteriophage phi X174 (Maratea, D., Young, K., and Young, R. (1985) Gene (Amst.) 40, 39-46). The slyD1 mutation, transduced into the normal phi X174 host, Escherichia coli C, confers an absolute block on the plaque-forming ability of the wild-type phage, indicating that slyD is required for E function rather than for expression from the plasmid vector. The cloning, sequencing, and deletion analysis of a 1-kilobase pair genomic fragment containing the slyD locus, mapping at 73.5', is reported. Three reading frames, orf72, orf159, and orf196, are contained within this fragment, with the latter two reading frames occupying the same DNA on opposite strands. Deletion analysis shows that the complementing activity is restricted to the orf159/orf196 DNA. Complementation of the SlyD phenotype was observed irrespective of the orientation of the orf159/orf196 DNA with respect to a vector promoter, indicating that a cryptic promoter serves slyD on this fragment. Using site-directed mutagenesis, nonsense mutations were created in each reading frame which were silent in the opposing frame. Both orf196 nonsense alleles failed to complement slyD1, whereas both orf159 nonsense alleles retained complementation, demonstrating rigorously that orf196 is slyD. A segment corresponding to the first 150 residues of the predicted SlyD protein has significant similarity throughout its length to the FKBP family of peptidyl-prolyl cis-trans-isomerases or rotamases. The COOH-terminal 46 codons of slyD encode a remarkable histidine-rich peptide sequence which is at least partly dispensable for slyD function in E-mediated lysis. Overexpression of slyD in E. coli is toxic. These findings are discussed in terms of a model for SlyD involvement in E function and in terms of a model for SlyD involvement of the ubiquitous FKBP rotamases.

Alleles↗
PubMed1994

Sequence divergence of the murB and rrfB genes from Escherichia coli and Salmonella typhimurium.

The murB gene of Salmonella typhimurium was cloned and found to be 75% and 82% identical to the DNA and protein sequences, respectively, of the same gene in Escherichia coli. These identities are among the lowest recorded between the two bacteria. Nevertheless, wild-type S. typhimurium murB complemented the known temperature-sensitive E. coli mutant, and wild-type E. coli murB complemented three temperature-sensitive mutants of S. typhimurium. The 5S rRNA gene, rrfB, and the region between murB and rrfB were also cloned and sequenced. The rrfB gene of S. typhimurium differs from rrfB of E. coli in only 2 of 120 nt, but the region between murB and rrfB has diverged greatly and includes a sequence that closely resembles a repetitive extragenic palindrome of the type normally associated with E. coli. Previous comparisons of gene divergence have suggested that the chromosomal mutation rate is lower in the vicinity of the origin of replication. However, the S. typhimurium murB gene, located 6 map minutes from the origin of replication, is highly substituted at synonymous sites and the sequence between murB and rrfB is significantly modified as well. Thus, murB is an exception to the general observation that genes near the origin of replication show less divergence than do genes elsewhere in the bacterial chromosome.

Amino Acid Sequence↗
PubMed1994

Artifactual processing of penicillin-binding proteins 7 and 1b by the OmpT protease of Escherichia coli.

Penicillin-binding proteins (PBPs) were visualized in strains of Escherichia coli that carried mutations in one or more of the following protease genes: tsp, degP, ptr, and ompT. In the absence of a functional ompT gene, PBPs 1b alpha and 7 were not processed to the shortened forms 1b beta and 8, respectively. Cleavage of PBPs 1b alpha and 7 could be restored by introduction of a plasmid carrying the wild-type ompT gene. These PBPs were processed only after cell lysis or after membrane perturbation of whole cells by freeze-thaw, suggesting that the cleavage was a nonspecific artifact due to contact with OmpT, an outer membrane protease, and that such processing was not biologically significant in vivo. The degradation of other PBPs during purification or storage may also be effected by OmpT.

Artifacts↗
PubMed1994

Characterization of the desulfurization genes from Rhodococcus sp. strain IGTS8.

Rhodococcus sp. strain IGTS8 possesses an enzymatic pathway that can remove covalently bound sulfur from dibenzothiophene (DBT) without breaking carbon-carbon bonds. The DNA sequence of a 4.0-kb BstBI-BsiWI fragment that carries the genes for this pathway was determined. Frameshift and deletion mutations established that three open reading frames were required for DBT desulfurization, and the genes were designated soxABC (for sulfur oxidation). Each sox gene was subcloned independently and expressed in Escherichia coli MZ1 under control of the inducible lambda pL promoter with a lambda cII ribosomal binding site. SoxC is an approximately 45-kDa protein that oxidizes DBT to DBT-5,5'-dioxide. SoxA is an approximately 50-kDa protein responsible for metabolizing DBT-5,5'-dioxide to an unidentified intermediate. SoxB is an approximately 40-kDa protein that, together with the SoxA protein, completes the desulfurization of DBT-5,5'-dioxide to 2-hydroxybiphenyl. Protein sequence comparisons revealed that the predicted SoxC protein is similar to members of the acyl coenzyme A dehydrogenase family but that the SoxA and SoxB proteins have no significant identities to other known proteins. The sox genes are plasmidborne and appear to be expressed as an operon in Rhodococcus sp. strain IGTS8 and in E. coli.

Amino Acid Sequence↗
PubMed1994

Effect of Donjoy Ankle Ligament Protector and Aircast Sport-Stirrup orthoses on functional performance.

Clinicians often are faced with the task of selecting an ankle orthosis that will provide protection against ankle sprain injury and will not have a deleterious effect on functional performance. The purpose of this study was to compare the effects of the DonJoy Ankle Ligament Protector (ALP) and the Aircast Sport-Stirrup (AS) on three functional performance tasks: the 40-m sprint run, the figure-of-eight run, and the standing vertical jump. Subjects were eight males and eight females who reported no history of ankle injury during the 6 months prior to testing; neurological condition; lower extremity surgery or pathology; or cardiac, pulmonary, vascular, or balance problems. Each subject performed all functional tasks on the first test day with both ankles unbraced and with one randomly selected ankle braced with one of the two orthoses. Each subject returned for testing on a second day and performed all tasks with both ankles unbranced and with the other orthosis on the previously selected ankle. Subjects also rated the orthoses for comfort and support. An ANOVA indicated that braced data were not significantly different than unbraced data and that data for the two orthoses did not differ significantly for any of the functional performance tasks. Seventy-five percent of the subjects reported the AS was more comfortable than the ALP, and 63% of the subjects indicated that the ALP provided more support than the AS. Neither orthosis had a deleterious effect on functional performance. The results may assist clinicians in selecting either of these orthoses for use in protection against ankle sprain injury.

Adolescent↗
PubMed1993

Identification and Cloning of Genes Involved in Specific Desulfurization of Dibenzothiophene by Rhodococcus sp. Strain IGTS8.

The gram-positive bacterium Rhodococcus sp. strain IGTS8 is able to remove sulfur from certain aromatic compounds without breaking carbon-carbon bonds. In particular, sulfur is removed from dibenzothiophene (DBT) to give the final product, 2-hydroxybiphenyl. A genomic library of IGTS8 was constructed in the cosmid vector pLAFR5, but no desulfurization phenotype was imparted to Escherichia coli. Therefore, IGTS8 was mutagenized, and a new strain (UV1) was selected that had lost the ability to desulfurize DBT. The genomic library was transferred into UV1, and several colonies that had regained the desulfurization phenotype were isolated, though free plasmid could not be isolated. Instead, vector DNA had integrated into either the chromosome or a large resident plasmid. DNA on either side of the inserted vector sequences was cloned and used to probe the original genomic library in E. coli. This procedure identified individual cosmid clones that, when electroporated into strain UV1, restored desulfurization. When the origin of replication from a Rhodococcus plasmid was inserted, the efficiency with which these clones transformed UV1 increased 20- to 50-fold and they could be retrieved as free plasmids. Restriction mapping and subcloning indicated that the desulfurization genes reside on a 4.0-kb DNA fragment. Finally, the phenotype was transferred to Rhodococcus fascians D188-5, a species normally incapable of desulfurizing DBT. The mutant strain, UV1, and R. fascians produced 2-hydroxybiphenyl from DBT when they contained appropriate clones, indicating that the genes for the entire pathway have been isolated.

Journal Article↗
PubMed1993

Metabolism of dibenzothiophene and naphthalene in Pseudomonas strains: complete DNA sequence of an upper naphthalene catabolic pathway.

From a soil isolate, Pseudomonas strain C18, we cloned and sequenced a 9.8-kb DNA fragment that encodes dibenzothiophene-degrading enzymes. Nine open reading frames were identified and designated doxABDEFGHIJ. Collectively, we refer to these genes as the DOX pathway. At the nucleotide level, doxABD are identical to the ndoABC genes that encode naphthalene dioxygenase of Pseudomonas putida. The DoxG protein is 97% identical to NahC (1,2-dihydroxynaphthalene dioxygenase) of P. putida. DoxE has 37% identity with cis-toluene dihydrodiol dehydrogenase. DoxF is similar to the aldehyde dehydrogenases of many organisms. The predicted DoxHIJ proteins have no obvious sequence similarities to known proteins. Gas chromatography with a flame ionization detector and mass spectroscopy confirmed that the DOX proteins convert naphthalene to salicylate and converting phenanthrene to 1-hydroxy-2-naphthoic acid. doxI mutants convert naphthalene to trans-o-hydroxybenzylidenepyruvate, indicating that the DoxI protein is similar to NahE (trans-o-hydroxybenzylidenepyruvate hydratase-aldolase). Comparison of the DOX sequence with restriction maps of cloned naphthalene catabolic pathway (NAH) genes revealed many conserved restriction sites. The DOX gene arrangement is identical to that proposed for NAH, except that the NAH equivalent of doxH has not been recognized. DoxH may be involved in the conversion of 2-hydroxy-4-(2'-oxo-3,5-cyclohexadienyl)-buta-2,4-dienoat e to cis-o-hydroxybenzylidenepyruvate. doxJ encodes an enzyme similar to NahD (isomerase). Our findings indicate that a single genetic pathway controls the metabolism of dibenzothiophene, naphthalene, and phenanthrene in strain C18 and that the DOX sequence encodes a complete upper naphthalene catabolic pathway similar to NAH.

Amino Acid Sequence↗
PubMed1991

Cell cycle-independent lysis of Escherichia coli by cefsulodin, an inhibitor of penicillin-binding proteins 1a and 1b.

Cefsulodin lyses actively growing Escherichia coli by binding specifically to penicillin-binding proteins (PBPs) 1a and 1b. Recent findings (F. García del Portillo, M. A. de Pedro, D. Joseleau-Petit, and R. D'Ari, J. Bacteriol. 171:4217-4221, 1989) have linked cefsulodin-induced lysis to septation during the first division cycle after a nutritional shift-up or chromosome replication realignment. We synchronized cells by membrane filtration to determine whether cefsulodin-induced lysis depended on septation in normally growing cells. Populations of newly divided cells were allowed to grow for variable lengths of time. Cefsulodin was added to these synchronous cultures, which represented points in two to three rounds of the cell cycle. Since the cell numbers were small, a new lysis assay was developed that was based on the release of DNA measured by fluorometry. Lysis occurred at a constant time after addition of the antibiotic, regardless of the time in the cell cycle at which the addition was made. Thus, cefsulodin-induced lysis is not linked to septation or to any other cell cycle-related event.

Bacterial Proteins↗
PubMed1991

Lysis of Escherichia coli by beta-lactams which bind penicillin-binding proteins 1a and 1b: inhibition by heat shock proteins.

The heat shock proteins (HSPs) of Escherichia coli were artificially induced in cells containing the wild-type rpoH+ gene under control of a tac promoter. At 30 degrees C, expression of HSPs produced cells that were resistant to lysis by cephaloridine and cefsulodin, antibiotics that bind penicillin-binding proteins (PBPs) 1a and 1b. This resistance could be reversed by the simultaneous addition of mecillinam, a beta-lactam that binds PBP 2. However, even in the presence of mecillinam, cells induced to produce HSPs were resistant to lysis by ampicillin, which binds all the major PBPs. Lysis of cells induced to produce HSPs could also be effected by imipenem, a beta-lactam known to lyse nongrowing cells. These effects suggest the existence of at least two pathways for beta-lactam-dependent lysis, one inhibited by HSPs and one not. HSP-mediated lysis resistance was abolished by a mutation in any one of five heat shock genes (dnaK, dnaJ, grpE, GroES, or groEL). Thus, resistance appeared to depend on the expression of the complete heat shock response rather than on any single HSP. Resistance to lysis was significant in the absence of the RelA protein, implying that resistance could not be explained by activation of the stringent response. Since many environmental stresses promote the expression of HSPs, it is possible that their presence contributes an additional mechanism toward development in bacteria of phenotypic tolerance to beta-lactam antibiotics.

Anti-Bacterial Agents↗
PubMed1990

Heterogeneity among membrane vesicles of Escherichia coli: effects of production and fractionation techniques.

We have previously shown that Escherichia coli membrane proteins are associated with subpopulations of membrane vesicles beyond the well-defined inner versus outer membrane classification. To separate these vesicles we used a shallow sucrose gradient which differed in many respects from established procedures. Here we compare this revised technique to the classical sucrose density centrifugation procedure. We found that certain manipulations common to the latter obscured heterogeneity among membrane vesicles. The following treatments degraded vesicle separation: growth in rich medium addition of EDTA to buffers, spheroplasting, sonication, pelleting of membranes prior to sucrose gradient centrifugation, overloading the gradient, and long centrifugation times. Some treatments, such as EDTA addition, affected only selected vesicles. When determining protein localization within bacterial membranes experiments should be designed to avoid or at least minimize these manipulations.

Cell Membrane↗
PubMed1989

Separation of Escherichia coli penicillin-binding proteins into different membrane vesicles by agarose electrophoresis and sizing chromatography.

Membrane vesicles from the envelope of Escherichia coli were separated by electrophoresis through dilute agarose and by sizing chromatography through Sephacryl S-1000. These techniques revealed that proteins were associated with different subsets of vesicles. In particular, dilute agarose electrophoresis clearly separated the inner membrane penicillin-binding proteins (PBPs) into different vesicle groups. Vesicles containing PBPs 4, 6, 7, and 8 migrated rapidly through agarose; vesicles with PBPs 1a, 1b, 2, 3, and 5 eluted later. With the exception of PBP 4, which migrated with PBPs 1 through 5, chromatography through Sephacryl S-1000 was able to distinguish the same two vesicle sets, though the extent of separation was poorer than with agarose. The existence of these associations among vesicles and proteins suggests that there is an organization to the inner membrane of E. coli which is not observed when membrane vesicles are separated solely on the basis of density in sucrose gradients.

Bacterial Proteins↗
PubMed1989

Lysis of Escherichia coli by the bacteriophage phi X174 E protein: inhibition of lysis by heat shock proteins.

Lysis of Escherichia coli by the cloned E protein of bacteriophage phi X174 was more rapid than expected when bacteria were shifted from 30 to 42 degrees C at the time of E induction. Since such treatment also induces the heat shock response, we investigated the effect of heat shock proteins on lysis. An rpoH mutant was more sensitive to lysis by E, but a secondary suppressor mutation restored lysis resistance to parental levels, which suggests that the sigma 32 subunit itself did not directly increase lysis resistance. At 30 degrees C, mutants in five heat shock genes (dnaK, dnaJ, groEL, groES, and grpE) were more sensitive to lysis than were their wild-type parents. The magnitude of lysis sensitivity varied with mutation and strain background, with dnaK, dnaJ, and groES mutants consistently exhibiting the greatest sensitivities. Extended protection against lysis occurred when overproduction of heat shock proteins was induced artificially in cells that contained a plasmid with the rpoH+ gene under control of the tac promoter. This protective effect was completely abolished by mutations in dnaK, dnaJ, or groES but not by grpE or groEL mutations. Altered membrane behavior probably explains the contradiction whereby an actual temperature shift sensitized cells to lysis, but production of heat shock proteins exhibited protective effects. The results demonstrate that E-induced lysis can be divided into two distinct operations which may now be studied separately. They also emphasize a role for heat shock proteins under non-heat-shock conditions and suggest cautious interpretation of lysis phenomena in systems where E protein production is under control of a temperature-sensitive repressor.

Bacterial Proteins↗
PubMed1988

Unequal distribution of penicillin-binding proteins among inner membrane vesicles of Escherichia coli.

Escherichia coli penicillin-binding proteins (PBPs) were associated only with inner membrane vesicles when separated on 30 to 65% or 19 to 49% (wt/wt) sucrose gradients. Fractionation of vesicles through the low-density gradient revealed at least two classes of PBP-inner membrane associations. The first class consisted of PBPs 1 through 4, and the second class consisted of PBPs 5 through 8. These classes were distinguished by the density of vesicles with which they were associated; class 1 PBPs migrated with vesicles of higher density than did class 2 PBPs. Such combinations suggest that PBPs are nonrandomly distributed within the inner membrane, implying potential functional relationships among the PBPs themselves and with particular membrane domains. In addition, in cell lysates and in vesicle fractions, a 60,000-dalton aztreonam-insensitive PBP or protein fragment was observed which could potentially be confused with PBP3.

Autoradiography↗
PubMed1983

The characteristics of inhibition of histamine release from human lung fragments by sodium cromoglycate, salbutamol and chlorpromazine.

1 Three drugs have been tested for activity against antigen-induced histamine release from passively sensitized human lung fragments after increasing periods of pre-incubation before challenge.2 After 30 s pre-incubation, sodium cromoglycate inhibited histamine release in the concentration range 0.2-200 muM, producing a maximum inhibition of 33.0%. As the pretreatment period was extended, tolerance developed in a dose-related manner, resulting in a 48.3% and 82.8% loss of activity of the 200 muM dose after 60 min and 19 h pre-incubation, respectively. Tolerance was independent of extracellular calcium and was poorly reversible. Lung tissue desensitized to cromoglycate was cross-tolerant to the related drug, bufrolin, but not to salbutamol or chlorpromazine.3 In acute studies, salbutamol (0.03-3.0 muM) produced dose-related inhibition of histamine release, with a maximum inhibition of 72.2%. The effect was blocked stereoselectively by 1 muM propranolol, suggesting that it occurred through an interaction with lung beta-adrenoceptors. Increasing the pre-incubation time with salbutamol from 30 s to 19 h did not produce tolerance. Inhibition produced by incubation with salbutamol for 19 h was totally prevented when propranolol was added at the beginning of the pre-incubation period, indicating that it resulted from stimulation of beta-receptors and not from a non-specific or toxic effect. However, studies of reversibility of effect through washing or late addition of propranolol did indicate some change in the nature of salbutamol inhibition with time.4 Chlorpromazine was a weak inhibitor of immunological histamine release. A 100 muM concentration was ineffective after 30 s pre-incubation but its activity increased with time. Pre-incubation of lung fragments with this concentration for 1 h or longer, or with a 1 mM dose for a shorter period, provoked histamine release in the absence of antigen. Effects of chlorpromazine were not reversed by washing.5 The different characteristics shown by sodium cromoglycate, salbutamol and chlorpromazine indicate that these drugs inhibit histamine release by interfering with the secretory mechanisms in different ways.

Albuterol↗
PubMed1983

Passive anaphylaxis in human lung fragments as a model for testing anti-allergic drugs: its variability and constraints.

Histamine release from human lung fragments, passively sensitized and challenged with antigen under standardized experimental conditions, varied between 0 and 41.2% (mean +/- SD 15.6 +/- 10.0%) in 89 experiments. Over 41 lungs, the mean coefficient of variation for release from 10 to 28 tissue replicates was 21.4% (range 7-44%). Sodium cromoglycate and chlorpromazine were both partial antagonists of histamine release producing, at best, 30-40% inhibition. The cromoglycate analogue, bufrolin, showed similar activity. There was considerable variation in the effects of these drugs both within and between experiments. Salbutamol was a more potent and more effective inhibitor of release but it, too, showed variability. Although theoretically a very appropriate model of allergic asthma, passive anaphylaxis in human lung fragments is quantitatively inconsistent and gives only a gross indication of drug efficacy.

Albuterol↗