Genomics and antimicrobial drug discovery.
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Anti-Bacterial Agents↗
Biomedical subjects
K J Shaw
Publications and source records attributed to K J Shaw.
Discovery of N-[2-[5-[Amino(imino)methyl]-2-hydroxyphenoxy]-3, 5-difluoro-6-[3-(4, 5-dihydro-1-methyl-1H-imidazol-2-yl)phenoxy]pyridin-4-yl]-N-methylgl y cine (ZK-807834): a potent, selective, and orally active inhibitor of the blood coagulation enzyme factor Xa.
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Administration, Oral↗
(Z,Z)-2,7-Bis(4-amidinobenzylidene)cycloheptan-1-one: identification of a highly active inhibitor of blood coagulation factor Xa.
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Amidines↗
The most frequent aminoglycoside resistance mechanisms--changes with time and geographic area: a reflection of aminoglycoside usage patterns? Aminoglycoside Resistance Study Groups.
The aminoglycoside resistance mechanisms revealed by two surveys in Europe and other countries have been compared to those revealed in earlier studies. Mechanisms have become more complex in all bacterial groups. In Providencia, Serratia, Pseudomonas, Acinetobacter, and Staphylococcus species isolates, genus-specific mechanisms were very common, and it was not possible to see differences between different geographic areas. In other Enterobacteriaceae, the increasing complexity of mechanisms was most often caused by combinations of gentamicin-modifying enzymes with AAC(6')-I, which acetylates amikacin but not gentamicin. The occurrence of these combinations varied by geographical region and among hospitals. The frequency of these combinations correlated with aminoglycoside usage in either the geographical regions or in individual hospitals. These broad-spectrum combinations occurred most frequently in Citrobacter, Enterobacter, and Klebsiella species but also occurred in Escherichia, Morganella, Proteus, Salmonella, and Shigella species. Often the only clinically available aminoglycoside that retained its normal activity was isepamicin.
Acinetobacter↗
Cloning and characterization of an aminoglycoside 6'-N-acetyltransferase gene from Citrobacter freundii which confers an altered resistance profile.
A novel gene encoding a 6'-N-aminoglycoside acetyltransferase, aac(6')-In, has been cloned and sequenced from Citrobacter freundii 13996-19, a clinical isolate from Venezuela. This gene mediates resistance to amikacin, 2'-N-ethylnetilmicin, isepamicin, kanamycin, netilmicin, and tobramycin. The aac(6')-In gene is 573 nucleotides in length and encodes a putative protein of 190 amino acids. AAC(6')-In is most closely related to AAC(6')-Im and AAC(6')-Ie, demonstrating 64.4% and 62.3% similarity, respectively, at the protein level, suggesting these proteins share a common ancestor. The aac(6')-In flanking sequences demonstrated homology to integron- and transposon-related elements which are often found associated with resistance determinants. Hybridization studies performed with an intragenic probe specific for aac(6')-In indicate that this gene is prevalent within Venezuela but has not been observed outside of the country. Furthermore, the aac(6)-In gene was found in 10 different species of gram-negative bacteria.
Acetyltransferases↗
A novel gentamicin resistance gene in Enterococcus.
Enterococcus gallinarum SF9117 is a veterinary isolate for which the MIC of gentamicin is 256 micrograms/ml. Time-kill studies with a combination of ampicillin plus gentamicin failed to show synergism against SF9117. A probe representing aac(6')-aph(2") did not hybridize to DNA from SF9117. A 3.2-kb fragment from plasmid pYN134 of SF9117 was cloned and conferred resistance to gentamicin in Escherichia coli DH5 alpha. Nucleotide sequence analysis revealed the presence of a 918-bp open reading frame whose deduced amino acid sequence had a region with homology to the C-terminal domain of the bifunctional enzyme AAC(6')-APH(2"). The gene is designated aph(2")-Ic, and its observed phosphotransferase activity is provisionally designated APH(2")-Ic. An intragenic probe hybridized to the genomic DNA from an Enterococcus faecium isolate from the peritoneal fluid of one patient and to the plasmid DNA of an Enterococcus faecalis isolate from the blood of another patient. An enterococcal isolate containing this novel resistance gene might not be readily detected in clinical laboratories that use gentamicin at 500 or 2,000 micrograms/ml for screening for high-level resistance.
Amino Acid Sequence↗
Human cardiotrophin-1: protein and gene structure, biological and binding activities, and chromosomal localization.
Cardiotrophin-1 (CT-1) is a new member of the interleukin-6 cytokine family that was identified from a mouse embryoid body cDNA library by expression cloning. Mouse CT-1 induces features of hypertrophy in neonatal rat cardiac myocytes and binds to and activates the leukaemia inhibitory factor/gp130 receptor complex. In this work we report the isolation and characterization of cDNA and genomic clones encoding human CT-1. These clones encode a 201 amino acid protein that is 80% identical to the mouse protein. Human CT-1 produced by transfection of the cDNA clones into mammalian cells induces the hypertrophy of neonatal rat cardiac myocytes. Human and mouse CT-1 bind to the leukaemia inhibitory factor receptor on both human and mouse cell lines indicating a lack of species specificity. No binding to the human oncostatin M specific receptor was detected. A 1.7 kb CT-1 mRNA is expressed in adult human heart, skeletal muscle, ovary, colon, prostate and testis and in fetal kidney and lung. The coding region of CT-1 is contained on three exons and is located on human chromosome 16p11.1-16p11.2.
Amino Acid Sequence↗
Disseminated intravascular coagulation and antithrombin III depression in acute fatty liver of pregnancy.
OBJECTIVE: Acute fatty liver of pregnancy has been associated with a syndrome of marked depression of antithrombin III and disseminated intravascular coagulation. We sought to identify the clinical importance of this accelerated coagulation. STUDY DESIGN: The medical records of patients with acute fatty liver of pregnancy identified during the period of 1982 to 1994 were retrospectively reviewed. RESULTS: Twenty-eight patients with acute fatty liver of pregnancy were identified for an incidence of 1:6692 births. Laboratory evidence of persistent disseminated intravascular coagulation was found in all patients tested. Six patients had clinical bleeding, all associated with genital tract injury. Twenty-three of twenty-three patients tested had markedly decreased antithrombin III levels (average 11%, normal range 80% to 100%). Seven patients received antithrombin III transfusions, which was associated with a significant transient rise in the plasma level. Compared with patients not transfused, however, there was a similar clinical outcome. CONCLUSION: Profoundly depressed antithrombin III levels and laboratory evidence of disseminated intravascular coagulation were present in all cases of acute fatty liver of pregnancy but rarely influenced clinical outcome unless there was concomitant genital tract injury. Antithrombin III transfusions increased plasma levels, but no definite clinical benefit was established in this series because of the small number of cases.
Acute Disease↗
Antibiotic susceptibility of multiply resistant Pseudomonas aeruginosa isolated from patients with cystic fibrosis, including candidates for transplantation.
Chronic lung disease caused by antibiotic-resistant Pseudomonas aeruginosa in patients with cystic fibrosis (CF) is difficult to treat, especially in those who are lung transplantation candidates. Analysis of antibiotic susceptibility and synergy studies of 1,296 isolates revealed that 172 (13.3%) were multiply resistant (i.e., resistant to two or more classes of anti-Pseudomonas agents). beta-Lactam agents (including imipenem and aztreonam) or aminoglycosides inhibited only 11% of the multiply resistant strains, while ciprofloxacin inhibited 34%. High concentrations of tobramycin and gentamicin (200 micrograms/mL), achievable by aerosol administration, inhibited 95% of isolates and overwhelmed permeability-resistance mechanisms. Antimicrobial pairs tested in checkerboard dilutions of clinically achievable drug concentrations inhibited 75% of the multiply resistant strains. On average, three additive and 2.4 synergistic pairs of antimicrobial agents had activity per strain. Transplantation candidates were older than nontransplantation candidates (P = .034), and isolates from transplantation candidates were less likely to be inhibited by antibiotic combinations (P < .001). Administration of aerosolized aminoglycosides and synergy testing of antimicrobial combinations may represent viable therapeutic options for patients with CF.
Adolescent↗
Radiologic studies in acute fatty liver of pregnancy. A review of the literature and 19 new cases.
OBJECTIVE: Radiologic findings thought to be characteristic of acute fatty liver of pregnancy have been described in a limited number of cases. We describe wide experience with liver imaging of this disease. STUDY DESIGN: A retrospective review was performed on charts from 1982 to 1994 to identify those patients with acute fatty liver of pregnancy who had radiologic studies. RESULTS: Twenty-eight patients with acute fatty liver of pregnancy were identified, for an incidence of 1/6,692 births. Nineteen patients underwent at least one imaging study, and seven had more than one. Findings consistent with fatty infiltration of the liver are found in 3/11 patients with ultrasound, 5/10 with computed tomography (CT) and 0/5 with magnetic resonance imaging (MRI). Three patients with normal ultrasound scans subsequently had evidence of fatty filtration on CT scan. CONCLUSION: Detection of fat in the liver of patients with acute fatty liver of pregnancy with current imaging techniques is limited and did not contribute to the management of patients in this series. Further studies are necessary to define to role of MRI--in particular, spectroscopy.
Adult↗
Isolation of cDNA clones encoding the mouse protein V-1.
The amino acid (aa) sequence of rat V-1, a developmentally regulated brain protein, was used to isolate clones encoding mouse V-1, a protein of 118 aa, from an embryoid body cDNA library. The aa sequences of rat and mouse V-1 are identical. V-1 shares several properties (including a 23 of 24 aa match of a tryptic peptide) with myotrophin, a protein that induces cardiac myocyte hypertrophy. Attempts to show that V-1 produced in Escherichia coli could induce the cardiac myocyte hypertrophy ascribed to myotrophin were unsuccessful.
Amino Acid Sequence↗
Cardiotrophin-1. Biological activities and binding to the leukemia inhibitory factor receptor/gp130 signaling complex.
Cardiotrophin-1 (CT-1) is a newly isolated cytokine that was identified based on its ability to induce cardiac myocyte hypertrophy. It is a member of the family of cytokines that includes interleukins-6 and -11, leukemia inhibitory factor (LIF), ciliary neurotrophic factor, and oncostatin M. These cytokines induce a pleiotropic set of growth and differentiation activities via receptors that use a common signaling subunit, gp130. In this work we determine the activity of CT-1 in six in vitro biological assays and examine the composition of its cell surface receptor. We find that CT-1 is inactive in stimulating the growth of the hybridoma cell line, B9 and inhibits the growth of the mouse myeloid leukemia cell line, M1. CT-1 induces a phenotypic switch in rat sympathetic neurons and promotes the survival of rat dopaminergic and chick ciliary neurons. CT-1 also inhibits the differentiation of mouse embryonic stem cells. CT-1 and LIF cross-compete for binding to M1 cells, Kd [CT-1] approximately 0.7 nM, and this binding is inhibited by an anti-gp130 monoclonal antibody. Both ligands can be specifically cross-linked to a protein on M1 cells with the mobility of the LIF receptor (approximately 200 kDa). In addition, CT-1 binds directly to a purified, soluble form of the LIF receptor in solution (Kd approximately 2 nM). These data show that CT-1 has a wide range of hematopoietic, neuronal, and developmental activities and that it can act via the LIF receptor and the gp130 signaling subunit.
Amino Acid Sequence↗
Expression cloning of cardiotrophin 1, a cytokine that induces cardiac myocyte hypertrophy.
Heart failure continues to be a leading cause of mortality worldwide. A hallmark of this disease is dilated cardiac hypertrophy, which is accompanied by a reactivation of genes expressed in fetal heart development. Reasoning that fetal or embryonic growth factors may mediate the onset of cardiac hypertrophy, we have coupled expression cloning with an embryonic stem cell-based model of cardiogenesis to isolate a 21.5-kDa protein, cardiotrophin 1, that potently induces cardiac myocyte hypertrophy in vitro. Amino acid similarity data indicate that cardiotrophin 1 is a member of the leukemia inhibitory factor/ciliary neurotrophic factor/oncostatin M/interleukin 6/interleukin 11 family of cytokines. Several members of this family that are known to signal through the transmembrane protein gp130 stimulate cardiac myocyte hypertrophy, like cardiotrophin 1, suggesting that the gp130 signaling pathway may play a role in cardiac hypertrophy. A 1.4-kb cardiotrophin 1 mRNA is expressed in the heart and several other mouse tissues.
Amino Acid Sequence↗
Cloning and characterization of a 3-N-aminoglycoside acetyltransferase gene, aac(3)-Ib, from Pseudomonas aeruginosa.
A novel gene encoding an aminoglycoside 3-N-acetyltransferase, which confers resistance to gentamicin, astromicin, and sisomicin, was cloned from Pseudomonas aeruginosa Stone 130. Its sequence was determined and found to show considerable similarity to an aac(3)-I gene previously cloned from R plasmids from Enterobacter, Pseudomonas, and Serratia spp. We have designated the genes from the R plasmids and this work aac(3)-Ia and aac(3)-Ib, respectively. The two aac(3)-I genes share 74% nucleotide identity, and their deduced protein products are 88% similar. These data suggest that the genes derive from a common ancestor. Homology between the flanking sequences of both aac(3)-I genes and other resistance determinants known to reside in integron environments was also observed. Intragenic probes specific for either aac(3)-Ia or aac(3)-Ib were used in hybridization studies with a series of gentamicin-, astromicin-, and sisomicin-resistant clinical isolates. Of 59 clinical isolates tested, no isolates hybridized with both probes, 30 (51%) hybridized with the aac(3)-Ia probe, 12 (20%) hybridized with the aac(3)-Ib probe, and 17 (29%) did not hybridize with either probe. These data suggest the existence of at least one other aac(3)-I gene.
Acetyltransferases↗
The changing nature of aminoglycoside resistance mechanisms and the role of isepamicin--a new broad-spectrum aminoglycoside. The Aminoglycoside Resistance Study Groups.
Aminoglycoside resistance mechanisms from recent studies were compared with those found in earlier studies in the USA and Europe for three pathogen groups. Among Citrobacter-Enterobacter-Klebsiella, four single mechanisms (AAc(3)-II, AAC(3)-I, ANT(2")-I and AAC(6')-I were found in all studies, but the most recent studies showed a significant increase in combinations of AAC(6')-I with the other common mechanisms. Since AAC(6')-I confers resistance to tobramycin, netilmicin and amikacin, combinations of it with the other gentamicin modifying enzymes conferred broad-spectrum resistance to all clinically available aminoglycosides except isepamicin. Similar changes occurred in Escherichia-Morganella-Proteus-Salmonella-Shigella except that the frequency of combinations was much lower and two additional single mechanisms - AAC(3)-IV and permeability - were also found frequently. Among aminoglycoside-resistant Pseudomonas, three mechanisms, AAC(6')-II, ANT(2")-I and permeability, were always common and remained common. However, combinations of the three mechanisms with each other and with other mechanisms were more common in the recent surveys. Different genes which produce different proteins with the same aminoglycoside-modifying activity are now known. The results of hybridisation studies with two aac(3)-I, 2 aac(6')-II and 4 aac(6')-I gene probes are presented. The most commonly occurring genes were: aac(3)-Ia, aac(3)-IIa, aac(6')-IIa, aac(6')-Ib and, in Serratia, aac(6')-Ic. The activity of isepamicin against amikacin resistant strain which produce AAC(6')-I can be related to differences in the structure of these two similar aminoglycosides at Position 3". Amikacin may form a stable complex with AAC(6')-I enzymes via binding interaction at Position 3 and 3". Isepamicin, which has a secondary amino group at Position 3", may only be able to interact at Position 3 and enzyme-isepamicin complexes are likely to be less stable.
Acetyltransferases↗
Proximal 2.6 kb of 5'-flanking DNA is insufficient for human renin promoter activity in renin-synthesizing chorio-decidual cells.
In order to determine the influence of proximal 5'-flanking DNA of the human renin gene (REN) in cells that express human renin, transient expression analyses were carried out in chorio-decidual cells. Constructs containing different lengths of REN promoter DNA, extending as far as 2595 bp upstream of the transcription start site, were unable to drive transcription of a chloramphenicol acetyl transferase reporter gene in chorio-decidual cells, nor in noncognate 293 or JEG-3 cells. The tk promoter was similarly inactive in constructs containing -2595 to -453 fragments of REN 5'-flanking DNA. In each cell type, the -2595 to -1300 DNA exerted a negative influence. Additional promoter- and cell type-dependent negative influences were noted for other regions of REN 5'-flanking DNA and the -453 to -145 DNA increased tk promoter activity 2.5-fold in chorio-decidual cells. By introducing the SV40 enhancer into constructs, a weak stimulation of the REN promoter was observed in chorio-decidual cells, but not in noncognate, JEG-3 cells, although the -2595 to -1300 DNA retained its negative influence in the cognate cell type. These results show that the proximal 2.6 kb of REN 5'-flanking DNA is unable to drive reporter gene activity in renin-synthesizing, chorio-decidual cells under basal conditions and suggest that trans-acting factors unique to at least this cell type, together with enhancer(s) located outside of the proximal 2.6 kb of REN promoter DNA tested, could be required for human renin promoter activity.
Chorion↗
Identification of cyclic AMP response element in the human renin gene.
In order to identify the mechanism by which cyclic AMP stimulates expression of the human renin gene (REN), the effect of forskolin was tested in transient expression analyses of REN 5'-flanking DNA-chloramphenicol acetyltransferase (CAT) reporter gene constructs in secondary cultures of human chorio-decidual cells, a major site of renin synthesis. Forskolin induced a mean 5-fold stimulation which was localized to DNA in the region -249 to -162 with respect to the transcription start site (+1). Such DNA also mediated a response to forskolin in heterologous (HSV thymidine kinase) promoter constructs. Strong cAMP-response element (CRE) homology at -222 to -218 resembled the target for members of the CRE binding protein (CREB) family. Gel shift assays demonstrated similarly migrating nucleoprotein complexes for oligonucleotides containing the putative REN CRE as for a canonical CRE, in chorio-decidual, JEG-3 and HeLa nuclear extracts. Mutation of residues critical for CREB attachment reduced binding. In conclusion, a CRE was identified at -222 to -218 that appears critical for cAMP-induced human renin gene transcription.
Animals↗
Analysis of beta-glucans and chitin in a Saccharomyces cerevisiae cell wall mutant using high-performance liquid chromatography.
We have previously shown that mutations in the yeast KNR4 gene resulted in pleiotropic cell wall defects, including resistance to killer 9 toxin, elevated osmotic sensitivity to SDS and increased resistance to zymolyase, a (1-->3)-beta-glucanase. In this report, we further demonstrated that knr4 mutant cells were more permeable to a chromogenic substrate, X-GAL, suggesting that the mutant cell walls were leakier to certain non-permeable molecules. To determine if these defects resulted from structural changes in the cell walls, we analysed the alkali-insoluble cell wall components using HPLC assays developed for this purpose. Comparative analysis using four isogenic strains from a 'knr4 disrupted' tetrad demonstrated that mutant cell walls contained much less (1-->3)-beta-glucan and (1-->6)-beta-glucan; however, the level of chitin, a minor cell wall component, was found to be five times higher in the mutant strains compared to the wild-type strains. The data suggested that the knr4 mutant cell walls were dramatically weakened, which may explain the pleiotropic cell wall defects.
Cell Wall↗