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K J Shaw

Publications and source records attributed to K J Shaw.

At least 55 records · Page 3Linked to original sources
PubMed1994

Cyclooxygenase gene expression in human endometrium and decidua.

The objective of this study was to determine if genes for cyclooxygenase (COX) and 5-lipoxygenase, key enzymes in the synthesis of eicosanoids, are expressed in human endometrium and to determine if the level of expression is affected by decidualization or preeclampsia, a form of pregnancy-induced hypertension. RT-PCR was used to detect mRNA in tissues obtained from various patients. Results demonstrated that COX and 5-lipoxygenase genes are expressed in endometrium and decidua. COX gene expression in endometrium is 2-3-fold greater than in decidua while 5-lipoxygenase gene expression is similar. Neither COX nor 5-lipoxygenase gene expression in decidua is altered by changes resulting from preeclampsia. Thus, genes for key enzymes in the synthesis of eicosanoids are expressed in human endometrium and decidua. Selective down-regulation is evident as decidualization results in a significant reduction in the gene expression of COX, while 5-lipoxygenase gene expression remains unchanged.

Arachidonate 5-Lipoxygenase↗
PubMed1994

Function of human renin proximal promoter DNA.

An understanding of the mechanisms involved in the control of the human renin promoter have been hampered and confounded in work to date because of deficiencies in material available and experimental design. The promoter appears to be weak and a good cell model is lacking. Chorio-decidual cultures have been used since these have high renin synthesis, are readily available and grow well in culture. They suffer, however, from phenotypic variability and do not transfect well in transient expression analyses. Recent evidence suggests that 2.6 kb of proximal 5'-flanking DNA is unable to induce native promoter activity under basal conditions. Experiments in which an exogenous enhancer was introduced have raised the possibility that an endogenous enhancer residing outside of the 2.6 kb 5'-flanking region could be required. Cell-type specific factors also appear to be needed. The proximal flanking DNA does, however, appear to be capable of conferring activity on the promoter in chorio-decidual cells under stimulated conditions, suggesting that factors so activated may have considerable importance. Evidence suggests that forskolin-responsive signal transduction pathways may lead cyclic AMP responsive element (CRE) binding protein (CREB) to act on a CRE at -222 in the proximal REN promoter DNA. Activation of the mouse promoter by cAMP appears to involve a different element, however. Furthermore, overall control of renin synthesis is likely to involve post-transcriptional mechanisms as well. Thus, despite being the first cardiovascular gene to be cloned, much more work is required before the control of the human renin gene is fully understood.

Animals↗
PubMed1994

Cloning and characterization of KNR4, a yeast gene involved in (1,3)-beta-glucan synthesis.

k9 killer toxin from Hansenula mrakii was used to select a number of resistant mutants from Saccharomyces cerevisiae. Preliminary biochemical and genetic studies showed that some of them acquired structural defects in the cell wall. One of these mutants, the knr4-1 mutant, displays a number of cell wall defects, including osmotic sensitivity; sensitivity to cercosporamide, a known antifungal agent; and resistance to Zymolyase, a (1,3)-beta-glucanase. We report here the isolation and analysis of the KNR4 gene. DNA sequence analysis revealed an uninterrupted open reading frame which contains five potential start codons. The longest coding template encodes a protein of 505 amino acids with a calculated molecular mass of 57,044 Da. A data base search revealed 100% identity with a nuclear protein, SMI1p. Disruption of the KNR4 locus does not result in cell death; however, it leads to reduced levels of both (1,3)-beta-glucan synthase activity and (1,3)-beta-glucan content in the cell wall. The gene was mapped to the right arm of chromosome VII.

Amino Acid Sequence↗
PubMed1993

Analysis of the aac(3)-VIa gene encoding a novel 3-N-acetyltransferase.

Biochemical analysis (G. A. Papanicolaou, R. S. Hare, R. Mierzwa, and G. H. Miller, abstr. 152, Program Abstr. 29th Intersci. Conf. Antimicrob. Agents Chemother., 1989) demonstrated the presence of a novel 3-N-acetyltransferase in Enterobacter cloacae 88020217. This organism was resistant to gentamicin, and the MIC of 2'-N-ethylnetilmicin for it was fourfold lower than that of 6'-N-ethylnetilmicin, a resistance pattern which suggested 2'-acetylating activity. However, high-pressure liquid chromatography analysis demonstrated that the enzyme acetylated sisomicin in the 3 position. We have cloned the structural gene for this enzyme from a large (> 70-kb) conjugative plasmid present in E. cloacae. Subcloning experiments have localized the aac(3)-VIa gene to a 2.1-kb Sau3A fragment. The deduced AAC(3)-VIa protein showed 48% amino acid identity to the AAC(3)-IIa protein and 39% identity to the AAC(3)-VII protein. Examination of the 5'-flanking sequences demonstrated that the aac(3)-VIa gene was located 167 bp downstream of the aadA1 gene and was present in an integron. In addition, the aac(3)-VIa gene is also downstream of a 59-base element often seen in an integron environment. Primer extension analysis has identified a promoter for the aac(3)-VIa gene downstream of both the aadA1 gene and a 59-base element.

Amino Acid Sequence↗
PubMed1993

Nucleotide sequence analysis and DNA hybridization studies of the ant(4')-IIa gene from Pseudomonas aeruginosa.

The ant(4')-IIa gene was previously cloned from Pseudomonas aeruginosa on a 1.6-kb DNA fragment (G. A. Jacoby, M. J. Blaser, P. Santanam, H. Hächler, F. H. Kayser, R. S. Hare, and G. H. Miller, Antimicrob. Agents Chemother. 34:2381-2386, 1990). In the current study, the ant(4')-IIa gene was localized by gamma-delta mutagenesis. A region of approximately 600 nucleotides which contained the ant(4')-IIa gene was identified, and DNA sequence analysis revealed two overlapping open reading frames (ORFs) within this region. Northern (RNA) blot analysis demonstrated expression of both ORFs in P. aeruginosa; therefore, site-directed mutagenesis was used to identify the ORF which encodes the ant(4')-IIa gene. No homology was found between ant(4')-IIa and ant(4')-Ia DNA sequences. Hybridization experiments confirmed that the ant(4')-Ia probe hybridized only to gram-positive presumptive ANT(4')-I strains and that the ant(4')-IIa probe hybridized only to gram-negative strains presumed to carry ANT(4')-II. Seven gram-negative strains which had been classified as having ANT(4')-II resistance profiles did not hybridize with probes for either ant(4')-Ia or ant(4')-IIa, suggesting that at least one additional ant(4') gene may exist. The predicted amino-terminal sequences of the ANT(4')-Ia and ANT(4')-IIa proteins showed significant sequence similarity between residues 38 and 63 of the ANT(4')-Ia protein and residues 26 and 51 of the ANT(4')-IIa protein.

Base Sequence↗
PubMed1993

Characterization and transcriptional regulation of the 2'-N-acetyltransferase gene from Providencia stuartii.

We have cloned the chromosomally encoded 2'-N-acetyltransferase gene [aac(2')-Ia] from Providencia stuartii. DNA sequence analysis of the cloned insert identified a single open reading frame, which is capable of encoding a protein with a predicted molecular mass of 20,073 Da. The deduced AAC(2')-Ia protein showed no significant homology to other proteins, including all of the AAC(3) and AAC(6') proteins. Primer extension analysis was used to identify the aac(2')-Ia promoter, which contained an unusual sequence (CTTTTT) at the -35 region. Expression of the aac(2')-Ia gene occurs at low levels in wild-type P. stuartii strains; therefore, they are aminoglycoside susceptible. We have isolated mutants with high-level AAC(2')-Ia expression at a frequency of 4.8 x 10(-6). Detailed analysis of one mutant demonstrated a 12.2-fold increase in the accumulation of aac(2')-Ia mRNA. In addition, the levels of beta-galactosidase expression from a plasmid-encoded aac(2')-lacZ transcriptional fusion were increased 11.5-fold in this mutant relative to those in an isogenic wild-type strain. These results suggested that a trans-acting factor, designated aar (for aminoglycoside acetyltransferase regulator), controls AAC(2')-Ia expression in P. stuartii.

Acetyltransferases↗
PubMed1993

Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes.

The three classes of enzymes which inactivate aminoglycosides and lead to bacterial resistance are reviewed. DNA hybridization studies have shown that different genes can encode aminoglycoside-modifying enzymes with identical resistance profiles. Comparisons of the amino acid sequences of 49 aminoglycoside-modifying enzymes have revealed new insights into the evolution and relatedness of these proteins. A preliminary assessment of the amino acids which may be important in binding aminoglycosides was obtained from these data and from the results of mutational analysis of several of the genes encoding aminoglycoside-modifying enzymes. Recent studies have demonstrated that aminoglycoside resistance can emerge as a result of alterations in the regulation of normally quiescent cellular genes or as a result of acquiring genes which may have originated from aminoglycoside-producing organisms or from other resistant organisms. Dissemination of these genes is aided by a variety of genetic elements including integrons, transposons, and broad-host-range plasmids. As knowledge of the molecular structure of these enzymes increases, progress can be made in our understanding of how resistance to new aminoglycosides emerges.

Acetyltransferases↗
PubMed1993

Presence of N-acyl and acetoxy derivatives of putrescine and cadaverine in the human gut.

N-acyl and acetoxy derivatives of putrescine and cadaverine have been found in the faeces of children and in cultures of isolates of gut bacteria. The evidence was accumulated from two dimensional, thin layer chromatography, field desorption mass spectrometry, and accurate mass measurement of the DANS derivatives of the amines. The acetoxy compounds of putrescine and cadaverine have not previously been reported.

Bacteria↗
PubMed1993

Successful delivery after age 50: a report of two cases as a result of oocyte donation.

BACKGROUND: Because of donor oocyte programs, women who previously were considered too old to successfully achieve conception and delivery can now bear children. To our knowledge, there have been no previous reports of pregnancy outcome in women over age 50 who conceived using donor oocytes. This study presents the pregnancy and delivery data on two women who delivered at age 52. CASES: Case 1 was a 51-year-old woman, gravida 3, para 3, whose three children had been conceived with her first husband more than 20 years previously. She had remarried 18 years before presentation and had been actively trying to conceive for the last 7 years. She was diagnosed as being in menopause based on elevated gonadotropins, amenorrhea, and failure to have progesterone-withdrawal menses. She conceived on her first embryo transfer cycle with embryos derived from donor oocytes and fertilized by her husband's sperm (oocytes were donated by a woman who was undergoing retrieval for in vitro fertilization). During pregnancy she remained healthy, but had uterine prolapse at 20 weeks. She delivered a normal healthy male at 40.5 weeks; cesarean was performed because of a presumptive diagnosis of fetal distress after 3 hours of labor, when monitoring revealed fetal heart decelerations. Case 2 was also a 51-year-old woman, gravida 6, para 4, who wished to conceive with her second husband's sperm through the donor oocyte program. She had amenorrhea of 2 years' duration and elevated gonadotropins. Conception occurred after fertilization of a donor oocyte by her husband's sperm. She had an uneventful pregnancy, but labor was induced at 38 weeks' gestation given the supposed high-risk status of this age group. Apgar scores were 8 and 9 at 1 and 5 minutes, respectively. CONCLUSION: Theoretically, the risks of pregnancy complications in older patients are magnified given the aging maternal cardiovascular system, which may predispose these women to placental insufficiency. These first two cases of donor oocyte pregnancies in women over age 50 found no maternal or fetal age-related complications. We hope these reports will encourage all researchers to share their findings so that prospective patients can make better, more informed decisions as to whether they want to participate in donor oocyte programs.

Embryo Transfer↗
PubMed1992

Cardiotonic agents. 7. Prodrug derivatives of 4-ethyl-1,3-dihydro- 5-[4-(2-methyl-1H-imidazol-1-yl)benzoyl]-2H-imidazol-2-one.

The cardiotonic agent 4-ethyl-1,3-dihydro-5-4-(2-methyl-1H-imidazol-1-yl)benzoyl]-2H- imidazol-2-one (1) was found to have low bioavailability when administered orally to rats and dogs. A series of N-acyl derivatives, an underutilized prodrug of acidic NH compounds, has been synthesized and tested for their ability to improve the oral bioavailability of 1. Reaction of the monosodium salt of 1 with various anhydrides afforded the N-1 monoacylimidazolones with surprisingly high regioselectivity. In addition to the prodrugs, acylation of 1 with propionic or phenylacetic anhydride led to the novel 3H-pyrrolo[1,2-c]imidazole-3,5(2H)-diones 6. The prodrugs showed a significant increase in the partition coefficients with a minor decrease in the aqueous solubility. The benzoyl derivative 4b exhibited the highest stability in both pH 1.5 and 7.4 buffer solutions. Further evaluation of 4b showed rapid conversion to 1 in canine plasma (t1/2 = 38 min), and human plasma (t1/2 = 10 min). Oral studies indicated that the bioavailability of 4b was increased to greater than 75% (compared to less than 20% for 1), and hemodynamic studies demonstrated that the selective inotropic profile of 1 was retained.

Animals↗
PubMed1992

Diversity of aminoglycoside resistance in Enterobacter cloacae in Greece.

Ninety Enterobacter cloacae strains isolated from 12 Greek hospitals were examined in terms of epidemiological types and resistance mechanisms. Using O serotyping 69% of the strains were assigned to a specific serotype and overall 16 different serotypes were identified. The combination of serotyping, phagetyping and biotyping efficiently discriminated most of the strains, indicating that single epidemic strains were not prevalent, although serotypes 3, 7, and group II predominated. Eight representative strains, all resistant to gentamicin, tobramycin, amikacin and netilmicin, were further examined for transferability and mechanisms of resistance. Aminoglycoside resistance was found to be transferable in most strains, and 13 R plasmids of 40-120 MDa molecular weight were detected. The enzymes detected consisted of three enzymes active against gentamicin [ANT(2h'), AAC(3)-I and AAC(3)-V]; three active against tobramycin [ANT(2"), AAC(3)-V and AAC(6')-I]; two active against netilmicin [AAC(3)-V and AAC(6')-I]; and one active against amikacin [AAC(6')-I]. APH(3') and ANT (3"), which modify neomycin and streptomycin plus spectinomycin respectively, were also found. Overall up to five aminoglycoside modifying enzymes were detected on the same R plasmid, AAC(6')-I plus ANT(2") being the most prevalent. The high incidence of multiresistance in Enterobacter cloacae and the fact that resistance is due to enzymatic inactivation of the antibiotics, indicate that in Greece this species might act as a gene pool for the spread of resistance to other bacteria of clinical relevance.

Aminoglycosides↗
PubMed1992

Cloning and DNA sequence analysis of an aac(3)-Vb gene from Serratia marcescens.

The AAC(3)-V resistance mechanism is characterized by high-level resistance to the aminoglycosides gentamicin, netilmicin, 2'-N-ethylnetilmicin, and 6'-N-ethylnetilmicin and moderate resistance levels to tobramycin. Serratia marcescens 82041944 contains an AA(3)-V resistance mechanism as determined from aminoglycoside resistance profiles. This strain, however, does not exhibit hybridization with a probe derived from the previously cloned aac(3)-Va gene, (R. Allmansberger, B. Bräu, and W. Piepersberg, Mol. Gen. Genet. 198:514-520, 1985). High-pressure liquid chromatography analysis of the acetylation products of sisomicin carried out by extracts of S. marcescens 82041944 have demonstrated the presence of an AAC(3) enzyme. We have cloned the gene encoding this acetyltransferase and have designated it aac(3)-Vb. Nucleotide sequence comparisons show that the aac(3)-Va and aac(3)-Vb genes are 72% identical. The predicted AAC(3)-Vb protein is 28,782 Da. Comparisons of the deduced amino acid sequences show 75% identity and 84% similarity between the AAC(3)-Va and AAC(3)-Vb proteins. The use of a DNA fragment internal to the aac(3)-Vb as a hybridization probe demonstrated that the aac(3)-Vb gene is very rare in clinical isolates possessing an AAC(3)-V mechanism.

Amino Acid Sequence↗
PubMed1992

Characterization of the chromosomal aac(6')-Ic gene from Serratia marcescens.

The DNA sequence of the chromosomal aac(6')-Ic gene from Serratia marcescens, which had been previously cloned (H. M. Champion, P. M. Bennett, D. A. Lewis, and D. S. Reeves, J. Antimicrob. Chemother. 22:587-596, 1988) was determined. High-pressure liquid chromatographic analysis of extracts prepared from Escherichia coli carrying the chromosomal aac(6')-Ic gene on a plasmid confirmed the presence of 6'-N-acetyltransferase activity in this strain, which was suggested by the aminoglycoside resistance profile. DNA sequence analysis of the cloned 2,057-bp PstI fragment revealed several regions of homology to previously characterized sequences from GenBank, including the rpoD and tRNA-2 genes of E. coli. Subcloning experiments confirmed the coding sequence of the aac(6')-Ic gene to be at positions 1554 to 1992. The predicted amino acid sequence of the AAC(6')-Ic protein suggested that it was the third member of a family of AAC(6') proteins which included a coding region identified between the aadB and aadA genes of Tn4000 and an AAC(6') protein encoded by pUO490, which was isolated from Enterobacter cloacae. Primer extension analysis suggested that the -35 region of the aac(6')-Ic promoter overlapped a large palindromic sequence which may be involved in the regulation of the aac(6')-Ic gene. Hybridization experiments utilizing a restriction fragment from the aac(6')-Ic gene showed that all S. marcescens organisms carried this gene whether or not the AAC(6')-I resistance profile was expressed. Organisms other than Serratia spp. did not hybridize to this probe.

Base Sequence↗
PubMed1992

Genetic analysis of bacterial acetyltransferases: identification of amino acids determining the specificities of the aminoglycoside 6'-N-acetyltransferase Ib and IIa proteins.

The aminoglycoside 6'-N-acetyltransferase [AAC(6')-I] and AAC(6')-II enzymes represent a class of bacterial proteins capable of acetylating tobramycin, netilmicin, and 2'-N-ethylnetilmicin. However, an important difference exists in their abilities to modify amikacin and gentamicin. The AAC(6')-I enzymes are capable of modifying amikacin. In contrast, the AAC(6')-II enzymes are capable of modifying gentamicin. Nucleotide sequence comparison of the aac(6')-Ib gene and the aac(6')-IIa gene showed 74% sequence identity (K. J. Shaw, C. A. Cramer, M. Rizzo, R. Mierzwa, K. Gewain, G. H. Miller, and R. S. Hare, Antimicrob. Agents Chemother. 33:2052-2062, 1989). Comparison of the deduced protein sequences showed 76% identity and 82% amino acid similarity. A genetic analysis of these two proteins was initiated to determine which amino acids were responsible for the differences in specificity. Results of domain exchanges, which created hybrid AAC(6') proteins, indicated that amino acids in the carboxy half of the proteins were largely responsible for determining specificity. Mutations shifting the specificity of the AAC(6')-Ib protein to that of the AAC(6')-IIa protein (i.e., gentamicin resistance and amikacin sensitivity) have been isolated. DNA sequence analysis of four independent isolates revealed base changes causing the same amino acid substitution, a leucine to serine, at position 119. Interestingly, this serine occurs naturally at the same position in the AAC(6')-IIa protein. Oligonucleotide-directed mutagenesis was used to construct the corresponding amino acid change, a serine to leucine, in the AAC(6')-IIa protein. This change resulted in the conversion of the AAC(6')-IIa substrate specificity to that of AAC(6')-Ib. Analysis of additional amino acid substitutions within this region of AAC(6')-Ib support the model that we have identified an aminoglycoside binding domain of these proteins.

Acetyltransferases↗
PubMed1992

Selective labor induction in postterm patients. Observations and outcomes.

Six hundred twenty-nine postterm patients with reliable dates were evaluated for expectant management. Patients with medical or obstetric complications, a Bishop score greater than 6 or abnormal antepartum testing were ineligible for such management. The incidences of macrosomia, fetal distress necessitating cesarean section and low five-minute Apgar scores were similar in patients with labor induction as compared to those managed expectantly. The cesarean section rate was significantly lower in patients who underwent induction for a Bishop score greater than 6 as compared to those followed expectantly (15% vs. 28%). The lowest cesarean section rate was seen in patients with Bishop scores greater than 8 (5%) versus 15% in patients with Bishop scores of 6-8.

Cesarean Section↗
PubMed1991

Synthesis and biological activity of angiotensin II analogues containing a Val-His replacement, Val psi[CH(CONH2)NH]His.

The dipeptide mimic Val psi[CH(CONH2)NH]His (4) was incorporated into angiotensin II (AII) analogues to provide an octapeptide saralasin derivative (29) as well as tetrapeptide analogue 19. Three C-terminal tetrapeptides (21, 25, and 28) were also prepared. All compounds were tested for their ability to displace 3H-AII from rabbit adrenal gland homogenate and as antagonists of AII and AI on guinea pig ileum. The octapeptide analogue 29 was 700 times less active than the parent peptide 30. All the C-terminal fragments 19, 21, 25, and 28 have no measurable AII antagonist activity. Of the four tetrapeptide fragments, only 21 showed any appreciable binding activity.

1-Sarcosine-8-Isoleucine Angiotensin II↗
PubMed1991

Correlation between aminoglycoside resistance profiles and DNA hybridization of clinical isolates.

DNA hybridization data and aminoglycoside resistance profiles (AGRPs) were determined for 4,088 clinical isolates from three studies (United States, Belgium, and Argentina). The correlation between susceptibility profiles and hybridization results was determined with nine DNA probes. For each of the seven aminoglycoside resistance profiles which we were able to test, the data suggested at least two distinct genes could encode enzymes which lead to identical resistance profiles. Furthermore, the DNA hybridization data showed that individual strains carried up to six unique aminoglycoside resistance genes. DNA hybridization revealed interesting differences in the frequencies of these genes by organism and by country.

Acinetobacter↗