PubMed Health⌕ Search

Biomedical subjects

B Cameron

Publications and source records attributed to B Cameron.

At least 37 records · Page 2Linked to original sources
PubMed1997

Glycosylated flavones as selective inhibitors of topoisomerase IV.

Three flavonoids which promoted Escherichia coli topoisomerase IV-dependent DNA cleavage were isolated from cottonseed flour and identified as quercetin 3-O-beta-D-glucose-[1,6]-O-alpha-L-rhamnose (rutin), quercetin 3-O-beta-D-galactose-[1,6]-O-alpha-L-rhamnose, and quercetin 3-O-beta-D-glucose (isoquercitrin). The most active one (rutin) also inhibited topoisomerase IV-dependent decatenation activity (50% inhibitory concentration, 64 microg/ml) and induced the SOS response of a permeable E. coli strain. Derivatives of quercetin glycosylated at position C-3 were shown to induce two site-specific DNA cleavages of pBR322 DNA, which were mapped by DNA sequence analysis to the gene encoding resistance to tetracycline. Cleavage at these sites was hardly detectable in cleavage reactions with quercetin or fluoroquinolones. None of the three flavonoids isolated from cottonseeds had any stimulatory activity on E. coli DNA gyrase-dependent or calf thymus topoisomerase II-dependent DNA cleavage, and they were therefore specific to topoisomerase IV. These results show that selective inhibitors of topoisomerase IV can be derived from the flavone structure. This is the first report on a DNA topoisomerase inhibitor specific for topoisomerase IV.

Base Sequence↗
PubMed1996

Biosynthesis of vitamin B12: the preparative multi-enzyme synthesis of precorrin-3A and 20-methylsirohydrochlorin (a 2,7,20-trimethylisobacteriochlorin).

The Bacillus subtilis genes hemB, hemC and hemD, encoding respectively the enzymes porphobilinogen synthase, hydroxymethylbilane synthase and uroporphyrinogen III synthase, have been expressed in Escherichia coli using a single plasmid construct. An enzyme preparation from this source converts 5-aminolaevulinic acid (ALA) preparatively and in high yield into uroporphyrinogen III. The Pseudomonas denitrificans genes cobA and cobI, encoding respectively the enzymes S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) and S-adenosyl-L-methionine:precorrin-2 methyltransferase (SP2MT), were also expressed in E. coli. When SUMT was combined with the coupled-enzyme system that produces uroporphyrinogen III, precorrin-2 was synthesized from ALA, and when SP2MT was also added the product from the coupling of five enzymes was precorrin-3A. Both of these products are precursors of vitamin B12, and they can be used directly for biosynthetic experiments or isolated as their didehydro octamethyl esters in > 40% overall yield. The enzyme system which produces precorrin-3A is sufficiently stable to allow long incubations on a large scale, affording substantial quantities (15-20 mg) of product.

Bacillus subtilis↗
PubMed1996

Triple test for prenatal detection of Edwards syndrome (trisomy 18).

OBJECTIVE(S): To demonstrate the limitation of complete reliance on computer generated interpretations and to highlight the need for understanding of pregnancy-related biochemistry when offering prenatal screening. METHODS: Four cases of cytogenetically confirmed trisomy 18 pregnancies are presented. All four cases underwent prenatal screening (Triple Test-AFP, uE3, t beta-hCG) at midgestation and risk assessment by the alpha algorithm. RESULTS: All four cases of trisomy 18 were assessed as being at low risk for DS and/or open NTD. Although marker levels were not consistent with either of these clinical situations, they were indicative of a compromised pregnancy. Circulating levels of trophoblast-derived antigens (uE3, t beta-hCG) were depressed (< or = 0.5 MoM) in all four cases. Further investigations (ultrasonography, amniocentesis) confirmed a trisomy 18 fetus. CONCLUSIONS: Risk assessment by computer based algorithms relies on maternal factors and specific DS/NTD marker profiles. Aberrant marker profiles are not distinguished from normal. Therefore, it is essential that prenatal screening is offered only by those competent in pregnancy biochemistry and able to identify these abnormal situations.

Adult↗
PubMed1996

Differential behaviors of Staphylococcus aureus and Escherichia coli type II DNA topoisomerases.

Staphylococcus aureus gyrA and gyrB genes encoding DNA gyrase subunits were cloned and coexpressed in Escherichia coli under the control of the T7 promoter-T7 RNA polymerase system, leading to soluble gyrase which was purified to homogeneity. Purified gyrase was catalytically indistinguishable from the gyrase purified from S. aureus and did not contain detectable amounts of topoisomerases from the E. coli host. Topoisomerase IV subunits GrlA and GrlB from S. aureus were also expressed in E. coli and were separately purified to apparent homogeneity. Topoisomerase IV, which was reconstituted by mixing equimolar amounts of GrlA and GrlB, had both ATP-dependent decatenation and DNA relaxation activities in vitro. This enzyme was more sensitive than gyrase to inhibition by typical fluoroquinolone antimicrobial agents such as ciprofloxacin or sparfloxacin, adding strong support to genetic studies which indicate that topoisomerase IV is the primary target of fluoroquinolones in S. aureus. The results obtained with ofloxacin suggest that this fluoroquinolone could also primarily target gyrase. No cleavable complex could be detected with S. aureus gyrase upon incubation with ciprofloxacin or sparfloxacin at concentrations which fully inhibit DNA supercoiling. This suggests that these drugs do not stabilize the open DNA-gyrase complex, at least under standard in vitro incubation conditions, but are more likely to interfere primarily with the DNA breakage step, contrary to what has been reported with E. coli gyrase. Both S. aureus gyrase-catalyzed DNA supercoiling and S. aureus topoisomerase IV-catalyzed decatenation were dramatically stimulated by potassium glutamate or aspartate (500- and 50-fold by 700 and 350 mM glutamate, respectively), whereas topoisomerase IV-dependent DNA relaxation was inhibited 3-fold by 350 mM glutamate. The relevance of the effect of dicarboxylic amino acids on the activities of type II topoisomerases is discussed with regard to the intracellular osmolite composition of S. aureus.

Anti-Bacterial Agents↗
PubMed1995

Didanosine compared with continued zidovudine therapy for HIV-infected patients with 200 to 500 CD4 cells/mm3. A double-blind, randomized, controlled trial. Canadian HIV Trials Network Protocol 002 Study Group.

OBJECTIVE: To compare the safety and efficacy of didanosine with that of continued zidovudine therapy in persons with human immunodeficiency virus (HIV) infection who had received zidovudine for at least 6 months and had CD4 cell counts of 200 to 500 CD4 cells/mm3. DESIGN: Double-blind, randomized controlled trial. SETTING: 10 Canadian university-affiliated specialty clinics. PATIENTS: 246 patients were assigned to receive standard doses of either zidovudine or didanosine. OUTCOME MEASURES: The primary clinical end point was the occurrence of a new, previously undiagnosed acquired immunodeficiency syndrome (AIDS)-defining illness or death. RESULTS: 245 of 246 patients were eligible (118 receiving didanosine and 127 receiving zidovudine). Sixty-six percent were asymptomatic, 30% had AIDS-related complex, and 4% had AIDS. The median baseline CD4 count was 320 cells/mm3. The median previous duration of zidovudine therapy was 471 days. Nine new AIDS-defining illnesses developed during the study; all but one were in the zidovudine group (relative risk, 7.9 [95% CI, 1.0 to 63.3; P = 0.02]). A change to didanosine led to a statistically significant increase in CD4 counts by week 2 that persisted until the end of the study at week 48 (P < or = 0.01). Viral sensitivity studies (done in 102 patients) showed that 28% of the zidovudine group and 21% of the didanosine group had high-level in vitro resistance to zidovudine (50% inhibitory concentration greater than 0.8 microM) at baseline (P = 0.49). Only one patient in the didanosine group developed high-level resistance to zidovudine during the study. In the zidovudine group, the cumulative probability of developing high-level resistance to zidovudine was 59% at 1 year (P = 0.01). Abdominal pain, leukopenia, and neutropenia were more frequent in the zidovudine group, and hyperuricemia was more frequent in the didanosine group (P < 0.05). CONCLUSION: In clinically stable patients with 200 to 500 CD4 cells/mm3 who had tolerated zidovudine for at least 6 months, a change to didanosine led to a decrease in the rate of disease progression, a sustained increase in CD4 counts, and a decrease in the chances of developing high-level resistance to zidovudine. Both drugs were generally well tolerated.

Adult↗
PubMed1995

Analysis of gyrA and grlA mutations in stepwise-selected ciprofloxacin-resistant mutants of Staphylococcus aureus.

Fluoroquinolone-resistant mutants were obtained in vitro from Staphylococcus aureus RN4220 by stepwise selection on increasing concentrations of ciprofloxacin. Results from sequence analysis of the quinolone resistance-determining region of GyrA and of the corresponding region of GrlA, the DNA topoisomerase IV subunit, showed an alteration of Ser-80 to Tyr (corresponding to Ser-83 of Escherichia coli GyrA) or Glu-84 to Lys in GrlA of both low- and high-level quinolone-resistant mutants. Second-step mutants were found to have, in addition to a mutation in grlA, reduced accumulation of norfloxacin or an alteration in GyrA at Ser-84 to Leu or Glu-88 to Lys. Third-step mutants derived from second-step mutants with reduced accumulation were found to have a mutation in gyrA. The results from this study demonstrated that mutations in gyrA or mutations leading to reduced drug accumulation occur after alteration of GrlA, supporting the previous findings (L. Ferrero, B. Cameron, B. Manse, D. Lagneaux, J. Crouzet, A. Famechon, and F. Blanche, Mol. Microbiol. 13:641-653, 1994) that DNA topoisomerase IV is a primary target of fluoroquinolones in S. aureus.

Ciprofloxacin↗
PubMed1995

Sensory interaction and descriptions of fabric hand.

82 subjects who viewed and felt fabrics (sensory interaction group) used different categories of terms to describe fabric hand than did 38 subjects who only felt the fabrics. Therefore, the methods used to measure fabric hand that isolate the senses may not accurately assess the way in which subjects describe fabric hand in nonlaboratory settings.

Adult↗
PubMed1994

Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target of fluoroquinolones.

A 4.6 kb Staphylococcus aureus DNA fragment containing DNA gyrase-like genes (grlA and grlB) was cloned and sequenced. The proteins GrlA and GrlB exhibit more than 30% identity with E. coli DNA topoisomerase IV subunits and with the gyrase subunits from S. aureus and Escherichia coli. The combined E. coli cell extracts of GrlA and GrlB overproducing strains catalysed ATP-dependent relaxation and decatenation specific to DNA topoisomerase IV. The temperature-sensitive phenotype of Salmonella typhimurium parC and parE mutants was complemented by the S. aureus grlA and grlB genes, when the two genes were co-expressed. These results show that GrlA and GrlB are the subunits of S. aureus DNA topoisomerase IV. The GyrA subunit of DNA gyrase has been previously defined as a primary target of quinolones based on genetic and biochemical experiments essentially carried out in E. coli. Single-point mutations occurring in the 'quinolone resistance-determining region' (QRDR) of GyrA were found in bacteria exhibiting quinolone resistance, the most common mutation being a substitution of Ser-83 on the E. coli GyrA sequence. We analysed eight S. aureus fluoroquinolone-resistant clinical isolates and observed that mutations in the QRDR of GyrA are not present in the low-quinolone-resistant isolates. In contrast, Ser-80 of GrlA, which corresponds to Ser-83 of E. coli GyrA, is substituted to Phe or Tyr in both high- and low-quinolone-resistant isolates. We propose that DNA topoisomerase IV is a primary target of fluoroquinolones in S. aureus.

Amino Acid Sequence↗
PubMed1994

IN VITRO INVESTIGATION OF THE FACTORS CONTRIBUTING TO THE UNIQUE CO2 TRANSPORT PROPERTIES OF BLOOD IN THE SEA LAMPREY (PETROMYZON MARINUS)

In vitro experiments were conducted to determine the factors contributing to the unusual distribution of CO2 in the blood of the sea lamprey. When rainbow trout red blood cells (RBCs) were equilibrated with a 3 % CO2:nitrogen mixture in either normal saline or sodium-free saline, the extracellular total carbon dioxide content (CCO2ext) was highly dependent upon the fraction of RBCs in the suspension. In contrast, when lamprey RBCs were equilibrated in normal saline, the CCO2ext decreased with increasing hematocrit. In the absence of extracellular sodium, however, the CCO2ext in the lamprey RBC suspension also became positively correlated with hematocrit. These results suggest that the membrane of sea lamprey RBCs may be somewhat permeable to bicarbonate, but that transmembrane bicarbonate movements may only be detectable in vitro when Na+/H+ exchange is inactivated. Also in contrast to the results for rainbow trout, the changes in CCO2 that occurred in lamprey RBC suspensions following a step increase in PCO2 were not associated with any change in RBC chloride concentration and were not markedly affected by the chloride/bicarbonate exchange inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). When lamprey RBCs in sodium-free saline were treated with an ionophore for anions, tributyl tin chloride (TBTC), however, the distribution of anions across the RBC membrane came to resemble that of the trout. Furthermore, the relationship between CCO2ext and hematocrit in suspensions of TBTC-treated lamprey RBCs also resembled that of trout in normal saline. Thus, these results demonstrate that both the presence of Na+/H+ exchange and the absence of significant anion exchange contribute to the unique CO2 transport properties of sea lamprey blood.

Journal Article↗
PubMed1994

The impact of pharmacy discharge planning on continuity of care.

Maintaining high quality patient care following hospital discharge is essential for complete recovery and continued well-being. Historically, pharmacist participation in discharge planning has been minimal and has been frequently limited to last minute patient counselling. Hospital pharmacists can contribute to the continuity of patient care by summarizing changes made to a patient's therapy, their rationale, and future considerations in a discharge report to the family physician and/or community pharmacist. In this study, pharmacy discharge summaries were prepared for inclusion in the discharge report to the family physician. Summaries were also forwarded to the community pharmacist, where appropriate. Two types of pharmacy summaries completed were "Rationale for Inpatient Changes" (RIC) and "Recommendations for Future Changes" (RFC) summaries. Evaluation forms accompanying the summaries elicited very favourable responses. An independent review group of two physicians and two pharmacists rated the potential for reduction of patient mortality/morbidity as either marked, modest, minor or negligible; most of the summaries were evaluated as having a "modest" impact. Workload associated with preparation of pharmacy summaries would require additional pharmacy staff. Direct and indirect cost savings, including decreased drug costs and avoidance of drug complications and hospital readmissions, are associated with this service.

Attitude of Health Personnel↗
PubMed1994

Expression of c-erb B-2 oncogene product in persistent gestational trophoblastic disease.

OBJECTIVE: Much debate exists on the initiation of chemotherapy for women at risk for persistent gestational trophoblastic disease. This is a result of a lack of early predictors for the development of persistent gestational trophoblastic disease after evacuation of a complete hydatidiform mole, because the only current reliable method of detection and diagnosis lies in persistent or rising postmolar beta-human chorionic gonadotropin values. We used immunocytochemical techniques to retrospectively study the expression of the c-erb B-2 oncogene product in formalin-fixed, paraffin-embedded trophoblastic tissues as a potential indicator of the development of persistent gestational trophoblastic disease. STUDY DESIGN: In this retrospective study 56 trophoblastic tumors were examined by means of immunocytochemical techniques to stain for the oncogene product for evidence of c-erb B-2 expression. Our 56 cases included original tissue from 20 cases of complete mole that progressed to persistent gestational trophoblastic disease, seven cases of choriocarcinoma after term pregnancy or abortion, and 29 cases of hydatidiform mole representing postevacuation, spontaneously regressing disease (including one partial mole). We also studied 11 cases of first-trimester trophoblast and 15 cases of term placenta as additional controls. RESULTS: Our results showed positive immunostaining for c-erb B-2 gene product in one case of persistent gestational trophoblastic disease, with negative staining in all other cases in the study groups and controls. CONCLUSION: Analysis for the significance of c-erb B-2 expression in persistent gestational trophoblastic disease showed that this correlation between c-erb B-2 expression and persistent gestational trophoblastic disease is not significant, suggesting that future efforts should be directed at the involvement of different oncoproteins.

Biomarkers, Tumor↗
PubMed1993

Biosynthesis of the corrin macrocycle of coenzyme B12 in Pseudomonas denitrificans.

Studies with cell-free protein preparations from a series of recombinant strains of Pseudomonas denitrificans demonstrated that precorrin-3 is converted into a further trimethylated intermediate, named precorrin-3B, along the pathway to coenzyme B12. It was then shown that the part of the pathway from precorrin-3 (called precorrin-3A hereafter) to precorrin-6x involves three intermediates, precorrin-3B, precorrin-4, and precorrin-5. Precorrin-3B was isolated in its native (reduced) as well as its oxidized (factor-IIIB) states, and precorrin-4 was isolated in its oxidized form only (factor-IV). Both factors were in vitro precursors of precorrin-6x. The synthesis of precorrin-6x from precorrin-3A was shown to be catalyzed by four enzymes, CobG, CobJ, CobM, and CobF, intervening in this order. They were purified to homogeneity. CobG, which converts precorrin-3A to precorrin-3B, was found to be an iron-sulfur protein responsible for the oxidation known to occur between precorrin-3A and precorrin-6x, and CobJ, CobM, and CobF are the C-17, C-11, and C-1 methylases, respectively. The acetate fragment is extruded after precorrin-4 formation. This study combined with our recent structural studies on factor-IV (D. Thibaut, L. Debussche, D. Fréchet, F. Herman, M. Vuilhorgne, and F. Blanche, J. Chem. Soc. Chem. Commun. 1993:513-515, 1993) and precorrin-3B (L. Debussche, D. Thibaut, M. Danzer, F. Debu, D. Fréchet, F. Herman, F. Blanche, and M. Vuilhorgne, J. Chem. Soc. Chem. Commun. 1993:1100-1103, 1993) provides a first step-by-step picture of the sequence of the enzymatic reactions leading to the corrin ring in P. denitrificans.

Carbon Isotopes↗
PubMed1992

Construction of a broad-host-range non-mobilizable stable vector carrying RP4 par-region.

Plasmid pXL1635 was constructed from the already segregationally stable incP-derived pRK290. Plasmid pXL1635 should be suitable for industrial and environmental uses in Gram- bacteria since (i) it contains the par fragment from RP4 which increases its stability in Pseudomonas denitrificans, a cobalamin-producing and industrially used bacterium, and (ii) the RK2 oriT has been deleted, leading to a non-mobilizable plasmid.

Base Sequence↗
PubMed1992

Assay, purification, and characterization of cobaltochelatase, a unique complex enzyme catalyzing cobalt insertion in hydrogenobyrinic acid a,c-diamide during coenzyme B12 biosynthesis in Pseudomonas denitrificans.

Hydrogenobyrinic acid a,c-diamide was shown to be the substrate of cobaltochelatase, an enzyme that catalyzes cobalt insertion in the corrin ring during the biosynthesis of coenzyme B12 in Pseudomonas denitrificans. Cobaltochelatase was demonstrated to be a complex enzyme composed of two different components of M(r) 140,000 and 450,000, which were purified to homogeneity. The 140,000-M(r) component was shown to be coded by cobN, whereas the 450,000-M(r) component was composed of two polypeptides specified by cobS and cobT. Each component was inactive by itself, but cobaltochelatase activity was reconstituted upon mixing CobN and CobST. The reaction was ATP dependent, and the Km values for hydrogenobyrinic acid a,c-diamide, Co2+, and ATP were 0.085 +/- 0.015, 4.2 +/- 0.2, and 220 +/- 36 microM, respectively. Spectroscopic data revealed that the reaction product was cob(II)yrinic acid a,c-diamide, and experiments with a coupled-enzyme incubation system containing both cobaltochelatase and cob(II)yrinic acid a,c-diamide reductase (F. Blanche, L. Maton, L. Debussche, and D. Thibaut, J. Bacteriol. 174:7452-7454, 1992) confirmed this result. This report not only provides the first evidence that hydrogenobyrinic acid and its a,c-diamide derivative are indeed precursors of adenosylcobalamin but also demonstrates that precorrin-6x, precorrin-6y, and precorrin-8x, three established precursors of hydrogenobyrinic acid (D. Thibaut, M. Couder, A. Famechon, L. Debussche, B. Cameron, J. Crouzet, and F. Blanche, J. Bacteriol. 174:1043-1049, 1992), are also on the pathway to cobalamin.

Amino Acid Sequence↗
PubMed1992

Precorrin-6x reductase from Pseudomonas denitrificans: purification and characterization of the enzyme and identification of the structural gene.

Precorrin-6x reductase, which catalyzes the NADPH-dependent reduction of precorrin-6x to a dihydro derivative named precorrin-6y, was purified 14,300-fold to homogeneity with an 8% yield from extracts of a recombinant strain of Pseudomonas denitrificans. Precorrin-6y was identified by fast atom bombardment-mass spectrometry. It was converted in high yield (90%) to hydrogenobyrinic acid by cell-free protein preparations from P. denitrificans. For the purification and characterization of precorrin-6x reductase, a coupled-enzyme radioenzymatic assay was developed in which precorrin-6y was methylated in situ by the cobL gene product (F. Blanche, A. Famechon, D. Thibaut, L. Debussche, B. Cameron, J. Crouzet, J. Bacteriol. 174:1050-1052, 1992) in the presence of [methyl-3H]S-adenosyl-L-methionine. Molecular weights of precorrin-6x reductase obtained by gel filtration (Mr congruent to 27,000) and by analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr congruent to 31,000) were consistent with the enzyme being a monomer. Km values of 3.6 +/- 0.2 microM for precorrin-6x and 23.5 +/- 3.5 microM for NADPH and a Vmax value of 17,000 U mg-1 were obtained at pH 7.7. The N-terminal sequence (six amino acids) and three internal sequences obtained after tryptic digestion of the enzyme were determined by microsequencing and established that precorrin-6x reductase is encoded by the cobK gene, located on a previously described 8.7-kb EcoRI fragment (J. Crouzet, B. Cameron, L. Cauchois, S. Rigault, M.-C. Rouyez, F. Blanche, D. Thibaut, and L. Debussche, J. Bacteriol. 172:5980-5990, 1990). However, the coding sequence was shown to be on the strand complementary to the one previously proposed as the coding strand.

Amino Acid Sequence↗
PubMed1992

The final step in the biosynthesis of hydrogenobyrinic acid is catalyzed by the cobH gene product with precorrin-8x as the substrate.

The final enzymatic reaction in the conversion of precorrin-6x to hydrogenobyrinic acid by cell-free protein preparations from Pseudomonas denitrificans was shown to be inhibited by hydrogenobyrinic acid. Use was made of this property to prepare the last biosynthetic precursor of hydrogenobyrinic acid, named precorrin-8x. Double-labeling experiments, mass spectrometry, and UV-visible light spectroscopy studies established that precorrin-8x was at the oxidation level of a corrin and differed from precorrin-6x by two additional methyl groups (presumably at C-5 and C-15) and decarboxylation of the acetic acid side chain at C-12. Precorrin-8x was not a corrin but had the same mass as hydrogenobyrinic acid, thus showing that this latter compound is synthesized from the former by a rearrangement. The enzyme catalyzing this rearrangement was purified 80-fold to homogeneity from a recombinant strain of P. denitrificans, sequenced at its N terminus, and shown to be encoded by the cobH gene. It was identical to the previously described hydrogenobyrinic acid-binding protein (F. Blanche, D. Thibaut, D. Frechet, M. Vuilhorgne, J. Crouzet, B. Cameron, G. Müller, K. Hlineny, U. Traub-Eberhard, and M. Zboron, Angew. Chem. Int. Ed. Engl. 29:884-886, 1990). This enzyme had a Km of 0.91 +/- 0.04 microM and a Vmax of 230 nmol h-1 mg-1 at pH 7.7 and was competitively inhibited by hydrogenobyrinic acid with a Ki of 0.17 +/- 0.01 microM. It is proposed that the cobH gene product is a mutase which transfers the methyl group from C-11 to C-12.

Amino Acid Sequence↗