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Robert Reynolds

Publications and source records attributed to Robert Reynolds.

17 recordsLinked to original sources
PubMed2006

Metabolic fate of administered [13C]galactose in tissues of galactose-1-phosphate uridyl transferase deficient mice determined by nuclear magnetic resonance.

The pattern of distribution of galactose and its metabolites was determined in tissues of mice deficient in galactose-1-phosphate uridyl transferase (G/G) 4 h after the administration of 1mg/g of [13C]galactose. Labeled galactose was found in all the tissues examined, the highest amounts in liver and kidney. Each of the tissues had its own pattern of labeling of galactose-1-phosphate (gal-1-P), galactitol and galactonate. [13C]gal-1-P and galactonate concentration was highest in liver while [13C]galactitol was higher in kidney and heart than in other tissues. Muscle had the lowest amounts of these compounds. In contrast, no galactose was found in tissues of normal mice (N/N) except for a minute amount in muscle. No [13C]gal-1-P was found in liver, kidney or brain and only minute amounts in heart and muscle of N/N animals. Barely detectible, labeled galactitol was observed in these tissues except liver, where none was found. [13C]Galactonate was formed in liver comparable to G/G mice. Almost all of the accumulating 13C isotope was found in liver and kidney glucose and lactate in the normal animals. [13C]Glucose and lactate was also found in liver of the G/G animals, but to a lesser extent than in normals, indicating the presence of a pathway in G/G animals for circumventing the block at GALT for the normal conversion of galactose to glucose.

Animals↗
PubMed2006

Mycobacterium tuberculosis cells growing in macrophages are filamentous and deficient in FtsZ rings.

FtsZ, a bacterial homolog of tubulin, forms a structural element called the FtsZ ring (Z ring) at the predivisional midcell site and sets up a scaffold for the assembly of other cell division proteins. The genetic aspects of FtsZ-catalyzed cell division and its assembly dynamics in Mycobacterium tuberculosis are unknown. Here, with an M. tuberculosis strain containing FtsZ(TB) tagged with green fluorescent protein as the sole source of FtsZ, we examined FtsZ structures under various growth conditions. We found that midcell Z rings are present in approximately 11% of actively growing cells, suggesting that the low frequency of Z rings is reflective of their slow growth rate. Next, we showed that SRI-3072, a reported FtsZ(TB) inhibitor, disrupted Z-ring assembly and inhibited cell division and growth of M. tuberculosis. We also showed that M. tuberculosis cells grown in macrophages are filamentous and that only a small fraction had midcell Z rings. The majority of filamentous cells contained nonring, spiral-like FtsZ structures along their entire length. The levels of FtsZ in bacteria grown in macrophages or in broth were comparable, suggesting that Z-ring formation at midcell sites was compromised during intracellular growth. Our results suggest that the intraphagosomal milieu alters the expression of M. tuberculosis genes affecting Z-ring formation and thereby cell division.

Bacterial Proteins↗
PubMed2005

Pathways of galactose metabolism by galactosemics: evidence for galactose conversion to hepatic UDPglucose.

To determine if classic galactosemics have residual galactose-1-phosphate uridyltransferase (GALT) activity to explain their considerable ability to oxidize galactose over 24 h, we devised a method for assessing their ability to form hepatic UDPglucose (UDPglu), an intermediate in the normal Leloir pathway of galactose metabolism. The protocol involved the single oral administration of 7 mg/kg [2-13C]galactose concomitant with multiple small doses of acetaminophen with measurement of the extent of labeling of urinary acetaminophen glucuronide, the glucuronide moiety being formed from hepatic UDPglu. We performed the study lasting 24 h in two normal subjects and three classic galactosemics, two homozygous for the Q188R mutation and one compound for the Q188R/K258N mutation. The labeling and total excretion of acetaminophen glucuronide was measured in urine by nuclear magnetic resonance techniques. Concomitant with determination of label in the glucuronide measurement was made of galactose oxidation to 13CO2 and the 13C enrichment of plasma glucose. All of the galactosemic patients formed 13C enriched acetaminophen glucuronide indicating that they had converted the labeled galactose to [13C]UDPglu and that residual GALT or another pathway that forms UDPglu is present in hepatic tissue. Compared to the normal whose glucuronide labeling was rapid and short-lived that of the galactosemics was delayed and extended for a long period over 10 h. The extent of isotopic enrichment of glucuronide by galactosemics was comparable to the normals, resulting in a much greater conversion of galactose to UDPglu by the galactosemics. The labeling of the UDPglu pool was reflected by the rate of 13CO2 formation being rapid in the normal with peak labeling at 2-3 h with total oxidation of over 70% in 24 h. The oxidation of the galactosemics was slow with a broad peak of 13CO2 at 10 h and a total excretion of 25-39% of the [13C]galactose administered. The normal subjects formed highly enriched plasma glucose within 30 min while no enrichment of plasma glucose was detected until after 300 min in galactosemics. The exact pathway(s) of galactose metabolism by galactosemics to UDPglu remain to be determined. Their delineation may contribute to new approaches to therapeutic strategies for this enigmatic disorder.

Acetaminophen↗
PubMed2005

Synthesis of novel 5-aryl-2-thio-1,3,4-oxadiazoles and the study of their structure-anti-mycobacterial activities.

The preparation of novel 5-aryl-2-thio-1,3,4-oxadiazoles 4a-41 and the computer-aided study of their in vitro anti-tubercular activity against Mycobacterium tuberculosis H37Rv (ATCC 27294) are reported. The average accuracy of the electronic-topological method and neural network methods applied to the activity prediction in leave-one-out cross validation is 80%.

Antitubercular Agents↗
PubMed2005

UDP-galactose pyrophosphorylase in mice with galactose-1-phosphate uridyltransferase deficiency.

UDP-glucose pyrophosphorylase (E.C. 2.7.7.9), encoded by ugp, provides UDP-glucose which is critical to the synthesis of glycogen, and also catalyzes the reaction between UTP and galactose-1-phosphate, yielding UDP-galactose. This activity of UDP-gal pyrophosphorylase (UDP-galPP) suggests a role in an alternate pathway for galactose metabolism in patients with deficiency of galactose-1-phosphate uridyltransferase (GALT). We examined the effects of GALT deficiency and dietary galactose on UDP-glucose pyrophosphorylase (UDP-gluPP) and UDP-galactose pyrophosphorylase activity and ugp expression in liver of mice with homozygous deletion of the critical regions of galt. Activity with glucose-1-phosphate as substrate was significantly higher than that with galactose-1-phosphate. In liver from mice with GALT deficiency (G/G), UDP-galPP activity appeared to be lower than that measured in liver from control (N/N) animals. This difference disappeared when the N/N tissue homogenate was dialyzed to remove residual UDP-glucose, confirming that careful elimination of residual GALT activity is necessary, since GALT has 1000-fold greater activity toward galactose-1-phosphate than that of UDP-galPP in liver homogenates. Prior exposure to conventional mouse chow, high galactose chow, and high glucose chow did not alter UDP-glu PP or UDP-galPP activity. Steady state UGP mRNA levels were determined in tissues from normal and G/G animals. UGP expression was highest in liver, and did not differ by genotype or exposure to high galactose chow. UDP-galPP activity may account for unexplained ability to oxidize galactose in animals with no GALT activity, but is insufficient to alter accumulation of galactose metabolites.

Animals↗
PubMed2005

Phase II trial of oral rubitecan in previously treated pancreatic cancer patients.

BACKGROUND: Additional systemic treatments for locally advanced or metastatic pancreatic cancer are needed, as current treatment options produce only modest survival benefits. Rubitecan (Orathecin; Supergen Inc., Dublin, CA, http://www.supergen.com) is an orally active camptothecin derivative with demonstrated responses in patients with pancreatic cancer in early clinical trials. This phase II, open-label trial was developed to assess the safety and efficacy of rubitecan in patients with locally advanced or metastatic pancreatic cancer refractory to conventional chemotherapy. METHODS: Fifty-eight patients with failed or relapsed advanced pancreatic cancer after receiving at least one prior chemotherapy regimen were enrolled to receive eight consecutive weeks of treatment with rubitecan at a dose of 1.5 mg/m2 orally on five consecutive days per week, followed by 2 days off therapy, repeatedly. The primary end point was response rate. Time to progression, overall survival, changes in CA19-9 levels, and the composite measure of clinical benefit response were evaluated as secondary end points. RESULTS: Among 43 patients with measurable disease, 7% (3/43) achieved partial responses and 16% (7/43) had disease stabilization for an overall response and disease stabilization rate of 23%. All responses were confirmed by independent radiology review. Median survival was longer in responding patients than in the overall study cohort (10 months versus 3 months). Gastrointestinal and hematologic toxicities were the most commonly reported adverse events. CONCLUSION: Oral rubitecan produced responses and was well tolerated by heavily pretreated patients with refractory pancreatic cancer. The overall risk-benefit profile of oral rubitecan appears promising, supporting further evaluation in phase III trials in patients with refractory and chemotherapy-naive pancreatic cancer.

Administration, Oral↗
PubMed2004

Defining the physical gate of a mechanosensitive channel, MscL, by engineering metal-binding sites.

The mechanosensitive channel of large conductance, MscL, of Escherichia coli is one of the best-studied mechanosensitive proteins. Although the structure of the closed or "nearly-closed" state of the Mycobacterium tuberculosis ortholog has been solved and mechanisms of gating have been proposed, the transition from the closed to the open states remains controversial. Here, we probe the relative position of specific residues predicted to line the pore of MscL in either the closed state or during the closed-to-open transition by engineering single-site histidine substitutions and assessing the ability of Ni2+, Cd2+ or Zn2+ ions to affect channel activity. All residues predicted to be within the pore led to a change in channel threshold pressure, although the direction and extent of this change were dependent upon the mutation and metal used. One of the MscL mutants, L19H, exhibited gating that was inhibited by Cd2+ but stimulated by Ni2+, suggesting that these metals bind to and influence different states of the channel. Together, the results derived from this study support the hypotheses that the crystal structure depicts a "nearly closed" rather than a "fully closed" state of MscL, and that a clockwise rotation of transmembrane domain 1 occurs early in the gating process.

Binding Sites↗
PubMed2004

Directly observed therapy for the management of HIV-infected patients in a methadone program.

The objective of this prospective, observational clinical study was to evaluate the safety and efficacy of once-daily and twice-daily directly observed therapy (DOT) in human immunodeficiency virus (HIV)-infected patients undergoing methadone treatment. Methadone and highly active antiretroviral therapy (HAART) were dispensed daily as DOT, with patients in the twice-daily HAART group self-administering the second dose. Clinical and laboratory end points were monitored, along with the impact of ongoing cocaine use. We studied 54 patients coinfected with HIV and hepatitis C virus. At baseline, the median virus load was 111,000 copies/mL, and the median CD4+ cell count was 165 cells/mm3. After a median of 24 months, 17 of 29 patients in the once-daily HAART group and 18 of 25 in the twice-daily HAART group had virus loads of <400 copies/mL, regardless of ongoing cocaine use. Thirty-two patients required methadone dose adjustment, which was managed without modification of HAART. Treatment-limiting hepatic toxicity was rare. A DOT program of coadministered methadone and HAART can be implemented with good results, even for patients who continue to use cocaine.

Anti-HIV Agents↗
PubMed2004

Galactitol and galactonate accumulation in heart and skeletal muscle of mice with deficiency of galactose-1-phosphate uridyltransferase.

Under conditions of dietary galactose loading, mice deficient in galactose-1-phosphate uridyltransferase (GALT) accumulate large amounts of galactitol and galactonate in heart and skeletal muscle. In contrast to liver, brain, and kidney, which form little galactitol when GALT-deficient animals (G/G) ingest a 40% galactose diet, heart and skeletal muscle galactitol reaches 22.90+/-1.62 (M+/-SE) and 38.88+/-2.62 micromol/g tissue, respectively, levels 40-100 times that of galactose-1-phosphate (Gal-1-P). Sixteen-day-old suckling G/G mice accumulate galactitol in heart and to a lesser extent, in skeletal muscle. Heart and skeletal muscle of G/G mice also form galactonate, with levels comparable to that of liver, which was presumed previously to be the only tissue capable of converting galactose to galactonate under conditions of loading. The data suggest that heart and skeletal muscle play a role in disposition of galactose when GALT activity is impaired, contributing a large share to urinary galactitol and galactonate excretion. The ability of heart and muscle to form galactonate may also contribute to the G/G mouse's ability to slowly oxidize galactose to CO2, since the compound is an intermediate in an alternate route for galactose disposition.

Animals↗
PubMed2004

International collaborative study of cardiovascular disease in Asia: design, rationale, and preliminary results.

OBJECTIVES: The overall objective of the International Collaborative Study of Cardiovascular Disease in Asia (InterASIA) was to estimate the prevalence and distribution of cardiovascular disease risk factors, including hypertension, hypercholesterolemia, diabetes, obesity, cigarette smoking, and physical inactivity, in the general population of both China and Thailand. METHODS AND RESULTS: A multistage cluster sampling method was used to select a nationally representative sample of 15,838 adults, aged 35-74 years, in China, and 5,350 similarly aged adults in Thailand. Data on medical history, cigarette smoking, alcohol consumption, diet, physical activity, and awareness and control of risk factors for cardiovascular disease, were collected. Blood pressure, body weight and height, and hip and waist circumferences, were measured using standard methods. Fasting blood specimens were collected to measure levels of total cholesterol, high-density lipoprotein cholesterol, triglycerides, glucose, and creatinine. All data were carefully collected by specially trained, experienced observers using standardized methods and a stringent level of quality control. The prevalence of self-reported cardiovascular disease in Chinese adults aged 35-74 years was 3.3% in men and 3.6% in women (representing 8,147,000 male and 8,270,000 female cases nationally). The prevalence of self-reported cardiovascular disease in Thai adults was 1.5% in men and 1.7% in women (representing 184,000 male and 226,000 female cases). CONCLUSIONS: The InterASIA study provides important information on the current prevalence and control of both cardiovascular disease and its risk factors in the region. These findings will be useful for developing national strategies to reduce the large, and increasing, burden of cardiovascular disease in China and Thailand.

Adult↗
PubMed2003

Single-nucleotide polymorphism genotyping on optical thin-film biosensor chips.

Single-nucleotide polymorphisms (SNPs) constitute the bulk of human genetic variation and provide excellent markers to identify genetic factors contributing to complex disease susceptibility. A rapid, sensitive, and inexpensive assay is important for large-scale SNP scoring. Here we report the development of a multiplex SNP detection system using silicon chips coated to create a thin-film optical biosensor. Allele-discriminating, aldehyde-labeled oligonucleotides are arrayed and covalently attached to a hydrazinederivatized chip surface. Target sequences (e.g., PCR amplicons) then are hybridized in the presence of a mixture of biotinylated detector probes, one for each SNP, and a thermostable DNA ligase. After a stringent wash (0.01 M NaOH), ligation of biotinylated detector probes to perfectly matched capture oligomers is visualized as a color change on the chip surface (gold to blue/purple) after brief incubations with an anti-biotin IgG-horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate. Testing of PCR fragments is completed in 30-40 min. Up to several hundred SNPs can be assayed on a 36-mm2 chip, and SNP scoring can be done by eye or with a simple digital-camera system. This assay is extremely robust, exhibits high sensitivity and specificity, and is format-flexible and economical. In studies of mutations associated with risk for venous thrombosis and genotyping/haplotyping of African-American samples, we document high-fidelity analysis with 0 misassignments in 500 assays performed in duplicate.

Biosensing Techniques↗
PubMed2003

Increasing the efficiency of fuzzy logic-based gene expression data analysis.

DNA microarray technology can accommodate a multifaceted analysis of the expression of genes in an organism. The wealth of spatiotemporal data generated by this technology allows researchers to potentially reverse engineer a particular genetic network. "Fuzzy logic" has been proposed as a method to analyze the relationships between genes and help decipher a genetic network. This method can identify interacting genes that fit a known "fuzzy" model of gene interaction by testing all combinations of gene expression profiles. This paper introduces improvements made over previous fuzzy gene regulatory models in terms of computation time and robustness to noise. Improvement in computation time is achieved by using a cluster analysis as a preprocessing method to reduce the total number of gene combinations analyzed. This approach speeds up the algorithm by a factor of 50% with minimal effect on the results. The model's sensitivity to noise is reduced by implementing appropriate methods of "fuzzy rule aggregation" and "conjunction" that produce reliable results in the face of minor changes in model input.

Animals↗
PubMed2003

Galactitol and galactonate in red blood cells of galactosemic patients.

The red blood cell (RBC) concentration of galactitol and galactonate was measured in 27 patients with galactose-1-phosphate uridyltransferase (GALT) deficiency galactosemia and 19 non-galactosemic subjects by a newly devised isotope dilution gas chromatography/mass spectrometry (GC/MS) method. The method utilizing UL[13C]galactitol and UL[13C]galactonate was reproducible with excellent precision and recovery of 99%. The RBC galactitol in galactosemic patients on galactose-restricted diets averaged 5.98+/-1.2 microM (M+/-SD) with a range of 3.54-8.81 microM. The mean in non-galactosemic patients was 0.73+/-0.31 microM with a range of 0.29-1.29 microM. The mean of RBC galactonate in the same galactosemic patients was 4.16+/-1.32 microM (M+/-SD) with a range of 0.68-6.47, while the mean in non-galactosemic subjects was 1.94+/-0.96 (M+/-SD) with a range of 0.69-3.84. In galactosemic RBC the galactitol was higher than galactonate while this was reversed in non-galactosemic cells. RBC galactose-1-phosphate (Gal-1-P) measured at the same time as galactitol and galactonate was 30 times the level of the other two metabolites. There was no relationship between RBC Gal-1-P and galactitol or galactonate. The ability to measure all three galactose metabolites in the same procedure offers the possibility of augmented monitoring of the galactose metabolic status of patients. The measurement of RBC galactitol and galactonate presents a new means of characterizing galactosemic patients and their levels monitored over time may provide new insight in the development of long-term complications observed in afflicted patients.

Adolescent↗
PubMed2003

Thiosemicarbazole (thiacetazone-like) compound with activity against Mycobacterium avium in mice.

In vitro screening of thiacetazone derivatives indicated that two derivatives, SRI-286 and SRI-224, inhibited a panel of 25 Mycobacterium avium complex (MAC) isolates at concentrations of 2 micro g/ml or lower. In mice, SRI-224 and thiacetazone had no significant activity against the MAC in livers and spleens, but treatment with SRI-286 resulted in significant reduction of bacterial loads in livers and spleens. A combination of SRI-286 and moxifloxacin was significantly more active than single drug regimens in liver and spleen.

Animals↗
PubMed2002

Metabolism of 13C galactose by lymphoblasts from patients with galactosemia determined by NMR spectroscopy.

In order to assess the pathways by which galactose is metabolized by galactose-1-phosphate uridyltransferase (GALT) deficient cells, lymphoblasts from 10 galactosemic patients with defined genotypes (six Q188R homozygotes, two S153L homozygotes, and two with homozygous deletions) were incubated with 1mM 1- or 2-13C galactose for 2.5 and 5 h. The 13C-labeled metabolites were identified and quantified using nuclear magnetic resonance and the results were compared to that obtained with cells from eight normal individuals. Cells from galactosemic patients formed two to three times the galactose-1-phosphate (Gal-1P) in normal cells, no difference being observed between the various genotypes. Galactitol formation was not significantly different from normal cells. No labeled galactonate was detected. Cells with the Q188R and S135L mutations formed both labeled uridine diphosphogalactose (UDPgal) and uridine diphosphoglucose (UDPglu), but to a lesser extent than normals, whereas cells with the GALT deletion did not. The pattern of 13C enrichment of the ribose carbons of adenosine monophosphate upon incubation of the normal cells with 1-13C galactose paralleled that found for incubations with 1-13C glucose, which is consistent with galactose disposition through the Leloir pathway to glucose and its subsequent metabolism to ribose. Cells with the GALT deletion formed no detectable labeled ribose, whereas cells from a patient homozygous for Q188R mutation formed labeled ribose in a pattern similar to normal albeit with lower enrichment. The results suggest that there is residual GALT activity and function of the Leloir pathway in the presence of the Q188R as well as S135L mutation.

Carbon Isotopes↗
PubMed2002

Erythrocyte galactose 1-phosphate quantified by isotope-dilution gas chromatography-mass spectrometry.

BACKGROUND: Measurements of alpha-D-galactose 1-phosphate (Gal-1-P) in erythrocytes are used to monitor the adequacy of dietary therapy in the treatment of galactosemia. We have devised a gas chromatography-mass spectrometry (GC/MS) isotope-dilution method for quantification of Gal-1-P. METHODS: We prepared trimethylsilyl (TMS) derivatives and used alpha-D-[2-(13)C]Gal-1-P as the internal standard for GC/MS. Results obtained with this method were compared with those determined by the established enzymatic method for samples from 23 healthy individuals (11 children and 12 adults), 9 suspected patients with galactosemia, 12 galactosemic patients on diet therapy, and 2 newly diagnosed toxic neonates. RESULTS: The method was linear up to 2.5 mmol/L with a lower limit of detection of 2.1 nmol (0.55 mg/L). Intra- and interassay imprecision (CVs) was 2.2-8.8%. In the 23 healthy individuals, values ranged from nondetectable to 9.2 micromol/L (2.4 mg/L of packed erythrocytes). Galactosemic patients on diet therapy had values of 10.9-45 mg/L of packed erythrocytes, whereas the newly identified patients had values of 166 and 373 mg/L. CONCLUSIONS: The GC/MS method is precise and useful over the wide range of concentrations needed to assess the galactose burden in patients with galactosemia.

Adult↗