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Biomedical subjects

M R Gray

Publications and source records attributed to M R Gray.

At least 55 records · Page 3Linked to original sources
PubMed1992

Rapid identification of deoxyribonucleic acid sequence differences in cytochrome P-450 21-hydroxylase (CYP21) genes with denaturing gradient gel blots.

Denaturing gradient gel electrophoresis was used to identify single-base differences in the cytochrome P-450 21-hydroxylase (CYP21) genes of 132 unrelated control individuals and family members of three unrelated patients with 21-hydroxylase deficiency. The salt-wasting variety was caused by gene deletion and gene conversion/deletion mutations in affected members of two families studied. The simple virilizing form, present in the third family, was caused by an apparent point mutation not detectable by routine Southern blots. We have detected many restriction fragment melting polymorphisms in the CYP21 genes of the members of both salt-wasting families and normal individuals with denaturing gradient gel electrophoresis. We also identified a restriction fragment melting polymorphism specific for the simple virilizing patient in the third family. The data demonstrate that the CYP21 genes are highly polymorphic and that denaturing gradient gel electrophoresis is useful for genomic deoxyribonucleic acid analysis of patients with 21-hydroxylase deficiency.

Adrenal Hyperplasia, Congenital↗
PubMed1992

Epithelial proliferation in Barrett's esophagus by proliferating cell nuclear antigen immunolocalization.

Proliferating cell nuclear antigen (PCNA) is an auxiliary protein to DNA polymerase delta and is an absolute requirement for cellular proliferation. Specialized-type Barrett's columnar-lined esophagus (CLE) is associated with adenocarcinomatous change. In the present study, the cellular proliferation of three histological types of CLE was assessed by semiquantitative evaluation of PCNA immunolocalization in 93 biopsy specimens from 45 patients using the murine monoclonal PC10. Statistical comparison was performed by the Mann-Whitney U test. Luminal surface cell labeling was uncommon in all histological types other than specialized CLE where 25 of the 43 biopsy specimens had at least occasional luminal surface cell labeling. Mean crypt labeling score of 4.06 for specialized type exceeded that for junctional (mean, 3.12; P < 0.001) and fundic types (mean, 1.6; P < 0.001). Gland cell PCNA staining scores for specialized-type CLE (mean, 3.18) exceeded that of junctional (mean, 1.97; P < 0.001) and fundic (mean, 1.04; P < 0.001). Summated PCNA scores for specialized-type, mean of 8.29, exceeded junctional mean score of 5.45 (P < 0.001) and fundic mean score of 2.76 (P < 0.001). PCNA immunolocalization reveals a high proportion of cells in cycle in the specialized-type CLE and expansion of the proliferative compartment, which may explain the association of specialized-type CLE with malignancy.

Antibodies, Monoclonal↗
PubMed1992

Models of hepatic drug elimination.

The liver is, by nature, heterogeneous. It contains a complex vascular network for blood flow and a stationary phase consisting of enzymes within parenchymal cells. Several physiological processes, therefore, may combine to give observed ranges in drug elimination. Net changes in concentration are a consequence of a series of steps: uptake of substrate into liver cells, enzymatic reactions within the cells, release of metabolites and unconverted substrate from the cells into the sinusoids, and the net flow of the perfusing medium in the vasculature. In addition, substrate binding to proteins in the blood and in the liver can influence hepatic elimination. An understanding of each of these processes is necessary to fully comprehend the overall process of drug elimination, and these processes must be accounted for, either individually or by grouping and approximation, if a model for drug elimination is to be developed. Existing models of hepatic elimination may be classified according to their treatment of mixing within the vasculature and whether or not the model explicitly accounts for mass transfer between the heterogeneous phases of the liver. Four major classes may be defined: 1. Nonparametric homogeneous models, which assume that either complete mixing or no mixing occurs within the vasculature of the organ. 2. Homogeneous mixing models, which allow for a range of mixing phenomena. 3. Heterogeneous micromixing models, which allow for mass transport between the cells and vasculature and describe mixing within the vasculature on a microscopic level. 4. Heterogeneous compartmental models, which also describe interphase mass transfer but assume complete mixing on a microscopic level, and therefore use a time and spatially averaged approach to model mixing. The utility of these models of hepatic elimination will be critically assessed based upon (1) their ability to account for the influence of the aforementioned physiological processes upon elimination; (2) the data requirements of the model, in addition to its mathematical complexity and ease of use; and (3) the range of compounds and metabolites which may be described using the model.

Animals↗
PubMed1992

Detection of DNA sequence polymorphisms in human genomic DNA by using denaturing gradient gel blots.

Denaturing gradient gel electrophoresis can detect sequence differences outside restriction-enzyme recognition sites. DNA sequence polymorphisms can be detected as restriction-fragment melting polymorphisms (RFMPs) in genomic DNA by using blots made from denaturing gradient gels. In contrast to the use of Southern blots to find sequence differences, denaturing gradient gel blots can detect differences almost anywhere, not just at 4-6-bp restriction-enzyme recognition sites. Human genomic DNA was digested with one of several randomly selected 4-bp recognition-site restriction enzymes, electrophoresed in denaturing gradient gels, and transferred to nylon membranes. The blots were hybridized with radioactive probes prepared from the factor VIII, type II collagen, insulin receptor, beta 2-adrenergic receptor, and 21-hydroxylase genes; in unrelated individuals, several RFMPs were found in fragments from every locus tested. No restriction map or sequence information was used to detect RFMPs. RFMPs can be used as genetic markers, because their alleles segregate in a Mendelian manner. Unlike most other methods for detecting DNA sequence polymorphisms, a genomic DNA blot made from one gel can be hybridized consecutively with many (30 or more) different probes.

Autoradiography↗
PubMed1992

Gonadotropin-releasing hormone-associated peptide gene sequences in women with hyperprolactinemia.

OBJECTIVE: To determine if mutations in the structural gene for gonadotropin-releasing hormone (GnRH)-associated peptide are present in women with hyperprolactinemia. DESIGN: Patients with hyperprolactinemia and controls were studied retrospectively for GnRH-associated peptide gene mutations. SETTINGS: Patients seen in a clinical setting were studied at a medical school laboratory setting. PATIENTS: Fifteen women with hyperprolactinemia and two fertile controls with normal prolactin levels were studied. INTERVENTIONS: Genomic deoxyribonucleic acid (DNA) was extracted from each patient and subjected to Southern blot analysis and polymerase chain reaction (PCR). For Southern blot analysis, DNA was digested with EcoRI, XbaI, BglII, PstI, and BamHI and hybridized to two DNA probes for GnRH-associated peptide. Exons II to IV, which encode for the structural gene, were amplified by PCR. MAIN OUTCOME MEASURES: Fragment sizes from autoradiographs were compared among patients and controls. Amplified PCR products of exons II to IV of the GnRH-associated peptide were also compared. RESULTS: No large deletions, insertions, or polymorphisms were identified in women with hyperprolactinemia or controls by Southern blotting. Each of the exons was present and of normal size by PCR in the study patients and controls. CONCLUSIONS: No large deletions of the GnRH-associated peptide gene appear to be present in our patients with hyperprolactinemia. Small deletions, insertions, or point mutations are not excluded by this analysis.

Adolescent↗
PubMed1991

Role of salivary epidermal growth factor in the pathogenesis of Barrett's columnar lined oesophagus.

Epidermal growth factor (EGF) is implicated in the regeneration of epithelial cells at the site of inflammation or ulceration in the gastrointestinal tract. Single parotid EGF concentration and production was studied in 64 patients with Barrett's columnar lined oesophagus (CLO), in 22 patients with severe reflux oesophagitis without columnar metaplasia and in 51 normal controls. In control patients, mean salivary EGF concentration was 2790 pg/ml (median 1450 pg/ml; range 450-16,500 pg/ml) and mean single parotid EGF production was 2550 pg/min (median 1750 pg/min; range 790-18,000 pg/min). Patients with severe reflux oesophagitis had a similar EGF concentration (mean 3112 pg/ml; median 1500 pg/ml; range 300-16,000 pg/ml) and production (mean 3100 pg/min; median 2200 pg/min; range 790-17,950 pg/min) to controls. Patients with CLO had a 62 per cent lower mean EGF concentration (mean 1197 pg/ml; median 640 pg/ml; range 233-4500 pg/ml) (P less than 0.001, Mann-Whitney U test) and a 60 per cent lower EGF production (mean 1254 pg/min; median 800 pg/min; range 170-3125 pg/min) (P less than 0.001, Mann-Whitney U test) than patients with severe reflux oesophagitis. A subpopulation with malignant change in CLO (n = 16) had a similar EGF concentration and production to the CLO group as a whole (mean 1240 and 1300 pg/min, respectively). Low salivary EGF levels are associated with Barrett's CLO but not with severe oesophagitis without columnar metaplasia. EGF levels do not identify those patients who will subsequently develop malignant change.

Adult↗
PubMed1991

Pharmacokinetics of drugs that inactivate metabolic enzymes.

The pharmacokinetics of drugs that cause inactivation of metabolic enzymes were derived for the single-compartment model with linear elimination kinetics. A simple mechanism for suicide inactivation of enzyme by its substrate was used to model the relationship between elimination of drug and enzyme activity as a function of time. Three modes of administration were considered: a single bolus dose, repetitive bolus dosing, and continuous administration. Unlike capacity-limited drug elimination, a bolus dose would always give apparent linear kinetics. The apparent terminal half-life after a bolus dose was a simple function of the remaining elimination activity. Repetitive dosing would give a gradual increase in half-life until the steady-state elimination activity was achieved. The time to achieve steady-state drug levels with continuous administration of drug, or repetitive dosing, was prolonged by the enzyme inactivation process, and was dependent on the rates of elimination, enzyme inactivation, and the dosing rate. Interactions between drugs would occur due to a reduction in elimination activity if they share the inactivated enzyme pathway, and the magnitude of the effect would be characterized by the shift in the terminal half-life of elimination.

Cytochrome P-450 Enzyme Inhibitors↗
PubMed1990

Diode array detection of low level co-eluting species in high-performance liquid chromatography.

A simple and sensitive method for detecting low levels (0.5 and 1.0%, w/w) of co-eluting species in HPLC has been developed. This method is based on the subtraction of normalized peak up-slope and down-slope spectra from that of the apex. Visual inspection of the resultant "difference spectra" allows for a qualitative judgement regarding the integrity of the peak under consideration.

Antazoline↗
PubMed1990

Quantification of three lidocaine metabolites and their conjugates.

A method has been developed to quantify three lidocaine metabolites, N-ethylglycyl-2,6-xylidide (MEGX), glycyl-2,6-xylidide (GX), and 4-hydroxy-2,6-xylidine (4-OH-XY), and their conjugates in pooled human urine using enzymic hydrolysis. The commonly used enzymes, pure beta-glucuronidase, sulfatase, and a mixture of the two, were tested for their efficiencies in hydrolyzing the conjugates. Initially, it was found that 4-OH-XY was highly unstable after it was released from conjugates by beta-glucuronidase and the enzyme mixture. This problem was corrected by purging the sample with nitrogen prior to incubation. It has been determined that 4-OH-XY is present in human urine exclusively as its glucuronide. The percentage of MEGX in free and in conjugated forms (glucuronide, sulfate, and others) are 44.9 +/- 6.8, 16.6 +/- 4.5, 6.6 +/- 1.8, and 31.9 +/- 4.4, respectively. GX was present mostly in the free form (90.6 +/- 10.5%).

Acetaminophen↗
PubMed1990

Effects of parenteral nutrition on hepatic elimination of lidocaine: a study using the isolated perfused rat liver.

The effects of acute and a short-term (7 days) parenteral nutrition (PN, consisting of 24.2% dextrose and 5.2% amino acids) on hepatic lidocaine elimination were studied using an isolated perfused rat liver preparation. In the acute study, lidocaine was coinfused with PN solution or normal saline at 0.123 ml/min for 80 min. There was no significant difference in hepatic lidocaine elimination between control and PN-treated groups (six per group). In the post-PN study, only lidocaine was infused for 80 min. Lidocaine hepatic clearance and extraction ratio were reduced by 29.8 and 31.1% (P less than .05), respectively, compared to what occurred in livers isolated from control animals with free access to standard laboratory rat-chow and water (six per group). Mass balance at steady state showed that recovery was higher for lidocaine (46.58 +/- 7.28% vs. 25.56 +/- 5.59%) and lower for N-(N,N-diethylglycyl)-2-hydroxymethyl-6-methylaniline (0.21 +/- 0.01% vs. 2.75 +/- 0.58%) in the PN group compared to chow-fed controls. Recoveries of other lidocaine metabolites: N-(N-ethylglycy)-2,6-xylidine, 3-hydroxylidocaine, 3-hydroxy-N-(N-ethylyglycyl)-2,6-xylidine and N-(N-ethylglycyl)-2-hydroxymethyl-6-methylaniline were similar in the PN- and chow-fed group. These findings suggest that acute PN infusion has no effect on lidocaine metabolism, and that the impaired hepatic lidocaine elimination after 7-days of PN occurs as a result of selective inhibition of hepatic enzyme activities such as those involved in aryl methyl hydroxylation and in other unidentified metabolic pathway(s).

Amino Acids↗
PubMed1989

Evidence for lidocaine-induced enzyme inactivation.

The effects of lidocaine on hepatic enzyme activity were studied using the isolated perfused rat liver. The in vivo liver activity was examined by infusing lidocaine via the jugular vein, followed by organ isolation and drug perfusion 24 h later. The liver was studied in vitro by perfusing the organ with lidocaine until steady state was reached, then allowing the drug and metabolites to wash out of the organ, followed by a second infusion of lidocaine to probe enzyme activity. In both types of experiments, pretreatment with lidocaine caused a reduction in deethylation, and led to a more rapid attainment of steady state. The experimental concentration-time profiles and literature data were successfully described by a mathematical model.

Animals↗
PubMed1989

Substrate inactivation of enzymes in vitro and in vivo.

Some enzymes are inactivated by their natural substrates during catalytic turnover, limiting the ultimate extent of reaction. These enzymes can be separated into three broad classes, depending on the mechanism of the inactivation process. The first type is enzymes which use molecular oxygen as a substrate. The second type is inactivated by hydrogen peroxide, which is present either as a substrate or a product, and are stabilized by high catalase activity. The oxidation of both types of enzymes shares common features with oxidation of other enzymes and proteins. The third type of enzyme is inactivated by non-oxidative processes, mainly reversible loss of cofactors or attached groups. Sub classes are defined within each broad classification based on kinetics and stoichiometry. Reaction-inactivation is in part a regulatory mechanism in vivo, because specific proteolytic systems give rapid turnover of such labelled enzymes. The methods for enhancing the stability of these enzymes under reaction conditions depends on the enzyme type. The kinetics of these inactivation reactions can be used to optimize bioreactor design and operation.

Journal Article↗
PubMed1988

Mitral incompetence associated with lipoma infiltrating the mitral valve.

A 54 year old man presented with presyncopal symptoms. Echocardiography and subsequently computed tomography showed a mass in the posterior mitral annulus causing incompetence of the valve. At operation a lipoma was found which could not be resected. Mitral incompetence was the result of chordal rupture where the lipoma had engulfed the papillary muscle. The valve was replaced and the patient made an uneventful recovery. This case report shows a potential danger of an otherwise benign lesion.

Heart Neoplasms↗
PubMed1988

A serologic survey of rice-field leptospirosis in Central Luzon, Philippines.

Although human cases of leptospirosis have been reported from the Philippines, there is a lack of data on its prevalence. We therefore surveyed three rice-farming villages for the presence of leptospiral antibody. Out of 155 sera tested, 63 (43.6%) tested positive using the standard microagglutination test. Antibodies were more frequent in men than women (48 vs. 31%, respectively, p less than 0.01), and less common in the elderly. Exposure to leptospires occurs frequently in rice farmers, and leptospirosis is likely to be an underdiagnosed cause of both mild and severe febrile illness in the Philippines.

Adolescent↗