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Biomedical subjects

M D Green

Publications and source records attributed to M D Green.

At least 73 records · Page 4Linked to original sources
PubMed1996

Folate and 10-formyltetrahydrofolate dehydrogenase in human and rat retina: relation to methanol toxicity.

Ocular toxicity, a well-recognized outcome of methanol poisoning in humans, correlates with formate accumulation in blood following the ingestion of methanol. Rodents, however, are a species known to be resistant to the toxicity of formate. The present study was designed to determine whether components of folate-dependent formate oxidation, e.g., folate and 10-CHO-H4-folate dehydrogenase (10-FDH), exist in retina and whether differences in these components might explain species-determined susceptibility to methanol intoxication. Total folate levels were determined in human and rat retinal tissues and were found to be much lower than the levels in liver. However, folate levels in human retina were only 14% of those determined in rat retina. Western blot analysis was used to quantify the amount of cytosolic and mitochondrial 10-FDH in retinal tissues. Results of these studies showed that comparable amounts of this enzyme were present in both cellular compartments in each species. However, the amount of 10-FDH in human retina was approximately three times the amount found in rat retina. In order to determine the cell type(s) within the retina that contained 10-FDH, immunohistochemical staining for 10-FDH was carried out. Confocal microscopic image analysis of human and rat retinae showed colocalization of 10-FDH primarily with Muller cell protein markers [glial fibrillary acidic protein (GFAP), vimentin, and carbonic anhydrase]. Therefore, 10-FDH was found to be preferentially localized in this cell type. Since Müller cells appear to represent the target for formate-induced ocular toxicity, our data suggest that formate oxidation reactions might serve two roles, first a protective role and then a role in methanol-induced toxicity in Müller cells.

Adenosine Triphosphate↗
PubMed1996

Automated system for detailed measurement of respiratory mechanics.

OBJECTIVE: The mechanical properties of the respiratory system (i.e., elastance and resistance) depend on the frequency, tidal volume, and shape of the flow waveform used for forcing. We developed a system to facilitate accurate measurements of elastance and resistance in laboratory and clinical settings at the frequencies and tidal volumes in the physiologic range of breathing. METHODS: A personal computer (PC) is used to drive a common clinically used ventilator while simultaneously collecting measurements of airway flow, airway pressure, and esophageal pressure from the experimental subject or animal at different frequencies and tidal volumes. Analysis analogous to discrete Fourier transform at the fundamental frequency (i.e., ventilator setting) is used to calculate elastances and resistances of the total respiratory system and its components, the lungs and the chest wall. We have shown that this analysis is independent of the high-frequency harmonics that are present in the waveform from clinical ventilators. RESULTS: The system has been used successfully to make measurements in anesthetized/paralyzed dogs and awake or anesthetized human volunteers in the laboratory, and in anesthetized human volunteers in the laboratory, and in anesthetized humans in the operating room and intensive care unit. Elastances and resistances obtained with this approach are the same as those obtained during more controlled conditions, e.g., sinusoidal forcing. CONCLUSIONS: Accurate, standardized measurements of lung and chest wall properties can be obtained in many settings with relative ease with the system described. These properties, and their frequency and tidal volume dependences in the physiologic range, provide important information to aid in the understanding of changes in respiratory function caused by day-to-day conditions, clinical intervention and pathologies.

Airway Resistance↗
PubMed1996

A phase II study in advanced breast cancer: ZD1694 ('Tomudex') a novel direct and specific thymidylate synthase inhibitor.

ZD1694 ('Tomudex'), a novel, direct and specific thymidylate synthase (TS) inhibitor, was developed in a collaborative research programme between Zeneca Pharmaceuticals and the Institute of Cancer Research (UK) and entered clinical trials in 1991; phase II studies began in 1992, using 3.0 mg m-2 every 3 weeks as a short 15 min infusion. Forty-six patients entered a phase II study of ZD1694 in advanced breast cancer. A total of 74% of patients had received prior systemic therapy (either as adjuvant cytotoxic or hormonal therapy or hormone therapy for advanced disease); 39% had received prior adjuvant cytotoxic chemotherapy. All patients had measurable disease and 50% had liver metastases. In all 43 patients were evaluable for response. Of these patients 26% achieved complete (CR) or partial response (PR) (95% Cl 14-42%). A response rate of 44% was seen in liver metastases. Two patients achieved CR of 265 and 301 days' duration respectively, one in locoregional disease, and one in liver metastases. The most common grade 3/4 adverse events were nausea and vomiting (11%), diarrhoea (11%) and leucopenia (20%). Grade 3/4, self-limited and reversible increases in transaminases were seen in 22% of patients. ZD1694 has useful single agent activity in patients with hormone-refractory advanced breast cancer, comparable with that reported for other anti-metabolites, with acceptable tolerability.

Adult↗
PubMed1996

Long-term malaria chemoprophylaxis with mefloquine in Dutch marines in Cambodia.

Three Dutch marine battalions (n=2289) serving in Western Cambodia during 1992-1993 used mefloquine as weekly malaria chemoprophylaxis. One battalion started with a loading dose. Full compliance with prophylaxis was reported by 86.3%, and possible mefloquine-related adverse events were reported by 30.2%. Sixty-four periods of malaria were diagnosed in 59 marines. During deployment, 31 Plasmodium falciparum and no Plasmodium vivax infections occurred. After return, there were 11 cases of falciparum malaria and 22 of vivax malaria, 16-72 days and 30-540 days, respectively, after stopping prophylaxis. Mefloquine-resistant parasites were isolated from 4 Dutch and 4 Khmer patients. Long-term mefloquine prophylaxis was well tolerated but not totally effective.

Adolescent↗
PubMed1996

A risk-benefit assessment of anthracycline antibiotics in antineoplastic therapy.

The anthracycline antibiotics comprise a group of cytotoxic compounds with wide-ranging activity against human malignancies. They are used extensively for curative, adjuvant and palliative therapy, both as single agents and in combination regimens. They produce a number of adverse effects, some of which are shared by other cytotoxic drugs. The most important adverse effect is cardiotoxicity, which is unique to this class of compounds. Strategies have been devised to circumvent these adverse effects, including the development of less toxic analogues, alterations in scheduling, the addition of cardioprotectant agents and methods of monitoring for cardiac abnormalities.

Antibiotics, Antineoplastic↗
PubMed1996

Field detection of sulfonamides in urine: the development of a new and sensitive test.

A new, field-adapted, colorimetric method for detecting sulfonamide drugs in urine is described. The method uses the color reagent, p-dimethylaminocinnamaldehyde, and has a detection limit of about 1 microgram/ml. Analysis of 35 samples collected in the field, comparing results obtained with the colorimetric field test with those obtained using high-performance liquid chromatography, indicated a calculated sensitivity value of 94% and a specificity value of 94% for the test to detect the presence of sulfonamides. The field test can be modified to allow quantitation of sulfonamides in urine in field situations, using a hand-held, portable photometer for measuring the absorbance of test solutions. For this test, calculated coefficients of variation for day to day reproducibility were < or = 5% at sulfonamide concentrations > or = 3 micrograms/ml. This new test for detecting the presence of sulfonamides in urine is more sensitive and reliable than the presently used Bratton-Marshall test.

Chromatography, High Pressure Liquid↗
PubMed1996

Glucuronidation of amines and hydroxylated xenobiotics and endobiotics catalyzed by expressed human UGT1.4 protein.

Glucuronide conjugation of tertiary amine xenobiotics represents a unique and important metabolic pathway for these compounds in humans. In this study, we show that human UDP-glucuronosyltransferase 1.4 protein, stably expressed in human embryonic kidney 293 cells, catalyzes the N-glucuronidation of primary, secondary, and tertiary amine substrates. In addition, the substrate specificity of the expressed enzyme toward many hydroxylated and carboxylic acid-containing compounds was examined. Of the hydroxylated compounds tested, only sapogenins gave glucuronidation rates comparable with those observed for amine substrates. The apparent KM and Vmax values for sapogenins were such that the efficiency of glucuronidation (Vmax/KM) for these compounds was higher than that determined for amine substrates. Human UDP-glucuronosyltranferase 1.4 also catalyzes the glucuronidation of monoterpenoid alcohols and simple phenolic compounds. The enzyme kinetic values determined for these substrates suggested that this enzyme may have relatively limited significance for the conjugation of these classes of compounds. Of the endobiotics tested, androstanediol and progestins were glucuronidated at high rates by expressed human UDP-glucuronosyltransferase 1.4 protein. The glucuronidation efficiency for 5alpha-pregnane-3beta,20alpha-diol was comparable with that determined for the sapogenins. Because UDP-glucuronosyltransferases are integral membrane proteins, the effects of different detergents on the catalytic activity of the expressed enzyme were determined. The results show that detergents (such as Lubrol PX, Emulgen 911, and Triton X-100) are inhibitory for the quaternary ammonium-linked glucuronidation of chlorpromazine and imipramine catalyzed by expressed human UDP-glucuronosyltransferase 1.4. In contrast, CHAPS and nonanoyl-N-methylglucamide are less inhibitory toward the glucuronidation of these compounds. The results suggest that human UDP-glucuronosyltransferase 1.4 may be an important enzyme for the detoxication of environmentally derived amines and sapogenins and for the conjugation of progestins.

Antipsychotic Agents↗
PubMed1995

Cloning and expression of a rat liver phenobarbital-inducible UDP-glucuronosyltransferase (2B12) with specificity for monoterpenoid alcohols.

A full-length cDNA, HBPA2, that encodes for a new rat hepatic UDP-glucuronosyltransferase protein, designated UGT2B12, was isolated from a rat liver cDNA library. The isolated clone contains a 1590-nucleotide open reading frame flanked by 2 and 252 base pairs of 5' and 3' noncoding sequences, respectively. Human embryonic kidney 293 cells transfected with UGT2B12 expressed a protein with a subunit molecular mass of 53 kDa. The expressed protein catalyzed the glucuronidation of monoterpenoid alcohols, such as (-)-borneol, (+)-menthol, and (-)-nopol. In addition, a number of simple phenolic compounds, such as hydroxybiphenyls, 7-hydroxylated coumarins, p-nitrophenol, and food-derived substances (e.g., naringenin and eugenol), were also substrates for the expressed enzyme. Northern blot analysis showed that treatment of rats with phenobarbital increased hepatic mRNA levels for UGT2B12 approximately twofold. In addition to liver, Northern blot analysis demonstrated that UGT2B12 mRNA is present in kidney and testis.

Alcohols↗
PubMed1995

HIV-1 protease specificity derived from a complex mixture of synthetic substrates.

A rapid and semiquantitative method is described for determining the relative kcat/Km for individual peptides in defined substrate mixtures. The method utilizes electrospray ionization/mass spectrometry alone to semiquantitatively determine relative peptide substrate turnover rates. Unlike previous studies, in which chromatographic separation of individual peptide species was required, this mass spectrometric-based method relies strictly on the ability to ionize and detect simultaneously all peptide species in a defined mixture. Differences in the ion intensities of the individual components before and after incubation with protease are used to semiquantitatively determine preferred substrates. This method was used to the identify preferred peptide substrates for HIV-1 protease. Optimal substrates were identified from a defined synthetic peptide substrate mixture based on Ser-Gln-Asn-Tyr-Pro-Ile-Val, where the P1' proline was substituted with 20 naturally occurring amino acids. The hydrophobic residues Leu, Ile, Val, Phe, and Tyr were preferred in addition to Pro at the P1' site. The results were corroborated by performing the more laborious HPLC/Frit-fast atom bombardment/MS analyses.

Amino Acid Sequence↗
PubMed1995

Chemiluminescent detection of artemisinin. Novel endoperoxide analysis using luminol without hydrogen peroxide.

A novel method for artemisinin quantitation employing high-performance liquid chromatography (HPLC) with chemiluminescence (CL) detection in the absence of hydrogen peroxide (H2O2), is reported. After elution from the HPLC column, artemisinin is combined with an alkaline solution of hematin and luminol. The resulting CL signal is detected by use of a spectrofluorometer with the excitation lamp disabled, and is proportional to artemisinin concentration. The CL method was optimized and applied to the analysis of artemisinin in spiked human serum. CL in the absence of H2O2 or other known oxidizing species is remarkable since such oxidizers are usually required to produce CL from luminol under alkaline conditions. Artemisinin, a naturally occurring sesquiterpene, is one of several natural products that contain an endoperoxide functional group. Since H2O2 is not needed in the analysis, the endoperoxide moiety on artemisinin is implicated as a contributing source of superoxide radicals required for the light-producing reaction with luminol.

Antimalarials↗
PubMed1995

Determination of sulfadoxine concentrations in whole blood using C18 solid-phase extraction, sodium dodecyl sulfate and dimethylaminocinnamaldehyde.

A simple method is described for the extraction and subsequent analysis of sulfadoxine in human whole blood using a solid-phase extraction technique and colorimetric reaction. This procedure utilizes the micellar properties of sodium dodecyl sulfate to: (1) extract sulfadoxine from a C18 solid-phase sample-preparation column; (2) enhance the colorimetric reaction produced by the addition of p-dimethylaminocinnamaldehyde (DMAC); and (3) provide stability to the coloured product generated by the reaction of sulfadoxine with DMAC. The intense, violet-red colour reaction can be conveniently used for qualitative and semiquantitative visual interpretations of sulfadoxine levels. Under the assay conditions, drug concentrations in the blood of subjects receiving sulfadoxine were determined from absorbance measurements. These results correlated well with the sulfadoxine levels determined from high-performance liquid chromatographic analysis. Important advantages of the procedure include the ability to evaluate small samples of whole blood (100 microliters), the minimal use of organic solvents, no sophisticated instrumentation, and formation of a stable, coloured reaction product. The method proved to be a suitable field assay for determining whole-blood levels of sulfonamides in the concentration range from 5 to 100 micrograms ml-1.

Chemistry Techniques, Analytical↗
PubMed1995

Cloning and stable expression of a cDNA encoding a rat liver UDP-glucuronosyltransferase (UDP-glucuronosyltransferase 1.1) that catalyzes the glucuronidation of opioids and bilirubin.

A chicken anti-rat polyclonal antibody to a purified rat liver UDP-glucuronosyltransferase (UGT) with catalytic activity toward opioid substrates was used to screen a liver cDNA library prepared from phenobarbital-treated Wistar rats. A number of positive clones were obtained, and one of these clones, pM1, was further characterized. Clone pM1 was found to be a full length cDNA coding for a member of the rat UGT1 gene family. Specifically, pM1 represents the full length homologue of the Gunn rat liver pseudo-gene product UGT1.1P and, therefore, has been designated UGT1.1r. The cDNA insert has an open reading frame of 1605 base pairs, which codes for a protein of 535 amino acids and is flanked by 2 and 632 base pairs of 5' and 3' noncoding sequence, respectively. The deduced amino acid sequence of pM1 contains amino acid sequences identical to the amino-terminal and internal peptides of the purified rat liver opioid UGT and to sequences reported for a rat liver bilirubin UGT [FEBS Lett. 299:183-186 (1992)]. Stable expression of UGT1.1r in human embryonic kidney 293 cells showed that a protein with a subunit molecular mass (56 kDa) identical to that of the purified protein was produced. Expressed UGT1.1r protein catalyzed the glucuronidation of buprenorphine and bilirubin at high rates. Other opioids, such as nalorphine and morphine, were also substrates for the expressed UGT1.1r protein. These results show that bilirubin and opioids can be conjugated by the same rat liver UGT.

Amino Acid Sequence↗
PubMed1995

Expressed human UGT1.4 protein catalyzes the formation of quaternary ammonium-linked glucuronides.

In humans, the metabolism of a number of tertiary amine-containing pharmacological agents to quaternary ammonium-linked glucuronides, catalyzed by UDP-glucuronosyltransferase (UGT), represents a unique and important metabolic pathway for these compounds. A full-length cDNA-encoding human UGT1.4 (the so-called "minor" human bilirubin UGT) was inserted into the expression vector pREP9 and transfected into human embryonic kidney 293 cells, and stable transfectants were obtained after geneticin selection. As expected, the expressed protein had low catalytic activity toward bilirubin. However, expressed human UGT1.4 protein exhibited glucuronidation activity toward tertiary amine substrates, such as imipramine, cyproheptadine, tripelennamine, and chlorpromazine, which form quaternary ammonium-linked glucuronides. Carcinogenic primary amines (beta-naphthylamine, benzidine, and 4-aminobiphenyl) also reacted with the expressed UGT1.4 protein at rates approximately 10-fold higher than the rates for quaternary ammonium glucuronide formation. Although a number of other UGT gene products are capable of catalyzing the glucuronidation of primary amine substrates, expressed human UGT1.4 protein is the only UGT isoform that has been shown to conjugate tertiary amine substrates, forming quaternary ammonium-linked glucuronides.

Carbohydrate Conformation↗
PubMed1994

A high throughput fluorogenic substrate for stromelysin (MMP-3).

Stromelysin, a member of the matrix metalloproteinase family of enzymes, has been implicated in the pathogenesis of tumor metastasis and inflammatory diseases such as rheumatoid arthritis. To screen prospective inhibitors of this protease, we developed a fluorogenic substrate with excitation and emission spectra compatible with commercially available 96-well plate readers. The substrate is based on the addition of 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino] hexanoic acid (NBD) (EX467/EM534) and 7-dimethylaminocoumarin-4-acetate (DMC) (EX368/EM459) to the previously reported peptide substrate for stromelysin, Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp-NH2. The new substrate, NBD-Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp-Lys-(DMC)-NH2 is 95% quenched and the fluorescent product, Nva-Trp-Lys(DMC)-NH2 is easily detected (EX350/EM465). In competition assays the new fluorogenic substrate has a relative kcat/Km that is one half that of the parent peptide. The fluorophores NBD and DMC were chosen based on the high fluorescence yield of DMC and the overlap of the emission spectrum of DMC and excitation spectrum of NBD which results in an efficient energy transfer system in the intact substrate. These characteristics make this an excellent substrate for routine determination of in vitro activities of stromelysin inhibitors.

Amino Acid Sequence↗
PubMed1994

Influence of waveform and analysis technique on lung and chest wall properties.

To test an approach for measuring respiratory system resistance (R) and elastance (E) during non-sinusoidal forcing, we measured airway and esophageal pressures and flow at the trachea of 9 anesthetized-paralyzed dogs during sinusoidal forcing (SF) and 4 types of non-sinusoidal forcings at 0.15 and 0.6 Hz and 300 ml tidal volume. During SF, calculations of E and R of the lungs, chest wall or total system from discrete Fourier transform (DFT) and two other widely used methods (multiple regression and volume-pressure loop analysis) did not differ from each other (P > 0.05). During forcing with sinusoidal or step inspiration with passive expiration (inspiratory to expiratory ratio, I/E, = 1:1), Es from any analysis method were within 10% of values during SF. Although Rs of the lungs, chest wall or total system were not affected by waveform shape with DFT (P > 0.05), the other analysis methods gave values for R during non-SF that differed (P < 0.05) from those during SF by up to 77%. If I/E was changed to 1:2, with or without an added 10% inspiratory pause, values for E and R differed least from values during SF if DFT was used. During severe pulmonary edema induced by infusion of oleic acid in the right atrium, results for lung properties were similar to controls, despite large increases in E and R of the lungs. We conclude that E and R of the lungs and chest wall can be measured by DFT using nonsinusoidal forcing waveforms available on most clinical ventilators, incurring only modest error.

Airway Resistance↗
PubMed1994

Lung and chest wall mechanical properties before and after cardiac surgery with cardiopulmonary bypass.

From measurements of airway and esophageal pressures and flow, we calculated the elastance and resistance of the total respiratory system (Ers and Rrs), chest wall (Ecw and Rcw), and lungs (EL and RL) in 11 anesthetized-paralyzed patients immediately before cardiac surgery with cardiopulmonary bypass and immediately after chest closure at the end of surgery. Measurements were made during mechanical ventilation in the frequency and tidal volume ranges of normal breathing. Before surgery, frequency and tidal volume dependences of the elastances and resistances were similar to those previously measured in awake seated subjects (Am. Rev. Respir. Dis. 145: 110-113, 1992). After surgery, Ers and Rrs increased as a result of increases in EL and RL (P < 0.05), whereas Ecw and Rcw did not change (P > 0.05). EL and RL exhibited nonlinearities (i.e., decreases with increasing tidal volume) that were not seen before surgery, and RL showed a greater dependence on frequency than before surgery. The changes in RL or EL after surgery were not correlated with the duration of surgery or cardiopulmonary bypass time (P > 0.05). We conclude that 1) frequency and tidal volume dependences of respiratory system properties are not affected by anesthesia, paralysis, and the supine posture, 2) open-chest surgery with cardiopulmonary bypass does not affect the mechanical properties of the chest, and 3) cardiac surgery involving cardiopulmonary bypass causes changes in the mechanical behavior of the lung that are generally consistent with those caused by pulmonary edema induced by oleic acid (J. Appl. Physiol. 73: 1040-1046, 1992) and decreases in lung volume.

Adult↗
PubMed1994

Comparative study of the pharmacokinetics and toxicity of high-dose epirubicin with or without dexrazoxane in patients with advanced malignancy.

PURPOSE: We evaluated the toxicity and pharmacokinetics of the combination of dexrazoxane with epirubicin at dexrazoxane/epirubicin dose ratios of 5 to 9:1 in a controlled, crossover phase I study in patients with advanced malignancy. PATIENTS AND METHODS: Thirty-eight patients with a variety of malignancies were enrolled. Assessable patients received two cycles of chemotherapy consisting of epirubicin alone and in combination with dexrazoxane. Comparisons were made between the toxicity and pharmacokinetics of epirubicin in the two treatment arms, using each patient as his or her own control. Dexrazoxane and epirubicin were delivered at dose levels of 600/120 mg/m2, 900/120 mg/m2, 900/135 mg/m2, 900/150 mg/m2, and 1,200/135 mg/m2, respectively. Twenty-six patients completed two cycles of chemotherapy and were therefore assessable. RESULTS: The maximum-tolerated doses (MTDs) of dexrazoxane/epirubicin were 1,200/135 mg/m2, with the dose-limiting toxicities being neutropenia, infection, and stomatitis. There was no difference in the nadir neutrophil or platelet counts between single-agent and combination treatment at any of the dose levels. Severe vomiting and stomatitis occurred less frequently following administration of epirubicin and dexrazoxane when compared with epirubicin alone (P = .01 and .02, respectively). Prior administration of higher doses (900 mg/m2 and 1,200 mg/m2) of dexrazoxane increased the systemic clearance of epirubicin, resulting in a decrease in the area under the curve (AUC). Elimination half-life, maximum plasma concentration (Cmax), and apparent volume of distribution of epirubicin were not significantly affected by dexrazoxane. Left ventricular ejection fraction (LVEF) decreased by greater than 10% in two patients, but neither developed clinical or radiologic evidence of cardiac failure. CONCLUSION: This study demonstrates that dexrazoxane can be safely combined with escalating doses of epirubicin at dose ratios of 5 to 9:1 without having an adverse impact on toxicity. Studies are need to determine the optimal dose ratio for cardioprotection and to explore further the pharmacokinetic interactions of the two drugs at increasing doses of epirubicin supported by hematopoietic growth factors.

Adult↗