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Biomedical subjects

M D Green

Publications and source records attributed to M D Green.

At least 19 recordsLinked to original sources
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

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

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↗
PubMed1994

Phase II study of autologous filgrastim (G-CSF)-mobilized peripheral blood progenitor cells to restore hemopoiesis after high-dose chemotherapy for lymphoid malignancies.

The hemopoietic growth factor filgrastim (r-metHu G-CSF) stimulates granulopoiesis after autologous BMT and can also be used as a peripheral blood progenitor cell (PBPC)-mobilizing agent. Rapid platelet recovery follows the addition of filgrastim-mobilized PBPC to autologous BMT. We have now studied 29 adults with malignant lymphoma, Hodgkin's disease or ALL to assess the ability of filgrastim-mobilized PBPC to rapidly and durably restore hemopoiesis without bone marrow (BM) infusion. Patients with a high yield of PBPC from three leukaphereses, defined as > 30 x 10(4)/kg GM-CFC, were eligible for PBPC transplant without BM. Patients with a low yield of GM-CFC received both PBPC and BM infusion. After filgrastim therapy 12 or 24 micrograms/kg/day by continuous sc infusion for 6 or 7 days, a high yield was obtained in 11 of 29 patients. Kinetics of recovery of both the platelet and neutrophil counts were more rapid in the high yield group than in the low yield group. The platelet count recovered to > 20 x 10(9)/l at a median of 9 days, to > 50 x 10(9)/l at 11 days and the neutrophil count to > 0.5 x 10(9)/l at 9 days in the high yield group compared with 12 days, 37 days and 10 days, respectively, in the low yield group (p = 0.028, p < 0.001 and p = 0.027). Fewer platelet transfusions were required in the high yield group (median 11 vs 29.5 units, p = 0.021).(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Cloning and characterization of rabbit liver UDP-glucuronosyltransferase cDNAs. Developmental and inducible expression of 4-hydroxybiphenyl UGT2B13.

A polyclonal antibody generated against rabbit liver p-nitrophenol UDP-glucuronosyltransferase (UGT) was used to screen a rabbit liver cDNA expression library constructed in lambda gt11. A 500-base pair cDNA clone, termed pPNP, generated a fusion protein that was antigenic with the antibody. Clone pPNP encoded the 3' region of a UGT. To identify larger recombinants, clone pPNP was used as a probe to screen a second cDNA library constructed in lambda ZAP. Two different cDNA clones were identified by DNA sequence analysis. Based upon their predicted amino acid sequence analysis, the clones encode transferases belonging to the UGT2 subfamily, and have been identified as UGT2B13 and UGT2B14. The predicted N-terminal sequence of UGT2B13 is identical to that determined for the purified rabbit liver estrone UGT. However, expression of the UGT2B13 cDNA in COS-1 cells displayed no activity in the presence of estrone but efficiently conjugated 4-hydroxybiphenyl. Results of Southern blot analysis using the 5' divergent region of the UGT2B13 cDNA that encodes exon 1 demonstrates that multiple genes share sequence homology to UGT2B13, an observation which indicates that the estrone UGT and UGT2B13 genes are encoded by separate alleles. When the 5' variable regions of the cDNAs where used in Northern blot analysis, the expression of UGT2B13 and UGT2B14 were shown to be expressed primarily in adult rabbits. However, when neonatal rabbits were treated with either dexamethasone or rifampicin, UGT2B13 mRNA levels were induced. The neonatal induction of UGT2B13 mRNA corresponded with similar increases in 4-hydroxybiphenyl UGT activity. The expression and induction of UGT2B13 paralleled that of the developmentally regulated rabbit liver progesterone 6 beta-hydroxylase P4503A6.

Amino Acid Sequence↗
PubMed1993

A high throughput fluorogenic substrate for interstitial collagenase (MMP-1) and gelatinase (MMP-9).

Two members of the matrix metalloproteinase family of enzymes, interstitial collagenase and 92-kDa gelatinase, have been implicated in the pathogenesis of rheumatoid arthritis and tumor metastasis. In order to characterize the activities of these enzymes, we have developed a fluorogenic peptide substrate which is efficiently hydrolyzed by both enzymes. This substrate was developed based on the addition of the fluorescent tag, N-methyl-anthranilic acid (Nma), to several previously synthesized substrates that had been evaluated with respect to their turnover by interstitial collagenase. One substrate, Dnp-Pro-Cha-Gly-Cys(Me)-His-Ala-Lys-(Nma)-NH2, had favorable solubility characteristics, was > 98% quenched, and produced a single cleavage product, Dnp-Pro-Cha-Gly, with a high fluorescence yield with both interstitial collagenase and 92-kDa gelatinase. Since the assay depends on measurement of increases in fluorescence, the position of the Nma group also proved to be important for optimization of the fluorescence signal. The assay is free from interference by organomercurial compounds and the cleavage product has excitation and emission spectra compatible with filters commonly available on commercial plate readers. The assay has been adapted to a 96-well format and provides a rapid screening protocol for the evaluation of inhibitors of these enzymes.

Amino Acid Sequence↗
PubMed1993

Effect of posture on lung and regional chest wall mechanics.

BACKGROUND: Little is known about the extent to which changes in postures in clinical situations affect respiratory mechanics, even in humans with healthy respiratory systems. This study tested the hypothesis that posture has only small effects on overall respiratory system mechanics in healthy subjects, despite changes in parts of the respiratory system in some postures. METHODS: Measurements were made of airway flow, airway and esophageal pressures, and rib cage and abdominal volume displacements (with inductance plethysmography) of awake, healthy subjects, relaxed at functional residual capacity, during external forcing at 0.2 Hz with a tidal volume of 8-10 ml/kg. From these measurements, discrete Fourier transform was used to calculate elastances (E) and resistances (R) of the total respiratory system, lungs, total chest wall, and compartments of the chest wall (rib cage, diaphragm-abdomen, and belly wall). Measurements were made while the subjects were in nine different postures: in six of these, the torso was straight; in three, the torso was bent or twisted. RESULTS: Although changes in mechanics of parts of the respiratory system were evident in certain postures, overall respiratory mechanics were not greatly affected by posture. Changing from sitting to supine decreased E and R of the diaphragm-abdomen about 50% (P < .05), but total chest wall E and R changed only slightly. Lung E increased 24% (P < .05), but total respiratory E did not change (P < .05). Lung and total respiratory R increased 40-50% (P < .05) with this same change in posture. As long as the torso was straight, however, changes in orientation of 30 degrees from the horizontal or a shift to lateral posture resulted in only minor changes in the variables measured. Postures in which the torso was twisted or bent increased E of the total chest wall 20-30% compared to supine (P < .05), due to increases in E of one or more compartments. Respiratory system E also increased, at most 14%. Although lung R decreased 30-45% (P < .05) in these postures compared to supine with a straight torso, chest wall and total respiratory R generally were unchanged. CONCLUSIONS: Changes in respiratory system mechanics over a wide range of postures that may be encountered clinically are relatively small in healthy awake subjects due to adaptability of total chest wall mechanical behavior.

Adult↗
PubMed1993

Primary cerebral lymphoma: an argument for the use of adjunctive systemic chemotherapy.

Eleven patients with primary cerebral lymphoma were treated at a single institution over a 5 year period. Patient characteristics were typical of this rare disease. One patient died prior to receiving treatment and of the remaining 10, all received cranial irradiation and in addition, five received systemic cyclophosphamide, adriamycin, vincristine and prednisolone (CHOP) chemotherapy. Of the six patients who are alive and disease-free, five received the combined modality therapy. The median survival for those patients receiving cranial irradiation alone was 18 months and for the combined modalities was 25+ months. Combination systemic chemotherapy, in addition to cerebral irradiation, may convey a survival benefit in patients with primary cerebral lymphoma but this requires further investigation with multicentre, prospective randomized trials.

Adolescent↗
PubMed1992

Delay by internists in obtaining diagnostic biopsies in patients with suspected cancer.

OBJECTIVE: To investigate the degree and type of delays in performing diagnostic biopsies in medical patients with suspected malignancy. DESIGN: Retrospective survey of clinical histories of patients referred between January 1985 and March 1989. SETTING: Inner city teaching hospital internal medicine (non-oncologic) services. PATIENTS: Patients with gastrointestinal and lung cancers, adenocarcinoma of unknown primary site, and lymphomas were referred as inpatients by internists. Two hundred fifty-five patients were eligible, and 177 were evaluable. MAIN OUTCOME MEASURES: The number, type, and results of tests done before and after biopsy were analyzed. RESULTS: In 67% of patients the biopsied lesion was detected by the second day of evaluation; however, there was an 8- to 10-day delay before a biopsy was done. This delay was consistent across the four malignancy groups studied. Although logistic and other unavoidable delays occurred in 40% of the cases, in 60% delays could only be attributed to continued, frequently low yield, noninvasive tests. An average of 3.3 tests were made per patient, with only 24% leading to a definitive biopsy. CONCLUSION: Because of the performance of many other tests, a substantial delay exists in proceeding to biopsy during the diagnosis of cancer by internists.

Adenocarcinoma↗
PubMed1992

Comparative study of doxorubicin, mitoxantrone, and epirubicin in combination with ICRF-187 (ADR-529) in a chronic cardiotoxicity animal model.

In this study doxorubicin, epirubicin, and mitoxantrone were compared for their cardiotoxic potential in a chronic mouse model in an effort to identify and compare their mechanism(s) of toxicity. In addition, the cardioprotective ability of ICRF-187 [(+/-)-1,2-bis(3,5-dioxopiperazinyl-1-yl)propane] with each anticancer drug was evaluated in this model. The antioxidant capacity (superoxide dismutase, reduced glutathione, catalase, and glutathione peroxidase) was assessed following drug treatment. Five-week-old BALB/c mice received weekly i.p. injections of each drug or the drug and ICRF-187 over a 3-month period. ICRF-187 was administered 30 min prior to the anticancer drug. The hearts were examined by electron and light microscopy to assess subcellular changes, and the cardiac and hepatic antioxidant levels were measured concurrently. Chronic treatment with these drugs or each combined with ICRF-187 did not change the antioxidant levels relative to the control values. However, all three drugs caused cardiac damage during chronic exposure. Both epirubicin and mitoxantrone caused less severe damage than doxorubicin, and epirubicin was the least cardiotoxic of the three. ICRF-187 was cardioprotective for epirubicin and doxorubicin but not for mitoxantrone. These results suggest epirubicin acts by a mechanism similar to that of doxorubicin that is probably mediated by oxygen-free radicals, while mitoxantrone acts by a different mechanism to cause cardiotoxicity.

Animals↗