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Biomedical subjects

M Lee

Publications and source records attributed to M Lee.

At least 559 records · Page 31Linked to original sources
PubMed1995

Sequence specificity of alkylation for a series of nitrogen mustard-containing analogues of distamycin of increasing binding site size: evidence for increased cytotoxicity with enhanced sequence specificity.

The covalent sequence specificity of a series of nitrogen mustard-containing analogues of distamycin was determined using modified sequencing techniques. The analogues tether benzoic acid mustard (BAM) and possess either one, two, or three pyrrole-amide units. Previous characterization of the biological profile of the series revealed an increase in cytotoxicity for each corresponding increase in the number of pyrrole units, while showing poor cross-link formation in isolated and cellular DNA. Examination of the sequence specificity revealed that BAM produced guanine-N7 lesions in similar manner to other conventional nitrogen mustards. The monopyrrole BAM conjugate also produced guanine-N7 alkylation in a similar pattern to BAM. However, alkylation of adenines was also seen that was found to be minor groove adenine-N3 lesions. The dipyrrole and tripyrrole conjugates did not produce detectable guanine-N7 alkylation but only alkylated in AT tracts. In addition, the tripyrrole conjugate preferentially alkylated only a subset of those sites alkylated by the monopyrrole and dipyrrole conjugates. Two sites, 5'-TTTTGG and 5'-TTTTGA, confirmed as guanine-N3 and adenine-N3 lesions, respectively, were strongly alkylated by the tripyrrole conjugate in preference to other similar sites including three occurrences of 5'-TTTTAA. Footprinting studies comparing distamycin and the tripyrrole conjugate showed identical non-covalent recognition of AT-rich sites. Hence, the drug that possessed the most enhanced sequence specificity for alkylation was also the most cytotoxic of this series.

Antineoplastic Agents, Alkylating↗
PubMed1995

Granulocyte colony-stimulating factor downregulates allogeneic immune responses by posttranscriptional inhibition of tumor necrosis factor-alpha production.

We report downregulatory effects of granulocyte colony-stimulating factor (G-CSF) on allogeneic immune responses in vitro. G-CSF did not affect the proliferative response of peripheral blood mononuclear cells (PBMC) against allogeneic Daudi cells but did inhibit tumor necrosis factor (TNF)-alpha secretion. In contrast with G-CSF, granulocyte-macrophage (GM)-CSF and interleukin (IL)-3 enhanced alloactivation-induced TNF-alpha production. G-CSF-mediated suppression of TNF-alpha production was not affected by fixation of stimulators. G-CSF did not inhibit TNF-alpha mRNA expression or accelerate mRNA degradation, whereas pentoxifylline inhibited the expression of TNF-alpha mRNA. These results indicate that G-CSF acts directly on responder cells and modulates TNF-alpha production at posttranscriptional levels. Suppression of TNF-alpha secretion was accompanied by an increase of intracellular cyclic adenosine monophosphate (cAMP) concentration in alloactivated PBMC. The cell-permeable cAMP analogue, dibutyryl cAMP, suppressed TNF-alpha secretion without affecting TNF-alpha mRNA expression. G-CSF showed an inhibitory effect on the development of cytotoxic effector cells against allogeneic Daudi cells. Anti-TNF-alpha monoclonal antibody (MoAb) also inhibited the induction of cytolytic activity, and the inhibitory effects of G-CSF and anti-TNF-alpha MoAb on killer activity generation were overcome by adding exogenous TNF-alpha. Hence, impaired generation of cytolytic effector cells by G-CSF is believed to be the result of reduced TNF-alpha production. Collectively, the results described above suggest that G-CSF downregulates allogeneic immune responses by posttranscriptionally inhibiting TNF-alpha production.

Antibodies, Monoclonal↗
PubMed1995

Transplantation of CD34+ peripheral blood progenitor cells after high-dose chemotherapy for patients with advanced multiple myeloma.

A major potential problem of autologous transplantation in the treatment of advanced malignancy is the infusion of tumor cells. A multi-institutional study of purified CD34-selected peripheral blood progenitor cell (PBPC) transplantation was conducted in 37 patients with advanced multiple myeloma receiving myeloablative chemotherapy. Fourteen days after intermediate-dose cyclophosphamide, prednisone, and granulocyte colony-stimulating factor (G-CSF), a median of 3 (range, 2 to 5) 10-L leukaphereses yielded 9.8 x 10(8)/kg (range, 3.7 to 28.3) mononuclear cells. The adsorbed (column-bound) fraction contained 5.9 x 10(6) cells/kg (range, 1.6 to 25.5) with 4.65 x 10(6) CD34 cells/kg (range, 1.2 to 23.3). Using Poisson distribution analysis of positive polymerase chain reactions with patient-specific complementarity-determining region 1 (CDR1) and CDR3 Ig-gene primers, tumor was detected in leukapheresis products from 8 to 14 unselected patients and ranged from 1.13 x 10(4) to 2.14 x 10(6) malignant cells/kg. After CD34 selection, residual tumor was detected in only three patients' products. Overall, a greater than 2.7- to 4.5-log reduction in contaminating multiple myeloma cells was achieved. CD34 PBPCs were infused 1 day after busulfan (14 mg/kg) and cyclophosphamide (120 mg/kg), and granulocyte-macrophage colony-stimulating factor was used until hematologic recovery. The median time to both neutrophil and platelet recovery was 12 days (range, 11 to 16 days and 9 to 52 days, respectively). The median number of erythrocyte and platelet transfusions was 7 (range, 2 to 37) and 3 (range, 0 to 85), respectively. Patients receiving fewer than 2 x 10(6) CD34 cells/kg had significantly prolonged neutropenia, thrombocytopenia, and an increased red blood cell and platelet transfusion requirement. Thus, CD34 selection of PBPCs markedly reduces tumor contamination in multiple myeloma and provides effective hematopoietic support for patients receiving myeloablative therapy.

Actuarial Analysis↗
PubMed1995

The effect of AT and GC sequence specific minor groove-binding agents on restriction endonuclease activity.

The ability of the naturally occurring A/T specific DNA minor groove binders netropsin and diastamycin A and two synthetic G/C selective oligopeptide analogues (1 and 2), to interfere with the catalytic activity of restriction endonucleases has been investigated. Enzymes were chosen to have A/T rich (EcoRI, EcoRV) or G/C rich (BalI, NruI) recognition sequences. An agarose gel assay was used to measure the cleavage of 32P-labelled DNA and ligand-DNA binding data was obtained using methidium-propyl EDTA footprinting. Netropsin and distamycin bind at the recognition sites, and dose-dependently inhibited cleavage by, EcoRI and EcoRV, (EcoRI > EcoRV). They were also more effective at inhibiting the catalytic activity of BalI than either 1 or 2. NruI was inhibited by distamycin and 2, but not by netropsin or 1. DNA footprinting revealed that neither 1 or 2 bound to the BalI or NruI recognition sequences under the conditions used whereas netropsin and distamycin footprint at adjacent sites. 1 binds to two of the three recognition sequences for the enzyme Fnu4HI (GCNGC) in the fragment studied and was shown to inhibit DNA cleavage only at these two sites. 2 binds strongly to two GGGCTC sequences which are recognition sites for the enzyme BanII. In this case a pronounced stimulation of cleavage was observed in the presence of 2 over a wide dose range. The results indicate that enzyme inhibition does not necessarily result from simultaneous occupancy of a common site, or at nearby flanking sequences, and in some circumstances, a pronounced stimulation of enzyme cleavage can occur.

Autoradiography↗
PubMed1995

Set up and run an objective structured clinical exam.

Objective structured clinical exams are increasingly used as a way of assessing a range of clinical skills at both undergraduate and postgraduate level. To those planning to introduce such assessments, this article provides basic guidance on their development and structure and the personnel required. For those already using the assessments, our article may provide new ideas or be the impetus for an exchange of ideas. For those who are facing such formal assessment as candidates, we hope this article shows the efforts that are made to achieve the necessary structure and objectivity in this type of examination.

Clinical Competence↗
PubMed1995

The thrombin receptor in human platelets is coupled to a GTP binding protein of the G alpha q family.

The thrombin receptor is a G protein-coupled receptor, but the G proteins functionally coupled to this receptor in human platelets are not yet definitively identified. Thrombin stimulation of platelets leads to phospholipase C-mediated increases in intracellular calcium, and previous studies have suggested that the thrombin receptor is coupled to members of the Gq family. We now demonstrate direct GTPase activation by thrombin receptor activation peptide (TRAP) in human platelet membranes, and specific inhibition of TRAP-activated GTPase by antibodies to Gq. These data demonstrate functional coupling of the thrombin receptor to a member of the Gq family.

Amino Acid Sequence↗
PubMed1995

Mutations associated with amyotrophic lateral sclerosis convert superoxide dismutase from an antiapoptotic gene to a proapoptotic gene: studies in yeast and neural cells.

Familial amyotrophic lateral sclerosis (FALS) is associated with mutations in SOD1, the gene encoding copper/zinc superoxide dismutase (CuZnSOD). However, the mechanism by which these mutations lead to amyotrophic lateral sclerosis is unknown. We report that FALS mutant SODs expressed in yeast lacking CuZnSOD are enzymatically active and restore the yeast to the wild-type phenotype. In mammalian neural cells, the overexpression of wild-type SOD1 inhibits apoptosis induced by serum and growth factor withdrawal or calcium ionophore. In contrast, FALS-associated SOD1 mutants promote, rather than inhibit, neural apoptosis, in a dominant fashion, despite the fact that these mutants retain enzymatic SOD activity both in yeast and in mammalian neural cells. The results dissociate the SOD activity of FALS-associated mutants from the induction of neural cell death, suggesting that FALS associated with mutations in SOD1 may not be simply the result of a decrease in the enzymatic function of CuZnSOD. Furthermore, the results provide an in vitro model that may help to define the mechanism by which FALS-associated SOD1 mutations lead to neural cell death.

Amyotrophic Lateral Sclerosis↗
PubMed1995

Cell-type and amyloid precursor protein-type specific inhibition of A beta release by bafilomycin A1, a selective inhibitor of vacuolar ATPases.

Treatment of human 293 cells transfected with amyloid precursor protein (APP)K595N,M596L (the "Swedish" mutation) with a specific inhibitor of the vacuolar H(+)-ATPases, bafilomycin A1 (baf A), leads to a potent inhibition of the release of the A beta peptide. This is accompanied by a selective inhibition of beta-secretase activity. Surprisingly, baf A did not inhibit the production of A beta from either wild-type APP (WT APP) or from APPv7171 (the "Hardy" mutation), expressed in the same cell type. In contrast, the robust production of A beta from a human neuroglioma-derived cell line (HS683) transfected with WT APP, or from primary human mixed brain cultures (HMBC) expressing genomic WT APP, were also effectively inhibited by baf A. The inhibition of A beta production from the HMBC was also accompanied by the inhibition of beta-s-APP release. No inhibition of alpha-s-APP release was seen in any of the cell types tested. These results indicate that intracellular acidic processes are rate-limiting for beta-secretase cleavage and A beta production from SW APP, but not WT APP, in the peripheral 293 cell line. Furthermore, such acidic processes also play a rate-limiting role in A beta release from human central nervous system-derived cells, including HMBC. Differential trafficking of the SW APP into an acidic compartment conducive to beta-secretase cleavage and A beta release could be one explanation for the increased production of A beta observed on expression of this mutation.

Adenosine Triphosphatases↗
PubMed1995

The power spectral analysis of heart rate variability in athletes during dynamic exercise--Part I.

In this study, the effects of long-term physical training on autonomic function in athletes and the response of the autonomic nervous system to dynamic exercise were investigated in nonathletes and athletes with power spectral analysis of heart rate variability (HRV). This study was performed on 13 healthy subjects (5 athletes and 8 non athletes). Electrocardiographic (ECG) signals were continuously recorded during (1) 15 min of rest in a sitting position on a bicycle ergometer, (2) the dynamic exercise test to the point of exhaustion, and (3) a 15 min postexercise period. After the recorded ECG signals were sampled at 500 samples/s, the instantaneous HRV signal was constructed from the detected R peaks and then resampled at 4 Hz in order to obtain an evenly spaced time series applicable to power spectral analysis. After linear trends were removed by the robust locally weighted regression algorithm, the power spectrum of HRV was estimated for contiguous records of 512 samples by Burg's maximum entrophy method. HRV was quantified by determining the spectral area (power) in two frequency bands, low-frequency power (LF power: 0.05-0.15 Hz) and high-frequency power (HF power: 0.15-0.8 Hz), and their ratio. The comparison between athletes and nonathlete was performed in terms of the above-mentioned parameters. Although both groups showed similar trends in heart rate (HR) at all stages of protocols, HR in athletes was significantly lower than that in nonathletes during rest and postexercise. In athletes and nonathletes, LF and HF powers gradually decreased with exercise. As recovery progressed, they continued to increase gradually, but remained below resting level. During rest and postexercise, HF power in athletes was significantly (p < 0.05) higher than than in nonathletes. Also, the recovery of HR and HF powers during early recovery (PO1) was more rapid in athletes than in nonathletes. Both groups showed an attenuation of LF and HF powers during dynamic exercise. It is likely that, in athletes, the lower HR during rest and the more rapid recovery of HR postexercise was due to a high level of HF power, indicating that vagal activity was enhanced by the adaptive changes in neural regulation produced by long-term physical training.

Adolescent↗
PubMed1995

Interactive effects of alcohol and diabetes during pregnancy on the rat fetus.

To determine the effect of maternal diabetes and alcohol intake, separately and in combination, on fetal growth and development, pregnant rats were divided into four groups: diabetic (D), diabetic plus alcohol (DA), control (C), and control plus alcohol (CA). Diabetes was induced by administration of streptozotocin before mating and alcohol was administered by gavage (2 g/kg body weight/day) on days 6-11 of gestation. Both diabetic groups (D and DA) had significantly lower weight gain during pregnancy compared to the controls (C and CA), despite the fact that the former consumed more food and water. Alcohol treatment resulted in reduced water and food intake and lower weight gain in the diabetic rats (DA), but not in the non-diabetic rats (CA), compared to their respective controls (D and C). On day 21 of gestation fetal body weights were significantly less and placental weights were significantly greater in the diabetic groups (D and DA) compared with the non-diabetic groups (C and CA). Differences in fetal and placental weights between rats exposed and not exposed to alcohol (C vs. CA and D vs. DA) were not significant. The number of fetuses with external malformations was significantly greater in the litters of alcohol exposed diabetic (DA) than non-alcohol exposed (D) animals. No external or skeletal malformations were observed in fetuses of non-diabetic rats regardless of whether or not they received alcohol (C or CA). The skeletal development of fetuses of diabetic rats, judged by the number and size of ossification centers on day 21 of gestation, was retarded when compared with fetuses of non-diabetic rats. Alcohol further retarded skeletal development of fetuses of diabetic animals (DA vs. D), but not of fetuses of non-diabetic rats (CA vs. C). It is concluded that maternal alcohol administration potentiates the effects of maternal diabetes on the incidence of fetal malformations and the retardation of skeletal development.

Abnormalities, Drug-Induced↗
PubMed1995

Delayed macular choriocapillary circulation in age-related macular degeneration.

PURPOSE: To investigate the macular choriocapillary circulation (MCC) in eyes with age-related macular degeneration (ARMD) and to correlate these findings with the associated clinical and angiographic drusen characteristics. METHODS: Scanning laser ophthalmoscope fluorescein videoangiography was performed on 34 eyes with age-related macular degeneration and eight age-matched normal volunteers. Drusen characteristics were assessed using the Wisconsin age-related maculopathy grading scale. RESULTS: A delayed macular choriocapillary circulation (DMCC) was defined as a macular choriocapillary filling time greater than 3 standard deviations from the normal mean (greater than 5 seconds). Nine (26%) of the 34 eyes with ARMD were found to have a DMCC. After age adjustment, eyes with DMCC were more likely to have geographic atrophy of the retinal pigment epithelium (p = 0.003) or choroidal neovascularization p = 0.07) than were eyes with a normal MCC. Regional differences in choriocapillary filling times were present in the eyes with a DMCC, including nasal-to-temporal, central-to-peripheral, and inferior-to-superior gradients of progressively less choriocapillary filling delay. The DMCC correlated with the location, number, size, confluence, and fluorescein staining characteristics of the associated drusen. CONCLUSION: DMCC occurs in some eyes with ARMD. This finding may not only assist in defining eyes at risk for progressive disease but may also help to elucidate the pathogenesis of age-related macular degeneration.

Aged↗
PubMed1995

Establishment of a colonic polyp registry in Rhode Island.

A colonic adenomatous polyp registry (PR) has been organized at the Roger Williams Medical Center whose main functions are to prevent the occurrence of colorectal cancer (CRC) in the enrollees, to provide a population of subjects for epidemiological and interventional studies, and to provide educational, including dietary, information to subjects and physicians. One hundred four and 202 patients with polyps, originally retrieved from the hospital pathology files, were enrolled in the 1984 and 1987 cohorts, respectively, of whom about 90% were followed for at least three years after polypectomy. Three carcinomas, all Dukes A, were found in the right colon in the follow-up period. New polyps identified in the first three years after polypectomy were generally small tubular adenomas with a greater predilection for the right colon than was found for the index polyps. Risk factors for new polyps included history of previous polyps and, probably, multiple index polyps. The use of colonoscopy for postpolypectomy surveillance increased between 1984 and 1987. About 25% of the subjects in each cohort were either lost to follow-up or received no endoscopic surveillance. On the other hand, some of those who were followed were probably subjected to excessive numbers of procedures. Defects in the PR include inadequacy of personal and family history data, and steady loss of patients during the three to six years after polypectomy. Despite the small size and limited resources of our hospital, its colonic polyp registry has already provided information that may help in the management of patients with this premalignant condition. The more widespread use of securely funded polyp registries would probably reduce the incidence of metachronous CRC in that population and would have significant epidemiological and educational functions.

Adenomatous Polyps↗
PubMed1995

A model of spine, ribcage and pelvic responses to a specific lumbar manipulative force in relaxed subjects.

One class of manipulative techniques commonly used during assessment and treatment of spinal disorders involves the patient lying face down while the therapist slowly applies a posteroanterior force to a selected vertebra. The aim of this investigation was to develop a model which was capable of predicting the vertebral displacements resulting from such a manipulative force, applied to the lumbar spine. A linear three-dimensional finite element model was generated using both previously published and original data to define the geometry and material properties. The complete model included the ribcage, thoraco-lumbar spine and pelvis with their associated soft tissues. The model simulated the relaxed state in a normal subject so the muscle forces were assumed to be negligible. Sensitivity analysis suggested that if the model was to be used to simulate the behaviour of individual subjects, then the model dimensions and pelvic constraints should be matched to the particular subject. The model validity was studied by comparing the predicted responses with those that have been observed in living human subjects. The model predictions were found to be in good agreement with the mean observed human responses, with predicted displacements being within one standard deviation of the mean observed values. This agreement suggests that the model is useful for predicting the linear region responses to slowly applied lumbar posteroanterior forces. The simulations predicted that appreciable global vertebral displacements (up to 1.5 mm) and rotations (up to 1 degree) occurred as far away as the middle and lower thoracic spine during low lumbar loading. Intervertebral translations were predicted to be 1 mm or more at up to four intervertebral joints away from the point of load application.(ABSTRACT TRUNCATED AT 250 WORDS)

Computer Simulation↗
PubMed1995

The mechanism of Escherichia coli tryptophan indole-lyase: substituent effects on steady-state and pre-steady-state kinetic parameters for aryl-substituted tryptophan derivatives.

We have examined the reaction of Escherichia coli tryptophan indole-lyase with fluoro, chloro, methyl and hydroxytryptophans using steady-state kinetics, rapid-scanning and single wavelength stopped-flow spectrophotometry, and rapid chemical quench methods. All of the 16 tryptophan derivatives examined are substrates for alpha, beta-elimination catalyzed by tryptophan indole-lyase. The steady-state kinetic parameter, kcat/Km, did not show a consistent trend with the steric bulk of the substituent, but Km increased for larger substituents. Rapid-scanning stopped-flow spectra show that all tryptophan analogues undergo covalent reaction with the pyridoxal-5'-phosphate cofactor to give equilibrating mixtures of external aldimine and quinonoid intermediates, but the relative amounts of each intermediate are strongly dependent on the nature and position of the substituent. The dissociation constants for external aldimine formation, Kd, obtained from single-wavelength stopped-flow experiments decreased for most substituted tryptophans, which suggests that part of the binding energy is derived from hydrophobic interactions between the enzyme and the indole ring of tryptophan. In contrast, the rate constants of quinonoid intermediate formation and reprotonation and of indole elimination were quite variable, depending on the position and the nature of the substituent. Overall, 6-substituted tryptophans have the most consistent reactivity, which indicates that there may be space in the enzyme active site near the 6-position. There is a good linear correlation between log (kcat/Km) and log (kf/Kd) (apparent second order rate constant for quinonoid intermediate formation), with a slope of 0.66. This suggests that quinonoid intermediate formation contributes only about 66% of the activation energy for the reaction, and thus a later step in the reaction must be partially rate-limiting. Rapid chemical quench experiments demonstrate a 'burst' of indole in the reaction of L-tryptophan under single turnover conditions, confirming that a step subsequent to the elimination is partially rate-determining. In contrast, 5-methyl-L-tryptophan does not exhibit a significant 'burst', suggesting that 5-methylindole elimination is nearly completely rate-determining. These results support the proposed mechanism and demonstrate that there are significant effects of aryl substituents on the distribution of covalent intermediates and on the rate-determining step in the alpha, beta-elimination reaction catalyzed by E. coli tryptophan indole-lyase.

Binding Sites↗