Biomedical subjects

D M Smith

Publications and source records attributed to D M Smith.

At least 253 records · Page 14Linked to original sources

PubMed1991

A comparison of pyridoxal 5'-phosphate dependent decarboxylase and transaminase enzymes at a molecular level.

Pyridoxal 5'-phosphate is a coenzyme for a number of enzymes which catalyse reactions at C alpha of amino acid substrates including transaminases, decarboxylases and serine hydroxymethyltransferase. Using the X-ray coordinates for a transaminase, aspartate aminotransferase, and the results of stereochemical and mechanistic studies for decarboxylases and serine hydroxymethyltransferase, an active-site structure for the decarboxylase group is constructed. The structure of the active-site is further refined through active-site pyridoxyllysine peptide sequence comparison and a 3-D catalytic mechanism for the L-alpha-amino acid decarboxylases is proposed. The chemistry of serine hydroxymethyltransferase is re-examined in the light of the proposed decarboxylase mechanism.

Amino Acid Sequence↗

PubMed1991

Manganese superoxide dismutase is induced by IFN-gamma in multiple cell types. Synergistic induction by IFN-gamma and tumor necrosis factor or IL-1.

We have previously characterized more than 20 proteins induced by the immunoregulatory lymphokine IFN-gamma in human fibroblasts by their m.w. and isoelectric points determined in two-dimensional gels. Some of these proteins are induced uniquely by IFN-gamma, whereas others are also induced by IFN-alpha, TNF, or IL-1. Recent technologic advances have allowed us to begin to rapidly identify proteins induced by IFN-gamma and other cytokines by sequencing the induced proteins from blots of preparative two-dimensional gels of total cell lysates. In this study, we show that the approximately 21 kDa, isoelectric point greater than 7 protein induced by IFN-gamma is manganese superoxide dismutase (Mn-SOD), a mitochondrial protective enzyme encoded by a nuclear gene. Mn-SOD is induced by IFN-gamma and also by TNF in all four human cell lines examined: HS153 fibroblasts, ACHN renal carcinoma, A549 lung carcinoma, and A375 melanoma. Induction of Mn-SOD mRNA is a primary, rapid, and dose-dependent response to IFN-gamma. In ACHN renal carcinoma cells, Mn-SOD mRNA and protein are induced synergistically by IFN-gamma in combination with either TNF or IL-1, and the induced protein is enzymatically active. IFN-gamma and TNF together induce Mn-SOD mRNA by more than 100-fold relative to its level in untreated ACHN cells. The induction of Mn-SOD by IFN-gamma and its synergistic induction by IFN-gamma in combination with TNF and IL-1 should protect healthy cells from the toxicity of O2- during an immune response, and may provide a mechanism for selective killing of infected cells.

Amino Acid Sequence↗

PubMed1991

Widespread dissemination of Pneumocystis carinii infection in a patient with acquired immune deficiency syndrome receiving long-term treatment with aerosolized pentamidine.

Patients with acquired immune deficiency syndrome (AIDS) may be infected with many opportunistic pathogens, the most common of which is Pneumocystis carinii. P. carinii infection typically presents as a subacute pneumonia. However, rare cases of localized, extrapulmonary, and disseminated disease have been described. Standard therapy for P. carinii is systemically administered trimethoprim-sulfamethoxazole or pentamidine. These agents, however, frequently are associated with serious adverse effects. More recently, aerosolized pentamidine has been proposed as an alternative treatment for those who cannot tolerate standard therapy and as primary and secondary prophylaxis. Inhaled pentamidine is effective, but it is not without hazards. The authors describe a patient with AIDS who received long-term treatment with aerosolized pentamidine and yet died as a result of widely disseminated P. carinii infection.

Acquired Immunodeficiency Syndrome↗

PubMed1991

Investigation and characterization of receptors for pituitary adenylate cyclase-activating polypeptide in human brain by radioligand binding and chemical cross-linking.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel peptide of hypothalamic origin which increases adenylate cyclase activity in rat anterior pituitary cell cultures. The 38-amino acid peptide shows a close sequence homology to vasoactive intestinal peptide (VIP). Binding sites for PACAP in membranes from postmortem human brain tissue were studied using [125I]PACAP27 as the radioligand. High specific binding sites (amount of specific binding measured at 0.25 nM [125I]PACAP27 in femtomoles per mg protein +/- SEM; n = 4) were present in hypothalamus (344.5 +/- 13.0), brain stem (343.0 +/- 29.3), cerebellum (292.0 +/- 21.1), cortex (259.6 +/- 19.8), and basal ganglia (259.2 +/- 50.3). Specific binding sites in pituitary, although present, were less abundant (35.0 +/- 8.9). Binding of [125I]PACAP27 was reversible and time, pH, and temperature dependent. Despite the homology with VIP, VIP was a poor inhibitor of [125I]PACAP27 binding (IC50, greater than 1 microM) compared with PACAP27 (IC50, 0.5-1.3 nM) and PACAP38 (IC50, 0.2-1.3 nM). Scatchard plots of [125I]PACAP27 binding showed the presence of both high and lower affinity sites. Chemical cross-linking of PACAP-binding sites revealed that [125I]PACAP27 was bound to polypeptide chains of 67,000 and 48,000 mol wt. Thus, we have demonstrated the presence of PACAP-specific receptors in human brain which are not VIP receptors. This opens the possibility of PACAP functioning as a novel neurotransmitter/neuromodulator in human brain.

Brain↗

PubMed1991

Nuclear localization of an O-glycosylated protein phosphotyrosine phosphatase from human cells.

Histochemical staining of immunoprecipitates of p65, a component of human M phase-promoting factor, identified the molecule as having phosphatase activity. The enzyme, purified 3400-fold from mitotic cell extracts by (NH4)2SO4 precipitation, DEAE chromatography, and immunoaffinity chromatography on immobilized anti-p65 IgG, was inhibited by Zn2- and Na3VO4 but not NaF or beta-glycerophosphate; 32P-labeled poly(Glu, Tyr) was more efficiently dephosphorylated than phosphorylated histone or phosphorylase a. Indirect immunofluorescence showed most of the phosphatase to be localized in the nucleus of interphase cells, with a fine, granular distribution unaltered by detergent extraction; in mitotic cells, p65 was localized on chromosomes. ELISA of subcellular fractions confirmed this localization. Immunoreactive p65 was recovered from immobilized wheat germ agglutinin (WGA) upon elution with N-acetylglucosamine; similarly, WGA recognized immunoaffinity-purified p65 on blots. Alkaline hydrolysis of blotted protein prevented WGA binding, indicating that phosphatase p65, like a small group of other nuclear proteins, contains O-linked carbohydrate terminating in N-acetylglucosamine.

Acetylglucosamine↗

PubMed1991

Action of pituitary adenylate cyclase-activating polypeptide and vasoactive intestinal polypeptide on the rat vascular system: effects on blood pressure and receptor binding.

The N-terminal fragment (PACAP 27) of the novel neuropeptide, pituitary adenylate cyclase-activating polypeptide 38 (PACAP 38), has 68% homology with vasoactive intestinal polypeptide (VIP). The administration of bolus doses of PACAP 38 and its 27 amino acid N-terminal fragment (PACAP 27) caused a rapid but transient dose-dependent hypotensive effect in the anaesthetized rat. The amplitude and duration of the response obtained by PACAP 38 was comparable with VIP whereas PACAP 27 was three times less potent than VIP. Furthermore, radioreceptor binding studies demonstrated that 125I-labelled PACAP 27 and 125I-labelled VIP bound to membranes prepared from blood vessels. Both PACAP 27 and VIP were capable of displacing the other from these binding sites. We propose that the hypotensive effect is via the same receptor type.

Animals↗

PubMed1991

Biochemical perturbations of BW 91Y (3-deazaadenosine) on human neutrophil chemotactic potential and lipid metabolism.

The studies reported here were designed to examine the chemotactic potential, arachidonic acid (AA) metabolism and phospholipid transmethylation in peripheral blood neutrophils from patients with rheumatoid arthritis (RA): a) prior to treatment with BW 91Y (3-deazaadenosine), b) after 4 weeks when half the patients were on active medication and half were on placebo, and c) after 4 weeks at which time all patients were on active medication. The authors demonstrate that BW 91Y in vitro at 600 pg/ml caused a decrease in chemotactic potential as measured by the leading front (LF) assay in neutrophils from both normal volunteers (p less than 0.025) and RA patients. They also demonstrate that BW 91Y caused a significant increase in production of [3H]LTB4 (LTB = leukotriene B) in ionophore-stimulated neutrophils from both normal (p less than 0.025) and RA patients (p less than 0.050) as compared to initial values. BW 91Y caused decreased incorporation and percent distribution of [3H]AA into phosphatidylcholine (PC), with a resultant increase in percent distribution into phosphatidylethanolamine (PE). There was also an increased release of [3H]AA from the PE fraction in BW 91Y-treated cells in response to ionophore stimulation. BW 91Y was found to exhibit a dose-dependent (10(-7) to 10(-4) g/ml) inhibition of the uptake and incorporation of L-[methyl-3H]methionine into the cellular lipids, while at low doses (10(-9) to 10(-5) g/ml) it stimulated the significant uptake and incorporation of [methyl-14C]choline chloride into PC. Although the total cellular content and percent composition of PC remained unchanged, it was found that BW 91Y caused a slight decrease in PC plasmalogens and an apparent increase in the 1,2-diacyl-glycerophosphatidylcholine (-GPC). BW 91Y was found, however, to have no effect on the amount or stimulated metabolism of the ether-linked 1-O-alkyl-2-acyl-GPC. As further evidence for this, the authors demonstrate that BW 91Y has no effect on the ionophore-stimulated production of [14C]acetate-labelled 1-O-alkyl-2-acetyl-GPC, or [14C]PAF.

Arachidonic Acids↗

PubMed1991

Addition of peripheral blood stem cells collected without mobilization techniques to transplanted autologous bone marrow did not hasten marrow recovery following myeloablative therapy.

A randomized prospective trial was conducted to determine if the addition of cryopreserved autologous peripheral blood stem cells (PBSC) collected without mobilization techniques to autologous cryopreserved bone marrow for patients receiving an autologous bone marrow transplant (ABMT) affected the time to marrow function recovery. Thirty-five evaluable patients with various malignancies were studied. Sixteen received PBSC + ABMT and 19 received ABMT alone. The PBSC were collected with 4 h leukapheresis procedures on 3 consecutive days. No manipulations to increase the number of circulating stem cells were used during the collections. The median time to recover 0.5 x 10(9)/l circulating granulocytes was 20 days after transplantation in the ABMT group and 27 days in the PBSC + ABMT group (p = 0.12). The median time to recover 20 x 10(9)/l platelets was 22 days after transplantation in the ABMT group and more than 27 days in the PBSC + ABMT group (p = 0.29). The day of discharge from the hospital was earlier for the ABMT group (median 29 days) than the PBSC + ABMT group (median 35 days, p = 0.03). We did not find that the addition of non-mobilized PBSC to infused autologous marrow accelerates marrow recovery.

Adolescent↗

PubMed1991

The preparticipation evaluation.

The preparticipation evaluation (PPE) is the first step toward meeting the responsibility of maintaining the health and safety of the athlete. The ideal timing for the PPE is 6 weeks prior to the competitive season to allow adequate time for treatment and rehabilitation of problems. It is suggested that a complete screening evaluation be performed prior to entrance into a new level of school with annual limited reevaluations thereafter. The annual reevaluation should focus on the medical history with the physical examination emphasizing the cardiovascular system and interim injuries and medical problems. Currently, there are no data to support routine screening laboratory, radiographic, or cardiovascular testing in the asymptomatic athlete with an unremarkable history and normal examination. Two different methods for performing the PPE can be used (station-type mass screening and office setting), with each having its own distinct advantages and disadvantages. The most important and difficult decision for the physician screening young athletes is determining clearance for activity. Guidelines based on the demands of the sport and specific medical problems have been established; however, the physician's clinical judgement should be used in interpreting these recommendations for each individual athlete. The PPE is an excellent opportunity for the physician to positively influence the health care and safety of a local sports program. Through genuine interest, thorough evaluation, and knowledgeable counseling, the athlete will develop confidence in the evaluating physician. This confidence can translate into a good relationship and hopefully this interaction will encourage the athlete to trust the physician's judgement and seek early evaluation and treatment for problems that arise.

Health Status↗

PubMed1990

Coordinated induction of autocrine tumor necrosis factor and interleukin 1 in normal human monocytes and the implications for monocyte-mediated cytotoxicity.

Cytokine production and cytotoxicity for tumor cells are two important aspects of monocyte function and the inflammatory response against tumors and infectious agents. In the present studies we provide direct evidence at the mRNA and protein levels for the existence of autocrine tumor necrosis factor (TNF) and its importance as a mediator of human monocyte cytotoxicity for WEHI-164 tumor cells. The induction of TNF and interleukin 1 beta (IL-1 beta) mRNA by exogenous TNF or IL-1 beta, as determined by Northern blot analysis, is time dependent in normal human monocytes isolated by countercurrent elutriation. With either TNF or IL-1 beta as the stimulus, TNF mRNA is induced first, peaks within 1-3 h, and declines to nearly undetectable levels by 9 h. TNF mRNA accumulation is enhanced in the presence of cycloheximide indicating that de novo protein synthesis is not required for maximal TNF mRNA induction. In contrast, IL-1 beta mRNA is induced later, peaks at 3-9 h, and remains considerably elevated at 18 h. IL-1 beta mRNA accumulation is partially suppressed in the presence of cycloheximide. TNF and IL-1 beta protein production as assayed by specific enzyme-linked immunosorbent assays correlates well with respective mRNA induction. Both TNF and IL-1 beta enhance monocyte cytotoxicity as single agents; however, their combined effect is less than additive. When both agents are combined, TNF mRNA levels, as assessed by densitometric analysis of slot blots, are approximately equal to those induced by TNF alone. In contrast, IL-1 beta mRNA levels are additive. Our studies provide evidence for highly coordinated and interrelated pathways of autocrine TNF and IL-1 induction in human monocytes and demonstrate the role of TNF and IL-1 in regulating monocyte-mediated cytotoxicity for tumor cells.

Autoradiography↗

PubMed1990

Separation and characterization of basal and secretory cells from the rat trachea by flow cytometry.

Basal and secretory cells have been separated as highly enriched viable populations from single-cell suspensions of rat tracheal epithelial cells. Isolation of the populations was achieved by preparation of a cell suspension and separation by flow cytometry using contour maps generated from 2 degrees and 90 degrees light scatter signals. Flow cytometric analysis of cells showed 10% of the whole preparation were cells in SG2M phase of the cell cycle. The secretory cells accounted for 86% of these cycling cells; the remainder were accounted for by the basal cells. Culture of sorted populations of basal and secretory cells in serum free defined medium showed that basal cells had a lower (0.6%) colony-forming efficiency than secretory cells (3.4%). Significant differences in blue auto-fluorescence, Hoechst 33342 uptake, and lectin staining were apparent between basal and secretory cells. These results suggest that the secretory cell rather than the basal cell is primarily the cell type involved in maintenance of the normal tracheal epithelium. Secretory cells are greater in number, have a higher proliferative potential, and greater metabolic capability. Because of these traits they may be a critical cell at risk from damage by environmental agents.

Animals↗

PubMed1990

Counterflow centrifugation apheresis for the collection of autologous peripheral blood stem cells from patients with malignancies: a comparison with a standard centrifugation apheresis procedure.

Two apheresis methods used to collect hematopoietic stem cells from peripheral blood were compared in eight patients with a variety of malignancies. The standard lymphocyte collection method was alternated with the counterflow centrifugation or lymphocyte surge protocol. The number of clonogenic cells (CFU-GM and BFU-E), the red cell volume, and the number of mononuclear cells in each collection were assessed as well as the changes in circulating leukocytes, platelets, and blood hemoglobin produced by each apheresis procedure. There was no statistically significant difference found in the number of clonogenic cells collected with either method, but the number of mononuclear cells collected with the standard procedure was significantly higher (P = 0.001). The red cell volume collected with the standard procedure was significantly higher, (P = 0.0001), but corrected for the number of mononuclear cells the difference was not significant. The counterflow centrifugation apheresis produced significantly less thrombocytopenia (P = 0.005). The counterflow centrifugation apheresis procedure used collected fewer mononuclear cells than the standard procedure, however, with less red cell contamination but a comparable number of CFU-GM and BFU-E in four hour apheresis procedures. Each collection method resulted in a comparable amount of anaemia and leukopenia but the lymphocyte surge method produced less thrombocytopenia following the collection.

Adult↗

PubMed1990

Evaluation of Tenidap (CP-66,248) on human neutrophil arachidonic acid metabolism, chemotactic potential and clinical efficacy in the treatment of rheumatoid arthritis.

Ten patients with rheumatoid arthritis (RA) were evaluated in a placebo-controlled, double-blind study examining the clinical efficacy of a novel nonsteroidal anti-inflammatory agent: Tenidap (CP-66,248). RA patients receiving active drug therapy (n = 6) demonstrated clinically significant improvements in observer assessment of pain (p less than 0.025), painful joint count (p less than 0.010), and overall clinical assessment as based on a modified rheumatoid activity index, MRAI (p less than 0.025). In parallel laboratory assays, Tenidap was found to exhibit a significant in vitro dose-dependent inhibition of ionophore-stimulated neutrophil production of the 5-lipoxygenase product: [3H]leukotriene B4 (LTB4). Although more importantly, Tenidap was also found to exhibit an in vitro dose-dependent inhibition (IC50 20 microM) of the ionophore-stimulated release (deacylation) of the precursor [3H]arachidonic acid (AA) from membrane phospholipids. In further studies, Tenidap did not have any effect on fMLP-induced neutrophil chemotactic response. These results suggest that one of the possible mechanisms for the clinical effectiveness of this agent, may be through its effect at inhibiting the release of free AA from membrane phospholipids and therefore limiting its further metabolism into certain biologically-active inflammatory lipids.

Anti-Inflammatory Agents, Non-Steroidal↗

PubMed1990

Synthesis and antihypertensive activity of 4-(substituted-carbonylamino)-2H-1-benzopyrans.

The synthesis and antihypertensive activity of a series of novel 4-(substituted-carbonylamino)-2H-1-benzopyran-3-ols, administered orally to conscious spontaneously hypertensive rats, are described. Optimum activity was observed for compounds with alkyl, amino, or aryl groups flanking the carbonyl group. Of the alkyl and amino series the most potent compounds contained the methyl and methylamino groups, respectively. Several analogues have been compared with cromakalim (1) for their effects on potassium ion efflux in the rabbit mesenteric artery using rubidium-86 as a marker. The ability of each compound to enhance rubidium-86 efflux is approximately parallelled by its blood pressure lowering activity, and thus these analogues, like compound (1), belong to the series of drugs which have been classified as potassium-channel activators.

Animals↗

PubMed1990

Antiviral and antiproliferative properties of liposome-associated human interferon-gamma.

Recombinant human interferon-gamma (rHuIFN-gamma) was associated with liposomes in an attempt to improve its therapeutic efficiency. It was associated with liposomes composed of phosphatidylserine (PS) and phosphatidylcholine (PC) at a ratio of 3:7, and of PS:PC and cholesterol (CHOL) at a ratio of 1:4:5 with efficiencies of 13% and 21%, respectively. The lipid composition influenced the antiviral activity of the liposome-complexed IFN-gamma tested against vesicular stomatitis virus. IFN associated with PS:PC liposomes was fully bioavailable and degraded by trypsin treatment. In contrast, PS:PC:CHOL-IFN was resistant to trypsin, and appeared latent as its full biological activity was seen only after disruption of the liposomes with detergent. Four human tumor cell lines were exposed to free and liposome-associated IFN-gamma. The growth of three solid tumor lines (colon, bladder, and lung) was inhibited by similar concentrations of free IFN and PS:PC-IFN. In contrast, less PS:PC-IFN than free IFN was needed to inhibit histiocytic lymphoma cells. Higher concentrations of PS:PC:CHOL-IFN than of free IFN were needed to inhibit growth of all four cell lines. The specificity of these effects of liposome-associated IFN-gamma were shown by their partial or complete neutralization by antibody to IFN-gamma. When liposome-IFN complexes of either type were stored at 4 degrees C, 30% of the IFN activity remained after 7 days; thereafter, decay was minimal over the next 3 weeks. These data show the formation of stable HuIFN-gamma-liposomes and indicate that the lipid components of these complexes influence their antiviral and antiproliferative activity for several different cell types.

Antineoplastic Agents↗

PubMed1990

Recurrent neurologic symptoms during peripheral stem cell apheresis in two patients with intracranial metastases.

Leukapheresis recently has been used to harvest circulating hematopoietic stem cells for autologous transplantation. This technique is especially useful when involvement of the bone marrow by tumor precludes its use as a source for stem cells. Thirty-five patients have had peripheral blood stem cells harvested for transplantation by this technique at our institution. Two patients with adenocarcinoma of the breast developed severe headaches during apheresis, and both were found to have extensive intracranial metastases. For one patient, the pain was excruciating and associated with hypotension at the end of each collection phase of the apheresis procedure and resolved approximately 5 minutes after reinfusion of the patient's red cells and plasma. Subsequently this patient had a documented subdural hydroma. Following an apheresis procedure, while receiving local low dose thrombolytic therapy for thrombosis of the brachiocephalic vein, she suffered a fatal intracerebral hemorrhage. The second patient had a severe exacerbation of a headache of recent onset. Leukapheresis was abandoned when extensive intracranial disease was documented. We conclude that recurrent neurologic symptoms, especially cranial pain, during the withdrawal phase of intermittent flow apheresis may predict intracranial pathology in patients. Peripheral stem cell apheresis and lymphocytapheresis are bringing patients with metastatic malignancies to the apheresis department with increasing frequency, and the apheresis staff must be alert to previously undescribed complications of apheresis.

Adenocarcinoma↗

PubMed1990

Dental screening of long stay geriatric patients in West Essex and recommendations for their care.

A dental screening programme at a West Essex Health Authority hospital provided data from 151 in-patients, most of whom were in long-stay geriatric care. 71 per cent were female and the mean age of the geriatric patients was 81 years. 61 subjects (40 per cent) suffered predominantly from mental illness. 107 subjects (71 per cent) were edentulous but a higher proportion of elderly mentally ill patients (42 per cent) had retained some of their natural teeth compared with 20 per cent of the others. 26 edentulous subjects were not in possession of any dentures and the standard of oral and denture hygiene was poor. 59 in-patients (39 per cent) had 'expressed' need for dental treatment and a further 51 (34 per cent) were unable or unwilling to communicate their need. An important aspect of their dental care was the provision and maintenance of a preventive dental programme for both dentate patients and denture wearers.

Aged↗

PubMed1989

Encouraging primary care physicians to help smokers quit. A randomized, controlled trial.

STUDY OBJECTIVE: To increase the effect that primary care physicians have on their patients who smoke. DESIGN: Randomized, controlled trial with 112 general internists and their patients who smoke. PATIENTS: Sample of 1420 patients from a general medicine clinic of a city-county teaching hospital, who smoke at least one cigarette a day and were recruited regardless of their interest in quitting smoking. INTERVENTIONS: Physicians were randomly assigned to one of four groups: participants who received a protocol for smoking management and a lecture on the consequences and management of smoking (control); in addition, had nicotine gum freely available to patients (gum); had stickers attached to their smokers' charts (reminder); or had both gum and reminders (both). MEASUREMENTS AND MAIN RESULTS: The percentage of patients with a return visit at 6 months who quit smoking (alveolar carbon monoxide of less than nine parts per million) was 1.3% (control), 7.7% (gum), 7.0% (reminders), and 6.3% (both). At 1 year the percentages were 2.7%, 8.8%, 15.0%, and 9.6%, respectively. Subsequent pairwise comparisons showed that the three intervention groups were not significantly different, but that each was significantly different from the control group (P less than 0.05). Physicians in all three intervention groups spent significantly more time than did the physicians in the control group counseling their patients about smoking. CONCLUSIONS: The availability of nicotine gum or labeling the charts of smokers can help primary care physicians increase their success rates two- to six-fold in helping patients quit smoking. If all primary care physicians used these procedures, they could help an additional 2 million smokers quit.

Adult↗