Biomedical subjects

R Mitra

Publications and source records attributed to R Mitra.

At least 55 records · Page 3Linked to original sources

PubMed1992

Isolated cardiomyocytes in conjunction with NMR spectroscopy techniques to study metabolism and ion flux.

To distinguish cellular from vascular responses to physiological and pathophysiological stimuli, we developed methods to perform NMR spectroscopy on isolated ventricular cardiomyocytes. Isolated adult rat cardiomyocytes, placed in agarose beads and superfused with phosphate-free buffer (Media 199 (GIBCO 400-1100) gassed with 95% O2, 5% CO2), were used to evaluate a variety of cellular processes during different pharmacological and physiological interventions. Bioenergetic function was monitored with 31P NMR. Intermediary metabolism, gluconeogenesis, and glycolysis were monitored with 13C NMR. Sodium flux was monitored with 23Na NMR. Calcium flux was monitored with 19F NMR in conjunction with an intracellular calcium-chelating agent, 5F-1,2-bis(2-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid. Creatine kinase kinetics (forward rate constant (Kf) and flux of phosphocreatine to ATP) were estimated with 31P NMR saturation transfer data. Various combinations of NMR parameters were monitored simultaneously so that the interaction of metabolism and ion flux could be evaluated. We have demonstrated that it is possible to simultaneously monitor a variety of cellular processes in intact heart cells in real time, without the confounding influences of perfusion, contractile function, and extrinsic blood-borne neurohumoral agents. This model will be useful for longitudinal studies of myocyte metabolism and ion flux.

Acetylcholine↗

PubMed1992

Serologic differentiation of human T lymphotropic virus type I from type II infection by synthetic peptide immunoassays.

Synthetic peptide-based serologic assays (Select-HTLV and SynthEIA) that distinguish the closely related human T-cell lymphotropic virus types I (HTLV-I) and II (HTLV-II) were tested blindly for their ability to correctly identify infection caused by either virus type. Of 57 HTLV-I and 38 HTLV-II specimens from individuals whose infections were confirmed by polymerase chain reaction, the Select-HTLV assay categorized 56 (98%) as HTLV-I, 36 (95%) as HTLV-II, and 3 (3%) as nontypeable. Similarly, the SynthEIA assay categorized 54 (95%) as HTLV-I, 29 (76%) as HTLV-II, and 12 (13%) as nontypeable. More importantly, no specimen was wrongly classified by either assay (100% specificity). Further, analysis of serial specimens from six patients also demonstrated concordant results with the PCR findings. Our results suggest that serotyping of HTLV infections can be achieved reliably by these peptide assays, thus the need for technically complex PCR-based HTLV typing, while not eliminated, can be greatly reduced.

Base Sequence↗

PubMed1990

Suppressed peripheral blood lymphocyte blastogenesis in pre- and postpartal sheep by chronic heat-stress, and suppressive property of heat-stressed sheep serum on lymphocytes.

Phytohemagglutinin (PHA) and concanavalin A (Con A)-induced blastogenesis of peripheral blood lymphocytes was examined in heat-stressed pre- and postpartal sheep. The peak responses of lymphocytes to PHA and Con A in heat-stressed sheep revealed significant reduction before and after parturition compared with those in the corresponding control animals kept under thermoneutral conditions. Furthermore, the effect of serum from control or heat-stressed sheep on PHA-induced lymphocyte blastogenesis was examined. Supplementation of serum from heat-stressed sheep significantly suppressed the blastogenesis of lymphocytes obtained from healthy sheep, bovine, and human donors. Unlike dexamethasone, heat-stressed sheep serum did not inhibit IL-2 production by PHA-stimulated human peripheral blood lymphocytes. These results indicate that the immunosuppression of heat-stressed sheep is in part mediated by serum factor(s) that can modulate T-cell function in a species nonspecific manner.

Animals↗

PubMed1990

All-trans retinoic acid stimulates growth and extracellular matrix production in growth-inhibited cultured human skin fibroblasts.

All-trans retinoic acid was examined for effects on human dermal fibroblast proliferation and for effects on fibroblast production and expression of non-collagenous and collagenous components of the extracellular matrix in vitro. Fibroblast proliferation was blocked when the cells were cultured in the presence of a serum-free culture medium containing epidermal growth factor, hydrocortisone, insulin, ethanolamine, phosphoethanolamine, and bovine pituitary extract as growth supplements and 0.15 mM Ca++. This level of extracellular Ca++ is lower than that needed to support fibroblast growth. Under these conditions, growth was stimulated by all-trans retinoic acid. Proliferation was also stimulated in the same basal medium without the growth supplements. Growth-promoting concentrations of all-trans retinoic acid ranged from 0.5-2.0 micrograms/ml (1.7-6.6 X 10(-6) M). Stimulation of proliferation was not seen at higher or lower concentrations. Concentrations of all-trans retinoic acid that stimulated proliferation also induced increased production of fibronectin as indicated by biosynthetic labeling/immunoprecipitation and by enzyme-linked immunosorbent assay. Increased production was associated with increased staining for fibronectin in the extracellular matrix. Increased production of two other non-collagenous extracellular matrix component, i.e., thrombospondin and laminin, also occurred in all-trans retinoic acid-treated cells. At 0.5 micrograms/ml, all-trans retinoic acid also stimulated production of type I collagen by the dermal fibroblasts, but at higher concentrations (2.5 micrograms/ml) production of type I collagen was inhibited. These data indicate that all-trans retinoic acid can induce changes in dermal fibroblasts in vitro (i.e., increased proliferation and extracellular matrix production) that mimic the major changes seen in the dermis after topical treatment with this agent.

Cell Division↗

PubMed1989

Monocyte killing of human squamous epithelial cells: role for thrombospondin.

Human peripheral blood monocytes maintained in culture for 18 h were examined for killing of normal human keratinocytes and squamous carcinoma cells. Keratinocytes grown under conditions which maintain the undifferentiated state were highly sensitive to killing but these cells became resistant to killing after induction of differentiation. A line of squamous carcinoma cells obtained from an undifferentiated tumor (designated as UM-SCC-11B) was sensitive to killing while a second line obtained from a more well-differentiated tumor (designated as UM-SCC-22B) was resistant. Several observations suggested that interaction of monocytes with the squamous epithelial cells was mediated, in part, through thrombospondin (TSP). Monocytes synthesized TSP and were positive by immunofluorescence for surface TSP. The normal and malignant squamous epithelial cells also expressed surface TSP as well as unoccupied TSP receptors and our previous studies have shown that both TSP and its receptor are much more prominently displayed on the undifferentiated cells than on the differentiated cells. A series of anti-TSP monoclonal antibodies inhibited killing. These included an antibody directed against the Mr 25,000 NH2-terminal region of the molecule which has heparin-binding activity and three antibodies the epitopes of which lie within the Mr 140,000 non-heparin-binding fragment of TSP. High concentrations of exogenously added TSP as well as the recombinant form of the heparin-binding domain from the TSP molecule also partially inhibited killing while laminin and fibronectin were ineffective. Taken together, these data suggest that TSP and TSP receptors on monocytes and squamous epithelial cells play a role in monocyte-mediated killing of the squamous epithelial cells.

Antibodies, Monoclonal↗

PubMed1988

Efficient isolation and propagation of human immunodeficiency virus on recombinant colony-stimulating factor 1-treated monocytes.

Monocytes were maintained in tissue culture for greater than 3 mo in media supplemented with rCSF-1. These cultures provided susceptible target cells for isolation and propagation of virus from PBMC of HIV-infected patients. HIV isolated into monocytes readily infected other rCSF-1-treated monocytes but only inefficiently infected PHA-stimulated lymphoblasts. Similarly, laboratory HIV strains passaged in T cell lines or virus isolated from patients' leukocytes into PHA-stimulated lymphoblasts inefficiently infected rCSF-1-treated monocytes. Persistent, low-level virion production was detected in macrophage culture fluids by reverse transcriptase activity or HIV antigen capture through 6-7 wk. Marked changes in cell morphology with cell death, syncytia, and giant cell formation were observed in monocyte cultures 2 wk after infection, but at 4-6 wk, all cells appeared morphologically normal. However, the frequency of infected cells in these cultures at 6 wk was 60-90% as quantified by in situ hybridization with HIV RNA probes or by immunofluorescence with AIDS patients' sera. Ultrastructural analysis by EM also showed a high frequency of infected cells; virtually all HIV budded into and accumulated within cytoplasmic vacuoles and virus particles were only infrequently associated with the plasma membrane. Retention of virus within macrophages and the macrophage tropism of HIV variants may explain mechanisms of both virus persistence and dissemination during disease.

Cells, Cultured↗

PubMed1988

Lamicel is more effective in preparing the cervix for midtrimester termination of pregnancy than Laminaria japonicum.

Laminaria tents are often used prior to prostaglandin termination of midtrimester pregnancy to reduce the induction-abortion interval. Natural laminaria suffers from a number of disadvantages and, recently, a synthetic form Lamicel (Cabot Medical, PA), has been produced. Uncontrolled evaluation has suggested that it is effective for dilating the cervix but scepticism remains because Lamicel does not exert radial force on the cervical canal. One hundred women who requested abortion between 12 and 24 weeks of pregnancy were studied. They were randomly divided into group A who had a Lamicel inserted for 6 hours and group B who had a laminaria tent inserted for 8 hours. The cervical dilatation was assessed at the end of this period and the patients were then given up to 6 injections of sulprostone at 4-hourly intervals. All patients in Group A aborted, but 7 of those in Group B did not. The mean cervical dilatation in Group A was 7.47 +/- 1.46 mm compared with 4.71 +/- 1.76 mm in Group B (p less than 0.001). The mean induction-abortion interval in Group A was also significantly shorter (p less than 0.05) than that in Group B: 11.79 +/- 7.24 hours compared with 12.51 +/- 6.52 hours in Group B (excluding 7 patients who failed to abort in Group B). The results of the study suggest that the degree of cervical dilatation that can be achieved using Lamicel is significantly greater than that by laminaria tents.

Abortifacient Agents, Nonsteroidal↗

PubMed1987

Purification and characterization of variant alcohol dehydrogenase isozymes from durum wheat.

Three alcohol dehydrogenase (ADH) isozymes from embryos of the durum wheat cultivar Bijaga Yellow having the variant Adh-Alb allele were purified using (NH4)2SO4 precipitation, gel filtration, and ion-exchange chromatography. ADH is a dimeric enzyme. The variant isozyme ADH-1-1, which is a homodimer composed of alpha b monomers, was compared with ADH-1-5 (homodimer composed of beta a monomers), the product of Adh-B1, and the ADH-1-3 isozyme (alpha b beta a heterodimer) on a number of parameters including Km, substrate specificities, and molecular weights. No appreciable differences among the three isozymes were found, except for the faster electrophoretic mobility of alpha b alpha b dimers (ADH-1-1). The results indicate that the variant isozyme is the result of a mutation altering only the charge of the isozyme.

Alcohol Dehydrogenase↗

PubMed1987

Activation properties of the inward-rectifying potassium channel on mammalian heart cells.

The early phase of activation of the inward-rectifying potassium channel is studied on single cells from guinea-pig heart. The current is quasi-instantaneous when it is outward, but activates with time when it is inward. This relaxation is exponential and its time-constant decreases with hyperpolarization. The I/V curve reflects a strong inward rectification and has a negative slope conductance on depolarization. Similar results were recorded in the absence of sodium, calcium, chloride ions and in isotonic potassium. Cesium slows down the phase of activation, and eventually appears to block the channels by suppression of the activation. Barium, conversely, does not affect the activation, but promotes an 'inactivation' of this current, which blocks it. These results are independent on the cells' dissociation method. They suggest that this current is the inward rectifier, called IK1 on heart. Its activation curve suggests that the inward and outward currents are flowing through the same channels. The inward rectifier is time- and voltage-dependent on heart as on other tissues. The effects of cesium and barium are also similar. The importance of its negative slope conductance is discussed.

Animals↗

PubMed1986

Two types of calcium channels in guinea pig ventricular myocytes.

In cardiac muscle, Ca2+ plays a key role in regulation of numerous processes, including generation of the action potential and development of tension. The entry of Ca2+ into the cell is regulated primarily by voltage-gated channels in the membrane. Until recently, it was felt that only one type of Ca2+ channel existed in cardiac ventricular muscle. Experiments reported here suggest that in isolated guinea pig ventricular myocytes, there are two distinct types of Ca2+ channels with markedly different activation thresholds, inactivation kinetics, and sensitivities to inorganic and organic Ca2+ channel blockers. The channels were also distinguished based on their response to increased frequency of clamping such that the current through the low-threshold channel decreased while that through the high-threshold channel increased. In a few cells, the current through both channels was enhanced by isoproterenol, a beta-adrenergic agonist, but only the high-threshold channel was enhanced by the Ca2+-channel agonist Bay K 8644. Thus, isolated guinea pig ventricular myocytes appear to have two types of Ca2+ channels distinguished by various criteria.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1986

Lactic dehydrogenase isozyme distributions in lymphocytes from normal and leukemic mice.

LDH isozyme distributions were studied in the thymocytes of normal and spontaneously leukemic mice. In the normal animals, H:M ratios were found to be higher for more mature cells of adult animals than for less mature thymocytes of the neonates. However, thymocytes from leukemic animals bearing mature phenotype displayed very low values of H:M ratios. From these results, the relationship between the changes in LDH-isozyme distributions in AKR thymocytes and their progressive maturation appears to be equivocal. Alterations in isozyme distribution patterns, reflecting a decrease in H:M ratios, on the other hand, appears to be a characteristic feature of terminal stages of this murine leukemia.

Animals↗

PubMed1986

Interlaboratory standardization of glycated hemoglobin determinations.

As the clinical utility of glycated hemoglobin (gHb) measurement increases, so does the need for standardization of values between different methods and different laboratories. Using three different methods, we examined the feasibility of interlaboratory standardization of gHb measurement. A liquid-chromatographic (HPLC) system from our research laboratory was designated the reference method. For gHb standards we used erythrocyte hemolysates prepared from blood samples from nondiabetic and diabetic subjects. Values assigned to each standard were based on the mean of multiple gHb determinations by the HPLC method. A clinical laboratory routinely prepared hemolysates and assayed gHb by commercially available ion-exchange ("mini column") and affinity chromatographic methods. For each assay a standard curve was constructed and gHb values were derived from these curves. Samples analyzed in the clinical laboratory were also analyzed in the research laboratory and the curve-derived values were compared with the HPLC-measured values, to determine the accuracy of our interlaboratory standardization procedure. Correlations were excellent (r = 0.99). The lack of significant differences between calculated and HPLC-measured values indicates that interlaboratory standardization is feasible.

Chromatography, Affinity↗

PubMed1985

Ca2+ and Ca2+-activated K+ currents in mammalian gastric smooth muscle cells.

Inward movement of calcium through voltage-dependent channels in muscle is thought to initiate the action potential and trigger contraction. Calcium-activated potassium channels carry large outward potassium currents that may be responsible for membrane repolarization. Calcium and calcium-activated potassium currents were identified in enzymatically isolated mammalian gastric myocytes. These currents were blocked by cadmium and nifedipine but were not substantially affected by diltiazem or D600. No evidence for a tetrodotoxin-sensitive sodium current or an inwardly rectifying potassium current was found.

Acetylcholine↗

PubMed1985

A uniform enzymatic method for dissociation of myocytes from hearts and stomachs of vertebrates.

A method is presented that consistently yields a large number of calcium-tolerant myocytes from mammalian, amphibian, and elasmobranch hearts and from mammalian stomach. The use of incubating solutions or cell harvesting techniques was not required. The time needed to isolate cells was shorter than previously reported values. Action potentials recorded from each cell type appear similar in configuration to that of the intact multicellular tissue. The isolated myocytes appear to tolerate long periods of electrophysiological experimentation using the "giga-seal" suction electrode technique of Hamill et al. (Pfluegers Arch. 391: 85-100, 1981). This method is ideally suited for comparative electrophysiological studies, since the procedure for cell isolation was not seriously modified according to the preparation or species used.

Animals↗

PubMed1984

A simplified micro ELISA procedure for the measurement of platelet-associated IgG (PAIgG).

A simplified micro ELISA procedure to measure platelet-associated IgG is described. The platelet-bound IgG first is extracted into the fluid phase by solubilizing washed platelets in 0.1% triton X-100. The solubilized IgG in the extract and IgG standards are incubated in microtiter wells previously coated with antihuman IgG. The IgG in the standards and extract bind to the solid phase antihuman IgG. The bound IgG then is measured by the addition of peroxidase labeled antihuman IgG and appropriate substrate. With this method platelets from normal controls were found to have 1.7 +/- 0.6 fg IgG/platelet (mean +/- SD). Platelets from patients with ATP had values that were two to seven times the control values. The relative advantage of this technic is discussed.

Autoantibodies↗

PubMed1983

Transcription and translation in the autonomous parvovirus KRV.

We describe here the isolation and partial characterization of at least five viral specific RNAs synthesized in the rat nephroma (RN) cell after infection with the parvovirus KRV. The RNAs have the approximate lengths of 4.7, 3.4, 3.0, 1.25, and 0.95 kilobases (kb) and are probably the functional messages. The 4.7-kb RNA would represent a transcript of 95 to 100% of the viral genome. The most abundant message, about 3.0 kb, represents over 50% of the viral genome and probably codes in the reticulocyte transcribing system for the most abundant viral protein (MW 68,000) which is the main viral capsid protein. This abundant RNA, on the basis of R loop data, has an origin of transcription about 0.38 to 0.42 map units from the 3' end of the single-stranded KRV genome.

Animals↗

PubMed1982

Variations in the levels of estrogen receptors in prolactin producing pituitary tumor cells.

The binding of [3H]-17 beta-estradiol in cells and cytoplasmic fractions of three different prolactin producing pituitary tumor lines was compared and found to vary widely. The concentration of estrogen receptors of the MtTW10 rat tumor line was high in early passages, but receptor levels decreased with subsequent passages in animals. Over a period of 12 months, the estrogen binding capacity in low speed supernatant fractions of cell homogenates decreased from 61.5 fmol [3H]-17 beta-estradiol per mg protein to less than 10 fmol [3H]-17 beta-estradiol per mg protein. A similar decrease in receptor concentration was found in MtTW10 cells which were adapted to in vitro culture. The concentration of receptor in low speed supernatant fractions of cultured GH3 rat tumor cells remained between 60 and 80 fmol [3H]-17 beta-estradiol per mg protein throughout the entire period of experimentation. In contrast, no high affinity receptors could be detected in similar fractions of the human pituitary cell line, 18-54, when cultured either in the presence or absence of serum. The Kd for the estradiol:receptor complex was determined to be 1.0 x 10(-10) M for receptors from MtTW10 cells and remained constant as the concentration of receptors declined. The receptors in both MtTW10 and GH3 cells were found to exist as 8s molecular species which are converted to 4s species by a temperature dependent process. The binding of estrogen to 8s and 4s receptors at 4 degrees C was shown to occur in the presence of 3 mg/ml of digitonin.

Animals↗

PubMed1982

Synthesis of mRNA by the parvovirus KRV in isolated nuclei.

We have synthesized vial-specific transcripts in nuclei isolated from cells infected with the parvovirus Kilham rat virus. Radioactive vial-specific RNA synthesized in the nuclei was extracted, hybridized to viral DNA, incubated in glyoxal, and electrophoresed in an agarose gel. At least five viral-specific RNAs were detected containing 4.6 Kb (25-26S), 3.2 Kb (major species, 20-22S), 2.9 Kb, 1.3 Kb, and 1.0 Kb pairs. The 4.6 Kb RNA represents a transcript of 95-100% of the viral genome. The major 3.2 and 2.9 Kb RNAs represent a transcript of 50-60% of the viral genome. Over 65% of the viral-specific RNA in the isolated nuclei is polyadenylylated on the 3' terminus. The 5' terminus of the RNA is capped in vitro by the sequence m7G(5')ppp(5')A. Incorporation of [beta-32p]ATP into the 5' cap sequence suggests that initiation of viral RNA synthesis may occur in the infected isolated nuclei.

Cell Nucleus↗