Exercise-induced myocardial ischemia in young patients of rheumatic mitral stenosis.
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Adult↗
Biomedical subjects
B Mittal
Publications and source records attributed to B Mittal.
Listeria monocytogenes intracellular migration: inhibition by profilin, vitamin D-binding protein and DNase I.
Infection of host cells by Listeria monocytogenes results in the recruitment of cytoplasmic actin into a tail-like appendage that projects from one end of the bacterium. Each filamentous actin tail progressively lengthens, providing the force which drives the bacterium in a forward direction through the cytoplasm and later results in Listeria cell-to-cell spread. Host cell actin monomers are incorporated into the filamentous actin tail at a discrete site, the bacterial-actin tail interface. We have studied the consequences of microinjecting three different actin monomer-binding proteins on the actin tail assembly and Listeria intracellular movement. Introduction of high concentrations of profilin (estimated injected intracellular concentration 11-22 microM) into infected PtK2 cells causes a marked slowing of actin tail elongation and bacterial migration. Lower intracellular concentrations of two other injected higher affinity monomer-sequestering proteins, Vitamin D-binding protein (DBP; 1-2 microM) and DNase I (6-7 microM) completely block bacterial-induced actin assembly and bacterial migration. The onset of inhibition by each protein is gradual (10-20 min) indicating that the mechanisms by which these proteins interfere with Listeria-induced actin assembly are likely to be complex. To exclude the possibility that Listeria recruits preformed actin filaments to generate the tails and that these monomer-binding proteins act by depolymerizing such performed actin filaments, living infected cells have been injected with fluorescently labeled phalloidin (3 microM). Although the stress fibers are labeled, no fluorescent phalloidin is found in the tails of the moving bacteria. These results demonstrate that Listeria-induced actin assembly in PtK2 cells is the result of assembly of actin monomers into new filaments and that Listeria's ability to recruit polymerization competent monomeric actin is very sensitive to the introduction of exogenous actin monomer-binding proteins.
Actinin↗
A Ca2+/calmodulin kinase inhibitor, KN-62, inhibits neurite outgrowth stimulated by CAMs and FGF.
We have used monolayers of parental 3T3 fibroblasts and 3T3 cells expressing transfected cell adhesion molecules (CAMs, NCAM, N-cadherin, or L1) as a culture substrate for cerebellar neurons. Previous studies suggest that the transfected CAMs promote neurite outgrowth by activating a second messenger pathway within the responding neuron that involves influx of calcium into neurons as a consequence of activation of an FGF receptor. The same neurite outgrowth response can be induced by FGF or a number of agents that directly activate defined steps in the CAM signaling pathway. In the present study we show that the neurite outgrowth stimulated by the above three CAMs, FGF, arachidonic acid (AA), and K+ depolarization can be abolished by the Ca2+/calmodulin-dependent (CaM) kinase inhibitor, KN-62. We also demonstrate that neurite outgrowth over astrocytes, which represent a more physiologically relevant cellular substrate, can be substantially inhibited by a number of agents that block the CAM signaling pathway, including KN-62. However, neurite outgrowth induced by activation of protein kinase A is unaffected by inhibition of CaM kinase activity as is basal neurite outgrowth over 3T3 monolayers or a polylysine/laminin substrate. These results suggest that CaM kinase activity is specifically required downstream of calcium influx in the CAM and FGF signaling pathway leading to axonal growth.
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
Significance of subxiphoid (high epigastric) pulsation in patients with acute myocardial infarction.
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Echocardiography↗
Infraspinatus muscle hypertrophy and wasting of axillary folds as the important signs in Duchenne muscular dystrophy.
Eighty-four patients with Duchenne muscular dystrophy (DMD) were examined clinically for hypertrophy and wasting in different muscles, parts of the muscles or muscle groups. Some muscles were examined under mild contraction to bring out any subclinical pseudohypertrophy. Findings revealed infraspinatus muscle hypertrophy to be significantly frequent (88%) and closely second to well known calf hypertrophy (94%). Infraspinatus hypertrophy was noted in 5 such DMD patients in whom calf hypertrophy was unremarkable. The wasting was consistently observed in the muscles forming anterior and posterior axillary folds. In conclusion, infraspinatus muscle hypertrophy and wasting of axillary folds are the important supportive clinical evidences during the examination of DMD patients.
Adolescent↗
Induction chemotherapy followed by concomitant chemoradiotherapy for advanced head and neck cancer: impact on the natural history of the disease.
PURPOSE: To determine survival rates and the pattern of failure in head and neck cancer patients treated with induction chemotherapy, limited surgery and concomitant chemoradiotherapy. PATIENTS AND METHODS: Three cycles of induction chemotherapy with cisplatin, fluorouracil (5-FU), leucovorin, and interferon alfa-2b (PFL-IFN) were followed by optional surgery, and seven or eight cycles of 5-FU, hydroxyurea, and concurrent radiation for 5 days (FHX) for a total radiation dose of 65 to 75 Gy. Surgical resection was performed with the intent to spare organ function. RESULTS: Seventy-one patients were treated at three institutions. Sixty-five patients (91%) had stage IV disease with N2/3 in 46. Thirty-three patients (51%; 95% confidence interval, 39% to 63%) achieved a clinical complete response (CR) to PFL-IFN. Local therapy consisted of surgery in 37 and/or FHX in 55 patients. With a median follow-up duration of 37 months, there have been 20 recurrences (15 local, four distant, and one both local and distant), and 29 deaths, 15 in patients with disease progression and 14 not directly related to the primary tumor. Four patients have developed second malignancies. At 3 years, 69% (+/- 6%) are progression-free and the overall survival rate is 60% (+/- 6%). Toxicity of PFL-IFN included severe or life-threatening mucositis (54%) and myelosuppression (60%). Five patients died of toxicity. During FHX, 70% of patients had grade 3 or 4 mucositis. CONCLUSION: PFL-IFN is highly active, producing clinical CRs in 51% of patients, and, when followed by FHX, resulting in high local and distant control and overall survival rates. Second malignancies and intercurrent medical disease emerge as major risks to long-term survival. In view of the high toxicity and long treatment duration, further modifications of this approach are required.
Adolescent↗
FGF inhibits neurite outgrowth over monolayers of astrocytes and fibroblasts expressing transfected cell adhesion molecules.
We have cultured cerebellar neurons on monolayers of cortical astrocytes in control medium or medium containing recombinant basic fibroblast growth factor (FGF). FGF was found to inhibit neurite outgrowth, with a significant effect seen at 0.5 ng/ml and a maximal effect at 10 ng/ml. FGF increased the production of arachidonic acid (AA) in cerebellar neurons, and when added directly to cultures or generated endogenously via activation of phospholipase A2 using melittin, this second messenger could mimic the inhibitory effect of FGF. FGF and AA could also specifically inhibit neurite outgrowth stimulated by three cell adhesion molecules (NCAM, N-cadherin and L1) expressed in transfected fibroblasts, or in the case of L1 bound to a tissue culture substratum. These data demonstrate that, in certain cellular contexts, FGF can act as an inhibitory cue for axonal growth and that arachidonic acid is the second messenger responsible for this activity. We discuss the possibility that arachidonic acid inhibits neurite outgrowth by desensitising the second messenger pathway underlying neuronal responsiveness to cell adhesion molecules.
3T3 Cells↗
Evaluation of antibody responses in Indian kala-azar by immunoblot.
When infected with Leishmania species, patients develop specific antibodies that constitute the basis of serodiagnosis. using Western blot analysis we studied the specificity of anti-leishmania donovani antibodies in patients with visceral leishmaniasis, healthy subjects living in an endemic and non-endemic areas, and patients of other infectious diseases like malaria, leprosy, tuberculosis and tropical splenomegaly. Sera from patients with kala-azar recognised numerous antigens that had a molecular weight of 150 KD, 145 KD, 120 KD, 92 KD, 87 KD, 72 KD, 65 KD, 56 KD, 50 KD, 40 KD, 26 KD, 21 KD, 14 KD, AND 12 KD. The 150, 145, 120, 92, 87, 81, 65, 25, 21, 14, and 12 KD antigens had the greatest specificity for kala-azar sera while the bands of molecular weights 72, 56, 50, and 40 KD were found to be cross reactive with sera of patients of other diseases.
Animals↗
Immunocytochemical and functional characterization of an immortalized type 1 astrocytic cell line.
The DI TNC1 cell line has been derived from cultures of rat brain astrocytes by targeted oncogenesis. Cultured astrocytes are known to promote neurite outgrowth via the production of adhesion molecules found either on the cell surface or in the extracellular matrix. We sought to investigate whether DI TNC1 cells retained the ability to produce such neurite-inducing molecules, and promote neurite growth. We found by immunofluorescence that DI TNC1 cells expressed laminin, N-CAM and 1A1. The latter is a cell adhesion molecule that is expressed exclusively on astrocytes of the type 1 lineage. In vitro neurite outgrowth assays were also used to assess the functional properties of these cells. Monolayers of DI TNC1 cells were almost as effective a substrate as monolayers of astrocytes purified from the neonatal rat brain in their ability to support neurite outgrowth. In addition, PC12 cells grown on extracellular matrix derived from either DI TNC1 cells or neonatal astrocytes displayed significantly more neurite growth than cells plated on plastic. This effect was partially inhibited by preincubation of the extracellular matrix with anti-laminin antibodies. Taken together, these results suggest that the immortalized DI TNC1 cells show many similarities to neonatal astrocytes. Given the heterogeneity of cultured astrocytes, this homogeneous cell line may prove to be particularly useful for future investigations on interactions between glia and neurons.
Animals↗
Occurrence of fibers and their association with talin in the cleavage furrows of PtK2 cells.
PtK2 cells of exceptionally large size were microinjected with fluorescently labeled probes for actin, myosin, filamin, and talin in order to follow the assembly of the contractile proteins into the cleavage furrows. Whereas in cells of normal size, there is usually a diffuse pattern of localization of proteins in the cleavage furrow, in these large, flat cells the labeled proteins localized in fibers in the cleavage furrow. Often, the fibers were striated in a pattern comparable to that measured in the stress fibers of the same cell type. The presence of talin in discrete plaques along fibers in the cleavage furrows of the large cells suggests a further similarity between cleavage furrow and stress fiber structure. The presence of filamin in the cleavage furrows also suggests the possibility of an overlapping mechanism in addition to that of a talin mediated mechanism for the attachment of actin filaments to the cell surfaces in the cleavage furrow. A model is presented that emphasizes the interrelationships between stress fibers, myofibrils, and cleavage furrows.
Actin Cytoskeleton↗
A monoclonal antibody that recognizes an adhesion molecule expressed by certain cells of neuroectodermal and mesenchymal origin.
A cell surface glycoprotein has been identified from detergent extracts of cultured astrocytes obtained from neonatal rat cerebral cortex using a mouse monoclonal antibody (MAb 1A1). This antibody inhibits neuron-astrocyte and astrocyte-astrocyte adhesion, as well as neurite outgrowth on astrocytes in vitro. The MAb 1A1 does not bind to tissue sections, but by indirect immunofluorescence of dissociated CNS cultures, the antibody labels subpopulations of astrocytes (flat, type 1 astrocytes and Bergmann glia) and cells derived from the mesenchyme (leptomeninges and fibroblasts). The latter cells are labeled only when grown to confluency. The 1A1 cell surface glycoprotein appears as a single band of approximately 135 kDa on both reduced and nonreduced SDS-PAGE. Based on its unique cell-type distribution, functional properties and biochemical analysis, this 135-kDa protein appears to be distinct from other known adhesion molecules expressed on astrocytes. This molecule, thus, belongs to the growing list of cell adhesion molecules that may play a role in histogenesis and axonal growth during development of the mammalian CNS.
Animals↗
The role of an astrocyte surface molecule in neuronal migration in the developing rat cerebellum.
We have shown previously that the MAb 1A1 recognizes a cell surface glycoprotein with adhesive properties expressed exclusively on astrocytes in the rat central nervous system (CNS). In this study, the role of this molecule in neuronal migration in the developing rat cerebellum was investigated by antibody perturbation experiments. The MAb 1A1 binds to the surface of cultured radial Bergmann glia, a subclass of astrocytes known to guide the migration of postmitotic neurons by cell-cell contact, but does not bind to neurons in the cerebellum. The antibody does not bind to tissue sections for immunohistochemical studies. However, by immunoprecipitation the level of this antigen increases by about twofold between Postnatal Day 1 (P1) and P20 followed by a decrease to the P1 level at P35. In addition, the number of 1A1+ Bergmann glia also increases from 33 to 78% between P1 and P7 in dissociated cell cultures. Monovalent fragments of the MAb 1A1 inhibit the adhesion of neurons to Bergmann glia by about 50% in dissociated cultures of the P7 cerebellum. In addition, the migration of neurons on Bergmann glia was assessed in microexplant cultures of the P7 cerebellar cortex after 3 days in vitro. In these experiments the MAb 1A1 blocks the migration of neurons by 60%. These findings suggest that the 1A1 molecule might play a role in glial-guided neuronal migration in the developing cerebellum.
Animals↗
Expression of desmin cDNA in PtK2 cells results in assembly of desmin filaments from multiple sites throughout the cytoplasm.
The assembly of intermediate filaments into a cytoplasmic network was studied by microinjecting into the nuclei and cytoplasms of PtK2 cells, plasmids that contained a full length desmin cDNA and an RSV promoter. Immunofluorescence was used to monitor the expression of desmin and its integration into the cells' vimentin intermediate filament network. We found that the expressed desmin co-localized with filaments of vimentin just as it does with fluorescently labelled desmin is microinjected into the cytoplasm of PtK2 cells. As early as two hours after microinjection of the plasmids, small discrete dots and short fragments of desmin could be detected throughout the cytoplasm of the cells. This initial distribution of desmin was superimposed on the filamentous pattern of vimentin in the cells. At 8 hours after microinjection of the plasmids, some of the desmin was present in long filaments that were coincident with vimentin filaments. By 18 hours, most of the desmin was in a filamentous network co-localizing with vimentin. There was no indication that desmin assembly began in the perinuclear region and proceeded toward the cell periphery. In some cells, excessively high levels of desmin were expressed. In these cases, overexpression led to clumping of desmin filaments as well as to an accumulation of diffusely distributed desmin protein in the center of the cells. This effect was apparent at approximately 18 hours after introduction of the plasmid. The native vimentin filaments in such cells were also aggregated around the nucleus, co-localizing with desmin. The microtubule networks in all injected cells appeared normal; microtubules were extended in typical arrays out to the periphery of the cells.
Animals↗
Detection of gene deletion in patients of Duchenne muscular dystrophy/Becker muscular dystrophy using polymerase chain reaction.
Polymerase chain reaction (PCR) was used to study the presence of gene deletion (the most prominent type of mutations) in some families afflicted by Duchenne muscular dystrophy/Becker muscular dystrophy (DMD/BMD). The results clearly demonstrate deletion in the central part of the DMD gene in two of the three families studied. This information can be useful for genetic counselling with particular reference to prenatal diagnosis and carrier analysis.
Base Sequence↗
Disruption of microfilament organization in living nonmuscle cells by microinjection of plasma vitamin D-binding protein or DNase I.
Plasma vitamin D-binding protein (DBP), which binds to monomeric actin, causes the breakdown of stress fibers when it is microinjected into nonmuscle cells. Disruption of the stress fiber network is also accompanied by shape changes in the cell that resemble those seen after cytochalasin treatment. When DBP was coinjected with fluorescently labeled alpha-actinin, no fluorescent stress fibers or attachment plaques were visible 30 min after injection. Twelve hours later the cells regained their flattened shape and their stress fibers. Fluorescently labeled DBP causes the same reversible changes in cell shape as the unlabeled protein. Upon injection, the labeled DBP diffuses throughout the cytoplasm, becoming localized by 12 hr in a punctate pattern, presumably due to lysozomal sequestration. Similar injections of DBP into skeletal myotubes and cardiac myocytes did not lead to shape changes or breakdown of nascent and/or fully formed myofibrils, even though DBP has a 2-fold higher binding affinity for muscle actin over that of the nonmuscle isoactins. Similar differential effects in nonmuscle cells were also observed after the microinjection of DNase I, another protein capable of binding monomer actin. The effects of these microinjected monomer actin-binding proteins imply that an accessible pool of monomer actin is needed to maintain stress fiber integrity in nonmuscle cells but not the integrity of the nascent or fully formed myofibrils in muscle cells.
Actin Cytoskeleton↗
Use of fluorescently labeled probes to analyze cell division in living cells.
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Actins↗
Visualization of intermediate filaments in living cells using fluorescently labeled desmin.
Fluorescently labeled desmin was incorporated into intermediate filaments when microinjected into living tissue culture cells. The desmin, purified from chicken gizzard smooth muscle and labeled with the fluorescent dye iodoacetamido rhodamine, was capable of forming a network of 10-nm filaments in solution. The labeled protein associated specifically with the native vimentin filaments in permeabilized, unfixed interphase and mitotic PtK2 cells. The labeled desmin was microinjected into living, cultured embryonic skeletal myotubes, where it became incorporated in straight fibers aligned along the long axis of the myotubes. Upon exposure to nocodazole, microinjected myotubes exhibited wavy, fluorescent filament bundles around the muscle nuclei. In PtK2 cells, an epithelial cell line, injected desmin formed a filamentous network, which colocalized with the native vimentin intermediate filaments but not with the cytokeratin networks and microtubular arrays. Exposure of the injected cells to nocadazole or acrylamide caused the desmin network to collapse and form a perinuclear cap that was indistinguishable from vimentin caps in the same cells. During mitosis, labeled desmin filaments were excluded from the spindle area, forming a cage around it. The filaments were partitioned into two groups either during anaphase or at the completion of cytokinesis. In the former case, the perispindle desmin filaments appeared to be stretched into two parts by the elongating spindle. In the latter case, a continuous bundle of filaments extended along the length of the spindle and appeared to be pinched in two by the contracting cleavage furrow. In these cells, desmin filaments were present in the midbody where they gradually were removed as the desmin filament network became redistributed throughout the cytoplasm of the spreading daughter cells.
Animals↗
Dynamics of the endoplasmic reticulum in living non-muscle and muscle cells.
The dynamic changes of the endoplasmic reticulum (ER) in interphase and mitotic cells was detected by the vital fluorescent dye 3,3'-dihexyloxacarbocyanine iodide. Two types of arrays characterize the continuous ER system in the non-muscle PtK2 cell: 1) a lacy network of irregular polygons and 2) long strands of ER that are found aligned along stress fibers. In cross-striated myotubes there was a periodic localization of fluorescence over each I-band corresponding to the positions of the terminal cisternae of the sarcoplasmic reticulum (SR). In contrast to the arrangement in muscle cells, the alignment of the long strands of ER alon stress fibers showed no strict periodicity that could be correlated with the sarcomeric units of the stress fibers. The ER and SR arrays seen in living cells were also detected in fixed cells stained with antibodies directed against proteins of the endoplasmic reticulum and sarcoplasmic reticulum, respectively. Observations of vitally stained PtK2 cells at 1 to 2 minute intervals using low light level video cameras and image processing techniques enabled us to see the polygonal ER units form and undergo changes in their shapes. During cell division, the ER, rhodamine 123-stained mitochondria, and phagocytosed fluorescent beads were excluded from the mitotic spindle while soluble proteins were not. No obvious concentration or alignment of membranes could be found associated with the contractile proteins in the cleavage furrow. After completion of cell division there was a redeployment of the ER network in each daughter cell.
Animals↗