Variational calculations for semiconductor superlattices and multilayer systems.
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Journal Article↗
Biomedical subjects
T Chakraborty
Publications and source records attributed to T Chakraborty.
The ActA protein of Listeria monocytogenes acts as a nucleator inducing reorganization of the actin cytoskeleton.
Listeria monocytogenes, a facultative intracellular pathogen, employs actin and other microfilament-associated proteins to move through the host cell cytoplasm. Isogenic mutants of L. monocytogenes lacking the surface-bound ActA polypeptide no longer interact with cytoskeletal elements and are, as a consequence, non-motile (Domann et al., 1992, EMBO J., 11, 1981-1990; Kocks et al., 1992, Cell, 68, 521-531). To investigate the interaction of ActA with the microfilament system in the absence of other bacterial factors, the listerial actA gene was expressed in eukaryotic cells. Immunofluorescence studies revealed that the complete ActA, including its C-terminally located bacterial membrane anchor, colocalized with mitochondria in transfected cells. When targeted to mitochondria, the ActA polypeptide recruited actin and alpha-actinin to these cellular organelles with concomitant reorganization of the microfilament system. Removal of the internal proline-rich repeat region of ActA completely abrogated interaction with cytoskeletal components. Our results identify the ActA polypeptide as a nucleator of the actin cytoskeleton and provide the first insights into the molecular nature of such controlling elements in microfilament organization.
Actinin↗
Naturally occurring virulence-attenuated isolates of Listeria monocytogenes capable of inducing long term protection against infection by virulent strains of homologous and heterologous serotypes.
Experimental infections of mice with strains of Listeria spp. isolated from contaminated food sources allowed discrimination of strains into those either exhibiting high, attenuated or low virulence. Compared to the highly virulent L. monocytogenes strain EGD, an attenuated strain such as L99 persisted for shorter times (5 versus 10 days) in the infected host. Using a tissue culture cell model of infection, we found that, although strain L99 was capable of accumulating actin like its virulent counterpart following invasion, it was unable to generate the polarized actin tails required for intracellular and cell-to-cell movement. Immunoblot analysis using specific antiserum to the ActA polypeptide, a molecule that is necessary for movement of the bacterium within the eucaryotic cell, indicated that a slightly truncated form of this polypeptide was produced in the L99 strain. Despite its reduced virulence, the attenuated strain L99 was just as effective in generating protection in immune mice as the highly virulent strains, albeit with a 1000-fold higher infective dose. Based on the results obtained from this study, we suggest that one of the mechanisms accounting for widespread resistance in humans to infection by Listeria may be due to asymptomatic infections by naturally occurring strains attenuated for virulence.
Animals↗
Mammalian cells transfected with the listeriolysin gene exhibit enhanced proliferation and focus formation.
Mouse 3T6 and 3T3 fibroblasts and rat epithelial L2 cells were transfected with recombinant plasmids containing the listeriolysin gene (hly) of Listeria monocytogenes. This bacterial gene (with and without the 5' signal sequence) was cloned under the control of a murine metallothionein promoter, resulting in elevated transcription of both forms of the hly gene after induction with ZnSO4. However, the gene product could be observed only when the listeriolysin gene lacking the 5' signal sequence was used. Intact listeriolysin could not be detected in the cytoplasm or in the supernatant of the hly-transfected cells. 3T6 and L2 cells transfected with the intact hly gene exhibited significantly increased cell proliferation and increased formation of actin microfilaments upon induction of hly expression with ZnSO4. Both cell types are not contact inhibited and formed large piles of spherical cells after transfection with hly. In contrast, contact-inhibited 3T3 cells transfected with the hly gene showed increased proliferation but no formation of such cell aggregates. When 3T6 fibroblasts were transfected with the hly gene without the 5' signal sequence, inhibition of growth, lack of cell layer confluency, and altered (spherical) cell morphology were observed.
3T3 Cells↗
Exploitation of microfilament proteins by Listeria monocytogenes: microvillus-like composition of the comet tails and vectorial spreading in polarized epithelial sheets.
Effective cell-to-cell spreading of the facultative intracellular pathogen Listeria monocytogenes requires the interaction between bacteria and the microfilament system of the host cell. By recruiting actin filaments into a 'comet tail' localized at one pole of the bacterial cell wall, Listeria become mobile and propel themselves through the cytoplasm. They create protrusions at the plasma membrane that can invaginate adjacent cells. In this work, we have analysed the structural composition of Listeria-recruited microfilaments in various epithelial cell lines by immunofluorescence microscopy. The microfilament-crosslinking proteins alpha-actinin, fimbrin and villin were localized around bacteria as soon as actin filaments could be detected on the bacterial surface. Surprisingly, the same was found for ezrin/radixin, proteins involved in linking microfilaments to the plasma membrane. We found that in a polarized cell line derived from brush border kidney epithelium (LLC-PK1), the actin filaments surrounding intracytoplasmic motile bacteria show the same immunoreactivity as the brush border-like microvilli, when analysed by a specific actin antibody. The successful invasion of polarized LLC-PK1 islets is vectorial, i.e. it progresses predominantly from the periphery of the islets towards the centre. Infection of the peripheral cells is sufficient for infiltration of the entire cellular islets, without any further contact with the extracellular milieu. This is in contrast to nonpolarized epithelial sheets, which can be invaded from the apical surface of any individual cell. The importance of active bacterial motility in this vectorial spreading is emphasized by our finding that an isogenic Listeria mutant that is unable to recruit actin filaments cannot colonize polarized epithelial layers but accumulates in the peripheral cells of the islets.
Actins↗
Human cyclic GMP-dependent protein kinase I beta overexpression increases phosphorylation of an endogenous focal contact-associated vasodilator-stimulated phosphoprotein without altering the thrombin-evoked calcium response.
The role of the cGMP-dependent protein kinase (cGK) and one of its major substrates, the vasodilator-stimulated phosphoprotein (VASP), in the regulation of a receptor-evoked calcium response was investigated. The human type I beta cGK was stably transfected in human embryonic kidney 293 cells and Swiss mouse 3T6 fibroblasts, which contained significant or no detectable levels of the focal adhesion protein VASP, respectively. Western blot analysis and protein kinase activity measurements demonstrated an 8-fold overexpression of cGK-I beta in 293 cells (7-fold in 3T6 cells), representing an intracellular cGK concentration of 0.33 microM. In experiments with intact 293 cells expressing cGK-I beta, beta-phenyl-1,N2-etheno-cGMP and 8-(p-chlorophenylthio)-cGMP were capable of converting up to 30-40% of the 46-kDa VASP to its 50-kDa phospho- form, equivalent to results observed with cGMP analogs that cause a marked inhibition of the stimulated Ca2+ transient in intact human platelets. In contrast to platelets, preincubation of fura-2-loaded 293 and 3T6 cells with 8-(p-chlorophenylthio)-cGMP did not significantly inhibit thrombin-evoked calcium transients, although sufficient cGK-mediated VASP phosphorylation was clearly detectable under these conditions in cGK-I beta-expressing 293 cells. These results demonstrate that cGK inhibition of agonist-evoked calcium mobilization is not a mechanism common to all cell types and that VASP phosphorylation may not be an essential or sufficient component of the cGK effect on calcium levels. In contrast, the observed VASP phosphorylation mediated by recombinant human cGK-I beta in intact 293 cells does support the hypothesis that focal adhesions and their associated proteins are important cellular sites of cGK action.
Blood Proteins↗
Collective excitations, pressure, and compressibility in multilayer systems.
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Journal Article↗
Effect of dexamethasone on the peptic activity of gastric lumen and mucosa.
Dexamethasone (9 alpha-fluoro-16 alpha methyl-11 beta,17 alpha,21-trihydroxy-1,4-pregnadiene-3,20-dione-21-phosphate), a synthetic glucocorticoid, has a dual role on pepsinogen content of the gastric lumen and mucosa as measured by its peptic activity. Following stimulation the luminal peptic activity gradually decreases after 6 hr, then returns to basal levels at 18 hr and by 24 hr is inhibited by 50%. The luminal peptic activity induced by the secretory compound mercaptomethylimidazole (MMI) is also decreased. Dexamethasone effect on both basal and MMI-induced peptic activity can be reproduced by cycloheximide or puromycin, translational blockers of protein synthesis. This drug also has an independent time and dose-dependent inhibitory effect on gastric mucosal peptic activity which does not correlate with increased peptic activity of the lumen. Dexamethasone appears to be more effective than hydrocortisone and corticosterone in inhibiting the basal peptic activity of both lumen and mucosa. The inhibitory effect of this drug on tissue peptic activity is not mediated through induction of any inhibitory protein as evidenced by the insensitivity of the effect to actinomycin D. Studies on [14C]phenylalanine incorporation into gastric protein indicate that the effect of dexamethasone on tissue pepsinogen content is not due to a generalized block of protein synthesis.
Animals↗
Listeria monocytogenes--a model system for studying the pathomechanisms of an intracellular microorganism.
Virulence of Listeria monocytogenes is determined by a cluster of five genes in the order plcA, hly, mpl, actA and plcB, which are coordinately regulated by a transcriptional activator, termed PrfA. The gene for PrfA is located in front of plcA. Mutations within each of these genes reduce the virulence considerably and render the mutants unable to properly multiply and/or spread within the infected host cells. Under growth-limiting conditions PrfA-dependent proteins are preferentially synthesised. These studies indicate the existence of additional PrfA-regulated proteins in L. monocytogenes. The synthesis of catalase, superoxide dismutase, LmaA and p60 is not under the control of PrfA. These proteins seem to be also associated with virulence of L. monocytogenes. P60-related proteins are found as major extracellular proteins in all Listeria species but only p60 of L. monocytogenes is able to restore the failure of R-mutants (exhibiting a drastically reduced synthesis of p60) to adhere to 3T6 mouse fibroblasts. Adherence of L. monocytogenes to the epithelial Caco-2 cells seem to be independent of p60. The p60 protein of L. monocytogenes differs characteristically from the p60-related proteins of the nonvirulent Listeria species.
Amino Acid Sequence↗
Localization of the ActA polypeptide of Listeria monocytogenes in infected tissue culture cell lines: ActA is not associated with actin "comets".
The ActA protein of the gram-positive pathogen Listeria monocytogenes is a 90-kDa polypeptide required for interaction of the bacteria with components of the host cell microfilament system to generate intra- and intercellular movement. To study the localization, distribution, and expression of the ActA polypeptide in L. monocytogenes grown either in broth culture or in infected tissue culture cells, we first isolated ActA by monoclonal antibody-based immunoaffinity chromatography. Polyclonal rabbit antisera raised against purified ActA revealed that ActA was associated with the cell wall and exposed on the surface of the bacteria, readily accessible to ActA antibodies. In contrast, a C-terminally truncated ActA1 polypeptide expressed by the isogenic actA1 mutant was detected only in the supernatant fluids. Immunofluorescence microscopy and electron microscopic studies using immunogold labeling showed that ActA was present on the surface of the bacteria infecting PtK2 and J774 cells at all stages of the infection cycle and was not found to be associated with the actin "tail" of individual bacteria. For the isogenic actA1 mutant strain, which grew as microcolonies within infected cells, only diffuse staining of the secreted ActA1 polypeptide in the host cytoplasm was observed. The ActA polypeptide therefore appears to be required in the initiation of actin accumulation by the bacterium and is apparently not directly involved in the generation of the actin tail. Analysis of strains of several L. monocytogenes serotypes indicated microheterogeneity in the molecular weights of the ActA polypeptides of individual strains and led to the detection of a serotype 3a strain that does not produce ActA.
Animals↗
Detection of a prfA-independent promoter responsible for listeriolysin gene expression in mutant Listeria monocytogenes strains lacking the PrfA regulator.
Expression of listeriolysin, a major virulence factor of pathogenic Listeria monocytogenes, is positively regulated by the pleiotropic virulence regulator PrfA. In this study, we demonstrate that L. monocytogenes strains lacking the prfA regulator gene produce listeriolysin in small, albeit detectable, amounts when analyzed in a hemolysin assay and by immunoblots with listeriolysin-specific monoclonal antibodies. Transcriptional analysis revealed the existence of a PrfA-independent promoter that was responsible for the hemolytic activity expressed by these strains.
Bacterial Proteins↗
Maternal deaths--a ten-year study.
In a 10-year retrospective study of maternal deaths at RG Kar Medical College, Calcutta there were 651 maternal deaths with 410 in first 5-year period and 241 in second 5-year period. The leading causes of deaths were due to abortion, toxaemia, haemorrhage (antepartum and postpartum) and jaundice in pregnancy. Direct causes were responsible for death in 77.6% cases, indirect causes in 17.5% cases and unrelated causes in 4.9% cases. Majority (557) were unbooked and mostly multigravida (434) cases. Most of them (251) were in the age group of 20-25 years. Haemorrhage was the single most common cause to claim 188 lives. It was also noted that 155 deaths occurred within 12 hours of hospital admission.
Adult↗
Analysis of the oligomerization of myogenin and E2A products in vivo using a two-hybrid assay system.
Members of the helix-loop-helix (HLH) family of proteins bind DNA and activate transcription as homo- and heterodimers. Myogenin is a muscle-specific HLH protein that binds DNA in vitro as a heterodimer with several widely expressed HLH proteins, such as the E2A gene products E12 and E47. We describe a method for detection of protein-protein interactions among HLH proteins in vivo in which dimerization through the HLH motif reconstructs a hybrid transcription factor containing the DNA-binding domain of yeast GAL4 linked to one HLH motif and the activation domain of VP-16 linked to another. We have used this assay to investiagate whether myogenin forms homomeric or heteromeric complexes in vivo and to determine whether growth factors and oncogenes that inhibit myogenesis influence myogenin's ability to dimerize. The results show that myogenin heterodimerizes with E12 and E47 in vivo, but it does not homodimerize to a measurable extent. Peptide growth factors, as well as the immediate early gene products c-Jun, v-Fos, and c-Myc, inhibit the activity of myogenin through a mechanism independent of its association with E2A products.
Adenovirus Early Proteins↗
Excitons in a parabolic quantum dot in magnetic fields.
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Journal Article↗
Effect of electron-electron interactions on the magnetization of quantum dots.
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Journal Article↗
A novel bacterial virulence gene in Listeria monocytogenes required for host cell microfilament interaction with homology to the proline-rich region of vinculin.
The ability of Listeria monocytogenes to move within the cytosol of infected cells and their ability to infect adjacent cells is important in the development of infection foci leading to systemic disease. Interaction with the host cell microfilament system, particularly actin, appears to be the basis for propelling the bacteria through the host cell cytoplasm to generate the membraneous protrusions whereby cell-to-cell spread occurs. The actA locus of L.monocytogenes encodes a 90 kDa polypeptide that is a key component of bacterium-host cell microfilament interactions. Cloning of the actA gene allowed the identification of its gene product and permitted construction of an isogenic mutant strain defective in the production of the ActA polypeptide. Sequencing of the region encoding the actA gene revealed that it was located region encoding the actA gene revealed that it was located between the metalloprotease (mpl) and phosphatidylcholine-specific phospholipase C (plcB) genes. Within the cytoplasm of the infected cells, the mutant strain grew as microcolonies, was unable to accumulate actin following escape from the phagocytic compartment and was incapable of infecting adjacent cells. It was also dramatically less virulent, demonstrating that the capacity to move intracellularly and spread intercellularly is a key determinant of L.monocytogenes virulence. Like all other virulence factors described for this microorganism, expression of the ActA polypeptide is controlled by the PrfA regulator protein. The primary sequence of this protein appeared to be unique with no extended homology to known protein sequences. However, an internal repeat sequence showed strong regional homology to a sequence from within the hinge region of the cytoskeletal protein vinculin.
Actin Cytoskeleton↗
Protective immunity and granulomatous inflammation is mediated in vivo by T cells reactive to epitopes common to avirulent and listeriolysin-negative mutants of Listeria monocytogenes.
The ability of several listeriolysin O-negative mutants of the EGD and NCTC 7973 strains of Listeria monocytogenes to activate specific T cell responses in vitro and in vivo was determined. T cell lines from different inbred mouse strains and derived T cell clones elicited by L. monocytogenes, strain EGD, which are able to adoptively transfer protection and granuloma formation were examined. Specificity testing revealed no differences between listeriolysin-positive and -negative strains to induce proliferation of the T cell lines and clones. Similar results were obtained when we examined CD4+ T cell-mediated granuloma formation in the livers of mice previously immunized with viable bacteria of the virulent strain. Granulomatous inflammation could be elicited by iv application of heat-killed bacteria of listeriolysin-positive and of -negative bacteria. Protective immunity to listerial infections and granulomatous inflammation therefore appears to be mediated by T cells recognizing epitopes on listerial antigens that are shared by both pathogenic and nonpathogenic Listeria strains.
Animals↗
Molecular studies on the aerolysin gene of Aeromonas species and discovery of a species-specific probe for Aeromonas trota species nova.
A large group of aeromonads and other enteric microorganisms were assayed for the presence of the aerolysin gene with use of DNA-DNA hybridization. Two DNA fragments corresponding to the regulatory region (aerC) and the structural gene (aerA) were used as probes for the detection of the aerolysin gene in these strains. Sequences corresponding to the aerolysin structural gene were widespread among Aeromonas isolates. In contrast, the aerC probe was much more selective, and sequences corresponding to the aerC region were detected in only a small subset of strains. Concurrent studies using numerical taxonomy and DNA hybridization with the aerC probe on a larger set of strains led to the identification of a distinct cluster of 14 presumed atypical Aeromonas sobria strains. These strains have recently been grouped into a new species designated Aeromonas trota. Hence, the DNA fragment aerC used in the study is a species-specific gene probe for A. trota. The ability of the aerC probe to detect strains belonging to a single species suggests that there is selection pressure to maintain the clonality of this species. These results have important implications with respect to the evolution of "pathogenic profiles" among these medically important bacteria.
Aeromonas↗