Biomedical subjects

A Topilko

Publications and source records attributed to A Topilko.

At least 19 recordsLinked to original sources

PubMed2000

Intimate adhesion of Neisseria meningitidis to human epithelial cells is under the control of the crgA gene, a novel LysR-type transcriptional regulator.

PilC1, a pilus-associated protein in Neisseria menin- gitidis, is a key element in initial meningococcal adhesion to target cells. A promoter element (CREN, contact regulatory element of Neisseria) is responsible for the transient induction of this gene upon cell contact. crgA (contact-regulated gene A) encodes a transcriptional regulator whose expression is also induced upon cell contact from a promoter region similar to the CREN of pilC1. CrgA shows significant sequence homologies to LysR-type transcriptional regulators. Its inactivation in meningococci provokes a dramatic reduction in bacterial adhesion to epithelial cells. Moreover, this mutant is unable to undergo intimate adhesion to epithelial cells or to provoke effacing of microvilli on infected cells. Purified CrgA is able to bind to pilC1 and crgA promoters, and CrgA seems to repress the expression of pilC1 and crgA. Our results support a dynamic model of bacteria-cell interaction involving a network of regulators acting in cascade. CrgA could be an intermediate regulator in such a network.

Bacterial Adhesion↗

PubMed2000

Upregulation by glucocorticoids of responses to eosinopoietic cytokines in bone-marrow from normal and allergic mice.

Since the production of eosinopoietic cytokines (GM-CSF, IL-3, IL-5) is inhibited by glucocorticoids, while responsiveness to these cytokines is enhanced in bone-marrow of allergic mice, we studied the ability of glucocorticoids to modulate murine bone-marrow eosinopoiesis. Progenitor (semi-solid) and/or precursor (liquid) cultures were established from bone-marrow of: (a) normal mice; (b) ovalbumin-sensitized and challenged mice or (c) dexamethasone (1-5 mg kg(-1)) injected mice. Cultures were established with GM-CSF (2 ng ml(-1)) or IL-5 (1 ng ml(-1)), respectively, alone or associated with dexamethasone, hydrocortisone or corticosterone. Total myeloid colony numbers, frequency and size of eosinophil colonies, and numbers of eosinophil-peroxidase-positive cells were determined at day 7. In BALB/c mice, dexamethasone (10(-7) M) increased GM-CSF-stimulated myeloid colony formation (P = 0.01), as well as the frequency (P=0.01) and size (P<0.01) of eosinophil colonies. Dexamethasone (10(-7) M) alone had no effect. Dexamethasone (10(-7)-10(-10) M) increased (P<0.002) eosinophil precursor responses to IL-5. Potentiation by dexamethasone was still detectable: (a) on low density, immature, nonadherent BALB/c bone-marrow cells, (b) on bone-marrow from other strains, and (c) on cells from allergic mice. Hydrocortisone and corticosterone had similar effects. Dexamethasone administered in vivo, 24 h before bone-marrow harvest, increased subsequent progenitor responses to GM-CSF (P = 0.001) and precursor responses to IL-5 (P<0.001). These effects were blocked by RU 486 (20 mg kg(-1), orally, 2 h before dexamethasone, or added in vitro at 10 microM, P<0.001). Glucocorticoids, acting in vivo or in vitro, through glucocorticoid receptors, enhance bone-marrow eosinopoiesis in naïve and allergic mice.

Administration, Intranasal↗

PubMed1999

Entry of human cytomegalovirus into retinal pigment epithelial and endothelial cells by endocytosis.

PURPOSE: Human retinal pigment epithelial (RPE) cells and endothelial cells (HUVECs) are targets of human cytomegalovirus (HCMV) infection in vivo with significantly protracted replication in vitro compared with that in fibroblasts. This study analyzes the kinetics and mechanisms of HCMV entry into both cell types. METHODS: RPE cells were obtained from donor eyes. HUVECs were isolated from human umbilical cords. HCMV entrance was followed by electron microscopy and immunofluorescence in the presence of lysosomotropic agents and cytochalasin B. RESULTS: Human cytomegalovirus entered into RPE cells and HUVECs as early as 5 minutes after virus- cell contact. Entry was mediated by endocytosis, whereas HCMV enters fibroblasts through fusion. Most internalized viral particles and dense bodies appeared to be degraded within vacuoles. Viral entry, transport of viral proteins to the nucleus, and onset of viral transcription (immediate early [IE] protein expression) were significantly blocked by cytochalasin B. Lysosomotropic agents did not significantly reduce IE expression in RPE cells or HUVECs. CONCLUSIONS: This study shows that HCMV penetrates these highly specialized relevant cells via endocytosis. The low level of infection and the delay in the onset of HCMV expression seen in these cells compared with fibroblasts may be related to the sequestration and degradation of incoming viral particles in endocytic vacuoles.

Ammonium Chloride↗

PubMed1997

Activation of the envelope proteins by a metalloproteinase enables attachment and entry of the hepatitis B virus into T-lymphocyte.

Previously, we identified an HBV binding factor (HBV-BF), a 50-kDa serum glycoprotein which interacts with HBV envelope proteins and which is also located in the membrane of normal human hepatocyte (A. Budkowska et al. (1993) J. Virol. 67, 4316). Here we show that HBV-BF is a neutral metalloproteinase which shares substrate specificity and properties with a newly described family of membrane type matrix metalloproteinases. HBV-BF treatment of the HBV resulted in the cleavage of the N-terminal part of the middle HBV envelope protein at the pre-S2(136-141) amino acid sequence VRGLYF/L (containing a single arginine cleavage site). HBV-BF affected the reactivity of the large HBV protein with pre-S1-specific MAbs, probably inducing the conformational change of the pre-S1 domain. The HBV-BF-digested virus remained morphologically intact with unchanged S antigenic determinants. The structural modifications of the viral envelope proteins induced by HBV-BF enabled cell membrane attachment and viral entry into the T-lymphocyte. Both processes were blocked by the metalloproteinase inhibitor 1,10 phenanthroline. Thus, the host-dependent proteolytic activation of the envelope proteins seems to be essential for the HBV entry into the cell. HBV-BF under a membrane bound or a secreted form could be (one of) the molecule(s) responsible for the HBV proteolytic activation.

Amino Acid Sequence↗

PubMed1996

Human cytomegalovirus carries serine/threonine protein phosphatases PP1 and a host-cell derived PP2A.

Human cytomegalovirus (CMV), a herpesvirus, is an important cause of morbidity and mortality in immunocompromised patients. When studying hyper-immediate-early events after contact between CMV virions and the cell membrane, we observed a hypophosphorylation of cellular proteins within 10 min. This can be explained in part by our finding that purified CMV contains serine/threonine protein phosphatase activities. Biochemical analyses indicate that this protein phosphatase activity has all characteristics of type 1 and 2A protein phosphatases (PP1 and PP2A). Specifically, PP1 accounts for approximately 30% and PP2A accounts for the remaining 70% of the phosphorylase phosphatase activity found. CMV produced in astrocytoma cells stably expressing an amino-terminally tagged PP2A catalytic subunit contained tagged enzyme, thus demonstrating the cellular origin of CMV-associated PP2A. PP2A is specifically found inside the virus, associated with the nucleocapsid fraction. Western blot (immunoblot) analysis of purified virus revealed the presence of the catalytic subunits of PP2A and PP1. Furthermore, the catalytic subunit of PP2A appears to be complexed to the regulatory subunits PR65 and PR55, which is also the most abundant configuration of this enzyme found in the host cells. Incubation of virus with okadaic acid before contact of CMV with cells prevented hypophosphorylation of cellular proteins, thus demonstrating the role of CMV-associated phosphatases in this phenomenon. CMV can thus transport an active enzyme from one cell to another.

Amino Acid Sequence↗

PubMed1996

Stably expressed antisense RNA to cytomegalovirus UL83 inhibits viral replication.

The human cytomegalovirus (HCMV) open reading frame UL83 encodes a phosphoprotein of 64 to 68kDa (pp65) which is a major constituent of this virion and dense bodies. To determine the importance of the HCMV gene in the virus cycle, we studied HCMV replication in astrocytoma cells stably transfected with a retroviral vector carrying an antisense UL83 cDNA. Reverse transcription-PCR detected antisense RNA in the cytoplasm. The steady-state level of a 4-kb RNA containing coding sequences for pp65 was significantly reduced after infection of antisense cells. Concomitant with this, levels of expression of pp65 and pp71 (UL82) were severely reduced. Extracellular HCMV production was almost completely blocked, irrespective of the multiplicity of infection or the time after infection studied. The block occurred at an early phase, since immediate-early protein synthesis occurred normally, while several late proteins (e.g., pp150 [ppUL32] and assembly protein [UL80]) were absent or strongly inhibited. Normal replication of herpes simplex virus and of a pp65 deletion mutant of HCMV (RVAd65), lacking target sequences of antisense RNA, demonstrated the specificity of the block for wild-type HCMV in the antisense-stabilized cells and indicated that the block was not due to indirect interference with cellular genes. Our results appear to contradict those of Schmolke et al (S. Schmolke, H.F. Kern, P. Drescher, G. Jahn, and B. Plachter, J. Virol. 69:5959-5968, 1995), which show that UL83 is a nonessential gene for HCMV replication in vitro. This contradiction is discussed in light of the fact that the 4-kb mRNA, which codes for pp65 and was targeted in UL83-antisense cell lines, may be a bicistronic mRNA which also codes for pp71 (UL82). Thus, interference of expression from the genes encoding pp65 and pp71 by blocking of this putative bicistronic message leads to severe impairment of viral replication.

Antiviral Agents↗

PubMed1996

Fusion of Leishmania amazonensis parasitophorous vacuoles with phagosomes containing zymosan particles: cinemicrographic and ultrastructural observations.

Studies on fixed preparations have shown that vacuoles containing zymosan (Z) particles internalized by infected macrophages can selectively fuse with the large parasitophorous vacuoles (PVs) that shelter Leishmania amazonensis. To examine the kinetics of vacuolar fusion in individual cells, particles were followed by time-lapse cinemicrography from their uptake to their entry in a PV. Newly formed Z-containing vacuoles moved centripetally and, if they contacted a PV, the two vacuoles remained closely apposed for variable, often extended, periods of time before they eventually fused. Transmission electron microscopy confirmed that the cytoplasm separating the partner vacuoles could be reduced to a very thin layer. Initiation of fusion was indicated by reduced refractility of the boundary between Z vacuoles and target PVs. Within a few minutes the PV enlarged and encompassed the Z particles, which remained immobile throughout. The interval between phagocytosis and fusion, 50 +/- 7.4 min (N = 17; range, 4 to 108 min), suggests that most but not all Z vacuoles underwent significant maturation by the time of fusion. Some particles were transferred singly, others entered PVs in groups of 2 or more, and additional clustered transfers to the same vacuole were also observed. These observations provide a baseline for studies of the biochemical mechanisms and the pharmacological control of the fusion of Leishmania PVs, and for the comparison of the fusion behavior of the PVs with that of other phagocytically derived vacuoles.

Animals↗

PubMed1988

Acetylcholinesterase and butyrylcholinesterase activities in human thyroid cancer cells.

The appearance and distribution of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in 12 human thyroid cancers and three normal thyroids were examined by electron microscopic study with indirect thiocholine method. The demonstration of AChE and BuChE activities in only two of nine cases of follicular and papillary carcinoma examined and none of the three cases of medullary carcinoma shows that the cholinesterases are not specific enzymes for the thyroid tumors. In normal thyroid tissue samples examined, no activities of AChE and BuChE were detected. On ultrastructural level AChE reaction product was revealed in the perinuclear space, in the endoplasmic reticulum, and in the Golgi complex of some but not in all cells in less-differentiated regions of the tumors. In contrast to the distribution of AChE, no staining for BuChE was noted in the Golgi elements. Ultrastructural localization of AChE activity in the thyroid cancer cells corresponds exactly to the current understanding of glycoproteins synthesis and processing in normal cells. The authors postulate that the copy of AChE gene suppressed in normal thyroid epithelium cells may be expressed in some follicular thyroid carcinoma cells. Their hypothesis is logical on the basis of recent finding of a significant homology between AChE and thyroglobulin.

Acetylcholinesterase↗

PubMed1985

Fine structural localization of acetylcholinesterase activity in rat submandibular gland.

Using the indirect thiocholine method, the ultrastructural localization of acetylcholinesterase (AChE) activity in the normal rat submandibular gland was studied. Cytochemical demonstration of AChE is based on coupling the hydrolysis of acetylthiocholine iodide to the precipitation of heavy metal salts. AChE-associated reaction product was selectively revealed in the perinuclear space and in the endoplasmic reticulum of the intercalated duct cells, in some cells of granular convoluted tubules, and in the striated duct epithelium, as well as in the myoepithelial cells. Although AChE activity generally occurred inside the cells, electron-dense precipitates were shown in intercellular space and in the stroma of the gland. Fine localization of AChE activity was also found in nerve bundles, predominantly between axons and between axons and Schwann cell. Our observations indicate that AChE is synthesized in the epithelium of the ducts and in the myoepithelial cells of the salivary gland. It is not known yet whether this enzyme is released from the intracytoplasmic membrane system into the extracellular space and then transported to the regions of the gland innervation. Conceivably AChE synthesized in the submandibular gland cells could also be considered an inhibitory modulator of the regulatory functions of biologically active polypeptides.

Acetylcholinesterase↗

PubMed1985

Acetylcholinesterase in human thymus cells.

Acetylcholinesterase (AChE) was long thought to be an enzyme found specifically at the sites of nerve synapses and neuromuscular junctions. It has also been found to occur, however, in cells that are not involved with neurotransmission. This study presents the ultrastructural localization of AChE activity in human thymus cells, using the indirect thiocholine method. Cytochemical demonstration of the enzyme was based on the coupling of acetylthiocholine iodide hydrolysis to the precipitation of heavy metal salts. AChE activity was selectively revealed in the perinuclear cisternae, within the endoplasmic reticulum, and in the Golgi complex of thymic lymphocytes and epithelial cells. Evidence of the presence of reaction product in the latter cells was also found in vesicles that opened into the extracellular space. This is the first demonstration of AChE in human thymus cells. Its possible physiologic role in the thymus gland is discussed.

Acetylcholinesterase↗

PubMed1984

Ultrastructural cytochemistry of intranuclear dense granules in nasopharyngeal angiofibroma.

Electron-dense round granules (ING) were found in the nuclei of fibroblastic cells of all the tumors examined by us (6 cases) and diagnosed clinically and histologically as nasopharyngeal angiofibroma (NPAF). The ING were consistently associated with this tumor and can be regarded as pathognomonic of NPAF. We studied at the ultrastructural level the composition of these particles using various cytochemical techniques. Enzymatic digestions carried out on thin sections as well as staining methods preferential or specific for nucleoproteins have revealed that ING represent tightly bound RNA-protein complexes and do not contain DNA. There are no data available that the ING takes part in the metabolism of NPAF fibroblastic cells.

Adolescent↗

PubMed1974