Synchronous appearance of fibronectin,integrin α5β1, vinculin and actin in epithelial cells and fibroblasts during rat tracheal wound healing

The distribution of integrin 51 (51) and associated components during wound healing was investigated in the rat trachea following mechanical injury. Under anesthesia, the ventral surface of the trachea was scratched, and tissue specimens were obtained from 6 h to 3 weeks after injury and studied using light and electron microscopy and immunohistochemistry. 51, vinculin and actin in regenerating epithelial cells and extracellular fibronectin appear virtually simultaneously after injury (from 12 h to 7 days) as do 51, vinculin and -smooth muscle actin in fibroblasts and cellular fibronectin in granulation tissue (from 3 to 10 days). Immunoelectron microscopy 2 days after injury showed that 51 and vinculin were localized on the basal and lateral surfaces of regenerating epithelial cells and fibroblast surfaces, and fibronectin was localized just under the regenerating epithelial cells, around collagen fibrils and sporadically around fibroblasts. Bromodeoxyuridine labeling showed that the appearance of these components was associated with the period of cell proliferation. The appearances of fibronectin, 51, vinculin and actin in regenerating epithelial cells and fibroblasts during tracheal wound healing are well coordinated. During the initial cell migration phase, plasma fibronectin may stimulate cell migration before cellular fibronectin is produced in situ, and regenerating epithelial cells appear to begin to migrate into the wound before cell proliferation starts.     

Transport and utilization of α-ketoglutarate by the rat kidney in vivo

In order to establish the characteristics of net renal transport and utilization of -ketoglutarate (-KG) in the rat, we have precisely quantified the renal blood flow, the urinary flow and the rates of -KG delivery, filtration, reabsorption or secretion, excretion, uptake or production by an in vivo rat kidney preparation. In normal rats, -KG uptake was higher than -KG reabsorption at both endogenous and elevated plasma -KG concentrations; thus, a net peritubular transport, which was the main supplier of -KG to the renal cells, took place. Saturation of reabsorption and peritubular transport of -KG occurred at blood -KG concentrations about 30 and 150 times above normal, respectively. Acute metabolic acidosis was found to have no effect on renal handling of -KG. At endogenous plasma -KG concentrations, alkalosis converted net renal uptake into net renal production of -KG resulting in addition of -KG by the renal cells both to blood and to the luminal fluid. Elevation of blood -KG concentration restored the renal uptake of -KG. This uptake, which was entirely accounted for by the peritubular transport of -KG, reached a maximum which was lower than that observed in normal and acidotic rats.     

Relationship between tenascin and α-smooth muscle actin expression in the developing human small intestinal mucosa

The expression of tenascin (Tn) and -smooth muscle actin (-SMA) was analyzed in the developing and adult human small intestine by means of double immunofluorescent staining with specific antibodies. By 7 weeks of gestation, the gut anlage has a simple tubular shape and is formed of a stratified undifferentiated epithelium surrounded by a poorly organized mesenchyme. Both Tn and -SMA were found exclusively at the periphery of the tissue, corresponding to the presumptive muscularis propria. By 9 weeks, villus rudiments had formed but Tn and -SMA remained restricted to the muscularis propria. Tn was first detected in the mesenchyme at 11 weeks. By 13 weeks, a preferential distribution of Tn in the subepithelial region of the mesenchyme was readily observed while -SMA was still absent. From this stage to 20 weeks, Tn gradually concentrated in this region that, as determined by -SMA detection, corresponded to the future muscularis mucosa area. As shown by double staining of Tn and -SMA, deposition of Tn also preceded the appearance of the other -SMA-expressing cells in the mucosa. These observations suggest that Tn could have a role in the differentiation of intestinal contractile cells.     

Comparison of the eIF-2α Homologous Proteins of Seven Ranaviruses (Iridoviridae)

The -subunit of the eukaryotic initiation factor 2 (eIF-2) is a key component of the translation machinery of the cell. In response to cellular stress such as viral infections, eIF-2 is phosphorylated by double-stranded RNA-dependent protein kinase (PKR) leading to the inhibition of cellular protein synthesis. The importance of eIF-2 as a regulatory mechanism for protein synthesis is illustrated by the wide variety of strategies employed by viruses to down-regulate PKR. Thus, Vaccinia virus encodes K3L protein, which resembles eIF-2 and acts as a pseudo-substrate inhibitor of PKR. Nucleotide sequencing of the genome of epizootic haematopoietic necrosis virus (EHNV), a member of the genus ranavirus of Iridoviridae, has revealed an eIF-2 equivalent gene. We have cloned and sequenced eIF-2 genes of several iridoviruses of fishes and frogs. The eIF-2 open reading frames and deduced proteins of the iridoviruses investigated exhibit a high degree of homology of both nucleotide and amino acid sequences. At the N-terminus, the iridoviral eIF-2 shows significant homology to the N-termini of cellular initiation factor 2- of various species, to full-length poxviral eIF-2 proteins, and to the S1 domain of ribosomal proteins. Comparison of amino acid sequences of corresponding iridoviral proteins with eIF-2 homologous proteins of poxviruses and eukaryotes has revealed a high conservation of motifs. A phylogenetic analysis of eukaryotic eIF-2 and poxvirus and iridovirus eIF-2 sequences has demonstrated the relationship of these iridoviruses. In order to investigate the role of the eIF-2 equivalent, respective genes have been expressed in prokaryotic and eukaryotic (insect, fish and chicken cell) systems. The iridoviral eIF-2 protein has a molecular weight of 31 kDa and is cytoplasmic. The cellular and viral protein synthesis of iridoviruses is probably regulated by a mechanism similar to that of Vaccinia virus. Frog-virus 3, the type species of the genus ranavirus of Iridoviridae, has a unique translational efficiency and, moreover, down-regulates the cellular protein synthesis of infected cells.     

An improved method for the quantitation of cellular migration: Role of αvβ3 integrin in endothelial and smooth muscle cell migration

The present study was undertaken to develop a simple and improvedmethod for the accurate quantitation of cellular migration and to examinethe role of v3 integrins in different cellular migration. Usingour newly developed micro-volume chemotaxis assay, we developed an improvedquantitative method to measure in vitro chemotaxis of smooth muscle orendothelial cells toward different extracellular matrix proteins. Theconvenience in setup and counting of migrated cells using this methodallows for large capacity screening and for various research applicationswith other cells as well. The signal. to noise ratios were in the range of10/1, along with about 10–20% intra- or inter-assayvariabilities. Using this method, we have determined that eithervitronectin at 0.4 µg/well or osteopontin at 0.4 µg/well areselective v3 chemoattractants for endothelial or smooth musclecells (0.5 × 105 cells/well). Additionally, a selective v3small molecule peptiddomimetic, monoclonal antibody LM609, or an anti-3 (v3/II3) anti-body, c7E3 demonstratedmaximal inhibition of cellular migration toward vitronectin or osteopontin.These data suggest the potential utility of this method in assessing therole of various mechanisms in cellular migration and also suggests the potential implication of an v3 antagonist in blocking pathologicalprocesses involving endothelial or smooth muscle cell adhesion/migration.     

Defective NF-κB Signaling in Dedifferentiated Hepatoma Cells

Dedifferentiated rat hepatoma cells contain defects that result in the loss of hepatic gene expression, including the liver-enriched HNF4/HNF1 pathway. We examined induction of NF-B, a key mediator of the inflammatory response, in hepatoma and dedifferentiated hepatoma cells. We show that exposure of dedifferentiated hepatoma cells, but not rat and human hepatoma cell lines, to proinflammatory cytokines or lipopolysaccharide resulted in rapid and sustained NF-B induction. IB- levels, but not NF-B subunit p65 or IB- levels, were elevated compared with those for parental hepatoma cells. Interestingly, LPS-mediated activation of NF-B was found to be independent of degradation of IB- or IB-. Thus, these results suggest that loci responsible for maintaining hepatic gene expression also influence cellular responses to inflammatory agents.     

Neutrophil elastase α1-proteinase inhibitor complexes in pleural effusions

Summary Polymorphonuclear (PMN) granulocyte derived neutrophil elastase (NE) is rapidly antagonized by ₁-proteinase inhibitor (₁ PI) in vivo. To determine the clinical value of elastase ₁-proteinase inhibitor complexes (E-₁ PI) in pleural effusions, fluid samples of 99 patients were examined. Fifty-six had malignant effusions, 30 had nonmalignant exudates (pleural protein above 3 g/dl) mainly of inflammatory origin, and 13 patients had low protein transudates (below 3 g/dl) due to congestive heart failure. Nonmalignant exudates showed significantly higher (P<0.001) concentrations of E-₁ PI compared with malignant effusions or low protein transudates (P<0.001). Malignant exudates secondary to lung cancer were characterized by higher (P<0.001) median pleural E-₁ PI concentrations compared to malignant exudates due to primarily extrathoracic malignancies. Total pleural leukocyte counts and pleural neutrophil counts were performed in 68 effusions. By this means no clear-cut differentiation between malignant and nonmalignant exudates seems possible except for marked empyema. In conclusion, E-₁ PI complexes in pleural fluid may better reflect the stage of inflammation of pleural effusions rather than mere pleural leukocyte counts. Low levels of E-₁ PI complexes (<75 ng/ml) in pleural exudates with protein values above 3 g/dl are characteristic of malignant exudates. Determination of E-₁ PI in pleural exudates may serve as a sensitive marker of inflammation and useful adjunct to pleural cytology in aspects of differential diagnosis of pleural effusions.Abbreviations E-₁ PI elastase ₁ proteinase inhibitor complexes - NE neutrophil elastase - PMN polymorphonuclear     

Transient Exposure of Human Bronchial Epithelial Cells to Cytokines Leads to Persistent Increased Expression of ICAM-1

Effects of several cytokines on kinetics of Intercellular Adhesion Molecule-1 (ICAM-1) and Vascular Cell Adhesion Molecule-1 (VCAM-1) expression were studied on a bronchial epithelial cell line (BEAS-2B). VCAM-1 was neither constitutively expressed on BEAS-2B cells nor induced by Interferon-gamma (IFN-), Tumor Necrosis Factor-alpha (TNF-), Interleukin-1beta (IL-1), IFN-, IL-4, IL-6, IL-8 or Granulocyte Macrophage-Colony Stimulating Factor (GM-CSF). ICAM-1 was constitutively expressed on BEAS-2B cells. IFN- and TNF- upregulated ICAM-1 expression on these cells. The functional importance of IFN- plus TNF- upregulation of ICAM-1 expression on BEAS-2B cells was demonstrated by neutrophil-BEAS-2B cell adhesion assays. Cytokines are rapidly released and cleared in animals. Therefore, transient cytokine(s) exposure might occur on the bronchial mucosa. Brief incubation of BEAS-2B cells with IFN- plus TNF- led initial upregulation of ICAM-1 expression followed by a protracted downregulation. Our findings stress the importance of studying the mechanism(s) controlling the persistent increased expression of ICAM-1 after brief cytokine(s) exposure.     

Immunogenicity and thymus-dependence of polymerized Clostridium perfringensα-toxoid

By condensing the -toxoid ofClostridium perfringens type A with glutaraldehyde a polymer of the -toxoid with molecular weight 450,000–600,000 was obtained. Experments on guinea pigs showed that the immunogenicity of both the monomer and the polymer of the -toxoid, when used in the adsorbed form is practically identical. On immunization with unadsorbed antigens the primary response to the polymer was 3 times greater than the immune response to the monomer. Polymerization of the -toxoid did not change its thymus dependence.Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 84, No. 12, pp. 695–698, December, 1977.