Unimpaired formation of hormone-stimulated inositol trisphosphate in human mesangial cells under hyperglycemic conditions.

The relationship between bulk cellular myo-inositol content and phosphatidylinositol metabolism was evaluated in a human mesangial cell line under euglycemic and hyperglycemic conditions. Mesangial cells maintained in high glucose medium displayed a concentration-dependent fall in myo-inositol as measured by gas-liquid chromatography. Measurements of phosphatidylinositol, phosphatidylinositol 4-monophosphate and phosphatidylinositol 4,5-bisphosphate mass revealed slight but statistically insignificant increases in cells exposed to high glucose containing medium. CDP-diacylglycerol: myo-inositol 3-phosphatidylinositol transferase activity, measured in plasma membranes from mesangial cells grown under control and hyperglycemic conditions, was kinetically similar with Michaelis constants (Km values) for myo-inositol of 2.9 and 2.1 mM, respectively. Finally, hormone-stimulated intracellular calcium mobilization and myo-inositol 1,4,5-trisphosphate mass was measured from mesangial cells grown under normal and hyperglycemic conditions. Both intracellular calcium and inositol trisphosphate formation were unchanged in cells previously exposed to high glucose conditions (400 mg/dl) compared to cells grown under normal glucose concentration (100 mg/dl). These data indicate that bulk changes in myo-inositol induced by hyperglycemia are neither associated with alterations in basal levels of inositol containing glycerolipids nor with changes in hormone-stimulated calcium mobilization and inositol trisphosphate formation under conditions of short term changes in extracellular glucose.     

Fractal analysis of protein chain conformation

This paper presents a simple practical method for characterizing conformation of protein chains. A single number Df, as the fractal dimension, is assigned to each chain. Df = Ln(N)/Ln(N.d/L), where N is the number of the amino acid residues in the chain, L and d are the total length and the planar diameter of the chain, respectively. In general, 1 less than Df less than or equal to 2, which is related to the shape of the protein chain. These values are different from those of Stapleton's group, but in agreement with computer simulations.     

Preliminary study of the distribution of the toxic elements As,Cd, and Hg in human hair and tissues by RNAA

In order to study the relationships between trace element concentrations of hair and internal body burdens, a radiochemical NAA technique has been used for determination of the elements As, Cd, and Hg in autopsy samples of liver, kidney-cortex, lung, and hair from 24 male persons who died by accident. High significant positive correlations were observed between the As concentration in hair and in kidney-cortex, and between Cd and Zn concentrations in kidney-cortex. The contents of Cd, both for lung and kidney-cortex, were related to the smoking habits of the subjects.

     

Role of viral antigens in destructive cellular immune responses to adenovirus vector-transduced cells in mouse lungs.

Adenoviruses missing E1 have been used as gene delivery vectors to the lungs for the treatment of cystic fibrosis. Transient expression of the recombinant gene and the development of inflammation have been two major limitations to the application of first-generation recombinant adenoviruses for gene therapy. Studies with mouse models of liver- and lung-directed gene therapy suggested that CD8+ cytotoxic T lymphocytes (CTLs) are effectors that contribute to extinction of transgene expression. The precise antigens responsible for activation of CTLs have not been identified. In this study, we examine the relative contributions of viral proteins versus the transgene product to the activation of CTLs which eliminate transgene-containing cells in mouse lungs. Instillation of a lacZ-expressing virus into the lungs of C57BL/6 mice elicited CTL responses to both viral proteins and the transgene product, beta-galactosidase, which collectively contribute to loss of trans-gene expression in mouse airways. Similar results were obtained in two experimental models in which the animals should be tolerant to the transgene, i.e., lacZ virus delivered to an animal transgenic for lacZ and a virus expressing the liver-specific enzyme ornithine transcarbamylase administered to the lungs of various strains of immune-competent mice. These data confirm the hypothesis that CTLs specific for viral antigens contribute to the problem of transgene instability in mouse lungs and indicate that CTLs specific for transgene product alone cannot account for the observed problem.     

Overexpressions of cDNAs for β-Amyloid Precursor Proteins 695, 751, and 770 Enhance the Secretion of β-Amyloid Precursor Protein Derivatives and the Survival of P19-Derived Neurons

Abstract: P19 is a C3H mouse-derived line of multipotent embryonic carcinoma cells that differentiate into neural cells. P19 cell clones overexpressing the three major forms of β-amyloid precursor protein from their cDNA constructs were established. Unlike a previous study in which P19-derived neurons had a limited α-secretase activity, all of these clones produced significant amounts of secreted β-amyloid precursor protein. When treated with retinoic acid, these transformed lines differentiated into neurons and survived better than did nontransformed parental P19 cells. Furthermore, P19-derived neurons survived better in medium conditioned by the transformed P19 line, and survival was reduced by immunoabsorption with an antibody to β-amyloid precursor protein. These results suggest neurotrophic effects of secreted β-amyloid precursor protein and contrast with a previous report in which overexpression of a full-length cDNA for β-amyloid precursor protein led to degeneration of P19-derived neurons. Western blot analysis suggested that this difference might result from different levels of expression of putative neurotoxic C-terminal fragments of β-amyloid precursor protein; moreover, P19-derived neurons differ from P19 stem cells in the processing of these C-terminal fragments.     

Inflammatory infiltration of the trigeminal ganglion after herpes simplex virus type 1 corneal infection.

Following herpes simplex virus type 1 (HSV-1) infection of the cornea, the virus is transmitted to the trigeminal ganglion, where a brief period of virus replication is followed by establishment of a latent infection in neurons. A possible role of the immune system in regulating virus replication and maintaining latency in the sensory neurons has been suggested. We have investigated the phenotype and cytokine pattern of cells that infiltrate the A/J mouse trigeminal ganglion at various times after HSV-1 corneal infection. HSV antigen expression in the trigeminal ganglion (indicative of the viral lytic cycle) increased until day 3 postinfection (p.i.) and then diminished to undetectable levels by day 7 p.i. The period of declining HSV antigen expression. was associated with a marked increase in Mac-1+ cells. These cells did not appear to coexpress the F4/80+ (macrophage) or the CD8+ (T cell) markers, and none showed polymorphonuclear leukocyte morphology, suggesting a possible early infiltration of natural killer cells. There was also a significant increase in the trigeminal ganglion of cells expressing the gamma delta T-cell receptor, and these cells were found almost exclusively in very close association with neurons. This period was also characterized by a rapid and equivalent increase in cells expressing gamma interferon and interleukin-4. The density of the inflammatory infiltrate in the trigeminal ganglion increased until days 12 to 21 p.i., when it was predominated by CD8+, Mac-1+, and tumor necrosis factor-expressing cells, which surrounded many neurons. By day 92 p.i., the inflammatory infiltrate diminished but was heaviest in mice with active periocular skin disease. Our data are consistent with the notion that gamma interferon produced by natural killer cells and/or gamma delta T cells may play an important role in limiting HSV-1 replication in the trigeminal ganglion during the acute stage of infection. In addition, tumor necrosis factor produced by CD8+ T cells and macrophages may function to maintain the virus in a latent state.     

The Epstein-Barr virus-encoded nuclear antigen EBNA-5 accumulates in PML-containing bodies.

EBNA-5 is one of the Epstein-Barr virus (EBV)-encoded nuclear proteins required for immortalization of human B lymphocytes. In the nuclei of EBV-transformed lymphoblastoid cell lines EBNA-5 is preferentially targetted to distinct nuclear foci. Previously we have shown (W.Q. Jiang, L. Szekely, V. Wendel-Hansen, N. Ringertz, G. Klein, and A. Rosen, Exp. Cell Res. 197:314-318, 1991) that the same foci also contained the retinoblastoma (Rb) protein. Using a similar double immunofluorescence technique, we now show that these foci colocalize with nuclear bodies positive for PML, the promyelocytic leukemia-associated protein. Artificial spreading of the chromatin by exposure to the forces of fluid surface tension disrupts this colocalization gradually, suggesting that the bodies consist of at least two subcomponents. Heat shock or metabolic stress induced by high cell density leads to the release of EBNA-5 from the PML-positive nuclear bodies and induces it to translocate to the nucleoli. In addition to their presence in nuclear bodies, both proteins are occasionally present in nuclear aggregates and doughnut-like structures in which PML is concentrated in an outer shell. Nuclear bodies with prominent PML staining are seen in resting B lymphocytes. This staining pattern does not change upon EBV infection. In freshly infected cells EBNA-5 antigens are first distributed throughout the nucleoplasm. After a few days intensely staining foci develop. These foci coincide with PML-positive nuclear bodies. At a later stage and in established lymphoblastoid cell lines EBNA-5 is almost exclusively present in the PML-positive nuclear foci. The colocalization is restricted to EBV-infected human lymphoblasts. The data presented indicate that the distinct EBNA-5 foci are not newly formed structures but the result of translocation of the viral protein to a specialized domain present already in the nuclei of uninfected cells.     

Physiological changes and alk gene instability in Pseudomonas oleovorans during induction and expression of alk genes.

The alk genes of Pseudomonas oleovorans, which is able to metabolize alkanes and alkenes, are organized in alkST and alkBFGHJKL clusters, in which the expression of alkBFGHJKL is positively regulated by AlkS. Growth of the wild-type strain GPo1 and P. oleovorans GPo12 alk recombinants on octane resulted in changes of cellular physiology and morphology. These changes, which included lower growth rates and a reduction of the number of CFU due to filamentation, were also seen when the cells were grown on aqueous medium, and the alk genes were induced with dicyclopropylketone, a gratuitous inducer of the alk genes. These effects were seen only for recombinants carrying both alkST and alkBFGHJKL operons. Deletion of parts of either alkB or alkJ, which encode two major Alk proteins located in the cytoplasmic membrane, modified but did not eliminate the effects described above, suggesting that they were due to induction and expression of several alk genes. Continuous growth of the cells in the presence of dicyclopropylketone for about 10 generations led to inactivation, but not elimination, of the alk genes. This resulted in a return of the recombinants to normal physiology and growth.