Plasma levels and gene expression of granulocyte colony-stimulating factor, tumor necrosis factor-alpha, interleukin (IL)-1beta, IL-6, IL-8, and soluble intercellular adhesion molecule-1 in neonatal early onset sepsis

We have previously reported that T lymphocytes proliferating in vitro to the hapten trinitrochlorobenzene (TNCB) exhibit a very restricted V beta gene usage and response to TNCB is limited to T-cell receptors (TCR) composed of V beta 8.2 in combination with V alpha 3.2, V alpha 8 and V alpha 10. This paper investigates the role played by T lymphocytes expressing the V beta 8.2 gene segment in the contact sensitivity (CS) reaction to TNCB in the intact mouse and in its passive transfer into naive recipient mice. Mice injected with monoclonal antibodies to V beta 8 are unable to develop CS upon immunization with TNCB and 4-day TNCB-immune lymph node cells from mice that had been depleted in vivo or in vitro of V beta 8+ T lymphocytes fail to transfer CS. However, when separated V beta 8+ and V beta 8- cells were used for passive transfer, it was found that V beta 8+ T lymphocytes failed to transfer CS when given alone to recipient mice and a V beta 8- population was absolutely required. Further analysis revealed that within the V beta 8- population, T lymphocytes expressing the gamma delta TCR were fundamental to allow transfer of the CS reaction. These gamma delta cells were found to be antigen non-specific, genetically unrestricted and to rearrange the V gamma 3 gene segment. This indicates that transfer of the CS reaction requires cross-talk between V beta 8+ and gamma delta+ T lymphocytes, thus confirming our previous results obtained using TNCB-specific T-cell lines. Time-course experiments showed that V beta 8+ lymphocytes taken 4-24 days after immunization with TNCB were able to proliferate and produce interleukin-2 (IL-2) in response to the specific antigen in vitro. Similar time-course experiments were then undertaken using the passive transfer of the CS reaction system. The results obtained confirm that TNCB-specific V beta 8+ T lymphocytes are present in the lymph nodes of immunized mice from day 4 to day 24, and reveal that gamma delta+ T lymphocytes are active for a very short period of time, i.e. days 4 and 5 after immunization. In fact, TNCB-specific V beta 8+ cells are able to transfer CS when taken 4-24 days after immunization, providing the accompanying V beta 8- or gamma delta+ T lymphocyte are obtained 4 days after immunization. In contrast, injection of V beta 8+ T lymphocytes together with V beta 8- or gamma delta+ T lymphocytes that had been taken 2 or 6 days after immunization, failed to transfer significant CS into recipient mice. Taken together, our results confirm that cross-talk between V beta 8+ and gamma delta+ T lymphocytes is necessary for full development of the CS reaction and may explain why the CS reaction in the intact mouse lasts up to 21 days after immunization while the ability of immune lymph node cells to transfer CS is limited to days 4 and 5 after immunization.     

Membrane association of FtsY, the E. coli SRP receptor

If a peptide hormone secreted from the gastroenteropancreatic (GEP) system is monitored by the hepatic vagal nerve, the nerve can signal the central nervous system and thereby exert control on its target organs. In this review, we offer a line of evidence for the hypothesis. When a physiological dose of somatostatin (SS), one of the GEP hormones, was injected into the rat portal vein, the spike discharge rate in the hepatic afferent vagus increased significantly. This SS-induced activation of the vagus was completely abolished by a prior administration of our monoclonal antibody to SS receptor into the portal vein. We further disclosed a morphological basis for this neural reception to SS in the hepatoportal area: the neural bodies, located beneath the endothelium of the rat portal vein, preferentially bound the exogenous SS injected intraportally as revealed immunohistologically. The bodies contained a structure of the nerve fiber arborizations resembling those of the afferent apparatus of Krause, on which the presence of SS receptor was confirmed histochemically using the anti-SS receptor antibody. These results provide a new insight into the receptor-mediated neural reception to GEP hormones in the hepatoportal area, implying the potential role of the reception in the GEP physiology.     

Evidence for cooperation between TCR V region and junctional sequences in determining a dominant cytotoxic T lymphocyte response to herpes simplex virus glycoprotein B

TCR repertoire availability has the potential to influence the immune response to foreign antigens. Here we have analysed how changes in V region availability influence the H-2b-restricted cytotoxic T lymphocyte (CTL) response to a dominant peptide determinant derived from the herpes simplex virus glycoprotein B (gB). We have previously shown that C57BL/6 mice mount a gB-specific, Kb-restricted CTL response which is dominated by a TCRBV10+ population and a TCRBV8S1+ subpopulation, both containing highly conserved CDR3 elements. We find that this dominant gB-specific CTL pool is lost in C57/L mice which have a different TCRBV haplotype. A population of CTL with diverse TCRBV and junctional sequence usage, which otherwise represents a minor subset in the gB-specific response, appears to emerge as a consequence of this TCRBV gene variation. The loss of preferential V region-encoded complementarity determining regions (CDR) 1- and/or CDR2-ligand interactions in this emerging population also results in a change in CDR3 sequence usage and a corresponding focusing of an otherwise promiscuous pattern of cross-reactivity with a panel of gB498-505 substitution analogues. This suggests that the difference between the two distinct TCR populations is the relative contributions of the CDR towards ligand recognition. Therefore, preferential V region-ligand interaction, at the expense of CDR3 peptide recognition, appears to control the dominant TCR selection in the C57BL/6 response to this peptide determinant.     

RNA polymerase III interferes with Ty3 integration

Many cytological processes such as cell proliferation, differentiation, transformation, apoptosis, etc., are accompanied by specific chromatin changes, usually identified on the basis of the relative content of euchromatin and heterochromatin. In order to achieve a quantitative, non-subjective evaluation of the chromatin pattern, two different approaches may be undertaken, one consisting in the analysis of the several morphological features of chromatin grains (size, shape, density, arrangement, and distribution), and the second consisting in the analysis of the chromatin globally considered as a coherent texture. Although the second approach appears to be simpler and more suitable, methods of texture analysis--including those specifically designed for the analysis of the chromatin pattern--are rarely applied due mainly to the unsuitability of sampling procedures and the excessive crypticism of results. As an alternative to traditional texture analysis, we suggest a method supported by a sound mathematical theory and approximately 30 years of applications in the field of geostatistics. The method, called variogram, analyzes the intrinsic structure of data sampled at different distance intervals and directions, and outputs easily understandable results. Recently, variogram analysis has successfully been exported from geostatistics to other fields (for example, ecology and epidemiology) that make use of spatially referenced variables. Based on the fact that pixels represent a perfect array of data ordered at regular distance intervals and directions, the variogram can be adopted to explore nuclear images and recognize chromatin patterns. Variograms of different nuclei can be summarized by multivariate methods without the need of previous standardization of data. This allows comparison and discrimination of chromatin patterns from mixed cell populations. Preliminary data obtained from young neurons undergoing massive apoptosis reveal a self-consistent map of nuclear changes correlated to the degenerative process.     

Susceptibility of human proximal tubular cells to hypoxia: effect of hypoxic preconditioning and comparison to glomerular cells

Three elements are crucial for the programmed frameshifting in translation of dnaX mRNA: a Shine-Dalgarno (SD)-like sequence, a double-shift site, and a 3' structure. The conformation of the mRNA containing these three elements was investigated using chemical and enzymatic probes. The probing data show that the structure is a specific stem-loop. The bottom half of the stem is more stable than the top half of the stem. The function of the stem-loop was further investigated by mutagenic analysis. Reducing the stability of the bottom half of the stem strongly effects frameshifting levels, whereas similar changes in the top half are not as effective. Stabilizing the top half of the stem gives increased frameshifting beyond the WT efficiency. The identity of the primary RNA sequence in the stem-loop is unimportant, provided that the overall structure is maintained. The calculated stabilities of the variant stem-loop structures correlate with frameshifting efficiency. The SD-interaction and the stem-loop element act independently to increase frameshifting in dnaX.     

Glutathione synthetase is dispensable for growth under both normal and oxidative stress conditions in the yeast Saccharomyces cerevisiae due to an accumulation of the dipeptide gamma-glutamylcysteine

Glutathione (GSH) synthetase (Gsh2) catalyzes the ATP-dependent synthesis of GSH from gamma-glutamylcysteine (gamma-Glu-Cys) and glycine. GSH2, encoding the Saccharomyces cerevisiae enzyme, was isolated and used to construct strains that either lack or overproduce Gsh2. The identity of GSH2 was confirmed by the following criteria: 1) the predicted Gsh2 protein shared 37-39% identity and 58-60% similarity with GSH synthetases from other eukaryotes, 2) increased gene dosage of GSH2 resulted in elevated Gsh2 enzyme activity, 3) a strain deleted for GSH2 was dependent on exogenous GSH for wild-type growth rates, and 4) the gsh2 mutant lacked GSH and accumulated the dipeptide gamma-Glu-Cys intermediate in GSH biosynthesis. Overexpression of GSH2 had no effect on cellular GSH levels, whereas overexpression of GSH1, encoding the enzyme for the first step in GSH biosynthesis, lead to an approximately twofold increase in GSH levels, consistent with Gsh1 catalyzing the rate-limiting step in GSH biosynthesis. In contrast to a strain deleted for GSH1, which lacks both GSH and gamma-Glu-Cys, the strain deleted for GSH2 was found to be unaffected in mitochondrial function as well as resistance to oxidative stress induced by hydrogen peroxide, tert-butyl hydroperoxide, and the superoxide anion. Furthermore, gamma-Glu-Cys was at least as good as GSH in protecting yeast cells against an oxidant challenge, providing the first evidence that gamma-Glu-Cys can act as an antioxidant and substitute for GSH in a eukaryotic cell. However, the dipeptide could not fully substitute for the essential function of GSH in the cell as shown by the poor growth of the gsh2 mutant on minimal medium. We suggest that this function may be the detoxification of harmful intermediates that are generated during normal cellular metabolism.