Regulators of G protein signaling (RGS proteins): novel central nervous system drug targets.

Many drugs of abuse signal through receptors that couple to G proteins (GPCRs), so the factors that control GPCR signaling are likely to be important to the understanding of drug abuse. Contributions by the recently identified protein family, regulators of G protein signaling (RGS) to the control of GPCR function are just beginning to be understood. RGS proteins can accelerate the deactivation of G proteins by 1000-fold and in cell systems they profoundly inhibit signaling by many receptors, including mu-opioid receptors. Coupled with the known dynamic regulation of RGS protein expression and function, they are of obvious interest in understanding tolerance and dependence mechanisms. Furthermore, drugs that could inhibit their activity could be useful in preventing the development of or in treating drug dependence.     

Molecular evolution of olfactomedin

Olfactomedin is a secreted polymeric glycoprotein of unknown function, originally discovered at the mucociliary surface of the amphibian olfactory neuroepithelium and subsequently found throughout the mammalian brain. As a first step toward elucidating the function of olfactomedin, its phylogenetic history was examined to identify conserved structural motifs. Such conserved motifs may have functional significance and provide targets for future mutagenesis studies aimed at establishing the function of this protein. Previous studies revealed 33% amino acid sequence identity between rat and frog olfactomedins in their carboxyl terminal segments. Further analysis, however, reveals more extensive homologies throughout the molecule. Despite significant sequence divergence, cysteines essential for homopolymer formation such as the CXC motif near the amino terminus are conserved, as is the characteristic glycosylation pattern, suggesting that these posttranslational modifications are essential for function. Furthermore, evolutionary analysis of a region of 53 amino acids of fish, frog, rat, mouse, and human olfactomedins indicates that an ancestral olfactomedin gene arose before the evolution of terrestrial vertebrates and evolved independently in teleost, amphibian, and mammalian lineages. Indeed, a distant olfactomedin homolog was identified in Caenorhabditis elegans. Although the amino acid sequence of this invertebrate protein is longer and highly divergent compared with its vertebrate homologs, the protein from C. elegans shows remarkable similarities in terms of conserved motifs and posttranslational modification sites. Six universally conserved motifs were identified, and five of these are clustered in the carboxyl terminal half of the protein. Sequence comparisons indicate that evolution of the N-terminal half of the molecule involved extensive insertions and deletions; the C-terminal segment evolved mostly through point mutations, at least during vertebrate evolution. The widespread occurrence of olfactomedin among vertebrates and invertebrates underscores the notion that this protein has a function of universal importance. Furthermore, extensive modification of its N-terminal half and the acquisition of a C-terminal SDEL endoplasmic-reticulum- targeting sequence may have enabled olfactomedin to adopt new functions in the mammalian central nervous system.      

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.     

ANALYSIS OF SPATIAL DISTRIBUTIONS OF CELLULAR MOLECULES DURING MECHANICAL STRESSING OF CELL SURFACE RECEPTORS USING CONFOCAL MICROSCOPY

Confocal laser scanning microscopy represents a suitable technique to study the localization of cellular components in three dimension. The authors used this technique to analyse cellular events related to mechanical stimulation of integrin receptors on the cell surface. By performing optical sections the distribution of integrin receptors on the apical surface of an osteoblastic cell was determined. Concerning intracellular compartimentalization of signal transduction events, it was demonstrated that mechanical stimulation of integrins induced their linkage to the cytoskeleton. Cytoskeletally associated proteins like vinculin and talin accumulated in the vicinity of the site where the mechanical stress was applied to integrins on the cell surface. Optical sections revealed that clustering of these proteins proceeded to the base of the cell with gradually decreasing extent. In summary, it was demonstrated that the local distribution of cellular components is an important factor in mechanically induced signal transduction.     

Reversal of chronic renal hypertension: role of salt and water excretion.

Rats with chronic one-kidney Goldlatt hypertension underwent an unclipping procedure with and without maintenance of body fluid volume through administration of iv salt solution. The blood pressure declined equally in the two groups. It is concluded that external loss of salt and water is not the mechanism for the reversal of this form of hypertension.     

How does a key fit a flexible lock? Structure and dynamics in receptor function

The preceding five years have brought remarkable advances in our understanding of the primary structure of drug receptors. The roles of certain amino acid residues in binding drugs and effecting receptor function have been proposed. As even more detailed structures become available, the goal of rational design of drug molecules based on predicted fits between the drug and its receptor will be near at hand. Although none of the classical receptors has yet yielded to X-ray crystallographic analysis, the methods of molecular biology facilitate the production of the large amounts of these rare proteins necessary for crystallization. Receptor proteins share one fundamental characteristic with allosterically regulated enzymes. Both have the structural flexibility that allows information to be transmitted to distant parts of the molecule. We will discuss recent observations about receptor structure and the dynamic nature of drug receptors, and pose questions about the significance of receptor dynamics for drug design.