Inhibition of sepsis-induced inflammatory response by ß1-adrenergic antagonists.

BACKGROUND: Although previous studies have described potential benefits of nonselective ß-adrenergic antagonist therapy in sepsis, there is a paucity of data on the use of ß1-selective antagonists (B1AA). The purposes of this study were to describe the effects of B1AA on survival in septic animals and to explore for molecular mechanisms of potential treatment benefit. METHODS: C57BL/6 male mice received intraperitoneal injection of lipopolysaccharide. Continuous infusion of a B1AA (esmolol) or an equal volume of saline (control) was initiated at 4 hours after injection. Kaplan-Meier survival analysis at 120 hours was used to explore for mortality differences. A subgroup of animals was sacrificed for microarray expression analysis. Top candidate genes were validated in vitro and in silico. Expression of our candidate genes in a human microarray database (GSE28750) was explored. RESULTS: B1AA infusion resulted in increased survival (p = 0.001) at 120 hours. Mean survival difference was 23.6 hours (p = 0.002). Hazard ratio for mortality with B1AA is 0.43 (95% confidence interval, 0.26-0.72). Immunologic disease (p = 0.0003-0.036) and cell death/survival (p = 0.0001-0.042) were significantly associated with improved survival in septic mice treated with B1AA. Further analysis of the gene structure revealed that eight genes shared common promoter activating sequence for NFKB and/or BRCA1 motifs. Analysis of a human sepsis database identified the up-regulation of CAMP (p = 0.032) and TNFSF10 (p = 0.001) genes in septic patients compared with healthy controls. CONCLUSION: Continuous infusion of a B1AA initiated after septic insult improves survival at 5 days in a murine model. Benefits may be caused by modulation of gene expression in immunologic pathways leading to an increase in CAMP and TNFSF10 expression. This observed effect may be explained by the activation of NFKB and BRCA1 genes involved in immune response and cell repair pathways. Our findings support further investigation of the use of B1AA in the treatment of sepsis.

Divergent and nonuniform gene expression patterns in mouse brain.

Considerable progress has been made in understanding variations in gene sequence and expression level associated with phenotype, yet how genetic diversity translates into complex phenotypic differences remains poorly understood. Here, we examine the relationship between genetic background and spatial patterns of gene expression across seven strains of mice, providing the most extensive cellular-resolution comparative analysis of gene expression in the mammalian brain to date. Using comprehensive brainwide anatomic coverage (more than 200 brain regions), we applied in situ hybridization to analyze the spatial expression patterns of 49 genes encoding well-known pharmaceutical drug targets. Remarkably, over 50% of the genes examined showed interstrain expression variation. In addition, the variability was nonuniformly distributed across strain and neuroanatomic region, suggesting certain organizing principles. First, the degree of expression variance among strains mirrors genealogic relationships. Second, expression pattern differences were concentrated in higher-order brain regions such as the cortex and hippocampus. Divergence in gene expression patterns across the brain could contribute significantly to variations in behavior and responses to neuroactive drugs in laboratory mouse strains and may help to explain individual differences in human responsiveness to neuroactive drugs.

Molecular cloning of four cDNAs encoding prepro-crustacean hyperglycemic hormone (CHH) from the eyestalk of the red rock crab Cancer productus: identification of two genetically encoded CHH isoforms and two putative post-translationally derived CHH variants.

Recently, we demonstrated that the four known sinus gland (SG) isoforms of Cancer productus crustacean hyperglycemic hormone precursor-related peptide (Capr-CPRP I-IV) are differentially distributed in conserved patterns among individual crabs. This finding strongly supported the presence of multiple prepro-crustacean hyperglycemic hormone (chh) transcripts in each crab, as well as the translation and processing of the encoded prepro-hormones. Whether these transcripts contained common or distinct isoforms of CHH remained unknown. To address this question, molecular analyses of the C. productus eyestalk prepro-chhs were undertaken. Using a PCR-based cloning strategy, four prepro-chh cDNAs were characterized: one encoding CPRP I, one encoding CPRP III (found to possess Ile(26) rather than Leu(26) as reported previously), and two encoding CPRP II. No cDNA encoding CPRP IV was identified. The deduced CHH present in the prepro-hormones containing CPRP I and III were identical (Capr-CHH I) and differed from that (Capr-CHH II) present in the two prepro-hormones containing Capr-CPRP II at a single residue, a Thr(5) for Ser(5) substitution. As both CHH isoforms possess Glu at position 1, a cyclization of this residue to pyroglutamine is likely as the peptides mature, as has been seen for the CHHs of other brachyuran species. Likewise, homology to other CHHs suggests all C. productus isoforms are C-terminally amidated. These post-translational modifications would result in four SG isoforms of CHH: Capr-CHH I, Capr-pyro-CHH I, Capr-CHH II, and Capr-pyro-CHH II. Southern blotting supported the hypothesis that at least three prepro-chh transcripts are present in each crab, while dual in situ hybridization-immunohistochemistry localized the transcripts to previously mapped CHH immunopositive somata in the X-organ, the major source of innervation to the SG.

Circadian desynchronization of core body temperature and sleep stages in the rat.

Proper functioning of the human circadian timing system is crucial to physical and mental health. Much of what we know about this system is based on experimental protocols that induce the desynchronization of behavioral and physiological rhythms within individual subjects, but the neural (or extraneural) substrates for such desynchronization are unknown. We have developed an animal model of human internal desynchrony in which rats are exposed to artificially short (22-h) light-dark cycles. Under these conditions, locomotor activity, sleep-wake, and slow-wave sleep (SWS) exhibit two rhythms within individual animals, one entrained to the 22-h light-dark cycle and the other free-running with a period >24 h (tau(>24 h)). Whereas core body temperature showed two rhythms as well, further analysis indicates this variable oscillates more according to the tau(>24 h) rhythm than to the 22-h rhythm, and that this oscillation is due to an activity-independent circadian regulation. Paradoxical sleep (PS), on the other hand, shows only one free-running rhythm. Our results show that, similarly to humans, (i) circadian rhythms can be internally dissociated in a controlled and predictable manner in the rat and (ii) the circadian rhythms of sleep-wake and SWS can be desynchronized from the rhythms of PS and core body temperature within individual animals. This model now allows for a deeper understanding of the human timekeeping mechanism, for testing potential therapies for circadian dysrhythmias, and for studying the biology of PS and SWS states in a neurologically intact model.

E74 exhibits stage-specific hormonal regulation in the epidermis of the tobacco hornworm, manduca sexta.

The transcription factor E74 is one of the early genes induced by ecdysteroids during metamorphosis of Drosophila melanogaster. Here, we report the cloning and hormonal regulation of E74 from the tobacco hornworm, Manduca sexta (MsE74). MsE74 is 98% identical to that of D. melanogaster within the DNA-binding ETS domain of the protein. The 5'-isoform-specific regions of MsE74A and MsE74B share significantly lower sequence similarity (30-40%). Developmental expression by Northern blot analysis reveals that, during the 5th larval instar, MsE74B expression correlates with pupal commitment on day 3 and is induced to maximal levels within 12h by low levels of 20-hydroxyecdysone (20E) and repressed by physiologically relevant levels of juvenile hormone I (JH I). Immunocytochemical analysis shows that MsE74B appears in the epidermis before the 20E-induced Broad transcription factor that is correlated with pupal commitment (Zhou and Riddiford, 2001). In contrast, MsE74A is expressed late in the larval and the pupal molts when the ecdysteroid titer has declined to low levels and in the adult molt just as the ecdysteroid titer begins to decline. This change in timing during the adult molt appears not to be due to the absence of JH as there was no change during the pupal molt of allatectomized animals. When either 4th or 5th instar larval epidermis was explanted and subjected to hormonal manipulations, MsE74A induction occurred only after exposure to 20E followed by its removal. Thus, MsE74B appears to have a similar role at the onset of metamorphosis in Manduca as it does in Drosophila, whereas MsE74A is regulated differently at pupation in Manduca than at pupariation in Drosophila.

The G protein-coupled receptor repertoires of human and mouse.

Diverse members of the G protein-coupled receptor (GPCR) superfamily participate in a variety of physiological functions and are major targets of pharmaceutical drugs. Here we report that the repertoire of GPCRs for endogenous ligands consists of 367 receptors in humans and 392 in mice. Included here are 26 human and 83 mouse GPCRs not previously identified. A direct comparison of GPCRs in the two species reveals an unexpected level of orthology. The evolutionary preservation of these molecules argues against functional redundancy among highly related receptors. Phylogenetic analyses cluster 60% of GPCRs according to ligand preference, allowing prediction of ligand types for dozens of orphan receptors. Expression profiling of 100 GPCRs demonstrates that most are expressed in multiple tissues and that individual tissues express multiple GPCRs. Over 90% of GPCRs are expressed in the brain. Strikingly, however, the profiles of most GPCRs are unique, yielding thousands of tissue- and cell-specific receptor combinations for the modulation of physiological processes.