Pegylated peptides. V. Carboxy-terminal PEGylated analogs of growth hormone-releasing factor (GRF) display enhanced duration of biological activity in vivo

In the present study, human growth hormone-releasing factor (hGRF) and analogs were successfully pegylated at the carboxy-terminus using a novel solid- and solution-phase strategy. Following synthesis, these pegylated hGRF analogs were evaluated for in vitro and in vivo biological activity. Specifically, hGRF (1-29)-NH2, [Ala15]-hGRF (1-29)-NH2, [desNH2Tyr1, D-Ala2, Ala15]-hGRF(1-29)-NH2 and [His1, Val2, Gln8, Ala15, Leu27]-hGRF(1-32)-OH were each C-terminally extended using a Gly-Gly-Cys-NH2 spacer (previously demonstrated not to alter intrinsic biological activity), and then monopegylated via coupling to an activated dithiopyridyl-PEG reagent. PEG moieties of 750, 2000, 5000 or 10,000 molecular weight (MW) were examined to determine the effect of polymer weight on activity. Initial biological evaluations in vitro revealed that all C-terminally pegylated hGRF analogs retained high growth hormone (GH)-releasing potencies, regardless of the MW of PEG polymer employed. Two of these pegylated hGRF analogs, [desNH2Tyr1, D-Ala2, Ala15]-hGRF (1-29)-Gly-Gly-Cys(NH2)-S-Nle-PEG5000 and [His1, Val2, Gln8, Ala15, Leu27]-hGRF(1-32)-Gly-Cys(NH2)-S-Nle-PEG5000, were subsequently evaluated in both pig and mouse models and found to be highly potent (in vivo potency range = 12-55-fold that of native hGRF). Relative to their non-pegylated counterparts, these two pegylated hGRF analogs exhibited enhanced duration of activity.     

Propagation of granulocytic Ehrlichia spp. from human and equine sources in HL-60 cells induced to differentiate into functional granulocytes

Ehrlichia spp. from human and equine sources in the northeastern Unites States were detected by PCR, isolated, and propagated in the HL-60 promyelocytic leukemia cell line. Growth of Ehrlichia from both equine and human sources was enhanced by addition of retinoic acid, which causes granulocytic differentiation of the HL-60 cells. DNA sequencing of a portion of the 16S rDNA gene supported the hypothesis that the same pathogen was responsible for both equine and human granulocytic ehrlichiosis.     

Histamine H3 receptors contribute to drinking elicited by eating in rats

A role for endogenous histamine and its H3 receptor subtype for mediating drinking elicited by eating was examined in adult male Sprague-Dawley rats. The i.p. injection of the H3 agonist R-alpha-methylhistamine (Ramh, 2.5 mg/kg) shortened the latency to initiate drinking and increased 1-h water intake in nondeprived rats freely eating pellets and drinking water. The ICV injection (through a surgically implanted chronic cannula) of 10 micrograms Ramh increased water intake; this Ramh-induced drinking was abolished by previous ICV injection of the H3 antagonist thioperamide (Th, 60 micrograms). For rats drinking and eating after 24-h food deprivation, s.c. Th inhibited drinking behavior: for example, 10 mg/kg Th s.c. delayed the latency to initiate drinking and inhibited 1-h water intake without inhibition of food intake. In contrast, 60 micrograms Th ICV failed to inhibit food-related drinking in rats eating after food deprivation. For nondeprived rats eating a small cracker, 10 mg/kg Th s.c. delayed the latency to initiate drinking and abolished water intake without effect of eating, and 60 micrograms Th ICV had similar effects upon drinking elicited by ingestion of cracker. The IG infusion (through a surgically implanted gastric catheter) of 2 ml 600 or 900 mOsm/kg NaCl, a treatment that is subthreshold for increase in systemic plasma osmolality at the initiation of drinking, elicited drinking that was abolished by 10 mg/kg Th s.c. and attenuated by 60 micrograms Th ICV. The IG infusion of 2 ml 1800 mOsm/kg NaCl, a treatment that is above threshold for increase in systemic plasma osmolality, elicited drinking that was attenuated by 10 mg/kg s.c. or 60 micrograms Th ICV. These results demonstrate that peripheral and central H3 receptors for histamine have a role in drinking elicited by eating and the postprandial gastrointestinal osmotic consequences of eating. These findings extend the evidence demonstrating a histaminergic contribution to food-related drinking in rats.     

Induction of c-fos and c-jun proto-oncogene expression by asbestos is ameliorated by N-acetyl-L-cysteine in mesothelial cells

Asbestos fibers cause dose-dependent, persistent increases in mRNA levels of c-jun and c-fos proto-oncogenes in rat pleural mesothelial (RPM) cells, the progenitor cells of asbestos-induced mesothelioma (N. Heintz, Y. M. W. Janssen, and B. T. Mossman. Proc. Natl. Acad. Sci. USA, 90: 3299-3303, 1993). Here we report that addition of N-acetyl-L-cysteine decreases asbestos-mediated induction of c-fos and c-jun mRNA levels in a dose-dependent fashion. Exposure of RPM cells to asbestos causes depletion of total cellular glutathione, a response that can be abolished by pretreatment with N-acetyl-L-cysteine. Pretreatment of cells with buthionine sulfoximine, an agent which diminishes glutathione pools, increases the magnitude of induction of c-fos and c-jun mRNA by asbestos. To determine whether asbestos-induced effects on proto-oncogene expression could be attributed to extracellular generation of active oxygen species (AOS), RPM cells were exposed to H2O2 or xanthine and xanthine oxidase, a generating system of AOS. These oxidant stresses did not decrease cellular glutathione levels nor alter mRNA levels of c-fos or c-jun. However, increased mRNA levels of manganese-containing superoxide dismutase and heme oxygenase were observed, indicating that RPM cells respond to AOS by increased expression of genes encoding antioxidant enzymes. These data indicate that the signaling pathways leading to c-fos/c-jun proto-oncogene induction by asbestos are not triggered directly by formation of extracellular AOS. However, intracellular thiol levels appear to influence the expression of c-fos and c-jun, suggesting a redox-sensitive component in the signaling cascade which modulates gene expression of c-fos and c-jun by asbestos.     

VH gene family utilization of anti-acetylcholine receptor antibodies in experimental autoimmune myasthenia gravis

The immunoglobulin heavy chain (VH) gene family usage in experimental autoimmune myasthenia gravis (EAMG) model was investigated by RNA slot blot hybridization using VH gene family specific probes. Anti-acetylcholine receptor (AChR) monoclonal antibodies (mAbs) isolated from susceptible C57BL/6 and resistant BALB/c mice were found to be encoded by VH genes from at least six different families. The Vgam3.8 family was overrepresented in alpha-bungarotoxin blocking mAbs. Expression of cross-reactive idiotypes by anti-AChR mAbs was irrespective of the VH gene family usage. Strain dependent differences in susceptibility for EAMG were not reflected in an aberrant VH gene family usage of anti-AChR mAbs.     

Plasma membrane cholesterol is a key molecule in shear stress-dependent activation of extracellular signal-regulated kinase

Shear stress, the dragging force generated by fluid flow, differentially activates extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) in bovine aortic endothelial cells (BAEC) (Jo, H., Sipos, K., Go, Y. M., Law, R., Rong, J., and McDonald, J. M. (1997) J. Biol. Chem. 272, 1395-1401). Here, we examine whether cholesterol-enriched compartments in the plasma membrane are responsible for such differential regulation. Pretreatment of BAEC with a cholesterol-binding antibiotic, filipin, did not inhibit shear-dependent activation of JNK. In contrast, filipin and other membrane-permeable cholesterol-binding agents (digitonin and nystatin), but not the lipid-binding agent xylazine, inhibited shear-dependent activation of ERK. The effect of cholesterol-binding drugs did not appear to be due to membrane permeabilization, since treatment of BAEC with a detergent, Triton X-100 which also permeabilizes membranes, did not inhibit shear-dependent activation of ERK. Furthermore, shear-dependent activation of ERK, but not JNK, was inhibited by cyclodextrin, a membrane-impermeable cholesterol-binding agent, which removes cell-surface cholesterol. Moreover, the effects of cyclodextrin were prevented by adding cholesterol during the incubation. These results indicate that cholesterol or cholesterol-sensitive compartments in the plasma membrane play a selective and essential role in activation of ERK, but not JNK, by shear stress. Although exposure to shear stress (1 h) increased the number of caveolae by 3-fold, treatment with filipin had no effect in either control or shear-exposed cells suggesting that caveolae density per se is not a crucial determinant in shear-dependent ERK activation. In summary, the current study suggests that cholesterol-sensitive microdomains in the plasma membrane, such as caveolae-like domains, play a critical role in differential activation of ERK and JNK by shear stress.