The alpha 2(VIII) collagen gene. A novel member of the short chain collagen family located on the human chromosome 1

Type VIII collagen is a major component of Descemet's membrane, the specialized basement membrane of corneal endothelial cells. Sequence analysis of a cDNA isolated from a library made with mRNA from rabbit corneal endothelial cells has indicated that type VIII molecules contain a polypeptide chain, alpha 1(VIII), consisting of a short triple-helical domain of 454 amino acid residues flanked by non-triple-helical domains of 117 and 173 amino acid residues at the amino and carboxyl ends, respectively (Yamaguchi, N., Benya, P. D., van der Rest, M., and Ninomiya, Y. (1989) J. Biol. Chem. 264, 16022-16029). The sequence of alpha 1(VIII) is strikingly similar to that of alpha 1(X) collagen, a product of hypertrophic chondrocytes. Also, characterization of the alpha 1(VIII) and alpha 1(X) collagen genes has shown that they are quite similar in their exon organization. It has been concluded, therefore, that they are homologous members of a distinct subclass of collagen genes (Yamaguchi, N., Mayne, R., and Ninomiya, Y. (1991) J. Biol. Chem. 266, 4508-4513). We have given this subclass the name short chain collagens because of the relatively small size of the triple-helical domain. In the present study, we report on the identification and characterization of a collagen gene encoding a polypeptide which is co-expressed with the alpha 1(VIII) chain in corneal endothelial cells. This collagen chain contains a triple-helical and a carboxyl non-triple-helical domain encoded by a single, large exon both in mice and humans. We conclude, therefore, that the genes encodes a novel member of the short chain collagen family, and we have given this chain the designation alpha 2(VIII) collagen. By in situ hybridization we demonstrate that the alpha 2(VIII) gene is located in the p32.3-p34.3 region of the short arm of chromosome 1.     

Central thyrotropin-releasing hormone increases hepatic cyclic AMP through vagal-cholinergic and prostaglandin-dependent pathways in rats

Central neuropeptides play roles in many physiologic regulations through the autonomic nervous system. We have demonstrated that central thyrotropin-releasing hormone (TRH), one of neuropeptides, induces a stimulation of hepatic proliferation through vagal-cholinergic pathways. Since cAMP is known to play an important role in the hepatic proliferation, effect of central TRH on hepatic cAMP was investigated. Rats were intracisternally injected with either a TRH analog, RX-77368 (1-100 ng), or saline. The liver was removed 2-72 h after the TRH analog and hepatic cAMP content was determined by radioimmunoassay. In some experiments, pretreatment with hepatic vagotomy, atropine methyl nitrate, or 6-hydroxydopamine (6-OHDA) was performed. Hepatic cAMP was dose-dependently increased by intracisternal TRH analog (5-100 ng) with a peak response occurring 12 h postinjection. The central TRH-induced increase in hepatic cAMP was abolished by vagotomy, atropine and indomethacin, but not by 6-OHDA. Intravenous injection of the TRH analog (10 ng) did not affect hepatic cAMP. These results demonstrate that TRH acts in the brain to increase hepatic cAMP through vagal-cholinergic and prostaglandin-dependent pathways, suggesting that central TRH modulates hepatic functions through cAMP-mediated signaling pathways.     

Thyrotropin-releasing hormone in the dorsal vagal complex stimulates pancreatic blood flow in rats

Central administration of thyrotropin-releasing hormone (TRH) enhanced pancreatic blood flow in animal models. TRH nerve fibers and receptors are localized in the dorsal vagal complex (DVC), and retrograde tracing techniques have shown that pancreatic vagal nerves arise from the DVC. However, nothing is known about the central sites of action for TRH to elicit the stimulation of pancreatic blood flow. Effect of microinjection of a TRH analog into the DVC on pancreatic blood flow was investigated in urethane-anesthetized rats. After measuring basal flow, a stable TRH analog (RX-77368) was microinjected into the DVC and pancreatic blood flow response was observed for 120 min by laser Doppler flowmetry. Vagotomy of the several portions, or pretreatment with atoropine methyl nitrate or N(G)-nitro-l-arginine-methyl ester was performed. Microinjection of RX-77368 (0.1-10 ng) into the left or right DVC dose-dependently increased pancreatic blood flow. The stimulation of pancreatic blood flow by RX-77368 microinjection was eliminated by the same side of cervical vagotomy as the microinjection site or subdiaphragmatic vagotomy, but not by the other side of cervical vagotomy. The TRH-induced stimulation of pancreatic blood flow was abolished by atropine or N(G)-nitro-l-arginine-methyl ester. These results suggest that TRH acts in the DVC to stimulate pancreatic blood flow through vagal-cholinergic and nitric oxide dependent pathways, indicating that neuropeptides may act in the specific brain nuclei to regulate pancreatic function.     

Protective effect of central thyrotropin-releasing hormone analog on cerulein-induced acute pancreatitis in rats

Central neuropeptides play a role in many physiological functions through the autonomic nervous system. We have recently demonstrated that central injection of a thyrotropin-releasing hormone (TRH) analog increases pancreatic blood flow through vagal and nitric oxide-dependent pathways. In this study, the central effect of a TRH analog on experimental acute pancreatitis was investigated in rats. Acute pancreatitis was induced by two intraperitoneal injections of cerulein (40 microg/kg) at 1-h interval. Either stable TRH analog, RX 77368 (5-100 ng), or saline was injected intracisternally 15 min before the first cerulein injection under ether anesthesia. Serum amylase level was measured before and 5 h after the first cerulein injection. Pancreatic wet/dry weight ratio and histological changes were also evaluated. Intracisternal TRH analog inhibited cerulean-induced elevation of serum amylase level, increase in pancreatic wet/dry weight ratio and pancreatic histological changes, such as interstitial edema, inflammation and vacuolization. The pancreatic cytoprotection induced by central TRH analog was abolished by subdiaphragmatic vagotomy and N(G)-nitro-L-arginine-methyl ester (L-NAME), but not by 6-hydroxydopamine (6-OHDA). Intravenous administration of the TRH analog did not influence cerulein-induced acute pancreatitis. These results indicate that the TRH analog acts in the central nervous system to protect against acute pancreatitis through vagal and nitric oxide-dependent pathways.     

Immunological properties of monoclonal antibodies to human and rat prolyl 4-hydroxylase

Monoclonal antibodies to human (8 clones) and rat (12 clones) prolyl 4-hydroxylase [EC 1.14.11.2] were prepared and characterized as regards subclass, subunit specificity, inhibition and crossreactivity. Among the antibodies to the human enzyme, four clones showed the IgG1 subclass, two IgA, one IgG2b, and one IgM. Four clones reacted with the alpha subunit of the enzyme, while the others reacted with the beta subunit. The enzymatic activity was inhibited by four clones. Five clones crossreacted with the rat enzyme. One clone inhibited the rat enzyme. Among the antibodies to the rat enzyme, seven clones showed the IgG1 subclass, four IgG2a and one IgG2b. Seven clones reacted with the alpha subunit, and four with the beta subunit. One reacted with neither subunit. The enzymatic activity was inhibited by seven clones. Seven clones crossreacted with the human enzyme. Three clones inhibited the human enzyme.     

Adenoviral gene transfer of BMP-7, Id2, or Id3 suppresses injury-induced epithelial-to-mesenchymal transition of lens epithelium in mice

We have examined the effect of adenovirus-mediated expression of bone morphogenic protein-7 (BMP-7) and inhibitors of differentiation 2 and 3 (Id2 and Id3) on injury-induced epithelial-to-mesenchymal transition (EMT) of lens epithelium in mice. Id2 and Id3 are known to be upregulated by BMP-7 and to antagonize Smad2/3 signaling. The Cre-LoxP system adenoviral gene transfer was used. Three microliters of adenoviral solution (2 x 10⁷ PFU/µl) were injected into the right lens of adult male C57BL/6 mice (n = 144) at the time of capsular injury induced using a hypodermic needle under both general and topical anesthesia. A mixture of Cre-adenovirus (Cre-Ad) and vector encoding mBMP-7, mId2, or mId3 was administered in a test group. Control lenses were treated with Cre-Ad alone. After healing intervals of 5 or 10 days, the animals were killed and then we performed histological processes or RNA extraction from the lens. RT-PCR, real-time RT-PCR, and immunohistochemistry showed expression of each introduced gene in the lens. Exogenous BMP-7 upregulated expression of Id2 and Id3 in injured lenses, and gene introduction of Id2 or Id3 also upregulated BMP-7 expression. Gene transfer of BMP-7, Id2, or Id3 delayed injury-induced EMT of the lens epithelial cells as evaluated by histology and expression patterns of -smooth muscle actin and collagens in association with reduction of Smad2 COOH-terminal phosphorylation. Gene transfer of BMP-7, Id2, or Id3 delayed injury-induced EMT of lens epithelial cells and subsequent sealing of the capsular break with fibrous tissue in mice. lens epithelial cell; bone morphogenic protein-7; inhibitor of differentiation; Smad     

Actin filaments function as a tension sensor by tension-dependent binding of cofilin to the filament

Intracellular and extracellular mechanical forces affect the structure and dynamics of the actin cytoskeleton. However, the underlying molecular and biophysical mechanisms, including how mechanical forces are sensed, are largely unknown. Actin-depolymerizing factor/cofilin proteins are actin-modulating proteins that are ubiquitously distributed in eukaryotes, and they are the most likely candidate as proteins to drive stress fiber disassembly in response to changes in tension in the fiber. In this study, we propose a novel hypothesis that tension in an actin filament prevents the filament from being severed by cofilin. To test this, we placed single actin filaments under tension using optical tweezers. When a fiber was tensed, it was severed after the application of cofilin with a significantly larger delay in comparison with control filaments suspended in solution. The binding rate of cofilin to an actin bundle decreased when the bundle was tensed. These results suggest that tension in an actin filament reduces the cofilin binding, resulting in a decrease in its effective severing activity.     

Involvement of endogenous CRF in carbon tetrachloride-induced acute liver injury in rats

Central neuropeptides play important roles in many physiological and pathophysiological regulation mediated through the autonomic nervous system. In regard to the hepatobiliary system, several neuropeptides act in the brain to regulate bile secretion, hepatic blood flow, and hepatic proliferation. Central injection of corticotropin-releasing factor (CRF) aggravates carbon tetrachloride (CCl4)-induced acute liver injury through the sympathetic nervous pathway in rats. However, still nothing is known about a role of endogenous neuropeptides in the brain in hepatic pathophysiological regulations. Involvement of endogenous CRF in the brain in CCl4-induced acute liver injury was investigated by centrally injecting a CRF receptor antagonist in rats. Male fasted Wistar rats were injected with CRF receptor antagonist alpha-helical CRF-(9-41) (0.125-5 microg) intracisternally just before and 6 h after CCl4 (2 ml/kg) administration, and blood samples were obtained before and 24 h after CCl4 injection for measurement of hepatic enzymes. The liver sample was removed 24 h after CCl4 injection, and histological changes were examined. Intracisternal alpha-helical CRF-(9-41) dose dependently (0.25-2 microg) reduced the elevation of alanine aminotransferase and aspartate aminotransferase levels induced by CCl4. Intracisternal alpha-helical CRF-(9-41) reduced CCl4-induced liver histological changes, such as centrilobular necrosis. The effect of central CRF receptor antagonist on CCl4-induced liver injury was abolished by sympathectomy and 6-hydroxydopamine pretreatment but not by hepatic branch vagotomy or atropine pretreatment. These findings suggest the regulatory role of endogenous CRF in the brain in experimental liver injury in rats.