NARCOBIT: a newly developed inhalational anesthesia system for mice.

NARCOBIT is the first anesthetic system for mice and rats to incorporate a ventilator. Therefore, it is expected to improve the reliability of mice and rat experiments by accurately controlling and maintaining the depth of anesthesia. In this study, we used NARCOBIT for inducing inhalational anesthesia in mice and evaluated the changes in their hemodynamic parameters. ICR mice were anesthetized with 5% isoflurane and room air, followed by endotracheal intubation. Subsequently, they were mechanically ventilated, and anesthesia was maintained by 2% isoflurane for a 60-min period (maintenance state) using NARCOBIT. In study 1, the heart rate (HR) and mean arterial blood pressure (MAP) were measured. The skin blood flow (SBF) from the hind legs was continuously measured during the maintenance state. Subsequently, the concentration-dependent effects of isoflurane on MAP were examined. In study 2, blood samples were obtained from the abdominal aorta for blood gas analysis. The HR and MAP decreased after anesthesia but were stable during the maintenance state. Decreased MAP and concentration-dependent effects of isoflurane were observed. The SBF increased slightly during the maintenance state but this increase was insignificant. The blood gas analysis showed neither hypoxia nor hypercapnia. Since the use of NARCOBIT enables the anesthetic concentration of isoflurane to be easily changed, a suitable anesthesia depth can be obtained for experimental purposes. Therefore, we conclude that NARCOBIT can be used for providing inhalational anesthesia to mice.     

Identification of oligopeptidase B in higher plants. Purification and characterization of oligopeptidase B from quiescent wheat embryo, Triticum aestivum

Proteolytic enzymes in general, and cysteine proteases in particular, play key roles in seed germination and early seedling growth. However, the precise mechanism by which the serine proteases are regulated remains unclear. Trypsin-like activity was detected in wheat germ (quiescent embryo) and this activity increased in the germinating embryo. In this work, a trypsin-like serine protease expressed in wheat germ was purified to homogeneity by chromatography through DEAE-cellulose, phenyl-Sepharose, Ultrogel AcA-34 and Blue-Sepharose. The molecular mass of the enzyme was estimated to be 81 kDa by SDS-PAGE under reducing conditions. Amino acid analysis of the peptides generated following digestion of the enzyme with lysyl endopeptidase indicated that the enzyme is a plant homologue of Escherichia. coli oligopeptidase B. The subsite specificity of the enzymes differ, although both enzymes hydrolyze synthetic substrates and model peptides at the carboxyl side of basic amino acids. The wheat enzyme is more sensitive to leupeptin and antipain than the E. coli emzyme. These results provide the basis for characterizing plant oligopeptidase B and contribute to our understanding of its role in the early development of seedlings.     

Solution structure of the SEA domain from the murine homologue of ovarian cancer antigen CA125 (MUC16)

Human CA125, encoded by the MUC16 gene, is an ovarian cancer antigen widely used for a serum assay. Its extracellular region consists of tandem repeats of SEA domains. In this study we determined the three-dimensional structure of the SEA domain from the murine MUC16 homologue using multidimensional NMR spectroscopy. The domain forms a unique alpha/beta sandwich fold composed of two alpha helices and four antiparallel beta strands and has a characteristic turn named the TY-turn between alpha1 and alpha2. The internal mobility of the main chain is low throughout the domain. The residues that form the hydrophobic core and the TY-turn are fully conserved in all SEA domain sequences, indicating that the fold is common in the family. Interestingly, no other residues are conserved throughout the family. Thus, the sequence alignment of the SEA domain family was refined on the basis of the three-dimensional structure, which allowed us to classify the SEA domains into several subfamilies. The residues on the surface differ between these subfamilies, suggesting that each subfamily has a different function. In the MUC16 SEA domains, the conserved surface residues, Asn-10, Thr-12, Arg-63, Asp-75, Asp-112, Ser-115, and Phe-117, are clustered on the beta sheet surface, which may be functionally important. The putative epitope (residues 58-77) for anti-MUC16 antibodies is located around the beta2 and beta3 strands. On the other hand the tissue tumor marker MUC1 has a SEA domain belonging to another subfamily, and its GSVVV motif for proteolytic cleavage is located in the short loop connecting beta2 and beta3.     

An autocrine function of nerve growth factor for cell cycle regulation of vascular endothelial cells

Nerve growth factor (NGF) regulates maintenance, survival, and function of not only neuronal cells but also various kinds of non-neuronal cells. Here we clearly demonstrated that mouse aortic endothelial cells (AEC) produced bioactive NGF, and the production was enhanced by a proinflammatory cytokine, interleukin (IL)-1beta. AEC expressed both high affinity (TrkA) and low affinity (p75(NGFR)) receptors for NGF. Exogenously added NGF induced rapid phosphorylation of TrkA tyrosine kinase. Addition of anti-NGF neutralizing antibody resulted in an increase in the proportion of AEC in S and G(2)/M phases and in a hypodiploid range. Since the vascular endothelium plays a pivotal role in inflammatory conditions, these results strongly suggest that NGF, whose production is enhanced at the affected site, may contribute to maintenance, survival, and function of vascular endothelial cells by autocrine and/or paracrine mechanisms.     

Leptin and insulin down-regulate angiopoietin-like protein 3, a plasma triglyceride-increasing factor

We reported previously that angiopoietin-like protein3 (ANGPTL3), a liver-specific secretory factor, increased plasma triglyceride (TG) via inhibition of lipoprotein lipase and free fatty acid (FFA) by activating adipose-lipolysis. The current study examined the regulation of Angptl3 by leptin and insulin, both of which are key players in the metabolic syndrome. Angptl3 expression and plasma ANGPTL3 levels were increased in leptin-resistant C57BL/6J(db/db) and -deficient C57BL/6J(ob/ob) mice, relative to the control. Leptin supplements decreased Angptl3 gene expression and plasma ANGPTL3 in C57BL/6J(ob/ob) mice. The changes of Angptl3 were associated with alterations of plasma TG and FFA levels. Leptin treatment directly suppressed Angptl3 gene expression in hepatocytes. Angptl3 gene expression and plasma protein levels were also increased in insulin-deficient streptozotocin-treated mice. Insulin treatment of hepatocytes decreased Angptl3 gene expression and protein secretion. Our results suggest that elevated ANGPTL3 by leptin- or insulin-resistance is attributed to increased plasma TG and FFA concentrations in obesity.     

Involvement of Ras in extraembryonic endoderm differentiation of embryonic stem cells

Embryonic stem (ES) cells, derived from the inner cell mass of blastocyst can differentiate into multiple cell lineages. In this study, we examined the possible involvement of Ras in ES cell differentiation. We found that Ras was activated upon formation of embryoid bodies (EBs), an initial step in ES cell differentiation. When expressed during EB differentiation, a dominant-negative mutant of Ras suppressed induction of marker genes for extraembryonic endoderm differentiation, including GATA-4, GATA-6, alpha-fetoprotein, and hepatocyte nuclear factor 3beta, while an activated mutant promoted their induction. Expression of a Ras mutant that selectively activates the Raf/MEK/Erk pathway also enhanced induction of extraembryonic endoderm markers, and treatment with a MEK inhibitor resulted in their decreased expression. In addition, Ras stimulated downregulation of Nanog, a suppressor of endoderm differentiation in ES cells. These data suggest that Ras activation during EB differentiation plays a crucial role in initiation of extraembryonic endoderm differentiation.