Lectin histochemical study on the olfactory organ of the newt,Cynops pyrrhogaster,revealed heterogeneous mucous environments in a single nasal cavity

Expression patterns of glycoconjugates were examined by lectin histochemistry in the nasal cavity of the Japanese red-bellied newt, Cynops pyrrhogaster. Its nasal cavity consisted of two components, a flattened chamber, which was the main nasal chamber (MNC), and a lateral diverticulum called the lateral nasal sinus (LNS), which communicated medially with the MNC. The MNC was lined with the olfactory epithelium (OE), while the diverticulum constituting the LNS was lined with the vomeronasal epithelium (VNE). Nasal glands were observed beneath the OE but not beneath the VNE. In addition, a secretory epithelium was revealed on the dorsal boundary between the MNC and the LNS, which we refer to as the boundary secretory epithelium (BSE) in this study. The BSE seemed to play an important role in the construction of the mucous composition of the VNE. Among 21 lectins used in this study, DBA, SBA and Jacalin showed different staining patterns between the OE and the VNE. DBA staining showed remarkable differences between the OE and the VNE; there was intense staining in the free border and the supporting cells of the VNE, whereas there was no staining or weak staining in the cells of the OE. SBA and Jacalin showed different stainings in the receptor neurons for the OE and the VNE. Furthermore, UEA-I and Con A showed different stainings for the nasal glands. UEA-I showed intense staining in the BSE and in the nasal glands located in the ventral wall of the MNC (VNG), whereas Con A showed intense staining in the BSE and in the nasal glands located in the dorsal and medial wall of the MNC (DMNG). The DMNG were observed to send their excretory ducts into the OE, whereas no excretory ducts were observed from the VNG to the OE or the VNE. These results suggested that the secretion by the supporting cells as well as the BSE and the DMNG establishes that there are heterogeneous mucous environments in the OE and the VNE, although both epithelia are situated in the same nasal cavity.     

Identification and characterization of temperature-sensitive mild mutations in three Japanese patients with nonsevere forms of very-long-chain acyl-CoA dehydrogenase deficiency

Very-long-chain acyl-CoA dehydrogenase (VLCAD) deficiency is clinically classified into severe, intermediate, and myopathic forms. We identified mutations in three unrelated Japanese patients with VLCAD deficiency: two with the myopathic form and one with the intermediate form, all compound heterozygotes of K264E/M437V, A416T/1798delA, and P89S/IVS16-3delAA, respectively. We characterized four missense mutations, K264E, M437V, A416T, and P89S, by transisent expression analysis, using SV40-transformed fibroblasts derived from a VLCAD-null patient, as recipient cells. In transient expression of the wild-type VLCAD cDNA, VLCAD activity at 30 degrees C was higher than at 37 degrees C. Moreover, this temperature-sensitive character is more evident in all the mutant proteins tested than in wild type. Based on characterization of the five missense mutations identified in four Japanese patients, including data on one patient with the myopathic form previously reported, patients with the nonsevere forms (intermediate or myopathic forms) have missense mutations with residual activities in at least one allele. Expression analysis at 30 degrees C may be more useful for evaluating these missense mutations, compared with that at 37 degrees C.     

ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats

Expansion of CAG trinucleotide repeats that encode polyglutamine is the underlying cause of at least nine inherited human neurodegenerative disorders, including Huntington's disease and spinocerebellar ataxias. PolyQ fragments accumulate as aggregates in the cytoplasm and/or in the nucleus, and induce neuronal cell death. However, the molecular mechanism of polyQ-induced cell death is controversial. Here, we show the following: (1) polyQ with pathogenic repeat length triggers ER stress through proteasomal dysfunction; (2) ER stress activates ASK 1 through formation of an IRE1-TRAF2-ASK1 complex; and (3) ASK1(-/-) primary neurons are defective in polyQ-, proteasome inhibitor-, and ER stress-induced JNK activation and cell death. These findings suggest that ASK1 is a key element in ER stress-induced cell death that plays an important role in the neuropathological alterations in polyQ diseases.     

Protective effect of recombinant neutrophil elastase inhibitor (R-020) on sepsis-induced organ injury in rat

Severe inflammatory responses after major surgeries, trauma, and infection develop multiple organ dysfunction. In the mechanisms of the pathogenesis of these responses, activated neutrophils are thought to be important in terms of their ability to produce various kinds of proteinases, which can degrade various proteins constructing human tissues. Among their proteinases, neutrophil elastase is the strongest serine proteinase secreted from activated neutrophils. Thus, we examined in this study the inhibitory effect and therapeutic efficacy of newly produced recombinant human Kunitz-type proteinase inhibitor (R-020), which coded the second domain of human urinary trypsin inhibitor. R-020 was effective in significantly improving the survival rate after induction of the rat lethal peritonitis model (cecal ligation and punctureinduced septic shock model). We suggest that various serine proteinases are implicated in the pathogenesis of neutrophil-related multiple organ failure and that recombinant human Kunitz-type proteinase inhibitor might be effective in the treatment of these kinds of organ dysfunction.     

Lipid components in the detergent-resistant membrane microdomain (DRM) obtained from the synaptic plasma membrane of rat brain

Lateral association of sphingolipids and cholesterol is considered to form membrane microdomains such as “lipid rafts” obtainable as a detergent-resistant membrane microdomain (DRM) fraction after solubilization with a non-ionic detergent and density gradient centrifugation. Since not only sphinogolipids and cholesterol, but also functional lipids such as phosphatidylinositol 4,5-bisphosphate (PIP₂) are reported to be localized in DRM prepared from several cultured cells, this domain is considered to be a platform mediating lipid-signaling. Although PIP₂ is considered to have pivotal roles in the nervous system, little information is available on the localization of PIP₂ in the DRM within the synaptic plasma membrane (SPM) obtained from matured rat brains. In this study, in order to know the localization of PIP₂ in SPM-derived DRM, we measured the amount of PIP₂ in SPM and SPM-derived DRM, by the thin-layer chromatography blotting method, using a GST-fusion protein of the pleckstrin-homology domain of phospholipase Cδ1 as a PIP₂ binding probe. About 10% of the PIP₂ in SPM was recovered in DRM. In contrast, over 40% recovery was observed for the membrane cholesterol and sphingomyelin, and about 30% recovery was observed for phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine in the DRM were detected using the thin-layer chromatography method. Since the recovery of proteins in DRM was about 10%, the result indicates that there occurs no enrichment of PIP₂ in DRM prepared from SPM.     

The inferior supernumerary renal arteries: A classification into three types

Among cases that had multiple renal arteries on one side, an inferior supernumerary renal artery was found in 24/270 cases (ca. 9%) on the right and in 19/270 cases (ca. 7%) on the left, together with the usual renal artery. We have noticed that there are correlations between their levels of origin from the aorta and their positional relation to the ureter and the inferior vena cava (IVC). An inferior supernumerary renal artery (InfRA) of lower origin passes in front of the IVC and behind the ureter. An InfRA of middle origin passes in front of both the IVC and the ureter. An InfRA of upper origin passes behind the IVC and in front of the ureter or renal pelvis. In addition there was a tendency for the lower origin type to have an ureteric branch, while the middle and upper origin types had a gonadal branch. These findings suggest that different derivations lead to the inferior supernumerary renal arteries.     

Peptide analysis,stability studies,and structural modeling explain contradictory peptide motifs and unique properties of the NOD mouse MHC class II molecule H2-A(g7)

The MHC class II molecule H2-A(g7) is the chief genetic determinant in insulin-dependent diabetes mellitus of the non-obese diabetic (NOD) mice. Poor peptide binding ability, as well as presentation of a unique subset of peptides by this molecule was suggested to promote autoimmunity in this strain. However, several laboratories have presented results in favor of an H2-A(g7) molecule that can avidly bind many different peptides. The crystal structures of H2-A(g7) in complex with two different peptides did not completely resolve this issue. To analyze the peptide binding capacity and the motif requirements of H2-A(g7), we eluted natural ligands from purified H2-A(g7) molecules isolated from the H2-A(g7)-transfected M12-C3 cells. A low peptide yield dominated by a few peptide ligands was found. Pool sequencing and alignment of individual ligands on the basis of molecular modeling revealed a peptide-binding motif with basic/aliphatic/small hydrophilic amino acids at relative position 1 (p1), aliphatic amino acids at p4, Ala at p6, and acidic amino acids and Ser/Gly at p9, as well as acidic residues at p10/11. Though weak, the binding of individual ligands, as well as the importance of an acidic C-terminal residue was confirmed by peptide binding studies to isolated H2-A(g7) molecules. Furthermore, the H2-A(g7) molecule incompletely dissociated into its constituent chains in SDS-electrophoresis under nonreducing conditions. This provides additional evidence of its weak affinity for peptides, which probably arises from the combination of beta56His/beta57Ser/beta78Ala and other unique H2-A(g7) residues in contact with the antigenic peptide. These results allow a better understanding of the role of this molecule in the development of autoimmunity and the identification of epitopes relevant to diabetes.     

Identification of fibroin-derived peptides enhancing the proliferation of cultured human skin fibroblasts

We previously reported that the fibroin of the silkworm Bombyx mori enhanced the proliferation of cultured human skin fibroblasts. In this work, the fibroin was digested by chymotrypsin, and the resulting peptide fragments were fractionated and assayed for their biological activity. Two peptides that promoted fibroblast growth were isolated and identified to be VITTDSDGNE and NINDFDED. Both sequences are found in the N-terminal region of the fibroin polypeptide and are thought to be the active principle of fibroblast growth-promoting activity.     

Neonatal impact of leukemia inhibitory factor on neurobehavioral development in rats

Cytokines have been implicated in the etiology or pathology of various psychiatric diseases of developmental origin such as autism and schizophrenia. Leukemia inhibitory factor (LIF) is induced by a variety of brain insults and known to have many influences on mature and immature nervous system. Here, we assessed the neurobehavioral and pathological consequences of peripheral administration of LIF in newborn rats. Subcutaneous LIF injection induced STAT3 phosphorylation in many brain regions and increased glial fibrillary acidic protein (GFAP) immunoreactivity in the neocortex, suggesting that LIF had direct effects in the central nervous system. The LIF-treated rats displayed decreased motor activity during juvenile stages, and developed abnormal prepulse inhibition in the acoustic startle test during and after adolescence. They displayed normal learning ability in active avoidance test, however. Brain neuronal structures and startle responses were grossly normal, except for the cortical astrogliosis during neonatal LIF administration. These results indicate that LIF induction in the periphery of the infant has a significant, but discrete impact on neurobehavioral development.