Distinct mechanisms of acid-induced HCO3- secretion in normal and slightly permeable stomachs

We investigated the regulatory mechanism of acid-induced HCO(3)(-) secretion in the slightly permeable rat stomach after an exposure to hyperosmolar NaCl. Under urethane anesthesia, a rat stomach was mounted on a chamber and perfused with saline, and the secretion of HCO(3)(-) was measured at pH 7.0 using a pH-stat method and by adding 2 mM HCl. Acidification of the normal stomach with 100 mM HCl increased HCO(3)(-) secretion, and this response was totally inhibited by pretreatment with indomethacin but not N(G)-nitro-l-arginine methyl ester (l-NAME) or chemical ablation of capsaicin-sensitive afferent neurons. Exposure of the stomach to 0.5 M NaCl deranged the unstirred mucus gel layer without damaging the surface epithelial cells. The stomach responded to 0.5 M NaCl by secreting slightly more HCO(3)(-), in an indomethacin-inhibitable manner, and responded to even 10 mM HCl with a marked rise in HCO(3)(-) secretion, although 10 mM HCl did not have an effect in the normal stomach. The acid-induced HCO(3)(-) response in the NaCl-treated stomach was significantly but partially attenuated by indomethacin, l-NAME, or sensory deafferentation and was totally abolished when these treatments were combined. These results suggest that gastric HCO(3)(-) secretion in response to acid is regulated by two independent mechanisms, one mediated by prostaglandins (PGs) and the other by sensory neurons and nitric oxide (NO). The acid-induced HCO(3)(-) secretion in the normal stomach is totally mediated by endogenous PGs, but, when the stomach is made slightly permeable to acid, the response is markedly facilitated by sensory neurons and NO.     

A novel method for construction of gene fragment library to searching epitopes

Identification of the epitope sequence or the functional domain of proteins is a laborious process but a necessary one for biochemical and immunological research. To achieve intensive and effective screening of these functional peptides in various molecules, we established a novel screening method using a phage library system that displays various lengths and parts of peptides derived from target protein. Applying this library for epitope mapping, epitope peptide was more efficiently identified from gene fragment library than conventional random peptide library. Our system may be a most powerful method for identifying functional peptides.     

Shootin1: A protein involved in the organization of an asymmetric signal for neuronal polarization

Neurons have the remarkable ability to polarize even in symmetrical in vitro environments. Although recent studies have shown that asymmetric intracellular signals can induce neuronal polarization, it remains unclear how these polarized signals are organized without asymmetric cues. We describe a novel protein, named shootin1, that became up-regulated during polarization of hippocampal neurons and began fluctuating accumulation among multiple neurites. Eventually, shootin1 accumulated asymmetrically in a single neurite, which led to axon induction for polarization. Disturbing the asymmetric organization of shootin1 by excess shootin1 disrupted polarization, whereas repressing shootin1 expression inhibited polarization. Overexpression and RNA interference data suggest that shootin1 is required for spatially localized phosphoinositide-3-kinase activity. Shootin1 was transported anterogradely to the growth cones and diffused back to the soma; inhibiting this transport prevented its asymmetric accumulation in neurons. We propose that shootin1 is involved in the generation of internal asymmetric signals required for neuronal polarization.     

Functional characterization of human and cynomolgus monkey UDP-glucuronosyltransferase 1A6 enzymes

UDP-glucuronosyltransferase 1A6 (UGT1A6) is a major isoform in the human liver that glucuronidates numerous drugs, environmental chemicals and endogenous substrates. In this study, human and cynomolgus monkey UGT1A6 cDNAs (humUGT1A6 and monUGT1A6, respectively) were cloned, and the corresponding proteins were heterologously expressed in yeast cells to identify the functions of primate UGT1A6s. The enzymatic properties of UGT1A6 proteins were characterized by the kinetic analysis of serotonin (5-hydroxytryptamine, 5-HT) and 4-methylumbelliferone (4-MU) glucuronidation. humUGT1A6 and monUGT1A6 showed 96% identity in their nucleotide and amino acid sequences. Immunoblotting analysis using an antibody raised against human UGT1A6 showed that protein staining intensities were different between human and cynomolgus monkey UGT1A6 enzymes in microsomal fractions from livers and yeast cells, although both enzymes were detectable. The apparent K(m) value (15 mM) for 5-HT glucuronidation of cynomolgus monkey liver microsomes was significantly higher than that (8.6mM) of human liver microsomes, whereas V(max) values were lower in cynomolgus monkeys (2.8 nmol/min/mg protein) than in humans (8.6 nmol/min/mg protein). No significant species difference was observed in K(m) (approximately 90 microM) or V(max) (approximately 25 nmol/min/mg protein) values for liver microsomal 4-MU glucuronidation. In yeast cell microsomes, K(m) values (approximately 6mM) for 5-HT glucuronidation by recombinant UGT1A6s were similar, while a V(max) value (0.1nmol/min/mg protein) of monUGT1A6 was significantly lower than that (0.7 nmol/min/mg protein) of humUGT1A6. In 4-MU glucuronidation, both K(m) (210 microM) and V(max) (3.5 nmol/min/mg protein) values of monUGT1A6 were significantly higher than those of humUGT1A6 (K(m), 110 microM; V(max), 1.5nmol/min/mg protein). These findings suggest that the enzymatic properties of UGT1A6 were extensively different between humans and cynomolgus monkeys, although humUGT1A6 and monUGT1A6 showed high homology at the amino acid level. The information gained in this study should help with in vivo extrapolation and to assess the toxicity of xenobiotics.     

Cephalometric study of elderly with nearly intact dental arches

Objective: The purpose of this study was to investigate the craniofacial morphology of elderly people with many remaining teeth using cephalometric analysis. Subjects and methods: The subjects were 30 Japanese elderly who participated in the ‘8020 campaign 2001’ in Bunkyo Ward, Tokyo, organised by The Dental Association of Tokyo, as well as 30 Japanese young adults with normal occlusion. Lateral cephalograms of all subjects were analysed using the Coben method. Results: In the female elderly group, the lower face depth was smaller than in the younger adults. In the male elderly group, the height and depth of both the total face and the lower face were longer than in the younger group. In comparing the 8020 achievers with the younger group, the proportion of the lower facial height was greater than the upper facial height, and this finding was more pronounced in women than in men. Conclusion: For the lateral facial pattern of the elderly, a reduction of lower facial height because of tooth occlusal reduction was not apparent. It was clear that there are age differences for males and females; in addition, differences in the total face and lower face area of the elderly group were due to their having many remaining teeth over a long time period. Also, these changes were more apparent in women than in men, and it is clear that there is a male–female difference in ageing.     

In vitro reconstitution of the GTPase-associated centre of the archaebacterial ribosome: the functional features observed in a hybrid form with Escherichia coli 50S subunits

We cloned the genes encoding the ribosomal proteins Ph (Pyrococcus horikoshii)-P0, Ph-L12 and Ph-L11, which constitute the GTPase-associated centre of the archaebacterium Pyrococcus horikoshii. These proteins are homologues of the eukaryotic P0, P1/P2 and eL12 proteins, and correspond to Escherichia coli L10, L7/L12 and L11 proteins respectively. The proteins and the truncation mutants of Ph-P0 were overexpressed in E. coli cells and used for in vitro assembly on to the conserved domain around position 1070 of 23S rRNA (E. coli numbering). Ph-L12 tightly associated as a homodimer and bound to the C-terminal half of Ph-P0. The Ph-P0.Ph-L12 complex and Ph-L11 bound to the 1070 rRNA fragments from the three biological kingdoms in the same manner as the equivalent proteins of eukaryotic and eubacterial ribosomes. The Ph-P0.Ph-L12 complex and Ph-L11 could replace L10.L7/L12 and L11 respectively, on the E. coli 50S subunit in vitro. The resultant hybrid ribosome was accessible for eukaryotic, as well as archaebacterial elongation factors, but not for prokaryotic elongation factors. The GTPase and polyphenylalanine-synthetic activity that is dependent on eukaryotic elongation factors was comparable with that of the hybrid ribosomes carrying the eukaryotic ribosomal proteins. The results suggest that the archaebacterial proteins, including the Ph-L12 homodimer, are functionally accessible to eukaryotic translation factors.     

Proteins associated with soluble adenylyl cyclase in sea urchin sperm flagella

Adenylyl cyclases (ACs) synthesize cAMP and are present in cells as transmembrane AC and soluble AC (sAC). In sperm, the cAMP produced regulates ion channels and it also activates protein kinase-A that in turn phosphorylates specific axonemal proteins to activate flagellar motility. In mammalian sperm, sAC localizes to the midpiece of flagella, whereas in sea urchin sperm sAC is along the entire flagellum. Here we show that in sea urchin sperm, sAC is complexed with proteins of the plasma membrane and axoneme. Immunoprecipitation shows that a minimum of 10 proteins is tightly associated with sAC. Mass spectrometry of peptides derived from these proteins shows them to be: axonemal dynein heavy chains 7 and 9, sperm specific Na+/H+ exchanger, cyclic nucleotide-gated ion channel, sperm specific creatine kinase, membrane bound guanylyl cyclase, cyclic GMP specific phosphodiesterase 5A, the receptor for the egg peptide speract, and alpha- and beta-tubulins. The sAC-associated proteins could be important in linking membrane signal transduction to energy utilisation in the regulation of flagellar motility.     

Functional consequences of stenophylly for leaf productivity: comparison of the anatomy and physiology of a rheophyte, Farfugium japonicum var. luchuence, and a related non-rheophyte, F. japonicum (Asteraceae)

We investigated the anatomical and physiological characteristics of stenophyllous leaves of a rheophyte, Farfugium japonicum var. luchuence, and sun and shade leaves of a non-rheophyte, F. japonicum, comparing three different populations from coastal, forest floor, and riparian habitats. Light adaptation resulted in smaller leaves, and riparian adaptation resulted in narrower leaves (stenophylly). The light-saturated rate of photosynthesis (P _max) per unit leaf area corresponded to the light availability of the habitat. Irrespective of leaf size, the P _max per unit leaf mass was similar for sun and shade leaves. However, the P _max per mass of stenophyllous leaves was significantly lower than that of sun and shade leaves. This was because the number and size of mesophyll cells were greater than that required for intercellular CO₂ diffusion, which resulted in a larger leaf mass per unit leaf area. Higher cell density increases contact between mesophyll cells and enhances leaf toughness. Stenophyllous leaves of the rheophyte are frequently exposed to a strong water flow when the water level rises, suggesting a mechanical constraint caused by physical stress.