Molecular clearance of ataxin-3 is regulated by a mammalian E4

Insoluble aggregates of polyglutamine-containing proteins are usually conjugated with ubiquitin in neurons of individuals with polyglutamine diseases. We now show that ataxin-3, in which the abnormal expansion of a polyglutamine tract is responsible for spinocerebellar ataxia type 3 (SCA3), undergoes ubiquitylation and degradation by the proteasome. Mammalian E4B (UFD2a), a ubiquitin chain assembly factor (E4), copurified with the polyubiquitylation activity for ataxin-3. E4B interacted with, and thereby mediated polyubiquitylation of, ataxin-3. Expression of E4B promoted degradation of a pathological form of ataxin-3. In contrast, a dominant-negative mutant of E4B inhibited degradation of this form of ataxin-3, resulting in the formation of intracellular aggregates. In a Drosophila model of SCA3, expression of E4B suppressed the neurodegeneration induced by an ataxin-3 mutant. These observations suggest that E4 is a rate-limiting factor in the degradation of pathological forms of ataxin-3, and that targeted expression of E4B is a potential gene therapy for SCA3.     

Phylogenetic conservation of a limb-specific, <Emphasis Type="Italic">cis</Emphasis>-acting regulator of Sonic hedgehog (<Emphasis Type="Italic">Shh</Emphasis>)

Polarized expression of the Sonic hedgehog (Shh) gene in the posterior mesenchyme is essential for pattern formation in the appendages of higher vertebrates, from teleost fins to tetrapod limb buds. We report on a sequence in intron 5 of the Lmbr1 gene, which resides approximately 1 Mb from the Shh coding region in the mouse genome and is highly conserved among teleost fishes and throughout the tetrapod lineage. Positional cloning revealed that two mouse mutations, Hx and M100081, characterized by mirror-image digit duplication and ectopic anterior Shh expression, have base substitutions in this sequence. Absence of the conserved sequence in limbless reptiles and amphibians and a cis-trans test using the Hx and Shh KO alleles suggest that the sequence is a cis-acting regulator that controls the polarized expression of Shh. The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession number: AB092986 to AB093004, AB093207, and AB114903.     

Effect of N-linked glycosylation on the aspartic proteinase porcine pepsin expressed from Pichia pastoris

A study was undertaken to examine the effects of N-linked glycosylation on the structure-function of porcine pepsin. The N-linked motif was incorporated into four sites (two on the N-terminal domain and two on the C-terminal domain), and the recombinant protein expressed using Pichia pastoris. All four N-linked recombinants exhibited similar secondary and tertiary structure to nonglycosylated pepsin, that is, wild type. Similar K(m) values were observed, but catalytic efficiencies were approximately one-third for all mutants compared with the wild type; however, substrate specificity was not altered. Activation of pepsinogen to pepsin occurred between pH 1.0 to 4.0 for wild-type pepsin, whereas the glycosylated recombinants activated over a wider range, pH 1.0 to 6.0. Glycosylation on the C-terminal domain exhibited similar pH activity profiles to nonglycosylated pepsin, and glycosylation on the N-domain resulted in a change in activity profile. Overall, glycosylation on the C-domain led to a more global stabilization of the structure, which translated into enzymatic stability, whereas on the N-domain, an increase in structural stability had little effect on enzymatic stability. Finally, glycosylation on the flexible loop region also appeared to increase the overall structural stability of the protein compared with wild type. It is postulated that the presence of the carbohydrate residues added rigidity to the protein structure by reducing conformational mobility of the protein, thereby increasing the structural stability of the protein.     

Tol1, a fission yeast phosphomonoesterase, is an in vivo target of lithium, and its deletion leads to sulfite auxotrophy

Lithium is the drug of choice for the treatment of bipolar affective disorder. The identification of an in vivo target of lithium in fission yeast as a model organism may help in the understanding of lithium therapy. For this purpose, we have isolated genes whose overexpression improved cell growth under high LiCl concentrations. Overexpression of tol1(+), one of the isolated genes, increased the tolerance of wild-type yeast cells for LiCl but not for NaCl. tol1(+) encodes a member of the lithium-sensitive phosphomonoesterase protein family, and it exerts dual enzymatic activities, 3'(2'),5'-bisphosphate nucleotidase and inositol polyphosphate 1-phosphatase. tol1(+) gene-disrupted cells required high concentrations of sulfite in the medium for growth. Consistently, sulfite repressed the sulfate assimilation pathway in fission yeast. However, tol1(+) gene-disrupted cells could not fully recover from their growth defect and abnormal morphology even when the medium was supplemented with sulfite, suggesting the possible implication of inositol polyphosphate 1-phosphatase activity for cell growth and morphology. Given the remarkable functional conservation of the lithium-sensitive dual-specificity phosphomonoesterase between fission yeast and higher-eukaryotic cells during evolution, it may represent a likely in vivo target of lithium action across many species.     

Development of a new instrument to measure oxygen saturation and total hemoglobin volume in local skin by near-infrared spectroscopy and its clinical application

The oxygen saturation (StO₂) and total hemoglobin volume in cutaneous blood are closely related to cutaneous metabolism and are important factors in determining the skin color. Most conventional apparatuses for the measurement of cutaneous metabolism have been designed to evaluate qualitative changes in the oxyhemoglobin volume, deoxyhemoglobin volume, and their sum (total Hb volume) relative to their baseline values. In this study, we developed an instrument for non-invasive evaluation of individual and regional differences in StO₂ and Hb volume, a system unaffected by melanin (Kao PSA system model III), and examined the validity of its application. First, changes in StO₂ and total Hb volume in the antebrachial region during venous occlusion and devascularization by compression of the brachial region were evaluated. Changes in total Hb volume following venous occlusion were found to reflect the cutaneous blood flow. Also, StO₂ was considered to reflect the state of oxygen consumption by the skin, because it was markedly reduced during devascularization. Next, the subjects were exposed to graded hypobaric conditions, and the relationships among StO₂, arterial blood oxygen saturation (SaO₂), and venous blood oxygen saturation (SvO₂) were studied. StO₂ showed significant positive correlations with SaO₂ (r=0.811, P<0.001) and SvO₂ (r=0.966, P<0.001), and its correlation with SvO₂ was particularly strong. Therefore, StO₂ was found to be closely dependent on SvO₂. Lastly, StO₂, total Hb volume, and other parameters were measured in healthy women (aged 20–69 years), and their regional differences and age- associated changes were evaluated. These regional differences (angle of mouth > cheek > forehead) and age-associated decreases in StO₂ are considered to be caused by the age-associated decreases in the cutaneous blood flow. Received: 21 May 1999 / Revised: 19 November 1999 / Accepted: 24 November 1999     

Diverse effects of hydrogen peroxide on cytosolic Ca2+ homeostasis in rat pancreatic beta-cells

Oxygen-free radicals are thought to be a major cause of beta-cell dysfunction in diabetic animals induced by alloxan or streptozotocin. We evaluated the effect of H2O2 on cytosolic Ca2+ concentration ([Ca2+]i) and the activity of ATP-sensitive potassium (K+ATP) channels in isolated rat pancreatic beta-cells using microfluorometry and patch clamp techniques. Exposure to 0.1 mM H2O2 in the presence of 2.8 mM glucose increased [Ca2+]i from 114.3+/-15.4 nM to 531.1+/-71.9 nM (n=6) and also increased frequency of K+ATP channel openings. The intensity of NAD(P)H autofluorescence was conversely reduced, suggesting that H2O2 inhibited the cellular metabolism. These three types of cellular parameters were reversed to the control level on washout of H2O2, followed by a transient increase in [Ca2+]i, the transient inhibition of K+ATP channels associated with action currents and increase of the NAD(P)H intensity with an overshoot. In the absence of external Ca2+, 0.1 mM H2O2 increased [Ca2+]i from 88.8+/-7.2 nM to 134.6+/-8.3 nM. Magnitude of [Ca2+]i increase induced by 0.1 mM H2O2 was decreased after treatment of cells with 0.5 mM thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ pump (45.8+/-4.9 nM vs 15.0+/-4.8 nM). Small increase in [Ca2+]i in response to an increase of external Ca2+ from zero to 2 mM was further facilitated by 0.1 mM H2O2 (330.5+/-122.7 nM). We concluded that H2O2 not only activates K+ATP channels in association with metabolic inhibition, but also increases partly the Ca2+ permeability of the thapsigargin-sensitive intracellular stores and of the plasma membrane in pancreatic beta-cells.     

Galanin-like peptide and ghrelin increase cytosolic Ca2+ in neurons containing growth hormone-releasing hormone in the arcuate nucleus

Galanin-like peptide (GALP), discovered in the porcine hypothalamus, is expressed predominantly in the arcuate nucleus (ARC), a feeding-controlling center. Intracerebroventricular injection of GALP has been shown to stimulate food intake in the rats. However, the mechanisms underlying the orexigenic effect of GALP are unknown. The present study aimed to determine the target neurons of GALP in the ARC. We investigated the effects of GALP on cytosolic free Ca2+ concentration ([Ca2+]i) in the neurons isolated from the rat ARC, followed by neurochemical identification of these neurons by immunocytochemistry using antisera against growth hormone-releasing hormone (GHRH), neuropeptide Y (NPY) and proopiomelanocortin (POMC), the peptides localized in the ARC. GALP at 10(-10) M increased [Ca2+]i in 11% of single neurons of the ARC, while ghrelin, an orexigenic and GH-releasing peptide, at 10(-10) M increased [Ca2+]i in 35% of the ARC neurons. Some of these GALP- and/or ghrelin-responsive neurons were proved to contain GHRH. In contrast, NPY- and POMC-containing neurons did not respond to GALP. These results indicate that GALP directly targets GHRH neurons, but not NPY and POMC neurons, and that ghrelin directly targets GHRH neurons in the ARC. The former action may be involved in the orexigenic effect of GALP and the latter in the GH-releasing and/or orexigenic effects ghrelin.     

Oleic acid interacts with GPR40 to induce Ca2+ signaling in rat islet beta-cells: mediation by PLC and L-type Ca2+ channel and link to insulin release

It has long been thought that long-chain free fatty acids (FFAs) stimulate insulin secretion via mechanisms involving their metabolism in pancreatic beta-cells. Recently, it was reported that FFAs function as endogenous ligands for GPR40, a G protein-coupled receptor, to amplify glucose-stimulated insulin secretion in an insulinoma cell line and rat islets. However, signal transduction mechanisms for GPR40 in beta-cells are little known. The present study was aimed at elucidating GPR40-linked Ca(2+) signaling mechanisms in rat pancreatic beta-cells. We employed oleic acid (OA), an FFA that has a high affinity for the rat GPR40, and examined its effect on cytosolic Ca(2+) concentration ([Ca(2+)](i)) in single beta-cells by fura 2 fluorescence imaging. OA at 1-10 microM concentration-dependently increased [Ca(2+)](i) in the presence of 5.6, 8.3, and 11.2 mM, but not 2.8 mM, glucose. OA-induced [Ca(2+)](i) increases at 11.2 mM glucose were inhibited in beta-cells transfected with small interfering RNA targeted to rat GPR40 mRNA. OA-induced [Ca(2+)](i) increases were also inhibited by phospholipase C (PLC) inhibitors, U73122 and neomycin, Ca(2+)-free conditions, and an L-type Ca(2+) channel blocker, nitrendipine. Furthermore, OA increased insulin release from isolated islets at 8.3 mM glucose, and it was markedly attenuated by PLC and L-type Ca(2+) channel inhibitors. These results demonstrate that OA interacts with GPR40 to increase [Ca(2+)](i) via PLC- and L-type Ca(2+) channel-mediated pathway in rat islet beta-cells, which may be link to insulin release.     

Serum evaluation of the balance between soluble interleukin-2 and interleukin-4 receptors

To elucidate the usefulness of the simultaneous analysis of the multiple kinds of soluble cytokine receptors, we determined both the soluble interleukin 2 receptor (sIL-2R, Th1-type cytokine receptor) and the soluble interleukin 4 receptor (sIL-4R, Th2-type cytokine receptor) levels in the sera of healthy subjects as reference values and preliminarily applied to evaluate the patients with diarrhea positive (D+) hemolytic uremic syndrome (HUS) as the diagnostic parameter of the severity. Both sIL-2R and sIL-4R levels in the sera of healthy children were significantly higher than those of healthy adults (p<0.01). The serum sIL-2R level of the patients with severe HUS (n=4) was higher than that of the patients with mild/moderate HUS (n=6) at the initial stage (p<0.01) or healthy children (n=51, p<0.01). Whereas, the serum sIL-4R level of both the severe and mild/moderate groups was lower than that of the healthy control children, although there was no significant difference among the three groups. Namely, the soluble receptor balance (sIL-2R/sIL-4R) in the patients with severe HUS may shift. We considered that the evaluation of the balance between soluble cytokine receptors might be informative for the evaluation of the immune states, as well as the conventional cytokine balance (Th1/Th2).     

Change in content of sugars and free amino acids in potato tubers under short-term storage at low temperature and the effect on acrylamide level after frying

Changes in the sugar and amino acid contents of potato tubers during short-term storage and the effect on the acrylamide level in chips after frying were investigated. The acrylamide content in chips began to increase after 3 days of storage at 2 degrees C in response to the increase of glucose and fructose contents in the tubers. There was strong correlation between the reducing sugar content and acrylamide level, R(2)=0.873 for fructose and R(2)=0.836 for glucose. The sucrose content had less correlation with the acrylamide content because of its decrease after 4 weeks of storage at 2 degrees C, while the reducing sugar in potato tubers and the acrylamide in chips continued to increase. The contents of the four amino acids, i.e., asparatic acid, asparagine, glutamic acid and glutamine, showed no significant correlation with the acrylamide level. These results suggest that the content of reducing sugars in potato tubers determined the degree of acrylamide formation in chips. The chip color, as evaluated by L* (lightness), was correlated well with the acrylamide content.