Proteomic identification of protein targets for 15-deoxy-Δ(12,14)-prostaglandin J2 in neuronal plasma membrane

15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) is one of factors contributed to the neurotoxicity of amyloid β (Aβ), a causative protein of Alzheimer's disease. Type 2 receptor for prostaglandin D(2) (DP2) and peroxysome-proliferator activated receptorγ (PPARγ) are identified as the membrane receptor and the nuclear receptor for 15d-PGJ(2), respectively. Previously, we reported that the cytotoxicity of 15d-PGJ(2) was independent of DP2 and PPARγ, and suggested that 15d-PGJ(2) induced apoptosis through the novel specific binding sites of 15d-PGJ(2) different from DP2 and PPARγ. To relate the cytotoxicity of 15d-PGJ(2) to amyloidoses, we performed binding assay [(3)H]15d-PGJ(2) and specified targets for 15d-PGJ(2) associated with cytotoxicity. In the various cell lines, there was a close correlation between the susceptibilities to 15d-PGJ(2) and fibrillar Aβ. Specific binding sites of [(3)H]15d-PGJ(2) were detected in rat cortical neurons and human bronchial smooth muscle cells. When the binding assay was performed in subcellular fractions of neurons, the specific binding sites of [(3)H]15d-PGJ(2) were detected in plasma membrane, nuclear and cytosol, but not in microsome. A proteomic approach was used to identify protein targets for 15d-PGJ(2) in the plasma membrane. By using biotinylated 15d-PGJ(2), eleven proteins were identified as biotin-positive spots and classified into three different functional proteins: glycolytic enzymes (Enolase2, pyruvate kinase M1 (PKM1) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)), molecular chaperones (heat shock protein 8 and T-complex protein 1 subunit α), cytoskeletal proteins (Actin β, F-actin-capping protein, Tubulin β and Internexin α). GAPDH, PKM1 and Tubulin β are Aβ-interacting proteins. Thus, the present study suggested that 15d-PGJ(2) plays an important role in amyloidoses not only in the central nervous system but also in the peripheral tissues.     

Cell membrane dynamics and the induction of apoptosis by lipid compounds

To investigate the induction of apoptosis by some lipid compounds which are a potent inducer of apoptosis, the plasma membrane fluidity of U937 cells was measured using the fluorescent probe, pyrene. The increase of the membrane fluidity was observed immediately after the treatment of cells with lipid inducers. We also found that the trigger of apoptosis was pulled within 30 min after treatment. Data from the dynamic light scattering experiment indicated that lipid inducers were dissolved to form the emulsion. At the very early stage of apoptosis, possibly, the well-controlled transfer of lipid inducers from the emulsion to the lipid layer of cells can bring about the increase of membrane dynamics which might lead to the induction of apoptosis.     

Geranylated flavanones from the secretion on the surface of the immature fruits of Paulownia tomentosa

Chemical investigation of the methanol extract of the viscous secretion on the surface of immature fruits of Paulownia tomentosa furnished nine geranylated flavanones, 6-geranyl-5,7-dihydroxy-3',4'-dimethoxyflavanone (1), 6-geranyl-3',5,7-trihydroxy-4'-methoxyflavanone (2), 6-geranyl-4',5,7-trihydroxy-3',5'-dimethoxyflavanone (3), 6-geranyl-4',5,5',7-tetrahydroxy-3'-methoxyflavanone (4), 6-geranyl-3,3',5,7-tetrahydroxy-4'-methoxyflavanone (5), 4',5,5',7-tetrahydroxy-6-[6-hydroxy-3,7-dimethyl-2(E),7-octadienyl]-3'-methoxyflavanone (6), 3,3',4',5,7-pentahydroxy-6-[6-hydroxy-3,7-dimethyl-2(E),7-octadienyl]flavanone (7), 3,3',4',5,7-pentahydroxy-6-[7-hydroxy-3,7-dimethyl-2(E)-octenyl]flavanone (8), and 3,4',5,5',7-pentahydroxy-3'-methoxy-6-(3-methyl-2-butenyl)flavanone (9), along with six known geranylated flavanones. Among these, compounds 4, 6-9 and the known 6-geranyl-3',4',5,7-tetraahydroxyflavanone (diplacone), 6-geranyl-3,3',4',5,7-pentahydroxyflavanone (diplacol) and 3',4',5,7-pentahydroxy-6-[7-hydroxy-3,7-dimethyl-2(E)-octenyl]flavanone showed potent radical scavenging effects towards DPPH radicals.     

The polyglycine and polyglutamine repeats in the androgen receptor gene in Japanese and Caucasian populations

Human androgen receptor (AR) gene contains two polymorphic trinucleotide repeats of CAG and GGC, which code for polyglutamine and polyglycine tracts in the N-terminal domain in which the receptor activity resides. Longer repeats induce decrease of transactivation function in the AR receptor, weaken an anti-proliferative effect on various steroid-related tissues, and may promote the carcinogenesis of these cancers, such as breast, endometrial, and ovarian cancers. However, the incidences of these steroid-related cancers are remarkably lower in Japanese than in Caucasians. We hypothesize that the GGC and CAG repeats in AR gene correspond to lower incidence of steroid-related cancers in the Japanese population. To test this hypothesis, these two polymorphic trinucleotide repeats in AR gene were genotyped in 221 Japanese and 177 Caucasians. The results of genotyping in these loci clearly show that the distribution of GGC repeat is significantly different between these populations (P<0.001). Japanese (73.7%) had 16 GGC repeats compared to 53.3% for Caucasians. Japanese (3.8%) also had 17 GGC repeats compared to 36.2% for Caucasians. No Japanese had more than 18 GGC repeats compared to 3.4% for Caucasians. The length of CAG repeats in the Japanese population was not significantly different than that of the Caucasian population, although the CAG repeats varied from 14 to 31 and 15 to 29 repeats in Japanese and German populations, respectively. This study demonstrates that the Japanese population has shorter GGC compared to the Caucasian population, which may explain the incidences of estrogen-related cancers in these populations.     

Localization of sepiapterin reductase in pigment cells of Oryzias latipes

Body colors of poikilothermal vertebrates are derived from three distinct types of pigment cells, melanophores, erythro/xanthophores and irido/leucophores. It is well known that melanin in melanophores is synthesized by tyrosinase within a specific organelle termed the melanosome. Although sepiapterin reductase (SPR) is an important enzyme involved in metabolizing biopterin and sepiapterin (a conspicuous pteridine as a coloring pigment in xanthophores) the distribution of SPR has not been shown in pigment cells. An antibody raised in rabbits against rat SPR was used to demonstrate the presence of SPR in pigment cells of Oryzias latipes. This study, which used immunohistochemistry with fluorescence or peroxidase/diaminobenzidine as markers, revealed that SPR could be detected readily in xanthophores, but only faintly in melanophores. These results suggest that sepiapterin is metabolized within xanthophores. Moreover, these experiments show that a protein sharing immunological cross-reactivity with rat SPR is located in teleost O. latipes xanthophores, which is significant considering the relationship of pteridine metabolism between poikilothermal vertebrates and mammals. Further progress in investigations of the roles of pteridines in vertebrates will be promoted by using these fish which can be bred in mass rather easily in the laboratory.     

Role of mitochondrial phosphate carrier in metabolism-secretion coupling in rat insulinoma cell line INS-1

In pancreatic β-cells, glucose-induced mitochondrial ATP production plays an important role in insulin secretion. The mitochondrial phosphate carrier PiC is a member of the SLC25 (solute carrier family 25) family and transports Pi from the cytosol into the mitochondrial matrix. Since intramitochondrial Pi is an essential substrate for mitochondrial ATP production by complex V (ATP synthase) and affects the activity of the respiratory chain, Pi transport via PiC may be a rate-limiting step for ATP production. We evaluated the role of PiC in metabolism-secretion coupling in pancreatic β-cells using INS-1 cells manipulated to reduce PiC expression by siRNA (small interfering RNA). Consequent reduction of the PiC protein level decreased glucose (10 mM)-stimulated insulin secretion, the ATP:ADP ratio in the presence of 10 mM glucose and elevation of intracellular calcium concentration in response to 10 mM glucose without affecting the mitochondrial membrane potential (Δψm) in INS-1 cells. In experiments using the mitochondrial fraction of INS-1 cells in the presence of 1 mM succinate, PiC down-regulation decreased ATP production at various Pi concentrations ranging from 0.001 to 10 mM, but did not affect Δψm at 3 mM Pi. In conclusion, the Pi supply to mitochondria via PiC plays a critical role in ATP production and metabolism-secretion coupling in INS-1 cells.     

Effects of Cl2MDP-encapsulating liposomes in a murine model of Pseudomonas aeruginosa-induced sepsis

Pseudomonas aeruginosa is a pathogen that frequently causes acute lung injury, bacteremia and sepsis in critically ill patients. As tissue macrophages are a major producer of inflammatory mediators that contribute to septic physiology, and are essential for eliminating bacteria from the circulation, we investigated the role of tissue macrophages in the generation of both inflammatory and anti-inflammatory cytokines in septic shock by using our mouse model of P. aeruginosa pneumonia. To see the effects of tissue macrophage depletion, we intravenously injected dichloromethylene-diphosphonate (Cl2MDP)-encapsulating liposomes in mice. Two days after the liposome injection, we instilled cytotoxic P. aeruginosa (PA103) into the lung that disseminates and causes septic shock. After the infection, we collected blood and bronchoalveolar lavage fluids. The samples were then analyzed for TNF-alpha, MIP-2, and IL-10 concentration. We compared these results to control mice that received either liposomes without Cl2MDP or phosphate buffered saline alone. Plasma TNF-alpha, MIP-2, and IL-10 levels were significantly decreased in the tissue macrophage-depleted mice compared to the control groups of mice. Although depletion of tissue macrophages by Cl2MDP-liposome administration did not affect the severity of bacteremia or the survival of infected mice, these results imply that tissue macrophages have a major role in the production of both proinflammatory and anti-inflammatory cytokines in the circulation and in the causing septic physiology associated with P. aeruginosa pneumonia.     

Design,synthesis, and conformation of a model peptide of endothelin with cystine-stabilized α-helix motif

A model 16-peptide of endothelin-1 (MET-1), which has the minimized sequence homology to the corresponding pan of endothelin-1 (ET-1), was designed to confirm the cystine-stabilized α-helix motif. The model structure consists of an extended structure, a β-turn part, and an α-helix structure that is stabilized by two disulfide bonds. The α-helix segment was designed to emphasize the amphiphilic nature. In order to combine the extended structure and the α-helix segment, a D -Ala-Pro sequence was selected to fix the β-turn. The model endothelin 16-peptide amide was synthesized by solid-phase synthesis on a 4-methylbenzhydrylamine resin. Its conformation was examined by CD and two-dimensional (2D) ¹H-nmr measurements. MET-1 showed similar CD patterns to ET-1 in both buffer and 50% aqueous trifluoroethanol solution. The 2D nmr experiments in 50% aqueous ethylene glycol revealed that MET-1 closely resembles the conformation of ET-1 with an extended structure, an α-helix, and a β-turn unit in the same position of the sequence. Furthermore, model peptides without disulfide bond(s) could not assume a stable structure in aqueous solution, while they did have similar α-helical content in 50% trifluoroethanol with MET-1. When the two disulfide bridges were simultaneously formed, the peptide with the correct disulfide bonds (MET-1) was obtained in threefold excess to the isomer (apamin type. MET-2). These findings obtained by the modeling of ET-1 showed an important role for the stabilization of peptide conformation with disulfide bonds. © 1994 John Wiley & Sons, Inc.     

Substrate Specificity of Nuclease P1

Nuclease P₁ cleaved substantially all phosphodiester bonds in rRNA, tRNA, poly(I), poly(U), poly(A), poly(C), poly(G), poly(I)·poly(C), native DNA and heat-denatured DNA to produce exclusively 5′-mononucleotides. Single-stranded polynucleotides were much more susceptible than double-stranded ones. Influence of pH and ionic strength on the hydrolysis rate significantly varied with the kind of polynucleotides. The enzyme also hydrolyzed 3′-phosphomonoester bonds in 3′-AMP, 3′-GMP, 3′-UMP, 3′-CMP, 3′-dAMP, 3′-dGMP, 3′-dCMP and 3′-dTMP. Ribonucleoside 3′-monophosphates were hydrolyzed 20 to 50 times faster than the corresponding 3′-deoxyribonucleotides. Base preference of the enzyme for 3′-ribonucleotides was in the order of G>A>C≧U, whereas that for 3′-deoxyribo-nucleotides was in the order of C≧T>A≧G. The 3′-phosphomonoester bonds in nucleoside 3′, 5′-diphosphates, coenzyme A and dinucleotides bearing 3′-phosphate were hydrolyzed at a rate similar to that for the corresponding 3′-mononucleotides. Adenosine 2′-monophosphate was highly resistant, being split at less than 1/3,000 the rate at which 3′-AMP was split.     

Normalization of phospholipids concentration of the gastric mucosa was observed in patients with peptic ulcer after eradication of Helicobacter pylori

Background. Phospholipids concentration in the gastric mucosa decreased in patients with Helicobacter pylori infection. The aim of this study is to examine the effects of eradication of H. pylori on decreasing the phospholipids concentration in the gastric mucosa in patients with gastric or duodenal ulcer. Materials and Methods. Phospholipids (phosphatidylcholine, phosphatidylethanolamine, and sphingonomyeline) were measured in biopsy specimens from the antrum and corpus using thin‐layer chromatography. In H. pylori positive patients with gastric ulcer (n = 26) and duodenal ulcer (n = 13), and H. pylori negative controls (n = 20), the biopsy specimens were obtained before and 3 months after eradication. Eradication was performed using lansoprazole, amoxycillin, and clarithromycin. Results. Compared with the H. pylori negative control group, the concentrations of phosphatidylcholine and phosphatidylethanolamine decreased significantly in the gastric ulcer group in both antrum and corpus mucosa, and in the duodenal ulcer group in antrum mucosa. This decrease returned to the control level after eradication. Conclusions. This study demonstrates that the eradication of H. pylori in patients with peptic ulcer normalized the decrease of phosphatidylcholine and phosphatidylethanolamine in the gastric mucosa.