BBA - General Subjects (v.1790, #1)

The S. cerevisiae α-factor receptor, Ste2p, is a G-protein coupled receptor that plays key roles in yeast signaling and mating. Oligomerization of Ste2p has previously been shown to be important for intracellular trafficking, receptor processing and endocytosis. However the role of ligand in receptor oligomerization remains enigmatic.Using functional recombinant forms of purified Ste2p, atomic force microscopy, dynamic light scattering and chemical crosslinking are applied to investigate the role of ligand in Ste2p oligomerization.Atomic force microscopy images indicate a molecular height for recombinant Ste2p in the presence of α-factor nearly double that of Ste2p alone. This observation is supported by complementary dynamic light scattering measurements which indicate a ligand-induced increase in the polydispersity of the Ste2p hydrodynamic radius. Finally, chemical cross-linking of HEK293 plasma membranes presenting recombinant Ste2p indicates α-factor induced stabilization of the dimeric form and higher order oligomeric forms of the receptor upon SDS-PAGE analysis.α-factor induces oligomerization of Ste2p in vitro and in membrane.These results provide additional evidence of a possible role for ligand in mediation of Ste2p oligomerization in vivo.
Keywords: G-protein coupled receptor; Ste2p; Ligand-induced oligomerization; Chemical crosslinking; Dynamic light scattering; Atomic force microscopy;

Prolonged high glucose suppresses phorbol 12-myristate 13-acetate and ionomycin-induced interleukin-2 mRNA expression in Jurkat cells by Koji Higai; Masatoshi Tsukada; Yumiko Moriya; Yutaro Azuma; Kojiro Matsumoto (8-15).
The disturbance of immunological responses is a complication of diabetes mellitus.We cultured Jurkat cells in 11.1 (normal) and 22.2 mmol/l (high) glucose for 12 weeks and stimulated them with 10 nmol/l phorbol 12-myristate 13-acetate (PMA) and 500 nmol/l ionomycin. RT-PCR revealed that induced interleukin (IL)-2 mRNA expression levels were suppressed in high glucose cultures compared to those in normal glucose. Promoter activities of IL-2, nuclear factor of activated T cells (NFAT), and activator protein-1 (AP-1), after 6 h stimulation with PMA and ionomycin, gradually decreased in high glucose cultures to approximately 20% of those in normal glucose at 12 weeks. The prolonged culture in high glucose increased inducible cAMP early repressor (ICER) II mRNA and protein levels, and overexpression of ICER II dose-dependently suppressed promoter activities of IL-2, NFAT, and AP-1. Moreover, ICER II mRNA expression was transiently induced by stimulation with PMA and ionomycin in normal glucose cultures; however, with high glucose, the induction disappeared.These results indicate that ICER II protein accumulates during prolonged culture in high glucose and suppresses IL-2 mRNA expression in Jurkat cells.
Keywords: Interleukin-2; Nuclear factor of activated T cells; Inducible cAMP early repressor; High concentration of glucose;

(5R⁎) and (5S⁎) diastereomers of 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxyhydantoin (5-OH-dHyd) and 1-[2-deoxy-β-d-erythro-pentofuranosyl]-5-hydroxy-5-methylhydantoin (5-OH-5-Me-dHyd) are major oxidation products of 2′-deoxycytidine and thymidine respectively. If not repaired, when present in cellular DNA, these base lesions may be processed by DNA polymerases that induce mutagenic and cell lethality processes.Synthetic oligonucleotides that contained a unique 5-hydroxyhydantoin (5-OH-Hyd) or 5-hydroxy-5-methylhydantoin (5-OH-5-Me-Hyd) nucleobase were used as probes for repair studies involving several E. coli, yeast and human purified DNA N-glycosylases. Enzymatic reaction mixtures were analyzed by denaturing polyacrylamide gel electrophoresis after radiolabeling of DNA oligomers or by MALDI-TOF mass spectrometry measurements. In vitro DNA excision experiments carried out with endo III, endo VIII, Fpg, Ntg1 and Ntg2, show that both base lesions are substrates for these DNA N-glycosylases. The yeast and human Ogg1 proteins (yOgg1 and hOgg1 respectively) and E. coli AlkA were unable to cleave the N-glycosidic bond of the 5-OH-Hyd and 5-OH-5-Me-Hyd lesions. Comparison of the k cat /K m ratio reveals that 8-oxo-7,8-dihydroguanine is only a slightly better substrate than 5-OH-Hyd and 5-OH-5-Me-Hyd. The kinetic results obtained with endo III indicate that 5-OH-Hyd and 5-OH-5-Me-Hyd are much better substrates than 5-hydroxycytosine, a well known oxidized pyrimidine substrate for this DNA N-glycosylase.The present study supports a biological relevance of the base excision repair processes toward the hydantoin lesions, while the removal by the Fpg and endo III proteins are effected at better or comparable rates to that of the removal of 8-oxoGua and 5-OH-Cyt, two established cellular substrates.The study provides new insights into the substrate specificity of DNA N-glycosylases involved in the base excision repair of oxidized bases, together with complementary information on the biological role of hydantoin type lesions.
Keywords: Oxidized DNA base lesion; Hydantoin nucleobase; DNA N-glycosylase; Base excision repair; Modified oligodeoxyribonucleotide;

Inhibitory activity of salicylic acid on lipoxygenase-dependent lipid peroxidation by Domenico Lapenna; Giuliano Ciofani; Sante Donato Pierdomenico; Matteo Neri; Chiara Cuccurullo; Maria Adele Giamberardino; Franco Cuccurullo (25-30).
Since iron is essential for lipoxygenase activity and salicylic acid (SA) can interact with the metal, possible lipoxygenase inhibition by SA was investigated.Kinetic spectrophotometric evaluation of enzymatic lipid peroxidation catalyzed by soybean lipoxygenase (SLO), rabbit reticulocyte 15-lipoxygenase (RR15-LOX), porcine leukocyte 12-lipoxygenase (PL12-LOX) and human recombinant 5-lipoxygenase (HR5-LOX) with and without SA.SA inhibited linoleic, arachidonic and docosahexaenoic acid or human lipoprotein peroxidation catalyzed by SLO with IC50 of, respectively, 107, 153, 47 and 108 μM. Using the same substrates, SA inhibited RR15-LOX with IC50 of, respectively, 49, 63, 27 and 51 μM. Further, arachidonic acid peroxidation catalyzed by PL12-LOX and HR5-LOX was inhibited by SA with IC50 of 101 and 168 μM, respectively. Enzymatic inhibition was complete, reversible and non-competitive. Conceivably due to its lower hydrophobicity, aspirin was less effective, indicating acetylation-independent enzyme inhibition. SA and aspirin were ineffective peroxyl radical scavengers but readily reduced Fe3+, i.e. FeCl3, to Fe2+, suggesting their capacity to reduce Fe3+ at the enzyme active site. Indeed, similar to the catecholic redox inhibitor nordihydroguaiaretic acid, SA inhibited with the same efficiency both ferric and the native ferrous SLO form, indicating that these compounds reduce the active ferric enzyme leading to its inactivation.SA can inhibit lipoxygenase-catalyzed lipid peroxidation at therapeutic concentrations, suggesting its possible inhibitory activity against enzymatic lipid peroxidation in the clinical setting.
Keywords: Salicylic acid; Aspirin; Lipoxygenase; Lipid peroxidation; Iron; Free radical; Atherosclerosis; Cancer; Inflammation; Pain;

Morelloflavone blocks injury-induced neointimal formation by inhibiting vascular smooth muscle cell migration by Decha Pinkaew; Sung Gook Cho; David Y. Hui; John E. Wiktorowicz; Nongporn Hutadilok-Towatana; Wilawan Mahabusarakam; Moltira Tonganunt; Lewis J. Stafford; Amornrat Phongdara; Mingyao Liu; Ken Fujise (31-39).
In-stent restenosis, or renarrowing within a coronary stent, is the most ominous complication of percutaneous coronary intervention, caused by vascular smooth muscle cell (VSMC) migration into and proliferation in the intima. Although drug-eluting stents reduce restenosis, they delay the tissue healing of the injured arteries. No promising alternative anti-restenosis treatments are currently on the horizon.In endothelium-denudated mouse carotid arteries, oral morelloflavone—an active ingredient of the Thai medicinal plant Garcinia dulcis—significantly decreased the degree of neointimal hyperplasia, without affecting neointimal cell cycle progression or apoptosis as evaluated by Ki-67 and TUNEL staining, respectively. At the cellular level, morelloflavone robustly inhibited VSMC migration as shown by both scratch wound and invasion assays. In addition, morelloflavone prevented VSMCs from forming lamellipodia, a VSMC migration apparatus. Mechanistically, the inhibition by morelloflavone of VSMC migration was through its negative regulatory effects on several migration-related kinases, including FAK, Src, ERK, and RhoA. Consistently with the animal data, morelloflavone did not affect VSMC cell cycle progression or induce apoptosis.These data suggest that morelloflavone blocks injury-induced neointimal hyperplasia via the inhibition of VSMC migration, without inducing apoptosis or cell cycle arrest.We propose morelloflavone to be a viable oral agent for the prevention of restenosis, without compromising effects on the integrity and healing of the injured arteries.
Keywords: Restenosis; Morelloflavone; Migration; Garcinia dulcis; Vascular smooth muscle cell;

Glycosaminoglycan affinity of the complete fibroblast growth factor family by Masahiro Asada; Michiyo Shinomiya; Masashi Suzuki; Emi Honda; Rika Sugimoto; Masahiko Ikekita; Toru Imamura (40-48).
Many fibroblast growth factor family proteins (FGFs) bind to the heparan sulfate/heparin (HP) subtypes of sulfated glycosaminoglycans (GAGs), and a few have recently been reported to also interact with chondroitin sulfate (CS), another sulfated GAG subtype.To gain additional insight into this interaction, we prepared all currently known FGFs (i.e., FGF1–FGF23) and assessed their affinity for HP, CS-B, CS-D and CS-E. In addition, midkine, hepatocyte growth factor and pleiotrophin were studied as other known HP-binding proteins.We found that members of the FGF19 subfamily (i.e., FGF15, 19, 21 and 23) had little or no affinity for HP; all of the other secretable growth factors tested had strong affinities for HP, as was indicated by the finding that their elution from HP-Sepharose columns required 1.0–1.5 M NaCl. We also found that FGF3, 6, 8 and 22 had strong affinities for CS-E, while FGF5 had a moderate affinity for CS-D. The interactions between FGFs and GAGs thus appear to be more diverse than previously understood.This is noteworthy, as the differential interactions of these growth factors with GAGs may be key determinants of their specific biological activities.
Keywords: FGF; Glycosaminoglycan; Heparan sulfate; Chondroitin sulfate;

Identification of aquaporin-5 and lipid rafts in human resting saliva and their release into cevimeline-stimulated saliva by Yan Pan; Fusako Iwata; Di Wang; Masahiro Muraguchi; Keiko Ooga; Yasukazu Ohmoto; Masaaki Takai; Gota Cho; Jinsen Kang; Masayuki Shono; Xue-jun Li; Ko Okamura; Toyoki Mori; Yasuko Ishikawa (49-56).
It is unknown whether AQP5 and lipid rafts are released into human unstimulated (resting) saliva and saliva in response to secretagogues.In order to quantitate the salivary concentration of AQP5, we produced a polyclonal antibody for human AQP5 and developed an enzyme-like immunosorbent assay (ELISA).AQP5 and lipid rafts were identified in human resting saliva. The amount of AQP5 in resting saliva showed a diurnal variation with high levels during waking hours, and an age-related decrease in AQP5 was coincident with the volume of resting saliva. Cevimeline, a muscarinic acetylcholine receptor (mAChR) agonist, induced the release of AQP5 with lipid rafts, amylase, mucin, and lysozyme. Changes in saliva AQP5 levels after cevimeline administration occurred simultaneously with changes in saliva flow rates. Confocal microscopy revealed that AQP5 was located in the apical plasma membrane and showed a diffuse pattern in parotid glands under resting conditions. Following cevimeline administration, AQP5 was predominantly associated with the APM and was localized in the lumen.AQP5 and lipid rafts were released with salivary proteins from human salivary glands by the stimulation of M3 mAChRs, and that changes in saliva AQP5 levels can be used as an indicator of salivary flow rate and also as a useful index of M3 mAChR agonist's action on human salivary glands.
Keywords: Aquaporin-5; Water channel; Lipid rafts; Human saliva; Biomarker; Salivary flow rate; Anti-human AQP5-antibody;

Atomic force microscopy, biochemical analysis of 3T3-L1 cells differentiated in the absence and presence of insulin by Vimal Pandey; Malepillil Vavachan Vijayakumar; Ruchika Kaul-Ghanekar; Hitesh Mamgain; Kishore Paknikar; Manoj Kumar Bhat (57-64).
There are ample evidences to demonstrate that differentiation of preadipocytes is associated with deposition of fat in cells. Still, it is unclear whether the differentiation process also alters membrane topology as well as cholesterol levels and whether insulin contributes to it.Membrane scanning of differentiated cells, along with freshly plated and 11 day preadipocytes, was performed using Atomic Force Microscopy (AFM) to gain qualitative information about cell surface properties as well as roughness. Moreover, glucose uptake, lipid analysis, expression profiling of transcription factors and signaling molecules involved in the process of differentiation was also performed.We report (i) differentiation in the presence of 500 μM isobutylmethylxanthine (IBMX), 0.25 μM dexamethasone (DEX) with or without 0.1 μM (0.57 μg/ml) insulin directly alters membrane topology. (ii) At nano-levels, addition of insulin maintains plasma membrane roughness during differentiation in comparison with IBMX and DEX only. (iii) At macro levels, decreased fat accumulation in preadipocytes exposed to insulin during the initial stages of differentiation is a result of reduced expression and nuclear localization of sterol regulatory element binding protein (SREBP)-1.This study reports a significant reduction of membrane cholesterol and total cholesterol (p  < 0.01) in cells differentiated in the presence of insulin.
Keywords: Atomic force microscopy; Insulin; Fat accumulation; Cholesterol;

Quantitative analysis of the bacteriophage Qβ infection cycle by Koji Tsukada; Misato Okazaki; Hiroshi Kita; Yoshio Inokuchi; Itaru Urabe; Tetsuya Yomo (65-70).
In this study, the infection cycle of bacteriophage Qβ was investigated. Adsorption of bacteriophage Qβ to Escherichia coli is explained in terms of a collision reaction, the rate constant of which was estimated to be 4 × 10− 10 ml/cells/min. In infected cells, approximately 130 molecules of β-subunit and 2 × 105 molecules of coat protein were translated in 15 min. Replication of Qβ RNA proceeded in 2 steps—an exponential phase until 20 min and a non-exponential phase after 30 min. Prior to the burst of infected cells, phage RNAs and coat proteins accumulated in the cells at an average of up to 2300 molecules and 5 × 105 molecules, respectively. An average of 90 infectious phage particles per infected cell was released during a single infection cycle up to 105 min.
Keywords: Qβ phage; RNA phage; Infection dynamics of bacteriophage;

Novel Ca2+/calmodulin-dependent protein kinase expressed in actively growing mycelia of the basidiomycetous mushroom Coprinus cinereus by Keisuke Kaneko; Yusuke Yamada; Noriyuki Sueyoshi; Akira Watanabe; Yasuhiko Asada; Isamu Kameshita (71-79).
We isolated cDNA clones for novel protein kinases by expression screening of a cDNA library from the basidiomycetous mushroom Coprinus cinereus. One of the isolated clones was found to encode a calmodulin (CaM)-binding protein consisting of 488 amino acid residues with a predicted molecular weight of 53,906, which we designated CoPK12. The amino acid sequence of the catalytic domain of CoPK12 showed 46% identity with those of rat Ca2+/CaM-dependent protein kinase (CaMK) I and CaMKIV. However, a striking difference between these kinases is that the critical Thr residue in the activating phosphorylation site of CaMKI/IV is replaced by a Glu residue at the identical position in CoPK12. As predicted from its primary sequence, CoPK12 was found to behave like an activated form of CaMKI phosphorylated by an upstream CaMK kinase, indicating that CoPK12 is a unique CaMK with different properties from those of the well-characterized CaMKI, II, and IV. CoPK12 was abundantly expressed in actively growing mycelia and phosphorylated various proteins, including endogenous substrates, in the presence of Ca2+/CaM. Treatment of mycelia of C. cinereus with KN-93, which was found to inhibit CoPK12, resulted in a significant reduction in growth rate of mycelia. These results suggest that CoPK12 is a new type of multifunctional CaMK expressed in C. cinereus, and that it may play an important role in the mycelial growth.
Keywords: Basidiomycete; Calmodulin; Ca2+/calmodulin-dependent protein kinase; Coprinus cinereus; Mushroom; Protein phosphorylation;