BBA - General Subjects (v.1723, #1-3)

Characterization of a novel Eph receptor tyrosine kinase, EphA10, expressed in testis by Hans-Christian Aasheim; Sebastian Patzke; Hanne Sagsveen Hjorthaug; Eivind Farmen Finne (1-7).
In mammals, 14 members of the Eph receptor tyrosine kinase family have been described so far. Here we present a not yet described member of this family denoted EphA10. We report the identification of three putative EphA10 isoforms: one soluble and two transmembrane isoforms. One of the latter isoforms lacked the sterile alpha motif commonly found in Eph receptors. The gene encoding EphA10 is located on chromosome 1p34 and expression studies show that EphA10 mRNA is mainly expressed in testis. Binding studies to ephrin ligands suggests that this receptor belongs to the EphA subclass of Eph receptors binding mainly to ephrin-A ligands.
Keywords: Eph receptor; Testis; Soluble isoform; Ephrin;

PSP/Lithostathine/PTP/regI, PAP/p23/HIP, reg1L, regIV and “similar to PAP” are the members of a multifunctional family of secreted proteins containing a C-type lectin-like domain linked to a short N-terminal peptide. The expression of this group of proteins is controlled by complex mechanisms, some members being constitutively expressed in certain tissues while, in others, they require activation by several factors. These members have several apparently unrelated biological effects, depending on the member studied and the target cell. These proteins may act as mitogenic, antiapoptotic or anti-inflammatory factors, can regulate cellular adhesion, promote bacterial aggregation, inhibit CaCO3 crystal growth or increase resistance to antitumoral agents. The presence of specific receptors for these proteins is suggested because biological effects were observed after the addition of purified protein to culture media or after systemic administration to animals, whereas other biological effects could be explained by their biochemical capacity to form homo or heteromers or to form insoluble fibrils at physiological pH.
Keywords: PAP/p23/xHIP; Similar to PAP; PSP/Lithostathine/reg/PTP; Reg1 like; RegIV; C-type lectin; CTLD;

A common genetically determined polymorphism in the human population leads to two distinct phenotypes in adults, lactase persistence and adult-type hypolactasia (lactase non-persistence). All healthy newborn children express high levels of lactase and are able to digest large quantities of lactose, the main carbohydrate in milk. Individuals with adult-type hypolactasia lose their lactase expression before adulthood and consequently often become lactose intolerant with associated digestive problems (e.g. diarrhoea). In contrast, lactase persistent individuals have a lifelong lactase expression and are able to digest lactose as adults. Lactase persistence can be regarded as the mutant phenotype since other mammals down-regulate their lactase expression after weaning (the postweaning decline). This phenomenon does not occur in lactase persistent individuals. The regulation of lactase expression is mainly transcriptional and it is well established that adult-type hypolactasia is inherited in an autosomal recessive manner, whereas persistence is dominant. The recent findings of single nucleotide polymorphisms associated with lactase persistence have made it possible to study the potential mechanisms underlying adult-type hypolactasia. This work has led to the identification of gene-regulatory sequences located far from the lactase gene (LCT). The present review describes the recent advances in the understanding of the regulation of lactase expression and the possible mechanisms behind adult-type hypolactasia.
Keywords: Gene regulation; Intestine; Enterocyte; Caco-2; Adult-type hypolactasia; Persistence; Lactose intolerance; Lactase; Digestion;

A hypomorphic allele of the first N-glycosylation gene, ALG7, causes mitochondrial defects in yeast by Richard D. Mendelsohn; Eva J. Helmerhorst; John F. Cipollo; Maria A. Kukuruzinska (33-44).
The modification of proteins at asparagine residues with oligosaccharides (N-glycans) plays critical roles in diverse cell functions. N-glycans originate from a common lipid-linked oligosaccharide (LLO) precursor whose synthesis is initiated by the Dol-P-dependent GlcNAc-1-P transferase (GPT) encoded by an essential ALG7 gene. To identify cellular processes affected by ALG7 and N-glycosylation, we replaced the genomic copy of ALG7 with its hypomorphic allele in two genetically distinct haploid yeast cells. We show that ALG7 knockdown gave rise to an unexpected phenotype of mitochondrial dysfunction. The alg7 mutants did not grow on glycerol and DNA arrays revealed the absence of mitochondrial genes' expression. Accordingly, the alg7 mutants displayed no detectable mtDNA and respiratory activity. Both mutants exhibited diminished abundance of LLO and under-glycosylation of carboxypeptidase Y (CPY). Moreover, another N-glycosylation mutant with a LLO defect, alg6, was respiratory deficient. Collectively, our studies provide evidence that the dysregulation of N-glycosylation in haploid yeast cells leads to mitochondrial dysfunction.
Keywords: N-glycosylation; ALG7 hypomorph; Mitochondrial gene; Respiration; Saccharomyces cerevisiae;

Interaction and electron transfer between the high molecular weight cytochrome and cytochrome c 3 from Desulfovibrio vulgaris Hildenborough: Kinetic, microcalorimetric, EPR and electrochemical studies by Marianne Guiral; Gisèle Leroy; Pierre Bianco; Philippe Gallice; Bruno Guigliarelli; Mireille Bruschi; Wolfgang Nitschke; Marie-Thérèse Giudici-Orticoni (45-54).
The complex formation between the tetraheme cytochrome c 3 and hexadecaheme high molecular weight cytochrome c (Hmc), the structure of which has recently been resolved, has been characterized by cross-linking experiments, EPR, electrochemistry and kinetic analysis, and some key parameters of the interaction were determined. The analysis of electron transfer between [Fe] hydrogenase, cytochrome c 3 and Hmc demonstrates a redox-shuttling role of cytochrome c 3 in the pathway from hydrogenase to Hmc, and shows an effect of redox state on the interaction between the two cytochromes. The role of polyheme cytochromes in electron transfer from periplasmic hydrogenase to membrane redox proteins is assessed.A model with cytochrome c 3 as an intermediate between hydrogenase and various polyheme cytochromes is proposed and its physiological consequences are discussed.
Keywords: Multiheme cytochrome; Cytochrome c 3; Hmc; Electron transfer; Desulfovibrio metabolism;

Salmonella enterica serovar Typhimurium normally salvage nucleobases and nucleosides by the action of nucleoside phosphorylases and phosphoribosyltransferases. In contrast to Escherichia coli, which catabolizes xanthosine by xanthosine phosphorylase (xapA), Salmonella cannot grow on xanthosine as the sole carbon and energy source. By functional complementation, we have isolated a nucleoside hydrolase (rihC) that can complement a xapA deletion in E. coli and we have overexpressed, purified and characterized this hydrolase. RihC is a heat stable homotetrameric enzyme with a molecular weight of 135 kDa that can hydrolyze xanthosine, inosine, adenosine and uridine with similar catalytic efficiency (k cat/Km=1 to 4×104 M−1s−1). Cytidine and guanosine is hydrolyzed with approximately 10-fold lower efficiency (k cat/Km=0.7 to 1.2×103 M−1s−1) while RihC is unable to hydrolyze the deoxyribonucleosides thymidine and deoxyinosine. The Km for all nucleosides except adenosine is in the mM range. The pH optimum is different for inosine and xanthosine and the hydrolytic capacity (k cat/Km) is 5-fold higher for xanthosine than for inosine at pH 6.0 while they are similar at pH 7.2, indicating that RihC most likely prefers the neutral form of xanthosine.
Keywords: Nucleoside hydrolase; Xanthosine metabolism; Purine salvage pathway; pH dependence; Salmonella typhimurium;

The anhydrofructose pathway describes the degradation of glycogen and starch to metabolites via 1,5-anhydro-d-fructose (1,5AnFru). Enzymes that form 1,5AnFru, ascopyrone P (APP), and ascopyrone M (APM) have been reported from our laboratory earlier. In the present study, APM formed from 1,5AnFru was found to be the intermediate to the antimicrobial microthecin. The microthecin forming enzyme from the fungus Phanerochaete chrysosporium proved to be aldos-2-ulose dehydratase (AUDH, EC 4.2.1.-), which was purified and characterized for its enzymatic and catalytic properties. The purified AUDH showing a molecular mass of 97.4 kDa on SDS-PAGE was partially sequenced. Total 332 amino acid residues in length were obtained, representing some 37% of the AUDH protein. The obtained amino acid sequences showed no homology to known proteins but to an unannotated DNA sequence in Scaffold 62 of the published genome of the fungus. The alignment revealed three introns of the identified AUDH gene (Audh; ph.chr), thus the first gene coding for a neutral sugar dehydratase is identified. AUDH was found to be a bi-functional enzyme, being able to dehydrate 1,5AnFru to APM and further isomerizing the APM formed to microthecin. The optimal pH for the formation of APM and microthecin was pH 5.8 and 6.8, respectively. AUDH showed 5 fold higher activity toward 1,5AnFru than toward its analogue glucosone, when tested at concentrations from 0.6 mM to 0.2 M. Based on the characteristic UV absorbance of microthecin (230 nm) and APM (262 nm) assay methods were developed for the microthecin forming enzymes.
Keywords: Aldos-2-ulose dehydratase; 1,5-Anhydro-d-fructose; Anhydrofructose pathway; Ascopyrone; Cortalcerone; Gracilariopsis; Microthecin; Morchella; Phanerochaete chrysosporium; Pyranosone dehydratase;

Ornithine decarboxylase activity is inhibited by the polyamine precursor amino acids at the protein stability level in Caco-2 cells by Hervé Chabanon; Corinne Aubel; Pierre Larvaron; Claude Villard; Valérie Carraro; Patrick Brachet (74-81).
High concentrations of certain amino acids are known to affect hormonal secretion, immune function, electrolyte balance or metabolic functions. However, there is a lack of knowledge regarding the molecular mechanisms responsible for these effects. We showed that, as well as spermidine transport, the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine biosynthesis, is decreased in human colon adenocarcinoma cells, Caco-2, following a 4-h supplementation with one of the two polyamine precursor amino acids, l-arginine or l-methionine. Dose-response assays indicated that the inhibitory effect of supplemental l-methionine was stronger than that of supplemental l-arginine. However, it was transient, being even replaced by ODC induction after 8 h, whereas the inhibitory effect of l-arginine lasted for at least 8 h. Unlike l-cysteine, neither l-methionine nor l-arginine could inhibit ODC activity in a crude acellular preparation of the enzyme. The inhibition of ODC activity in cells exposed to l-methionine or l-arginine was due to a decreased abundance of ODC protein without change at the mRNA level and each of these amino acids could counteract ODC induction by a glycine supplement. Contrary to the latter, supplemental l-methionine or l-arginine induced a marked decrease in ODC half-life, concomitantly with an increase in the activity of antizyme, an ODC inhibitory protein. Thus, depending on their nature, amino acids can up- or downregulate ODC activity at the protein stability level.
Keywords: Polyamine; l-Arginine; l-Methionine; Post-translational regulation; Antizyme;

New GlcNAc/GalNAc-specific lectin from the ascidian Didemnum ternatanum by Valentina Molchanova; Irina Chikalovets; Wei Li; Stanislav Kobelev; Svetlana Kozyrevskaya; Raisa Bogdanovich; Eric Howard; Natalia Belogortseva (82-90).
Previously we isolated GlcNAc-specific lectin (DTL) from the ascidian Didemnum ternatanum by affinity chromatography on cross-linked ovalbumin. Here we report the purification and characterization of new D-GlcNAc/D-GalNAc-specific lectin DTL-A from the same ascidian. This lectin was isolated from non-bound cross-linked ovalbumin fraction and further was purified by gel filtration on Sepharose CL-4B, affinity chromatography on GlcNAc-agarose and gel filtration on Superdex 200. SDS-polyacrylamide gel electrophoresis and gel filtration of purified lectin on Sepharose CL-4B indicates that it exists as large aggregates in the native state. Investigations of the carbohydrate specificity of DTL-A by enzyme-linked lectin assay suggest the multi-specificity of this lectin. DTL-A binds BSM, asialo-BSM as well as heparin and dextran sulfate. The binding of DTL-A to BSM was inhibited by monosaccharides D-GlcNAc and D-GalNAc, their α- but not β-anomers. Among polysaccharides and glycoconjugates, DTL-A binding to BSM was effectively inhibited by BSM, asialo-BSM, pronase-treated BSM and synthetic α-D-GalNAc-PAA. Fetuin and asialofetuin showed a much lower inhibitory potency, heparin and dextran sulfate were noninhibitory. On the other hand, DTL-A binding to heparin was effectively inhibited by dextran sulfate, fucoidan, whereas BSM showed insignificantly inhibitory effect. DTL-A binding to heparin was not inhibited by D-GlcNAc and D-GalNAc.
Keywords: Invertebrate; Didemnum ternatanum; GlcNAc/GalNAc-specific lectin; Glycoprotein; Sulfated polysaccharide;

Expression of the standard scorpion alpha-toxin AaH II and AaH II mutants leading to the identification of some key bioactive elements by Christian Legros; Brigitte Céard; Hélène Vacher; Pascale Marchot; Pierre E. Bougis; Marie-France Martin-Eauclaire (91-99).
The AaH II toxin from the scorpion Androctonus australis Hector is considered to be the standard α-toxin because it selectively binds with the highest known affinity to site 3 of mammalian voltage-activated Na+ channels (Nav) on rat brain synaptosomes but does not bind to insect synaptosomes. We generated two different constructs in pMALp allowing us to produce AaH II fused with the maltose-binding protein (MBP) in E. coli. We obtained reasonable amounts of recombinant AaH II after cleavage by enterokinase at the site DDDDK. We show that the introduction of a net negative charge at the C-terminus by the suppression of H64 amidation and the addition of an extra residue to the C-terminus (G65) led to fully active AaH II mutants, exhibiting exactly the same affinity as the native toxin for its target on rat brain synaptosomes. In contrast, the mutation of residue K58 into V, I or E residues drastically reduced toxin activity.
Keywords: Scorpion; Alpha-toxin; Voltage-activated sodium channel; Heterologous expression;

We have measured the lateral mobility of individual α5 integrin molecules in ventral plasma membranes of fibroblasts, which were prepared by removal of apical surfaces and nuclei followed by elimination of actin filaments with gelsolin, an actin-severing protein. The cytoplasmic domain of individual integrin molecules was tagged with 100 nm fluorescent polystyrene bead, and motion of the bead was observed and video-recorded. Position of the bead in each frame was determined from the centroid of the fluorescence image, from which plots of the mean-square displacement against time intervals were derived. Within short intervals of time (<100 ms) the mean-square displacement was proportional to the time interval, and the averaged translational diffusion coefficient of (5.3±4.4)×10−10 cm2/s was obtained with a broad distribution of (1.3–20)×10−10 cm2/s. The broad distribution might reflect the oligomerized state of integrin. The largest diffusion coefficient was comparable to that of lipid molecules previously measured in cells and probably represented the diffusion of a single integrin molecule in the presence of little interference of actin cytoskeleton or extracellular matrix. In longer time intervals (>100 ms) the motion of the bead was confined in an area, the average diameter of which was 410±160 nm. This was similar to the values described in previous reports, in which the motion of other membrane receptors labeled on their extracellular domain was measured in living cells.
Keywords: α5β1 integrin; Plasma membrane; Single particle tracking; Translational diffusion coefficient; Actin cytoskeleton; Extracellular matrix;

Thermostabilized ovalbumin that occurs naturally during development accumulates in embryonic tissues by Hiroshi Shinohara; Tsukasa Iwasaki; Yukiko Miyazaki; Kanako Matsuo; Takayoshi Aoki; Mitsuharu Matsumoto; Tatsuzo Oka; Jun-ichi Kurisaki; Koko Mizumachi; Takahiro Kusakabe; Katsumi Koga; Yasushi Sugimoto (106-113).
We have reported that ovalbumin accumulates without digestion in various tissues during embryonic development of the chicken. There are different types of ovalbumin with respect to thermal stability and one of them, which was named “HS-ovalbumin” in the present study, was found to have a T m value of 83 °C and to be present dominantly in albumen, egg yolk, amniotic fluid, and serum of fertilized eggs. HS-ovalbumin, arising physiologically from its native form (N-ovalbumin), is reminiscent of the previously described intermediate form appearing during the production processes of the so-called S-ovalbumin, which disappeared shortly in fertilized eggs. We showed that HS-ovalbumin is distinguishable from S-ovalbumin by a monoclonal antibody and also from N-ovalbumin by the stability to heating. At the late stages of development, ovalbumin of amniotic fluid seems to be swallowed through pharynx, carried in the intestine through stomach, and absorbed in the blood. Analyses by monoclonal antibody and heat treatment indicated that the HS-form occupies the largest fraction of ovalbumin that accumulates in the embryonic tissues. The current findings suggest that HS-ovalbumin is crucial for embryogenesis.
Keywords: Ovalbumin; S-ovalbumin; Serpin; Thermostability; Embryogenesis; Gallus gallus;

“In vitro” protection of DNA from Fenton reaction by plant polyphenol verbascoside by Chenyang Zhao; Guy Dodin; Chenshan Yuan; Haifeng Chen; Rongliang Zheng; Zhongjian Jia; Bo-Tao Fan (114-123).
The protection effect of verbascoside (Ver) against Fenton reaction on plasmid pBR322 DNA was studied using agarose gel electrophoresis and UV-visible spectroscopy. The pBR322 plasmid DNA is damaged by hydroxyl radical (OH) generated from the Fenton reaction with H2O2 and Fe(II) or Fe(III). This DNA damage is characterized by the diminution of supercoiled DNA forms or by the increase of relaxed or linear DNA forms after oxidative attack. The UV spectrum study showed that verbascoside can form complexes with Fe(II) or Fe(III), and the complexation can be reversed by the addition of EDTA. The formation constants of verbascoside–Fe complexes were estimated as 1021.03 and 1031.94 M−2 for Fe(II) and Fe(III) respectively. The inhibition of Fenton reaction by verbascoside could be partially explained by the sequestration of Fe ions.
Keywords: Verbascoside; Antioxidant activity; Complexation; Oxidative DNA damage; Fenton reaction;

Exercise might favor reverse cholesterol transport and lipoprotein clearance: Potential mechanism for its anti-atherosclerotic effects by Chen Wei; Meera Penumetcha; Nalini Santanam; Ya-Guang Liu; Mahdi Garelnabi; Sampath Parthasarathy (124-127).
Livers of C57 BL/6 mice exercised for 2 weeks showed a dramatic increase in scavenger receptor B1 (SR-B1), CD36 and low density lipoprotein (LDL) receptor and a decrease in acetyl LDL receptor gene expression. These effects on lipoprotein receptors are reminiscent of the effects mediated by peroxisome proliferator-activated receptor (PPARγ) ligands.
Keywords: Exercise; Scavenger receptors; Atherosclerosis; PPARs;

Glutathione depletion and anaesthesia in mice alter heme and drug metabolising enzymes by Ana Maria Buzaleh; Alcira Maria Del Carmen Batlle (128-134).
The effects of enflurane and isoflurane on heme metabolism, its regulation, and on some parameters involved in the hepatic drug metabolising system in animals under GSH depletion were investigated.A single dose of the anaesthethics (1 ml kg−1, i.p.) was administered to control and GSH depleted mice, animals were sacrificed 20 min after.As a consequence of GSH depletion, a significant inhibition in δ-Aminolevulinic acid synthetase activity, the first enzyme of heme bioxynthesis, and a striking induction in Heme oxygenase activity, the main enzyme of heme metabolism, were observed. Cytochrome P-450 levels and the activities of P-4502E1 and glutathione S-transferase were increased. These changes in heme metabolism and drug metabolising enzyme system were not altered further by the administration of enflurane or isoflurane.These findings would indicate that the status of oxidative stress produced by GSH depletion could not be affected by these anaesthetics and/or that disturbances in heme metabolism were already too important to undergo further variations.
Keywords: Enflurane; Isoflurane; GSH; Heme metabolism; Drug metabolising enzyme system;

Encapsulation of hemoglobin (Hb) within red blood cells (RBCs) preserves nitric oxide (NO) bioactivity. With encapsulation, millimolar concentrations of Hb quench only a fraction of NO bioactivity, whereas mere micromolar concentrations of cell-free Hb completely quench NO bioactivity. A submembrane cytoskeletal barrier has been hypothesized to account for the lowered quenching of NO bioactivity. In order to substantiate this hypothesis, here, the underlying submembrane cytoskeletal barrier was physically reduced and the rate of NO entry into the modified RBC measured. The submembrane cytoskeletal barrier of normal and depleted RBCs was characterized using atomic force microscopy and the lipid to protein ratio measured. The reduction in the submembrane cytoskeletal barrier resulted in an increase in the rate of NO entry. We suggest that the underlying submembrane cytoskeleton may be a key component of RBC mediated regulation of NO bioavailability.
Keywords: Nitric oxide; Erythrocyte; Red blood cell; Cytoskeleton; Membrane;

CD39 is a membrane-bound ecto-nucleoside triphosphate diphosphohydrolase that is involved in the regulation of purinergic signaling. It has been previously reported that N-linked glycosylation is essential for the surface localization of CD39 and for its cellular activity. Here we have addressed the roles of different stages of N-linked glycosylation on CD39's activity and surface expression by using various glycosylation inhibitors, glycosylation deficient CHO cells, and oligosaccharide removal enzymes. The results demonstrate that endoplasmic reticulum glycosylation is required for protein folding and essential for functional surface expression of CD39, while Golgi glycosylation is less important. The study has also shown that N-linked glycosylation of CD39 is dispensable for the activity after the protein is properly folded and targeted.
Keywords: Endoplasmic reticulum; Golgi; N-linked glycosylation; Surface expression; Ecto-nucleoside triphosphate diphosphohydrolase; Protein folding;

Isolation and spectroscopic characterization of the membrane-bound nitrate reductase from Pseudomonas chlororaphis DSM 50135 by Dora Pinho; Stéphane Besson; Pedro J. Silva; Baltazar de Castro; Isabel Moura (151-162).
A nitrate reductase was solubilized with Triton X-100 from the membranes of Pseudomonas chlororaphis DSM 50135 grown microaerobically in the presence of nitrate. Like other membrane-bound nitrate reductases, it contains three subunits, of 129, 66 (64) and 24 kDa, referred to in the literature as α, β and γ, respectively. Electrocatalytic studies revealed that only the membrane-bound, not the solubilized form of the enzyme, can accept electrons from a menaquinone analog, menadione, whereas both forms can accept electrons from methylviologen. The isolated enzyme possesses several iron–sulfur clusters and a molybdopterin guanine dinucleotide active center. The iron–sulfur clusters can be grouped in two classes according to their redox properties, the high-potential and low-potential clusters. In the as-isolated enzyme, two forms of the molybdenum center, high- and low-pH, are detectable by electron paramagnetic resonance spectroscopy. The low-pH form shows a hyperfine splitting due to a proton, suggesting the presence of an –OHx ligand. Dithionite reduces the Mo(V) center to Mo(IV) and subsequent reoxidization with nitrate originates a new Mo(V) signal, identical to the oxidized low-pH form but lacking its characteristic hyperfine splitting. The isolated preparation also contains heme c (in a sub-stoichiometric amount) with the ability to relay electrons to the molybdenum center, suggesting that this nitrate reductase may contain heme c instead of the heme b usually found in this class of enzymes.
Keywords: Nitrate reductase; EPR; Molybdenum; Iron–sulfur centers; Heme c;

Mitogenic and anti-proliferative activity of a lectin from the tubers of Voodoo lily (Sauromatum venosum) by Jagmohan SinghBains; Jatinder Singh; Sukhdev Singh Kamboj; Kamaljeet Kaur Nijjar; Javed N. Agrewala; Vinod Kumar; Ashok Kumar; A.K. Saxena (163-174).
A new lectin with the potent mitogenic and in vitro anti-proliferative activity was isolated from the tubers of a wild monocotyledonous plant Sauromatum venosum (Schott), from the family Araceae, by affinity chromatography on the asialofetuin linked amino-activated silica beads. The apparent native molecular mass of S. venosum lectin (SVL), as determined by gel filtration chromatography, was 54 kDa. In HPLC, size exclusion and cation exchange chromatography, SVL gave a single peak and also a single band of 13.5 kDa in SDS-PAGE, pH 8.3, under reducing and non-reducing conditions, indicating that the lectin is composed of four identical subunits. S. venosum lectin agglutinated rabbit, rat, sheep and guinea pig erythrocytes but reacted with goat erythrocytes after the neuraminidase treatment. However, SVL was unable to agglutinate human ABO blood group erythrocytes even after treatment with neuraminidase. SVL was inhibited by N-acetyl-d-Lactosamine (LacNAc), which is an important marker in various carcinomas and a complex desialylated glycoprotein, asialofetuin. The amino acid composition showed that lectin contained a high amount of aspartic acid and glycine but totally devoid of cysteine. However, trace amounts of methionine was present. The lectin showed a potent mitogenic response towards BALB/c splenocytes and human lymphocytes. As the mitogenic stimulation was more than that of Con A, a standard well-known plant mitogen and the response of this lectin was almost double than that of Con A. This lectin is endowed with proliferation of T cells as revealed by IL-2 bioassay but showed no production of immunoglobulins thus indicating the non-stimulation of B cells. SVL significantly inhibited the proliferation of murine cancer cell-lines, i.e., WEHI-279 to 84.6%, J774 to 81%, P388D1 to 74% and A-20 to 47%. In addition, the in vitro anti-proliferative activity of SVL was also evaluated against nine human cancer cell lines representing different organs and tissues namely, T-47D (breast), SiHa (cervix), SK-N-MC (CNS), SK-N-SH (CNS), SW-620 (colon), HT-29 (colon), HEP-2 (liver), OVCAR-5 (ovary) and PC-3 (prostate). SVL showed a significant inhibition towards the entire cell lines except the cell lines from CNS, which showed partial response in comparison to a standard anticancer drug adriamycin which was used at a concentration of 5×10−5 M. Thus the anti-proliferative ability of SVL may be helpful in identification of new lectin probes that can lead to better understanding in the detection and study of certain types of cancer.
Keywords: Mitogenic; In vitro anti-proliferative; Tuber lectin; Murine cancer cell line; Human cancer cell line;

It has been proposed that various urinary proteins interact specifically with different calcium oxalate hydromorphs and these interactions have important implications regarding the understanding of the onset and progress of kidney stone disease. Calcium oxalate monohydrate and dihydrate crystals were grown and characterised thoroughly to establish sample purity. These crystals were then incubated in artificial urine samples containing isolated urinary macromolecules. Crystal growth was prevented by saturating the incubation mix with calcium oxalate, and this was confirmed through electron microscopy and calcium measurements of the incubation mix. The surface interactions between the different calcium oxalate hydrates and urinary proteins were investigated by the use of Western blots and immunoassays. The same proteins, notably albumin, Tamm–Horsfall protein, osteopontin and prothrombin fragment 1, associated with both hydrates. There was a trend for more protein to associate with calcium oxalate dihydrate, and greater quantities of different proteins associated with both hydrates when Tamm–Horsfall protein was removed from the incubation mix. There is no evidence from this study to indicate that particular proteins interact with specific calcium oxalate hydrates, which in turn suggests that these protein–mineral interactions are likely to be mediated through non-specific charge interactions.
Keywords: Biomineralisation; Calcium oxalate; Urolithiasis; Crystal matrix; Protein matrix;

Differential scanning calorimetric studies of a Bacillus halodurans α-amylase by Suhaila O. Hashim; Rajni-Hatti Kaul; Maria Andersson; Francis J. Mulaa; Bo Mattiasson (184-191).
The thermal unfolding of Amy 34, a recombinant α-amylase from Bacillus halodurans, has been investigated using differential scanning calorimetry (DSC). The denaturation of Amy 34 involves irreversible processes with an apparent denaturation temperature (T m) of 70.8 °C at pH 9.0, with four transitions, as determined using multiple Gaussian curves. The T m increased by 5 °C in the presence of 100-fold molar excess of CaCl2 while the aggregation of Amy 34 was observed in the presence of 1000-fold molar excess of CaCl2. Increase in the calcium ion concentration from 1- to 5-fold molar excess resulted in an increase in calorimetric enthalpy (ΔH cal), however, at higher concentrations of CaCl2 (up to 100-fold), ΔH cal was found to decrease, accompanied by a decrease in entropy change (ΔS), while the T m steadily increased. The presence of 100-fold excess of metal chelator, EDTA, resulted in a decrease in T m by 10.4 °C. T m was also decreased to 61.1 °C and 65.9 °C at pH 6.0 and pH 11.0, respectively.
Keywords: α-Amylase; Bacillus halodurans; Calcium; Differential scanning calorimetry;

When pea lectin was exposed to a low pH range, it was found that the secondary structure of the lectin resisted conformational changes to a large extent up to pH 2.4 and below this pH, a sharp transition was observed which could be due to the presence of 27 acidic amino acid residues present in the protein.The effects of 1,1,1,3,3,3 hexafluoro-isopropanol (HFIP) and 2,2,2-Trifluoroethanol (TFE) on the conformation of pea lectin at pH 2.4 were studied using circular dichroism and fluorescence spectroscopy. Analysis varying the TFE concentration showed that up to 80% TFE (v/v) protein retained the residual β-structure accompanied by a loss in tertiary structure. A similar conformation is presumed to exist at 4% HFIP (v/v), with an increase in HFIP concentration structural rearrangements occurred and a transition from β-structure to α-helical structure started from 12% HFIP which completed at 30% HFIP. Our studies show the occurrence of a common intermediate in the folding pathway of pea lectin induced by two different fluoroalcohols, which differ in their mode of action to stabilize the secondary structure of a given protein. While TFE was not found to induce any α-helical structure, HFIP caused the transition of pea lectin, which is predominantly a β-sheet protein, to a structure rich in α-helical contacts. Thus, our results also point out the possibility of a non-hierarchical model of protein folding in lectins.
Keywords: Pea lectin; Molten globule state; Circular dichroism; Acid unfolding; Fluorescence;

Biochemical characterisation of the trehalase of thermophilic fungi: An enzyme with mixed properties of neutral and acid trehalase by Agda Karina B. Lúcio-Eterovic; João A. Jorge; Maria de Lourdes T.M. Polizeli; Héctor F. Terenzi (201-207).
The trehalases from some thermophilic fungi, such as Humicola grisea, Scytalidium thermophilum, or Chaetomium thermophilum, possess mixed properties in comparison with those of the two main groups of trehalases: acid and neutral trehalases. Such as acid trehalases these enzymes are highly thermostable extracellular glycoproteins, which act at acidic pH. However, these enzymes are activated by calcium or manganese, and as a result inhibited by chelators and by ATP, properties typical of neutral trehalases. Here we extended the biochemical characterisation of these enzymes, by assaying their activity at acid and neutral pH. The acid activity (25–30% of total) was assayed in McIlvaine buffer at pH 4.5. Under these conditions the enzyme was neither activated by calcium nor inhibited by EDTA or ATP. The neutral activity was estimated in MES buffer at pH 6.5, after subtracting the activity resistant to EDTA inhibition. The neutral activity was activated by calcium and inhibited by ATP. On the other hand, the acid activity was more thermostable than the neutral activity, had a higher temperature optimum, exhibited a lower K m, and different sensitivity to several ions and other substances. Apparently, these trehalases represent a new class of trehalases. More knowledge is needed about the molecular structure of this protein and its corresponding gene, to clarify the structural and evolutionary relationship of this trehalase to the conventional trehalases.
Keywords: Scytalidium thermophilum; Humicola grisea; Trehalase; Trehalose; Fungi;

Rhabdomyolysis-induced myoglobinuric acute renal failure accounts for about 10–40% of all cases of acute renal failure (ARF). Nitric oxide and reactive oxygen intermediates play a crucial role in the pathogenesis of myoglobinuric acute renal failure (ARF). This study was designed to investigate the effect of molsidomine and l-arginine in glycerol induced ARF in rats. Six groups of rats were employed in this study, group I served as control, group II was given 50% glycerol (8 ml/kg, intramuscularly), groups III and IV were given glycerol plus molsidomine (5 mg/kg, and 10 mg/kg p.o. route respectively) 60 min prior to the glycerol injection, group V animals were given glycerol plus l-arginine (125 mg/kg, p.o.) 60 min prior to the glycerol injection, and group VI received l-NAME (10 mg/kg, i.p.) along with glycerol 30 min prior to glycerol administration. Renal injury was assessed by measuring plasma creatinine, blood urea nitrogen, creatinine and urea clearance. The oxidative stress was measured by renal malondialdehyde levels, reduced glutathione levels and by enzymatic activity of catalase, reduced glutathione and superoxide dismutase. Tissue and urine nitrite levels were measured as an index of total nitric oxide levels. Glycerol treatment resulted in a marked decrease in tissue and urine nitric oxide levels, renal oxidative stress and significantly deranged the renal functions along with deterioration of renal morphology. Pre-treatment of animals with molsidomine (10 mg/kg) and l-arginine 60 min prior to glycerol injection markedly attenuated fall in nitric oxide levels, renal dysfunction, morphological alterations, reduced elevated TBARS and restored the depleted renal antioxidant enzymes. The animals treated with l-NAME along with glycerol further worsened the renal damage observed with glycerol. As a result, our results indicate that molsidomine and l-arginine may have beneficial effects in myoglobinuric ARF.
Keywords: Rhabdomyolysis; Glycerol; Acute renal failure; Molsidomine; l-arginine; l-NAME; Nitric oxide; Oxidative stress;

Selenium binding to human hemoglobin via selenotrisulfide by Mamoru Haratake; Katsuyoshi Fujimoto; Masahiro Ono; Morio Nakayama (215-220).
Selenotrisulfide (e.g., glutathione selenotrisulfide (GSSeSG)) is an important intermediate in the metabolism of selenite. However, its reactivity with biological substances such as peptides and proteins in the subsequent metabolism is still far from clearly understood, because of its chemical instability under physiological conditions. Penicillamine (Pen) is capable of generating a chemically stable and isolatable selenotrisulfide, PenSSeSPen. To explore the metabolic fate of selenite in red blood cells (RBC), we investigated the reaction of selenotrisulfide with human hemoglobin (Hb) using PenSSeSPen as a model. PenSSeSPen rapidly reacted with Hb under physiological conditions. From the analysis of selenium binding using the Langmuir type binding equation, the apparent binding number of selenium per Hb tetramer almost corresponded to the number of reactive thiol groups of Hb. The thiol group blockade of Hb by iodoacetamide treatment completely inhibited the reaction of PenSSeSPen with Hb. In addition, MALDI-TOF mass spectrometric analysis of the selenium-bound Hb revealed that PenSSe moiety binds to the β subunits of Hb. Overall, the reaction of PenSSeSPen with Hb appears to involve the thiol exchange between Pen and the cysteine residues on the β subunit of Hb.
Keywords: Hemoglobin; Red blood cell; Selenite metabolism; Selenotrisulfide;

Mechanism of horseradish peroxidase-catalyzed heme oxidation and polymerization (β-hematin formation) by Vishal Trivedi; Prem Chand; Prakas R. Maulik; Uday Bandyopadhyay (221-228).
Horseradish peroxidase (HRP) catalyzes the polymerization of free heme (β-hematin formation) through its oxidation. Heme when added to HRP compound II (Fe IV=O) causes spectral shift from 417 nm (Compound II) to 402 nm (native, Fe III) indicating that heme may be oxidized via one-electron transfer. Direct evidence for one-electron oxidation of heme by HRP intermediates is provided by the appearance of an E.s.r signal of a 5,5-dimethyl-1-pyrroline N-oxide (spin trap)-heme radical adduct (a 1 H=14.75 G, a 2 H=4.0 G) in E.s.r studies. Heme-polymerization by HRP is inhibited by spin trap indicating that one-electron oxidation product of heme ultimately leads to the formation of heme-polymer. HRP, when incubated with diethyl pyrocarbonate (DEPC), a histidine specific reagent, shows concentration dependent loss of heme-polymerization indicating the role of histidine residues in the process. We suggest that HRP catalyzes the formation of heme-polymer through one-electron oxidation of free heme.
Keywords: Heme; Oxidation; Free radical; Horseradish peroxidase; H2O2; Spin trap;

New features of site-specific horseradish peroxidase (HRP) glycosylation uncovered by nano-LC-MS with repeated ion-isolation/fragmentation cycles by Manfred Wuhrer; Crina I.A. Balog; Carolien A.M. Koeleman; André M. Deelder; Cornelis H. Hokke (229-239).
Horseradish peroxidase (HRP) is widely used in biomedical research as a reporter enzyme in diagnostic assays. In addition, it is of considerable interest as a model glycoprotein with core-xylosylated and -(α1-3)-fucosylated N-glycans that form antigenic elements of plant allergens and parasitic helminths.Using a combination of techniques comprising (1) nano-liquid chromatography (LC)-mass spectrometry (MS)/MS with multiple selection/fragmentation cycles of HRP tryptic (glyco-)peptides, (2) nano-electrospray MS of intact HRP, and (3) carbohydrate linkage analysis, it was revealed that most of the HRP N-glycosylation sites can be occupied with an alternative Fuc(1-3)GlcNAc-disaccharide. Two main variants of HRP occur: The major population (approximately 60%) has eight glycosylation sites carrying core(1-3)fucosylated, xylosylated, trimannosyl N-glycans, with the ninth potential N-glycosylation site Asn316 not occupied. Another group of HRP carries seven of the above-mentioned N-glycans, with an eighth N-glycosylation site carrying the alternative Fuc(1-3)GlcNAc-unit (approximately 35%). In addition, minor subsets of HRP were found to contain a xylosylated, trimannosyl N-glycan lacking core-fucosylation as a ninth N-glycan attached to Asn316, which has hitherto been assumed to be unoccupied. The finding of these new features of glycosylation of an already exceptionally well-studied glycoprotein underscores the potential of the nano-LC-MS n based analytical approach followed.
Keywords: Disaccharide; Ion trap; Multistage MS/MS; Plant glycosylation;

Multimerization of expressed protein-arginine methyltransferases during the growth and differentiation of rat liver by Yongchul Lim; Young-Ho Kwon; Nam Hee Won; Bon-Hong Min; In-Sun Park; Woon Ki Paik; Sangduk Kim (240-247).
Protein-arginine methylation is a posttranslational modification which yields monomethyl and dimethyl (asymmetric or symmetric) arginines in proteins. We investigated the expressions of PRMT1 and PRMT5 in relation to their catalytic activities in rat liver during growth and differentiation as well as in the pancreas. Western immunoblot analysis revealed that both PRMT1 and PRMT5 proteins were expressed in the cytosol of liver and pancreas with molecular mass of about 42 kDa and 72 kDa, respectively. However, on molecular sieve chromatography, the enzyme activities were eluted at about 500 kDa for PRMT5 and 440 kDa for PRMT1, indicating that the multimer complex of these expressed monomers were catalytically active. While the 500 kDa complex methylated predominantly myelin basic protein (MBP), the 440 kDa complex methylated hnRNP A1 protein. In fetal rat liver, the amount of expressed 42 kDa PRMT1 protein and the enzyme activity to methylate hnRNPA1 protein were 2- to 3-fold and 4- to 5-fold higher, respectively, than those of post-natal livers. While the 72 kDa PRMT5 protein was consistently expressed, its activity varied only about 2-fold. However, PRMT5 to methylate MBP showed one distinct peak at around the 20th day post-natal. Furthermore, while the PRMT1 enzyme activity increased more than 10-fold after 3 days of 70% partial hepatectomy, the amount of expressed PRMT1 protein was only about 3.2-fold higher than the control livers. In summary, we observed that PRMTs are catalytically active only in the form of multimers, but not as a dimer or tetramer of the expressed subunit. Furthermore, the amount of expressed PRMT protein, determined by Western immunoblot, did not correlate with the amount of their catalytic activity, and thus, some uncharacterized additional factor(s) may multimerize PRMTs to express catalytic activities in vivo.
Keywords: Multimer of PRMT; Rat liver development; Regenerating rat liver;

Connections between integrins and Rac GTPase pathways control gonad formation and function in C. elegans by Myeongwoo Lee; Bing Shen; Jean E. Schwarzbauer; Jeong Ahn; John Kwon (248-255).
The integrins are a family of αβ heterodimeric transmembrane receptors that link extracellular matrix (ECM) proteins to the cytoskeleton and orchestrate cell behaviors. It's been suggested that integrins interact with Rho family small GTPases, such as Rho and Rac. We took advantage of a C. elegans nematode line expressing HA-βtail, a β integrin transgene inhibiting the functions of endogenous integrins, to determine the combined effects of reducing PAT-3 β integrin and Rac pathway activities. Double mutants of HA-βtail and unc-73, a guanine nucleotide exchange factor GEF for MIG-2/Rac, had body wall and vulval muscle abnormalities. On the other hand, HA-βtail combined with mutant CED-5, another Rac interacting protein, showed ovulation defects and sterility. RNA-mediated interference (RNAi) of pat-3 on Rac mutant backgrounds also affected gonad structure and function. These results show a functional link between integrins and Rac signaling in muscles and gonads. Furthermore, data showing distinct phenotypes of HA-βtail with unc-73 versus ced-5 suggest some tissue-specificity in the usage of Rac signaling pathways.
Keywords: Integrin; Rac GTPase; Gonad; Cell migration; Fertility; Muscle;

Genes of the thymidine salvage pathway: Thymine-7-hydroxylase from a Rhodotorula glutinis cDNA library and iso-orotate decarboxylase from Neurospora crassa by Jeffrey A. Smiley; Melisa Kundracik; Daniel A. Landfried; Vincient R. Barnes; Armend A. Axhemi (256-264).
Genes for two enzymes in the thymidine salvage pathway, thymine-7-hydroxylase (THase; official name thymine dioxygenase) and iso-orotate decarboxylase (IDCase) have been isolated from fungal sources. THase was isolated from a Rhodotorula glutinis cDNA library using a degenerate oligonucleotide based on the published amino acid sequence. The coding sequence was transferred to an Escherichia coli expression system, from which recombinant THase activity was measured using 14C-labeled thymine. The THase sequence shows an almost complete avoidance of codons ending in A or T: 95.8% GC content is present in the third position of codons. A connection between this codon bias and the role of the thymidine salvage pathway in pyrimidine metabolism is proposed. The THase sequence is similar to Group I Fe+2-dependent, αKG-dependent dioxygenases. The R. glutinis THase gene was used to locate the probable THase genes in the sequenced genomes of Neurospora crassa and Aspergillus nidulans. The genes neighboring THase in these two genomes are similar to each other, and are similar to the mammalian 2-amino-3-carboxymuconate-6-semialdhyde decarboxylase (ACMSD), leading to their identification as IDCase genes. The N. crassa version was isolated by PCR of genomic DNA, and IDCase activity was measured in recombinant E. coli carrying this gene. A new family of decarboxylases, using similar substrates, is identified by virtue of the protein sequence similarity.
Keywords: Thymine dioxygenase; Uracil-5-carboxylate decarboxylase; Group I Fe+2, αKG-dependent dioxygenase; Rhodotorula glutinis; Neurospora crassa; 2-Amino-3-carboxymuconate-6-semialdhyde decarboxylase; Codon bias; Enzyme families; Pyrimidine metabolism;

PCR-based unidirectional deletion method for creation of comprehensive cDNA libraries by Stephanie Pollock; David Y. Thomas; Gregor Jansen (265-269).
A new strategy for the rapid creation of DNA deletion libraries using a simple PCR-based method is presented. Unidirectional deletion fragments are created and may be cloned into any vector system without the constraint of using restriction enzymes. Our strategy combines methodologies from DNA sequencing, PCR, and homologous recombination (either in vivo or in vitro) to allow for the creation of a library containing fragments representing all possible deletions of a given cDNA. Using this strategy we have successfully constructed a deletion library of the cDNA encoding for the lumenal domain of yeast Ire1p, and have shown that resulting fragments range from 100 bp to the full length cDNA (1557 bp). This method is simple, inexpensive, and can easily be adapted for automated high-throughput research.
Keywords: Cloning; PCR; Recombination; Deletion library;

Theaflavin derivatives in black tea and catechin derivatives in green tea inhibit HIV-1 entry by targeting gp41 by Shuwen Liu; Hong Lu; Qian Zhao; Yuxian He; Jinkui Niu; Asim K. Debnath; Shuguang Wu; Shibo Jiang (270-281).
Theaflavin derivatives and catechin derivatives are the major polyphenols in black tea and green tea, respectively. Several tea polyphenols, especially those with galloyl moiety, can inhibit HIV-1 replication with multiple mechanisms of action. Here we showed that the theaflavin derivatives had more potent anti-HIV-1 activity than catechin derivatives. These tea polyphenols could inhibit HIV-1 entry into target cells by blocking HIV-1 envelope glycoprotein-mediated membrane fusion. The fusion inhibitory activity of the tea polyphenols was correlated with their ability to block the formation of the gp41 six-helix bundle, a fusion-active core conformation. Computer-aided molecular docking analyses indicate that these tea polyphenols, theaflavin-3,3′-digallate (TF3) as an example, may bind to the highly conserved hydrophobic pocket on the surface of the central trimeric coiled coil formed by the N-terminal heptad repeats of gp41. These results indicate that tea, especially black tea, may be used as a source of anti-HIV agents and theaflavin derivatives may be applied as lead compounds for developing HIV-1 entry inhibitors targeting gp41.
Keywords: Tea polyphenol; HIV entry inhibitors; HIV-1 gp41; Six-helix bundle; Theaflavin;

Biochemical and molecular characterization of Staphylococcus xylosus lipase by Habib Mosbah; Adel Sayari; Hafedh Mejdoub; Hafedh Dhouib; Youssef Gargouri (282-291).
The Staphylococcus xylosus strain secretes a non-induced lipase in culture medium: S. xylosus lipase (SXL). Pure SXL is a monomeric protein (43 kDa).The 23 N-terminal amino acid residues were sequenced. This sequence is identical to that of Staphylococcus simulans lipase (SSL); in addition, it exhibits a high degree of homology with Staphylococcus aureus lipase (SAL NCTC 8530) sequences. The cloning and sequencing of gene part encoding the mature lipase shows one nucleotide difference with SSL, which corresponds to the change of one residue at a position 311.The lipase activity is maximal at pH 8.2 and 45 °C. SXL is able to hydrolyse triacylglycerols without chain length specificity. The specific activity of about 1900 U/mg was measured using tributyrin or triolein as substrate at pH 8.2 and at 45 °C in the presence of 2 mM CaCl2. In contrast to some previously characterized staphylococcal lipases, Ca2+ is not required to trigger the activity of SXL. SXL was found to be stable between pH 5 and pH 8.5. The enzyme maintains 50% of its activity after a 15-min incubation at 60 °C.Using tripropionin or vinyl esters as substrates, SXL does not present the interfacial activation phenomenon.Unlike many lipases, SXL is able to hydrolyse its substrate in the presence of bile salts or amphiphilic proteins. SXL is a serine enzyme, which is inhibited by THL.
Keywords: Staphylococcus xylosus lipase; Purification; Interfacial activation; Amphiphile; PCR; Sequencing;

Nardilysin facilitates complex formation between mitochondrial malate dehydrogenase and citrate synthase by K. Martin Chow; Zhangliang Ma; Jian Cai; William M. Pierce; Louis B. Hersh (292-301).
Gel filtration chromatography showed that nardilysin activity in a rat testis or rat brain extract exhibited an apparent molecular weight of ∼300 kDa compared to ∼187 kDa for the purified enzyme. The addition of purified nardilysin to a rat brain extract, but not to an E. coli extract, produced the higher molecular species. The addition of a GST fusion protein containing the acidic domain of nardilysin eliminated the higher molecular weight nardilysin forms, suggesting that oligomerization involves the acidic domain of nardilysin. Using an immobilized nardilysin column, mitochondrial malate dehydrogenase (mMDH) and citrate synthase (CS) were isolated from a fractionated rat brain extract. Porcine mMDH, but not porcine cytosolic MDH, was shown to form a heterodimer with nardilysin. Mitochondrial MDH increased nardilysin activity about 50%, while nardilysin stabilized mMDH towards heat inactivation. CS was co-immunoprecipitated with mMDH only in the presence of nardilysin showing that nardilysin facilitates complex formation.
Keywords: Acidic domain; Protein–protein interaction; Metabolon; N-arginine dibasic convertase; Centaurin-alpha; Tyrosyl-tRNA ligase;

Quantitative change in mitochondrial DNA content in various mouse tissues during aging by Mika Masuyama; Reiko Iida; Hisakazu Takatsuka; Toshihiro Yasuda; Takasumi Matsuki (302-308).
In order to systematically characterize age-related changes in the mtDNA content of various tissues during aging, we analyzed the mtDNA content of eight tissues from mice at five different ages from young to senescent by quantitative real-time PCR analysis. Obvious variations of mtDNA content among the tissues were detected: There was a 20-fold range in 2-week-old mice and a 50-fold range in 15-month-old mice. The mtDNA contents of the heart, lung, kidney, spleen and skeletal muscle increased gradually with age, whereas those of bone marrow and brain showed no age-related pattern. The expression patterns of mitochondrial transcription factor A (mtTFA) and mitochondrial single-strand DNA binding protein (mtSSB), possible regulatory factors of the mtDNA copy number, were not necessarily linked with the age-related pattern of the mtDNA content, suggesting the existence of other factors that affect the mtDNA content. The Western blot analysis of mtDNA-encoded cytochrome c oxidase subunit III (MTCO3) demonstrated that the expression levels of this protein in the heart and skeletal muscle increase with age in parallel with the mtDNA content. These findings confirm that the mtDNA content of tissues changes during aging.
Keywords: Mitochondrial DNA; Aging; Real-time PCR; Gene expression; Mouse tissue;

Seventeen new genes encoding 24 kDa family dimeric α-amylase inhibitors had been characterized from cultivated wheat and its diploid putative progenitors. And the different α-amylase inhibitors in this family, which were determined by coding regions single nucleotide polymorphisms (cSNPs) of their genes, were investigated. The amino acid sequences of 24 kDa α-amylase inhibitors shared very high coherence (91.2%). It indicated that the dimeric α-amylase inhibitors in the 24 kDa family were derived from common ancestral genes by phylogenetic analysis. Eight α-amylase inhibitor genes were characterized from one hexaploid wheat variety, and clustered into four subgroups, indicating that the 24 kDa dimeric α-amylase inhibitors in cultivated wheat were encoded by multi-gene. Forty-five cSNPs, including 35 transitions and 10 transversions, were found, and resulted in a total of ten amino acid changes. The cSNPs at the first site of a codon cause much more nonsynonymous (92.9%) than synonymous mutations, while nonsynonymous and synonymous mutations were almost equal when the cSNPs were at the third site. It was observed that there was Ile105 instead of Val105 at the active region Val104-Val105-Asp106-Ala107 of the α-amylase inhibitor by cSNPs in some inhibitors from Aegilops speltoides, diploid and hexaploid wheats.
Keywords: Single nucleotide polymorphism; 24 kDa dimeric α-amylase inhibitor; Triticum; Aegilops;

Aorta coarctation results in hypertension (HTN) in the arterial tree proximal to stenosis and, as such, provides an ideal model to discern the effects of different levels of blood pressure on the vascular tissue in the same animal. Compelling evidence has emerged supporting the role of oxidative stress as a cause of HTN. However, whether or not HTN (independent of the circulating humoral factors) can cause oxidative stress is less certain. NAD(P)H oxidase isoforms are the main source of reactive oxygen species (ROS) in the vascular tissues. We therefore compared the expressions of NOX-I, gp91phox and the regulatory subunits of the enzyme in the aorta segments residing above and below coarctation in rats with abdominal aorta banding. Rats were studied 4 weeks after aorta banding above the renal arteries or sham operation. Subunits of NAD(P)H oxidase and its NOX-I isoform as well as endothelial NO synthase (eNOS) and nitrotyrosine (footprint of NO oxidation by superoxide) were measured in the aorta segments above and below coarctation. The gp91phox, p47phox, and p67phox subunits of NAD(P)H oxidase, NOX-I isoform, eNOS and nitrotyrosine were markedly increased in the aorta segment above coarctation (hypertensive zone), but were virtually unchanged in the segment below coarctation. Since, excepting blood pressure, all other conditions were constant, the upregulation of NAD(P)H oxidase isoforms and the increased NO oxidation in the aorta segment above, but not below, coarctation prove that HTN, per se, independent of circulating mediators can cause oxidative/nitrosative stress in the arterial wall. These observations suggest that HTN control may represent a specific form of antioxidant therapy for hypertensive disorders.
Keywords: Hypertension; Oxidative stress; NADPH oxidase; NOX-I; Nitric oxide; Nitric oxide synthase; Aorta coarctation; Superoxide dismutase;