BBA - General Subjects (v.1724, #1-2)

The influence of extracellular pH (pHo) on low-voltage-activated calcium channels of acutely isolated DRG neurons of rats was examined using the whole cell patch-clamp technique. It has been found that in the neurons of middle size with capacitance C  = 60 ± 4.8 pF (mean ± S.E., n  = 8) extracellular acidification from pHo 7.35 to pHo 6.0 significantly and reversibly decreased LVA calcium current densities by 75 ± 3.7%, shifted potential for half-maximal activation to more positive voltages by 18.7 ± 0.6 mV with significant reduction of its voltage dependence. The half-maximal potential of steady-state inactivation shifted to more positive voltages by 12.1 ± 1.7 mV (n  = 8) and also became less voltage dependent. Dose–response curves for the dependence of maximum values of LVA currents on external pH in neurons of middle size have midpoint pK a  = 6.6 ± 0.02 and hill coefficient h  = 0.94 ± 0.04 (n  = 5). In small cells with capacitance C  = 26 ± 3.6 pF (n  = 5), acidosis decreased LVA calcium current densities only by 15.3 ± 1.3% and shifted potential for half-maximal activation by 5.5 ± 1.0 mV with reduction of its voltage dependence. Half-maximal potential of steady-state inactivation shifted to more positive voltages by 10 ± 1.6 mV (n  = 4) and also became less voltage dependent. Dose–response curves for the dependence of maximum values of LVA currents on external pH in neurons of small size have midpoint pK a  = 7.9 ± 0.04 and hill coefficient h  = 0.25 ± 0.1 (n  = 4). These two identified types of LVA currents besides different pH sensitivity demonstrated different kinetic properties. The deactivation of LVA currents with weak pH sensitivity after switching off depolarization to −30 mV had substantially longer decay time than do currents with strong pH sensitivity (τ d ∼5 ms vs. 2 ms respectively). It was found that the prolongation of depolarization steps slows the subsequent deactivation of T-type currents in small DRG neurons. Deactivation traces in these neurons were better described by the sum of two exponentials. Thus, we suppose that T-type channels in small DRG neurons are presented mostly by α1I subunit. We suggest that these two types of LVA calcium channels with different sensitivity to external pH can be differently involved in the origin of neuropathic changes.
Keywords: Dorsal root ganglion; T-type Ca2+ channels; Acidosis; pH sensitivity; Neuropathy;

Effects of a time-varying strong magnetic field on transient increase in Ca2+ release induced by cytosolic Ca2+ in cultured pheochromocytoma cells by Toshitaka Ikehara; Hisao Yamaguchi; Keiko Hosokawa; Hitoshi Houchi; Ki Ho Park; Kazuo Minakuchi; Hideki Kashimoto; Mitsuo Kitamura; Yohsuke Kinouchi; Kazuo Yoshizaki; Hiroshi Miyamoto (8-16).
Exposure of pheochromocytoma (PC 12) cells to a time-varying 1.51 T magnetic field inhibited an increase in the intracellular Ca2+ concentration ([Ca2+]i) induced by addition of caffeine to Ca2+-free medium. This inhibition occurred after a 15-min exposure and was maintained for at least 2 h. [Ca2+]i sharply increased in cells loaded with cyclic ADP-ribose, and 2-h exposure significantly suppressed the increase. Addition of ATP induced a transient increase in intracellular Ca2+ release mediated by IP3 receptor, and this increase was strongly inhibited by the exposure. Results indicated that the magnetic field exposure strongly inhibited Ca2+ release mediated by both IP3 and ryanodine receptors in PC 12 cells. However, thapsigargin-induced Ca2+ influx (capacitative Ca2+ entry) across the cell membrane was unaffected. The ATP content was maintained at the normal level during the 2-h exposure, suggesting that ATP hydrolysis was unchanged. Therefore, Mg2+ which is known to be released by ATP hydrolysis and inhibit intracellular Ca2+ release may not relate the exposure-caused inhibition. Eddy currents induced in culture medium appear to change cell membrane properties and indirectly inhibit Ca2+ release from endoplasmic reticulum and other Ca2+ stores in PC 12 cells.
Keywords: Ryanodine receptor; Caffeine; Cyclic ADP-ribose; Time-varying magnetic field; Eddy currents;

Mechanism of neopterin-induced myocardial dysfunction in the isolated perfused rat heart by Agnes Balogh; Markus Mittermayr; Andreas Schlager; Doris Balogh; Wolfgang Schobersberger; Dietmar Fuchs; Josef Margreiter (17-22).
Neopterin is a sensitive marker for diseases involving increased activity of the cellular immune system in humans. Many studies, however, provide evidence for neopterin not only as a marker, but also for its characteristic effects. Recently, we were able to demonstrate a considerable influence of exogenous neopterin at a concentration of 100 μmol/l on cardiac performance in the Langendorff model of isolated perfused rat hearts. The present study was designed to investigate its possible mechanism.During co-infusion of neopterin at a concentration of 100 μmol/l with the unspecific nitric oxide synthase inhibitor N G-monomethyl-l-arginine monoacetate, the nitric oxide donor PAPA NONOate, the free radical scavenger N-acetylcysteine, or the pro-inflammatory cytokine tumor necrosis factor-α the effects on cardiac contractility parameters and coronary vascular resistance were studied in 67 male Sprague–Dawley rats. The temperature-controlled and pressure-constant Langendorff apparatus was used with retrograde perfusion of the aorta and a Krebs–Henseleit buffer.Neither the unspecific nitric oxide synthase inhibitor nor the nitric oxide donor excludes nitric oxide from playing a mechanistic role in our perfusion studies. Tumor necrosis factor-α was without any synergistic or antagonistic effects when co-treated with neopterin. N-acetylcysteine was most effective in abolishing neopterin-dependent effects on cardiac function.The negative effects of neopterin on cardiac performance might be due to an enhancement of oxidative stress by neopterin that can be attenuated by the antioxidant N-acetylcysteine. Neopterin has to be considered a pathogenic factor in the development of cardiac dysfunction in chronic disease states with high neopterin levels secondary to activation of the immune system.
Keywords: Neopterin; Oxidant stress; Myocardial contractility; Langendorff; Rat heart;

Glucose induces complex patterns of oscillations in intracellular Ca2+ concentration ([Ca2+]i), metabolism and secretion in islets of Langerhans including “slow” and “fast” pulses with period of 2–5 min and 10–20 s respectively. In an effort to elucidate the origin of slow oscillations, individual mouse islets were exposed to different fuels including glyceraldehyde, pyruvate, methyl pyruvate and α-ketoisocaproate (KIC), all of which bypass key steps of glycolytic metabolism, while monitoring [Ca2+]i, oxygen consumption and secretion. Glyceraldehyde gave rise to slow oscillations only when substimulatory glucose was also added to the media. Glucosamine, an inhibitor of glucokinase, blocked these slow oscillations. KIC, pyruvate, and methyl pyruvate did not give rise to slow oscillations alone or with glucose present. The addition of glucose to islets bathed in nutrient-rich cell culture media accelerated metabolism and initiated slow oscillations while glyceraldehyde did not. It is concluded that glucose has a special role in accelerating metabolism and generating slow oscillations in isolated islets of Langerhans from mice. Combined with previous observations of Ca2+ dependency for all oscillations in islets, we propose that interactions between Ca2+ influx and glycolysis are responsible for the slow oscillations. In contrast, fast oscillations can occur independent of glycolytic flux.
Keywords: Islets of Langerhans; Oscillation; Secretion; Calcium; Oxygen; Metabolism;

Site-directed mutagenesis was used to study the role of histidine residues located in domain I in the neutral-to-basic (N–B) transition of human serum albumin (HSA). Based on a previous study of the N–B transition by means of proton NMR, the following recombinant HSA species were synthesized in the yeast species, Pichia pastoris: H9F, H9S, H39F, H39S, H67F, H67S, H105F, H105S, H128F, H128S, H146F, H146S, and wild type HSA. By monitoring the fluorescent intensity of warfarin bound to the above recombinant human serum albumin species as a function of pH, the mutational effect of individual histidine residues on the N–B transition was examined. While H9, H67, H105, H128 and H146 contribute to the transition significantly, H39 appears to have virtually no contribution to the transition. Based on the X-ray crystallographic structure, it is suggested that electrostatic interactions are the principal factor in determining the histidine pK shifts.
Keywords: Human serum albumin; Neutral-to-basic transition; Histidine residues; Site-directed mutagenesis; pK shift;

Coenzyme Q2 induced p53-dependent apoptosis by Yuki Esaka; Yukitoshi Nagahara; Yoshimune Hasome; Reiji Nishio; Masahiko Ikekita (49-58).
Coenzyme Q functions as an electron carrier and reversibly changes to either an oxidized (CoQ), intermediate (CoQ·), or reduced (CoQH2) form within a biomembrane. The CoQH2 form also acts as an antioxidant and prevents cell death, and thus has been successfully used as a supplement. On the other hand, the value of the CoQ/CoQH2 ratio has been shown to increase in a number of diseases, presumably due to an anti-proliferative effect involving CoQ. In the present study, we examined the effect of CoQ and its isoprenoid side chain length variants on the growth of cells having different p53 statuses. Treatment with CoQs having shorter isoprenoid chains, especially CoQ2, induced apoptosis in p53-point mutated BALL-1 cells, whereas treatment with longer isoprenoid chains did not. However, CoQ2 did not induce apoptosis in either a p53 wild-type cell line or a p53 null mutant cell line. These results indicated that the induction of apoptosis by CoQ2 was dependent on p53 protein levels. Moreover, CoQ2 induced reactive oxygen species (ROS) and the phosphorylation of p53. An antioxidant, l-ascorbic acid, inhibited CoQ2-induced p53 phosphorylation and further apoptotic stimuli. Overall, these results suggested that short tail CoQ induces ROS generation and further p53-dependent apoptosis.
Keywords: Coenzyme Q; p53; Apoptosis; Isoprenoid side chain; ROS;

Saccharomyces cerevisiae ribosomal stalk consists of five proteins: P0 protein, with molecular mass of 34 kDa, and four small, 11 kDa, P1A, P1B, P2A and P2B acidic proteins, which form a pentameric complex P0–(P1A–P2B)/(P1B–P2A). This structure binds to a region of 26S rRNA termed GTPase-associated domain and plays a crucial role in protein synthesis. The consecutive steps leading to the formation of the stalk structure have not been fully elucidated and the function of individual P-proteins in the assembling of the stalk and protein synthesis still remains elusive. We applied an integrated approach in order to examine all the P-proteins with respect to stalk assembly. Several in vitro methods were utilized to mimic protein self-organization in the cell. Our efforts resulted in reconstitution of the whole recombinant stalk in solution as well as on the ribosomal particle. On the basis of our analysis, it can be inferred that the P1A–P2B protein complex may be regarded as the key element in stalk formation, having structural and functional importance, whereas P1B–P2A protein complex is implicated in regulation of stalk function. The mechanism of quaternary structure formation could be described as a sequential co-folding/association reaction of an oligomeric system with P0–(P1A–P2B) protein complex as an essential element in the acquisition of a stable quaternary structure of the ribosomal stalk. On the other hand, the P1B–P2A complex is not involved in the cooperative stalk formation and our results indicate an increased rate of protein synthesis due to the latter protein pair.
Keywords: Ribosome; Ribosomal stalk; Acidic ribosomal P protein;

Regulation of SNARK activity in response to cellular stresses by Diana L. Lefebvre; Cheryl F. Rosen (71-85).
SNARK is a member of the AMPK subfamily of serine/threonine protein kinases. In this study, we examined the regulation of SNARK activity in kidney (BHK, HEK293), pancreatic β-cell insulinoma (INS-1), hepatocarcinoma (H4IIE) and keratinocyte (NRKC)-derived cell lines in response to diverse cellular stresses. We show that SNARK activity is regulated by glucose- or glutamine-deprivation, induction of endoplasmic reticulum stress by homocysteine or DTT, elevation of cellular AMP and/or depletion of ATP, hyperosmotic stress, salt stress, ultraviolet B radiation and oxidative stress caused by hydrogen peroxide. Moreover, the regulation of SNARK activity in response to cellular stresses depends greatly upon cell type. Furthermore, SNARK activity is downregulated by metformin in a dose- and time-dependent manner in H4IIE cells. These observations support a role for SNARK as a molecular component of the cellular stress response.
Keywords: Protein kinase; SNARK; AMPK; stress; Metformin;

Purification and characterization of a lectin from the white shrimp Litopenaeus setiferus (Crustacea decapoda) hemolymph by Juan Alpuche; Ali Pereyra; Concepción Agundis; Carlos Rosas; Cristina Pascual; Marie-Christine Slomianny; Lorena Vázquez; Edgar Zenteno (86-93).
A 291-kDa lectin (LsL) was purified from the hemolymph of the white shrimp Litopenaeus setiferus by affinity chromatography on glutaraldehyde-fixed stroma from rabbit erythrocytes. LsL is a heterotetramer of two 80-kDa and two 52-kDa subunits, with no covalently-liked carbohydrate, and mainly composed by aspartic and glutamic acids, glycine and alanine, with relatively lower methionine and cysteine contents. Edman degradation indicated that the NH2-terminal of the 80-kDa subunit is composed DASNAQKQHDVNFLL, whereas the NH2-terminal of the 52-kDa subunit is blocked. The peptide mass fingerprint of LsL was predicted from tryptic peptides from each subunit by MALDI-TOF, and revealed that each subunit showed 23 and 22%, respectively, homology with the hemocyanin precursor from Litopenaeus vannamei. Circular dichroism analysis revealed beta sheet and alpha helix contents of 52.7 and 6.1%, respectively. LsL agglutinate at higher titers guinea pig, murine, and rabbit erythrocytes its activity is divalent cation-dependent. N-acetylated sugars, such as GlcNAc, GalNAc, and NeuAc, were the most effective inhibitors of the LsL hemagglutinating activity. Sialylated O-glycosylated proteins, such as bovine submaxillary gland mucin, human IgA, and fetuin, showed stronger inhibitory activity than sialylated N-glycosylated proteins, such as human orosomucoid, IgG, transferrin, and lactoferrin. Desialylation of erythrocytes or inhibitory glycoproteins abolished their capacity to bind LsL, confirming the relevance of sialic acid in LsL–ligand interactions.
Keywords: Lectins; Crustaceans; Litopenaeus setiferus; MALDI-TOF; Hemocyanin; Sialic acid; Specific lectin;

Hyaluronan-binding motif identified by panning a random peptide display library by Kana Amemiya; Tatsuya Nakatani; Akio Saito; Atsuo Suzuki; Hiroshi Munakata (94-99).
The glycosaminoglycan hyaluronan (HA) is involved in a variety of functions such as cell migration, adhesion, activation of intracellular signaling, metastasis, inflammation and wound repair. These functions of HA are mediated via HA-binding proteins (HABPs). To derive details of the HA-binding site in HABPs, here, we panned a random peptide display library expressed on the E. coli flagellin protein using HA-coated plates. Using this random peptide display library, 40 positive clones were obtained and the nucleotide sequences were determined. As a result, an Arg–Arg sequence, in addition to the known B-X7-B motif, was found to bind to HA. A binding experiment using the IAsys resonant mirror biosensor verified that a peptide containing an Arg–Arg sequence binds to HA.
Keywords: Hyaluronan; Binding motif; Random peptide display library;

Reactivity towards singlet oxygen of propofol inside liposomes and neuronal cells by B. Heyne; D. Brault; M.-P. Fontaine-Aupart; S. Kohnen; F. Tfibel; A. Mouithys-Mickalad; G. Deby-Dupont; P. Hans; M. Hoebeke (100-107).
Singlet oxygen (1O2), a reactive oxygen species, has been found to be implicated in many cellular events and pathological disorders. Herein, we investigated the reactivity of 1O2 towards the anaesthetic agent propofol (PPF) encapsulated within DMPC liposomes. By time resolved luminescence, the rate constant of 1O2 quenching by PPF was evaluated, depending on the location of the sensitizer, with following values: 1.35 ± 0.05 × 107 M−1 s−1 for deuteroporphyrin (as embedded source) and 0.8 ± 0.04 × 107 M−1 s−1 for uroporphyrin (as external source), respectively. The nature of the oxidation product, resulting from the reaction of 1O2 with PPF, was determined using absorption and HPLC techniques. Finally, the in vitro protective effect of PPF towards the 1O2-induced neuronal cell toxicity was evaluated in terms of cell viability.
Keywords: Propofol; Singlet oxygen; Antioxidant; Phenoxyl radical; Liposome; Neuro-2a;

Modification of photosynthetic regulation in tomato overexpressing glutathione peroxidase by Stephane Herbette; Aline Le Menn; Patrick Rousselle; Thierry Ameglio; Zehava Faltin; Gérard Branlard; Yuval Eshdat; Jean-Louis Julien; Joël R. Drevet; Patricia Roeckel-Drevet (108-118).
To investigate the function of glutathione peroxidase (GPX) in plants, we produced transgenic tomato plants overexpressing an eukaryotic selenium-independent GPX (GPX5). We show here that total GPX activity was increased by 50% in transgenic plants, when compared to control plants transformed with the binary vector without the insert (PZP111). A preliminary two-dimensional electrophoretic protein analysis of the GPX overexpressing plants showed notably a decrease in the accumulation of proteins identified as rubisco small subunit 1 and fructose-1,6-bisphosphate aldolase, two proteins involved in photosynthesis. These observations, together with the fact that in standard culture conditions, GPX-overexpressing plants were not phenotypically distinct from control plants prompted us to challenge the plants with a chilling treatment that is known to affect photosynthesis activity. We found that upon chilling treatment with low light level, photosynthesis was not affected in GPX-overexpressing plants while it was in control plants, as revealed by chlorophyll fluorescence parameters and fructose-1,6-biphosphatase activity. These results suggest that overexpression of a selenium-independent GPX in tomato plants modifies specifically gene expression and leads to modifications of photosynthetic regulation processes.

Sulfotransferase 2B1b (SULT2B1b) is a member of SULT 2 gene family. SULT2B1a and SULT2B1b are transcribed from the same gene using different transcriptional start sites and contain different first exons as the result of alternative splicing. SULT2B1a and SULT2B1b are 350 and 365 AA in length, respectively. Northern blot analysis and SULT2B1 isoform specific RT-PCR detected only SULT2B1b message in human lung tissue. Immunoblot analysis of human lung tissue with a specific rabbit anti-SULT2B1 polyclonal antibody detected only SULT2B1b immunoreactive protein in human lung cytosol. Immunoprecipitation and MALDI mass spectroscopic analysis verified that the immunoreactive protein was SULT2B1b. Immunohistochemical localization of SULT2B1b in human tissues showed expression in the cytoplasm of ciliated columnar or cuboidal epithelial cells in terminal bronchia. No staining was observed in alveolar cells. SULT2B1b is selective for the sulfation of 3β-hydroxysteroids such as dehydroepiandrosterone and pregnenolone as well as cholesterol. The presence of SULT2B1b in lung tissues suggests a role in the regulation of local steroid hormone synthesis and metabolism.
Keywords: Sulfotransferase; Lung; SULT2B1b; Sulfation; Immunohistochemical localization;

An improved RNA amplification procedure results in increased yield of autologous RNA transfected dendritic cell-based vaccine by Jason Harris; Tamara Monesmith; Alicia Ubben; Marcus Norris; Jonathan H. Freedman; Irina Tcherepanova (127-136).
Use of antigen encoding RNA transfected Dendritic cells in the field of cancer immunotherapy has been well established. The use of RNA overcomes limitations inherent to other autologous DC-based vaccines as it does not require specific HLA haplotypes, identification and characterization of antigens, and captures the broadest antigen repertoire. RNA offers yet another advantage—it could be amplified minimizing the requirement of tumor mass for autologous vaccine production, and will afford the opportunity to treat patients with minimal tumor burden. The original procedure described for RNA amplification resulted in a proportion of RNA transcribed in the antisense orientation. This study also demonstrates that the presence of double-stranded RNA correlates with the presence of antisense RNA. Alternative design of oligonucleotides that removes sequence redundancy eliminates the formation of both antisense and double-stranded RNA species. We provide further evidence that amplified RNA containing antisense and double-stranded RNA species results in lower recovery of DCs post-transfection and maturation, presumably through sequence-specific gene silencing. The removal of the double-stranded species from amplified RNA results in higher recovery of mature autologous amplified RNA transfected dendritic cells. Higher DC yield will allow for reduction of cost of vaccine manufacturing and prolonged treatment of a patient.
Keywords: Amplified RNA; Dendritic cell vaccine; Antisense RNA; Immunotherapy;

Purification of a lectin from the marine red alga Gracilaria ornata and its effect on the development of the cowpea weevil Callosobruchus maculatus (Coleoptera: Bruchidae) by Yáskara Fabíola Monteiro Marques Leite; Luana Maria Castelo Melo Silva; Rodrigo César das Neves Amorim; Eder Almeida Freire; Daniel Macedo de Melo Jorge; Thalles Barbosa Grangeiro; Norma Maria Barros Benevides (137-145).
A lectin from the marine red alga Gracilaria ornata (Gracilariaceae, Rodophyta) was purified and characterized. The purification procedure consisted of extracting soluble proteins in 0.025 M Tris–HCl buffer, pH 7.5, followed by ammonium sulfate precipitation (70% saturation), ion exchange chromatography on DEAE-cellulose and affinity chromatography on mucin-Sepharose 4B. The purified G. ornata lectin (GOL) showed a single protein band with an apparent molecular mass of 17 kDa when submitted to SDS-polyacrylamide gel electrophoresis under reducing conditions. The native molecular mass of GOL determined by gel filtration on a Sephadex G-100 column was 17.4 kDa and its carbohydrate content was estimated to be 2.9%. Therefore, GOL is a monomeric glycoprotein. The purified lectin agglutinated trypsin-treated erythrocytes from rabbit and chicken but not from human. Its activity was not inhibited by any of the mono- and disaccharides tested but by the complex glycoproteins porcine stomach mucin, lactotransferrin, asialofetuin and bovine and porcine thyroglobulins. Isoelectric focusing showed that GOL is an acidic protein with a pI of 5.4 with analysis of its amino acid composition revealing high contents of Asx, Glx, Ser, Glu, Ala and Cys. When incorporated in artificial seeds, GOL significantly affected the development of Callosobruchus maculatus larvae, indicating the possibility of using this lectin in a biotechnological strategy for insect management of stored cowpea seeds.
Keywords: Lectin; Alga; Gracilaria ornata; Characterization; Callosobruchus maculatus; Insecticidal;

Engineering dihydropteroate synthase (DHPS) for efficient expression on M13 phage by Eeva-Christine Brockmann; Urpo Lamminmäki; Petri Saviranta (146-154).
Phage display is a commonly used selection technique in protein engineering, but not all proteins can be expressed on phage. Here, we describe the expression of a cytoplasmic homodimeric enzyme dihydropteroate synthetase (DHPS) on M13 phage, established by protein engineering of DHPS. The strategy included replacement of cysteine residues and screening for periplasmic expression followed by random mutagenesis and phage display selection with a conformation-specific anti-DHPS antibody. Cysteine replacement alone resulted in a 12-fold improvement in phage display of DHPS, but after random mutagenesis and three rounds of phage display selection, phage display efficiency of the library had improved 280-fold. Most of the selected clones had a common Asp96Asn mutation that was largely responsible for the efficient phage display of DHPS. Asp96Asn affected synergistically with the cysteine replacing mutations that were needed to remove the denaturing effect of potential wrong disulfide bridging in phage display. Asp96Asn alone resulted in a 1.8-fold improvement in phage display efficiency, but in combination with the cysteine replacing mutations, a total of 130-fold improvement in phage display efficiency of DHPS was achieved.
Keywords: Dihydropteroate synthase; Phage display; Protein expression; Protein engineering; Cysteine residue;

Isolation of the receptor for the Amaranthus leucocarpus lectin from human T lymphocytes by Flor Porras; Francisco Urrea; Blanca Ortiz; Salvador Martínez-Cairo; Stéphane Bouquelet; Gisela Martínez; Ricardo Lascurain; Edgar Zenteno (155-162).
Amaranthus leucocarpus lectin (ALL) is specific for GalNAc, and recognizes human T cells. The receptor for ALL was purified from T cells using biotin-labeled lectin and avidin-agarose as affinity matrix. It is a 70-kDa glycoprotein, constituted mainly by serine, glycine, and glutamic acid; its glycosidic portion contains mainly GalNAc; galactose, sialic acid, mannose, and GlcNAc were identified at a lower proportion. By ionic strength chromatography, as well as double dimension electrophoresis, we identified four isoforms of the ALL-receptor. N-terminal amino acid was blocked both in the ALL-receptor and its isoforms, therefore, tryptic peptides of ALL-receptor, analyzed through MALDI-TOF, were compared with the relative values obtained from the NCBInr (ProFound 2004/06/01) database. Our results indicated that the tryptic peptides obtained showed 54% homology with a DnaK-core molecular chaperone, 47% with human KIAA protein, and 44% with heat shock protein 8. The most frequent phenotype of the CD4 or CD8 ALL+ T cells was CD45RA+ CD27+; 26% of ALL+ T cells were CD25+ and 13% were CD69+, indicating that the glycoprotein recognized by ALL is present mainly on naive or quiescent T cells.
Keywords: T-cells; Glycoproteins; Lectins; Amaranthus leucocarpus; Ontogeny; O-glycans;

Effect of arylformamidase (kynurenine formamidase) gene inactivation in mice on enzymatic activity, kynurenine pathway metabolites and phenotype by Vasily N. Dobrovolsky; John F. Bowyer; Michael K. Pabarcus; Robert H. Heflich; Lee D. Williams; Daniel R. Doerge; Björn Arvidsson; Jonas Bergquist; John E. Casida (163-172).
The gene coding for arylformamidase (Afmid, also known as kynurenine formamidase) was inactivated in mice through the removal of a shared bidirectional promoter region regulating expression of the Afmid and thymidine kinase (Tk) genes. Afmid/Tk -deficient mice are known to develop sclerosis of glomeruli and to have an abnormal immune system. Afmid-catalyzed hydrolysis of N-formyl-kynurenine is a key step in tryptophan metabolism and biosynthesis of kynurenine-derived products including kynurenic acid, quinolinic acid, nicotinamide, NAD, and NADP. A disruption of these pathways is implicated in neurotoxicity and immunotoxicity. In wild-type (WT) mice, Afmid-specific activity (as measured by formyl-kynurenine hydrolysis) was 2-fold higher in the liver than in the kidney. Formyl-kynurenine hydrolysis was reduced by ∼50% in mice heterozygous (HZ) for Afmid/Tk and almost completely eliminated in Afmid/Tk knockout (KO) mice. However, there was 13% residual formyl-kynurenine hydrolysis in the kidney of KO mice, suggesting the existence of a formamidase other than Afmid. Liver and kidney levels of nicotinamide plus NAD/NADP remained the same in WT, HZ and KO mice. Plasma concentrations of formyl-kynurenine, kynurenine, and kynurenic acid were elevated in KO mice (but not HZ mice) relative to WT mice, further suggesting that there must be enzymes other than Afmid (possibly in the kidney) capable of metabolizing formyl-kynurenine into kynurenine. Gradual kidney deterioration and subsequent failure in KO mice is consistent with high levels of tissue-specific Afmid expression in the kidney of WT but not KO mice. On this basis, the most significant function of the kynurenine pathway and Afmid in mice may be in eliminating toxic metabolites and to a lesser extent in providing intermediates for other processes.
Keywords: Arylformamidase; Formyl-kynurenine; Kynurenine; Kynurenic acid; Kynurenine formamidase; Tryptophan;

The control system of sperm motility in the fishes with internal fertilization still remains to be studied. In the present study, we examined the sperm activation in the viviparous fish guppy Poecilia reticulata as a function of solute composition. The degree of sperm activation by inorganic and organic solutes correlated well with the lyotropic or Hofmeister series. The solutes that induced sperm activation corresponded to those that have been termed “chaotropes” or macromolecule-destabilizing solutes. In addition to this Hofmeister rule, multivalent cations and anions had valency-dependent activating effects at low concentrations. We investigated the cell signaling triggered by chaotropes and divalent cations and found an intracellular cAMP concentration increase upon the activation of sperm motility. From the present findings, we propose a novel regulation mechanism of sperm motility in the viviparous fish.
Keywords: Hofmeister series; Sperm motility; Viviparous fish; Chaotropic ion; Guppy Poecilia reticulata;

Glucose-derived Amadori compounds of glutathione by Mikhail D. Linetsky; Ekaterina V. Shipova; Roy D. Legrand; Ognyan O. Argirov (181-193).
Under the chromatographic conditions used in these studies we observed time- and concentration-dependent formation of N-1-Deoxy-fructos-1-yl glutathione as the major glycation product formed in the mixtures of GSH with glucose. N-1-Deoxy-fructos-1-yl glutathione had a characteristic positively charged ion with m/z  = 470 Th in its LC-MS spectra. Mixtures of glutathione disulfide and glucose generated two compounds: N-1-Deoxy-fructos-1-yl GSSG (m/z  = 775 Th) as major adduct and bis di-N, N′-1-Deoxy-fructos-1-yl GSSG (m/z  = 937 Th) as the minor one. All three compounds showed a resonance signal at 55.2 ppm in the 13C-NMR spectra as C(1) methylene group of deoxyfructosyl, which represents direct evidence that they are Amadori compounds. All three compounds purified from GSSG/Glc or GSH/Glc mixtures also showed LC-MS/MS fragmentation patterns identical to those of the synthetically synthesized N-1-Deoxy-fructos-1-yl glutathione, N-1-Deoxy-fructos-1-yl GSSG and bis di-N, N′-1-Deoxy-fructos-1-yl GSSG. N-1-Deoxy-fructos-1-yl glutathione was shown to be a poor substrate for glutathione peroxidase (6.7% of the enzyme's original specific activity) and glutathione-S-transferase (25.7% of the original enzyme's specific activity). Glutathione reductase failed to recycle the disulfide bond within the structure of di-substituted bis di-N, N′-1-Deoxy-fructos-1-yl GSSG. It showed only 1% of the original enzyme's specific activity, but retained its ability to reduce the disulfide bond within the structure of N-1-Deoxy-fructos-1-yl GSSG by 57% of its original specific activity. Since the GSH concentration in diabetic lens is significantly decreased and the glucose concentration can increase 10-fold and higher, the formation of Amadori products of the different forms of glutathione with this monosaccharide may be favored under these conditions and could contribute to a lowering of glutathione levels and an increase of oxidative stress observed in diabetic lens.
Keywords: Non-enzymatic glycation; Glutathione; Glucose; Amadori compounds;

Two metalloproteinases, a 24-kDa P-I EoVMP1 and a 56-kDa P-III EoVMP2, have recently been isolated from the venom of the West African saw-scaled viper Echis ocellatus. We now reveal a new 65-kDa haemorrhagic group P-III metalloproteinase which we have designated EoVMP3. The aim of this study was to determine whether these three snake venom metalloproteinases (SVMPs) affect platelets and blood coagulation. EoVMP1 had no effect on the aggregation of washed human platelets, whereas EoVMP2 inhibited collagen-induced platelet aggregation. In contrast, EoVMP3 did not inhibit the aggregation of platelets by collagen but instead activated platelets in the absence of any additional co-factors. All three SVMPs were capable of activating prothrombin to varying degrees and can therefore be described as procoagulants. EoVMP1, EoVMP2 and EoVMP3 share sequence identity with other members of the reprolysin family, but differ greatly in their effects on some of the components that control haemostasis.
Keywords: Echis; Venom; Metalloproteinase; Platelet; Coagulation; Inhibition;

We describe the synthesis of new perfluorinated dimerizable detergents which contain a tricationic or tetracationic (linear or branched spermine, respectively) polar head, and report on their cmc, their ability to condense DNA into cationic monomolecular DNA nanoparticles as well as on the in vitro transfection efficiency of these nanoparticles. Such cationic nanoparticles were prone to display efficient cell transfection properties as a result of increased contact to the anionic cell surface and internalization by endocytosis, low size compatible with improved intracellular diffusion and nuclear pore crossing, and the presence of amine function of low pK a for their endosomal escape. The challenge was to design polymerizable polycationic detergents that display a cmc high enough for the monomer to perform monomolecular DNA condensation (as cationic particles) and low enough for the dimer to form stable nanoparticles capable of efficient cell transfection. Although we succeeded in formulating small-sized cationic monomolecular DNA nanoparticles (<40 nm) with these dimerizable perfluorinated spermine-based detergents for N/P ratios of up to 5 (N  = number of detergent amine equivalents /  P  = number of DNA phosphate equivalents), these small-sized cationic nanoparticles proved to be poor non-specific transfection agents in vitro, even in the presence of chloroquine. Their poor transfection potential could be due more likely to Brownian motion which prevents these very small-sized particles from sedimentation and adsorption onto the adherent cell monolayer, and, consequently, from proteoglycan-triggered endocytosis.
Keywords: Gene transfer; DNA complex; Nonviral vector; Synthetic vector;

Hesperetin (5,7,3′-trihydroxyl-4′-methoxyl-flavanone) is an important bioactive compound in Chinese traditional medicine and has multiple biological and pharmacological activities. The interaction of hesperetin with human serum albumin (HSA) has been investigated by UV absorption, fluorescence and Fourier transformed infrared spectrometry. Fluorescence results showed that one molecule of protein combined with one molecule of drug at the molar ratio of drug to HSA ranging from 0.3 to 7 and the binding affinity (K A) was 8.11 × 104 M−1. The primary binding site was most likely located on subdomain IIA. The binding ability of the drug to protein decreased from pH 6.4 to 8.4 in the drug to protein molar ratio of 1. Combining the curve-fitting results of infrared amide I band in D2O and H2O phosphate buffers, the alterations of protein secondary structure after drug complexation were estimated. With increasing the drug concentration, the percentage of protein α-helix structure decreased gradually. The reduction of protein α-helix structure reached about 7–9% after the protein interacted with hesperetin in D2O and H2O buffer solution at pH 7.4 when the drug to protein molar ratio was 10. This indicated a partial unfolding of HSA in the presence of the drug. From the results of UV absorption, fluorescence and Fourier transformed infrared spectrometry, the binding mode was discussed. The main mechanism of protein fluorescence quenching was a static quenching process and the hydroxyl groups of the drug in its neutral part played an important role in the binding process.
Keywords: Hesperetin; Human serum albumin; Fourier transform infrared spectroscopy; Fluorescence spectroscopy; UV absorption; Secondary structure.;