Black tea extract, thearubigin fraction, counteracts the effect of tetanus toxin in mice

The aim of this study was to find an inactivating substance for tetanus toxin in natural foodstuff. Tetanus toxin (4 micrograms/ml) abolished indirect twitches in In vitro mouse phrenic nerve-diaphragm preparations within 2.5 hr. Hot water infusion of black tea mixed with tetanus toxin blocked the inhibitory effect of the toxin. Mixing the toxin with thearubigin fraction extracted from black tea infusion produced an identical result. Furthermore, thearubigin fraction mixed with the toxin protected against the in vivo paralytic effect of the toxin. Thearubigin fraction had no protective effect on other toxins, such as tetrodotoxin and saxitoxin. The specific binding of [125I]tetanus toxin to rat cerebrocortical synaptosomes was inhibited by mixing iodinated toxin with thearubigin fraction. These results imply that thearubigin fraction counteracts the effect of tetanus toxin by binding with toxin, and also suggest that this fraction may be able to apply for prophylaxis of tetanus.     

LOX-1 inhibition in myocardial ischemia-reperfusion injury: modulation of MMP-1 and inflammation

A recently identified lectin-like oxidized low-density lipoprotein receptor (LOX-1) mediates endothelial cell injury and facilitates inflammatory cell adhesion. We studied the role of LOX-1 in myocardial ischemia-reperfusion (I/R) injury. Anesthetized Sprague-Dawley rats were subjected to 60 min of left coronary artery (LCA) ligation, followed by 60 min of reperfusion. Rats were treated with saline, LOX-1 blocking antibody JXT21 (10 mg/kg), or nonspecific anti-goat IgG (10 mg/kg) before I/R. Ten other rats underwent surgery without LCA ligation and served as a sham control group. LOX-1 expression was markedly increased during I/R (P < 0.01 vs. sham control group). Simultaneously, the expression of matrix metalloproteinase-1 (MMP-1) and adhesion molecules (P-selectin, VCAM-1, and ICAM-1) was also increased in the I/R area (P < 0.01 vs. sham control group). There was intense leukocyte accumulation in the I/R area in the saline-treated group. Treatment of rats with the LOX-1 antibody prevented I/R-induced upregulation of LOX-1 and reduced MMP-1 and adhesion molecule expression as well as leukocyte recruitment. LOX-1 antibody, but not nonspecific IgG, also reduced myocardial infarct size (P < 0.01 vs. saline-treated I/R group). To explore the link between LOX-1 and adhesion molecule expression, we measured expression of oxidative stress-sensitive p38 mitogen-activated protein kinase (p38 MAPK). The activity of p38 MAPK was increased during I/R (P < 0.01 vs. sham control), and use of LOX-1 antibody inhibited p38 MAPK activation (P < 0.01). These findings indicate that myocardial I/R upregulates LOX-1 expression, which through p38 MAPK activation increases the expression of MMP-1 and adhesion molecules. Inhibition of LOX-1 exerts an important protective effect against myocardial I/R injury.     

Transcriptional regulation of nuclear orphan receptor,NOR-1, by Ca(2+)/calmodulin-dependent protein kinase cascade

It has been proposed that the REV1 protein plays an important role in the induced-mutagenesis pathway. We show that purified REV1 protein inserts dCMP opposite template G, A, T and C, and dGMP and dTMP opposite template G in the presence of magnesium, while in the presence of manganese the specificity for dCMP was found to be relaxed and the REV1 protein acquired the ability to insert dCMP, dGMP, dAMP and dTMP opposite templates G, A, T, and C. Kinetic analysis provided evidence for high affinity for dCTP with template G, suggesting that the REV1 protein is specialized for dCTP and template G.     

Effect of carnosine and related compounds on the inactivation of human Cu,Zn-superoxide dismutase by modification of fructose and glycolaldehyde

Glycolaldehyde, an intermediate of the Maillard reaction, and fructose, which is mainly derived from the polyol pathway, rapidly inactivate human Cu,Zn-superoxide dismutase (SOD) at the physiological concentration. We employed this inactivation with these carbonyl compounds as a model glycation reaction to investigate whether carnosine and its related compounds could protect the enzyme from inactivation. Of eight derivatives examined, histidine, Gly-His, carnosine and Ala-His inhibited the inactivation of the enzyme by fructose (p<0.001), and Gly-His, Ala-His, anserine, carnosine, and homocarnosine exhibited a marked protective effect against the inactivation by glycolaldehyde (p<0.001). The carnosine-related compounds that showed this highly protective effect against the inactivation by glycolaldehyde had high reactivity with glycolaldehyde and high scavenging activity toward the hydroxyl radical as common properties. On the other hand, the carnosine-related compounds that had a protective effect against the inactivation by fructose showed significant hydroxyl radical-scavenging ability. These results indicate that carnosine and such related compounds as Gly-His and Ala-His are effective anti-glycating agents for human Cu,Zn-SOD and that the effectiveness is based not only on high reactivity with carbonyl compounds but also on hydroxyl radical scavenging activity.     

Regulation of galectin-9 expression and release in Jurkat T cell line cells

Ecalectin/galectin-9 was recently described as a novel eosinophil chemoattractant highly expressed in immune tissues. We investigated the regulation of galectin-9 expression and release in Jurkat (a T cell line) cells. We demonstrated that medium and long-sized galectin-9 isoforms were constitutively expressed, and phorbol 12-myriastate 13-acetate (PMA) upregulated the level of galectin-9 mRNA in Jurkat cells. Western blotting and flow cytometry analyses revealed that PMA stimulation resulted in the upregulation of both intracellular and surface galectin-9 protein. The stimulated Jurkat cells simultaneously released evident eosinophil chemoattractant activity (ECA). Main ECA was adsorbed by both lactose and anti-galectin-9 antibody affinity column, suggesting that the ECA was ascribed to galectin-9. When Jurkat cells were stimulated with PMA in the presence of a BB94, a matrix metalloproteinase (MMP) inhibitor, but not tissue inhibitor of metalloproteinase-1 (TIMP-1), the release of galectin-9 was suppressed in a dose-dependent manner. We further found that calphostin c, a protein kinase c (PKC) inhibitor, weakly but significantly suppressed the release of galectin-9. The present data suggested that galectin-9 production in Jurkat cells is provoked by the stimulation with PMA and that some MMP and PKC is, at least, partly involved in the release of galectin-9 from Jurkat cells.     

Synthesis of DNA conjugates by solid phase fragment condensation

Development of a novel method for the synthesis of DNA conjugates is described. Oligonucleotides were successfully conjugated with a variety of functional molecules on a solid phase (Solid Phase Fragment Condensation) using an amino, a hydroxyl, a thiol, and a carboxyl group. DNA-peptide conjugate was obtained as a pure from by a single RPHPLC purification approximately in 20% yield. Moreover, it was demonstrated that the present method was effective for the preparation of conjugate molecules, DNA-sugar, DNA-polyamine, DNA-lipid and so on. The study to create new intelligent DNAs by accumulation various biofunctions on the molecule by SPFC is now in progress in our laboratory.     

Tissue distribution,molecular cloning,and gene expression of cytosolic glutathione peroxidase in Japanese monkey

Cytosolic glutathione peroxidase (GPX-1) is an important antioxidant enzyme that scavange hydrogen peroxide in mammalian cells. The level of GPX-1 activity in Japanese monkey (Macaca fuscata) tissues was determined and it was found to be high in the liver, kidney, and adrenal gland followed by the small intestine. We also cloned the GPX-1 cDNA that included the whole protein-coding region. The active-site selenocysteine was assumed to be encoded by a TGA codon. Compared to the GPX-1s of other mammalian species, essential residues in catalysis were well conserved in monkey GPX-1. Amino acid substitutions were frequent in the N- and C-terminal regions which are less essential in catalysis. Expression of GPX-1 mRNA was found to be high in the liver, kidney, and adrenal gland, in consistence with the tissue distribution of GPX-1 activity.     

Upregulation of LOX-1 expression in aorta of hypercholesterolemic rabbits: modulation by losartan

Angiotensin-II (Ang-II) enhances the modification of LDL and the expression of its lectin-like receptor (LOX-1) by activating type 1 (AT(1)) receptors. This study was designed to determine the effect of hypercholesterolemia on LOX-1 expression in aorta and its modulation by the AT(1) receptor blocker losartan. Male New Zealand White rabbits were fed regular chow (Control group), chow with 1% cholesterol and 4% peanut oil (HC-diet group), or 1% cholesterol and 4% peanut oil diet plus losartan (25 mg/kg/day) (Losartan + HC-diet group) for 10 weeks. Animal body weight, serum cholesterol levels, and arterial blood pressure were measured. Aortic intimal thickening was quantitated in H&E-stained segments. LOX-1 expression in aortas was examined by immunohistochemistry and semi-quantitative RT-PCR. High-cholesterol diet did not affect body weight, but induced hypercholesterolemia and extensive intimal thickening. Aortas of rabbits in the control group showed a modest LOX-1 expression in the endothelium. Aortic intimal proliferation in HC-diet group was associated with a marked increase in LOX-1 expression (protein and mRNA) in the endothelium and neointima. Treatment with losartan attenuated aortic intimal proliferation and markedly decreased the enhanced LOX-1 expression. Thus high-cholesterol diet induces the upregulation of LOX-1 expression in neointima of aortas of rabbits. Treatment with losartan, an AT(1) blocker, markedly decreases this enhanced LOX-1 expression.     

Excision repair of uracil-containing DNA in a mitochondrial extract of Xenopus oocytes

A mitochondrial extract of Xenopus oocytes was prepared to characterize DNA repair reactions operating in mitochondria. We asked whether and how uracils, spontaneously formed in DNA as a result of cytosine deamination, would be repaired in the extract. When a 40-mer oligonucleotide duplex with a single deoxyuridine at position 21 was incubated in the extract, incision took place at the 5' side of the lesion, and the uracil-containing strand was mostly cleaved within 15 min. Subsequent repair DNA synthesis produced DNA fragments of various sizes. Analysis of these repair intermediates indicated that the 3' border of the repair patches was at position 22. Completion of the repair reaction however was rare in this system, due in part to insufficient ligation activity of the extract.     

Biosynthesis and post-translational processing of lectin-like oxidized low density lipoprotein receptor-1 (LOX-1). N-linked glycosylation affects cell-surface expression and ligand binding

LOX-1 (lectin-like oxidized low density lipoprotein receptor-1) is a type II membrane protein belonging to the C-type lectin family that can act as a cell-surface receptor for atherogenic oxidized low density lipoprotein (Ox-LDL) and may play crucial roles in atherogenesis. In this study, we show, by pulse-chase labeling and glycosidase digestion, that LOX-1 is synthesized as a 40-kDa precursor protein with N-linked high mannose carbohydrate chains (pre-LOX-1), which is subsequently further glycosylated and processed into the 48-kDa mature form within 40 min. Furthermore, when treated with an N-glycosylation inhibitor, tunicamycin, both tumor necrosis factor-alpha-activated bovine aortic endothelial cells and CHO-K1 cells stably expressing bovine LOX-1 (BLOX-1-CHO) exclusively produced a 32-kDa deglycosylated form of LOX-1. Cell enzyme-linked immunosorbent assay, flow cytometry, and immunofluorescence confocal microscopy demonstrated that the deglycosylated form of LOX-1 is not efficiently transported to the cell surface, but is retained in the endoplasmic reticulum or Golgi apparatus in tumor necrosis factor-alpha-activated bovine aortic endothelial cells, but not in BLOX-1-CHO cells. Radiolabeled Ox-LDL binding studies revealed that the deglycosylated form of LOX-1 expressed on the cell surface of BLOX-1-CHO cells has a reduced affinity for Ox-LDL binding. Taken together, N-linked glycosylation appears to play key roles in the cell-surface expression and ligand binding of LOX-1.