In Vitro Antiviral Activity of Mycophenolic Acid and Its Reversal by Guanine-Type Compounds

With the agar diffusion test and BS-C-1 cells, mycophenolic acid was found to give a straight-line dose-response activity in inhibiting the cytopathic effects of vaccinia, herpes simplex, and measles viruses. Plaque tests have shown 100% reduction of virus plaques by mycophenolic acid over drug ranges of 10 to 50 mug/ml and virus input as high as 6,000 plaque-forming units (PFU) per flask. Back titration studies with measles virus inhibited by mycophenolic acid have indicated that extracellular virus titers were reduced by approximately 3 logs(10) and total virus was reduced by 1 log(10). The agar diffusion test system lends itself readily to drug reversal studies. Mycophenolic acid incorporated into agar at 10 mug/ml gave 100% protection to virus-infected cells. Filter paper discs impregnated with selected chemical agents at concentrations of 1,000 mug/ml (20 mug per filter paper disc) were placed on the agar surface. Reversal of the antiviral activity of mycophenolic acid was indicated by virus breakthrough in those cells in close proximity to the filter paper disc. Chemicals showing the best reversal of the antiviral activity of mycophenolic acid were guanine, guanosine, guanylic acid, deoxyguanylic acid, and 2,6-diaminopurine. The reversal of antiviral activity was confirmed by titrations of virus produced with various amounts of both mycophenolic acid and guanine present and by isotope tracer methods with uptakes of labeled uridine, guanine, leucine, and thymidine in treated and nontreated, infected and noninfected cells as parameters. All antiviral effects of mycophenolic acid at 10 mug/ml could be reversed to the range shown by untreated controls by the addition of 10 mug/ml of those chemicals exhibiting reversal activity.     

Influence of Ovarian Hormones on Cortical Spreading Depression and Its Suppression by L-kynurenine in Rat

Migraine is sexually dimorphic and associated in 20–30% of patients with an aura most likely caused by cortical spreading depression (CSD). We have previously shown that systemic L-kynurenine (L-KYN), the precursor of kynurenic acid, suppresses CSD and that this effect depends on the stage of the estrous cycle in female rats. The objectives here are to determine the influence of ovarian hormones on KCl-induced CSD and its suppression after L-KYN by directly modulating estradiol or progesterone levels in ovariectomized rats. Adult female rats were ovariectomized and subcutaneously implanted with silastic capsules filled with progesterone or 17β-estradiol mixed with cholesterol, with cholesterol only or left empty. Two weeks after the ovariectomy/capsule implantation, the animals received an i.p. injection of L-KYN (300 mg/kg) or NaCl as control. Thirty minutes later CSDs were elicited by applying KCl over the occipital cortex and recorded by DC electrocorticogram for 1 hour. The results show that both estradiol and progesterone increase CSD frequency after ovariectomy. The suppressive effect of L-KYN on CSD frequency, previously reported in normal cycling females, is not found anymore after ovariectomy, but reappears after progesterone replacement therapy. Taken together, these results emphasize the complex role of sex hormones on cortical excitability. The CSD increase by estradiol and, more surprisingly, progesterone may explain why clinically migraine with aura appears or worsens during pregnancy or with combined hormonal treatments.     

Oleic Acid Alleviates Cadmium-Induced Oxidative Damage in Rat by Its Radicals Scavenging Activity

Toxic heavy metal cadmium wildly pollutes the environment and threats the human health. Effective treatment of cadmium-induced toxicity and organ damage is an important issue. Cadmium causes organ damage through inducing oxidative stress. Our previous study also found oleic acid (OA) synthesis-related gene can confer resistance to cadmium and alleviate cadmium-induced stress in yeast. However, its alleviation mechanism on cadmium stress especially in animals is still unclear. In this study, the alleviative effects of OA on cadmium and cadmium-induced oxidative stress in rats were investigated. Oral administration of 10, 20, and 30 mg/kg/day OA can significantly increase the survival rate of rats intraperitoneally injected with 30 mg/kg/day cadmium continuously for 7 days. Similar to ascorbic acid (AA), OA can significantly reduce the cadmium-induced lipid peroxidation in multiple organs of rats. The investigation of OA on superoxide dismutase (SOD) activity showed that OA increased the SOD activity of cadmium-treated rat organs. More important, OA reduced the level of superoxide radical O²⁻ of cadmium-treated rat organs. And OA exhibited a strong DPPH radicals scavenging activity at dose of 10, 20 and 30 mg/mL, which may contributed to alleviating cadmium-induced oxidative stress. This study revealed that OA could significantly alleviate cadmium stress via reducing cadmium-induced lipid peroxidation and SOD activity inhibition through its radicals scavenging activity.

     

Convergent Synthesis of the Rat Galanin and Study of Its Biological Activity

Russian Journal of Bioorganic Chemistry - The full-length rat galanin (GWTLNSAGYLLGPHAIDNHRSFSDKHGLT-NH2, G29) was prepared by the solid phase peptide synthesis using the Fmoc-strategy. The peptide...     

Phosphorylation of Rat Brain Choline Acetyltransferase and Its Relationship to Enzyme Activity

Abstract— Choline acetyltransferase catalyzes the formation of acetylcholine from choline and acetyl-CoA in cholin-ergic neurons. The present study examined conditions for modulation of kinase-mediated phosphorylation of this enzyme. By using a monospecific polyclonal rabbit anti-human choline acetyltransferase antibody to immunoprecipi-tate cytosolic and membrane-associated subcellular pools of enzyme from rat hippocampal synaptosomes, we determined that only the cytosolic fraction of the enzyme (67,000 ± 730 daltons) was phosphorylated under basal, unstimulated conditions. The quantity of this endogenous phosphoprotein was dependent, in part, upon the level of intracellular calcium, with ³²P_i incorporation into the enzyme in nerve terminals incubated in nominally calcium-free medium only 43 ± 7% of control. The corresponding enzymatic activity of cytosolic choline acetyltransferase did not appear to be altered by lowered cytosolic calcium, whereas membrane-associated choline acetyltransferase activity was decreased to 58 ± 11 % of control. Depolarization of synaptosomes with 50 μ M veratridine neither altered the extent of phosphorylation or specific activity of cytosolic choline acetyltransferase, nor induced detectable phosphorylation of membrane-associated choline acetyltransferase, although the specific activity of the membrane-associated enzyme was increased to 132 ± 5% of control. In summary, phosphorylation of choline acetyltransferase does not appear to regulate cholinergic neurotransmission by a direct action on catalytic activity of the enzyme.     

CONTACT-MEDIATED REVERSIBLE SUPPRESSION OF MYOGENESIS : II. Reversal of Suppression by Bromodeoxyuridine

Chondrocytes from the vertebral columns of 11-day chick embryos were cultured in the continuous presence of 5-bromodeoxyuridine (BUdR) Under these conditions the cells form multilayers but synthesize little extracellular matrix as determined by toluidine blue metachromasia or sulfate-³⁵S incorporation into polysaccharide. Myogenic cells from the breast muscles of 11-day chick embryos formed myotubes when plated into BUdR-treated chondrocyte cultures. When plated on untreated chondrocyte multilayers or on multilayers which had been permitted to recover from BUdR treatment for 3 days, myogenic cells failed to form myotubes Since extracellular matrix is present in untreated chondrocyte cultures and reappears in multilayers recovering from BUdR treatment, it is suggested that extracellular matrix is the active agent in the suppression of myogenesis An attempt was made to duplicate the suppressing activity of multilayer cultures by using ion exchange resins as substrates for myogenic cells Myotubes formed on acidic and basic resin particles If extracellular matrix is the active suppressing agent, it may have to fulfill certain spatial distributional requirements before its activity is expressed     

Suppression of Anti-Candida Activity of Murine and Human Neutrophils by Glucocorticoids

Effects of glucocorticoid (GC) compounds on inhibitory activity of neutrophils to mycelial growth of Candida albicans were examined by in vitro crystal violet staining method with 14 hr co-culture. Both GC hormones (hydrocortisone ≥6 × 10^–7 m and corticosterone ≥10^–6 m ) and anti-inflammatory GC agents (prednisolone ≥10^–7 m and dexamethasone ≥10^–8 m ) significantly suppressed anti-Candida activity of murine casein-induced neutrophils. Anti-Candida activity of human neutrophils prepared from peripheral blood was also suppressed by hydrocortisone (≥6 × 10^–7 m ). These GC compounds did not affect the Candida growth in the absence of neutrophils. Steroidal compounds without anti-inflammatory activity, cholesterol, cholic acid, aldosterone did not suppress neutrophil activity. These results suggest that GCs at their physiological or clinical concentration may suppress anti-Candida activity of neutrophils in vivo.     

Suppression of Membrane Phospholipase C Activity by Synthetic Autoinhibitor Peptides

It has been demonstrated that the phospholipase C-γ (PLCγ) molecule possesses within it a phospholipase C inhibitor (PCI) region and that synthetic peptides based on the sequence of the PCI region suppress the enzymatic activity of PLC isoforms [Y. Homma and T. Takenawa (1992) J. Biol. Chem.267, 21884–218891. For the present study, the applicability of these peptides as inhibitors of PLC activity In plasma membranes was examined. Synthetic peptides, the original PCI peptide (24-mer) and a minimum octamer (YRKMRLRY), inhibit Ca²⁺-inducible PLC activation in digitonine-permeabilized cells, while a dodecamer sequence within the PCI region (SYYEKHALYRKM) does not. Similar results were obtained in both agonist- and GTP-binding protein-inducible PLC activation systems using purified plasma membranes. The inhibitory effect described here appears to reflect the inhibitory potency of the peptides against purified PLC isoforms. Therefore, these inhibitor peptides could provide an excellent tool for analyzing protein–protein interactions and resulting PLC activation.     

Stabilization of Acetylcholine Receptors by Exogenous ATP and Its Reversal by cAMP and Calcium

Innervation of the neuromuscular junction (nmj) affects the stability of acetylcholine receptors (AChRs). A neural factor that could affect AChR stabilization was studied using cultured muscle cells since they express two distinct populations of AChRs similar to those seen at the nmjs of denervated muscle. These two AChR populations are (in a ratio of 9 to 1) a rapidly degrading population (Rr) with a degradation half-life of ~1 d and a slowly degrading population (Rs) that can alternate between an accelerated form (half-life ~3–5 d) and a stabilized form (half-life ~10 d), depending upon the state of innervation of the muscle.

Previous studies have shown that elevation of intracellular cAMP can stabilize the Rs, but not the Rr. We report here that in cultured rat muscle cells, exogenous ATP stabilized the degradation half-life of Rr and possibly also the Rs. Furthermore, pretreatment with ATP caused more stable AChRs to be inserted into the muscle membrane. Thus, in the presence of ATP, the degradation rates of the Rr and Rs overlap. This suggests that ATP released from the nerve may play an important role in the regulation of AChR degradation. Treatment with either the cAMP analogue dibutyryl-cAMP (dB-cAMP) or the calcium mobilizer ryanodine caused the ATP-stabilized Rr to accelerate back to a half-life of 1 d. Thus, at least three signaling systems (intracellular cAMP, Ca²⁺, and extracellular ATP) have the potential to interact with each other in the building of an adult neuromuscular junction.

     

Induction of Salt Sensitivity in Ophiostoma and Its Reversal by Imidazole Derivatives

Cells of the ascomycete Ophiostoma multiannulatum became sensitive to inorganic salts after a heat shock or a treatment with 2,4-dinitrophenol. The induced sensitivity to chloride and bromide ions could be largely reversed by histidine and certain other imidazole derivatives. No other organic compounds tested except imidazoles possessed this ability. The sensitization is interpreted as a consequence of an injury to cellular membranes, in particular those of the mitochondria, and to an impaired oxidative phosphorylation.     

Growth Inhibition by D-Mannosamine and Its Reversal by Glucose and Mannose in Bacillus subtilis

Growth of the Bacillus subtilis wild type strain was severelyinhibited by mannosamine. This inhibitory effect was reversedby the addition of glucose or mannose. Growth in peptone mediumwas accompanied by the consumption of the added glucose andby the accumulation of acetoin. Glucose utilization and acetoinproduction were markedly repressed in the cells grown in thepresence of mannosamine. Growth in the presence of mannosaminewas accompanied by a relative decrease in a protein with themolecular weight of 70,000 as revealed by SDS polyacrylamidegel electrophoresis. Our results indicate that mannosamine'sinhibition of growth is due primarily to low glucose utilizationalthough other cellular metabolic activity also may be involved. (Received August 15, 1983; Accepted April 2, 1984)     

Reversal of the Antimicrobial Activity of Trimethoprim by Thymidine in Commercially Prepared Media

The ability of a potent dihydrofolate reductase inhibitor, trimethoprim, to inhibit the growth of Escherichia coli B in vitro is dependent on the composition of the medium in which the cells are grown. The inhibition observed in minimal broth could be partially reversed by the addition of thymidine, ribonucleosides, amino acids, and vitamins. No reversal occurred in the absence of thymidine. In a number of commercially prepared media, the inhibitory activity of trimethoprim correlated inversely with the amount of thymidine found to be present by microbiological assay. The significance of these findings for the routine testing of new, synthetic antibacterial agents is discussed.     

Induced Salt Sensitivity in Fungal Cells and Its Reversal by Imidazole Derivatives

The sensitivity to Cl(-) and Br(-) induced by heat shock of cells of Ophiostoma multiannulatum and of Rhodotorula glutinis could be partially reversed by histidine and some other imidazole derivatives.     

雌激素拮抗剂对胎盘EGF受体及其基因表达的影响

中期孕鼠在他莫昔芬作用下,其颌下腺,血清中ＥＧＦ含量下降,胎盘中ＥＧＦ受体结合位点数下降以及它的ｍＲＮＡ表达受到抑制,再次证实了他莫昔芬抑制雌激素诱导ＥＧＦ受体ｍＲＮＡ的表达。从而使ＥＧＦ受体结合位点数减少,因此,他莫昔芬对孕鼠胚胎生长发育有不可忽视的影响。     

应用抑制性消减杂交技术克隆大鼠肝再生过程中特异表达基因

应用抑制性消减杂交技术成功地构建了高消减效率的正向消减cDNA文库,从随机挑取的50个克隆中有31个均检出了60~400bp插入片段,对这些插入cDNA片段进行测序后经Genbank同源性检索,表明其中7个片段为未知新序列。大鼠肝切除后肝再生cDNA正向消减文库的建立为进一步大批量筛选、克隆肝再生特异性表达的未知新基因奠定了基础,初步筛选出的特异性表达的序列标记为进一步研究肝再生中基因的功能提供了依据。 Abstract:The cDNA from rat regenerating liver tissue was used as the tester and that from normal liver was used as the driver.A highly efficient subtractive cDNA library was constructed by suppression subtractive hybridization(SSH).After screening,31 clones from 50 clones which were derived from the cDNA library were inserted by 60~400bp cDNA fragments.24 cDNA fragments corresponded to known genes and 7 cDNA fragments were unknown sequences(GenBank accession number:BG447490~447496).     

大鼠再生肝中表达上调基因的筛选与鉴定

采用新发展的抑制差减杂交技术（suppression subtractive hybridization,SSH）在基因组水平筛选再生肝中高表达基因。大鼠肝部分切除后24h的再生杆组织来源的cDNA作为受检者（tester）,正常肝组织的cDNA作为驱动者（driver）,进行差减杂交,获得一900个克隆的差减杂交库,随后对差减克隆进行了差异筛选,得到50个在再生肝中高表达的强阳性克隆,序列测定和同源比较表明这些克隆代表了37个基因,其中13个与已报道的肝再生相关的基因同源,15个为忆知基因但首次发现与肝再生相关,9个为新的基因（EST）已被GenBank收录。制备了标准化RNA点杂交膜,通过对上述部分基因的RNA点杂交分析,不但确认了这些基因在再生肝中表达水平的升高,同时发现它们在肝再生过程中有不同的表达模式。实验结果提示这些基因在肝再生过程中具有重要功能。     

Reversal of Cadmium-Induced Thyroid Dysfunction by Selenium, Zinc, or Their Combination in Rat

The aim of this study was to evaluate the potential benefit of combined treatment with zinc (Zn) and selenium (Se) in reversing cadmium (Cd)-induced thyroid dysfunction compared to Se or Zn treatment alone in rats exposed to Cd. For this purpose, 30 adult male Wistar albino rats were equally divided into control and four treated groups receiving either 200 ppm Cd (as CdCl2), 200 ppm Cd + 500 ppm Zn (as ZnCl2), 200 ppm Cd + 0.1 ppm Se (as Na2SeO3), or 200 ppm Cd + 500 ppm Zn + 0.1 ppm Se in their drinking water for 35 days. The results showed that Cd exposure increased significantly the relative thyroid weight (RTW), the thyroid Cd concentration, and the serum thyroid stimulating hormone (TSH) level, whereas the serum thyroxine (T4) level was decreased compared to control rats. The treatment of Cd-exposed rats with Se alone only partially protected from the Cd-induced decrease in serum T4 level. The treatment of Cd-exposed animals with Zn alone partially protected against Cd-induced thyroid dysfunction by maintaining normal RTW and by decreasing Cd concentration in the thyroid. It also partially prevents Cd-induced decrease in serum T4 level. The combined treatment of Cd-exposed animals with Se and Zn induced a more significant decrease in the thyroid Cd concentration than the Zn supplement and a total correction of the RTW. This treatment was also more effective than that with Se or Zn alone in reversing Cd-induced decrease in serum T4 level and Cd-induced increase in serum TSH level. Se and Zn can have a synergistic role against Cd-induced thyroid dysfunction.     

Ethanol Inhibition of N-Methyl-D-Aspartate-Stimulated Endogenous Dopamine Release from Rat Striatal Slices: Reversal by Glycine

N-Methyl-D-aspartate stimulated a concentration-dependent release of endogenous dopamine from rat striatal slices. The threshold for activation was between 10 and 25 microM and reached a maximum at 1 mM. Release was completely blocked by magnesium or tetrodotoxin. Ethanol (10-200 mM) significantly inhibited the N-methyl-D-aspartate-stimulated release of dopamine by 20-45%, with half-maximal inhibition occurring at approximately 21 mM. Addition of ethanol plus increasing concentrations of magnesium resulted in a greater inhibition of N-methyl-D-aspartate-stimulated dopamine release than that observed with magnesium alone. However, this effect appeared to be due to a noninteractive additive effect of the two antagonists, as the IC50 value for magnesium inhibition was not significantly altered by ethanol. Glycine, which had no effect on dopamine release by itself, completely reversed the inhibitory effects of ethanol (25 mM) at low micromolar concentrations. These results suggest that ethanol may produce its effects in striatal slices by interfering with a glycine modulatory site of the N-methyl-D-aspartate receptor-ionophore complex.