Inhibition of cytomegalovirus infection by lactoferrin in vitro and in vivo

Lactoferrin is an antimicrobial agent, that, amongst other viruses, inhibits cytomegalovirus (CMV). In this study, we addressed the mechanism(s) by which lactoferrin interacts with CMV and its target cells to inhibit infection. We also studied the antiviral activity of lactoferrin in vivo in rat CMV models with and without immune suppression. We cationized a protein of similar molecular weight, i.e. human serum albumin (HSA), as well as a protein with a smaller molecular weight (beta-lactoglobulin). While HSA itself displayed no anti-CMV activity in vitro, cationic HSA inhibited CMV replication to a similar extent as lactoferrin. Time-of-addition assays indicated that all cationic proteins interacted with an early event in the infection and pre-incubation of cells rather than of virus significantly reduced CMV replication. Rats were treated with lactoferrin (4, 40 or 160 mg/kg, intravenously), beginning at 6h after CMV administration. Subsequently, the rats were treated three times a week. As a positive control, CMV-infected rats were treated with cidofovir, and this agent proved to be highly active in the rat models for CMV. Treatment with lactoferrin was beneficial when infection was initiated with cell-free virus, but not with virus-infected leukocytes. Lactoferrin treatment led to a 10-fold reduction in the final virus titers (salivary glands) at 4 weeks after infection in the immunocompromised rats. Lactoferrin exerted its effects via inhibition of cell entry rather than via stimulation of the immune system.     

格尔德霉素体内外抑制单纯疱疹病毒l型的复制

格尔德霉素(GA)是一种抗生素,它的作用靶点是热休克蛋白Hsp90 N末端的ATP/ADP结合位点.在我们对抗病毒的抗生素筛选中,发现格尔德霉素显著抗单纯疱疹病毒1型(HSV一1).体外在Vero细胞内GA显著抑制HSv-1的复制,IC50为0,093μmol/L,GA对Vero细胞的毒性CC50为350μmol/L,治疗指数可达3763.HSV-1腹腔(ip)感染1h后腹腔(ip)注射GA(0.093～0.37mg/kg)可以将存活率增加到67%～100%,皮下(sc)给药(0.37mg/kg)的存活率为43.8%,都明显高于生理盐水对照组(ipP＜0.001.scP＜0.05).GA对小白鼠的急性LD50为15.5mg/kg(ip).格尔德霉素不影响病毒的吸附、穿人.由于GA在体内外都能抑制单纯疱疹病毒1型,GA可以成为新的抗单纯疱疹病毒感染的药物.     

格尔德霉素体内外抑制单纯疱疹病毒1型的复制

格尔德霉素 ( GA)是一种抗生素 ,它的作用靶点是热休克蛋白 Hsp90 N末端的 ATP/ ADP结合位点。在我们对抗病毒的抗生素筛选中 ,发现格尔德霉素显著抗单纯疱疹病毒 1型 ( HSV- 1)。体外在 Vero细胞内 GA显著抑制 HSV- 1的复制 ,IC50 为0 .0 93μmol/ L,GA对 Vero细胞的毒性 CC50 为 35 0 μmol/ L,治疗指数可达 376 3。HSV- 1腹腔 ( ip)感染 1h后腹腔 ( ip)注射 GA( 0 .0 93～ 0 .37mg/ kg)可以将存活率增加到 6 7%～ 10 0 % ,皮下 ( sc)给药 ( 0 .37mg/ kg)的存活率为 4 3.8% ,都明显高于生理盐水对照组 ( ipP<0 .0 0 1,sc P<0 .0 5 )。GA对小白鼠的急性 L D50 为 15 .5 mg/ kg( ip)。格尔德霉素不影响病毒的吸附、穿入。由于 GA在体内外都能抑制单纯疱疹病毒 1型 ,GA可以成为新的抗单纯疱疹病毒感染的药物     

人阴道乳酸杆菌对大肠杆菌的体外拮抗作用

目的探讨生殖道不同产过氧化氢（H2O2）乳酸杆菌对大肠杆菌的拮抗作用，筛选出作用较强菌株，以治疗女性泌尿生殖道感染。方法从阴道分泌物中分离筛选出产H2O2乳酸杆菌，采用打孔法测量共培养24h后抑制大肠杆菌的抑菌圈直径。结果分离筛选出4株产H2O2乳酸杆菌，它们所产生的抑菌圈大小不同，其中L．acidophilus 1周围产生的抑菌圈最大，L．crispatus次之。结论阴道产H2O2乳酸杆菌对大肠杆菌的拮抗作用不同，L．acidophilus 1和L．crispatus作用较强。     

Inhibition of histamine synthesis in vitro and in vivo by S-alpha-fluoromethylhistidine

(S)-alpha-Fluoromethylhistidine (alpha-FMH) is a Kcat or "suicide-substrate" inhibitor of partially purified mammalian histidine decarboxylase; i.e. the agent is converted enzymatically to a more active form which effects a time-dependent, irreversible inhibition. Incubation of a alpha-FMH[4-3H] with enzyme and pyridoxal phosphate resulted in an apparently irreversible labeling of protein, with no demonstratable formation of free-amine product, suggesting a very low to non-existent turnover ratio. alpha-FMH was accumulated in isolated mastocytoma cells and effected a time-dependent inhibition of the conversion histidine[3H]----histamine[3H], the latter product having a markedly different distribution between cells and medium than the pre-existing histamine pool. Inhibition of whole-body histidine decarboxylase activity, as specifically measured by alpha-methylhistidine-14COOH----14CO2, was also time dependent. Concomitant reduction in histamine levels was seen only in the rapidly turning-over pools of stomach and brain. However, over the course of 13 weeks of chronic treatment, depletion of the relatively inert mast-cell histamine pool(s) was seen as well.     

Inhibition by picotamide of thromboxane production in vitro and ex vivo

Summary The effect of picotamide on platelet function has been studied in vitro and ex vivo.Picotamide at micromolar concentrations inhibited platelet aggregation induced by ADP, arachidonic acid and collagen, and it also inhibited the production of thromboxane A₂ (TxA₂). Unlike aspirin, picotamide did not affect the synthesis of prostacyclin by blood vessels.In eight healthy subjects who took picotamide 1200 mg/d platelet aggregation and TxA₂ production were inhibited.Picotamide appears to be an antiplatelet drug that reduces TxA₂ synthesis without affecting cyclooxygenase activity.     

Inhibition of HSV cell-to-cell spread by lactoferrin and lactoferricin

The milk protein lactoferrin (Lf) has multiple functions, including immune stimulation and antiviral activity towards herpes simplex virus 1 and 2 (HSV-1 and HSV-2); antiviral activity has also been reported for the N-terminal pepsin-derived fragment lactoferricin (Lfcin). The anti-HSV mode of action of Lf and Lfcin is assumed to involve, in part, their interaction with the cell surface glycosaminoglycan heparan sulfate, thereby blocking of viral entry. In this study we investigated the ability of human and bovine Lf and Lfcin to inhibit viral cell-to-cell spread as well as the involvement of cell surface glycosaminoglycans during viral cell-to-cell spread. Lf and Lfcin from both human and bovine origin, inhibited cell-to-cell spread of both HSV-1 and HSV-2. Inhibition of cell-to-cell spread by bovine Lfcin involved cell surface chondroitin sulfate. Based on transmission electron microscopy studies, human Lfcin, like bovine Lfcin, was randomly distributed intracellularly, thus differences in their antiviral activity could not be explained by differences in their distribution. In contrast, the cellular localization of iron-saturated (holo)-Lf appeared to differ from that of apo-Lf, indicating that holo- and apo-Lf may exhibit different antiviral mechanisms.     

Inhibition of glycogen breakdown by imino sugars in vitro and in vivo

The imino sugar N-butyldeoxynojirimycin (NB-DNJ) is a glucose analogue which inhibits the glycoprotein N-glycan processing enzymes alpha-glucosidases I and II and the ceramide glucosyltransferase that catalyses the first step of glycosphingolipid biosynthesis. This and other N-alkylated DNJ compounds have the potential to inhibit other glucosidase, including acid alpha-glucosidase and alpha-1,6-glucosidase, enzymes involved in glycogen breakdown. We have investigated the effect of NB-DNJ and N-nonyldeoxynojirimycin (NN-DNJ) on glycogen catabolism. Both NB-DNJ and NN-DNJ were potent inhibitors of acid alpha-glucosidase and alpha-1,6-glucosidase in vitro. NB-DNJ and NN-DNJ inhibited liver glycogen breakdown in vivo in fasting mice. Inhibition of glycogen catabolism occurred in the cytosol and lysosomes. The liver glycogen breakdown inhibition was only induced at high doses of NB-DNJ, whereas NN-DNJ caused glycogen accumulation at lower doses. The in vivo effect of NB-DNJ on liver glycogen was transient as there was no inhibition of breakdown after 90 days of treatment. The inhibition by NN-DNJ, was more pronounced, reached a plateau at 50 days and then remained unchanged. Increased glycogen was also observed in skeletal muscle in NB-DNJ- and NN-DNJ-treated mice. Since the effects on glycogen metabolism by NB-DNJ are transient and only occur at high concentrations, it is not predicted that glycogen breakdown will be impaired in patients receiving NB-DNJ therapy. NN-DNJ is the prototype of long alkyl chain derivatives of DNJ that are entering pre-clinical development as potential hepatitis B/hepatitis C (HBV/HCV) therapeutics. Depending on the dose of these compounds used, there is the potential for glycogen catabolism to be partially impaired in experimental animals and man.     

Inhibition of ossification in vivo and differentiation of osteoblasts in vitro by tributyltin

Tributyltin is ubiquitous in the environment and an endocrine disruptor for many wildlife species. However, minimal information is available regarding the effect of this chemical on bone formation. When tributyltin chloride (TBT) (1mg/kg body weight) was administered subcutaneously to pregnant mice at 10, 12, and 14 days post coitus (dpc), fetuses at 17.5 days post coitus revealed the inhibition of calcification of supraoccipital bone. In contrast, 1mg/kg body weight monobutyltin trichloride (MBT) did not affect the fetal skeleton. Therefore, we examined the effects of TBT and its metabolites (dibutyltin dichloride, DBT, and MBT) on bone metabolism using rat calvarial osteoblast-like cells (ROB cells). The viability of ROB cells was not affected by the exposure of the cells to 10(-10) to 10(-7)M TBT. However, TBT reduced the activity of alkaline phosphatase (ALPase) and the rate of deposition of calcium of ROB cells. In addition, the expression levels of mRNA for ALPase and osteocalcin, which are markers of osteoblastic differentiation, were depressed by the treatment with TBT. TBT inhibited ALPase activity and the deposition of calcium to a greater extent than did DBT. MBT had no effect on the osteoblast differentiation of ROB cells. Tributyltin is known to inhibit the activity of aromatase. However, the aromatase inhibitor aminoglutethimide did not reproduce the inhibitory effects of TBT on osteoblast differentiation. Our findings indicate that TBT might have critical effects on the formation of bone both in vivo and in vitro although its action mechanism is not clarified.     

Inhibition of platelet aggregation by verapamil: quantification by in vivo and in vitro techniques

Platelet aggregation appears to play a prominent role in myocardial ischemia. Verapamil, a slow-channel blocking agent with important antiarrhythmic and vasodilating actions, has been shown to inhibit in vitro platelet aggregation. We used an electronic particle size analyzer to evaluate the effects of verapamil on platelet aggregation in vitro and in vivo in 88 rats. The intravenous injection of verapamil (0.4 mg/kg) did not change the platelet count compared to control animals receiving an equal volume of normal saline (verapamil, 1.1 +/- 0.04 x 10(6)/mm3, vs. control, 1.2 +/- 0.09 x 10(6)/mm3, (p greater than 0.05). The mean size of platelet aggregates induced by adenosine diphosphate (0.2 microM), was reduced by verapamil (verapamil, 15.3 +/- 1.2 x 10(3) micron3 vs. control 24.4 +/- 2.7 x 10(3) micron; p less than 0.01). Platelet aggregates induced in vivo, following a standardized technique of extravasation of right iliac artery blood into the peritoneal cavity, were also smaller following verapamil infusion (verapamil, 12.6 +/- 1.1 x 10(3)micron3, vs control, 17.3 +/- 0.9 x 10(3) micron3 p less than 0.001). We conclude that verapamil exerts and inhibitory effect on platelet aggregation both in vitro and in vivo. This property may add an important new dimension to its potential therapeutic usefulness in ischemic heart disease.     

Inhibition of cytochrome P450 isozymes by curcumins in vitro and in vivo.

To study the mechanism(s) of turmeric-mediated chemoprevention and to compare the chemopreventive efficacy of turmeric/curcumin(s) against benzo[a]pyrene (B(a)P) and 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK, a tobacco-specific carcinogen), the effects of turmeric/curcumin (C), demethoxycurcumin (dmC), bis-demethoxycurcumin (bdmC) and phenyl and phenethyl-isothiocyanates (PITC and PEITC) on the dealkylation of ethoxyresorufin (ER), methoxyresorufin (MR) and pentoxyresorufin (PR) by rat liver microsomes (in vitro) were studied. These reactions are predominantly mediated by cytochrome P450 (CYP450) isozymes 1A1, 1A2 and 2B1, respectively. In vitro incubation of rat liver microsomes with each of the compounds--C, dmC, bdmC, PITC and PEITC--showed a dose-dependent decrease in carbon monoxide binding to microsomes and also showed a dose-dependent inhibition of CYP 1A1, 1A2 and 2B1 activity, as judged by a decrease in formation of resorufin from respective biochemical probes used. Both the isothiocyanates inhibited activity of CYP 2B1 more readily than that of CYP 1A1/1A2. Significantly lower concentrations of curcumin(s) than isothiocyanates achieved 50% inhibition of activity of CYP 1A1 and 1A2, while concentrations of C (4 microM), bdmC (2.5 microM) required to inhibit CYP 2B1 were slightly higher than that of PEITC (1.3 microM), suggesting curcumin(s) to be effective inhibitors of CYP 2B1 as well. Pretreatment of rats with 1% turmeric through the diet resulted in a significant decrease in induction of B(a)P-induced CYP 1A1 and 1A2 and phenobarbitone (PB)-induced CYP 2B1 in liver, lung and stomach, although the extent of the decrease was different. These results suggest that turmeric/curcumin(s) as in the case of isothiocyanate, PEITC, are likely to inhibit activation of carcinogens metabolized by CYP450 isozymes, namely, CYP 1A1, 1A2 and 2B1.     

Inhibition of Hepatitis B virus replication by phospholipid scramblase 1 in vitro and in vivo

Human Phospholipid scramblase 1 (PLSCR1) is an α/β interferon-inducible protein that mediates antiviral activity against RNA viruses including vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV). In the present study, we investigated the antiviral activity of PLSCR1 protein against HBV (Hepatitis B virus). Firstly, PLSCR1 mRNA and protein expression was found to be downregulated in HepG2 cells after HBV infection. Then by performing co-transient-transfection experiments in cells and hydrodynamics-based transfection experiments in mice using a HBV expression plasmid and a PLSCR1 expression plasmid, we found that PLSCR1 inhibited HBV replication in vitro and in vivo through a significant reduction in the synthesis of viral proteins, DNA replicative intermediates and HBV RNAs. We also demonstrated that the antiviral action of PLSCR1 against HBV occurs, partly at least, by activating the Jak/Stat pathway. In conclusion, our results suggest that the expression of PLSCR1 is involved in HBV replication and that PLSCR1 has antiviral activity against HBV.     

Inhibition of HIV infection by bicyclams, highly potent and specific CXCR4 antagonists

The bicyclams represent a new entity of low-molecular weight molecules that inhibit human immunodeficiency virus (HIV) infection through a specific blockade of CXCR4 (fusin), the receptor for the CXC chemokine SDF-1 (soluble-derived factor), which is also used as coreceptor by T-lymphotropic HIV strains to enter their target cells. The bicyclam AMD3100 or 1,1'-[1,4-phenylenebis(methylene)]-bis-1,4, 8,11-tetraazacyclotetradecane octahydrochloride dihydrate, is able to block the CXCR4 receptor and to inhibit HIV replication at nanomolar concentrations while not being toxic to the host cells at 100,000-fold higher concentrations. It is the most specific and most potent CXCR4 antagonist that has been described to date.     

In vivo and in vitro Inhibition of the Metabolism of N-Alkyl-substituted Amphetamines in Rat by Ferrocenylisopropylamine

Abstract

1. Ferrocenylisopropylamine (FIPA) inhibits the elimination of amphetamines in rat. The half-life of isopropylamphetamine was increased from approx. 30 to 85–100 min after administration of FIPA.

2. With isolated, perfused, rat liver, the half-lives of isopropylamphetamine, biamphetamine and benzylamphetamine were increased from 5–20 min to about 200 min by equimolar amounts of FIPA, indicating that the prolonging effect of FIPA is due to interference at the metabolic level.

3. Experiments with hepatic microsomal suspensions demonstrated that FIPA competitively inhibits the oxidative N-dealkylation of isopropylamphetamine; the K_i of FIPA is 4·1 × 10^?6 M.

4. Binding of isopropylamphetamine and FIPA to cytochrome P-450 was studied using hepatic microsomes of phenobarbital-treated rats. Isopropylamphetamine caused a type I, and FIPA a type II difference spectrum; FIPA showed a much higher binding affinity (K_s = 1·24 × 10^?2 M) than isopropylarnphetamine (K_s = 0·96 × 10^?3 M). FIPA acts as a modifier of the spectral changes induced by isopropylamphetamine.

5. Results suggest that the competitive inhibition of the N-dealkylation of N-alkylamphetamines, and thus the prolonging of their action, by FIPA is related to competition for binding to cytochrome P-450.     

Inhibition of hepatic mixed-function oxidase activity in vitro and in vivo by various thiono-sulfur-containing compounds.

Ten thiono-sulfur-containing compounds of varying structure were administered by intraperitoneal injection to untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated adult male rats. Six hr later, the concentration of hepatic cytochrome P-450 and the ability of the hepatic microsomes to metabolize benzphetamine were examined. In the untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated groups, two, four and four compounds, respectively, significantly decreased the concentration of cytochrome P-450 in the hepatic microsomes. A similar effect on benzphetamine metabolism was also seen. When examined 48 hr after the administration of the ten thiono-sulfurcontaining compounds, four, five and seven of the compounds decreased both the levels of hepatic cytochrome P-450 and the rate of benzphetamine metabolism in the untreated, phenobarbital-pretreated and 3-methylcholanthrene-pretreated animals respectively. Eight of the thiono-sulfur-containing compounds were incubated in the presence of NADPH with hepatic microsomes isolated from untreated, phenobarbital-pretreated or 3-methylcholanthrene-pretreated animals. All of the compounds examined significantly decreased the concentration of cytochrome P-450 in the microsomes from each treatment group. Similar reductions in benzphetamine metabolism were also seen. When these same compounds were incubated with microsomes in the absence of NADPH, no significant reduction of cytochrome P-450 or benzphetamine metabolism was seen. When the oxygen analogs of six of the thiono-sulfur compounds were administered in vivo or incubated with hepatic microsomes either in the presence or absence of NADPH, no significant reduction of cytochrome P-450 or benzphetamine metabolism was seen.