Fibrinolytic and fibrinogenolytic effects of acyl urokinase and urokinase combinations in vitro

The in vitro thrombolytic and side effects of double-strand urokinase-type plasminogen activator (tcu-PA), p-guanidinobenzoyl tcu-PA (GB-tcu-PA), and their combinations were compared. The reversible blocking of the active site stabilizes GB-tcu-PA in human plasma and results in longer thrombolysis and lesser side effects than those of tcu-PA. However, the acylated activator displays a marked lag period of thrombolytic action. GB-tcu-PA and tcu-PA combinations (molar ratios 4 : 1 and 1 : 1) induce a prolonged and effective thrombolysis without the initial lag period at a low level of plasminogen, fibrinogen, and alpha2-antiplasmin depletion in the plasma surrounding the clot.     

Construction, expression, and characterization of a recombinant annexin B1-low molecular weight urokinase chimera in Escherichia coli

Thrombolytic therapy has been a major advance in thtreatment of myocardial infarction over the last twdecades. Urokinase (UK), streptokinase (SK), and tissuplasminogen activator (t-PA) are the common availablthrombolytic agents. These agents, however, hav…     

Interaction between kringle and growth-factor-like domains in the urokinase molecule: Possible role in stimulation of chemotaxis

The results presented in this paper suggest the presence of an interaction between the kringle- and the growth-factor-like urokinase domains. This interaction regulates chemotactic properties of urokinase. We also show that interaction of urokinase with its "classical" receptor (uPAR) has a "permissive" effect on the interactions between the kringle domain and other targets on the cell surface. On the basis of our data we can suggest that uPAR serves as an "adaptor" for urokinase, and the binding of urokinase kringle domain to its receptor causes immediate activation of intracellular signaling and induction of cell migration.     

Activity and interactions of antibiotic and phytochemical combinations against Pseudomonas aeruginosa in vitro

In this study the in vitro activities of seven antibiotics (ciprofloxacin, ceftazidime, tetracycline, trimethoprim, sulfamethoxazole, polymyxin B and piperacillin) and six phytochemicals (protocatechuic acid, gallic acid, ellagic acid, rutin, berberine and myricetin) against five P. aeruginosa isolates, alone and in combination are evaluated. All the phytochemicals under investigation demonstrate potential inhibitory activity against P. aeruginosa. The combinations of sulfamethoxazole plus protocatechuic acid, sulfamethoxazole plus ellagic acid, sulfamethoxazole plus gallic acid and tetracycline plus gallic acid show synergistic mode of interaction. However, the combinations of sulfamethoxazole plus myricetin shows synergism for three strains (PA01, DB5218 and DR3062). The synergistic combinations are further evaluated for their bactericidal activity against P. aeruginosa ATCC strain using time-kill method. Sub-inhibitory dose responses of antibiotics and phytochemicals individually and in combination are presented along with their interaction network to suggest on the mechanism of action and potential targets for the phytochemicals under investigation. The identified synergistic combinations can be of potent therapeutic value against P. aeruginosa infections. These findings have potential implications in delaying the development of resistance as the antibacterial effect is achieved with lower concentrations of both drugs (antibiotics and phytochemicals).     

Hormonal effects on insects and other endoparasites in vitro

Summary Metamorphic and reproductive events in vertebrates and invertebrates are under endocrine control and are often correlated with developmental, behavioral, or reproductive changes in the parasites living in or on these hosts. This paper reviews selected examples ofa) host hormone mediated influences on endoparasites in vivo,b) host hormone effects in vitro on protozoan, helminth, and insect endoparasites, andc) identifies possible relationships in hormone effects across parasite taxa. The significance of studies on endoparasites in vitro in relation to the impact of host hormones, antihelminthic, and prophylactic drugs on parasite growth and proliferation will also be addressed. A review of the literature indicates only limited studies have been done in vitro in an attempt to elucidate the bases of reported host hormone influences on endoparasites in vivo. Steroid hormones of hosts seem to stimulate growth, molting or encystment or both of helminth, insect, and protozoan parasites. Vertebrate steroids such as estrogen, testosterone, and progesterone had primarily reproduction- or growth-promoting effects or both on protozoan and nematode parasites. Insect ecdysteroids such as ecdysone, 20-hydroxyecdysone, and makisterone were the most widely studied steroids in vitro and induced growth or molting or both of cestode, nematode, and insect parasite larvae. Although juvenile hormone (JH III) stimulated growth in the protozoan and nematode parasites tested, the analogue methoprene and JH precursors, farnesal, farnesol, and farnesol methyl ether had various effects. Biogenic amines also varied in their effects on the nematode parasites tested, while the peptide hormone, insulin, stimulated growth in the protozoans tested. The evidence for in vitro effects of host hormones on their natural endoparasites is patchy at best. Additional studies are needed to identify the biochemical bases for the numerous host hormone mediated effects on parasites. This work was supported by grant DCB 8502235 from the National Science Foundation, Washington, DC, GAM 8700433 from the United States Department of Agriculture, Washington, DC. This work was supported by grant DCB 8502235 from the National Science Foundation, Washington, DC, GAM 8700433 from the United States Department of Agriculture, Washington, DC.     

Anomalous antioxidant effects in selenium- and vitamin E-deficient liver mitochondria

In this study, we investigate the mechanisms of two anomalous protective effects of exogenous vitamin E that had previously been postulated to involve either a specific antioxidant effect or a non-antioxidant function of the vitamin. These atypical vitamin E effects were observed during the prevention of NAD-induced respiratory decline occurring in homogenates and mitochondria prepared from vitamin E- and selenium-deficient rat liver. The study showed neither hypothesis to be true; rather, the two effects, one in homogenates and the other in isolated mitochondria, were explained by other mechanisms. The protective effect against respiratory decline in homogenates was found to result from interference in the thiobarbituric acid assay for lipid peroxidation by ethanol (the conventional solvent for vitamin E addition). With other non-interfering solvents, inhibition of lipid peroxidation by vitamin E, in contrast to previous studies, correlated perfectly with prevention of respiratory decline. The atypical vitamin E effect occurring in isolated mitochondria—and consisting of a requirement for cytosol proteins for the prevention of respiratory decline by exogenous vitamin E—was found to be caused by the prevention of adverse glass effects and not by the action of vitamin E-specific binding proteins. Frequent failures in the combined protective effect of vitamin E and cytosol, which had been a major complication of respiratory decline studies, were found to be caused by phospholipase activity generated during isolation procedures. Irreversible deactivation of respiratory enzymes by lipid peroxidation was found not to be involved in the respiratory decline mechanism. In memoriam: Klaus Schwarz, MD, 1914–1978.     

In vivo and in vitro effects of aluminum treatment on rat liver mitochondrial function

This study examines the effect on mitochondrial respiration and permeability of in vivo and in vitro aluminium (Al) exposure. Rats were treated intraperitoneally with AlCl₃ to achieve serum and liver Al concentrations comparable to those seen in Al-related disorders. Mitochondria isolated from Al-treated rats had higher (p<0.01) Al concentration, lower (p<0.05) state 3 respiration, respiratory control (RCR), and ADP/O ratio (succinate substrate), and greater passive swelling in 100 mM KCl or 200 mM NH₄NO₃ than controls. The in vitro addition of Al (0–180 μM) to mitochondria from normal rats also decreased (p<0.01) state 3 respiration, RCR, and ADP/O and stimulated passive swelling in KCl and NH₄NO₃ at 42–180 μM Al. These studies show that Al depresses mitochondrial energy metabolism and increases membrane permeability. The toxicity associated with Al may be related to its effect on mitochondria.     

The antifungal drug voriconazole is an efficient inhibitor of brain cholesterol 24S-hydroxylase in vitro and in vivo

Cholesterol 24S-hydroxylase (CYP46A1) is of key importance for cholesterol homeostasis in the brain. This enzyme seems to be resistant toward most regulatory factors and at present no drug effects on its activity have been described. The crystal structures of the substrate-free and substrate-bound CYP46A1 were recently determined (Mast et al., Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principal cholesterol hydroxylase in the brain. Proc. Natl. Acad. Sci. USA. 2008. 105: 9546–9551). These structural studies suggested that ligands other than sterols can bind to CYP46A1. We show here that the antifungal drug voriconazole binds to the enzyme in vitro and inhibits CYP46A1-mediated cholesterol 24-hydroxylation with a Ki of 11 nM. Mice treated with daily intraperitoneal injections of voriconazole for 5 days had high levels of voriconazole in the brain and significantly reduced brain levels of 24S-hydroxycholesterol. The levels of squalene, lathosterol, and HMG-CoA reductase mRNA were reduced in the brain of the voriconazole-treated animals as well, indicating a reduced cholesterol synthesis. Most of this effect may be due to a reduced utilization of cholesterol by CYP46A1. One of the side-effects of voriconazole is visual disturbances. Because CYP46A1 is also expressed in the neural retina, we discuss the possibility that the inhibition of CYP46A1 by voriconazole contributes to these visual disturbances.     

Pseudo-cyclic oligonucleotides: in vitro and in vivo properties

We have designed and studied antisense oligodeoxynucleotides (oligonucleotides; oligos) which we call ‘pseudo-cyclic oligonucleotides’ (PCOs). PCOs contain two oligonucleotide segments attached through their 3′-3′- or 5′-5′-ends. One of the segments of the PCO is an antisense oligo complementary to a target mRNA, and the other is a short protective oligo that is 5–8 nucleotides long and complementary to the 3′- or 5′-end of the antisense oligo. As a result of complementarity between the antisense and protective oligo segments, PCOs form intramolecular pseudo-cyclic structures in the absence of the target RNA. The antisense oligo segment of PCOs used for the studies described here is complementary to an 18-nucleotide-long site on the mRNA of the protein kinase A regulatory subunit RI (PKA-RI). Thermal melting studies of PCOs in the absence and presence of the complementary RNA suggest that the pseudo-cyclic structures formed in the absence of the target RNA dissociate, bind to the target RNA, and form heteroduplexes. The results of RNase H cleavage assays suggest that PCOs bind to complementary RNA and activate RNase H in a manner similar to that of an 18-mer conventional antisense PS-oligo. In snake venom (a 3′-exonuclease) or spleen (a 5′-exonuclease) phosphodiesterase digestion studies, PCOs are more stable than conventional antisense oligos because of the presence of 3′-3′- or 5′-5′-linkages and the formation of intramolecular pseudo-cyclic structures. PCOs with a phosphorothioate antisense oligo segment inhibited cell growth of MDA-MB-468 and GEO cancer cell lines similar to that of the conventional antisense PS-oligo, suggesting efficient cellular uptake and target binding. The nuclease stability studies in mice suggest that PCOs have higher in vivo stability than antisense PS-oligos. The studies in mice showed similar pharmacokinetic and tissue distribution profiles for PCOs to those of antisense PS-oligos in general, but rapid elimination from selected tissues.     

Growth-promoting effects of gastrin on mouse colon cancer cells in vitro: Absence of autocrine effects

Summary We have previously demonstrated trophic effects of gastrin on mouse colon cancer (MC-26) cells, in vivo, and demonstrated the presence of gastrin receptors (GR) on these cells. The cellular and intracellular mechanism by which gastrin expresses trophic effects on colon cancer cells is, however, as yet unknown. For us to start investigating the possible mechanisms involved, it was important that we first develop an in vitro model, in which gastrin expresses its trophic effects directly on the MC-26 cells. The growth-promoting effects of gastrin on the MC-26 cells were examined in various in vitro culture models, in terms of [³H]thymidine incorporation and cell number. A significant trophic effect of gastrin could be demonstrated on quiescent cells in culture, in the absence of serum. The optimal cell-culture conditions for observing trophic effects of gastrin were defined and included a 24-h period of rapid growth of MC-26 cells in serum-supplemented normal growth medium, followed by a 24-h period of culture in serum-free medium containing an optimal dose (1.0 mM) of thymidine, to achieve growth-arrest of the cells. Addition of gastrin (0.5 to 25 nM) to the quiescent, growth-arrested cells resulted in significant dose-dependent increases in both the incorporation of [³H]thymidine uptake by the cells, and a significant increase in cell number. The concentration of GR on the growth-arrested quiescent MC-26 cells in culture was significantly increased compared to the GR concentration on the control, asynchronized cells. The increased presence of GR on the growth-arrested, synchronized MC-26 cells may have allowed us to observe a significant trophic effect of gastrin on the MC-26 cells, in vitro itself. To determine if gastrin was functioning as an autocrine growth factor for MC-26 cells, we examined the effect of gastrin antibodies on the growth of MC-26 cells; no significant effect of the antigastrin IgG on the growth of MC-26 cells was observed. Supported by grants from the National Institutes of Health, Bethesda, MD (PO1 DK 35608 and CA 38651, and a grant from the American Cancer Society, Washington, DC (PDT-220).     

胶红酵母CYJ03的体外体内抗氧化活性研究

旨在深入研究海洋源胶红酵母CYJ03的抗氧化能力,挖掘其开发成抗氧化产品的潜力.通过测定CYJ03的过氧化氢耐受性,及其发酵上清液、完整细胞和色素提取物3种不同组分的还原力、自由基清除能力和Fe2+螯合能力来评价该菌株的体外抗氧化能力;通过在罗非鱼日粮中添加CYJ03,测定罗非鱼的生长性能以及血清和肝脏的总抗氧化能力(...     

The EZMTT cell proliferation assay provides precise measurement for drug combinations and better correlation between in vitro and in vivo efficacy

The rate of drug-induced proliferation (DIP) has been proposed as an unbiased alternative drug effect metric. However, current assays are not easy and precise enough to track minor changes in cell growth. Here, we report the optimized EZMTT based detection method which can continuously measure time-dependent growth after drug treatment and reliably detect partial drug resistance for cancer cells. Importantly, tracking time-dependent growth after drug treatment demonstrated that a KGA allosteric inhibitor alone failed to completely inhibit cancer cell growth, but a drug combination was able to provide complete inhibition in cell-based assays that translated well in in vivo animal experiments. In conclusion, this simple EZMTT method provided precise measurement of loss of susceptibility after drug treatment and has great potential to be developed for drug efficacy and drug combination studies to solve the unmet medical need.     

The antitheilerial effects of Theileria parva parva reaction and recovery sera in vitro

Peripheral blood leucocytes (PBL) of cattle were infected in vitro with the sporozoites of Theileria parva spp. The transformed cell lines were adapted to grow in sera from the PBL donors. The cattle were then infected with T. p. parva stabilate and either treated with parvaquone or the disease allowed to run its course. Sera harvested during severe disease reaction or early recovery were substituted for pre infection sera and caused the intracellular degeneration of the Theileria macroschizonts. Cell lines passaged in these sera died out as the parasites were eliminated. The antiparasitic effects of sera were short lived and were neither host nor parasite isolate restricted.     

‘神马’菊花的离体保存及遗传稳定性

用添加不同蔗糖与甘露醇配比的培养基对切花菊品种‘神马’(Jinba)试管苗进行离体保存,并对保存材料再生后代的遗传稳定性进行了研究。结果表明,在温度(23±2)℃、光照强度2 000~3 000 lx、光照时间12 h/d的培养条件下,MS 0.3 mg.L-16-BA 0.1 mg.L-1NAA培养基中分别添加10 g.L-1蔗糖和10 g.L-1甘露醇、10 g.L-1蔗糖和20 g.L-1甘露醇、20 g.L-1蔗糖和20 g.L-1甘露醇或30 g.L-1蔗糖和10 g.L-1甘露醇均能使菊花试管苗连续保存12个月,其中10 g.L-1蔗糖和20 g.L-1甘露醇、20 g.L-1蔗糖和20 g.L-1甘露醇组合处理保存12个月后成活率较高,分别达到50.00%和60.00%,且均保持最高的增殖倍数2.67。保存12个月的试管苗在增殖、生根培养基上均能正常恢复生长,且其再生后代的田间生物学性状、过氧化物酶(POD)及酯酶(EST)同工酶酶谱、ISSR扩增图谱与对照株无差异,保持了良好的遗传稳定性,说明利用上述方法保存‘神马’种质是可行的。     

Procedures for the characterisation of bioaerosol particles. Part I: aerosolisation and recovery agent effects

The sampling and assay of bioaerosols are important ina number of industrial and health-care applications. Airborne microorganisms are notoriously difficult toenumerate accurately under such conditions and nosingle procedure is suitable for all applications. Problems are compounded by the differences in assaymethod or sampler type selected, making theinterpretation of results difficult.Understanding the airborne behaviour of microorganismsover a range of environmental conditions is vital ifprocedures are to be defined and recommended for theassessment of bioaerosols. Microorganisms that arerobust over a wide range of conditions are ideal astracer particles. Unfortunately, the large majorityof non-fungal bioaerosols are susceptible to damage. A predictable assessment procedure is required whichwill not affect the viability of the collectedsample.This paper examines how aerosolisation may affect the characteristics of two speciesof microorganism (Pseudomonas fluorescens andMS2 coliphage). It forms part of a larger programmeto develop standards for the assessment of biologicalparticles. The aim of the work was to develop procedures toexamine the effects of aerosolisation onmicroorganisms, with particular reference topre-aerosolisation protocol (spray suspension age) andpost-sampling handling protocol (aerosol age incollection solution). These procedures were then usedto examine the effect of recovery agents, addedto the spray suspension prior to aerosolisation, onthe culturability of E.coli.Aerosolisation reduces the culturability of P. fluorescensand the viability of viability of MS2coliphage. Pre-sampling and post-collection handlingand storage of these aerosolised microorganisms werealso found to have an effect. This and earlierstudies have shown that the culturable fraction ofmicroorganisms can be affected by the same factorsdescribed above. Of five microorganisms tested so farin the main programme, only Penicillium expansumspores were shown to be robust and stable with aconstant culturable fraction. Therefore, recommendinga particular microorganism (apart from P. expansum) as an airborne biological standard foraerosol studies is not advised. It is recommendedthat a microorganism, representative of the envisagedapplication, be characterised it in terms of theaerosolisation parameters, storage time and conditionsin the manner reported in this study. This can beachieved using the experimental equipment described.The addition of 0.1 mM concentrations of the sugarsinositol, trehalose and raffinose to spray suspensionsof Escherichia coli, prior to aerosolisation,made no significant difference to the culturablefraction of the aerosol.     

The in vitro screening of methylated 4-oxypiperidine compounds for inhibition of protein synthesis in a rabbit reticulocyte cell-free translation system

A variety of methylated 4-oxypiperidine derivatives were tested for their ability to inhibit protein synthesis in vitro. A direct correlation was found between the extent of methylation of these compounds and their inhibitory activity in a rabbit reticulocyte lysate cell-free translation system.Abbreviation IC₅₀ 50% inhibitory concentration     

风信子品种间杂交与幼胚离体培养研究

选用5个风信子品种进行花粉生命力测定,对完全双列杂交获得的杂种种子剥胚进行离体培养.试验表明:(1)风信子不同品种花粉萌发率差别较大,其中Fondante的花粉萌发率最高为74.45%,Amsterdam的最低为12.68%,其他3个品种的花粉萌发率介于二者之间.(2)品种间自交杂交亲和力存在一定差异,结实率大于30%的组合从高到低依次为Fondante×Atlantic(61.2%)、Fondante×Fondante(54.0%)、Carnegie×Carnegie(42.6%)、JanBos×JanBos(36.0%)、Carnegie×Atlantic(32.7%)、Atlantic×Fondante(30.0%),组合Atlantic×Amster-dam、Carnegie×JanBos、Amsterdam×Fondante、Amsterdam×JanBos、JanBos×Amsterdam未得到果实,其余组合结实率介于0～30%之间.(3)适宜风信子杂交幼胚离体培养的初代培养基为MS+100 mg/L谷氨酰胺,其次为MS+0.2 mg/LIBA+0.02 mg/L6-BA+100 mg/L谷氨酰胺...     

Postantibiotic effects of rifampin, amikacin, clarithromycin and ethambutol used alone or in various two-, three- and four-drug combinations against Mycobacterium avium

The postantibiotic effects (PAEs) of rifampin, amikacin, clarithromycin, and ethambutol were determined radiometrically against five AIDS-associated isolates of Mycobacterium avium. and were found to be 20.8+/-3.4. 18.4+/-2.5, 11.8+/-1.7. and 2.4+/-0.9 h, respectively. Various two-, three- or four-drug combinations were also screened: the PAEs for a two-drug combination were generally longer than individual drugs (mean PAE of 13.8+/-1.5 to 29.2+/-7.4 h instead of 2.4+/-0.9 to 18.4+/-2.5 h for single drugs). The addition of a third drug further increased the mean PAE to a range of 21.0+/-2.6 to 32.4+/-6.1 h. Both rifampin+clarithromycin and rifampin+amikacin were the most potent two-drug combinations resulting in longer PAEs than individual drugs, whereas rifampin+amikacin+clarithromycin was the most potent three-drug combination. Parallel viable count determinations showed a good correlation between the PAE results obtained by the radiometric method or by bacterial viability assessment. These results are useful in planning future clinical investigations to clarify the possible implication of PAE in drug schedule and dosage, a line of information that is urgently needed to guide the drug administration in M. avium-infected AIDS patients, who are presently over-burdened with the administration of too many drugs for HIV-treatment and opportunistic infections.