Plasmid DNA is protected against ultrasonic cavitation-induced damage when complexed to cationic liposomes

Cationic liposomes bound to plasmid DNA are currently used for in vitro and in vivo gene therapy applications, but such complexes readily form large, heterogeneous aggregates that are not appropriate for pharmaceutical development. More importantly, size heterogeneity makes studies focused on optimizing gene transfer to cells difficult to conduct or understand. For this reason we have evaluated the effect of microprobe sonication on these complexes in an effort to achieve process-controlled size homogeneity. Complexes were prepared using a 7.2 kb reporter plasmid and the following liposomal lipid combinations: DDAB/DOPE (50:50 mol %), DDAB/DOPE/PEG-PE (50:45:5 mol %), DDAB/EPC (50:50 mol %), DDAB/EPC/PEG-PE (50:45:5, 50:40:10, 50:35:15 mol %), DODAC/DOPE (50:50 mol %), and DODAC/EPC (50:50 mol %) (DDAB, dimethyldioctadecylammonium bromide; DOPE, dioleoylphosphatidylethanolamine; PEG-PE, monomethoxypolyethylene glycol2000 succinate- distearoylphosphatidylethanolamine; EPC, egg phosphatidylcholine; DODAC, dioleoyldimethylammonium chloride). The influence of complex composition and lipid:DNA ratio was evaluated. Particle size was determined before and after complexation and again after sonication using the quasi-elastic light scattering technique. DNA integrity was assessed via agarose gel electrophoresis. Finally, gene transfection was evaluated using CHO cells that were transfected in vitro with sonicated and unsonicated complexes. It is established in this study that size reduction can occur, but this is dependent on cationic and neutral lipid composition and, in some cases, lipid:DNA ratio. Surprisingly, the process of sonication leaves a significant percentage of the plasmid DNA intact and capable of in vitro transfection. This study shows that plasmid DNA can be protected from damage due to sonication by liposome complex formation. This may indicate that more common pharmaceutical methods for size reduction which subject particles to mechanical stress may be applicable in preparation of liposome/DNA formulations for in vivo application.     

Cytotoxic effects of topotecan combined with various anticancer agents in human cancer cell lines

BACKGROUND: Topotecan (TPT) is a topoisomerase I poison that exhibits antineoplastic activity. Analysis of the cytotoxic effects of combinations of TPT and other anticancer agents has been limited. PURPOSE: We assessed the cytotoxic effects produced by combinations of TPT and other antineoplastic agents in experiments involving multiple human cancer cell lines of diverse histologic origins. METHODS: The cytotoxic effects of various antimetabolites (fluorouracil, methotrexate, or cytarabine), antimicrotubule agents (vincristine or paclitaxel [Taxol]), DNA alkylating agents (melphalan, bis[chloroethyl]nitrosourea [BCNU], or 4-hydroperoxycyclophosphamide [4HC]), and a DNA-platinating agent (cisplatin), alone and in combination with TPT, were measured in clonogenic (i.e., colony-forming) assays. HCT8 ileocecal adenocarcinoma, A549 non-small-cell lung carcinoma, NCI-H82ras(H) lung cancer, T98G glioblastoma, and MCF-7 breast cancer cell lines were used in these assays. The data were analyzed by the median effect method, primarily under the assumption that drug mechanisms of action were mutually nonexclusive (i.e., completely independent of one another). For each level of cytotoxicity (ranging from 5% to 95%), a drug combination index (CI) was calculated. A CI less than 1 indicated synergy (i.e., the effect of the combination was greater than that expected from the additive effects of the component agents), a CI equal to 1 indicated additivity, and a CI greater than 1 indicated antagonism (the effect of the combination was less than that expected from the additive effects of the component agents). RESULTS: When the mechanisms of drug action were assumed to be mutually nonexclusive, virtually all CIs for combinations of TPT and either antimetabolites or antimicrotubule agents revealed cytotoxic effects that were less than additive. The CIs calculated at low-to-intermediate levels of cytotoxicity for combinations of TPT and the DNA alkylating agents melphalan, BCNU, and 4HC also showed drug effects that were less than additive; in most cases, however, nearly additive or even synergistic effects were observed with these same drug combinations at high levels of cytotoxicity (i.e., at > or = 90% inhibition of colony formation). Results obtained with combinations of TPT and cisplatin varied according to the cell line examined. With A549 cells, less than additive effects were seen at low-to-intermediate levels of cytotoxicity, and more than additive effects were seen at high levels of cytotoxicity. With NCI-H82ras(H) cells, synergy was observed over most of the cytotoxicity range. CONCLUSIONS AND IMPLICATIONS: TPT cytotoxicity appears to be enhanced more by combination with certain DNA-damaging agents than by combination with antimetabolites or antimicrotubule agents. Interactions between TPT and other drugs can vary depending on the cell type examined. Further investigation is required to determine the basis of the observed effects and to determine whether these in vitro findings are predictive of results obtained in vivo.     

一种摩托车用ABS的试验研究

本文详细介绍了一种摩托车用ABS的结构，设计了其输入输出特性的试验方案，并测定了输入输出特性。根据结构和输入输出特性分析了该装置的工作原理，认为该ABS实际上只是一个限压装置，以用来防止抱死，有很大的局限性。     

3-Chloro-1,2-Propanediol Derived Amino Alcohol in Protein Hydrolysates

In model aqueous solutions containing 3-chIoro-l,2-propanediol (arising by the interaction of hydrochloric acid with lipids in protein hydrolysate and ammonia, 3-amino-1,2-propanediol was found as the main reaction product together with smaller amounts of glycerol. The reaction mechanism of formation of these two compounds was outlined and discussed as well as the possibilities of formation of some other amino analogues of glycerol chlorohydrins. 3-Amino-1,2-pro-panediol was also identified in commercially available seasonings (hydrolyzed vegetable protein) being present in concentrations of 30 mg kg^-1.     

Evidence for an endogenous cAMP-adenosine pathway in the rat kidney

In the rat kidney, exogenous adenosine-3'-5'-monophosphate (cAMP) is converted to adenosine via the metabolism of cAMP to adenosine-5'-monophosphate by phosphodiesterase and adenosine-5'-monophosphate to adenosine by 5'-nucleotidase. Our purpose was to investigate whether in the rat kidney adenosine is synthesized from endogenous cAMP via the same pathway. Rat kidneys were perfused with Tyrode's solution, and stabilized for 3 hr to minimize basal renal purine secretion. In control experiments (n = 6), the renal venous secretion rate of adenosine, inosine, hypoxanthine and Sigmapurines (adenosine + inosine + hypoxanthine) did not change over the two 10-min experimental periods. In contrast, the beta adrenoceptor agonist (+/-)-isoproterenol (1 and 10 microM added to the perfusate) caused a significant (1-factor analysis of variance with repeated measures; n = 31) increase in the renal venous secretion of adenosine (P <.0001), inosine (P <.0007), hypoxanthine (P <.0007) and Sigmapurines (P <.0001) as measured by high-performance liquid chromatography with ultraviolet detection. The Sigmapurines was the most discriminating index of isoproterenol-induced changes in purine release, and the renal venous secretion of Sigmapurines was significantly (2-factor analysis of variance with repeated measures) attenuated by inhibition of beta adrenoceptors with propranolol (.1 microM, n = 6; P <.05), phosphodiesterase with 3-isobutyl-1-methylxanthine (1 mM, n = 5; P <.002) and 5'-nucleotidase with alpha, beta-methyleneadenosine-5'-diphosphate (0.1 mM, n = 5; P <.03). Our data indicate that activation of beta adrenoceptors increases purine biosynthesis in the rat kidney via a mechanism that involves phosphodiesterase and 5'-nucleotidase. These results support the existence of an endogenous cAMP-adenosine pathway in the rat kidney.     

In vitro activities of novel antifolate drug combinations against Plasmodium falciparum and human granulocyte CFUs

The potency of antimalarial dihydrofolate reductase inhibitors, alone and in synergistic combination with dihydropteroate synthetase inhibitors, against the Kenyan K39 strain of Plasmodium falciparum (pyrimethamine resistant) and against normal replicating human bone marrow cells in in vitro culture has been studied. Therapeutic indices and rank order of synergistic potency were derived. Trimethoprim, pyrimethamine, and the quinazolines WR159412 and WR158122 had the smallest therapeutic indices (1.39, 4.38, 2.56, and 90.0, respectively), while the three triazines clociguanil, WR99210, and chlorcycloguanil had the largest (3,562, 3,000, and 2,000, respectively). In rank order of decreasing activity against P. falciparum, the six most potent drug combinations were WR99210-dapsone, chlorcycloguanil-dapsone, WR158122-dapsone, WR159412-dapsone, WR159412-sulfamethoxazole, and chlorcycloguanil-sulfamethoxazole; pyrimethamine-sulfadoxine was the least potent combination. These experiments form a basis for the selection of rapidly eliminated antifolate combinations for further clinical testing.     

A pilot experiment to compare regression of transplants of two different human tumors in athymic nude mice following single exposures to 60co radiation

Human tumor transplants were grown in a thymus defective nude mouse mutant (nu/nu BALB/c/A/Bom). Procedures for local irradiation of the tumor with 60Co-radiation are described. An introductory test of the usefulness of these transplants in studies of radiation effects on human tumors is performed by investigation of the time course of regression of adenocarcinoma transplants in six mice following single exposures of 375, 750, 1180 and 1575 rd, respectively, and malignant melanoma transplants in two mice following single exposures to 1475 and 2420 rd, respectively. The higher radioresistance of the malignant melanomas relative to that of the ovarian tumor is expected on the basis of clinical experience and indicates that radiation effects on human tissue implants in nude mice mirror some of the fundamental features of the in situ situation.     

The acute effects of amitriptyline, iprindole and trazodone on blood pressure and heart rate in rats

The cardiovascular effects of the tricyclic anti-depressant amitriptyline, a monoamine uptake inhibitor, and iprindole and trazodone, two novel anti-depressants of unknown mechanism, were monitored in urethane anesthetized rats following intravenous (IV) or intracerebroventricular (IVT) injection. Amitriptyline (2 mg IV or 0.25 mg IVT) produced hypotension that might reflect an action of norepinephrine on the anterior hypothalamus. Iprindole (2 mg IV) produced hypertension and (0.25 mg IVT) tachycardia that is consistent with a partial beta-agonist mechanism. Trazodone (1 mg IV or 0.25 mg IVT) produced hypotension and bradycardia that is consistent with the activation of noradrenergic neurons in the anterior hypothalamus perhaps as a result of trazodone acting on presynaptic alpha 2 receptors or on presynaptic serotonin receptors to increase the release of norepinephrine. All three of these anti-depressants have the potential to precipitate cardiovascular complications, particularly in patients with pre-existing cardiovascular abnormalities.