Broad-host-range cloning vectors derived from the W-plasmid Sa

Four nonconjugative broad-host-range cloning vectors were derived from the W-plasmid Sa. They are small (M_r 5.6?7.2 × 10⁶), carry several drug-resistance markers, and allow constructing and screening for recombinant plasmids generated by the restriction enzymes EcoRI, PstI, BglII, HindIII, BamHI and SalI,     

Liposome-cell interactions A study of the interactions of liposomes containing entrapped anti-cancer drugs with the EMT6, S49 and AE1 (transport-deficient) cell lines

A study has been made to determine if the cytotoxicity observed when cells in culture were exposed to liposome-entrapped cytotoxic drugs was liposome mediated or resulted from leakage of drug from the liposomes with subsequent uptake of free drug by the cells. In preliminary experiments with the EMT6 cell line in monolayer culture, the cytotoxicity observed when the cells were exposed to a range of concentrations of liposome-entrapped methotrexate, actinomycin D and cytosine arabinoside for a variety of liposome compositions was somewhat less than that observed when the cells were exposed to similar concentrations of free drug. We suspected that the cytotoxicity was mediated via uptake of free drug leaked from liposomes. This was confirmed in experiments involving the EMT6 and S49 cell lines in monolayer or suspension culture, respectively, in the absence and presence of the nucleoside transport inhibitor, 6-((4-nitrobenzyl)thio)-9-β-d-ribofuranosylpurine. Additional experiments were performed on a transport-deficient mutant of the S49 cell line, the AE₁ cell line. No evidence for liposome-mediated cell death could be found in these cell lines when tubercidin 5′-monophosphate was entrapped in either large or small unilamellar liposomes composed of egg phosphatidylcholine/ cholesterol (2:1), bovine brain phosphatidylserine/egg phosphatidylcholine/ cholesterol (8:2:5) or egg phosphatidylcholine/stearylamine/cholesterol (10:1:5). Considerable toxicity due to empty liposomes of a variety of compositions was observed in the S49 cell line at high lipid concentrations.     

Trypanosoma brucei: An evaluation of salicylhydroxamic acid as a trypanocidal drug

The chemotherapeutic potential of salicylhydroxamic acid (SHAM) was studied in adult rats infected with a strain of Trypanosoma brucei that kills the rats in about 100 hr. The median lethal dose, administered intraperitoneally in a carboxymethyl-cellulose suspension, is approximately 820 mg/kg body weight for male and 850 mg/kg for female rats. The apparent cause of death is severe depression of the central nervous system.Half-maximal inhibition of O₂ uptake by trypanosomes in vitro requires 15 μM SHAM, whereas 100 μM inhibits over 90%. This inhibitory effect on trypanosome respiration was used as a biological assay for the effective SHAM concentration in rat plasma. After administration of a sublethal SHAM dose to rats, the effective plasma SHAM concentration rose rapidly to about 500 μM and then fell to about 10 μM at 4 hr. Nevertheless, this dose did not significantly affect the survival time of rats infected with T. brucei. Even if, by repeated SHAM administration, the plasma SHAM concentration was kept at around 100 μM for more than 4 hr, no therapeutic effect was observed.These results show that O₂ uptake is not essential for the survival of trypanosomes in rats and they support the idea that bloodstream trypanosomes have an alternative pathway for glycolysis, allowing energy production in the absence of respiration.The possibility that SHAM or other inhibitors of trypanosome respiration could stilll be trypanocidal if used in conjunction with another inhibitor of glycolysis is discussed.     

A proteomic and phosphoproteomic landscape of KRAS mutant cancers identifies combination therapies

1. Download : Download high-res image (203KB)
2. Download : Download full-size image

     

n-Dodecyl β-D-maltoside specifically competes with general anesthetics for anesthetic binding sites

We recently demonstrated that the anionic detergent sodium dodecyl sulfate (SDS) specifically interacts with the anesthetic binding site in horse spleen apoferritin, a soluble protein which models anesthetic binding sites in receptors. This raises the possibility of other detergents similarly interacting with and occluding such sites from anesthetics, thereby preventing the proper identification of novel anesthetic binding sites. n-Dodecyl β-D-maltoside (DDM) is a non-ionic detergent commonly used during protein-anesthetic studies because of its mild and non-denaturing properties. In this study, we demonstrate that SDS and DDM occupy anesthetic binding sites in the model proteins human serum albumin (HSA) and horse spleen apoferritin and thereby inhibit the binding of the general anesthetics propofol and isoflurane. DDM specifically interacts with HSA (K_d?=?40?μM) with a lower affinity than SDS (K_d?=?2?μM). DDM exerts all these effects while not perturbing the native structures of either model protein. Computational calculations corroborated the experimental results by demonstrating that the binding sites for DDM and both anesthetics on the model proteins overlapped. Collectively, our results indicate that DDM and SDS specifically interact with anesthetic binding sites and may thus prevent the identification of novel anesthetic sites. Special precaution should be taken when undertaking and interpreting results from protein-anesthetic investigations utilizing detergents like SDS and DDM.     

Brown drug substance color investigation in cell culture manufacturing using chemically defined media: A case study

Drug substance (DS) color is an important quality attribute for release, stability and comparability studies of biologics. With the increase of DS concentrations and biologics pipelines made in chemically defined media, atypical DS color other than colorless or pale yellow has been recently reported in the biopharmaceutical industry. We recently observed a brown DS color in manufacturing. Although analytical characterization data indicated that the brown color DS had no major quality issue, it is necessary to find the root cause and reduce DS color to ease placebo design for clinical use. It was demonstrated that the brown color was caused by the chemically defined basal medium containing high levels of iron and vitamin B12 (VB12) regardless of cell lines. Iron caused tryptophan oxidation in the protein to form N-formylkynurenine and kynurenine products, which likely contributed to a yellow DS color. A pink DS color was caused by the residual VB12 bound to DS. The brown color was the result of the combinatory effect of yellow and pink colors. Finally a modified basal medium was developed to produce a pale yellow DS in manufacturing.