Summary Solid tumours develop an acidic extracellular environment with high concentration of lactic acid, and lactic acid produced by glycolysis has been assumed to be the major cause of tumour acidity. Experiments using lactate dehydrogenase (LDH)-deficient rastransfected Chinese hamster ovarian cells have been undertaken to address directly the hypothesis that lactic acid production is responsible for tumour acidification. The variant cells produce negligible quantities of lactic acid and consume minimal amounts of glucose compared with parental cells. Lactate-producing parental cells acidified lightly-buffered medium but variant cells did not. Tumours derived from parental and variant cells implanted into nude mice were found to have mean values of extracellular pH (pHe) of 7.03 ± 0.03 and 7.03 ± 0.05, respectively, both of which were significantly lower than that of normal muscle (pHe = 7.43 ± 0.03; P < 0.001). Lactic acid concentration in variant tumours (450 ± 90 ,ug g-1 wet weight) was much lower than that in parental tumours (1880 ± 140 gg/g-1) and similar to that in serum (400 ± 35 ,ug/g-1).These data show discordance between mean levels of pHe and lactate content in tumours; the results support those of Newell et al (1993) and suggest that the production of lactic acid via glycolysis causes acidification of culture medium, but is not the only mechanism, and is probably not the major mechanism responsible for the development of an acidic environment within solid tumours.
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Holo-neocarzinostatin (holo-NCS) is a complex protein carrying the anti-tumor active enediyne ring chromophore by a scaffold consisting of an immunoglobulinlike seven-stranded anti-parallel -barrel. Because of the labile chromophore reflecting its extremely strong DNA cleavage activity and complete stabilization in the complex, holo-NCS has attracted much attention in clinical use as well as for drug delivery systems. Despite many structural analyses for holo-NCS, the chromophore-releasing mechanism to trigger prompt attacks on the target DNA is still unclear. We determined the three-dimensional structure of the protein and the internal motion by multinuclear NMR to investigate the releasing mechanism. The internal motion studied by 13 C NMR methine relaxation experiments showed that the complex has a rigid structure for its loops as well as the -barrel in aqueous solution. This agrees with the refined NMR solution structure, which has good convergence in the loop regions. We also showed that the chromophore displayed a similar internal motion as the protein moiety. The structural comparison between the refined solution structure and x-ray crystal structure indicated characteristic differences. Based on the findings, we proposed the chromophore-releasing mechanism by a three-state equilibrium, which sufficiently describes both the strong binding and the prompt releasing of the chromophore. We demonstrated that we could bridge the dynamic properties and the static structure features with simple kinetic assumptions to solve the biochemical function.Holo-neocarzinostatin (holo-NCS) 1 (molecular mass 12 kDa, 113 amino acid residues ϩ chromophore) is a prominent member of the strongest anti-tumor reagent chromoprotein family (Fig. 1, B and D) (1). It is a non-covalent complex of an enediyne ring chromophore and its carrier protein isolated from Streptomyces carzinostaticus (2-4). The chromophore, which has a selective bulged DNA-cleaving activity (5), is easily inactivated by light, heat, and molecular oxygen because of its labile structure. Despite its intrinsic instability in the free state, the chromophore in the complex is stable even in vivo. It is thought that holo-NCS penetrates into the target cell carrying the chromophore and promptly releases it at the cell nucleus to kill the cell. Therefore, the holo-NCS family has attracted much attention in clinical use as well as for use in targeting drug delivery systems (6 -14). The three-dimensional structures of holo-NCS and the chromoprotein family have been investigated by solution NMR (12, 15-19) and x-ray diffraction (20), elucidating an immunoglobulin-like sevenstranded anti-parallel -barrel structure (Fig. 1B) (21).Based on the previous NMR and x-ray studies for holo-NCS, several mechanisms of the chromophore stabilization have been proposed. These mechanisms were dominated by hydrophobic interactions with the binding pocket. However, the mechanism of releasing and binding of the chromophore remains unclear, even though the process has attracted much atte...
Conjugal transferability of drug resistance was examined, in eleven Pseudomonas aeruginosa strains which were isolated in Frankfurt. Four R factors were demonstrated from three strains using P. aeruginosa as recipients but they were nontransferable to Escherichia coli K12. Two R factors, i.e., Rms146 and Rms147, mediated resistances to tetracycline (TC), streptomycin (SM), sulfanilamide (SA), kanamycin (KM), lividomycin (LV), gentamicin C complex (GM) and 3′,4′‐dideoxykanamycin B (DKB). They mediated the formation of aminoglycoside‐inactivating enzymes, i.e., SM phosphotransferase, SM adenylyltransferase, KM and LV phosphotransferase 1, and GM and DKB 6′‐N‐acetyltransferase. TC resistance conferred by these R factors was due to impermeability of the drug. P. aeruginosa Ps 142 carried two kinds of R factor in one cell, Rms148 (SM) and Rms149 (SM·SA·GM·CPC) (CPC, carbenicillin). Rms148 (SM) was transferable at a high frequency of 10–1 and mediated the formation of SM phosphotransferase. Rms149 mediated the formation of drug‐inactivating enzymes, i.e., GM 3‐N‐acetyltransferase and β‐lactamase, but did not inactivate SM. SM resistance was probably due to impermeability of the drug.
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