We report the stepwise and quantitative transformation of the Zr(μ-O)(μ-OH)(HCO) nodes in Zr-BTC (MOF-808) to the [Zr(μ-O)(μ-OH)Cl] nodes in ZrCl-BTC, and then to the organometallic [Zr(μ-O)(μ-OLi)R] nodes in ZrR-BTC (R = CHSiMe or Me). Activation of ZrCl-BTC with MMAO-12 generates ZrMe-BTC, which is an efficient catalyst for ethylene polymerization. ZrMe-BTC displays unusual electronic and steric properties compared to homogeneous Zr catalysts, possesses multimetallic active sites, and produces high-molecular-weight linear polyethylene. Metal-organic framework nodes can thus be directly transformed into novel single-site solid organometallic catalysts without homogeneous analogs for polymerization reactions.
The sensitivity of Candida albicans, grown in batch culture at 37 "C, to amphotericin methyl ester (AME), judged by the concentration of AME required to induce a standard rate of leakage of K+ from suspensions of organisms, decreased with the time of growth. Organisms in exponential growth were sensitive to 0.1 to 0~2 p g AME/ml while organisms in the stationary phase were resistant to 4 to 60 pg AME/ml, depending on the initial concentration of glucose in the medium and the length of time for which incubation had been continued. When the initial concentration of glucose was low (0.1 %, w/v), the AME resistance rose during the early stationary phase and then, after 40 h incubation at 37 "C, decreased again. Sphaeroplasts were prepared from organisms at different phases of growth and did not show these changes in AME sensitivity, but remained highly sensitive for growth up to 40 h.Sphaeroplasts were prepared by treating suspensions of organisms with mercaptoethanol and then digesting with Streptomyces enzyme preparation. Addition of the material extracted by the digestion to suspensions of exponential-phase organisms or sphaeroplasts increased their AME resistance. Fractionation of the digest showed that the antagonistic material was contained in the neutral lipid fraction. Pure lipids fell into the following order of decreasing antagonism to AME when added together with the antibiotic to suspensions of exponential-phase organisms : sterol esters (ergosterol esters > cholesterol esters ; unsaturated fatty acid esters > saturated fatty acid esters), sterols, triglycerides, unsaturated fatty acids, saturated fatty acids. The amount of antagonistic material released from stationary Organisms was not markedly greater than that from exponential-phase organisms and analysis of the lipid content of wall preparations showed that the content of total lipid, neutral lipid and triglyceride of 40 h organisms was not more than 75,25 and 30 04, respectively, greater than that of exponential-phase organisms.The AME resistance of stationary-phase organisms decreased rapidly if suspensions were incubated with glucose or mercaptoethanol. The decrease in the presence of glucose was prevented by metabolic inhibitors, especially SH binding agents. Treatment of organisms with either iodoacetamide or N-ethylmaleimide gave a rapid increase in AME resistance, amounting in some cases to 5-to 15-fold. The effect of iodoacetamide decreased as the organisms passed into the stationary phase and their intrinsic resistance increased. Evidence is presented which suggests that the degree of reduction of SH groups in the cell surface is an important factor in determining AME resistance.
The development of resistance to amphotericin methyl ester, measured in terms of the amount of drug required to induce a standard rate of release of K+ from suspensions of washed organisms, has been followed in Candida dbicans in starved cultures under controlled conditions of aeration, stirring and temperature. Resistance develops at a rate which increases with the rate of aeration, limited by the onset of damage due to turbulence. Resistance decreases rapidly if gassing with Nz is substituted for aeration, but sensitivity does not reach that of exponentially growing cells. Resumption of aeration is followed by a slow recovery of resistance.The addition of inhibitors of protein synthesis (trichodermin, verrucarin) or uncoupling agents (2,4-dinitrophenolY sodium azide) at the beginning of starvation results in an increased rate of development of resistance. Adding inhibitors at a later stage, when resistance has developed after 72 h aeration, does not affect the decrease in resistance produced by gassing with N2 but the presence of trichodermin or verrucarin delays the recovery of resistance on resumption of aeration. I N T R O D U C T I O NThe sensitivity of Candida albicans, grown in batch culture at 37 "C, to polyene antibiotics varies markedly with the growth phase (Gale, 1974; Hammond & Kliger, 1974; Hammond, Lambert & Kliger, 1974). Judged by the concentration of amphotericin methyl ester (AME) required to induce a standard rate of leakage of K+, the sensitivity of Candida dbicans decreases from 0.1 pg AME ml-l for organisms in the exponential phase of growth to 10 to 60 pg AME ml-l for organisms in the late-stationary phase (Gale et al., 1975). Gale et al. (1975) found that protoplasts of stationary phase organisms had the same sensitivity to AME as those from exponentially growing organisms and that lipid material antagonistic to the antibiotic could be extracted from cell walls. The amount of antagonistic material released from stationary phase organisms was not markedly greater than that from exponential phase organisms and it seemed improbable that the large difference in AME sensitivity of the two cultures was due to changes in the lipid content. The AME resistance of stationary phase organisms decreased rapidly on incubation with mercaptoethanol or glucose, the latter effect being prevented by the presence of metabolic inhibitors. Further treatment of such 'reduced' cells with oxygen or oxidizing agents resulted in increased AME resistance. Treatment of organisms with the SH-binding agents, iodoacetamide or N-ethylmaleimide, gave a rapid and marked increase in AME resistance ; this resistance was not affected by further treatment with reducing agents. It was suggested that the degree of reduction of SH groups in the cell surface is an important factor in determining AME sensitivity. The present paper shows that the development of AME resistance in starved Candida albicans is dependent on the rate of aeration of the culture, and that
The interaction of amphotericin B methyl ester (AME) with protoplasts of Candida albicans was measured indirectly by following the incorporation of [U-l*C]phenylalanine into the acid-insoluble material. The inhibitory effects of AME at the minimum inhibitory concentration were prevented by the addition of 85 mM-KC1 and 45 mM-MgCl,, as shown by Liras & Lampen (1974) for Saccharomyces cerevisiae. In C. albicans, pretreatment of the yeast before antibiotic addition was unnecessary. KCl and MgCl, did not prevent AME from binding to the protoplast membrane. This interaction was reversed by incubating the protoplasts in the presence of the protecting salts.
The development of resistance to amphotericin B methyl ester (AME), measured in terms of the amount (pg AME ml-l = s.r.c.) of the antibiotic required to induce a standard rate of release of K+ from suspensions of washed organisms, has been followed in Candida albicans in starved cultures under controlled conditions of aeration, pH, stirring and temperature for periods up to 120 h, and the amino acid content of the pool and the carbohydrate content of the organisms have been determined. The soluble glucan fraction of the cells decreased during the first 24 h starvation but no significant changes were observed in other carbohydrate or lipid fractions. At pH 3, the glutamate content of the pool decreased rapidly during the starvation period but other amino acids showed a general pattern of increase during the first 24 to 72 h and decrease during the later stages of starvation. Increasing the glutamate content of the pool by growth in media supplemented with sodium glutamate delayed the onset of resistance, and, in all experiments, high resistance did not develop until the glutamate content of the pool had fallen below 10 nmol (mg dry wt organisms)-l. There was a highly significant correlation between the level of resistance and (i) the glutamate concentration in the pool and (ii) the rate of disappearance of glutamate from the pool.Treatment of starved organisms with N-ethylmaleimide (NEM) increased the resistance, the increase ranging from 10-fold for cultures starved for 24 h to 2-fold or less for cultures starved for 120 h. The resistance of NEM-treated organisms increased with the time of starvation. Treatment of organisms with P-mercaptoethanol (ESH) decreased resistance ; for organisms after 24 to 96 h starvation, possessing moderate resistance (s.r.c. 15 to 20), treatment with ESH reduced this resistance to a value approximating to that of growing organisms (s.r.c. 0.1). In organisms starved for longer periods and with higher resistance, the effect of ESH was smaller and the level of ESH-insensitive resistance increased rapidly with time. Resistance thus developed in two stages; first, a stage where reduction of some components of the cell decreased resistance and, later, a stage of high resistance unaffected by reduction. Changes, similar in quality but smaller in quantity, were found in the development of resistance in organisms grown and starved at pH 7.The activities of glutamate : NAD+ oxidoreductase (EC 1 .4.1.2) and glutamate : NADP+ oxidoreductase (EC 1 .4.1.3) have been estimated in extracts from organisms during growth and starvation ; the NAD-linked enzyme activity remained relatively unchanged during the starvation period of 96 h while the NADP-linked enzyme activity decreased markedly during the first 24 h starvation. Estimation of the degree of reduction of the cellular NAD during the starvation period showed that AME resistance rose as the ratio of cellular NADH/NAD+ decreased.It is suggested that the first stage of phenotypic resistance can be attributed to a reducible factor in the cell wall...
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