Novel NADP-linked isocitrate dehydrogenase present in peroxisomes of n-alkane-utilizing yeast,Candida tropicalis: comparison with mitochondrial NAD-linked isocitrate dehydrogenase

Peroxisomal NADP-linked isocitrate dehydrogenase (Ps-NADP-IDH) was purified for the first time from Candida tropicalis cells grown on n-alkane as a carbon source, which was effective in proliferation of peroxisomes. The properties of Ps-NADP-IDH were compared with those of mitochondrial NAD-linked isocitrate dehydrogenase (Mt-NAD-IDH) purified from the cells grown on acetate, in which peroxisomes did not proliferate. Ps-NADP-IDH was a homodimer of identical subunits (45 kDa), while Mt-NAD-IDH was suggested to be a heterooctamer composed of two types of subunits with different molecular masses (41 and 38 kDa). Kinetic studies revealed that Ps-NADP-IDH gave Michaelis-Menten saturation curves against isocitrate and NADP concentrations, whereas Mt-NAD-IDH was an allosteric enzyme regulated by ATP, AMP, and citrate. Inhibition by 2-oxoglutarate, a precursor of glutamate, was observed only for Ps-NADP-IDH. Both enzymes were inhibited by concomitant addition of oxalacetate and glyoxylate. The function of Ps-NADP-IDH seems to be completely discriminated from that of Mt-NAD-IDH as reflected by their distinct subcellular localizations. Furthermore, the properties of Ps-NADP-IDH were also compared with those of other mitochondrial and cytosolic IDHs from sources reported previously.     

Interspecific hybridisation between Candida albicans and Candida tropicalis

Abstract Protoplasts from auxotrophic mutants of Candida albicans and Candida tropicalis were produced by snail enzyme treatment and their fusion was induced with polyethylene glycol (PEG). During selective regeneration, nutritionally complemented interspecific hybrids were obtained. Their cells contained one nucleus, and the DNA content per cell was higher than in the parents. The isoenzymic and sugar assimilation patterns of the mutants, and those of the hybrids and the products after their haploidisation, were also analysed. The results indicated that the hybrids were partial alloploids containing the total chromosomal set of either of the parental species and one or a few chromosomes of the other.     

侵袭性热带念珠菌感染流行病学及药物敏感性

全球范围内,随着抗肿瘤药物、免疫抑制剂和广谱抗菌药物的使用,真菌感染的发病率显著提高,其中念珠菌感染占到绝大多数。目前热带念珠菌已经成为非白念珠菌中最常见的病原菌。我国热带念珠菌的临床分离率及对氟康唑及伏立康唑的耐药率都明显高于世界平均水平。但是,相比于白念珠菌,关于热带念珠菌的研究及相关临床信息相对较少。该文就侵袭性热带念珠菌感染危险因素、流行病学以及药物敏感性进行全面的综述。     

Characterization of the intron-containing citrate synthase gene from the alkanotrophic yeast Candida tropicalis: cloning and expression in Saccharomyces cerevisiae

Citrate synthase, an essential enzyme of the tricarboxylic acid cycle in mitochondria, was purified from acetate-grown Candida tropicalis. Results from SDS-PAGE and gel filtration showed that this enzyme was a dimer composed of 45-kDa subunits. A citrate synthase cDNA fragment was amplified by the 5′-RACE method. Nucleotide sequence analysis of this cDNA fragment revealed that the deduced amino acid sequence contained an extended leader sequence which is suggested to be a mitochondrial targeting signal, as judged from helical wheel analysis. Using this cDNA probe, one genomic citrate synthase clone was isolated from a yeast λEMBL3 library. The nucleotide sequence of the gene encoding C. tropicalis citrate synthase, CtCIT, revealed the presence of a 79-bp intron in the N-terminal region. Sequences essential as yeast splicing motifs were present in this intron. When the CtCIT gene including its intron was introduced into Saccharomyces cerevisiae using the promoter UPR-ICL, citrate synthase activity was highly induced, which strongly indicated that this intron was correctly spliced in S. cerevisiae. Received: 20 November 1996 / Accepted: 25 February 1997     

Effect of culture conditions on the isocitrate dehydrogenase and isocitrate lyase activities in Chlamydomonas reinhardtii

In the green alga Chlamydomonas reinhardtii , nitrogen staravation induced a reversible increase (2-fold) in NAD-isocitrate dehydrogenase (NAD-IDH; EC 1.1.1.41) and NADP-isocitrate dehydrogenase (NADP-IDH; EC 1.1.1.42) activities. Both enzymes were not affected by the concentration of CO₂, the dark or the nature of the nitrogen source (nitrate, nitrite, or ammonium). When cells growing autotrophically were transferred to heterotrophic conditions, a 40% reduction of the NAD-IDH activity was detected, a 2-fold increase of NADP-IDH was observed and isocitrate lyase (ICL; EC 4.1.3.1) activity was induced. The replacement of autotrophic conditions led to the initial activity levels. NAD- and NADP-IDH activities showed markedly different patterns of increase in synchronous cultures of this alga obtained by 12 h light/12 h dark transitions. While NAD-IDH increased in the last 4 h of the dark period, NADP-IDH increased during the last 4 h of the light period, remaining constant for the rest of the cycle.     

Biogenesis of peroxisomes and mitochondria: linked by division

Peroxisomes and mitochondria are metabolically linked organelles, which are crucial to human health and development. The search for components involved in their dynamics and maintenance led to the interesting finding that mitochondria and peroxisomes share components of their division machinery. Recently, it became clear that this is a common strategy used by mammals, fungi and plants. Furthermore, a closer interrelationship between peroxisomes and mitochondria has been proposed, which might have an impact on functionality and disease conditions. Here, we briefly highlight the major findings, views and open questions concerning peroxisomal formation, division, and interrelationship with mitochondria. Presented at the 50th Anniversary Symposium of the Society for Histochemistry, Interlaken, Switzerland, October 1–4, 2008.     

Subcellular localization of long-chain alcohol dehydrogenase and aldehyde dehydrogenase in n-alkane-grown Candida tropicalis

Long-chain alcohol dehydrogenase and longchain aldehyde dehydrogenase were induced in the cells of Candida tropicalis grown on n-alkanes. Subcellular localization of these dehydrogenases, together with that of acyl-CoA synthetase and glycerol-3-phosphate acyltransferase, was studied in terms of the metabolism of fatty acids derived from n-alkane substrates. Both longchain alcohol and aldehyde dehydrogenases distributed in the fractions of microsomes, mitochondria and peroxisomes obtained from the alkane-grown cells of C. tropicalis. Acyl-CoA synthetase was also located in these three fractions. Glycerol-3-phosphate acyltransferase was found in microsomes and mitochondria, in contrast to fatty acid -oxidation system localized exclusively in peroxisomes. Similar results of the enzyme localization were also obtained with C. lipolytica grown on n-alkanes. These results suggest strongly that microsomal and mitochondrial dehydrogenases provide long-chain fatty acids to be utilized for lipid synthesis, whereas those in peroxisomes supply fatty acids to be degraded via -oxidation to yield energy and cell constituents.     

产核酸和乳糖酶的热带假丝酵母QZN0209培养条件研究

经筛选得到一株具有一定核酸含量和乳糖酶活力的热带假丝酵母（Candida tropicalis)QZN0209,以综合利用酵母为目的,通过着重优化该菌株产核酸的培养条件。将其摇瓶产核酸水平从4.16%提高至13.94%;同时改变乳糖诱导方法将其乳糖酶活力提高约4倍,达到10.56ONPG单位/g湿菌体。     

Diclofenac exhibits synergism with azoles against planktonic cells and biofilms of Candida tropicalis

Abstract

This study aimed to evaluate the effect of diclofenac on minimum inhibitory concentrations of antifungals against planktonic cells and biofilms of Candida tropicalis. Susceptibility testing of planktonic cells was evaluated using the broth microdilution assay and checkerboard method. Biofilm formation by C. tropicalis in the presence of diclofenac, alone or in combination with antifungals, was also evaluated, and scanning electron microscope (SEM) and confocal microscope (CLSM) analyses were performed. Diclofenac showed an MIC of 1024?μg?ml^?1 against planktonic cells. The MICs of fluconazole and voriconazole against azole-resistant isolates were reduced 8- to 32-fold and 16- to 256-fold, respectively, when in combination with diclofenac. When in combination with fluconazole or voriconazole, diclofenac reduced the antifungal concentration necessary to inhibit C. tropicalis biofilm formation. In conclusion, diclofenac presents synergism with fluconazole and voriconazole against resistant C. tropicalis strains and improves the activity of these azole drugs against biofilm formation.     

Knockdown of both mitochondrial isocitrate dehydrogenase enzymes in pancreatic beta cells inhibits insulin secretion

Background

There are three isocitrate dehydrogenases (IDHs) in the pancreatic insulin cell; IDH1 (cytosolic) and IDH2 (mitochondrial) use NADP(H). IDH3 is mitochondrial, uses NAD(H) and was believed to be the IDH that supports the citric acid cycle.

Methods

With shRNAs targeting mRNAs for these enzymes we generated cell lines from INS-1 832/13 cells with severe (80%–90%) knockdown of the mitochondrial IDHs separately and together in the same cell line.

Results

With knockdown of both mitochondrial IDH's mRNA, enzyme activity and protein level, (but not with knockdown of only one mitochondrial IDH) glucose- and BCH (an allosteric activator of glutamate dehydrogenase)-plus-glutamine-stimulated insulin release were inhibited. Cellular levels of citrate, α-ketoglutarate, malate and ATP were altered in patterns consistent with blockage at the mitochondrial IDH reactions. We were able to generate only 50% knockdown of Idh1 mRNA in multiple cell lines (without inhibition of insulin release) possibly because greater knockdown of IDH1 was not compatible with cell line survival.

Conclusions

The mitochondrial IDHs are redundant for insulin secretion. When both enzymes are severely knocked down, their low activities (possibly assisted by transport of IDH products and other metabolic intermediates from the cytosol into mitochondria) are sufficient for cell growth, but inadequate for insulin secretion when the requirement for intermediates is certainly more rapid. The results also indicate that IDH2 can support the citric acid cycle.

General significance

As almost all mammalian cells possess substantial amounts of all three IDH enzymes, the biological principles suggested by these results are probably extrapolatable to many tissues.     

Characterization of NADP+-dependent isocitrate dehydrogenase isozymes from a psychrophilic bacterium,Colwellia psychrerythraea strain 34H

NADP⁺-dependent isocitrate dehydrogenase (IDH) isozymes of a psychrophilic bacterium, Colwellia psychrerythraea strain 34H, were characterized. The coexistence of monomeric and homodimeric IDHs in this bacterium was confirmed by Western blot analysis, the genes encoding two monomeric (IDH-IIa and IDH-IIb) and one dimeric (IDH-I) IDHs were cloned and overexpressed in Escherichia coli, and the three IDH proteins were purified. Both of the purified IDH-IIa and IDH-IIb were found to be cold-adapted enzymes while the purified IDH-I showed mesophilic properties. However, the specific activities of IDH-IIa and IDH-IIb were lower even at low temperatures than that of IDH-I. Therefore, IDH-I was suggested to be important for the growth of this bacterium. The results of colony formation of E. coli transformants carrying the respective IDH genes and IDH activities in their crude extracts indicated that the expression of the IDH-IIa gene is cold-inducible in the E. coli cells.     

Optimization of fed-batch fermentation for xylitol production by Candida tropicalis

Xylitol, a functional sweetener, was produced from xylose by biological conversion using Candida tropicalis ATCC 13803. Based on a two-substrate fermentation using glucose for cell growth and xylose for xylitol production, fed-batch fermentations were undertaken to increase the final xylitol concentration. The effects of xylose and xylitol on xylitol production rate were studied to determine the optimum concentrations for fed-batch fermentation. Xylose concentration in the medium (100 g l⁻¹) and less than 200 g l⁻¹ total xylose plus xylitol concentration were determined as optimum for maximum xylitol production rate and xylitol yield. Increasing the concentrations of xylose and xylitol decreased the rate and yield of xylitol production and the specific cell growth rate, probably because of an increase in osmotic stress that would interfere with xylose transport, xylitol flux to secretion to cell metabolism. The feeding rate of xylose solution during the fed-batch mode of operation was determined by using the mass balance equations and kinetic parameters involved in the equations in order to increase final xylitol concentration without affecting xylitol and productivity. The optimized fed-batch fermentation resulted in 187 g l⁻¹ xylitol concentration, 0.75 g xylitol g xylose⁻¹ xylitol yield and 3.9 g xylitol l⁻¹ h⁻¹ volumetric productivity. Journal of Industrial Microbiology & Biotechnology (2002) 29, 16–19 doi:10.1038/sj.jim.7000257 Received 15 October 2001/ Accepted in revised form 30 March 2002     

A subpopulation of Candida albicans and Candida tropicalis biofilm cells are highly tolerant to chelating agents

Many Candida spp. produce surface-adherent biofilm populations that are resistant to antifungal compounds and other environmental stresses. Recently, certain chelating agents have been recognized as having strong antimicrobial activity against biofilms of Candida species. This study investigated and characterized the concentration- and time-dependent killing of Candida biofilms by the chelators tetrasodium EDTA and sodium diethyldithiocarbamate. Here, Candida albicans and Candida tropicalis biofilms were cultivated in the Calgary Biofilm Device and then exposed to gradient arrays of these agents. Population survival was evaluated by viable cell counting and by confocal laser scanning microscopy (CLSM) in conjunction with fluorescent viability staining. At concentrations of > or =2 mM, both EDTA and diethyldithiocarbamate killed c. 90-99.5% of the biofilm cell populations. Notably, a small fraction (c. 0.5-10%) of biofilm cells were able to withstand the highest concentrations of these antifungals that were tested (16 and 32 mM for EDTA and diethyldithiocarbamate, respectively). Interestingly, CLSM revealed that these surviving cells were irregularly distributed throughout the biofilm community. These data suggest that the use of chelating agents against biofilms of Candida spp. may be limited by the refractory nature of a variant cell subpopulation in the surface-adherent community.     

Semi-continuous and continuous riboflavin production by calcium-alginate-immobilized Candida tropicalis in concentrated rectified grape must

Candida tropicalis cells were immobilized in calcium alginate. Gel beads were used to inoculate a medium containing concentrated rectified grape must as the sole carbohydrate source for both semi-continuous and continuous riboflavin production, in a 1litre glass stirred-tank fermenter. In semi-continuous mode, vitamin B₂ concentrations in culture broth ranged from approximately 350 to 650mg.l^-1 in successive runs. In continuous mode, concentrations of about 400–600mg.l^-1 were observed in the effluent for more than 1monthwithout significant loss of flavinogenic activity. The highest value of both vitamin B₂ production and sugar bioconversion occurred at the lowest dilution rate (0.008h^-1).     

一株耐高盐热带假丝酵母菌降解苯酚的研究

从含酚废水处理池污泥中驯化分离得到一株能以苯酚为唯一碳源的菌株FD-1。经18SrDNA和ITS序列的BLAST比对及系统发育分析,鉴定FD-1为热带假丝酵母(Candida tropicalis)的近缘种。FD-1对苯酚的降解能力较强,能够完全降解浓度为1 000mg·L-1的苯酚溶液。初步确定了FD-1在降解苯酚溶液时的最适温度为30~35℃,pH为6.0~7.0,并且通过探讨加入无机盐、培养基原料以及改变接种量三个因素对苯酚降解的影响,其耐受盐的浓度可达5%,对实践中应用微生物降解含酚废水具有积极的意义。     

High level expression of isocitrate lyase gene of n-alkane-utilizing yeast Candida tropicalis in Sacchromyces cerevisiae

The genomic DNA of peroxisomal isocitrate lyase (ICL) isolated from an n-alkane-assimilating yeast, Candida tropicalis, was truncated to utilize the original open reading frame under the control of the GAL7 promoter and was expressed in Saccharomyces cerevisiae. The recombinant ICL was synthesized as a functionally active enzyme with a specific activity similar to the enzyme purified from C. tropicalis, and was accounted for approximately 30% of the total extractable proteins in the yeast cells. This recombinant enzyme was easily purified to homogeneity. N-Terminal amino acid sequence, molecular masses of native form and subunit, amino acid composition, peptide maps, and kinetic parameters of the recombinant ICL were essentially the same as those of ICL purified from C. tropicalis. From these facts, S. cerevisiae was suggested to be an excellent microorganism to highly express the genes encoding peroxisomal proteins of C. tropicalis.Abbreviations ICL isocitrate lyase - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

Synergistic effects of geldanamycin with fluconazole are associated with reactive oxygen species in Candida tropicalis resistant to azoles and amphotericin B

With a significant increase in the incidence of system invasive fungal infections, the limited antifungal drugs and increased frequency of cross-resistance make it necessary to explore new and effective therapeutic strategies. Combination drug therapy has become one widely used choice to alleviate this problem. Geldanamycin (GdA), as an inhibitor of Hsp90, displayed broad antifungal activity when combined with fluconazole. However, due to its cytotoxicity, the dose and duration of GdA is limited. In this study, we observed the effect of fluconazole plus GdA on Candida tropicalis resistant to azoles and amphotericin B. The results showed that this synergism led to a decrease in growth and survival rate. In addition, fluconazole combined with GdA caused mitochondrial depolarisation, disruption of plasma membrane integrity and multinucleated morphology. However, the supplement of a reactive oxygen species (ROS) scavenger, N-acetylcysteine (NAC), rescued the above phenotypes. This study indicated that the oxidative stress mediated by fluconazole plus GdA played an important role in the antifungal activity, and targeting oxidative stress might extend target choices to treat fungal infections.     

Role of metal cations in the regulation of NADP-linked isocitrate dehydrogenase from porcine heart

The regulatory role of divalent metal cations in the NADP-linked isocitrate dehydrogenase (EC 1.1.1.42) from porcine heart was analysed. Saturation curves with respect to the substrate threo-Ds-isocitrate complexed with the metals including manganous, cadmium, cobaltous and zinc ions showed sigmoid relationships characteristic of allosteric enzymes. The Hill's interaction coefficients were 1.90, 1.75, 1.28 and 1.12, respectively. Saturation kinetics of the substrate-metal complexes including magnesium, ferrous and nickel ions exhibited normal hyperbolic curves with Hill's coefficients of 1. The ionic radii of metal cations were closely correlated with the maximal velocity, the enzyme affinity and the Hill's n values for the substrate-metal complexes. Cooperative interactions of metal-substrate complexes with NADP-isocitrate dehydrogenase are discussed in relation to the sites of the enzyme for the binding of the metal-substrate complex.