<Emphasis Type="Italic">In vivo</Emphasis> studies with a <Emphasis Type="Italic">Candida tropicalis</Emphasis> isolate exhibiting paradoxical growth <Emphasis Type="Italic">in vitro</Emphasis> in the presence of high concentration of caspofungin

We investigated the activity of caspofungin against a Candida tropicalis clinical isolate showing paradoxical growth in vitro. BALB/c mice immunosuppressed by cyclophosphamide were infected intraperitoneally using 10⁷ CFU/mouse. Caspofungin was administered intraperitoneally once daily for 5 days or as a single dose using the following doses: 0.12, 0.25, 1, 2, 3, 5, and 15 mg/kg. The single dose of caspofungin was effective only at 5 and 15 mg/kg concentrations (100% survival). Five-day caspofungin treatment led to 100% survival at doses of 1 mg/kg or higher. Caspofungin treatment significantly decreased the number of viable yeasts in the peritoneal lavage samples as well as in the infected abscesses at doses 1, 3, 5, and 15 mg/kg caspofungin as compared to the untreated control (P<0.001 in all cases), and even to the group treated with 0.12 mg/kg caspofungin (P<0.05 in all cases). At 2 mg/kg caspofungin dose, sterilization of the internal organs was reproducibly incomplete, suggesting that the role of paradoxical growth in the late clinical failure cannot be excluded.     

Low abundance does not mean less importance in cysteine metabolism

The cysteine molecule plays an essential role in cells because it is part of proteins and because it functions as a reduced sulfur donor molecule. In addition, the cysteine molecule may also play a role in the redox signaling of different stress processes. Even though the synthesis of cysteine by the most abundant of the isoforms of O-acetylserine(thiol) lyase in the chloroplast, the mitochondria and the cytosol is relatively well-understood, the role of the other less common isoforms homologous to O-acetylserine(thiol)lyase is unknown. Several studies on two of these isoforms, one located in the cytosol and the other one in the chloroplast, have shown that while one isoform operates with a desulfhydrase activity and is essential to regulate the homeostasis of cysteine in the cytosol, the other, located in the chloroplast, synthesizes S-sulfocysteine. This metabolite appears to be essential for the redox regulation of the chloroplast under certain lighting conditions.Key words: cysteine, S-sulfocysteine, desulfhydrase, sulfur metabolism, redox regulation, ArabidopsisCysteine occupies a central position in the plant primary and secondary metabolism due to its biochemical functions. Cysteine is the first organic compound with reduced sulfur synthesized by the plant in the photosynthetic primary sulfate assimilation. The importance of cysteine for plants derives from its role as an amino acid in proteins but also because of its functions as a precursor for a huge number of essential bio-molecules, such as many plant defense compounds formed in response to different environmental adverse conditions.^1,2 All of these bio-molecules contain sulfur moieties that act as functional groups and are derived from cysteine, and therefore, are intimately linked via their biosynthetic pathways.In addition to the final destination of the reduced sulfur atom in the primary and secondary metabolism of cells, the thiol residue of the cysteine molecule is a functional group that translates the physico-chemical signal (redox) of ROS and RNS into a functional signal, altering the properties of small molecules such as GSH or proteins whose enzymatic or functional properties depend on the redox state of its cysteine residues.³Sulfate is the major sulfur form available to plants. Sulfate is taken up to plant cells through specific sulfate transporters and is activated to adenosine 5′-phosphosulfate (APS). The reduction of the activated sulfate form, APS, is linked to plastids and the photosynthetic activity; therefore, APS is reduced to sulfite by the APS reductase using two GSH molecules as donors of the two electrons required in this step. Sulfite is further reduced to sulfide by the sulfite reductase that uses photosynthetically reduced ferredoxine (Fd) as an electron donor of the six required electrons. The biosynthesis of cysteine is further accomplished by the sequential reaction of two enzymes: First, the serine acetyltransferase (SAT) synthesizes the intermediary product, O-acetylserine (OAS), from acetyl-CoA and serine; and second, the O-acetylserine(thiol)lyase (OASTL) incorporates the sulfide to OAS producing the cysteine. Recently, much progress has been made toward understanding the action of the O-acetylserine(thiol)lyase (OASTL) enzyme, one of the enzymes responsible for the biosynthesis of cysteine, using as a model system the plant Arabidopsis thaliana. The focus of the research has been mainly placed on the most abundant enzymes based on their involvement in the primary sulfate assimilation pathway. Biochemical and molecular analysis of the major OASTL knockout mutants in Arabidopsis thaliana revealed that part of the produced sulfide is incorporated to O-acetylserine to form cysteine in the chloroplast with the assistance of the OAS-B isoform. However, most of the chloroplastic sulfide overflows and escapes into the cytosol and the mitochondria, where it is also assimilated into cysteine by the OAS-A1 and OAS-C isoforms, respectively.^4–6The three major OASTL isoforms seem to be redundant under normal growth conditions. However, our investigations on the major cytosolic isoform, the OAS-A1, revealed new insights on the function of this enzyme as a determinant of the antioxidative capacity of the cytosol.⁷ The OASTL homolog, CYS-C1, exhibits OASTL activity, but in fact, it is a β-cyanoalanine synthase enzyme that uses cysteine to detoxify cyanide within the mitochondria.⁸ Furthermore, Arabidopsis cells contain four additional O-acetylserine(thiol)lyase isoforms encoded by the CYS-D1 (At3g04940), CYS-D2 (At5g28020), CS26 (At3g03630) and CS-LIKE (At5g28030) genes with unknown function. Are these four isoforms authentic OASTL and are, therefore, redundant enzymes or do they have different activities and, therefore, different functions?Our recent research on the less-common isoforms, CS-like and CS26, shed light on this issue, and we are decoding two important aspects of the sulfur metabolism in plants.^9,10 The CS-LIKE protein was identified by sequence homology upon the completion of the sequencing of the Arabidopsis genome. Because of its cytosolic localization, it is thought to have an auxiliary function with respect to the major cytosolic isoform, the OAS-A1. The characterization of the purified recombinant protein has shown that the CS-LIKE isoform catalyzes the desulfuration of L-cysteine to sulfide plus ammonia and pyruvate; thus, CS-LIKE is a novel L-cysteine desulfhydrase (EC 4.4.1.1), and it is designated as DES1 (Fig. 1). This enzyme is important for maintaining the homeostasis of cysteine in the cell, and the loss of function of this protein in knockout mutant plants results in higher levels of cysteine and glutathione. This increased level of soluble thiols results also in a higher antioxidant capacity of the plant, which, in turn, becomes more resistant to abiotic stress phenomena such as the presence of heavy metals or hydrogen peroxide. This observation may indicate that the regulation of this enzyme may be a key component of the plant physiological processes that involve redox reactions. Cytosolic cysteine degrading enzymes with desulfhydrase activity has been found in plants, but the protein responsible for this activity remained unisolated until now that it is revealed with our investigation on DES1.¹¹ From the standpoint of biotechnology, plants with this modified enzyme may result in abiotic stress-resistant lines that deserve to be studied.Open in a separate windowFigure 1Biosynthesis of cysteine and S-sulfocysteine in the chloroplast and cytosol of Arabidopsis and subcellular localization of the responsible enzymes. The cytosolic and plastidial O-acetylserine(thiol)lyase, L-cysteine desulfhydrase and S-sulfocysteine synthase are shown in red. A single representative of a grana thylakoid is shown as a grey oval compartment.The other less common enzyme studied, called CS26 and localized in the chloroplast, has proved to be an enzyme with S-sulfocysteine synthase activity.¹⁰ This enzyme synthesizes the incorporation of thiosulfate to O-acetylserine to form S-sulfocysteine (RSSO₃⁻). This activity, discovered for the first time in plants, was previously reported in bacteria where the biosynthesis of cysteine can be accomplished by two enzymes encoded by the cysK and cysM genes.^12,13 This enzyme activity is essential for the chloroplast function under long-day growing conditions but seems to be superfluous under short-day conditions. Morphologic and biochemical phenotype comparisons of the knockout oas-b and cs26 highlight the importance of the metabolite S-sulfocysteine and not the cysteine in the redox control of the chloroplast. Under long-day growth conditions, the cs26 mutants exhibit a reduction in size and show leaf paleness, have reductions in the chlorophyll content and photosynthetic activity, and are not able to properly detoxify reactive oxygen species, which are accumulated to high levels. None of these changes are observed in the oas-b mutant.Although we do not know the function of the S-sulfocysteine molecule in the chloroplast, two aspects are important to note. On the one hand, the enzyme CS26 can be located in the chloroplast''s lumen in opposition to the enzyme OAS-B, which is located in the stroma. The second aspect is the difference in chemical reactivity of S-sulfocysteine and cysteine. The S-sulfocysteine has two sulfur atoms with different degrees of oxidation, −1 and +5; therefore, it may act as an oxidant molecule by reacting with reduced thiols forming a disulfide bridge and releasing sulfite.¹⁴ We have suggested that a putative target of S-sulfocysteine can be the STN7 kinase, which contains a transmembrane region that separates its catalytic kinase domain on the stromal side from its N-terminal end in the thylakoid lumen with two conserved cysteines that are critical for its activity. A disulfide bridge between these two cysteines is required for the kinase activity, but how the redox states of these two cysteines are regulated in the lumen remains an open question.¹⁵ In general, how the thiol oxidation of proteins located in the thylakoid lumen takes place is still unclear because no sulfhydryl oxidases have been identified in this compartment. In fact, this process is highly important because the chaperones and peptidyl-prolyl cis-trans isomerases, such as the AtFKBP13, need to be oxidized in order to be functional in the lumen and to regulate the folding of the Rieske protein.^16–18     

Functional analysis of MUTYH mutated proteins associated with familial adenomatous polyposis

The MUTYH DNA glycosylase specifically removes adenine misincorporated by replicative polymerases opposite the oxidized purine 8-oxo-7,8-dihydroguanine (8-oxoG). A defective protein activity results in the accumulation of G > T transversions because of unrepaired 8-oxoG:A mismatches. In humans, MUTYH germline mutations are associated with a recessive form of familial adenomatous polyposis and colorectal cancer predisposition (MUTYH-associated polyposis, MAP). Here we studied the repair capacity of the MUTYH variants R171W, E466del, 137insIW, Y165C and G382D, identified in MAP patients. Following expression and purification of human proteins from a bacterial system, we investigated MUTYH incision capacity on an 8-oxoG:A substrate by standard glycosylase assays. For the first time, we employed the surface plasmon resonance (SPR) technology for real-time recording of the association/dissociation of wild-type and MUTYH variants from an 8-oxoG:A DNA substrate. When compared to the wild-type protein, R171W, E466del and Y165C variants showed a severe reduction in the binding affinity towards the substrate, while 137insIW and G382D mutants manifested only a slight decrease mainly due to a slower rate of association. This reduced binding was always associated with impairment of glycosylase activity, with adenine removal being totally abrogated in R171W, E466del and Y165C and only partially reduced in 137insIW and G382D. Our findings demonstrate that SPR analysis is suitable to identify defective enzymatic behaviour even when mutant proteins display minor alterations in substrate recognition.     

Prevalence of radiographic detectable intervertebral disc calcifications in Dachshunds surgically treated for disc extrusion

Background  

An association between the occurrence of calcified discs, visible on radiographic examination (CDVR), and disc extrusions has been suggested in published literature over the past 10-20 years, mainly from Nordic countries. It has also been postulated that dogs without CDVR would not develop disc extrusions. Furthermore, inheritance of CDVR has been calculated and it has been postulated that, by selecting dogs for breeding with few, or no CDVR, the prevalence of disc extrusions in the Dachshund population may be reduced.     

Bone Mass and the CAG and GGN Androgen Receptor Polymorphisms in Young Men

Background

To determine whether androgen receptor (AR) CAG (polyglutamine) and GGN (polyglycine) polymorphisms influence bone mineral density (BMD), osteocalcin and free serum testosterone concentration in young men.

Methodology/Principal Findings

Whole body, lumbar spine and femoral bone mineral content (BMC) and BMD, Dual X-ray Absorptiometry (DXA), AR repeat polymorphisms (PCR), osteocalcin and free testosterone (ELISA) were determined in 282 healthy men (28.6±7.6 years). Individuals were grouped as CAG short (CAG_S) if harboring repeat lengths of ≤21 or CAG long (CAG_L) if CAG >21, and GGN was considered short (GGN_S) or long (GGN_L) if GGN ≤23 or >23. There was an inverse association between logarithm of CAG and GGN length and Ward''s Triangle BMC (r = −0.15 and −0.15, P<0.05, age and height adjusted). No associations between CAG or GGN repeat length and regional BMC or BMD were observed after adjusting for age. Whole body and regional BMC and BMD values were similar in men harboring CAG_S, CAG_L, GGN_S or GGN_L AR repeat polymorphisms. Men harboring the combination CAG_L+GGN_L had 6.3 and 4.4% higher lumbar spine BMC and BMD than men with the haplotype CAG_S+GGN_S (both P<0.05). Femoral neck BMD was 4.8% higher in the CAG_S+GGN_S compared with the CAG_L+GGN_S men (P<0.05). CAG_S, CAG_L, GGN_S, GGN_L men had similar osteocalcin concentration as well as the four CAG-GGN haplotypes studied.

Conclusion

AR polymorphisms have an influence on BMC and BMD in healthy adult humans, which cannot be explained through effects in osteoblastic activity.     

When the optimal is not the best: parameter estimation in complex biological models

Background

The vast computational resources that became available during the past decade enabled the development and simulation of increasingly complex mathematical models of cancer growth. These models typically involve many free parameters whose determination is a substantial obstacle to model development. Direct measurement of biochemical parameters in vivo is often difficult and sometimes impracticable, while fitting them under data-poor conditions may result in biologically implausible values.

Results

We discuss different methodological approaches to estimate parameters in complex biological models. We make use of the high computational power of the Blue Gene technology to perform an extensive study of the parameter space in a model of avascular tumor growth. We explicitly show that the landscape of the cost function used to optimize the model to the data has a very rugged surface in parameter space. This cost function has many local minima with unrealistic solutions, including the global minimum corresponding to the best fit.

Conclusions

The case studied in this paper shows one example in which model parameters that optimally fit the data are not necessarily the best ones from a biological point of view. To avoid force-fitting a model to a dataset, we propose that the best model parameters should be found by choosing, among suboptimal parameters, those that match criteria other than the ones used to fit the model. We also conclude that the model, data and optimization approach form a new complex system and point to the need of a theory that addresses this problem more generally.     

MicroRNA-210 Regulates Mitochondrial Free Radical Response to Hypoxia and Krebs Cycle in Cancer Cells by Targeting Iron Sulfur Cluster Protein ISCU

Background

Hypoxia in cancers results in the upregulation of hypoxia inducible factor 1 (HIF-1) and a microRNA, hsa-miR-210 (miR-210) which is associated with a poor prognosis.

Methods and Findings

In human cancer cell lines and tumours, we found that miR-210 targets the mitochondrial iron sulfur scaffold protein ISCU, required for assembly of iron-sulfur clusters, cofactors for key enzymes involved in the Krebs cycle, electron transport, and iron metabolism. Down regulation of ISCU was the major cause of induction of reactive oxygen species (ROS) in hypoxia. ISCU suppression reduced mitochondrial complex 1 activity and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. Cancers with low ISCU had a worse prognosis.

Conclusions

Induction of these major hallmarks of cancer show that a single microRNA, miR-210, mediates a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation.