Multiple forms of xylose reductase in Candida intermedia: comparison of their functional properties using quantitative structure-activity relationships, steady-state kinetic analysis, and pH studies

The xylose-fermenting yeast Candida intermedia produces two isoforms of xylose reductase: one is NADPH-dependent (monospecific xylose reductase; msXR), and another is shown here to prefer NADH approximately 4-fold over NADPH (dual specific xylose reductase; dsXR). To compare the functional properties of the isozymes, a steady-state kinetic analysis for the reaction d-xylose + NAD(P)H + H(+) <--> xylitol + NAD(P)(+) was carried out and specificity constants (k(cat)/K(aldehyde)) were measured for the reduction of a series of aldehydes differing in side-chain size as well as hydrogen-bonding capabilities with the substrate binding pocket of the enzyme. dsXR binds NAD(P)(+) (K(iNAD+) = 70 microM; K(iNADP+) = 55 microM) weakly and NADH (K(i) = 8 microM) about as tightly as NADPH (K(i) = 14 microM). msXR shows uniform binding of NADPH and NADP(+) (K(iNADP+) approximately K(iNADPH) = 20 microM). A quantitative structure-activity relationship analysis was carried out by correlating logarithmic k(cat)/K(aldehyde) values for dsXR with corresponding logarithmic k(cat)/K(aldehyde) values for msXR. This correlation is linear with a slope of approximately 1 (r (2) = 0.912), indicating that no isozyme-related pattern of substrate specificity prevails and aldehyde-binding modes are identical in both XR forms. Binary complexes of dsXR-NADH and msXR-NADPH show the same macroscopic pK of approximately 9.0-9.5, above which the activity is lost in both enzymes. A lower pK of 7.4 is seen for dsXR-NADPH. Specificity for NADH and greater binding affinity for NAD(P)H than NAD(P)(+) are thus the main features of enzymic function that distinguish dsXR from msXR.     

The effects of temperature and pressure on the characteristics of the extracts from high-pressure CO(2) extraction of Majorana hortensis Moench

The aim of this work is to assess the influence of temperature and pressure (solvent density) on the characteristics of the essential oil obtained from high-pressure carbon dioxide extraction of marjoram (commercial marjoram samples available in the free market and Majorana hortensis Moench, cultivated in South Brazil under rigorous agronomic conditions). The extracts were analyzed in terms of the liquid yield (extract/raw material, wt/wt) and distribution of volatile chemical components. The experiments were performed in a laboratory-scale unit using the dynamic method in the temperature range of 293.15-313.15 K, from 100 to 200 bar in pressure. Chemical analyses were carried out in a GC/MSD. Results show that an increase in temperature leads to a rise in the extract liquid yield despite large changes in solvent density. Chromatographic analyses permitted the identification of cis-sabinene hydrate, terpineol-4, alpha-terpineol, and cis-sabinene hydrate acetate as the main volatile compounds present in both commercial and cultivated samples.     

Meiotic behavior as a selection tool in the breeding of <Emphasis Type="Italic">Brachiaria humidicola</Emphasis> (Poaceae)

Brachiaria humidicola is a tropical grass that grows in seasonally swampy grasslands in Africa. In Brazil, two apomictic cultivars (2n = 54) of this species are widely used as pastures in poorly drained soils. The recent discovery of a sexual polyploid accession (2n = 36) in the germplasm collection at the Embrapa Beef Cattle Research Center allowed intraspecific hybridization with the objective of broadening the genetic variability and selection of superior genotypes in this species. Hybridization, however, depends on accessions with the same ploidy level. Cytological analyses of 55 accessions revealed that 19 apomictic accessions also presented 2n = 36 chromosomes. Chromosome pairing in hexavalent association at diakinesis and metaphase I suggested that the basic chromosome number for this species is x = 6. Cytological analysis revealed abnormalities in variable frequencies in the meiosis of these hexaploid (2n = 6x = 36) accessions. The most common were those related to irregular chromosome segregation which led to unbalanced gamete formation, but chromosome stickiness was also recorded. These results clearly demonstrate the value of cytogenetics in the choice of genitors and for superior hybrids to be obtained in the breeding of this species. For that both the ploidy level and the frequency of abnormalities need to be considered, besides other favorable agronomic characteristics.     

Determination of pesticides and PCBs in virgin olive oil by multicolumn solid-phase extraction cleanup followed by GC-NPD/ECD and confirmation by ion-trap GC-MS

A multicolumn solid-phase extraction cleanup for the determination of organophosphorus (OP) and organochlorine (OC) pesticides plus PCB congeners in virgin olive oil is presented. The method involves dissolution of the olive oil in hexane, followed by a cleanup system using a diatomaceous earth column (Extrelut-QE) with reversed (C(18)) and normal (alumina) phase SPE columns. Determination of OPs was by GC-NPD, while the OCs and PCBs were analyzed using GC-ECD. Recovery assays for OPs varied from 81.7% to 105.3%, for OCs ranged between 74.3% and 99.4%, while for PCBs were from 60.1% to 119.2%. Quantitation limits ranged from 10 to 25 microg/kg olive oil for OPs, and from 1 to 6 microg/kg olive oil for OCs and PCBs. In the case of positive samples, the confirmation of pesticide identity was performed by ion-trap GC-MS/MS. The applicability of the method was assayed with 19 virgin olive oil samples collected from different olive mills of Aragón (Spain). Only one OP pesticide (acephate) was detected in one sample at a concentration of 10 microg/kg. Organochlorine pesticides were found in 5-47% of samples at very low levels ranging from 1.5 to 5.2 microg/kg. PCBs were found in 20-90% of samples, showing concentrations between 2.3 and 17.3 microg/kg.     

Interaction of curcumin and bixin with β-cyclodextrin: complexation methods, stability, and applications in food

This work aimed to compare methods for the formation of complexes of bixin and curcumin with β-cyclodextrin (β-CD) and to evaluate the stability of the complexes formed by these methods and their food applications. The stoichiometric relationship between curcumin and β-CD was 1:2 and that between bixin and β-CD was 1:1. Curcumin-β-CD and bixin-β-CD complexes formed by kneading, coprecipitation, and simple mixing were evaluated by differential scanning calorimetry (DSC), thermogravimetry analysis (TGA), or nuclear magnetic resonance (NMR-H). For both curcumin and bixin, the best method of complexation was coprecipitation. Complexation of colorants with β-CD promoted an intensification of color and increased water solubility; however, stabilization in the presence of light occurred only for bixin. Application of curcumin-β-CD in cheese and yogurt and bixin-β-CD in the curd did not alter the initial characteristics of the products, which were sensorialy well accepted. Therefore, the complexation of these natural colorants with β-CD favors their use in low-fat foods, broadening the field of industrial application.     

The challenge of sustainable tourism development in the Maldives: Understanding the social and political dimensions of sustainability

Issues of power and politics are central to the development of the tourism sector and its prospects for contributing to sustainable development. This is demonstrated through a case study of the evolution of tourism in the Maldives, a luxury tourism destination where the government has followed a consistent policy of ‘quality tourism’ that has often been cited as a prime example of sustainable tourism. However, recently concerns have been raised about environmental degradation, human rights abuses, connections between the political and economic elite, and huge economic disparities associated with tourism here. Research on sustainable tourism needs to recognise the state's pivotal role in directing tourism development and consider how states balance the competing interests of other powerful tourism stakeholders.     

Chronic "Candidatus Mycoplasma turicensis" infection

"Candidatus Mycoplasma turicensis" infects felids. The pathogenesis of "Candidatus M. turicensis" chronic infection is poorly understood. The goals of the present study were to (1) induce reactivation of the infection in chronic carrier cats by attempted immunosuppression, (2) identify potential tissue sequestration using real-time TaqMan^® PCR and (3) monitor the humoral immune response by DnaK enzyme-linked immunosorbent assay (ELISA). Ten specified pathogen-free cats that had ostensibly recovered from experimental "Candidatus M. turicensis" infection were used: five cats (group 1) received high dose methylprednisolone (attempted immunosuppression), while five cats served as untreated controls (group 2). Besides weekly blood samples, tissue samples were collected from bone marrow, kidney, liver and salivary glands at selected time points. The cats in group 1 had significantly lower lymphocyte counts and higher blood glucose levels after methylprednisolone administration than the controls. After methylprednisolone administration one blood and three tissue samples from cats in group 1 tested PCR-positive; before the administration, only one sample was positive. All other samples tested PCR-negative. All cats stayed seropositive; the antibody levels of the cats in group 1 showed a significant transient decrease after methylprednisolone administration. This is the first study to report the presence of "Candidatus M. turicensis" in tissues of chronically infected cats and the persistence of anti-feline hemoplasma antibodies in the absence of detectable bacteremia. Methylprednisolone administration did not lead to a significant reactivation of the infection. Our results enhance the knowledge of "Candidatus M. turicensis" infection pathogenesis and are clinically relevant to the prognosis of hemoplasma-infected cats.     

Dissemination of antimicrobial resistant strains of Campylobacter coli and Campylobacter jejuni among cattle in Washington State and California

The purpose of this study was to investigate the genetic similarity of Campylobacter jejuni and Campylobacter coli with similar antimicrobial resistance phenotypes, isolated from cattle on different farms and at different times, in order to evaluate the possible existence of disseminated antimicrobial resistant clones. PFGE after SmaI and KpnI restriction identified 23 and 16 distinct PFGE patterns among 29 C. jejuni and 66 C. coli isolates, respectively. In C. coli, 51 (77%) of the resistant isolates demonstrated one of the four indistinguishable PFGE patterns, whereas only 24% doxycycline resistant C. jejuni shared one of the two indistinguishable PFGE patterns. The genetic mechanisms of resistance were homogeneous within and between these clonal types. Genetically indistinguishable (clonal) groups of C. coli accounted for most Campylobacter sp. with multiple antimicrobial resistance observed in this study, consistent with a role for clonal dissemination in the epidemiology of resistance in this species.     

From Haemobartonella to hemoplasma: molecular methods provide new insights

Hemotropic mycoplasmas (aka hemoplasmas) are the causative agents of infectious anemia in numerous mammalian species. Originally known as Haemobartonella and Eperythrozoon species, these organisms have been reclassified within the genus Mycoplasma. The development of new molecular assays has expanded our knowledge of this heterogeneous group of agents and allowed us to study their epidemiology and pathogenesis. The present review summarizes recently gained insights into feline hemotropic mycoplasmas, formerly known as Haemobartonella felis. Besides the two initially identified feline hemoplasma species, Mycoplasma haemofelis and Candidatus Mycoplasma haemominutum, we discovered a third novel hemoplasma in a Swiss pet cat; preliminary results suggest that the pathogenic potential of the latter agent depends on cofactors. In applying PCR-based assays, feline hemoplasma infections have been documented in domestic cats and wild felids worldwide. Differences between the three hemoplasmas in regard to response to antibiotic treatment and establishment of a carrier status have been reported. Additionally, besides an ostensible vector-borne transmission, direct transmission by aggressive interaction of cats or interspecies transmission might play a role in the epidemiology of these organisms. Based on a potential vector-borne and interspecies transmission, a zoonotic potential of hemoplasmas should be further investigated.