Micro/nanostructured carbon composite modified with a hybrid redox mediator and enzymes as a glucose biosensor

A carbon micro/nanostructured composite based on cup-stacked carbon nanotubes (CSCNTs) grown onto a carbon felt has been found to be an efficient matrix for enzyme immobilization and chemical signal transduction. The obtained CSCNT/felt was modified with a copper hexacyanoferrate/polypyrrole (CuHCNFe/Ppy) hybrid mediator, and the resulting composite electrode was applied to H(2)O(2) detection, achieving a sensitivity of 194 +/- 15 mu A mmol(-1) L. The results showed that the CSCNT/felt matrix significantly increased the sensitivity of CuHCNFe/Ppy-based sensors compared to those prepared on a felt unrecovered by CSCNTs. Our data revealed that the improved sensitivity of the as-prepared CuHCNFe/Ppy-CSCNT/felt composite electrode can be attributed to the electronic interactions taking place among the CuHCNFe nanocrystals, Ppy layer and CSCNTs. In addition, the presence of CSCNTs also seemed to favor the dispersion of CuHCNFe nanocrystals over the Ppy matrix, even though the CSCNTs were buried under the conducting polymer layer. The CSCNT/felt matrix also enabled the preparation of a glucose biosensor whose sensitivity could be tuned as a function of the number of glucose oxidase (GOx) layers deposited through a Layer-by-Layer technique with an sensitivity of 11 +/- 2 mu A mmol(-1) L achieved at 15 poly(diallyldimethylammoniumchloride)/GOx bilayers. (C) 2011 Elsevier Ltd. All rights reserved.

Electrochemical Behavior of biosensor by determination the catechol and phenolic compounds using the enzyme PPO

An electrochemical biosensor using poly-phenol oxidasa (PPO) was constructed for the determination of phenolic compounds. The PPO employed with enzyme, it was obtained from Archontophoenix Cunninghamiana. The biosensor showed range of linearity in the range of 1 x 10(-3) to 1 x 10(-4) mol/L and a detection limit of 1 x 10(-4) mol/L. The optimal pH was 6,7 in medium phosphate buffer. The lifetime of the biosensors was 1 months, stored in phosphate buffer solution 0.1 mol/L to ambient temperature.

Development of Highly Selective Enzymatic Devices Based on Deposition of Permselective Membranes on Aligned Nanowires

We describe a simple and efficient strategy to fabricate enzymatic devices based on the deposition of glucose oxidase on aligned and highly oriented CoNiMo metallic nanowires. CoNiMo nanowires with an average diameter of 200 nm and length of 50 mu m were electrodeposited on Au-covered alumina substrates via electrodeposition, using alumina membranes as templates. Enzyme-modified electrodes were fabricated via enzyme immobilization using a cross-linker. To minimize nonspecific reactions in the presence of interfering agents, a permselective membrane composed of poly(vinylsulfonic acid) and polyamidoamine dendrimer was deposited via electrostatic interaction. The formation of hydrogen peroxide as a product of the enzymatic reaction was monitored at low overpotential, 0.0 V (vs Ag/AgCl). The detection limit was estimated at 22 mu M under an applied potential of 0.0 V. The apparent Michaelis-Menten constant determined from the Lineweaver-Burke plot was 2 mM.

Copper hexacyanoferrate nanoparticles modified electrodes: A versatile tool for biosensors

Copper hexacyanoferrate nanoparticles of about 30 nm in size have been prepared by the sonochemical irradiation of a mixture of aqueous potassium ferricyanide and copper chloride solutions. The nanoparticles were immobilized onto fluorine doped tin oxide (FTO) electrodes by using the electrostatic deposition layer-by-layer technique (LbL), obtaining electroactive films with electrocatalytic properties towards H2O2 reduction, providing higher currents than those observed for electrodeposited bulk material, even in electrolytes containing NH4+, Na+ and K+. The nanoparticles assembly was used as mediator in a glucose biosensor by immobilizing glucose oxidase enzyme by both, cross-linking and LbL. techniques. Sensitivities obtained were dependent on the immobilization method ranging from 1.23 mu A mmol(-1) L cm(-2) for crosslinking to 0.47 mu A mmol(-1) L cm(-2) for LbL; these values being of the same order than those obtained with electrodes where the amount of enzyme used is much higher. Moreover, the linear concentration range where the biosensors can operate was 10 times higher for electrodes prepared with the LbL immobilization method than with the conventional crosslinking one. (C) 2008 Elsevier B.V. All rights reserved.

Immobilization of Catalysts of Biological Interest on Porous Oxidized Silicon Surfaces

The present paper deals with the immobilization of redox mediators and proteins onto protected porous silicon surfaces to obtain their direct electrochemical reactions and to retain their bioactivities. This paper shows that MP-11 and viologens are able to establish chemical bonds with 3-aminopropyltriethoxylsilane-modified porous silicon surface. The functionalization of the surfaces have been fully characterized by energy dispersive X-ray analysis (EDX) and X-ray photoelectron spectroscopy (XPS) to examine the immobilization of these mediators onto the solid surface. Amperometric and open circuit potential measurements have shown the direct electron transfer between glucose oxidase and the electrode in the presence of the viologen mediator covalently linked to the 3-aminopropyltriethoxylsilane (APTES)-modified porous silicon surfaces.

Copper in disorders with neurological symptoms: Alzheimer`s, Menkes, and Wilson Diseases

Copper is an essential element for the activity of a number of physiologically important enzymes. Enzyme-related malfunctions may contribute to severe neurological symptoms and neurological diseases: copper is a component of cytochrome c oxidase, which catalyzes the reduction of oxygen to water, the essential step in cellular respiration. Copper is a cofactor of Cu/Zn-superoxide-dismutase which plays a key role in the cellular response to oxidative stress by scavenging reactive oxygen species. Furthermore, copper is a constituent of dopamine-β-hydroxylase, a critical enzyme in the catecholamine biosynthetic pathway. A detailed exploration of the biological importance and functional properties of proteins associated with neurological symptoms will have an important impact on understanding disease mechanisms and may accelerate development and testing of new therapeutic approaches. Copper binding proteins play important roles in the establishment and maintenance of metal-ion homeostasis, in deficiency disorders with neurological symptoms (Menkes disease, Wilson disease) and in neurodegenerative diseases (Alzheimer’s disease). The Menkes and Wilson proteins have been characterized as copper transporters and the amyloid precursor protein (APP) of Alzheimer’s disease has been proposed to work as a Cu(II) and/or Zn(II) transporter. Experimental, clinical and epidemiological observations in neurodegenerative disorders like Alzheimer’s disease and in the genetically inherited copper-dependent disorders Menkes and Wilson disease are summarized. This could provide a rationale for a link between severely dysregulated metal-ion homeostasis and the selective neuronal pathology.

Copper-dependent iron assimilation pathway in the model photosynthetic eukaryote chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is a valuable model for studying metal metabolism in a photosynthetic background. A search of the Chlamydomonas expressed sequence tag database led to the identification of several components that form a copper-dependent iron assimilation pathway related to the high-affinity iron uptake pathway defined originally for Saccharomyces cerevisiae. They include a multicopper ferroxidase (encoded by Fox1), an iron permease (encoded by Ftr1), a copper chaperone (encoded by Atx1), and a copper-transporting ATPase. A cDNA, Fer1, encoding ferritin for iron storage also was identified. Expression analysis demonstrated that Fox1 and Ftr1 were coordinately induced by iron deficiency, as were Atx1 and Fer1, although to lesser extents. In addition, Fox1 abundance was regulated at the posttranscriptional level by copper availability. Each component exhibited sequence relationship with its yeast, mammalian, or plant counterparts to various degrees; Atx1 of C. reinhardtii is also functionally related with respect to copper chaperone and antioxidant activities. Fox1 is most highly related to the mammalian homologues hephaestin and ceruloplasmin; its occurrence and pattern of expression in Chlamydomonas indicate, for the first time, a role for copper in iron assimilation in a photosynthetic species. Nevertheless, growth of C. reinhardtii under copper- and iron-limiting conditions showed that, unlike the situation in yeast and mammals, where copper deficiency results in a secondary iron deficiency, copper-deficient Chlamydomonas cells do not exhibit symptoms of iron deficiency. We propose the existence of a copper-independent iron assimilation pathway in this organism.

Utility of mitochondrial DNA sequences from four gene regions for systematic studies of Australian freshwater crayfish of the Genus Cherax (Decapoda: Parastacidae)

One of the most important requirements for systematic and phylogenetic studies is the identification of gene regions with the appropriate level of variation for the question of interest. Molecular phylogenetic and systematic studies of freshwater crayfish have made use of DNA sequences mainly from ribosomal genes, especially the 16S rRNA gene region. Thus, little information is available on other potentially useful mitochondrial gene regions for systematic studies in these animals. In this study, we look at nucleotide variation and phylogenetic relations within and between four species of freshwater crayfish of the genus Cherax from the southwest of Western Australia using four fragments amplified from the 16S rRNA, 12S rRNA, Cytochrome Oxidase I (COI), and Cytochrome b (Cyt b) gene regions. Samples of Engaeus strictifrons, Euastacus bispinosus, and Geocharax falcata were also sequenced for comparative purposes. The size of the fragments varied from 358 bp to 600 bp. Across all samples, the four fragments showed significant phylogenetic signal and showed similar proportions of variable sites (28.81–37.33%). Average divergence within species for the mitochondrial gene regions varied from 1.18% to 4.91%, with the 16S rRNA being the least variable and Cyt b the most variable. Average divergence between species ranged 7.63–15.53%, with 16S rRNA being the least variable and COI the most variable. At the generic level, average divergence ranged 17.21–23.82%. Phylogenetic analyses of the 16S rRNA, 12S rRNA, and COI regions generated four clades consistent with the presence of four species previously identified on the basis of allozyme and morphological studies. The relationships among samples were largely congruent across the data set, although some relationships remained unresolved. Not all samples could be amplified using the Cyt b primers, and some of those that were showed quite anomalous relationships, suggesting that one or more Cyt b pseudogenes were being amplified.

Molecular phylogeny of the Cheilodactylidae and Latridae (Perciformes: Cirrhitoidea) with notes on taxonomy and biogeography

Phylogenetic relationships among cheilodactylid and latrid fishes were estimated from cytochrome oxidase I and cytochrome b mitochondrial DNA sequences. Two South African cheilodactylids, Cheilodactylus fasciatus and Cheilodactylus pixi, were divergent from the remaining members of their genus and family, and the monophyly of these groups was rejected based on parametric bootstrap analysis. As C. fasciatus is the nominal species for the genus and family, widespread taxonomic reassignment is implicated for the remaining 12 and 17 members of these groups, respectively. As these 17 cheilodactylids are not genetically or morphologically distinct from the latrids, it is proposed that the Latridae should be expanded to encompass them. The inferred relationships among those Cheilodactylus requiring generic reassignment were largely unresolved, and hence few recommendations can be made regarding their placement. Divergence time estimates indicate that chance oceanic dispersal subsequent to Gondwanan fragmentation best explains the Southern Hemisphere radiation of cheilodactylids.

Phylogeny of the Australian freshwater crayfish Cherax destructor-complex (Decapoda:Parastacidae) inferred from four mitochondrial gene regions

The phylogenetic relationships among 32 individuals of Australian freshwater crayfish belonging to the Cherax destructor-complex were investigated using a dataset comprising sequences from four mitochondrial gene regions: the large subunit rRNA (16S rRNA), cytochrome oxidase I (COI), adenosine triphosphatase 6 (ATPase 6), and cytochrome oxidase III (COIII). A total of 1602 bp was obtained, and a combined analysis of the data produced a tree with strong support (bootstrap values 94–100%) for three divergent lineages, verifying the phylogenetic hypotheses of relationships within the C. destructor species-complex suggested in previous studies. Overall, sequences from the 16S rRNA gene showed the least variation compared to those generated from protein coding genes, which presented considerably greater levels of divergence. The level of divergence within C. destructor was found to be greater than that observed in other species of freshwater crayfish, but interspecific variation among species examined in the present study was similar to that reported previously.

Heterozygous tx mice have an increased sensitivity to copper loading: implications for Wilson's Disease carriers

Wilson's disease carriers constitute 1% of the human population. It is unknown whether Wilsons disease carriers are at increased susceptibility to copper overload when exposed to chronically high levels of ingested copper. This study investigated the effect of chronic excess copper in drinking water on the heterozygous form of the Wilson’s disease mouse model – the toxic milk (tx) mouse. Mice were provided with drinking water containing 300 mg/l copper for 4–7, 8–11, 12–15 or 16–20 months. At the completion of the study liver, spleen, kidney and brain tissue were analyzed by atomic absorption spectroscopy to determine copper concentration. Plasma ceruloplasmin oxidase activity and liver histology were also assessed. Chronic copper loading resulted in significantly increased liver copper in both tx heterozygous and tx homozygous mice, while wild type mice were resistant to the effects of copper loading. Copper loading effects were greatest in tx homozygous mice, with increased extrahepatic copper deposition in spleen and kidney – an effect absent in heterozygote and wild type mice. Although liver histology in homozygous mice was markedly abnormal, no histological differences were noted between heterozygous and wild type mice with copper loading. Tx heterozygous mice have a reduced ability to excrete excess copper, indicating that half of the normal liver Atp7b copper transporter activity is insufficient to deal with large copper intakes. Our results suggest that Wilsons disease carriers in the human population may be at increased risk of copper loading if chronically exposed to elevated copper in food or drinking water.

Nitric oxide protects against mitochondrial permeabilizastion induced by gluthathione depletion: role of S-nitrosylation?

Nitric oxide (NO) is known to mediate a multitude of biological effects including inhibition of respiration at cytochrome c oxidase (COX), formation of peroxynitrite (ONOO⁻) by reaction with mitochondrial superoxide (O2^{• −}), and S-nitrosylation of proteins. In this study, we investigated pathways of NO metabolism in lymphoblastic leukemic CEM cells in response to glutathione (GSH) depletion. We found that NO blocked mitochondrial protein thiol oxidation, membrane permeabilization, and cell death. The effects of NO were: (1) independent of respiratory chain inhibition since protection was also observed in CEM cells lacking mitochondrial DNA (ρ⁰) which do not possess a functional respiratory chain and (2) independent of ONOO⁻ formation since nitrotyrosine (a marker for ONOO⁻ formation) was not detected in extracts from cells treated with NO after GSH depletion. However, NO increased the level of mitochondrial protein S-nitrosylation (SNO) determined by the Biotin Switch assay and by the release of NO from mitochondrial fractions treated with mercuric chloride (which cleaves SNO bonds to release NO). In conclusion, these results indicate that NO blocks cell death after GSH depletion by preserving the redox status of mitochondrial protein thiols probably by a mechanism that involves S-nitrosylation of mitochondrial protein thiols.

Elucidation of bucephalid parasite life-cycles using mutation scanning analysis

Nucleotide variation in a portion of the mitochondrial cytochrome c oxidase subunit1 (cox1) gene from asexual stages of bucephalids of southern Australian scallops (Chlamys asperrima, Chlamys bifrons and Pecten fumatus) was investigated using a mutation scanning–sequencing approach. Single-strand conformation polymorphism (SSCP) analysis revealed three main profile types (A, B and C) for parasites isolated from scallops. Sequence analysis revealed that samples represented by profiles B and C had a high degree (97.3%) of sequence similarity, whereas they were ~21% different in sequence from those represented by profile A. These findings suggested that at least two types or species (represented by profile A, or profile B or C) of bucephalid infect scallops, of which both were detected in South Australia, while only one was found in Victoria. The prevalence of bucephalids (and their SSCP haplotypes) appeared to differ among the three species of scallop in South Australia as well as between the two scallop species in Victoria, indicating a degree of host specificity. Adult bucephalids were collected from Eastern Australian Salmon (Arripis trutta), in an attempt to match them with the asexual stages from the scallop hosts. Neither of the two taxa of adult bucephalid (Telorhynchus arripidis and an un-named Telorhynchus species) shared SSCP profiles with the bucephalids from scallops, but were genetically similar, suggesting that the asexual stages from scallops may represent the genus Telorhynchus. This study, which assessed nucleotide sequence variation in a portion of the mitochondrial cox1 gene for bucephalids found in scallops and arripid fish, illustrates the usefulness of the mutation scanning approach to elucidate complex life-cycles of marine parasites.

New gall midge taxa (Diptera : Cecidomyiidae) from Australian Chenopodiaceae

Five new species and a new genus of gall midge are described from flower galls on native chenopod plants in Eyre Peninsula, South Australia. Asphondylia vesicaria sp. n. induces galls on Atriplex vesicaria; A. mcneilli sp. n. on Sclerolaena diacantha; and A. tonsura sp. n. on Enchylaena tomentosa. Infested flowers develop into galls and produce no seeds. DNA analysis of part of the cytochrome-c oxidase subunit I mitochondrial gene supported the morphological and biological differences between each of the new species and the previously described A. floriformis (Veenstra-Quah & Kolesik) and A. sarcocorniae (Veenstra-Quah & Kolesik) that induce galls on leaves and branches, respectively, of Sarcocornia quinqueflora (Chenopodiaceae) in Australian salt marshes. A new genus, Dactylasioptera gen. n. and two new species of Lasiopterini, D. adentata sp. n. and D. dentata sp. n. are described – both were reared from galls of A. mcneilli and A. tonsura.

Undernutrition during suckling in rats elevates plasma adiponectin and its receptor in skeletal muscle regardless of diet composition : a protective effect?

Objective:
Nutrition during critical periods in early life may increase the subsequent risk of obesity, hypertension and metabolic diseases in adulthood. Few studies have focused on the long-term consequences of poor nutrition during the suckling period on the susceptibility to developing obesity when exposed to a palatable cafeteria-style high-fat diet (CD) after weaning.

Design:
This study examined the impact of early undernutrition, followed by CD exposure, on blood pressure, hormones and genes important for insulin sensitivity and metabolism and skeletal muscle mRNA expression of adiponectin receptor 1 (AdipoR1), carnitine palmitoyl-transferase I (CPT-1), cytochrome c oxidase 4 (COX4) and peroxisome proliferator-activated receptor alpha (PPARalpha). Following normal gestation, Sprague–Dawley rat litters were adjusted to 18 (undernourished) or 12 (control) pups. Rats were weaned (day 21) onto either palatable CD or standard chow.

Results:
Early undernourished rats were significantly lighter than control by 17 days, persisting into adulthood only when animals were fed chow after weaning. Regardless of litter size, rats fed CD had doubled fat mass at 15 weeks of age, and significant elevations in plasma leptin, insulin and adiponectin. Importantly, undernutrition confined to the suckling period, elevated circulating adiponectin regardless of post-weaning diet. Blood pressure was reduced in early undernourished rats fed chow, and increased by CD. Early undernutrition was associated with long-term elevations in the expression of AdipoR1, CPT-1, COX4 and PPARalpha in skeletal muscle.

Conclusion:
This study demonstrates the important role of early nutrition on body weight and metabolism, suggesting early undernourishment enhances insulin sensitivity and fatty-acid oxidation. The long-term potential benefit of limiting nutrition in the early postnatal period warrants further investigation.