Highly sensitive and selective hydrogen peroxide biosensor based on hemoglobin immobilized at multiwalled carbon nanotubes-zinc oxide composite electrode

A highly sensitive and selective amperometric hydrogen peroxide (H(2)O(2)) biosensor based on immobilization of hemoglobin (Hb) at multiwalled carbon nanotubes-zinc oxide (MWCNT/ZnO) composite modified glassy carbon electrode (GCE) is reported. ZnO microsponges were electrochemically grown on MWCNT surface by the simple, cost-effective, green, electrochemical method at room temperature. The MWCNT/ZnO/Hb composite film showed a pair of well-defined, quasi-reversible redox peaks with a formal potential (E°') of -0.336V, characteristic features of heme redox couple of Hb. The electron transfer rate constant (k(s)) of immobilized Hb was 1.26s(-1). The developed biosensor showed a very fast response (>2s) toward H(2)O(2) with good sensitivity, wide linear range, and low detection limit of 0.02μM. The fabricated biosensor showed interesting features, including high selectivity, acceptable stability, good reproducibility, and repeatability along with excellent conductivity, facile electron mobility of MWCNT, and good biocompatibility of ZnO. The fabrication method of this biosensor is simple and effective for determination of H(2)O(2) in real samples with quick response, good sensitivity, high selectivity, and acceptable recovery.     

Activation of calcineurin expression in ischemia-reperfused rat heart and in human ischemic myocardium

Calcineurin (CaN) has been reported as a critical mediator for cardiac hypertrophy and cardiac myocyte apoptosis. In the present study, we investigated the activity and expression of CaN and the effect of calpain in rat heart after ischemia and reperfusion. Rat ischemic heart showed significant increase in CaN activity. Western blot analysis of normal rat heart extract with a polyclonal antibody raised against bovine CaN indicated a prominent immunoreactive band of 60 kDa (CaN A). In ischemic-reperfused hearts, the expression of CaN A was significantly low and immunoreactivity was observed in proteolytic bands of 46 kDa. This may be due to the proteolytic degradation of CaN A in ischemic tissues by m-calpain. We also noticed in vitro proteolysis of bovine cardiac CaN A by m-calpain. Immunohistochemical studies showed strong staining of immunoreactivity in rat hearts that had gone under 30 min ischemia followed by 30 min reperfusion similar to that found in human ischemic heart. Ischemia is associated with multiple alterations in the extracellular and intracellular signaling of cardiomyocytes and may act as an inducer of apoptosis. The increase in CaN activity and strong immunostaining observed in ischemic/perfused rat heart may be due to the calpain-mediated proteolysis of this phosphatase.     

Killer Ig-like receptor haplotype analysis by gene content: evidence for genomic diversity with a minimum of six basic framework haplotypes,each with multiple subsets

Killer Ig-like receptor (KIR) genes constitute a multigene family whose genomic diversity is achieved through differences in gene content and allelic polymorphism. KIR haplotypes containing a single activating KIR gene (A-haplotypes), and KIR haplotypes with multiple activating receptor genes (B-haplotypes) have been described. We report the evaluation of KIR gene content in extended families, sibling pairs, and an unrelated Caucasian panel through identification of the presence or absence of 14 KIR genes and 2 pseudogenes. Haplotype definition included subtyping for the expressed and nonexpressed KIR2DL5 variants, for two alleles of pseudogene 3DP1, and for two alleles of 2DS4, including a novel 2DS4 allele, KIR1D. KIR1D appears functionally homologous to the rhesus monkey KIR1D and likely arose as a consequence of a 22 nucleotide deletion in the coding sequence of 2DS4, leading to disruption of Ig-domain 2D and a premature termination codon following the first amino acid in the putative transmembrane domain. Our investigations identified 11 haplotypes within 12 families. From 49 sibling pairs and 17 consanguineous DNA samples, an additional 12 haplotypes were predicted. Our studies support a model for KIR haplotype diversity based on six basic gene compositions. We suggest that the centromeric half of the KIR genomic region is comprised of three major combinations, while the telomeric half can assume a short form with either 2DS4 or KIR1D or a long form with multiple combinations of several stimulatory KIR genes. Additional rare haplotypes can be identified, and may have arisen by gene duplication, intergenic recombination, or deletions.     

Cytotoxic and topographical properties of 6-arylidene-2-dimethylaminomethylcyclohexanone hydrochlorides and related compounds

A number of 2-arylidenecyclohexanones (1a-h) were converted into the corresponding Mannich bases (2a-h) and (3a,f). Evaluation against murine L1210 cells as well as human Molt 4/C8 and CEM T-lymphocytes revealed the marked cytotoxicity of the Mannich bases and also the fact that almost invariably these compounds were more potent than the precursor enones (1a-h). Further evaluation of most of the Mannich bases towards a panel of nearly 60 human tumour cell lines confirmed their utility as potent cytotoxins. In this assay, the compounds showed growth-inhibiting properties greater than the anticancer alkylator melphalan. QSAR studies revealed that in some cell lines compounds possessing small electron-attracting aryl substituents showed the greatest potencies. Molecular modeling and X-ray crystallography demonstrated that various interatomic distances and torsion angles correlated with cytotoxicity. A representative compound (2a) demonstrated weak inhibiting properties towards human N-myristoyltransferase and stimulated a tyrosine protein kinase. A single dose of 100 mg/kg of most of the compounds did not prove to be lethal in mice.     

Protective effect of DL-alpha-lipoic acid on cyclophosphamide induced oxidative cardiac injury

Cyclophosphamide (CP), one of the most widely prescribed antineoplastic drugs could cause a lethal cardiotoxicity. The present study is aimed at evaluating the role of DL-alpha-lipoic acid (LA) in oxidative cardiac damage induced by CP. Adult male Wistar rats were divided into four treatment groups. Two groups received single intraperitoneal injection of CP (200 mg/kg BW) to induce cardiotoxicity, one of these groups received LA treatment (25 mg/kg BW for 10 days). A vehicle treated control group and a LA drug control were also included. Cardiotoxicity, evident from increased activities of serum creatine phosphokinase, lactate dehydrogenase, aspartate transaminase and alanine transaminase in CP administered rats, was reversed by LA treatment. CP administered rats showed abnormal levels of enzymic (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glutathione-S-transferase) and non-enzymic antioxidants (glutathione, vitamin C and vitamin E) along with high malondialdehyde levels. However, normalized lipid peroxidation and antioxidant defenses were reported in the LA treated rats. These findings highlight the efficacy of LA as a cytoprotectant in CP induced cardiotoxicity.     

Expression of methionine aminopeptidase 2, N-myristoyltransferase, and N-myristoyltransferase inhibitor protein 71 in HT29

Protein myristoylation is a co-translational process, catalyzed by N-myristoyltransferase (NMT) that occurs after the initiating methionine is removed by methionine aminopeptidase (MetAP). The enzymes NMT and MetAP play a major role in the process of myristoylation of oncoproteins including the c-src family. In this study, we examined the levels of expression of MetAP2, NMT, and NMT inhibitor protein 71 (NIP71) in human colon cancer cell lines (HCCLs). We examined the influence of cell density on the expression of the above proteins in HT29 cells. Western blot analysis of MetAP2 and NMT demonstrated higher levels of protein expression in low density of HT29 while low expression in high density was observed. In addition, we observed that NIP71 and pp60(c-src) expressions were dependent on the cell density of HT29. This is the first study demonstrating the expression of MetAP2, NMT, pp60(c-src), and NIP71 in HCCLs.     

Beneficial effects of DL-alpha-lipoic acid on cyclophosphamide-induced oxidative stress in mitochondrial fractions of rat testis

The present study investigated the protective efficacy of dl-alpha-lipoic acid on the peroxidative damage and abnormal antioxidant levels in the mitochondrial fraction of testis in cyclophosphamide (CP) administered rats. Male Wistar rats of 140+/-20 g were categorized into four groups. Two groups were administered CP (15 mg/kg body weight once a week for 10 weeks by oral gavage) to induce testicular toxicity; one of these groups received lipoic acid treatment (35 mg/kg body weight intraperitoneally once a week for 10 weeks, 24 h prior to CP administration). A vehicle-treated control group and a lipoic acid drug control group were also included. The mitochondrial fraction of untreated CP-exposed testis showed 1.84-fold increase in lipid peroxidation, along with a significant (P<0.001) increase in hydrogen peroxide levels. In CP-exposed rats, we observed abnormal changes in the activities/levels of mitochondrial enzymic (superoxide dismutase, glutathione peroxidase and glutathione reductase) and non-enzymic (reduced glutathione, ascorbate and alpha-tocopherol) antioxidants. CP-treated rats also showed decline in the activities of mitochondrial enzymes such as succinate dehydrogenase, malate dehydrogenase and isocitrate dehydrogenase. In contrast, rats pretreated with lipoic acid showed normal lipid peroxidation and antioxidant defenses, thereby showing the protection rendered by lipoic acid.     

Molecular cloning of bovine cardiac muscle heat-shock protein 70 kDa and its phosphorylation by cAMP-dependent protein kinase in vitro

The 70-kDa heat-shock protein (Hsp70) has been cloned and sequenced from bovine cardiac muscle. On the basis of sequence features, the gene corresponds to the cytoplasmic form of Hsp70. This cardiac Hsp70 cDNA clone has an open reading frame of 1926 bp coding for 641 amino acids and a predicted molecular mass of 70.25 kDa. Comparison of the amino acid sequence revealed an extensive sequence identity with other species of Hsp70. Escherichia coli expressed cardiac Hsp70 stimulated a 2-fold increase in calcineurin (CaN) activity. Notably, we observed that Hsp70 directly interacts with CaN using a pull-down assay. Furthermore, expressed cardiac-specific Hsp70 was phosphorylated in vitro by cAMP-dependent protein kinase. Phosphorylation resulted in the incorporation of 0.1 mol of phosphate per mol of Hsp70. The phosphorylated Hsp70 was unable to activate the phosphatase activity of CaN. This is the first demonstration that Hsp70 is phosphorylated by cAMP-dependent protein kinase and provides an on/off switch for the regulation of CaN signaling by Hsp70.     

Ultrastructure of Spermatozoa in a Freshwater Fairy Shrimp, <Emphasis Type="Italic">Streptocephalus dichotomus</Emphasis> (Crustacea: Anostraca)

Ultrastructure of spermatozoa in fairy shrimp Streptocephalus dichotomus revealed that they are amoeboid type with no acrosome and flagella. Surface topography of the spermatozoon is smooth with occasional pseudopodial projections. Transmission electron micrographs of spermatozoa show organelle and the mitochondria which is not fused to form the so called ‘Nebenkern’. The testicular lumen reveals spermatozoa in varying sizes and shapes.     

Structure of canine tracheobronchial mucin glycoprotein

Canine tracheal mucin glycoprotein was isolated from beagle dogs fitted with tracheal pouches. Following exclusion chromatography on Sepharose CL-4B, noncovalently associated proteins were further resolved by dissociative density gradient centrifugation in CsBr-guanidinium chloride, and the mucin was then extracted with chloroform-methanol. The delipidated high-density product obtained had a nominal molecular weight of about 10(6) and an overall composition characteristic for a mucin glycoprotein, viz., a high content of serine and threonine, about 80% carbohydrate by weight, the absence of mannose or uronic acid, measurable ester sulfate, and a Pronase-resistant domain of molecular weight (1.75-3.0) X 10(5) which contains essentially all of the saccharide residues. Noncovalently bound lipid amounted to 6-10% by weight and was primarily cholesterol and cholesteryl esters. Cleavage of disulfide bonds by performic acid oxidation resulted in the release of a protein (Mr 65,000) not otherwise resolved by sodium dodecyl sulfate gel electrophoresis or the purification scheme.