Differential influence of tacrolimus and sirolimus on mitochondrial-dependent signaling for apoptosis in pancreatic cells

Molecular and Cellular Biochemistry - To examine and compare the mitochondria-related cellular mechanisms by which tacrolimus (TAC) or sirolimus (SIR) immunosuppressive drugs alter the pancreatic...     

Improvement of efficient in vitro regeneration potential of mature callus induced from Malaysian upland rice seed (Oryza sativa cv. Panderas)

A new and rapid protocol for optimum callus production and complete plant regeneration has been assessed in Malaysian upland rice (Oryza sativa) cv. Panderas. The effect of plant growth regulator (PGR) on the regeneration frequency of Malaysian upland rice (cv. Panderas) was investigated. Mature seeds were used as a starting material for callus induction experiment using various concentrations of 2,4-D and NAA. Optimal callus induction frequency at 90% was obtained on MS media containing 2,4-D (3 mg L⁻¹) and NAA (2 mg L⁻¹) after 6 weeks while no significant difference was seen on tryptophan and glutamine parameters. Embryogenic callus was recorded as compact, globular and light yellowish in color. The embryogenic callus morphology was further confirmed with scanning electron microscopy (SEM) analysis. For regeneration, induced calli were treated with various concentrations of Kin (0.5–1.5 mg L⁻¹), BAP, NAA and 0.5 mg L⁻¹ of TDZ. The result showed that the maximum regeneration frequency (100%) was achieved on MS medium containing BAP (0.5 mg L⁻¹), Kin (1.5 mg L⁻¹), NAA (0.5 mg L⁻¹) and TDZ (0.5 mg L⁻¹) within four weeks. Developed shoots were successfully rooted on half strength MS free hormone medium and later transferred into a pot containing soil for acclimatization. This cutting-edge finding is unique over the other existing publishable data due to the good regeneration response by producing a large number of shoots.Abbreviations: 2,4-D, 2,4-dichlorophenoxyacetic acid; NAA, naphthaleneacetic acid; Kin, kinetin; MS, Murashige and Skoog; BAP, benzylaminopurine; TDZ, thidiazuron     

Pathway Network Analyses for Autism Reveal Multisystem Involvement,Major Overlaps with Other Diseases and Convergence upon MAPK and Calcium Signaling

We used established databases in standard ways to systematically characterize gene ontologies, pathways and functional linkages in the large set of genes now associated with autism spectrum disorders (ASDs). These conditions are particularly challenging—they lack clear pathognomonic biological markers, they involve great heterogeneity across multiple levels (genes, systemic biological and brain characteristics, and nuances of behavioral manifestations)—and yet everyone with this diagnosis meets the same defining behavioral criteria. Using the human gene list from Simons Foundation Autism Research Initiative (SFARI) we performed gene set enrichment analysis with the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database, and then derived a pathway network from pathway-pathway functional interactions again in reference to KEGG. Through identifying the GO (Gene Ontology) groups in which SFARI genes were enriched, mapping the coherence between pathways and GO groups, and ranking the relative strengths of representation of pathway network components, we 1) identified 10 disease-associated and 30 function-associated pathways 2) revealed calcium signaling pathway and neuroactive ligand-receptor interaction as the most enriched, statistically significant pathways from the enrichment analysis, 3) showed calcium signaling pathways and MAPK signaling pathway to be interactive hubs with other pathways and also to be involved with pervasively present biological processes, 4) found convergent indications that the process “calcium-PRC (protein kinase C)-Ras-Raf-MAPK/ERK” is likely a major contributor to ASD pathophysiology, and 5) noted that perturbations associated with KEGG’s category of environmental information processing were common. These findings support the idea that ASD-associated genes may contribute not only to core features of ASD themselves but also to vulnerability to other chronic and systemic problems potentially including cancer, metabolic conditions and heart diseases. ASDs may thus arise, or emerge, from underlying vulnerabilities related to pleiotropic genes associated with pervasively important molecular mechanisms, vulnerability to environmental input and multiple systemic co-morbidities.     

Spray‐Cast Multilayer Organometal Perovskite Solar Cells Fabricated in Air

Spray‐coating is a versatile coating technique that can be used to deposit functional films over large areas at speed. Here, spray‐coating is used to fabricate inverted perovskite solar cell devices in which all of the solution‐processible layers (PEDOT:PSS, perovskite, and PCBM) are deposited by ultrasonic spray‐casting in air. Using such techniques, all‐spray‐cast devices having a champion power conversion efficiency (PCE) of 9.9% are fabricated. Such performance compares favorably with reference devices spin‐cast under a nitrogen atmosphere that has a champion PCE of 12.8%. Losses in device efficiency are ascribed to lower surface coverage and reduced uniformity of the spray‐cast perovskite layer.     

Screening methods used to determine the anti-microbial properties of Aloe vera inner gel

The emergence of antibiotic resistant bacterial strains is a growing problem and is an important concern for patients, physicians, healthcare managers, and policymakers as it results in poorer health and economic outcomes. This has led to an urgent global call for new antimicrobial drugs, particularly from natural resources. We have been studying the antimicrobial properties of the inner leaf gel component of Aloe barbadensis Miller and have used a number of different, simple in vitro assays to establish a scientific basis for the potential use of Aloe vera on a range of clinically relevant bacteria. The bacteria used include Shigella flexneri, Methicillin-Resistant Staphylococcus aureus (MRSA), Enterobacter cloacae and Enterococcus bovis. In this paper, we compare standard methods recommended by the Clinical and Laboratory Standards Institute (CLSI) with a microtitre assay using a metabolic colour indicator Alamar blue. All the techniques described have shown that Aloe vera has an antimicrobial effect, however, the microtitre assay enables high throughput screening, under similar conditions and is less wasteful of plant material.     

Simulated hyperglycemia in rat cardiomyocytes: a proteomics approach for improved analysis of cellular alterations

Diabetic hyperglycemia can lead to stress-related cellular apoptosis of cardiac tissue. However, the mechanism by which hyperglycemia inflicts this damage on the structure and function of the heart is unclear. In this study, we examined the relationship between proteome alterations, mitochondrial function, and major biochemical and electrophysiological changes affecting cardiac performance during simulated short-term hyperglycemia. Two-dimensional comparative proteomics analysis of rat hearts perfused with glucose at high (30 mM) or control (5.5 mM) levels revealed that glucose loading alters cardiomyocyte proteomes. It increased expression levels of initial enzymes of the tricarboxylic acid cycle, and of enzymes of fatty acid beta-oxidation, with consequent up-regulation of enzymes of mitochondrial electron transport. It also markedly decreased expression of enzymes of glycolysis and the final steps of the tricarboxylic acid cycle. Glucose loading increased the rate of Bax-independent apoptosis. High glucose increased the duration of the action potential and elevated level of intracellular cytoplasmic calcium. Surprisingly, glucose loading did not influence levels of nitric oxide or mitochondrial superoxide in isolated cardiomyocytes. In summary, short-term simulated hyperglycemia attenuated expression of many anti-apoptotic proteins. This effect was apparently mediated via alterations in multiple biochemical pathways that collectively increased apoptotic susceptibility.     

Structure-function analysis of the C-terminal domain of LcrV from Yersinia pestis

LcrV, a multifunctional protein, acts as a positive regulator of effector protein secretion for the type III secretion system (T3SS) in Yersinia pestis by interaction with the negative regulator LcrG. In this study, LcrV was analyzed to identify regions required for LcrG interaction. Random-linker insertion mutagenesis, deletion analysis, and site-directed mutagenesis of hydrophobic amino acids between residues 290 and 311 allowed the isolation of an LcrV mutant (LcrV L291R F308R) defective for LcrG interaction. The new residues identified in LcrG interaction lie in helix 12 of LcrV; residues in helix 7 of LcrV are known to be involved in LcrG interaction. Helix 7 and helix 12 of LcrV interact to form an intramolecular coiled coil; these new results suggest that the intramolecular coiled coil in LcrV is required for LcrG interaction and activation of the T3SS.     

Stabilization of hyperdynamic microtubules is neuroprotective in amyotrophic lateral sclerosis

Mutations in copper/zinc superoxide dismutase 1 (SOD1), a genetic cause of human amyotrophic lateral sclerosis, trigger motoneuron death through unknown toxic mechanisms. We report that transgenic SOD1G93A mice exhibit striking and progressive changes in neuronal microtubule dynamics from an early age, associated with impaired axonal transport. Pharmacologic administration of a microtubule-modulating agent alone or in combination with a neuroprotective drug to symptomatic SOD1G93A mice reduced microtubule turnover, preserved spinal cord neurons, normalized axonal transport kinetics, and delayed the onset of symptoms, while prolonging life by up to 26%. The degree of reduction of microtubule turnover was highly predictive of clinical responses to different treatments. These data are consistent with the hypothesis that hyperdynamic microtubules impair axonal transport and accelerate motor neuron degeneration in amyotrophic lateral sclerosis. Measurement of microtubule dynamics in vivo provides a sensitive biomarker of disease activity and therapeutic response and represents a new pharmacologic target in neurodegenerative disorders.     

Differential association of hemoglobin with proinflammatory high density lipoproteins in atherogenic/hyperlipidemic mice. A novel biomarker of atherosclerosis

Studies in both mice and humans suggest that the anti- or proinflammatory nature of high density lipoprotein (HDL) may be a more sensitive predictor of risk for coronary heart disease events. In this study, we report the identification and characterization of two proteins (m/z 14,900 and 15,600) that are most dramatically associated with HDL in mouse models of atherosclerosis. Mass spectral analyses of proinflammatory HDL identified the two peaks to be hemoglobin (Hb) alpha and beta chains, respectively, with no apparent post-translational modification. Biochemical analysis confirmed the differential association of Hb with HDL from hyperlipidemic mice. We further show that HDL-associated Hb is predominantly in the oxyHb form with distinct physical and chemical properties. Furthermore oxyHb-containing proinflammatory HDL potently consumed nitric oxide and contracted arterial vessels ex vivo. Moreover Hb also was found differentially associated with HDL from coronary heart disease patients compared with healthy controls. Our data suggest that Hb contributes to the proinflammatory nature of HDL in mouse and human models of atherosclerosis and may serve as a novel biomarker for atherosclerosis.     

Measurement of very low rates of cell proliferation by heavy water labeling of DNA and gas chromatography/pyrolysis/isotope ratio-mass spectrometric analysis

DNA replication during S-phase represents a biochemical metric of cell division. We present here a protocol for measuring very low rates of cell proliferation, on the basis of the incorporation of deuterium ((2)H) from heavy water ((2)H(2)O) into the deoxyribose moiety of purine deoxyribonucleotides in DNA of dividing cells, by use of gas chromatography/pyrolysis/isotope ratio-mass spectrometry (GC/P/IRMS). Very low levels of label incorporation (>or=0.002% atom percent excess (2)H) can be quantified by GC/P/IRMS. This protocol thereby permits shorter periods or lower amounts of (2)H(2)O administration than would be required using standard GC/MS techniques for measuring cell proliferation kinetics (see accompanying protocol in this issue). A disadvantage of this approach compared to GC/MS is the requirement for larger numbers of cells (> approximately 10(7)). This protocol enables definitive in vivo studies of the fraction or absolute number of newly divided cells and their subsequent survival kinetics in animals and humans, even when turnover rates are very low. Indolent hematologic malignancies, such as chronic lymphocytic leukemia, and other relatively quiescent cells represent promising areas of application.