Asymmetric distribution of cooperativity in the binding cascade of normal human hemoglobin. 1. Cooperative and noncooperative oxygen binding in Zn-substituted hemoglobin

The complete binding cascade of human hemoglobin consists of eight partially ligated intermediates and 16 binding constants. Each intermediate binding constant can be evaluated via dimer-tetramer assembly when ligand configurations within the tetramer are fixed through the use of hemesite analogs. The Zn/Fe analog, in which the nonbinding Zn2+ heme substitutes for deoxy Fe2+ heme, also permits direct measurement of O2 binding to the remaining Fe2+ hemesites within the symmetrically ligated Hb tetramers. Measurement of O2 binding over a range of Zn/Fe Hb concentrations to both alpha-subunits (species 23) or to both beta-subunits (species 24) shows noncooperative binding and incomplete saturation of the available Fe2+ hemesites. In contrast, the asymmetrically ligated Zn/FeO2 species 21, in which both oxygens are bound to one of the dimers within the tetramer, exhibits positive cooperativity and >90% ligation under atmospheric conditions. These properties are confirmed in the present study by measurement of the rate constant for tetramer dissociation to free dimer. The binding constants thus derived for these partially ligated intermediates are consistent with the stoichiometric constants measured for native hemoglobin by standard O2 binding techniques, providing additional evidence that Zn2+-heme substitution provides an excellent deoxy hemoglobin analog. There is no evidence that Zn-substitution stabilizes a low-affinity form of the tetramer, as previously suggested. These characterizations demonstrate distinct, nonadditive physical properties of the doubly ligated tetrameric species, yielding an asymmetric distribution of cooperativity within the cascade of O2 binding by human hemoglobin.     

"Plasmo2D": an ancillary proteomic tool to aid identification of proteins from Plasmodium falciparum

Bioinformatics tools to aid gene and protein sequence analysis have become an integral part of biology in the post-genomic era. Release of the Plasmodium falciparum genome sequence has allowed biologists to define the gene and the predicted protein content as well as their sequences in the parasite. Using pI and molecular weight as characteristics unique to each protein, we have developed a bioinformatics tool to aid identification of proteins from Plasmodium falciparum. The tool makes use of a Virtual 2-DE generated by plotting all of the proteins from the Plasmodium database on a pI versus molecular weight scale. Proteins are identified by comparing the position of migration of desired protein spots from an experimental 2-DE and that on a virtual 2-DE. The procedure has been automated in the form of user-friendly software called "Plasmo2D". The tool can be downloaded from http://144.16.89.25/Plasmo2D.zip.     

Analysis of variance components in gene expression data

MOTIVATION: A microarray experiment is a multi-step process, and each step is a potential source of variation. There are two major sources of variation: biological variation and technical variation. This study presents a variance-components approach to investigating animal-to-animal, between-array, within-array and day-to-day variations for two data sets. The first data set involved estimation of technical variances for pooled control and pooled treated RNA samples. The variance components included between-array, and two nested within-array variances: between-section (the upper- and lower-sections of the array are replicates) and within-section (two adjacent spots of the same gene are printed within each section). The second experiment was conducted on four different weeks. Each week there were reference and test samples with a dye-flip replicate in two hybridization days. The variance components included week-to-week, animal-to-animal and between-array and within-array variances. RESULTS: We applied the linear mixed-effects model to quantify different sources of variation. In the first data set, we found that the between-array variance is greater than the between-section variance, which, in turn, is greater than the within-section variance. In the second data set, for the reference samples, the week-to-week variance is larger than the between-array variance, which, in turn, is slightly larger than the within-array variance. For the test samples, the week-to-week variance has the largest variation. The animal-to-animal variance is slightly larger than the between-array and within-array variances. However, in a gene-by-gene analysis, the animal-to-animal variance is smaller than the between-array variance in four out of five housekeeping genes. In summary, the largest variation observed is the week-to-week effect. Another important source of variability is the animal-to-animal variation. Finally, we describe the use of variance-component estimates to determine optimal numbers of animals, arrays per animal and sections per array in planning microarray experiments.     

High-performance liquid chromatography method for the determination and quantitation of arabinosylguanine triphosphate and fludarabine triphosphate in human cells

A gradient anion-exchange high-performance liquid chromatographic assay was developed for the simultaneous determination and quantitation of the cytotoxic triphosphates of arabinosylguanine (ara-GTP) and fludarabine (F-ara-ATP). The method was validated with respect to selectivity, recovery, linearity, precision, and accuracy using authentic standards. To test this assay in a more complex biological matrix, perchloric acid extracts of circulating human leukemia cells spiked with known concentrations of ara-GTP and F-ara-ATP were examined. Finally, to assess the clinical utility of our method, perchloric acid extracts of circulating human leukemia cells isolated from patients treated with fludarabine and nelarabine were analyzed. The range of quantitation was 0.0125–10 nmol for the ara- and native NTPs in cellular extracts. This assay should be helpful in establishing the mechanistic rationales for drug scheduling and combinations of nelarabine and fludarabine, and for correlating the therapeutic efficacy and levels of the cytotoxic triphosphates in target cells.     

Life Cycle Analysis of Kidney Gene Expression in Male F344 Rats

Age is a predisposing condition for susceptibility to chronic kidney disease and progression as well as acute kidney injury that may arise due to the adverse effects of some drugs. Age-related differences in kidney biology, therefore, are a key concern in understanding drug safety and disease progression. We hypothesize that the underlying suite of genes expressed in the kidney at various life cycle stages will impact susceptibility to adverse drug reactions. Therefore, establishing changes in baseline expression data between these life stages is the first and necessary step in evaluating this hypothesis. Untreated male F344 rats were sacrificed at 2, 5, 6, 8, 15, 21, 78, and 104 weeks of age. Kidneys were collected for histology and gene expression analysis. Agilent whole-genome rat microarrays were used to query global expression profiles. An ANOVA (p<0.01) coupled with a fold-change>1.5 in relative mRNA expression, was used to identify 3,724 unique differentially expressed genes (DEGs). Principal component analyses of these DEGs revealed three major divisions in life-cycle renal gene expression. K-means cluster analysis identified several groups of genes that shared age-specific patterns of expression. Pathway analysis of these gene groups revealed age-specific gene networks and functions related to renal function and aging, including extracellular matrix turnover, immune cell response, and renal tubular injury. Large age-related changes in expression were also demonstrated for the genes that code for qualified renal injury biomarkers KIM-1, Clu, and Tff3. These results suggest specific groups of genes that may underlie age-specific susceptibilities to adverse drug reactions and disease. This analysis of the basal gene expression patterns of renal genes throughout the life cycle of the rat will improve the use of current and future renal biomarkers and inform our assessments of kidney injury and disease.     

Intermolecular interaction of voriconazole analogues with model membrane by DSC and NMR,and their antifungal activity using NMR based metabolic profiling

The development of novel antifungal agents with high susceptibility and increased potency can be achieved by increasing their overall lipophilicity. To enhance the lipophilicity of voriconazole, a second generation azole antifungal agent, we have synthesized its carboxylic acid ester analogues, namely p-methoxybenzoate (Vpmb), toluate (Vtol), benzoate (Vbz) and p-nitrobenzoate (Vpnb). The intermolecular interactions of these analogues with model membrane have been investigated using nuclear magnetic resonance (NMR) and differential scanning calorimetric (DSC) techniques. The results indicate varying degree of changes in the membrane bilayer’s structural architecture and physico-chemical characteristics which possibly can be correlated with the antifungal effects via fungal membrane. Rapid metabolite profiling of chemical entities using cell preparations is one of the most important steps in drug discovery. We have evaluated the effect of synthesized analogues on Candida albicans. The method involves real time ¹H NMR measurement of intact cells monitoring NMR signals from fungal metabolites which gives Metabolic End Point (MEP). This is then compared with Minimum Inhibitory Concentration (MIC) determined using conventional methods. Results indicate that one of the synthesized analogues, Vpmb shows reasonably good activity.     

30-Year Trends in Stroke Rates and Outcome in Auckland,New Zealand (1981-2012): A Multi-Ethnic Population-Based Series of Studies

Background

Insufficient data exist on population-based trends in morbidity and mortality to determine the success of prevention strategies and improvements in health care delivery in stroke. The aim of this study was to determine trends in incidence and outcome (1-year mortality, 28-day case-fatality) in relation to management and risk factors for stroke in the multi-ethnic population of Auckland, New Zealand (NZ) over 30-years.

Methods

Four stroke incidence population-based register studies were undertaken in adult residents (aged ≥15 years) of Auckland NZ in 1981–1982, 1991–1992, 2002–2003 and 2011–2012. All used standard World Health Organization (WHO) diagnostic criteria and multiple overlapping sources of case-ascertainment for hospitalised and non-hospitalised, fatal and non-fatal, new stroke events. Ethnicity was consistently self-identified into four major groups. Crude and age-adjusted (WHO world population standard) annual incidence and mortality with corresponding 95% confidence intervals (CI) were calculated per 100,000 people, assuming a Poisson distribution.

Results

5400 new stroke patients were registered in four 12 month recruitment phases over the 30-year study period; 79% were NZ/European, 6% Māori, 8% Pacific people, and 7% were of Asian or other origin. Overall stroke incidence and 1-year mortality decreased by 23% (95% CI 5%-31%) and 62% (95% CI 36%-86%), respectively, from 1981 to 2012. Whilst stroke incidence and mortality declined across all groups in NZ from 1991, Māori and Pacific groups had the slowest rate of decline and continue to experience stroke at a significantly younger age (mean ages 60 and 62 years, respectively) compared with NZ/Europeans (mean age 75 years). There was also a decline in 28-day stroke case fatality (overall by 14%, 95% CI 11%-17%) across all ethnic groups from 1981 to 2012. However, there were significant increases in the frequencies of pre-morbid hypertension, myocardial infarction, and diabetes mellitus, but a reduction in frequency of current smoking among stroke patients.

Conclusions

In this unique temporal series of studies spanning 30 years, stroke incidence, early case-fatality and 1-year mortality have declined, but ethnic disparities in risk and outcome for stroke persisted suggesting that primary stroke prevention remains crucial to reducing the burden of this disease.     

Different Munc13 Isoforms Function as Priming Factors in Lytic Granule Release from Murine Cytotoxic T Lymphocytes

In order to fuse lytic granules (LGs) with the plasma membrane at the immunological synapse, cytotoxic T lymphocytes (CTLs) have to render these LGs fusion‐competent through the priming process. In secretory tissues such as brain and neuroendocrine glands, this process is mediated by members of the Munc13 protein family. In human CTLs, mutations in the Munc13‐4 gene cause a severe loss in killing efficiency, resulting in familial hemophagocytic lymphohistiocytosis type 3, suggesting a similar role of other Munc13 isoforms in the immune system. Here, we investigate the contribution of different Munc13 isoforms to the priming process of murine CTLs at both the mRNA and protein level. We demonstrate that Munc13‐1 and Munc13‐4 are the only Munc13 isoforms present in mouse CTLs. Both isoforms rescue the drastical secretion defect of CTLs derived from Munc13‐4‐deficient Jinx mice. Mobility studies using total internal reflection fluorescence microscopy indicate that Munc13‐4 and Munc13‐1 are responsible for the priming process of LGs. Furthermore, the domains of the Munc13 protein, which is responsible for functional fusion, could be identified. We conclude from these data that both isoforms of the Munc13 family, Munc13‐1 and Munc13‐4, are functionally redundant in murine CTLs .