Isolation of a novel chromium(III) binding protein from bovine liver tissue after chromium(VI) exposure

In the ongoing investigation into the biological importance and toxicity issues surrounding the bioinorganic chemistry of chromium, the accepted literature procedure for the isolation of the biological form of chromium, low molecular weight chromium binding protein (LMWCr) or chromodulin, was investigated for its specificity. When chromium(VI) is added to bovine liver homogenate, results presented here indicate at least four chromium(III) binding peptides and proteins are produced and that the process is non-specific for the isolation of LMWCr. A novel trivalent chromium containing protein (1) has been isolated to purity and initial characterization is reported here. Chromium(III) identification was determined by optical spectroscopy and diphenylcarbazide testing. This chromium binding protein has a molecular weight of 15.6kDa, which was determined from both gel-electrophoresis and mass spectrometry. The protein is comprised primarily of Asx, Glx, His, Gly/Thr, Ala, and Lys in a 1.00:2.51:0.37:2.09:0.39:1.17 ratio and is anionic at pH 7.4. In addition, the protein binds approximately 2.5 chromium(III) ions per molecule.     

Fat Storage-inducing Transmembrane Protein 2 Is Required for Normal Fat Storage in Adipose Tissue

Triglycerides within the cytosol of cells are stored in a phylogenetically conserved organelle called the lipid droplet (LD). LDs can be formed at the endoplasmic reticulum, but mechanisms that regulate the formation of LDs are incompletely understood. Adipose tissue has a high capacity to form lipid droplets and store triglycerides. Fat storage-inducing transmembrane protein 2 (FITM2/FIT2) is highly expressed in adipocytes, and data indicate that FIT2 has an important role in the formation of LDs in cells, but whether FIT2 has a physiological role in triglyceride storage in adipose tissue remains unproven. Here we show that adipose-specific deficiency of FIT2 (AF2KO) in mice results in progressive lipodystrophy of white adipose depots and metabolic dysfunction. In contrast, interscapular brown adipose tissue of AF2KO mice accumulated few but large LDs without changes in cellular triglyceride levels. High fat feeding of AF2KO mice or AF2KO mice on the genetically obese ob/ob background accelerated the onset of lipodystrophy. At the cellular level, primary adipocyte precursors of white and brown adipose tissue differentiated in vitro produced fewer but larger LDs without changes in total cellular triglyceride or triglyceride biosynthesis. These data support the conclusion that FIT2 plays an essential, physiological role in fat storage in vivo.     

Toxoplasma gondii Is Dependent on Glutamine and Alters Migratory Profile of Infected Host Bone Marrow Derived Immune Cells through SNAT2 and CXCR4 Pathways

The obligate intracellular parasite, Toxoplasma gondii, disseminates through its host inside infected immune cells. We hypothesize that parasite nutrient requirements lead to manipulation of migratory properties of the immune cell. We demonstrate that 1) T. gondii relies on glutamine for optimal infection, replication and viability, and 2) T. gondii-infected bone marrow-derived dendritic cells (DCs) display both “hypermotility” and “enhanced migration” to an elevated glutamine gradient in vitro. We show that glutamine uptake by the sodium-dependent neutral amino acid transporter 2 (SNAT2) is required for this enhanced migration. SNAT2 transport of glutamine is also a significant factor in the induction of migration by the small cytokine stromal cell-derived factor-1 (SDF-1) in uninfected DCs. Blocking both SNAT2 and C-X-C chemokine receptor 4 (CXCR4; the unique receptor for SDF-1) blocks hypermotility and the enhanced migration in T. gondii-infected DCs. Changes in host cell protein expression following T. gondii infection may explain the altered migratory phenotype; we observed an increase of CD80 and unchanged protein level of CXCR4 in both T. gondii-infected and lipopolysaccharide (LPS)-stimulated DCs. However, unlike activated DCs, SNAT2 expression in the cytosol of infected cells was also unchanged. Thus, our results suggest an important role of glutamine transport via SNAT2 in immune cell migration and a possible interaction between SNAT2 and CXCR4, by which T. gondii manipulates host cell motility.     

Proliferating cell nuclear antigen (PCNA) as a proliferative marker during embryonic and adult zebrafish hematopoiesis

We investigated the expression of proliferative cell nuclear antigen (PCNA) in zebrafish to delineate the proliferative hematopoietic component during adult and embryonic hematopoiesis. Immunostaining for PCNA and enhanced green fluorescence protein (eGFP) was performed in wild-type and fli1-eGFP (endothelial marker) and gata1-eGFP (erythroid cell marker) transgenic fish. Expression of PCNA mRNA was examined in wild-type and chordin morphant embryos. In adult zebrafish kidney, the renal tubules are surrounded by endothelial cells and it is separated into hematopoietic and excretory compartments. PCNA was expressed in hematopoietic progenitor cells but not in mature neutrophils, eosinophils or erythroid cells. Some PCNA+ cells are scattered in the hematopoietic compartment of the kidney while others are closely associated with renal tubular cells. PCNA was also expressed in spermatogonial stem cells and intestine crypts, consistent with its role in cell proliferation and DNA synthesis. In embryos, PCNA is expressed in the brain, spinal cord and intermediate cell mass (ICM) at 24 h-post fertilization. In chordin morphants, PCNA is significantly upregulated in the expanded ICM. Therefore, PCNA can be used to mark cell proliferation in zebrafish hematopoietic tissues and to identify a population of progenitor cells whose significance would have to be further investigated.     

Impact of temperature on Na2Sr(PO4)F:Eu3+ phosphor

In this article, we report the synthesis of Na₂Sr_1‐x(PO₄)F:Eu_x phosphor via a combustion method. The influence of different annealing temperatures on the photoluminescence properties was investigated. The phosphor was excited at both 254 and 393 nm. Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphors emit strong orange and red color at 593 and 612 nm, respectively, under both excitation wavelengths. Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphors annealed at 1050°C showed stronger emission intensity compared with 600, 900 and 1200°C. Moreover, Na₂Sr_1‐x(PO₄)F:Eu_x³⁺ phosphor was found to be more intense when compared with commercial Y₂O₃:Eu³⁺ phosphor. Copyright © 2013 John Wiley & Sons, Ltd.     

Selection of suitable housekeeping genes for expression analysis in glioblastoma using quantitative RT-PCR

Background  

Considering the broad variation in the expression of housekeeping genes among tissues and experimental situations, studies using quantitative RT-PCR require strict definition of adequate endogenous controls. For glioblastoma, the most common type of tumor in the central nervous system, there was no previous report regarding this issue.     

New Candidate Biomarkers in the Female Genital Tract to Evaluate Microbicide Toxicity

Vaginal microbicides hold great promise for the prevention of viral diseases like HIV, but the failure of several microbicide candidates in clinical trials has raised important questions regarding the parameters to be evaluated to determine in vivo efficacy in humans. Clinical trials of the candidate microbicides nonoxynol-9 (N9) and cellulose sulfate revealed an increase in HIV infection, vaginal inflammation, and recruitment of HIV susceptible lymphocytes, highlighting the need to identify biomarkers that can accurately predict microbicide toxicity early in preclinical development and in human trials. We used quantitative proteomics and RT-PCR approaches in mice and rabbits to identify protein changes in vaginal fluid and tissue in response to treatment with N9 or benzalkonium chloride (BZK). We compared changes generated with N9 and BZK treatment to the changes generated in response to tenofovir gel, a candidate microbicide that holds promise as a safe and effective microbicide. Both compounds down regulated mucin 5 subtype B, and peptidoglycan recognition protein 1 in vaginal tissue; however, mucosal brush samples also showed upregulation of plasma proteins fibrinogen, plasminogen, apolipoprotein A-1, and apolipoprotein C-1, which may be a response to the erosive nature of N9 and BZK. Additional proteins down-regulated in vaginal tissue by N9 or BZK treatment include CD166 antigen, olfactomedin-4, and anterior gradient protein 2 homolog. We also observed increases in the expression of C-C chemokines CCL3, CCL5, and CCL7 in response to treatment. There was concordance in expression level changes for several of these proteins using both the mouse and rabbit models. Using a human vaginal epithelial cell line, the expression of mucin 5 subtype B and olfactomedin-4 were down-regulated in response to N9, suggesting these markers could apply to humans. These data identifies new proteins that after further validation could become part of a panel of biomarkers to effectively evaluate microbicide toxicity.     

Genomic analysis of the relationship between gene expression variation and DNA polymorphism in Drosophila simulans

Background

Understanding how DNA sequence polymorphism relates to variation in gene expression is essential to connecting genotypic differences with phenotypic differences among individuals. Addressing this question requires linking population genomic data with gene expression variation.

Results

Using whole genome expression data and recent light shotgun genome sequencing of six Drosophila simulans genotypes, we assessed the relationship between expression variation in males and females and nucleotide polymorphism across thousands of loci. By examining sequence polymorphism in gene features, such as untranslated regions and introns, we find that genes showing greater variation in gene expression between genotypes also have higher levels of sequence polymorphism in many gene features. Accordingly, X-linked genes, which have lower sequence polymorphism levels than autosomal genes, also show less expression variation than autosomal genes. We also find that sex-specifically expressed genes show higher local levels of polymorphism and divergence than both sex-biased and unbiased genes, and that they appear to have simpler regulatory regions.

Conclusion

The gene-feature-based analyses and the X-to-autosome comparisons suggest that sequence polymorphism in cis-acting elements is an important determinant of expression variation. However, this relationship varies among the different categories of sex-biased expression, and trans factors might contribute more to male-specific gene expression than cis effects. Our analysis of sex-specific gene expression also shows that female-specific genes have been overlooked in analyses that only point to male-biased genes as having unusual patterns of evolution and that studies of sexually dimorphic traits need to recognize that the relationship between genetic and expression variation at these traits is different from the genome as a whole.