Accumulation of heme oxygenase-1 (HSP32) in Xenopus laevis A6 kidney epithelial cells treated with sodium arsenite, cadmium chloride or proteasomal inhibitors

The present study examined the effect of sodium arsenite, cadmium chloride, heat shock and the proteasomal inhibitors MG132, withaferin A and celastrol on heme oxygenase-1 (HO-1; also known as HSP32) accumulation in Xenopus laevis A6 kidney epithelial cells. Immunoblot analysis revealed that HO-1 accumulation was not induced by heat shock but was enhanced by sodium arsenite and cadmium chloride in a dose- and time-dependent fashion. Immunocytochemistry revealed that these metals induced HO-1 accumulation in a granular pattern primarily in the cytoplasm. Additionally, in 20% of the cells arsenite induced the formation of large HO-1-containing perinuclear structures. In cells recovering from sodium arsenite or cadmium chloride treatment, HO-1 accumulation initially increased to a maximum at 12h followed by a 50% reduction at 48 h. This initial increase in HO-1 levels was likely the result of new synthesis as it was inhibited by cycloheximide. Interestingly, treatment of cells with a mild heat shock enhanced HO-1 accumulation induced by low concentrations of sodium arsenite and cadmium chloride. Finally, we determined that HO-1 accumulation was induced in A6 cells by the proteasomal inhibitors, MG132, withaferin A and celastrol. An examination of heavy metal and proteasomal inhibitor-induced HO-1 accumulation in amphibians is of importance given the presence of toxic heavy metals in aquatic habitats.

Regenerative silver nanoparticles for SERRS investigation of metmyoglobin with conserved heme pocket

Shell isolated silver nanoparticles with an ultrathin silica layer (Ag@SiO2NPs) are used as a surface-enhanced resonance Raman scattering (SERRS) substrate for probing metmyoglobin (metMb) in aqueous solution. The ultrathin silica layer protects metMb from reaching the bare silver surface and conserves the heme pocket during SERRS analysis with a Raman enhancement factor (EFSERS) of 4.78 × 104. In spite of the good SERRS enhancement, the interaction between the protein and Ag@SiO2NPs is weak enough to separate them by centrifugation in such a way that both are regenerated in their original form and can be reused. Using Ag@SiO2NPs as the SERRS substrate, the lowest detection limit of 2 nM was achieved for metMb whilst conserving the native structure of the heme centre.

Neutrophil activation, antioxidant supplements and exercise-induced oxidative stress

Neutrophils produce free radicals known as reactive oxygen species (ROS), which assist in the clearance of damaged host tissue. Tissue damage may occur during exercise due to muscle damage, thermal stress and ischaemia/reperfusion. When produced in excess, neutrophil-derived ROS may overwhelm the body's endogenous antioxidant defence mechanisms, and this can lead to oxidative stress. There is increasing evidence for links between oxidative stress and a variety of pathological disorders such as cardiovascular diseases, cancer, chronic inflammatory diseases and post-ischaemic organ injury. A small number of studies have investigated whether there is a link between neutrophil activation and oxidative stress during exercise. In this review, we have summarised the findings of these studies. Exercise promotes the release of neutrophils into the circulation, and some evidence suggests that neutrophils mobilised after exercise have an enhanced capacity to generate some forms of ROS when stimulated in vitro. Neutrophil activation during exercise may challenge endogenous antioxidant defence mechanisms, but does not appear to increase lipid markers of oxidative stress to any significant degree, at least in the circulation. Antioxidant supplements such as N-acetylcysteine are effective at attenuating increases in the capacity of neutrophils to generate ROS when stimulated in vitro, whereas vitamin E reduces tissue infiltration of neutrophils during exercise. Free radicals generated during intense exercise may lead to DNA damage in leukocytes, but it is unknown if this damage is the result of neutrophil activation. Exercise enhances the expression of inducible haem (heme)-oxygenase (HO-1) in neutrophils after exercise, however, it is uncertain whether oxidative stress is the stimulus for this response.

The highly abundant urinary metabolite urobilin interferes with the bicinchoninic acid assay

Estimation of total protein concentration is an essential step in any protein- or peptide-centric analysis pipeline. This study demonstrates that urobilin, a breakdown product of heme and a major constituent of urine, interferes considerably with the bicinchoninic acid (BCA) assay. This interference is probably due to the propensity of urobilin to reduce cupric ions (Cu2+) to cuprous ions (Cu1+), thus mimicking the reduction of copper by proteins, which the assay was designed to do. In addition, it is demonstrated that the Bradford assay is more resistant to the influence of urobilin and other small molecules. As such, urobilin has a strong confounding effect on the estimate of total protein concentrations obtained by BCA assay and thus this assay should not be used for urinary protein quantification. It is recommended that the Bradford assay be used instead.

Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants

Background We describe novel plasmid vectors for transient gene expression using Agrobacterium, infiltrated into Nicotiana benthamiana leaves. We have generated a series of pGreenII cloning vectors that are ideally suited to transient gene expression, by removing elements of conventional binary vectors necessary for stable transformation such as transformation selection genes. Results We give an example of expression of heme-thiolate P450 to demonstrate effectiveness of this system. We have also designed vectors that take advantage of a dual luciferase assay system to analyse promoter sequences or post-transcriptional regulation of gene expression. We have demonstrated their utility by co-expression of putative transcription factors and the promoter sequence of potential target genes and show how orthologous promoter sequences respond to these genes. Finally, we have constructed a vector that has allowed us to investigate design features of hairpin constructs related to their ability to initiate RNA silencing, and have used these tools to study cis-regulatory effect of intron-containing gene constructs. Conclusion In developing a series of vectors ideally suited to transient expression analysis we have provided a resource that further advances the application of this technology. These minimal vectors are ideally suited to conventional cloning methods and we have used them to demonstrate their flexibility to investigate enzyme activity, transcription regulation and post-transcriptional regulatory processes in transient assays.

Cytochrome p450 1b1, a new keystone in gene-environment interactions related to human head and neck cancer?

Alcohol consumption and tobacco smoking are major causes of head and neck cancers, and regional differences point to the importance of research into gene-environment interactions. Much interest has been focused on polymorphisms of CYP1A1 and of GSTM1 and GSTT1, but a number of studies have not demonstrated significant effects. This has mostly been ascribed to small sample sizes. In general, the impact of polymorphisms of metabolic enzymes appears inconsistent, with some reports of weak-to-moderate associations, and with others of no elevation of risks. The classical cytochrome P450 isoenzyme considered for metabolic activation of polycyclic aromatic hydrocarbons (PAH) is CYP1A1. A new member of the CYP1 family, CYP1B1, was cloned in 1994, currently representing the only member of the CYP1B subfamily. A number of single nucleotide polymorphisms of the CYP1B1 gene have been reported. The amino acid substitutions Val432Leu (CYP1B1*3) and Asn453Ser (CYP1B1*4), located in the heme binding domain of CYP1B1, appear as likely candidates to be linked with biological effects. CYP1B1 activates a wide range of PAH, aromatic and heterocyclic amines. Very recently, the CYP1B1 codon 432 polymorphism (CYP1B1*3) has been identified as a susceptibility factor in smoking-related head-and-neck squamous cell cancer. The impact of this polymorphic variant of CYP1B1 on cancer risk was also reflected by an association with the frequency of somatic mutations of the p53 gene. Combined genotype analysis of CYP1B1 and the glutathione transferases GSTM1 or GSTT1 has pointed to interactive effects. This provides new molecular evidence that tobacco smoke-specific compounds relevant to head and neck carcinogenesis are metabolically activated through CYP1B1 and is consistent with a major pathogenetic relevance of PAH as ingredients of tobacco smoke.

Haemoglobin expression in human endometrium

BACKGROUND: The general concept that haemoglobin is only a carrier protein for oxygen and carbon dioxide is challenged since recent studies have shown haemoglobin expression in non-erythroid cells and the protection of haemoglobin against oxidative and nitrosative stress. Using microarrays, we previously showed expression of haemoglobins alpha, beta, delta and gamma and the haeme metabolizing enzyme, haeme oxygenase (HO)-1 in human endometrium. METHODS: Using real-time quantitative PCR, haemoglobin alpha, beta, delta and gamma, and HO-1 mRNA levels were assessed throughout the menstrual cycle (n = 30 women). Haemoglobin and HO-1 protein levels in the human endometrium were assessed with immunohistochemistry. For steroid responsiveness, menstrual and late proliferative-phase endometrial explants were cultured for 24 h in the presence of vehicle (0.1% ethanol), estradiol (17 beta-E-2, 1 nM), progestin (Org 2058, 1 nM) or 17 beta-E-2+Org 2058 (1 nM each). RESULTS: All haemoglobins and the HO-1 were expressed in normal human endometrium. Haemoglobin mRNA and protein expression did not vary significantly during the menstrual cycle. Explant culture with Org 2058 or 17 beta-E-2+Org 2058 increased haemoglobin gamma mRNA expression (P < 0.05). HO-1 mRNA levels, and not protein levels, were significantly higher during the menstrual (M)-phase of the cycle (P < 0.05), and were down-regulated by Org 2058 in M-phase explants and by 17 beta-E-2+Org 2058 in LP-phase explants, versus control (P < 0.05). CONCLUSIONS: The haemoglobin-HO-1 system may be required to ensure adequate regulation of the bioavailability of haeme, iron and oxygen in human endometrium.

Plasmon-tuned silver colloids for SERRS analysis of methemoglobin with preserved nativity

Optically tuned silver nanoparticles (AgNP's) functionalized with ω-mercaptoalkanoic acids are synthesized and used as a signal amplifier for the surface-enhanced resonance Raman scattering (SERRS) study of heme cofactor in methemoglobin (metHb). Even though both mercaptopropionic acid (MPA)- and mercaptononanoic acid (MNA)-functionalized AgNP's exemplify vastly enhanced SERRS signal of metHb, MNA-AgNP's amplify the SERRS signal amid preservation of the nativity of the heme pocket, unlike MPA-AgNP's. The electrostatic interaction between MNA-AgNP's and metHb leads to instant signal enhancement with a Raman enhancement factor (EF(SERS)) of 4.2 × 10(3). Additionally, a Langmuir adsorption isotherm has been employed for the adsorption of metHb on the MNA-AgNP surface, which provides the real surface coverage and equilibrium constant (K) of metHb as 139 nM and 3.6 × 10(8) M(-1), respectively. The lowest detection limit of 10 nM for metHb has been demonstrated using MNA-AgNP's besides retaining the nativity of the heme pocket.

Complementary surface-enhanced resonance raman spectroscopic biodetection of mixed protein solutions by chitosan- and silica-coated plasmon-tuned silver nanoparticles

Silver nanoparticles with identical plasmonic properties but different surface functionalities are synthesized and tested as chemically selective surface-enhanced resonance Raman (SERR) amplifiers in a two-component protein solution. The surface plasmon resonances of the particles are tuned to 413 nm to match the molecular resonance of protein heme cofactors. Biocompatible functionalization of the nanoparticles with a thin film of chitosan yields selective SERR enhancement of the anionic protein cytochrome b5, whereas functionalization with SiO2 amplifies only the spectra of the cationic protein cytochrome c. As a result, subsequent addition of the two differently functionalized particles yields complementary information on the same mixed protein sample solution. Finally, the applicability of chitosan-coated Ag nanoparticles for protein separation was tested by in situ resonance Raman spectroscopy.

Functionalized Ag nanoparticles with tunable optical properties for selective protein analysis

We present a preparation procedure for small sized biocompatibly coated Ag nanoparticles with tunable surface plasmon resonances. The conditions were optimised with respect to the resonance Raman signal enhancement of heme proteins and to the preservation of the native protein structure....

Whole-Body Imaging with Single-Cell Resolution by Tissue Decolorization

The development of whole-body imaging at single-cell resolution enables system-level approaches to studying cellular circuits in organisms. Previous clearing methods focused on homogenizing mismatched refractive indices of individual tissues, enabling reductions in opacity but falling short of achieving transparency. Here, we show that an aminoalcohol decolorizes blood by efficiently eluting the heme chromophore from hemoglobin. Direct transcardial perfusion of an aminoalcohol-containing cocktail that we previously termed CUBIC coupled with a 10 day to 2 week clearing protocol decolorized and rendered nearly transparent almost all organs of adult mice as well as the entire body of infant and adult mice. This CUBIC-perfusion protocol enables rapid whole-body and whole-organ imaging at single-cell resolution by using light-sheet fluorescent microscopy. The CUBIC protocol is also applicable to 3D pathology, anatomy, and immunohistochemistry of various organs. These results suggest that whole-body imaging of colorless tissues at high resolution will contribute to organism-level systems biology.

Biliverdin modulates the expression of C5aR in response to endotoxin in part via mTOR signaling

Macrophages play a crucial role in the maintenance and resolution of inflammation and express a number of pro- and anti-inflammatory molecules in response to stressors. Among them, the complement receptor 5a (C5aR) plays an integral role in the development of inflammatory disorders. Biliverdin and bilirubin, products of heme catabolism, exert anti-inflammatory effects and inhibit complement activation. Here, we define the effects of biliverdin on C5aR expression in macrophages and the roles of Akt and mammalian target of rapamycin (mTOR) in these responses. Biliverdin administration inhibited lipopolysaccharide (LPS)-induced C5aR expression (without altering basal expression), an effect partially blocked by rapamycin, an inhibitor of mTOR signaling. Biliverdin also reduced LPS-dependent expression of the pro-inflammatory cytokines TNF-alpha and IL-6. Collectively, these data indicate that biliverdin regulates LPS-mediated expression of C5aR via the mTOR pathway, revealing an additional mechanism underlying biliverdin's anti-inflammatory effects.