MicroRNA profiling using µParaflo microfluidic array technology.

The diverse functions of microRNA (miRNA) molecules have drawn broad and intensive interest in various biological fields, biomedical applications, and technology development. Which are endogeneous cellular short RNA molecules found in the cytoplasm as well as in various serum fluids. miRNAs are transcriptional and translational regulatory molecules active in cell division, growth, and apoptosis (1). Dysregulated expression of miRNAs has been implicated in various disease states and has been tested as biomarker candidates (2-4). miRNAs are endogeneous cellular short RNA molecules found in the cytoplasm as well as in various serum fluids. miRNAs are transcriptional and translational regulatory molecules active in cell division, growth, and apoptosis (Bartel, Cell 116:281-97, 2004). Dysregulated expression of miRNAs has been implicated in various disease states and has been tested as biomarker candidates (He et al., Nature 435:828-833, 2005; Lu et al., Nature 435:834-838, 2005; O'Donnell, et al., Nature 435:839-843, 2005). In this chapter, we describe the methods using µParaflo(®) microfluidic oligonucleotide microarray technology for applications in miRNA profiling. One unique feature of this technology is the flexibility that provides users with the freedom to select sequence content either for focused studies wherein only the most relevant sequences are included or for discovery studies wherein the most updated sequence content such as those newly derived from deep sequencing. This chapter provides detailed information from experimental design to sample preparation, as well as data analysis for a miRNA array experiment.

Nonalcoholic steatohepatitis is associated with altered hepatic MicroRNA expression.

UNLABELLED: The expression of microRNA in nonalcoholic steatohepatitis (NASH) and their role in the genesis of NASH are not known. The aims of this study were to: (1) identify differentially expressed microRNAs in human NASH, (2) tabulate their potential targets, and (3) define the effect of a specific differentially expressed microRNA, miR-122, on its targets and compare these effects with the pattern of expression of these targets in human NASH. The expression of 474 human microRNAs was compared in subjects with the metabolic syndrome and NASH versus controls with normal liver histology. Differentially expressed microRNAs were identified by the muParaflo microRNA microarray assay and validated using quantitative real-time polymerase chain reaction (PCR). The effects of a specific differentially expressed miRNA (miR-122) on its predicted targets were assessed by silencing and overexpressing miR-122 in vitro. A total of 23 microRNAs were underexpressed or overexpressed. The predicted targets of these microRNAs are known to affect cell proliferation, protein translation, apoptosis, inflammation, oxidative stress, and metabolism. The miR-122 level was significantly decreased in subjects with NASH (63% by real-time PCR, P < 0.00001). Silencing miR-122 led to an initial increase in mRNA levels of these targets (P < 0.05 for all) followed by a decrease by 48 hours. This was accompanied by an increase in protein levels of these targets (P < 0.05 for all). Overexpression of miR-122 led to a significant decrease in protein levels of these targets. CONCLUSIONS: NASH is associated with altered hepatic microRNA expression. Underexpression of miR-122 potentially contributes to altered lipid metabolism implicated in the pathogenesis of NASH.

A metal-ligand-mediated intersubunit allosteric switch in related SmtB/ArsR zinc sensor proteins.

The origin of metal ion selectivity by members of the SmtB/ArsR family of bacterial metal-sensing transcriptional repressors and the mechanism of negative allosteric regulation of DNA binding is poorly understood. Here, we report that two homologous zinc sensors, Staphylococcus aureus CzrA and cyanobacterial SmtB, are "winged" helix homodimeric DNA-binding proteins that bind Zn(II) to a pair of tetrahedral, interhelical binding sites, with two ligands derived from the alpha5 helix of one subunit, Asp84 O(delta1) (Asp104 in SmtB), His86 N(delta1) (His106), and two derived from the alpha5 helix of the other, His97' N(delta1) (His117') and His100' N(epsilon2) (Glu120'). Formation of the metal chelate drives a quaternary structural switch mediated by an intersubunit hydrogen-binding network that originates with the non-liganding N(epsilon2) face of His97 in CzrA (His117 in SmtB) that stabilizes a low-affinity, DNA-binding conformation. The structure of the Zn(1) SmtB homodimer shows that both metal-binding sites of the dimer must be occupied for the quaternary structural switch to occur. Thus, a critical zinc-ligating histidine residue obligatorily couples formation of the metal-sensing coordination chelate to changes in the conformation and dynamics of the putative DNA-binding helices.

Comparative modeling of the latent form of a plant catechol oxidase using a molluskan hemocyanin structure.

The structure of the precursor form of catechol oxidase from sweet potatoes (Ipomoea batatas) has been modeled on the basis of the 3D structural data of mature catechol oxidase [Nat. Struct. Biol. 5 (1998) 1084] and of hemocyanin from giant octopus (Octopus dofleini) [J. Mol. Biol. 278 (1998) 855]. A C-terminal extension peptide is found in the cDNA sequence but not in the purified, mature form of catechol oxidase. Superimposition of the 3D structures of the native hemocyanin and catechol oxidase reveals a close relationship except for an additional C-terminal domain only found in the hemocyanin structure. As sequence alignment shows good homology this domain of the hemocyanin structure was used as a template to model the 3D structure of the C-terminal extension peptide of catechol oxidase. As hemocyanins show no or only weak catecholase activity due to this domain this indicates an inhibitory function of this extension peptide. Beside this possible shielding function for the precursor form, evidence for a function in copper-uptake also increases due to the location of three histidine residues in the model.

The crystal structure of catechol oxidase: new insight into the function of type-3 copper proteins.

The crystal structure of catechol oxidase reveals new insight into the functional properties of the type-3 copper proteins. This class of proteins includes the closely related and better-known tyrosinase as well as hemocyanin, an oxygen transport protein. All these proteins have a dinuclear copper center, have similar spectroscopic behaviors, and show close evolutionary and functional relationships. Comparison between the 3D structures of catechol oxidase and hemocyanins reveals the structural reasons for the divergence in function.

Crystal structure of Lyme disease variable surface antigen VlsE of Borrelia burgdorferi.

VlsE is an outer surface lipoprotein of Borrelia burgdorferi that undergoes antigenic variation through an elaborate gene conversion mechanism and is thought to play a major role in the immune response to the Lyme disease borellia. The crystal structure of recombinant variant protein VlsE1 at 2.3-A resolution reveals that the six variable regions form loop structures that constitute most of the membrane distal surface of VlsE, covering the predominantly alpha-helical, invariant regions of the protein. The surface localization of the variable amino acid segments appears to protect the conserved regions from interaction with antibodies and hence may contribute to immune evasion.