Kinetic isotope effects of nucleoside hydrolase from Escherichia coli

rihC is one of a group of three ribonucleoside hydrolases found in Escherichia coli (E. coli). The enzyme catalyzes the hydrolysis of selected nucleosides to ribose and the corresponding base. A family of Vmax/Km kinetic isotope effects using uridine labeled with stable isotopes, such as 2H, 13C, and 15N, were determined by liquid chromatography/mass spectrometry (LC/MS). The kinetic isotope effects were 1.012+/-0.006, 1.027+/-0.005, 1.134+/-0.007, 1.122+/-0.008, and 1.002+/-0.004 for [1'-13C], [1-15N], [1'-2H], [2'-2H], and [5'-2H2] uridine, respectively. A transition state based upon a bond-energy bond-order vibrational analysis (BEBOVIB) of the observed kinetic isotope effects is proposed. The main features of this transition state are activation of the heterocyclic base by protonation of/or hydrogen bonding to O2, an extensively broken C-N glycosidic bond, formation of an oxocarbenium ion in the ribose ring, C3'-exo ribose ring conformation, and almost no bond formation to the attacking nucleophile. The proposed transition state for the prokaryotic E. coli nucleoside hydrolase is compared to that of a similar enzyme isolated from Crithidia fasciculata (C. fasciculata).     

2'-fluoro-4'-thioarabino-modified oligonucleotides: conformational switches linked to siRNA activity

The synthesis of oligonucleotides containing 2′-deoxy-2′-fluoro-4′-thioarabinonucleotides is described. 2′-Deoxy-2′-fluoro-5-methyl-4′-thioarabinouridine (4′S-FMAU) was incorporated into 18-mer antisense oligonucleotides (AONs). 4′S-FMAU adopts a predominantly northern sugar conformation. Oligonucleotides containing 4′S-FMAU, unlike those containing FMAU, were unable to elicit E. coli or human RNase H activity, thus corroborating the hypothesis that RNase H prefers duplexes containing oligonucleotides that can adopt eastern conformations in the antisense strand. The duplex structure and stability of these oligonucleotides was also investigated via circular dichroism (CD)- and UV- binding studies. Replacement of the 4′-oxygen by a sulfur atom resulted in a marked decrease in melting temperature of AON:RNA as well as AON:DNA duplexes. 2′-Deoxy-2′-fluoro-4′-thioarabinouridine (4′S-FAU) was incorporated into 21-mer small interfering RNA (siRNA) and the resulting siRNA molecules were able to trigger RNA interference with good efficiency. Positional effects were explored, and synergy with 2′F-ANA, which has been previously established as a functional siRNA modification, was demonstrated.     

MicroRNAs in breast cancer: Roles,functions, and mechanism of actions

Breast cancer is one of the most lethal malignancies in women in the world. Various factors are involved in the development and promotion of the malignancy; most of them involve changes in the expression of certain genes, such as microRNAs (miRNAs). MiRNAs can regulate signaling pathways negatively or positively, thereby affecting tumorigenesis and various aspects of cancer progression, particularly breast cancer. Besides, accumulating data demonstrated that miRNAs are a novel tool for prognosis and diagnosis of breast cancer patients. Herein, we will review the roles of these RNA molecules in several important signaling pathways, such as transforming growth factor, Wnt, Notch, nuclear factor-κ B, phosphoinositide-3-kinase/Akt, and extracellular-signal-regulated kinase/mitogen activated protein kinase signaling pathways in breast cancer.     

Chitooligosaccharide-mediated neuroprotection is associated with modulation of Hsps expression and reduction of MAPK phosphorylation

There is mounting evidence implicating the role of oxidative stress induced by reactive oxygen species (ROS) in neurodegenerative disease, including Alzheimer's disease. In this study we aimed to investigate the possible protective effect of chitooligosaccharide (COS), an antioxidant oligosaccharide, on hydrogen peroxide induced apoptosis in NGF-differentitated rat pheochromocytoma (PC12) cells. COS treatment reversed the decrease of cell viability induced by H(2)O(2) and this was associated with diminished intracellular ROS and decreased level of cytosolic Ca(2+). Additionally, COS contributed to up-regulation of Bcl-2, down regulation of Bax protein and reduction of cleaved Caspase-3 protein. COS treatment stabilized Nrf2 in nucleus and increased the Hsp70 level within cell while down-regulated Hsp90 expression. Moreover, COS could inhibit the phosphorylation of different mitogen activated protein kinases (MAPKs), whose aberrant phosphorylation has been implicated in AD. Our findings suggest that heat shock response and MAPK cascades are both involved in cell survival, and by concomitantly regulating both pathways, COS can be a promising agent in treating neurodegenerative diseases.     

Role of annexin 1 gene expression in mouse craniofacial bone development

BACKGROUND: Annexin 1 is a 37-kDa protein that has complex intra- and extracellular effects. To discover whether the absence of this protein alters bone development, we monitored this event in the annexin-A1 null mice in comparison with littermate wild-type controls. METHODS: Radiographic and densitometry methods were used for the assessment of bone in annexin-A1 null mice at a gross level. We used whole-skeleton staining, histological analysis, and Western blotting techniques to monitor changes at the tissue and cellular levels. RESULTS: There were no gross differences in the appendicular skeleton between the genotypes, but an anomalous development of the skull was observed in the annexin-A1 null mice. This was characterized in the newborn annexin-A1 null animals by a delayed intramembranous ossification of the skull, incomplete fusion of the interfrontal suture and palatine bone, and the presence of an abnormal suture structure. The annexin-A1 gene was shown to be active in osteocytes during this phase and COX-2 was abundantly expressed in cartilage and bone taken from annexin-A1 null mice. CONCLUSIONS: Expression of the annexin-A1 gene is important for the normal development of the skull in mice, possibly through the regulation of osteoblast differentiation and a secondary effect on the expression of components of the cPLA2-COX-2 system.     

Large‐scale identification of membrane proteins with properties favorable for crystallization

Membrane protein crystallography is notoriously difficult due to challenges in protein expression and issues of degradation and structural stability. We have developed a novel method for large‐scale screening of native sources for integral membrane proteins that have intrinsic biochemical properties favorable for crystallization. Highly expressed membrane proteins that are thermally stable and nonaggregating in detergent solutions were identified by mass spectrometry from Escherichia coli, Saccharomyces cerevisiae, and Sus scrofa cerebrum. Many of the membrane proteins identified had been crystallized previously, supporting the promise of the approach. Most identified proteins have known functions and include high‐value targets such as transporters and ATPases. To validate the method, we recombinantly expressed and purified the yeast protein, Yop1, which is responsible for endoplasmic reticulum curvature. We demonstrate that Yop1 can be purified with the detergent dodecylmaltoside without aggregating.     

Response of genetically obese Zucker rats to ciprofibrate,a hypolipidemic agent,with peroxisome proliferation activity as compared to Zucker lean and Sprague-Dawley rats

Summary— Genetically obese Zucker (fa/fa) rats were used as an experimental model to study the effects of hypolipidemic agents on peroxisome proliferation; comparison was made with Zucker lean phenotype (Fa/?) and Sprague-Dawley strain/phenotype. The pharmacokinetics of a single administration of ciprofibrate (1 or 3 mg/kg), appeared to be similar in all strains/phenotypes. After a 2-week oral administration at the same dosages, there were dosage-related increases in hepatocellular peroxisomal yield and in the hepatic enzymes' cyanide-insensitive acyl-CoA oxidase and catalase. The peroxisomal yield was less increased in Zucker than in Sprague-Dawley rats, while the enzyme activities were similarly increased. Although the absolute specific activity of microsomal ω-lauryl hydroxylase (cytochrome P4504A1) was lower in Zucker rats, it was increased more in this strain than in Sprague-Dawley rats in response to drug exposure. The hypolipidemic effect (cholesterol and triglyceride reduction) was more pronounced in Zucker obese rats. Based on biochemical and morphological results, no major differences between strains/phenotypes in terms of peroxisome proliferation were observed following a 2-week administration of ciprofibrate.     

Purification of RanGDP, RanGTP, and RanGMPPNP by ion exchange chromatography

Ran is a small GTPase that cycles between a guanosine diphosphate (GDP)-bound form (RanGDP) and a guanosine triphosphate (GTP)-bound form (RanGTP) and plays important roles in nuclear transport and mitosis. For studies of Ran function and its interactions with partner proteins, pure RanGDP and RanGTP complexes are critical. Ran complexed with the nonhydrolyzable GTP analog, GMPPNP (RanGMPPNP), is used instead of RanGTP when inhibition of hydrolysis is required. In this study, we demonstrate that the binding of Ran to a UNO Q ion exchange column is remarkably sensitive to small shifts in MgCl(2) concentration, and we use this property to purify recombinant RanGTP, RanGMPPNP, and RanGDP complexes. At 10 mM MgCl(2), Ran was found predominantly in the flow-through and, thus, was separated from the vast majority of bacterial proteins. After reducing the concentration of MgCl(2) to 5 mM, further purification of RanGTP, RanGMPPNP, and RanGDP was achieved by loading onto ion exchange columns and elution with an NaCl gradient. Purity of the resulting preparations was confirmed by releasing the bound nucleotide and checking it against a known nucleotide by high-performance liquid chromatography (HPLC). To further confirm the purity and function of the Ran preparations, appropriate protein-binding, enzymatic, and nuclear import assays were carried out. These methods should facilitate studies of cellular processes involving Ran by providing pure functional Ran-nucleotide complexes.