Rac1 GTPase is activated by hepatitis B virus replication--involvement of HBX

Hepatitis B virus (HBV) is a causative agent for liver diseases including hepatocellular carcinoma. Understanding its interactions with cellular proteins is critical in the elucidation of the mechanisms of disease progression. Using a cell-based HBV replication system, we showed that HBV replication in HepG2 cells resulted in a cellular morphological changes displaying membrane rufflings and lamellipodia like structures reminiscent of cells expressing constitutively activated Rac1. We also showed that activated Rac1 resulted in increased viral replication. HBV replication specifically activated wild type Rac1, but not Cdc42. The Rac1 activation by HBV replication also resulted in the phosphorylation of ERK1/2 and AKT, the downstream targets of Rac1 signaling cascade. The smallest HBV viral protein, HBX, was able to activate the endogenous Rac1 and induce membrane ruffling when transfected into cells. Significantly, HBX was found to directly interact with a Rac1 nucleotide exchange factor (betaPIX) through a SH3 binding motif. Taken together, we have shown the interaction of HBV with the Rho GTPase, affecting cell morphology through the Rac1 activation pathway. HBV may possibly make use of an activated Rac1 signaling pathway for increased replication and resultant metastatic effects.     

Evolution of rDNA in Nicotiana allopolyploids: a potential link between rDNA homogenization and epigenetics

Background: The evolution and biology of rDNA have interested biologistsfor many years, in part, because of two intriguing processes:(1) nucleolar dominance and (2) sequence homogenization. Wereview patterns of evolution in rDNA in the angiosperm genusNicotiana to determine consequences of allopolyploidy on theseprocesses. Scope: Allopolyploid species of Nicotiana are ideal for studying rDNAevolution because phylogenetic reconstruction of DNA sequenceshas revealed patterns of species divergence and their parents.From these studies we also know that polyploids formed overwidely different timeframes (thousands to millions of years),enabling comparative and temporal studies of rDNA structure,activity and chromosomal distribution. In addition studies onsynthetic polyploids enable the consequences of de novo polyploidyon rDNA activity to be determined. Conclusions: We propose that rDNA epigenetic expression patterns establishedeven in F₁ hybrids have a material influence on the likely patternsof divergence of rDNA. It is the active rDNA units that arevulnerable to homogenization, which probably acts to reducemutational load across the active array. Those rDNA units thatare epigenetically silenced may be less vulnerable to sequencehomogenization. Selection cannot act on these silenced genes,and they are likely to accumulate mutations and eventually beeliminated from the genome. It is likely that whole silencedarrays will be deleted in polyploids of 1 million years of ageand older.     

LRRK2 R1628P increases risk of Parkinson’s disease: replication evidence

We showed that the frequency of a LRRK2 variant (c.4883G > C, R1628P) was higher in Parkinson’s disease (PD) compared to controls (8.4 vs. 3.4%, P = 0.046, OR 2.5, 95% CI 1.1–5.6). In the multivariate logistic regression (with adjustments made for the effect of age, age of onset, and gender), the heterozygous R1628P genotype was associated with an increased risk of PD compared to controls (OR 3.3, 95% CI 1.4– 7.9, P = 0.007). We provided an independent confirmation that the R1628P variant increases the risk of PD among Chinese.     

Common physical basis of macromolecule-binding sites in proteins

Protein–DNA/RNA/protein interactions play critical roles in many biological functions. Previous studies have focused on the different features characterizing the different macromolecule-binding sites and approaches to detect these sites. However, no common unique signature of these sites had been reported. Thus, this work aims to provide a ‘common’ principle dictating the location of the different macromolecule-binding sites founded upon fundamental principles of binding thermodynamics. To achieve this aim, a comprehensive set of structurally nonhomologous DNA-, RNA-, obligate protein- and nonobligate protein-binding proteins, both free and bound to their respective macromolecules, was created and a novel strategy for detecting clusters of residues with electrostatic or steric strain given the protein structure was developed. The results show that regardless of the macromolecule type, the binding strength and conformational changes upon binding, macromolecule-binding sites are energetically less stable than nonmacromolecule-binding sites. They also reveal new energetic features distinguishing DNA- from RNA-binding sites and obligate protein- from nonobligate protein-binding sites in both free/bound protein structures.     

Human amnion-derived mesenchymal stem cells are a potential source for uterine stem cell therapy

Abstract. Objectives: Human amnion is an easy‐to‐obtain novel source of human mesenchymal stem cells, which poses little or no ethical dilemmas. We have previously shown that human amnion‐derived mesenchymal (HAM) cells exhibit certain mesenchymal stem cell‐like characteristics with respect to expression of stem cell markers and differentiation potentials. Materials and methods: In this study, we further characterized HAM cells’ potential for in vivo therapeutic application. Results: Flow cytometric analyses of HAM cells show that they express several stem cell‐related cell surface markers, including CD90, CD105, CD59, CD49d, CD44 and HLA‐ABC, but not CD45, CD34, CD31, CD106 or HLA‐DR. HAM cells at the 10th passage showed normal karyotype. More interestingly, the AbdB‐like HOXA genes HOXA9, HOXA10 and HOXA11 that are expressed in the mesenchyme of the developing female reproductive tract and pregnant uteri are also expressed in HAM cells, suggesting similarities between these two mesenchymal cell types. Progesterone receptor is also highly expressed in HAM cells and expression of genes or proteins in HAM cells could be manipulated with the aid of lentivirus technology or cell‐permeable peptides. To test potentials of HAM cells for in vivo application, we introduced enhanced green fluorescence protein (EGFP)‐expressing HAM cells to mice by intrauterine infusion (into uteri) or by intravenous injection (into the circulation). Presence of EGFP‐expressing cells within the uterine mesenchyme after intrauterine infusion or in lungs after intravenous injection was noted within 1–4 weeks. Conclusions: Collectively, these results suggest that HAM cells are a potential source of mesenchymal stem cells with therapeutic potential.     

Synthesis, SAR, and X-ray structure of human BACE-1 inhibitors with cyclic urea derivatives

We describe synthesis and evaluation of a series of cyclic urea derivatives with hydroxylethylamine isostere. Modification of P3, P1, and P2′ and combination of SAR display a >100-fold increase in potency with good cellular activity (IC₅₀ = 0.15 μM) relative to the previously reported compound 3.     

Further evidence supporting an inner sphere mechanism in the NO reduction of the copper(II) complex Cu(dmp)2 (dmp = 2,9-dimethyl-1,10-phenanthroline)

Described are further studies directed towards elucidating the mechanism of the nitric oxide reduction of the copper(II) model system, Cu(dmp)2(2+) (I, dmp=2,9-dimethyl-1,10-phenanthroline). The reaction of I with NO in methanol results in the formation of Cu(dmp)2+ (II) and methyl nitrite (CH3ONO), with a second order rate constant kNO=38.1 M-1 s-1 (298K). The activation parameters for this reaction in buffered aqueous medium were measured to be DeltaH(double dagger)=41.6 kJ/mol and DeltaS(double dagger)=-82.7 kJ/mol deg. The addition of azide ion (N3-) as a competing nucleophile results in a marked acceleration in the rate of the copper(II) reduction. Analysis of the kinetics for the NO reduction of the bulkier Cu(dpp)(2)2+ (IV, dpp=2,9-diphenyl-1,10-phenanthroline) and the stronger oxidant, Cu(NO2-dmp)2(2+) (V, NO2-dmp=5-nitro-2,9-dimethyl-1,10-phenanthroline), gave the second order rate constants kNO=21.2 and 29.3 M-1 s-1, respectively. These results argue against an outer sphere electron transfer pathway and support a mechanism where the first step involves the formation of a copper-nitrosyl (Cu(II)-NO or Cu(I)-NO+) adduct. This would be followed by the nucleophilic attack on the bound NO and the labilization of RONO to form the nitrite products and the cuprous complex.     

Human VEGF165-myoblasts produce concomitant angiogenesis/myogenesis in the regenerative heart

Bioengineering the regenerative heart may provide a novel treatment for heart failure. On May 14, 2002, a 55-year-old man suffering from ischemic myocardial infarction received 25 injections carrying 465 million cGMP-produced pure myoblasts into his myocardium after coronary artery bypass grafting. As on August 28, 2002, his EKG was normal and showed no arrhythmia. His ejection fraction increased by 13%. He no longer experienced shortness of breath and angina as he did before the treatment. Three myogenesis mechanisms were elucidated with 17 human/porcine xenografts using cyclosporine as immunosuppressant. Some myoblasts developed to become cardiomyocytes. Others transferred their nuclei into host cardiomyocytes through natural cell fusion. As yet others formed skeletal myofibers with satellite cells. De novo production of contractile filaments augmented the heart contractility. Human myoblasts transduced with VEGF₁₆₅ gene produced six times more capillaries in porcine myocardium than in placebo. Xenograft rejection was not observed for up to 20 weeks despite cyclosporine discontinuation at 6 weeks. Pros and cons of autografts vs. allografts are compared to guide future development of heart cell therapy. (Mol Cell Biochem 263: 173–178, 2004)