Solution structure of the c-terminal dimerization domain of SARS coronavirus nucleocapsid protein solved by the SAIL-NMR method

The C-terminal domain (CTD) of the severe acute respiratory syndrome coronavirus (SARS-CoV) nucleocapsid protein (NP) contains a potential RNA-binding region in its N-terminal portion and also serves as a dimerization domain by forming a homodimer with a molecular mass of 28 kDa. So far, the structure determination of the SARS-CoV NP CTD in solution has been impeded by the poor quality of NMR spectra, especially for aromatic resonances. We have recently developed the stereo-array isotope labeling (SAIL) method to overcome the size problem of NMR structure determination by utilizing a protein exclusively composed of stereo- and regio-specifically isotope-labeled amino acids. Here, we employed the SAIL method to determine the high-quality solution structure of the SARS-CoV NP CTD by NMR. The SAIL protein yielded less crowded and better resolved spectra than uniform ¹³C and ¹⁵N labeling, and enabled the homodimeric solution structure of this protein to be determined. The NMR structure is almost identical with the previously solved crystal structure, except for a disordered putative RNA-binding domain at the N-terminus. Studies of the chemical shift perturbations caused by the binding of single-stranded DNA and mutational analyses have identified the disordered region at the N-termini as the prime site for nucleic acid binding. In addition, residues in the β-sheet region also showed significant perturbations. Mapping of the locations of these residues onto the helical model observed in the crystal revealed that these two regions are parts of the interior lining of the positively charged helical groove, supporting the hypothesis that the helical oligomer may form in solution.     

Kidney Function,Endothelial Activation and Atherosclerosis in Black and White Africans with Rheumatoid Arthritis

Objective

To determine whether kidney function independently relates to endothelial activation and ultrasound determined carotid atherosclerosis in black and white Africans with rheumatoid arthritis (RA).

Methods

We calculated the Jelliffe, 5 Cockcroft-Gault equations, Salazar-Corcoran, Modification of Diet in Renal Disease (MDRD) and Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) estimated glomerular filtration rate (EGFR) equations in 233 (112 black) RA patients.

Results

The CKD-EPI eGFR was <90 ml/min/1.73m² in 49.1% and 30.6% of black and white patients, respectively (odds ratio (95% confidence interval) = 2.19 (1.28–3.75), p = 0.004). EGFRs were overall consistently associated with monocyte chemoattractant protein-1 and angiopoietin 2 concentrations in white patients, and with carotid intima-media thickness and plaque in black participants. Amongst black patients, plaque prevalence was 36.7% and the area under the curve (AUC) of the receiver operating characteristic (ROC) curve was not associated with plaque presence for the MDRD equation (p = 0.3), whereas the respective relationship was significant or borderline significant (p = 0.003 to 0.08) and of similar extent (p>0.1 for comparisons of AUC (SE)) for the other 8 equations. Based on optimal eGFR cutoff values with sensitivities and specificities ranging from 42 to 60% and 70 to 91% respectively, as determined in ROC curve analysis, a low eGFR increased the odds ratio for plaque 2.2 to 4.0 fold.

Conclusion

Reduced kidney function is independently associated with atherosclerosis and endothelial activation in black and white Africans with RA, respectively. CKD is highly prevalent in black Africans with RA. Apart from the MDRD, eGFR equations are useful in predicting carotid plaque presence, a coronary heart disease equivalent, amongst black African RA patients.     

Lipopolysaccharide-mediated reactive oxygen species and signal transduction in the regulation of interleukin-1 gene expression

Lipopolysaccharide (LPS) stimulates macrophages to release inflammatory cytokines, interleukin-1 beta (IL-1), and tumor necrosis factor (TNF). LPS-induced TNF suppresses scavenger receptor functions in macrophages (van Lenten, B. J., and Fogelman, A. M. (1992) J. Immunol. 148, 112-116), which is regulated by TNF-mediated protein kinases (Hsu, H. Y., and Twu, Y. C. (2000) J. Biol. Chem. 275, 41035-41048). To examine the molecular mechanism for LPS induction of IL-1 in macrophages, we demonstrated that LPS quickly stimulated reactive oxygen species (ROS), and 3 h later induced prointerleukin-1 beta (pro-IL-1, precursor of IL-1) production and IL-1 secretion. LPS stimulated pro-IL-1 message/protein between 3 and 10 h; however, there was a 40% reduction of pro-IL-1 in preincubation of the antioxidant, N-acetylcysteine (NAC). Moreover, NAC moderated LPS-induced IL-1 secretion partially via interleukin 1-converting enzyme. The maximal activity of LPS-induced ERK, JNK, and p38 was 12- (30 min), 5- (30 min), and 16-fold (15 min), respectively. In contrast, NAC reduced ERK activity to 60% and decreased p38 activity to the basal level, but JNK activity was induced 2-fold. Furthermore, the pharmacological antagonists LY294002, SB203580, curcumin, calphostin C, and PD98059 revealed the diverse roles of LPS-mediated protein kinases in pro-IL-1. On the other hand, NAC and diphenyleneiodonium chloride partially inhibited LPS-induced Rac activity and protein-tyrosine kinase (PTK), indicating that LPS-mediated ROS and NADPH oxidase correspond to Rac activation and IL-1 expression. Our findings establish for the first time that LPS-mediated PTK/phosphatidylinositol 3-kinase/Rac/p38 pathways play a more important role than pathways of PTK/PKC/MEK/ERK and of PTK/phosphatidylinositol 3-kinase/Rac/JNK in the regulation of pro-IL-1/IL-1. The findings also further elucidate the critical role of LPS-mediated ROS in signal transduction pathways. Our results suggest that understanding LPS-transduced signals in IL-1 induction upon the antibacterial action of macrophages should provide a therapeutic strategy for aberrant inflammatory responses leading to severe cellular injury or concurrent multiorgan septic damage.     

Yohimbine increases plasma insulin concentrations of dogs

Recent evidence suggests that catecholamines inhibit insulin release by stimulating alpha 2-adrenoreceptors in beta-cells of the pancreatic islets. In the present study, iv injections of 0.1 and 0.3 mg/kg of yohimbine, an alpha 2-adrenoreceptor antagonist, resulted in increased plasma insulin and decreased plasma glucose concentrations in the dog. The use of alpha 2-adrenoreceptor antagonists may be of value in non-insulin-dependent diabetic patients by counteracting the inhibitory influence of endogenous catecholamines.     

Nuclear magnetic resonance studies on the solution conformation of histone IV fragments obtained by cyanogen bromide cleavage.

Two histone IV fragments obtained by cleavage at Met-84 by cyanogen bromide have been examined by proton magnetic resonance (PMR) spectroscopy as a function of temperature, peptide concentration, ionic strength, and pD. Sedimentation and gel electrophoresis studies on these peptides have also been carried out. The 220-MHz PMR spectrum of the N-peptide in both the high- and low-field regions was shown to be almost identical with that calculated for an extended coil N-peptide monomer. The calculated random coil and experimental C-peptide spectra, on the other hand, differ in many respects. Evidence was obtained for the presence of rigid secondary structure in the C-peptide. In addition, the Val, Leu, Ile CH3 resonance displays a prominent high-field satellite band which shifts downfield with increasing temperature. Sedimentation studies on the N-peptide reveal the formation of extremely large, remarkably homogeneous aggregates at ionic strengths larger than or equal to 0.01. The C-peptide, on the other hand, does not appear to form aggregates of sufficient size to be detectable in velocity sedimentation studies of a few hours duration. The relative area changes which have previously been noted in the PMR spectrum of histone IV with increasing ionic strength were also observed for the N-peptide but not the C-peptide. Interpretation of these relative area changes has been made in terms of the amino acid sequence of histone IV, and an effort was made to identify that segment of the polypeptide which undergoes secondary structural change with increasing ionic strength.     

Effect of a sputum digestant on the viabiltiy of Mycobacterium fortuitum.

A microcolony technique has been demonstrated as being useful for the rapid determination of the viabilities of single cells of Myocbacterium fortuitum. Cultures of M. fortuitum grown to early logarithmic phase in broth were treated with the sputum digestant N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) for periods of 10 to 40 s. After growth for three generations (7.5 h) on agar films, viabilities were determined by counting under a phase contrast microscope. The viable mycobacteria grew into microcolonies that exhibited extensive branching, whereas the nonviable mycobacteria remained as single cells. Sputum was mixed with some broth cultures before treatment to stimulate the digestion process in a clinical laboratory. When broth cultures were treated with sputum digestant for 40 s, only 2.8% of the cells remained viable. When the broth cultures were mixed in a ratio of 1:4 with sputum and then treated for 40 s, 16.4% of the cells remained viable. The results also indicate M. fortuitum is very sensitive to the digestant. The results also indicate that a microcolony technique could be used for the assessment of the viability of mycobacteria.     

Chronic Administration of Cyclosporine A Changes Expression of BDNF and TrkB in Rat Hippocampus and Midbrain

Neurotrophins, including the brain-derived neurotrophic factor (BDNF), are essential for regulating neuronal differentiation in developing brains. BDNF and its receptor tyrosine kinase receptor B (TrkB) are involved in neuronal signaling, survival and plasticity. Cyclosporine A (CsA) is a potent immunosuppressive agent which prevents allograft rejection in organ transplantation and various immunological diseases. We investigated whether chronic administration of CsA decreases BDNF gene expression in rats, and the influence of CsA on mRNA levels of TrkB receptors was also examined. For 30 days of CsA (10 mg/kg/day) administration, the expression of BDNF and TrkB mRNA was significantly decreased in the hippocampus and midbrain, but there was no significant difference in the cortex. CsA (0, 1, 5 10, 15 ug/ml) down-regulated BDNF and TrkB gene expression through cultured SH-SY5Y cells, as did all-trans retinoic acid (ATRA), and there was no effect on cell viability. These experimental results indicate that suppression of the BDNF and TrkB mRNA, protein level of BDNF expression in the hippocampus and midbrain may be related to altered behavior observed following chronic administration of CsA. A common mechanism of adverse effects of CsA induced depressive symptoms may involve neurotoxicity mediated by down-regulation of brain BDNF and TrkB.     

Donor–Acceptor–Acceptor's Molecules for Vacuum‐Deposited Organic Photovoltaics with Efficiency Exceeding 9%

Three vacuum‐deposited donor–acceptor–acceptor (d–a–a') small molecule donors are studied with different side chains attached to an asymmetric heterotetracene donor block for use in high efficiency organic photovoltaics (OPVs). The donor with an isobutyl side chain yields the highest crystal packing density compared to molecules with 2‐ethylhexyl or n‐butyl chains, leading to the largest absorption coefficient and short circuit current in an OPV. It also exhibits a higher fill factor, consistent with its preferred out‐of‐plane molecular π–π stacking arrangement that facilitates charge transport in the direction perpendicular to the substrate. A power conversion efficiency of 9.3 ± 0.5% is achieved under 1 sun intensity, AM 1.5 G simulated solar illumination, which is significantly higher than 7.5 ± 0.4% of the other two molecules. These results indicate that side chain modification of d–a–a' small molecules offers an effective approach to control the crystal packing configuration, thereby improving the device performance.