Chromophore structure in lumirhodopsin and metarhodopsin I by time-resolved resonance Raman microchip spectroscopy.

Time-resolved resonance Raman microchip flow experiments have been performed on the lumirhodopsin (Lumi) and metarhodopsin I (Meta I) photointermediates of rhodopsin at room temperature to elucidate the structure of the chromophore in each species as well as changes in protein-chromophore interactions. Transient Raman spectra of Lumi and Meta I with delay times of 16 micros and 1 ms, respectively, are obtained by using a microprobe system to focus displaced pump and probe laser beams in a microfabricated flow channel and to detect the scattering. The fingerprint modes of both species are very similar and characteristic of an all-trans chromophore. Lumi exhibits a relatively normal hydrogen-out-of-plane (HOOP) doublet at 951/959 cm(-1), while Meta I has a single HOOP band at 957 cm(-1). These results suggest that the transitions from bathorhodopsin to Lumi and Meta I involve a relaxation of the chromophore to a more planar all-trans conformation and the elimination of the structural perturbation that uncouples the 11H and 12H wags in bathorhodopsin. Surprisingly, the protonated Schiff base C=N stretching mode in Lumi (1638 cm(-1)) is unusually low compared to those in rhodopsin and bathorhodopsin, and the C=ND stretching mode shifts down by only 7 cm(-1) in D2O buffer. This indicates that the Schiff base hydrogen bonding is dramatically weakened in the bathorhodopsin to Lumi transition. However, the C=N stretching mode in Meta I is found at 1654 cm(-1) and exhibits a normal deuteration-induced downshift of 24 cm(-1), identical to that of the all-trans protonated Schiff base. The structural relaxation of the chromophore-protein complex in the bathorhodopsin to Lumi transition thus appears to drive the Schiff base group out of its hydrogen-bonded environment near Glu113, and the hydrogen bonding recovers to a normal solvated PSB value but presumably a different hydrogen bond acceptor with the formation of Meta I.     

Preparation and characterization of radioactive castanospermine

A procedure for the preparation of tritiated castanospermine is described. The tritiated alkaloid was shown to be chromatographically identical to the native material and exhibited the same inhibitory properties. Radiolabeled castanospermine tightly bound to purified intestinal sucrase. Following gel chromatography, each mole of enzyme was shown to have bound 1 mol of the radioactive alkaloid. Cultured MDCK cells were also shown to take up the labeled castanospermine. This compound should be a useful tool in the investigation of enzymes that are responsible for the processing of glycoprotein oligosaccharides.     

An iso-random Bi Bi mechanism for adenylate kinase.

An iso-random Bi Bi mechanism has been proposed for adenylate kinase. In this mechanism, one of the enzyme forms can bind the substrates MgATP and AMP, whereas the other form can bind the products MgADP and ADP. In a catalytic cycle, the conformational changes of the free enzyme and the ternary complexes are the rate-limiting steps. The AP(5)A inhibition equations derived from this mechanism show theoretically that AP(5)A acts as a competitive inhibitor for the forward reaction and a mixed noncompetitive inhibitor for the backward reaction.     

MSI4/FVE is required for accumulation of 24-nt siRNAs and DNA methylation at a subset of target regions of RNA-directed DNA methylation

DNA methylation is an important epigenetic mark. In plants, de novo DNA methylation occurs mainly through the RNA-directed DNA methylation (RdDM) pathway. Researchers have previously inferred that a flowering regulator, MULTICOPY SUPPRESSOR OF IRA1 4 (MSI4)/FVE, is involved in non-CG methylation at several RdDM targets, suggesting a role of FVE in RdDM. However, whether and how FVE affects RdDM genome-wide is not known. Here, we report that FVE is required for DNA methylation at thousands of RdDM target regions. In addition, dysfunction of FVE significantly reduces 24-nucleotide siRNA accumulation that is dependent on factors downstream in the RdDM pathway. By using chromatin immunoprecipitation and sequencing (ChIP-seq), we show that FVE directly binds to FVE-dependent 24-nucleotide siRNA cluster regions. Our results also indicate that FVE may function in RdDM by physically interacting with RDM15, a downstream factor in the RdDM pathway. Our study has therefore revealed that FVE, by associating with RDM15, directly regulates DNA methylation and siRNA accumulation at a subset of RdDM targets.     

Compositional Characterization and Pyrolysis of Loblolly Pine and Douglas-fir Bark

Two potential biofuel resources, Douglas-fir and Loblolly pine bark, were subjected to extensive chemical and compositional analysis. The barks were initially extracted with dichloromethane, and the resulting extracted compounds were characterized by gas chromatography coupled with mass spectrometric analysis. Characterization of the major bark biocomponents indicated that Douglas-fir and Loblolly pine bark contained 22.5 and 13.2 % tannins, 44.2 and 43.5 % lignin, 16.5 and 23.1 % cellulose, and 7.6 and 14.1 % hemicellulose, respectively. Of particular interest is the high content of tannins and lignin, which make these barks excellent potential precursors for bio-oils and/or other value-added chemicals. ¹³C nuclear magnetic resonance (NMR) was used to characterize the chemical structure of the lignin and tannins. These samples were also analyzed by ³¹P NMR after phosphitylation of the hydroxyl groups in lignin and tannins. The NMR spectral data indicated that the lignin in both barks contained p-hydroxyphenyl (h) and guaiacyl (g) of lignin monomers with an h/g ratio of 10:90 and 22:78 for Douglas-fir and Loblolly pine bark, respectively. Gel permeation chromatography was used to analyze the molecular weight distributions of extracted tannins, isolated cellulose, and ball-milled lignin. The pyrolysis of Douglas-fir and pine bark at 500°C in a tubular reactor generated 48.2 and 45.2 % of total oil, of which the light oil contents are 14.1 and 20.7 % and heavy oil are 34.1 and 24.4 %. Similarly, fast pyrolysis at 375°C yielded 56.1 and 49.8 % of total oil for Douglas-fir and pine bark, respectively.     

Impact of Water Chemistry,Pipe Material and Stagnation on the Building Plumbing Microbiome

A unique microbiome establishes in the portion of the potable water distribution system within homes and other buildings (i.e., building plumbing). To examine its composition and the factors that shape it, standardized cold water plumbing rigs were deployed at the treatment plant and in the distribution system of five water utilities across the U.S. Three pipe materials (copper with lead solder, CPVC with brass fittings or copper/lead combined pipe) were compared, with 8 hour flush cycles of 10 minutes to simulate typical daily use patterns. High throughput Illumina sequencing of 16S rRNA gene amplicons was employed to profile and compare the resident bulk water bacteria and archaea. The utility, location of the pipe rig, pipe material and stagnation all had a significant influence on the plumbing microbiome composition, but the utility source water and treatment practices were dominant factors. Examination of 21 water chemistry parameters suggested that the total chlorine concentration, pH, P, SO₄ ^2- and Mg were associated with the most of the variation in bulk water microbiome composition. Disinfectant type exerted a notably low-magnitude impact on microbiome composition. At two utilities using the same source water, slight differences in treatment approaches were associated with differences in rare taxa in samples. For genera containing opportunistic pathogens, Utility C samples (highest pH of 9–10) had the highest frequency of detection for Legionella spp. and lowest relative abundance of Mycobacterium spp. Data were examined across utilities to identify a true universal core, special core, and peripheral organisms to deepen insight into the physical and chemical factors that shape the building plumbing microbiome.     

Novel miR-sc4 regulates the proliferation and migration of Schwann cells by targeting Cdk5r1

Clinical and experimental studies have shown an association between intracellular free Zn²⁺ ([Zn²⁺]_i)-dyshomeostasis and cardiac dysfunction besides [Ca²⁺]_i-dyshomeostasis. Since [Zn²⁺]_i-homeostasis is regulated through Zn²⁺-transporters depending on their subcellular distributions, one can hypothesize that any imbalance in Zn²⁺-homeostasis via alteration in Zn²⁺-transporters may be associated with the induction of ER stress and apoptosis in hypertrophic heart. We used a transverse aortic constriction (TAC) model to induce hypertrophy in young male rat heart. We confirmed the development of hypertrophy with a high ratio of heart to body weight and cardiomyocyte capacitance. The expression levels of ER stress markers GRP78, CHOP/Gadd153, and calnexin are significantly high in TAC-group in comparison to those of controls (SHAM-group). Additionally, we detected high expression levels of apoptotic status marker proteins such as the serine kinase GSK-3β, Bax-to-Bcl-2 ratio, and PUMA in TAC-group in comparison to SHAM-group. The ratios of phospho-Akt to Akt and phospho-NFκB to the NFκB are significantly higher in TAC-group than in SHAM-group. Furthermore, we observed markedly increased phospho-PKCα and PKCα levels in TAC-group. We, also for the first time, determined significantly increased ZIP7, ZIP14, and ZnT8 expressions along with decreased ZIP8 and ZnT7 levels in the heart tissue from TAC-group in comparison to SHAM-group. Furthermore, a roughly calculated total expression level of ZIPs responsible for Zn²⁺-influx into the cytosol (increased about twofold) can be also responsible for the markedly increased [Zn²⁺]_i detected in hypertrophic cardiomyocytes. Taking into consideration the role of increased [Zn²⁺]_i via decreased ER-[Zn²⁺] in the induction of ER stress in cardiomyocytes, our present data suggest that differential changes in the expression levels of Zn²⁺-transporters can underlie mechanical dysfunction, in part due to the induction of ER stress and apoptosis in hypertrophic heart via increased [Zn²⁺]_i- besides [Ca²⁺]_i-dyshomeostasis.     

Simultaneous determination of Zn,Cd, Pb,and Cu in urine of patients with blackfoot disease using anodic stripping voltammetry

Blackfoot disease (BFD) is an endemic peripheral vascular disorder resulting in gangrene of the lower extremities, especially the feet, among residents in a limited area on the southwest coast of Taiwan. In the present study, the concentrations of zinc, cadmium, lead, and copper in urine of BFD patients with matched normal controls are investigated by differential pulse anodic stripping voltammetry (DPASV) on a hanging mercury drop electrode (HMDE). The analytical results indicate that urinary copper, cadmium, and lead of the BFD patients are significantly higher than those of the controls. In addition, the patients showed a significantly lower concentration of zinc in the urine than the normal controls. The possible connection of these elements with the etiology of the disease is discussed.