Hydrogen bonding in a model bacteriochlorophyll-binding site drives assembly of light harvesting complex

In this study, the contribution of intramembrane hydrogen bonding at the interface between polypeptide and cofactor is explored in the native lipid environment by use of model bacteriochlorophyll proteins. In the peripheral antenna complex, LH2, large portions of the transmembrane helices, which make up the dimeric bacteriochlorophyll-binding site, are replaced by simplified, alternating alanine-leucine stretches. Replacement of either one of the two helices with the helices containing the model sequence at a time results in the assembly of complexes with nearly native light harvesting properties. In contrast, replacement of both helices results in the loss of antenna complexes from the membrane. The assembly of such doubly modified complexes is restored by a single intramembrane serine residue at position -4 relative to the liganding histidine of the alpha-subunit. In situ analysis of the spectral properties in a series of site-directed mutants reveals a critical dependence of the model complex assembly on the side chain of the residue at this position in the helix. A hydrogen bond between the hydroxy group of the serine and the 13(1) keto group of one of the central bacteriochlorophylls of the complexes is identified by Raman spectroscopy in the model antenna complex containing one of the alanine-leucine helices. The additional OH group of the serine residue, which participates in hydrogen bonding, increases the thermal stability of the model complexes in the native membrane. Intramembrane hydrogen bonding is thus shown to be a key factor for the binding of bacteriochlorophyll and assembly of this model cofactor-polypeptide site.     

Specific excision of the selenocysteine tRNA[Ser]Sec (Trsp) gene in mouse liver demonstrates an essential role of selenoproteins in liver function

Selenium is essential in mammalian embryonic development. However, in adults, selenoprotein levels in several organs including liver can be substantially reduced by selenium deficiency without any apparent change in phenotype. To address the role of selenoproteins in liver function, mice homozygous for a floxed allele encoding the selenocysteine (Sec) tRNA([Ser]Sec) gene were crossed with transgenic mice carrying the Cre recombinase under the control of the albumin promoter that expresses the recombinase specifically in liver. Recombination was nearly complete in mice 3 weeks of age, whereas liver selenoprotein synthesis was virtually absent, which correlated with the loss of Sec tRNA([Ser]Sec) and activities of major selenoproteins. Total liver selenium was dramatically decreased, whereas levels of low molecular weight selenocompounds were little affected. Plasma selenoprotein P levels were reduced by about 75%, suggesting that selenoprotein P is primarily exported from the liver. Glutathione S-transferase levels were elevated in the selenoprotein-deficient liver, suggesting a compensatory activation of this detoxification program. Mice appeared normal until about 24 h before death. Most animals died between 1 and 3 months of age. Death appeared to be due to severe hepatocellular degeneration and necrosis with concomitant necrosis of peritoneal and retroperitoneal fat. These studies revealed an essential role of selenoproteins in liver function.     

Phylogenetic analysis of archaea in three fractions of cow rumen based on the 16S rDNA sequence

Phylogenetic analysis of archaea in the rumen ecosystem was analysed by PCR of 16S rDNA from the bovine rumen using archaea-specific primers. The libraries were constructed from rumen fluid (AF), rumen solid (AS), and rumen epithelium (AE) from a rumen-fistulated Korean cow (Hanwoo). The 45 AF clones could be divided into three groups and the largest group was affiliated with the Methanomicrobiaceae family (96% of clones). The AF clones contained a high proportion of unidentifiable clones (67%). The 39 AE clones could be divided into two groups and the largest group was also affiliated with the Methanomicrobiaceae family (95% of clones). The AE clones contained a low proportion of unidentifiable clones (5%). The 20 AS clones could be divided into two groups that were affiliated with either the Methanobacteriaceae family (55%) or the Methanomicrobiaceae family (45%). The AS clones contained a moderate proportion of unidentifiable clones (40%). The predominant family of whole rumen archaea was found to belong to the Methanomicrobiaceae (85%). Methanomicrobiaceae were predominant in the rumen epithelium and the rumen fluid while Methanobacteriaceae were predominant in the rumen solid. One clone from the rumen fluid and two clones from the rumen epithelium contained rDNA sequences of Non-Thermophilic-Crenarchaeota (NTC) and Thermophilic-Crenarchaeota (TC), respectively, which have not previously been described from the rumen.     

Syntheses, characterization and crystal structures of copper(I) o-(diphenylphosphino)benzaldehyde complexes

The complexes [Cu(PCHO)₂(NCMe)][BF₄] (1) and [Cu(PCHO)₃][BF₄] (2) have been prepared by treating [Cu(NCMe)₄][BF₄] with two and three equivalents of Ph₂P(o-C₆H₄)C(O)H (abbreviated as PCHO) at room temperature, respectively. The reaction of 1 and (Ph₂PC₅H₄)₂Fe (abbreviated as DPPF) affords [Cu(PCHO)(DPPF)][BF₄] (3). The molecular structures of 1-3 have been determined by an X-ray diffraction study. The aldehyde groups in 1 are pendant, while one of the formyl groups in 2 is weakly coordinated to the copper ion through the oxygen atom. On the other hand, the copper atom in 3 is strongly chelated by both DPPF and PCHO ligands.     

Fishing for biomarkers: analyzing mass spectrometry data with the new ClinProTools software

Recently, applications of mass spectrometry in the field of clinical proteomics have gained tremendous visibility in the scientific and clinical community. One major objective is the search for potential biomarkers in complex body fluids like serum, plasma, urine, saliva, or cerebral spinal fluid. For this purpose, efficient visualization of large data sets derived from patient cohorts is crucial to provide clinical experts an interactive impression of the data quality. Additionally, it is necessary to apply statistical analysis and pattern matching algorithms to attain validated signal patterns that may allow for later applications in sample classification. We introduce the new ClinProTools bioinformatics software, which performs all major steps of profiling, screening, and monitoring applications in clinical proteomics. ClinProTools is the data interpretation software of the mass spectrometry-based ClinProt solutions for biomarker analysis. ClinProTools performs data pretreatment, visualization, statistics, pattern determination, pattern evaluation, and classification of spectra. This article will focus on ClinProTool's powerful and intuitive visualization options for clinical proteomics applications.     

Purification and characterization of nitric oxide synthase from Staphylococcus aureus

We previously reported the presence of nitric oxide synthase (NOS) in Staphylococcus aureus ATCC6538P whose activity was induced by methanol. In the present study, the methanol-induced NOS was purified 900-fold from S. aureus by means of Mono Q ion exchange column, 2',5'-ADP-agarose affinity column, and Superdex 200HR gel permeation column chromatography. The purified bacterial NOS showed two protein bands with 67 and 64 kDa molecular mass on SDS-PAGE. However, the molecular mass of the NOS was 135 kDa on Superdex 200HR gel permeation column chromatography, indicating that the native enzyme exists as a heterodimer. This bacterial NOS had K(m) value of 13.4x10(-6) M for L-arginine and V(max) of 35.3 nmol min(-1) mg(-1) protein. In addition, reduced nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin, calmodulin and Ca(2+) were required as cofactors in the conversion of L-arginine to L-citrulline, and NOS inhibitors selectively inhibited the activity of the purified NOS.     

Distribution,levels, and activation of MEK1 in Alzheimer's disease

Extracellular-signal-regulated kinase (ERK) has been implicated in the pathogenesis of Alzheimer's disease (AD), but the upstream cascade leading to ERK activation has not been elucidated. In this study, we focused on one of the physiological activators of ERK, mitogen-activated protein kinase (MAPK)/ERK kinase 1 (MEK1). Although there was no significant difference in the level and distribution of total MEK1 between AD and age-matched control cases, increased levels of activated phospho-MEK1 were specifically localized to neuronal intracytoplasmic granular structures in severe AD (Braak stage V-VI). The considerable overlap between MEK1 and its downstream effector, phospho-ERK, suggests both a functional and mechanistic link. Nuclear localization of phospho-MEK1 was a prominent feature in both mild AD cases (Braak stage III-IV) and control cases with limited pathology (Braak stage I-II). Since MEK1 is normally cytoplasmic due to the active export from nucleus because of the presence of nuclear export signal in its amino-terminus, we suspect that the apparent nuclear accumulation of phospho-MEK1 in AD patients at early stages suggests that abnormal nuclear trafficking may contribute to the pathogenesis of AD. By immunoblot analyses, phospho-MEK1 was significantly increased in AD over control cases. Together, these findings lend further credence to the notion that the ERK pathway is dysregulated in AD and also indicate an active role for this pathway in disease pathogenesis.     

Interfering with disease: opportunities and roadblocks to harnessing RNA interference

RNA interference (RNAi) is an evolutionarily conserved mechanism for silencing gene expression by targeted degradation of mRNA. Short double-stranded RNAs, known as small interfering RNAs (siRNA), are incorporated into an RNA-induced silencing complex that directs degradation of RNA containing a homologous sequence. RNAi has been shown to work in mammalian cells, and can inhibit viral infection and control tumor cell growth in vitro. Recently, it has been shown that intravenous injection of siRNA or of plasmids expressing sequences processed to siRNA can protect mice from autoimmune and viral hepatitis. RNAi could provide an exciting new therapeutic modality for treating infection, cancer, neurodegenerative disease and other illnesses.