Lhca5 – an LHC-Type Protein Associated with Photosystem I

The light-harvesting antenna of higher plant photosystem (PS) I is known to be composed of four different types of light-harvesting complex (LHC) proteins (Lhca1-4). However, the genomic sequence of Arabidopsis thaliana contains open reading frames coding for two additional LHC type proteins (Lhca5-6) that are presumably associated with PSI. While Lhca6 might not be expressed at all, ESTs have been detected for the Lhca5 gene in Arabidopsis and a number of other plant species. Here we demonstrate the presence of the Lhca5 gene product in the thylakoid membrane of Arabidopsis as an additional type of Lhca-protein associated with PSI. Lhca5 seems to be regulated differently from the other LHC proteins since Lhca5 mRNA levels increase under high light conditions. Analyses reported here of Lhca5 in plants lacking individual Lhca1-4 proteins show that it is more abundant in plants lacking Lhca1/4, and suggest that it interacts in a direct physical fashion with Lhca2 or Lhca3. We propose that Lhca5 binds chlorophylls in a similar fashion to the other Lhca proteins and is associated with PSI only in sub-stoichiometric amounts.     

Binding differences of human and amphibian corticotropin-releasing factor type 1 (CRF(1)) receptors: identification of amino acids mediating high-affinity astressin binding and functional antagonism

The corticotropin-releasing factor (CRF) type 1 receptors (CRF(1)) from human (hCRF(1)) and Xenopus (xCRF(1)) differ from one another by their agonist- and antagonist-binding preference. While the agonist-binding site of the xCRF(1) receptor has been mapped, the amino acids that mediate binding of the potent peptide antagonist astressin are unknown. By constructing receptor chimeras followed by site-directed mutagenesis, the astressin-binding site of the xCRF(1) receptor was located between residues 76 and 83. This region partially overlaps with the agonist-selective domain of the xCRF(1) receptor (residues 76-89). Mutagenesis of the amphibian residues Gln(76), Gly(81) and Val(83) to the human sequence (Arg(76)Asn(81)Gly(83)) generated a receptor mutant that bound astressin with even higher affinity than the native hCRF(1) receptor. An amino acid doublet (Glu(70)Tyr(71)) that is conserved in the xCRF(1) and hCRF(2(a)) receptor after incorporation into the hCRF(1) receptor sequence was found to facilitate antagonist binding up to 15-fold higher. In agreement with the binding data, astressin was a more potent functional antagonist at receptors expressing the Glu(70)Tyr(71) motif. These data show that the agonist- and antagonist-binding sites of the hCRF(1) receptor partially overlap and that two amino acids within the N terminus of the hCRF(1) receptor negatively influence binding and functional antagonism of astressin.     

Taurine kinetics assessed using [1,2-13C2]taurine in healthy adult humans

To assess the dynamics of taurine metabolism in vivo, two sets of studies were carried out in healthy volunteers. First, pilot studies were carried in a single human subject to determine the time course of plasma and whole blood isotope enrichment over the course of an 8-h, unprimed continuous infusion of [1,2-(13)C(2)]taurine. Second, five healthy adult males received two tracer infusions on separate days and in randomized order: 1) a 6-h continuous infusion of [1,2-(13)C(2)]taurine (3.1 +/- 0.2 micromol x kg(-1) x h(-1)) and 2) a bolus injection of [(13)C(2)]taurine (3.0 +/- 0.1 micromol/kg). Isotope enrichments in plasma and whole blood taurine were determined by gas chromatography-mass spectrometry. The pilot experiments allowed us to establish that steady-state isotope enrichment was reached in plasma and whole blood by the 5th h of tracer infusion. The plateau enrichment reached in whole blood was lower than that obtained in plasma taurine (P < 0.02). In the second set of studies, the appearance rate (R(a)) of plasma taurine, determined from continuous infusion studies was 31.8 +/- 3.1 micromol x kg(-1) x h(-1). After a bolus injection of tracer, the enrichment decay over the subsequent 2 h was best fitted by a two-exponential curve. Taurine R(a) was approximately 85% higher when determined using the bolus injection technique compared with continuous infusion of tracer. We conclude that 1) taurine R(a) into plasma is very low in healthy postabsorptive humans, and, due to taurine compartmentation between the extra- and intracellular milieus, may represent only interorgan taurine transfer and merely a small fraction of whole body taurine turnover; and 2) the bolus injection technique may overestimate taurine appearance into plasma. Further studies are warranted to determine whether alterations in bile taurine dynamics affect taurine R(a).     

Neutrophils augment recovery of porcine ischemia-injured ileal mucosa by an IL-1beta- and COX-2-dependent mechanism

Polymorphonuclear neutrophils (PMNs) play a critical role in intestinal mucosal injury and repair. To study effects of PMNs on acutely injured mucosa, we applied PMNs isolated from circulation or peritoneal fluid from animals with chemically induced peritonitis to ischemia-injured porcine ileal mucosa. In preliminary experiments, PMNs enhanced recovery of transepithelial electrical resistance (TER), and this action was inhibited by pretreatment with the nonselective cyclooxygenase (COX) inhibitor indomethacin. Because COX-2 is upregulated by inflammatory mediators such as IL-1beta, which is released by PMNs, we postulated that PMNs enhance recovery of ischemia-injured mucosa by a pathway involving IL-1beta and COX-2. Application of 5 x 10(6) PMNs to the serosal surface of ischemia-injured mucosa significantly enhanced recovery of TER (P < 0.05), an effect that was inhibited by the selective COX-2 inhibitor NS-398 (5 microM) and by an IL-1beta receptor antagonist (0.1 mg/ml). Addition of 10 ng/ml IL-1beta to the serosal surface of injured tissues caused a significant increase in TER (P < 0.05) that was inhibited by pretreatment with NS-398. Western blot analysis of mucosal homogenates revealed dramatic upregulation of COX-2 in response to IL-1beta or peritoneal PMNs, and the latter was inhibited by an IL-1beta receptor antagonist. Real-time PCR revealed that increased mRNA COX-2 expression preceded increased COX-2 protein expression in response to IL-1beta. We concluded that PMNs augment recovery of TER in ischemia-injured ileal mucosa via IL-1beta-dependent upregulation of COX-2.     

Dosimetric analysis of the carousel setup for the exposure of rats at 1.62 GHz

The so-called carousel setup has been widely utilized for testing the hypotheses of adverse health effects on the central nervous system (CNS) due to mobile phone exposures in the frequency bands 800-900 MHz. The objectives of this article were to analyze the suitability of the setup for the upper mobile frequency range, i.e., 1.4-2 GHz, and to conduct a detailed experimental and numerical dosimetry for the setup at the IRIDIUM frequency band of 1.62 GHz. The setup consists of a plastic base on which ten rats, restrained in radially positioned tubes, are exposed to the electromagnetic field emanating from a sleeved dipole antenna at the center. Latest generation miniaturized dosimetric E field and temperature probes were used to measure the specific absorption rate (SAR) inside the brain of three rat cadavers of the Lewis strain and two rat cadavers of the Fisher 344 strain. A numerical analysis was conducted on the basis of three numerical rat phantoms with voxel sizes between 1.5 and 0.125 mm3 that are based on high resolution MRI scans of a 300 g male Wistar rat and a 370 g male Sprague-Dawley rat. The average of the assessed SAR values in the brain was 2.8 mW/g per W antenna input power for adult rats with masses between 220 and 350 g and 5.3 mW/g per W antenna input power for a juvenile rat with a mass of 95 g. The strong increase of the SAR in the brain with decreasing animal size was verified by simulations of the absorption in numerical phantoms scaled to sizes between 100 and 500 g with three different scaling methods. The study also demonstrated that current rat phantom models do not provide sufficient spatial resolution to perform absolute SAR assessment for the brain tissue. The variation of the SAR(brain)(av) due to changes in position was assessed to be in the range from +15% to -30%. A study on the dependence of the performance of the carousel setup on the frequency revealed that efficiency, defined as SAR(brain)(av) per W antenna input power, and the ratio between SAR(brain)(av) and SAR(body)(av) are optimal in the mobile communications frequency range, i.e., 0.8-3 GHz.     

Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation

An 11.7-A-resolution cryo-EM map of the yeast 80S.eEF2 complex in the presence of the antibiotic sordarin was interpreted in molecular terms, revealing large conformational changes within eEF2 and the 80S ribosome, including a rearrangement of the functionally important ribosomal intersubunit bridges. Sordarin positions domain III of eEF2 so that it can interact with the sarcin-ricin loop of 25S rRNA and protein rpS23 (S12p). This particular conformation explains the inhibitory action of sordarin and suggests that eEF2 is stalled on the 80S ribosome in a conformation that has similarities with the GTPase activation state. A ratchet-like subunit rearrangement (RSR) occurs in the 80S.eEF2.sordarin complex that, in contrast to Escherichia coli 70S ribosomes, is also present in vacant 80S ribosomes. A model is suggested, according to which the RSR is part of a mechanism for moving the tRNAs during the translocation reaction.     

Mannopeptimycin esters and carbonates, potent antibiotic agents against drug-resistant bacteria

A series of ester and carbonate derivatives of the glycopeptide mannopeptimycin alpha (1) with potent activity against G+ bacteria, including the methicillin-resistant staphylococci and vancomycin-resistant enterococci, was synthesized. The SAR data obtained from natural and semisynthetic compounds demonstrated the importance of a hydrophobic group in the terminal mannosyl moiety for antibacterial activity.     

SAR and factor IXa crystal structure of a dual inhibitor of factors IXa and Xa

Modifications to the P4 moiety and pyrazole C3 substituent of factor Xa inhibitor SN-429 provided several new compounds, which are 5-10nM inhibitors of factor IXa. An X-ray crystal structure of one example complexed to factor IXa shows that these compounds adopt a similar binding mode to that previously observed with pyrazole inhibitors in the factor Xa active site both with regard to how the inhibitor binds and the position of Tyr99.