Synthesis of protein kinase Cδ C1b domain by native chemical ligation methodology and characterization of its folding and ligand binding

The C1b domain of protein kinase Cδ (PKCδ), a potent receptor for ligands such as diacylglycerol and phorbol esters, was synthesized by utilizing native chemical ligation. With this synthetic strategy, the domain was efficiently constructed and shown to have high affinity ligand binding and correct folding. The C1b domain has been utilized for the development of novel ligands for the control of phosphorylation by PKC family members. This strategy will pave the way for the efficient construction of C1b domains modified with fluorescent dyes, biotin, etc. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Persistent Overexpression of Phosphoglycerate Mutase,a Glycolytic Enzyme,Modifies Energy Metabolism and Reduces Stress Resistance of Heart in Mice

Background

Heart failure is associated with changes in cardiac energy metabolism. Glucose metabolism in particular is thought to be important in the pathogenesis of heart failure. We examined the effects of persistent overexpression of phosphoglycerate mutase 2 (Pgam2), a glycolytic enzyme, on cardiac energy metabolism and function.

Methods and Results

Transgenic mice constitutively overexpressing Pgam2 in a heart-specific manner were generated, and cardiac energy metabolism and function were analyzed. Cardiac function at rest was normal. The uptake of analogs of glucose or fatty acids and the phosphocreatine/βATP ratio at rest were normal. A comprehensive metabolomic analysis revealed an increase in the levels of a few metabolites immediately upstream and downstream of Pgam2 in the glycolytic pathway, whereas the levels of metabolites in the initial few steps of glycolysis and lactate remained unchanged. The levels of metabolites in the tricarboxylic acid (TCA) cycle were altered. The capacity for respiration by isolated mitochondria in vitro was decreased, and that for the generation of reactive oxygen species (ROS) in vitro was increased. Impaired cardiac function was observed in response to dobutamine. Mice developed systolic dysfunction upon pressure overload.

Conclusions

Constitutive overexpression of Pgam2 modified energy metabolism and reduced stress resistance of heart in mice.     

Recruitment of the autophagic machinery to endosomes during infection is mediated by ubiquitin

Although ubiquitin is thought to be important for the autophagic sequestration of invading bacteria (also called xenophagy), its precise role remains largely enigmatic. Here we determined how ubiquitin is involved in this process. After invasion, ubiquitin is conjugated to host cellular proteins in endosomes that contain Salmonella or transfection reagent–coated latex (polystyrene) beads, which mimic invading bacteria. Ubiquitin is recognized by the autophagic machinery independently of the LC3–ubiquitin interaction through adaptor proteins, including a direct interaction between ubiquitin and Atg16L1. To ensure that invading pathogens are captured and degraded, Atg16L1 targeting is secured by two backup systems that anchor Atg16L1 to ubiquitin-decorated endosomes. Thus, we reveal that ubiquitin is a pivotal molecule that connects bacteria-containing endosomes with the autophagic machinery upstream of LC3.     

P-glycoprotein Mediates Drug Resistance via a Novel Mechanism Involving Lysosomal Sequestration

Localization of the drug transporter P-glycoprotein (Pgp) to the plasma membrane is thought to be the only contributor of Pgp-mediated multidrug resistance (MDR). However, very little work has focused on the contribution of Pgp expressed in intracellular organelles to drug resistance. This investigation describes an additional mechanism for understanding how lysosomal Pgp contributes to MDR. These studies were performed using Pgp-expressing MDR cells and their non-resistant counterparts. Using confocal microscopy and lysosomal fractionation, we demonstrated that intracellular Pgp was localized to LAMP2-stained lysosomes. In Pgp-expressing cells, the Pgp substrate doxorubicin (DOX) became sequestered in LAMP2-stained lysosomes, but this was not observed in non-Pgp-expressing cells. Moreover, lysosomal Pgp was demonstrated to be functional because DOX accumulation in this organelle was prevented upon incubation with the established Pgp inhibitors valspodar or elacridar or by silencing Pgp expression with siRNA. Importantly, to elicit drug resistance via lysosomes, the cytotoxic chemotherapeutics (e.g. DOX, daunorubicin, or vinblastine) were required to be Pgp substrates and also ionized at lysosomal pH (pH 5), resulting in them being sequestered and trapped in lysosomes. This property was demonstrated using lysosomotropic weak bases (NH₄Cl, chloroquine, or methylamine) that increased lysosomal pH and sensitized only Pgp-expressing cells to such cytotoxic drugs. Consequently, a lysosomal Pgp-mediated mechanism of MDR was not found for non-ionizable Pgp substrates (e.g. colchicine or paclitaxel) or ionizable non-Pgp substrates (e.g. cisplatin or carboplatin). Together, these studies reveal a new mechanism where Pgp-mediated lysosomal sequestration of chemotherapeutics leads to MDR that is amenable to therapeutic exploitation.     

Prevention of biofilm formation on titanium surfaces modified with conjugated molecules comprised of antimicrobial and titanium-binding peptides

Specific binding of antimicrobial peptides to titanium (Ti) surfaces may serve to prevent biofilm formation, leading to a reduction in peri-implantitis. This study evaluated the binding behavior of conjugated molecules consisting of antimicrobial and hexapeptidic Ti-binding peptides (minTBP-1) using the quartz crystal microbalance (QCM-D) technique, and investigated the effect of modification of Ti surfaces with these peptides on the bioactivity of Porphyromonas gingivalis. Four kinds of peptide were prepared: histatin 5 (DSHAKRHHGYKRKFHEKHHSHRGY), minTBP-1 + histatin 5 (RKLPDAPDSHAKRHHGYKRKFHEKHHSHRGY), lactoferricin (FQWQRNMRKVR), and minTBP-1 + lactoferricin (RKLPDAPGGFQWQRNMRKVR). The QCM-D analysis demonstrated that significantly larger increases in peptide adsorption were observed in the conjugated peptides than in antimicrobial peptides alone. In addition, ATP activity in P. gingivalis in peptide-modified specimens significantly decreased compared to that in the Ti control. These results indicate that surface modification with conjugated molecules consisting of antimicrobial and Ti-binding peptides is a promising method for reduction of biofilm formation on Ti surfaces.     

Studies on Nitrogen Metabolism in Tobacco Plants,B

Nitrate reductase from the leaves of Burley 21 tobacco (Nicotiana tabacum L.) was partially purified by ammonium sulfate fractionation, protamine sulfate treatment and calcium-phosphate gel adsorption.

The enzyme has optimum pH at 7.4 and is specific for reduced diphosphopyridine nucleotide (DPNH) as the electron donor. The nitrite formed increased in proportion to the rate at which DPNH disappeared in the reaction mixtures. Addition of flavin adenine dinucleo-tide (FAD) to the assay system enhanced the activity. FAD content in the “highly purified” enzyme was also determined. The enzyme was sensitive to heavy metals and SH-group inhibitors.

Discussions are presented on the metal and the properties of the enzyme in comparison to those published on other higher plants.     

Studies on Mannan in Wood Pulp

The glucomannan isolated from holocellulose pulp of Akamatsu (Pinus densiflora Sieb. et Zucc.) as its triacetate was methylated and the following methylated sugars were obtained by hydrolysis: the 2,3,4,6-tetra-O-methyl ethers of d-glucose and d-mannose (I part) and the 2,3,6-tri-O-methyl ethers of d-mannose and d-glucose (34–37 parts). Periodate oxidation of the glueomannan showed that 1.00 mole of periodate was consumed per mole of hexose unit and 3 moles of formic acid liberated for every 33 hexose units.     

Structure-activity Relationship of Herbicidal 2,3-Dicyano-5-Substituted Pyrazines

Sixty six 2,3-dicyano-5-substituted pyrazines were synthesized and their herbicidal activities against barnyard grass were measured in pot tests to clarify the relationship between chemical structure and activity. The activity of 59 derivatives was related parabolically to the hydrophobic substituent parameter at the 5-position of the pyrazine ring.     

Heme Requirement of a Novel Yeast-lysing Bacterium

A novel yeast-lysing bacterium (YLM-1) grew on nutrient agar containing yeast cell-free extract, but not on nutrient agar. We searched for the growth factor for YLM-1, which turned out to be hemin or hemoproteins, catalase or horseradish peroxidase. Also, the effects of growth accelerators and inhibitors on H₂O₂ metabolism were discussed.