A novel conus peptide ligand for K+ channels

Voltage-gated ion channels determine the membrane excitability of cells. Although many Conus peptides that interact with voltage-gated Na(+) and Ca(2+) channels have been characterized, relatively few have been identified that interact with K(+) channels. We describe a novel Conus peptide that interacts with the Shaker K(+) channel, kappaM-conotoxin RIIIK from Conus radiatus. The peptide was chemically synthesized. Although kappaM-conotoxin RIIIK is structurally similar to the mu-conotoxins that are sodium channel blockers, it does not affect any of the sodium channels tested, but blocks Shaker K(+) channels. Studies using Shaker K(+) channel mutants with single residue substitutions reveal that the peptide interacts with the pore region of the channel. Introduction of a negative charge at residue 427 (K427D) greatly increases the affinity of the toxin, whereas the substitutions at two other residues, Phe(425) and Thr(449), drastically reduced toxin affinity. Based on the Shaker results, a teleost homolog of the Shaker K(+) channel, TSha1 was identified as a kappaM-conotoxin RIIIK target. Binding of kappaM-conotoxin RIIIK is state-dependent, with an IC(50) of 20 nm for the closed state and 60 nm at 0 mV for the open state of TSha1 channels.     

Feedback regulation of the Bradyrhizobium japonicum nodulation genes

Lipochitin Nod signals are produced by rhizobia and are required for the establishment of a nitrogen-fixing symbiosis with a legume host. The nodulation genes encode products required for the synthesis of this signal and are induced in response to plant-produced flavonoid compounds. The addition of chitin and lipo-chitin oligomers to Bradyrhizobium japonicum cultures resulted in a significant reduction in the expression of a nod–lacZ fusion. Intracellular expression of NodC, encoding a chitin synthase, also reduced nod gene expression. In contrast, expression of the ChiB chitinase increased nod gene expression. The chain length of the oligosaccharide was important in feedback regulation, with chitotetraose molecules the best modulators of nod gene expression. Feedback regulation is mediated by the induction of nolA by chitin, resulting in elevated levels of the repressor protein, NodD2.     

Biotransformation kinetics of Pseudomonas putida for cometabolism of phenol and 4-chlorophenol in the presence of sodium glutamate

A kinetic model to describe the degradation of phenol and cometabolictransformation of 4-chlorophenol (4-cp) in the presence of sodium glutamate(SG) has been developed and validated experimentally. The integrated modelaccounts for cell growth, toxicity of 4-cp, cross-inhibitions among the threesubstrates, and the different roles of the specific growth substrate (phenol)and the conventional carbon source (SG) in the cometabolism of 4-cp. In thisternary substrate system, the overall phenol degradation and 4-cp transformation rates are greatly enhanced by the addition of SG since SG is able to attenuate the toxicity of 4-cp and therefore increase the cell growth rate. Model analysis indicates that the maximum specific degradation rate of phenol (0.819 mg (mg.h)^-1) is lowered by SG by up to 46% whereas the specific transformation rate of 4-cp is notdirectly affected by the presence of SG. The competitive inhibition coefficient of 4-cp to phenol degradation (K_i,cp) and that of phenol to 4-cp transformation (K_i,ph) were determined to be 6.49 mg l^-1 and 0.193 mg l^-1, respectively, indicatingthat phenol imposes much larger competitive inhibition to 4-cp transformation than the converse. The model developed can simultaneously predict phenol degradation and 4-cp transformation, and is useful for dealing with cometabolism involving multiple substrates.     

Carboxypeptidase E, a prohormone sorting receptor, is anchored to secretory granules via a C-terminal transmembrane insertion.

Carboxypeptidase E (CPE) is a sorting receptor that directs the prohormone pro-opiomelanocortin (POMC) to the regulated secretory pathway, and is also a prohormone processing enzyme in neuro/endocrine cells. It has been suggested that the 25 C-terminal amino acids are necessary for the binding of CPE to secretory granule membranes, but its orientation in the membrane is not known. In this study, we examined the structure and orientation of the membrane-binding domain at the C-terminus of CPE. In vitro experiments using model membranes demonstrated that the last 22 amino acids of CPE (CP peptide) insert in a shallow orientation into lipid bilayers at low pH. Circular dichroism analysis indicated that the CP peptide adopts a partial alpha-helical configuration at low pH, and helix content increases when it is bound to lipid. Protease protection experiments, immunolabeling, and immunoisolation of intact secretory granules with a C-terminal antibody revealed a cytoplasmic domain in CPE, consistent with a transmembrane orientation of this protein. We conclude that the membrane-binding domain of CPE must adopt an alpha-helical configuration to bind to lipids, and that CPE may require another integral membrane "chaperone" protein to insert through the lipid bilayer in a transmembrane fashion.     

Silver staining of proteins on electroblotting membranes and intensification of silver staining of proteins separated by polyacrylamide gel electrophoresis

A fast and convenient method for silver staining of proteins on electroblotting membranes was developed based on Gallyas' histochemical intensifier and applied to human endothelial cell proteins separated by one- and two-dimensional electrophoresis and electroblotted to polyvinyl difluoride membranes. The method allowed detection of proteins on membranes with a sensitivity equal to the sensitivity of the most sensitive silver-staining protocols for electrophoresis gels. Also, the method was compatible with preceding immunostaining on the same membrane. Furthermore, an intensifying method for proteins in silver-stained SDS-PAGE gels was developed based on Gallyas' histochemical intensifier. This method was applied to proteins separated by one- and two-dimensional gel electrophoresis and visualized by one of several silver-staining methods. Maximal intensification was achieved for the less sensitive but fast acidic silver-staining protocols, but even for the very sensitive alkaline protocols a significant increase in signal to noise ratio was obtained. In particular, negatively stained or invisible proteins on the silver-stained gels were found to be visualized by the Gallyas stain. Proteins from silver-stained and Gallyas-stained gels were identified by mass spectrometry, and the intensification procedure was fully compatible with mass spectrometry.     

Iron stress responses in the cyanobacterium Synechococcus sp. PCC7942

In the present study, we describe the sequential events by which the cyanobacterium Synechococcus sp. PCC 7942 adapts to iron deficiency. In doing so, we have tried to elucidate both short and long-term acclimation to low iron stress in order to understand how the photosynthetic apparatus adjusts to low iron conditions. Our results show that after an initial step, where CP43' is induced and where ferredoxin is partly replaced by flavodoxin, the photosynthetic unit starts to undergo major rearrangements. All measured components of Photosystem I (PSI), PSII and cytochrome (Cyt) ƒ decrease relative to chlorophyll (Chl) a . The photochemical efficiencies of the two photosystems also decline during this phase of acclimation. The well-known drop in phycobilisome content measured as phycocyanin (PC)/Chl was not due to an increased degradation, but rather to a decreased rate of synthesis. The largest effects of iron deficiency were observed on PSI, the most iron-rich structure of the photosynthetic apparatus. In the light of the recent discovery of an iron deficiency induced CP43' ring around PSI a possible dual function of this protein as both an antenna and a quencher is discussed. We also describe the time course of a blue shift in the low temperature Chl emission peak around 715 nm, which originates in PSI. The shift might reflect the disassembly and/or degradation of PSI during iron deficiency and, as a consequence, PSI might under these conditions be found predominantly in a monomeric form. We suggest that the observed functional and compositional alterations represent cellular acclimation enabling growth and development under iron deficiency, and that growth ceases when the acclimation capacity is exhausted. However, the cells remain viable even after growth has ceased, since they resumed growth once iron was added back to the culture.