Identification of bikunin as an endogenous inhibitor of dynorphin convertase in human cerebrospinal fluid

Dynorphin-converting enzymes constitute a group of peptidases capable of converting dynorphins to enkephalins. Through the action of these enzymes, the dynorphin-related peptides bind to delta-opioid instead of kappa-opioid receptors, leading to a change in the biological function of the neuropeptides. In this article, we describe the identification of the protein bikunin as an endogenous, competitive inhibitor of a dynorphin-converting enzyme in human cerebrospinal fluid. This protein is present together with its target enzyme in the same body fluids. The K(M) value of the convertase was found to be 9 microm, and the K(i) value of the inhibitor was 1.7 nm. The finding indicates that bikunin may play a significant role as a regulatory mechanism of neuropeptides, where one bioactive peptide is converted to a shorter sequence, which in turn, can affect the action of its longer form.     

The effect of aromatase inhibitor, fadrozole, on sGnRHa stimulated LH secretion in goldfish (Carassius auratus) and common carp (Cyprinus carpio)

The aromatase inhibitor, fadrozole, was applied to common carp and goldfish in order to examine its ability to increase the spontaneous and sGnRHa stimulated LH secretion. First, trials in goldfish in 2003 showed fadrozole's moderate ability to potentiate sGnRHa-stimulated LH secretion. However, this ability was much weaker than that obtained with dopamine antagonist, pimozide. There was no ovulation in fadrozole-treated fish. Several experiments on goldfish and carp during the two consecutive years with the different treatment regimes and doses of fadrozole did not confirm the optimistic results obtained in 2003. This shows that fadrozole is unable to replace the antidopaminergic drugs being used in fisheries practice.     

A Minimal Cysteine Motif Required to Activate the SKOR K+ Channel of Arabidopsis by the Reactive Oxygen Species H2O2

Reactive oxygen species (ROS) are essential for development and stress signaling in plants. They contribute to plant defense against pathogens, regulate stomatal transpiration, and influence nutrient uptake and partitioning. Although both Ca²⁺ and K⁺ channels of plants are known to be affected, virtually nothing is known of the targets for ROS at a molecular level. Here we report that a single cysteine (Cys) residue within the Kv-like SKOR K⁺ channel of Arabidopsis thaliana is essential for channel sensitivity to the ROS H₂O₂. We show that H₂O₂ rapidly enhanced current amplitude and activation kinetics of heterologously expressed SKOR, and the effects were reversed by the reducing agent dithiothreitol (DTT). Both H₂O₂ and DTT were active at the outer face of the membrane and current enhancement was strongly dependent on membrane depolarization, consistent with a H₂O₂-sensitive site on the SKOR protein that is exposed to the outside when the channel is in the open conformation. Cys substitutions identified a single residue, Cys¹⁶⁸ located within the S3 α-helix of the voltage sensor complex, to be essential for sensitivity to H₂O₂. The same Cys residue was a primary determinant for current block by covalent Cys S-methioylation with aqueous methanethiosulfonates. These, and additional data identify Cys¹⁶⁸ as a critical target for H₂O₂, and implicate ROS-mediated control of the K⁺ channel in regulating mineral nutrient partitioning within the plant.     

Loss of stearoyl-CoA desaturase 1 rescues cardiac function in obese leptin-deficient mice

The heart of leptin-deficient ob/ob mice is characterized by pathologic left ventricular hypertrophy along with elevated triglyceride (TG) content, increased stearoyl-CoA desaturase (SCD) activity, and increased myocyte apoptosis. In the present study, using an ob/ob;SCD1^−/− mouse model, we tested the hypothesis that lack of SCD1 could improve steatosis and left ventricle (LV) function in leptin deficiency. We show that disruption of the SCD1 gene improves cardiac function in ob/ob mice by correcting systolic and diastolic dysfunction without affecting levels of plasma TG and FFA. The improvement is associated with reduced expression of genes involved in FA transport and lipid synthesis in the heart, as well as reduction in cardiac FFA, diacylglycerol, TG, and ceramide levels. The rate of FA β-oxidation is also significantly lower in the heart of ob/ob;SCD1^−/− mice compared with ob/ob controls. Moreover, SCD1 deficiency reduces cardiac apoptosis in ob/ob mice due to increased expression of antiapoptotic factor Bcl-2 and inhibition of inducible nitric oxide synthase and caspase-3 activities. Reduction in myocardial lipid accumulation and inhibition of apoptosis appear to be one of the main mechanisms responsible for improved LV function in ob/ob mice caused by SCD1 deficiency.     

Enzymatic microreactors in chemical analysis and kinetic studies

The fields of application of microreactors are becoming wider every year. A considerable number of papers have been published recently reporting successful application of enzymatic microreactors in chemistry and biochemistry. Most are devices with enzymes immobilized on beads or walls of microfluidic channels, whilst some use dissolved enzymes to run a reaction in the microfluidic system. Apart from model systems, mostly with glucose oxidase, horseradish peroxidase and alkaline phosphatase, the principal fields of application of microreactors are tryptic digestion of proteins and polymerase chain reaction in automated analyses of proteomic and genetic material, respectively. Enzymatic microreactors also facilitate characterization of enzyme activity as a function of substrate concentration, and enable fast screening of new biocatalysts and their substrates. They may constitute key parts of lab-on-a-chip and muTAS, assisting the analysis of biomolecules. This review provides systematic coverage of examples of reports on enzymatic microreactors published recently, as well as relevant older papers.     

Will my protein crystallize? A sequence‐based predictor

We propose a machine-learning approach to sequence-based prediction of protein crystallizability in which we exploit subtle differences between proteins whose structures were solved by X-ray analysis [or by both X-ray and nuclear magnetic resonance (NMR) spectroscopy] and those proteins whose structures were solved by NMR spectroscopy alone. Because the NMR technique is usually applied on relatively small proteins, sequence length distributions of the X-ray and NMR datasets were adjusted to avoid predictions biased by protein size. As feature space for classification, we used frequencies of mono-, di-, and tripeptides represented by the original 20-letter amino acid alphabet as well as by several reduced alphabets in which amino acids were grouped by their physicochemical and structural properties. The classification algorithm was constructed as a two-layered structure in which the output of primary support vector machine classifiers operating on peptide frequencies was combined by a second-level Naive Bayes classifier. Due to the application of metamethods for cost sensitivity, our method is able to handle real datasets with unbalanced class representation. An overall prediction accuracy of 67% [65% on the positive (crystallizable) and 69% on the negative (noncrystallizable) class] was achieved in a 10-fold cross-validation experiment, indicating that the proposed algorithm may be a valuable tool for more efficient target selection in structural genomics. A Web server for protein crystallizability prediction called SECRET is available at http://webclu.bio.wzw.tum.de:8080/secret.     

Effect of selected factors on the effectiveness ofCapsicum annuum L. anther culture

The primary aim of the study was to establish the effectiveness of induced androgenesis in in vitro anther culture of two pepper (Capsicum annuum L.) breeding lines--ATZ1 and PO, and a hybrid between these two lines (ATZ1 x PO)F1. Anther culture was maintained according to the method developed by Dumas de Vaulx et al. (1981) with some modifications. The experiment revealed that the effectiveness of androgenesis ranged from 4 %; for the ATZ1 line to 1.5 %; for the (ATZ1 x PO)F1 and strongly depended on the developmental stage of flower buds, as well as the conditions for anther culture maintenance. The development of androgenic embryos was successfully induced only in anthers which originated from the flower buds with petals equal or slightly longer than sepals and there was a clear relationship between the length of the period of anther induction on CP medium and the level of kinetin in R1 regeneration medium.     

Diffraction to study protein and peptide assemblies

Proteins and peptides are able to self-assemble in vivo and in vitro. In vitro, this ability can be exploited to make bionanomaterials with many potential uses. Peptides are capable of forming a wide range of structures including fibres, tubules and scaffolds. In vivo, proteins assemble to form cellular fibrous proteins, as well as being involved in protein misfolding in disease. Recent advances using X-ray diffraction have highlighted the internal structure of self-assembled proteins and peptides, showing packing of side chain residues and have enabled a deeper understanding of mechanisms of assembly.