The proteins of the ejaculatory bulbs in different species of Drosophila

A comparative electrophoretic study or ejaculatory bulb proteins in 29 different Drosophila species has been carried out. In all analyzed species, ejaculatory bulb contains a major component (designated as PEB). It has molecular mass of 61-65 kDa in the species of virilis group, 33-36 kDa in species of obscura group, and 34-56 kDa in species of melanogaster group. Using immunoblotting technique, we have demonstrated that PEB is introduced into organs of female sex tract during mating. The nature and significance of revealed interspecific differences in PEB proteins has been discussed.     

A comparative study of cyclization strategies applied to the synthesis of head-to-tail cyclic analogs of a viral epitope.

A family of head-to-tail cyclic peptide models of the antigenic site A (G-H loop of viral protein 1) of foot-and-mouth disease virus has been designed on the basis of the three-dimensional structure adopted by the linear peptide YTASARGDLAHLTTT upon binding to neutralizing monoclonal antibodies. Three different methods of cyclization have been examined to access the peptides. Solution cyclization of a minimally protected linear precursor provided the expected products but required several purification steps that lowered the yields to approximately 10%. The two other approaches relied on side-chain anchoring of the peptide through the Asp residue and cyclization on the solid phase. A synthetic scheme combining Fmoc, tBu and OAI protections was practicable but inefficient when scaled-up. The combination of Boc, Bzl and OFm protections was more promising, but suffered from high epimerization during the initial esterification of Boc-Asp-OFm to benzyl alcohol-type resins. This problem was solved by performing the esterification via the cesium salt of Boc-Asp-OFm. With this improvement, the Boc/Bzl/OFm has become the method of choice for the preparation of cyclic head-to-tail peptides in satisfactory yields and with minimal purification.     

Role of Ca(2+) fluctuations in L6 myotubes in the regulation of the hexokinase II gene.

Expression of the hexokinase (HK) II gene in skeletal muscle is upregulated by electrically stimulated muscle contraction and moderate-intensity exercise. However, the molecular mechanism by which this occurs is unknown. Alterations in intracellular Ca(2+) homeostasis accompany contraction and regulate gene expression in contracting skeletal muscle. Therefore, as a first step in understanding the exercise-induced increase in HK II, the ability of Ca(2+) to increase HK II mRNA was investigated in cultured skeletal muscle cells, namely L6 myotubes. Exposure of cells to the ionophore A-23187 resulted in an approximately threefold increase in HK II mRNA. Treatment of cells with the extracellular Ca(2+) chelator EGTA did not alter HK II mRNA, nor was it able to prevent the A-23187-induced increase. Treatment of cells with the intracellular Ca(2+) chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester (BAPTA-AM) also resulted in an approximately threefold increase in HK II mRNA in the absence of ionophore, which was similar to the increase in HK II mRNA induced by the combination of BAPTA-AM and A-23187. In summary, a rise in intracellular Ca(2+) is not necessary for the A-23187-induced increase in HK II mRNA, and increases in HK II mRNA occur in response to treatments that decrease intracellular Ca(2+) stores. Depletion of intracellular Ca(2+) stores may be one mechanism by which muscle contraction increases HK II mRNA.     

A para-nitrophenol phosphonate probe labels distinct serine hydrolases of Arabidopsis

Activity-based protein profiling represents a powerful methodology to probe the activity state of enzymes under various physiological conditions. Here we present the development of a para-nitrophenol phosphonate activity-based probe with structural similarities to the potent agrochemical paraoxon. We demonstrate that this probes labels distinct serine hydrolases with the carboxylesterase CXE12 as the predominant target in Arabidopsis thaliana. The designed probe features a distinct labeling pattern and therefore represents a promising chemical tool to investigate physiological roles of selected serine hydrolases such as CXE12 in plant biology.     

Computational Investigations of the Chalcogen-Substituted Carboxylic Acids RC(=O)XH and RC(=X)OH and their Dimers [RC(=O)XH]2 and [RC(=X)OH]2 (X=S, Se, Te)

Semiempirical and ab initio theoretical methods have been used to investigate molecular structures of the chalcogen-substituted carboxylic acid isomers RC(=O)XH (chalcogenol acid) and RC(=X)OH (chalcogenon acid). A recent experimental report suggests that the chalcogenon isomers, although less stable at room temperature, predominate at low temperature in polar solvents and that there is only a small barrier to isomerization between the isomers. Theoretical calculations have been used to locate minimum energy structures of chalcogen-substituted carboxylic acid isomers and to calculate energy differences between pairs of isomers. Carboxylic acids are well known to dimerize, especially in the gas phase and in non-polar solvents. We have, therefore, also calculated energies of dimerization of the chalcogen-substituted acids by optimizing the geometries of the symmetric dimers. We note that the PM3 level of theory is only qualitatively correct for sulfur- and selenium-containing species but fails even qualitatively for the tellurium-containing compounds. Ab initio results confirm the experimental observations and provide good estimates of both isomerization and dimerization energies. We conclude that for many functional groups with tautomers RC(=X)YH and RC(=Y)XH, the more acidic tautomer is the one with the acid proton on the smaller, more electronegative atom, although in many cases this may not be the more stable tautomer.Electronic Supplementary Material available.     

Simulation methods with extended stability for stiff biochemical Kinetics

Background  

With increasing computer power, simulating the dynamics of complex systems in chemistry and biology is becoming increasingly routine. The modelling of individual reactions in (bio)chemical systems involves a large number of random events that can be simulated by the stochastic simulation algorithm (SSA). The key quantity is the step size, or waiting time, τ, whose value inversely depends on the size of the propensities of the different channel reactions and which needs to be re-evaluated after every firing event. Such a discrete event simulation may be extremely expensive, in particular for stiff systems where τ can be very short due to the fast kinetics of some of the channel reactions. Several alternative methods have been put forward to increase the integration step size. The so-called τ-leap approach takes a larger step size by allowing all the reactions to fire, from a Poisson or Binomial distribution, within that step. Although the expected value for the different species in the reactive system is maintained with respect to more precise methods, the variance at steady state can suffer from large errors as τ grows.     

Typing of Genetic Markers Involved in Stress Response by Fluorescent cDNA-Amplified Fragment Length Polymorphism Technique

Identification of genetic markers involved in stress response to physical factors or chemical substances in organisms is a challenging task. Typing of upregulated gene expression due to selective antibacterial pressure is a promising approach in the search of molecular mechanisms responsible for development of resistance. cDNA-Fluorescent Amplified Fragment Length Polymorphism (cDNA-FAFLP) strategy was developed and applied in the search of antimycotic drug resistance marker(s) in medically important fungi as an alternative method to microarray analysis. We compared differential gene expression of two sensitive Candida albicans reference strains (ATCC 10231 and ATCC 60133) and two of their paired resistant to fluconazole and itraconazole mutants. Resistant mutants Candida albicans FLC-R, resistant to fluconazole (MIC > 128 μg/ml) and Candida albicans ICZ-R, resistant to itraconazole (MIC > 4 μg/ml) were obtained in subcultures with gradual increase of the antifungal in the culture medium. cDNA-AFLP profile in both itraconazole resistant mutants showed specific spectrophotometric peaks with 5–6-fold RNA overexpression product of 500 bp length compared to the sensitive strains. Fluconazole mutants do not reveal RNA level changes under tested by us typing conditions. These results indicate that the cDNA-FAFLP strategy is a relatively rapid, simple, and reliable method for simultaneous typing of both constitutive and induced differences in expression of host genes providing insight into the biological processes involved in response to drugs in bacteria and fungi. Moreover, this methodology could be tested for typing of the genome response of any organism to physical or chemical stress factors.