The action of catecholamine-synthesis inhibitors and of spiperone on sea urchin and mouse embryos

We studied the effects of three inhibitors of catecholamine synthesis on the development of sea urchins Sphaerechinus granularis and Paracentrotus lividus. These drugs affected the early embryogenesis, which was expressed in inhibition of the cleavage divisions, appearance of abnormal embryos, and developmental arrest. The addition of arachidonic acid amide and dopamine to the incubation medium weakened the effects of the inhibitors. Spiperone induced developmental defects in preimplantation mouse embryos and sea urchin embryos. Arachidonic acid amide with dopamine exerted a protective effect against spiperone when introduced to sea urchin embryos at the blastula or late gastrula stages, rather than after fertilization. In murine embryos, this amide induced developmental defects and arrest itself and its effect was reversible. Possible mechanisms underlying the effects of these drugs are discussed.     

Effect of inhibitors of synthesis of dopamine and its antagonists on Hydra attenuata regeneration

Obvious inhibition of the hydra regeneration with no subsequent morphological abnormalities, was shown to occur when using alpha-methylthyrosine and 3 Jthyrosine. alpha-Methyldopa induced a slight inhibition but a considerable morphological change: ectopic tentacles, projections, bipolar forms in the gastric fragment. The apical and basal fragments did not suffer. The role of neurotransmitters in the hydra morphogenesis is discussed.     

A genetically encoded indicator for monitoring intracellular chloride

Inhibitory drives in the nervous system are provided by synapses operating mostly through Cl⁻ ion channels. We describe a novel CFP-YFP-based fluorescence Cl⁻ indicator, Cl-sensor, characterized by a high sensitivity, which can be successfully used as a tool for monitoring intracellular Cl⁻ in various biological preparations. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 380–381, July–October, 2007.     

Kinetic constant variability in bacterial oxidation of elemental sulfur

Wide ranges of growth yields on sulfur (from 2.4 x 10(10) to 8.1 x 10(11) cells g(-1)) and maximum sulfur oxidation rates (from 0.068 to 1.30 mmol liter(-1) h(-1)) of an Acidithiobacillus ferrooxidans strain (CCM 4253) were observed in 73 batch cultures. No significant correlation between the constants was observed. Changes of the Michaelis constant for sulfur (from 0.46 to 15.5 mM) in resting cells were also noted.     

Tyrosine phosphorylation regulates maturation of receptor tyrosine kinases

Constitutive activation of receptor tyrosine kinases (RTKs) is a frequent event in human cancer cells. Activating mutations in Fms-like tyrosine kinase 3 (FLT-3), notably, internal tandem duplications in the juxtamembrane domain (FLT-3 ITD), have been causally linked to acute myeloid leukemia. As we describe here, FLT-3 ITD exists predominantly in an immature, underglycosylated 130-kDa form, whereas wild-type FLT-3 is expressed predominantly as a mature, complex glycosylated 150-kDa molecule. Endogenous FLT-3 ITD, but little wild-type FLT-3, is detectable in the endoplasmic reticulum (ER) compartment. Conversely, cell surface expression of FLT-3 ITD is less efficient than that of wild-type FLT-3. Inhibition of FLT-3 ITD kinase by small molecules, inactivating point mutations, or coexpression with the protein-tyrosine phosphatases (PTPs) SHP-1, PTP1B, and PTP-PEST but not RPTPalpha promotes complex glycosylation and surface localization. However, PTP coexpression has no effect on the maturation of a surface glycoprotein of vesicular stomatitis virus. The maturation of wild-type FLT-3 is impaired by general PTP inhibition or by suppression of endogenous PTP1B. Enhanced complex formation of FLT-3 ITD with the ER-resident chaperone calnexin indicates that its retention in the ER is related to inefficient folding. The regulation of RTK maturation by tyrosine phosphorylation was observed with other RTKs as well, defines a possible role for ER-resident PTPs, and may be related to the altered signaling quality of constitutively active, transforming RTK mutants.     

All three Ca2+-binding loops of photoproteins bind calcium ions: the crystal structures of calcium-loaded apo-aequorin and apo-obelin

The crystal structures of calcium-loaded apo-aequorin and apo-obelin have been determined at resolutions 1.7A and 2.2 A, respectively. A calcium ion is observed in each of the three EF-hand loops that have the canonical calcium-binding sequence, and each is coordinated in the characteristic pentagonal bipyramidal configuration. The calcium-loaded apo-protein retain the same compact scaffold and overall fold as the unreacted photoproteins containing the bound substrate, 2-hyroperoxycoelenterazine, and also the same as the Ca2+-discharged obelin bound with product, coleneteramide. Nevertheless, there are easily discerned shifts in both helix and loop regions, and the shifts are not the same between the two proteins. It is suggested that these photoproteins to sense Ca2+ concentration transients and to produce their bioluminescence response on the millisecond timescale. A mechanism of intrastructural transmission of the calcium signal is proposed.     

The development of a continuous isothermal titration calorimetric method for equilibrium studies

A continuous isothermal titration calorimetry (cITC) method for microcalorimeters has been developed. The method is based on continuous slow injection of a titrant into the calorimetric vessel. The experimental time for a cITC binding experiment is 12-20 min and the number of data points obtained is on the order of 1000. This gives an advantage over classical isothermal titration calorimetry (ITC) binding experiments that need 60-180 min to generate 20-30 data points. The method was validated using two types of calorimeters, which differ in calorimetric principle, geometry, stirring, and way of delivering the titrant into the calorimetric vessel. Two different experimental systems were used to validate the method: the binding of Ba(2+) to 18-crown-6 and the binding of cytidine 2'-monophosphate to RNAse A. Both systems are used as standard test systems for titration calorimetry. Computer simulations show that the dynamic range for determination of equilibrium constants can be increased by three orders of magnitude compared to that of classical ITC, making it possible to determine high affinities. Simulations also show an improved possibility to elucidate the actual binding model from cITC data. The simulated data demonstrate that cITC makes it easier to discriminate between different thermodynamic binding models due to the higher density of data points obtained from one experiment.     

Variation in seed protein and isoenzyme patterns in Cucurbita cultivars

The genetic variability in the seed proteins and the enzyme alcohol dehydrogenase (ADH) in representative species of the genus Cucurbita was studied. The banding patterns were obtained by means of vertical block electrophoresis in polyacrylamide gel. A specific protein components and ADH isoenzymes were established in the polymorphic banding patterns which can be applied individually or in combination as potential biochemical markers for breeding purposes.     

Changes in the connexin 26 (GJB2) gene in Russian patients with hearing disorders: results of long-term molecular diagnostics of hereditary nonsyndromic deafness

Search for mutations in the connexin 26 gene (GJB2) is a routine molecular-genetic analysis ofthe hereditary deafness worldwide. However, till now there is no assessment of the diagnostic significance of this analysis for Russian patients, and there are difficulties in interpretation of the results of DNA diagnostics. In the present study, a sample of 705 patients with nonsyndromic autosomal recessive deafness from different regions of Russian Federation was investigated. A portion of deafness like DFNB1 caused by mutations in the GJB2 gene among the sample was 46%. The frequency of deafness of such genetic type was 1:1000, that is, the frequency of isolated autosomal recessive deafness was 1:500 in the population. It was found that each sixteenth individual in Russia is a heterozygous carrier of the mutation in the GJB2gene. Totally, 20 pathological GJB2 alleles were detected; among them, a c.35delG mutation with the allelic frequency 81% prevails. Six most frequent mutations (c.35delG, c.313_326de114, c.-23+1G>A (IVS1+1G>A), c.235delC, c.167delT, and p.Glul20del), which account for 95% of pathological GJB2 alleles, were detected. Mutations previously not described in the GJB2 gene (c.129delG, p.Gly200Arg, and c[Arg127His, Gly160Ser]) were found. An optimal algorithm of molecular investigation of Russian patients which detects up to 100% of mutations in the GJB2 gene was suggested. Data concerning a clinical significance of p.Met34Thr and p.Va137Ile mutations are confirmed in the study. Eight polymorphic substitutions in the GJB2gene which do not have clinical significance (p.Va127Ile, c.*3C>A, p.Va115311e, p.Gly160Ser, c.Arg127His, p.Glull4Gly (c.341A>G), c.-45C>A, and p.Ala149Thr) were also detected.