Mitochondrial DNA and bindin gene sequence evolution among allopatric species of the sea urchin genus Arbacia

Sea urchins of the genus Arbacia (order Stirodonta) have discontinuous allopatric distributions ranging over thousands of kilometers. Mitochondrial DNA (mtDNA) sequences were used to reconstruct phylogenetic relationships of four Arbacia species and their geographic populations. There is little evidence of genetic structuring of populations within species, except in two cases at range extremes. The mtDNA sequence differentiation between species suggests that divergence occurred about 4-9 MYA. Gene sequences encoding the sperm protein bindin and its intron were obtained and compared with the mtDNA phylogeny. Sea urchins among the well-studied echinoid order Camarodonta, with degrees of mtDNA divergence similar to those of Arbacia species, are known to have remarkable variation in bindin. However, in Arbacia, little variation in deduced amino acid sequences of bindin was found, indicating that purifying selection acts on the protein. In contrast, bindin intron sequences showed much differentiation, including numerous insertion/deletions. Fertilization experiments performed between a divergent pair of Arbacia species from the Atlantic and Pacific Oceans revealed no evidence of blocks to gamete recognition. In Arbacia, fertilization specificities may have evolved relatively slowly as a result of extensive gene flow within species, greater functional constraint on the bindin polypeptide, or reduced selective pressure for species recognition in singly occurring species.      

Females of the sea urchin Strongylocentrotus purpuratus differ in the structures of their egg jelly sulfated fucans

The egg jelly coats of sea urchins contain sulfated fucans which bind to a sperm surface receptor glycoprotein to initiate the signal transduction events resulting in the sperm acrosome reaction. The acrosome reaction is an ion channel regulated exocytosis which is an obligatory event for sperm binding to, and fusion with, the egg. Approximately 90% of individual females of the sea urchin Strongylocentrotus purpuratus spawned eggs having only one of two possible sulfated fucan electrophoretic isotypes, a slow migrating (sulfated fucan I), or a fast migrating (sulfated fucan II) isotype. The remaining 10% of females spawned eggs having both sulfated fucan isotypes. The two sulfated fucan isotypes were purified from egg jelly coats and their structures determined by NMR spectroscopy and methylation analysis. Both sulfated fucans are linear polysaccharides composed of 1-->3-linked alpha-L-fucopyranosyl units. Sulfated fucan I is entirely sulfated at the O -2 position but with a heterogeneous sulfation pattern at O -4 position. Sulfated fucan II is composed of a regular repeating sequence of 3 residues, as follows: [3-alpha-L-Fuc p - 2,4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)-1-->3-alpha-L-Fuc p -4(OSO3)- 1]n. Both purified sulfated fucans have approximately equal potency in inducing the sperm acrosome reaction. The significance of two structurally different sulfated fucans in the egg jelly coat of this species could relate to the finding that the sperm receptor protein which binds sulfated fucan contains two carbohydrate recognition modules of the C-type lectin variety which differ by 50% in their primary structure.      

Effect of thallium of ATP-ase activity and transmembrane potential in bacteria

Effect of thallium (TlNO3) on the ATPase activity and transmembrane potential (Dj) of bacteria with different levels of resistance to this metal has been studied. The hypothesis has been made that the resistance biochemical mechanism is based on the energy transformation systems in the cell.     

Effect of n-chloromercuribenzoate and albumin on Mg2+, Ca2+-ATPase activity of plasma membranes of smooth muscle cells

It is shown that Mg2+, Ca2+-ATPase activity of plasma membrane fragments from the rat small intestine myocytes is inhibited by p-chloromercuribenzoate and dithionitrobenzoate (50 and 90%, respectively). The effect of p-chloromercuribenzoate inhibition is removed by serum albumin promoting a rise in the ATPase activity of the plasma membranes and shifting the temperature maximum point up to 32 degrees C (in the norm the maximum is observed at 36 degrees C). According to the data presented, albumin changes the composition and phase state of the lipid surrounding of the membrane enzymes.     

Selection of the optimal conditions for the procurement and regeneration of protoplasts of the industrial strain of Streptomyces rimosus, the producer of oxytetracycline, and the effect of the protoplasting process on the antibiotic activity]

Optimal conditions for protoplasting of the Streptomyces rimosus industrial strain No. 1 producing oxytetracycline were developed. Observation of the early stages of the protoplast regeneration in microchambers showed that there were two regeneration types: normal and anomalous. The latter was likely defined by the glycine effect on cell wall synthesis. It was accompanied by the stage in which the protoplasts had the form of multiplying protoplast-like cells. The protoplasting of the S. rimosus culture producing oxytetracycline resulted in an increase in the variability of an antibiotic producing property and the frequency of low active variants.     

Obtaining of bacterial bioluminescent strain Protobacterium phosphoreum B7071 (lux+) for the determination of zinc ion concentration

The transconjugate containing hybrid plasmid (Te(R)Zn(R)lux+) was obtained using the conjugation method on Pseudomonasfragi T2(5) (Te(R)ZnR) strain and bioluminescent strain Protobacterium phosphoreum B7071 (lux+). The expression regulation of lux-genes on the obtained plasmid is carried out by promotor-operational area conjointly with zinc resistance genes. The cells of the obtained genetically modified bacteria have the ability to specific induced luminescence, which is a respond to zinc ions' presence in the measuring medium. It was shown that the cells' bioluminescence intensity of trans-conjugate is linearly dependent on zinc ions' concentration within the range of 1-100 microM, that provides the opportunity of using biosensor as a strain for qualitative and quantitative detection of the metal. The low sensitivity limit of this method is 0.5 microM for the metal. Besides having high sensitivity, the developed lux-biosensor is highly specified.     

Extraction of plasma membranes from smooth muscle cells of the rabbit small intestine

A method is offered for isolation of subcellular fractions from small intestinal smooth muscle cells enriched by plasma membranes (PM). The method is based on differential centrifugation over sucrose density gradient. According to the localization of marker enzymes, the membrane fraction obtained with the use of 30% sucrose is considered to be optimal. The PM fraction is superior to the homogenate 10-fold on the average in the magnitude of Na, K-ATPase, 17-fold in Mg2+-ATPase, and 15-fold in that of 5'-nucleotidase activity. ATPase of PM is activated by Ca2+ in micro- and millimolar concentrations. It is suggested that Mg2+-dependent Ca-activated ATPase of PM is related to the Ca2+ content control in the cell.     

Effect of Arsenic on Bacterial Growth and Plasma Membrane ATPase Activity

The effects of arsenic in the forms of arsenite and arsenate on bacterial growth and plasma membranes' ATPase activity was studied. Correlation of the rate of ATP hydrolysis was found to be correlated with bacterial resistance to toxic arsenic ions. Detoxification of arsenate by resistant cultures of bacteria was suggested to be related to an increase in bacterial ATPase activity and the degree of ATPase mobilization.     

Inhibitory effect of lead ions on cells of some strains of Pseudomonas species bacteria

The inhibition effect of ionic lead on membrane ATPase activity, transmembrane potential (delta psi) and permeability level of the Pb-sensitive P. fluorescens B894 and Pb-resistant P. fluorescens B4252 bacteria cells have been studied. It have been shown that decreasing ATPase activity and transmembrane potential values and the increasing of permeability by lead are higher for Pb-sensitive strain then for Pb-resistant. It is suggested that mechanism of the ionic lead toxic effect deals with plasma membrane biochemical parameters (ATPase activity, value of delta psi) alterations and interruption of it barrier function.