Heavy metals in phyto- and zooplankton of Lake Kenon (Transbaikalia)

The content and accumulation of particular heavy metals in phyto- and zooplankyon from the technogenically transformed Lake Kenon (Chita, Transbaikalia) have been studied. Algae contains the highest concentrations of Zn and Sr. Zooplankton is characterized by concentrations of Ni, Cu, Cd, and Hg either below or equivalent to those known for other water bodies. It has been shown that the distribution of heavy metals in the studied water body is differentiated. Their greatest concentration has been registered in the plankton of technogenically contaminated sites—CHPP and Oil Tank Farm.     

The effect of antibodies against Ca2(+)-ATPase and its hydrophobic fragment on the activity of this enzyme and on the passive transport of Ca2(+)]

It is shown that both Ca(2+)-ATPase and its hydrophobic fragment are immunogenic factors. A region between hydrophobic and hydrophilic parts of Ca(2+)-ATP-ase is immunogenic. These antibodies clearly inhibit inflow and outflow of Ca2+ through a hydrophobic fragment reconstructed into liposomes and do not influence Ca(2+)-ATPase activity.     

An improved real time PCR method for simultaneous detection of C282Y and H63D mutations in the HFE gene associated with hereditary hemochromatosis

HFE-linked hereditary hemochromatosis (HH) is one of the most common inherited diseases among individuals of Northern European ancestry. Two sites of point mutations in the HFE gene--C282Y and H63D--are associated with greater than 90% of HH cases. We have developed a sensitive real time PCR (TaqMan) 5'-nuclease assay for single nucleotide polymorphism (SNP) detection using novel DNA chemistry, and successfully applied this method to detect these mutations. Fluorogenic PCR probes, chemically modified with a minor groove binding agent to increase duplex stability, were used in single and multiplex probe closed tube formats. The probes were tested in two commercially available thermocycling fluorimeters (the Light Cycler and the ABI Prism 7700). Comparison of the results obtained from the analysis of 43 samples showed no discrepancies between our 5' nuclease assay and the restriction length polymorphism analysis, which is routinely used in hospitals. The reported real time PCR technology is ideal for the clinical setting as it is sensitive, eliminates the labor and supply costs of post-PCR steps, reduces the risk of crossover contamination, minimizes sources of error, and can be fully automated.     

Effect of synthetic cationic peptides on activation of the adenylyl cyclase signaling system by biogenic amines in muscle tissue of molluscs and rats

The hormone-sensitive adenylyl cyclase signaling system (ACS), made of serpentine receptor, heterotrimeric G-protein and enzyme adenylyl cyclase (AC), regulates a wide spectrum of growth and metabolic processes in the cell. Molecular mechanisms of functional coupling of ACS components still remain obscure. We examined the influence of synthetic cationic peptides Ac-Ala-His(Ala)2-His-Ala-NH2 (I), Ac-Ala-His-(Ala)3-His-(Ala)2-His-Ala-NH2 (II), and Ac-(Pro)2-His-(Ala)2-His-(Ala)3-His-(Ala)2-His-Ala-NH2 (III) on the basal AC activity and that stimulated by nonhormonal (NaF) and hormonal reagents (serotonin--molluscs, beta-isoproterenol--rats) in smooth muscles of the freshwater bivalve molluscs Anodonta cygnea and in skeletal muscles of rats. Peptides II and III (the latter more effective) were shown to decrease hormone-stimulated AC activity in both tissues, in a dose-dependent manner. Peptide III strongly reduced NaF stimulating effect to AC, which suggests the involvement of this peptide in the functional coupling of both receptors with G-proteins, and of G-proteins with AC. A correlation was found between the efficacy of peptide action on the functional activity of ACS components and peptide length. As shown by IR-spectroscopy, in water all peptides can form helical structures. However, alpha-helicity of peptides I and II was higher than that of peptide III, which does not conform to a power series in efficacy of these peptides. Thus, it is the length of cationic peptides that plays a key role in hormonal regulation of the functional activity of ACS, especially on the step of receptor-G-protein coupling.     

Primers with 5' flaps improve real-time PCR

Primers that contain portions noncomplementary to the target region are usually used to add to the PCR product a utility sequence such as a restriction site or a universal probe binding site. We have demonstrated that primers with short 5'AT-rich overhangs increase real-time PCR fluorescent signal. The improvement is particularly significant for difficult to amplify templates, such as highly variable viral sequences or bisulfite-treated DNA.     

Antibodies to Streptococcus pneumoniae antigens in newborn infants

The levels of antibodies to capsular polysaccharide antigens of pneumococci (serotypes 1, 3, 6B, 8, 9N, 15F, 23F), C-polysaccharide and protein antigen of pneumococci in the blood sera of 38 newborn infants at the moment of their birth (umbilical blood) and on the 5th or 6th day of their life, in their mothers' blood sera, as well as in the colostrum and milk of 48 nursing women, have been studied by means of the enzyme immunoassay. The study showed that in the normal course of pregnancy antibodies to pneumococci were transferred transplacentally from the mother to the fetus. Though in most cases their content in the blood of newborn infants was lower than that in maternal blood, it exceeded the average level of antipneumococcal antibodies in children aged 3-12 months. In the milk of nursing mothers considerable amount of IgA antibodies to pneumococci was detected, which might be an additional protective factor with respect to pneumococcal infection in infants.     

Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder.

The tripeptide 1,2-dihydro-(3H)-pyrrolo[3,2-e]indole-7-carboxylate (CDPI3) binds to the minor groove of DNA with high affinity. When this minor groove binder (MGB) is conjugated to the 5'-end of short oligodeoxynucleotides (ODNs), the conjugates form unusually stable hybrids with complementary DNA in which the tethered CDPI3group resides in the minor groove. We show that these conjugates can be used as PCR primers. Due to their unusually high binding affinity, conjugates as short as 8-10mers can be used to amplify DNA with good specificity and efficiency. The reduced length primers described here might be appropriate for the PCR amplification of viral sequences which possess a high degree of variability (e.g., HPV, HIV) or for recent techniques such as gene hunting and differential display which amplify multiple sequences using short primer pairs.     

Sequence-specific targeting and covalent modification of human genomic DNA.

We compare two techniques which enable selective, nucleotide-specific covalent modification of human genomic DNA, as assayed by quantitative ligation- mediated PCR. In the first, a purine motif triplex-forming oligonucleotide with a terminally appended chlorambucil was shown to label a target guanine residue adjacent to its binding site in 80% efficiency at 0.5 microM. Efficiency was higher in the presence of the triplex-stabilizing intercalator coralyne. In the second method, an oligonucleotide targeting a site containing all four bases and bearing chlorambucil on an interior base was shown to efficiently react with a specific nucleotide in the target sequence. The targeted sequence in these cases was in the DQbeta1*0302 allele of the MHC II locus.