A competition binding assay for determination of the inositol (1,4,5)-trisphosphate content of human leucocytes

We developed a competition binding assay for estimation of the intracellular inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) and optimalized it for the measurement of the Ins(1,4,5)P3 content of human blood leucocytes. The present method is considerably cheaper and requires five times fewer cells than the commercial Ins(1,4,5)P3 kit. The mean Ins(1,4,5)P3 content of human blood monocytes, granulocytes, and lymphocytes amounted to 3.3 +/- 1.2 microM, 3.1 +/- 1.4 microM, and 4.6 +/- 1.5 microM, respectively. After stimulation with formyl-methionyl-leucyl-phenylalanine (f-MLP) the Ins(1,4,5)P3 content of human granulocytes and monocytes increased 2-3 times within 10 sec and then gradually decreased, returning to basal values at 60 sec. Lymphocytes did not respond to f-MLP with an increase in their Ins(1,4,5)P3 content.     

C-C bond formation at the 3'-position of nucleosides by the use of their enol esters

Nucleosides having an enol ester structure in the sugar portion were synthesized and their reaction with several types of electrophiles were carried out. This furnished a new method for constructing C-C bond at the 3'-position.     

Preservation of plastic properties of synaptic transmission in long-lasting hippocampal slices under the effects of a peptide analog of piracetam, L-pGlu-D-Ala-NH2

The tetanic stimulation of the Schaffer collaterals (SC) in rat hippocamp slices after 6 hrs in vitro conditions did not produce long-term potentiation (LTP) of the field response amplitude in the CA1 pyramidal cell layer. In contrast, LTP after the late tetanization was well preserved in the slices that were perfused for 20 minutes with 0.5 mkM L-pGlu-D-Ala-NH2 (PGAA) after 4-4.5 hrs in vitro. There were no significant reactivity changes during the perfusion of the slices with this drug concentration. Two other drugs with nootropic activity, piracetam (100 mkM) and gamma-hydroxybutyrate (100 mkM, Na-salt) did not prevent the disappearance of LTP in the late period in vitro, while enhanced the reactivity during perfusion period. The maintenance of the plastic properties of the SC-CA1 synaptic transmission under the influence of PGAA is thought to be the result of some specific interaction of the drug with LTP induction mechanisms. LTP damaged in the late period in vitro might be a new model of memory disturbances and this model can turn out to be useful for the comparative estimation of the effectiveness of the drugs with proposed nootropic activity and for the analysis of the possible mechanisms of their action.     

Interactions of lipid micelles with blood proteins

Interaction of lipid micelles (LM), containing cholesterol and hydroxycholesterol, with human serum lipoproteins was investigated. It was shown that cholesterol-containing LM interact with low density lipoproteins (LDL). Selectivity of LM-LDL interaction depended on the cholesterol content of micelles and almost did not depend on the composition of LM core. Up to 90% of LDL were bound with cholesterol-saturated LM. By means of gel chromatography it was shown that interaction of cholesterol- and 7-hydroxycholesterol-containing micelles with serum led to the partial fusion of LDL with LM and LDL-LM complex formation, as well as to the cholesterol and 7-hydroxycholesterol transfer from micelles to LDL. The obtained results indicate that cholesterol-containing LM can be used for the delivery of oxidized cholesterol to cells involving LDL and receptor-dependent pathway of their capture by peripheral cells.     

The determination of the oxidation phenotype in inbred C57Bl/6 and BALB/c mice

Antipyrine oxidation was studied in C57BL/6 and BALB/c inbred mice. It was found that C57BL/6 are weak oxidant but BALB/c are strong oxidants of antipyrine. Animals F1 hybrids inherited the high capacity of antipyrine oxidation.     

Detection of a large cation-selective channel in nuclear envelopes of avian erythrocytes.

To determine whether the nuclear envelope of eukaryotic cells has the capability to regulate ion fluxes, we have used the patch-clamp technique to detect ion channels in this membrane system. Since possible sites for ion channels in the nuclear envelope include not only the nuclear pores, but also both the inner and outer nuclear membranes, we have patched giant liposomes composed of phosphatidylcholine and nuclear envelope fragments isolated from mature avian erythrocytes. A large, cation-selective channel with a maximum conductance of approximately 800 pS in symmetrical 100 mM KCl was detected. This channel is a possible candidate for a nuclear pore.     

Immunoassay employing surface-enhanced Raman spectroscopy

Surface-enhanced Raman scattering (SERS) was used to measure binding between biomolecules with mutual affinity, including antigen-antibody interactions. The conjugation of nitro groups onto bovine serum albumin enhanced their specific SERS activity 10(4)-fold. A dye, 2-[4'-hydroxyphenylazo]benzoic acid (HABA), with a major absorption at the Raman excitation frequency, demonstrated surface-enhanced resonance Raman scattering (SERRS) when captured from solution by avidin-coated silver films. Individual peak intensities showed a logarithmic relationship to the HABA concentration in solution over the range 10(-8) to 10(-5) M. Another resonance dye, p-dimethylaminoazobenzene (DAB) was covalently attached to an antibody directed against human thyroid stimulating hormone (TSH), without loss of antibody activity. The resultant conjugate was used in a sandwich immunoassay for TSH antigen: silver surfaces coated with anti-TSH antibody captured TSH antigen which in turn captured the DAB-anti-TSH antibody conjugate. A linear relationship was observed between the intensity of the resultant SERRS signals and the TSH antigen concentration over a range of from 4 to 60 microIU/ml. These results demonstrate the potential utility of the SERRS effect as a readout in a one-step, no wash immunoassay system.     

Quantification of the paternal allele bias for new germline mutations in the retinoblastoma gene

New germline mutations in the human retinoblastoma gene are known to arise preferentially on paternally derived chromosomes, but the magnitude of that bias has not been measured. We evaluated 49 cases with a new germline mutation and found that in 40 cases (82%) the mutation arose on the paternally derived allele. We also evaluated 48 cases likely to have a somatic initial mutation; in this group the initial mutation arose on paternal or maternal chromosomes with approximately equal frequency. There was no statistically significant difference in the average age of fathers of children with new paternal germline mutations from the average age of fathers of children with new maternal germline mutations or somatic initial mutations. Combining the data with that from previous reports from other groups, the proportion of new germline mutations arising on a paternally derived allele is 85% (based on 72 cases; 95% confidence interval = 76–93%). This number can be useful in the genetic counseling of some families with retinoblastoma. Received: 18 December 1996 / Accepted: 30 April 1997