Molecular cloning of an amino acid-regulated mRNA (amino acid starvation-induced) in rat hepatoma cells

Using the combination of a subtracted library and differential hybridization, a 409-base pair cDNA was identified that corresponds to a mRNA that is induced 2-3-fold when rat Fao hepatoma cells are subjected to amino acid starvation for 12 h. While this mRNA species was induced during starvation, others such as beta-actin, Cu-Zn superoxide dismutase, glyceraldehyde-3-P, and histone H4 were decreased in abundance to 25-50% of their original levels. The induction of the amino acid starvation-induced (ASI) mRNA was repressed when starved cells were returned to a medium supplemented with amino acids. Tissue distribution analysis showed the ASI mRNA, approximately 650 base pairs in length, to be present in every rat tissue tested. The cDNA clone has been sequenced and appears to correspond to the 3'-most end of the mRNA. The cDNA sequence includes the poly(A) tail, two potential polyadenylation signal sequences, and an open reading frame that we presume to be a portion of the coding sequence. The ASI cDNA will be used to investigate the molecular mechanisms for amino acid-dependent regulation of protein expression by mammalian cells.     

Analysis of shear wave propagation in skin; application to an experimental procedure

An analysis of an experimental procedure used to determine age-related changes in the skin was performed. The experiment is based on the propagation of a shear wave through the skin. The analysis of the experiment was used to determine the effects of parameters such as properties of the skin and subcutaneous fat, skin thickness and experimental conditions on the propagation. Computer simulations showed that at lower frequencies (less than 1000 Hz), measured results depended strongly on skin thickness, while at higher frequencies (2000 Hz) the results were relatively insensitive to both skin thickness and the properties of the subcutaneous fat. Results also depended upon the distance between the applied shear disturbance and the measurement point. The analysis suggests that higher frequency (2000 Hz) experimental results are more indicative of mechanical properties of skin than lower frequency (less than 1000 Hz) results.     

Activation of endogenous lipid peroxidation in the brain in oxidation stress caused by administration of iron and its prevention by vitamin E

Looking for an appropriate model of accelerated aging in vivo we investigated the content of endogenous products of lipid peroxidation (LP) in the rat brain after single or 4 day-lasting intramuscular injection of complex-bind iron (ferum Hausman, 50 mg/kg body weight) like promoter of LP. We found that the single administration of this iron complex fails to induce endogenous LP; after 4 day-application of iron we observed significant increase in content of primary (lipid peroxides) and final (fluorescent) products of LP. Iron-promoted activation of endogenous LP could be abolished by animal pretreatment with the natural antioxidant alpha-tocopherol. The calcium antagonist nifedipine didn't affect the content of endogenous LP products neither alone nor in combination with alpha-tocopherol.     

Side reaction in peptide synthesis. Formation of oxazolidone derivatives

2-Oxazolidone derivatives formed through an intramolecular reaction in the process of alkaline treatment of urethane-type N-protected peptides of which the N-terminal residues were Ser or Thr having unprotected hydroxyl groups. In oder to avoid this side reaction, the esters of these peptides could be cleaved by enzymatic hydrolyses instead of saponification.     

A radioimmunoprecipitation method in the serodiagnosis of HIV infection: the development of the optimal conditions for its performance and the evaluation of the possibilities for its use

The optimum conditions for using the method of radioimmunoprecipitation (RIP) for the detection of human immunodeficiency virus (HIV) in serum samples have been established. Out of several available cell lines persistently infected with HIV, specially selected line 17 has been chosen. The characteristic feature of this is the high and stable (under the conditions of prolonged cultivation) accumulation of virus-specific proteins in infected cells. The optimum conditions for making the test and its evaluation have also been established. The data of literature on the advantages of the method of RIP over such traditional methods as the enzyme immunoassay and immunoblotting have been confirmed. Thus, the presence of specific antibodies in several serum samples registered as false negative has been established. The intertypical reactivity of two serotypes of the virus, HIV-1 and HIV-2, has been studied. Cross reactivity of antibodies with respect to the HIV gene gag, but not with respect to viral glycoproteids, has been established. Ideas on the expediency and prospects of using RIP for the serological control of HIV infection are presented.     

Liquid-liquid partition chromatography of biopolymers in aqueous two-phase systems.

The theoretical and practical principles of liquid-liquid partition chromatography (LLPC) applying aqueous two-phase polymer systems are presented. The method is based on support materials which bind one of the two aqueous phases with high preference and reject the other. This selectivity is obtained by making use of incompatibilities between polymers grafted on support particles and polymers in solution. Applications of the separation technique to the fractionation of protein and nucleic acid mixtures are shown. For the DNA-fractionation according to base composition an affinity partition chromatography using polyethylene glycol-bound base-specific complexing agents has been developed which exhibits a resolution superior to all other methods known.     

A novel method of preparing totally alpha-deuterated amino acids for selective incorporation into proteins. Application to assignment of 1H resonances of valine residues in dihydrofolate reductase

The pyridoxal/2H2O exchange reaction of the alpha-CH of amino acids is known to be accompanied by racemisation: Thus by using a D-amino acid as the starting material any L-amino acid formed in the reaction will be essentially fully deuterated at its alpha-position. We have used this method to prepare alpha-deuterated L-valine and incorporated this biosynthetically into L. casei dihydrofolate reductase. A comparison of the alpha CH-NH fingerprint regions of COSY spectra of deuterated and normal DHFR complexes allows one to identify cross-peaks from 15 of the 16 valine residues.