Molecular extinction coefficients of lead sulfide and polymerized diaminobenzidine as final reaction products of histochemical phosphatase reactions.

Molar extinction coefficients of precipitated lead sulfide (PbS) and polymerized diaminobenzidine (polyDAB) have been determined at wavelengths of 450 nm and 480 nm, respectively, for quantitative histochemical analysis of phosphatase reactions. These values are essential for the conversion of cytophotometric (mean integrated) absorbance values to absolute units of substrate converted per unit time and volume of tissue. This conversion allows direct comparison of histochemical and biochemical data. The molar extinction coefficient of PbS at 450 nm was found to be 3,800 and therefore, per mole phosphate liberated, the molar extinction coefficient is 5,700 because 3 moles phosphate are captured by 2 moles lead at neutral or alkaline pH. Parallel experiments with the cerium-DAB method revealed that the molar extinction coefficient of polyDAB at 480 nm is 5,500 with respect to liberated phosphate. The molar extinction coefficients were applied for comparison of data from biochemical and histochemical assays of glucose-6-phosphatase activity in rat livers. A significant correlation was found between both sets of data. The values were in the same order of magnitude with histochemical values approximately 1.4 times higher than biochemical values.     

Intercalation of anthracyclines into living cell DNA analyzed by flow cytometry.

Anthracyclines (ANT) are used in the treatment of leukemia and other cancers. These drugs have been shown to intercalate between the strands of DNA. In the present study, we show that the amount of ANT intercalated into DNA can be determined by measuring the fluorescence resonance energy transfer (FRET) between Hoechst 33342 (H33342) and ANT bound to DNA. The transfer efficiency was found to depend on the amount of disposable ANT but was independent of the amount of H33342 bound to DNA over a wide range of H33342 concentrations. The method was adapted for flow cytometric measurement of FRET in whole living cells and was used to evaluate the degree of intercalation of daunorubicin (DAU) and idarubicine (IDA) into DAU-sensitive and DAU-resistant leukemic cell lines. ANT intercalation into DNA was affected by factors which modify the intracytoplasmic concentration of ANT, and it was shown that the action of ANT and the resistance to ANT could not be attributed solely to the intercalative effect of the drugs. The method has advantages over previously described methods and represents a useful complementary tool in studies on the mode of action of ANT and the mechanisms of chemoresistance.     

Catalytic effect of gamma-thrombin on synthetic low molecular weight peptide substrates]

Hydrolysis and respective catalytic parameters of hydrolysis of ester peptide substrates that contain residues of hydrophobic and nonpolar amino acids in P2, P3 subsites have been studied. It is shown that efficiency of hydrolysis by thrombin is determined by the length of polypeptide chains and by the nature of the amino acids in P2, P3 subsites in the substrate. In spite of the fact that gamma-thrombin retains the conformation activity of the catalytic centre the local conformation changes of the second binding region of the enzyme have been discovered.     

Biochemical bases of prevention of the cytotoxic effect of xenobiotics]

Possible sources of cytotoxicity of xenobiotics and main mechanisms of realization of their damaging effect are briefly considered. Protective system of a cell is characterized with regard for the peculiarities essential for development of principles of alimentary prophylaxis of cytotoxic effects of foreign compounds. Modern approaches to alimentary modification of the processes of biotransformation of xenobiotics and promotion of protective capacities of a cell are discussed.     

Effect of stress factors on carboxypeptidase H activity in areas of the rat brain]

It is established that carboxypeptidase H activity increases as affected by various stress factors in the rat brain departments. The increase of the enzyme activity because of the emotional-pain stress is of continuous character. Possible role of carboxypeptidase H in the development of the stress response is discussed.     

Ventral root afferent and dorsal root efferent fibres in dog and human lower sacral nerve roots.

Two pairs of wire electrodes were used to record single afferent action potentials from ventral roots and single efferent action potentials from dorsal roots of dogs and humans. A human lower sacral ventral root contained about 20 to 30% afferents among fibres with a diameter larger than 5 microns; a comparable ventral root of a dog contained about 1% afferents. Human S3, S4 and S5 dorsal roots contained 3, 18, and 20 to 30% efferent fibres respectively; a comparable dorsal root of the dog contained less than 1% efferent fibres. Primary and secondary muscle spindle afferents, Golgi tendon organ afferents, and afferents from the mechanoreceptors of the urinary bladder and anal canal mucosa were activated in a dog ventral root by pulling bladder and anal catheters. Their peak group conduction velocities were 82, 57, 71 and 18 m/s at 34 degrees C respectively. The dog afferents conducted more than 30% faster than did comparable human nerve fibres. By strongly pulling the bladder catheter, the static human dorsal root gamma 21-motoneurons increased their activity for about 7 s which in turn strongly increased the dorsal root spindle afferent activity for more than 10 min; the human static intrafusal gamma-motoneurons seemed to show cumulative properties.     

Prevention of early inflammatory complications after transthoracic interventions and estimation of the degree of immunologic deficiency]

An analysis of the results of the examination and treatment of 379 patients undergoing transthoracic interventions showed that the use of the target-aimed selective immunomodulation, short-term antibacterial prophylaxis, inhibitors of arachidonic acid metabolites, active elimination of the circulating immune complexes and medium-weight molecular peptides and adequate local analgesia after the interventions providing normalization of the laryngeal reflex and early activation of the patients permitted the incidence of the pulmonary complications to be decreased by 2.5 times, pleural complications by 1.7 times, suppuration of the postoperative wound by 1.8 times and the total expenses by 25.6 per cent.     

Cytoplasmic delivery of ribozymes leads to efficient reduction in alpha-lactalbumin mRNA levels in C127I mouse cells.

Ribozymes targeted to five sites along the alpha-lactalbumin (alpha-lac) mRNA were delivered to the cytoplasm of mouse C127I mammary cells using the T7-vaccinia virus delivery system and the amount of alpha-lac mRNA was monitored 24-48 h post-transfection. Three target sites were selected in the alpha-lac coding region (nucleotides 15, 145 and 361) and two were located in the 3' non-coding region (nucleotides 442 and 694). Acting in trans and at a target:ribozyme ratio of 1:1000, ribozymes targeting sites 361 and 694 reduced alpha-lac mRNA by > 80%; another two ribozymes (targeting nucleotides 442 and 145) reduced mRNA levels by 80 and 60% respectively; the fifth ribozyme (targeting nucleotide 15, near the AUG) was largely ineffective. The kinetic activity (kcat) of each ribozyme in vitro was somewhat predictive of the activity of the two ribozymes that targeted nucleotides 361 and 694, but was not predictive of the in vivo activity of the other three ribozymes. Down-regulation of the intracellular levels of alpha-lac paralleled the ribozyme-dependent reduction achieved for mRNA. For site 442, the reduction in both mRNA and protein was attributed to the catalytic activity of the ribozyme rather than to the antisense effects of the flanking arms, because delivery of an engineered (catalytically-inactive) variant had no effect on mRNA levels and a minimal effect on the level of alpha-lac present in the cell.     

Structure and evolution of insulins: implications for receptor binding.

Insulin is a member of a family of hormones, growth factors and neuropeptides which are found in both vertebrates and invertebrates. A common 'insulin fold' is probably adopted by all family members. Although the specificities of receptor binding are different, there is a possibility of co-evolution of polypeptides and their receptors.     

Additional binding sites in lysozyme. X-ray analysis of lysozyme complexes with bromophenol red and bromophenol blue.

The binding sites in hen egg-white lysozyme for neutral bromophenol red (BPR) and ionized bromophenol blue (BPB) have been characterized at 2 A resolution. In either case, the dye-bound enzyme is active against the polysaccharide, but not against the cell wall. Both binding sites are outside, but close to, the hexasaccharide binding cleft in the enzyme. The binding site of BPR made up of Arg5, Lys33, Phe34, Asn37, Phe38, Ala122, Trp123 and possibly Arg125, is close to subsite F while that of BPB made up of Tyr20, Arg21, Asn93, Lys96, Lys97 and Ser100, is close to subsites A and B. The binding sites of the neutral dye and the ionized dye are thus spatially far apart. The peptide component of the bacterial cell wall probably interacts with these cells during enzyme action. Such interactions are perhaps necessary for appropriately positioning the enzyme molecule on the bacterial cell wall.