Solid phase ligation of synthetic DNA fragments

A synthetic oligodeoxyribonucleotide (oligo) covalently bound by an internucleotide linkage to the succinylated Sephacryl S-500 support through 1.9-diaminononane spacer was used as starting compound to assemble the E. coli rec A promoter DNA fragment from synthetic oligos by means of T4 DNA ligase. The solid-phase assembly of the designed DNA was performed by two ways: stepwise ligation of two pairs of oligos (2 dyads) or simultaneous ligation of four oligos (tetrad). Both ways gave equal results with some preference in the tetrad case. The reliability of E. coli promoter DNA fragment assembly was demonstrated by cloning it in a plasmid vector and sequencing the cloned DNA by the solid-phase Maxam--Gilbert technique.     

Site-specific endonucleases LplI and AagI

New site-specific endonucleases LplI and AagI have been isolated from the Lactobacillus plantarum and Achromobacter agile cells, respectively. The enzymes' purification stages included treatment of cell-free extracts with polyethylenimine, fractionation in two-phase system by Albertsson's method, chromatography on blue Sepharose and DEAE-cellulose. The results of cleavage of a 5'-32P-labelled oligodeoxynucleotide duplex by restriction endonucleases LplI and AagI indicate that these enzymes recognize and cut the sequence AT decreases CGAT, being therefore true isoschizomers of the ClaI restriction endonuclease from Caryophanon latum. The L. plantarum strain has 400 fold endonuclease productivity as compared with the ClaI producent and is perspective for preparative isolation of LplI.     

Photometric method of determination of the amidase activity of proteinases using 4-methylcoumaryl-7-amide substrates

It was shown that 7-amino-4-methylcoumarin (MC-amine), resulted from the enzymatic hydrolysis of 4-methylcoumaryl-7-amide (MC-amide) peptide substrates, may be estimated not only fluorometrically but also photometrically. A photometric method for estimating activity of tissue kallikrein (EC 3.4.21.35) and urokinase (EC 3.4.21.31) is suggested using Z-Phe-Arg-NHMC and Z-Gly-Gly-Arg-NHMC, respectively, as substrates. Kinetic parameters of the enzymatic hydrolysis, as obtained by photometric and fluorometric detection of the MC-amine formed, were in good agreement. The differential coefficient of molar extinction of the substrates and MC-amine at 360 nm was found to be 10,800 M-1 cm-1.     

Pentose phosphate pathway in rat colonic epithelium

The colonic cells of the large intestine are one of the most proliferative tissues of the animal body. The pentose pathway has an essential role in cell division and growth being the only pathway forming ribose 5-P necessary for all nucleotide and nucleic acid sunthesis. The pentose pathway may also provide reducing potential as NADPH for biosynthesis and C-3- C-8 glycolyl compounds. The maximum catalytic capacities of the reactions of the non-oxidative pentose pathway for the conversion of ribose 5-P to hexose and triose phosphates by the proximal and distal colon under feeding and starvation regimes are among the highest in the animal body. The qualitative presence of the oxidative pentose pathway was assessed by measurement of the C-1/C-6 ratio value of 1.67-1.82. Enzymes of the F-type and L-type pentose pathways are present in colonocytes and their maximum catalytic activities in colonocyte cytosol are reported. The contribution of the F-type pentose cycle to the total glucose metabolism of colonocytes, measured by the specific yield method, is negligibly low (approximately 1.5%). Colonic epithelial cells use glucose at a high rate (7.1 +/- 0.33 mumol min-1g-1 dry wt) and 79% of the glucose is converted to lactate. Arabinose 5-P has an intermediary role in the formation of keto pentose, sedoheptulose and hexose phosphates from ribose 5-P by colonocyte cytosol. The intermediary and reaction products of [1-13C] ribose 5-P dissimilation by colonocytes is investigated by 13C NMR spectroscopy. The 13C positional isotope distributions show labelling of C-1 and C-3 of hexose 6-phosphates consistent with either the theoretical predictions of the F-type pentose pathway or of the activities of exchange reactions catalysed by transketolase and/or transaldolase. Measurements of exchange reactions showed that the C-1/C-3 labelling of these compounds is mostly, if not wholly, attributable to exchange catalysis by these group transferring enzymes. The results suggest that the F-type PC has little role in the glucose metabolism of colonocytes and pentose phosphate formation may thus occur by a contribution (approx 20% of the total glucose metabolism) by the alternate L-type pathway.     

Global change: Translating plant ecophysiological responses to ecosystems

The physiological responses of plants to elevated CO(2) have not been incorporated into most models of ecosystem function under changed climate. These responses are now well documented, and recent work demonstrates that they can be readily included in ecosystem models. Simulations show that the effects of elevated CO(2) levels on transpiration and gas exchange will increase the sensitivity of community structure (particularly of forests) to climate change.     

Biosynthesis of immunoglobulin G-binding factor in cattle lymphocytes

Synthesis of the IgG-binding factor was estimated by the amount of labeled [14C]hydrolysate of proteins in the total synthesized de novo protein. The obtained data showed that the IgG binding factor which was synthesized by blood lymphocytes of the normal cattle and that suffered from chronic leukemia had a molecular weight of 72 kDa and was revealed before and after restoration of S-S bonds as one peptide.     

Phagocytic activity of peripheral blood monocytes and peritoneal macrophages in the progeny of animals with experimental chronic liver disease

Peripheral blood monocytes and peritoneal macrophages (phagocytic index, phagocytosis intensity, metabolic level) in the offspring of mice with chronic experimental autoimmune liver affection have been studied for different parameters of their phagocytic properties. The obtained results testify to absorption and bactericidal activity disturbance of mononuclears studied in this group of animals.     

Conformational state of thin myofilament proteins in normal and chronically failing heart

Circular dichroism (CD) spectra of myocardial G-actin significantly differ from those of F-actin, and the spectra of G- and F-actins differ from those of myocardial tropomyosin, native tropomyosin and alpha-actinin. In heart failure in man and experimental animals, characterized by a significantly decreased ability of the contractile protein system to generate force, considerable changes in the tertiary structure of Straub G-actin are observed. During polymerization a monomer of this actin is included in F-actin as a promoter without corresponding conformational changes of a part of G-actin globule; G-actin from the failing myocardium loses its conformational mobility. According to CD data the secondary protein structure is not altered. CD spectra analysis with regard to the regions of aromatic amino acid residue localization in active sites of actin suggests that the sites of actin-myosin and actin-actin interactions do not assume the conformation necessary for normal functioning of thin filaments.     

Nephrotoxic effect of the specific brain serotonin depletor para-chlorophenylalanine

Brain serotonin depletion induced by peripheral parachlorophenylalanine (pCPA) is frequently used to evaluate the role of the central serotoninergic system in the regulation of a number of physiological functions, including the secretion of renin by the kidney. We found that due to the treatments applied in the protocol used for the investigation of pCPA effect on renin and vasopressin secretion in rats (300 mg/kg i.p. 64 and 40 h before sacrifice) renal injury was induced as well. Typical changes indicating acute renal failure were observed--an initial polyuria, natriuresis and body mass loss, succeeded by oliguria, decreased glomerular filtration rate, and salt and creatinine retention. Morphological changes in the glomeruli included a thickening of the basal membranes, a confluence and a reduced number of podocyte pedicles. A slight to moderate granular degeneration was observed in epithelial cells of the proximal convoluted tubule, combined with mitochondrial changes--an increase in number, matrix disorganization, and myelin degeneration. In conclusion, the renal function changes after i.p. pCPA may be due not to brain serotonin depletion-alone, but also to nephrotoxic effect.     

Taxonomic spectrum of pathogens of mixed infections in oncology patients and principles of their antibacterial therapy

Mixed infections were observed in oncological patients irrespective of the tumor or infectious process localization. 25-30 per cent of the pathological materials from such patients were contaminated with bacterial associations. The number of the associates ranged from 2 to 5 depending on the pathological material tested. Therapy of infectious complications due to pathogen associations should stem from analysis of many factors, i.e. properties of the isolated or likely pathogens, individual characteristics of the patients, compatibility of the drugs used and their pharmacokinetic features.