Increase of seizure threshold and resistance to stress in rats after immunization against fragment of diazepam-binding inhibitor]

White rats were immunized against fragment of diazepam-binding inhibitor octadecaneuropeptide (ODN) with conjugate ODN bovine serum albumin. This rats have reduced reactions of fear and anxiety in stress model of "open field" and in conflict Vogel test; their pain sensitivity ("tail flick" test) was lowered. The number and intensity of generalized seizure reactions after injection of pentylenetetrazole were decreased. The results show that active immunization to endogenous ODN has stress--protective and anti-seizure effects.     

Differences in anti-oncogene p53 expression in human monocytes and lymphocytes in vitro]

The p53 gene has been associated with malignant transformation as well as "anti-oncogene" activity. In the present report expression of p53 in resting and activated human blood monocytes and lymphocytes is analyzed. It is found that human monocytes freshly isolated by continuous percoll gradient centrifugation contained detectable level of p53 mRNA. Stimulation of monocytes by potent activation inducer Staphylococcus Aureus Cowan I for 3-5 hr caused disappearance of r53 mRNA. In contrast, induction of high level of TNF-alpha mRNA was detected. Addition of cycloheximide had no effect on p53 mRNA content in stimulated monocytes, and caused disappearance of mRNA in resting cells. In lymphocytes cultures p53 mRNA was absent in freshly isolated cells and in resting lymphocytes cultured for 20 hr. Activation of lymphocytes by lectin caused accumulation of p53 mRNA. We suggest that r53 gene regulation and functions might be different in human monocytes and lymphocytes.     

A simple and efficient method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded plasmid DNA.

A method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded DNA without the necessity for phenotypic selection is described. Plasmids denatured with alkali and purified by adsorption to and elution from nitrocellulose have single-stranded regions where primers can hybridize and serve as templates for a T7 DNA polymerase-catalyzed synthesis of complementary mutant DNA strands. When this procedure was carried out such that the original nonmutant strand contained uracil [method of Kunkel, Proc. Natl. Acad. Sci. USA 82(1985)488-492], mutation frequencies of between 30% and 40% were obtained. The technique has been used to generate mutant genes in plasmids of a wide variety of sizes. The largest plasmid manipulated and successfully mutagenized was 22 kb. The method is rapid and efficient and is not dependent upon either f1 phage vectors or the presence of restriction sites in the vicinity of the sequence targeted for mutation.     

Enzymes of a bacteriolytic lysoamidase preparation. Various properties of bacteriolytic protease L2]

Bacteriolytic proteinase L2 is able to cleave fluorogenic synthetic tripeptide anthranoyl-alanyl-alanyl-phenylalanyl-nitroanilide (Abz-Ala-Ala-Phe-pNA) at the bond between phenylalanine and p-nitroaniline. Optimal conditions of the tripeptide cleavage have been determined: pH 6.7 + 0.1; mu = 2 (by NaCl); t = 40 degrees C; KM = 2.6 x 10(-5) M. Metal cations reduced the enzyme activity. The enzyme was inhibited by EDTA, p-CMB, DIF. The synthetic tripeptide can be used to determine the activity of the L2 enzyme.     

New principles of ion exchange preparative chromatography and their applications for isolation, purification and super purification of antibiotics]

New types of structurally segregated heteronetwork biosorbents with given parameters of heterogeneity and porosity have been developed. Physico-chemical characteristics of the biosorbents on the basis of which one can predict optimal structures of ion exchangers to be used in preparative chromatography of biologically active compounds were studied. A new principle of sequential displacement of ions of organic compounds, in particular antibiotics, adsorbed on selective biosorbents with a high adsorption capacity was developed, which enables purification and superpurification of the desired compound. The method is based on the effect of small thermodynamic shifts in physico-chemical parameters of the elution system, which results in preparative separation of substances with close properties and in purification of the desired compound from microadmixtures. The "small shift effect" is realized in the case of limiting thermodynamic and kinetic parameters of heterogeneous mass exchange and with biosorbents possessing a highly selective adsoprtion capacity.     

The effect of age on the response of the detrusor to intracellular mechanical stimulus: DNA replication and the cell actin matrix.

Benign prostatic hypertrophy and posterior urethral valves present at both extremes of the age spectrum. Both disease processes can obstruct the urinary stream and ultimately have pathophysiological effects on detrusor structure and function. The mechanisms regulating the structural reorganization of the detrusor to a mechanical outflow obstruction are not known. In an attempt to identify maturational differences in myocyte ultrastructure and consequent effects these might have in modifying the response of the detrusor to mechanical stimulus, we studied differences in dynamic nuclear-cytoskeletal interactions in detrusor tissue in an animal model. Using a drug which specifically severs actin, cytochalasin D (CD), as an intracellular mechanical stimulus, we measured changes in nuclear area and the rate of DNA synthesis in detrusor myocytes from young (2-3 week) and old (8-12 mon) guinea pigs. We found that there were age specific differences to intracellular mechanical stimuli in detrusor muscle. Nuclei of myocytes from young animals showed elastic recoil on severing the cell actin matrix and the tissue from young animals increased replicative DNA synthesis with an intracellular stimulus. In contrast, nuclear shape changes in myocytes from old animals suggested less elasticity, and there was no increase in DNA synthesis with disruption of the cell actin matrix. Anti-alpha-smooth muscle actin antibody and rhodamine phalloidin staining of actin in cytochalasin D treated primary explants of detrusor myocytes showed dose dependent disruption of the actin component of the cytoskeleton. These results suggest that there are fundamental modifications in detrusor myocyte ultrastructure with age. These maturational changes might result in differences in the pathophysiological and structural reorganization of the detrusor in response to outflow obstruction in infancy and adulthood. Furthermore, they suggest that 1) a tensile equilibrium exists between the myocyte nucleus and cytoskeleton; 2) there appears to be a decrease in myocyte nuclear elasticity with ageing; 3) release of nuclear template restrictions increases activity of DNA polymerase alpha in young, but not old, detrusor myocytes; and 4) mechanico-chemical signal transduction in detrusor myocytes may be mediated via the cytoskeleton. In addition, based on previous reports of actin within the nucleus, the results suggest that 1) nuclear actin may have a homeostatic structural role, maintaining the tensile equilibrium between nucleus and cytoskeleton, and 2) integrity of nuclear actin may function to maintain the spatial template restriction on DNA polymerase alpha activity.     

Immunochemical and biological analysis of pheromone biosynthesis activating neuropeptide in Heliothis peltigera.

This study describes the preparation and characterization of a highly specific antiserum to Helicoverpa zea pheromone biosynthesis activating neuropeptide (Hez-PBAN), and the use of this antiserum, in an enzyme linked immunosorbent assay (ELISA), to determine: a) the content of endogenous PBAN in head extracts of male and female Heliothis peltigera; b) the level of PBAN at different developmental stages; and c) the content of PBAN in four different moth species. Cross-reactivity studies revealed that the antiserum is directed mainly toward the N-terminal region of the neuropeptide, and that it exhibits similar binding affinities toward the oxidized and reduced forms of PBAN. Analysis of PBAN content in head extracts of male and female H. peltigera, at scotophase, revealed the presence of 4.97 and 4.58 pmol, respectively, in 3-day-old moths, and 5.33 and 4.78 pmol, respectively, in 7-day-old moths. The similarity in the content of PBAN at both ages and sexes was in accordance with the amount of pheromonotropic activity in these extracts which stimulated pheromone biosynthesis to a similar level. Analysis of PBAN-like immunoreactivity (IR) in head extracts of H. peltigera larvae and pupae demonstrated the existence of the neuropeptide in the 4th larval instar and continued to increase as a function of development. No IR could be detected in the first three larval instars. The larval and pupal extracts also exerted pheromonotropic activity which followed a similar pattern. The activity in these extracts, however, was considerably lower than that found in adult male and female heads. IR was also detected in head extracts of three other Noctuidae moths: Helicoverpa armigera, Cornutiplusia circumflexa and Spodoptera littoralis, indicating a high degree of chemical and structural similarity of PBAN in these moths.     

Changes in the phase response curve of the circadian clock to a phase-shifting stimulus.

Experiments were conducted in hamsters to determine whether the phase response curve (PRC) to injections of the short-acting benzodiazepine triazolam is a fixed or a labile property of the circadian clock. The results indicated that (1) both the shape and the amplitude of the PRC to triazolam generated on the first day of transfer from a light-dark cycle (LD 14:10) to constant darkness (DD) (i.e., PRCLD) were different from those of the PRC generated after many days in DD (PRCDD); and (2) the phase-shifting effects of triazolam on the activity rhythms of hamsters transferred from LD 14:10 or 12:12 to DD changed dramatically within the first 8-9 days spent in DD. In an attempt to accelerate the resynchronization of the circadian clock of hamsters subjected to an 8-hr advance in the LD cycle, triazolam was given to the animals at a time selected on the basis of the characteristics of PRCLD. The activity rhythms of five of eight triazolam-treated animals were resynchronized to the new LD cycle within 2-4 days after the shift, whereas those of most of the control animals were resynchronized 21-29 days after the shift. These findings suggest that attempts to use pharmacological or nonpharmacological tools to phase-shift circadian clocks under entrained conditions should take into account information derived from PRCs generated at the time of transition from entrained to free-running conditions.