Identification of differentially expressed genes by restriction endonuclease-based gene expression fingerprinting.

A novel method for identification of differentially expressed genes has been developed. It is based on the consecutive restriction digestions of 3' terminal cDNA fragments to produce a fingerprint of gene expression. cDNA molecules are synthesized using a biotinylated oligo(dT) primer, digested with a frequently cutting restriction endonuclease and the 3'-terminal restriction fragments are isolated using streptavidin microbeads. After amplification by PCR, cDNA fragments are immobilized again on streptavidin beads, radiolabeled and treated sequentially with a set of restriction endonucleases. The products of individual enzymatic reactions from two or more different RNA populations are resolved by polyacrylamide gel electrophoresis and compared to reveal differentially expressed genes. This strategy enabled us to identify and clone the fragments of five genes expressed differentially in murine thymus and spleen. One of the genes was found to encode terminal deoxynucleotidyl transferase; others are apparently previously unknown genes.     

A study of pathogenic factors of Streptococcus pneumoniae strains causing meningitis

Abstract Pneumococcal meningitis in St. Petersburg in the period 1985–1991 occurred in 1.7–2.3 children per 100 000 annually. The most common serotypes among pneumococcal strains isolated from patients with meningitis were 19, 1, 6, 15, and 2, whereas, among the capsulated strains isolated from carriers, type 3 predominated. Only one third of strains from cases of meningitis were highly virulent for mice (types 1, 2, 3). Hyaluronidase was produced by all the 39 studied strains, 22 (84.6±7.1%) out of 26 strains from patients with otitis media, and only by 15 (11.5±2.8%) out of 130 strains isolated from carriers. Non-capsulated strains lacked this enzyme. Results of intranasal inoculation of pneumococcal strains with different hyaluronidase activity and addition of exogenous hyaluronidase to strains which did not produce the enzyme confirm the hypothesis that this enzyme plays an important role in bacterial dissemination and breaching of the blood brain barrier by pneumococci. It was concluded that high hyaluronidase activity, presence of capsule, and pneumolysin or serotype (1, 2, and 19) despite hyaluronidase titer, are the most important factors contributing to the development of pneumococcal meningitis. The role of the mouse toxic factor is unclear.     

Cortical motor potential associated with voluntary termination of a movement

The data are presented on potentials revealed by averaging buman EEG sections recorded during termination of a motor act. The potential consisted of two components: fast positivity and slow negative wave. The complex under study was named "final" or "relaxation" potential. The latency of the "final" potential and that of the muscle relaxation were linearily related. The potential was recorded only in cases when the relaxation of all muscles involved was necessary for a motor task solving at movement termination. If some groups of muscles relaxed and the other ones contracted the potential was not recorded.     

Conditioned reflex transformation of unconditioned flexion of the limbs of the dog into extension

In response to electro-skin stimulation of the left forelimb eliciting its flexion,--an escape instrumental reflex was elaborated in five dogs in the form of extension of the stimulated limb (pressure on the support), i.e. a reaction antagonistic to the inborn one. The reaction was elaborated due to conditioned transformation of the unconditioned flexion: the flexion became gradually inhibited and the new form of reaction, previously not inherent in the animal increased and became stable. Such transformation of one reaction into another is connected with the reorganization of inborn coordinations.     

Glucoamylase immobilization on silochrome using gossypol

The paper is concerned with conditions of glucoamylase binding with silanized silochrome using gossypol, dialdehyde isolated from cotton-plant. Kinetic properties of the immobilized enzyme are studied. The enzyme pH optimum does not change with immobilization and the temperature optimum is shifted from 50 degrees to 60 degrees C; a certain increase of the seeming Km is also observed. A high yield of the enzyme activity in immobilization evidences for the possibility of using gossypol as a binding agent in glucoamylase immobilization.     

Effect of colchicine on polarization of cells on narrow strips of an adherent substrate

Effect of antitubulin colcemide on polarization of mouse fibroblast-like cells on anisotropic substrate was studied. Such a substrate was obtained by scratching narrow strips in lipid films, adsorbed on the glass. The control cells were seen spread only along the strips, and in 4-6 hours they approached the length of120-150 mcm. In colcemide-containing media, the cells remain in an unspread state for a long time; they extrude their outgrowths both along the strip and perpendicularly to it. Due to frequent refractions of outgrowths, two thirds of colcemide-treated cells were detached from the substrate. Possible mechanisms of these effects of antitubulins are discussed.     

Effect of colcemid on the radial spreading of fibroblasts in culture

Effect of colcemide upon the spreading of mouse embryo fibroblast-like cells on the substrate was studied with the aid of time-lapse microcinematography and scanning electron microscopy. On the glass, colcemide did not prevent the transition of cells into a well-attached state, however, the time needed for this transition was seen considerably increased as compared with the control cultures. Intermediate stages of spreading on flat glass had the following abnormal features in colcemide-containing medium: a) shapes of cytoplasmic outgrowths formed by the cell were altered and their distribution along the cell border appeared less regular; b) partial detachments of the attached parts of cells occurred very frequently; c) the spreading of various parts of the cells was not correlated. Possible mechanisms of colcemide action on the cell spreading are discussed, and it is suggested that intracellular structures sensitive to colcemide are essential for coordination of reactions that occur in various parts of the cell in the course of spreading.     

State of the hypothalamo-hypophyseal neurocirculatory system of the gerbil, Rhombomys opimus, during dehydration

Big gerbils, inhabitants of desert, were found to have indices of an increased functional activity of HHNS: low content of neurosecretory material all over the system, relatively large volumes of neurosecretory cell nuclei and nucleoli, hyperemia. Under the condition of a seven-day dehydration, the neurosecretory material reduction in all links of HHNS, enlargement of neurosecretory cell nuclei and nucleoli, intensification of hyperemia were found. With the increase in dehydration time to 23 days a progressive reduction of neurosecretory material in cell processes and hypophysis are seen. However, the decrease in nuclear size takes place under the condition of continuing increase in nucleolar size both in supraoptic and paraventricular nuclei. Extremely intensive hyperemia of all regions of HHNS is seen. The activation of HHNS under the water-salt regime change, seen in big gerbils, is not as pronounced as in albino rats. This is, evidently, due to a specificity of the function and their osmoregulating system.     

Interaction of cholesterol-conjugated alkylating oligonucleotide derivatives with cellular biopolymers.

Interactions of oligonucleotide derivatives with mammalian cells and cellular biopolymers have been investigated. The derivatives were oligonucleotides bearing an alkylating 2-chloroethylamino group at the 3'-end and a cholesterol residue at the 5'-terminal phosphate. These compounds are readily taken up by cells and react with cellular DNA, RNA and some proteins which may play a role in delivery of the compounds into cells.     

Stability of the cardiovascular functions of different genetic strains of rats subjected to emotional stress

The level of catecholamines was measured in various brain parts in rats of August and Wistar lines, characterized by different stability of cardio-vascular functions in emotional stress. In norm, the August line has a higher content of dopamine (DA), in the mid-brain in particular; the Wistar line has a higher content of noradrenaline (NA) in the hypothalamus. In susceptible animals, which showed different cardio-vascular disorders during 30 hours of immobilization (48% of the Wistar line and 90% of the August), and in animals resistent against emotional stress, the changes in the initial level of catecholamines were observed, both common and specific for two lines. An assumption is made that the stability of cardio-vascular functions in the emotional stress is provided for by an intensive metabolism of the NA-synthesizing neurones in the brain isthmus, and by a moderate activity of the DA-synthesizing neurones of the mid-brain; the susceptibility to cardio-vascular disorders in the emotional stress is due to increase in the activity of the DA-synthesizing mid-brain neurones, the decrease in the activity of the DA-hydroxilase and the exhaustion of NA in the hypothalamus.