Development of rational methods for purifying restriction endonucleases

The work deals with the search for rational schemes of the purification of endonucleases, suitable for the isolation of different enzymes. The scheme using the combination of Blue Sepharose and phosphocellulose has proved to be most universal. The expediency of starting the purification of Haemophilus enzymes on biogel A-0.5m has been established.     

Development of a diagnostic test system for detecting soluble staphylococcal antigens by an immunoenzyme method

ELISA is used for detecting the soluble staphylococcal antigen in patients with purulent septic infections. The optimum conditions for the assay have been established: the dose of staphylococcal gamma globulin for plate sensitization should be 5.0-10.0 micrograms/ml, the pH of the buffer solution 9.6-10.0, the time and temperature of incubation 18-20 hours at 4 degrees C or 5 hours at 37 degrees C. The possibility of using plates manufactured in the USSR has been shown. The sensitivity of the above diagnostic test system is 0.005 microgram/ml.     

Action of heparin on protein fractions of isolated nuclei and on their DNA content.

Isolated nuclei of rat hepatocytes were incubated with 0.05% sodium heparinate for 2 to 10 min. Alterations in the nuclei were controlled biochemically, determining the amounts of DNA and histones, and by cytophotometric methods determining the amounts of total and nonhistone proteins and DNA. Under the selected experimental conditions 95% of histones are bound already after 5-min incubation with heparin; nonhistone proteins of cell nuclei remain unchanged. The blockade of histones is followed by DNA diffusion into the incubation medium. Experiments with nuclear staining with alcian blue proved the specificity of heparin binding with histones and showed that heparin-histone complex remains in the nuclei, and its histones lose their extractability with 0.25 n HCl.     

Radiodiagnosis of traumatic injuries of the kidneys in children

Altogether 35 children (21 boys and 14 girls) with traumatic renal lesions ages 5 to 14 were examined, of them 23 were subjected to operation 2-13 h after injury, 12 patients received conservative therapy. The patients were divided into 3 groups with relation to a degree of severity of injury: mild (12 patients), moderate (20 patients) and grave (3 patients). All the children were given combined clinical, x-ray and radiological examination. Normally the most informative diagnostic method was excretory urography, particularly its infusion variant. Renography, renoscanning or dynamic renoscintigraphy were employed for observation over time to reduce radiation exposure.     

Effect of trehalose on the phase properties of hydrated and lyophilized dipalmitoylphosphatidylcholine multilayers

The structure and thermal behavior of hydrated and lyophilized dipalmitoylphosphatidylcholine (DPPC) multilayers in the presence of trehalose were investigated by differential scanning calorimetry and X-ray diffraction methods. Trehalose enters the aqueous space between hydrated bilayers and increases the interbilayer separation (from 0.36 to 1.37 nm in the different DPPC phases at 1 M trehalose). It does not affect the lipid chain packing and also the slow isothermal conversion at 4 degrees C of the metastable L beta' phase into the equilibrium crystalline Lc phase. Addition of trehalose leads to a slight upward shift (about 1 degrees C at 1 M trehalose) of the three phase transitions (sub-, pre-, and main transition) in fully hydrated DPPC while their other properties (enthalpy, excess specific heat, and transition width) remain unchanged. The effect of trehalose on the thermal behavior of DPPC multilayers freeze-dried from an initially completely hydrated state is qualitatively similar to that of water. These data support the "water replacement" hypothesis about trehalose action. It is suggested that trehalose prevents the formation of direct interbilayer hydrogen bonds in states of low hydration.     

Direct tRNA-protein interactions in ribosomal complexes.

Nucleotide residues in E. coli tRNA(Phe) interacting directly with proteins in pre- and posttranslocated ribosomal complexes have been identified by UV-induced cross-linking. In the tRNA(Phe) molecule located in the Ab-site (pretranslocated complex) residues A9, G18, A26 and U59 are cross-linked with proteins S10, L27, S7 and L2, respectively. In tRNA(Phe) located in the Pt-site (posttranslocated complex) residues C17, G44, C56 and U60 are cross-linked with proteins L2, L5, L27 and S9, respectively. The same cross-links (except for G44-L5) have been found for tRNA in the Pb-site of the pretranslocated ribosomal complex. None of the tRNA(Phe) residues cross-linked with proteins in the complexes examined by us are involved in the stabilization of the secondary structure, but residues A9, G18, A26, G44 and C56 participate in stabilization of tRNA tertiary structure. Since translocation of tRNA(Phe) from Ab- to P-site is accompanied by changes of tRNA contacts with proteins L2 and L27, we postulate that this translocation is coupled with tRNA turn around the axis joining the anticodon loop with the CCA-end of the molecule. This is in agreement with the idea about the presence of a kink in mRNA between codons located in the ribosomal A- and P-sites. In all E. coli tRNAs with known primary structure positions 18 and 56, interacting with L27 protein, when tRNA is located either in A- or P-site, are invariant, whereas positions 17 and 60, interacting with proteins only when tRNA is in the P-site, are strongly conserved. In positions 9, 26 and 59 purines are the preferred residues. In most E. coli tRNAs deviations from the consensus in these three positions is strongly correlated.     

A rapid coagglutination test for the detection and serogrouping of Legionellae

The applicability of coagglutination for the rapid detection and serogrouping of Legionellae has been investigated. The coagglutination reaction is carried out with the aid of self-made preparations of protein A containing staphylococci, sensitized with specific antibodies against the antigens of L. pneumophila (serogroups I to 6), L. bozemanii and L. micdadei. Preliminary heating of Legionella suspensions at 100% C for 15 min was used to prevent cross coagglutination reactions and ensure greater safety of laboratory personnel during the performance of the test. The results obtained demonstrate a high specificity of coagglutination. With the aid of the coagglutination reactions it has been shown that L. pneumophila strains isolated in Bulgaria belong to serogroup I. The coagglutination method is characterized by its rapidity, simplicity and feasibility. It is a useful and convenient means for the rapid detection and serogrouping of Legionellae.     

Intracellular Synthesis of Myxovirus Neuraminidase in Chick Embryo Cell Monolayer Culture I. Neuraminidase Activity, Hemagglutinin Synthesis, and Content of Cell-bound Sialic Acid in Newcastle Disease Virus-infected Cells

Neuraminidase (Nase) activity of chick embryo monolayer cell homogenates was determined by its rate of splitting of neuraminlactose, free neuraminic acid (NA) being determined by the thiobarbituric acid assay. Noninfected cells were found to have no detectable amount of Nase activity. Newcastle disease virus (NDV)-infected cells (multiplicity of infection, 20 to 75 plaque-forming units per cell) displayed a high level of Nase synthesis, the rate of synthesis being parallel to that of hemagglutinin (HA) synthesis (with a 1.5 hr delay in the latter). An "eclipse" of the Nase and HA activities associated with the virus that was adsorbed onto cells was observed. The data provide evidence that the Nase is not incorporated into the viral envelope from a pre-existing cell supply but that its synthesis is coded by the viral genome. The content of cell-bound sialic acid, determined simultaneously in infected-cell homogenates, showed characteristic features allowing certain conclusions concerning the renewal of NA-terminating cell receptors during the course of infection, and the intracellular action of the Nase of the virus introduced into cells by the inoculum and that of the newly synthesized Nase at different stages of infection.