Adenosine triphosphate hydrolysis in rat dental tissues. A histochemical study to differentiate the enzymes involved.

The purpose of this study was to try to differentiate histochemically between the various enzymes which may catalyze the hydrolysis of ATP in developing rat dental tissues. Freeze cut and freeze dried sections of molar and incisor teeth were incubated in lead capture-based media at pH 5.0, 7.2 or 9.4 with one of the following substrates: beta-glycerophosphate, AMP, ADP, ATP, AMP-PNP and tetrasodium pyrophosphate. To establish the enzymatic nature of the hydrolysis parallel sections were incubated after prior fixation in either formaldehyde or glutaraldehyde. By comparing the enzymatic stainings obtained with the various substrates and at the different pH:s, it was concluded that ATP can be visibly hydrolyzed in rat dental tissues by alkaline phosphatase (stratum intermedium, apical part of maturation ameloblasts, basal part of all ameloblasts, odontoblasts and subodontoblastic layer), specific ATPase (apical and basal parts of secretory ameloblasts) and ATP pyrophosphatase and/or adenylate cyclase (stratum intermedium, odontoblasts). Acid phosphatase, specific ADPase, 5'-nucleotidase, inorganic pyrophosphatase, 3':5'-cyclic-AMP-phosphodiesterase and adenylate kinase on the other hand, seem not to be engaged in the ATP hydrolysis to such a degree as to complicate the interpretation of the histochemical staining. The alkaline phosphatase part of the ATP hydrolysis appeared to be rather insensitive to aldehyde fixation, while the hydrolysis effected by specific ATPase and ATP pyrophosphatase and/or adenylate cyclase was extinguished after fixation with formaldehyde for 4 h or glutaraldehyde for 10 min.     

Incorporation of tritiated amino acids into the ultrastructure of the cerebral cortex following phenamine stimulation]

The method of electronoscopic autoradiography has revealed an increased biosynthesis of protein by neuronal and glial cells of the motor cortex of rats under phenamine stimulation. The increased level of incorporation of labelled precursors in the molecular layer of the cortex is likely to be associated with increased accumulation of newly synthetized protein in axo-dendritic synapses due to stimulation of the rapid component of the axonal transport of macromolecules. At the same time, participation of the autonomic protein-synthetizing system in the biosynthesis of synaptic proteins is not excluded. The labelled products were found to localize in the field of the thorn apparatus in processes of the "structural-functional" adaptation of synaptic entrances (A. A. Manina, 1972).     

Purification and some properties of a neutral protease from human leukocyte granules and its comparison with pancreatic elastase.

1. A cationic protease has been purified from the granule fraction of blood-donor leukocytes by a preparative method including precipitation by acetone and chromatography on Bio-Gel A 1.5 m, CM-Sephadex C-50 and Sephadex G-G-75. 2. The pH optimum against denatured bovine hemoglobin is 7.4. Gel chromatography indicated a molecular weight close to 23 000. 3. This neutral protease (EC 3.4.-.-) is able to split the synthetic esters Z-Ala-NPh and AcAla3OMe, its activity on the former substrate being 2.2 times greater than that of pancreatic elastase, on the latter the same. It differs crucially from pancreatic elastase in having small elastinolytic activity. 4. In cationic disk electrophoresis, neutral protease resolves into three protein bands with lower mobility than lysozyme: all bands exhibit esterolytic activity against 2-acetoxy-3-naphthoic acid o-toluidide, strongly suggesting that they represent isoenzymes. 5. The enzyme is completely inhibited by iPr2P-F, partially so by soybean trypsin inhibitor and Trasylol. Cysteine, EDTA and TosLysCH2Cl have no effect. 6. During chromatography on CM-Sephadex C-50 a more positively charged enzyme(s) was identified. This had hemoglobinolytic activity at pH 7.4 but only a small esterolytic effect on Z-Ala-NPh; it showed only traces of activity against AcAla3OMe.     

5-Formyl-2'-deoxyuridine: cytostatic and antiviral properties and possible modes of action.

5-Formyl-2'-deoxyuridine (fdUrd) was prepared by a new method starting from thymidine and investigated for its influence both on proliferation of cultured mammalian cells and virus replication in vitro. The compound was found to have strong cytostatic and antiviral properties: 50% inhibition of proliferation of BHK 21/C13 cells or Ehrlich ascites tumour cells (EAT) was obtained at 4 - 10(-6) and 6 - 10(-6) M, respectively, while the treatment of pseudorabies virus with the same concentration resulted in about 1.5 log reduction of virus yield. A concentration of 1 - 10(-4) M inhibited cell proliferation by 80 to 100% while the virus yield was reduced by more than 3 orders of magnitude. All inhibitions can be prevented by thymidine.--DNA synthesis of EAT cells in vitro, as estimated by incorporation of [32P]-phosphate or low concentrations of [3H]-thymidine, was inhibited. Further biochemical experiments have provided indirect evidence that the compound is phosphorylated by thymidine and thymidylate phosphorylating enzymes. An inhibition of cell free DNA synthesis was found to be depending on a given period of preincubation with the compound (supposed to be needed for the formation of fdUrd 5'-triphosphate). This suggests that the 5'-triphosphate of fdUrd is an inhibitor of DNA polymerases and--by analogy with experiments with 5-formyluridine-5'-triphosphate and RNA polymerases [14]--may be used as an affinity label for this group of enzymes. It is concluded that the described cytostatic and antiviral effects of fdUrd are due to an intracellular "lethal" synthesis of the relevant phosphates which inhibit thymidylate synthetase (as had been found earlier to occur with the chemically prepared nucleotide in cell free extracts [1, 2]) and DNA synthesizing enzymes.     

Characterization of surface glycoproteins of mouse lymphoid cells

We have labeled exposed surface glycoproteins of mouse lymphoid cells by the galactose oxidase-tritated sodium borohydride technique. The labeled glyco-proteins were separated by polyacrylamide slab gel electrophoresis and visualized by autoradiography (fluorography). The major thymocyte surface proteins have molecular weights of 170,000 and 125,000. Thymocytes from TL antigen-positive mouse strains showed an additional band with a molecular weight of 27,000. Highly purified T lymphocytes contain two major surface glycoproteins with molecular weights of 180,000 and 125,000. Purified B lymphocytes have one major surface glycoprotein with a molecular weight of 210,000. When T lymphocytes are stimulated in vitro by concanavalin A or phytohemag-glutinin, the major proteins characteristic of T cells are relatively weakly labeled, but new components of lower molecular weights appear on the cell surface. A similar change is seen in B lymphocytes stimulated by Escherichia coli lipopolysaccharide. T lymphoblasts isolated from mixed lymphocyte cultures show a slightly different surface glycoprotein pattern. A polypeptide with a molecular weight of 57,000, which was labeled without enzymatic treatment by tritiated sodium borohydride alone, is strongly labeled in proliferating cells.     

Tumor-associated blocking factors: isolation from sera of tumor-bearing mice.

An isolation and partial purifications of tumor-associated blocking factors from the sera of tumor-bearing mice is described. Columns for affinity chromatography were prepared by coupling syngeneic tumor-immune antibodies to Sepharose 4B. Passage of serum through such immunoadsorbent columns removed all blocking activity from tumor-bearers' sera; subsequent elution of the absorbent with 3 M NaSCN recovered the activity. The blocking material was further purified on Sephadex G-200. The data provide evidence for the presence of antigen in tumor-associated blocking factors and are compatible with the hypothesis that blocking factors often consist of antigen and antibodies in the form of immune complexes.     

The effect of splenectomy, produced chemically by means of ethylpalmitate, on the formation of heteroagglutinins in the rat]

The formation of heteroagglutinins against human O-erythrocytes was pursued in Wistar rats splenectomized "chemically" by means of intravenous injections of ethyl palmitite. Contrary to some data in literature the initial inhibition in the formation of antibodies was replaced by a significant titre increase ranging from the 8th to the 14th day following immunization. The tween 20 contained in the emulsion of ethyl palmitic did not influence the formation of heteroagglutinins. The examinations show that a possible use of palmitic ethyl for immunosuppression is questionable.     

Bone marrow ribonucleic acid polymerase. Effect of testosterone on nucleotide incorporation into nuclear RNA.

The incorporation of 3H-UTP into RNA by isolated rat bone marrow nuclei is stimulated by testosterone. This effect is hormone and tissue specific. Using alpha-amanitine and different ionic strength conditions it was found that testosterone enhances preferentially RNA polymerase I activity. The sedimentation pattern of RNA isolated from bone marrow nuclei shows that the synthesis of RNA species within the 14-30 S range is mainly stimulated by the hormone.     

Changes in fibrinolysis components during short-term adaptation to high altitude]

In studying hemocoagulation in dogs under conditions of Frunze (760 m above the sea level) and Tuya-Ashu (3200 m above the sea level) it was shown that in the "emergency" phase of adaptation (the first three days) there was seen activation of fibrinolysin and profibrinolysin with depression of antifibrinolysins and inhibitors of profibrinolysin activators. The concentration of plasma fibrinogen at that period decreased by 100 mg%, which could promote an increase in the vascular permeability and improvement of oxygen approach to the tissues. Later, along with elevation of fibrinolysin and profibrinolysin activators there was a marked increase in the level of fibrinolysis inhibitors. Correlation of all the fibrinolysis components was established at a new level.