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.     

Prostaglandins,indomethacin and dysmenorrhea

Uterine contractility was recorded during the period of menstruation in six dysmenorrheic women. A variable high tonus was observed in each case. Uterine recordings were repeated during the subsequent menstruation following pre-treatment with indomethacin at an oral dose of 75 mg or 200 mg per day beginning one day before the expected onset of menstruation. A lower uterine tonus was found in all indomethacin-treated cycles. Complete alleviation of spasmodic pain was obtained in the six subjects. The endogenous concentration of 15-keto-13,14-dihydro PGF_2α was determined by the gas chromatography-mass spectrometry method and observed to be relatively high in women with dysmenorrhea.     

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.     

Proton NMR bandshape studies of lamellar liquid crystals and gel phases containing lecithins and cholesterol.

Proton NMR spectra for gel and liquid crystalline samples, composed of dimyristoyl and/or dipalmitoyl lecithin, cholesterol and water, can be consistently interpreted in terms of mesophase symmetry and molecular diffusion according to a model proposed by Wennerstrom (Wennerstrom, H. (1973) Chem. Phys. Lett. 18, 41-44). It is shown by computer simulation that the characteristic "super-lorentzian" bandshape of the lamellar mesophase can be described by the superposition of three gaussian curves. The NMR signal of the gel phase can be simulated by the superposition of two gaussian curves with widths at half height of 2.5 kHz and 19 kHz. An upper limit of the lateral diffusion coefficient of the lecithin molecules in the gel phase is calculated to be about 5-10(-15) m-2/s. It is therefore concluded that the static intermolecular dipolar couplings average to zero in the lamellar mesophase. An estimation of the order parameter of the liquid crystalline phase is made from experimental data and a calculated "rigid lattice" linewidth. A two phase system is shown to exist in the temperature range 28-34 degrees C for a mesophase of a mixture of dimyristoyl and dipalmitoyl lecithin. The presence of cholesterol results in enhanced lateral diffusion of the lecithin molecules at temperatures below the Chapman transition point.     

Genetic and physiological analysis of an envB spherelike mutant of Escherichia coli K-12 and characterization of its transductants.

The envB1 mutation mediating a distorted cell morphology of Escherichia coliK-12 was cotransducible with strA, aroE, aspB, and argG. The mapping data is consistent with a gene location for envB around 62.5 min. In partial diploids envB1 was recessive to its wild-type allele. The original envB mutant contained a second mutation in a locus denoted sloB close to strA. The following gene order is suggested: sloB-strA-aroE-envB-aspB-argG. The sloB1 mutation caused a marked reduction in the growth rate of both envB and envB+ strains. Moreover, this mutation in the presence of envB1 appears to increase the ratio between deoxyribonucleic acid and protein in cells growing in rich medium. The phenotypic properties of envB1, sloB+ and envB+ transductants were characterized. Cells with envB1, sloB+ genotype were hypersensitive to several penicillins including the beta-lactam compound, amidino penicillin. Penicillin hypersensitivity could not be explained by increased outer membrane penetrability. The original envB mutant (envB1,SLOB1), as well as envB1, sloB1 or envB+, SLOB1 transductants were resistant to amidino penicillin. Resistance was explained by the slow growth rate mediated by the sloB1 mutation. The similarity between envB cells and wild-type cells treated with sublethal concentrations of amidino penicillin was emphasized.     

UPTAKE OF 3-O-METHYL-d-GLUCOSE INTO CULTURED HUMAN GLIOMA CELLS

Human glioma cells (138 MG) were found to take up 3-O-methyl-d -glucose (3-OMG) by a saturable low affinity transport system with a K_m of 20 mm and a V_max of 500 nmol/mg protein/min. About 20 per cent of the total uptake was due to passive diffusion. d -Glucose was a competitive inhibitor with a K_i of 10 mm . Follow-up experiments indicated that the same transport mechanism is involved in the uptake of n-glucose and 3-OMG. Phloretin (0·02 mm ) and cytochalasin B (0·002 mm ) strongly inhibited the uptake of 3-OMG, whereas phlorizin (0·02 mm ), ouabain (0·1 mm ), NaCN (0·5 mm ) and iodoacetic acid (1·0 mm ) had no effect. The data suggest that 3-OMG and d -glucose enter 138 MG cells mainly by a Na⁺-independent passive carrier-mediated transport system. Serum-deprivation doubled the population doubling time (T_d) without affecting the total uptake of 3-OMG. An increase in the non-specific (diffusional) uptake was balanced by a decrease in the specific (carrier-medíated) uptake. After addition of dibutyryl cyclic AMP (dbcAMP, 0·25 mm ) the cells attained a morphology characteristic of differentiated glia cells. T_d was maintained unchanged. The non-specific uptake of 3-OMG was not affected in cells grown in serum-containing medium plus dbcAMP, whereas the specific uptake increased by 40 per cent and there-fore also the total uptake. Similar, but more pronounced, changes were observed if serum-deprived cells were treated with dbcAMP.     

Binding of bile salts to pancreatic colipase and lipase.

The binding of conjugated bile salts to pancreatic colipase and lipase has been studied by equilibrium dialysis and gel filtration. The results indicate that at physiological ionic strength and pH, conjugated bile salts bind as micelles to colipase: 12-15 moles/mole of colipase for the dihydroxy conjugates and 2-4 for the trihydroxy conjugates. No binding of bile salt takes place from monomeric solutions. Under the same experimental conditions, only 1-2 moles of conjugated dihydroxy bile salts bind to pancreatic lipase.     

Cyclization of 13beta-ethyl-3-methoxy-17beta-ol-8,14-seco-1,3,5(10),9-gonatetraen-14-one and its 17 acetate derivative.

13beta-Ethyl-3-methoxy-17beta-ol-8,14-seco-1,3,5(10),8-gonatetraen-14-one (IIIa) was isolated and its participation in the well-known acidic cyclization process was established.     

The protein-A plaque assay: a new system for the detection of cells secreting a given idiotype

With a modification of the protein-A plaque assay, cells secreting a given idiotype could be detected. Different anti-idiotypic antisera raised against the M-component of macroglobulinemia Waldenstr?m patients were used. The antibodies did not cross-react with cell clones of other patients or normal controls. Spontaneous plaques, without prior cultivation, were mainly shown to be of IgM class, the majority being idiotype specific, in blood and bone marrow lymphocytes from these patients. This is in contrast to the predominance of Ig or IgA found in normal blood donors. A low but recognizable stimulation of the malignant clone may be observed when cells were stimulated by certain polyclonal B cell activators.     

Quantitative Properties of Random Amplified Polymorphic DNA (RAPD)

Random Amplified Polymorphic DNA analysis (RAPD) is a methodology that has been used as a tool for monitoring microbial communities. To be useful in this application RAPD, and any other methodology, must show properties that allows for the detection of quantitative changes in composition of the microbiota. Therefore, the objective of this study was to establish whether RAPD possesses such properties. The strategy was to use genomic DNA, extracted from a set of tertiary bacterial mixtures defined according to an experimental mixture design, and containing varying proportions of Escherichia coli, Bacillus subtilis, and Pseudomonas CF600. RAPD-PCR was performed on the mixed DNA extracts and the amplified DNA fragments were separated on sequencing gels to produce genomic fingerprints that were digitized and modeled by Partial Least Squares regression (PLS). Significant predictions were obtained using an external test set for validation, with Root Mean Square Error of Predictions (RMSEP) of 0.21, 0.19 and 0.20 for the proportion of E. coli, B. subtilis and Pseudomonas CF600 respectively. Taken together, the results showed that RAPD patterns quantitatively represented the initial mixture proportions. Therefore, the view that RAPD could be useful for whole microbial community monitoring was strengthened.