Changes in lipid composition of tritosomes during lysis

Membrane phospholipids and free fatty acids were analyzed after lysis of lysosomes which had engulfed Triton WR 1339 (tritosomes). Significant elevations of lysophosphatidyl choline and lysophosphatidyl ethanolamine were seen. Phosphatidyl choline, phosphatidyl ethanolamine and sphingomyelin decreased significantly. Triglyceride also fell. The released free fatty acids paralleled closely the liberation of β-glucuronidase from the tritosomes. Thus, lipases hydrolyzed endogenous phospholipids and neutral lipids of tritosomes during incubation at 37°C at pH 5.0 to produce significant elevations of lysophospholipids and free fatty acids. The known surface-active properties of these latter compounds raise the possibility of their participation in the process of lysis of the tritosomes.     

Epithelial cell proliferation and migration in the developing rat gastric mucosa

To investigate cell proliferation in developing gastric antrum and fundus, proliferating gastric epithelial cells were labeled in fetal rats by intravenously injecting mothers with [³H]thymidine. In addition 14-day postnatal (dPN) rats were given [³H]thymidine intraperitoneally. Tissue was removed 1.5 hr later, and autoradiographs were prepared to identify proliferating cells. Total epithelial labeling indices (L.I.) reached a peak at 20 days gestation (dG), coincident with the appearance of pit/glands, then declined again by 22 dG (gestation end). At 18 dG, labeled cells were distributed throughout all levels of the stratified epithelia. Between 20 and 22 dG, as pit/gland development proceeded, labeled cells became concentrated in the gland bases and were only rarely seen on the surface (L.I. of surface cells <1% at 22 dG). By 14 dPN, proliferating cells were entirely absent from the epithelial surface. Approximately 15% of the endocrine cells were labeled at 22 dG, compared to only 2% at 14 dPN; zymogen cells were labeled (～6%) at 14 dPN; parietal cells did not label at any age studied. In addition, cell migration to the epithelial surface was studied in rats labeled at 22 dG, 14 dPN, or 28 dPN, and killed 1–20 days later. Migration times were slightly shorter in the 28 dPN group and were longer in fundus than antrum in all groups.     

A microassay for the determination of monoamine oxidase activity using electron capture gas chromatography

A sensitive and specific assay for determining monoamine oxidase (MAO) activity in serum and platelets is described. The procedure employs m-iodobenzylamine as substrate. The product, m-iodobenzaldehyde, is separated on an OV-17 column and measured by electron capture. Gas chromatography offers the advantage of analytical specificity without the need for additional procedural steps to eliminate potential interferences. Electron capture detection of the iodinated aldehyde is sufficiently sensitive to allow routine analysis on platelet samples of less than 15 μg of protein and serum aliquots of 50 μl or less. Analysis of approximately 80 samples per day may be accomplished by a single worker by employing an automatic sampling system for gas chromatographic injection. This fact, in addition to the small sample size, makes the method particularly suitable for the determination of large numbers of clinical samples.     

Chemical quantitation of hemoglobin glycosylation: fluorometric detection of formaldehyde released upon periodate oxidation of glycoglobin

A sensitive fluorometric method for the quantitation of hemoglobin glycosylation, based upon periodate oxidation of the carbohydrate moieties present on both the α- and ?-amino groups of globin is described. The formaldehyde product is measured as the fluorescent 3,5-diacetyl-1,4-dihydrolutidine formed from the condensation of formaldehyde with acetylacetone and ammonia.This method is rigorously designed to assay glycosylated hemoglobin levels and to give a direct measure of the number of glycogroups per milligram of hemoglobin. It requires only 1 mg of protein and may also be used to determine the extent of the nonenzmatic glycosylation of other proteins.     

Capillary gas chromatography of methylhexitol acetates obtalned upon methylation of N-glycosidically linked glycoprotein oligosaccharides

The 30 O-methylated hexitol and 2-deoxy-2-(N-methyl)acetamidohexitol acetates which commonly may be obtained during methylation analysis of N-glycosidically linked glycoprotein oligosaccharides or their biosynthetic precursors, were subjected to chromatography through glass capillary columns, wall-coated with either Silar 9CP, Dexsil 410, SE-30, or OV 101. All 22 methylhexitol acetates (e.g., 1,5-di-O-acetyl-2,3,4,6-tetra-O-methylglucitol and -mannitol) were optimally resolved on the (most polar) Silar column, whereas the 8 aminohexitol derivatives (as well as the corresponding unmethylated hexitol and aminohexitol acetates) were best separated on either Dexsil 410 or OV 101.     

Temperature-dependent inactivation of nucleic acid binding and aggregation of the 1,25-dihydroxyvitamin D3 receptor

The interaction of the 1α,25-dihydroxyvitamin D₃ receptor with immobilized calf thymus DNA has been compared with its sedimentation properties on hypotonic sucrose gradients. Forty to sixty percent of total hormone:receptor complexes formed at 4 °C were retained by DNA-cellulose and could be eluted by 0.18 to 0.2 m KCl. In contrast, heating preparations to 25 °C rapidly and irreversibly converted receptor to a form which bound hormone and DEAE-cellulose normally, but was unable to associate with DNA. Similarly, the ability of receptor to aggregate to a 6 S species was labile at 25 °C. Stabilization of receptor in the DNA binding aggregating form was accomplished using Ca²⁺, Mg²⁺, Mn²⁺, or Na₂MoO₄ while several protease and phosphatase inhibitors were ineffective. An examination of DNA binding properties of aggregating and nonaggregating receptor forms revealed that only receptor competent to enter into aggregates could bind DNA suggesting that a functional nucleic acid binding site, and, hence, a nucleic acid interaction is necessary for aggregate formation. Consistent with this view, an RNA:receptor interaction appears to be involved in formation of the 6 S complex since removal of RNA by ribonuclease treatment or purification of receptor reduced aggregation, an effect that could be reversed by addition of purified RNA.     

The synthesis and characterization of nucleotide complexes involving an asymmetric metal

The synthesis of cobalt and chromium complexes of H₄ATP and H₄GTP in which the metal is asymmetric are reported. These compounds were characterized by visible spectroscopy, fast atom bombardment mass spectroscopy (FAB MS), and ³¹P NMR. The mass spectral data allow identification of the complexes to be made from ions in the molecular weight region. The effect of an asymmetric metal greatly alters the appearance of the ³¹P NMR spectra in comparison to complexes which do not have this feature. Complexes of uridine diphosphoglucose, UDPG, are also reported. The effect of an asymmetric metal ion on the chromatographic and spectral properties of the complexes are discussed.     

Ligand-affinity sedimentation equilibrium using an air-driven ultracentrifuge

An air-driven ultracentrifuge has been used to study the distribution of radioactive ligands at sedimentation equilibrium. In the presence of a suitable acceptor and under conditions where the ligand is essentially all bound the distribution of ligand can be analyzed to yield the molecular weight of the acceptor molecule. Suitable conditions can be chosen either experimentally by measuring the ratio of bound ligand compared to unbound ligand or theoretically for systems in which the ligand-binding affinity and number of acceptor binding sites is known. The method is applicable to the molecular characterization of binding proteins in crude mixtures and results are presented for the binding of various fatty acids to serum albumin samples.     

Studies on 125I-labeled proteins in rat plasma using an air-driven ultracentrifuge: protein-protein interactions and nonideality

The molecular weights of a number of ¹²⁵I-labeled plasma proteins have been determined from an analysis of their sedimentation equilibrium behavior in an air-driven ultracentrifuge. The values obtained agree well with results obtained by other methods. Molecular weights obtained for ¹²⁵I-labeled bovine serum albumin and the rat serum proteins albumin, α₁-acid glycoprotein, and major acute-phase α₁-protein were unaffected by the addition of 7% rat plasma. Direct evidence for protein-protein interactions was obtained for mixtures of ¹²⁵I-labeled rat α₁-acid glycoprotein and the plant lectin concanavalin A and for mixtures of ¹²⁵I-labeled protein A from Staphylococcus aureus and 7% rat plasma. Interactions of a different type were observed when the sedimentation equilibrium profiles of ¹²⁵I-labeled proteins were determined in concentrated solutions of other proteins. Under these conditions the effects of molecular exclusion or nonideality became significant and low estimates were obtained for the molecular weights of the labeled proteins. Analysis of the data obtained for ¹²⁵I-labeled bovine serum albumin in concentrated solutions of bovine serum albumin (20–80 mg/ ml) yielded nonideality coefficients in good agreement with literature values. Analysis of the behavior of ¹²⁵I-labeled rat serum albumin, transferrin, and α₁-acid glycoprotein yielded nonideality coefficients and hence activities of these proteins in undiluted rat plasma.     

Chromosome segregation in an asymmetrically dividing bacterium,Caulobacter crescentus

The mode of chromosome segregation in an asymmetrically dividing bacterium, Caulobacter crescentus, was studied by examining the fate of labeled DNA strands. Swarmer cells (one type of Caulobacter daughter cell), in which single strands of DNA had been labeled with [³H]thymidine during the previous round of chromosome replication, were grown synchronously in a non-radioactive medium for two generations. The distribution of radioactivity among the cells was visualized by autoradiography under a phase-contrast microscope. The labeled DNA strands in each cell were found to consist of two conserved units. From this, we propose a model in which the swarmer cell has two identical chromosomes, which are segregated into the progeny swarmer cell and the progeny stalked cell after chromosome replication.     

Competition equilibria of metal ions with nucleobases: an ESR study

An Electron Paramagnetic Relaxation study of the competitive equilibria of Cu(II) and Mn(II) towards imidazole and methyl-imidazole was carried out. ΔH, a, I and lineshape variations were studied in order to define the extent and the limits of the metal ions interaction with nucleobases. The EPR evidence of a major affinity of Cu(II) with respect to Mn(II) in binding to nucleobases was demonstrated.     

Enantiomeric cisplatin analogues: an investigation on their activity towards tumors in mice

A series of enantiomeric cisplatin analogues of formula [diamPtCl₂] (diam = chiral chelating diamine) and the corresponding sulfato derivatives was prepared and tested for activity against tumors in mice, particularly P 388 leukemia. The configuration of the diamines has practically no influence on the antitumor activity. The effects of the leaving group and of the nature of the diamines are briefly discussed.     

Characterization of an ATP-Mg2+-dependent guanine nucleotide-stimulated adenylate cyclase from Neurospora crassa

A novel adenylate cyclase activity was found in crude homogenates of Neurospora crassa. The adenylate cyclase had substantial activity with ATP-Mg2+ as substrate differing significantly from the strictly ATP-Mn2+-dependent enzyme characterized previously. Additionally, the ATP-Mg2+-dependent activity was stimulated two- to fourfold by GTP or guanyl-5'-yl-imido-diphosphate (Gpp(NH)p). We propose that the ATP-Mg2+-dependent, guanine nucleotide-stimulated activity is due to a labile regulatory component (G component) of the adenylate cyclase which was present in carefully prepared extracts. The adenylate cyclase had a pH optimum of 5.8 and both the catalytic and G component were particulate. The Km for ATP-Mg2+ was 2.2 mM in the presence of 4.5 mM excess Mg2+. Low Mn2+ concentrations had no effect on adenylate cyclase activity whereas high concentrations of Mn2+ or Mg2+ stimulated the enzyme. Maximal Gpp(NH)p stimulation required preincubation of the enzyme in the presence of the guanine nucleotide and the K1/2 for Gpp(NH)p stimulation was 110 nM. Neither fluoride nor any of a variety of glycolytic intermediates or hormones, including glucagon, epinephrine, and dopamine, had an effect on ATP-Mg2+-dependent adenylate cyclase activity. However, the enzymatic activity was stimulated not only by GTP but also by 5'-AMP and was inhibited by NADH.     

Effects of stress on release of dopamine and serotonin in the striatum of spontaneously hypertensive rats: an in vivo voltammetric study

By use of in vivo voltammetry technique, in vivo release of dopamine and serotonin in the striatum under stress was found to be more prominent in spontaneously hypertensive rats (SHR) at 4 weeks of age than in control Wistar-Kyoto rats (WKY). Simultaneously, a greater activation of tyrosine hydroxylase in the striatum was demonstrated in SHR than in WKY. These results indicate that SHR is more susceptible to stress in the central monoaminergic neurons than WKY.