Purification and characterization of a tuberculin-active substance from Mycobacterium bovis BCG

A new tuberculin-active substance, designated TAS-1D3, has been purified from the extract of Mycobacterium bovis BCG by precipitation at pH 4.2, ethanol fractionation, and column chromatography involving CM-cellulose, QAE-Sephadex A-25, Sephadex G-100, and Sephadex G-75. TAS-1D3 was homogeneous in polyacrylamide gel electrophoresis and positive in both Coomassie brilliant blue and periodic acid-Shiff staining, suggesting that TAS-1D3 is a glycoprotein. The molecular weight of TAS-1D3 was estimated to be 26,000 by gel filtration. In amino acid analysis, TAS-1D3 was distinctive in having proline as a dominant amino acid, and in that it lacked basic amino acids, sulfur-containing amino acids and aromatic amino acids. Moreover, TAS-1D3 was almost devoid of absorption at around 280 nm. In guinea pigs sensitized with BCG vaccine, the tuberculin activity of TAS-1D3 was about forty times more potent than that of purified protein derivative (PPD).     

Characterization of a chloroplast DNA sequence from Chlorella ellipsoidea that promotes autonomous replication in yeast

Summary An EcoRI 2.7 kbp fragment from Chlorella ellipsoidea chloroplast DNA (cpDNA) cloned in YIp5 was shown to promote autonomous replication in Saccharomyces cerevisiae. The fragment was localized in the small single copy region close to the inverted repeat. The ARS activity (autonomously replicating sequences in yeast) was found to be confined within a subclone of a ca. 300 bp HindIII fragment. Sequence analysis of this fragment revealed its high AT content and the presence of several direct and inverted repeats and a few elements that were related to the yeast ARS consensus sequence. Electron microscopic studies revealed that this sequence did not coincide with the primary replication origin of chloroplast DNA. The functioning of this sequence as a possible origin of plasmid replication in vivo is discussed. This is the first report on Chlorella cpDNA sequence. re]19850821 rv]19851211 ac]19851216     

Immunohistochemical detection of bromodeoxyuridine in formalin-fixed tissues

Bromodeoxyuridine (BrdUrd) immunohistochemistry has recently been introduced for the visualization of DNA-synthesizing nuclei. In order to detect the BrdUrd incorporated into nuclear DNA in formalin-fixed, paraffin-embedded tissues, we tested several different pretreatment procedures including digestion with proteinase and hydrolysis with HCl, prior to immunoperoxidase staining. In order to determine the optimal conditions for detecting nuclear BrdUrd, mice were given BrdUrd and 3H-thymidine simultaneously, and the autoradiographic and immunohistochemical results obtained in BrdUrd-stained sections were compared. It was found that digestion with 0.05% proteinase at 37 degrees C for 20 min and hydrolysis with 1N HCl at 37 degrees C for 20 min was sufficient to detect BrdUrd immunoreactivity in 3H-thymidine-labelled nuclei, the results being virtually unaffected by the orders in which the two pretreatments were performed. Our method extends the range of application for BrdUrd immunohistochemistry in cell-kinetic studies.     

Quantitative detection of DNA damage in the neuronal cells of the cerebellum and cerebrum by the analysis of Feulgen hydrolysis curves

Summary Touch smears of the cerebellum and cerebrum of ageing rats were fixed with methanol, hydrolyzed with 2N HCl at various temperatures and for various periods, and stained with pararosaniline-Schiff reagent. The hydrolysis curves were determined by fluorescence cytophotometry and were computer fitted to the Bateman function to determine the kinetic parameters, the initial yield of apurinic acid or single-stranded DNA (y ₀), and the rate constants for depurination or denaturation (k ₁) and depolymerization (k ₂). The values for k ₁ (1/k ₁ is correlated with the degree of chromatin condensation) and k ₂ (which reflects the degree of DNA instability) steadily increased with age. The values for y ₀, which may indicate the degree of DNA denaturation or damage present before acid hydrolysis, also increased with age in both the cerebellum and cerebrum; however, this value was lower in the cerebellum untill 15 weeks, with the situation being reversed after 35 weeks, the cross-over time being at about 25 weeks. The values of lnk ₁ and lnk ₂ were plotted as the function of the reciprocal of the absolute temperature (T) (Arrhenius plot) for both the cerebellum and cerebrum of 15- and 74-week-old rats, and the activation energies (E) for depurination and depolymerization were calculated from the slopes. In particular, the values of E for k ₂ decreased much more quickly with age and were smaller in cerebellum. In conclusion, the degree of DNA damage and DNA instability steadily increases in both the cerebellum and cerebrum of ageing rats, and this process is much faster in the cerebellum.In honour of Prof. P. van Duijn     

Neuromedin K: a novel mammalian tachykinin identified in porcine spinal cord

A new peptide, designated "neuromedin K" has been discovered and isolated from porcine spinal cord by using bioassays for a tachykinin-like effect on the contractility of smooth muscle preparation from guinea-pig ileum. Porcine neuromedin K has been identified by microsequencing as: Asp-Met-His-Asp-Phe-Phe-Val-Gly-Leu-Met-NH2. The sequence thus determined has been confirmed by synthesis. Neuromedin K has been found to have not only a remarkable sequence homology to kassinin and substance P, but also a prompt stimulant activity on guinea-pig ileum in a manner similar to that of substance P, suggesting that neuromedin K may be involved in neural transmission.     

Regulation by divalent cations of 3H-baclofen binding to GABAB sites in rat cerebellar membranes

When investigating the effects of divalent cations (Mg2+, Ca2+, Sr2+, Ba2+, Mn2+ and Ni2+) on 3H-baclofen binding to rat cerebellar synaptic membranes, we found that the specific binding of 3H-baclofen was not only dependent on divalent cations, but was increased dose-dependently in the presence of these cations. The effects were in the following order of potency: Mn2+ congruent to Ni2+ greater than Mg2+ greater than Ca2+ greater than Sr2+ greater than Ba2+. Scatchard analysis of the binding data revealed a single component of the binding sites in the presence of 2.5 mM MgCl2, 2.5 mM CaCl2 or 0.3 mM MnCl2 whereas two components appeared in the presence of 2.5 mM MnCl2 or 1 mM NiCl2. In the former, divalent cations altered the apparent affinity (Kd) without affecting density of the binding sites (Bmax). In the latter, the high-affinity sites showed a higher affinity and lower density of the binding sites than did the single component of the former. As the maximal effects of four cations (Mg2+, Ca2+, Mn2+ and Ni2+) were not additive, there are probably common sites of action of these divalent cations. Among the ligands for GABAB sites, the affinity for (-), (+) and (+/-) baclofen, GABA and beta-phenyl GABA increased 2-6 fold in the presence of 2.5 mM MnCl2, in comparison with that in HEPES-buffered Krebs solution (containing 2.5 mM CaCl2 and 1.2 mM MgSO4), whereas that for muscimol was decreased to one-fifth. Thus, the affinity of GABAB sites for its ligands is probably regulated by divalent cations, through common sites of action.