Stimulation of intermolecular ligation with E. coli DNA ligase by high concentrations of monovalent cations in polyethylene glycol solutions.

In the presence of high concentrations of the nonspecific polymer polyethylene glycol (PEG), intermolecular cohesive-end ligation with the DNA ligase from Escherichia coli was stimulated by high salt concentrations: 200 mM NaCl or 300 mM KCl in 10% (w/v) PEG 6000 solutions, and 100-200 mM NaCl or 150-300 mM KCl in 15% PEG 6000 solutions. Intermolecular blunt-end ligation with this ligase was also stimulated at 100-150 mM NaCl or 150-250 mM KCl in 15% PEG 6000 solutions. The extent of such intermolecular ligation increased and the salt concentrations at which ligation was stimulated extended to lower concentrations when we raised the temperature from 10 to 37 degrees C.     

A simple procedure for the isolation of highly purified lysosomes from normal rat liver

A procedure for the isolation of highly purified lysosomes from normal rat liver is described. The method depends on the swelling of mitochondria when the postnuclear supernatant fraction is incubated with 1 mM Ca2+. The lysosomes can then be separated from the swollen mitochondria by Percoll density gradient centrifugation. The lysosomal fraction obtained by our method was enriched more than 120-fold in terms of the marker enzymes with a yield of 25%. The electron microscopic examination and the measurement of the activities of marker enzymes for various subcellular organelles indicated that our lysosomal preparation was essentially free from contamination by other organelles.     

Photochemically induced nuclear polarization study of exposed tyrosines, tryptophans, and histidines in postsynaptic neurotoxins and in membranotoxins of elapid and hydrophid snake venoms

The accessibility of surface tyrosines, histidines, and tryptophans in snake venom neurotoxins (short and long) and in membranotoxins to excited triplet 10-(carboxyethyl)-flavin was studied by photochemically induced dynamic nuclear polarization at 270 MHz. Trp-29 is accessible in the short neurotoxins--erabutoxins a, b, and c and cobrotoxin--and also in the long neurotoxins--alpha-cobratoxin and alpha-bungarotoxin. Tyr-25 is practically inaccessible in all neurotoxins. Tyr-39 in cobrotoxin and Tyr-55 in alpha-bungarotoxin are accessible. His-6 (revised sequence) is inaccessible in the erabutoxins while His-26 is only very weakly accessible. His-22 of alpha-cobratoxin is inaccessible as are His-4 and -68 in alpha-bungarotoxin and His-4 of cobrotoxin. His-33 of cobrotoxin is accessible. The rigidity order alpha-bungarotoxin greater than or equal to alpha-cobratoxin greater than or equal to erabutoxins, with respect to the unfolding effect of 7 M urea, was deduced in this study. In the membranotoxins studied (cardiotoxin and its analogues I, II, and IV as well as cytotoxin I and II), the two tyrosines Tyr-25 and Tyr-58 are only weakly accessible. Tyr-14 is completely accessible and so is in all probability Tyr-29. These studies allow deductions to be made about the accessibilities in analogous systems. Thus, the accessibility of His-33 and the inaccessibility of His-4 in cobrotoxin can be used to deduce the conformations of these residues in a large group of neurotoxins including the alpha-toxin of Naja nigricollis, neurotoxin II of Naja naja oxiana, and neurotoxins I and III of Naja mossambica mossambica.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of micronucleus formation in relation to chromosomal aberrations in mouse bone marrow

A simulation analysis of the kinetics of micronucleus formation in polychromatic erythrocytes in mouse bone marrow was performed after a single administration of 3 chemicals--mitomycin C (MMC), 6-mercaptopurine (6-MP) and 1-beta-D-arabinofuranosylcytosine (Ara-C)--with different modes of action. The time-response patterns in the incidence of chromosomal aberrations and micronuclei after treatment with each chemical were compared and subjected to the simulation study with 3 parameters. Two of them, the time between the final mitotic metaphase of the erythroid series and nucleus expulsion (T1), and the duration of the polychromatic erythrocyte (PCE) stage in the bone marrow (T2), were almost identical for the 3 chemicals. However, the coefficients of formation rate of micronucleated cells resulting from cells with chromosomal aberration(s) (k) differed: Ara-C differed from the other two. These results indicate that chromosomal aberrations, especially chromatid breaks and probably gaps, induced by this chemical, effectively contribute to micronucleus formation. The DNA content of micronuclei was also compared to the length of acentric fragments induced by Ara-C and it was found that their distributions were comparable. These findings strongly suggest that chromosomal aberrations induced by chemicals are essential events for the induction of micronuclei in the PCE of bone marrow.     

The reaction of superoxide radical with catalase. Mechanism of the inhibition of catalase by superoxide radical

We have studied the time course of the absorption of bovine liver catalase after pulse radiolysis with oxygen saturation in the presence and absence of superoxide dismutase. In the absence of superoxide dismutase, catalase produced Compound I and another species. The formation of Compound I is due to the reaction of ferric catalase with hydrogen peroxide, which is generated by the disproportionation of the superoxide anion (O-2). The kinetic difference spectrum showed that the other species was neither Compound I nor II. In the presence of superoxide dismutase, the formation of this species was found to be inhibited, whereas that of Compound I was little affected. This suggests that this species is formed by the reaction of ferric catalase with O-2 and is probably the oxy form of this enzyme (Compound III). The rate constant for the reaction of O-2 and ferric catalase increased with a decrease in pH (cf. 4.5 X 10(4) M-1 s-1 at pH 9 and 4.6 X 10(6) M-1 s-1 at pH 5.). The pH dependence of the rate constant can be explained by assuming that HO2 reacts with this enzyme more rapidly than O-2.     

Determination of 9α,11β-prostaglandin F2 by stereospecific antibody in various rat tissues

In view of the recent finding that prostaglandin D₂ is stereospecifically converted to 9α,11β-prostaglandin F₂, an isomer of prostaglandin F₂α, a highly specific and sensitive radioimmunoassay for 9α,11β-prostaglandin F₂ was developed and applied to determine the content of this prostaglandin in various rat tissues. Antisera against 9α-11β-prostaglandin F₂ were raised in rabbits immunized with the bovine serum albumin conjugate, and [³H]9α,11β-prostaglandin F₂ was enzymatically prepared from [³H]prostaglandin D₂. The assay detected 9α,11β-prostaglandin F₂ over the range of 20 pg to 1 ng, and the antiserum showed less than 0.04% cross-section with prostaglandin F₂α, prostaglandin F₂β and 9β,11β-prostaglandin F₂. To avoid postmortem changes, tissues were frozen in liquid nitrogen immediately after removal. The basal level of 9α,11β-prostaglandin F₂ was hardly detectable in various tissues of the rat examined, including spleen, lung, liver and brain; although it was found to be 0.31 ± 0.06 ng/g wet weight in the small intestine. During convulsion induced by pentylenetetrazole, enormous amounts of prostaglandin D₂ (ca. 180 ng/g wet weight) and prostaglandin F₂α (ca. 70 ng/g) were produced in the brain; however, 9α,11β-prostaglandin F₂ was detected neither there nor in the blood. This result demonstrates that the conversion to 9α,11β-prostaglandin F₂ is a minor pathway, if one at all, of prostaglandin D₂ metabolism in the rat brain.     

A variant in the restriction endonuclease cleavage pattern of mitochondrial DNA in the domestic fowl, Gallus gallus domesticus

The cleavage patterns of mitochondrial DNAs (mtDNAs) were investigated from 15 lines of domestic fowls, Gallus gallus domesticus, using 11 restriction endonucleases. The cleavage patterns with 10 restriction endonucleases were identical in all the lines. A variant was found in a line of White Leghorn in the pattern with MspI digestions. Cleavage patterns of the red jungle fowl, Gallus gallus gallus, were identical to the common patterns shown by the 14 lines of domestic fowls.     

Development of human pancreas

The developmental sequence of human pancreatic secretory proteins has not previously been studied in detail. We applied immunohistochemistry to study 20 fetal and neonatal pancreas' (8th to 39th gestational weeks) using antisera against the following pancreatic secretory proteins: pancreatic secretory trypsin inhibitor (PSTI), serine proteinases (trypsin, chymotrypsin, and elastase I), and amylase. PSTI was first detected in developing buds of the pancreas during the 8th gestational week, and proteinases were observed in acinar cells during the 14th week of gestation. Immunoreactivity for both PSTI and proteinases was found in most acinar cells soon after their appearance. Immunoreactivity for amylase could not be detected in fetal or neonatal pancreas tissue. PSTI was also found in developing islets during the 14th gestational week, but the number of immunoreactive cells had decreased by term. Cells positive for serine proteinases were occasionally in contact with islets in second-trimester fetuses. In discussing these results, we give particular attention to the nonparallel appearance of secretory products in the fetal pancreas, and the significance of cells immunoreactive for secretory proteins in endocrine islets.     

Amylase in human lungs and the female genital tract. Histochemical and immunohistochemical localization

We investigated the localization of amylase in normal human lungs and the female genital tract using immunohistochemical and histochemical methods. Immunohistochemical procedures were applied to formaldehyde-fixed, paraffin-embedded specimens as well as to cryostat sections of periodate/lysine/paraformaldehyde (PLP)-fixed tissues. The starch-substrate-film method was used for the histochemical investigation of unfixed frozen sections. Amylase immunoreactivity was observed in ciliated epithelial cells of the bronchus and in serous cells of the bronchial glands but not in the alveolar epithelium. Immunoreactive amylase was also found in the cytoplasm of the ciliated epithelium of the fallopian tubes, especially in the apical part of the cytoplasm and in ciliary vesicles. Immunoreactive amylase was also found to be present in the surface epithelial cells and glands of the uterine cervix, as well as in the superficial part of the endometrial glands. The distribution of amylase activity revealed using histochemistry was similar to that observed in cryostat sections of PLP-fixed tissues after immunohistochemical staining. Amylase antigenicity was better preserved in cryostat sections of PLP-fixed materials than in formaldehyde-fixed, paraffin-embedded specimens. The results are discussed in relation to pulmonary and female-genital-tract diseases.