Histochemical detection of horseradish peroxidase activity in the rat by the vibratome-paraplast method

A method is described for the histochemical detection of horseradish peroxidase in Paraplast Plus embedded brain sections. The procedure uses 150-micron-thick Vibratome-cut slices of glutaraldehyde-paraformaldehyde-fixed brain tissue. Tetramethylbenzidine stabilized by diaminobenzidine/cobalt/H2O2 is used as chromogen. The Vibratome-cut slices are dehydrated through a graded series of acetone, cleared in toluol and flat-embedded in Paraplast Plus embedding medium. Serial sections can be cut as thin as 5-7 micron. The method is universal in its application and permits optimal visualization of labeled neurons with great morphological detail at the light-microscopic level.     

Change of hepatitis B virions (Dane particles) phosphorylation pattern by human hepatoma cell particulate fraction

Protein kinase activity has been found in hepatitis B virions (Dane particles) purified from the plasma of hepatitis B surface antigen carriers [Albin, C., and Robinson, W.S. (1980) J. Virol. 34, 297-302]. Dane particles were purified from the pooled, HBeAg-positive plasma. When this preparation was incubated with [gamma 32P]ATP in the presence of 10mM MnCl2 and 0.5% NP-40 for 15 seconds at 30 degrees C, several phosphorylated polypeptides of 20,000, 42,000, 48,000, 50,000 and 56,000 daltons were detected in sodium dodecyl sulfate-polyacrylamide gels. When the Dane particles were incubated with [gamma 32P]ATP, 10 mM MnCl2, and 0.5% NP-40 in the presence of human hepatoma cell (J-5) particulate fraction at 30 degrees C, 15 seconds, the 42,000, 48,000 and 50,000 daltons phosphorylated polypeptides were not found. When human peripheral blood lymphocytes particulate fraction was incubated with Dane particles under the same conditions, no change of Dane particle phosphorylated polypeptides was detected. Previous publications [Albin, C., and Robinson, W.S. (1980) J. Virol. 34, 297-302; Gerlich, W.H. et al. (1982) J. Virol. 42, 761-766] showed that when hepatitis B core particles purified from hepatoma tissues contained protein kinase activity, only phosphorylated polypeptide was 20,000 daltons. Our data suggested that when Dane particles were put in an environment of hepatoma cells (or tissues), the protein kinase could only phosphorylate selected polypeptides in these particles.     

Formation of Virus Assembly Intermediate Complexes in the Cytoplasm by Wild-Type and Assembly-Defective Mutant Human Immunodeficiency Virus Type 1 and Their Association with Membranes

We have previously identified two distinct forms of putative viral assembly intermediate complexes, a detergent-resistant complex (DRC) and a detergent-sensitive complex (DSC), in human immunodeficiency virus type 1 (HIV-1)-infected CD4(+) T cells (Y. M. Lee and X. F. Yu, Virology 243:78-93, 1998). In the present study, the intracellular localization of these two viral assembly intermediate complexes was investigated by use of a newly developed method of subcellular fractionation. In wild-type HIV-1-infected H9 cells, the DRC fractionated with the soluble cytoplasmic fraction, whereas the DSC was associated with the membrane fraction. The DRC was also detected in the cytoplasmic fraction in H9 cells expressing HIV-1 Myr- mutant Gag. However, little of the unmyristylated Gag and Gag-Pol proteins was found in the membrane fraction. Furthermore, HIV-1 Gag proteins synthesized in vitro in a rabbit reticulocyte lysate system in the absence of exogenous lipid membrane were able to assemble into a viral Gag complex similar to that of the DRC identified in infected H9 cells. The density of the viral Gag complex was not altered by treatment with the nonionic detergent Triton X-100, suggesting a lack of association of this complex with endogenous lipid. Formation of the DRC was not significantly affected by mutations in assembly domains M and L of the Gag protein but was drastically inhibited by a mutation in the assembly I domain. Purified DRC could be disrupted by high-salt treatment, suggesting electrostatic interactions are important for stabilizing the DRC. The Gag precursor proteins in the DRC were more sensitive to trypsin digestion than those in the DSC. These findings suggest that HIV-1 Gag and Gag-Pol precursors assemble into DRC in the cytoplasm, a process which requires the protein-protein interaction domain (I) in NCp7; subsequently, the DRC is transported to the plasma membrane through a process mediated by the M domain of the matrix protein. It appears that during this process, a conformational change might occur in the DRC either before or after its association with the plasma membrane, and this change is followed by the detection of virus budding structure at the plasma membrane.     

Lipase catalyzed reactions of aliphatic and arylaliphatic carbonic acid esters

Symmetrical dialkyl carbonates and dibenzyl carbonates reacted with various nucleophiles in the presence of Candida antarctica lipase B in organic solvents. For example, reaction of dibutyl and dibenzyl carbonate with an alcohol gave a mixture of the mono- and disubstituted products. Aminolysis, however, afforded only the carbamates, without subsequent reaction to the ureum derivatives. The reaction rates were rather low compared with carboxylic esters; the reactivity increased in the order dimethyl相似文献   

Cloning and sequence analysis of two embryonic beta-like globin cDNAs (y and z) of hamster.

A cDNA library was prepared from poly(A) mRNA extracted from 9-day hamster-yolk-sac erythroid cells. Two clones containing inserts coding for embryonic beta-like z or y globin-chains were isolated. Their identity was confirmed by (a) translation of hybrid selected mRNAs and (b) nucleotide sequence analysis of the inserts and comparison to the embryonic beta-like globin genes of Balb/c mouse. Availability of sequences for embryonic and adult globin cDNAs will aid in investigations of the molecular mechanisms of the globin switch in hamster YSEC.     

Sequences related to the major subunit gene fedA of F107 fimbriae in porcine Escherichia coli strains that express adhesive fimbriae

Abstract Porcine Escherichia coli strains isolated from cases fo postweaning diarrhea or edema disease were analysed for the presence of fedA , the major subunit gene of F107 fimbriae. The E. coli isolates were known to contain colonisation factor '8813', or to express F107, 2134P or other fimbriae, different from F4, F5, F6, and F41. PCR with fedA -specific primers, restriction enzyme digestion of the PCR product, and nucleotide sequence analysis demonstrated that 2134P pili, colonisation factor '8813' and fimbriae identified on Australian strains of the O141 serotype belong to one family of F107 fimbrial antigens.     

Reconstitution of the fusogenic activity of vesicular stomatitis virus.

Enveloped virus glycoproteins exhibit membrane fusion activity. We have analysed whether the G protein of vesicular stomatitis virus, reconstituted into liposomes, is able to fuse nucleated cells in a pH-dependent fashion. Proteoliposomes produced by octylglucoside dialysis did not exhibit cell fusion activity of the G protein. However, by making use of n-dodecyl octaethylene monoether (C12E8) as the solubilizing agent and by removal of the detergent in two steps, we were able to produce fusogenic G protein liposomes. These G protein liposomes fuse to the BHK-21 cell surface at pH 5.7-6.0 with an efficiency of fusion comparable with that of the parent virus. Physical and chemical analysis revealed that the fusogenic liposomes exhibited a protein to lipid weight ratio of 0.67 and showed an average diameter of 130 nm.     

Identification of porphyrin present in apo-cytochrome c oxidase of copper-deficient yeast cells

Heme a was not detected either in mitochondria isolated from copper-deficient yeast or in the intact cells. Nevertheless, the intracellular concentration of free porphyrins indicated that the pathway of porphyrin and heme synthesis was not impaired in copper-deficient cells. The immunoprecipitated apo-oxidase from copper-deficient cells revealed an absorption spectrum with maxima at 645, 592, 559, 519 and 423 nm, similar to that of purified porphyrin a. When solubilized mitochondria from [³H]leucine and δ-amino[¹⁴C]levulinic acid-labeled copper-deficient yeast cells were incubated with rabbit antiserum against cytochrome c oxidase, a precipitate was obtained. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of this immunoprecipitate showed [³H]leucine associated with six bands and δ-amino[¹⁴C]levulinic acid resolved in a single band. HCl fractionation of copper-deficient mitochondria labeled with δ-amino[¹⁴C]levulinic acid showed a high specific radioactivity in the fraction extracted by 20% HCl, a solvent which extracts porphyrin a. Thinlayer chromatography of the radioactivity found in 20% HCl showed an R_F value identical to that of purified porphyrin a. When δ-amino[³H]levulinic acid-labeled, copper-deficient yeast cells are grown in copper-supplemented medium, the porphyrin a accumulated in copper-deficient cells wa converted into heme a, and this conversion was prevented by cycloheximidine.These observations suggest that porphyrin a is present in the apo-oxidase of copper-deficient cells, but that the conversion to heme a does not occur. This conversion reaction appears to be a point in the biosynthetic pathway of cytochrome c oxidase which is blocked by copper deficieny.     

Lymphatic drainage in patients after replantation of extremities

Lymph drainage was studied by means of lymph scintigraphy in eight patients in whom successful replantation of a totally or subtotally amputated extremity had been performed. Scintigrams were made after subcutaneous injection of technetium-99m in the replanted part of the patient and the contralateral, normal extremity. In all scintigrams, axillary or inguinal lymph node activity is seen, implying drainage of lymph by means of the lymph vessels. Retention of colloid in the replanted part (79 to 94 percent) shows no significant difference with the contralateral, normal side (86 to 94 percent). Unquestionable evidence of regeneration of lymphatics in humans is delivered in the three patients, in whom lymph node activity and normal retention percentages are seen on the scintigrams after total amputation of an extremity followed by replantation without anastomosing of interrupted lymph vessels.