Expression and localization of bacterial luciferase determined by immunogold labeling

Using a polyclonal antibody raised against purified luciferase of Vibrio harveyi and immunogold labeling on thin sections, the amounts and cellular localization of luciferase were examined during the growth of the bacteria. Cells harvested at different times during cultivation in liquid medium at 22°C were fixed, either chemically or by fast freeze fixation followed by freeze substitution, and embedded in LR White. Concomitant measures of bioluminescence, both in vitro and in vivo,showed the classical curve of autoinduction. The number of gold particles per cell area showed a similar pattern. Their localization was always cytoplasmic, with no indication of special periplasmic or membrane associations.     

Ultrastructural localization of osteocalcin in rat tooth germs by immunogold staining

Summary Osteocalcin was localized by indirect immunogold staining of thin frozen sections of rat tooth germs which had been fixed by different methods. Acrolein fixation proved to be satisfactory considering the preservation of fine structure and antigenicity. In odontoblasts, osteocalcin was found to be localized in the cisternae of the rough endoplasmic reticulum and Golgi apparatus. Few positive transport vesicles were found. Staining for osteocalcin in odontoblastic processes was only observed after strong fixation and was intense in odontoblasts engaged in early dentine formation. Predentine was slightly positive in the neighbourhood of positive processes. Matrix vesicles were negative and strong osteocalcin labeling of dentine seemed to appear after the onset of mineralization.     

Ultrastructural localization of osteocalcin in rat tooth germs by immunogold staining

Osteocalcin was localized by indirect immunogold staining of thin frozen sections of rat tooth germs which had been fixed by different methods. Acrolein fixation proved to be satisfactory considering the preservation of fine structure and antigenicity. In odontoblasts, osteocalcin was found to be localized in the cisternae of the rough endoplasmic reticulum and Golgi apparatus. Few positive transport vesicles were found. Staining for osteocalcin in odontoblastic processes was only observed after strong fixation and was intense in odontoblasts engaged in early dentine formation. Predentine was slightly positive in the neighbourhood of positive processes. Matrix vesicles were negative and strong osteocalcin labeling of dentine seemed to appear after the onset of mineralization.     

Apolipoprotein E localization in rat hepatocytes by immunogold labeling of cryothin sections

The distribution of apolipoprotein (apo) E in rat hepatocytes was investigated with an affinity-purified polyclonal antibody raised against apoE isolated from hepatogeneous very low density lipoproteins (VLDL). The distribution of this antibody was visualized with colloidal gold complexed to anti-rabbit IgG. By epipolarization microscopy, apoE was found uniformly along the basolateral surfaces of all hepatic parenchymal cells, showing a striking intensity along the sinusoidal front. Punctate deposits of colloidal gold appeared to be randomly distributed within all hepatocytes. Widely scattered Kupffer cells also stained for apoE. Electron microscopic examination of immunogold-labeled cryothin sections showed that hepatocytic microvilli projecting into the space of Disse consistently contained clusters of immunogold. The gold particles were variably associated with evident lipoprotein particles, raising the possibility that apoE alone may bind to receptors or other macromolecules at the surface of hepatocytes. Endosomes near the sinusoidal front and multivesicular bodies in the Golgi/biliary area labeled intensely for apoE, consistent with a high content of apoE associated with triglyceride-rich lipoprotein remnants contained within these organelles. Some but not all nascent VLDL particles within putative forming Golgi secretory vesicles were labeled, but many other Golgi vesicles and cisternae that lacked evident VLDL particles were also labeled. These results suggest that at least some apoE associates with nascent VLDL in forming Golgi secretory vesicles. Unexpectedly, the matrix of all hepatocytic peroxisomes was heavily labeled. Immunoblots with the affinity-purified anti-rat apoE IgG against proteins from highly purified peroxisomes isolated from rat hepatocytes revealed a protein with an apparent molecular mass of 34.5 kDa, similar to that of rat apoE in rat blood plasma. In addition, gold was sometimes found in the area either adjacent to peroxisomes or between multivesicular bodies and the bile canaliculus not evidently associated with a membranous compartment. These observations suggest that apoE may participate in interorganellar cholesterol transport within hepatocytes.     

Ultrastructural localization of dentine phosphoprotein in rat tooth germs by immunogold staining

Summary Dentine phosphoprotein (DPP) was localized on thin frozen sections of fixed rat tooth germs by indirect immunogold staining. Antisera were directed against DPP and against glutaraldehyde-treated DPP and were characterized by immuno-electroblotting. In odontoblasts, DPP was found to be localized in the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus and in Golgi-associated vesicles. Odontoblastic processes were moderately positive for DPP and dentine was intensely labeled on frozen sections of unfixed tissue. Predentine showed a slight immunoreactivity. These results indicate the synthesis of DPP in the RER, its accumulation in the Golgi apparatus and its vesicular transport and secretion via the odontoblastic processes into dentine. The close association of the gold particles with the dentinal collagen fibres makes a role of DPP in linking mineral to collagen conceivable. Matrix vesicles were negative for DPP, suggesting that the protein is not present at the sites of matrix vesicleassociated nucleation.     

免疫胶体金及其在植物激素定位中的应用

植物激素对于调节植物的各种生长发育过程和环境的应答具有十分重要的作用。国内外关于植物激素的生物学效应已有了大量的研究,但对于它们在植物体内的分布和转运却报道很少,究其原因主要是缺乏准确和灵敏的定位手段。新近发展起来的免疫胶体金技术为植物激素的原位分析提供了有效手段。本文介绍了免疫胶体金技术原理和发展历程,综述了其在植物激素定位研究上的应用,并对存在的问题和进一步的发展进行了讨论。     

Ultrastructural localization of dentine phosphoprotein in rat tooth germs by immunogold staining

Dentine phosphoprotein (DPP) was localized on thin frozen sections of fixed rat tooth germs by indirect immunogold staining. Antisera were directed against DPP and against glutaraldehyde-treated DPP and were characterized by immuno-electroblotting. In odontoblasts, DPP was found to be localized in the cisternae of the rough endoplasmic reticulum (RER) and the Golgi apparatus and in Golgi-associated vesicles. Odontoblastic processes were moderately positive for DPP and dentine was intensely labeled on frozen sections of unfixed tissue. Predentine showed a slight immunoreactivity. These results indicate the synthesis of DPP in the RER, its accumulation in the Golgi apparatus and its vesicular transport and secretion via the odontoblastic processes into dentine. The close association of the gold particles with the dentinal collagen fibres makes a role of DPP in linking mineral to collagen conceivable. Matrix vesicles were negative for DPP, suggesting that the protein is not present at the sites of matrix vesicle-associated nucleation.     

Electron-microscopical localization of chloroplast proteins by immunogold labelling on cryo-embedded spinach leaves

Cryo-substituted spinach leaf pieces, embedded in LR-White resin by chemical polymerization at low temperature, were used to localize enzymes within chloroplasts by immuno-electron microscopy. Employing monospecific antibodies and protein A-gold as label, the spatial distribution of six chloroplast enzymes was investigated. Statistical methods were used to determine whether each enzyme was bound to the thylakoids. By this means the coupling factor 1 (CF1), ferredoxin-NADP⁺ reductase, sedoheptulosebisphosphatase, and ribulose-5-phosphate kinase were found to be membrane-associated. In case of glyceraldehyde-3-phosphate dehydrogenase the label was only weak and an unequivocal determination of its location was not possible. In contrast, ribulosebisphosphate carboxylase was randomly distributed throughout the chloroplast.     

Pregnenolone 16 alpha-carbonitrile-inducible P-450 gene family: gene conversion and differential regulation.

A cytochrome P-450 cDNA clone, designated pP450PCN2, homologous to the previously characterized pregnenolone 16 alpha-carbonitrile (PCN)-induced P-450 cDNA (pP450PCN1; F. J. Gonzalez, D. W. Nebert, J. P. Hardwick, and C. B. Kasper, J. Biol. Chem. 260:7435-7441), was isolated from a rat liver cDNA expression library by use of a polyclonal anti-P450PCN1 antibody. This P-450 cDNA contains 2,014 base pairs and yields an open reading frame of a protein consisting of 504 amino acids (Mr = 57,760). P450PCN2 cDNA and protein shared 90% nucleotide and 89% amino acid similarity with P450PCN1 cDNA and protein, respectively. The 5' untranslated, coding, and 3' untranslated regions between the two cDNAs share 94, 93, and 79% similarities, respectively. Nucleotide differences in the coding regions, however, are not evenly distributed. Complete homology exists between the two mRNAs for 425 nucleotides (positions 346 through 771). Other regions of 93 nucleotides containing only one difference and 147 nucleotides containing two differences exist toward the 3' end of the coding regions. These data suggest the possibility that a gene conversion event(s) have occurred subsequent to duplication of the ancestral P450PCN gene. Oligonucleotide probes unique for P450PCN1 and P450PCN2 cDNAs were used to examine the levels of their respective mRNAs in noninduced and PCN-induced liver cells and in male and female rats of various ages. P450PCN1 mRNA was not detectable in either male or female rats at any ages. In contrast, P450PCN2 mRNA was present at a low level in newborn rats and became elevated in both males and females at 1 week of age. Levels of p450PCN2 mRNA continued to increase in males until 12 weeks, whereas the mRNA in females reached peak levels at 2 weeks of age but declined continuously at the onset of puberty (between 4 and 12 weeks). These levels of P45PCN2 mRNA closely parallel the increases in testosterone 6 beta-hydroxylase activity and P450PCN2 protein level, as analyzed by Western blots. P450PCN1 mRNA was induced by PCN, dexamethasone, and phenobarbital in both male and female rats. P450PCN2 mRNA was not significantly induced by PCN or dexamethasone but was readily induced by phenobarbital. Testosterone 6 beta-hydroxylase activity was also induced severalfold by PCN, dexamethasone, and phenobarbital. These data demonstrate that P450PCN1 and P450PCN2 genes are differentially regulated during development and after administration of inducing compounds and furthermore suggest that both enzymes possess testosterone 6 beta-hydroxylase activity.     

Asparagine synthetases of Klebsiella aerogenes: properties and regulation of synthesis

We isolated pleiotropic mutants of Klebsiella aerogenes with the transposon Tn5 which were unable to utilize a variety of poor sources of nitrogen. The mutation responsible was shown to be in the asnB gene, one of two genes coding for an asparagine synthetase. Mutations in both asnA and asnB were necessary to produce an asparagine requirement. Assays which could distinguish the two asparagine synthetase activities were developed in strains missing a high-affinity asparaginase. The asnA and asnB genes coded for ammonia-dependent and glutamine-dependent asparagine synthetases, respectively. Asparagine repressed both enzymes. When growth was nitrogen limited, the level of the ammonia-dependent enzyme was low and that of the glutamine-dependent enzyme was high. The reverse was true in a nitrogen-rich (ammonia-containing) medium. Furthermore, mutations in the glnG protein, a regulatory component of the nitrogen assimilatory system, increased the level of the ammonia-dependent enzyme. The glutamine-dependent asparagine synthetase was purified to 95%. It was a tetramer with four equal 57,000-dalton subunits and catalyzed the stoichiometric generation of asparagine, AMP, and inorganic pyrophosphate from aspartate, ATP, and glutamine. High levels of ammonium chloride (50 mM) could replace glutamine. The purified enzyme exhibited a substrate-independent glutaminase activity which was probably an artifact of purification. The tetramer could be dissociated; the monomer possessed the high ammonia-dependent activity and the glutaminase activity, but not the glutamine-dependent activity. In contrast, the purified ammonia-dependent asparagine synthetase, about 40% pure, had a molecular weight of 80,000 and is probably a dimer of identical subunits. Asparagine inhibited both enzymes. Kinetic constants and the effect of pH, substrate, and product analogs were determined. The regulation and biochemistry of the asparagine synthetases prove the hypothesis strongly suggested by the genetic and physiological evidence that a glutamine-dependent enzyme is essential for asparagine synthesis when the nitrogen source is growth rate limiting.     

Mitochondrial dolichyl-phosphate mannose synthase. Purification and immunogold localization by electron microscopy.

Mitochondrial dolichyl-phosphate mannose synthase has been purified to homogeneity using an original procedure, reconstitution into specific phospholipid vesicles and sedimentation on a sucrose gradient as final step. The enzyme has an apparent molecular mass of 30 kDa on an SDS/polyacrylamide gel. Increased enzyme activity could be correlated with this polypeptide band. A specific antibody was raised in rabbits against this transferase. Specific IgG obtained from the immune serum removed enzymatic activity from a detergent extract of mitochondrial outer membrane and reacted specifically with the 30-kDa band on immunoblots. Furthermore, an immunocytochemical experiment proved the localization of dolichyl-phosphate mannose synthase on the cytosolic face of the outer membrane of mitochondria.