Electron microscopy of hepatitis B virus core antigen expressing yeast cells by freeze-substitution fixation

We have used the freeze-substitution fixation technique for electron microscopy of yeast cells that express the hepatitis B virus core antigen (HBcAg) following transformation with the cloned gene. Abundant spherical particles were found within the transformed cells. These particles had a uniform size and shape, measured about 21 nm in diameter, had electron-lucent centers, and consisted of many subunits. They were localized in both the cytoplasm and the nucleus. None of these particles was found in the cells of the parent strain. Comparison of the HBcAg particles isolated from the yeast cells and the particles within the yeast cells demonstrated that the 21-nm particles were in fact ultrastructurally superimposable on HBcAg. Thus, the HBcAg particles within the yeast cells were similar to the HBcAg particles in human liver tissues infected with hepatitis B virus, not only in their size and appearance, but also in their intracellular localization. These results suggest that the yeast cell has the same machinery for synthesis and intracellular translocation of the HBcAg polypeptides as the human cell.     

Isolation and Characterization of a Photosystem II Core Complex Depleted in the 43 kDa-Chlorophyll-Binding Subunit

The photosystem II core complex purified from digitonin extractsof spinach chloroplasts was resolved into two chlorophyll-proteincomplexes by digitonin polyacrylamide gel electrophoresis aftertreatment with 1 M potassium thiocyanate. One of the chlorophyll-proteincomplexes resolved consisted of 47, 32, 30 and 9 kDa polypeptidesand the other was complementally composed of only the 43 kDapolypeptide. The former complex was highly active in the photoreductionof 2, 6-dichlorophenol indophenol by 1,5-diphenylcarbazide andretained all of the components responsible for the electrontransport from the secondary electron donor (Z) to the primaryelectron acceptor (Q_A). EPR signal II_fast and II_slow were alsopreserved in this complex although their hyperfine structureswere largely modified. The complex was estimated to contain1.8 molecules of plastoquinone A as well as 1.5, 3.7 and 3.9molecules of cytochrome b559, pheophytin and ß-carotene,respectively, per Q_A. These results indicate that potassiumthiocyanate specifically removes the 43 kDa polypeptide fromthe PS II core complex leaving the electron transport systemin an almost intact state. (Received June 17, 1987; Accepted October 23, 1987)     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens I. Auxin and Cytokinin Contents of Cultured Tissues Transformed with Wild-Type and Mutant Ti Plasmids

Carrot and tobacco plants were transformed with Agrobacteriumtumefaciens harboring wild-type, aux^– or cyt^– Tiplasmids. In tobacco, these wild and mutant Ti plasmids inducedthe formation of non-morphogenic galls, galls with shoots, andgalls with roots, respectively. In carrot, however, transformationwith any of these plasmids resulted in only the formation ofamorphous tumors. Determination of IAA and cytokinin contentsshowed that in tobacco, significantly high amounts of cytokininsor IAA are present in the cells transformed with Ti plasmidspossessing cytokinin or IAA biosynthetic genes, respectively.In carrot, cytokinin contents were also high in the cells transformedwith Ti plasmids having cytokinin biosynthetic genes, whereasIAA contents of the cells were similar regardless of the plasmidsused for transformation. These results suggest that the mechanism regulating IAA metabolismmay be different in tobacco and carrot. (Received June 25, 1987; Accepted February 1, 1988)     

The primary structure of the mitochondrial energy-linked nicotinamide nucleotide transhydrogenase deduced from the sequence of cDNA clones

The amino acid sequence of the bovine mitochondrial nicotinamide nucleotide transhydrogenase, which catalyzes hydride ion transfer between NAD(H) and NADP(H) coupled to proton translocation across the mitochondrial inner membrane, has been deduced from the corresponding cDNA. Two clones were isolated by screening a bovine lambda gt10 cDNA library, using two synthetic oligonucleotides and a cDNA restriction fragment as probes. The inserts together covered 3,105 base pairs of coding sequence, corresponding to 1.035 amino acid residues. However, the reading frame at the 5' end was still open. N-terminal sequence analysis of the isolated enzyme indicated the presence of 8 additional residues. Thus, the mature transhydrogenase appeared to have 1,043 amino acid residues and a calculated molecular weight of 109,212. The deduced amino acid sequence of the transhydrogenase contained the sequences of four tryptic peptides that had been isolated from the enzyme. Two of these were the peptides that had been used for construction of the oligonucleotide probes. The other two were tryptic peptides isolated after labeling the NAD-binding site of the transhydrogenase once with [3H]p-fluorosulfonylbenzoyl-5'-adenosine (FSBA), and another time with [14C]N,N'-dicyclohexylcarbodiimide. The FSBA-labeled peptide was found to be located immediately upstream of the [14C]N,N'-dicyclohexylcarbodiimide-labeled peptide, about 230 residues from the N terminus. One of the tryptic peptides used for oligonucleotide probe construction was the same as that labeled with [3H]FSBA when the NAD-binding site was protected from FSBA attack. This peptide, which might be at the NADP-binding site of the transhydrogenase, was located very near the C terminus of the enzyme. The central region of the transhydrogenase (residues 420-850) is highly hydrophobic and appears to comprise about 14 membrane-spanning segments. By comparison, the N- and the C-terminal regions of the enzyme, which contain the NAD- and the putative NADP-binding sites, respectively, are relatively hydrophilic and are probably located outside the mitochondrial inner membrane on the matrix side. There is considerable homology between the bovine enzyme and the Escherichia coli transhydrogenase (two subunits, alpha with Mr = 54,000 and beta with Mr = 48,700), whose amino acid sequence has been determined from the genes (Clarke, D.M., Loo, T.W., Gillam, S., and Bragg, P.D. (1986) Eur. J. Biochem. 158, 647-653).     

Calcium-binding protein regucalcin increases calcium-independent proteolytic activity in rat liver cytosol

The effect of regucalcin, isolated from rat liver cytosol, on neutral proteolytic activity in the hepatic cytosol was investigated. The Ca²⁺-requiring proteinase required 5–10 µM Ca²⁺ for maximal activity in the presence of a protein substrate (globin). The proteinase activity was markedly elevated by the addition of regucalcin (0.25–2.0 µM) in the absence or presence of Ca²⁺ (5.0 µM) added. The effect of regucalcin, however, was the greater in the absence of Ca²⁺ than that in the presence. The pronounced effect of regucalcin on the proteinase activity was also seen in the presence of 1.0 mM EGTA with or without Ca²⁺ (5.0 µM). In the absence of Ca²⁺, the regucalcin-increased proteinase activity was clearly inhibited by the presence of anti-regucalcin antiserum (diluted to 240-fold), leupeptin (20 and 200 µg/ml), and heavy metals (25 µM cadmium or 25 µM zinc), although the inhibition was not complete at the concentration used. The present findings suggest that regucalcin increases proteolytic activity in rat liver cytosol, and that regucalcin may activate Ca²⁺-independent neutral cysteinyl-proteinase.     

Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells

Summary Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1^-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.     

New preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for fatty acids and derivatives

A preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for carboxylic acids is described. 9-Anthraldehyde hydrazone is oxidized with an organic oxidant, N-chlorosuccinimide, in an organic solvent such as ethyl acetate to give ADAM, and then the reaction mixture is directly used as the reagent solution for the derivatization of carboxylic acids. Both the oxidation and the derivatization reaction are carried out at room temperature, and an aliquot of the derivatization mixture is directly injected into a chromatograph. 9-Anthrylmethyl ester derivatives formed from ADAM and various carboxylic acids are sufficiently separated on a reversed-phase column and are sensitively detected fluorometrically. The present method was applied to the high-performance liquid chromatographic determination of long and short chain fatty acids, keto acids, and hydroxy acids.