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.     

Structure of the Bombyx mori fibroin light-chain-encoding gene: upstream sequence elements common to the light and heavy chain.

Two overlapping genomic clones containing the fibroin light-chain (Fib-L)-encoding gene (Fib-L) were obtained from the cosmid library of the silkworm, Bombyx mori J-139, by hybridization with the Fib-L cDNA clone. Sequencing of the 14.6-kb region revealed that Fib-L was 13472 bp long containing seven exons, and that the gene contained a large first intron which occupied about 60% of the gene. Comparison of restriction patterns of the J-139 Fib-L with those of eight other B. mori breeds producing normal-level fibroin demonstrated that considerable restriction-fragment length polymorphisms were present in regions containing the first intron and the 3′-flanking sequence. However, sizes of the Fib-L mRNA and the Fib-L polypeptide were very similar among the nine breeds tested, suggesting that the exon sequences and the splice signals were all well conserved. 5′-Flanking regions of Fib-L and the fibroin heavy-chain (Fib-H)-encoding gene (Fib-H) compared in this study contained three 18-30-bp sequences of high similarity and many 8-10-bp common elements, six of which coincided with the binding sites of homeodomain proteins. Gel retardation assays with the nuclear extracts of the posterior and middle silk glands suggested that protein factors present in the posterior silk-gland nuclei could bind to a set of those common upstream elements.     

Induction of Cytoplasmic Streaming and Movement of Chloroplast Induced by L-Histidine and its Derivatives in Leaves of Egeria densa

Rotational cytoplasmic streaming in leaves of Egeria densa wasinduced by light as well as by L-histidine (L-His). During bothtreatement with light and with L-His chloroplasts on the periclinalface were dislodged and moved to the anticlinal face where rotationalcytoplasmic streaming occurred. The effective concentrationof L-His was about 0.01 mM and the effect was almost saturatedat 0.1 mM. A derivative of L-His, 3-methyl-L-histidine, wasslightly less effective than L-His. By contrast, 1-methyl-L-histidinewas almost ineffective for induction of streaming, not onlyin Egeria but also in Vallisneria. Our resutlts are in markedcontrast to Fitting's result (1936) that 1-M-L-His is more effectivethan L-His. In Egeria, 1-methyl-L-His counteracted the stimulativeeffect of L-His. 1-Methyl-L-His penetrated into leaf cells ofEgeria to the same extent as 3-methyl-L-His and to a greaterextent than L-His. This observation excludes the possibilitythat the impermeability of leaves to 1-M-L-His might be responsiblefor its ineffectiveness. 1-M-L-His did not interfere with photodinesis.Differences in the mechanism of induction of rotational streamingby L-His and by light are discussed. ⁴ Present address: Fukui Institute of Technology, Gakuen, Fukui,910 Japan (Received July 16, 1990; Accepted December 20, 1990)     

Cytoplasmic Alkalization and Cytoplasmic Streaming Induced by Light and Histidine in Leaf Cells of Egeria densa: in vivo 31P-NMR study

Rotational streaming of the cytoplasm including chloroplastswas induced by L-histidine, as well as by light, on the anticlinalface of leaf cells of Egeria densa. In the case of treatmentwith L-histidine some of the chloroplasts remained stationaryon the periclinal face of cells after rotational cytoplasmicstreaming was initiated. However, these chloroplasts were easilydislodged and translocated to the centrifugal end of the histidine-treatedcells by application of a centrifugal force that barely affectedthe location of chloroplasts in cells incubated in the darkwithout L-histidine. This result indicates that the anchoringof chloroplasts was weakened by L-histidine. Thus only the releaseof chloroplasts from anchoring was not enough for initiationof their streaming. The cytoplasmic pH (pH_c) and vacuolar pH(pH_v) were noninvasively monitored by in vivo ³¹P-nuclear magneticresonance (NMR) spectroscopy. Compared with the dark controlvalue, both illumination and treatment with L-histidine increasedthe pH_c by 0.3 units. In contrast, pH_v changed only a littlewith both illumination and treatment with L-histidine. Releaseof chloroplasts from anchoring and initiation of cytoplasmicstreaming are discussed in relation to the increase in pH_c inducedby both light and L-histidine. ⁴ Present address: Department of Cell Biology, National Instituteof Agrobiological Resources, Kannondai, Tsukuba, Ibaraki, 305Japan ⁵ Present address: Marine Biotechnology Institute Co., Ltd.,Head Office, 2-35-10 Hongo, Bunkyo-ku, Tokyo, 113 Japan (Received July 16, 1990; Accepted December 20, 1990)     

Incorporation of fatty acids by Streptococcus mutans

In a series of investigations into the cariogenicity of Streptococcus mutans, we studied the incorporation of exogenous fatty acids with reference to glucosyltransferase secretion and membrane fatty acid changes. When cells were grown with different fatty acids, both saturated and unsaturated fatty acids were readily incorporated into the membrane lipids and were biotransformed and elongated preferentially to the longer 16- and 18-carbon-chain fatty acids. This incorporation and chain-elongation led to significant changes in fatty acids composition. By adding fatty acids to the medium, it was possible to appropriately modify the degree of unsaturation and the relative ratio between specific fatty acids in the membrane lipids of S. mutans.     

Preparation of colloidal gold for staining proteins electrotransferred onto nitrocellulose membranes

This paper describes a simple method of preparing colloidal gold for staining protein blots. Colloidal gold was prepared from 0.005 or 0.01% HAuCl4 by the addition of formalin as a reductant and potassium hydroxide. Staining of small cell carcinoma tissue extract blotted onto nitrocellulose membranes with this colloidal gold solution resulted in the appearance of a large number of clear wine-red bands. The sensitivity of gold staining was 60 times higher than that of Coomassie brilliant blue staining and almost comparable to that of silver staining of proteins in polyacrylamide gel. The sensitivity of this method was also satisfactory in comparison with that of enzyme immunoblotting. The colloidal gold prepared by this method is usable for routine work.     

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.