Desaturation of Oleic and Linoleic Acids by Leaves of Dark- and Light-grown Maize Seedlings

Oleate and linoleate desaturation in leaves of maize seedlings was largely independent of previous light treatment of the seedlings; there was no evidence of light-induced desaturase activities. These results are in sharp contrast to those observed with developing cucumber cotyledons in which pronounced increase in desaturation occurs after exposure of tissue to light. The rates of desaturation of oleate were about four times those of linoleate in both etiolated and 16-hour greened maize leaves. In both etiolated and greened tissues, about two-thirds of the label from oleate was esterified after 4 hours, half of which was in phosphatidylcholine. Phosphatidylcholine and diglyceride contained large proportions of [¹⁴C]linoleate formed from [¹⁴C]oleate but not [¹⁴C]linolenate. In monogalactolipid, about two-thirds of the labeled fatty acids were linolenate. In vivo desaturase activity was present in tissue of widely different levels of differentiation and chlorophyll content obtained from light-grown maize seedlings.     

An Evaluation of Various Fixing and Staining Procedures for Mitochondria in Plant Root Tissues

Mitochondria were stained intensely by a Regaud iron-hematoxylin procedure for roots fixed in formalin-sublimate or in Helly's fluid. Formalin-sublimate fixation required iodization during the staining sequence, but roots fixed in Helly's fluid were best iodized to remove mercurial precipitates before embedding in paraffin. Both methods required treatment with 1% KOH before immersion in the staining solution to remove RNA and produce pale cytoplasm. A third successful method was to postosmicate methacrylate-embedded roots after fixation in Hermann's fluid. Blackened mitochondria were produced by the postosmication and further staining was unnecessary. Fixation in Regaud's fluid did not give successful stains in any of the three methods tested. A prefixation treatment in quinone did not aid in obtaining sharply stained mitochondria of roots fixed in Bouin's fluid and stained with Heiden-hain's iron-hematoxylin.     

铬在南瓜体内的分布与积累

采用^51Cr同位素标记法检测富铬南瓜品种‘永安2号’在不同发育时期对^51Cr的吸收和分配的结果表明：苗期（8片叶）,南瓜地上各部分^51Cr放射性强度的相对比例分别为：叶64．4％,茎30．2％,花5．4％,主要分布在距根部较近的叶片和茎中,幼叶中含量很低;幼果期分别为：果肉51．1％,叶片41．6％,茎4．8％,花2.4％,主要分布在果肉和中部叶片中。     

Non-Enzymatic Reduction of Nitrite by Means of Ascorbic Acid in Citrus and Other Higher Plant Tissues

It has been proved that the nitrite reduction in the leaves and other plant tissues of citrus and other green plants is partly or mainly a non-enzymatic chemical process, and a heat-stable factor present in these tissues is responsible for this reduction. It is suggested that ascorbic acid plays a major role in this chemical reaction since the reduction is inhibited by ascorbic acid oxidase. A significant association was also found between the ascorbic acid content and the nitrite reduction capacity of citrus leaves. Evidence has been presented that this non-enzymatic chemical reduction of nitrite occurs also in vivo as undetached citrus leaves on branches placed in NaNO₂ solution have shown diminution of their ascorbic acid content along with the absorption of nitrite. Stronger accumulation of nitrite in these leaf tissues was observed under dark conditions, apparently due to the inhibition of the biosynthesis of the ascorbic acid.     

Effect of Salicylhydroxamic Acid on Respiration, Photosynthesis, and Peroxidase Activity in Various Plant Tissues

Earlier reports from our laboratory described salicylhydroxamicacid (SHAM) stimulation of O₂ uptake by expanded soybean leavesor older green cotyledons. This stimulation could not be interpretedin terms of engagement or capacity of the cytochrome and alternativerespiratory pathways. In this report, we tested the possibilitythat a soluble peroxidase, which can be easily eluted from soybeanleaves and cotyledons, might be responsible for SHAM stimulationin whole tissue. The peroxidase catalyzes oxidation of NADHby O₂, is strongly stimulated by SHAM and benzhydroxamic acid(BHAM) and inhibited by KCN, propyl gallate and gentisic acid.This peroxidase, however, does not seem to be responsible forSHAM-stimulated O₂ uptake in whole, green tissue. In our earlier work reporting SHAM-stimulated respiration ingreen tissue, the samples had not been shielded from room light(10–20 µmol photons m^–2.s^–1). In thisreport, we show that O²-uptake rates of controls measured indarkness were always greater than those measured in room light.SHAM stimulation was not observed in the dark or in tissue withoutchlorophyll. We also found that CO₂ uptake of whole leafletsin saturating light was completely inhibited by SHAM fed throughthe transpiration stream. SHAM, therefore, is a potent inhibitorof photosynthesis. We conclude that the SHAM-stimulated respirationof green tissues we reported earlier likely was due to verylow rates of photosynthesis occurring under room light. ³Present address: SANDOZ Ltd., Agrobiological Research Station,4108 Witterswil, Switzerland⁴Present address: WTC 1A3, Weyerhaeuser Co., Tacoma, WA 98477,U.S.A. (Received June 23, 1989; Accepted October 20, 1989)     

Sequence comparison of the 63-, 61-, and 59-kDa calmodulin-dependent cyclic nucleotide phosphodiesterases.

Partial protein sequences from the 59-kDa bovine heart and the 63-kDa bovine brain calmodulin-dependent phosphodiesterases (CaM-PDEs) were determined and compared to the sequence of the 61-kDa isozyme reported by Charbonneau et al. [Charbonneau, H., Kumar, S., Novack, J. P., Blumenthal, D. K., Griffin, P. R., Shabanowitz, J., Hunt, D. F., Beavo, J. A. & Walsh, K. A. (1991) Biochemistry (preceding paper in this issue)]. Only a single segment (34 residues) at the N-terminus of the 59-kDa isozyme lacks identity with the 61-kDa isozyme; all other assigned sequence is identical in the two isozymes. Peptides from the 59-kDa isozyme that correspond to residues 23-41 of the 61-kDa protein bind calmodulin with high affinity. The C-terminal halves of these calmodulin-binding peptides are identical to the corresponding 59-kDa sequence; the N-terminal halves differ. The localization of sequence differences within this single segment suggests that the 61- and 59-kDa isozymes are generated from a single gene by tissue-specific alternative RNA splicing. In contrast, partial sequence from the 63-kDa bovine brain CaM-PDE isozyme displays only 67% identity with the 61-kDa isozyme. The differences are dispersed throughout the sequence, suggesting that the 63- and 61-kDa isozymes are encoded by separate but homologous genes.     

Molecular cloning and characterization of the Cl(-) pump-associated 55-kDa protein in rat brain.

The Cl(-)-ATPase/pump in the plasma membrane of the rat brain is a candidate for active outwardly directed Cl(-) translocating systems. We recently isolated a Cl(-) pump, 520- or 580-kDa protein complex, which consisted of 51-, 55-, 60-, and 62-kDa proteins. In this study, we cloned a cDNA encoding a 55-kDa glycoprotein, designated as ClP55, which contained an open reading frame of 1512 base pairs encoding a protein of 504 amino acids including a signal peptide of 28 amino acids. Northern and Western blot analyses demonstrated expression of ClP55 mainly in the cerebrum. Application of antisense phosphorothioate oligonucleotides to cultured neurons resulted in a marked increase in the intracellular Cl(-) concentration ([Cl(-)](i)). Immunohistochemical analysis indicated that ClP55 was localized to the plasma membranes of neurons such as hippocampal pyramidal neurons and cerebellar Purkinje cells. Taken together, these results suggest that ClP55 is one of the Cl(-) pump subunits responsible for Cl(-) pump activity.     

Free Sugars and Organic Acids in the Leaves of Various Plant Species and their Compartmentation Between the Tissues4

The distribution of free sugars and organic acids between theepidermis and mesophyll of Tulipa gesneriana L., Vicia fabaL., and Commelina communis L. leaves was studied using mainlygas-liquid chromatography. Fructose, glucose, sucrose, and myo-inositol were found in theepidermis and mesophyll of all three species. In T. geenerianaleaf tissues arabinose (trace levels), stachyose, tuliposidesA and B (mainly in the mesophyll), and xylose (trace levelsalso in V. faba tissues) were also detected. The acids were more difficult to detect and identify, beingat considerably lower concentrations than the sugars in bothtissues. Fumaric, citric, malic, ascorbic (trace levels), andan unidentified acid were common to the epidermis and mesophyllof all three species. Of special interest was the detectionof large amounts of glyceric acid in the epidermis and mesophyllof V. faba; this acid was not detected in the tissues from theother species. Fumaric acid was also very abundant in the epidermisof V.faba. A special study was made of the compartmentation of acids andsugars between the epidermis and mesophyll of T. geenerianaleaves after light and dark treatments. No changes in free acidor sugar levels were detected in the epidermis or mesophyllafter these treatments. Except for suceinic acid (P < 0·05),there were no statistically significant differences in acidlevels between the epidermis and mesophyll but for most of thesugars (myo-inositol, arabinose, and xylose being exceptions)differences were highly significant (P < 0·001), highestlevels occurring in the mesophyll. The differences in sugarlevels and the similarity in acid levels between epidermis andmesophyll of tulip leaves were considered to be essentiallydue to the different CO₂ fixing mechanisms and capacities ofthe two tissues. The energy source for the essentially non-greenepidermal tissue was discussed.     

Cholinesterases from Plant Tissues: III. Distribution and Subcellular Localization in Phaseolus aureus Roxb

The distribution and localization of cholinesterase in Phaseolus aureus, Glycine max, and Pisum sativum is described. The enzyme is present in roots, leaves, stems, root callus tissue, root cells suspension cultures, and root nodules. Cholinesterase in roots is found primarily in the cell wall. In cell fractionation experiments, at least 95% of the cholinesterase activity is associated with cell wall material. The enzyme can be solubilized by salt solutions, whereas Triton X-100 and sodium deoxycholate solubilize relatively small amounts of the enzyme. Cytochemical techniques have been employed to show the presence of cholinesterase activity at the cell surface and in the cell wall of certain cells of the root.     

Distribution of Protein Phosphatase Inhibitor-1 in Brain and Peripheral Tissues of Various Species: Comparison with DARPP-32

The distribution of inhibitor-1, a cyclic AMP-regulated inhibitor of protein phosphatase-1, was analyzed in various brain regions and peripheral tissues of various species by immunolabeling of sodium dodecyl sulfate-poly-acrylamide gel transfers using specific antibodies. The distribution of inhibitor-1 was directly compared to that of DARPP-32, a structurally related cyclic AMP-regulated inhibitor of protein phosphatase-1. In rat CNS, a single immunoreactive protein of M(r) 30,000, identified as inhibitor-1, was widely distributed. In contrast, DARPP-32 was highly concentrated in the basal ganglia. Inhibitor-1 was detected in brain tissue from frog (M(r) 27,000), turtle (M(r) 29,000/33,000), canary (M(r) 26,000), pigeon (M(r) 28,000), mouse (M(r) 30,500), rabbit (M(r) 26,500), cow (M(r) 27,000), and monkey (M(r) 27,500), but not from goldfish. Inhibitor-1 was detected at various levels in most peripheral tissues of the species studied; however, it was not detectable in certain tissues of particular species (e.g., rat and cow liver). DARPP-32 was detected in brain tissue of all the species tested except frog and goldfish, but was not detectable in most peripheral tissues. Both inhibitor-1 and DARPP-32 were concentrated in the cytosol and synaptosomal cytosol of rat striatum. The developmental expressions of inhibitor-1 and DARPP-32 in rat striatum differed: the level of inhibitor-1 peaked in the first postnatal week and then declined by the third postnatal week, whereas the level of DARPP-32 increased to a peak level by the third postnatal week and remained elevated thereafter.(ABSTRACT TRUNCATED AT 250 WORDS)     

The production of 53-55-kDa isoforms is not required for rat L-histidine decarboxylase activity

Post-translational processing of the histamine-producing enzyme, L-histidine decarboxylase (HDC), leads to the formation of multiple carboxyl-truncated isoforms. Nevertheless, it has been widely reported that the mature catalytically active dimer is dependent specifically on the production of carboxyl-truncated 53-55-kDa monomers. Here we use transiently transfected COS-7 cells to study the properties of carboxyl-truncated rat HDC isoforms in the 52-58-kDa size range. Amino acid sequences important for the production of a 55-kDa HDC isoform were identified by successive truncations through amino acids 502, 503, and 504. Mutating this sequence in the full-length protein prevented the production of 55-kDa HDC but did not affect enzymatic activity. Further truncations to amino acid 472 generated an inactive 53-kDa HDC isoform that was degraded by the proteasome pathway. These results suggested that processed isoforms, apart from 53-55-kDa ones, contribute toward histamine biosynthesis in vivo. This was confirmed in physiological studies where regulated increases in HDC activity were associated with the expression of isoforms that were greater than 55 kDa in size. We provide evidence to show that regulation of HDC expression can be achieved by the differential production or differential stabilization of multiple enzyme isoforms.     

Distribution and functional analysis of a 120- to 130-kDa T-cell surface antigen

A monoclonal antibody (mAb), SPV-L14, was raised that detected a human T-cell surface antigen with a molecular weight (MW) of 120 kDa on resting and phytohemagglutinin-activated peripheral blood T lymphocytes (PBL). An additional band with a MW of 130 kDa could be precipitated with variable intensities from thymocytes, neoplastic T cells, and CD4+- or CD8+ T-cell clones. Based on their reactivity with SPV-L14 and a mAb directed against CD3, four subpopulations of CD2+ lymphocytes could be detected and their existence was confirmed at the clonal level. The majority (95%) of the CD3+ cells were SPV-L14+, whereas 5% were CD3+, SPV-L14-. Among cloned cell lines CD3-,SPV-L14- and CD3-,SPV-L14+ cells were found to exist. The CD3-,SPV-L14- and CD3-,SPV-L14+ clones were shown to have NK cell activity, indicating that the 120- to 130-kDa antigen is expressed heterogeneously on CD3- NK cell clones. In addition, neoplastic T cells representing these four subpopulations were shown to exist. Although the tissue distribution and the MW of the SPV-L14 target antigen strongly suggest that SPV-L14 reacts with an epitope on CD6, the SPV-L14 mAb did not react with resting or activated B cells or with malignant B cells. Blocking studies showed that SPV-L14 inhibited the proliferative response of PBL, induced by anti-CD3 mAb, but that SPV-L14 did not affect the proliferation induced by phytohemagglutinin. These results suggest that the 120- to 130-kDa MW antigen is associated with T-cell proliferation, depending on the mode of activation.     

Regional Distribution of Catalase in the Adult Rat Brain

Catalase activity was measured in 11 areas of perfused adult rat brain. The hypothalamus and substantia nigra contained the highest activities. The corpus callosum. a white-matter structure, contained intermediate activity. The caudate-putamen and frontal cortex contained the lowest activities. Regional catalase bears some relationship to the reported distribution of microperoxisomes, but considerable activity is present in areas with few microperoxisomes. Catalase may function as one of the systems detoxifying H₂O₂ formed in CNS amine metabolism.     

A Study of Peroxidase and Catalase Distribution in the Potato Tuber

The activities of peroxidase and catalase were determined inconsecutive segments from cores struck from heel to rose endsof potato tubers, cv. Majestic, which had been grown in plotsof soil having nominal pH values of 4.5, 5.0, 5.5, 6.0, 6.5,7.0 and 7.5. Gradients of activity were computed and shown tobe parabolic upwards for peroxidase and parabolic downwardsfor catalase at the higher soil pH levels but both tended toshow linear trends in the more acid soils. The ratios of theactivities of peroxidase to catalase were at a maximum betweenpH 5.5 and 6.0 and decreased towards either end of the pH range. Solanum tuberosum, potato, tuber, enzyme activity, peroxidase, catalase     

Autoradiography of Water-Soluble Materials in Plant Tissues

Samples of tissue with water-soluble substances are lyophilized and doubly-infiltrated with parlodion and paraffin. Sections are mounted on an adhesive-coated cover slip with chloroform. The reverse side of the cover slip is coated successively with a thin layer of Harleco resin and a thick layer of Kodak “Opaque”. A corner of the cover slip is broken off as a marker. The cover slip is assembled with a covering glass slide on a nuclear track plate (Kodak NTB-2) with protective coating, the section being in contact with the emulsion, and held in place during radioactive exposure. Before development, the cover slip and plate are exposed briefly to light and then disassembled. Following development of the plate and removal of the layer of resin and opaque from the cover slip, staining of the section is optional. Lining up of the section with its autoradiograph can be accurate within 1-5μ.