Phytochrome-mediated effects on extracellular peroxidase activity, lignin content and bending resistance in etiolated Vicia faba epicotyls

Etiolated Vicia faba seedlings were exposed to continuous red light to investigate whether changes in extracellular peroxidase activity were correlated in time and localization with changes in extension growth and/or lignin content in the subapical region of the epicotyl. Continuous red light: (a) increased extracellular peroxidase activity after a lag of ca 0.5 h, followed by a maximum peak after 2.5 h due to slightly acidic isoforms (pI = 6–6.5, according to isoelectrofocusing gels), a minimum after 4 h and a second maximum after 8 h due to acidic isoforms (pI=4–5), (b) increased lignin content and epicotyl resistance to bending after a lag of ca 4 h, i.e. simultaneously with changes in acidic extracellular peroxidase activity, and (c) reduced extension growth to a stable rate after a lag of ca 1 h, not coinciding with the kinetics of any of the extracellular peroxidase isoforms. These effects of continuous red light were at least partially mediated by phytochrome. Tissue printing and anatomical studies revealed red light effects on extracellular peroxidase activity and lignin content mainly in the outer cortical parenchyma. The results are consistent with the involvement of phyto-chrome-mediated effects on extracellular peroxidases (acidic isoforms) in the transduction chain leading to lignin responses to red light.     

Tissue and subcellular immunolocalisation of enzymes of lignin synthesis in differentiating and wounded hypocotyl tissue of French bean (Phaseolus vulgaris L.)

Antisera raised againstl-phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), and a cationic cell-wall peroxidase, which had all been purified from suspension-cultured cells of French bean, have been used to carry out immunogold localisations in the growing plant. Immunoglobulin-G fractions were prepared from each antiserum and used to study the distribution of the enzymes in differentiating and wounded hypocotyls by immunogold techniques and visualisation by both light and electron microscopy. Following silver enhancement to amplify the signal, proteins were detected by confocal microscopy in both developing (pre-xylem/ phloem) and later metaxylem stelar tissue.l-Phenylalanine ammonia-lyase and C4H also accumulated in cells adjacent to metaxylem, presumably involved in maintaining a supply of phenylpropanoid precursors to the enucleated xylem for further lignin synthesis. In these cells, PAL subunits were cytosolic although some were associated with endomembrane. Cinnamate-4-hydroxylase was wholly associated with membrane and particularly high concentrations were found in the Golgi bodies. The cationic peroxidase accumulated in xylem at sites of secondary thickening and in the middle lamella. The three proteins are also involved in defensive lignification. Thus when visualised by light microscopy, PAL and C4H were seen to accumulate to high levels throughout the cell types in wound sites and especially in the epidermal cells. An even more intense general distribution was found upon hyperinduction of wounded cells with-aminooxy--phenylpropionic acid. At the subcellular level, PAL was found to be localised in the cytosol in the wounded cells; however, because of the loss of membrane through mechanical damage, association with membrane structures, particularly endoplasmic reticulum, in unwounded cells is not entirely ruled out. Cinnamate-4-hydroxylase was associated with membranes when these were preserved. In wounded tissue, the peroxidase was found at the growing edges of tylose-like structures in the vascular xylem.Abbreviations AOPP -aminooxy--phenylpropionic acid - C4H cinnamic acid-4-hydroxylase - CHS chalcone synthase - GRP glycine-rich glycoprotein - HRGP hydroxyproline-rich glycoprotein - Ig immunoglobulin - PAL phenylalanine ammonia-lyase G.P.B. thanks the Agicultural and Food Research Council for support.     

Identification of aneuploid cells in cytological specimens by combined in situ hybridization and immunocytochemistry

The purpose of our study was the application of non-isotopic in situ hybridization with chromosome-specific repetitive DNA probes for the determination of cytogenetically aberrant cells in routine cytological materials, such as cervical smears and breast tumour aspirates. Hyperdiploid cells in fine needle aspirates (FNA) of breast tumours could be visualized by in situ hybridization with a chromosome l-specific repetitive DNA probe. However, for the evaluation of a specific cell type in heterogeneous cell populations, i.e. cervical smears, a procedure combining immunocytochemistry and in situ hybridization can be required. Therefore, we developed a combination protocol using β-galactosidase/ ferri-ferrocyanide (blue-green) for immunocytochemistry and peroxidase/DAB (brown-black) for detection of the DNA probe. the described protocol enabled us to distinguish squamous epithelial cells within heterogeneous cell populations. By combining the chromosome 1 DNA probe with a specific cytokeratin marker it was possible to identify the chromosomal abnormal cells within cervical smears.     

A surface mutant (G82R) of a human α-glutathione S-transferase shows decreased thermal stability and a new mode of molecular association in the crystal

A chimeric enzyme (GST121) of the human α-glutathione S-transferases GST1-1 and GST2-2, which has improved catalytic efficiency and thermostability from its wild-type parent proteins, has been crystallized in a space group that is isomorphous with that reported for crystals of GST1-1. However, a single-site (G82R) mutant of GST121, which exhibits a significant reduction both in vitro and in vivo in protein thermostability, forms crystals that are not isomorphous with GST1-1. The mutant protein crystallizes in space group P2₁2₁2₁, with cell dimensions a = 49.5, b = 92.9, c = 115.9 Å, and one dimer per asymmetric unit. Preliminary crystallographic results show that a mutation of the surface residue Gly 82 from a neutral to a charged residue causes new salt bridges to be formed among the GST dimers, suggesting that the G82R mutant might aggregate more readily than does GST121 in solution resulting in a change of its solution properties. © 1994 Wiley-Liss, Inc.     

On the use of the quenching of luminol luminescence to evaluate sod activity

Addition of horseradish peroxidase to a luminol solution (pH = 9.4) produces a burst of light followed by a steady luminescence that lasts for several minutes. This steady-state luminescence is readily quenched by SOD, with a concentration (the additive concentration needed to decrease by one-half the emitted luminescence intensity) of c.a. 4 ng/ml (14 mU/ml). The luminescence intensity decrease can then be employed to evaluate SOD activity in SOD-containing samples. However, the light intensity can also be quenched by additives, such as Trolox, that are able to trap luminol-derived intermediates. It is proposed that double quenching experiments must be performed in order to be able to relate the observed effect of an additive to its SOD-like activity.     

Weak antioxidant defenses make the heart a target for damage in copper-deficient rats

Copper deficiency causes more salient pathologic changes in the heart than in the liver of rats. Although oxidative stress has been implicated in copper deficiency-induced pathogenesis, little is known about the selective toxicity to the heart. Therefore, we examined the relationship between the severity of copper deficiency-induced oxidative damage and the capacity of antioxidant defense in heart and liver to investigate a possible mechanism for the selective cardiotoxicity. Weanling rats were fed a purified diet deficient in copper (0.4 μg/g diet) or one containing adequate copper (6.0 μg/g diet) for 4 weeks. Copper deficiency induced a 2-fold increase in lipid peroxidation in the heart (thiobarbituric assay) but did not alter peroxidation in the liver. The antioxidant enzymatic activities of superoxide dismutase, catalase, and glutathione peroxidase were, respectively, 3-, 50- and 1.5-fold lower in the heart than in the liver, although these enzymatic activities were depressed in both organs by copper deficiency. In addition, the activity of glutathione reductase was 4 times lower in the heart than in the liver. The data suggest that a weak antioxidant defense system in the heart is responsible for the relatively high degree of oxidative damage in copper-deficient hearts.     

Chiral copper(I)—thiolate clusters in metallothionein and glutathione

Metallothionein (MT) is a ubiquitous mammalian protein comprising 61 or 62 nonaromatic amino acids of which 20 are cysteine residues. The high sulfhydryl content imparts to this protein a unique and remarkable ability to bind multiple metal ions in structurally significant metal–thiolate clusters. MT can bind seven divalent metal ions per protein molecule in two domains with exclusive tetrahedral metal coordination. The domain stoichiometries for the M₇S₂₀ structure are M₄(S_cys)₁₁ (α domain) and M₃(S_cys)₉ (β domain). Up to 12 Cu(I) ions can displace the 7 Zn²⁺ ions bound per molecule in Zn₇–MT. The incoming Cu(I) ions adopt a trigonal planar geometry with domain stoichiometries for the Cu₁₂S₂₀ structure of Cu₆(S_cys)₁₁ and Cu₆(S_cys)₉ for the α and β domains, respectively. The circular dichroism (CD) spectra recorded as Cu⁺ is added to Zn₇–MT to form Cu₁₂–MT directly report structural changes that take place in the metal binding region. The spectrum arises under charge transfer transitions between the cysteine S and the Cu(I); because the Cu(I)–thiolate cluster units are located within the chiral binding site, intensities in the CD spectrum are directly related to changes in the binding site. The CD technique clearly indicates stoichiometries of several Cu(I)–MT species. Model Cu(I)–thiolate complexes, using the tripeptide glutathione as the sulfhydryl source, were examined by CD spectroscopy to obtain transition energies and the Cu(I)–thiolate coordination geometries which correspond to these bands. Possible structures for the Cu(I)–thiolate clusters in the α and β domains of Cu₁₂–MT are proposed. © 1994 Wiley-Liss, Inc.     

Guanine nucleotide regulatory protein alterations in young Milan hypertensive strain rats

Vascular smooth muscle cell membranes from prehypertensive rats of the Milan hypertensive strain (MHS) were used to examine adenylyl cyclase activity and its regulation by guanine nucleotide regulatory proteins (G-proteins). Basal adenylyl cyclase activity was similar in MHS and Milan normontensive strain (MNS) membranes. Forsokolin (10^?4 M) produced a significantly greater stimulatory response in MHS membranes, but this was not observed with NaF (10^?2 M). Isoporterenol (10^?4 M) caused a significantly decreased stimulation of adenylyl cyclase activity in MHS membranes, while prostaglandin E₁ (10^?5 M) produced similar responses in the two strains. G_i function and GTP responses, as observed by biphasic effects of GTP on isoproterenol-stimulated membranes, were similar in both strains. The levels of G_i2α and G_qα/G₁₁α were similar in the two strains, while the levels of G_sα (44 and 42 kDa forms) and the β-subunit were significantly reduced by ～20% in MHS membranes. The α-subunit of G_i3 was dramatically reduced by ～80% in MHS membranes. The affinities of β-adrenergic receptors for the antagonist, cyanophindolol, were similar in the two strains; however, the number of β-adrenoceptors was substantially reduced in MHS membranes. These findings may be of relevance to altered vascular reactivity and transmembrane ion distribution observed in the MHS.     

胎盘型谷胱甘肽S－转移酶基因在胃癌中的表达

用Ｄｉｇ－ＧＳＴ－πｃＤＮＡ探针分子杂交方法,检测了正常胃组织,胃癌及相应癌旁正常组织中ＧＳＴ－πＤＮＡ和ＧＳＴ－πＲＮＡ水平,发现ＧＳＴ－πＤＮＡ水平没有明显变化,而ＧＳＴ－πＲＮＡ在８例胃癌组织中有６例高于正常胃组织,在１２例低分化腺癌中有７例癌旁正常组织高于相应癌组织,表明ＧＳＴ－π基因表达增加与胃癌有关,而且早于细胞形态的变化。