Detection of cytokinins and auxin in plant tissues using histochemistry and immunocytochemistry

We report a new method for histochemical localization of cytokinins (CKs) in plant tissues based on bromophenol blue/silver nitrate staining. The method was validated by immunohistochemistry using anti-trans-zeatin riboside antibody. Indole-3-acetic acid (auxin, IAA) was localized by anti-IAA antibody in plant tissues as a proof for IAA histolocalization. We used root sections, because they are major sites of CKs synthesis, and insect galls of Piptadenia gonoacantha that accumulate IAA. Immunostaining confirmed the presence of zeatin and sites of accumulation of IAA indicated by histochemistry. The colors developed by histochemical reactions in free-hand sections of plant tissues were similar to those obtained by thin layer chromatography (TLC), which reinforced the reactive sites of zeatin. The histochemical method for detecting CKs is useful for galls and roots, whereas IAA detection is more efficient for gall tissues. Therefore, galls constitute a useful model for validating histochemical techniques due to their rapid cell cycles and relatively high accumulation of plant hormones.     

Electrochemical, kinetic, and circular dichroic consequences of mutations at position 82 of yeast iso-1-cytochrome c

Replacement of Phe-82 in yeast iso-1-cytochrome c with Tyr, Leu, Ile, Ser, Ala, and Gly produces a gradation of effects on (1) the reduction potential of the protein, (2) the rate of reaction with Fe(EDTA)2-, and (3) the CD spectra of the ferricytochromes in the Soret region under conditions where contributions from the alkaline forms of these proteins are absent. The reduction potential of cytochrome c is lowered by as little as 10 mV (Tyr-82) or by as much as 43 mV (Gly-82; pH 6.0) as the result of these substitutions. The second-order rate constants for reduction of these cytochromes range from a low of 6.20 (2) x 10(4) for the Tyr-82 variant to a high of 14.8 x 10(4) M-1 s-1 for the Ser-82 variant [pH 6.0, 25 degrees C, mu = 0.1 M (sodium phosphate)]. Analysis of these rates by use of relative Marcus theory produces values of k11corr that range from 10.9 M-1 s-1 for the wild-type protein to 190 M-1 s-1 for the Gly-82 mutant [25 degrees C, mu = 0.1 M, pH 6.0 (sodium phosphate)]. Reinvestigation of the effect of substituting Phe-82 by a Tyr residue on the CD spectrum of the protein now reveals little alteration of the intense, negative Cotton effect in the Soret CD spectrum of ferricytochrome c. On the other hand, substitution of nonaromatic residues of various sizes at this position results in loss of this spectroscopic feature, consistent with previous findings.(ABSTRACT TRUNCATED AT 250 WORDS)     

Small substrates and cytochrome c are oxidized at different sites of cytochrome c peroxidase.

Modeling studies suggest that electrons are transferred from cytochrome c to cytochrome c peroxidase (CcP) with cytochrome c predominantly bound at a site facing the gamma-meso edge of the CcP prosthetic heme group (Poulos, T.L., and Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330). As shown here, guaiacol and ferrocyanide are oxidized at a different site of CcP. Thus, the oxidations of cytochrome c and guaiacol are differentially inactivated by phenylhydrazine and sodium azide. The loss of guaiacol oxidation activity correlates with covalent binding of 1 equivalent of [14C]phenylhydrazine to the protein, whereas the slower loss of cytochrome c activity correlates with the appearance of a 428-nm absorbance maximum attributed to the formation of a sigma-phenyl-iron heme complex. The delta-meso-phenyl and 8-hydroxymethyl derivatives of heme are formed as minor products. Catalytic oxidation of azide to the azidyl radical results in inactivation of CcP and formation of delta-meso-azidoheme. Reconstitution of apo-CcP with delta-meso-azido-, -ethyl-, and -(2-phenylethyl)heme yields holoproteins that give compound I species with H2O2 and exhibit 80, 59, and 31%, respectively, of the control kcat value for cytochrome c oxidation but little or no guaiacol or ferrocyanide oxidizing activity. Conversely, CcP reconstituted with gamma-meso-ethylheme is fully active in the oxidation of guaiacol and ferrocyanide but only retains 27% of the cytochrome c oxidizing activity. These results indicate that guaiacol and ferrocyanide are primarily oxidized near the delta-meso-heme edge rather than, like cytochrome c, at a surface site facing the gamma-meso edge.     

Change in charge of an unvaried heme contact residue does not cause a major change of conformation in cytochrome c.

The structure of the Ala38 variant of yeast iso-1-cytochrome c, in which the previously unchanged Arg38 has been replaced, has been characterised by NMR. The NMR data indicate that the structure of the Ala38 variant is very similar to that of the wild type protein. In particular, the heme environment and interactions of the heme macrocycle are shown to be preserved. Analysis of the chemical shift perturbations to the resonances of Ile35 is shown to be consistent with the change in charge at position 38. The only significant area of conformational change detected was at residues 39 and 58, close to the site of modification. Therefore the redox potential change accompanying the modification [1988, Biochemistry 28, 3188-3197] appears to be a direct consequence of the altered side-chain of residue 38 and not a result of secondary conformational changes induced by the modification.     

Phospholipid membranes affect tertiary structure of the soluble cytochrome b5 heme-binding domain

The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b₅ has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b₅vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b₅ upon the interaction with its redox partners is discussed.     

Functional comparison of specifically cross-linked hemoglobins biased toward the R and T states.

Selected functional and spectroscopic properties of two human hemoglobin (HbA0) derivatives that were site-specifically cross-linked in the cleft between beta-chains where 2, 3-bisphosphoglycerate normally binds have been determined to assess the effects of the cross-linking on the behavior of the protein. Trimesoyl tris(3,5-dibromosalicylate) (TTDS) cross-links Hb between beta82Lys residues. The resulting TTDS-Hb exhibits a slower rate of oxygen dissociation and an increased rate of carbon monoxide association than observed for HbA0. The electron paramagnetic resonance (EPR) spectrum of TTDS-HbNO does not exhibit the hyperfine structure that is indicative of significant conformational change despite the fact that the 2,3-bisphosphoglycerate binding site is occupied by the cross-linking reagent. The reactivity of the beta93Cys residues of TTDS-Hb is only slightly decreased relative to that of HbA0. On the other hand, cross-linking Hb between Lys82 and the amino-terminal beta1Val group with trimesoyl tris(methyl phosphate) (TMMP) increases the rate of oxygen dissociation and reduces the rate of CO association relative to the rates observed for HbA0. In addition, the EPR spectrum of the TMMP-HbNO exhibits the three-line hyperfine structure that results from disruption of the proximal His-Fe bond of the alpha-chains, and the accessibility of the betaCys93 residues in this derivative is decreased fourfold. The present results are consistent with the conclusion that the quaternary structure of TTDS-Hb is shifted toward the R state whereas the quaternary structure of TMMP-Hb is shifted toward the T state and provides additional evidence that the identity of the residues involved in intramolecular cross-linking of hemoglobin within the 2,3-bisphosphoglycerate binding site between beta-chains can have a significant influence on the conformational and functional properties of the protein.     

Alterations in hippocampal serotonergic and INSR function in streptozotocin induced diabetic rats exposed to stress: neuroprotective role of pyridoxine and <Emphasis Type="Italic">Aegle marmelose</Emphasis>

Diabetes and stress stimulate hippocampal 5-HT synthesis, metabolism and release. The present study was carried out to find the effects of insulin, Aegle marmelose alone and in combination with pyridoxine on the hippocampal 5-HT, 5-HT_2A receptor subtype, gene expression studies on 5-HT_2A, 5-HTT, INSR, immunohistochemical studies and elevated plus maze in streptozotocin induced diabetic rats. 5-HT content showed a significant decrease (p < 0.001) and a significant increase (p < 0.001) in 5-HIAA in hippocampus of diabetic rats compared to control. 5-HT receptor binding parameters B_max and K_d showed a significant decrease (p < 0.001) whereas 5-HT_2A receptor binding parameters B_max showed a significant decrease (p < 0.001) with a significant increase (p < 0.05) in K_d in hippocampus of diabetic rats compared to control. Gene expression studies of 5-HT_2A, 5-HTT and INSR in hippocampus showed a significant down regulation (p < 0.001) in diabetic rats compared to control. Pyridoxine treated in combination with insulin and A. marmelose to diabetic rats reversed the 5-HT content, B_max , K_d of 5-HT, 5-HT_2A and gene expression of 5-HT_2A, 5-HTT and INSR in hippocampus to near control. The gene expression of 5-HT_2A and 5-HTT were confirmed by immunohistochemical studies. Behavioural studies using elevated plus maze showed that serotonin through its transporter significantly increased (p < 0.001) anxiety-related traits in diabetic rats which were corrected by combination therapy. Our results suggest that pyridoxine treated in combination with insulin and A. marmelose has a role in the regulation of insulin synthesis and release, normalising diabetic related stress and anxiety through hippocampal serotonergic function. This has clinical significance in the management of diabetes.     

Stable copper-nitrosyl formation by nitrite reductase in either oxidation state

Nitrite reductase (NiR) is an enzyme that uses type 1 and type 2 copper sites to reduce nitrite to nitric oxide during bacterial denitrification. A copper-nitrosyl intermediate is a proposed, yet poorly characterized feature of the NiR catalytic cycle. This intermediate is formally described as Cu(I)-NO+ and is proposed to be formed at the type 2 copper site after nitrite binding and electron transfer from the type 1 copper site. In this study, copper-nitrosyl complexes were formed by prolonged exposure of exogenous NO to crystals of wild-type and two variant forms of NiR from Alcaligenes faecalis (AfNiR), and the structures were determined to 1.8 A or better resolution. Exposing oxidized wild-type crystals to NO results in the reverse reaction and formation of nitrite that remains bound at the active site. In a type 1 copper site mutant (H145A) that is incapable of electron transfer to the type 2 site, the reverse reaction is not observed. Instead, in both oxidized and reduced H145A crystals, NO is observed bound in a side-on manner to the type 2 copper. In AfNiR, Asp98 forms hydrogen bonds to both substrate and product bound to the type 2 Cu. In the D98N variant, NO is bound side-on but is more disordered when observed for the wild-type enzyme. The solution EPR spectra of the crystallographically characterized NiR-NO complexes indicate the presence of an oxidized type 2 copper site and thus are interpreted as resulting from stable copper-nitrosyls and formally assigned as Cu(II)-NO-. A reaction scheme in which a second NO molecule is oxidized to nitrite can account for the formation of a Cu(II)-NO- species after exposure of the oxidized H145A variant to NO gas.     

Induction of eosinophil apoptosis by hydrogen peroxide promotes the resolution of allergic inflammation

Eosinophils are effector cells that have an important role in the pathogenesis of allergic disease. Defective removal of these cells likely leads to chronic inflammatory diseases such as asthma. Thus, there is great interest in understanding the mechanisms responsible for the elimination of eosinophils from inflammatory sites. Previous studies have demonstrated a role for certain mediators and molecular pathways responsible for the survival and death of leukocytes at sites of inflammation. Reactive oxygen species have been described as proinflammatory mediators but their role in the resolution phase of inflammation is poorly understood. The aim of this study was to investigate the effect of reactive oxygen species in the resolution of allergic inflammatory responses. An eosinophilic cell line (Eol-1) was treated with hydrogen peroxide and apoptosis was measured. Allergic inflammation was induced in ovalbumin sensitized and challenged mouse models and reactive oxygen species were administered at the peak of inflammatory cell infiltrate. Inflammatory cell numbers, cytokine and chemokine levels, mucus production, inflammatory cell apoptosis and peribronchiolar matrix deposition was quantified in the lungs. Resistance and elastance were measured at baseline and after aerosolized methacholine. Hydrogen peroxide accelerates resolution of airway inflammation by induction of caspase-dependent apoptosis of eosinophils and decrease remodeling, mucus deposition, inflammatory cytokine production and airway hyperreactivity. Moreover, the inhibition of reactive oxygen species production by apocynin or in gp91^phox−/− mice prolonged the inflammatory response. Hydrogen peroxide induces Eol-1 apoptosis in vitro and enhances the resolution of inflammation and improves lung function in vivo by inducing caspase-dependent apoptosis of eosinophils.Eosinophils express numerous receptors and secrete a wide variety of inflammatory mediators that influence many innate and adaptive immune responses. These multifunctional cells are important in the defense against helminth infection and are involved in the pathogenesis of many eosinophilic dominant allergic diseases.¹ High levels of eosinophil granule proteins (such as major basic protein (MBP)) have been found in bronchoalveolar lavage fluid from patients with asthma and evidence indicates that high-concentration granule products have contributed to the development of airway hyperreactivity (AHR), a cardinal feature of asthma.² Asthma is an inflammatory disease of the airways with participation of many cell types including leukocytes especially eosinophils and lymphocytes.^{3, 4} Activation of these cells (mainly lymphocytes) leads to the release of proinflammatory mediators and cytokines such as leukotriene B₄, interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-9 (IL-9), interleukin-13 (IL-13) and colony-stimulating factor granulocyte-macrophage (GM-CSF).^{3, 5, 6, 7} Investigations using preclinical animal models of asthma and clinical studies in patients with asthma have demonstrated that the presence of eosinophils in the lungs are associated with epithelial damage, goblet cell hyperplasia, smooth muscle hypertrophy and airway hyperresponsiveness resulting in airflow limitation which can be fatal.^{3, 8, 9, 10} Recently, anti-IL-5 treatment has been shown to ameliorate lung function in patients with eosinophilic asthma.¹¹Apoptosis of leukocytes is regarded as an important process for the successful resolution of inflammatory responses. Reduced eosinophil apoptosis in bronchoalveolar lavage (BAL) fluid has been shown to correlate positively with severity of asthma.^{3, 12, 13, 14} Indeed, defective leukocyte apoptosis and subsequent removal of apoptotic cells by phagocytes is thought to be important for the initiation and propagation of chronic inflammatory diseases such as asthma.¹⁵ Therefore, a balance in the tissue microenvironment between pro- and antiapoptotic signals is likely to greatly influence the load of eosinophils in the asthmatic lung.¹⁶ Thus, there is a great interest in understanding the mechanisms responsible for the elimination of eosinophils and other leukocytes and inactivation of proinflammatory mediators in inflammatory sites.¹⁷Several molecular pathways have been shown to modulate the survival and death of leukocytes at sites of inflammation, including reactive oxygen species (ROS).¹⁸ ROS are a family of molecules containing oxygen and includes hydrogen peroxide (H₂O₂), superoxide O₂⁻, hydroxyl radical (OH) and nitric oxide (NO).¹⁹ In inflammatory conditions, ROS are increased as they help in neutralizing invading organisms during infection either directly or indirectly by formation of extracellular traps (ETs).²⁰ ROS have traditionally been regarded as quintessentially proinflammatory. However, evidence for ROS-mediated anti-inflammatory actions has been described.²¹ The importance for ROS production in the context of infection can be exemplified in patients with chronic granulomatous disease (CGD) where defective production in ROS results in multiple infections and often early death.^{22, 23} Furthermore, studies in mouse models have shown that NADPH oxidase is key for regulating lung inflammation and injury as well as NF-κB activation and downstream cytokine production in response to LPS.²⁴ More recently, our group has demonstrated that NADPH oxidase-derived H₂O₂ is directly linked to induction of apoptosis of neutrophils and resolution of inflammation in a model of antigen-induced arthritis.¹⁸ However, the role of ROS in the context of the resolution of allergic inflammation is still unknown.Here, we evaluated whether H₂O₂ drives apoptosis of eosinophils and thereby influences the resolution of established eosinophilic inflammation and reduction of airflow obstruction. Our study provides evidence that H₂O₂ is released during allergic inflammation in a gp91^phox−/−-dependent manner and induces a caspase-dependent proapoptotic effect in eosinophils, thus having a crucial role in the resolution of allergic inflammation.