Putrescine protects hulless barley from damage due to UV-B stress via H<Subscript>2</Subscript>S- and H<Subscript>2</Subscript>O<Subscript>2</Subscript>-mediated signaling pathways

Key message

In hulless barley, H ₂ S mediated increases in H ₂ O ₂ induced by putrescine, and their interaction enhanced tolerance to UV-B by maintaining redox homeostasis and promoting the accumulation of UV-absorbing compounds.

Abstract

This study investigated the possible relationship between putrescence (Put), hydrogen sulfide (H₂S) and hydrogen peroxide (H₂O₂) as well as the underlying mechanism of their interaction in reducing UV-B induced damage. UV-B radiation increased electrolyte leakage (EL) and the levels of malondialdehyde (MDA) and UV-absorbing compounds but reduced antioxidant enzyme activities and glutathione (GSH) and ascorbic acid (AsA) contents. Exogenous application of Put, H₂S or H₂O₂ reduced some of the above-mentioned negative effects, but were enhanced by the addition of Put, H₂S and H₂O₂ inhibitors. Moreover, the protective effect of Put against UV-B radiation-induced damage to hulless barley was diminished by dl-propargylglycine (PAG, a H₂S biosynthesis inhibitor), hydroxylamine (HT, a H₂S scavenger), diphenylene iodonium (DPI, a PM-NADPH oxidase inhibitor) and dimethylthiourea (DMTU, a ROS scavenger), and the effect of Put on H₂O₂ accumulation was abolished by HT. Taken together, as the downstream component of the Put signaling pathway, H₂S mediated H₂O₂ accumulation, and H₂O₂ induced the accumulation of UV-absorbing compounds and maintained redox homeostasis under UV-B stress, thereby increasing the tolerance of hulless barley seedlings to UV-B stress.

     

Different pathways of radical translocation in yeast cytochrome <Emphasis Type="Italic">c</Emphasis> peroxidase and its W191F mutant on reaction with H<Subscript>2</Subscript>O<Subscript>2</Subscript> suggest an antioxidant role

In the absence of exogenous donors, turnover of 10 molar equivalents of H(2)O(2) by wild-type recombinant cytochrome c peroxidase [CCP(MI)] and its W191F mutant at pH 7.0 occurs by oxidation of endogenous donors on the polypeptide. No O(2) evolution was observed with either enzyme on reaction with 10 molar equivalents of H(2)O(2), eliminating catalase-like activity, but O(2) evolution was observed when 100 molar equivalents of H(2)O(2) were added to the enzymes. Protein dimers were observed by SDS-PAGE following H(2)O(2) turnover by the peroxidases, and dimeric forms of CCP(MI) and CCP(W191) were isolated by gel-permeation chromatography. LC-ESI-MS analysis of the tryptic digests of the dimers revealed the previously reported T(6)-T(6) crosslink and a new crosslink between T(6)-T(26), but no T(26)-T(26) crosslink. The crosslinked tryptic peptides contain the exposed tyrosine residues Tyr36, Tyr39 and Tyr42 (T(6)), and Tyr229 and Tyr236 (T(26)). Addition of a spin trap, 2-methyl-2-nitrosopropane (MNP), to the CCP(MI)/H(2)O(2) reaction resulted in MNP labeling of peptides T(6), T(21) (which contains Tyr153) and T(26). MNP labeling of Tyr236 was found by sequencing peptide T(26). MNP labeling did not compete with dimerization of H(2)O(2)-oxidized CCP(W191F), suggesting that dityrosine formation in this mutant is very rapid owing to the high reactivity of radicals formed on T(6). H(2)O(2)-dependent formation of CCP-cytochrome c heterodimers was observed for both CCP(MI) and W191F in the presence of ferricytochrome c, the oxidized form of CCP's donor substrate. Interestingly, no H(2)O(2)-dependent cytochrome crosslinking to the W51F mutant was observed, even though this mutant underwent extensive homocrosslinking. The translocation of oxidizing equivalents from the heme to the surface residues of CCP is discussed in terms of an antioxidant role for CCP.     

H<Subscript>2</Subscript>S biotreatment with sulfide-oxidizing heterotrophic bacteria

Many industrial activities produce H₂S, which is toxic at high levels and odorous at even very low levels. Chemolithotrophic sulfur-oxidizing bacteria are often used in its remediation. Recently, we have reported that many heterotrophic bacteria can use sulfide:quinone oxidoreductase and persulfide dioxygenase to oxidize H₂S to thiosulfate and sulfite. These bacteria may also potentially be used in H₂S biotreatment. Here we report how various heterotrophic bacteria with these enzymes were cultured with organic compounds and the cells were able to rapidly oxidize H₂S to zero-valence sulfur and thiosulfate, causing no apparent acidification. Some also converted the produced thiosulfate to tetrathionate. The rates of sulfide oxidation by some of the tested bacteria in suspension, ranging from 8 to 50 µmol min^?1 g^?1 of cell dry weight at pH 7.4, sufficient for H₂S biotreatment. The immobilized bacteria removed H₂S as efficiently as the bacteria in suspension, and the inclusion of Fe₃O₄ nanoparticles during immobilization resulted in increased efficiency for sulfide removal, in part due to chemical oxidation H₂S by Fe₃O₄. Thus, heterotrophic bacteria may be used for H₂S biotreatment under aerobic conditions.     

Electrostatic attraction between cytochrome <Emphasis Type="Italic">bc</Emphasis><Subscript>1</Subscript> and cytochrome <Emphasis Type="Italic">c</Emphasis> affects kinetics of cytochrome <Emphasis Type="Italic">c</Emphasis> reduction

The kinetics of the ubiquinol-cytochrome c reductase reaction was examined using membrane fragments and purified bc(1) complexes derived from a wild-type (WT) and a newly constructed mutant (MUT) strains of Paracoccus denitrificans. The cytochrome c(1) of the WT samples possessed an additional stretch of acidic amino acids, which was lacking in the mutant. The reaction was followed with positively charged mitochondrial and negatively charged bacterial cytochromes c, and specific activities, apparent k(cat) values, and first-order rate constant values were compared. These values were distinctly lower for the MUT fractions using mitochondrial cytochrome c but differed only slightly with the bacterial species. The MUT preparations were less sensitive to changes of ionic strength of the reaction media and showed pure first-order kinetics with both samples of cytochrome c. The reaction of the WT enzyme was first order only with bacterial cytochrome c but proceeded with a non-linear profile with mitochondrial cytochrome c. The analysis of the reaction pattern revealed a rapid onset of the reaction with a successively declining rate. Experiments performed in the absence of an electron donor indicated that electrostatic attraction could directly participate in cytochrome c reduction.     

Cyanobacterial Photosystem I lacks specificity in its interaction with cytochrome c<Subscript>6</Subscript> electron donors

In cyanobacteria, plastocyanin and cytochrome c ₆, the alternate donor proteins to Photosystem I, can be acidic, neutral or basic; the role of electrostatics in their interaction with photosystem I varies accordingly. In order to elucidate whether these changes in the electron donors’ properties correlate with complementary changes in the docking site of the corresponding photosystem, we have investigated the kinetics of reactions between three cytochrome c ₆ with isoelectric points of 5.6, 7.0 and 9.0, with Photosystem I particles from the same three genera of cyanobacteria which provided the cytochromes. The model systems compared here thus sample the full range of charge properties observed in cytochromes c ₆: acidic, basic and neutral. The rate constants and dependence on ionic strength for photosystem I reduction were distinctive for each cytochrome c ₆, but independent of Photosystem I. We conclude that the specific structural features of each cytochrome c ₆ dictate their different kinetic behaviours, whereas the three photosystems are relatively indiscriminate in docking with the electron donors.     

Effect of Prostaglandins E<Subscript>1</Subscript> and F<Subscript>1α</Subscript> on Biosynthesis of Collagen

THE prostaglandins (PG) are possible mediators of inflammation. Prostaglandins E and F are present in inflammatory exudates^1–3 and could be related to the increase of collagen biosynthesis associated with inflammation. Vane and his colleagues^4–6 recently observed that indomethacin, aspirin and sodium salicylate potently block the biosynthesis of prostaglandins. These anti-inflammatory drugs are also inhibitors of collagen biosynthesis^7,8. Morphological studies⁹ have revealed increased deposition of collagen or collagen-related elements in organ cultures of chick embryo skin containing prostaglandins E₁ and B₁. We report here results which indicate stimulation of collagen biosynthesis by prostaglandins E₁ and F_1α evaluated by hydroxylation of proline and lysine and glycosylation of hydroxylysine in 10 day chick embryo tibiae.     

Gum arabic modified Fe<Subscript>3</Subscript>O<Subscript>4</Subscript> nanoparticles cross linked with collagen for isolation of bacteria

Background  

Multifunctional magnetic nanoparticles are important class of materials in the field of nanobiotechnology, as it is an emerging area of research for material science and molecular biology researchers. One of the various methods to obtain multifunctional nanomaterials, molecular functionalization by attaching organic functional groups to nanomagnetic materials is an important technique. Recently, functionalized magnetic nanoparticles have been demonstrated to be useful in isolation/detection of dangerous pathogens (bacteria/viruses) for human life. Iron (Fe) based material especially FePt is used in the isolation of ultralow concentrations (< 10² cfu/ml) of bacteria in less time and it has been demonstrated that van-FePt may be used as an alternative fast detection technique with respect to conventional polymerase chain reaction (PCR) method. However, still further improved demonstrations are necessary with interest to biocompatibility and green chemistry. Herein, we report the synthesis of Fe₃O₄ nanoparticles by template medication and its application for the detection/isolation of S. aureus bacteria.     

Structures of EccB<Subscript>1</Subscript> and EccD<Subscript>1</Subscript> from the core complex of the mycobacterial ESX-1 type VII secretion system

Background

The ESX-1 type VII secretion system is an important determinant of virulence in pathogenic mycobacteria, including Mycobacterium tuberculosis. This complicated molecular machine secretes folded proteins through the mycobacterial cell envelope to subvert the host immune response. Despite its important role in disease very little is known about the molecular architecture of the ESX-1 secretion system.

Results

This study characterizes the structures of the soluble domains of two conserved core ESX-1 components – EccB₁ and EccD₁. The periplasmic domain of EccB₁ consists of 4 repeat domains and a central domain, which together form a quasi 2-fold symmetrical structure. The repeat domains of EccB₁ are structurally similar to a known peptidoglycan binding protein suggesting a role in anchoring the ESX-1 system within the periplasmic space. The cytoplasmic domain of EccD₁has a ubiquitin-like fold and forms a dimer with a negatively charged groove.

Conclusions

These structures represent a major step towards resolving the molecular architecture of the entire ESX-1 assembly and may contribute to ESX-1 targeted tuberculosis intervention strategies.

     

The influence of Sc doping on structural,electronic and optical properties of Be<Subscript>12</Subscript>O<Subscript>12</Subscript>, Mg<Subscript>12</Subscript>O<Subscript>12</Subscript> and Ca<Subscript>12</Subscript>O<Subscript>12</Subscript> nanocages: a DFT study

Density functional theory (DFT) calculations were used to study the effect of scandium doping on the structural, energetic, electronic, linear and nonlinear optical (NLO) properties of Be₁₂O₁₂, Mg₁₂O₁₂ and Ca₁₂O₁₂ nanoclusters. Scandium (Sc) doping on nanoclusters leads to narrowing of their E _g, which enhances their conductance greatly. Also, the polarizability (α) and first hyperpolarizability (β₀) of nanoclusters were dramatically increased as Be, Mg or Ca atoms are substituted with a Sc atom. Among all clusters, α and β₀ values for Sc-doped Ca₁₂O₁₂ were the largest. Consequently, the effect of the doping atom, as well as of cluster size, on electronic and optical properties was explored. Time dependent (TD)-DFT calculations were also carried out to confirm the β₀ values; the results show that the higher value of first hyperpolarizability belongs to Sc-doped Ca₁₂O₁₂, which has the smallest transition energy (ΔEgn). The results obtained show that these clusters can be candidates for using in electronic devices and NLO materials in industry.     

Occurrence of fumonisins (B<Subscript>1</Subscript>, B<Subscript>2</Subscript>, B<Subscript>3</Subscript>) in maize-based food and feed samples from Indonesia

A survey to evaluate the contamination level of total fumonisins in maize-based foodstuffs, maize and feed from Indonesia is described. The analyses were carried out by enzyme-linked immunosorbent assay (ELISA). Samples were collected from local retail stores around Yogyakarta, Indonesia between February and May 2001. The 101 samples were classified into six categories, i.e. industrially-produced food (n=24), products of small food manufacturers (n=17), maize flour (n=4), maize for food (n=9), maize for feed (n17), and formulated feed (n30). Control of the method showed that the detection limit was 8.7 μg/kg and repeatability is shown by relative standard deviation (RSD) of analyses of contaminated maize (n=5) of 10 %. Results of analyses indicate that 80 samples analysed were contaminated over a large range from 10.0-3307 pg/kg, and the concentration of fumonisins depended on the type of sample. Of four samples of maize flour, none were contaminated (below detection limit). Of 24 samples of industrially produced food, 14 were contaminated in the range 22.8 - 105 μg/kg and 18 of 19 food samples from small manufacturers were contaminated ranging from 12.9 to 234 μg/kg. The highest contamination was observed in maize samples: six of ten samples of maize for food were contaminated between 68.0 - 2471 μg/kg and 16 of 17 samples for feed contained fumonisins over a large range from 17.6 to 3306 μg/kg.     

C<Subscript>3</Subscript> and C<Subscript>4</Subscript> photosynthetic pathways and life form types for native species from agro-forestry region,Northeastern China

Of the total 570 species, 194 species in 116 genera and 52 families were found with C₃ photosynthesis, 24 species in 17 genera and 6 families with C₄ photosynthesis, and 2 species in 1 genera and 1 family with CAM photosynthesis. 90 % of the total species can be found in Changbai Mountain flora, more a half (69 %) in North China flora, and about 1/3 in Mongolian flora and Xinan flora, respectively. The occurrence of C₄ species was not as common as that in adjacent grasslands and deserts, but relatively more than in the adjacent forests. Of the total 24 C₄ species, 63 % C₄ species (15 of 24) was found in Gramineae. Nine life form types can be found, reflecting the moist climate in the region, especially the occurrence of epiphyte and liana forms. Relatively more geophyte life form plants suggested the winter in the region was much colder than in grasslands. These indicated that both ecological studies and land management decisions must take into account plant photosynthetic pathway and life form patterns, for both of them are closely related to climatic changes and land use.     

Resolution-optimized NMR measurement of <Superscript>1</Superscript>D<Subscript>CH</Subscript>, <Superscript>1</Superscript>D<Subscript>CC</Subscript> and <Superscript>2</Superscript>D<Subscript>CH</Subscript> residual dipolar couplings in nucleic acid bases

New methods are described for accurate measurement of multiple residual dipolar couplings in nucleic acid bases. The methods use TROSY-type pulse sequences for optimizing resolution and sensitivity, and rely on the E.COSY principle to measure the relatively small two-bond ²D_CH couplings at high precision. Measurements are demonstrated for a 24-nt stem-loop RNA sequence, uniformly enriched in ¹³C, and aligned in Pf1. The recently described pseudo-3D method is used to provide homonuclear ¹H-¹H decoupling, which minimizes cross-correlation effects and optimizes resolution. Up to seven ¹H-¹³C and ¹³C-¹³C couplings are measured for pyrimidines (U and C), including ¹D_C5H5, ¹D_C6H6, ²D_C5H6, ²D_C6H5, ¹D_C5C4, ¹D_C5C6, and ²D_C4H5. For adenine, four base couplings (¹D_C2H2, ¹D_C8H8, ¹D_C4C5, and ¹D_C5C6) are readily measured whereas for guanine only three couplings are accessible at high relative accuracy (¹D_C8H8, ¹D_C4C5, and ¹D_C5C6). Only three dipolar couplings are linearly independent in planar structures such as nucleic acid bases, permitting cross validation of the data and evaluation of their accuracies. For the vast majority of dipolar couplings, the error is found to be less than ±3% of their possible range, indicating that the measurement accuracy is not limiting when using these couplings as restraints in structure calculations. Reported isotropic values of the one- and two-bond J couplings cluster very tightly for each type of nucleotide.     

Characterization of Myelin Sheath F<Subscript>o</Subscript>F<Subscript>1</Subscript>-ATP Synthase and its Regulation by IF<Subscript>1</Subscript>

F_oF₁-ATP synthase is the nanomotor responsible for most of ATP synthesis in the cell. In physiological conditions, it carries out ATP synthesis thanks to a proton gradient generated by the respiratory chain in the inner mitochondrial membrane. We previously reported that isolated myelin vesicles (IMV) contain functional F_oF₁-ATP synthase and respiratory chain complexes and are able to conduct an aerobic metabolism, to support the axonal energy demand. In this study, by biochemical assay, Western Blot (WB) analysis and immunofluorescence microscopy, we characterized the IMV F_oF₁-ATP synthase. ATP synthase activity decreased in the presence of the specific inhibitors (olygomicin, DCCD, FCCP, valynomicin/nigericin) and respiratory chain inhibitors (antimycin A, KCN), suggesting a coupling of oxygen consumption and ATP synthesis. ATPase activity was inhibited in low pH conditions. WB and microscopy analyses of both IMV and optic nerves showed that the Inhibitor of F₁ (IF₁), a small protein that binds the F₁ moiety in low pH when of oxygen supply is impaired, is expressed in myelin sheath. Data are discussed in terms of the role of IF₁ in the prevention of the reversal of ATP synthase in myelin sheath during central nervous system ischemic events. Overall, data are consistent with an energetic role of myelin sheath, and may shed light on the relationship among demyelination and axonal degeneration.     

Computational synthesis of hydrogenated fullerenes from C<Subscript>60</Subscript> to C<Subscript>60</Subscript>H<Subscript>60</Subscript>

Hydrogenation from C₆₀ to C₆₀H₆₀ was studied by an unrestricted broken spin symmetry Hartree–Fock approach implemented in semiempirical codes based on the AM1 technique. The calculations focused on the successive addition of hydrogen molecules to the fullerene cage following the identification of the cage target atoms by calculating the highest atomic chemical susceptibility at each step. The results obtained are analyzed from energy, symmetry, and composition perspectives.     

Aflatoxins B<Subscript>1</Subscript> and M<Subscript>1</Subscript>: risks related to milk produced in Brazil

This work shows data on the occurrence of aflatoxins in milk produced in Brazil. A review of the literature on this contamination. Several studies carried out in Brazil show that levels of aflatoxin M₁ in milk are higher than the ones established by the legislation, an evidence of the lack of control and inspection of these mycotoxins. Taking into account that milk has been widely consumed as an important source of nutrients, mainly by children, it is fundamental to carry out a thorough study of the occurrence of aflatoxins and take measures to mitigate milk contamination.     

Synthesis of Protein-Inorganic Nanohybrids with Improved Catalytic Properties Using Co<Subscript>3</Subscript>(PO<Subscript>4</Subscript>)<Subscript>2</Subscript>

In the present study, a method for easy and rapid synthesis of lipase nanohybrids was evaluated using cobalt chloride as an encapsulating agent. The synthesized nanohybrids exhibited higher activity (181%) compared to free lipase and improved catalytic properties at higher temperature and in harsh conditions. The nanohybrids retained 84% of their residual activity at 25 °C after 10 days. In addition, these nanohybrids also exhibited high storage stability and reusability. Collectively, the synthesis of carrier-free immobilized biocatalysts was performed rapidly within 24 h at 4 °C. Their high reusability and catalytic activities highlight the broad applicability of this method for catalysis in organic and aqueous media.     

Components of CO<Subscript>2</Subscript> exchange in leaves of C<Subscript>3</Subscript> species with different ability of starch accumulation

Using a radiogasometric method the rates of photorespiratory and respiratory decarboxylations of primary and stored photosynthates in the leaves of two groups of C₃ species, differing in the ability of starch accumulation, were determined. One group included starch-accumulating (SA) species with rates of starch synthesis on the average 38 % the rate of photosynthesis [Solanum tuberosum L., Arabidopsis thaliana (L.) Heynh, Helianthus annuus L., and Plantago lanceolata L.]. The second group represented starch-deficient (SD) species with rates of starch synthesis less than 8 % the rate of photosynthesis (Secale cereale L., Triticum aestivum L., Hordeum vulgare L., and Poa trivialis L.). In SA species the rate of respiration in the dark was significantly higher than in SD species. No differences were found in the rates of photosynthesis, photorespiration, and respiration under irradiation. Thus, the degree of inhibition of respiration by irradiation was in SA species higher than in SD species. It is concluded that starch does not provide substrates for respiratory and photorespiratory decarboxylations in irradiated photosynthesizing leaves.     

ADP-Inhibition of H<Superscript>+</Superscript>-F<Subscript>O</Subscript>F<Subscript>1</Subscript>-ATP Synthase

H⁺-F_OF₁-ATP synthase (F-ATPase, F-type ATPase, F_OF₁ complex) catalyzes ATP synthesis from ADP and inorganic phosphate in eubacteria, mitochondria, chloroplasts, and some archaea. ATP synthesis is powered by the transmembrane proton transport driven by the proton motive force (PMF) generated by the respiratory or photosynthetic electron transport chains. When the PMF is decreased or absent, ATP synthase catalyzes the reverse reaction, working as an ATP-dependent proton pump. The ATPase activity of the enzyme is regulated by several mechanisms, of which the most conserved is the non-competitive inhibition by the MgADP complex (ADP-inhibition). When ADP binds to the catalytic site without phosphate, the enzyme may undergo conformational changes that lock bound ADP, resulting in enzyme inactivation. PMF can induce release of inhibitory ADP and reactivate ATP synthase; the threshold PMF value required for enzyme reactivation might exceed the PMF for ATP synthesis. Moreover, membrane energization increases the catalytic site affinity to phosphate, thereby reducing the probability of ADP binding without phosphate and preventing enzyme transition to the ADP-inhibited state. Besides phosphate, oxyanions (e.g., sulfite and bicarbonate), alcohols, lauryldimethylamine oxide, and a number of other detergents can weaken ADP-inhibition and increase ATPase activity of the enzyme. In this paper, we review the data on ADP-inhibition of ATP synthases from different organisms and discuss the in vivo role of this phenomenon and its relationship with other regulatory mechanisms, such as ATPase activity inhibition by subunit ε and nucleotide binding in the noncatalytic sites of the enzyme. It should be noted that in Escherichia coli enzyme, ADP-inhibition is relatively weak and rather enhanced than prevented by phosphate.