Three-dimensional organization of the archaeal A1-ATPase from Methanosarcina mazei Gö1

A modified isolation procedure provides a homogeneous A(1)-ATPase from the archaeon Methanosarcina mazei G?1, containing the five subunits in stoichiometric amounts of A(3):B(3):C:D:F. A(1) obtained in this way was characterized by three-dimensional electron microscopy of single particles, resulting in the first three-dimensional reconstruction of an A(1)-ATPase at a resolution of 3.2 nm. The A(1) consists of a headpiece of 10.2 nm in diameter and 10.8 nm in height, formed by the six elongated subunits A(3) and B(3). At the bottom of the A(3)B(3) complex, a stalk of 3.0 nm in length can be seen. The A(3)B(3) domain surrounds a large cavity that extends throughout the length of the A(3)B(3) barrel. A part of the stalk penetrates inside this cavity and is displaced toward an A-B-A triplet. To investigate further the topology of the stalk subunits C-F in A(1), cross-linking has been carried out by using dithiobis[sulfosuccinimidylpropionate] (DSP) and 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (EDC). In experiments where DSP was added the cross-linked products B-F, A(x)-D, A-B-D, and A(x)-B(x)-D were formed. Subunits B-F, A-D, A-B-D, and A-B-C-D could be cross-linked by EDC. The subunit-subunit interaction in the presence of DSP was also studied as a function of nucleotide binding, demonstrating movements of subunits C, D, and F during ATP cleavage. Finally, the three-dimensional organization of this A(1) complex is discussed in terms of the relationship to the F(1)- and V(1)-ATPases at a resolution of 3.2 nm.     

Defective pro-IL-1β responses in macrophages from aged mice

Background

Cytokines regulated by the inflammasome pathway have been extensively implicated in various age-related immune pathologies. We set out to elucidate the contribution of the nod-like receptor protein 3 (NLRP3) inflammasome pathway to the previously described deficiencies in IL-1β production by macrophages from aged mice. We examined the production of pro-IL-1β and its conversion into IL-1β as two separate steps and compared these cytokine responses in bone marrow derived macrophages from young (6–8 weeks) and aged (18–24 months) C57BL/6 mice.

Findings

Relative to macrophages from young mice, macrophages from aged mice produced less pro-IL-1β after TLR4 stimulation with LPS. However upon activation of the NLRP3 inflammasome with ATP, macrophages from young and aged mice were able to efficiently convert and secrete intracellular pro-cytokines as functional cytokines.

Conclusions

Lower levels of IL-1β production are a result of slower and lower overall production of pro-IL-1β in macrophages from aged mice.

     

Four new β-lactones from the endophytic Streptomyces sp. T1B1

The detailed investigation of endophytic Streptomyces sp. T1B1 was performed during a search for new structural and active compounds. The strain T1B1 was isolated from the old bast tissue of Taxus yunnanensis and determined to be a member of Streptomyces, according to the 16S rRNA analysis. The extracts from the PDA solid fermentation media of Streptomyces sp. T1B1 were purified and four β-lactones were isolated. They were identified as 4α-(3,5-dihydroxy hexyl)-3α-methyl-2-oxetanone (1), 4α-(3-methyl-4-formyloxy-hexyl)-3α-methyl-2-oxetanone (2), 4α-(3,5-dihydroxy-heptyl)-3α-methyl-2-oxetanone (3) and 4α-(3-methyl-4-formyloxy-heptyl)-3α-methyl-2-oxetanone (4) on the basis of spectral data.     

β-N-Acetylglucosaminidase MthNAG from Myceliophthora thermophila C1, a thermostable enzyme for production of N-acetylglucosamine from chitin

Applied Microbiology and Biotechnology - Thermostable enzymes are a promising alternative for chemical catalysts currently used for the production of N-acetylglucosamine (GlcNAc) from chitin. In...     

Peli1 induction impairs cardiac microvascular endothelium through Hsp90 dissociation from IRE1α

Ameliorating cardiac microvascular injury is the most effective means to mitigate diabetes-induced cardiovascular complications. Inositol-requiring 1α (IRE1α), a sensor of endoplasmic reticulum stress, is activated by Toll like receptors (TLRs), and then promotes cardiac microvascular injury. Peli1 is a master regulator of TLRs and activates IRE1α. This study aims to investigate whether Peli1 in endothelial cells promotes diabetes-induced cardiac microvascular injury through activating IRE1α. Here we found that Peli1 was markedly up-regulated in cardiac endothelial cells of both diabetic mice and in AGEs-treated cardiac microvascular endothelial cells (CMECs). Peli1 deficiency in endothelial cells significantly alleviated diabetes-induced cardiac microvascular permeability, promoted microvascular regeneration, and suppressed apoptosis, accompanied by the attenuation of adverse cardiac remodeling. Furthermore, Peli1 deletion in CMECs ameliorated AGEs-induced damages in vitro. We identified heat shock protein 90 (Hsp90) as a potential binding partner for Peli1, and the Ring domain of Peli1 directly bound with Hsp90 to enhance IRE1α phosphorylation. Our study suggests that blocking Peli1 in endothelial cells may protect against diabetes-induced cardiac microvascular injury by restraining ER stress.     

Meprin A and meprin α generate biologically functional IL-1β from pro-IL-1β

The present study demonstrates that both oligomeric metalloendopeptidase meprin A purified from kidney cortex and recombinant meprin α are capable of generating biologically active IL-1β from its precursor pro-IL-1β. Amino-acid sequencing analysis reveals that meprin A and meprin α cleave pro-IL-1β at the His¹¹⁵-Asp¹¹⁶ bond, which is one amino acid N-terminal to the caspase-1 cleavage site and five amino acids C-terminal to the meprin β site. The biological activity of the pro-IL-1β cleaved product produced by meprin A, determined by proliferative response of helper T-cells, was 3-fold higher to that of the IL-1β product produced by meprin β or caspase-1. In a mouse model of sepsis induced by cecal ligation puncture that results in elevated levels of serum IL-1β, meprin inhibitor actinonin significantly reduces levels of serum IL-1β. Meprin A and meprin α may therefore play a critical role in the production of active IL-1β during inflammation and tissue injury.     

Purification and Characterization of β-Fructofuranosidase from Aspergillus japonicus TIT-KJ1

A β-fructofuranosidase (EC 3.2.1.26) was purified to homogeneity from Aspergillus japonicus TIT-KJ1. The enyme had an optimum pH for activity of 5.4 and pH stability at 7.0–8.4. The optimum temperature at pH 5.4 was 60°C. The enzyme had a molecular weight of 236,000 with two subunits and an isoelectric point of pH 4.0. The enzyme was inactivated by 5 mM Hg^{2 +} and Ag⁺. The enzyme had a high transfructosylating activity. Treatment of 50% (w/v) sucrose with the enzyme under optimum conditions afforded more than 55% fructooligosaccharides.     

An origin of DNA replication from streptomycete phage ΦU1

A DNA fragment from phage ΦU1 containing an origin of DNA replication was identified. This fragment, designatedori, was able to support the maintenance inStreptomyces lividans of a plasmid lacking a functional Gram-positiveori. The sequence of the minimalori fragment was determined and analyzed. The minimal fragment conferring replication origin function contained a number of direct and inverted repeats. The absence of an open reading frame in thisori fragment indicates that host factors alone were sufficient to initiate replication atori.     

Catalytic properties of β subunit isolated from chloroplast coupling factor 1

The chloroplast coupling factor (CF₁) was dissociated into subunits by the freezing-thawing procedure in the presence of 0.5 M NaBr and the subunit was purified by ion-exchange chromatography on a DEAE-cellulose column. The subunit did not catalyze ATP hydrolysis either in the presence or in the absence of reagents known to activate Mg²⁺-dependent ATPase activity of CF₁. However, it manifested appreciable adenylate kinase-like and ATP-ADP -phosphate exchange activities. The adenylate kinase-like activity only slightly depended on Mg²⁺ ions. Ethanol, and especially diadenosine pentaphosphate, inhibited the reaction effectively. In contrast, the ATP-ADP exchange activity was Mg²⁺-dependent. Ethanol and diadenosine pentaphosphate were poor inhibitors. Sulfite, the CF₁-ATPase activator, and quercetin, its inhibitor, had a minor effect on catalytic activity of the subunit.Abbreviations CF chloroplast coupling factor 1 - RBP carboxylase-ribulose-1,5-bisphosphate carboxylase - TLC thin layer chromatography - MES morpholinoethane sulfonic acid - PMSF phenylmethylsulfonyl fluoride - AP₅A diadenosine pentaphosphate, P¹, P⁵-bis(5-adenosyl)pentaphosphate - DCCD N₁N-dicyclohexylcarbodiimide - SDS sodium dodecylsulfate - PAAG polyacrylamide gel     

Bioenergy from permanent grassland – A review: 1. Biogas

Grassland biomass is suitable in numerous ways for producing energy. It is well established as feedstock for biogas production. The aim of this review is to summarize current knowledge on suitability and sustainability of grassland biomass for anaerobic digestion. In the first section grassland management for biogas feedstock as well as specifics of harvest, postharvest and digestion technology are described. Methane yields from grass are influenced by many factors. While the effects of some parameters such as grass species, cutting period and management intensity can be regarded as well known, other parameters such as preservation and processing still need investigation. In the second section economic aspects and environmental impacts are discussed. Profitability can be achieved depending on grass silage supply costs and the concept of anaerobic digestion and energy use. Grassland biomass for biogas production competes with other feedstock and other forms of grassland use, in particular animal husbandry. In developed countries a growing production of milk and meat is achieved with decreasing ruminant numbers, resulting in an increasing amount of surplus grassland with a remarkable bioenergy potential. In emerging and developing countries a rapidly rising demand for and production of milk and meat induce growing pressure on grasslands, so that their use for animal feed presumably will take priority over use for bioenergy. Grasslands provide a variety of essential environmental benefits such as carbon storage, habitat function, preservation of ground and surface water quality. When producing biogas from grassland these benefits will remain or even grow, providing appropriate grassland management is implemented. In particular, greenhouse gas emissions can be considerably reduced.     

A novel ginsenoside Rb1-hydrolyzing β-d-glucosidase from Cladosporium fulvum

A novel β-glucosidase (G-II) was purified to homogeneity from a culture filtrate of the phytopathogenic fungus Cladosporium fulvum (syn. Fulvia fulva). G-II specifically cleaved the β-(1 → 6)-glucosidic linkage at the C-20 site of ginsenoside Rb₁ to produce ginsenoside Rd, but did not hydrolyze the other β-d-glucosidic linkages in protopanaxadiol-type ginsenosides. In specificity tests, G-II was active against pNPG and disaccharides such as cellobiose and gentiobiose, but exhibited very low activities against other aryl-glycosides and methyl-α-glycosides. G-II consisted of two identical subunits with a native molecular mass of 180 kDa and a pI of 4.4. The optimal pH of G-II was pH 5.5, and the enzyme was highly stable over a range of pH 5.0–11.0. The optimal temperature was 45 °C, and the enzyme became unstable at temperatures above 40 °C. The K_m and V_max values against pNPG were 0.19 mM and 57.7 μmol/(min mg), respectively. The enzyme was inhibited by Zn²⁺, Cu²⁺ (over 50 mM) and SDS (250 mM). However, the inhibition by SDS was partially reversed by 10 mM dithiothreitol. Three oligopeptide fragments obtained after enzymatic digestion of G-II were sequenced by nanoESI-MS/MS. The amino acid sequence homology analysis showed that G-II possessed significant homology with the family 3 β-glucosidases.     

Amyloid-β triggers the release of neuronal hexokinase 1 from mitochondria

Brain accumulation of the amyloid-β peptide (Aβ) and oxidative stress underlie neuronal dysfunction and memory loss in Alzheimer's disease (AD). Hexokinase (HK), a key glycolytic enzyme, plays important pro-survival roles, reducing mitochondrial reactive oxygen species (ROS) generation and preventing apoptosis in neurons and other cell types. Brain isozyme HKI is mainly associated with mitochondria and HK release from mitochondria causes a significant decrease in enzyme activity and triggers oxidative damage. We here investigated the relationship between Aβ-induced oxidative stress and HK activity. We found that Aβ triggered HKI detachment from mitochondria decreasing HKI activity in cortical neurons. Aβ oligomers further impair energy metabolism by decreasing neuronal ATP levels. Aβ-induced HKI cellular redistribution was accompanied by excessive ROS generation and neuronal death. 2-deoxyglucose blocked Aβ-induced oxidative stress and neuronal death. Results suggest that Aβ-induced cellular redistribution and inactivation of neuronal HKI play important roles in oxidative stress and neurodegeneration in AD.     

Structural implications of mutations assessed by molecular dynamics: Vpu1–32 from HIV-1

Structural pore models are generated for Vpu_1–32WT from HIV-1 as well as for three mutants W23L, S24L and R31V. A computational methodology is employed which samples the whole conformational space of the pentameric assemblies of Vpu. The analysis of the related energy landscape reveals a small set of reasonable pore models, which are thoroughly investigated regarding their structural properties as well as their putative stability under native-like conditions. The models are also discussed in respect of earlier experimental findings about their channel activities. The study proposes functional pores reflecting the experimentally found conductance states of Vpu and its mutants.     

Structural Insights into the A1 ATPase from the archaeon, Methanosarcina mazei Gö1

The low-resolution structure and overall dimensions of the A(3)B(3)CDF complex of the A(1) ATPase from Methanosarcina mazei G?1 in solution is analyzed by synchrotron X-ray small-angle scattering. The radius of gyration and the maximum size of the complex are 5.03 +/- 0.1 and 18.0 +/- 0.1 nm, respectively. The low-resolution shape of the protein determined by two independent ab initio approaches has a knob-and-stalk-like feature. Its headpiece is approximately 9.4 nm long and 9.2 nm wide. The stalk, which is known to connect the headpiece to its membrane-bound A(O) part, is approximately 8.4 nm long. Limited tryptic digestion of the A(3)B(3)CDF complex was used to probe the topology of the smaller subunits (C-F). Trypsin was found to cleave subunit C most rapidly at three sites, Lys(20), Lys(21), and Arg(209), followed by subunit F. In the A(3)B(3)CDF complex, subunit D remained protected from proteolysis.     

Do microbial protein elicitors PeaT1 obtained from Alternaria tenuissima and PeBL1 from Brevibacillus laterosporus enhance defense response against tomato aphid (Myzus persicae)?

Tomato aphid (Myzus persicae) is a destructive insect pest of tomato responsible for huge losses in the production as well in the vegetable industry. In the present in vitro study two protein elicitors, PeaT1 and PeBL1 were considered to study their efficacies to exhibit defense response against tomato aphid. Three different concentrations of both protein elicitors were applied on the tomato seedlings. After the application of PeaT1 and PeBL1, population growth rates of tomato aphid were decreased as compared to the control treatment. In host preference assay, the tomato aphid showed a preference to build a colony on the control as compared to the treated tomato plant, because tomato leaves provided hazardous surface for aphid after the formation of wax and trichome. The concentrations of protein showed significant (p < 0.05) results in life-history traits of the aphid. Jasmonic acid (JA), salicylic acid (SA) and ethylene (ET) showed significant accumulation in tomato seedlings treated with PeaT1 and PeBL1. Elicitors treated plants produced resistance against M. persicae. Our finding suggests that PeaT1 and PeBL1 have shown high potentials against the damage of M. persicae, and both elicitors could be used as novel biological tools against tomato aphid.