Characterization of a novel xylanase from Armillaria gemina and its immobilization onto SiO2 nanoparticles

Enhanced catalytic activities of different lignocellulases were obtained from Armillaria gemina under statistically optimized parameters using a jar fermenter. This strain showed maximum xylanase, endoglucanase, cellobiohydrolase, and β-glucosidase activities of 1,270, 146, 34, and 15 U mL^?1, respectively. Purified A. gemina xylanase (AgXyl) has the highest catalytic efficiency (k _cat/K _m?=?1,440 mg?mL^?1?s^?1) ever reported for any fungal xylanase, highlighting the significance of the current study. We covalently immobilized the crude xylanase preparation onto functionalized silicon oxide nanoparticles, achieving 117 % immobilization efficiency. Further immobilization caused a shift in the optimal pH and temperature, along with a fourfold improvement in the half-life of crude AgXyl. Immobilized AgXyl gave 37.8 % higher production of xylooligosaccharides compared to free enzyme. After 17 cycles, the immobilized enzyme retained 92 % of the original activity, demonstrating its potential for the synthesis of xylooligosaccharides in industrial applications.     

Glucocerebrosidase inhibition causes mitochondrial dysfunction and free radical damage

Mutations of the gene for glucocerebrosidase 1 (GBA) cause Gaucher disease (GD), an autosomal recessive lysosomal storage disorder. Individuals with homozygous or heterozygous (carrier) mutations of GBA have a significantly increased risk for the development of Parkinson’s disease (PD), with clinical and pathological features that mirror the sporadic disease. The mechanisms whereby GBA mutations induce dopaminergic cell death and Lewy body formation are unknown. There is evidence of mitochondrial dysfunction and oxidative stress in PD and so we have investigated the impact of glucocerebrosidase (GCase) inhibition on these parameters to determine if there may be a relationship of GBA loss-of-function mutations to the known pathogenetic pathways in PD. We have used exposure to a specific inhibitor (conduritol-β-epoxide, CβE) of GCase activity in a human dopaminergic cell line to identify the biochemical abnormalities that follow GCase inhibition. We show that GCase inhibition leads to decreased ADP phosphorylation, reduced mitochondrial membrane potential and increased free radical formation and damage, together with accumulation of alpha-synuclein. Taken together, inhibition of GCase by CβE induces abnormalities in mitochondrial function and oxidative stress in our cell culture model. We suggest that GBA mutations and reduced GCase activity may increase the risk for PD by inducing these same abnormalities in PD brain.     

LOX-1 deletion and macrophage trafficking in atherosclerosis

Background

Atherosclerosis is associated with macrophage accumulation. LOX-1 has been shown to induce macrophage attachment, and its deletion (LOX-1 knockout, KO) reduces atherosclerosis in LDLr KO mice fed a high cholesterol diet. We examined differences in macrophage trafficking in age-matched wild type, LOX-1 KO, LDLr KO, and LDLr/LOX-1 double KO mice.

Methods

Sections of aortas of mice fed high cholesterol diet were collected at weeks 0, 4, 8, 12 and 19 and analyzed by immunohistochemistry and flow cytometry.

Results

In the LDLr KO mice aorta, CD68 positivity (macrophage accumulation) increased over time up to 12 weeks, and then the accumulation fell modestly but significantly. The periaortal fat and adventitia showed more CD68 positivity than the media and intima. This pattern was also evident in the non-atherosclerotic areas. Importantly, LOX-1 KO and LDLr–LOX-1 double KO mice showed diminished CD68 positivity in comparison to wild type and LDLR KO mice, respectively. Further, macrophages from LOX-1 KO mice revealed a marked reduction in migration (vs. macrophages from wild type mice) in in vitro migration assay.

Conclusions

LOX-1 deletion translates into reduction in macrophage trafficking in the aorta of LDLr KO mice. Most of the macrophage trafficking appears in the subadventitial regions.     

Design and synthesis of (4E)-4-(4-substitutedbenzylideneamino)-3-substituted-2,3-dihydro-2-thioxothiazole-5-carbonitrile as novel A2A receptor antagonists

Novel 2-thioxothiazole derivatives (6–19) as potential adenosine A_2A receptor (A_2AR) antagonists were synthesized. The strong interaction of the compounds (6–19) with A_2AR in docking study was confirmed by high binding affinity with human A_2AR expressed in HEK293T cells using radioligand-binding assay. The compound 19 demonstrated very high selectivity for A_2AR as compared to standard A_2AR antagonist SCH58261. Decrease in A_2AR-coupled release of endogenous cAMP in treated HEK293T cells demonstrated in vitro A_2AR antagonist potential of the compound 19. Attenuation in haloperidol-induced impairment (catalepsy) in Swiss albino male mice pre-treated with compound 19 is evocative to explore its prospective in therapy of PD.     

Changes in litter decomposition and soil organic carbon in a reforested tropical deciduous cover (India)

Soil organic carbon (SOC) up to 1 m depth originates from contemporary vegetation cover dating from past millennia. Deforestation and reforestation with economically important species is influencing soil carbon sequestration. An attempt has been made in this study to evaluate the impact of vegetation cover change (due to replacement of natural heterogeneous cover by teak and bamboo) on SOC using carbon isotopes (δ¹³C, ¹⁴C) in a tropical system (India). A litter decomposition study was carried out to understand the impact of differences in vegetation characteristics (specifically of leaves) on decomposition. Both experiments were carried out to look at the impact of changes in vegetation characteristics (specifically of leaves) on litter decomposition, and how these influence near term litter decomposition rates (k values) and long-term SOC content of the soil system beneath. Leaves of teak, bamboo and eight other species were selected for this study. The proportion of structural carbohydrates (lignin and cellulose) in leaves significantly (at 5 % level) influenced k values. The SOC and carbon isotope data collected in this study indicate that C₃ vegetation cover in the study area could be contemporary and dominant for the past few centuries. This can be extended up to ~2,200 years from the recorded ¹⁴C values of teak cover. The study confirms that k values of leaf litter influence SOC present beneath the vegetation cover at the decadal/century time scale.     

Antioxidant efficacy and adhesion rescue by a recombinant mussel foot protein‐6

Mytilus foot protein type 6 (mfp‐6) is crucial for maintaining the reducing conditions needed for optimal wet adhesion in marine mussels. In this report, we describe the expression and production of a recombinant Mytilus californianus foot protein type 6 variant 1 (rmfp‐6.1) fused with a hexahistidine affinity tag in Escherichia coli and its purification by affinity chromatography. Recombinant mfp‐6 showed high purification yields of 5–6 mg L^?1 cell culture and excellent solubility in low pH buffers that retard oxidation of its many thiol groups. Purified rmfp‐6.1 protein showed high 2,2‐diphenyl‐1‐picrylhydrazyl radical scavenging activity when compared with vitamin C. Using the highly sensitive surface forces apparatus (SFA) technique to measure interfacial surface forces in the nano‐Newton range, we show that rmfp‐6.1 is also able to rescue the oxidation‐dependent adhesion loss of mussel foot protein 3 (mfp‐3) at pH 3. The adhesion rescue is related to a reduction of dopaquinone back to 3,4‐dihydroxyphenyl‐l ‐alanine in mfp‐3, which is the reverse reaction observed during the detrimental enzymatic browning process in fruits and vegetables. Broadly viewed, rmfp‐6.1 has potential as a versatile antioxidant for applications ranging from personal products to antispoilants for perishable foods during processing and storage. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1587–1593, 2013     

Iron Is a Sensitive Biomarker for Inflammation in Multiple Sclerosis Lesions

MRI phase imaging in multiple sclerosis (MS) patients and in autopsy tissue have demonstrated the presence of iron depositions in white matter lesions.The accumulation of iron in some but not all lesions suggests a specific, potentially disease-relevant process, however; its pathophysiological significance remains unknown.Here, we explore the role of lesional iron in multiple sclerosis using multiple approaches: immunohistochemical examination of autoptic MS tissue, an in vitro model of iron-uptake in human cultured macrophages and ultra-highfield phase imaging of highly active and of secondary progressive MS patients.Using Perls'' stain and immunohistochemistry, iron was detected in MS tissue sections predominantly in non-phagocytosing macrophages/microglia at the edge of established, demyelinated lesions. Moreover, iron-containing macrophages but not myelin-laden macrophages expressed markers of proinflammatory (M1) polarization.Similarly, in human macrophage cultures, iron was preferentially taken up by non-phagocytosing, M1-polarized macrophages and induced M1 (super) polarization. Iron uptake was minimal in myelin-laden macrophages and active myelin phagocytosis led to depletion of intracellular iron.Finally, we demonstrated in MS patients using GRE phase imaging with ultra-highfield MRI that phase hypointense lesions were significantly more prevalent in patients with active relapsing than with secondary progressive MS.Taken together, our data provide a basis to interpret iron-sensitive GRE phase imaging in MS patients: iron is present in non-phagocytosing, M1-polarized microglia/macrophages at the rim of chronic active white matter demyelinating lesions. Phase imaging may therefore visualize specific, chronic proinflammatory activity in established MS lesions and thus provide important clinical information on disease status and treatment efficacy in MS patients.