Topiramate Improves Neuroblast Differentiation of Hippocampal Dentate Gyrus in the <Emphasis Type="SmallCaps">d</Emphasis>-Galactose-Induced Aging Mice via Its Antioxidant Effects

Some anticonvulsant drugs are associated with cognitive ability in patients; Topiramate (TPM) is well known as an effective anticonvulsant agent applied in clinical settings. However, the effect of TPM on the cognitive function is rarely studied. In this study, we aimed to observe the effects of TPM on cell proliferation and neuronal differentiation in the dentate gyrus (DG) of the d-galactose-induced aging mice by Ki-67 and doublecortin (DCX) immunohistochemistry. The study is divided into four groups including control, d-galactose-treated group, 25 and 50 mg/kg TPM-treated plus d-galactose-treated groups. We found, 50 mg/kg (not 25 mg/kg) TPM treatment significantly increased the numbers of Ki-67⁺ cells and DCX immunoreactivity, and improved neuroblast injury induced by d-galactose treatment. In addition, we also found that decreased immunoreactivities and protein levels of antioxidants including superoxide dismutase and catalase induced by d-galactose treatment were significantly recovered by 50 mg/kg TPM treatment in the mice hippocampal DG (P < 0.05). In conclusion, our present results indicate that TPM can ameliorate neuroblast damage and promote cell proliferation and neuroblast differentiation in the hippocampal DG via increasing SODs and catalase levels in the d-galactose mice.     

Protective Effect of Hyperbaric Oxygen on Cognitive Impairment Induced by <Emphasis Type="SmallCaps">d</Emphasis>-Galactose in Mice

Memory decline is characteristic of aging and age-related neurodegenerative disorders. This study was designed to investigate the protective effect of hyperbaric oxygen (HBO) against cognitive impairment induced by d-galactose (d-gal) in mice. d-gal was intraperitoneally injected into mice daily for 8 weeks to establish the aging model. HBO was simultaneously administered once daily. The results indicate that HBO significantly reversed D-gal-induced learning and memory impairments. Studies on the potential mechanisms of this action showed that HBO significantly reduced oxidative stress by increasing superoxide dismutase, glutathione peroxidase, and catalase levels, as well as the total anti-oxidation capability, while decreasing the content of malondialdehyde, nitric oxide, and nitric oxide synthase in the hippocampal CA1 region. HBO also inhibited advanced glycation end-product formation and decreased levels of tumor necrosis factor-α and interleukin-6. Moreover, HBO significantly attenuated d-gal-induced pathological injury in the hippocampus, as well as β-amyloid protein_1?42 expression and retained BDNF expression. Furthermore, HBO decreased p16, p21 and p53 gene and protein expression in the hippocampus of d-gal-treated mice. In conclusion, the protective effect of HBO against d-gal-induced cognitive impairment was mainly due to its ability to reduce oxidative damage, suppress inflammatory responses, and regulate aging-related gene expression.     

Ginsenoside Rg1 Decreases Oxidative Stress and Down-Regulates Akt/mTOR Signalling to Attenuate Cognitive Impairment in Mice and Senescence of Neural Stem Cells Induced by <Emphasis Type="SmallCaps">d</Emphasis>-Galactose

Adult hippocampal neurogenesis plays a pivotal role in learning and memory. The suppression of hippocampal neurogenesis induced by an increase of oxidative stress is closely related to cognitive impairment. Neural stem cells which persist in the adult vertebrate brain keep up the production of neurons over the lifespan. The balance between pro-oxidants and anti-oxidants is important for function and surviving of neural stem cells. Ginsenoside Rg1 is one of the most active components of Panax ginseng, and many studies suggest that ginsenosides have antioxidant properties. This research explored the effects and underlying mechanisms of ginsenoside Rg1 on protecting neural stem cells (NSCs) from oxidative stress. The sub-acute ageing of C57BL/6 mice was induced by subcutaneous injection of d-gal (120 mg kg^?1 day^?1) for 42 day. On the 14th day of d-gal injection, the mice were treated with ginsenoside Rg1 (20 mg kg^?1 day^?1, intraperitoneally) or normal saline for 28 days. The study monitored the effects of Rg1 on proliferation, senescence-associated and oxidative stress biomarkers, and Akt/mTOR signalling pathway in NSCs. Compared with the d-gal group, Rg1 improved cognitive impairment induced by d-galactose in mice by attenuating senescence of neural stem cells. Rg1 also decreased the level of oxidative stress, with increased the activity of superoxide dismutase and glutathione peroxidase in vivo and in vitro. Rg1 furthermore reduced the phosphorylation levels of protein kinase B (Akt) and the mechanistic target of rapamycin (mTOR) and down-regulated the levels of downstream p53, p16, p21 and Rb in d-gal treated NSCs. The results suggested that the protective effect of ginsenoside Rg1 on attenuating cognitive impairment in mice and senescence of NSCs induced by d-gal might be related to the reduction of oxidative stress and the down-regulation of Akt/mTOR signaling pathway.     

Synthesis and preliminary biological evaluation of <Emphasis Type="Italic">S</Emphasis>-<Superscript>11</Superscript>C-methyl-<Emphasis Type="SmallCaps">d</Emphasis>-cysteine as a new amino acid PET tracer for cancer imaging

S-¹¹C-methyl-l-cysteine (LMCYS) is an attractive amino acid tracer for clinical tumor positron emission tomography (PET) imaging. d-isomers of some radiolabeled amino acids are potential PET tracers for tumor imaging. In this work, S-¹¹C-methyl-d-cysteine (DMCYS), a d-amino acid isomer of S-¹¹C-methyl-cysteine for tumor imaging was developed and evaluated. DMCYS was prepared by ¹¹C-methylation of the precursor d-cysteine, with an uncorrected radiochemical yield over 50 % from ¹¹CH₃I within a total synthesis time from ¹¹CO₂ about 12 min. In vitro competitive inhibition studies showed that DMCYS uptake was primarily transported through the Na⁺-independent system L, and also the Na⁺-dependent system B^0,+ and system ASC, with almost no system A. In vitro incorporation experiments indicated that almost no protein incorporation was found in Hepa 1–6 hepatoma cell lines. Biodistribution studies demonstrated higher uptake of DMCYS in pancreas and liver at 5 min post-injection, relatively lower uptake in brain and muscle, and faster radioactivity clearance from most tissues than those of l-isomer during the entire observation time. In the PET imaging of S180 fibrosarcoma–bearing mice and turpentine-induced inflammatory model mice, 2-¹⁸F-fluoro-2-deoxy-d-glucose (FDG) exhibited significantly high accumulation in both tumor and inflammatory lesion with low tumor-to-inflammation ratio of 1.40, and LMCYS showed low tumor-to-inflammation ratio of 1.64 at 60 min post-injection. By contrast, DMCYS showed moderate accumulation in tumor and very low uptake in inflammatory lesion, leading to relatively higher tumor-to-inflammation ratio of 2.25 than ¹¹C-methyl-l-methionine (MET) (1.85) at 60 min post-injection. Also, PET images of orthotopic transplanted glioma models demonstrated that low uptake of DMCYS in normal brain tissue and high uptake in brain glioma tissue were observed. The results suggest that DMCYS is a little better than the corresponding l-isomers as a potential PET tumor-detecting agent and is superior to MET and FDG in the differentiation of tumor from inflammation.     

The occurrence and accumulation of <Emphasis Type="SmallCaps">d</Emphasis>-pinitol in fenugreek (<Emphasis Type="Italic">Trigonella foenum graecum</Emphasis> L.)

This article presents changes in concentrations of d-pinitol (and other cyclitols as well as low molecular weight carbohydrates) in vegetative and reproductive organs of fenugreek (Trigonella foenumgraecum L.) during an entire plant growing period. d-Pinitol was the major cyclitol in all tested organs, representing 43–94% of total cyclitols and 2–77% of total soluble carbohydrates. The highest concentration of d-pinitol was found in pods (14–23 mg g^?1 of dry weight, DW), lower in leaves and stems (5–20 and 9–10 mg g^?1 DW, respectively), and the lowest in maturing seeds (2–5 mg g^?1 DW). Although maturing seeds accumulate α-d-galactosides of d-pinitol (galactosyl pinitols, up to 6.6 mg g^?1 DW), the major storage sugars were raffinose family oligosaccharides (RFOs, 65.37 mg g^?1 DW). Both RFOs and galactosyl pinitols are hydrolyzed during seed germination, releasing sucrose and d-pinitol, respectively. Accumulation of free galactose was not detected. Owing to the high concentration of d-pinitol (up to 23.70 mg g^?1 DW) and low concentration of soluble sugars, developing pods seem to be the best source of d-pinitol.     

Intrathecal Delivery of ssAAV9-DAO Extends Survival in SOD1<Superscript>G93A</Superscript> ALS Mice

Amyotrophic lateral sclerosis (ALS) is an adult-onset, irreversible neurodegenerative disease that leads to progressive paralysis and inevitable death 3–5 years after diagnosis. The mechanisms underlying this process remain unknown, but new evidence indicates that accumulating levels of d-serine result from the downregulation of d-amino acid oxidase (DAO) and that this is a novel mechanism that leads to motoneuronal death in ALS via N-methyl-d-aspartate receptor-mediated cell toxicity. Here, we explored a new therapeutic approach to ALS by overexpressing DAO in the lumbar region of the mouse spinal cord using a single stranded adeno-associated virus serotype 9 (ssAAV9) vector. A single intrathecal injection of ssAAV9-DAO was made in SOD1^G93A mice, a well-established mouse model of ALS. Treatment resulted in moderate expression of exogenous DAO in motorneurons in the lumbar spinal cord, reduced immunoreactivity of d-serine, alleviated motoneuronal loss and glial activation, and extended survival. The potential mechanisms underlying these effects were associated with the down-regulation of NF-κB and the restoration of the phosphorylation of Akt. In conclusion, administering ssAAV9-DAO may be an effective complementary approach to gene therapy to extend lifespans in symptomatic ALS.     

Minocycline ameliorates <Emphasis Type="SmallCaps">d</Emphasis>-galactose-induced memory deficits and loss of Arc/Arg3.1 expression

Dysfunction of learning and memory is widely found in many neurological diseases. Understanding how to preserve the normal function of learning and memory will be extremely beneficial for the treatment of these diseases. However, the possible protective effect of minocycline in memory impairment is unknown. We used the well-established d-galactose rat amnesia model and two behavioral tasks, the Morris water maze and the step-down task, for memory evaluation. Western blot and PCR were used to examine the protein and mRNA levels of Arc/Arg3.1. We report that minocycline supplementation ameliorates both the spatial and fear memory deficits caused by d-galactose. We also found that Arc/Arg3.1, c-fos, and brain-derived neurotrophic factor levels are decreased in the d-galactose animal model, and that minocycline reverses the protein and mRNA levels of Arc in the hippocampus, suggesting the potential role of Arc/Arg3.1 in minocycline’s neuroprotective mechanism. Our study strongly suggests that minocycline can be used as a novel treatment for memory impairment in neurological diseases.     

Cytotoxicity and Glycan-Binding Profile of a <Emphasis Type="SmallCaps">d</Emphasis>-Galactose-Binding Lectin from the Eggs of a Japanese Sea Hare (<Emphasis Type="Italic">Aplysia kurodai</Emphasis>)

A divalent cation-independent 16 kDa d-galactose binding lectin (AKL-2) was isolated from eggs of sea hare, Aplysia kurodai. The lectin recognized d-galactose and d-galacturonic acid and had a 32 kDa dimer consisting of two disulfide-bonded 16 kDa subunits. Eighteen N-terminus amino acids were identified by Edman degradation, having unique primary structure. Lectin blotting analysis with horseradish peroxidase-conjugated lectins has shown that AKL-2 was a glycoprotein with complex type oligosaccharides with N-acetyl d-glucosamine and mannose at non-reducing terminal. Two protein bands with 38 and 36 kDa in the crude extract of sea hare eggs after purification of the lectin was isolated by AKL-2-conjugated Sepharose column and elution with 0.1 M lactose containing buffer. It suggested that the lectin binds with an endogenous ligand in the eggs. AKL-2 kept extreme stability on haemagglutination activity if it was treated at pH 3 and 70 °C for 1 h. Glycan binding profile of AKL-2 by frontal affinity chromatography technology using 15 pyridylamine labeled oligosaccharides has been appeared that the lectin uniquely recognized globotriose (Galα1-4Galβ1-4Glc; Gb3) in addition to bi-antennary complex type N-linked oligosaccharides with N-acetyllactosamine. Surface plasmon resonance analysis of AKL-2 against a neo-glycoprotein, Gb3-human serum albumin showed the k _ass and k _diss values are 2.4 × 10³ M^?1 s^?1 and 3.8 × 10^?3 s^?1, respectively. AKL-2 appeared cytotoxicity against both Burkitt’s lymphoma Raji cell and erythroleukemia K562. The activity to Raji by the lectin was preferably cancelled by the co-presence of melibiose mimicing Gb3. On the other hand, K562 was cancelled effectively by lactose than melibiose. It elucidated that AKL-2 had cytotoxic ability mediated glycans structure to cultured cells.     

Metabolomic and proteomic analysis of <Emphasis Type="SmallCaps">d</Emphasis>-lactate-producing <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> under various fermentation conditions

As an important feedstock monomer for the production of biodegradable stereo-complex poly-lactic acid polymer, d-lactate has attracted much attention. To improve d-lactate production by microorganisms such as Lactobacillus delbrueckii, various fermentation conditions were performed, such as the employment of anaerobic fermentation, the utilization of more suitable neutralizing agents, and exploitation of alternative nitrogen sources. The highest d-lactate titer could reach 133 g/L under the optimally combined fermentation condition, increased by 70.5% compared with the control. To decipher the potential mechanisms of d-lactate overproduction, the time-series response of intracellular metabolism to different fermentation conditions was investigated by GC–MS and LC–MS/MS-based metabolomic analysis. Then the metabolomic datasets were subjected to weighted correlation network analysis (WGCNA), and nine distinct metabolic modules and eight hub metabolites were identified to be specifically associated with d-lactate production. Moreover, a quantitative iTRAQ–LC–MS/MS proteomic approach was employed to further analyze the change of intracellular metabolism under the combined fermentation condition, identifying 97 up-regulated and 42 down-regulated proteins compared with the control. The in-depth analysis elucidated how the key factors exerted influence on d-lactate biosynthesis. The results revealed that glycolysis and pentose phosphate pathways, transport of glucose, amino acids and peptides, amino acid metabolism, peptide hydrolysis, synthesis of nucleotides and proteins, and cell division were all strengthened, while ATP consumption for exporting proton, cell damage, metabolic burden caused by stress response, and bypass of pyruvate were decreased under the combined condition. These might be the main reasons for significantly improved d-lactate production. These findings provide the first omics view of cell growth and d-lactate overproduction in L. delbrueckii, which can be a theoretical basis for further improving the production of d-lactate.     

Biosynthesis of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from lignocellulosic biomass

d-lactic acid is a versatile and important industrial chemical that can be applied in the synthesis of thermal-resistant poly-lactic acid. Biosynthesis of d-lactic acid can be achieved by a variety of microorganisms, including lactic acid bacteria, yeast, and fungi; however, the final product yield, optical purity, and the utilization of both glucose and xylose are restricted. Consequently, engineered microbial systems are essential to attain high titer, productivity, and complete utilization of sugars. Herein, we critically evaluate the promising wild-type microorganisms, as well as genetically modified microorganisms to produce enantiomerically pure d-lactic acid, particularly from renewable lignocellulosic biomass. In addition, innovative bioreactor operation, metabolic flux analysis, and recent genetic engineering methods for targeted microbial d-lactic acid synthesis will be discussed.     

Cerebralcare Granule<Superscript>®</Superscript>, a Chinese Herb Compound Preparation,Attenuates <Emphasis Type="SmallCaps">d</Emphasis>-Galactose Induced Memory Impairment in Mice

Cerebralcare granule^® (CG) is a preparation of Traditional Chinese Medicine that widely used in China. It was approved by the China State Food and Drug Administration for treatment of headache and dizziness associated with cerebrovascular diseases. In the present study, we aimed to investigate whether CG had protective effect against d-galactose (gal)-induced memory impairment and to explore the mechanism of its action. d-gal was administered (100 mg/kg, subcutaneously) once daily for 8 weeks to induced memory deficit and neurotoxicity in the brain of aging mouse and CG (7.5, 15, and 30 g/kg) were simultaneously administered orally. The present study demonstrates that CG can alleviate aging in the mouse brain induced by d-gal through improving behavioral performance and reducing brain cell damage in the hippocampus. CG prevents aging mainly via suppression of oxidative stress response, such as decreasing NO and MDA levels, renewing activities of SOD, CAT, and GPx, as well as decreasing AChE activity in the brain of d-gal-treated mice. In addition, CG prevents aging through inhibiting NF-κB-mediated inflammatory response and caspase-3-medicated neurodegeneration in the brain of d-gal treated mice. Taken together, these data clearly demonstrates that subcutaneous injection of d-gal produced memory deficit, meanwhile CG can protect neuron from d-gal insults and improve memory ability.     

Increased productivity of <Emphasis Type="SmallCaps">l</Emphasis>-2-aminobutyric acid and total turnover number of NAD<Superscript>+</Superscript>/NADH in a one-pot system through enhanced thermostability of <Emphasis Type="SmallCaps">l</Emphasis>-threonine deaminase

Objective

To strengthen NADH regeneration in the biosynthesis of l-2-aminobutyric acid (l-ABA).

Results

l-Threonine deaminase (l-TD) from Escherichia coli K12 was modified by directed evolution and rational design to improve its endurance to heat treatment. The half-life of mutant G323D/F510L/T344A at 42 °C increased from 10 to 210 min, a 20-fold increase compared to the wild-type l-TD, and the temperature at which the activity of the enzyme decreased by 50% in 15 min increased from 39 to 53 °C. The mutant together with thermostable l-leucine dehydrogenase from Bacillus sphaericus DSM730 and formate dehydrogenase from Candida boidinii constituted a one-pot system for l-ABA biosynthesis. Employing preheat treatment in the one-pot system, the biosynthesis of l-ABA and total turnover number of NAD⁺/NADH were 0.993 M and 16,469, in contrast to 0.635 M and 10,531 with wild-type l-TD, respectively.

Conclusions

By using the engineered l-TD during endured preheat treatment, the one-pot system has achieved a higher productivity of l-ABA and total turnover number of coenzyme.

     

Highly efficient production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from chicory-derived inulin by <Emphasis Type="Italic">Lactobacillus bulgaricus</Emphasis>

Inulin is a readily available feedstock for cost-effective production of biochemicals. To date, several studies have explored the production of bioethanol, high-fructose syrup and fructooligosaccharide, but there are no studies regarding the production of d-lactic acid using inulin as a carbon source. In the present study, chicory-derived inulin was used for d-lactic acid biosynthesis by Lactobacillus bulgaricus CGMCC 1.6970. Compared with separate hydrolysis and fermentation processes, simultaneous saccharification and fermentation (SSF) has demonstrated the best performance of d-lactic acid production. Because it prevents fructose inhibition and promotes the complete hydrolysis of inulin, the highest d-lactic acid concentration (123.6 ± 0.9 g/L) with a yield of 97.9 % was obtained from 120 g/L inulin by SSF. Moreover, SSF by L. bulgaricus CGMCC 1.6970 offered another distinct advantage with respect to the higher optical purity of d-lactic acid (>99.9 %) and reduced number of residual sugars. The excellent performance of d-lactic acid production from inulin by SSF represents a high-yield method for d-lactic acid production from non-food grains.     

UDP-<Emphasis Type="SmallCaps">d</Emphasis>-galactose synthesis by UDP-glucose 4-epimerase 4 is required for organization of the <Emphasis Type="Italic">trans</Emphasis>-Golgi network/early endosome in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> root epidermal cells

Endomembrane organization is essential for cell physiology. We previously identified an Arabidopsis thaliana mutant in which a plasma membrane (PM) marker GFP-NIP5;1 and trans-Golgi network/early endosome (TGN/EE) markers were accumulated in intracellular aggregates in epidermal cells of the root elongation zone. The mutant was identified as an allele of UDP-glucose epimerase 4 (UGE4)/root hair defective 1/root epidermal bulgar 1, which was previously described as a mutant with swollen root epidermal cells and has an altered sugar composition in cell wall polysaccharides. Importantly, these defects including aggregate formation were restored by supplementation of d-galactose in the medium. These results suggested that UDP-d-galactose synthesis by UGE4 is important for endomembrane organization in addition to cell wall structure. Here, we further investigated the nature of the aggregates using various markers of endomembrane compartments and BOR1-GFP, which traffics from PM to vacuole in response to high-B supply. The markers of multi-vesicular bodies/late endosomes (MVB/LEs) and BOR1-GFP were strongly accumulated in the intracellular aggregates, while those of the endoplasmic reticulum (ER), the vacuolar membrane, and the Golgi were only slightly affected in the uge4 mutant. The abnormal localizations of these markers in the uge4 mutant differed from the effects of inhibitors of actin and microtubule polymerization, although they also affected endomembrane organization. Furthermore, electron microscopy analysis revealed accumulation of abnormal high-electron-density vesicles in elongating epidermal cells. The abnormal vesicles were often associated or interconnected with TGN/EEs and contained ADP-ribosylation factor 1, which is usually localized to the Golgi and the TGN/EEs. On the other hand, structures of the ER, Golgi apparatus, and MVB/LEs were apparently normal in uge4 cells. Together, our data indicate the importance of UDP-d-galactose synthesis by UGE4 for the organization and function of endomembranes, especially TGN/EEs, which are a sorting station of the secretory and vacuolar pathways.     

Characterization of aspartate kinase and homoserine dehydrogenase from <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> IWJ001 and systematic investigation of <Emphasis Type="SmallCaps">l</Emphasis>-isoleucine biosynthesis

Previously we have characterized a threonine dehydratase mutant TD^F383V (encoded by ilvA1) and an acetohydroxy acid synthase mutant AHAS^{P176S, D426E, L575W} (encoded by ilvBN1) in Corynebacterium glutamicum IWJ001, one of the best l-isoleucine producing strains. Here, we further characterized an aspartate kinase mutant AK^A279T (encoded by lysC1) and a homoserine dehydrogenase mutant HD^G378S (encoded by hom1) in IWJ001, and analyzed the consequences of all these mutant enzymes on amino acids production in the wild type background. In vitro enzyme tests confirmed that AK^A279T is completely resistant to feed-back inhibition by l-threonine and l-lysine, and that HD^G378S is partially resistant to l-threonine with the half maximal inhibitory concentration between 12 and 14 mM. In C. glutamicum ATCC13869, expressing lysC1 alone led to exclusive l-lysine accumulation, co-expressing hom1 and thrB1 with lysC1 shifted partial carbon flux from l-lysine (decreased by 50.1 %) to l-threonine (4.85 g/L) with minor l-isoleucine and no l-homoserine accumulation, further co-expressing ilvA1 completely depleted l-threonine and strongly shifted carbon flux from l-lysine (decreased by 83.0 %) to l-isoleucine (3.53 g/L). The results demonstrated the strongly feed-back resistant TD^F383V might be the main driving force for l-isoleucine over-synthesis in this case, and the partially feed-back resistant HD^G378S might prevent the accumulation of toxic intermediates. Information exploited from such mutation-bred production strain would be useful for metabolic engineering.     

Effect of Active Site Pocket Structure Modification of <Emphasis Type="SmallCaps">d</Emphasis>-Stereospecific Amidohydrolase on the Recognition of Stereospecific and Hydrophobic Substrates

d-Stereospecific amidohydrolase (DAH) from Streptomyces sp. 82F2 has potential utility for the synthesis of d/l configuration dipeptides by an aminolysis reaction. Structural comparison of DAH with substrate-bound d-amino acid amidase revealed that three residues located in the active site pocket of DAH (Thr145, Ala267, and Gly271) might be involved in interactions with d-phenylalanine substrate. We substituted Ala267 and Gly271, which are located at the bottom of the hydrophobic pocket of DAH, with Phe and observed changes in the stereoselectivity and specific activity toward the free and acetylated forms of d/l-Phe-methyl esters. In contrast, the mutation of Thr145, which likely supplies negative charge for recognition of the amino group of the substrate, hardly affected the stereoselectivity of the enzyme. A similar effect was observed in an investigation of hydrolysis and aminolysis reactions using the acetylated forms of d/l-Phe-methyl esters and 1,8-diaminooctane as an acyl-donor and acyl-acceptor, respectively. Substrate binding by DAH was disrupted by the mutation of Ala267 to Val or Trp and kinetic analysis showed that the hydrophobicity of the bottom of the active site pocket (Ala267 and Gly271) is important for both stereoselectivity and recognition of hydrophobic substrates.     

Selenite and ebselen supplementation attenuates <Emphasis Type="SmallCaps">d</Emphasis>-galactose-induced oxidative stress and increases expression of SELR and SEP15 in rat lens

Selenite and ebselen supplementation has been shown to possess anti-cataract potential in some experimental animal models of cataract, however, the underlying mechanisms remain unclear. The present study was designed to evaluate the anti-cataract effects and the underlying mechanisms of selenite and ebselen supplementation on galactose induced cataract in rats, a common animal model of sugar cataract. Transmission electron microscopy images of lens fiber cells (LFC) and lens epithelial cells (LEC) were observed in d-galactose-induced experimental cataractous rats treated with or without selenite and ebselen, also redox homeostasis and expression of proteins such as selenoprotein R (SELR), 15kD selenoprotein (SEP15), superoxide dismutase 1 (SOD1), catalase (CAT), β-crystallin protein, aldose reductase (AR) and glucose-regulated protein 78 (GRP78) were estimated in the lenses. The results showed that d-galactose injection injured rat lens and resulted in cataract formation; however, selenite and ebselen supplementation markedly alleviated ultrastructural injury of LFC and LEC. Moreover, selenite and ebselen supplementation could mitigate the oxidative damage in rat lens and increase the protein expressions of SELR, SEP15, SOD1, CAT and β-crystallin, as well as decrease the protein expressions of AR and GRP78. Taken together, these findings for the first time reveal the anti-cataract potential of selenite and ebselen in galactosemic cataract, and provide important new insights into the anti-cataract mechanisms of selenite and ebselen in sugar cataract.     

Selenium nanoparticles-loaded chitosan/citrate complex and its protection against oxidative stress in <Emphasis Type="SmallCaps">d</Emphasis>-galactose-induced aging mice

Background

Selenium (Se) is an indispensable trace element required for animals and humans, and extra Se-supplement is necessary, especially for those having Se deficiency. Recently, selenium nanoparticles (SeNPs), as a special form of Se supplement, have attracted worldwide attention due to their distinguished properties and excellent bioactivities. In this present study, an eco-friendly and economic way to prepare stable SeNPs was introduced. SeNPs were synthesized in the presence of chitosan (CTS) and then embedded into chitosan/citrate gel, generating selenium nanoparticles-loaded chitosan/citrate complex (SeNPs-C/C). Additionally, the clinical potential of SeNPs-C/C was evaluated by using d-galactose (d-gal)-induced aging mice model.

Results

SeNPs in high uniform with an average diameter of around 50 nm were synthesized in the presence of chitosan, and reversible ionic gelation between chitosan and citrate was utilized to load SeNPs. Subsphaeroidal SeNPs-C/C microspheres of 1–30 μm were obtained by spay-drying. Single SeNPs were physically separated and embedded inside SeNPs-C/C microparticles, with excellent stability and acceptable release. Acute fetal test showed SeNPs-C/C was safer than selenite, with a median lethal dose (LD₅₀) of approximately 4-fold to 11-fold of that of selenite. Oral administration of SeNPs-C/C remarkably retarded the oxidative stress of d-gal in Kunming mice by enhancing the activity of antioxidase, as evidenced by its significant protection of the growth, liver, Se retention and antioxidant bio-markers of mice against d-gal.

Conclusions

The design of SeNPs-C/C opens a new path for oral delivery of SeNPs with excellent stability, energy-conservation and environment-friendliness. SeNPs-C/C, as a novel supplement of Se, could be further developed to defend the aging process induced by d-gal.

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Discovery of a RuBisCO-like Protein that Functions as an Oxygenase in the Novel <Emphasis Type="SmallCaps">d</Emphasis>-Hamamelose Pathway

Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is the key catalyst of CO₂ fixation in nature. RuBisCO forms I, II, and III catalyze CO₂ fixation reactions, whereas form IV, also called the RuBisCO-like protein (RLP), is known to have no carboxylase or oxygenase activities. Here, we describe an RLP in Ochrobactrum anthropi ATCC 49188 (Oant_3067; HamA) that functions as an oxygenase in the metabolism of d-hamamelose, a branched-chain hexose found in most higher plants. The d-hamamelose pathway is comprised of five previously unknown enzymes: d-hamamelose dehydrogenase, d-hamamelono-lactonase, d-hamamelonate kinase, d-hamamelonate-2′,5-bisphosphate dehydrogenase (decarboxylating), and the RLP 3-keto-d-ribitol-1,5-bisphosphate (KRBP) oxygenase, which converts KRBP to 3-d-phosphoglycerate and phosphoglycolate. HamA represents the first RLP catalyzing the O₂-dependent oxidative C–C bond cleavage reaction, and our findings may provide insights into its applications in oxidative cleavage of organic molecules.