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.     

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.     

Comparison of N-Methyl-d-aspartate Receptor Subunit 1 and 4-Hydroxynonenal in the Hippocampus of Natural and Chemical-Induced Aging Accelerated Mice

In this study, we compared N-methyl-d-aspartate receptor type 1 (NMDAR1) and 4-hydroxynonenal (4-HNE) in the hippocampus of d-galactose (d-gal)-induced and naturally aging models of mice. These markers represent general phenotypes in aging, and they allowed us to examine the possibility of d-gal as a chemical model agent for aging. We observed an age-dependent reduction of NMDAR1 and an increase in 4-HNE in the dentate gyrus, CA1, and CA3 regions of the hippocampus via immunohistochemistry and western blot analyses. In the d-gal-induced chemical aging model, we observed similar changes in NMDAR1 and 4-HNE although the degree of reduction/increase in NMDAR1/4-HNE was not as severe as that in the naturally aged mice. These results suggest that the d-gal-induced aging model is comparable to naturally aged mice and may be useful for studies of the aging hippocampus.     

Puerarin and Amlodipine Improvement of d-Galactose-Induced Impairments of Behaviour and Neurogenesis in Mouse Dentate Gyrus: Correlation with Glucocorticoid Receptor Expression

Glucocorticoid receptors (GRs) exert actions on the hippocampus that are important for memory formation. There are correlations between vascular dysfunctions and GR-related gene expression. Both vascular dysfunction and GR gene expression decline occur during the ageing process. Therefore, hypotensors, which have effects on improving vascular dysfunction, may be able to ameliorate GR gene expression decline in ageing mice and improve ageing-mediated memory deficits. In this study, we hypothesized that hypotensors could alleviate the decline of GR gene expression and ameliorate age-induced learning and memory deficits in a d-gal-induced ageing mice model. In line with our hypothesis, we found that chronic d-gal treatment decreased GR and DCX expression in the hippocampus, leading to learning and memory deficits. Amlodipine (AM) and puerarin (PU) treatment improved GR gene expression decline in the hippocampus and ameliorated the learning and memory deficits of d-gal-treated mice. These changes correlated with enhanced DCX expression and brain-derived neurotrophic factor (BDNF) expression in the hippocampus. Furthermore, PU treatment conveyed better effects than AM treatment, but combination therapy did not enhance the effects on improving GR expression. However, we did not find evidence of these changes in non-d-gal-treated mice that lacked GR gene expression decline. These results suggest that AM and PU could improve d-gal-induced behavioural deficits in correlation with GR gene expression.

     

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.

Open image in new window

     

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.     

Application and microbial preparation of <Emphasis Type="SmallCaps">d</Emphasis>-valine

d-Valine is an important organic chiral source and has extensive industrial application, which is used as intermediate for the synthesis of agricultural pesticides, semi-synthetic veterinary antibiotics and pharmaceutical drugs. Its derivatives have shown great activity in clinical use, such as penicillamine for the treatment of immune-deficiency diseases, and actinomycin D for antitumor therapy. Fluvalinate, a pyrethroid pesticide made from d-valine, is a broad-spectrum insecticide with low mammalian toxicity. Valnemulin, a semi-synthetic pleuromutilin derivative synthesized from d-valine, is an antibiotic for animals. Moreover, d-valine is also used in cell culture for selectively inhibiting fibroblasts proliferation. Due to its widespread application, d-valine is gaining more and more attention and some approaches for d-valine preparation have been investigated. In comparison with other approaches, microbial preparation of d-valine is more competitive and promising because of its high stereo selectivity, mild reaction conditions and environmental friendly process. So far, microbial preparation of d-valine can be mainly classified into three categories: microbial asymmetric degradation of dl-valine, microbial stereoselective hydrolysis of N-acyl-dl-valine by d-aminoacylase, and microbial specific hydrolysis of dl-5-isopropylhydantoin by d-hydantoinase coupled with d-carbamoylase. In this paper, the industrial application of d-valine and its microbial preparation are reviewed.     

Ectopic expression of sorbitol-6-phosphate 2-dehydrogenase gene from <Emphasis Type="Italic">Haloarcula marismortui</Emphasis> enhances salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

d-Sorbitol-6-phosphate 2-dehydrogenase (S6PDH, E.C. 1.1.1.140) catalyzes the NADH-dependent conversion of d-fructose 6-phosphate (F6P) to d-sorbitol 6-phosphate (S6P). In this work, recombination and characterization of Haloarcula marismortui d-sorbitol-6-phosphate 2-dehydrogenase are reported. Haloarcula marismortui d-sorbitol-6-phosphate 2-dehydrogenase was expressed in P. pastoris and Arabidopsis thaliana. Enzyme assay indicated that HmS6PDH catalyzes the reduction of d-fructose 6-phosphate to d-sorbitol 6-phosphate and HmS6PDH activity was enhanced by NaCl. Furthermore, transgenic A. thaliana ectopic expressing HmS6PDH accumulate more sorbitol under salt stress. These results suggest that the ectopic expression of HmS6PDH in plants can facilitate future studies regarding the engineering and breeding of salt-tolerant crops.     

Engineering an ATP-dependent <Emphasis Type="SmallCaps">d</Emphasis>-Ala:<Emphasis Type="SmallCaps">d</Emphasis>-Ala ligase for synthesizing amino acid amides from amino acids

We successfully engineered a new enzyme that catalyzes the formation of d-Ala amide (d-AlaNH₂) from d-Ala by modifying ATP-dependent d-Ala:d-Ala ligase (EC 6.3.2.4) from Thermus thermophilus, which catalyzes the formation of d-Ala-d-Ala from two molecules of d-Ala. The new enzyme was created by the replacement of the Ser293 residue with acidic amino acids, as it was speculated to bind to the second d-Ala of d-Ala-d-Ala. In addition, a replacement of the position with Glu performed better than that with Asp with regards to specificity for d-AlaNH₂ production. The S293E variant, which was selected as the best enzyme for d-AlaNH₂ production, exhibited an optimal activity at pH 9.0 and 40 °C for d-AlaNH₂ production. The apparent K _m values of this variant for d-Ala and NH₃ were 7.35 mM and 1.58 M, respectively. The S293E variant could catalyze the synthesis of 9.3 and 35.7 mM of d-AlaNH₂ from 10 and 50 mM d-Ala and 3 M NH₄Cl with conversion yields of 93 and 71.4 %, respectively. This is the first report showing the enzymatic formation of amino acid amides from amino acids.     

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.     

Metabolomics of groundnut (<Emphasis Type="Italic">Arachis hypogaea</Emphasis> L.) genotypes under varying temperature regimes

Various metabolites were analyzed in groundnut genotypes grown under varying temperature regimes (based on date of sowing). Four contrasting groundnut genotypes viz. ICGS44 (high-temperature tolerant), AK159 and GG7 (moderately-high-temperature tolerant), and DRG1 (high-temperature sensitive) were grown at three different temperature regimes i.e., low (early date of sowing), normal (normal date of sowing) and high temperature (late date of sowing) under field conditions. Untargeted metabolomic analysis of leaf tissue was performed by GC–MS, while targeted metabolite profiling was carried out by HPLC (polyamines) and UPLC-MS/MS (phenolics) at both the pegging and pod filling stages. Untargeted metabolomic profiling revealed exclusive expression/induction of beta-d-galactofuranoside, l-threonine, hexopyranose, d-glucopyranose, stearic acid, 4-ketoglucose, d-gulose, 2-o-glycerol-alpha-d-galactopyranoside and serine in ICGS44 during the pegging stage under high-temperature conditions. During the pod filling stage at higher temperature, alpha-d-galactoside, dodecanedioic acid, 1-nonadecene, 1-tetradecene and beta-d-galactofuranose were found to be higher in both ICGS44 and GG7. Moreover, almost all the metabolites detected by GC–MS were found to be higher in GG7, except beta-d-galactopyranoside, beta-d-glucopyranose, inositol and palmitic acid. Accumulation of putrescine was observed to be higher during low-temperature stress, while agmatine showed constitutive expression in all the genotypes, irrespective of temperature regime and crop growth stage. Interestingly, spermidine was observed only in the high-temperature tolerant genotype ICGS44. In our study, we found a higher accumulation of cinnamic acid, caffeic acid, salicylic acid and vanillic acid in ICGS44 compared to that of other genotypes at the pegging stage, whereas catechin and epicatechin were found during the pod filling stage in response to high-temperature stress, suggesting their probable roles in heat-stress tolerance in groundnut.     

Stereoisomeric Prodrugs to Improve Corneal Absorption of Prednisolone: Synthesis and <Emphasis Type="Italic">In Vitro</Emphasis> Evaluation

A series of stereoisomeric prodrugs have been designed to examine efficacy in generating higher corneal absorption relative to prednisolone. Prodrugs have been studied and identified with LC/MS/MS and NMR analyses. Prodrugs have been characterized for aqueous solubility, buffer stability, and cytotoxicity. Cellular uptake and permeability studies have been conducted across MDCK-MDR1 cells to determine prodrug affinity towards P-glycoprotein (P-gp) and peptide transporters. Enzyme-mediated degradation of prodrugs has been determined using Statens Seruminstitut rabbit cornea (SIRC) cell homogenates. Prodrugs exhibited higher aqueous solubility relative to prednisolone. Prodrugs circumvented P-gp-mediated cellular efflux and were recognized by peptide transporters. Prodrugs (DP, DDP) produced with d-isomers (d-valine) were significantly stable against both chemical and enzymatic hydrolyses. The order of degradation rate constants observed in chemical and enzymatic hydrolyses were in the same order, i.e., l-valine-l-valine-prednisolone (LLP)?>?l-valine-d-valine-prednisolone (LDP)?>?d-valine-l-valine-prednisolone (DLP)?>?d-valine-d-valine-prednisolone (DDP). Results obtained from this study clearly suggest that stereoisomeric prodrug approach is an effective strategy to overcome P-gp-mediated efflux and improve transcorneal permeability of prednisolone following topical administration.     

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.     

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.     

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.     

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.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-alanine from <Emphasis Type="SmallCaps">dl</Emphasis>-alanine using immobilized cells of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> HLZ-68

Immobilized cells of Bacillus subtilis HLZ-68 were used to produce d-alanine from dl-alanine by asymmetric degradation. Different compounds such as polyvinyl alcohol and calcium alginate were employed for immobilizing the B. subtilis HLZ-68 cells, and the results showed that cells immobilized using a mixture of these two compounds presented higher l-alanine degradation activity, when compared with free cells. Subsequently, the effects of different concentrations of polyvinyl alcohol and calcium alginate on l-alanine consumption were examined. Maximum l-alanine degradation was exhibited by cells immobilized with 8% (w/v) polyvinyl alcohol and 2% (w/v) calcium alginate. Addition of 400 g of dl-alanine (200 g at the beginning of the reaction and 200 g after 30 h of incubation) into the reaction solution at 30 °C, pH 6.0, aeration of 1.0 vvm, and agitation of 400 rpm resulted in complete l-alanine degradation within 60 h, leaving 185 g of d-alanine in the reaction solution. The immobilized cells were applied for more than 15 cycles of degradation and a maximum utilization rate was achieved at the third cycle. d-alanine was easily extracted from the reaction solution using cation-exchange resin, and the chemical and optical purity of the extracted d-alanine was 99.1 and 99.6%, respectively.     

Change of the <Emphasis Type="Italic">N</Emphasis>-terminal codon bias combined with tRNA supplementation outperforms the selected fusion tags for production of human <Emphasis Type="SmallCaps">d</Emphasis>-amino acid oxidase as active inclusion bodies

Objectives

To optimize the production of active inclusion bodies (IBs) containing human d-amino acid oxidase (hDAAO) in Escherichia coli.

Results

The optimized initial codon region combined with the coexpressed rare tRNAs, fusion of each of the N-terminal partners including cellulose-binding module, thioredoxin, glutathione S-transferase and expressivity tag, deletion of the incorporated linker, and improvement of tRNA abundance affected the production and activity for oxidizing d-alanine of the hDAAO in IBs. Compared with the optimized fusion constructs and expression host, IBs yields and activity were increased to 2.6- and 2.8-fold respectively by changing the N-terminal codon bias of the hDAAO. The insoluble hDAAO codon variant displayed the same substrate specificity as the soluble one for oxidizing d-alanine, d-serine and d-aspartic acid. The freshly prepared hDAAO codon variant was used for analyzing the l-serine racemization activity of the bacterially expressed maize serine racemase.

Conclusions

Optimization of the N-terminal codon bias combined with the coexpression of rare tRNAs is a novel and efficient approach to produce active IBs of the hDAAO.

     

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.     

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.