Safety of long-term dietary supplementation with <Emphasis Type="SmallCaps">l</Emphasis>-arginine in rats

This study was conducted with rats to determine the safety of long-term dietary supplementation with l-arginine. Beginning at 6 weeks of age, male and female rats were fed a casein-based semi-purified diet containing 0.61 % l-arginine and received drinking water containing l-arginine-HCl (0, 1.8, or 3.6 g l-arginine/kg body-weight/day; n = 10/group). These supplemental doses of l-arginine were equivalent to 0, 286, and 573 mg l-arginine/kg body-weight/day, respectively, in humans. After a 13-week supplementation period, blood samples were obtained from rats for biochemical analyses. Supplementation with l-arginine increased plasma concentrations of arginine, ornithine, proline, homoarginine, urea, and nitric oxide metabolites without affecting those for lysine, histidine, or methylarginines, while reducing plasma concentrations of ammonia, glutamine, free fatty acids, and triglycerides. l-Arginine supplementation enhanced protein gain and reduced white-fat deposition in the body. Based on general appearance, feeding behavior, and physiological parameters, all animals showed good health during the entire experimental period; Plasma concentrations of all measured hormones (except leptin) did not differ between control and arginine-supplemented rats. l-Arginine supplementation reduced plasma levels of leptin. Additionally, l-arginine supplementation increased l-arginine:glycine amidinotransferase activity in kidneys but not in the liver or small intestine, suggesting tissue-specific regulation of enzyme expression by l-arginine. Collectively, these results indicate that dietary supplementation with l-arginine (e.g., 3.6 g/kg body-weight/day) is safe in rats for at least 91 days. This dose is equivalent to 40 g l-arginine/kg body-weight/day for a 70-kg person. Our findings help guide clinical studies to determine the safety of long-term oral administration of l-arginine to humans.     

<Emphasis Type="SmallCaps">l</Emphasis>-Serine overproduction with minimization of by-product synthesis by engineered <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

The direct fermentative production of l-serine by Corynebacterium glutamicum from sugars is attractive. However, superfluous by-product accumulation and low l-serine productivity limit its industrial production on large scale. This study aimed to investigate metabolic and bioprocess engineering strategies towards eliminating by-products as well as increasing l-serine productivity. Deletion of alaT and avtA encoding the transaminases and introduction of an attenuated mutant of acetohydroxyacid synthase (AHAS) increased both l-serine production level (26.23 g/L) and its productivity (0.27 g/L/h). Compared to the parent strain, the by-products l-alanine and l-valine accumulation in the resulting strain were reduced by 87 % (from 9.80 to 1.23 g/L) and 60 % (from 6.54 to 2.63 g/L), respectively. The modification decreased the metabolic flow towards the branched-chain amino acids (BCAAs) and induced to shift it towards l-serine production. Meanwhile, it was found that corn steep liquor (CSL) could stimulate cell growth and increase sucrose consumption rate as well as l-serine productivity. With addition of 2 g/L CSL, the resulting strain showed a significant improvement in the sucrose consumption rate (72 %) and the l-serine productivity (67 %). In fed-batch fermentation, 42.62 g/L of l-serine accumulation was achieved with a productivity of 0.44 g/L/h and yield of 0.21 g/g sucrose, which was the highest production of l-serine from sugars to date. The results demonstrated that combined metabolic and bioprocess engineering strategies could minimize by-product accumulation and improve l-serine productivity.     

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.

     

Genome shuffling and high-throughput screening of <Emphasis Type="Italic">Brevibacterium flavum</Emphasis> MDV1 for enhanced <Emphasis Type="SmallCaps">l</Emphasis>-valine production

l-valine is an essential branched-amino acid that is widely used in multiple areas such as pharmaceuticals and special dietary products and its use is increasing. As the world market for l-valine grows rapidly, there is an increasing interest to develop an efficient l-valine-producing strain. In this study, a simple, sensitive, efficient, and consistent screening procedure termed 96 well plate-PC-HPLC (96-PH) was developed for the rapid identification of high-yield l-valine strains to replace the traditional l-valine assay. l-valine production by Brevibacterium flavum MDV1 was increased by genome shuffling. The starting strains were obtained using ultraviolet (UV) irradiation and binary ethylenimine treatment followed by preparation of protoplasts, UV irradiation inactivation, multi-cell fusion, and fusion of the inactivated protoplasts to produce positive colonies. After two rounds of genome shuffling and the 96-PH method, six l-valine high-yielding mutants were selected. One genetically stable mutant (MDVR2-21) showed an l-valine yield of 30.1 g/L during shake flask fermentation, 6.8-fold higher than that of MDV1. Under fed-batch conditions in a 30 L automated fermentor, MDVR2-21 accumulated 70.1 g/L of l-valine (0.598 mol l-valine per mole of glucose; 38.9% glucose conversion rate). During large-scale fermentation using a 120 m³ fermentor, this strain produced?>?66.8 g/L l-valine (36.5% glucose conversion rate), reflecting a very productive and stable industrial enrichment fermentation effect. Genome shuffling is an efficient technique to improve production of l-valine by B. flavum MDV1. Screening using 96-PH is very economical, rapid, efficient, and well-suited for high-throughput screening.     

Enhanced biocatalytic production of <Emphasis Type="SmallCaps">l</Emphasis>-cysteine by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. B-3 with in situ product removal using ion-exchange resin

Bioconversion of dl-2-amino-Δ²-thiazoline-4-carboxylic acid (dl-ATC) catalyzed by whole cells of Pseudomonas sp. was successfully applied for the production of l-cysteine. It was found, however, like most whole-cell biocatalytic processes, the accumulated l-cysteine produced obvious inhibition to the activity of biocatalyst and reduced the yield. To improve l-cysteine productivity, an anion exchange-based in situ product removal (ISPR) approach was developed. Several anion-exchange resins were tested to select a suitable adsorbent used in the bioconversion of dl-ATC for the in situ removal of l-cysteine. The strong basic anion-exchange resin 201 × 7 exhibited the highest adsorption capacity for l-cysteine and low adsorption for dl-ATC, which is a favorable option. With in situ addition of 60 g L^?1 resin 201 × 7, the product inhibition can be reduced significantly and 200 mmol L^?1 of dl-ATC was converted to l-cysteine with 90.4 % of yield and 28.6 mmol L^?1 h^?1 of volumetric productivity. Compared to the bioconversion without the addition of resin, the volumetric productivity of l-cysteine was improved by 2.27-fold using ISPR method.     

Investigations on in vitro anti-carcinogenic potential of <Emphasis Type="SmallCaps">l</Emphasis>-carnosine in liver cancer cells

This study was carried out to investigate the anti-carcinogenic effect of l-carnosine in human carcinoma cells (SNU-423). The SNU-423 cancer cells were cultured at a density of 2 × 10⁴ cells/well in Dulbecco modified Eagle medium. After 24 h of adherence, the cells were treated with l-carnosine (0.2 and 1 mg/mL) for 48 h. Then, cell viability was assessed by sulforhodamine assay, while mitochondrial dysfunction was measured by fluorescence microscopy using chromatin-specific dye Hoechst 33258. Intracellular levels of ROS were assayed by fluorescence spectroscopy with 2′,7′-dichlorofluorescein diacetate (DCFDA). l-Carnosine significantly inhibited the growth of the SNU-423 cells (p < 0.05). The inhibitory effect of l-carnosine was confirmed by results from mitochondrial fragmentation assay. The relative fluorescent unit was increased in a dose-dependent manner by l-carnosine, with values of 79.43, 186.87 and 400.89 for 0.6, 0.8 and 1 mg/mL of l-carnosine, respectively (p < 0.05). These results demonstrate that l-carnosine exerts anti-carcinogenic effects in human liver cancer cells.     

Interactions of amino acids with aluminum octacarboxyphthalocyanine hydroxide. Experimental and DFT studies

The influence of albumin and amino acids (l-serine, glycine, l-histidine, l-tryptophan, l-cysteine) on the properties of aluminum octacarboxyphthalocyanine hydroxide (Al(OH)PcOC) was investigated in a phosphate buffer (pH 8.0). Particular attention was paid to the spectroscopic properties and photostability of Al(OH)PcOC. The effect of albumin or amino acids on the photodegradation of Al(OH)PcOC was examined in water using red light: 685 nm and daylight irradiation. Analysis of kinetic curves indicated that interaction with those molecules increases the photostability of Al(OH)PcOC. The molecular structure of Al(OH)PcOC complexes (in vacuum and in water) with axially or equatorially coordinated amino acids was studied by the B3LYP/6-31G* method, and the effects on molecular structure and electronic absorption spectrum were investigated on the basis of the density functional theory. The calculation results revealed that axial coordination significantly reduces the non-planarity of the phthalocyanine ring, and, thus, alters the electronic structure. On the other hand, hydrogen bonding of phthalocyanine side COOH groups with amino acids, in equatorial complexes, does not change the structure within the center of the phthalocyanine, and causes only a slight increase in UV–vis bands intensity, which is in perfect agreement with experimental data.

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Graphical abstract Structure of equatorial complex of Al(OH)PcOC with l-histidine calculated byB3LYP/6-31G(d) method. Dotted lines H-bonds

     

Isolation and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-glutamate oxidase with strict substrate specificity from <Emphasis Type="Italic">Streptomyces diastatochromogenes</Emphasis>

Objectives

To find an l-glutamate oxidase (LGox), to be used for the quantitative analysis of l-glutamic acid, an lgox gene encoding LGox from Streptomyces diastatochromogenes was isolated, cloned and characterized.

Results

The gene had an ORF of 1974 bp encoding a protein of 657 amino acid residues. In comparison to the LGox precursor, the proteinase K-treated enzyme exhibited improved affinity to substrate and with a K _m of 0.15 mM and V _max of 62 μmol min^?1 mg^?1. The 50% thermal inactivation temperature of the proteinase K treated enzyme was increased from 50 to 70 °C. The enzyme exhibited strict specificity for l-glutamate.

Conclusions

LGox treated by proteinase K exhibited strict specificity for l-glutamate, good thermostability and high substrate affinity.

     

Functional expression of a human GDP-<Emphasis Type="SmallCaps">l</Emphasis>-fucose transporter in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Objectives

To investigate the translocation of nucleotide-activated sugars from the cytosol across a membrane into the endoplasmatic reticulum or the Golgi apparatus which is an important step in the synthesis of glycoproteins and glycolipids in eukaryotes.

Results

The heterologous expression of the recombinant and codon-adapted human GDP-l-fucose antiporter gene SLC35C1 (encoding an N-terminal OmpA-signal sequence) led to a functional transporter protein located in the cytoplasmic membrane of Escherichia coli. The in vitro transport was investigated using inverted membrane vesicles. SLC35C1 is an antiporter specific for GDP-l-fucose and depending on the concomitant reverse transport of GMP. The recombinant transporter FucT1 exhibited an activity for the transport of ³H-GDP-l-fucose with a V_max of 8 pmol/min mg with a K_m of 4 µM. The functional expression of SLC35C1 in GDP-l-fucose overproducing E. coli led to the export of GDP-l-fucose to the culture supernatant.

Conclusions

The export of GDP-l-fucose by E. coli provides the opportunity for the engineering of a periplasmatic fucosylation reaction in recombinant bacterial cells.

     

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.     

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.     

An optimized fed-batch culture strategy integrated with a one-step fermentation improves <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid production by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

In previous work, we proposed a novel modified one-step fermentation fed-batch strategy to efficiently generate l-lactic acid (l-LA) using Rhizopus oryzae. In this study, to further enhance efficiency of l-LA production through one-step fermentation in fed-batch cultures, we systematically investigated the initial peptone- and glucose-feeding approaches, including different initial peptone and glucose concentrations and maintained residual glucose levels. Based on the results of this study, culturing R. oryzae with initial peptone and glucose concentrations of 3.0 and 50.0 g/l, respectively, using a fed-batch strategy is an effective approach of producing l-LA through one-step fermentation. Changing the residual glucose had no obvious effect on the generation of l-LA. We determined the maximum LA production and productivity to be 162 g/l and 6.23 g/(l·h), respectively, during the acid production stage. Compared to our previous work, there was almost no change in l-LA production or yield; however, the productivity of l-LA increased by 14.3%.     

The decreased expression of Stat3 and p-Stat3 in preeclampsia-like rat placenta

This study aims to investigate the expression of Stat3 and p-Stat3 in the placenta of a preeclampsia-like rat model induced by Nωnitro-l-arginine methyl ester (l-NAME). Two-to three-month-old (20 males, 40 females) Sprague–Dawley rats were used in this study. After conception was confirmed by vaginal smears, on the thirteenth day of pregnancy, the animals were allocated into two groups: control (0.9% NaCl administered) group and l-NAME (75 mg/kg) group. After the treatment of l-NAME, there was a significant increase in systolic blood pressure (SBP) levels in the l-NAME group (148.5?±?5.71 mmHg) on day 21 compared to the SBPs in the control group (117.5?±?4.57 mmHg) (P?<?0.001). There was also an increase in total proteinuria on day 21 in the l-NAME group (766.57?±?17.7 mg/L), when compared to the control group (459.89?±?20.1 mg/L) (P?<?0.001). Moreover, we also found a decrease in fetal numbers and fetal weight in the PE group in comparison to the control group. After the rats were sacrificed, the placentas were obtained from both groups. We found that the l-NAME group exhibited fewer placentas compared with the control group. Furthermore, the immunohistochemistry (IHC) and Western blot results showed that decreased expression of Stat3 and p-Stat3 were detected in the placenta of the preeclampsia-like rat model compared to Stat3 and p-Stat3 in the control group. We found the expression and activation of Stat3 and p-Stat3 were decreased in the placenta of the l-NAME-induced preeclampsia rat model.     

Engineering rTCA pathway and C4-dicarboxylate transporter for <Emphasis Type="SmallCaps">l</Emphasis>-malic acid production

l-Malic acid is an important component of a vast array of food additives, antioxidants, disincrustants, pharmaceuticals, and cosmetics. Here, we presented a pathway optimization strategy and a transporter modification approach to reconstruct the l-malic acid biosynthesis pathway and transport system, respectively. First, pyruvate carboxylase (pyc) and malate dehydrogenase (mdh) from Aspergillus flavus and Rhizopus oryzae were combinatorially overexpressed to construct the reductive tricarboxylic acid (rTCA) pathway for l-malic acid biosynthesis. Second, the l-malic acid transporter (Spmae) from Schizosaccharomyces pombe was engineered by removing the ubiquitination motification to enhance the l-malic acid efflux system. Finally, the l-malic acid pathway was optimized by controlling gene expression levels, and the final l-malic acid concentration, yield, and productivity were up to 30.25 g L^?1, 0.30 g g^?1, and 0.32 g L^?1 h^?1 in the resulting strain W4209 with CaCO₃ as a neutralizing agent, respectively. In addition, these corresponding parameters of pyruvic acid remained at 30.75 g L^?1, 0.31 g g^?1, and 0.32 g L^?1 h^?1, respectively. The metabolic engineering strategy used here will be useful for efficient production of l-malic acid and other chemicals.     

Increased production of plumbagin in <Emphasis Type="Italic">Plumbago indica</Emphasis> root cultures by biotic and abiotic elicitors

Objectives

To evaluate the effects of 12 biotic and abiotic elicitors for increasing the production of plumbagin in Plumbago indica root cultures.

Results

Most elicitors showed minimal effects on the root dry weight, except for 250 mg chitosan l^?1 and 10 mM l-alanine that markedly decreased root biomass by about 40 % compared to the untreated root cultures (5 g l^?1). Treatments with 100 µM AgNO₃ significantly increased intracellular plumbagin production by up to 7.6 mg g^?1 DW that was 4-fold more than the untreated root cultures (1.9 mg g^?1 DW). In contrast, treatments with 150 mg chitosan l^?1, 5 mM l-alanine, and 50 µM 1-naphthol significantly enhanced the extracellular secretion of plumbagin by up to 10.6, 6.9, and 5.7 mg g^?1 DW, respectively, and increased the overall production of plumbagin by up to 12.5, 12.5, and 9.4 mg g^?1 DW, respectively.

Conclusions

Chitosan (150 mg l^?1), l-alanine (5 mM), and 1-naphthol (50 µM) were the best elicitors to enhance plumbagin production in P. indica root cultures.

     

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.     

Photobiomodulation Therapy Improves Acute Inflammatory Response in Mice: the Role of Cannabinoid Receptors/ATP-Sensitive K<Superscript>+</Superscript> Channel/p38-MAPK Signalling Pathway

Although photobiomodulation therapy (PBM) has been applied clinically for the treatment of pain and inflammation, wound healing, sports and soft tissue injuries, as well as to repair injured spinal cords and peripheral nerves, it remains unclear which molecular substrates (receptor) are implicated in the cellular mechanisms of PBM. Here, we reported that PBM (660 nm, 30 mW, 0.06 cm², 50 J/cm², plantar irradiation) significantly inhibited carrageenan-induced paw oedema, but not noxious thermal response, through positive modulation to both CB1 and CB2 cannabinoid receptors. The use of CB1 antagonist AM281 or CB2 antagonist AM630 significantly reversed the anti-inflammatory effect of PBM. Analysis of signalling pathway downstream of cannabinoid receptors activation reveals that anti-inflammatory effects of PBM depend, in great extent, on its ability to activate ATP-dependent K⁺ channels and p38 mitogen-activated protein kinase. Moreover, PBM therapy significantly reduced the levels of pro-inflammatory cytokine IL-6 in both paw and spinal cord, and restored the reduction of the level of anti-inflammatory cytokine IL-10 in spinal cord after carrageenan injection. Unlike the potent cannabinoid receptor agonist (WIN 55212-2), PBM did not exert any CNS-mediated effects in the tetrad assay. Finally, PBM does not reduce inflammation and noxious thermal response induced by LPS and zymosan, a TLR4 and TLR2/dectin-1 ligand, respectively. Thus, cannabinoid receptors and, possibly, the endocannabinoid system, represent an important site of action of PBM that opens the possibility of complementary and nonpsychotropic therapeutic interventions in clinical practice.

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Graphical Abstract ?

     

Rational design and analysis of an <Emphasis Type="Italic">Escherichia coli</Emphasis> strain for high-efficiency tryptophan production

l-tryptophan (l-trp) is a precursor of various bioactive components and has great pharmaceutical interest. However, due to the requirement of several precursors and complex regulation of the pathways involved, the development of an efficient l-trp production strain is challenging. In this study, Escherichia coli (E. coli) strain KW001 was designed to overexpress the l-trp operator sequences (trpEDCBA) and 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase (aroG ^fbr ). To further improve the production of l-trp, pyruvate kinase (pykF) and the phosphotransferase system HPr (ptsH) were deleted after inactivation of repression (trpR) and attenuation (attenuator) to produce strain KW006. To overcome the relatively slow growth and to increase the transport rate of glucose, strain KW018 was generated by combinatorial regulation of glucokinase (galP) and galactose permease (glk) expression. To reduce the production of acetic acid, strain KW023 was created by repressive regulation of phosphate acetyltransferase (pta) expression. In conclusion, strain KW023 efficiently produced 39.7 g/L of l-trp with a conversion rate of 16.7% and a productivity of 1.6 g/L/h in a 5 L fed-batch fermentation system.     

The modulation of NMDA receptors and <Emphasis Type="SmallCaps">l</Emphasis>-arginine/nitric oxide pathway is implicated in the anti-immobility effect of creatine in the tail suspension test

The modulation of N-methyl-D-aspartate receptor (NMDAR) and l-arginine/nitric oxide (NO) pathway is a therapeutic strategy for treating depression and neurologic disorders that involves excitotoxicity. Literature data have reported that creatine exhibits antidepressant and neuroprotective effects, but the implication of NMDAR and l-arginine/nitric oxide (NO) pathway in these effects is not established. This study evaluated the influence of pharmacological agents that modulate NMDAR/l-arginine-NO pathway in the anti-immobility effect of creatine in the tail suspension test (TST) in mice. The NOx levels and cellular viability in hippocampal and cerebrocortical slices of creatine-treated mice were also evaluated. The anti-immobility effect of creatine (10 mg/kg, po) in the TST was abolished by NMDA (0.1 pmol/mouse, icv), d-serine (30 µg/mouse, icv, glycine-site NMDAR agonist), arcaine (1 mg/kg, ip, polyamine site NMDAR antagonist), l-arginine (750 mg/kg, ip, NO precursor), SNAP (25 μg/mouse, icv, NO donor), L-NAME (175 mg/kg, ip, non-selective NOS inhibitor) or 7-nitroindazole (50 mg/kg, ip, neuronal NOS inhibitor), but not by DNQX (2.5 µg/mouse, icv, AMPA receptor antagonist). The combined administration of sub-effective doses of creatine (0.01 mg/kg, po) and NMDAR antagonists MK-801 (0.001 mg/kg, po) or ketamine (0.1 mg/kg, ip) reduced immobility time in the TST. Creatine (10 mg/kg, po) increased cellular viability in hippocampal and cerebrocortical slices and enhanced hippocampal and cerebrocortical NO _x levels, an effect potentiated by l-arginine or SNAP and abolished by 7-nitroindazole or L-NAME. In conclusion, the anti-immobility effect of creatine in the TST involves NMDAR inhibition and enhancement of NO levels accompanied by an increase in neural viability.     

Production of tartaric acid using immobilized recominant <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase

Objective

To investigate the expression and immobilization of recombinant cis-epoxysuccinate hydrolase (ESH), and its application in the biological production of l-(+)-tartaric acid.

Results

E. coli BL21 (DE3)/pET11a-ESH (His) was engineered to express recombinant ESH. The enzyme had an activity of 262 U mg^?1. The recombinant ESH was immobilized on agarose Ni-IDA matrix with metal ion affinity interaction to improve its thermostability and pH stability. The immobilization efficiency and activity yield were 94 and 95%, respectively. The specific catalytic efficiency of immobilized ESH was 104 mg U^?1 h^?1 during the continuous enzymatic production process.

Conclusion

ESH with a histidine tag was immobilized and used for the continuous production of l-(+)-tartaric acid.