Development of a whole-cell biosensor for detecting organophosphorus pesticide methyl parathion in the farmland soil / 生物工程学报

The evaluation of the bioavailability of pollutants in soil is crucial to accurately assess the pollution risk, and whole-cell biosensor is one of the important tools for such evaluation. This study aimed to develop a novel whole-cell biosensor for the detection of methyl parathion in soil using. First, a whole-cell biosensor was constructed by the screened methyl parathion hydrolase mpd gene, the existing specific induction element pobR, and the pUC19 plasmid skeleton. Then, the detection method of methyl parathion in soil extracts was established using 96-well microtiter plate as carrier and five whole-cell biosensors as indicator. The method was applied in the detection of methyl parathion in tested and field soil extracts. Taking E. coli DH5α/pMP-AmilCP with the best detection performance as an example, this biosensor had a detection limit of 6.21-6.66 µg/L and a linear range of 10-10 000 µg/L for methyl parathion in four soil extracts. E. coli DH5α/pMP-RFP and E. coli DH5α/pMP-AmilCP methods have good detection performance for the analysis of methyl parathion in soil extract samples. This biosensor method can help to quickly assess the bioavailability of methyl parathion in soil, and thus help to understand the risk of soil pollution caused by organophosphorus pesticide methyl parathion.

Efficient biosynthesis of γ-aminobutyric acid by rationally engineering the catalytic pH range of a glutamate decarboxylase from Lactobacillus plantarum / 生物工程学报

γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGADS24R/D88R/Y309K was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and LpgadS24R/D88R/Y309K genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD600) 20, 100 g/L l-glutamic acid substrate and 100 μmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.

Opinión de un panel de expertos sobre la protección contra la tos ferina en México: el papel de las vacunas acelulares y de células enteras / An expert panel opinion on protection against pertussis in Mexico: The role of acellular and whole-cell vaccines

Resumen La tos ferina es una enfermedad causada por Bordetella pertussis. Aunque es altamente contagiosa, puede ser prevenible por vacunación. Existen dos tipos de vacunas: las de células enteras y las acelulares. La tos ferina ha resurgido en algunos países debido a que su control a escala mundial es heterogéneo. Esta reemergencia se ha relacionado con diversos factores: mayor sensibilidad hacia la infección, mejor detección de la enfermedad, problemas para obtener adecuadas coberturas de vacunación, incremento en los sujetos susceptibles (especialmente menores de 6 meses), pérdida de la inmunidad en los adolescentes y adultos jóvenes, y probables cambios genéticos y adaptativos de B. pertussis. En este documento se analizan las características, las ventajas y las desventajas de las vacunas de células enteras y de las vacunas acelulares. Se presentan las recomendaciones internacionales y se ofrece el posicionamiento de los participantes con respecto a la influencia del uso de vacunas acelulares y las desventajas potenciales de volver a utilizar vacunas de células enteras, en especial por su reactogenicidad. Por último, se analizan las estrategias para lograr un mejor control de la tos ferina en México.

Abstract Pertussis is a highly contagious disease caused by Bordetella pertussis, which may be preventable by vaccination. There are two types of vaccines: whole-cell vaccines and acellular vaccines. Since pertussis control worldwide is heterogeneous, re-emergence of whooping cough has been observed in some countries. This re-emergence has been related to several factors: increased susceptibility to infection, better detection of disease, problems in obtaining adequate vaccination coverage, increase in susceptible subjects (mainly under 6 months of age), loss of immunity in adolescents and young adults, and likely genetic and adaptive B. pertussis changes. This paper discusses whole-cell and acellular vaccines' characteristics, advantages, and disadvantages. International recommendations are presented, and the participants' position is offered regarding the influence of the use of acellular vaccines and the potential disadvantages of reintroducing whole-cell vaccines, mainly due to their reactogenicity. Finally, strategies to achieve better control of pertussis in Mexico are discussed.

Effect of Sodium Salicylate on Calcium Currents and Exocytosis in Cochlear Inner Hair Cells: Implications for Tinnitus Generation / 神经科学通报·英文版

Sodium salicylate is an anti-inflammatory medication with a side-effect of tinnitus. Here, we used mouse cochlear cultures to explore the effects of salicylate treatment on cochlear inner hair cells (IHCs). We found that IHCs showed significant damage after exposure to a high concentration of salicylate. Whole-cell patch clamp recordings showed that 1-5 mmol/L salicylate did not affect the exocytosis of IHCs, indicating that IHCs are not involved in tinnitus generation by enhancing their neuronal input. Instead, salicylate induced a larger peak amplitude, a more negative half-activation voltage, and a steeper slope factor of Ca2+ current. Using noise analysis of Ca2+ tail currents and qRT-PCR, we further found that salicylate increased the number of Ca2+ channels along with CaV1.3 expression. All these changes could act synergistically to enhance the Ca2+ influx into IHCs. Inhibition of intracellular Ca2+ overload significantly attenuated IHC death after 10 mmol/L salicylate treatment. These results implicate a cellular mechanism for tinnitus generation in the peripheral auditory system.

Characterization of highly active tyrosine ammonia lyase and its application in biosynthesis of p-coumaric acid / 生物工程学报

p-coumaric acid is one of the aromatic compounds that are widely used in food, cosmetics and medicine due to its properties of antibacterium, antioxidation and cardiovascular disease prevention. Tyrosine ammonia-lyase (TAL) catalyzes the deamination of tyrosine to p-coumaric acid. However, the lack of highly active and specific tyrosine ammonia lyase limits cost-effective microbial production of p-coumaric acid. In order to improve biosynthesis efficiency of p-coumaric acid, two tyrosine ammonia-lyases, namely Fc-TAL2 derived from Flavobacterium columnare and Fs-TAL derived from Flavobacterium suncheonense, were selected and characterized. The optimum temperature (55 ℃) and pH (9.5) for Fs-TAL and Fc-TAL2 are the same. Under optimal conditions, the specific enzyme activity of Fs-TAL and Fc-TAL2 were 82.47 U/mg and 13.27 U/mg, respectively. Structural simulation and alignment analysis showed that the orientation of the phenolic hydroxyl group of the conserved Y50 residue on the inner lid loop and its distance to the substrate were the main reasons accounting for the higher activity of Fs-TAL than that of Fc-TAL2. The higher activity and specificity of Fs-TAL were further confirmed via whole-cell catalysis using recombinant Escherichia coli, which could convert 10 g/L tyrosine into 6.2 g/L p-coumaric acid with a yield of 67.9%. This study provides alternative tyrosine ammonia-lyases and may facilitate the microbial production of p-coumaric acid and its derivatives.

Isolation, identification and electrophysiological activity of BmK M2 from Buthus martensii Karsch venom / 中国药科大学学报

@#This study aimed to isolate and identify novel toxin peptides targeting voltage-gated sodium channels (VGSGs) from the venom of the Buthus martensii Karsch (BmK) scorpion. Using G50-gel filtration, HPLC, peptide fingerprinting and amino acid sequencing, a novel sodium channel modulator, BmK M2, was identified from BMK scorpion. BmK M2 is a relatively abundant long chain polypeptide toxin in BmK scorpion venom with a molecular weight of 7 235.59, consisting of 64 amino acids and 4 pairs of disulfide bonds.Sequence alignment showed that the amino acid sequence of BmK M2 had high sequence and structural similarity to that of the discovered sodium channel toxins of BmK M1, BmK M3 and BmK M9, etc.BmK M2 is a potential new sodium channel modulator.Electrophysiological results revealed that BmK M2 can significantly enhance the activation, delay the steady-state inactivation and closed-state inactivation of Nav1.7, but has no activity on Nav1.8.BmK M2 can be used as a novel peptide probe for the study of the structure and function of Nav1.7 and the development of drugs targeting Nav1.7.

Rational design of the C-terminal Loop region of leucine dehydrogenase and cascade biosynthesis L-2-aminobutyric acid / 生物工程学报

Leucine dehydrogenase (LDH) is the key rate-limiting enzyme in the production of L-2-aminobutyric acid (L-2-ABA). In this study, we modified the C-terminal Loop region of this enzyme to improve the specific enzyme activity and stability for efficient synthesis of L-2-ABA. Using molecular dynamics simulation of LDH, we analyzed the change of root mean square fluctuation (RMSF), rationally designed the Loop region with greatly fluctuated RMSF, and obtained a mutant EsLDHD2 with a specific enzyme activity 23.2% higher than that of the wild type. Since the rate of the threonine deaminase-catalyzed reaction converting L-threonine into 2-ketobutyrate was so fast, the multi-enzyme cascade catalysis system became unbalanced. Therefore, the LDH and the formate dehydrogenase were double copied in a new construct E. coli BL21/pACYCDuet-RM. Compared with E. coli BL21/pACYCDuet-RO, the molar conversion rate of L-2-ABA increased by 74.6%. The whole cell biotransformation conditions were optimized and the optimal pH, temperature and substrate concentration were 7.5, 35 °C and 80 g/L, respectively. Under these conditions, the molar conversion rate was higher than 99%. Finally, 80 g and 40 g L-threonine were consecutively fed into a 1 L reaction mixture under the optimal conversion conditions, producing 97.9 g L-2-ABA. Thus, this strategy provides a green and efficient synthesis of L-2-ABA, and has great industrial application potential.

Biosynthesis of 2,5-dimethylpyrazine from L-threonine by whole-cell biocatalyst of recombinant Escherichia coli / 生物工程学报

2,5-dimethylpyrazine (2,5-DMP) is of important economic value in food industry and pharmaceutical industry, and is now commonly produced by chemical synthesis. In this study, a recombinant Escherichia coli high-efficiently converting L-threonine to 2,5-DMP was constructed by combination of metabolic engineering and cofactor engineering. To do this, the effect of different threonine dehydrogenase (TDH) on 2,5-DMP production was investigated, and the results indicate that overexpression of EcTDH in E. coli BL21(DE3) was beneficial to construct a 2,5-DMP producer with highest 2,5-DMP production. The recombinant strain E. coli pRSFDuet-tdh(Ec) produced (438.3±23.7) mg/L of 2,5-DMP. Furthermore, the expression mode of NADH oxidase (NoxE) from Lactococcus cremoris was optimized, and fusion expression of EcTDH and LcNoxE led to balance the intracellular NADH/NAD⁺ level and to maintain the high survival rate of cells, thus further increasing 2,5-DMP production. Finally, the accumulation of by-products was significantly decreased because of disruption of shunt metabolic pathway, thereby increasing 2,5-DMP production and the conversion ratio of L-threonine. Combination of these genetic modifications resulted in an engineered E. coli Δkbl ΔtynA ΔtdcB ΔilvA pRSFDuet-tdhEcnoxELc-PsstT (EcΔkΔAΔBΔA/TDH(Ec)NoxE(Lc)-PSstT) capable of producing (1 095.7±81.3) mg/L 2,5-DMP with conversion ratio of L-threonine of 76% and a yield of 2,5-DMP of 28.8% in 50 mL transformation system with 5 g/L L-threonine at 37 °C and 200 r/min for 24 h. Therefore, this study provides a recombinant E. coli with high-efficiently catalyzing L-threonine to biosynthesize 2,5-DMP, which can be potentially used in biosynthesis of 2,5-DMP in industry.

Growth Differentiation Factor-15 Produces Analgesia by Inhibiting Tetrodotoxin-Resistant Nav1.8 Sodium Channel Activity in Rat Primary Sensory Neurons / 神经科学通报·英文版

Growth differentiation factor 15 (GDF-15) is a member of the transforming growth factor-β superfamily. It is widely distributed in the central and peripheral nervous systems. Whether and how GDF-15 modulates nociceptive signaling remains unclear. Behaviorally, we found that peripheral GDF-15 significantly elevated nociceptive response thresholds to mechanical and thermal stimuli in naïve and arthritic rats. Electrophysiologically, we demonstrated that GDF-15 decreased the excitability of small-diameter dorsal root ganglia (DRG) neurons. Furthermore, GDF-15 concentration-dependently suppressed tetrodotoxin-resistant sodium channel Nav1.8 currents, and shifted the steady-state inactivation curves of Nav1.8 in a hyperpolarizing direction. GDF-15 also reduced window currents and slowed down the recovery rate of Nav1.8 channels, suggesting that GDF-15 accelerated inactivation and slowed recovery of the channel. Immunohistochemistry results showed that activin receptor-like kinase-2 (ALK2) was widely expressed in DRG medium- and small-diameter neurons, and some of them were Nav1.8-positive. Blockade of ALK2 prevented the GDF-15-induced inhibition of Nav1.8 currents and nociceptive behaviors. Inhibition of PKA and ERK, but not PKC, blocked the inhibitory effect of GDF-15 on Nav1.8 currents. These results suggest a functional link between GDF-15 and Nav1.8 in DRG neurons via ALK2 receptors and PKA associated with MEK/ERK, which mediate the peripheral analgesia of GDF-15.

Growth Differentiation Factor-15 Produces Analgesia by Inhibiting Tetrodotoxin-Resistant Nav1.8 Sodium Channel Activity in Rat Primary Sensory Neurons / 神经科学通报·英文版

Growth differentiation factor 15 (GDF-15) is a member of the transforming growth factor-β superfamily. It is widely distributed in the central and peripheral nervous systems. Whether and how GDF-15 modulates nociceptive signaling remains unclear. Behaviorally, we found that peripheral GDF-15 significantly elevated nociceptive response thresholds to mechanical and thermal stimuli in naïve and arthritic rats. Electrophysiologically, we demonstrated that GDF-15 decreased the excitability of small-diameter dorsal root ganglia (DRG) neurons. Furthermore, GDF-15 concentration-dependently suppressed tetrodotoxin-resistant sodium channel Nav1.8 currents, and shifted the steady-state inactivation curves of Nav1.8 in a hyperpolarizing direction. GDF-15 also reduced window currents and slowed down the recovery rate of Nav1.8 channels, suggesting that GDF-15 accelerated inactivation and slowed recovery of the channel. Immunohistochemistry results showed that activin receptor-like kinase-2 (ALK2) was widely expressed in DRG medium- and small-diameter neurons, and some of them were Nav1.8-positive. Blockade of ALK2 prevented the GDF-15-induced inhibition of Nav1.8 currents and nociceptive behaviors. Inhibition of PKA and ERK, but not PKC, blocked the inhibitory effect of GDF-15 on Nav1.8 currents. These results suggest a functional link between GDF-15 and Nav1.8 in DRG neurons via ALK2 receptors and PKA associated with MEK/ERK, which mediate the peripheral analgesia of GDF-15.

Integrative analysis of in vivo recording with single-cell RNA-seq data reveals molecular properties of light-sensitive neurons in mouse V1

Vision formation is classically based on projections from retinal ganglion cells (RGC) to the lateral geniculate nucleus (LGN) and the primary visual cortex (V1). Neurons in the mouse V1 are tuned to light stimuli. Although the cellular information of the retina and the LGN has been widely studied, the transcriptome profiles of single light-stimulated neuron in V1 remain unknown. In our study, in vivo calcium imaging and whole-cell electrophysiological patch-clamp recording were utilized to identify 53 individual cells from layer 2/3 of V1 as light-sensitive (LS) or non-light-sensitive (NS) by single-cell light-evoked calcium evaluation and action potential spiking. The contents of each cell after functional tests were aspirated in vivo through a patch-clamp pipette for mRNA sequencing. Moreover, the three-dimensional (3-D) morphological characterizations of the neurons were reconstructed in a live mouse after the whole-cell recordings. Our sequencing results indicated that V1 neurons with a high expression of genes related to transmission regulation, such as Rtn4r and Rgs7, and genes involved in membrane transport, such as Na/K ATPase and NMDA-type glutamatergic receptors, preferentially responded to light stimulation. Furthermore, an antagonist that blocks Rtn4r signals could inactivate the neuronal responses to light stimulation in live mice. In conclusion, our findings of the vivo-seq analysis indicate the key role of the strength of synaptic transmission possesses neurons in V1 of light sensory.

Integrative analysis of in vivo recording with single-cell RNA-seq data reveals molecular properties of light-sensitive neurons in mouse V1

Vision formation is classically based on projections from retinal ganglion cells (RGC) to the lateral geniculate nucleus (LGN) and the primary visual cortex (V1). Neurons in the mouse V1 are tuned to light stimuli. Although the cellular information of the retina and the LGN has been widely studied, the transcriptome profiles of single light-stimulated neuron in V1 remain unknown. In our study, in vivo calcium imaging and whole-cell electrophysiological patch-clamp recording were utilized to identify 53 individual cells from layer 2/3 of V1 as light-sensitive (LS) or non-light-sensitive (NS) by single-cell light-evoked calcium evaluation and action potential spiking. The contents of each cell after functional tests were aspirated in vivo through a patch-clamp pipette for mRNA sequencing. Moreover, the three-dimensional (3-D) morphological characterizations of the neurons were reconstructed in a live mouse after the whole-cell recordings. Our sequencing results indicated that V1 neurons with a high expression of genes related to transmission regulation, such as Rtn4r and Rgs7, and genes involved in membrane transport, such as Na/K ATPase and NMDA-type glutamatergic receptors, preferentially responded to light stimulation. Furthermore, an antagonist that blocks Rtn4r signals could inactivate the neuronal responses to light stimulation in live mice. In conclusion, our findings of the vivo-seq analysis indicate the key role of the strength of synaptic transmission possesses neurons in V1 of light sensory.

Production of L-2-aminobutyric acid from L-threonine using a trienzyme cascade / 生物工程学报

L-2-aminobutyric acid (L-ABA) is an important chemical raw material and chiral pharmaceutical intermediate. The aim of this study was to develop an efficient method for L-ABA production from L-threonine using a trienzyme cascade route with Threonine deaminase (TD) from Escherichia. coli, Leucine dehydrogenase (LDH) from Bacillus thuringiensis and Formate dehydrogenase (FDH) from Candida boidinii. In order to simplify the production process, the activity ratio of TD, LDH and FDH was 1:1:0.2 after combining different activity ratios in the system in vitro. The above ratio was achieved in the recombinant strain E. coli 3FT+L. Moreover, the transformation conditions were optimized. Finally, we achieved L-ABA production of 68.5 g/L with a conversion rate of 99.0% for 12 h in a 30-L bioreactor by whole-cell catalyst. The environmentally safe and efficient process route represents a promising strategy for large-scale L-ABA production in the future.

Evaluation of different total Leishmania amazonensis antigens for the development of a first-generation vaccine formulated with a Toll-like receptor-3 agonist to prevent cutaneous leishmaniasis

BACKGROUND Unfortunately, no any vaccine against leishmaniasis has been developed for human use. Therefore, a vaccine based on total Leishmania antigens could be a good and economic approach; and there are different methodologies to obtain these antigens. However, it is unknown whether the method to obtain the antigens affects the integrity and immune response caused by them. OBJECTIVES to compare the protein profile and immune response generated by total L. amazonensis antigens (TLA) produced by different methods, as well as to analyse the immune response and protection by a first-generation vaccine formulated with sonicated TLA (sTLA) and polyinosinic:polycytidylic acid [Poly (I:C)]. METHODS TLA were obtained by four different methodologies and their integrity and immune response were evaluated. Finally, sTLA was formulated with Poly (I:C) and their protective immune response was measured. FINDINGS sTLA presented a conserved protein profile and induced a strong immune response. In addition, Poly (I:C) improved the immune response generated by sTLA. Finally, sTLA + Poly (I:C) formulation provided partial protection against L. amazonensis infection. MAIN CONCLUSIONS The protein profile and immune response depend on the methodology used to obtain the antigens. Also, the formulation sTLA + Poly (I:C) provides partial protection against cutaneous leishmaniasis in mice.

Morphology and electrophysiological properties of neurons in the inner nuclear layer of the rat retina slices prepared by reformed method / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the morphology and electrophysiological properties of neurons in the inner nuclear layer(INL)of the adult rat retina slices. The retinal slices were prepared by low-melt agarose embedding and then cutted by vibratome. <p>METHODS: Whole cell patch clamp and intracellular staining with Lucifer Yellow were used in this study to study the morphology and electrophysiological properties of the INL neurons in retinal slices.<p>RESULTS: Retinal slices prepared in this method possessed a very smooth surface. The cells on the retinal slices maitained very good vitality, and some of the cells even retained their dendritic connections with other cells on the slice. According to the size and location of the cell bodies, neurons in the INL were easy to differentiate. Luciifer Yellow contained in the intracellular solution revealed the morphology of the recorded cell very well. Bipolar cells possessed elongated cell bodies and their processes mainly extended along the vertical direction. Horizontal cells and amacrine cells possessed much bigger and round cell bodies, resided in the outermost and inner most of the INL, respectively. The rest membrane potential and membrane capacitance of horizontal cell and amacrine cell were much higher than that of bipolar cells. Under a voltage step from -60mV to +40mV, 10mV per step, 41.7% of the cone bipolar cells and 64.7% of the amacrine cells exhibited inward sodium current and outward potassium current. Other cells only possessed outward potassium current. <p>CONCLUSION: The method of preparing retinal slices was very simple, and the viability of the slices were stable. These facilitated the patch-clamp recording of all the neurons in the INL including horizontal cells. Further investigation of the electrophysiological properties of the neurons in the INL was essential in revealing the mechanism of vision.

Qualitative anti-tubercular activity of synthetic ethyl 7-acetyl2-substituted-3-(4-substituted benzoyl) indolizine-1-carboxylate analogues

Both the emergence of multidrug-resistant and extensively drug-resistant tuberculosis (TB) are currently the majorchallenges in the treatment of TB. Only delamanid and bedaquiline have been recently approved as anti-TB drugs inthe past 40 years. In an attempt to search for active anti-TB compounds against the sensitive strain of Mycobacteriumtuberculosis, H37Rv—a series of synthetic ethyl 7-acetyl-2-substituted-3-(4-substituted benzoyl)indolizine-1-carboxylates (2a–r)—have been screened for in vitro qualitative anti-TB activity using an agar dilution method. Itwas found that compounds 2a, 2b, 2c, 2f, 2g, 2i, 2j, 2l, 2o, 2p, and 2r, which have various functional groups on theindolizine nucleus, were active against the H37Rv strain.

Heterologous expression of Streptomyces coelicolor trehalose synthase and whole-cell biocatalyst production of trehalose in Escherichia coli / 生物工程学报

The trehalose synthase (ScTreS) gene from Streptomyces coelicolor was successfully cloned and heterologously expressed in Escherichia coli BL21(DE3). The protein purified by Ni-NTA affinity column showed an apparent molecular weight (MW) of 62.3 kDa analyzed by SDS-PAGE. The optimum temperature of the enzyme was 35 °C and the optimum pH was 7.0; the enzyme was sensitive to acidic conditions. By homologous modeling and sequence alignment, the enzyme was modified by site-directed mutagenesis. The relative activities of the mutant enzymes K246A and A165T were 1.43 and 1.39 times that of the wild type, an increased conversion rate of 14% and 10% respectively. To optimize the synthesis conditions of trehalose, the mutant strain K246A was cultivated in a 5-L fermentor and used for whole-cell transformation. The results showed that with the substrate maltose concentration of 300 g/L at 35 °C and pH 7.0, the highest conversion rate reached 71.3%, and the yield of trehalose was 213.93 g/L. However, when maltose concentration was increased to 700 g/L, the yield of trehalose can reach 465.98 g/L with a conversion rate of 66%.

Biocatalytic strategies in producing ginsenoside by glycosidase-a review / 生物工程学报

Panax ginseng is a traditional Chinese medicine with significant pharmaceutical effects and wide application. Through orientational modification and transformation of ginsenoside glycosyl, rare ginsenosides with high antitumor activities can be generated. Traditional chemical methods cannot be applied in clinic. because of extremely complex preparation technologies and very high cost Transformations using microorganisms and their enzymatic systems provide the most feasible methods for solving the main problems. At present, the key problems in enzymatic synthesis of ginsenosides include low specific enzyme activities, identity of enzymes involved in the enzymatic synthesis, and their catalytic mechanisms, as well as nonsystematic studies on structural bioinformatics; specificity of enzymatic hydrolysis for saponin glycosyl has been rarely studied. Many reviews have been reported on glycosidase molecular recognition, immobilization, and biotransformation in ionic liquids (ILs), whereas ginsenoside transformation and application have not been systematically studied. To evaluate theoretical and applied studies on ginsenoside-oriented biotransformation, by reviewing the latest developments in related fields and evaluating the widely applied biocatalytic strategy, this review aims to evaluate the ginsenoside-oriented transformation method with improved product specificity, increased biocatalytic efficiency, and industrial application prospect based on the designed transformations of enzyme and solvent engineering of ILs. Therefore, useful theoretical and experimental evidence can be obtained for the development of ginsenoside anticancer drugs, large-scale preparation, and clinical applications in cancer therapy.

Effects of daidzein on Inain rat ventricular myocytes / 中国药理学通报

Aim To study the effects of daidzein on sodium channel current ( /Na) in ventricular myocytes of rats and the mechanism of its antiarrhythmia. Methods The effect of daidzein on the viability of ventricular myocytes was delected by MTT assay; single ventricular myocytes from rats were isolated by single enzymatic hydrolysis; the changes of /N, and its dynamic characteristics in rat ventricular myocytes before and after administration of daidzein were observed, recorded and analyzed by cell patch clamp technique. Results MTT experiments showed that the ICjo of daidzein was 30 to 100 jjimol • L"1 ,so the concentration of 0. 3 - 10 jimol • L"1 was chosen for the subsequent experiments. When daidzein was given 0. 3,1,3,10 pjnol • L"',the /Nb amplitude of ventricular myocytes in rats showed a concentration-dependent inhibition. The concentration of daidzein 0. 3 imol • L"1 also had certain effect on the time course of /Nt. The /Nl, peak decreased gradually over time. The 1,3,10 jimol • L"1 daidzein raised the I-U curve obviously. Under the same condition, the activation curve moved to the direction of depolarization. The steady-state inactivation curve shifted toward hyperpolarization, and the t value of the recovery curve was prolonged after inactivated state. Conclusions Daidzein significantly inhibited the Na∗ channel of ventricular myocardium in rats, which may l)e one of its mechanisms of anti-arrhythmia.

Inhibitory effect of monoclonal antibody NCX-2D2 on isoproterenol-induced arrhythmias in adult rat hearts / 中国药理学通报

Aim To investigate the effects of mono-clonal antibody NCX-2D2 on isoproterenol-induced ar-rhythmias in rat hearts, and to explore the electrophys-iological mechanism. Methods Using isoproterenol to establish in vitro and in vivo arrhythmic rat models to observe the effect of NCX-2D2 antibody on ventricular arrhythmias in rats. The whole-cell patch clamp tech-nique was used to investigate the effects of NCX-2D2 antibody on INa/Ca, ICa-Lat voltage-clamp mode and on DADs at current-clamp mode in single rat ventricular myocytes. Results 10 mg·L-1NCX-2D2 antibody significantly inhibited cardiac arrhythmias induced by ISO in vitro ( P<0.01) . 80 μg·kg-1NCX-2D2 anti-body markedly inhibit the occurrence of arrhythmias in ISO-induced anesthetized rats in vivo ( P <0.01 ) . 5 mg·L-1NCX-2D2 antibody partially inhibited the in-crease of INa/Ca(P<0.01) and the increase of ICa-L(P<0.01 ) , and could effectively inhibit ISO-induced DADs in rat ventricular myocytes ( P <0.05 ) . Con-clusions The sodium-calcium exchanger monoclonal antibody NCX-2D2 significantly inhibits isoproterenol-induced ventricular arrhythmias in rats. The mecha-nism against ventricular arrhythmias is mainly due to its inhibition of cardiomyocyte sodium-calcium exchanger and L-type calcium channel and marked suppression of DADs in rat ventricular myocytes.