PPM1F regulates ovarian cancer progression by affecting the dephosphorylation of ITGB1.

PPM1F has been shown to play diverse biological functions in the progression of multiple tumors. PPM1F controls the T788/T789 phosphorylation switch of ITGB1 and regulates integrin activity. However, the impacts of PPM1F and ITGB1 on ovarian cancer (OV) progression remain unclear. Whether there is such a regulatory relationship between PPM1F and ITGB1 in ovarian cancer has not been studied. Therefore, the purpose of this study is to elucidate the function and the mechanism of PPM1F in ovarian cancer. The expression level and the survival curve of PPM1F were analyzed by databases. Gain of function and loss of function were applied to explore the function of PPM1F in ovarian cancer. A tumor formation assay in nude mice showed that knockdown of PPM1F inhibited tumor formation. We tested the effect of PPM1F on ITGB1 dephosphorylation in ovarian cancer cells by co-immunoprecipitation and western blotting. Loss of function was applied to investigate the function of ITGB1 in ovarian cancer. ITGB1-mut overexpression promotes the progression of ovarian cancer. Rescue assays showed the promoting effect of ITGB1-wt on ovarian cancer is attenuated due to the dephosphorylation of ITGB1-wt by PPM1F. PPM1F and ITGB1 play an oncogene function in ovarian cancer. PPM1F regulates the phosphorylation of ITGB1, which affects the occurrence and development of ovarian cancer.

Construction of Membraneless and Multicompartmentalized Coacervate Protocells Controlling a Cell Metabolism-like Cascade Reaction.

In recent years, there has been growing attention to designing synthetic protocells, capable of mimicking micrometric and multicompartmental structures and highly complex physicochemical and biological processes with spatiotemporal control. Controlling metabolism-like cascade reactions in coacervate protocells is still challenging since signal transduction has to be involved in sequential and parallelized actions mediated by a pH change. Herein, we report the hierarchical construction of membraneless and multicompartmentalized protocells composed of (i) a cytosol-like scaffold based on complex coacervate droplets stable under flow conditions, (ii) enzyme-active artificial organelles and a substrate nanoreservoir capable of triggering a cascade reaction between them in response to a pH increase, and (iii) a signal transduction component based on the urease enzyme capable of the conversion of an exogenous biological fuel (urea) into an endogenous signal (ammonia and pH increase). Overall, this strategy allows a synergistic communication between their components within the membraneless and multicompartment protocells and, thus, metabolism-like enzymatic cascade reactions. This signal communication is transmitted through a scaffold protocell from an "inactive state" (nonfluorescent protocell) to an "active state" (fluorescent protocell capable of consuming stored metabolites).

The mechanism of VCP-mediated metastasis of osteosarcoma based on cell autophagy and the EMT pathway.

OBJECTIVE: Study of the molecular mechanisms of metastasis is still the research focus for osteosarcoma (OS) prevention. This study investigates the mechanism of valosin-containing protein (VCP) promoting OS metastasis in vitro through autophagy and epithelial-mesenchymal transition (EMT). METHODS: Different cell lines of osteosarcoma (143B and MG63) were adopted in this study. The level of VCP expression in osteosarcoma cells was changed, and the level of autophagy and the progression of the epithelial-mesenchymal transition (EMT) were observed. Then autophagy and EMT in OS cells were changed artificially, and proliferation and migration ability were observed. RESULTS: The expression of LC3II/I was decreased, but the insolubilized P62 protein expression was increased in the VCP inhibiting group and the autophagy inhibitor treatment group. Simultaneously, E-cadherin protein expression increased while N-cadherin protein expression decreased in the VCP inhibiting group but increased in the TGF-ß1 treatment group. In addition, suppressing VCP can cause a decrease in Transforming Growth Factor ß1 (TGF-ß1), smad2, smad3, phosphorylated smad2 (p-smad2), and phosphorylated smad3 (p-smad3). Autophagy inhibitors and agonists have no significant effect on the migration and invasion of OS cells but can significantly affect the ability of cells to resist anoikis. EMT inhibitors and agonists have a proportional effect on the migration and invasion of OS cells. CONCLUSION: VCP is likely to promote the migration and invasion of OS cells by inducing EMT, possibly via TGF-ß1/smad2/3 signaling pathway. In this process, VCP-mediated autophagy may contribute to successful distant metastasis of tumor cells indirectly.

Neuronal GRK2 regulates microglial activation and contributes to electroacupuncture analgesia on inflammatory pain in mice.

BACKGROUND: G protein coupled receptor kinase 2 (GRK2) has been demonstrated to play a crucial role in the development of chronic pain. Acupuncture is an alternative therapy widely used for pain management. In this study, we investigated the role of spinal neuronal GRK2 in electroacupuncture (EA) analgesia. METHODS: The mice model of inflammatory pain was built by subcutaneous injection of Complete Freund's Adjuvant (CFA) into the plantar surface of the hind paws. The mechanical allodynia of mice was examined by von Frey test. The mice were subjected to EA treatment (BL60 and ST36 acupuncture points) for 1 week. Overexpression and downregulation of spinal neuronal GRK2 were achieved by intraspinal injection of adeno associated virus (AAV) containing neuron-specific promoters, and microglial activation and neuroinflammation were evaluated by real-time PCR. RESULTS: Intraplantar injection with CFA in mice induced the decrease of GRK2 and microglial activation along with neuroinflammation in spinal cord. EA treatment increased the spinal GRK2, reduced neuroinflammation, and significantly decreased CFA-induced mechanical allodynia. The effects of EA were markedly weakened by non-cell-specific downregulation of spinal GRK2. Further, intraspinal injection of AAV containing neuron-specific promoters specifically downregulated neuronal GRK2, and weakened the regulatory effect of EA on CFA-induced mechanical allodynia and microglial activation. Meanwhile, overexpression of spinal neuronal GRK2 decreased mechanical allodynia. All these indicated that the neuronal GRK2 mediated microglial activation and neuroinflammation, and subsequently contributed to CFA-induced inflammatory pain. CONCLUSION: The restoration of the spinal GRK2 and subsequent suppression of microglial activation and neuroinflammation might be an important mechanism for EA analgesia. Our findings further suggested that the spinal GRK2, especially neuronal GRK2, might be the potential target for EA analgesia and pain management, and we provided a new experimental basis for the EA treatment of pain.

Neuronal GRK2 regulates microglial activation and contributes to electroacupuncture analgesia on inflammatory pain in mice

BACKGROUND: G protein coupled receptor kinase 2 (GRK2) has been demonstrated to play a crucial role in the development of chronic pain. Acupuncture is an alternative therapy widely used for pain management. In this study, we investigated the role of spinal neuronal GRK2 in electroacupuncture (EA) analgesia. METHODS: The mice model of inflammatory pain was built by subcutaneous injection of Complete Freund's Adjuvant (CFA) into the plantar surface of the hind paws. The mechanical allodynia of mice was examined by von Frey test. The mice were subjected to EA treatment (BL60 and ST36 acupuncture points) for 1 week. Overexpression and down-regulation of spinal neuronal GRK2 were achieved by intraspinal injection of adeno associated virus (AAV) containing neuron-specific promoters, and microglial activation and neuroinflammation were evaluated by real-time PCR. RESULTS: Intraplantar injection with CFA in mice induced the decrease of GRK2 and microglial activation along with neuroinflammation in spinal cord. EA treatment increased the spinal GRK2, reduced neuroinflammation, and significantly decreased CFA-induced mechanical allodynia. The effects of EA were markedly weakened by non-cell-specific downregulation of spinal GRK2. Further, intraspinal injection of AAV containing neuron-specific promoters specifically downregulated neuronal GRK2, and weakened the regulatory effect of EA on CFA-induced mechanical allodynia and microglial activation. Meanwhile, overexpression of spinal neuronal GRK2 decreased mechanical allodynia. All these indicated that the neuronal GRK2 mediated microglial activation and neuroinflammation, and subsequently contributed to CFA-induced inflammatory pain. CONCLUSION: The restoration of the spinal GRK2 and subsequent suppression of microglial activation and neuroinflammation might be an important mechanism for EA analgesia. Our findings further suggested that the spinal GRK2, especially neuronal GRK2, might be the potential target for EA analgesia and pain management, and we provided a new experimental basis for the EA treatment of pain.

A Adiponectina Previne a Reestenose pela Inibição da Proliferação Celular em um Modelo de Enxerto Venoso em Ratos / Adiponectin Prevents Restenosis Through Inhibiting Cell Proliferation in a Rat Vein Graft Model

Resumo Fundamento: O enxerto de bypass na artéria coronária (CABG) continua a ser eficiente como tratamento para pacientes portadores de doença arterial coronariana; entretanto, o enxerto venoso tende a apresentar reestenose ou oclusão. A adiponectina (ADP) é uma proteína hormonal plasmática com a função de regular a proliferação celular. Objetivo: Foram utilizadas duas doses diferentes da proteína ADP em um modelo de enxerto venoso em ratos para estimular a alteração do enxerto venoso. O objetivo deste estudo foi investigar o efeito da ADP sobre a reestenose em enxerto venoso. Métodos: Veias jugulares autólogas foram implantadas como enxertos interposicionais de carótida pela técnica de anastomose de manga em ratos Sprague Dawley. A adiponectina (2,5 μg e 7,5 μg) foi entregue ao enxerto venoso por bypass de forma perivascular, suspensa em gel Pluronic-F127 a 30%. O grupo tratado apenas com bypass e o grupo tratado com gel veículo carregado apenas com Pluronic funcionaram como controle. Foram feitas comparações com análise de via única de variância e teste post-hoc, com p <0,05 sendo considerado significativo. Resultados: A proliferação celular (índice de PCNA) foi significativamente baixa no grupo tratado com adiponectina em comparação com o grupo de controle e o grupo tratado com o gel veículo na íntima e na adventícia dos enxertos a partir do dia 3 (p <0,01). VCAM-1 e ICAM-1 avaliados por imuno-histoquímica diminuíram significativamente em enxertos venosos tratados com adiponectina na quarta semana (p <0,01). O tratamento de enxertos venosos com gel carregado com adiponectina reduziu a espessura da íntima, da média e da adventícia, em comparação com os enxertos de controle e tratados com gel veículo no dia 28 (p <0,01). Conclusões: Este estudo oferece evidências adicionais do possível papel terapêutico da adiponectina na modulação de lesão vascular e seu reparo.

Abstract Background: Coronary artery bypass grafting (CABG) continues to be an effective therapy for coronary artery disease patients, but the vein graft is prone to restenosis or occlude. Adiponectin (ADP) is a plasma hormone protein with the function of regulating cell proliferation. Objective: This study used two different doses of ADP protein in a rat vein graft model to stimulate vein graft change. The aim of our study was to investigate the effect of ADP on vein graft restenosis. Methods: Autologous jugular veins were implanted as carotid interposition grafts through the anastomotic cuff technique in Sprague Dawley rats. Adiponectin (2.5 μg and 7.5 μg) was delivered to the vein bypass grafts in a perivascular fashion, suspended in a 30% Pluronic-F127 gel. No treatment (bypass only) and vehicle loaded Pluronic gel served as controls. Comparisons were made with one-way analysis of variance and a post-hoc test, with p < 0.05 considered significant. Results: Cell proliferation (PCNA index) was significantly low in adiponectin-treated versus control and vehicle-gel-treated grafts, both in intima and adventitia, as of day 3 (p < 0.01). VCAM-1 and ICAM-1 evaluated by immunohistochemistry significantly down-regulated in the adiponectin-treated vein grafts in the fourth week (p <0.01). Treatment of vein grafts with adiponectin-loaded gels reduced intimal, media, and adventitia thickness when compared with the control and vehicle-gel-treated vein grafts at day 28 (p < 0.01). Conclusions: Our studies provide further support for the potential therapeutic role of adiponectin in modulating vascular injury and repair.

Structural characterization of two novel polysaccharides from Gastrodia elata and their effects on Akkermansia muciniphila.

Two homogeneous polysaccharides, GEP-3 and GEP-4, were purified from Gastrodia elata, a precious traditional Chinese medicine. Their structural characteristics were obtained using HPGPC, PMP-HPLC, LC/MS, FT-IR, NMR, and SEM methods. GEP-3 was 1,4-glucan with molecular weight of 20 kDa. Interestingly, GEP-4 comprised of a backbone of →[4)-α-Glcp-(1]10→[4)-α-Glcp-(1→]5[6)-ß-Glcp-(1]11→6)-α-Glcp-(3→ and two branches of ß-Glcp and p-hydroxybenzyl alcohol citrate, with repeating p-hydroxybenzyl alcohol attached to the backbone chain at O-6 position of →4,6)-α-Glcp-(1→ and O-1 position of →3,6)-α-Glcp-(1→. GEP-4 is a novel polysaccharide obtained and characterized for the first time. Bioactivity test indicated that both of them significantly promote the growth of Akkermansia muciniphila (Akk. muciniphila). Furthermore, GEP-3 and GEP-4 promoted the growth of Akk. muciniphila from high-fat diet (HFD) fecal microbiota. These results indicated that GEP-3 and GEP-4 were potential Akk. muciniphila growth promoters.

Hermansky-Pudlak Syndrome and Lung Disease: Pathogenesis and Therapeutics.

Hermansky-Pudlak Syndrome (HPS) is a rare, genetic, multisystem disorder characterized by oculocutaneous albinism (OCA), bleeding diathesis, immunodeficiency, granulomatous colitis, and pulmonary fibrosis. HPS pulmonary fibrosis (HPS-PF) occurs in 100% of patients with subtype HPS-1 and has a similar presentation to idiopathic pulmonary fibrosis. Upon onset, individuals with HPS-PF have approximately 3 years before experiencing signs of respiratory failure and eventual death. This review aims to summarize current research on HPS along with its associated pulmonary fibrosis and its implications for the development of novel treatments. We will discuss the genetic basis of the disease, its epidemiology, and current therapeutic and clinical management strategies. We continue to review the cellular processes leading to the development of HPS-PF in alveolar epithelial cells, lymphocytes, mast cells, and fibrocytes, along with the molecular mechanisms that contribute to its pathogenesis and may be targeted in the treatment of HPS-PF. Finally, we will discuss emerging new cellular and molecular approaches for studying HPS, including lentiviral-mediated gene transfer, induced pluripotent stem cells (iPSCs), organoid and 3D-modelling, and CRISPR/Cas9-based gene editing approaches.

Adiponectin Prevents Restenosis Through Inhibiting Cell Proliferation in a Rat Vein Graft Model. / A Adiponectina Previne a Reestenose pela Inibição da Proliferação Celular em um Modelo de Enxerto Venoso em Ratos.

BACKGROUND: Coronary artery bypass grafting (CABG) continues to be an effective therapy for coronary artery disease patients, but the vein graft is prone to restenosis or occlude. Adiponectin (ADP) is a plasma hormone protein with the function of regulating cell proliferation. OBJECTIVE: This study used two different doses of ADP protein in a rat vein graft model to stimulate vein graft change. The aim of our study was to investigate the effect of ADP on vein graft restenosis. METHODS: Autologous jugular veins were implanted as carotid interposition grafts through the anastomotic cuff technique in Sprague Dawley rats. Adiponectin (2.5 µg and 7.5 µg) was delivered to the vein bypass grafts in a perivascular fashion, suspended in a 30% Pluronic-F127 gel. No treatment (bypass only) and vehicle loaded Pluronic gel served as controls. Comparisons were made with one-way analysis of variance and a post-hoc test, with p < 0.05 considered significant. RESULTS: Cell proliferation (PCNA index) was significantly low in adiponectin-treated versus control and vehicle-gel-treated grafts, both in intima and adventitia, as of day 3 (p < 0.01). VCAM-1 and ICAM-1 evaluated by immunohistochemistry significantly down-regulated in the adiponectin-treated vein grafts in the fourth week (p <0.01). Treatment of vein grafts with adiponectin-loaded gels reduced intimal, media, and adventitia thickness when compared with the control and vehicle-gel-treated vein grafts at day 28 (p < 0.01). CONCLUSIONS: Our studies provide further support for the potential therapeutic role of adiponectin in modulating vascular injury and repair.

FUNDAMENTO: O enxerto de bypass na artéria coronária (CABG) continua a ser eficiente como tratamento para pacientes portadores de doença arterial coronariana; entretanto, o enxerto venoso tende a apresentar reestenose ou oclusão. A adiponectina (ADP) é uma proteína hormonal plasmática com a função de regular a proliferação celular. OBJETIVO: Foram utilizadas duas doses diferentes da proteína ADP em um modelo de enxerto venoso em ratos para estimular a alteração do enxerto venoso. O objetivo deste estudo foi investigar o efeito da ADP sobre a reestenose em enxerto venoso. MÉTODOS: Veias jugulares autólogas foram implantadas como enxertos interposicionais de carótida pela técnica de anastomose de manga em ratos Sprague Dawley. A adiponectina (2,5 µg e 7,5 µg) foi entregue ao enxerto venoso por bypass de forma perivascular, suspensa em gel Pluronic-F127 a 30%. O grupo tratado apenas com bypass e o grupo tratado com gel veículo carregado apenas com Pluronic funcionaram como controle. Foram feitas comparações com análise de via única de variância e teste post-hoc, com p <0,05 sendo considerado significativo. RESULTADOS: A proliferação celular (índice de PCNA) foi significativamente baixa no grupo tratado com adiponectina em comparação com o grupo de controle e o grupo tratado com o gel veículo na íntima e na adventícia dos enxertos a partir do dia 3 (p <0,01). VCAM-1 e ICAM-1 avaliados por imuno-histoquímica diminuíram significativamente em enxertos venosos tratados com adiponectina na quarta semana (p <0,01). O tratamento de enxertos venosos com gel carregado com adiponectina reduziu a espessura da íntima, da média e da adventícia, em comparação com os enxertos de controle e tratados com gel veículo no dia 28 (p <0,01). CONCLUSÕES: Este estudo oferece evidências adicionais do possível papel terapêutico da adiponectina na modulação de lesão vascular e seu reparo.

A feasible classification method of wet masses to predict pellet formation of powdered herbal slices

The aim of the current study was to explore the correlation between physical properties of wet masses and pellet quality by using powdered herbal slices as model drugs. Wet masses with 100 formulations were prepared by taking 20 kinds of powdered herbal slices as model drugs, microcrystalline cellulose as pelletization aid and five levels of added water as liquid binder. Physical properties of the wet masses such as hardness, adhesiveness, springiness, cohesiveness, chewiness, and resilience were measured by a texture analyzer. Meanwhile, the moisture retention capacities (MRC) of powdered herbal slices and wet masses were determined. Particles were classified after they were produced during spheronization. Principal component analysis, factor analysis and classification analysis were performed on the data. Wet masses could be classified into three groups by taking Ha as the first classification index and Ha/Sp as the second classification index. The correct rate of the classification was 91.00%. If Ha value of wet masses was greater than 15610 g, pellets of type ① would form, otherwise, pellets of type ② or type ③ would form based on Ha/Sp value. Then a classification plot of wet masses was developed to predict pellet formation of powdered herbal slices. Meanwhile, the probable mechanism of pellets formation during spheronisation was concluded in this study, which provided useful information to improve pellet quality

Design, synthesis and anti-tumor activities of carbamate derivatives of cinobufagin.

A series of cinobufagin-3-yl nitrogen-containing-carbamate derivatives were designed, synthesized, and evaluated for their proliferation inhibition activities. The structure-activity relationships suggested that the substituents at C-16 was a crucial factor for the potency and that follows this trends: acetic ester â‰« benzoic ester ≈ hydroxy > carbamate. Compounds 3f, 3g, 3h, and 3i exhibited significant in vitro antiproliferative activities against the eight tested tumor cell lines, with IC50 values ranging from 8.1 to 237.4 nM. Furthermore, 3g tartrate (3g-TA) significantly inhibited tumor growth by 64.5%, 83.9%, and 93.0% at a doses of 4, 6, 8 mg/kg/qod by ip, respectively.

Association between genotype and drug resistance profiles of Mycobacterium tuberculosis strains circulating in China in a national drug resistance survey.

We describe the population structure of a representative collection of 3,133 Mycobacterium tuberculosis isolates, collected within the framework of a national resistance survey from 2007 in China. Genotyping data indicate that the epidemic strains in China can be divided into seven major complexes, of which 92% belonged to the East Asian (mainly Beijing strains) or the Euro-American lineage. The epidemic Beijing strains in China are closely related to the Beijing B0/W148 strain earlier described in Russia and a large cluster of these strains has spread national wide. The density of Beijing strains is high in the whole of China (average 70%), but the highest prevalence was found North of the Yellow river. The Euro-American lineage consists of three sublineages (sublineage_1, 2, and 3) and is more prevalent in the South. Beijing lineage showed the highest cluster rate of 48% and a significantly higher level of resistance to rifampicin (14%, p<0.001), ethambutol (9%, p = 0.001), and ofloxacin (5%, p = 0.011). Within the Euro-American Lineage, sublineage_3 revealed the highest cluster rate (28%) and presented a significantly elevated level of resistance to streptomycin (44%, p<0.001). Our findings suggest that standardised treatment in this region may have contributed to the successful spread of certain strains: sublineage_3 in the Euro-American lineage may have thrived when streptomycin was used without rifampicin for treatment, while later under DOTS based treatment, in which rifampicin plays a key role, Beijing lineage appears to be spreading.

The effect of capsaicin on expression patterns of CGRP in trigeminal ganglion and trigeminal nucleus caudalis following experimental tooth movement in rats

ABSTRACT Objectives The aim of this study was to explore the effect of capsaicin on expression patterns of calcitonin gene-related peptide (CGRP) in the trigeminal ganglion (TG) and trigeminal subnucleus caudalis (Vc) following experimental tooth movement. Material and Methods Male Sprague-Dawley rats were used in this study and divided into small-dose capsaicin+force group, large-dose capsaicin+force group, saline+force group, and no force group. Closed coil springs were used to mimic orthodontic forces in all groups except for the no force group, in which springs were inactivated. Capsaicin and saline were injected into periodontal tissues. Rats were euthanized at 0 h, 12 h, 1 d, 3 d, 5 d, and 7 d following experimental tooth movement. Then, TG and Vc were obtained for immunohistochemical staining and western blotting against CGRP. Results Immunohistochemical results indicated that CGRP positive neurons were located in the TG, and CGRP immunoreactive fibers were distributed in the Vc. Immunohistochemical semiquantitative analysis and western blotting analysis demonstrated that CGRP expression levels both in TG and Vc were elevated at 12 h, 1 d, 3 d, 5 d, and 7 d in the saline + force group. However, both small-dose and large-dose capsaicin could decrease CGRP expression in TG and Vc at 1 d and 3 d following experimental tooth movement, as compared with the saline + force group. Conclusions These results suggest that capsaicin could regulate CGRP expression in TG and Vc following experimental tooth movement in rats.

The effect of capsaicin on expression patterns of CGRP in trigeminal ganglion and trigeminal nucleus caudalis following experimental tooth movement in rats.

Objectives: The aim of this study was to explore the effect of capsaicin on expression patterns of calcitonin gene-related peptide (CGRP) in the trigeminal ganglion (TG) and trigeminal subnucleus caudalis (Vc) following experimental tooth movement. Material and Methods: Male Sprague-Dawley rats were used in this study and divided into small-dose capsaicin+force group, large-dose capsaicin+force group, saline+force group, and no force group. Closed coil springs were used to mimic orthodontic forces in all groups except for the no force group, in which springs were inactivated. Capsaicin and saline were injected into periodontal tissues. Rats were euthanized at 0 h, 12 h, 1 d, 3 d, 5 d, and 7 d following experimental tooth movement. Then, TG and Vc were obtained for immunohistochemical staining and western blotting against CGRP. Results: Immunohistochemical results indicated that CGRP positive neurons were located in the TG, and CGRP immunoreactive fibers were distributed in the Vc. Immunohistochemical semiquantitative analysis and western blotting analysis demonstrated that CGRP expression levels both in TG and Vc were elevated at 12 h, 1 d, 3 d, 5 d, and 7 d in the saline + force group. However, both small-dose and large-dose capsaicin could decrease CGRP expression in TG and Vc at 1 d and 3 d following experimental tooth movement, as compared with the saline + force group. Conclusions: These results suggest that capsaicin could regulate CGRP expression in TG and Vc following experimental tooth movement in rats.

Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori.

Alcohol dehydrogenases (ADH) are a class of enzymes that catalyze the reversible oxidation of alcohols to corresponding aldehydes or ketones, by using either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), as coenzymes. In this study, a short-chain ADH gene was identified in Bombyx mori by 5'-RACE PCR. This is the first time the coding region of BmADH has been cloned, expressed, purified and then characterized. The cDNA fragment encoding the BmADH protein was amplified from a pool of silkworm cDNAs by PCR, and then cloned into E. coli expression vector pET-30a(+). The recombinant His-tagged BmADH protein was expressed in E. coli BL21 (DE3), and then purified by metal chelating affinity chromatography. The soluble recombinant BmADH, produced at low-growth temperature, was instrumental in catalyzing the ethanol-dependent reduction of NAD(+), thereby indicating ethanol as one of the substrates of BmADH.

Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori

Alcohol dehydrogenases (ADH) are a class of enzymes that catalyze the reversible oxidation of alcohols to corresponding aldehydes or ketones, by using either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), as coenzymes. In this study, a short-chain ADH gene was identified in Bombyx mori by 5'-RACE PCR. This is the first time the coding region of BmADH has been cloned, expressed, purified and then characterized. The cDNA fragment encoding the BmADH protein was amplified from a pool of silkworm cDNAs by PCR, and then cloned into E. coli expression vector pET-30a(+). The recombinant His-tagged BmADH protein was expressed in E. coli BL21 (DE3), and then purified by metal chelating affinity chromatography. The soluble recombinant BmADH, produced at low-growth temperature, was instrumental in catalyzing the ethanol-dependent reduction of NAD+, thereby indicating ethanol as one of the substrates of BmADH.