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Nicotinamide mononucleotide (NMN) is one of the key precursors of coenzyme Ⅰ (NAD+). NMN exists widely in a variety of organisms, and β isomer is its active form. Studies have shown that β-NMN plays a key role in a variety of physiological and metabolic processes. As a potential active substance in anti-aging and improving degenerative and metabolic diseases, the application value of β-NMN has been deeply explored, and it is imminent to achieve large-scale production. Biosynthesis has become the preferred method to synthesize β-NMN because of its high stereoselectivity, mild reaction conditions, and fewer by-products. This paper reviews the physiological activity, chemical synthesis as well as biosynthesis of β-NMN, highlighting the metabolic pathways involved in biosynthesis. This review aims to explore the potential of improving the production strategy of β-NMN by using synthetic biology and provide a theoretical basis for the research of metabolic pathways as well as efficient production of β-NMN.
Subject(s)
Nicotinamide Mononucleotide/metabolism , NAD/metabolismABSTRACT
Abstract This study showed the synthesis of Glass ionomer cements (GIC) modified with calcium phosphate nanoparticles (nCaP). The nCaP/GIC were submitted to mechanical compression and diametral tensile tests. The biocomposite were characterized by scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD) and Fourier-transform infrared spectroscopy (FTIR). Cytotoxicity and cell viability tests were performed on the human bone marrow mesenchymal stem cells using a 3-(4,5-dimethylthiazol-2yl)2,5-diphenyl- tetrazolium-bromide assay and LIVE/DEAD assays. Statistically significant differences were observed for mechanical properties (Kruskal-Wallis, p<0.001), nCaP/GIC showed higher resistance to compression and diametral traction. The SEM analyses revealed a uniform distribution nCaP in the ionomer matrix. The EDX and XRD results indicated that hydroxyapatite and calcium β-triphosphate phases. The FTIR spectra revealed the asymmetric band of ν3PO43- between 1100-1030cm-1 and the vibration band associated with ν1PO43- in 963cm-1 associated with nCaP. The nCaP/GIC presented response to adequate cell viability and non-cytotoxic behavior. Therefore, the new nCaP/GIC composite showed great mechanical properties, non-cytotoxic behavior, and adequate response to cell viability with promising dental applications.
Resumo Este estudo apresenta a síntese de cimentos de ionômero de vidro (GIC) modificados com nanopartículas de fosfato de cálcio (nCaP). Os nCaP / GIC foram submetidos a ensaios mecânicos de compressão e tração diametral. Os biocompósitos foram caracterizados por microscopia eletrônica de varredura (MEV), espectroscopia de energia dispersiva de raios-X (EDX), difração de raios-X (XRD) e espectroscopia de infravermelho com transformada de Fourier (FTIR). Os testes de citotoxicidade e viabilidade celular foram realizados em células-tronco mesenquimais da medula óssea humana usando um ensaio de 3- (4,5-dimetiltiazol-2-il) 2,5-difeniltetrazólio-brometo e ensaios LIVE / DEAD. Diferenças estatisticamente significativas foram observadas para as propriedades mecânicas (Kruskal-Wallis, p <0,001), nCaP / GIC apresentou maior resistência à compressão e tração diametral. As análises de SEM revelaram uma distribuição uniforme de nCaP na matriz do ionômero. Os resultados de EDX e DRX indicaram fases de hidroxiapatita e β-trifosfato de cálcio. Os espectros de FTIR revelaram a banda assimétrica de ν3PO4 3- entre 1100-1030cm-1 e a banda de vibração associada a ν1PO4 3- em 963cm-1 associada a nCaP. O nCaP / GIC apresentou resposta adequada à viabilidade celular e comportamento não citotóxico. Portanto, o novo compósito nCaP / GIC apresentou ótimas propriedades mecânicas, comportamento não citotóxico e resposta adequada à viabilidade celular com promissoras aplicações odontológicas.
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Objetivo: Analizar los indicadores hematológicos en pacientes con infección por SARS COV-2. Método: Observacional de tipo descriptiva. Resultados: Se ha encontrado la existencia de un patrón común de exámenes anormales del recuento leucocitario con Neutrófilos elevados en 43.69 % (neutrofilia) y los linfocitos, pero por debajo de los valores normales (linfopenia) en un 20.16 %. Además de monocitopenia en un 13.44%. Conclusión: La detección de Proteína C reactiva elevada y neutrofilia se relaciona con casos severos de COVID-19 en pacientes adultos. Ambas pruebas combinadas tienen una alta especificidad y sensibilidad para predicción temprana de casos se veros de COVID -19.
Objective: To analyze hematological indicators in patients with SARS COV-2 infection. Methods: Descriptive observational study. Results: A common pattern of abnormal leukocyte count tests was found with elevated neutrophils in 43.69% (neutrophilia) and lymphocytes, but below normal values (lymphopenia) in 20.16%. In addition to monocytopenia in 13.44%. Conclusion: The detection of elevated C-reactive protein and neutrophilia is associated with severe cases of COVID-19 in adult patients. Both tests combined have high specificity and sensitivity for early prediction of severe cases of COVID -19.
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Most bacterial cell surface glycans are structurally unique, and have been considered as ideal target molecules for the developments of detection and diagnosis techniques, as well as vaccines. Chemical synthesis has been a promising approach to prepare well-defined oligosaccharides, facilitating the structure-activity relationship exploration and biomedical applications of bacterial glycans. L-Galactosaminuronic acid is a rare sugar that has been only found in cell surface glycans of gram-negative bacteria. Here, an orthogonally protected L-galactosaminuronic acid building block was designed and chemically synthesized. A synthetic strategy based on glycal addition and TEMPO/BAIB-mediated C6 oxidation served well for the transformation of commercial L-galactose to the corresponding L-galactosaminuronic acid. Notably, the C6 oxidation of the allyl glycoside was more efficient than that of the selenoglycoside. In addition, a balance between the formation of allyl glycoside and the recovery of selenoglycoside was essential to improve efficiency of the NIS/TfOH-catalyzed allylation. This synthetically useful L-galactosaminuronic acid building block will provide a basis for the syntheses of complex bacterial glycans.
Subject(s)
Carbohydrates , Glycosides , Oligosaccharides , Oxidation-Reduction , Polysaccharides/chemistryABSTRACT
Bacterial surface glycans perform a diverse and important set of biological roles, and have been widely used in the treatment of bacterial infectious diseases. The majority of bacterial surface glycans are decorated with diverse rare functional groups, including amido, acetamidino, carboxamido and pyruvate groups. These functional groups are thought to be important constituents for the biological activities of glycans. Chemical synthesis of glycans bearing these functional groups or their variants is essential for the investigation of structure-activity relationships by a medicinal chemistry approach. To date, a broad choice of synthetic methods is available for targeting the different rare functional groups in bacterial surface glycans. This article reviews the structures of naturally occurring rare functional groups in bacterial surface glycans, and the chemical methods used for installation of these groups.
Subject(s)
Humans , Bacterial Infections , Polysaccharides/chemistry , Structure-Activity RelationshipABSTRACT
Objective:To establish an optimized automatic synthesis method of 18F-fallypride, and evaluate its biodistribution and microPET/CT characteristics. Methods:18F-fallypride was automatically prepared by AIO synthesis module and disposable cassette & reagents kit. The crude product was purified by a dedicated coupled column (HLB+ Alumin-N) to obtain the final product. Radiochemical purity and radiolabeling yield were determined. Parkinson′s disease (PD) model mice and rats were established. The radioactive distribution of different organs of PD model mice ( n=24) were monitored. The distribution process of the agent in the SD rat brain (PD model, n=6; normal rat, n=6) were evaluated by microPET/CT imaging. Results:The radiochemical yield of 18F-fallypride synthesized by automatic synthesis module was stable at (10±1)% ( n=5, no decay corrected). The total synthesis time was about 40 min. The radiochemical purity of 18F-fallypride was more than 95%, and the radiochemical purities were also over 95% after being stored in saline and serum for 120 min at room temperature. 18F-fallypride was mainly excreted by the kidneys, and it was less radioactive intake in the liver and spleen in PD mice. MicroPET/CT imaging showed that higher accumulation of 18F-fallypride was noted in corpus striatum and the SUV ratio of PD group was lower than that of control group (5.00±0.93 vs 6.53±1.96). Conclusion:18F-fallypride can be successfully prepared automatically by improved multifunctional module, with the advantages of convenient preparation, stable radiochemical yield, satisfying purity and quality control, so it can be used in the follow-up standardized production of Good Manufacture Practice (GMP) system.
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Indanomycins are a class of secondary metabolites of microorganisms with a trans-tetrahydroindan (indane) ring. These compounds generally have good antibacterial, insecticidal and antitumor biological activities, which have caused wide interest for medicinal chemists and biologists. This review summarizes the research progress of the discovery, biological activity, chemical synthesis and biosynthesis of indanomycin compounds since 1979 and provides scientific reference for the research and development of indanomycin antibiotics.
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OBJECTIVE@#To improve the methods to synthesize and purify of optical-magnetic bimodal molecular probe of Gd-[4, 7-Bis-carboxymethyl-10-(2-fluorescein thioureaethyl)-1, 4, 7, 10-tetraaza-cyclododec-1-yl]-acetic acid complexes.@*METHODS@#Target compound (7), optical-magnetic bimodal molecular molecular probe, was synthesized by the use of 1, 4, 7, 10-tetraazacyclododecane (1) as starting material via substitution reaction, hydrolysis reaction, coupling reaction and complexation reaction with metal.@*RESULTS@#The synthetic route of Gd-[4, 7-Bis-carboxymethyl-10-(2-fluoresceinthioureaethyl)-1, 4, 7, 10-tetraaza-cyclododec-1-yl]-acetic acid complexes was improved. The optical-magnetic bimodal molecular probes were synthesized by substitution reaction, hydrolysis reaction, coupling reaction and complex reaction with metal respectively. For the improved route, the total yield could reach 34.6% which was higher than the original route (18.0%). The structures of those compounds were identified by 1H nuclear magnetic resonance, 13C nuclear magnetic resonance, and mass spectrometry. The improved route could avoid the uncontrollable disadvantage of the substitution reaction, this process could reduce the formation of impurities and made the purification process easier, and in the aspect of purification and separation, the preparative high-performance liquid chromatography with less sample loading and high cost was improved to a column chromatography with many sample loads and being easy to operate. Therefore, the use of column chromatography could be more conducive to mass production of the optical-magnetic bimodal molecular molecular probe.@*CONCLUSION@#The improved synthetic route improves the controllability of the reaction conditions and makes it easier to purify and separate the compounds. At the same time, the improved synthetic route can increase the total yield significantly. The optical-magnetic bimodal molecular probe can combine the living magnetic resonance imaging with the in vitro optical imaging to realize the dual synchronous detection of magneto-optics, so that the detection results of the living magnetic resonance imaging and the in vitro optical imaging are mutually verified. In other words, this synthetic optical-magnetic bimodal molecular probe will make the experimental results more accurate and reliable. In subsequent biological experimental studies, the optical-magnetic bimodal molecular probe can be applied to related research of brain structure and function, and the probe can be used for the brain-related diseases researches, such as brain tumors. after intravenous administration, and thus the optical-magnetic bimodal molecular probe can play an important role in medical treatment of brain tumors and cerebrovascular diseases.
Subject(s)
Acetic Acid , Brain , Magnetic Resonance Imaging , Magnetic Resonance Spectroscopy , Molecular ProbesABSTRACT
Objective@#To synthesize and analyze 18F-prostate specific membrane antigen (PSMA)-1007, followed by its imaging in prostate cancer.@*Methods@#Based on one-step method, 18F-PSMA-1007 was produced by the CFN-MPS-200 automatic synthesis module and its quality analysis was conducted. 18F-PSMA-1007 PET/CT imaging was performed in a prostate cancer patient (66 years old).@*Results@#The synthesis time of 18F-PSMA-1007 was about 50 min, and the radiochemical yield was (25.0±5.0) % (attenuation correction, n=3). The radiochemical purity was above 99.0% and was above 98.0% after 6 h. The product was colorless transparent solution with pH value of 7.0-7.5, and the specific activity was (410.0±11.0) MBq/ml and the radioactive nuclear purity was above 99.0%. PET/CT imaging in the patient showed that 18F-PSMA-1007 was highly concentrated in prostate cancer with maximum standardized uptake value (SUVmax) of 40.9.@*Conclusion@#Based on CFN-MPS-200 multifunction synthesis module, 18F-PSMA-1007 can be stably synthesized with high radiochemical yield and can be concentrated in prostate cancer.
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Objective To synthesize and analyze 18 F-prostate specific membrane antigen ( PSMA)-1007, followed by its imaging in prostate cancer. Methods Based on one-step method, 18 F-PSMA-1007 was produced by the CFN-MPS-200 automatic synthesis module and its quality analysis was conducted. 18 F-PSMA-1007 PET/CT imaging was performed in a prostate cancer patient (66 years old). Results The syn-thesis time of 18F-PSMA-1007 was about 50 min, and the radiochemical yield was (25.0±5.0) ％ (attenua-tion correction, n=3). The radiochemical purity was above 99.0％ and was above 98.0％ after 6 h. The product was colorless transparent solution with pH value of 7.0-7.5, and the specific activity was (410.0± 11.0) MBq/ml and the radioactive nuclear purity was above 99.0％. PET/CT imaging in the patient showed that 18 F-PSMA-1007 was highly concentrated in prostate cancer with maximum standardized uptake value ( SUVmax ) of 40.9. Conclusion Based on CFN-MPS-200 multifunction synthesis module, 18 F-PSMA-1007 can be stably synthesized with high radiochemical yield and can be concentrated in prostate cancer.
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Objective@#To synthesis 177Lu-prostate specific membrane antigen (PSMA)-I&T with automated module, evaluate the biodistribution and pharmacokinetics in mice and study the targeting property in human prostate cancer cell line LNCaP Clone FGC.@*Methods@#The iQS-TS automated module was applied in labeling 177Lu-PSMA-I&T. Radiochemical purity and stability were determined with high performance liquid chromatography (HPLC). The biodistribution was observed in normal ICR mice and U-SPECT/CT imaging was performed in LNCaP Clone FGC tumor-bearing mice. Independent-sample t test was used to analyze the data.@*Results@#177Lu-PSMA-I&T was stable in vitro and in vivo, with the radiolabeled yield of (91.5±4.9)% and radiochemical purity >99%. The half maximal inhibitory concentration (IC50) of 177Lu-PSMA-I&T binding to LNCaP Clone FGC cells was (26.74±3.53) nmol/L. The uptake of 177Lu-PSMA-I&T by LNCaP Clone FGC cells increased with time and significantly decreased after the inhibitor addition (t values: 4.301-27.483, all P<0.05). 177Lu-PSMA-I&T was cleared from blood rapidly and predominantly excreted by kidneys. Significant radioactive uptake was observed in tumors with a long retention time.@*Conclusion@#177Lu-PSMA-I&T can be produced in a convenient and efficient procedure using iQS-TS automated module, with good biological properties and excellent affinity and targeting property towards prostate cancer cells, which making it a potential radiopharmaceutical for prostate cancer therapy.
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Bacterial resistance is an increasingly serious problem.Therefore,it is necessary to find new antibacterial drugs.Flavonoids are a class of polyphenolic chemicals with low molecular weight,which exhibit many beneficial properties for human health by the interaction of cellular targets,such as the key cell signaling pathways.Flavonoids have been extensively studied as one of the important sources of antimicrobial compounds.At present,antibacterial flavonoids can be divided into two categories.One is the natural,extracted from the plant or microbial secondary metabolites.The other is the product of chemical synthesis,i.e.the derivatives of flavonoids or chalcone.In this paper,the progress of antibacterial flavonoids by natural extraction and chemical synthesis were summarized.
ABSTRACT
Bacterial resistance is an increasingly serious problem.Therefore,it is necessary to find new antibacterial drugs.Flavonoids are a class of polyphenolic chemicals with low molecular weight,which exhibit many beneficial properties for human health by the interaction of cellular targets,such as the key cell signaling pathways.Flavonoids have been extensively studied as one of the important sources of antimicrobial compounds.At present,antibacterial flavonoids can be divided into two categories.One is the natural,extracted from the plant or microbial secondary metabolites.The other is the product of chemical synthesis,i.e.the derivatives of flavonoids or chalcone.In this paper,the progress of antibacterial flavonoids by natural extraction and chemical synthesis were summarized.
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La biopelícula como un mecanismo de virulencia en Staphylococcus involucrada en infecciones intrahospitalarias es regulada por un represor negativo icaR, responsable de la transcripción completa del operón icaADBC. La búsqueda de dominios funcionales por modulación computacional de icaR permitió hallar las secuencias peptídicas con actividad biológica análoga a la proteína icaR. Mediante biología computacional se diseñaron péptidos empleando el programa de predicción AntiBP (http://www.imtech.res.in/raghava/antibp/); la síntesis química se hizo por Nα-Fmoc y se caracterizaron y purificaron tres moléculas por RP-HPLC y MALDI-TOF. Se evaluó su seguridad biológica mediante ensayo de actividad citotóxica realizada sobre macrófagos murinos de la línea J774 y la actividad hemolítica se determinó mediante el uso de glóbulos rojos. Los tres péptidos caracterizados IR1, IR2 e IR3, presentaron estructura secundaria predominantemente alfa helicoidal, alto grado de pureza y alto score antimicrobiano; además, mostraron baja toxicidad, evidenciada por la actividad citotóxica y hemolítica en las concentraciones ensayadas y en comparación con los controles usados, que permitiría su potencial uso como moléculas candidatas o principios activos con actividad análoga al represor nativo icaR, frente a la biopelícula de los Staphylococcus sp.
Staphylococcus sp. biofilm, formed as a mechanism of virulence that is involved in hospital acquired infections, is regulated by a negative repressor icaR, which is responsible for the full transcription of the operon icaADBC. This study, through functional commands by computational modulation of icaR, allowed to find peptide sequences with similar biological activity to the icaR protein. Peptides were designed by means of computational biology using the prediction program AntiBP (http://www.imtech.res.in/raghava/antibp/). The chemical synthesis of peptides was performed by Nα-Fmoc. The purification and characterization of three molecules were carried out using RP-HPLC and MALDI-TOF. Biological safety of peptides was evaluated by tests of cytotoxic activity on murine macrophage cells line J774, and their hemolytic activity was determined by using red cells. The three characterized peptides IR1, IR2 and IR3 presented a predominantly secondary alpha helical structure with a high degree of purity and high antimicrobial scores. In addition, the peptides exhibited low toxicity, proved by their low cytotoxic and hemolytic activity in the tested concentrations and in comparison to the standards used. These results allow the potential use of these peptides as candidate molecules or active principles with similar activity to the native repressor icaR against the Staphylococcus biofilm.
O biofilme formado como um mecanismo de virulência em Staphylococcus sp., que está envolvido com infecções intra-hospitalares, é regulado por um repressor negativo icaR, o qual é responsável pela plena transcrição do operão icaADBC. Por tanto, o presente estudo, avaliando a segurança biológica de moléculas, concebeu peptídeos antibiofilme semelhantes ao repressor icaR. Por meio da biologia computacional foram concebidos peptídeos usando o programa de predição AntiBP (http://www.imtech.res.in/raghava/antibp/) para identificar as sequências com uma atividade biologicamente similar à da proteína icaR. A Síntese química dos peptídeos se fez pelo Nα-Fmoc e foram caracterizadas e purificadas três moléculas por RP-HPLC e MALDI-TOF. O ensaio de atividade citotóxica foi realizado nos macrófagos murinos da linha J774 e a atividade hemolítica foi determinada por meio do uso de glóbulos vermelhos. Foram caraterizados três peptídeos IR1, IR2 e IR3, além de mostrarem uma estrutura secundaria predominantemente alfa helicoidal, com alto grau de pureza, alto score antimicrobiano e baixa toxicidade, estes podem ser postulados como moléculas candidatas ou princípios ativos com uma atividade similar ao repressor nativo icaR frente ao biofilme dos Staphylococcus sp.
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Polypyrrole (PPy) and polypyrrole/polyethylene glycol (PPy/PEG) implants synthesized by chemical, electro-chemical, and plasma polymerization methods were implanted into the injured spinal cord of rats to determine their effect on motor function recovery. Before implantation, the materials were characterized by infrared (IR) spectroscopy. An experimental model of traumatic spinal cord injury (TSCI) by complete transection at thoracic level 9, in rats was used. The polymer implants were inserted immediately after transection. Motor function recovery was evaluated once a week during 5 weeks using the Basso, Beattie and Bresnahan (BBB) motor scale. Histological evaluation was done at the end of the recovery evaluation period using hematoxylin/eosin stain. Results showed that animals implanted with polymers synthesized by plasma had a better integration into the nerve tissue, less inflammatory response and a better functional recovery than animals implanted with polymers synthesized by chemical or electrochemical methods.
En el presente trabajo se comparó el efecto de implantes poliméricos derivados del pirrol (polipirrol o PPy) y del copolímero polipirrol/polietilenglicol (PPy/PEG), obtenidos por diferentes métodos de síntesis: químico, electroquímico y polimerización por plasma con el propósito de determinar si el método de síntesis puede influir sobre el efecto que producen al ser implantados después de una lesión traumática de la médula espinal de ratas. Antes de realizar el implante, las características químicas y estructurales de los polímeros fueron analizadas por espectroscopia de infrarrojo (IR). Se utilizó un modelo experimental de lesión traumática de médula espinal (LTME) por sección completa en ratas. La LTME se realizó a nivel torácico 9 y el polímero fue implantado de inmediato en la zona de lesión. La recuperación de la función motora se evaluó mediante la escala Basso, Beattie y Bresnahan (BBB) una vez por semana durante 5 semanas. La evaluación histológica se realizó al término del seguimiento con la tinción de hematoxilina/eosina. Los resultados muestran que los animales implantados con polímeros sintetizados por plasma se integraron mejor al tejido nervioso, redujeron la respuesta inflamatoria y favorecieron una mayor recuperación funcional en comparación con los animales implantados con materiales sintetizados por métodos químicos o electroquímicos.
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Background:The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin.Methods:The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider Brachypelma albiceps.It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a+. Each vector was transfected into E. coli M15 and BL21 cells, respectively, and expressed under induction with isopropyl thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer.Results:Both expression systems pQE40 and pET28a+ expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 μg/L and 900 μg/L of culture medium. HisrBa1 was reduced and folded under in vitroconditions. The in vitro folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand, Ba1 was chemically synthesized (sBa1) with a yield of 11 mg per 0.1 mmol of amino acid assembly.Conclusions:The two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.(AU)
Subject(s)
Animals , Spiders , Protein Isoforms , Isopropyl Thiogalactoside , Neurotoxins , In Vitro Techniques , Polymerase Chain ReactionABSTRACT
Background: The choice between heterologous expression versus chemical synthesis for synthesizing short cysteine-rich insecticidal peptides from arthropods may impact the obtainment of yields and well-folded bioactive molecules for scientific research. Therefore, two recombinant expression systems were compared to that of chemical synthesis for producing Ba1, a cysteine-rich spider neurotoxin. Methods: The transcription of the insecticidal neurotoxin Ba1 was obtained from a cDNA library of venom glands of the spider Brachypelma albiceps.It was cloned into the pCR®2.1-TOPO® cloning vector and then introduced in two different expression vectors, pQE40 and pET28a+. Each vector was transfected into E. coli M15 and BL21 cells, respectively, and expressed under induction with isopropyl thiogalactoside (IPTG). The chemical synthesis of Ba1 was performed in an Applied Biosystems 433A peptide synthesizer. Results: Both expression systems pQE40 and pET28a+ expressed the His-tagged recombinant protein products, HisrDFHRBa1 and HisrBa1, respectively, as inclusion bodies. The recombinant proteins HisrDFHRBa1 and HisrBa1 presented respective molecular masses of 28,289 and 8274.6 Da, and were not biologically active. These results suggested that both HisrDFHRBa1 and HisrBa1 were oxidized after cell extraction, and that their insecticidal activities were affected by their N-terminal pro-peptides and different disulfide bridge arrangements. The respective protein expression yields for HisrDFHRBa1 and HisrBa1 were 100 μg/L and 900 μg/L of culture medium. HisrBa1 was reduced and folded under in vitroconditions. The in vitro folding of HisrBa1 produced several isoforms, one of which, after removing its N-terminal pro-peptide by enzymatic cleavage, presented elevated insecticidal activities compared to the native Ba1. Furthermore, the His-tagged protein HisrDFHRBa1 underwent enzymatic cleavage to obtain recombinant Ba1 (rBa1). As expected, the molecular mass of rBa1 was 4406.4 Da. On the other hand, Ba1 was chemically synthesized (sBa1) with a yield of 11 mg per 0.1 mmol of amino acid assembly. Conclusions: The two recombinant insecticidal peptides and the one synthesized chemically were as active as the native Ba1; however, toxin yields differed drastically.
Subject(s)
Animals , Spiders , Cysteine , Insecticides , PeptidesABSTRACT
Objective To develop an efficient and rapid method to synthesize 68 Ga?DOTA?TATE, and to perform PET/CT imaging on neuroendocrine tumor (NET) bearing mice and one patient. Methods 68GaCl3 was eluted by 0.05 mol/L HCl in 68 Ge?68 Ga generator, then added to pre?adjusted DOTA?TATE buffer, and heated for 15 min at 85 ℃, then filtered with 0.22 μm microfiltration membrane. The radiochemical purity and in vitro stability of 68 Ga?DOTA?TATE were analyzed with radio?HPLC. The biodistribution and PET/CT imaging on HT?29 colon cancer xenografts mice model were performed. After approved by Peking University Cancer Hospital Ethics Committee, a case of NET patient received 148 MBq 68 Ga?DOTA?TATE intravenous?ly, followed by a whole?body scan at 60 min post?injection. Results 68 Ga?DOTA?TATE was prepared in high radiochemical purity (>98%) and with specific activity of 55 GBq/μmol in 25 min. The tracer showed excellent in vitro stability and no acute toxicity. PET/CT imaging on mice model showed positive uptake in tumor tissues. PET/CT imaging on one NET patient showed positive uptake of 68 Ga?DOTA?TATE in primary and metastases lesions. Conclusions A rapid, high radiolabeling yield and high specific activity method to prepare 68 Ga?DOTA?TATE is established. 68 Ga?DOTA?TATE could be specifically uptaken by somatostatin receptor ( SSTR) positive NET with high affinity. It could serve as a useful tool for early diagnosis of SSTR positive NET and has clinical prospective for somatostatin?mediated targeting therapy.
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Maca (Lepidium meyenii Walp.) has been used as both food and traditional medicine for centuries for its high nutritional value and multi-medicinal properties. Macamides, the marker compounds of maca, are a series of nonpolar, long chain fatty acid N-benzylamides. Most of them have neuroprotective and neuroregulation effects. In this paper, we reviewed the structures of macamides, methods of chemical synthesis, and quantitative determination, and their bioactivities to provide a reference for further research and development.
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Objective To synthesize 11 C-methyldopamine (MDA) and to explore its feasibility as an agent for cardiac sympathetic nerve imaging.Methods 11 C-MDA was synthesized by direct N-methylation method and purified by semi-preparation reverse HPLC.Thirty Kunming mice were divided into five groups by random number table.The mice were respectively sacrificed at 2,5,10,20 and 30 min after injection of 7.4 MBq 11C-MDA.The lung,liver,spleen,kidney,stomach,intestine,brain,muscle,bone tissues and blood of mice were removed and weighed before radioactive γ-counting.The ％ID/g was calculated.Six Chinese mini-swine were divided into normal group (n=3) and inhibition group (n =3) for myocardial imaging.Mini-swine of inhibition group were injected with 10 mg/kg imipramine hydrochloride at 30 min before 11C-MDA (370 MBq) injection.The data were analyzed with SPSS 15.0 software.Results The synthesis of 11 C-MDA took 45 min with radiochemical yields of (20±3)％.The solution of11 C-MDA was colorless and the pH value was 6.5.The radiochemical purity was more than 98％ and the specific activity was 50 GBq/mmol.The myocardial uptake reached the peak value of (8.78± 1.18) ％ID/g at 2 min after injection of 11 C-MDA in mice.11C-MDA was mainly metabolized through liver and kidney.PET/CT imaging showed that 11 C-MDA was highly uptaken in swine myocardium and could be blocked by imipramine hydrochloride.Conclusions 11C-MDA can be synthesized by a simple and economic method.The high uptake rate of 11 C-MDA in the heart suggests it may be a potential agent for cardiac nerve imaging.