RESUMEN

Vglycin, a novel natural polypeptide isolated from pea seeds, possesses antidiabetic properties. Our previous studies have shown that Vglycin can induce the differentiation of human colon adenocarcinoma cells. We aimed to determine the anticancer activity of Vglycin against colon cancer cells and to elucidate related apoptosis-inducing mechanisms. Treatment with purified Vglycin significantly reduced growth, viability, and colony formation of CT-26, SW480, and NCL-H716 colon cancer cells in a dose-dependent manner while down-regulating the expression of proliferating cell nuclear antigen. Mouse xenograft studies showed a 38% inhibition of colon cancer growth in mice treated with Vglycin (20 mg/kg/day) at day 21. Furthermore, the potential mechanisms involved in Vglycin-induced cell apoptosis were examined using cell cycle studies, ultrastructural examination, as well as apoptosis-associated pathway analysis. The results showed that Vglycin significantly promoted apoptosis and G1/S phase cell cycle arrest. As revealed by Western blot, the expression of CDK2 and Cyclin D1 was down-regulated in all three Vglycin-treated colon cancer cells, indicating that the CDK2/Cyclin D1 cell cycle pathway involved in the initiation and progression of colon cancer. Moreover, the inhibition of Vglycin-induced cell proliferation in colon cancer cells was accompanied by alteration of the expression levels of the apoptosis-related proteins Bax, Bcl-2 and Mcl-1, and an increase of caspase-3 activity. Together, our results suggest that Vglycin may be another plant-derived peptide that suppresses colon cancer, supporting the continued investigation of Vglycin as therapeutic agent for colon cancer. Impact statement The antidiabetic properties and the capability of inducing differentiation of human colon adenocarcinoma cells of Vglycin have been reported in our previous studies. However, the anticancer potential of Vglycin on colon cancer cells and its possible related mechanisms were still unknown. In this study, we found that Vglycin could reduce growth, viability, and colony formation or colony size of CT-26, SW480, and NCL-H716 colon cancer cells. Moreover, Vglycin decreased tumor volume by 38% in xenograft mice transplanted with CT-26 cells. The mechanisms of these phenomena may be due to the down-regulated CDK2 and Cyclin D1, G1/S phase cell cycle arrest, and the dysregulated expression of Bax, Bcl-2, and Mcl-1. The findings highlight the anticancer potential of Vglycin against colon cancer cells, and suggest Vglycin may be another colon cancer potential suppressive component of plant-derived peptides.

Asunto(s)

Antineoplásicos/farmacología , Neoplasias del Colon/tratamiento farmacológico , Células Epiteliales/efectos de los fármacos , Proteínas de Soja/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Células Epiteliales/fisiología , Xenoinjertos/patología , Humanos , Ratones , Resultado del Tratamiento

RESUMEN

Allograft inflammatory factor-1 (AIF-1) plays an important role in various inflammatory conditions. Our previous study demonstrated that AIF-1 was over-expressed in the liver of BALB/c mice infected with Schistosoma japonicum and played significant role in the pathogenesis of schistosomiasis. The aim of this study was to focus on the effect of AIF-1 treatment on liver fibrosis and necrosis of BALB/c mice infected with S. japonicum. Seventy-two BALB/c mice were infected with cercariae of S. japonicum and then divided into three groups: AIF-1-treated group, saline-treated group, and control group. The vital signs, liver function, egg load, and hepatic pathological changes of the mice were assessed, and the levels of AIF-1 and TNF-α in the liver and spleen were measured at 5, 8, and 14 weeks postinfection. The treatment of AIF-1 on the mice infected with S. japonicum suppressed the expression of TNF-α and increased the effectiveness of AIF-1 in the liver and spleen at 14 weeks postinfection. Histopathological analysis and Masson trichrome staining for the liver tissues showed that the liver fibrosis and necrosis were alleviated previously compared with other infected mice at 14 weeks postinfection. The treatment of AIF-1 on the mice infected with S. japonicum can alleviate hepatic fibrosis and necrosis which indicate that AIF-1 use may prevent and cure the liver fibrosis.

Asunto(s)

Proteínas de Unión al Calcio/metabolismo , Proteínas de Unión al ADN/farmacología , Cirrosis Hepática/tratamiento farmacológico , Hígado/efectos de los fármacos , Proteínas de Microfilamentos/metabolismo , Esquistosomiasis Japónica/tratamiento farmacológico , Animales , Proteínas de Unión al ADN/genética , Femenino , Expresión Génica , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/metabolismo , Cirrosis Hepática/mortalidad , Cirrosis Hepática/parasitología , Ratones , Ratones Endogámicos BALB C , Recuento de Huevos de Parásitos , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Schistosoma japonicum/efectos de los fármacos , Schistosoma japonicum/crecimiento & desarrollo , Schistosoma japonicum/patogenicidad , Esquistosomiasis Japónica/metabolismo , Esquistosomiasis Japónica/mortalidad , Esquistosomiasis Japónica/parasitología , Bazo/efectos de los fármacos , Bazo/metabolismo , Bazo/patología , Análisis de Supervivencia , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/biosíntesis

RESUMEN

Direct metal-free C-4-selective indolation of pyridines is achieved for the first time using TEMPO and (Boc)2O. A variety of substituents on both indoles and pyridines are tolerated to give 3-(pyridin-4-yl)-1H-indole derivatives in moderate to excellent yields. This finding provides a novel approach for developing metal-free C-H functionalization of pyridines.

Asunto(s)

Carbonatos/química , Óxidos N-Cíclicos/química , Indoles/química , Metales/química , Piridinas/química , Alquilación , Oxidación-Reducción , Estereoisomerismo

RESUMEN

An 84-residue bactericidal peptide, PSK, was purified from a Chrysomya megacephala fly larvae preparation. Its amino acid sequence is similar to that of a previously reported larval peptide of the Drosophila genus (SK84) noticed for its anticancer and antimicrobial properties. The PSK sequence is also homologous to mitochondrial ATPase inhibitors from insects to humans (35-65% sequence identity), indicating an intracellular protein target and possible mechanism for PSK. It contains a cluster of six glycine residues, and has several two- and three-residue repeats. It is active against both Gram-positive and Gram-negative bacteria via a mechanism apparently involving cell membrane disintegration and inhibition of ATP hydrolysis. In addition, PSK induces an inward cationic current in pancreatic ß cells. Together, the findings identify a bioactive peptide of the ATPase inhibitor family with specific effects on both prokaryotic and mammalian cells.

Asunto(s)

Antibacterianos/farmacología , Fenómenos Fisiológicos Bacterianos/efectos de los fármacos , Dípteros/metabolismo , Células Secretoras de Insulina/fisiología , Activación del Canal Iónico/fisiología , Proteínas/farmacología , Secuencia de Aminoácidos , Animales , Antibacterianos/química , Línea Celular , Humanos , Hidrólisis , Células Secretoras de Insulina/efectos de los fármacos , Activación del Canal Iónico/efectos de los fármacos , Larva/metabolismo , Datos de Secuencia Molecular , Proteínas/química , Inhibidores de la Bomba de Protones/química , Inhibidores de la Bomba de Protones/farmacología , Relación Estructura-Actividad , Proteína Inhibidora ATPasa

RESUMEN

A general and efficient method for the cross-coupling of indoles with ß-keto esters by using TEMPO/CuSO4·5H2O in air as oxidant has been developed. This reaction features high functional-group compatibility and an excellent selectivity. This methodology provides an alternative approach for the ketonization-olefination of indoles in moderate to good yields.

Asunto(s)

Alquenos/síntesis química , Cobre/química , Indoles/química , Cetonas/síntesis química , Alquenos/química , Catálisis , Óxidos N-Cíclicos/química , Ésteres/química , Cetonas/química , Estructura Molecular , Estereoisomerismo

RESUMEN

Allograft Inflammatory Factor-1 (AIF-1) is a 17kDa cytoplasmic, calcium-binding, inflammation-responsive scaffold protein that is mainly expressed in immunocytes. AIF-1 influences the immune system at several key points and thus modulates inflammatory diseases. AIF-1 boosts the expression of inflammatory mediators such as cytokines, chemokines, inducible nitric oxide synthase (iNOS) and promotes inflammatory cell proliferation and migration. Here we provide an overview of the different pathological processes regulated by AIF-1 mainly including allograft rejection, autoimmune diseases, central nervous system (CNS) injury, vasculopathy and cancer et al.

Asunto(s)

Enfermedades Autoinmunes/inmunología , Proteínas de Unión al ADN/inmunología , Rechazo de Injerto/inmunología , Inflamación/inmunología , Secuencia de Aminoácidos , Animales , Enfermedades Autoinmunes/genética , Secuencia de Bases , Proteínas de Unión al Calcio , Proteínas de Unión al ADN/genética , Rechazo de Injerto/genética , Humanos , Inflamación/genética , Proteínas de Microfilamentos , Datos de Secuencia Molecular , Alineación de Secuencia

RESUMEN

Free heme is potentially cytotoxic, particularly in the presence of oxidants or activated phagocytes. Daintain/AIF-1 (allograft inflammatory factor-1) is a macrophage factor that has been implicated in the regulation of inflammation. In the present study, daintain/AIF-1 was found to induce cytolysis of erythrocytes, resulting in heme release in vitro. Furthermore, the interacting protein of daintain/AIF-1 was purified by daintain/AIF-1-6 histidine antigen fusion protein nickel affinity chromatography. MALDI-TOF-MS analysis identified hemoglobin subunit beta-1 as an interacting protein of daintain/AIF-1.These data suggest that daintain/AIF-1 may be involved in heme-associated diseases.

Asunto(s)

Proteínas de Unión al Calcio/fisiología , Eritrocitos/metabolismo , Hemo/metabolismo , Hemoglobinas/metabolismo , Proteínas de Microfilamentos/fisiología , Animales , Cromatografía de Afinidad , Ratones , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción

RESUMEN

The title compound, C(18)H(14)O(2), is almost planar with a dihedral angle of 1.24 (2)° between the phenyl-ethynyl and styryl groups. The acet-oxy group is tilted by 82.46 (2) and 82.26 (3)° with respect to the benzene ring planes.

RESUMEN

A large body of experimental evidence suggests that cytokines trigger pancreatic ß-cell death in type 1 diabetes mellitus. Daintain/AIF-1 (Allograft Inflammatory Factor-1), a specific marker for activated macrophages, is accumulated in the pancreatic islets of pre-diabetic BB rats. In the present study, we demonstrate that daintain/AIF-1 is released into blood and the levels of daintain/AIF-1 in the blood of type 1 diabetes-prone non-obese diabetic (NOD) mice suffering from insulitis are significantly higher than that in healthy NOD mice. When injected intravenously into NOD mice, daintain/AIF-1 stimulates white blood cell proliferation, increases the concentrations of blood glucose, impairs insulin expression, up-regulates nitric oxide (NO) production in pancreases and accelerates diabetes in NOD mice, while the antibody against daintain/AIF-1 delays or prevents insulitis in NOD mice. These results imply daintain/AIF-1 triggers type 1 diabetes probably via arousing immune cells activation and induction of NO production in pancreas of NOD mice.

Asunto(s)

Proteínas de Unión al Calcio/metabolismo , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Tipo 1/inmunología , Células Secretoras de Insulina/inmunología , Ratones Endogámicos NOD/inmunología , Proteínas de Microfilamentos/metabolismo , Animales , Glucemia , Proteínas de Unión al Calcio/sangre , Proteínas de Unión al Calcio/farmacología , Diabetes Mellitus Tipo 1/sangre , Femenino , Recuento de Leucocitos , Ratones , Ratones Endogámicos NOD/sangre , Proteínas de Microfilamentos/sangre , Proteínas de Microfilamentos/farmacología , Óxido Nítrico/biosíntesis

RESUMEN

A simple, convenient and efficient metal-free catalyzed oxidative trimeric reaction of indoles toward a variety of 2-(1H-indol-3-yl)-2,3'-biindolin-3-one derivatives in moderate to excellent yields has been developed. This transformation proceeds via a tandem oxidative homocoupling reaction by using TEMPO in air as an environmentally benign oxidant. This methodology provides an alternative approach for the direct generation of all-carbon quaternary centers at the C3 position of indoles.

Asunto(s)

Biomimética/métodos , Óxidos N-Cíclicos/química , Indoles/química , Oxidantes/química , Catálisis , Indoles/síntesis química , Modelos Moleculares , Oxidación-Reducción

RESUMEN

We investigated the effect of 17ß-estradiol (E2) on the expression of daintain/AIF-1, a marker of activated macrophages, in RAW264.7. E2 upregulated the protein and mRNA levels of daintain/AIF-1 in similar manners under physiological concentrations of 10(-11) M to 10(-7) M. The application of ICI 182,780, an estrogen receptor (ER) antagonist, attenuated E2-induced daintain/AIF-1 production, suggesting the involvement of ER in this process.

Asunto(s)

Proteínas de Unión al Calcio/metabolismo , Estradiol/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Animales , Línea Celular , Relación Dosis-Respuesta a Droga , Ratones , Proteínas de Microfilamentos , Receptores de Estrógenos/metabolismo

RESUMEN

We report herein the isolation and characterization of a novel glycine-rich antimicrobial peptide purified from the larvae of Drosophila virilis. A range of chromatographic methods was used for isolation and its antibacterial activity against Bacillus subtilis was employed to screen for the most active fractions. The peptide, termed SK84 due to its N-terminal serine, C-terminal lysine and a total of 84 residues, was completed sequenced using RT-PCR cDNA cloning. SK84 contains a high level of glycine (15.5%) and a hexaglycine cluster motif in the N-terminal part. SK84 displayed antibacterial activity against the tested Gram-positive bacteria (B. subtilis, Bacillus thuringiensis and Staphylococcus aureus), but had no effect on Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli) and fungi (Saccharomyces cerevisiae, Candida albicans). SK84 had specific inhibitory effects on the proliferation of several cancer cell lines (Human leukemia THP-1, liver cancer HepG2, and breast cancer MCF-7 cells), but no hemolytic activity. The results from scanning electron microscopy observations revealed that SK84 killed THP-1 cells by destroying the cell membranes. Alignment results show that SK84 is a mature protein processed from the pseudoprotein GJ19999 from D. virilis, and is very similar to several pseudoproteins from different Drosophila species. Our results show that SK84 represents a novel glycine-rich peptide family in Drosophila species with antimicrobial and anti-cancer cell activities.

Asunto(s)

Antibacterianos , Péptidos Catiónicos Antimicrobianos , Antineoplásicos , Proteínas de Drosophila/química , Proteínas de Drosophila/aislamiento & purificación , Proteínas de Drosophila/farmacología , Drosophila , Glicina/metabolismo , Proteínas de Insectos , Secuencia de Aminoácidos , Animales , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/farmacología , Antineoplásicos/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Bacillus subtilis/efectos de los fármacos , Secuencia de Bases , Línea Celular Tumoral , Membrana Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Drosophila/química , Drosophila/embriología , Glicina/química , Bacterias Grampositivas/efectos de los fármacos , Hemólisis/efectos de los fármacos , Humanos , Proteínas de Insectos/química , Proteínas de Insectos/aislamiento & purificación , Proteínas de Insectos/farmacología , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Alineación de Secuencia

RESUMEN

Daintain/AIF-1 was identified from injured rat carotid arteries and porcine intestine in the mid 1990s. It is involved in autoimmune disorders, chronic rejection of allografts, gliomas, and breast cancer. Since it is convenient and economical to obtain such a peptide biologically, in this study, we describe the expression, purification, and characterization of recombinant human daintain/AIF-1 (rhdaintain/AIF-1). The backbone of vector pET32a, a high-level expression plasmid, was used to construct the pET32a-daintain/AIF-1 plasmid for daintain/AIF-1 expression in Escherichia coli. The recombinant daintain/AIF-1 protein was solubly expressed in the BL21 (DE3) strain and was purified by Ni2+ affinity chromatography. After purification, the recombinant protein showed the expected size of 18 kDa on Tricine-SDS-PAGE gels which was further confirmed by Western blotting. A total of 34.0 mg of high purity (over 98%) rhdaintain/AIF-1 was obtained from 1 L culture. The recombinant peptide was able to increase blood glucose elimination rates and enhance the proliferation of human MCF-7 cells. These results suggest that biological activity of the recombinant peptide was preserved after purification.

Asunto(s)

Proteínas de Unión al ADN/genética , Escherichia coli/genética , Animales , Secuencia de Bases , Western Blotting , Proteínas de Unión al Calcio , Línea Celular Tumoral , Cartilla de ADN , Proteínas de Unión al ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas de Microfilamentos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa

RESUMEN

Pea albumin 1F (PA1F), a plant peptide isolated from pea seeds, can dramatically increase blood glucose concentration by subcutaneous injection with a dosage of 5 or 10 microg/g (body weight) in normal and type II diabetic mice (KK/upj-Ay). The voltage-dependent anion channel 1 (VDAC-1) has been identified as the PA1F binding protein from mice pancreatic cell membrane, which may be involved in the regulation of enhancing blood glucose in response to PA1F binding. The results clearly show that peptide-signaling molecules from plants can affect mammalian physiological functions, especially, in association with glucose metabolism.

Asunto(s)

Albúminas/farmacología , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Proteínas de Plantas/farmacología , Albúminas/administración & dosificación , Albúminas/química , Albúminas/aislamiento & purificación , Albúminas/metabolismo , Animales , Técnicas Biosensibles , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/genética , Relación Dosis-Respuesta a Droga , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos , Pisum sativum/química , Extractos Vegetales/química , Extractos Vegetales/aislamiento & purificación , Extractos Vegetales/farmacología , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo

RESUMEN

Recent research indicates that inflammatory factors play important roles in the initiation and progression of cancers, including breast cancer. Daintain/allograft inflammatory factor-1 (AIF-1) is a crucial mediator in the inflammatory response, but it has not yet been reported whether daintain/AIF-1 is involved in the development of breast cancers. In this study, immunohistochemical analysis found strong positive expression of daintain/AIF-1 in breast ductal tumor epithelia, but only weakly positive or negative expression in the adjacent histologically normal ductal epithelia. Then, the effect of daintian/AIF-1 on the proliferation of the breast cancer cell line MDA-MB-231 was explored via transduction of the daintian/AIF-1 gene into the cells, and via inhibition of the expression of daintain/AIF-1 through short interference RNA. The results demonstrated that up-regulation and down-regulation of daintain/AIF-1 expressions promoted and inhibited the proliferation of MDA-MB-231, respectively. More interestingly, daintain/AIF-1 overexpression facilitated tumor growth in female nude mice. Furthermore, we found that daintain/AIF-1 overexpression up-regulated the expression of cyclin D1 and enhanced the transcriptional activity of nuclear factor-kappa B (NF-kappaB), a regulator of cyclin D1 expression. In contrast, the down-regulation of daintain/AIF-1 expression decreased cyclin D1 expression and inhibited the transcriptional activity of NF-kappaB. These results strongly suggest that daintain/AIF-1 can promote the growth of breast tumors via activating NF-kappaB signaling, which consequently up-regulates the expression of cyclin D1, implying that daintain/AIF-1 may be a novel target molecule for the prognosis and therapy of breast cancer.

Asunto(s)

Neoplasias de la Mama/metabolismo , Ciclina D1/metabolismo , Proteínas de Unión al ADN/metabolismo , FN-kappa B/metabolismo , Animales , Proteínas de Unión al Calcio , Línea Celular Tumoral , Proliferación Celular , Ciclina D1/genética , Humanos , Ratones , Ratones Desnudos , Proteínas de Microfilamentos , FN-kappa B/genética , Transducción de Señal , Transcripción Genética , Transfección , Regulación hacia Arriba

RESUMEN

Thioredoxin (Trx) is a member of the thioredoxin protein family and has a conserved catalytic domain (-Trp-Cys-Gly-Pro-Cys-Lys-) with reduction/oxidation (redox) activity. There are two main members in this family, Trx-1, a cytosolic and nuclear form, and Trx -2, a mitochondrial form. Trx-1 is a 104 amino acid multifunctional protein that has been extensively studied. Here we report the preparation of monoclonal antibodies (MAb) against recombinant human Trx-1(hTrx). To enhance its immunogenicity, Trx-1 was coupled to carrier protein bovine serum albumin by a two-step glutaraldehyde method. Using conventional procedures, we prepared three stable hybridoma cell lines that can produce and secret anti-Trx MAbs. We further analyzed their isotypes, titer, and affinity and found that those MAbs belong to the G1 subclass with kappa light chains, respectively. The MAbs were capable of recognizing hTrx-1, as determined by Western blotting.

Asunto(s)

Anticuerpos Monoclonales/biosíntesis , Proteínas Recombinantes/inmunología , Tiorredoxinas/inmunología , Animales , Bovinos , Fusión Celular , Línea Celular , Línea Celular Tumoral , Humanos , Hibridomas , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular

RESUMEN

Different cell line with increased thioredoxin-1 (Trx-1) showed a decreased or increased sensitivity to cell killing by cisplatin. Recently, several studies found that the subcellular localization of Trx-1 is closely associated with its functions. In this study, we explored the association of the nuclear Trx-1 with the cisplatin-mediated apoptosis of breast cancer cells MCF-7. Firstly, we found that higher total Trx-1 accompanied by no change of nuclear Trx-1 can not influence apoptosis induced by cisplatin in MCF-7 cells transferred with Trx-1 cDNA. Secondly, higher nuclear Trx-1 accompanied by no change of total Trx-1 can protect cells from apoptosis induced by cisplatin. Thirdly, high nuclear Trx-1 involves in the cisplatin-resistance in cisplatin-resistive cells. Meanwhile, we found that the mRNA level of p53 is closely correlated with the level of nuclear Trx-1. In summary, we concluded that the nuclear Trx-1 is required to resist apoptosis of MCF-7 cells induced by cisplatin, probably through up-regulating the anti-apoptotic gene, p53.

Asunto(s)

Apoptosis/efectos de los fármacos , Núcleo Celular/metabolismo , Cisplatino/farmacología , Tiorredoxinas/metabolismo , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/efectos de la radiación , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de la radiación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Rayos Ultravioleta

RESUMEN

Monoclonal antibodies (MAbs) against insulin are useful for insulin assays because of their specificity and plentiful supply. We have produced four monoclonal cell strains stably secreting the monoclonal antibodies against insulin; further established subpopulation, titer, affinity; and identified the antibodies as being of subclass IgG(2b)(kappa), one strain, or IgG(1)(kappa), three strains. The smallest detectable level of human insulin by ELISA using this IgG(1) was 1.25 microg/L. The monoclonal antibodies have been used for analyzing insulin content from the sera of type 2 diabetes patients and normal subjects.

Asunto(s)

Anticuerpos Monoclonales/biosíntesis , Insulina/inmunología , Animales , Bovinos , Diabetes Mellitus Tipo 2/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Hibridomas/inmunología , Inmunoglobulina G/biosíntesis , Insulina/sangre , Ratones , Ratones Endogámicos BALB C

RESUMEN

A 37 residue peptide, aglycin, has been purified from porcine intestine. The sequence is identical to that of residues 27-63 of plant albumin 1 B precursor (PA1B, chain b) from pea seeds. Aglycin resists in vitro proteolysis by pepsin, trypsin and Glu-C protease, compatible with its intestinal occurrence and an exogenous origin from plant food. When subcutaneously injected into mice (at 10 microg.g(-1) body weight), aglycin has a hyperglycemic effect resulting in a doubling of the blood glucose level within 60 min. Using surface plasmon resonance biosensor technology, an aglycin binding protein with an apparent molecular mass of 34 kDa was detected in membrane protein extracts from porcine and mice pancreas. The polypeptide was purified by affinity chromatography and identified through peptide mass fingerprinting as the voltage-dependent anion-selective channel protein 1. The results indicate that aglycin has the potential to interfere with mammalian physiology.

Asunto(s)

Glucemia/análisis , Péptidos/química , Proteínas de Plantas/química , Animales , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Ratones , Peso Molecular , Páncreas/química , Pisum sativum/química , Mapeo Peptídico , Péptidos/metabolismo , Péptidos/farmacología , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacología , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Resonancia por Plasmón de Superficie/métodos , Porcinos , Canal Aniónico 1 Dependiente del Voltaje/metabolismo

RESUMEN

The title compound, C(21)H(22)N(2)O(3), was synthesized by the reaction of tert-butyl-hydrazine with phthalic anhydride and further O-benzoyl-ation of the resulting inter-mediate by 3,5-dimethyl-benzoyl chloride. Inter-molecular C-H⋯O=C inter-actions link the mol-ecules into layers.