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1.
Eur Rev Med Pharmacol Sci ; 28(12): 3808, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38946390

RESUMO

The article "Roles of the Nrf2/HO-1 pathway in the anti-oxidative stress response to ischemia-reperfusion brain injury in rats", by L.-J. Jiang, S.-M. Zhang, C.-W. Li, J.-Y. Tang, F.-Y. Che, Y.-C. Lu, published in Eur Rev Med Pharmacol Sci 2017; 21 (7): 1532-1540-PMID: 28429353 has been retracted by the Editor in Chief. Following some concerns raised on PubPeer (link: https://pubpeer.com/publications/4C502B6EB4FCA59AC9F42A8278A3D4), the Editor in Chief has started an investigation to assess the validity of the results as well as possible figure manipulation. The authors have been informed about the journal's investigation but remained unresponsive and have not provided the study's raw data. The journal investigation revealed several figure duplications and manipulations in Figures 3 and 6. Consequently, the Editor in Chief mistrusts the results presented and has decided to retract the article. This article has been retracted. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/12521.

2.
Zhonghua Zhong Liu Za Zhi ; 45(8): 642-650, 2023 Aug 23.
Artigo em Chinês | MEDLINE | ID: mdl-37580268

RESUMO

Objective: To investigate the effects of regenerating islet-derived protein 3A (REG3A) on the proliferation and invasion of glioma cells and its molecular mechanism. Methods: Five low-grade, five high-grade glioma tissues and ten adjacent tissues from glioma patients who underwent surgery at Linyi People's Hospital from October 17, 2017 to October 18, 2018 were collected. Human glioma cell lines (SF295, U251, TG905, A172, CRT) and a primary glioma cell line PT-1 were cultured in vitro. The protein and mRNA expressions of REG3A in these tissues and glioma cell lines were detected by Western blot and reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). SF295 cells were infected with lentivirus and labeled as REG3A plasmid transfection group, and the TG905 cells were transfected with si-REG3A by liposome transfection reagent and labeled as si-REG3A transfection group. At the same time, the empty transfection control and blank control groups were set up. Glioma cells were treated with REG3A recombinant protein alone or in combination with Akt1/2 inhibitors. Cell counting kit-8 (CCK-8) and cell scratch assay were used to detect cell proliferation and invasion, respectively. Western blot was used to detect the protein expression of N-cadherin, vimentin and phosphorylation of Akt (p-Akt) in REG3A overexpressed and knockdown glioma cells. Results: RT-qPCR results showed that the mRNA expression levels of REG3A in glioma cells in each group were U251 (2.129±0.13), TG905 (2.22±0.59), CRT (5.02±0.31), A172 (6.62±1.34) and PT-1 (9.18±0.61), respectively, higher than its expression in SF295 cells (1.00±0.18, P<0.001). The mRNA expression level of REG3A in high-grade glioma tissue samples (3.18±2.92) was higher than that in the control group (1.00±1.14, P=0.031) and low-grade glioma group (0.90±0.67, P=0.014). The results of western blot and immunohistochemical staining were consistent with that of RT-qPCR. The migration rate of cells in si-REG3A transfection group [(60.57±5.30)%] was lower than that of the empty transfection group [(84.18±13.63)% (P=0.038)] and blank control group [(79.65±12.09)% (P=0.076)]. The results of the scratch experiment showed that the migration rate of cells in REG3A plasmid transfected cells in the SF295 group was (96.05±6.41)%, which was significantly higher than that of empty transfected cells [(74.47±8.23)%, P=0.021)]. REG3A recombinant protein could up-regulate the expression of N-cadherin, vimentin and p-Akt in SF295 cells. Compared with the control group [(100.00±2.53)%], the proliferation rate in the REG3A recombinant protein group [(117.70±10.24)%] was significantly up-regulated, and the proliferation rate in the REG3A recombinant protein+ Akt inhibitor group [(98.31±3.64)%] was significantly lower than that of the REG3A recombinant protein group (P=0.017). The migration rate of the REG3A recombinant protein+ Akt inhibitor group was (63.35±4.06)%, which was significantly lower than (89.26±11.07)% of the REG3A recombinant protein group (P=0.019). Conclusion: REG3A can promote the proliferation and invasion of human glioma cells by activating the PI3K/Akt signaling pathway.


Assuntos
Glioma , Proteínas Proto-Oncogênicas c-akt , Humanos , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Glioma/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais , Vimentina/metabolismo
3.
Zhonghua Er Ke Za Zhi ; 58(2): 101-106, 2020 Feb 02.
Artigo em Chinês | MEDLINE | ID: mdl-32102145

RESUMO

Objective: To analyze the genetic characteristics of a five generations pedigree with homozygous familial hypercholesterolemia (HoFH). Methods: Prospective study. Twenty family members included a proband diagnosed as familial hyperlipidemia at the cardiology Department of Xi'an Children's Hospital in October 2018 were research object. Clinical data were collected. Genome DNAs were extracted. Whole exons sequencing was performed on the proband using target capture next generation sequencing. Candidate gene mutation sites identified by bioinformatics were verified by Sanger sequencing in the family members. The genotype-phenotype correlation of the pedigree was analyzed between heterozygous mutation carriers and non-carriers. Results: The proband was a 7-years and 10-month-old boy. He was born with a roundgreen bean size yellow skin protuberance in the skin of the coccyx. Since the age of 3-4 years old, xanthoma-like lesions with a diameter of 0.5-1.5 cm gradually appeared in the skin of bilateral elbow joints, knee joints and Achilles tendon. The height, weight and intellectual development of the child were the same as those of normal children at the same age. No similar xanthoma-like lesion was found in the other family members. The proband's total cholesterol (TC) reached 18.16-21.24 mmol/L, and his low density lipoproteincholesterol (LDL-C) was 14.08-15.51 mmol/L. Carotid ultrasonography showed diffuse sclerotic plaques in bilateral carotid and vertebral arteries, and color Doppler echocardiography revealed aortic valve thickening and calcification. Gene testing identified that the proband carried a homozygous mutation C. 418G>A (p. E140K) in LDLR gene inherited from his parents who had a consanguineous marriage and carried a heterozygous mutation of LDLR-E140K, respectively.The TC, LDL-C and apolipoproteinB (ApoB) of LDLR-E140K gene heterozygous carriers ((8.40±0.13), (6.79±0.01) and (1.95±0.05) mmol/L, respectively) were significantly higher than those of non-carriers ((4.59±0.28), (3.35±0.39) and (0.86±0.10) mmol/L, t=7.269, 4.595, 6.311, respectively, P<0.05). Conclusions: LDLR-E140K gene homozygous mutation is first reported to be associated with most severe phenotype HoFH. The genotype-phenotype analysis of the pedigree shows that the clinical phenotype of the proband with homozygous mutation is the most serious, and all the heterozygous mutation carriers present with hypercholesterolemia phenotype. The investigation confirms that LDLR-E140K is the pathogenic variation of familial hyperlipidemia.


Assuntos
LDL-Colesterol/sangue , DNA/genética , Hiperlipoproteinemia Tipo II/genética , Hiperlipoproteinemia Tipo I/genética , Receptores de LDL/genética , Valva Aórtica/diagnóstico por imagem , Sequência de Bases , Criança , Pré-Escolar , Análise Mutacional de DNA , Ecocardiografia Doppler , Feminino , Predisposição Genética para Doença , Heterozigoto , Homozigoto , Humanos , Hiperlipoproteinemia Tipo I/diagnóstico , Hiperlipoproteinemia Tipo II/sangue , Hiperlipoproteinemia Tipo II/diagnóstico , Lactente , Lipoproteínas HDL/sangue , Lipoproteínas LDL/sangue , Masculino , Proteínas de Membrana , Mutação , Linhagem , Fenótipo , Estudos Prospectivos
4.
Genet Mol Res ; 16(2)2017 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-28510254

RESUMO

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that affects motor neurons and lacks an effective treatment. The disease pathogenesis has not been clarified at present. Pathological transactive response DNA-binding protein 43 (TDP-43) plays an important role in the pathogenesis of ALS. Nuclear translocation of nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) is found in a mutant TDP-43 transgenic cell model, but its downstream antioxidant enzyme expression is decreased. To elucidate the specific mechanism of Nrf2/ARE (antioxidant responsive element) signaling dysfunction, we constructed an ALS cell model with human mutant TDP-43 using the NSC-34 cell line to evaluate the impact of the TDP-43 mutation on the Nrf2/ARE pathway. We found the nuclear translocation of Nrf2, but the expression of total Nrf2, cytoplasmic Nrf2, and downstream phase II detoxifying enzyme (NQO1) was decreased in NSC-34 cells transfected with the TDP-43-M337V plasmid. Besides, TDP-43-M337V plasmid-transfected NSC-34 cells were rounded with reduced neurites, shortened axons, increased levels of intracellular lipid peroxidation products, and decreased viability, which suggests that the TDP-43-M337V plasmid weakened the antioxidant capacity of NSC-34 cells and increased their susceptibility to oxidative damage. We further showed that expression of the MafK protein and the Jun dimerization protein 2 (JDP2) was reduced in TDP-43-M337V plasmid-transfected NSC-34 cells, which might cause accumulation of Nrf2 in nuclei but a decrease in NQO1 expression. Taken together, our results confirmed that TDP-43-M337V impaired the Nrf2/ARE pathway by reducing the expression of MafK and JDP2 proteins, and provided information for further research on the molecular mechanisms of TDP-43-M337V in ALS.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição MafK/metabolismo , Mutação de Sentido Incorreto , Fator 2 Relacionado a NF-E2/metabolismo , Proteínas Repressoras/metabolismo , Elementos de Resposta , Animais , Linhagem Celular , Proteínas de Ligação a DNA/genética , Fator de Transcrição MafK/genética , Camundongos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Neurônios/metabolismo , Estresse Oxidativo , Proteínas Repressoras/genética
5.
Eur Rev Med Pharmacol Sci ; 21(7): 1532-1540, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-28429353

RESUMO

OBJECTIVE: The aim of this study was to investigate the roles of the Nrf2/HO-1 pathway in the responses to the oxidative stress created by ischemia-reperfusion brain injury in rats. MATERIALS AND METHODS: 54 healthy, adult, male SD rats were included in the study. Eighteen (18) rats were placed in the sham group. The ischemia-reperfusion model was created in the other 36 rats, among which 18 received injections of Nrf2 agonist before the surgery. The suture method was used to create artery occlusions in the right brain of the rats; and reperfusion was done after 90-minute ischemia (MCAO); while no suture was inserted in the sham group. At 3, 6, 12, 24, 48 and 72 hours after the modeling, their neurological functions were evaluated. Also, at different time points, rats were decapitated, and their fresh brain tissues were used to detect the infarct volume percentages by TTC staining and the brain water contents by the dry-wet weight method. The SOD contents in the brain tissue were measured by Xanthine oxidase assay. RT-PCR was used to detect the mRNA expression of HO-1 in the brain tissues, and western blot method was used to detect the expression level of HO-1 and Nrf-2. RESULTS: The rats in the sham group had no obvious neurological defects; while those in the MCAO group showed significant neurological defects at all time points. The MCAO group had higher neurological evaluation scores than the sham group. TTC staining showed that infarct in the MCAO group kept increasing over time and peaked at 24h. Measurements of SOD found that the sham group had the highest SOD among the three groups, and showed no significant fluctuation over time. The MCAO group had much lower SOD activities than the sham group at all the time points. The higher the level of HO-1mRNA and protein expression in the brain tissue of rats in each group, the higher the degree of brain injury, but the lower the level of Nrf2 protein expression and the lower degree of brain injury. Nrf2 agonist markedly improved all these indicators in the rats which underwent the MCAO surgery. CONCLUSIONS: The expression of HO-1 after ischemia-reperfusion brain injury may contribute to the increased infarct volume. Activation of Nrf2 could improve the prognosis of ischemia-reperfusion brain injury.


Assuntos
Fator 2 Relacionado a NF-E2 , Estresse Oxidativo/efeitos dos fármacos , Animais , Lesões Encefálicas/tratamento farmacológico , Isquemia Encefálica/tratamento farmacológico , Masculino , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/tratamento farmacológico
6.
Genet Mol Res ; 14(4): 13572-81, 2015 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-26535670

RESUMO

The primary aims of this study were to investigate mitochondrial metabolism during experimental allergic encephalomyelitis (EAE) animal model axonal injury and to determine the correlation among neurological function scores, pathological changes, and the activities of the BB isoenzyme of creatine kinase (CK-BB), catalase (CAT), and calpain in the brain tissues of EAE rats. Another goal was to preliminarily define the mechanism of mitochondrial metabolism resulting from the effect of beta 2 adrenergic agonists in the process of EAE animal model axonal damage. EAE was induced in specific pathogen free Wistar rats by guinea pig spinal cord homogenate, complete Freund's adjuvant, and pertussis vaccine. We recorded the behavioral change in EAE rats, detected pathological changes in central nervous tissue, and observed the changes of the CK-BB, CAT, and calpain in the EAE rat brain and spinal cord. The results indicated that the average neurologic function score increased in the EAE group compared to that of the controls (P < 0.01). In addition, CAT and CK-BB activities significantly decreased and the calpain activity significantly increased compared with those of the control group (P < 0.05). The decrease of the activity of central nervous CK-BB and CAT content, as well as the increase of calpain activity at the highest time point were considered to be the consequences of EAE. Furthermore, the results revealed that use of salbutamol could alleviate disease symptoms and reduce the recurrence of the EAE disease.


Assuntos
Agonistas de Receptores Adrenérgicos beta 2/uso terapêutico , Axônios/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Animais , Calpaína/metabolismo , Catalase/metabolismo , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Cobaias , Ratos , Ratos Wistar
7.
Cell Mol Biol (Noisy-le-grand) ; 49(5): 713-22, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-14528907

RESUMO

We previously described a method of quantitating levels of peptides in Cpe(fat)/Cpe(fat) mice using affinity chromatography to isolate peptide-processing intermediates and differential isotopic labeling/mass spectrometry. In the present study, we compared two different isotopic labels, acetic anhydride and succinic anhydride for detection and quantitation of peptides in wild type mice. As previously found for acetic anhydride, succinic anhydride efficiently labels all primary amines in various peptides. Of these two reagents, succinic anhydride provides better resolution between the heavy and light peaks of the labelled peptides due to a greater mass difference between the deuterated (heavy) and non-deuterated (light) form of this label (4 Da for succinate, 3 Da for acetate). Using succinic anhydride labeling, the accuracy of measuring 1:1 and 1:2 ratios of peptides in pituitary extracts was within 5% of the theoretical value for most peptides. The accuracy with succinic anhydride is comparable to the accuracy of acetic anhydride and more peptides could be detected and quantitated with succinic anhydride. The two labels were then used to examine pituitary peptides in mice with a defect in copper transport (Atp7a mice) vs wild type mice. Using succinic anhydride, 13 peptides could be detected, 12 of which matched the theoretical mass of known pituitary peptides. Five of the six peptides which contain C-terminal amide groups were significantly decreased in the Atp7a mice relative to wild type mice, whereas only one non-amidated peptide was significantly decreased in Atp7a mice. With acetic anhydride, only five peptides could be quantitated. The three peptides which contain C-terminal amide groups were decreased approximately 30% in the Atp7a mice. The selective decrease in amidated peptides in Atp7a mice is consistent with the copper-requirement of the enzyme that forms C-terminal amides.


Assuntos
Adenosina Trifosfatases/deficiência , Proteínas de Transporte de Cátions/deficiência , Hipófise/química , Proteômica/métodos , Proteínas Recombinantes de Fusão/deficiência , Anidridos Acéticos/química , Acetilação , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/fisiologia , Animais , Antígenos de Diferenciação de Linfócitos B/análise , Antígenos de Diferenciação de Linfócitos B/fisiologia , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/fisiologia , Cobre/metabolismo , ATPases Transportadoras de Cobre , Deutério/química , Feminino , Antígenos de Histocompatibilidade Classe II/análise , Antígenos de Histocompatibilidade Classe II/fisiologia , Cadeias J de Imunoglobulina/análise , Cadeias J de Imunoglobulina/fisiologia , Marcação por Isótopo/métodos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Peptídeos/análise , Peptídeos/isolamento & purificação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/fisiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Anidridos Succínicos/química , Vasopressinas/análise , Vasopressinas/fisiologia , alfa-MSH/análise , alfa-MSH/fisiologia , beta-Lipotropina/análise , beta-Lipotropina/fisiologia
8.
Proc Natl Acad Sci U S A ; 98(17): 9971-6, 2001 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-11481435

RESUMO

Cpe(fat)/Cpe(fat) mice have a naturally occurring point mutation within the carboxypeptidase E gene that inactivates this enzyme, leading to an accumulation of many neuroendocrine peptides containing C-terminal basic residues. These processing intermediates can be readily purified on an anhydrotrypsin affinity resin. Using MS to obtain molecular mass and partial sequence information, more than 100 peptides have been identified. These peptides represent fragments of 16 known secretory pathway proteins, including proenkephalin, proopiomelanocortin, protachykinins A and B, chromogranin A and B, and secretogranin II. Many of the identified peptides represent previously uncharacterized fragments of the precursors. For example, 12 of the 13 chromogranin B-derived peptides found in the present study have not been previously reported. Of these 13 chromogranin B-derived peptides, only five contain consensus cleavage sites for prohormone convertases at both the C and N termini. Two distinct chromogranin B-derived peptides result from cleavage at Trp-Trp bonds, a site not typically associated with neuropeptide processing. An RIA was used to confirm that one of these peptides, designated WE-15, exists in wild-type mouse brain, thus validating the approach to identify peptides in Cpe(fat)/Cpe(fat) mice. These "orphan" peptides are candidate ligands for orphan G protein-coupled receptors. In addition, the general technique of using affinity chromatography to isolate endogenous substrates from a mutant organism lacking an enzyme should be applicable to a wide range of enzyme-substrate systems.


Assuntos
Química Encefálica , Carboxipeptidases/deficiência , Cromatografia de Afinidade/métodos , Camundongos Mutantes/metabolismo , Neuropeptídeos/metabolismo , Fragmentos de Peptídeos/análise , Hipófise/química , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Animais , Carboxipeptidase H , Carboxipeptidases/genética , Cromatografia Líquida de Alta Pressão , Sequência Consenso , Furina , Ligantes , Camundongos , Camundongos Mutantes/genética , Dados de Sequência Molecular , Neuropeptídeos/química , Neuropeptídeos/isolamento & purificação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Mutação Puntual , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Subtilisinas/metabolismo , Técnica de Subtração , Tripsina/química
9.
Biotechniques ; 30(6): 1272-6, 1278, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11414218

RESUMO

Here, we describe a simple and sensitive method that allows fluorescent detection of glycoproteins on polyvinylidene difluoride (PVDF) membrane. We used periodic acid oxidation of carbohydrate chains of glycoproteins and fluorescent labeling with 8-aminonaphthalene-1,3,6-trisulfonate (AN-TS) by reductive amination. We developed an additional method to enhance the ability of PVDF to absorb glycoproteins by using non-glycoprotein lectin, such as wheat germ agglutinin (WGA), as a link between the PVDF membrane and glycoproteins, resulting in considerably increased detection sensitivity to glycoproteins.


Assuntos
Corantes Fluorescentes/análise , Fluorometria/métodos , Glicoproteínas/análise , Membranas Artificiais , Naftalenos/análise , Polivinil/química , Coloração e Rotulagem/métodos , Absorção , Animais , Western Blotting , Eletroforese em Gel de Poliacrilamida , Fluorometria/economia , Glicosilação , Humanos , Immunoblotting , Lectinas/química , Oxirredução , Ácido Periódico/química , Processamento de Proteína Pós-Traducional , Sensibilidade e Especificidade , Coloração e Rotulagem/economia , Aglutininas do Germe de Trigo/química
10.
J Cell Sci ; 114(Pt 2): 311-22, 2001 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11148133

RESUMO

Carboxypeptidase D (CPD) is a transmembrane protein that processes proteins in the trans-Golgi network (TGN). A 20-residue region within the cytoplasmic tail of CPD binds protein phosphatase 2A (PP2A). PP2A also binds to the cytoplasmic tails of other secretory pathway proteins: peptidylglycine-(amino)-amidating mono-oxygenase, the cation-independent mannose-6-phosphate receptor and TGN38. The CPD tail is phosphorylated on Thr residues in the AtT-20 cell line. The CPD tail can also be phosphorylated by purified protein kinase A, protein kinase C and casein kinase II. Both the in vitro and the in vivo phosphorylated CPD tail can be dephosphorylated by purified PP2A. The binding of CPD tail peptide to PP2A does not influence phosphatase activity. The rate of transport of CPD from the TGN to the cell surface of AtT-20 cells is decreased 45% by okadaic acid, a PP2A inhibitor. Microinjection of the CPD tail into AtT-20 cells inhibits the transition of CPD from endosomal compartments to the TGN. However, okadaic acid does not affect the rate of budding of CPD from the TGN into nascent vesicles or the rate of uptake from the cell surface into endosomal compartments. These results are consistent with the model that PP2A is involved in the trafficking of proteins between a TGN recycling loop and a cell-surface recycling loop, but is not involved in the individual recycling loops.


Assuntos
Encéfalo/enzimologia , Carboxipeptidases/química , Carboxipeptidases/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Rede trans-Golgi/fisiologia , Sequência de Aminoácidos , Animais , Sítios de Ligação , Carboxipeptidases/genética , Linhagem Celular , Membrana Celular/enzimologia , Glutationa Transferase/metabolismo , Camundongos , Microssomos/enzimologia , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fosfatos/metabolismo , Fosforilação , Proteína Fosfatase 2 , Transporte Proteico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
11.
J Biol Chem ; 276(9): 6207-13, 2001 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-11094058

RESUMO

ProSAAS is a newly discovered protein with a neuroendocrine distribution generally similar to that of prohormone convertase 1 (PC1), a peptide-processing endopeptidase. Several proSAAS-derived peptides were previously identified in the brain and pituitary of the Cpe(fat)/Cpe(fat) mouse based on the accumulation of C-terminally extended peptides due to the absence of enzymatically active carboxypeptidase E, a peptide-processing exopeptidase. In the present study, antisera against different regions of proSAAS were used to develop radioimmunoassays and examine the processing profile of proSAAS in wild type and Cpe(fat)/Cpe(fat) mouse tissues following gel filtration and reverse phase high performance liquid chromatography. In wild type mouse brain and pituitary, the majority of proSAAS is processed into smaller peptides. These proSAAS-derived peptides elute from the reverse-phase column in the same positions as synthetic peptides that correspond to little SAAS, PEN, and big LEN. Mass spectrometry revealed the presence of peptides with the expected molecular masses of little SAAS and big LEN in the fractions containing immunoreactive peptides. The processing of proSAAS is slightly impaired in Cpe(fat)/Cpe(fat) mice, relative to wild-type mice, leading to the accumulation of partially processed peptides. One of these peptides, the C-terminally extended form of PEN, is known to inhibit PC1 activity and this could account for the reduction in enzymatically active PC1 seen in Cpe(fat)/Cpe(fat) mice. The observation that little SAAS and big LEN are the major forms of these peptides produced in mouse brain and pituitary raises the possibility that these peptides function as neurotransmitters or hormones.


Assuntos
Encéfalo/metabolismo , Carboxipeptidases/fisiologia , Neuropeptídeos/metabolismo , Hipófise/metabolismo , Precursores de Proteínas/metabolismo , Animais , Carboxipeptidase H , Carboxipeptidases/genética , Cromatografia Líquida de Alta Pressão , Soros Imunes/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neuropeptídeos/análise , Fragmentos de Peptídeos/análise , Precursores de Proteínas/análise
12.
Electrophoresis ; 20(14): 2930-7, 1999 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-10546830

RESUMO

A simple and highly sensitive capillary electrophoresis (CE) method for determining the content of N-acetylneuraminic acid (Neu5Ac) in glycoproteins was developed. Neu5Ac was derivatized with 2-aminoacridone (AMAC) by reductive amination, and the AMAC-Neu5Ac adduct could be readily separated from the other 11 AMAC-derivatized neutral and acidic monosaccharides usually present in glycoproteins by CE in a 0.3 mol/L borate buffer, pH 10.5, and detected at 260 nm. The derivatization of Neu5Ac was achieved at 55 degrees C for 4 h. AMAC-Neu5Ac was stable at 20 degrees C in the dark for at least 12 h while at room temperature it spontaneously converted into another substance with a lower electrophoretic mobility, which was identified as decarboxylated AMAC-Neu5Ac by high performance liquid chromatography - ion trap mass spectrometry (HPLC-ITMS). Concentration and mass of Neu5Ac as low as 1 micromol/L and 35 fmol could be detected. The linear correlation coefficient between the ratio of peak area to migration time of AMAC-Neu5Ac and the concentration of Neu5Ac ranging from 10 to 120 micromol/L was 0.9978 (n=8). This method was successfully applied to the analysis of sialic acid in human urinary trypsin inhibitor (hu-UTI), bovine alpha1-acid glycoprotein (alpha1-AGP) and recombinant human erythropoietin (rhu-EPO). By combination of CE and HPLC-ITMS we found that N-glycolylneuraminic acid (Neu5Gc) was present in bovine alpha1-AGP in addition to Neu5Ac, with a quantity comparable to that of the latter.


Assuntos
Aminoacridinas , Glicoproteínas/química , Ácidos Siálicos/química , Animais , Bovinos , Cromatografia Líquida , Eletroforese Capilar , Humanos , Espectrometria de Massas
13.
J Chromatogr A ; 858(2): 229-38, 1999 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-10551355

RESUMO

Dextran was partially hydrolyzed with 0.1 mol/l HCl and the hydrolysate was derivatized with 8-aminonaphthalene-1,3,6-trisulfonate (ANTS) by reductive amination. The derivatized-oligosaccharide mixture was separated by capillary electrophoresis (CE) in a buffer of 1% HAc-NH4OH, pH 3.4, and the separated components were detected on-line by electrospray ionization quadrupole ion trap mass spectrometry (ESI-QIT-MS) in the negative ion mode. A mass accuracy lower than 0.01% could be achieved and as low as 1.6 pmol of detxran octaose could be detected. ANTS-derivatized dextran oligosaccharide with a degree of polymerization (DP) lower than 6 produced both [M-H]- and [M-2H]2- ions, whereas those with a DP of 6 or higher than 6 produced only [M-2H]2- ion. As 1< or =DP< or =6, the percentage of [M-2H]2- ion in the total ions of [M-H]- and [M-2H]2- was found to be a linear function of the logarithmic DP. Molecular mass determination with ESI-QIT-MS strengthens the power of CE analysis of oligosaccharides.


Assuntos
Eletroforese Capilar/métodos , Espectrometria de Massas/métodos , Naftalenos/química , Oligossacarídeos/análise , Oligossacarídeos/química , Espectrofotometria Ultravioleta
14.
J Chromatogr A ; 849(2): 599-608, 1999 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-10457454

RESUMO

Two commercial turkey egg ovalbumins (TEOs) with different quantities of mannose, were further purified by reversed-phase high-performance liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis for either of the purified glycoproteins showed one big wide band and one close small band. Capillary electrophoresis was used for the investigation of the separation of glycoforms of both glycoproteins. The best resolution of the glycoforms was obtained, reproducibly, with 100 mM borate, 1.8 mM 1,4-diaminobutane and pH 8.6 electrophoretic buffer. At least 13 glycoform peaks could be separated for either of the two glycoproteins. Their glycoform patterns were highly similar except for the conspicuous decrease in quantity of four glycoforms in the ovalbumin containing less mannose, compared to that of the other with more mannose. Coinjection electrophoresis of the two glycoproteins indicated that almost every glycoform peak of the former exactly overlapped with its corresponding glycoform peak of the latter. These results clearly indicated that the two TEOs possessed the same glycoform patterns but differed in quantity at least four glycoforms. It was found that the glycoform patterns were remarkably different between TEO and chicken egg ovalbumin.


Assuntos
Eletroforese Capilar/métodos , Ovalbumina/análise , Animais , Boratos/química , Soluções Tampão , Galinhas , Cromatografia Líquida de Alta Pressão/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Glicoproteínas/análise , Glicosilação , Concentração Osmolar , Fosfatos/química , Putrescina/química , Perus
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