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1.
Acta Physiologica Sinica ; (6): 544-554, 2023.
Article in Chinese | WPRIM | ID: wpr-1007770

ABSTRACT

Titin, the largest known protein in the body expressed in three isoforms (N2A, N2BA and N2B), is essential for muscle structure, force generation, conduction and regulation. Since the 1950s, muscle contraction mechanisms have been explained by the sliding filament theory involving thin and thick muscle filaments, while the contribution of cytoskeleton in force generation and conduction was ignored. With the discovery of insoluble protein residues and large molecular weight proteins in muscle fibers, the third myofilament, titin, has been identified and attracted a lot of interests. The development of single molecule mechanics and gene sequencing technology further contributed to the extensive studies on the arrangement, structure, elastic properties and components of titin in sarcomere. Therefore, this paper reviews the structure, isforms classification, elastic function and regulatory factors of titin, to provide better understanding of titin.


Subject(s)
Connectin/genetics , Muscle Proteins/metabolism , Protein Isoforms/genetics , Sarcomeres/metabolism , Muscle Fibers, Skeletal/metabolism
2.
Int. j. morphol ; 40(3): 728-734, jun. 2022. ilus, tab
Article in English | LILACS | ID: biblio-1385653

ABSTRACT

SUMMARY: The main objective of this study was to analyze by real-time quantitative polymerase chain reaction (RT-qPCR) the expression patterns of the myosin heavy chain (MHC) isoforms (MHC-I, MHC-IIa, MHC-IIx) in the sphenomandibularis portion of the temporalis muscle. We expected to find differences between the sphenomandibularis and the other portions of the temporalis that could be related to the functional characteristics of the sphenomandibularis identified by electromyography. We dissected the right temporalis muscle of ten adult human individuals (five men and five women). Samples of the anterior and posterior temporalis and of the sphenomandibularis portion were obtained from each dissected muscle. These samples were analyzed by RT-qPCR to determine the percentages of expression of the MHC-I, MHC-IIa and MHC-IIx isoforms. No significant differences were identified between the anterior and the posterior temporalis in the expression patterns of the MHC-I, MHC-IIa and MHC-IIx isoforms. However, there were significant differences between the sphenomandibularis and the anterior temporalis. Specifically, the sphenomandibularis portion had a higher percentage of expression of the MHC-I isoform (P=0.04) and a lower percentage of expression of the MHC-IIx isoform (P=0.003). The pattern of expression that we observed in the sphenomandibularis reflects a greater resistance to fatigue, a lower contraction speed, and a lower capacity of force generation in the sphenomandibularis compared to the anterior temporalis. These characteristics are consistent with electromyographic findings on the functional differences between these two portions.


RESUMEN: El principal objetivo de este estudio fue analizar mediante real-time quantitative polymerase chain reaction (RT-qPCR) los patrones de expresión de las isoformas de la cadena pesada de la miosina (MHC-I, MHC-IIa y MHC-IIx) en la porción esfenomandibular del músculo temporal. Se esperó encontrar diferencias entre el esfenomandibular y las otras porciones del músculo temporal que se pudieran relacionar con las características funcionales del esfenomandibular, identificadas mediante electromiografía. Para obtener estos resultados, se diseccionó el músculo temporal derecho en diez humanos adultos (cinco hombres y cinco mujeres) y se obtuvieron muestras de la porción anterior y posterior del músculo temporal y de su porción esfenomandibular. Estas muestras fueron analizadas mediante RT-qPCR para determinar los porcentajes de expresión de las isoformas MHC-I, MHC- IIa y MHC-IIx. No se identificaron diferencias significativas de los patrones de expresión entre la porción anterior y la porción posterior del músculo temporal, pero sí que se observaron diferencias significativas entre la porción anterior del músculo temporal y su porción esfenomandibular. Concretamente, la porción esfenomandibular presentó un mayor porcentaje de expresión de la isoforma MHC-I (P=0.04) y un menor porcentaje de expresión de la isoforma MHC-IIx (P=0.003). El patrón de expresión que hemos observado en la porción esfenomandibular del músculo temporal refleja una mayor resistencia a la fatiga, una velocidad de contracción más lenta y una menor capacidad de generar fuerza si se compara esta porción con la porción anterior del músclo temporal. Estas características son consistentes con las diferencias funcionales que presentan estas dos porciones, que han sido descritas mediante electromiografía.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Aged, 80 and over , Temporal Muscle/metabolism , Myosin Heavy Chains/metabolism , Sphenoid Bone , RNA, Messenger/metabolism , Immunohistochemistry , Protein Isoforms , Electromyography , Real-Time Polymerase Chain Reaction
3.
Acta Physiologica Sinica ; (6): 59-66, 2022.
Article in Chinese | WPRIM | ID: wpr-927581

ABSTRACT

Vascular endothelial growth factor-A (VEGF-A) is a critical angiogenic factor which is mainly secreted from podocytes and epithelial cells in kidney and plays an important role in renal pathophysiology. In recent years, functions of different isoforms of VEGF-A and the new secretion approach via extracellular vesicles (EVs) have been identified. Thus, further understanding are needed for the role of VEGF-A and its isoforms in renal injury and repair. In this review, we summarized the expression, secretion and regulation of VEGF-A, its biological function, and the role of different isoforms of VEGF-A in the development of different renal diseases. Meanwhile, the research progress of VEGF-A as diagnostic marker and therapeutic target for renal diseases were discussed.


Subject(s)
Humans , Kidney/metabolism , Kidney Diseases , Protein Isoforms/metabolism , Vascular Endothelial Growth Factor A/physiology
4.
J. venom. anim. toxins incl. trop. dis ; 28: e20210042, 2022. graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1360568

ABSTRACT

Spider venoms induce different physio-pharmacological effects by binding with high affinity on molecular targets, therefore being of biotechnological interest. Some of these toxins, acting on different types of ion channels, have been identified in the venom of spiders of the genus Phoneutria, mainly from P. nigriventer. In spite of the pharmaceutical potential demonstrated by P. nigriventer toxins, there is limited information on molecules from venoms of the same genus, as their toxins remain poorly characterized. Understanding this diversity and clarifying the differences in the mechanisms of action of spider toxins is of great importance for establishing their true biotechnological potential. This prompted us to compare three different venoms of the Phoneutria genus: P. nigriventer (Pn-V), P. eickstedtae (Pe-V) and P. pertyi (Pp-V). Methods: Biochemical and functional comparison of the venoms were carried out by SDS-PAGE, HPLC, mass spectrometry, enzymatic activities and electrophysiological assays (whole-cell patch clamp). Results: The employed approach revealed that all three venoms had an overall similarity in their components, with only minor differences. The presence of a high number of similar proteins was evident, particularly toxins in the mass range of ~6.0 kDa. Hyaluronidase and proteolytic activities were detected in all venoms, in addition to isoforms of the toxins Tx1 and Tx2-6. All Tx1 isoforms blocked Nav1.6 ion currents, with slight differences. Conclusion: Our findings showed that Pn-V, Pe-V and Pp-V are highly similar concerning protein composition and enzymatic activities, containing isoforms of the same toxins sharing high sequence homology, with minor modifications. However, these structural and functional variations are very important for venom diversity. In addition, our findings will contribute to the comprehension of the molecular diversity of the venoms of the other species from Phoneutria genus, exposing their biotechnological potential as a source for searching for new active molecules.(AU)


Subject(s)
Animals , Mass Spectrometry/instrumentation , Spider Venoms/analysis , Spiders , Protein Isoforms/biosynthesis , Hyaluronoglucosaminidase , Pharmaceutical Preparations
5.
Braz. J. Pharm. Sci. (Online) ; 58: e19704, 2022. tab, graf
Article in English | LILACS | ID: biblio-1384007

ABSTRACT

Abstract Due to the fact that different isoforms of carbonic anhydrase play distinct physiological roles, their diseases/disorders involvement are different as well. Involvement in major disorders such as glaucoma, epilepsy, Alzheimer's disease, obesity and cancers, have turned carbonic anhydrase into a popular case study in the field of rational drug design. Since carbonic anhydrases are highly similar with regard to their structures, selective inhibition of different isoforms has been a significant challenge. By applying a proteochemometrics approach, herein the chemical interaction space governed by acyl selenoureido benzensulfonamides and human carbonic anhydrases is explored. To assess the validity, robustness and predictivity power of the proteochemometrics model, a diverse set of validation methods was used. The final model is shown to provide valuable structural information that can be considered for new selective inhibitors design. Using the supplied information and to show the applicability of the constructed model, new compounds were designed. Monitoring of selectivity ratios of new designs shows very promising results with regard to their selectivity for a specific isoform of carbonic anhydrase.


Subject(s)
Selenium/agonists , Drug Design , Carbonic Anhydrases/analysis , Carbonic Anhydrases/adverse effects , Protein Isoforms , Epilepsy/pathology , Alzheimer Disease/pathology , Neoplasms/pathology
6.
Repert. med. cir ; 30(2): 134-141, 2021. tab.
Article in English, Spanish | LILACS, COLNAL | ID: biblio-1362701

ABSTRACT

Introducción: la apolipoproteína E (APOE) es una glicoproteína implicada en el transporte de moléculas lipídicas. Se han descrito tres alelos del gen APOE: Ɛ2, Ɛ3 y Ɛ4. Varios estudios demuestran asociación de la isoforma APOE4 con Alzheimer de inicio tardío. Objetivos: determinar las frecuencias genotípicas y alélicas del gen APOE en una muestra de adultos en Bogotá. Materiales y métodos: estudio observacional descriptivo de corte transversal. A partir de una muestra de sangre periférica se extrajo ADN genómico y se realizó PCR-Tetraprimer para la determinación de los alelos de APOE. Resultados: se incluyeron 1.254 sujetos, 942 mujeres (75%) y 312 hombres (25%) con edades entre 40 y 100 años. El alelo más frecuente fue el Ɛ3 (85%), seguido por Ɛ4 (11%) y Ɛ2 (2%). De la población que manifestó tener ascendencia cundiboyacense, 567 sujetos (74.6%) presentaban el genotipo Ɛ3/Ɛ3, mientras que 156 (20.4%) el Ɛ3/Ɛ4, 23 (3%) el Ɛ2/Ɛ3, 11 (1.5%) el Ɛ4/Ɛ4y 4 (0.5%) el Ɛ2/Ɛ4. Los individuos con genotipoƐ2/Ɛ2 manifestaron no conocer el dato de ascendencia. Conclusiones: las frecuencias alélicas y genotípicas de APOE varían según el origen étnico, sin embargo es posible la identificación de sujetos con el genotipo menos frecuente (Ɛ2/Ɛ2) al analizar muestras de mayor tamaño. En los reportes previos en el país no se ha descrito el genotipo Ɛ2/Ɛ2, el cual fue identificado en la presente muestra como el de menor proporción.


Introduction:apolipoprotein E (APOE) is a glycoprotein involved in the transport of lipid molecules. Three alleles of the APOE gene have been described: Ɛ2, Ɛ3 and Ɛ4. Several studies show an association of the APOE isoform with late-onset Alzheimer ́s disease. Objectives: to determine the genotypic and allelic frequencies of the APOE gene in an adult sample in Bogotá. Materials and Methods: a cross-sectionalobservational descriptive study. Genomic DNA was extracted from a peripheral blood sample and APOE alleles and genotypes were determined using the PCR tetra-primer method. Results:we included 1254 subjects, 942 women (75%) and 312 men (25%) aged between 40 and 100 years. The most frequent allele was Ɛ3 (85%), followed by Ɛ4 (11%) and Ɛ2 (2%). Of the population that declared to have Cundinamarca and Boyacá sub-regions ancestry, 567 subjects (74.6%) had genotype Ɛ3/Ɛ3, while 156 (20.4%) hadƐ3/Ɛ4, 23 (3%) Ɛ2/Ɛ3, 11 (1.5%) Ɛ4/Ɛ4and 4 (0.5%) had genotype Ɛ2/Ɛ4.The individuals with genotype Ɛ2/Ɛ2 declared not to know the data on their ancestry. Conclusions: the allelic and genotypic frequencies of APOE vary according to ethnic origin. However identifying subjects with the less frequent genotype (Ɛ2/Ɛ2) is possible when analyzing larger samples. In previous reports in the country, genotype Ɛ2/Ɛ2, has not been described and was identified in the present sample as the one with the lowest proportion.


Subject(s)
Humans , Male , Female , Middle Aged , Apolipoproteins E , Polymerase Chain Reaction , Alzheimer Disease , Protein Isoforms
7.
Article in Chinese | WPRIM | ID: wpr-941993

ABSTRACT

OBJECTIVE@#To explore the clinical value of serum isoform [-2] proprostate-specific antigen (p2PSA) and its derivatives %p2PSA and prostate health index (PHI) in predicting aggressive prostate cancer (PCa).@*METHODS@#The pre-operation serum and basic clinical data of 322 patients with PCa (including 143 patients diagnosed with PCa by transrectal ultrasound-guided prostate biopsy and 179 patients undergoing radical prostatectomy) in Peking University First Hospital were collected from August 2015 to May 2018. Serum total prostate-specific antigen (tPSA), free prostate antigen (fPSA) and fPSA/tPSA (f/t) and the p2PSA level of all these patients were measured on automatic immune analyzers DxI800, and then %p2PSA and PHI were calculated. The prostate pathologic result was considered as the gold standard to evaluate the Gleason score of the patients with PCa. Receiver operator curves (ROC) were used to assess the ability of p2PSA, %p2PSA and PHI to predict aggressive PCa (pathologic Gleason score≥7) compared with those traditional markers tPSA, fPSA and f/t.@*RESULTS@#Among these patients, the p2PSA, %p2PSA and PHI median levels were significantly higher in patients with pathologic Gleason score≥7 than those with Gleason score<7 (p2PSA: 30.22 ng/L vs. 18.33 ng/L; %p2PSA: 2.50 vs. 1.27; PHI: 91.81 vs. 35.44; all P<0.01). The area under curve (AUC) of %p2PSA and PHI (0.770, 0.760) in predicting Gleason score≥7 were higher than those of the traditional indicators tPSA, fPSA and f/t (AUC were 0.648, 0.536 and 0.693, respectively). Among those patients diagnosed with PCa by transrectal ultrasound-guided prostate biopsy, the AUC of %p2PSA and PHI (AUC were 0.808 and 0.801, respectively) in predicting Gleason score≥7 were higher than those of the traditional indicators tPSA, fPSA and f/t (AUC were 0.729, 0.655 and 0.665 respectively). Among those patients undergoing radical prostatectomy, PHI and %p2PSA also had the trend of higher predictive value than those of the traditional indicators. The AUC of %p2PSA and PHI were 0.798 and 0.744, respectively while the AUC of tPSA, fPSA and f/t were 0.625, 0.507 and 0.697, respectively.@*CONCLUSION@#Compared with traditional markers tPSA, fPSA and f/t, %p2PSA and PHI had much higher predictive value for aggressive PCa, which may help clinicians to evaluate the therapeutic regime and make more appropriate management plan for the patients.


Subject(s)
Humans , Male , Neoplasm Grading , Prostate-Specific Antigen , Prostatectomy , Prostatic Neoplasms , Protein Isoforms , ROC Curve
8.
Experimental Neurobiology ; : 658-669, 2019.
Article in English | WPRIM | ID: wpr-785791

ABSTRACT

Anoctamin1 (ANO1) also known as TMEM16A is a transmembrane protein that functions as a Ca²⁺ activated chloride channel. Recently, the structure determination of a fungal Nectria haematococca TMEM16 (nhTMEM16) scramblase by X-ray crystallography and a mouse ANO1 by cryo-electron microscopy has provided the insight in molecular architecture underlying phospholipid scrambling and Ca²⁺ binding. Because the Ca²⁺ binding motif is embedded inside channel protein according to defined structure, it is still unclear how intracellular Ca²⁺ moves to its deep binding pocket effectively. Here we show that EF-hand like region containing multiple acidic amino acids at the N-terminus of ANO1 is a putative site regulating the activity of ANO1 by Ca²⁺ and voltage. The EF-hand like region of ANO1 is highly homologous to the canonical EF hand loop in calmodulin that contains acidic residues in key Ca²⁺-coordinating positions in the canonical EF hand. Indeed, deletion and Ala-substituted mutation of this region resulted in a significant reduction in the response to Ca²⁺ and changes in its key biophysical properties evoked by voltage pulses. Furthermore, only ANO1 and ANO2, and not the other TMEM16 isoforms, contain the EF-hand like region and are activated by Ca²⁺. Moreover, the molecular modeling analysis supports that EF-hand like region could play a key role during Ca²⁺ transfer. Therefore, these findings suggest that EF-hand like region in ANO1 coordinates with Ca²⁺ and modulate the activation by Ca²⁺ and voltage.


Subject(s)
Animals , Mice , Amino Acids, Acidic , Calcium , Calmodulin , Chloride Channels , Cryoelectron Microscopy , Crystallography, X-Ray , EF Hand Motifs , Models, Molecular , Mutagenesis , Nectria , Protein Isoforms
9.
Article in English | WPRIM | ID: wpr-786127

ABSTRACT

BACKGROUND: In the present multi-institutional study, the prevalence and clinicopathologic characteristics of non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) were evaluated among Korean patients who underwent thyroidectomy for papillary thyroid carcinoma (PTC).METHODS: Data from 18,819 patients with PTC from eight university hospitals between January 2012 and February 2018 were retrospectively evaluated. Pathology reports of all PTCs and slides of potential NIFTP cases were reviewed. The strict criterion of no papillae was applied for the diagnosis of NIFTP. Due to assumptions regarding misclassification of NIFTP as non-PTC tumors, the lower boundary of NIFTP prevalence among PTCs was estimated. Mutational analysis for BRAF and three RAS isoforms was performed in 27 randomly selected NIFTP cases.RESULTS: The prevalence of NIFTP was 1.3% (238/18,819) of all PTCs when the same histologic criteria were applied for NIFTP regardless of the tumor size but decreased to 0.8% (152/18,819) when tumors ≥1 cm in size were included. The mean follow-up was 37.7 months and no patient with NIFTP had evidence of lymph node metastasis, distant metastasis, or disease recurrence during the follow-up period. A difference in prevalence of NIFTP before and after NIFTP introduction was not observed. BRAF(V600E) mutation was not found in NIFTP. The mutation rate for the three RAS genes was 55.6% (15/27).CONCLUSIONS: The low prevalence and indolent clinical outcome of NIFTP in Korea was confirmed using the largest number of cases to date. The introduction of NIFTP may have a small overall impact in Korean practice.


Subject(s)
Humans , Carcinoma, Papillary , Diagnosis , Follow-Up Studies , Genes, ras , Hospitals, University , Korea , Lymph Nodes , Mutation Rate , Neoplasm Metastasis , Pathology , Prevalence , Protein Isoforms , Recurrence , Retrospective Studies , Thyroid Gland , Thyroid Neoplasms , Thyroidectomy
10.
Neuroscience Bulletin ; (6): 497-506, 2019.
Article in English | WPRIM | ID: wpr-775419

ABSTRACT

Neuroligins (NLs) are postsynaptic cell-adhesion proteins that play important roles in synapse formation and the excitatory-inhibitory balance. They have been associated with autism in both human genetic and animal model studies, and affect synaptic connections and synaptic plasticity in several brain regions. Yet current research mainly focuses on pyramidal neurons, while the function of NLs in interneurons remains to be understood. To explore the functional difference among NLs in the subtype-specific synapse formation of both pyramidal neurons and interneurons, we performed viral-mediated shRNA knockdown of NLs in cultured rat cortical neurons and examined the synapses in the two major types of neurons. Our results showed that in both types of neurons, NL1 and NL3 were involved in excitatory synapse formation, and NL2 in GABAergic synapse formation. Interestingly, NL1 affected GABAergic synapse formation more specifically than NL3, and NL2 affected excitatory synapse density preferentially in pyramidal neurons. In summary, our results demonstrated that different NLs play distinct roles in regulating the development and balance of excitatory and inhibitory synapses in pyramidal neurons and interneurons.


Subject(s)
Animals , Cell Adhesion Molecules, Neuronal , Physiology , Cells, Cultured , Cerebral Cortex , Embryology , Physiology , GABAergic Neurons , Physiology , Interneurons , Physiology , Membrane Proteins , Physiology , Nerve Tissue Proteins , Physiology , Protein Isoforms , Physiology , Pyramidal Cells , Physiology , Rats, Sprague-Dawley , Synapses , Physiology
11.
Article in English | WPRIM | ID: wpr-776717

ABSTRACT

DNA double-stranded break (DSB) is one of the most catastrophic damages of genotoxic insult. Inappropriate repair of DNA DSBs results in the loss of genetic information, mutation, and the generation of harmful genomic rearrangements, which predisposes an organism to immunodeficiency, neurological damage, and cancer. The tumor repressor p53 plays a key role in DNA damage response, and has been found to be mutated in 50% of human cancer. p53, p63, and p73 are three members of the p53 gene family. Recent discoveries have shown that human p53 gene encodes at least 12 isoforms. Different p53 members and isoforms play various roles in orchestrating DNA damage response to maintain genomic integrity. This review briefly explores the functions of p53 and its isoforms in DNA DSB repair.


Subject(s)
Animals , Humans , Mice , DNA Breaks, Double-Stranded , DNA Repair , Protein Isoforms , Physiology , Tumor Protein p73 , Physiology , Tumor Suppressor Protein p53 , Genetics , Physiology
12.
Article in English | WPRIM | ID: wpr-763149

ABSTRACT

PURPOSE: Pim kinases are highly conserved serine/threonine kinases, and different expression patterns of each isoform (Pim-1, Pim-2, and Pim-3) have been observed in various types of human cancers, including gastric cancer. AZD1208 is a potent and selective inhibitor that affects all three isoforms of Pim. We investigated the effects of AZD1208 as a single agent and in combination with an Akt inhibitor in gastric cancer cells. MATERIALS AND METHODS: The antitumor activity of AZD1208 with/without an Akt inhibitor was evaluated in a large panel of gastric cancer cell lines through growth inhibition assays. The underlying mechanism was also examined by western blotting, immunofluorescence assay, and cell cycle analysis. RESULTS: AZD1208 treatment decreased gastric cancer cell proliferation rates and induced autophagy only in long-term culture systems. Light chain 3B (LC3B), a marker of autophagy, was increased in sensitive cells in a dose-dependent manner with AZD1208 treatment, which suggested that the growth inhibition effect of AZD1208 was achieved through autophagy, not apoptosis. Moreover, we found that cells damaged by Pim inhibition were repaired by activation of the DNA damage repair pathway, which promoted cell survival and led the cells to become resistant to AZD1208. We also confirmed that the combination of an Akt inhibitor with AZD1208 produced a highly synergistic effect in gastric cancer cell lines. CONCLUSION: Treatment with AZD1208 alone induced considerable cell death through autophagy in gastric cancer cells. Moreover, the combination of AZD1208 with an Akt inhibitor showed synergistic antitumor effects through regulation of the DNA damage repair pathway.


Subject(s)
Humans , Apoptosis , Autophagy , Blotting, Western , Cell Cycle , Cell Death , Cell Line , Cell Proliferation , Cell Survival , DNA Damage , Fluorescent Antibody Technique , Phosphotransferases , Protein Isoforms , Stomach Neoplasms
13.
Article in English | WPRIM | ID: wpr-764308

ABSTRACT

Peroxiredoxins (Prxs) are antioxidant enzymes that protect cells from oxidative stress by reducing intracellular accumulation of reactive oxygen species (ROS). In mammalian cells, the six Prx isoforms are ubiquitously expressed in diverse intracellular locations. They are involved in the regulation of various physiological processes including cell growth, differentiation, apoptosis, immune response and metabolism as well as intracellular ROS homeostasis. Although there are increasing evidences that Prxs are involved in carcinogenesis of many cancers, their role in cancer is controversial. The ROS levels in cancer cells are increased compared to normal cells, thus promoting cancer development. Nevertheless, for various cancer types, an overexpression of Prxs has been found to be associated with poor patient prognosis, and an increasing number of studies have reported that tumorigenesis is either facilitated or inhibited by regulation of cancer-associated signaling pathways. This review summarizes Prx isoforms and their basic functions, the relationship between the expression level and the physiological role of Prxs in cancer cells, and their roles in regulating cancer-associated signaling pathways.


Subject(s)
Humans , Apoptosis , Carcinogenesis , Homeostasis , Metabolism , Oxidative Stress , Peroxiredoxins , Physiological Phenomena , Prognosis , Protein Isoforms , Reactive Oxygen Species
14.
Chinese Journal of Biotechnology ; (12): 159-168, 2019.
Article in Chinese | WPRIM | ID: wpr-771390

ABSTRACT

Two proteins of similar molecular weight (named as ASPR-C-1 and ASPR-C-2) from the crude drug of Angelica sinensis were purified and characterized by 80% ammonium sulfate precipitation, Sephadex G-50 gel filtration chromatography, and DEAE-Sepharose anion exchange chromatography. The molecular weight of ASPR-C-1 and ASPR-C-2 on SDS-PAGE was 17.33 kDa and 17.18 kDa, respectively. They were mainly monomeric in solution, but partially formed dimers and they were glycoproteins with glycosyl content of 2.6% and 8.2%, respectively. Both ASPR-C-1 and ASPR-C-2 were identified to be members of pathogenesis-related 10 family of proteins by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and have ribonuclease activities with the specific activity of 73.60 U/mg and 146.76 U/mg, respectively. The optimum pH of the two isoforms was similar, at about 5.6, while their optimum temperatures were different. The optimum temperature of ASPR-C-1 was 50 ℃, and that of ASPR-C-2 was 60 ℃. Both isoforms presented highest thermal stability at 60 ℃. However, ASPR-C-2 was more thermotolerant than ASPR-C-1. The latter was rapidly inactivated and retained only about 20% residual activity while the former still maintained about 80% of its original activity at a higher treatment temperature (80 to 100 ℃). In addition, Fe²⁺ had an activating effect on the ribonuclease activities of two isoforms while Ca²⁺, Mg²⁺, Zn²⁺, Mn²⁺, Ag⁺, Cu²⁺, EDTA (Elhylene diamine tetraacetic acid), dithiothreitol and sodium dodecylsulphate showed different degrees of inhibition of the enzyme activities. Our findings provide a foundation for further research on the biological function of PR-10 protein from Angelica sinensis.


Subject(s)
Angelica sinensis , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Hydrogen-Ion Concentration , Kinetics , Molecular Weight , Protein Isoforms , Temperature
15.
Article in English | WPRIM | ID: wpr-719421

ABSTRACT

PURPOSE: The p38 mitogen-activated protein kinase (MAPKs) play a crucial role in the production of pro-inflammatory cytokines and over-expression of it increase cytokines which promote cancer. Among four isoforms, p38α has been well studied in head and neck squamous cell carcinoma (HNSCC) and other cancers as a therapeutic target. p38δ has recently emerged as a potential disease-specific drug target. Elevated serum p38α level in HNSCC was reported earlier from our lab. This study aims to estimate the levels of p38 MAPK-isoforms in the serum of HNSCC and design peptide inhibitor targeting the same. MATERIALS AND METHODS: Levels of p38 MAPK isoforms in the serum of HNSCC and healthy controls were quantified by surface plasmon resonance technology. The peptide inhibitor for p38 MAPK was designed by molecular modeling using Grid-based Ligand Docking with Energetics tools and compared with known specific inhibitors. RESULTS: We have observed highly elevated levels of all four isoforms of p38 MAPK in serum of HNSCC patients compared to the control group. Further, serum p38α, p38β, and p38δ levels were down regulated after therapy in follow-up patients, while p38γ showed no response to the therapy. Present study screened designed peptide WFYH as a specific inhibitor against p38δ. The specific inhibitor of p38δ was found to have no effect on p38α due to great structural difference at ATP binding pocket. CONCLUSION: In this study, first time estimated the levels of p38 MAPK isoforms in the serum of HNSCC. It can be concluded that p38 MAPK isoforms can be a diagnostic and prognostic marker for HNSCC and p38δ as a therapeutic target.


Subject(s)
Humans , Adenosine Triphosphate , Carcinoma, Squamous Cell , Cytokines , Epithelial Cells , Follow-Up Studies , Head , Models, Molecular , Neck , p38 Mitogen-Activated Protein Kinases , Protein Isoforms , Protein Kinases , Surface Plasmon Resonance
16.
Immune Network ; : e17-2019.
Article in English | WPRIM | ID: wpr-764014

ABSTRACT

Galectin-4 (Gal-4) is a β-galactoside-binding protein mostly expressed in the gastrointestinal tract of animals. Although intensive functional studies have been done for other galectin isoforms, the immunoregulatory function of Gal-4 still remains ambiguous. Here, we demonstrated that Gal-4 could bind to CD14 on monocytes and induce their differentiation into macrophage-like cells through the MAPK signaling pathway. Gal-4 induced the phenotypic changes on monocytes by altering the expression of various surface molecules, and induced functional changes such as increased cytokine production and matrix metalloproteinase expression and reduced phagocytic capacity. Concomitant with these changes, Gal-4-treated monocytes became adherent and showed elongated morphology with higher expression of macrophage markers. Notably, we found that Gal-4 interacted with CD14 and activated the MAPK signaling cascade. Therefore, these findings suggest that Gal-4 may exert the immunoregulatory functions through the activation and differentiation of monocytes.


Subject(s)
Animals , Lipopolysaccharide Receptors , Cell Differentiation , Galectin 4 , Galectins , Gastrointestinal Tract , Macrophages , Monocytes , Protein Isoforms
17.
Article in Chinese | WPRIM | ID: wpr-774367

ABSTRACT

OBJECTIVE@#To explore the possible molecular mechanism of Ikaros regulation on FUT4 expression by analyzing the correlation of the functional state of Ikaros with level of FUT4 expression, so as to provide the theoretical basis for personalized treatment in children with ALL.@*METHODS@#The subtypes of Ikaros were identified by nested PCR and sequencing. The expression level of FUT4 was detected by quantitative PCR and analyzed by ΔΔCt method in the early stage of treatment, remission and relapse of ALL.@*RESULTS@#Ik1 and Ik2 were the main functional subtypes, and the dominant negative Ikaros was Ik6; the Ik6 was detected in 23 patients with ALL. It was found that 2.73% patients expressing Ik6 alone and 18.18% patients with heterozygous expression were detected. The expression of FUT4 in the newly diagnosed ALL was higher than that in the control group, and the functional Ikaros negatively correlated with the FUT4 expression(r=-0.6329).@*CONCLUSION@#Dominant negative Ikaros closely correlated with the relapse of acute lymphoblastic leukemia in children. The functional Ikaros negatively correlated with FUT4 expression. Ikaros inhibit the transcriptional activity of FUT4, that may be the molecular mechanism of Ikaros regulating the expression of FUT4.


Subject(s)
Child , Humans , Acute Disease , Fucosyltransferases , Metabolism , Ikaros Transcription Factor , Metabolism , Lewis X Antigen , Metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma , Protein Isoforms , Recurrence
18.
Article in Chinese | WPRIM | ID: wpr-771890

ABSTRACT

OBJECTIVE@#To establish a method for detecting the exosomal PML-RARA fusion gene expression by droplet digital PCR (ddPCR).@*METHODS@#By using Taqman probe-based ddPCR technique, the method that able to detect both long and short isoforms of PML-RARA fusion gene transcripts was established. RNA from PML-RARA negative cell line HL-60 as negative control was used to set the limit of blank (LOB), while the RNA from PML-RARA positive cell line NB4 and the recombinant plasmid pSG5-PML-RARA(S) were used to set the limit of detection (LOD) for long and short PML-RARA transcripts, respectively. Furtherly, the expression of exosomal PML-RARA fusion gene in NB4 cell culture supernatant and serum of patients with acute promyelocytic leukemia (APL) was analyzed by ddPCR technique.@*RESULTS@#The LOB of ddPCR assay for long and short PML-RARA transcripts were 0.0725 and 0.083 copies per microliter of PCR reaction system, respectively, while the LOD of long and short PML-RARA transcripts were 0.19 and 0.21 copies per microliter of PCR reaction system, respectively. In addition, the expression of exosomal PML-RARA fusion gene derived from both NB4 cell culture supernatant and serum of APL patients was successfully detected.@*CONCLUSION@#A ddPCR-based technique for detecting fusion gene transcripts has been established, which can be used to analyze absolute quantification in the minimal quantity of PML-RARA transcripts derived from exosomes. It suggests the possibility of this technique to non-invasively and dynamicly monitore the exosomal PML-RARA transcripts from APL patients' serum.


Subject(s)
Humans , Exosomes , Gene Expression , Leukemia, Promyelocytic, Acute , Oncogene Proteins, Fusion , Polymerase Chain Reaction , Protein Isoforms
19.
Article in English | WPRIM | ID: wpr-761745

ABSTRACT

Steroids are commonly used in patients with eosinophilic meningitis caused by A. cantonensis infections. The mechanism steroids act on eosinophilic meningitis remains unclear. In this mouse experiments, expressions of 14-3-3 isoform β and γ proteins significantly increased in the CSF 2–3 weeks after the infection, but not increasedin the dexamethasone-treated group. Expression of 14-3-3 β, γ, ɛ, and θ isoforms increased in brain meninges over the 3-week period after infection and decreased due to dexamethasone treatment. In conclusion, administration of dexamethasone in mice with eosinophilic meningitis decreased expressions of 14-3-3 isoform proteins in the CSF and in brain meninges.


Subject(s)
Animals , Humans , Mice , Angiostrongylus cantonensis , Angiostrongylus , Brain , Dexamethasone , Eosinophils , Meninges , Meningitis , Protein Isoforms , Steroids
20.
Rev. bras. ginecol. obstet ; 40(12): 763-770, Dec. 2018. tab, graf
Article in English | LILACS | ID: biblio-977813

ABSTRACT

Abstract Objective The aim of the present study was to provide a better understanding of the specific action of two follicle-stimulating hormone (FSH) isoforms (β-follitropin and sheep FSH) on the membrane potential of human cumulus cells. Methods Electrophysiological data were associated with the characteristics of the patient, such as age and cause of infertility. The membrane potential of cumulus cells was recorded with borosilicate microelectrodes filled with KCl (3 M) with tip resistance of 15 to 25 MΩ. Sheep FSH and β-follitropin were topically administered onto the cells after stabilization of the resting potential for at least 5 minutes. Results In cumulus cells, the mean resting membrane potential was - 34.02 ± 2.04 mV (n = 14). The mean membrane resistance was 16.5 ± 1.8 MΩ (n = 14). Sheep FSH (4 mUI/mL) and β-follitropin (4 mUI/mL) produced depolarization in the membrane potential 180 and 120 seconds after the administration of the hormone, respectively. Conclusion Both FSH isoforms induced similar depolarization patterns, but β-follitropin presented a faster response. A better understanding of the differences of the effects of FSH isoforms on cell membrane potential shall contribute to improve the use of gonadotrophins in fertility treatments.


Resumo Objetivo O objetivo do presente estudo foi fornecer uma melhor compreensão da ação específica de duas isoformas de hormônio folículo estimulante (FSH, sigla em inglês) (β-folitropina e FSH ovino) no potencial de membrana de células do cumulus oophorus humanas. Métodos Dados eletrofisiológicos foram associados às características da paciente, como idade e causa da infertilidade. O potencial de membrana das células do cumulus foi registrado com microeletrodos de borossilicato preenchidos com KCl (3 M) com uma resistência de 15 a 25 MΩ. O FSH ovino e a β-folitropina foram administrados topicamente nas células após a estabilização do potencial de repouso durante pelo menos 5 minutos. Resultados Nas células do cumulus, o potencial médio de membrana em repouso foi de -34,02 ± 2,04 mV (n = 14). A resistência média da membrana foi de 16,5 ± 1,8 MΩ (n = 14). O FSH ovino (4 mUI/mL) e a β-folitropina (4 mUI/mL) produziram despolarização no potencial de membrana 180 e 120 segundos após a aplicação do hormônio, respectivamente. Conclusão Ambas as isoformas de FSH induzem padrões de despolarização semelhantes, mas a β-folitropina apresentou uma resposta mais rápida. Uma melhor compreensão das diferenças dos efeitos das isoformas do FSH no potencial da membrana celular contribuirá para aprimorar o uso das gonadotrofinas no estímulo ovariano controlado e em protocolos de maturação oocitária in vitro.


Subject(s)
Humans , Female , Adult , Cumulus Cells/physiology , Follicle Stimulating Hormone/physiology , Cells, Cultured , Protein Isoforms , Electrophysiological Phenomena
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