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Objective To investigate the shedding of CAV2-ΔE3-CGS after immunization and the background of canine adenovirus (CAV) infection, and to establish a dual fluorescent quantitative PCR detection method for rabies virus (RV) and canine adenovirus type 2 (CAV2). Methods A dual fluorescent quantitative PCR detection method was established by designing specific primers and probes for E1 gene of CAV and G gene of RV for the detection of CAV2-ΔE3-CGS. Oral swabs, anal swabs and environmental samples of stray dogs from experimental animal farm and dog detention center were tested. Results The standard curves generated by this method were Y=-3.351 × logX + 44.895, R2 = .999 and Y=-3.413 × logX + 45.192, R2=0.996, respectively. The linear relationships were good, and the minimum detection limits were both 102 copies/μL. CAV2-ΔE3-CGS was not detected in experimental animal farm. CAV was detected in dog detention center, and the positive rates were 5.88% (5/85) in oral swabs, 8.24% (7/85) in anal swabs, and 4% (1/25) in environmental samples. Conclusion The dual fluorescent quantitative PCR method can be used for the detection of CAV2-ΔE3-CGS after immunization and the investigation of CAV infection. The present study has shown that no CAV2-ΔE3-CGS has been detected after immunization and CAV infection rate of stay dogs is low in Shanghai. CAV2-ΔE3-CGS oral immunization meets requirement and is applicable.
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Neuropathic pain is a chronic disease that severely afflicts the life and emotional status of patients, but currently available treatments are often ineffective. Novel therapeutic targets for the alleviation of neuropathic pain are urgently needed. Rhodojaponin VI, a grayanotoxin from Rhododendron molle, showed remarkable antinociceptive efficacy in models of neuropathic pain, but its biotargets and mechanisms are unknown. Given the reversible action of rhodojaponin VI and the narrow range over which its structure can be modified, we perforwmed thermal proteome profiling of the rat dorsal root ganglion to determine the protein target of rhodojaponin VI. N-Ethylmaleimide-sensitive fusion (NSF) was confirmed as the key target of rhodojaponin VI through biological and biophysical experiments. Functional validation showed for the first time that NSF facilitated trafficking of the Cav2.2 channel to induce an increase in Ca2+ current intensity, whereas rhodojaponin VI reversed the effects of NSF. In conclusion, rhodojaponin VI represents a unique class of analgesic natural products targeting Cav2.2 channels via NSF.
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One of the important causes of developmental epileptic encephalopathy (DEE) is the mutation of ion channel genes, including the mutation of the CACNA1E gene. CACNA1E-related DEE cases were first reported in 2018.The mutation types include new missense mutations, nonsense mutations and frameshift mutations, but the correlation between mutation sites and types with the phenotype of DEE is not clear.This review aims to summarize the reported CACNA1E-related DEE cases, and explore the correlation between the clinical phenotype of CACNA1E-related DEE and gene mutation sites and mutation types.Meanwhile, possible pathogenesis of CACNA1E-related DEE and the progress of drug intervention were reviewed to provide references for the diagnosis and precise treatment of DEE.
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[Objective]To study the association of CAV1/CAV2 gene polymorphisms with type 2 diabetes in Chinese Han population.[Methods]14 single nucleotide polymorphisms(SNPs)of CAV1/CAV2 gene were genotyped in 272 pa-tients with type 2 diabetes mellitus(T2DM group)and 287 subjects with normal glucose tolerance(control group)by ma-trix-assisted laser desorption ionization-time of flight-mass spectrometry(MALDI-TOF-MS).Waist circumference,body mass index,plasma glucose,serum insulin and lipid profiles were measured.Homeostatic model assessment of insulin resis-tance(HOMA-IR)and β-cell function(HOMA-β)were calculated.[Results]The minor allele frequency(MAF)distri-butions of CAV1 rs926198,CAV2 rs2270188,and rs1052990 were significantly different between T2DM group and con-trol group(P=0.008,0.021,and 0.045,respectively). After adjusting for age,gender,and BMI,logistic regression analysis showed that minor allele carriers(CC/CT genotype)of CAV1 rs926198 displayed a particularly increased risk of developing T2DM compared to major allele homozygotes(TT genotype)(OR=2.240,95% CI=1.415-3.544,P=0.001). GG/GA genotype carriers of CAV1 rs3807986 had lower odds for T2DM than that of AA genotype(OR=0.640,95% CI=0.449-0.913,P=0.014). Compared with TT genotype,GG/GT genotype of CAV2 rs2270188 was a protective factor for T2DM(OR=0.616,95% CI=0.432-0.878,P=0.007). Significant genotype association with T2DM was also identified in CAV2 rs1052990(GG/GT versus TT genotype:OR=0.658,95% CI=0.453-0.956,P=0.028). Multiple linear regression showed that minor allele C of SNP rs926198 was associated with an increased level of HOMA-IR(beta=1.010,P<0.001) and minor allele G of SNP rs2270188 was associated with a decreased level of HOMA-IR(beta=-0.379,P=0.023). No significant association was identified between any SNP and HOMA-β.Allele G of CAV1 rs3807986 and CAV2 rs2270188 were also associated with a decreased level of LDL-C(P=0.033 and 0.030,respectively).[Conclusion]CAV1/CAV2 locus might be the candidate genes for conferring susceptibility to T2DM in the Chinese Han population.SNP rs926198, rs3807986,rs2270188,and rs1052990 in CAV1/CAV2 locus were associated with T2DM risk perhaps through insulin resistance pathway.
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Dentre as propriedades biológicas da própolis, a atividade antimicrobiana tem merecido destacada atenção. Neste artigo, descreve-se a atividade antiviral de dois extratos etanólicos de própolis (EP1 e EP2) frente aos vírus: calicivírus felino (FCV), adenovírus canino tipo 2 (CAV-2) e vírus da diarréia viral bovina (BVDV). Um dos extratos (EP1) foi obtido por extração etanólica de própolis obtida da região central do Estado do Rio Grande do Sul e o segundo (EP2), obtido comercialmente de uma empresa de Minas Gerais. A análise dos extratos de própolis através da cromatografia líquida de alta eficiência (CLAE) identificou a presença de flavonóides como: rutina, quercetina e ácido gálico. A atividade antiviral bem como a citotoxicidade dos extratos aos cultivos celulares foram avaliadas através do MTT [3- (4,5 dimetiltiazol-2yl)-2-5-difenil-2H tetrazolato de bromo]. Ambos os extratos evidenciaram atividade antiviral frente ao BVDV e CAV-2 quando acrescidos ao cultivo celular anteriormente à inoculação viral. Os extratos foram menos efetivos contra o FCV em comparação aos resultados obtidos com os outros vírus, e a atividade antiviral neste caso foi observada apenas quando a própolis estava presente após a inoculação viral. O extrato obtido no laboratório (EP1) apresentou valores mais altos de índice de seletividade (IS=CC50/ CE50), quando comparado à outra amostra (EP2). Em resumo, a própolis apresentou atividade antiviral frente a três diferentes vírus, o que a torna alvo para o desenvolvimento de novos compostos naturais com atividade antiviral.
Propolis is a resinous substance produced by bees for which several biological activities have been attributed. In this article, the antiviral activity of two propolis extracts was tested against bovine viral diarrhea virus (BVDV), canine adenovirus type 2 (CAV-2), and feline calicivirus (FCV). One of the extracts was obtained by ethanolic extraction of propolis from the Santa Maria (RS) region, while the other was bought from a Minas Gerais industry. The high efficiency liquid cromatography (HPLC) analysis detected the presence of some flavonoids like rutin, quercetin, and gallic acid. The MTT test was applied in order to detect the citotoxicity and also the antiviral activity. Both extracts showed antiviral activity against BVDV and CAV-2 when incubated with the cell cultures before viral inoculation. The extracts were less effective against FCV comparing to the results for the other viruses and, the antiviral activity was observed only when the própolis was present after virus inoculation The extract obtained in the lab showed the highest selectivity index (SI= CC50/ EC50). Thus, propolis showed antiviral activity against three different viruses, making it a target for the development of new natural compounds with antiviral activity.
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R-type Cav2.3 high voltage-activated Ca2+ channels in peripheral sensory neurons contribute to pain transmission. Recently we have demonstrated that, among the six Cav2.3 isoforms (Cav2.3a~Cav2.3e), the Cav2.3e isoform is primarily expressed in trigeminal ganglion (TG) nociceptive neurons. In the present study, we further investigated expression patterns of Cav2.3 isoforms in the dorsal root ganglion (DRG) neurons. As in TG neurons, whole tissue RT-PCR analyses revealed the presence of two isoforms, Cav2.3a and Cav2.3e, in DRG neurons. Single-cell RT-PCR detected the expression of Cav2.3e mRNA in 20% (n=14/70) of DRG neurons, relative to Cav2.3a expression in 2.8% (n=2/70) of DRG neurons. Cav2.3e mRNA was mainly detected in small-sized neurons (n=12/14), but in only a few medium-sized neurons (n=2/14) and not in large-sized neurons, indicating the prominence of Cav2.3e in nociceptive DRG neurons. Moreover, Cav2.3e was preferentially expressed in tyrosine-kinase A (trkA)-positive, isolectin B4 (IB4)-negative and transient receptor potential vanilloid 1 (TRPV1)-positive neurons. These results suggest that Cav2.3e may be the main R-type Ca2+ channel isoform in nociceptive DRG neurons and thereby a potential target for pain treatment, not only in the trigeminal system but also in the spinal system.
Subject(s)
Animals , Rats , Calcium Channels, R-Type , Diagnosis-Related Groups , Ganglia, Spinal , Lectins , Neurons , Nociceptors , Protein Isoforms , RNA, Messenger , Sensory Receptor Cells , Spinal Nerve Roots , Trigeminal GanglionABSTRACT
The organic anion transporters (OAT) have recently been identified. Although the some transport properties of OATs in the kidney have been verified, the regulatory mechanisms for OAT's functions are still not fully understood. The rat OAT1 (rOAT1) transports a number of negatively charged organic compounds between the cells and their extracellular milieu. Caveolin (Cav) also plays a role in membrane transport. Therefore, we investigated the protein-protein interactions between rOAT1 and caveolin-2. In the rat kidney, the expressions of rOAT1 mRNA and protein were observed in both the cortex and the outer medulla. With respect to Cav-2, the expressions of mRNA and protein were observed in all portions of the kidney (cortex < outer medulla = inner medulla). The results of Western blot analysis using the isolated caveolae-enriched membrane fractions or the immunoprecipitates by respective antibodies from the rat kidney showed that rOAT1 and Cav-2 co-localized in the same fractions and they formed complexes each other. These results were confirmed by performing confocal microscopy with immunocytochemistry using the primary cultured renal proximal tubular cells. When the synthesized cRNA of rOAT1 along with the antisense oligodeoxynucleotides of Xenopus Cav-2 were co-injected into Xenopus oocytes, the [14C]p-aminohippurate and [3H]methotrexate uptake was slightly, but significantly decreased. The similar results were also observed in rOAT1 over-expressed Chinese hamster ovary cells. These findings suggest that rOAT1 and caveolin-2 are co-expressed in the plasma membrane and rOAT1's function for organic compound transport is upregulated by Cav-2 in the normal physiological condition.
Subject(s)
Animals , Rats , Biological Transport, Active/physiology , CHO Cells , Caveolins/metabolism , Cell Membrane/metabolism , Cells, Cultured , Cricetinae , Immunoprecipitation , Kidney Tubules, Proximal/metabolism , Methotrexate/metabolism , Microscopy, Confocal , Oligonucleotides, Antisense/pharmacology , Oocytes/metabolism , Organic Anion Transport Protein 1/antagonists & inhibitors , RNA, Complementary/metabolism , RNA, Messenger/genetics , Xenopus laevis/metabolism , p-Aminohippuric Acid/metabolismABSTRACT
The CaV 2.3 encoded Ca2+ channel is one of the least well-known voltage-gated calcium channels in terms of physiology, pharmacology and clinical relevance. Epilepsy is a family of neurological disorders that are common and harmful to human health. More and more studies such as gene knock-out experiment have demonstrated that the channel is related to epileptic seizure, including participating to plateau potential generation, regulating intracellular Ca2+ concentration, CaV 2.3 splice variant, interacting with some muteins. In addition, some antiepileptic drugs inhibit the epileptiform discharges by targeting CaV 2.3 voltage-gated calcium channel.