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In order to screen African swine fever virus (ASFV) diagnostic antigen with the best enzyme linked immunosorbent assay (ELISA) reactivity. By establishing the ELISA method, the diagnostic antigen of ASFV p30 protein expressed by baculovirus-insect cell expression system as reference, we explored the antigenic properties and diagnostic potential of ASFV p35 protein expressed by prokaryotic expression system as a diagnostic antigen. The results of Western blotting and immunofluorescence show that the molecular weight of the recombinant p35 protein and p30 protein obtained was 40 kDa and 30 kDa, respectively, and these two proteins had good immuno-reactivity with ASFV positive serum. Recombinant p30 and p35 proteins were used as diagnostic antigens to establish ELISA, and the sensitivity and repeatability of these methods were tested. The results show that although the detection sensitivity of the p30-ELISA established in this study was higher than that of the p35-ELISA, the sensitivity of p35-ELISA was 95.8%, and variations in intra- and inter-assay repeatability of the two methods were less than 10%. The coincidence rate between the p35-ELISA and the imported kit was 97.2%. Results show that p35-ELISA was sensitive and stable, and could detect specific antibodies against ASFV.
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Animais , Febre Suína Africana/diagnóstico , Vírus da Febre Suína Africana/genética , Anticorpos Antivirais , Ensaio de Imunoadsorção Enzimática , Proteínas Recombinantes/genética , SuínosRESUMO
Objective:To observe the effect of electroacupuncture at Baihui (DU20) and Shenshu (BL23) acupoints on learning-memory ability and expression of the relative protein of P35/P25-cyclin-dependent kinase 5 (CDK5)-Tau phosphorylation signaling pathway in the prefrontal cortex (PFC) in rats with Alzheimer's disease (AD), so as to reveal its potential mechanisms in treating AD. Methods:Male adult Sprague-Dawley rats were randomly divided into normal control group, sham group, model group and treatment group with six rats in each group. The AD model was constructed by bilateral hippocampal injection of Aβ25-35 in latter two groups. Equal amount of normal saline was injected into the sham group. The treatment group was acupunctured at Baihui and Shenshu once a day for ten days. All the rats were tested with Morris Water Maze. Immunohistochemistry staining and Western blotting were used to detect the related protein of P35/P25-CDK5-Tau protein phosphorylation in the PFC. Results:Compared with the normal control group and the sham group, the escape latency and escape length increased (P < 0.05) and the times crossing the platform reduced (P < 0.05) in the model group; compared with the model group, the escape latency and escape length reduced (P < 0.05), and the times crossing the platform increased (P < 0.05) in the treatment group. The optical density of P35/P25 and CDK5 were significantly higher in the model group than in the normal control group and the sham group (P < 0.01), and they were lower in the treatment group than in the model group (P < 0.001). The relative expression of P35/P25, CDK5, Tau[pS199] and Tau[pS202] were higher in the model group than in the normal control group and the sham group (P < 0.05), and the expression of the above proteins was lower in the treatment group than in the model group (P < 0.05). Conclusion:Electroacupuncture could improve the learning-memory and spatial exploration ability, which associate with inhabiting the P35/P25-CDK5-Tau protein phosphorylation signaling pathway in the PFC to delay the development of AD.
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OBJECTIVE: To investigate the effect of electroacupuncture on the P35/P25-cyclin-dependent kinase 5 (CDK5)-Tau pathway in rats with Alzheimer's disease (AD), as well as the mechanism of electroacupuncture in the prevention and treatment of AD. METHODS: Sprague-Dawley rats were randomly divided into control group, sham-operation group, model group, and electroacupuncture treatment group, with 12 rats in each group. A rat model of AD was established by injection of Aβ25-35 into the bilateral hippocampus. The rats in the electroacupuncture treatment group were given electroacupuncture at "Baihui" (GV20) and "Shenshu" (BL23) once a day, 15 min each time, for 10 days. Morris water maze was used to evaluate learning and memory abilities, immunohistochemistry was used to measure the distribution and expression of P35/P25, CDK5, and Tau5 in the hippocampus, and Western blot was used to measure the expression of the above mentioned proteins, phosphory-lated Tau(Ser199, Ser202). RESULTS: In the visual platform test, there were no significant differences in escape latency and search path between groups (P>0.05). In the hidden platform test, there were no significant differences in escape latency and search path between the control group and the sham-operation group (P>0.05); the model group had significantly longer escape latency and search path than the control group and the sham-operation group (P0.05). The model group had significantly higher protein expression of phosphorylated Tau(Ser199, Ser202) in the hippocampus than the control group and the sham-operation group (P<0.01, P<0.05). The electroacupuncture treatment group had significantly lower protein expression of phosphorylated Tau(Ser199，Ser202) than the model group (P<0.05, P<0.01). CONCLUSION: Electroacupuncture may delay the progression of AD by affecting the expression of proteins involved in the P35/P25-CDK5-Tau pathway in the hippocampus of rats.
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Objective: To investigate the expressions and possible roles of cyclin dependent kinase 5 (CDK5), p35 and P25 in the blood of patients with vascular cognitive impairment (VCI). Methods: Totally 91 cases of VCI were recruited as the study group, including 49 cases of non-dementia vascular cognitive impairment (VCIND) and 42 cases of vascular dementia (VAD), and 30 cases of cerebral apoplexy cognitive function (NC) and healthy control (N) group, respectively. RT-qPCR was used to detect the relative expression of CDK5 mRNA in the blood. Western blot method was used to detect the protein expressions of CDK5, p35 and p25 in each group. Results: The relative expression of CDK5 mRNA and the expression levels of CDK5, p35 and p25 protein in the four groups: VAD>VCIND>NC>N respectively, with significant differences among the groups (P<0.05). Conclusion: CDK5, p35 and p25 may be involved in the occurrence of VCI, and their expression levels are positively correlated with VCI.
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Objective To study the expression of p35 and p25 in rat after focal cerebral contusion and to provide experimental data for estimating brain injury time. Methods Fifty adult male SD rats were randomly divided into 0 h, 6 h, 12 h, 24 h, 3 d, 5 d, 7 d, 10 d after focal cerebral contusion, control and sham-operated groups (5 rats each group). The focal cerebral contusion rat model was established. The expression of p35 and p25 protein of the damage peripheral zone in brain were detected by HEstain-ing, immunohistochemistry and western blotting at different injury time. Results Alarge number of p35 protein and a small amount of p25 protein were expressed in control group and sham-operated group. After focal cerebral contusion, p35 presented unimodal change with time and p25 presented bimodal changes with time. Conclusion Expression of p35 and p25 showed different regularity with good time correlation, which could help to estimate the brain injury time.
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Abstract] Objective To study the roles of IL-12 and IL-23 in the development of protective im-munity and pathological changes during chlamydial urogenital infection.Methods C57BL/6J wild type (wt) mice and mice deficient in IL-12p35 (IL-12p35 KO) or IL-12p40 (IL-12p40 KO)were inoculated in-travaginally with 1×104 IFU of live Chlamydia muridarum ( C.muridarum) organisms.Half mice of each group were reinfected on day 114 after primary infection.Vaginal swabs were taken every 3 or 4 days to mo-nitor live organism shedding.The mice were sacrificed after 114 or 143 days of primary infection and the va-ginal tract and kidney samples were collected for pathological analysis.The numbers of chlamydial inclusion bodies and bacteria in kidney homogenates were titrated after 100 days of primary infection.Results The infection time courses of mice deficient in either IL-12p35 or IL-12p40 were similar after primary infection, but were prolonged as compared with the wild type mice.All mice regardless of genotypes developed severe pathological damages in upper genital tracts with no significant difference among different groups.Almost all IL-12p40 KO mice and some IL-12p35 KO mice showed pathological changes in kidney samples.No obvious abnormality was observed in any of the kidneys from wild type mice.Neither the age-matched IL-12p35 KO nor IL-12p40 KO mice developed any gross pathological changes in kidney in the absence of chlamydial in-fection.C.muridarum inclusions were detected in kidney samples with gross pathological damages from IL-12p35 KO mice and IL-12p40 KO mice.No inclusions were ever detected in kidneys from the wild type mice.The numbers of chlamydial inclusions in the IL-12p40 KO mice were much higher than those of the IL-12p35 KO mice.Live bacteria were detected in mice deficient in either IL-12p35 or IL-12p40, but not in the wild type mice.No significant difference with the number of live bacteria was found between IL-12p35 KO mice and IL-12p40 KO mice.Conclusion IL-12 and IL-23 could inhibit the spread of C.muridarum in-fection from genital tract to kidney.The deficiency of IL-12 or IL-23 might relate to the renal lesions induced by Chlamydia infection.
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Objective To investigate the impact of celastrol on cognitive function and the ex-pression of Cdk5/p25 in hippocampus in APPswe/PS1dE9 double transgenic mouse after partial hepa-tectomy.Methods Three-month-old APPswe/PS1dE9 double transgenic mice (n=96)were randomly divided into surgery group (partial hepatectomy,group S),celastrol group (celastrol and partial hepa-tectomy,group C)and DMSO group (DMSO and partial hepatectomy,group O).Eight mice were se-lected randomly in each group and were Morris-water maze trained for 5 continuous days.Their learn-ing and memory abilities were evaluated at 1,3,7 and 14 d after the surgery,respectively.For the remaining mice in each group,the hippocampus were collected and the changes of Cdk5,p35 and p25 in hippocampus were measured by western blot at the time 1,3,7,14 d after partial hepatectomy. Six mice were killed at each time for data collection.Results The average escape latency of group C was significantly shorter than those of groups S and O at 3,7 and 14 d after partial hepatectomy (P <0.05).The percentages of time in target quadrant of groups S and O decreased significantly than that in group C (P <0.05).Western blot showed the expression of Cdk5 in group C was significantly low-er than that in groups S and O at 3,7 and 14 d after partial hepatectomy (P <0.05),and the same re-sult was also found in the expression of p25 at 1,3,7 and 14 d after partial hepatectomy (P <0.05). There was no significant difference in the expression of p35 between each group.Conclusion Celas-trol can improve the learning and memory ability in APPswe/PS1dE9 the double transgenic mouse, with the mechanism may be related to the decrease of Cdk5 and p25 in hippocampus.
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Background Retinitis pigmentosa (RP)is a common hereditary blinding eye disease in ophthalmology.Current researches documented that RP may have the common pathophysiologic basis to Alzheimer disease and chronic neurodegenerative disease.Understanding this mechanism will offer a new therapeutic target for RP.Objective The purpose of the present study was to investigate the roles of cyclin-dependent kinase 5 (Cdk5)/P25 activation in the apoptosis of retinal neural cells of RCS rats.Methods Eighteen SPF RCS rats and 18 RCS-rdy+ rats were randomized into 17-,25-and 35-day groups respectively and 6 rats for each.The rats were sacrificed at corresponding time points and retinal hemogenete was prepared.Expressions of CdkS,P35,P25 and tau phosphorylation in the retinas were detected by Western blot,and the kinase activity of Cdk5/P25 was analyzed by quantitative colorimetric assay.Results The expressing level of P35 protein(A340) in the retinas of 17-day-old RCS rats was near that of 17-day-old RCS-rdy+ rats(t =0.52,P＞0.05).In 25-and 35-day-old RCS rats,the expressing levels of P35 protein were 2.20±0.48 and 1.23±0.14,which were higher than those of RCS-rdy+ rats(1.43±0.13 and 0.93±0.10),showing significant differences between them(t =3.78,4.28,P＜0.05).The expression of P25 was undetectable at postnatal 17 days in RCS rats and RCS-rdy+ rats,but it showed significantly higher in RCS rats(0.300±0.003 and 0.230±0.004) than that in RCS-rdy+ rats(0.040±0.004 and 0.070±0.004) at postnatal 25 days and 35 days(t=121.81,77.51,P＜0.01).No significant difference was found in the expression of Cdk5 in RCS rats and RCS-rdy+ rats at different ages (t =-0.60,0.19,1.62,P＞ 0.05).The kinase activity of Cdk5/P25 did not show significantly different between RCS and RCS-rdy+ rats at postnatal 17 days(t =0.19,P＞0.05),but significantly higher kinase activity of Cdk5/P25 was seen in RCS rats (0.0058 ±0.0005 and 0.0056±0.0004) than that in RCS-rdy+ rats(0.0038±0.0003 and 0.0032 ±0.0007) at postnatal 25 days and 35 days (t =8.07,5.97,P＜ 0.01).No expression of tau phosphorylation was detected in RCS rats at postnatal 17 days,but significantly higher tau phosphorylation level was seen in RCS rats at postnatal 25 days and 35 days(1.80±0.22 and 1.23±0.17),which were significant different in comparison with RCS-rdy+ rats at postnatal 25 days and 35 days(1.60 ±0.20 and 1.04 ±0.12)(t=4.71,3.17,P＜0.05).Conclusions The Cdk5/P25 kinase activity shows a consistent trend with theexpressions of P25 and tau phosphorylation in the RCS rats,indicating that the upregulation of P25 induces the enhance of enzyme activity of Cdk5,which phosphorylate its substrates to result in more apoptosis of retinal neural cells.
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IL-12 is a secretory heterodimeric cytokine composed of p35 and p40 subunits. IL-12 p35 and p40 subunits are sometimes produced as monomers or homodimers. IL-12 is also produced as a membrane-bound form in some cases. In this study, we hypothesized that the membrane-bound form of IL-12 subunits may function as a costimulatory signal for selective activation of TAA-specific CTL through direct priming without involving antigen presenting cells and helper T cells. MethA fibrosarcoma cells were transfected with expression vectors of membrane-bound form of IL-12p35 (mbIL-12p35) or IL-12p40 subunit (mbIL-12p40) and were selected under G418-containing medium. The tumor cell clones were analyzed for the expression of mbIL-12p35 or p40 subunit and for their stimulatory effects on macrophages. The responsible T-cell subpopulation for antitumor activity of mbIL-12p35 expressing tumor clone was also analyzed in T cell subset-depleted mice. Expression of transfected membrane-bound form of IL-12 subunits was stable during more than 3 months of in vitro culture, and the chimeric molecules were not released into culture supernatants. Neither the mbIL-12p35-expressing tumor clones nor mbIL-12p40-expressing tumor clones activated macrophages to secrete TNF-alpha. Growth of mbIL-12p35-expressing tumor clones was more accelerated in the CD8+ T cell-depleted mice than in CD4+ T cell-depleted or normal mice. These results suggest that CD8+ T cells could be responsible for the rejection of mbIL-12p35-expressing tumor clone, which may bypass activation of antigen presenting cells and CD4+ helper T cells.
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Animais , Camundongos , Células Apresentadoras de Antígenos , Células Clonais , Corynebacterium , Fibrossarcoma , Interleucina-12 , Subunidade p35 da Interleucina-12 , Subunidade p40 da Interleucina-12 , Macrófagos , Rejeição em Psicologia , Linfócitos T , Linfócitos T Auxiliares-Indutores , Fator de Necrose Tumoral alfaRESUMO
The anti-allodynic effect of NMDA receptor antagonist and acupuncture treatments were explored through spinal p35 regulation of diabetic neuropathic rat. We evaluated the change over time of p35/p25 protein levels in the spinal cord compared with behavioral responses to thermal and mechanical stimulation in streptozotocin (STZ)-induced diabetic rats. Additionally, we studied p35 expression when electroacupuncture (EA) and a sub-effective dose of NMDA (N-methyl-D-aspartate) receptor antagonist (MK-801) were used to treat hyperalgesia in the diabetic neuropathic pain (DNP). Thermal paw withdrawal latency (PWL) and mechanical paw withdrawal threshold (PWT) were significantly decreased in the early stage of diabetes in rats. p35 expression after STZ injection gradually decreased from 1 week to 4 weeks compared to normal controls. p25 expression in 4-week diabetic rats was significantly higher than that of 2-week diabetic rats, and thermal PWL in 4-week diabetic rats showed delayed responses to painful thermal stimulation compared with those at 2 weeks. EA applied to the SP-9 point (2 Hz frequency) significantly prevented the thermal and mechanical hyperalgesia in the DNP rat. Additionally, EA combined with MK-801 prolonged anti-hyperalgesia, increased p35 expression, and decreased the cleavage of p35 to p25 during diabetic neuropathic pain. In this study we show EA combined with a sub-effective dose of MK-801 treatment in DNP induced by STZ that is related to p35/p25 expression in spinal cord.
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Animais , Ratos , Acupuntura , Neuropatias Diabéticas , Maleato de Dizocilpina , Eletroacupuntura , Hiperalgesia , N-Metilaspartato , Neuralgia , Medula Espinal , EstreptozocinaRESUMO
Baculoviruses are used as microbial insecticides, protein expression vectors, epitope display platforms, and most recently as vectors for gene therapy. Understanding the mechanisms that control baculovirus host-range and tissue tropisms are important for assessing their safety and for improving their properties for these biotechnology applications. In the past two decades some progress has been made and several baculovirus genes that influence host-range have been identified.Despite this progress, our understanding of the underlying mechanisms that restrict baculovirus host-range is still limited. Here we review what is currently known about baculovirus genes that influence virus host-range.
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Baculovirus has many advantages as vectors for gene transfer. We demonstrated that recombinant baculovirus vectors expressing p35 (Ac-CMV-p35) and eGFP (Ac-CMV-GFP) could be transduced into human kidney 293 cells efficiently. The level of transgene expression was viral dose dependent and high-level expression of the target gene could be achieved under the heterogonous promoter. MTT assay suggested that both Ac-CMV-p35 and Ac-CMV-GFP did not have cytotoxic effect on human embryo kidney 293 cells. Cell growth curve showed the Ac-CMV-p35 and Ac- CMV-GFP transduced and non-transduced cells had similar proliferation rate, so baculovirus-mediated p35expression had no adverse effect on cell proliferation. In addition, baculovirus-mediated p35 gene expression protected human embryo kidney 293 cells against apoptosis induced by various apoptosis inducers such as Actinomycin D, UV or serum-free media. These results suggested that the baculovirus vector mediated p35 gene expression was functional and it could be widely used in molecular research and even gene therapy.
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In order to investigate the mRNA expression and function of interleukin-23 (p19/p40) and interleukin-12 (p35/p40) in the psoriatic lesion, no-lesion and normal human skin, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to detect the expression of IL-23 (p19/p40) and IL-12 (p35/p40). The results showed that the expression of IL-23p19 mRNA and p40 (IL-12/IL-23)mRNA were higher in psoriatic lesion than those of non-lesional skin and normal skin. The levels of IL-23p19 mRNA and p40 (IL-12/IL-23) mRNA were higher in psoriatic non-lesional skin than normal skin. However, no significant difference was found in the level of IL-12p35 mRNA among the psoriatic lesional skin, non-lesional skin and normal skin. It was suggested that IL-23 might be more important in the pathogenesis of psoriasis than IL-12.
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Cyclin-dependent kinase 5 (cdk5) is essential for brain development and p35 and p67 are the regulatory molecules for cdk5. In this study, we have investigated the expression of cdk5, p35, and p67 mRNAs in the developing rat brain with in situ hybridization histochemistry. The expression of cdk5 mRNA was already observed in embryonic day 12 (E12), start point of neurogenesis in rat brain, throughout the brain and gradually increased until postnatal day 3 (P3). At this period, strong expression of cdk5 mRNA was observed in the cerebral cortex, hippocampus, dentate gyrus, thalamus, hypothalamus, and inferior colliculus. High level of cdk5 expression was maintained in the postnatal rat brain and prominent expression was observed in the hippocampus, dentate gyrus, cerebellum, and choroid plexus of adult rat brain. Strong expression of p35 mRNA was observed between E16 and E20 in the cerebral cortex, hippocampus, dentate gyrus, thalamus, hypothalamus, and inferior colliculus as like as cdk5. After birth, the expression of p35 mRNA was gradually decreased and significant differences in the expression of cdk5 and p35 were observed in the thalamus, hypothalamus, midbrain, and cerebellum. In the embryonic period, the expression pattern of p67 was very similar with that of p35 but expression level was lower than p35. After birth, strong expression of p67 was observed in the areas where the expression of cdk5 was high. From these results, it is suspected that p35 may function in neuronal migration, and p67 in differentiation and maturation, as a major regulator for cdk5 in developing rat brain.