Cyclin-dependent kinase 5 is required for suppressing D1-dependent signaling mediated through muscarinic 4 in isolated medium spiny neurons

OBJECTIVE Previous studies have demonstrated acetylcholine muscarinic 4(M4) receptor regulates DARPP-32 phosphorylation at Thr75 in isolated medium spiny neurons(MSNs),indicating antagonistic mechanism with D1 dependent signal cascade,but the exact molecular mechanisms remain unclearly.In this study,we investigated the roles of M4 receptor in modulation D1 dependent signal to integrate striatal DA inputs in isolated MSNs.METHODS(1)Lentivirus technology was employed to genetically knock down the M4 receptor of MSNs;(2) Apomorphine(APO),acts as a dopamine receptor agonist,while SCH23390,acts as a selective antagonist for D1,were used to study the pharmacologically profiles with D1 receptor stimulation or blockade,respectively.Then the no subtype-selective muscarinic agonist oxotremorine M(OX) were used to show that mAchRs activation,in order to dissect the particular function of M4,a selective M4 antagonist,MT3 was used;(3) Intracellular cAMP production of MSNs was measured by using time resolved fluorescence resonance energy transfer detection method;(4) Laser confocal was used to explore the expression of M4 and D1 in MSNs;(5) Immunofluorescence cytochemistry and Western blotting were used to confirm the alteration of signaling molecular including P-CREB,DARPP-32 P-Thr34,DARPP-32 P-Thr75,cyclin-dependent kinase 5(CDK5) as wel as p25/35,which are involved in DA-dependent signaling modulations.RESULTS Firstly,TR-FRET assay revealed APO(10-2 mol·L^(-1))significantly increased the level of intracellular cAMP(vs control,n=3,P<0.01),also Western blotting results showed that APO(10-6 mol · L^(-1))increased DARPP-32 Thr34 phosphorylation(vs control,n=3,P<0.01),and these effect were reversed by D1 receptor antagonist SCH23390(vs APO,n=3,P<0.01).Interestingly,we confirmed that OX(10-6 mol · L^(-1)) down-regulated APO-induced DARPP-32 Thr34 phosphorylation(vs APO,n=3,P<0.01),due to its effects on DARPP-32 phosphorylation at Thr75.The results presented the antagonistic mechanism of mAchRs stimulation with D1 dependent signal cascade in MSNs.Meanwhile,OX(10-7,10-6 and10^(-5) mol·L^(-1)) stimulated DARPP-32 phosphorylation at Thr75,and simultaneously up regulated P25/35 and CDK5 activity(vs control,n=3,P<0.01) by using Western blotting assay.Furthermore,roscovitine(10^(-5) mol · L^(-1)),acts as a CDK5 inhibitor,suppressed CDK5 activity(vs control,n=10,P<0.01),and fully inhibited OX-induced DARPP-32 Thr75 phosphorylation(vs OX,n=10,P<0.01).More important,pretreated with roscovitine(10^(-5) mol·L^(-1)),the effect of APO on DARPP-32 Thr34 phosphorylation was potentiated(vs APO,n=3,P<0.05).The result presented CDK5 is required in suppression of APO on DARPP-32 Thr34 phosphorylation mediated through mAchRs stimulation.In addition,laser confocal results showed that the CDK5 up-regulation was mostly confined to MSNs co-expressing M4,which means that M4 participated in CDK5-mediated phosphorylation of DARPP-32 at Thr75.Consistently,immunofluorescence and Western blotting results confirmed that both genetic knockdown and pharmacologic inhibition of M4 receptors with MT3(10-7 mol · L^(-1)) down-regulated the OX-induced the expression of CDK5(vs OX,n=3,P<0.01) and P25/35(vs OX,n=3,P<0.01)in isolated MSNs.CONCLUSION M4 receptor may play an important role in antagonistic regulation D1 dependent signaling,in which CDK5 is required for suppressing D1-DARPP-32 Thr34 phosphorylation in isolated medium spiny neurons.

Crosslink among cyclin-dependent kinase 9,ATP binding cassette transporter G2 and Beclin 1 in colorectal cancer

Colorectal cancer(CRC)ranks third in the number of cancers mainly because of the inability to diagnose it at an early stage.The pathogenesis of CRC is complicated,which is the result of the complex interaction of multiple genetic and environmental factors.Currently,one of the main treatments for CRC is chemotherapy.But the primary cause of CRC treatment failure is drug resistance.The expression of cyclin-dependent kinase 9(CDK9)was correlated with elevated autophagy levels in colon cancer,and high expression of CDK9 indicates a poor prognosis in CRC.The incidence of autophagy and the expressions of Beclin 1 and ATP binding cassette transporter G2 are different in left and right colon cancer,and autophagy may be involved in the occurrence of chemotherapy resistance.In this article,the roles of CDK9,ATP binding cassette transporter G2 and Beclin 1 in CRC were elucidated,emphasizing the linkages among them and providing potential therapeutic targets of CRC.

Expression of cyclin-dependent protein kinase 5 in the hippocampus of vascular dementia mice after cerebral ischemia and reperfusion

BACKGROUND： The p25-activated cyclin-dependent protein kinase 5 （Cdk5） may induce neuronal cell death and cause the development of dementia following cerebral ischemia and reperfusion. OBJECTIVE： To observe changes in the expression of Cdk5 and p25 in hippocampal tissue of vascular dementia mice at different time points following cerebral ischemia and reperfusion. DESIGN, TIME AND SETTING： A randomized, controlled animal experiment was performed in the clinical trial center of Hebei Provincial People＇s Hospital between September 2007 and October 2008. MATERIALS： Cdk5 rabbit anti-mouse polyclonal antibody, p35 rabbit anti-mouse polyclonal antibody, and β-actin mouse monoclonal antibody were purchased from Santa Cruz Biotechnology, Inc., USA; horseradish peroxidase-labeled goat anti-rabbit IgG and horseradish peroxidase-labeled goat anti-mice IgG were offered by Beijing Zhongshan Geldenbridye Biotechnology Co.,Ltd., China; the protein quantitative kit was produced by Applygen Gene Technology Corp., Beijing, China; cDNA reverse transcription and PCR amplification reagents were products of TianGen＆ Biotech （Beijing） Co.,Ltd., China. METHODS： One hundred and sixty male Kunming mice were randomly divided into two groups： a sham-operated group （n = 65） and a model group （n = 95）. Vascular dementia was induced with three periods of transient ischemia and reperfusion of the bilateral common carotid arteries. In the sham-operated group, the bilateral common carotid arteries were not blocked. MAIN OUTCOME MEASURES： Behavioral tests were done at four and six weeks post surgery. Pathological changes in the hippocampal CA1 region were observed with hematoxylin-eosin staining Cdk5 mRNA expression was examined by RT-PCR, and Western blots were used to evaluate Cdk5 and p25 expression. Learning and memory performance were assayed using the Morris water maze. RESULTS： Vascular dementia reduced learning and memory performance at 4 and 6 weeks post surgery. Vascular dementia also caused severe, time-dependent neuronal damage and death in the hippocampal CA1 region. Dementia induction also increased mRNA and protein expression of Cdk5 and p25 at both 4 and 6 weeks after surgery. CONCLUSION： Cdk5/p25 is involved in the development of vascular dementia in mice following cerebral ischemia and reperfusion.

Expression of cyclin-dependent kinase inhibitor 2A 16,tumour protein 53 and epidermal growth factor receptor in salivary gland carcinomas is not associated with oncogenic virus infection

It is known that human papillomavirus （HPV） infection can cause squamous cell neoplasms at several sites, such as cervix uteri carcinoma and oral squamous carcinoma. There is little information on the expression of HPV and its predictive markers in tumours of the major and minor salivary glands of the head and neck. We therefore assessed oral salivary gland neoplasms to identify associations between HPV and infection-related epidermal growth factor receptor （EGFR）, cyclin-dependent kinase inhibitor 2A （CDKN2A/p16） and tumour protein p53 （TP53）. Formalin-fixed, paraffin-embedded tissue samples from oral salivary gland carcinomas （n=51） and benign tumours （n=26） were analysed by polymerase chain reaction （PCR） analysis for several HPV species, including high-risk types 16 and 18. Evaluation of EGFR, CDKN2A, TP53 and cytomegalovirus （CMV） was performed by immunohistochemistry. Epstein-Barr virus （EBV） was evaluated by EBV-encoded RNA in situ hybridisation. We demonstrated that salivary gland tumours are not associated with HPV infection. The expression of EGFR, CDKN2A and TP53 may be associated with tumour pathology but is not induced by HPV. CMV and EBV were not detectable. In contrast to oral squamous cell carcinomas, HPV, CMV and EBV infections are not associated with malignant or benign neoplastic lesions of the salivary glands.

The Neuroprotective Effects of Cyclin-dependent Kinase-5 Inhibition in Mice with Niemann-Pick Disease Type C

In order to investigate the neuroprotective effects of cyclin-dependent kinase-5 （cdk-5） inhibition in mice with Niemarm-Pick disease type C （NPC） （npc^-/-）, recombinant adeno-associated virus （rAAV） carrying the small interfering RNA （siRNA） specific for cdk-5 gene was injected into 3-day-old npc^-/- mice intracerebroventricularly. The rAAV-GFP-injected age-matched npc^-/- mice and non-surgery age-matched npc^-/- mice were employed as controls （n=6-10/group）. From the 4th to 8th week after the treatment, mice were weighed, and evaluated for limb motor activity by using the coat hanger test once a week. Eight-week-old npc^-/- mice were sacrificed by decapitation, and brains were quickly dissected and halved sagittally. Immunohistochemistry, Western blotting, and HE staining were used to evaluate the neuropathology in npc^-/- mice. The results showed that rAAV-cdk-5-siRNA-GFP significantly reduced the number of axonal spheroids, delayed the death of Purkinje neurons, ameliorated motor defects in npc^-/- mice, and significantly attenuated the hyperphosphorylation oftau proteins. These data suggested that inhibition of cdk-5 activity has neuroprotective effect on neurons in NPC mice.

Can cyclin-dependent kinase 4/6 inhibitors convert inoperable breast cancer relapse to operability? A case report

BACKGROUND Pathological complete response(pCR) is rare in hormone receptor-positive(HR+)HER2-negative breast cancer(BC) treated with either endocrine therapy(ET) or chemotherapy. Radical resection of locoregional relapse, although potentially curative in some cases, is challenging when the tumor invades critical structures.The oral cyclin-dependent kinase 4/6 inhibitor palbociclib in combination with ET has obtained a significant increase in objective response rates and progression-free survival in patients with advanced BC and is now being evaluated in the neoadjuvant setting. We present a clinical case of a patient with an inoperable locoregional relapse of HR+ HER2-negative BC who experienced p CR after treatment with palbociclib.CASE SUMMARY We report the clinical case of a 60-year-old patient who presented with an inoperable locoregional relapse of HR+, HER2-negative BC 10 years after the diagnosis of the primary tumor. During a routine follow-up visit, breast magnetic resonance imaging and positron emission tomography/computed tomography revealed a 4-cm lesion in the right subclavicular region, infiltrating the chest wall and extending to the subclavian vessels, but without bone or visceral involvement. Treatment was begun with palbociclib plus letrozole, converting the disease to operability over a period of 6 mo. Surgery was performed and a p CR achieved. Of note, during treatment the patient experienced a very uncommon toxicity characterized by burning tongue and glossodynia associated with dysgeusia, paresthesia, dysesthesia, and xerostomia. A reduction in the dose of palbociclib did not provide relief and treatment with the inhibitor was thus discontinued, resolving the tongue symptoms. Laboratory exams were unremarkable. Given that this was a late relapse, the tumor was classified asendocrine-sensitive, a condition associated with high sensitivity to palbociclib.CONCLUSION This case highlights the potential of the cyclin-dependent kinase 4/6 inhibitor plus ET combination to achieve pCR in locoregional relapse of BC, enabling surgical resection of a lesion initially considered inoperable.

Expression of cyclin-dependent kinases in HL-60 cells during differentiation induced by retinoic acid

This study was designed to investigate the relationship of the expression of cyclin-dependent kinases (CDKs) with theeffects of all-trans retinoic acid (ATRA) on the proliferation of HL-cells. HL-60 cells were treated with ATRA for 1-4 d. Then thecapacity of DNA Synthesis was evaluated with 3H-TdR incorporation and the expression of cyclin E, cyclin D, CDK2 and CDK4protein determined with immunocytochemical staining. In addition, the expression Of CDC2, CDK2 and CDK4 mRNA was deter-mined with in situ hybridization. It was found that ATRA suppressed the proliferation of HL-60 cells and decreased their capacityof DNA synthesis to result in a down-regulation of the expression of cyclin E, cyclin D and CDC2 without comcomittant suppressionon the expression of CDK2 and CDK4. It is concluded that the effects of ATRA on the proliferation of HL-60 cells may be relatedto the down-regulation of the expression of cyclin E, cyclin D and CDC2.

Phylogenetic relationships of 18 passerines based on Adenylate Kinase Intron 5 sequences

The 18 species of bird studied originally are known to belong to muscicapids, robins and sylviids of passerines, but some dis- putations are always present in their classification systems. In this experiment, phylogenetic relationships of 18 species of passerines were studied using Adenylate Kinase lntron 5 （AKS） sequences and DNA techniques. Through sequences analysis in comparison with each other, phylogenetic tree figures of 18 species of passerines were constructed using Neighbor-Joining （N J） and Maximum-Parsimony （MP） meth- ods . The results showed that sylviids should be listed as an independent family, while robins and flycatchers should be listed into Musci- capidae. Since the phylogenetic relationships between long-tailed tits and old world warblers are closer than that between long-tailed tits and parids, the long-tailed tits should be independent of paridae and be categorized into aegithalidae. Muscicapidae and Paridae are known to be two monophylitic families, but Sylviidae is not a monophyletic group. AK5 sequences had better efficacy in resolving close relationships of interspecies among intrageneric groups.

Chromodomain-helicase-DNA binding protein 5, 7 and pronecrotic mixed lineage kinase domain-like protein serve as potential prognostic biomarkers in patients with resected pancreatic adenocarcinomas

Pancreatic cancer is one of the deadliest cancers with a very poor prognosis. Recently, there has been a significant increase in research directed towards identifying potential biomarkers that can be used to diagnose and provide prognostic information for pancreatic cancer. These markers can be used clinically to optimize and personalize therapy for individual patients. In this review, we focused on 3 biomarkers involved in the DNA damage response pathway and the necroptosis pathway: Chromodomainhelicase-DNA binding protein 5, chromodomain-helicaseDNA binding protein 7, and mixed lineage kinase domain-like protein. The aim of this article is to review present literature provided for these biomarkers and current studies in which their effectiveness as prognostic biomarkers are analyzed in order to determine their future use as biomarkers in clinical medicine. Based on the data presented, these biomarkers warrant further investigation,and should be validated in future studies.

Role of Activator Protein-1 in the Transcription of Interleukin-5 Gene Regulated by Protein Kinase C Signal in Asthmatic Human TLymphocytes

Summary: In order to explore the role of activator protein-1 (AP-1) in the transcription of interleukin-5 (IL-5) gene regulated by protein kinase C (PKC) signal in peripheral blood T lymphocytes from asthmatic patient, T lymphocytes were isolated and purified from peripheral blood of each asthmatic patient. The T lymphocytes were randomly divided into 4 groups: group A (blank control), group B (treated with PKC agonist phorbol 12-myristate 13-acetate (PMA)), Group C (treated with PMA and AP-1 cis-element decoy oligodeoxynucleotides (decoy ODNs)), and group D (treated with PMA and AP-1 mutant decoy ODNs). The ODNs were transfected into the T cells of group C and D by cation liposome respectively. Reverse transcription-polymerase chain reaction (RT-PCR) was employed to assess IL-5 mRNA expression, and electrophoretic mobility shift assays (EMSA) for the activation of AP-1. The results showed that the activation of AP-1 (88 003.58±1 626.57) and the expression of IL-5 mRNA (0.8300±0.0294) in T lymphocytes stimulated with PMA were significantly higher than these in blank control (20 888.47±1103.56 and 0.3050±0.0208, respectively, P< 0.01), while the indexes (23 219.83±1 024.86 and 0.3425±0.0171 respectively) of T lymphocytes stimulated with PMA and AP-1 decoy ODNs were significantly inhibited, as compared with group B (P< 0.01). The indexes (87 107.41±1 342.92 and 0.8225±0.0222, respectively) in T lymphocytes stimulated with PMA and AP-1 mutant decoy ODNs did not exhibit significant changes, as compared with group B (P>0.05). The significant positive correlation was found between the activation of AP-1 and the expression of IL-5 mRNA (P< 0.01). It was concluded that AP-1 might participate in the signal transduction of PKC-triggered transcription of IL-5 gene in asthmatic T lymphocytes. This suggests the activation of PKC/AP-1 signal transduction cascade of T lymphocytes may play an important role in the pathogenesis of asthma.

Dual targeting of Polo-like kinase 1 and baculoviral inhibitor of apoptosis repeat-containing 5 in TP53-mutated hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma(HCC),often diagnosed at advanced stages without curative therapies,is the fifth most common malignant cancer and the second leading cause of cancer-related mortality.Polo-like kinase 1(PLK1)is activated in the late G2 phase of the cell cycle and is required for entry to mitosis.Interestingly,PLK1 is overexpressed in many HCC patients and is highly associated with poor clinical outcome.Baculoviral inhibitor of apoptosis repeatcontaining 5(BIRC5)is also highly overexpressed in HCC and plays key roles in this malignancy.AIM To determine the expression patterns of PLK1 and BIRC5,as well as their correlation with p53 mutation status and patient clinical outcome.METHODS The expression patterns of PLK1 and BIRC5,and their correlation with p53 mutation status or patient clinical outcome were analyzed using a TCGA HCC dataset.Cell viability,cell apoptosis,and cell cycle arrest assays were conducted to investigate the efficacy of the PLK1 inhibitors volasertib and GSK461364 and the BIRC5 inhibitor YM155,alone or in combination.The in vivo efficacy of volasertib and YM155,alone or in combination,was assessed in p53-mutated Huh7-derived xenograft models in immune-deficient NSIG mice.RESULTS Our bioinformatics analysis using a TCGA HCC dataset revealed that PLK1 and BIRC5 were overexpressed in the same patient subset and their expression was highly correlated.The overexpression of both PLK1 and BIRC5 was more frequently detected in HCC with p53 mutations.High PLK1 or BIRC5 expression significantly correlated with poor clinical outcome.PLK1 inhibitors(volasertib and GSK461364)or a BIRC5 inhibitor(YM155)selectively targeted Huh7 cells with mutated p53,but not HepG2 cells with wild-type p53.The combination treatment of volasertib and YM155 synergistically inhibited the viability of Huh7 cells via apoptotic pathway.The efficacy of volasertib and YM155,alone or in combination,was validated in vivo in a Huh7-derived xenograft model.CONCLUSION PLK1 and BIRC5 are highly co-expressed in p53-mutated HCC and inhibition of both PLK1 and BIRC5 synergistically compromises the viability of p53-mutated HCC cells in vitro and in vivo.

白藜芦醇激活细胞外信号调节激酶5信号蛋白促进小鼠MC3T3-E1细胞增殖

背景:细胞外信号调节激酶5信号蛋白对生物体的存活不可或缺,白藜芦醇能通过多种途径促进成骨细胞增殖,但其是否能通过细胞外信号调节激酶5信号蛋白调控成骨细胞功能还需进一步验证。目的:探究细胞外信号调节激酶5对MC3T3-E1细胞增殖以及相关分泌蛋白的调控作用,进一步验证白藜芦醇通过激活细胞外信号调节激酶5完成上述过程。方法:小鼠MC3T3-E1前成骨细胞分别用完全培养基、XMD8-92(细胞外信号调节激酶5抑制剂)、表皮生长因子(细胞外信号调节激酶5激活剂)和白藜芦醇单独干预及XMD8-92+表皮生长因子、白藜芦醇+XMD8-92干预后,通过Western blot检测各组细胞内细胞外信号调节激酶5、磷酸化细胞外信号调节激酶5蛋白,增殖相关蛋白Cyclin D1、CDK4、PCNA,以及成骨细胞分泌蛋白骨保护素、核因子κB受体活化因子配体的表达情况,使用细胞免疫荧光染色检测各组细胞外信号调节激酶5、骨保护素和核因子κB受体活化因子配体荧光强度,使用EdU染色检测各组细胞增殖情况。白藜芦醇干预MC3T3-E1细胞的适宜浓度及时间由细胞形态学观察和CCK-8实验确定。结果与结论:①细胞外信号调节激酶5信号蛋白的激活能有效促进MC3T3-E1细胞增殖、上调骨保护素/核因子κB受体活化因子配体比值;②白藜芦醇干预MC3T3-E1细胞的适宜浓度及时间为5μmol/L,24 h;③白藜芦醇可以激活细胞外信号调节激酶5信号蛋白,进而促进成骨细胞增殖,并上调骨保护素/核因子κB受体活化因子配体比值;④研究结果表明,白藜芦醇可以通过激活细胞外信号调节激酶5信号蛋白促进MC3T3-E1细胞增殖,并通过激活细胞外信号调节激酶5信号蛋白上调骨保护素/核因子κB受体活化因子配体比值。

Suppressing high mobility group box-1 release alleviates morphine tolerance via the adenosine5'-monophosphate-activated protein kinase/heme oxygenase-1 pathway

Opioids,such as morphine,are the most potent drugs used to treat pain.Long-term use results in high tolerance to morphine.High mobility group box-1(HMGB1) has been shown to participate in neuropathic or inflammatory pain,but its role in morphine tolerance is unclear.In this study,we established rat and mouse models of morphine tolerance by intrathecal injection of morphine for 7 consecutive days.We found that morphine induced rat spinal cord neurons to release a large amount of HMGB1.HMGB1 regulated nuclear factor κB p65 phosphorylation and interleukin-1β production by increasing Toll-like receptor 4receptor expression in microglia,thereby inducing morphine tolerance.Glycyrrhizin,an HMGB1 inhibito r,markedly attenuated chronic morphine tole rance in the mouse model.Finally,compound C(adenosine 5’-monophosphate-activated protein kinase inhibitor) and zinc protoporphyrin(heme oxygenase-1 inhibitor)alleviated the morphine-induced release of HMGB1 and reduced nuclear factor κB p65 phosphorylation and interleukin-1β production in a mouse model of morphine tolerance and an SH-SY5Y cell model of morphine tole rance,and alleviated morphine tolerance in the mouse model.These findings suggest that morphine induces HMGB1 release via the adenosine 5’-monophosphate-activated protein kinase/heme oxygenase-1 signaling pathway,and that inhibiting this signaling pathway can effectively reduce morphine tole rance.

Inositol 1,4,5-trisphosphate 3-kinases:functions and regulations

Inositol 1,4,5-trisphosphate 3-kinase (IP3 3-kinase/IP3K) plays an important role in signal transduction in animal cellsby phosphorylating inositol 1,4,5-trisphosphate (IP3) to inositol 1,3,4,5-tetrakisphosphate (IP4). Both IP3 and IP4 arecritical second messengers which regulate calcium (Ca2+) homeostasis. Mammalian IP3Ks are involved in many biologicalprocesses, including brain development, memory, learning and so on. It is widely reported that Ca2+ is a canonicalsecond messenger in higher plants. Therefore, plant IP3K should also play a crucial role in plant development. Recently,we reported the identification of plant IP3K gene (AtIpk2β/AtIP3K) from Arabidopsis thaliana and its characterization.Here, we summarize the molecular cloning, biochemical properties and biological functions of IP3Ks from animal, yeastand plant. This review also discusses potential functions of IP3Ks in signaling crosstalk, inositol phosphate metabolism,gene transcriptional control and so on.

In Vitro Functional Study of Rice Adenosine 5'-Phosphosulfate Kinase

Sulfate can be activated by ATP sulfurylase and adenosine 5'-phosphosulfate kinase(APSK) in vivo. Recent studies suggested that APSK in Arabidopsis thaliana regulated the partition between APS reduction and phosphorylation and its activity can be modulated by cellular redox status. In order to study regulation of APSK in rice(Os APSK), Os APSK1 gene was cloned and its activity was analyzed. Os APSK1 C36 and C69 were found to be the conserved counterparts of C86 and C119, which involved in disulfide formation in At APSK. C36A/C69 A Os APSK1 double mutation was made by site directed mutagenesis. Os APSK1 and its mutant were prokaryotically over-expressed and purified, and then assayed for APS phosphorylation activity. Os APSK1 activity was depressed by oxidized glutathione, while the activity of its mutant was not. Further studies in the case that oxidative stress will fluctuate in vivo 3'-phosphoadenosine-5'-phosphosulfate content, and all APSK isoenzymes have similar regulation patterns are necessary to be performed.

Levetiracetam induces tyrosine kinase receptor B expression in SH-SY5Y cells

Tyrosine kinase receptor B （TrkB） plays an important role in long-term potentiation and memory formation.The present study used all-trans retinoic acid to induce TrkB expression in SH-SY5Y cells,and observed the effects of levetiracetam （LEV） on TrkB expression.Following exposure to 10,50,and 100 μg/mL LEV,the number of TrkB-positive cells,and average absorbance value were increased.Results demonstrated that LEV can induce TrkB expression in SH-SY5Y cells.

Role of doublecortin-like kinase 1 and leucine-rich repeat-containing G-protein-coupled receptor 5 in patients with stage Ⅱ/Ⅲ colorectal cancer:Cancer progression and prognosis

BACKGROUND Cancer stem cells(CSCs)are a subpopulation of cancer cells with the potential of self-renewal and differentiation.CSCs play critical roles in tumorigenesis,recurrence,metastasis,radiation tolerance and chemoresistance.AIM To assess the expression patterns and clinical potential of doublecortin-like kinase 1(DCLK1)and leucine-rich repeat-containing G-protein-coupled receptor 5(Lgr5),as prognostic CSC markers of colorectal cancer(CRC).METHODS The expression of DCLK1 and Lgr5 in CRC tissue sections from 92 patients was determined by immunohistochemistry.Each case was evaluated using a combined scoring method based on signal intensity staining(scored 0-3)and the proportion of positively stained cancer cells(scored 0-3).The final staining score was calculated as the intensity score multiplied by the proportion score.Low expression of DCLK1 and Lgr5 was defined as a score of 0-3;high expression of DCLK1 and Lgr5 was defined as a score of≥4.Specimens were categorized as either high or low expression,and the correlation between the expression of DCLK1 or Lgr5 and clinicopathological factors was investigated.RESULTS DCLK1 and Lgr5 expression levels were significantly positively correlated.CRC patients with high DCLK1,Lgr5 and DCLK1/Lgr5 expressions had poorer progression-free survival and overall survival.Moreover,high expression of DCLK1 was an independent prognostic factor for recurrence and overall survival in patients with CRC by multivariate analysis(P=0.026 and P=0.049,respectively).CONCLUSION DCLK1 may be a potential CSC marker for the recurrence and survival of CRC patients.

Polyglutamylase activity of tubulin tyrosine ligase-like 4 is negatively regulated by the never in mitosis gene A family kinase never in mitosis gene A-related kinase 5

BACKGROUND Tubulins,building blocks of microtubules,are modified substrates of diverse post-translational modifications including phosphorylation,polyglycylation and polyglutamylation.Polyglutamylation of microtubules,catalyzed by enzymes from the tubulin tyrosine ligase-like(TTLL)family,can regulate interactions with molecular motors and other proteins.Due to the diversity and functional importance of microtubule modifications,strict control of the TTLL enzymes has been suggested.AIM To characterize the interaction between never in mitosis gene A-related kinase 5(NEK5)and TTLL4 proteins and the effects of TTLL4 phosphorylation.METHODS The interaction between NEK5 and TTLL4 was identified by yeast two-hybrid screening using the C-terminus of NEK5(a.a.260–708)as bait and confirmed by immunoprecipitation.The phosphorylation sites of TTLL4 were identified by mass spectrometry and point mutations were introduced.RESULTS Here,we show that NEK5 interacts with TTLL4 and regulates its polyglutamylation activity.We further show that NEK5 can also interact with TTLL5 and TTLL7.The silencing of NEK5 increases the levels of polyglutamylation of proteins by increasing the activity of TTLL4.The same effects were observed after the expression of the catalytically inactive form of NEK5.This regulation of TTLL4 activity involves its phosphorylation at Y815 and S1136 amino acid residues.CONCLUSION Our results demonstrate,for the first time,the regulation of TTLL activity through phosphorylation,pointing to NEK5 as a potential effector kinase.We also suggest a general control of tubulin polyglutamylation through NEK family members in human cells.

Cloning of the Full-length Gene for Tobacco Ethylene Receptor NTHK2 and Characterization of Its Kinase Domain

Previously the partial sequence of an ethylene receptor gene NTHK2 was isolated from tobacco (Nicotiana tabacum L. var. Xanthi) plants and it was wound and drought inducible. In the present study full-length cDNA of NTHK2 was cloned by 5'-RACE method. NTHK2 gene has 3 216 bp, with 509 bp of 5'-non-coding region and 427 lip of 3'-non-coding region, and encodes an ethylene-receptor homolog of 760, amino acids. NTHK2 protein has a putative signal peptide, three transmembrane domains, a histidine kinase domain and a receiver domain. In the putative histidine kinase domain, the histidine at the phosphorylation site was replaced by an asparagine. To study the biochemical property of NTHK2, its kinase domain was expressed as a fusion protein with glutathione S-transferase (GST) using yeast Schizzosaccharomyces pombe as an expression system. In vitro kinase assay showed that NTHK2 kinase domain can autophosphorylate in the presence of Mg2+, indicating that NTHK2 may function as a kinase. Further studies will elucidate the function of NTHK2 in plant.

Inhibition of Cyclin F Promotes Cellular Senescence through Cyclin-dependent Kinase 1-mediated Cell Cycle Regulation

Objective Kidney renal clear cell carcinoma(KIRC)is a common renal malignancy that has a poor prognosis.As a member of the F box family,cyclin F(CCNF)plays an important regulatory role in normal tissues and tumors.However,the underlying mechanism by which CCNF promotes KIRC proliferation still remains unclear.Methods Bioinformatics methods were used to analyze The Cancer Genome Atlas(TCGA)database to obtain gene expression and clinical prognosis data.The CCK8 assay,EdU assay,and xenograft assay were used to detect cell proliferation.The cell senescence and potential mechanism were assessed by SA-β-gal staining,Western blotting,as well as ELISA.Results Our data showed that CCNF was highly expressed in KIRC patients.Meanwhile,downregulation of CCNF inhibited cell proliferation in vivo and in vitro.Further studies showed that the reduction of CCNF promoted cell senescence by decreasing cyclin-dependent kinase 1(CDK1),increasing the proinflammatory factors interleukin(IL)-6 and IL-8,and then enhancing the expression of p21 and p53.Conclusion We propose that the high expression of CCNF in KIRC may play a key role in tumorigenesis by regulating cell senescence.Therefore,CCNF shows promise as a new biomarker to predict the clinical prognosis of KIRC patients and as an effective therapeutic target.