Decreased levels of phosphorylated synuclein in plasma are correlated with poststroke cognitive impairment

Poststro ke cognitive impairment is a major secondary effect of ischemic stroke in many patients;however,few options are available for the early diagnosis and treatment of this condition.The aims of this study were to(1)determine the specific relationship between hypoxic andα-synuclein during the occur of poststroke cognitive impairment and(2)assess whether the serum phosphorylatedα-synuclein level can be used as a biomarker for poststro ke cognitive impairment.We found that the phosphorylatedα-synuclein level was significantly increased and showed pathological aggregation around the cerebral infa rct area in a mouse model of ischemic stroke.In addition,neuronalα-synuclein phosphorylation and aggregation were observed in the brain tissue of mice subjected to chronic hypoxia,suggesting that hypoxia is the underlying cause ofα-synuclein-mediated pathology in the brains of mice with ischemic stroke.Serum phosphorylatedα-synuclein levels in patients with ischemic stroke were significantly lower than those in healt hy subjects,and were positively correlated with cognition levels in patients with ischemic stroke.Furthermore,a decrease in serum high-density lipoprotein levels in stroke patie nts was significantly correlated with a decrease in phosphorylatedα-synuclein levels.Although ischemic stroke mice did not show significant cognitive impairment or disrupted lipid metabolism 14 days after injury,some of them exhibited decreased cognitive function and reduced phosphorylatedα-synuclein levels.Taken together,our results suggest that serum phosphorylatedα-synuclein is a potential biomarker for poststroke cognitive impairment.

Phosphorylated cellulose nanofibers establishing reliable ion-sieving barriers for durable lithium-sulfur batteries

The shuttle effect is among the most characteristic and formidable challenges in the pursuit of high-performance lithium-sulfur(Li-S)batteries.Herein,phosphorylated cellulose nanofibers(pCNF)are intentionally engineered to establish an ion-sieving barrier against polysulfide shuttling and thereby improve battery performance.The phosphorylation,involving the grafting of phosphate groups onto the cellulose backbone,imparts an exceptional electronegativity that repels the polysulfide anions from penetrating through the separator.Moreover,the electrolyte wettability and Li^(+)transfer can be significantly promoted by the polar nature of pCNF and the facile Li^(+)disassociation.As such,rational ion management is realized,contributing to enhanced reversibility in both sulfur and lithium electrochemistry.As a result,Li-S cells equipped with the self-standing pCNF separator demonstrate outstanding long-term cyclability with a minimum fading rate of 0.013%per cycle over 1000 cycles at 1 C,and a decent areal capacity of 5.37 mA h cm^(-2) even under elevated sulfur loading of 5.0 mg cm^(-2) and limited electrolyte of 6.0 mL g^(-1).This work provides a facile and effective pathway toward the well-tamed shuttle effect and highly durable Li-S batteries.

Apoptosis of colon cancer CT-26 cells induced polysaccharide from Cyclocarya paliurus and its phosphorylated derivative via intrinsic mitochondrial passway

In this study,the antitumor properties and the possible molecular mechanisms of Cyclocarya paliurus polysaccharide(CP)and its phosphorylated derivative(P-CP)on CT-26 mouse colon carcinoma cells were investigated.Results found that CP had high inhibition ratio against CT-26 cells.The flow cytometry results found that CP treatment could cause the intracellular acidification,arrest the cell cycle in the S phase and increase reactive oxygen species generation.Additionally,CP treatment triggered mitochondrial membrane potential depolarization and Ca^(2+)overloading,and broke down the balance of antioxidant system,Na^(+)/K^(+)-ATPase and Ca^(2+)-ATPase.Further analysis found CP induced cell apoptosis through improving the activities of caspase-3 and caspase-9,and increasing the level of cytochrome C.Furthermore,the comparative study of antitumor effect on CT-26 cells displayed that the phosphorylation enhanced antitumor activities of polysaccharides.These results suggest CP is a potential natural therapeutic agent for colon cancer and phosphorylation represents an effective method of enhancing the antitumor activity of CP.

Raf kinase inhibitor protein combined with phosphorylated extracellular signal-regulated kinase offers valuable prognosis in gastrointestinal stromal tumor

BACKGROUND Gastrointestinal stromal tumors(GISTs)are the most common mesenchymal tumors of the gastrointestinal tract.Tyrosine kinase inhibitors,such as imatinib,have been used as first-line therapy for the treatment of GISTs.Although these drugs have achieved considerable efficacy in some patients,reports of resistance and recurrence have emerged.Extracellular signal-regulated kinase 1/2(ERK1/2)protein,as a member of the mitogen-activated protein kinase(MAPK)family,is a core molecule of this signaling pathway.Nowadays,research reports on the important clinical and prognostic value of phosphorylated-ERK(P-ERK)and phosphorylated-MAPK/ERK kinase(P-MEK)proteins closely related to raf kinase inhibitor protein(RKIP)have gradually emerged in digestive tract tumors such as gastric cancer,colon cancer,and pancreatic cancer.However,literature on the expression of these downstream proteins combined with RKIP in GIST is scarce.This study will focus on this aspect and search for answers to the problem.AIM To detect the expression of RKIP,P-ERK,and P-MEK protein in GIST and to analyze their relationship with clinicopathological characteristics and prognosis of this disease.Try to establish a new prognosis evaluation model using RKIP and PERK in combination with analysis and its prognosis evaluation efficacy.METHODS The research object of our experiment was 66 pathologically diagnosed GIST patients with complete clinical and follow-up information.These patients received surgical treatment at China Medical University Affiliated Hospital from January 2015 to January 2020.Immunohistochemical method was used to detect the expression of RKIP,PERK,and P-MEK proteins in GIST tissue samples from these patients.Kaplan-Meier method was used to calculate the survival rate of 63 patients with complete follow-up data.A Nomogram was used to represent the new prognostic evaluation model.The Cox multivariate regression analysis was conducted separately for each set of risk evaluation factors,based on two risk classification systems[the new risk grade model vs the modified National Institutes of Health(NIH)2008 risk classification system].Receiver operating characteristic(ROC)curves were used for evaluating the accuracy and efficiency of the two prognostic evaluation systems.RESULTS In GIST tissues,RKIP protein showed positive expression in the cytoplasm and cell membrane,appearing as brownish-yellow or brown granules.The expression of RKIP was related to GIST tumor size,NIH grade,and mucosal invasion.P-ERK protein exhibited heterogeneous distribution in GIST cells,mainly in the cytoplasm,with occasional presence in the nucleus,and appeared as brownish-yellow granules,and the expression of P-ERK protein was associated with GIST tumor size,mitotic count,mucosal invasion,and NIH grade.Meanwhile,RKIP protein expression was negatively correlated with P-ERK expression.The results in COX multivariate regression analysis showed that RKIP protein expression was not an independent risk factor for tumor prognosis.However,RKIP combined with P-ERK protein expression were identified as independent risk factors for prognosis with statistical significance.Furthermore,we establish a new prognosis evaluation model using RKIP and P-ERK in combination and obtained the nomogram of the new prognosis evaluation model.ROC curve analysis also showed that the new evaluation model had better prognostic performance than the modified NIH 2008 risk classification system.CONCLUSION Our experimental results showed that the expression of RKIP and P-ERK proteins in GIST was associated with tumor size,NIH 2008 staging,and tumor invasion,and P-ERK expression was also related to mitotic count.The expression of the two proteins had a certain negative correlation.The combined expression of RKIP and P-ERK proteins can serve as an independent risk factor for predicting the prognosis of GIST patients.The new risk assessment model incorporating RKIP and P-ERK has superior evaluation efficacy and is worth further practical application to validate.

Localization of Phosphorylated Histone H3 at Mitosis and Meiosis in Wheat

One of the prominent cell cycle related modifications of histone proteins, whose function is correlated with chromosome condensation, is the phosphorylation of histone H3. Wheat (Triticum aestivum L.) mitotic and meiotic cells were analyzed with indirect immunoflurorescence labeling with an antibody recognizing histone H3 phosphorylated at Serine 10 to study the localization of phosphorylated histone H3 at mitosis and meiosis. Our results showed that, during mitotic division, the phosphoryiation of H3 started from early prophase and vanished at telophase, remaining mainly in the pericentromeric regions at metaphase and anaphase. During meiotic division, phosphorylation of H3 initiated at the transition from leptotene to zygotene and remained uniform, along the chromosomes from prophase I until telophase whereas it showed slightly stronger in the pericentromeric regions than along the chromosome arms from metaphase II until Lelophase II The different patterns of H3 phophorylation at mitosis and meiosis in wheat suggested that this evolutionarily conserved post-translational chromatin modification might be involved in more roles besides chromosome condensation.

Casein kinase 2 interacts with and phosphorylates ataxin-3

Objective Machado-Joseph disease （MJD）/Spinocerebellar ataxia type 3 （SCA3） is an autosomal dominant neurodegenerative disorder caused by an expansion of polyglutamine tract near the C-terminus of the MJD1 gene product, ataxin-3. The precise mechanism of the MJD/SCA3 pathogenesis remains unclear. A growing body of evidence demonstrates that phosphorylation plays an important role in the pathogenesis of many neurodegenerative diseases. However, few kinases are known to phosphorylate ataxin-3. The present study is to explore whether ataxin-3 is a substrate of casein kinase 2 （CK2）. Methods The interaction between ataxin-3 and CK2 was identified by glutathione S-transferase （GST） pull-down assay and co-immunoprecipition assay. The phosphorylation of ataxin-3 by CK2 was measured by in vitro phosphorylation assays. Results （1） Both wild type and expanded ataxin-3 interacted with CK2α and CK2β in vitro. （2） In 293 cells, both wild type and expanded ataxin-3 interacted with CK2β, but not CK2α. （3） CK2 phosphorylated wild type and expanded ataxin-3. Conclusion Ataxin-3 is a substrate of protein kinase CK2.

Dynamic distribution of Ser-10 phosphorylated histone H3 in cytoplasm of MCF-7 and CHO cells during mitosis

The dynamic distribution of phosphorylated Histone H3 on Ser10 (phospho-H3) in cells was investigated to determineits function during mitosis. Human breast adenocarcinoma cells MCF-7, and Chinese hamster cells CHO were analyzedby indirect immunofluorescence staining with an antibody against phospho-H3. We found that the phosphorylationbegins at early prophase, and spreads throughout the chromosomes at late prophase. At metaphase, most of the phospho-H3 aggregates at the end of the condensed entity of chromosomes at equatorial plate. During anaphase and telophase,the fluorescent signal of phospho-H3 is detached from chromosomes into cytoplasm. At early anaphase, phospho-H3shows ladder bands between two sets of separated chromosome, and forms “sandwich-like structure” when the chro-mosomes condensed. With the cleavage progressing, the “ladders” of the histone contract into a bigger bright dot. Thenthe histone aggregates and some of compacted microtubules in the midbody region are composed into a “bar-like”complex to separate daughter cells. The daughter cells seal their plasma membrane along with the ends of the “bar”,inside which locates microtubules and modified histones, to finish the cytokinesis and keep the “bar complex” out of thecells. The specific distribution and kinetics of phospho-H3 in cytoplasm suggest that the modified histones may takepart in the formation of midbody and play a crucial role in cytokinesis.

Icariin upregulates phosphorylated cyclic adenosine monophosphate response element binding protein levels in the hippocampus of the senescence-accelerated mouse

At 8 weeks after intragastric administration of icariin to senescence-accelerated mice （P8 strain）, Morris water maze results showed that escape latency was shortened, and the number of platform crossings was increased. Immunohistochemical staining and western blot assay detected significantly increased levels of cyclic adenosine monophosphate response element binding protein These results suggest that icariin upregulates phosphorylated cyclic adenosine monophosphate response element binding protein levels and improves learning and memory functions in hippocampus of the senescence-accelerated mouse.

Resveratrol-downregulated Phosphorylated Liver Kinase B1 Is Involved in Senescence of Acute Myeloid Leukemia Stem Cells

Summary： Senescence is an important obstacle to cancer development. Engaging a senescent response may be an effective way to cure acute myeloid leukemia （AML）. The aim of this study was to examine the effect of resveratrol-downregulated phosphorylated liver kinase B1 （pLKB1） on the senescence of acute myeloid leukemia （AML） stem cells. The protein expressions of pLKB 1 and Sirtuin 1 （SIRT1）, a regulator ofpLKB1, were measured in CD34＋CD38-KGla cells treated with resveratrol （40 μmol/L） or not by Western blotting. Senescence-related factors were examined, including p21 mRNA tested by real-time PCR, cell morphology by senescence-associated β-galactosidase （SA-β-gal） staining, cell pro- liferation by MTT assay and cell cycle by flow cytometry. Besides, apoptosis was flow cytometrically determined. The results showed that pLKB1 was highly expressed in CD34＋CD38- KGla cells, and resveratrol, which could downregulate pLKB1 through activation of SIRT1, induced senescence and apoptosis of CD34＋CD38- KGla cells. It was concluded that resveratrol-downregulated pLKB1 is in- volved in the senescence of AML stem cells.

Phosphorylated tau as a toxic agent in synaptic mitochondria: implications in aging and Alzheimer’s disease

During normal aging,there is a decline in all physiological functions in the organism.One of the most affected organs is the brain,where neurons lose their proper synaptic function leading to cognitive impairment.Aging is one of the main risk factors for the development of neurodegenerative diseases,such as Alzheimer’s disease.One of the main responsible factors for synaptic dysfunction in aging and neurodegenerative diseases is the accumulation of abnormal proteins forming aggregates.The most studied brain aggregates are the senile plaques,formed by Aβpeptide;however,the aggregates formed by phosphorylated tau protein have gained relevance in the last years by their toxicity.It is reported that neurons undergo severe mitochondrial dysfunction with age,with a decrease in adenosine 5′-triphosphate production,loss of the mitochondrial membrane potential,redox imbalance,impaired mitophagy,and loss of calcium buffer capacity.Interestingly,abnormal tau protein interacts with several mitochondrial proteins,suggesting that it could induce mitochondrial dysfunction.Nevertheless,whether tau-mediated mitochondrial dysfunction occurs indirectly or directly is still unknown.A recent study of our laboratory shows that phosphorylated tau at Ser396/404(known as PHF-1),an epitope commonly related to pathology,accumulates inside mitochondria during normal aging.This accumulation occurs preferentially in synaptic mitochondria,which suggests that it may contribute to the synaptic failure and cognitive impairment seen in aged individuals.Here,we review the main tau modifications promoting mitochondrial dysfunction,and the possible mechanism involved.Also,we discuss the evidence that supports the possibility that phosphorylated tau accumulation in synaptic mitochondria promotes synaptic and cognitive impairment in aging.Finally,we show evidence and argue about the presence of phosphorylated tau PHF-1 inside mitochondria in Alzheimer’s disease,which could be considered as an early event in the neurodegenerative process.Thus,phosphorylated tau PHF-1 inside the mitochondria could be considered such a potential therapeutic target to prevent or attenuate age-related cognitive impairment.

Phosphorylated vasodilator-stimulated phosphoprotein is localized on mitotic spindles of the gastric cancer cell line SGC-7901

AIM： To elucidate the localization of vasodilator stimulated phosphoprotein （VASP）, a cytoskeletal organizing protein and a substrate of protein kinases A and G in mitotic gastric cancer cells. METHODS： Immunofluorescence microscopy was used to observe the localization of α-tubulin, VASP and Ser157 phosphorylated VASP （p-VASP） in interphase of mitotic gastric cancer of the cell line SGC-7901. RESULTS： Immunofluorescence staining showed that p-VASP but not VASP was co-localized with α-tubulin on spindle poles and fibers in prophase, metaphase and anaphase of the mitotic process of the gastric cancer cell line SGC-7901. H89, an inhibitor of protein kinases A and G, had no effect on the localization of p-VASP on the spindles. CONCLUSION： VASP may play a role in assembling and stabilizing the mitotic spindle of cells, and phosphorylation of the protein is the precondition for it to exert this function.

Phosphorylated mesoporous carbon as effective catalyst for the selective fructose dehydration to HMF

Phosphorylated mesoporous carbons （PMCs） were investigated as catalysts in the dehydration of fructose to hydroxymethylfurfural （HMF）. The acidic PMCs show better selectivity to HMF compared to sulfonated carbon catalyst （SC） despite lower activity. The concentration of P-O groups on the PMC was correlated with the activity/selectivity of the catalysts; the higher the P-O concentration, the higher the activity. However, the higher the P-O content, the lower the selectivity to HME Indeed, a lower concentration of the P-O groups minimized the degradation of HMF to levulinic acid and the formation of by-products, such as humines. Stability tests showed that these systems deactivate due to the formation of humines and water insoluble by-products derived from the dehydration of fructose which blocked the catalytically active sites.

Nanocomposite membranes with high-charge and size-screened phosphorylated nanocellulose fibrils for CO_(2)separation

In this study,cellulose nanofibrils(CNF)of high charge(H-P-CNF)and screened size(H-P-CNF-S)were fabricated by increasing the charge of phosphorylated cellulose nanofibrils(P-CNFs)during the pre-treatment step of CNF production.Results show that the H-P-CNF have a significantly higher charge(3.41 mmol g^(-1))compared with P-CNF(1.86 mmol g^(-1)).Centrifugation of H-P-CNF gave a supernatant with higher charge(5.4 mmol g^(-1))and a reduced size(H-P-CNF-S).These tailored nanocelluloses were added to polyvinyl alcohol(PVA)solutions and the suspensions were successfully coated on porous polysulfone(PSf)supports to produce thin-film nanocomposite membranes.The humid mixed gas permeation tests show that CO_(2)permeability increases for membranes with the addition of H-P-CNF-S by 52%and 160%,compared with the P-CNF/PVA membrane and neat PVA membrane,respectively.

REINFORCEMENT OF CALCIUM PHOSPHATE CEMENTS WITH PHOSPHORYLATED CHITIN

Phosphorylated chitins (P-chitins) as the additives of calcium phosphate cements (CPCs) were prepared by the phosphorylation of chitin with phosphorus pentoxide in methanesulfonic acid. Their physical properties and effects on CPCs from monocalcium phosphate monohydrate (MCPM) and calcium oxide (CaO) or dicalcium phosphate dihydrate (DCPD) and calcium hydroxide [Ca(OH)(2)] were investigated. Addition of P-chitin (M-w = 2.60 x 10(4); degree of substitution, DS = 0.68) to the liquid phase in amounts up to 3 wt.% for MCPM and CaO cements or 1.5 wt.% for DCPD and Ca(OH)(2) cements could enhance the mechanical strength considerably, while little influence on the setting time was observed. However, further addition of P-chitin will cause no setting.

Role of phosphorylated Smad3 signal components in intraductal papillary mucinous neoplasm of pancreas

Background:Malignant intraductal papillary mucinous neoplasm(IPMN)has poor prognosis.The carcinogenesis of IPMN is not clear.The aim of this study was to clarify transitions in phosphorylated Smad3 signaling during IPMN carcinogenesis.Methods:By using immunohistochemistry,we examined the expression of pSmad3C and pSmad3L from 51 IPMN surgical specimens resected at our institution between 2010 and 2013.We also examined the expression of Ki-67,c-Myc and p-JNK.Results:The median immunostaining index of pSmad3C was 79.2%in low-grade dysplasia,74.9%in highgrade dysplasia,and 42.0%in invasive carcinoma(P<0.01),whereas that of pSmad3L was 3.4%,4.3%,and 42.4%,respectively(P<0.01).There was a negative relationship between the expression of pSmad3C and c-Myc(P<0.001,r=-0.615)and a positive relationship between the expression of pSmad3L and c-Myc(P<0.001,r=0.696).Negative relationship between the expression of pSmad3C and Ki-67(P<0.01,r=-0.610)and positive relationship between the expression of pSmad3L and Ki-67(P<0.01,r=0.731)were confirmed.p-JNK-positive cells were frequently observed among pSmad3L-positive cancer cells.The median of pSmad3L/pSmad3C ratio in the non-recurrence group and the recurrence group were 0.58(range,0.05–0.93),3.83(range,0.85–5.96),respectively(P=0.02).The median immunostaining index of c-Myc in the non-recurrence group and the recurrence group were 2.91(range,0–36.9)and 82.1(range,46.2–97.1),respectively(P=0.02).The median immunostaining index of Ki-67 in the non-recurrence group and the recurrence group were 12.9(range 5.7–30.8)and 90.9(range 52.9–98.5),respectively(P=0.02).Conclusions:pSmad3L was upregulated in malignant IPMN.pSmad3L/pSmad3C ratio may be a useful prognostic factor in IPMN.

Overexpression of tyrosine phosphorylated proteins in reproductive tissues of polycystic ovary syndrome rats induced by letrozole

Objective:To identify the alteration of tyrosine phosphorylated protein expression in rats with polycystic ovary syndrome(PCOS).Methods:Sixteen female Sprague-Dawley rats were divided into the control and letrozole-induced PCOS groups.The oestrus cycle of rats was performed by vaginal smear.Sex hormones and morphology of the ovary,oviduct,and uterus were observed.Expressions and intensity of androgen receptor and tyrosine phosphorylated proteins of reproductive organs were investigated by Western blot.Results:Various polycysts and increased androgen receptor expression were present in the ovary of the PCOS group.The levels of follicle-stimulating hormone and testosteone were significantly higher in the PCOS group while progesterone and estradiol levels were significantly decreased as compared with the control group(P<0.05).Only the size of uterus in the PCOS group was significantly smaller than the control group.However,the density of collagen fibers observed in PCOS uterus was greater than the control group.Moreover,tyrosine phosphorylated proteins were significantly overexpressed in ovary(52,42,and 28 kDa),oviduct(72,56,42,and 28 kDa),and uterus(53 and 42 kDa)of the PCOS group compared to the control group.Conclusions:Presence of tyrosine phosphorylated proteins in the ovary,oviduct and uterus suggests that overexpression of tyrosine phosphorylated proteins may be involved in potential mechanism of female infertility especially in PCOS.

Distribution of phosphorylated Elk-1 in rat brain after Y-maze active avoidance training in a temporal manner

BACKGROUND： Elk-1 mRNA distributes extensively in the neurons of mice, rat and human brains, and the Elk-1 expression may be correlated with the synaptic plasticity, learning and memory. OBJECTIVE： To observe the distribution of phosphorylated Elk-1 （pEIk-1） in whole brain of rats received Y-maze active avoidance training and the changes of pEIk-1 expression at different time points after training. DESIGN ： A randomized controlled study SETTING ： Research Room of Neurobiology, the Second Affiliated Hospital of Southern Medical University MATERIALS ： Fifty-five male clean-degree SD rats of 3-4 months old, weighing 200-250 g, were provided by the Experimental Animal Center of Southem Medical University. The rabbit anti-monoclonal pEIk-1 antibody was purchased from Cell Signal Transduction Company, and ABC kit from Vector Company. METHODS ： The experiments were carried out in the Research Room of Neurobiology, Second Affiliated Hospital of Southern Medical University from September 2004 to February 2005. ① Grouping： The rats were randomly divided into training group （n = 25）, sham-training group （n = 25） and normal control group （n = 5）, and the training and sham-training groups were observed at 0, 1, 3, 6 and 24 hours after training, which represented the five phases in the process of leaming and memory. ② Y-maze training： The rats were preconditioned in the electrical Y-maze apparatus, 20 minutes a day for 3 days continuously, and training began from the 4^th day. In the training group, the rats were trained with the combination of light and electddty. Each rat repeated for 60 times in each training, and the correct times were recorded, those correct for less than 25 times were taken as unqualified, and excluded from the training group, and supplemented by other rats in time. In the sham-training group, there was no fixed correlation between the application of light and electricity. The rats in the normal contrel group were given not any training. ③Detection of pEIk-1 expression： The rats were anesthetized after Y-maze training, brain tissue was removed to prepare coronal freezing sections, and the pEIk-1 expression was detected with routine ABC method. MATN OUTCOME MEASURES： ① Distribution of pEIk-1 immuno-positive neurons in whole brain of rats in the normal control group. ②Comparison of the expression of pEIk-1 immuno-positive neurons in whole brain at different time points after training between the training group and sham-training group. RESULTS ： All the 55 rats were involved in result analysis. ③ Distribution of pEIk-1 immuno-positive neurons in the whole brain of rats in the normal control group： Strong expressions of pEIk-1 immuno-positive neurons were observed in prefrontal lobe, granular layer of olfactory bulbs, Purkinje cell layer and granular layer of cerebellum, whole stdate cortex, temporal cortex, pre-pyriform cortex, hypothalamic supraoptic nucleus, hypothalamic paraventricular nucleus and periventricular nucleus, thalamic paraventricular nucleus, pronucleus and postnucleus of amygdala cortex, central nucleus of amygdala, medial amygdaloid nucleus, entorhinal cortex, hippocampal dentate gyros, CA1-4 regions, caudate-putamen, material division, brain stem spinal nucleus of trigeminal nerve, and superior olivary nucleus, and those in hippocampal dentate gyrus and CA1 region were the strongest.② Distribution of pEIk-1 immuno-positive neurons in the whole brain of rats at different time points after training in the training group and sham-training group： In the training group, the expressions were obviously enhanced in caudate-putamen of striatum, material division, most cortexes, hippocampal dentate gyrus, hippocampal CA regions, nucleus amygdalae, thalamic paraventricular nucleus, Purkinje cell layer of cerebellum, entorhinal cortex, hypothalamic supraoptic nucleus, hypothalamic paraventricular nucleus, and periventricular nucleus at 0 hour after training, and the enhancement lasted for 6 hours at least, and those at 24 hours were decreased to normal. In the sham-training group, obvious enhanced expressions of pEIk-1 immuno-positive neurons could be observed in most cortexes, nucleus amygdalae, entorhinal cortex, hypothalamic supraoptic nucleus, hypothalamic paraventdoular nucleus and periventricular nucleus, brain stem spinal nucleus of trigeminal nerve, Purkinje cell layer and granular layer of cerebellum at O, 1, 3 and 6 hours, and decreased to normal after 24 hours. The expressions in material division, caudate-putamen of striatum, hippocampus were not obviously enhanced as compared with those in the normal control group, but significantly different from those in the training group （0, 1, 3, 6 hours after training, material division： F= 0.576, 0.023, 0.116, 8.873, P〈 0.01; caudate-putamen： F= 0.157, 0.427, 0.030, 0.001, P〈 0.01; hippocampus： F= 6.716, 2.405, 14.137, 1.416, P 〈 0.05-0.01 ）. CONCLUSION： The expression of activated pEIk-1 can be detected in the learning related brain areas under normal status, and the perk-1 expression in the brain areas dynamically changed in a time-dependent manner after Y-maze training, and it is indicated that pEIk-1 is involved in the learning and memory process in Y-maze related brain areas.

Tau protein,phosphorylated tau protein,and beta-amyloid 42 levels in patients with neurodegenerative diseases complicated by cognitive deficits A non-randomized,concurrent,case-control investigation

BACKGROUND： The differential diagnosis of many neurodegenerative disorders depends primarily on clinical symptoms together with imaging methods. Recently, increased importance has been placed on the use of biomarkers for diagnosing various neurodegenerative disorders. OBJECTIVE： To assess the feasibility of tau-protein, phosphorylated tau-protein, beta-amyloid 42 （Aβ42）, and 14-3-3 protein as biomarkers for diagnosing several neurodegenerative diseases complicated by cognitive deficits. DESIGN, TIME AND SETTING： A non-randomized, concurrent, case-control investigation was performed in three medical centers in the Czech Republic （Department of Neurology at the University Hospital in Hradec Kralove, Department of Neurology at the 2rd Medical Faculty, and the University Hospital Motol） between October 2000 and November 2006. PARTICIPANTS： Eighteen patients with probable AIzheimer＇s disease, 4 patients with Creutzfeldt-Jakob disease, 10 patients with frontotemporal dementia, 9 patients with clinically isolated syndrome suggestive of multiple sclerosis, and 7 patients with multiple sclerosis, as well as 38 race-, nationality-, and age-matched cognitively intact controls, were included in the study. Diagnoses were established based on the following criteria： the criteria for Alzheimer＇s disease proposed by the National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer＇s Disease and Related Disorders Association, WHO criteria for Creutzfeldt-Jakob disease, Neary criteria for frontotemporal dementia, and McDonald＇s criteria for multiple sclerosis. All included patients were confirmed to suffer from various degrees of dementia. METHODS： Enzyme-linked immunosorbent assay was used to measure concentrations of tau-protein, phosphorylated tau-protein, and Aβ42 in cerebrospinal fluid （CSF） samples collected by standard lumbar puncture from each patient. Moreover, 14-3-3 protein was assessed by Western blot in CSF of Creutzfeldt-Jakob disease patients. Cognitive status was assessed using the Mini Mental Scale Examination （MMSE） in all subjects. MAIN OUTCOME MEASURES： Establishment of biomarkers with greatest specificity and sensitivity for the investigated disorders according to Receiver Operating Characteristic curves, which were based on values from patients and controls; correlation between concentrations of given biomarkers and demographic parameters, diagnosis, duration of disease, and level of cognitive deficit. RESULTS： Increased concentrations of total tau protein and phosphorylated tau protein, and decreased levels of Aβ42, in CSF of Alzheimer＇s disease patients reached the required sensitivity/specificity ratio of 80% or greater. A marked elevation in CSF concentrations of total tau protein showed even greater sensitivity than 14-3-3 protein in Creutzfeldt-Jakob disease. There was no association between selected biomarkers and frontotemporal dementia or multiple sclerosis. Phosphorylated tau-protein was the only biomarker that noticeably correlated with MMSE scores for Alzheimer＇s disease.CONCLUSION： Levels of total tau protein, phosphorylated tau protein, and A!342 in the CSF could differentiate patients with Alzheimer＇s disease and Creutzfeldt-Jakob disease from healthy controls and patients with other neurodegenerative disorders. The diversity of absolute values demonstrates the necessity to establish a specific standard for each laboratory.

Proteomic Analysis of Nuclear Phosphorylated Proteins in Dairy Cow Mammary Epithelial Cells Treated with Prolactin

Prolactin （PRL） is a versatile signaling molecule and regulates a variety of physiological processes, including mammary gland growth and differentiation and the synthesis of milk proteins. While PRL is known to be necessary for high levels of milk protein expression, the mechanism by which the synthesis of milk proteins is stimulated at the transcript level is less known. A major modification in the transcript level is protein phosphorylation. To gain additional insights into the molecular mechanisms at the transcript level underlying PRL action on the dairy cow mammary epithelial cells （DCMECs）, nuclear phosphoproteins whose expression distinguishes proliferating regulated by PRL in DCMECs were identified. A phosphoprotein-enriched fraction from nuclear proteins was obtained by affinity chromatography, and a two-dimensional gel electrophoresis （2-DE） and matrix assisted laser desorption/ionization time of matrix-assisted laser desorption/ionization/time of flight mass spectrometry （MALDI-TOF MS） were used to identify the changes of nuclear phosphoproteins in DCMECs treated with prolactin. Seven proteins displaying~〉2-fold difference in abundance upon PRL treatment in DCMECs were identified by MALDI-TOF MS. The protein-GARS （GlyRS）, which belonged to the class-II aminoacyl-tRNA synthetase family, played a global role in the milk protein synthesis. SERPINH1 （Heat shock protein 47）, which was the first heat shock protein found to be a member of the serpin superfamily, regulated physiologic functions, such as complement activation, programmed cell death, and inflammatory processes. PRDX3, which belonged to a family of antioxidant enzymes, played an important role in scavenging intracellular reactive oxygen species （ROS）. ACTR1A, belonged to the actin family, which was associated with transport of p53 to the nucleus. Annexin A2, a Ca2＋-dependent phospholipid-binding protein, maintained the viability and cell cycle regulation of DCMECs. PSMB2 and PSMD10, which belonged to ubiquitin-proteasome system, were involved in several cellular processes, including cell cycle control, cellular stress response, intracellular signaling. This screening revealed that prolactin influenced the level of nuclear phosphoproteins in DCMECs. This result opens new avenues for the study of the molecular mechanism linked to the synthesis of milk proteins.

Cyclin A-Cdk2 Phosphorylates BH3 only Protein Bad in vitro and in vivo

Increasing evidence suggests that Cyclin A-Cdk2 activity is required in the apoptosis process induced by various stimuli.To determine a specific substrate of Cyclin A-Cdk2 for apoptosis,in this study,we carried out an in vitro kinase assay using immunoprecipitated complex Cyclin A-Cdk2 as an enzyme source,and recombinant protein GST-Bad as a substrate.Our study showed that Bad was clearly phosphorylated by Cyclin A-Cdk2 in vitro.To examine whether protein Bad can also be phosphorylated by Cyclin A-Cdk2 kinase in vivo,we transiently overexpressed protein Bad with Cyclin A or Cdk2-dn,a dominant negative version of Cdk2,in Hela cells and determined the phosphorylation status of protein Bad.The test showed that protein Bad was clearly phosphorylated in Cyclin A overexpressed cells,but not in Cdk2-dn or mock transfectent.Moreover,etoposide also caused the phosphorylation of endogenetic Bad.In conclusion,here we provide first time evidence that protein Bad can be a substrate of Cyclin A-Cdk2 apoptosis for in vitro and in vivo.