Plasma Trace Elements, Vitamin B12, Folate, and Homocysteine Levels in Cirrhotic Patients Compared to Healthy Controls

Increased serum homocysteine (Hcy) can induce liver diseases and can play a remarkable role in hepatic disorders. The purpose of the present study therefore was to investigate the relationship between serum vitamin B(12), folate, zinc and copper, cysteine, and Hcy level differences between cirrhotic patients and healthy subjects. We studied 32 cirrhotic patients (12 females and 20 males) aged 45 +/- 11 years and 32 control subjects (12 females and 20 males) aged 39 +/- 9 years. There was an inverse correlation between Hcy and vitamin B(12) in controls (r = -0.442, p < 0.011) but not in cirrhotic patients (r = -0.147, not significant). Also, mean plasma folate was decreased in cirrhotic patients compared to controls (p < 0.001). Copper increased whereas zinc decreased significantly in cirrhotic patients. A positive correlation was seen between the Cu/Zn ratio and Cu in controls (r = 0.690, p < 0.01), but the correlation between the Cu/Zn ratio and Cu was not significant in the cirrhotic group. Negative correlations were seen between plasma concentration of zinc and the Cu/Zn ratio in controls and cirrhotic patients (r = -0.618, p < 0.01 and r = -0.670, p < 0.01, respectively). Cirrhotic patients displayed multiple abnormalities, including changes in cysteine metabolism and in zinc and copper levels. Although hyperhomocysteinemia is known as an atherogenic and thrombogenic risk factor for cardiovascular disease, it might also be a risk factor for cirrhotic patients. Plasma Hcy, vitamin B(12), and folic acid measurement may be useful in the evaluation of cirrhotic patients.     

Folate deficiency and homocysteine induce toxicity in cultured dorsal root ganglion neurons via cytosolic calcium accumulation

Folate deficiency induces neurotoxicity by multiple routes, including increasing cytosolic calcium and oxidative stress via increasing levels of the neurotoxin homocysteine (HC), and inducing mitochondrial and DNA damage. Because some of these neurotoxic effects overlap with those observed in motor neuron disease, we examined the impact of folate deprivation on dorsal root ganglion (DRG) neurons in culture. Folate deprivation for 2 h increased cytosolic calcium and reactive oxygen species (ROS) and impaired mitochondrial function. Treatment with nimodipine [an L voltage-sensitive calcium channel (LVSCC) antagonist], MK-801 (an NMDA channel antagonist) and thapsigarin (an inhibitor of efflux of calcium from internal stores) indicated that folate deprivation initially induced calcium influx via the LVSCC, with subsequent additional calcium derived from NMDA channels and internal stores. These compounds also reduced ROS and mitochondrial degeneration, indicating that calcium influx contributed to these phenomena. Calcium influx was prevented by co-treatment with 3-deaza-adenosine, which inhibits HC formation, indicating that HC mediated increased cytosolic calcium following folate deprivation. Nimodipine, MK-801 and thapsigargin had similar effects following direct treatment with HC as they did following folate deprivation. These findings support the idea that folate deprivation and HC treatment can compromise the health of DRG neurons by perturbing calcium homeostasis.     

Identification of new tyrosine phosphorylated proteins in rat brain mitochondria

Reversible protein-phosphorylation is emerging as a key player in the regulation of mitochondrial functions. In particular tyrosine phosphorylation represents a promising field to highlight new mechanisms of bioenergetic regulation. Utilizing immunoaffinity enrichment of phosphotyrosine-containing peptides coupled to mass spectrometric analysis we detected new tyrosine phosphorylated proteins in rat brain mitochondria after peroxovanadate treatment. By bioinformatic predictions we provide suggestions about the potential role of tyrosine phosphorylation in mitochondrial physiology. Our results indicate a primary role of tyrosine phosphorylation in regulating energy production at the mitochondrial level. Moreover, tyrosine phosphorylation might regulate the mitochondrial membrane permeability targeting protein complexes containing ADP/ATP translocase, VDAC, creatine kinase and hexokinase.     

5,10‐Methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C polymorphisms: genotype frequency and association with homocysteine and folate levels in middle‐southern Italian adults.

Two genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) gene (C677T and A1298C) can influence the plasma homocysteine (Hcy) levels, especially in the presence of an inadequate folate status. The aim of this study was to evaluate the frequencies of C677T and of A1298C MTHFR polymorphisms and their correlation with Hcy and serum folate concentrations in a population of blood donors living in a region of middle‐southern Italy (the Molise Region). One hundred ninety seven blood donors were studied for total plasma Hcy, serum folate and C677T and A1298C MTHFR genotypes. The frequency of C677T genotypes was 20.8% (CC), 49.8% (CT) and 29.4% (TT); for the A1298C genotypes: 48.7% (AA), 43.7% (AC) and 7.6% (CC). Hcy and serum folate concentrations were significantly different among genotypes of the C677T polymorphism (CC versus CT versus TT: <0.0001 both for Hcy and folate), with Hcy values increasing, and serum folate decreasing, from CC to TT subjects. Regarding to A1298C polymorphism, the difference among genotypes (AA versus AC versus CC; p: 0.026 for Hcy and 0.014 for serum folate), showed an opposite trend for both parameters, with Hcy higher in the wild‐type and lower in the homozygotes and serum folate higher in CC than in AA subjects. In conclusion, we found a high frequency of MTHFR allele associated with high level of Hcy and low levels of folate in an Italian southern population. Copyright © 2013 John Wiley & Sons, Ltd.     

Acute anoxia induces tau dephosphorylation in rat brain slices and its possible underlying mechanisms

Abnormal phosphorylation of microtubule-associated protein tau plays a critical role in Alzheimer's disease (AD), together with a distinct decrease of energy metabolism in the affected brain regions. To explore the effect of acute energy crisis on tau phosphorylation and the underlying mechanisms, we incubated rat brain slices in artificial cerebrospinal fluid (aCSF) at 37 degrees C with or without an oxygen supply, or in aCSF with low glucose concentrations. Then, the levels of total, phosphorylated and unphosphorylated tau, as well as the activities and levels of protein phosphatase (PP)-1, PP-2A, glycogen synthase kinase 3 (GSK-3), extracellular signal-regulated protein kinase (ERK) and C-jun amino terminal kinase (JNK), were measured. It was found, unexpectedly, that tau was significantly dephosphorylated at Ser396/Ser404 (PHF-1), Ser422 (R145), Ser199/Ser202 (Tau-1), Thr181 (AT270), Ser202/Thr205 (AT8) and Thr231 (AT180) by acute anoxia for 30 min or 120 min. The activity of PP-2A and the level of dephosphorylated PP-2A catalytic subunit at tyrosine 307 (Tyr307) were simultaneously increased. The active forms of ERK1/2 and JNK1/2 were decreased under anoxic incubation. The PP-2A inhibitor, okadaic acid (OA, 0.75 microm), completely prevented tau from acute anoxia-induced dephosphorylation and restored the active forms of ERK1/2 and JNK1/2 to the control level. The activities and protein levels of GSK-3 and PP-1 showed no change during acute anoxia. These data suggest that acute anoxia induces tau dephosphorylation, and that PP-2A may play a key role in tau dephosphorylation induced by acute anoxia.     

还原叶酸载体基因（RFC1）与神经管和颅面畸形病因学关系的研究进展

神经管畸形和颅面畸形是最常见的出生缺陷,由遗传和环境因素共同作用所致,大规模的人群流行病学研究已证实,叶酸能降低发生这类畸形的危险。叶酸缺乏是神经管和颅面畸形发生的主要环境因素,但其机制尚不清楚,通过对与叶酸代谢有关的还原叶酸载体（reduced folate carrier,RFC）的生化特点、生理功能、还原叶酸载体基因（RFC1）结构功能、调控、表达及其与叶酸水平和神经管颅面畸形的关系等研究进展进行综述,从而为神经管和颅面畸形的病因学研究提出可能的候选基因。 Abstract: Neural tube and craniofacial defects are common birth defects which are ascribed to the combination of genetic and environmental factors. The population epidemiological studies suggested that periconceptional use of multivitamins containing folic acid can reduce a woman’s risk of having a child with neural tube and craniofacial defects. It’s a major environmental factor that periconceptinal women with deficiency of folic acid may increase their risk for delivering babies with neural tube and craniofacial defects, but the mechanism by which folic acid facilitated this risk rediction is unknown. This paper reviews folate transport carrier, Reduced Folate Carrier(RFC)’s characteristics in biological chemistry, physiological function, the folate transport mechanism, structure, function, regulation and expression of reduced folate carrier gene(RFC1), and the relationship between RFC1 with plasm or erythrocyte folate level and neural tube defects, et al. It is suggested a etiologic hypothesis in investigation of candidate gene encoding specific folat-related pathways of neural tube and craniofacial defects.     

PRAS40(Ser183)磷酸化多克隆抗体的制备及检测

PRAS40为富含脯氨酸、分子量为40kD的Akt底物蛋白,能够与雷怕霉素哺乳动物细胞靶点复合物1(mTORC1)结合,其苏氨酸183位点(Ser183)可被mTORC1磷酸化。为了制备PRAS40(Ser183)磷酸化多克隆抗体,本实验通过蛋白疏水性抗原性分析设计多肽抗原,用其免疫家兔获得抗血清,ELISA检测其效价为1:10000;Western blotting法检测发现,通过rProtein A Sepharose亲和层析纯化并经非磷酸化的抗原条吸附处理后的抗体可以明显提高磷酸化抗体的特异性;用PRAS40抗体及PRAS40(Ser183)磷酸化抗体对正常细胞HL7702、HEK293及肿瘤细胞HepG2、A549、S180的检测显示:磷酸化的Ser183在不同细胞中表达差异不显著,而在经细胞饥饿处理的HEK293细胞中却明显观察到了S183磷酸化水平随氨基酸含量降低而减弱的现象。因此,本实验所制备的抗体可用于PRAS40(Ser183)磷酸化位点的功能研究。     

Melatonin reduces protein and lipid oxidative damage induced by homocysteine in rat brain homogenates

Numerous data indicate that hyperhomocysteinemia is a risk factor for cardio- and cerebrovascular diseases. At least in part, homocysteine (HCY) impairs cerebrovascular function because it generates large numbers of free radicals. Since melatonin is a well-known antioxidant, which reduces oxidative stress and decreases HCY concentrations in plasma, the aim of this study was to investigate the effect of melatonin in preventing HCY-induced protein and lipid oxidation in rat brain homogenates. Brain homogenates were obtained from Sprague-Dawley rats and were incubated with or without HCY (0.01-5 mM) or melatonin (0.01-3 mM). Carbonyl content of proteins, and malondialdehyde (MDA) and 4-hydroxyalkenals (4-HDA) concentrations in the brain homogenates were used as an index of protein and lipid oxidation, respectively. Under the experimental conditions used, the addition of HCY (0.01-5 mM) to the homogenates enhanced carbonyl protein and MDA+4-HDA formation. Melatonin reduced, in a concentration-dependent manner, protein and lipid oxidation due to HCY in the brain homogenates. These data suggest that preserving proteins from oxidative insults is an additional mechanism by which melatonin may act as an agent in potentially decreasing cardiovascular and cerebrovascular diseases related to hyperhomocysteinemia.     

The cytoplasmic domain of Alzheimer's amyloid precursor protein is phosphorylated at Thr654, Ser655, and Thr668 in adult rat brain and cultured cells.

BACKGROUND: The cytoplasmic domain of the Alzheimer's disease amyloid precursor protein (APP) is phosphorylated in vitro at Thr654 and Ser655, and both in vitro and in intact cells at Thr668 (numbering for APP695 isoform). MATERIALS AND METHODS: We have developed phosphorylation state-specific antibodies to each of the sites, and we have used these to analyze the phosphorylation of APP in adult rat brain and in cultured cell lines. RESULTS: We demonstrate that all three sites in APP are phosphorylated in adult rat brain. Phosphorylation at Thr654, Ser655, and Thr668 was also observed in several cultured cell lines. In PC12 cells, phosphorylation at Ser655 was increased more than 10-fold by treatment with okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, but was not affected by activators of protein kinase C. In HeLa cells, phosphorylation at Thr668 was regulated in a cell cycle-dependent manner with near-stoichiometric phosphorylation being observed at the G2/M phase of the cell cycle. In general, phosphorylation at Ser655 was found to be highest in mature APP isoforms, whereas phosphorylation of Thr668 was highest in immature APP isoforms in cultured cells. CONCLUSIONS: The results demonstrate that phosphorylation of the cytoplasmic domain of APP occurs at Thr654, Ser655, and Thr668 under physiological conditions. The further characterization of APP phosphorylation using phosphorylation-specific antibodies may help in the elucidation of the biological function of APP.     

Stopped-Flow Kinetic Studies of the Interaction of Bovine Folate Binding Protein (FBP) and Folate

The kinetics of the interaction of bovine folate binding protein and folate at pH 7.4 and 5.0 were followed by measuring the changes of the intrinsic protein fluorescence intensity using the stopped-flow technique, which enables the study of reactions from the millisecond time-range. Our results immediately reject a simple one-step binding model, which requires a linear dependence of the observed rate constant on the concentration of the ligand. Thus, we are able to conclude that at pH 5.0 the interaction occurs in two steps and at pH 7.4 in three steps. Changes of fluorescence spectra at equilibrium were used to estimate the overall binding constants. Comparative studies on the binding of folate to human albumin are also reported.     

低氧对小鼠学习记忆力及脑中tau蛋白磷酸化的影响

目的：研究不同低氧暴露对小鼠学习记忆及脑中tau蛋白磷酸化的影响。方法：雄性昆明小鼠40只,随机分为4组（n=10）：对照组（control）、8h低氧暴露组（8h）、7d低氧暴露组（7d）和28d低氧暴露组（28d）。将低氧暴露模型组置于模拟高原海拔5500m的低压氧舱,每天低氧暴露8h,避暗和旷场实验检测其活动能力及学习记忆能力：免疫印迹技术检测小鼠海马和皮层中tau蛋白磷酸化水平。结果：随着低氧时间的增加,小鼠短期学习记忆力及活动能力下降程度增大,28d低氧暴露后其下降程度最大;海马中tau蛋白多个位点的磷酸化水平呈现升高趋势,28d时tau蛋白磷酸化程度最高（P〈0．05）;皮层中的磷酸化水平在低氧暴露7d时达到最高,低氧暴露28d时略有降低,但与control组相比仍有明显差异（P〈0．05）。结论：慢性间歇性低氧可导致小鼠学习记忆能力下降,其机制可能与tau蛋白过度磷酸化相关。     

Immunoreactivity of the phosphorylated axonal neurofilament H subunit (pNF-H) in blood of ALS model rodents and ALS patients: evaluation of blood pNF-H as a potential ALS biomarker

Levels of neurofilament subunits, potential biomarkers of motor axon breakdown, are increased in amyotrophic lateral sclerosis (ALS) patient's CSF but data on blood are not available. We measured blood levels of the phosphorylated axonal form of neurofilament H (pNF-H) by ELISA in transgenic rodent models of superoxide dismutase 1 (SOD1) ALS, and in 20 ALS patients and 20 similar aged controls monthly for 4 months. All symptomatic rodent ALS models showed robust levels of blood pNF-H, while control rodents or mice transgenic for unmutated SOD1 showed no detectable blood pNF-H. Average pNF-H levels in the G93A SOD1 mouse progressively increased from day 74 through death (day ∼130). Median blood pNF-H level in ALS patients was 2.8-fold higher than controls ( p  < 0.001). Median ALSFRS-R declined a median of 0.8 pt/month ( p  < 0.001); higher baseline pNF-H level appeared to be associated with faster ALSFRS-R decline over 4 months ( p  = 0.087). The median rate of decline in ALSFRS-R was 1.9 pt/month in patients with baseline pNF-H levels above the median pNF-H value of 0.53 ng/mL; ALSFRS-R declined at a median of 0.6 pt/month in patients below this level. The pNF-H levels were relatively stable month to month in individual patients, raising questions regarding the molecular pathogenesis of ALS. Baseline control human pNF-H levels were higher in men than women and increased minimally over time. These data suggest that blood pNF-H can be used to monitor axonal degeneration in ALS model rodents and support further study of this protein as a potential biomarker of disease prognosis in ALS patients.     

Synapsin I is phosphorylated at Ser603 by p21-activated kinases (PAKs) in vitro and in PC12 cells stimulated with bradykinin

The function of synapsin I is regulated by phosphorylation of the molecule at multiple sites; among them, the Ser(603) residue (site 3) is considered to be a pivotal site targeted by Ca(2+)/calmodulin-dependent kinase II (CaMKII). Although phosphorylation of the Ser(603) residue responds to several kinds of stimuli, it is unlikely that many or all of the stimuli activate the CaMKII-involved pathway. Among the several stimulants tested in PC12 cells, bradykinin evoked the phosphorylation of Ser(603) without inducing the autophosphorylation of CaMKII, which was determined using phosphorylation site-specific antibodies against phospho-Ser(603)-synapsin I (pS603-Syn I-Ab) and phospho-Thr(286/287)-CaMKII. The bradykinin-evoked phosphorylation of Ser(603) was not suppressed by the CaMKII inhibitor KN62, whereas high KCl-evoked phosphorylation was accompanied by CaMKII autophosphorylation and inhibited by KN62. Thus, we attempted to identify Ser(603) kinase(s) besides CaMKII. We consequently detected four and three fractions with Ca(2+)/calmodulin-independent Ser(603) kinase activity on the DEAE column chromatography of bovine brain homogenate and PC12 cell lysate, respectively, two of which were purified and identified by amino acid sequence of proteolytic fragments as p21-activated kinase (PAK) 1 and PAK3. The immunoprecipitants from bovine brain homogenate with anti-PAK1 and PAK3 antibodies incorporated (32)P into synapsin I in a Cdc42/GTPgammaS-dependent manner, and its phosphorylation site was confirmed as Ser(603) using pS603-Syn I-Ab. Additionally, recombinant GST-PAK2 could phosphorylate the Ser(603) residue in the presence of Cdc42/GTPgammaS. Finally, we confirmed by immunocytochemical analysis that the transfection of constitutively active rat alphaPAK (PAK1) in PC12 cells evokes the phosphorylation of Ser(603) even in the resting mutant cells and enhances it in the bradykinin-stimulated cells, whereas that of dominant-negative alphaPAK quenches the phosphorylation. These results raise the possibility that Ser(603) on synapsin I is alternatively phosphorylated by PAKs, not only by CaMKII, in neuronal cells in response to some stimulants.     

GRP94 (endoplasmin) co-purifies with and is phosphorylated by Golgi apparatus casein kinase

Certain picornaviruses encode proteinases which cleave the translation initiation factor eIF4G, a member of the eIF4F complex which recruits mRNA to the 40S ribosomal subunit during initiation of protein synthesis in eukaryotes. We have compared the efficiency of eIF4G cleavage in rabbit reticulocyte lysates during translation of mRNAs encoding the foot-and-mouth disease virus leader proteinase (L^pro) or the human rhinovirus 2A^pro. Under standard translation conditions, L^pro cleaved 50% of eIF4G within 4 min after initiation of protein synthesis, whereas 2A^pro required 15 min. At these times, the molar ratios of proteinase to eIF4G were 1:130 for L^pro and 1:12 for 2A^pro, indicating a much more efficient in vitro cleavage than previously observed. The molar ratios are similar to those observed during viral infection in vivo.     

Depleted uranium is not toxic to rat brain endothelial (RBE4) cells

Studies on Gulf War veterans with depleted uranium (DU) fragments embedded in their soft tissues have led to suggestions of possible DU-induced neurotoxicity. We investigated DU uptake into cultured rat brain endothelial cells (RBE4). Following the determination that DU readily enters RBE4 cells, cytotoxic effects were analyzed using assays for cell volume increase, heat shock protein 90 (Hsp90) expression, 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) reduction, and lactate dehydrogenase (LDH) activity. The results of these studies show that uptake of the U₃O₈ uranyl chloride form of DU into RBE4 cells is efficient, but there are little or no resulting cytotoxic effects on these cells as detected by common biomarkers. Thus, the present experimental paradigm is rather reassuring and provides no indication for overt cytotoxicity in endothelial cells exposed to DU.     

The 12‐15‐lipoxygenase is a modulator of Alzheimer's‐related tau pathology in vivo

12/15‐lipoxygenase (12‐15LO) is a lipid‐peroxidizing enzyme widely expressed in the central nervous system where it has been involved in the neurobiology of Alzheimer's disease (AD) because it modulates amyloid beta (Aβ) and APP processing. However, its biological effect on tau protein is unknown. We investigated the effect of 12‐15LO on tau levels and metabolism in vivo and in vitro and the mechanism involved by using genetic and pharmacologic approaches. While no significant differences were observed in the levels of total tau for both groups, compared with controls, Tg2576 mice overexpressing 12‐15LO had elevated levels of phosphorylated tau at two specific epitopes, Ser 202/Thr 205 and Ser 396. In vitro and in vivo studies show that 12‐15LO modulates tau metabolism specifically via the cdk5 kinase pathway. Associated with these changes were biochemical markers of synaptic pathology. Finally, 12‐15LO‐dependent alteration of tau metabolism was independent from an effect on Aβ. Our findings reveal a novel pathway by which 12‐15LO modulates endogenous tau metabolism making this protein an appealing pharmacologic target for treatment of AD and related tauopathies.