Changes of voltage-dependent anion-selective channel proteins VDAC1 and VDAC2 brain levels in patients with Alzheimer's disease and Down syndrome

Voltage-dependent anion-selective channel proteins (VDACs) are pore-forming proteins found in the other mitochondrial membrane of all eukaryotes and in brain postsynaptic membranes. VDACs regulate anion fluxes of a series of metabolites including ATP, thus regulating mitochondrial metabolic functions. We determined protein levels of VDACs in individual post-mortem brain regions of patients with Down Syndrome (DS) and Alzheimer's disease (AD) using two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-mass spectroscopy (MALDI-MS). VDAC1 (SWISS-PROT accession number P21796) and VDAC2 (P45880) were unambiguously identified and quantified, but VDAC3 was not found. The spots representing VDAC1 were separated with different p/s (p/7.5, 8.5, and 10.0) probably caused by post-translational modifications as, e.g., phosphorylation. In DS cerebellum, total VDAC1 protein was elevated significantly whereas VDAC2 did not show any significant alterations. In AD brains, VDAC1 p/10.0 was significantly reduced in temporal, frontal, and occipital cortex with the p/7.5 form elevated in occipital cortex. Total VDAC1 was significantly decreased in frontal cortex and thalamus. VDAC2 was significantly elevated in temporal cortex only. The biological meaning of our results may be derangement of voltage-dependent anion-selective channel function and reflecting impaired glucose, energy, and intermediary metabolism as well as apoptotic mechanisms.     

Conformational studies of the robust 2-Cys peroxiredoxin Salmonella typhimurium AhpC by solution phase hydrogen/deuterium (H/D) exchange monitored by electrospray ionization mass spectrometry

This is the first comprehensive HX-MS study of a "robust" 2-Cys peroxiredoxin (Prx), namely Salmonella typhimurium AhpC (StAhpC). Prx proteins control intracellular peroxide levels and are abundant antioxidant proteins in eukaryotes, archaea and bacteria. Crystal structural analyses and structure/activity studies of several bacterial and mammalian 2-Cys Prxs have revealed that the activity of 2-Cys Prxs is regulated by redox-dependent oligmerization and a sensitivity of the active site cysteine residue to overoxidation. The propensity to overoxidation is linked to the conformational flexibility of the peroxidatic active site loop. The HX-MS results emphasize the modulation of the conformational motility of the active site loop by disulfide formation. To obtain information on the conformational impact of decamer formation on the active site loop motility, mutants with Thr77 substituted by Ile, a decamer-disrupting mutation or by Val, a decamer-stabilizing mutation, were studied. For the isoleucine mutant, enhanced mobility was observed for regions encompassing the α4 helix located in the dimer-dimer interface and regions surrounding the peroxidatic loop. In contrast, the T77V mutation resulted in an increase in conformational stability in most regions of the protein except for the active site loop and the region encompassing the resolving cysteine.     

Evidence for the relation of herpes simplex virus type 1 to Down syndrome and Alzheimer's disease

The peripheral and central nervous system are harbouring herpes simplex virus type 1 (HSV-1) and this virus has been proposed to be implicated in the aetiology of Alzheimer's disease (AD). We tested whether the HSV-1 genome is found indeed in the brain of controls, patients with AD and Down syndrome (DS) and whether HSV-1 infectious proteins in brain were induced. Moreover, we tested whether interleukin (IL)-6, a marker for neuroinflammation, is found in brains of AD and DS. HSV-1 glycoprotein D gene, as well as viral phosphoprotein and glycoprotein were detected in all brain samples. IL-6 was detectable in seven out of the eight AD and all of the eight DS patients, but only three out of ten controls in the frontal cortex. IL-6 in cerebellum was detectable in all AD and DS patients, but only three out of nine controls. In conclusion, we propose that the detection of HSV-1 genome and HSV-1 inducible protein IL-6 not only shows the presence in human brain, but may indicate a role for HSV-1 in the process of neuroinflammation and apoptosis, known to occur in both neurodegenerative disorders, AD and DS.     

Differential expression of molecular chaperones in brain of patients with Down syndrome

Heat shock proteins (HSPs) in their molecular capacity as chaperones have been reported to regulate the apoptotic pathway and also play a critical role in protein conformational diseases such as Alzheimer's disease (AD). As all Down syndrome (DS) brains display AD-like neuropathology, neuronal loss in DS was shown to be mediated by apoptosis. We decided to investigate the expression patterns of HSPs in seven brain regions of adults with DS using two-dimensional polyacrylamide gel electrophoresis (2-DE). Following 2-DE, approximately 120 protein spots were successfully identified by matrix-assisted laser desorption/ionization--mass spectrometry (MALDI-MS) followed by quantification of the identified proteins. We unambiguously identified and quantified nine different chaperone proteins. Accordingly, all but three chaperone proteins did exhibit a significant change in expression. HSP 70 RY, heat shock cognate (HSC) 71 and glucose-regulated protein (GRP) 75 showed a significant decrease (P < 0.05) in DS temporal cortex whereas HSP 70.1 and GRP 78 were significantly increased (P<0.05) in cerebellum. Whilst T-complex 1 (TCP-1) epsilon subunit showed a significant decrease (P< 0.05) in parietal cortex, a similar extent of increase (P<0.05) as that observed in cerebellum was obtained in parietal levels of GRP 78. Alpha-crystallin B, HSP 60 and GRP 94 did not show any detectable changes in expression patterns. This report presents the first approach to quantify nine different chaperones simultaneously at the protein level in different brain regions and provides evidence for aberrant chaperone expression patterns in DS. The relevance of this aberrant expression patterns are discussed in relation to the biochemical and neuropathological abnormalities in DS brain.     

Structural Properties of Macrodontain I,a Cysteine Protease from <Emphasis Type="Italic">Pseudananas macrodontes</Emphasis> (Morr.) Harms (Bromeliaceae)

The primary structure of macrodontain I, a peptidase from Pseudananas macrodontes fruits, was determined using Edman’s degradation. The enzyme is a non-glycosylated peptidase composed by 213 amino acids with a calculated molecular weight of 23,486.18 Da, pI value 6.99, and a molar extinction coefficient at 280 nm of 61,685 M^?1 cm^?1. The alignment of the sequence of macrodontain I with those cysteine peptidases from species belonging to the family Bromeliaceae showed the highest identity degree (87.74%) against fruit bromelain. A remarkable fact is that all these peptidase sequences show two Met contiguous residues (Met121 and 122) and the nonapeptide VPQSIDWRD located in the mature N-terminal region. Residues Cys26 and His159, which constitute the catalytic dyad in all cysteine peptidases, as well as active site residues Gln20 and Asn176, characteristic of Clan C1A, are conserved in macrodontain I. The 3-D model suggests that the enzyme belongs to the α?+?β class of proteins, with two disulfide bridges (Cys23-Cys63 and Cys57-Cys96) in the α domain, while the β domain is stabilized by another disulfide bridge (Cys153-Cys201). Further, we were able to establish that the cysteine peptidases from P. macrodontes are involved in the anti-inflammatory activity.     

Natural products triptolide,celastrol, and withaferin A inhibit the chaperone activity of peroxiredoxin I

Peroxiredoxin I (Prx I) plays an important role in cancer development and inflammation. It is a dual-functional protein which acts as both an antioxidant enzyme and a molecular chaperone. While there have been intensive studies on its peroxidase activity, Prx I''s chaperone activity remains elusive, likely due to the lack of chaperone inhibitors. Here we report that natural product triptolide selectively inhibits the chaperone activity of Prx I, but not its peroxidase activity. Through direct interaction with corresponding cysteines, triptolide triggers dissociation of high-molecular-weight oligomers of Prx I, and thereby inhibits its chaperone activity in a dose-dependent manner. We have also identified celastrol and withaferin A as novel Prx I chaperone inhibitors that are even more potent than triptolide in the chaperone activity assay. By revealing the exact molecular mechanisms of interaction and inhibition, the current study provides the first Prx I chaperone inhibitors as promising pharmacological tools for modulating and dissecting the chaperone function of Prx I.     

卒中后癫痫的静息态脑功能磁共振成像研究

本文探讨了静息态脑功能磁共振成像(rs-fMRI)检查在评价卒中后癫痫患者静息态脑功能改变中的应用价值。选择2017年5月~2019年5月我院收治的80例卒中后癫痫患者作为观察组,另选择同期健康体检者53例作为对照组。两组均完成rs-fMRI检查,采集相关数据,计算并比较两组被试全脑低频振幅(ALFF)值。分别以大脑(小脑)ALFF差异区作为种子区,计算并比较其与小脑(大脑)所有体素之间静息态功能连接(rsFC)。与对照组比较,卒中后癫痫患者右侧楔前叶、左侧额中回的ALFF明显升高,而右侧海马、右侧小脑脚2区、左侧小脑4/5区的ALFF明显降低(P<0.05)。以各大脑ALFF差异区(右侧海马、右侧楔前叶、左侧额中回)作为种子区域,卒中后癫痫患者依次对应在左侧小脑8区、右侧小脑4/5区的rsFC值明显升高(P<0.05);以各小脑ALFF差异区(右侧小脑脚2区、左侧小脑4/5区)作为种子区域,卒中后癫痫患者依次对应在左侧额下回、右侧颞中回的rsFC值明显降低(P<0.05)。结果表明,卒中后癫痫患者大脑和小脑之间多个脑区功能连通性发生改变,rs-fMRI检查可客观评价患者静息态脑功能变化,为患者的临床诊疗及预后评估提供影像学依据。     

Agave fructans and oligofructose decrease oxidative stress in brain regions involved in learning and memory of overweight mice

Obesity is currently a public health problem worldwide. Recently, non-reducing carbohydrates, that include β(2→1) and β(2→6) linkages in their structure, have been of particular interest in the field of obesity because they are involved in lipid metabolism. Some of these are agave fructans (AF) and oligofructose (OF). In this study, we evaluated both AF and OF on oxidative stress (OS) markers in the brain of overweight mice (OM). AF and OF decreased TBARS levels and carbonyls at different levels in hippocampus (HP), frontal cortex (FC) and cerebellum (CB) of OM. The results indicated that fructans may have anti-oxidative potential and can be used as an alternative treatment for the prevention of the consequences of this pathology.     

Decreased l-tryptophan concentration in distinctive brain regions of mice treated repeatedly with phencyclidine

It has been reported that repeated phencyclidine (PCP) treatment induces schizophrenia-like behavior in mice. l-Tryptophan (Trp) concentrations in brain tissues of control (n?=?8) and PCP-treated mice (10 mg/kg/day, s.c., 14 days, n?=?10) were determined using high-performance liquid chromatography (HPLC) with fluorescence detection. The HPLC method involved pre-column fluorescence derivatization with (R)-(?)-4-(N,N-dimethylaminosulfonyl)-7-(3-isothiocyanatopyrrolidin-1-yl)-2,1,3-benzoxadiazole (DBD-PyNCS). Eight different parts of the brain, namely, the frontal cortex, nucleus accumbens, striatum, hippocampus, amygdala, thalamus, hypothalamus, and cerebellum, of both groups were investigated. A significant decrease in the l-Trp concentration in the nucleus accumbens (p?=?0.024) and hippocampus (p?=?0.027) was observed in PCP-treated mice, suggesting that alteration of the l-Trp metabolism might occur in these brain parts.     

An important role for peroxiredoxin II in survival of A549 lung cancer cells resistant to gefitinib

Redox adaptation is an important concept that explains the mechanisms by which cancer cells survive under persistent endogenous oxidative stress and become resistant to certain anticancer agents. To investigate this concept, we determined the expression levels of peroxiredoxins (Prxs), antioxidant enzymes in drug-resistant non-small cell lung carcinoma cells. Prx II was remarkably increased only in A549/GR (gefitinib-resistant) cells compared with A549 cells, consistent with methylation/demethylation. Prx II was highly methylated in the A549 cells but was demethylated in the A549/GR cells. The elevated expression of Prx II resulted in the downregulation of reactive oxygen species (ROS) and cell death and upregulation of cell cycle progression in the A549/GR cells. When Prx II mRNA in the A549/GR cells was knocked down, the levels of ROS and apoptosis were significantly recovered to the levels of the controls. In addition, signaling molecules involved in apoptosis were increased in the A549/GR-shPrx II cells. There was no difference in the expression of MAPK/ERK between the A549/GR cells and A549/GR-shPrx II cells, but the phosphorylation of JNK was increased in the A549/GR cells and was markedly decreased in the A549/GR-shPrx II cells. Colony number and tumor growth were significantly decreased in the A549/GR-shPrx II cells compared with the A549/GR cells. Our findings suggest that Prx II has an important role in cancer cell survival via the modulation of signaling molecules involved in apoptosis and the phosphorylation of JNK by the downregulation of ROS levels in A549/GR cells.     

Cloning, Sequencing, and Identification Using Proteomic Tools of a Protease from Bromelia hieronymi Mez

Fruits of Bromelia hieronymi, a tropical South American plant, possess a high content of peptidases with potential biotechnological uses. Total RNA was extracted from unripe fruits and peptidase cDNA was obtained by 3'RACE-PCR. The consensus sequence of the cysteine peptidase cDNA contained 875 bp, the 690 first ones codifying for a hypothetical polypeptide chain of the mature peptidase, named Bh-CP1 (molecular mass 24.773 kDa, pI 8.6, extinction molar coefficient 58,705 M(-1) cm(-1)). Bh-CP1 sequence shows a high percentage of identity with those of other cysteine plant proteases. The presence of highly preserved residues is observed, like those forming the catalytic site (Gln19, Cys25, His159, and Asn175, papain numbering), as well as other six Cys residues, involved in the formation of disulfide bounds. Molecular modeling results suggest the enzyme belongs to the α?+?β class of proteins, with two disulfide bridges (Cys23-Cys63 and Cys57-Cys96) in the α domain, while the β domain is stabilized by another disulfide bridge (Cys153-Cys203). Additionally, peptide mass fingerprints (PMFs) of the three peptidases previously isolated from B. hieronymi fruits (namely hieronymain I, II, and III) were performed and compared with the theoretical fingerprint of PMF of Bh-CP1, showing a partial matching between the in silico-translated protein and hieronymain II.     

Exploration of Potential Tumor Markers for Lung Adenocarcinomas by Two‐Dimensional Gel Electrophoresis Coupled with Nano‐LC/MS/MS

To explore the potential tumor markers for lung adenocarcinoma, two‐dimensional gel electrophoresis (2DE) coupled with nano‐LC/MS/MS was used to analyze the differentially expressed proteins in 10 surgical resected lung adenocarcinoma tissues. 16 proteins were significantly different between the cancer tissue and adjacent normal tissue. Galectin‐1, peroxiredoxin II (Prx II), proapolipoprotein, glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), aldolase A, enolase 1, neuropolypeptide h3, Prx V, cyclophilin A, vimentin, protein disulfide isomerase (PDI), tropomyosin 3 (TPM 3), glutathione S‐transferase Pi (GST‐Pi), manganese superoxide dismutase (MnSOD), and cofilin 1 were up‐regulated in the cancer tissue. On the other hand, profilin was down‐regulated in the cancer tissue. Among these proteins, six proteins were validated by Western blot analysis. The identified proteins contributing to the spectrum of cancer progression may be used as potential diagnostic biomarkers for lung adenocarcinomas.     

Trace element concentration differences in regions of human brain by INAA

Studies have shown that there is a potential relationship between the levels of trace elements in cerebral tissues and neurological disorders. However, there are few publications available on the elemental composition of these tissues as well as for different regions of the brain. The aim of this study was to investigate trace element differences in various regions of the human brain from an elderly population of normal individuals. Brain samples from 31 individuals of both genders, aged 51–95 years were provided by the Brain Bank of the Brazilian Aging Study Group of the São Paulo University, Medical School. The tissues from the regions of the hippocampus, cerebellum and frontal, parietal, temporal, occipital cortex were dissected using a titanium knife, ground, freeze-dried and then analyzed by instrumental neutron activation analysis (INAA). Samples and element standards were irradiated with a neutron flux at the IEA-R1 nuclear research reactor for Br, Fe, K, Na, Rb, Se and Zn determinations. One-way ANOVA test (p < 0.05) was used to compare the results which showed significant differences for several elements among the brain regions. Most of our brain analysis results agreed with the literature data. The results were also submitted for brain region classification by cluster analysis.     

Proteome analysis of mouse brain: two-dimensional electrophoresis profiles of tissue proteins during the course of aging

Mouse brain proteins were isolated from five regions (cerebellum, cerebral cortex, hippocampus, striatum, and cervical spinal cord) at five ages from the 10th week to the 24th month, and separated by two-dimensional gel electrophoresis (2-DE). 2-DE was carried out with an immobilized pH gradient bar in the first dimension, and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Over one thousand protein spots were visualized by silver staining and quantified by image processing. In the analyses, 58 protein spots were distinguishable among the above five brain regions, and 17 proteins were shown to be varied in quantity in the course of aging. Partial amino-terminal sequences and/or internal sequences for a total of 301 protein spots were analyzed. One hundred and eighty proteins appeared to have blocked N-termini and 122 proteins were identified. Twenty-seven new proteins were identified by sequence homology search. A mouse brain proteome database was constructed, which consists of the 2-DE map images and the respective spot data files with 15 related references.     

Preparation and reactivity of metal-containing monomers

We have observed the phenomenon of thermally initiated frontal polymerization of acrylamide complexes of nitrates of Mn(II), Co(II), Ni(II), and Zn(II). The velocity of propagation of the polymerization front when using different complexes is (2–9)·10^–2 cm/sec and decreases in the sequence of nitrates: Co(II) > Ni(II) > Mn(II) > Zn(II). Analogous complexes of Cu(II) and Fe(III) under the studied conditions are not polymerized. We have shown that the frontal polyerization products are three-dimensional polymers which are insoluble in organic solvents and slowly swell in water.Translated from Izvestiya Akademii Nauk SSSR, Seriya Khimicheskaya, No. 4, pp. 768–772, April, 1990.We thank I. N. Ivlev for making the magnetic susceptibility measurements, and T. I. Ponomarev for studying the thermomechanical properties of the frontal polymerization products; and we acknowledge S. P. Davtyan for discussion of the results obtained.     

Antidementia Effects of Alternanthera philoxeroides in Ovariectomized Mice Supported by NMR-Based Metabolomic Analysis

The crude ethanol extract of the whole plant of Alternanthera philoxeroides (Mart.) Griseb was investigated for its potential as antidementia, induced by estrogen deprivation, based on in vitro antioxidant activity, β-amyloid aggregation inhibition and cholinesterase inhibitory activity, as well as in vivo Morris water maze task (MWMT), novel object recognition task (NORT), and Y-maze task. To better understand the effect of the extract, oxidative stress-induced brain membrane damage through lipid peroxidation in the whole brain was also investigated. Additionally, expressions of neuroinflammatory cytokines (IL-1β, IL-6 and TNF-α) and estrogen receptor-mediated facilitation genes such as PI3K and AKT mRNA in the hippocampus and frontal cortex were also evaluated. These effects were confirmed by the determination of its serum metabolites by NMR metabolomic analysis. Both the crude extract of A. philoxeroides and its flavone constituents were found to inhibit β-amyloid (Aβ) aggregation.     

High-performance liquid chromatography-fluorescent assay of catechol-O-methyltransferase activity in rat brain

A method to measure catechol- O-methyltransferase (COMT) activity using high performance liquid chromatography-fluorescence detection with norepinephrine (NE) as a natural substrate was optimized for both soluble (S-) and membrane-bound (MB-) COMT activities in rat brain areas, cerebral cortex, cerebellum, hippocampus, brain stem, hypophysis, and hypothalamus. The highest S-COMT activity in Sprague-Dawley rat brain was found in hippocampus. MB-COMT activities in all brain areas were about 3-8 times lower than S-COMT activities. However, considering Vmax/ Km values, specificity constants for NE to S- and MB-COMT contributes mainly to the metabolism of NE in cerebral cortex and cerebellum.     

Metal components analysis of metallothionein-III in the brain sections of metallothionein-I and metallothionein-II null mice exposed to mercury vapor with HPLC/ICP-MS

Mercury vapor is effectively absorbed via inhalation and easily passes through the blood–brain barrier; therefore, mercury poisoning with primarily central nervous system symptoms occurs. Metallothionein (MT) is a cysteine-rich metal-binding protein and plays a protective role in heavy-metal poisoning and it is associated with the metabolism of trace elements. Two MT isoforms, MT-I and MT-II, are expressed coordinately in all mammalian tissues, whereas MT-III is a brain-specific member of the MT family. MT-III binds zinc and copper physiologically and is seemed to have important neurophysiological and neuromodulatory functions. The MT functions and metal components of MTs in the brain after mercury vapor exposure are of much interest; however, until now they have not been fully examined. In this study, the influences of the lack of MT-I and MT-II on mercury accumulation in the brain and the changes of zinc and copper concentrations and metal components of MTs were examined after mercury vapor exposure by using MT-I, II null mice and 129/Sv (wild-type) mice as experimental animals. MT-I, II null mice and wild-type mice were exposed to mercury vapor or an air stream for 2 h and were killed 24 h later. The brain was dissected into the cerebral cortex, the cerebellum, and the hippocampus. The concentrations of mercury in each brain section were determined by cold vapor atomic absorption spectrometry. The concentrations of mercury, copper, and zinc in each brain section were determined by inductively coupled plasma mass spectrometry (ICP-MS). The mercury accumulated in brains after mercury vapor exposure for MT-I, II null mice and wild-type mice. The mercury levels of MT-I, II null mice in each brain section were significantly higher than those of wild-type mice after mercury vapor exposure. A significant change of zinc concentrations with the following mercury vapor exposure for MT-I, II null mice was observed only in the cerebellum analyzed by two-way analysis of variance. As for zinc, the copper concentrations only changed significantly in the cerebellum. Metal components of metal-binding proteins of soluble fractions in the brain sections were analyzed by size-exclusion high-performance liquid chromatography (HPLC) connected with ICP-MS. From the results of HPLC/ICP-MS analyses, it was concluded that the mercury components of MT-III and high molecular weight metal-binding proteins in the cerebellum of MT-I, II null mice were much higher than those of wild-type mice. It was suggested that MT-III is associated with the storage of mercury in conditions lacking MT-I, and MT-II. It was also suggested that the physiological role of MT-III and some kind of high molecular weight proteins might be impaired by exposure to mercury vapor and lack of MT-I and MT-II.     

Identification of differentially expressed proteins in the heart of translationally controlled tumor protein over-expressing transgenic mice

We previously reported that transgenic (TG) mice over-expressing translationally controlled tumor protein (TCTP) developed systemic arterial hypertension at about 6 weeks after birth. In the present study, we identified, using proteomics technologies, 24 other proteins that were differentially expressed in the heart of TCTP over-expressing TG mice. These 24 proteins are involved in a variety of biological processes such as reactive oxygen species metabolism, cytoskeleton organization, fatty acid metabolism, amino acid metabolism and energy metabolism. We determined protein expression levels of the peroxiredoxin (Prx)2, Prx3, myosin light chain 1, stress protein (heat shock protein) 25K, and T-complex protein 1 alpha subunit by western blot analysis. Over-expression of TCTP probably regulates the expression of other proteins which play a pivotal role in a variety of cellular functions in TCTP over-expressing TG mice.     

Brain site-specific proteome changes in aging-related dementia

This study is aimed at gaining insights into the brain site-specific proteomic senescence signature while comparing physiologically aged brains with aging-related dementia brains (for example, Alzheimer''s disease (AD)). Our study of proteomic differences within the hippocampus (Hp), parietal cortex (pCx) and cerebellum (Cb) could provide conceptual insights into the molecular mechanisms involved in aging-related neurodegeneration. Using an isobaric tag for relative and absolute quantitation (iTRAQ)-based two-dimensional liquid chromatography coupled with tandem mass spectrometry (2D-LC-MS/MS) brain site-specific proteomic strategy, we identified 950 proteins in the Hp, pCx and Cb of AD brains. Of these proteins, 31 were significantly altered. Most of the differentially regulated proteins are involved in molecular transport, nervous system development, synaptic plasticity and apoptosis. Particularly, proteins such as Gelsolin (GSN), Tenascin-R (TNR) and AHNAK could potentially act as novel biomarkers of aging-related neurodegeneration. Importantly, our Ingenuity Pathway Analysis (IPA)-based network analysis further revealed ubiquitin C (UBC) as a pivotal protein to interact with diverse AD-associated pathophysiological molecular factors and suggests the reduced ubiquitin proteasome degradation system (UPS) as one of the causative factors of AD.