A differential proteomic approach identifies structural and functional components that contribute to the differentiation of brain capillary endothelial cells

When in the vicinity of astrocytes, brain capillary endothelial cells (BCECs) develop the characteristic structural and functional features of the blood-brain barrier (BBB). The latter has low cellular permeability and restricts various compounds from entering the brain. We recently reported that the cytoskeleton-related proteins actin, gelsolin and filamin-A undergo the largest quantitative changes in bovine BCECs after re-induction of BBB functions by co-culture with glial cells. In the present study, we used an in-depth, proteomic approach to quantitatively compare differences in Triton-X-100-solubilized proteins from bovine BCECs with limited or re-induced BBB functions (i.e. cultured in the absence or presence of glial cells, respectively). The 81 protein spots of differing abundance were linked to 55 distinct genes. According to the Protein ANalysis THrough Evolutionary Relationships classification system and an Ingenuity Pathway Analysis, these quantitative changes mainly affected proteins involved in (i) cell structure and motility and (ii) protein metabolism and modification. The fold-changes affecting HSPB1, moesin and ANXA5 protein levels were confirmed by western blot analysis but were not accompanied by changes in the corresponding mRNA expression levels. Our results reveal that the bovine BCECs' phenotype adaptation to variations in their environment involves the reorganization of the actin cytoskeleton.     

miRNA profiling of bilateral rat hippocampal CA3 by deep sequencing

Brain capillary endothelial cells (BCECs) form blood brain barrier (BBB) to maintain brain homeostasis. Cell turnover of BCECs by the balance of cell proliferation and cell death is critical for maintaining the integrity of BBB. Here we found that stimuli with tunicamycin, endoplasmic reticulum (ER) stress inducer, up-regulated inward rectifier K⁺ channel (K_ir2.1) and facilitated cell death in t-BBEC117, a cell line derived from bovine BCECs. The activation of K_ir channels contributed to the establishment of deeply negative resting membrane potential in t-BBEC117. The deep resting membrane potential increased the resting intracellular Ca²⁺ concentration due to Ca²⁺ influx through non-selective cation channels and thereby partly but significantly regulated cell death in t-BBEC117. The present results suggest that the up-regulation of K_ir2.1 is, at least in part, responsible for cell death/cell turnover of BCECs induced by a variety of cellular stresses, particularly ER stress, under pathological conditions.     

In vivo delivery of small interfering RNA targeting brain capillary endothelial cells

Brain capillary endothelial cells (BCECs) play an important role in blood-brain barrier (BBB) functions and pathophysiologic mechanisms in brain ischemia and inflammation. We try to suppress gene expression in BCECs by intravenous application of small interfering RNA (siRNA). After injection of large dose siRNA with hydrodynamic technique to mouse, suppression of endogenous protein and the BBB function of BCECs was investigated. The brain-to-blood transport function of organic anion transporter 3 (OAT3) that expressed in BCECs was evaluated by Brain Efflux Index method in mouse. The siRNA could be delivered to BCECs and efficiently inhibited endogenously expressed protein of BCECs. The suppression effect of siRNA to OAT3 is enough to reduce the brain-to-blood transport of OAT3 substrate, benzylpenicillin at BBB. The in vivo siRNA-silencing method with hydrodynamic technique may be useful for the study of BBB function and gene therapy targeting BCECs.     

[HCO<Subscript>3</Subscript><Superscript>-</Superscript>]-regulated expression and activity of soluble adenylyl cyclase in corneal endothelial and Calu-3 cells

Background  

Bicarbonate activated Soluble Adenylyl Cyclase (sAC) is a unique cytoplasmic and nuclear signaling mechanism for the generation of cAMP. HCO₃ ^- activates sAC in bovine corneal endothelial cells (BCECs), increasing [cAMP] and stimulating PKA, leading to phosphorylation of the cystic fibrosis transmembrane-conductance regulator (CFTR) and increased apical Cl^- permeability. Here, we examined whether HCO₃ ^- may also regulate the expression of sAC and thereby affect the production of cAMP upon activation by HCO₃ ^- and the stimulation of CFTR in BCECs.     

Protein solubility and differential proteomic profiling of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> overexpressing double-tagged fusion proteins

Background  

Overexpression of recombinant proteins usually triggers the induction of heat shock proteins that regulate aggregation and solubility of the overexpressed protein. The two-dimensional gel electrophoresis (2-DE)-mass spectrometry approach was used to profile the proteome of Escherichia coli overexpressing N-acetyl-D-glucosamine 2-epimerase (GlcNAc 2-epimerase) and N -acetyl-D-neuraminic acid aldolase (Neu5Ac aldolase), both fused to glutathione S-transferase (GST) and polyionic peptide (5D or 5R).     

Evolution of phage with chemically ambiguous proteomes

Background  

The widespread introduction of amino acid substitutions into organismal proteomes has occurred during natural evolution, but has been difficult to achieve by directed evolution. The adaptation of the translation apparatus represents one barrier, but the multiple mutations that may be required throughout a proteome in order to accommodate an alternative amino acid or analogue is an even more daunting problem. The evolution of a small bacteriophage proteome to accommodate an unnatural amino acid analogue can provide insights into the number and type of substitutions that individual proteins will require to retain functionality.     

An interactomics overview of the human and bovine milk proteome over lactation

Background

Milk is the most important food for growth and development of the neonate, because of its nutrient composition and presence of many bioactive proteins. Differences between human and bovine milk in low abundant proteins have not been extensively studied. To better understand the differences between human and bovine milk, the qualitative and quantitative differences in the milk proteome as well as their changes over lactation were compared using both label-free and labelled proteomics techniques. These datasets were analysed and compared, to better understand the role of milk proteins in development of the newborn.

Methods

Human and bovine milk samples were prepared by using filter-aided sample preparation (FASP) combined with dimethyl labelling and analysed by nano LC LTQ-Orbitrap XL mass spectrometry.

Results

The human and bovine milk proteome show similarities with regard to the distribution over biological functions, especially the dominant presence of enzymes, transport and immune-related proteins. At a quantitative level, the human and bovine milk proteome differed not only between species but also over lactation within species. Dominant enzymes that differed between species were those assisting in nutrient digestion, with bile salt-activated lipase being abundant in human milk and pancreatic ribonuclease being abundant in bovine milk. As lactation advances, immune-related proteins decreased slower in human milk compared to bovine milk. Notwithstanding these quantitative differences, analysis of human and bovine co-expression networks and protein-protein interaction networks indicated that a subset of milk proteins displayed highly similar interactions in each of the different networks, which may be related to the general importance of milk in nutrition and healthy development of the newborn.

Conclusions

Our findings promote a better understanding of the differences and similarities in dynamics of human and bovine milk proteins, thereby also providing guidance for further improvement of infant formula.

     

Functional relevance of carnitine transporter OCTN2 to brain distribution of l-carnitine and acetyl-l-carnitine across the blood–brain barrier

Transport of L-[3H]carnitine and acetyl-L-[3H]carnitine at the blood-brain barrier (BBB) was examined by using in vivo and in vitro models. In vivo brain uptake of acetyl-L-[3H]carnitine, determined by a rat brain perfusion technique, was decreased in the presence of unlabeled acetyl-L-carnitine and in the absence of sodium ions. Similar transport properties for L-[3H]carnitine and/or acetyl-L-[3H]carnitine were observed in primary cultured brain capillary endothelial cells (BCECs) of rat, mouse, human, porcine and bovine, and immortalized rat BCECs, RBEC1. Uptakes of L-[3H]carnitine and acetyl-L-[3H]carnitine by RBEC1 were sodium ion-dependent, saturable with K(m) values of 33.1 +/- 11.4 microM and 31.3 +/- 11.6 microM, respectively, and inhibited by carnitine analogs. These transport properties are consistent with those of carnitine transport by OCTN2. OCTN2 was confirmed to be expressed in rat and human BCECs by an RT-PCR method. Furthermore, the uptake of acetyl-L-[3H]carnitine by the BCECs of juvenile visceral steatosis (jvs) mouse, in which OCTN2 is functionally defective owing to a genetical missense mutation of one amino acid residue, was reduced. The brain distributions of L-[3H]carnitine and acetyl-L-[3H]carnitine in jvs mice were slightly lower than those of wild-type mice at 4 h after intravenous administration. These results suggest that OCTN2 is involved in transport of L-carnitine and acetyl-L-carnitine from the circulating blood to the brain across the BBB.     

Interleukin-25 Expressed by Brain Capillary Endothelial Cells Maintains Blood-Brain Barrier Function in a Protein Kinase C?-dependent Manner

Interleukin (IL)-25, a member of the IL-17 family of cytokines, is expressed in the brains of normal mice. However, the cellular source of IL-25 and its function in the brain remain to be elucidated. Here, we show that IL-25 plays an important role in preventing infiltration of the inflammatory cells into the central nervous system. Brain capillary endothelial cells (BCECs) express IL-25. However, it is down-regulated by inflammatory cytokines, including tumor necrosis factor (TNF)-α, IL-17, interferon-γ, IL-1β, and IL-6 in vitro, and is also reduced in active multiple sclerosis (MS) lesions and in the inflamed spinal cord of experimental autoimmune encephalomyelitis, an animal model of MS. Furthermore, IL-25 restores the reduced expression of tight junction proteins, occludin, junction adhesion molecule, and claudin-5, induced by TNF-α in BCECs and consequently repairs TNF-α-induced blood-brain barrier (BBB) permeability. IL-25 induces protein kinase Cϵ (PKCϵ) phosphorylation, and up-regulation of claudin-5 is suppressed by PKCϵ inhibitor peptide in the IL-25-stimulated BCECs. These results suggest that IL-25 is produced by BCECs and protects against inflammatory cytokine-induced excessive BBB collapse through a PKCϵ-dependent pathway. These novel functions of IL-25 in maintaining BBB integrity may help us understand the pathophysiology of inflammatory brain diseases such as MS.     

Identification and bioinformatic analysis of the membrane proteins of synechocystis sp. PCC 6803

Background  

The membranes of Synechocystis sp. PCC 6803 play a central role in photosynthesis, respiration and other important metabolic pathways. Comprehensive identification of the membrane proteins is of importance for a better understanding of the diverse functions of its unique membrane structures. Up to date, approximately 900 known or predicted membrane proteins, consisting 24.5% of Synechocystis sp. PCC 6803 proteome, have been indentified by large-scale proteomic studies.     

Proteome characterization of cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis> Crantz) somatic embryos,plantlets and tuberous roots

Background  

Proteomics is increasingly becoming an important tool for the study of many different aspects of plant functions, such as investigating the molecular processes underlying in plant physiology, development, differentiation and their interaction with the environments. To investigate the cassava (Manihot esculenta Crantz) proteome, we extracted proteins from somatic embryos, plantlets and tuberous roots of cultivar SC8 and separated them by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).     

A novel transgenic zebrafish model for blood-brain and blood-retinal barrier development

Background

Development and maintenance of the blood-brain and blood-retinal barrier is critical for the homeostasis of brain and retinal tissue. Despite decades of research our knowledge of the formation and maintenance of the blood-brain (BBB) and blood-retinal (BRB) barrier is very limited. We have established an in vivo model to study the development and maintenance of these barriers by generating a transgenic zebrafish line that expresses a vitamin D-binding protein fused with enhanced green fluorescent protein (DBP-EGFP) in blood plasma, as an endogenous tracer.

Results

The temporal establishment of the BBB and BRB was examined using this transgenic line and the results were compared with that obtained by injection of fluorescent dyes into the sinus venosus of embryos at various stages of development. We also examined the expression of claudin-5, a component of tight junctions during the first 4 days of development. We observed that the BBB of zebrafish starts to develop by 3 dpf, with expression of claudin-5 in the central arteries preceding it at 2 dpf. The hyaloid vasculature in the zebrafish retina develops a barrier function at 3 dpf, which endows the zebrafish with unique advantages for studying the BRB.

Conclusion

Zebrafish embryos develop BBB and BRB function simultaneously by 3 dpf, which is regulated by tight junction proteins. The Tg(l-fabp:DBP-EGFP) zebrafish will have great advantages in studying development and maintenance of the blood-neural barrier, which is a new application for the widely used vertebrate model.     

The Impact of Stannous,Fluoride Ions and Its Combination on Enamel Pellicle Proteome and Dental Erosion Prevention

Objectives

To compare the effects of stannous (Sn) and fluoride (F) ions and their combination on acquired enamel pellicle (AEP) protein composition (proteome experiment), and protection against dental erosion (functional experiment).

Methods

In the proteome experiment, bovine enamel specimens were incubated in whole saliva supernatant for 24h for AEP formation. They were randomly assigned to 4 groups (n=10), according to the rinse treatment: Sn (800ppm/6.7mM, SnCl₂), F (225ppm/13mM, NaF), Sn and F combination (Sn+F) and deionized water (DIW, negative control). The specimens were immersed 3× in the test rinses for 2min, 2h apart. Pellicles were collected, digested, and analyzed for protein content using liquid chromatography electrospray ionization tandem mass spectrometry. In the functional experiment, bovine enamel specimens (n=10) were similarly treated for pellicle formation. Then, they were subjected to a five-day erosion cycling model, consisting of 5min erosive challenges (15.6 mM citric acid, pH 2.6, 6×/d) and 2min treatment with the rinses containing Sn, F or Sn+F (3×/d). Between the treatments, all specimens were incubated in whole saliva supernatant. Surface loss was determined by profilometry.

Results

Our proteome approach on bovine enamel identified 72 proteins that were common to all groups. AEP of enamel treated with Sn+F demonstrated higher abundance for most of the identified proteins than the other groups. The functional experiment showed reduction of enamel surface loss for Sn+F (89%), Sn (67%) and F (42%) compared to DIW (all significantly different, p<0.05).

Conclusion

This study highlighted that anti-erosion rinses (e.g. Sn+F) can modify quantitatively and qualitatively the AEP formed on bovine enamel. Moreover, our study demonstrated a combinatory effect that amplified the anti-erosive protection on tooth surface.     

Disruption of Four Kinesin Genes in <Emphasis Type="Italic">Dictyostelium</Emphasis>

Background  

Kinesin and dynein are the two families of microtubule-based motors that drive much of the intracellular movements in eukaryotic cells. Using a gene knockout strategy, we address here the individual function(s) of four of the 13 kinesin proteins in Dictyostelium. The goal of our ongoing project is to establish a minimal motility proteome for this basal eukaryote, enabling us to contrast motor functions here with the often far more elaborate motor families in the metazoans.     

Studies on substantially increased proteins in follicular fluid of bovine ovarian follicular cysts using 2-D PAGE and MALDI-TOF MS

Background  

The objective of this study was to identify substantially increased proteins in bovine cystic follicular fluid (FF) in order to clarify the pathology and etiology of bovine ovarian follicular cysts (BOFC).     

Sufficient virus-neutralizing antibody in the central nerve system improves the survival of rabid rats

Background

Rabies is known to be lethal in human. Treatment with passive immunity for the rabies is effective only when the patients have not shown the central nerve system (CNS) signs. The blood–brain barrier (BBB) is a complex functional barrier that may compromise the therapeutic development in neurological diseases. The goal of this study is to determine the change of BBB integrity and to assess the therapeutic possibility of enhancing BBB permeability combined with passive immunity in the late stage of rabies virus infection.

Methods

The integrity of BBB permeability in rats was measured by quantitative ELISA for total IgG and albumin levels in the cerebrospinal fluid (CSF) and by exogenously applying Evans blue as a tracer. Western blotting of occludin and ZO-1, two tight junction proteins, was used to assess the molecular change of BBB structure.The breakdown of BBB with hypertonic arabinose, recombinant tumor necrosis factor-alpha (rTNF-γ), and focused ultrasound (FUS) were used to compare the extent of BBB disruption with rabies virus infection. Specific humoral immunity was analyzed by immunofluorescent assay and rapid fluorescent focus inhibition test. Virus-neutralizing monoclonal antibody (mAb) 8-10E was administered to rats with hypertonic breakdown of BBB as a passive immunotherapy to prevent the death from rabies.

Results

The BBB permeability was altered on day 7 post-infection. Increased BBB permeability induced by rabies virus infection was observed primarily in the cerebellum and spinal cord. Occludin was significantly decreased in both the cerebral cortex and cerebellum. The rabies virus-specific antibody was not strongly elicited even in the presence of clinical signs. Disruption of BBB had no direct association with the lethal outcome of rabies. Passive immunotherapy with virus-neutralizing mAb 8-10E with the hypertonic breakdown of BBB prolonged the survival of rabies virus-infected rats.

Conclusions

We demonstrated that the BBB permeability was altered in a rat model with rabies virus inoculation. Delivery of neutralizing mAb to the infected site in brain combined with effective breakdown of BBB could be an aggressive but feasible therapeutic mode in rabies when the CNS infection has been established.     

Fingolimod Prevents Blood-Brain Barrier Disruption Induced by the Sera from Patients with Multiple Sclerosis

Objective

Effect of fingolimod in multiple sclerosis (MS) is thought to involve the prevention of lymphocyte egress from lymphoid tissues, thereby reducing autoaggressive lymphocyte infiltration into the central nervous system across blood-brain barrier (BBB). However, brain microvascular endothelial cells (BMECs) represent a possible additional target for fingolimod in MS patients by directly repairing the function of BBB, as S1P receptors are also expressed by BMECs. In this study, we evaluated the effects of fingolimod on BMECs and clarified whether fingolimod-phosphate restores the BBB function after exposure to MS sera.

Methods

Changes in tight junction proteins, adhesion molecules and transendothelial electrical resistance (TEER) in BMECs were evaluated following incubation in conditioned medium with or without fingolimod/fingolimod-phosphate. In addition, the effects of sera derived from MS patients, including those in the relapse phase of relapse-remitting (RR) MS, stable phase of RRMS and secondary progressive MS (SPMS), on the function of BBB in the presence of fingolimod-phosphate were assessed.

Results

Incubation with fingolimod-phosphate increased the claudin-5 protein levels and TEER values in BMECs, although it did not change the amount of occludin, ICAM-1 or MelCAM proteins. Pretreatment with fingolimod-phosphate restored the changes in the claudin-5 and VCAM-1 protein/mRNA levels and TEER values in BMECs after exposure to MS sera.

Conclusions

Pretreatment with fingolimod-phosphate prevents BBB disruption caused by both RRMS and SPMS sera via the upregulation of claudin-5 and downregulation of VCAM-1 in BMECs, suggesting that fingolimod-phosphate is capable of directly modifying the BBB. BMECs represent a possible therapeutic target for fingolimod in MS patients.     

Proteomic analysis of acidic chaperones, and stress proteins in extreme halophile Halobacterium NRC-1: a comparative proteomic approach to study heat shock response

Background  

Halobacterium sp. NRC-1 is an extremely halophilic archaeon and has adapted to optimal growth under conditions of extremely high salinity. Its proteome is highly acidic with a median pI of 4.9, a unique characteristic which helps the organism to adapt high saline environment. In the natural growth environment, Halobacterium NRC-1 encounters a number of stressful conditions including high temperature and intense solar radiation, oxidative and cold stress. Heat shock proteins and chaperones play indispensable roles in an organism's survival under many stress conditions. The aim of this study was to develop an improved method of 2-D gel electrophoresis with enhanced resolution of the acidic proteome, and to identify proteins with diverse cellular functions using in-gel digestion and LC-MS/MS and MALDI-TOF approach.     

Polyamine Modification Increases the Permeability of Proteins at the Blood-Nerve and Blood-Brain Barriers

Abstract: The permeability of the blood-nerve barrier (BNB) and the blood-brain barrier (BBB) to superoxide dismutase (SOD), insulin, albumin, and IgG in normal adult rats was quantified by measuring the permeability coefficient-surface area product (PS) with the intravenous bolus injection technique before and after covalent protein modification with the naturally occurring polyamines—putrescine (PUT), spermidine (SPD), and spermine (SPM). The PS value of the BNB for PUT-SOD was 21.1-fold greater than the native SOD, and the PS values of the BBB for PUT-SOD ranged from 17.6-fold greater for the thalamus to 23.6-fold greater for the caudate-putamen compared with native SOD. In a similar manner, polyamine-modified insulin showed a 1.7–2.0-fold increase in PS of the BNB and BBB compared with the high values of native insulin. Polyamine-modified albumin showed a remarkable 54–165-fold increase in PS of the BNB and BBB compared with native albumin, whereas PUT-IgG resulted in an even higher increase in the PS that ranged from 111- to 349-fold for nerve and different brain regions compared with native IgG. Polyamine modification of proteins, therefore, can dramatically increase the permeability at the BNB and BBB of a variety of proteins with widely differing M_r and function. It is surprising that the PS values of the BNB and BBB decreased with the increasing number of positive charges of the protonated amino groups on the polyamines (PUT > SPD > SPM). Although cationic proteins are known to interact with fixed anionic charges on the lumen of the microvascular endothelium, this observation of decreased permeability with increased positive charge distribution along the aliphatic carbon chain of the polyamines implies mechanisms other than simple electrostatic interaction involving charge density. It is suggested that the polyamine transporter may be responsible for the transport of these polyamine-modified proteins. Systemic administration of polyamine-modified peptides and proteins might prove to be an efficient approach to deliver therapeutic agents into the CNS and PNS for the treatment of a variety of neurological diseases.