bluuebunny/crossref_metadata_2025 huggingface dataset:
Establishment of a human in vitro model of the outer blood–retinal barrier
Authors: Hamilton R. D. , Foss A. J. , Leach L.
Published: October 2007

The outer blood–retinal barrier is composed of a monolayer of retinal pigment epithelium, Bruch's membrane and the choriocapillaris which is fenestrated. Endothelial proliferation and breaching of Bruch's membrane leads to the neovascular form of age‐related macula degeneration (ARMD). The aim of this study was to generate an in vitro model that mimics more faithfully the phenotype of the choriocapillaris and the trilayer architecture in vitro. A trilayer culture model was generated with retinal pigment epithelium (ARPE‐19) cell cultures on the epithelial surface of amniotic membrane and with human umbilical vein‐derived endothelial cells on the other surface. A control model for the effect of retinal pigment epithelium on endothelial changes was generated with corneal epithelial cells replacing the ARPE‐19. Both human umbilical vein‐derived endothelial and ARPE‐19 cells formed confluent monolayers on respective surfaces of the amnion. The human umbilical vein‐derived endothelial cells in the trilayer became fenestrated when co‐cultured with the ARPE‐19 cells, but not with corneal epithelial cells, or when grown as monolayers on the amnion, showing a loss of fidelity of origin in the presence of ARPE‐19 cells. These cells also revealed VE‐cadherin and ZO‐1 at cell–cell contacts from 24 h in the trilayer. The tight junctional molecules, occludin and ZO‐1, were localized to cell–cell contact regions in the retinal pigment epithelium, both in the monolayer and in the trilayer system. Permeability of the trilayer was tested by using fluorescein and fluorescein‐conjugated tracers under flow. At 72 h the trilayer severely restricted transfer of sodium fluorescein (NaF) (ten‐fold reduction) whilst transfer of a 4 kDa FITC‐conjugated dextran was virtually occluded, confirming a restrictive barrier. Ultrastructural studies showed the retinal pigment epithelium monolayer was polarized with microvilli present on the apical surface. Paracellular clefts showed numerous tight junctional‐like appositions, similar to that seen on amnion alone. This study demonstrates that ARPE‐19 and human umbilical vein‐derived endothelial cells can be co‐cultured on the amniotic membrane and that the resultant cross‐talk leads to formation of a fenestrated endothelium, whilst maintaining a polarized restrictive epithelial layer. The fenestrated endothelial phenotype achieved in this human in vitro trilayer model is a first and offers an outer‐retinal barrier which approaches the in vivo state and has potential for studies into induced junctional disruption, endothelial proliferation and migration: features of ARMD.
PACAP‐38 induces neuronal differentiation of human SH‐SY5Y neuroblastoma cells via cAMP‐mediated activation of ERK and p38 MAP kinases1
Authors: Monaghan T. K. , MacKenzie C. J. , Plevin R. , Lutz E. M.
Published: October 2007

The intracellular signaling pathways mediating the neurotrophic actions of pituitary adenylate cyclase‐activating polypeptide (PACAP) were investigated in human neuroblastoma SH‐SY5Y cells. Previously, we showed that SH‐SY5Y cells express the PAC1and VIP/PACAP receptor type 2 (VPAC2) receptors, and that the robust cAMP production in response to PACAP and vasoactive intestinal peptide (VIP) was mediated by PAC1receptors (Lutzet al.2006). Here, we investigated the ability of PACAP‐38 to differentiate SH‐SY5Y cells by measuring morphological changes and the expression of neuronal markers. PACAP‐38 caused a concentration‐dependent increase in the number of neurite‐bearing cells and an up‐regulation in the expression of the neuronal proteins Bcl‐2, growth‐associated protein‐43 (GAP‐43) and choline acetyltransferase: VIP was less effective than PACAP‐38 and the VPAC2receptor‐specific agonist, Ro 25‐1553, had no effect. The effects of PACAP‐38 and VIP were blocked by the PAC1receptor antagonist, PACAP6‐38. As observed with PACAP‐38, the adenylyl cyclase activator, forskolin, also induced an increase in the number of neurite‐bearing cells and an up‐regulation in the expression of Bcl‐2 and GAP‐43. PACAP‐induced differentiation was prevented by the adenylyl cyclase inhibitor, 2′,5′‐dideoxyadenosine (DDA), but not the protein kinase A (PKA) inhibitor, H89, or by siRNA‐mediated knock‐down of the PKA catalytic subunit. PACAP‐38 and forskolin stimulated the activation of extracellular signal‐regulated kinase (ERK), mitogen‐activated protein kinase (MAP; p38 MAP kinase) and c‐Jun N‐terminal kinase (JNK). PACAP‐induced neuritogenesis was blocked by the MEK1 inhibitor PD98059 and partially by the p38 MAP kinase inhibitor SB203580. Activation of exchange protein directly activated by cAMP (Epac) partially mimicked the effects of PACAP‐38, and led to the phosphorylation of ERK but not p38 MAP kinase. These results provide evidence that the neurotrophic effects of PACAP‐38 on human SH‐SY5Y neuroblastoma cells are mediated by the PAC1receptor through a cAMP‐dependent but PKA‐independent mechanism, and furthermore suggest that this involves Epac‐dependent activation of ERK as well as activation of the p38 MAP kinase signaling pathway.
Over‐expression of GTP‐binding proteins and GTPase activity in mouse astrocyte membranes in response to Theiler’s murine encephalomyelitis virus infection
Authors: Rubio Nazario , Gonzalez‐Tirante Maria , Arevalo Maria‐Angeles , Aranguez Isabel
Published: October 2007

Intracerebral infection with Theiler’s murine encephalomyelitis virus (TMEV) induces a demyelinating disease that resembles human multiple sclerosis. In order to delineate the early events in this virus‐induced neuroinflammatory disease, we have analyzed global GTPases gene activation following TMEV infection of murine brain astrocytes. DNA hybridization microchip analysis demonstrated that 10 sequences described as GTPbinding proteins and GTPases in different protein databases were over‐expressed, in response to this infectious agent in astroglial cells. We have first characterized both the GTP‐binding and GTPase activities in uninfected astrocyte membranes from a biochemical point of view. The increase in such activities was further validated in TMEV‐infected astrocytes, peaking 2–4 h after infection. Over‐expression is also induced by the inflammation‐related chemokines interleukin‐6 and interferon‐γ but not by interleukin‐1α or tumor necrosis factor‐α. From the many GTPases that could be over‐expressed we have studied two, because of its biological significance; Ras p21 and the subunit αi2 of G proteins. Western blots revealed increases in both proteins after infection with TMEV, in accordance with the previous enzymologic results. An increase in the active form of Ras (the GTP bound form) in cell lysates was also confirmed by affinity binding to a glutathione‐S‐transferase‐fusion protein, following TMEV infection. A final demonstration of physiological up‐regulation is provided by UV cross‐linking of membrane proteins with the hydrolysis‐resistant GTP agonist GTP [γ‐35S]. This technique allow us to detect, after SDS‐PAGE, the increase of two further majoritary GTPbinding proteins with MW of 62 and 49 KDa. A quantitative analysis of four selected genes coding for p21 ras, Gαi2 subunit of protein G, Munc‐18 and protein interacting with C kinase 1, was performed by real‐time RT‐PCR to verify the microarray results. The study of GTPase activity and of the above genes by RT‐PCR in brains of sick mice, demonstrated a significative increase in mRNA coding for p21ras and protein interacting with C kinase 1 in vivo. Here we demonstrate that one of the mechanisms triggered by TMEV infection of astrocytes is the up‐regulation of proteins related to GTP metabolism, one important signal transduction system in mammalian cells.
Localization of organic cation/carnitine transporter (OCTN2) in cells forming the blood–brain barrier
Authors: Miecz Dorota , Januszewicz Elżbieta , Czeredys Magdalena , Hinton Barry T. , Berezowski Vincent , Cecchelli Roméo , Nałęcz Katarzyna A.
Published: October 2007

Carnitine β‐hydroxy‐γ‐(trimethylammonio)butyrate – a compound necessary in the peripheral tissues for a transfer of fatty acids for their oxidation within the cell, accumulates in the brain despite low β‐oxidation in this organ. In order to enter the brain, carnitine has to cross the blood–brain barrier formed by capillary endothelial cells which are in close interaction with astrocytes. Previous studies, demonstrating expression of mRNA coding two carnitine transporters – organic cation/carnitine transporter 2 (OCTN2) and B0,+in endothelial cells, did not give any information on carnitine transporters polarity in endothelium. Therefore more detailed experiments were performed on expression and localization of a high affinity carnitine transporter OCTN2 in anin vitromodel of the blood–brain barrier by real‐time PCR, western blot analysis, and immunocytochemistry. The amount of mRNA was comparable in endothelial cells and kidney, when referred to house‐keeping genes, it was, however, significantly lower in astrocytes. Polarity of OCTN2 localization was further studied in anin vitromodel of the blood–brain barrier with use of anti‐OCTN2 antibodies.Z‐axis analysis of the confocal microscope pictures of endothelial cells, with anti‐P‐glycoprotein antibodies as the marker of apical membrane, showed OCTN2 localization at the basolateral membrane and in the cytoplasmic region in the vicinity of nuclei. Localization of OCTN2 suggest that carnitine can be also transported from the brain, playing an important role in removal of certain acyl esters.
Microtubule‐associated STOP protein deletion triggers restricted changes in dopaminergic neurotransmission
Authors: Bouvrais‐Veret Caroline , Weiss Stéphanie , Hanoun Naima , Andrieux Annie , Schweitzer Annie , Job Didier , Hamon Michel , Giros Bruno , Martres Marie‐Pascale
Published: October 2007

The microtubule‐associated stable tubule only polypeptide (STOP) protein plays a key‐role in neuron architecture and synaptic plasticity. Recent studies suggest that schizophrenia is associated with alterations in the synaptic connectivity. Mice invalidated for the STOP gene display phenotype reminiscent of some schizophrenic‐like symptoms, such as behavioral disturbances, dopamine (DA) hyper‐reactivity, and possible hypoglutamatergia, partly improved by antipsychotic treatment. In the present work, we examined potential alterations in some DAergic key proteins and behaviors in STOP knockout mice. Whereas the densities of the DA transporter, the vesicular monoamine transporter and the D1 receptor were not modified, the densities of the D2 and D3 receptors were decreased in some DAergic regions in mutant versus wild‐type mice. Endogenous DA levels were selectively decreased in DAergic terminals areas, although the in vivo DA synthesis was diminished both in cell bodies and terminal areas. The DA uptake was decreased in accumbic synaptosomes, but not significantly altered in striatal synaptosomes. Finally, STOP knockout mice were hypersensitive to acute and subchronic locomotor effects of cocaine, although the drug equally inhibited DA uptake in mutant and wild‐type mice. Altogether, these data showed that deletion of the ubiquitous STOP protein elicited restricted alterations in DAergic neurotransmission, preferentially in the meso‐limbic pathway.
New insight into the molecular pathways of metallothionein‐mediated neuroprotection and regeneration
Authors: Chung R. S. , Hidalgo J. , West A. K.
Published: October 2007

There is a large body of evidence demonstrating that metallothioneins (MTs) expressed in astrocytes following CNS injury, exhibit both neuroprotective and neuroregenerative properties and are critical for recovery outcomes. As these proteins lack signal peptides, and have well characterized free radical scavenging and heavy metal binding properties, the neuroprotective functions of MTs have been attributed to these intracellular roles. However, there is an increasing realization that the neuroprotective functions of MTs may also involve an extracellular component. In this issue of Journal of Neurochemistry, Ambjørn et al. reveal considerable insight into this novel function of MTs. In this review, we examine the seminal work of Ambjørn et al. in the context of our current understanding of the role of MT in astrocyte‐neuron interactions in the injured brain, and also discuss the significant therapeutic potential of their work.
Tyr‐701 is a new regulatory site for neurotrophin receptor TrkA trafficking and function
Authors: De Pablo Yolanda , Pérez‐García M. José , Georgieva Maya V. , Sanchis Daniel , Lindqvist Niclas , Soler Rosa M. , Comella Joan X. , Llovera Marta
Published: October 2007

Tropomyosin‐related kinase A (TrkA) receptor mediates the effects exerted by nerve growth factor on several subpopulations of neuronal cells. Ligand binding to TrkA induces receptor autophosphorylation on several tyrosine residues and the activation of signaling cascades. In this study, we describe a new site relevant for TrkA regulation, the tyrosine 701 (Y701), which is important for receptor trafficking and activation. Y701 replacement by aspartate or phenylalanine reduces receptor internalization rate and decreases the colocalization and association of TrkA with clathrin heavy chain, demonstrating that Y701 constitutes a YxxΦ (YRKF701–704) trafficking motif relevant for the regulation of receptor endocytosis. In accordance with this hypothesis, the colocalization of Y701 mutant receptors with a lysosomal marker is also reduced giving support to the involvement of the YRKF701–704 motif in the lysosomal targeting of TrkA receptors. Contrary to what was expected, substitution of Y701 for an Asp in order to mimic phosphorylation, impairs TrkA ability to mediate nerve growth factor‐induced differentiation, although the mutant receptor retains its in vitro kinase activity. This is the first evidence that a Tyr residue can simultaneously regulate TrkA receptor trafficking and activity.
Crustacean dopamine receptors: localization and G protein coupling in the stomatogastric ganglion
Authors: Clark Merry C. , Khan Reesha , Baro Deborah J.
Published: October 2007

Neuromodulators, such as dopamine (DA), control motor activity in many systems. To begin to understand how DA modulates motor behaviors, we study a well‐defined model: the crustacean stomatogastric nervous system (STNS). The spiny lobster STNS receives both neuromodulatory and neurohormonal dopaminergic input, and extensive background information exists on the cellular and network effects of DA. However, there is a void of information concerning the mechanisms of DA signal transduction in this system. In this study, we show that Gs, Gi, and Gq are activated in response to DA in STNS membrane preparations from five crustacean species representing distant clades in the order Decapoda. Three evolutionarily conserved DA receptors mediate this response in spiny lobsters: D1αPan, D1βPan and D2αPan. G protein coupling for these receptors can vary with the cell type. In the native membrane, the D1αPan receptor couples with Gs and Gq, the D1βPan receptor couples with Gs, and the D2αPan receptor couples with Gi. All three receptors are localized exclusively to the synaptic neuropil and most likely generate global biochemical signals that alter ion channels in distant compartments, as well as local signals.
Cellular uptake and lysosomal delivery of galactocerebrosidase tagged with the HIV Tat protein transduction domain
Authors: Zhang Xian‐Yang , Dinh Annie , Cronin James , Li Su‐Chen , Reiser Jakob
Published: October 2007

A number of studies have shown that a short peptide, the protein transduction domain (PTD) derived from the HIV‐1 Tat protein (Tat‐PTD) improved cellular uptake in vitro and distribution in vivo of recombinant proteins bearing such PTDs when administered systemically. To investigate the effects of Tat‐PTD addition on the subcellular localization of the lysosomal enzyme galactocerebrosidase (GALC, EC 3.2.2.46) and with a view towards designing improved therapeutic strategies for Krabbe disease (globoid cell leukodystrophy), mouse GALC was tagged C‐terminally with the Tat‐PTD. Compared with unmodified GALC, GALC bearing a Tat‐PTD, a myc epitope and 6 consecutive His residues [GALC‐TMH (Tat‐PTD, a myc epitope and 6 consecutive His residues)] was found to be secreted more efficiently. Also, GALC‐TMH was found to be taken up by cells both via mannose‐6‐phosphate receptor (M6PR)‐mediated endocytosis as well as by M6PR‐independent mechanisms. GALC‐TMH displayed increased M6PR‐independent uptake in fibroblasts derived from twitcher mice (a murine model of globoid cell leukodystrophy) and in neurons derived from the mouse brain cortex compared with GALC lacking a Tat‐PTD. Immunocytochemical analyses revealed that Tat‐modified GALC protein co‐localized in part with the lysosome‐associated membrane protein‐1. Complete correction of galactosylceramide accumulation was achieved in twitcher mouse fibroblasts lacking GALC activity following addition of GALC‐TMH. Therefore, GALC‐TMH not only maintained the features of the native GALC protein including enzymatic function, intracellular transport and location, but also displayed more efficient cellular uptake.
The C‐terminus of the metabotropic glutamate receptor 1b regulates dimerization of the receptor
Authors: Remelli Rosaria , Robbins Melanie J. , McIlhinney R. A. Jeffrey
Published: October 2007

The Group C G protein‐coupled receptors include the metabotropic glutamate receptors (mGluRs), the GABAB receptor, the calcium sensor and several taste receptors, most of which are obligate dimers, indeed recent work has shown that dimerization is necessary for the activation of these receptors. Consequently factors that regulate their ability to homo‐ or heterodimerize are important. The Group 1 mGluRs include mGluR1 and mGluR5 both of which have splice variants with altered C‐termini. In this study, we show that mGluR1b is a dimer and that it does not efficiently heterodimerize with mGluR1a, unlike the two splice variants of mGluR5 that can heterodimerize. Mutation of a positively charged motif (RRKK) at the C‐terminus of the mGluR1b tail permits mGluR1b to heterodimerize with mGluR1a. Co‐expression of mGluR1a and mGluR1b in COS‐7 cells results in the accumulation of mGluR1b in intracellular inclusions that do not contain mGluR1a. This behaviour is mimicked by a chimera of the lymphocyte antigen CD2 with the C‐terminus of mGluR1b (pCD1b) and depends on the presence of the RRKK motif. These accumulations are immunoreactive for endoplasmic reticulum (ER) markers, but not Golgi and ERGIC markers. This segregation of mGluR1b from other ER proteins may contribute to its failure to dimerize with mGluR1a.
Ca2+/calmodulin‐dependent protein kinase IIα clusters are associated with stable lipid rafts and their formation traps PSD‐95
Authors: Suzuki Tatsuo , Du Feng , Tian Qing‐Bao , Zhang Jingping , Endo Shogo
Published: October 2007

Relatively large number of post‐synaptic density (PSD) proteins, including Ca2+/calmodulin‐dependent protein kinase II (CaMKII), have the potential to associate with lipid rafts. We in this study demonstrate that the CaMKIIα clusters induced by ionomycin in human embryonic kidney 293 cells, as well as unclustered CaMKIIα (Du F., Saitoh F., Tian Q. B., Miyazawa S., Endo S. and Suzuki T, 2006, Biochem. Biophys. Res. Commun 347, 814–820), were associated with lipid rafts. The CaMKIIα clusters associated with lipid raft fraction became resistant to treatment with methyl‐β‐cyclodextrin and subsequent cold Triton X‐100, which suggests the stabilization of CaMKIIα cluster‐associated lipid rafts. Next, we found that PSD‐95, which is also a component of lipid raft fraction and does not interact directly with CaMKII, was trapped by stable CaMKIIα cluster‐containing structure. Association of PSD‐95 with CaMKIIα clusters was also observed in cultured neuronal cells. These results suggest the CaMKIIα clusters associated with the lipid rafts in the cytoplasmic region play a role in the assembly and stabilization of certain PSD proteins that have the potential to associate with lipid rafts.
Delta‐like 1 participates in the specification of ventral midbrain progenitor derived dopaminergic neurons
Authors: Bauer Matthias , Szulc Jolanta , Meyer Morten , Jensen Charlotte Harken , Terki Toufik Abbas , Meixner Andrea , Kinkl Norbert , Gasser Thomas , Aebischer Patrick , Ueffing Marius
Published: October 2007

Delta‐like 1 (Dlk1), a member of the Delta/Notch protein family, is expressed in the mouse ventral midbrain (VM) as early as embryonic day 11.5 (E11.5) followed by exclusive expression in tyrosine 3‐monooxygenase (TH) positive neurons from E12.5 onwards. To further elucidate the yet unknown function of Dlk1 in VM neuron development, we investigated the effect of soluble Dlk1 protein as well as the intrinsic Dlk1 function in the course of VM progenitor expansion and dopaminergic (DA) neuron differentiation in vitro. Dlk1 treatment during expansion increased DA progenitor proliferation and the proportion of NR4A2+ neurons expressing TH after differentiation, whereas Dlk1 treatment during the course of DA precursor differentiation did not alter TH+ neuron counts. In contrast, silencing of endogenously expressed Dlk1 prior to DA precursor differentiation partially prevented the expression of DA neuron markers, which was not accompanied with alteration of overall or local proliferation. Due to the latter finding in combination with the absence of Dlk1 negative DA neurons in differentiated cultures, we suggest that Dlk1 expression might have a permissive effect on DA neuron differentiation in vitro. The study presented here is the first publication identifying Dlk1 effects on ventral midbrain‐derived DA precursor differentiation.
Variable phenotype of Alzheimer’s disease with spastic paraparesis
Authors: Karlstrom Helena , Brooks William S , Kwok John B. J , Broe G. Anthony , Kril Jillian J , McCann Heather , Halliday Glenda M , Schofield Peter R.
Published: October 2007

Pedigrees with familial Alzheimer’s disease (AD) show considerable phenotypic variability. Spastic paraparesis (SP), or progressive spasticity of the lower limbs is frequently hereditary and exists either as uncomplicated (paraparesis alone) or complicated (paraparesis and other neurological features) disease subtypes. In some AD families, with presenilin‐1 (PSEN1) mutations, affected individuals also have SP. These PSEN1 AD pedigrees frequently have a distinctive and variant neuropathology, namely large, non‐cored plaques without neuritic dystrophy called cotton wool plaques (CWP). The PSEN1 AD mutations giving rise to CWP produce unusually high levels of the amyloid β peptide (Aβ) ending at position 42 or 43, and the main component of CWP is amino‐terminally truncated forms of amyloid β peptide starting after the alternative β‐secretase cleavage site at position 11. This suggests a molecular basis for the formation of CWP and an association with both SP and AD. The SP phenotype in some PSEN1 AD pedigrees also appears to be associated with a delayed onset of dementia compared with affected individuals who present with dementia only, suggesting the existence of a protective factor in some individuals with SP. Variations in neuropathology and neurological symptoms in PSEN1 AD raise the prospect that modifier genes may underlie this phenotypic heterogeneity.
Rapid purification of active γ‐secretase, an intramembrane protease implicated in Alzheimer’s disease
Authors: Cacquevel Matthias , Aeschbach Lorene , Osenkowski Pamela , Li Dongyang , Ye Wenjuan , Wolfe Michael S. , Li Huilin , Selkoe Dennis J. , Fraering Patrick C.
Published: October 2007

γ‐Secretase is an unconventional aspartyl protease that processes many type 1 membrane proteins within the lipid bilayer. Because its cleavage of amyloid‐β precursor protein generates the amyloid‐β protein (Aβ) of Alzheimer’s disease, partially inhibiting γ‐secretase is an attractive therapeutic strategy, but the structure of the protease remains poorly understood. We recently used electron microscopy and single particle image analysis on the purified enzyme to generate the first 3D reconstruction of γ‐secretase, but at low resolution (15 Å). The limited amount of purified γ‐secretase that can be produced using currently available cell lines and procedures has prevented the achievement of a high resolution crystal structure by X‐ray crystallography or 2D crystallization. We report here the generation and characterization of a new mammalian cell line (S‐20) that overexpresses strikingly high levels of all four γ‐secretase components (presenilin, nicastrin, Aph‐1 and Pen‐2). We then used these cells to develop a rapid protocol for the high‐grade purification of proteolytically active γ‐secretase. The cells and purification methods detailed here provide a key step towards crystallographic studies of this ubiquitous enzyme.
Minoxidil prevents 3,4‐methylenedioxymethamphetamine‐induced serotonin depletions: role of mitochondrial ATP‐sensitive potassium channels, Akt and ERK
Authors: Goñi‐Allo Beatriz , Puerta Elena , Ramos María , Lasheras Berta , Jordán Joaquín , Aguirre Norberto
Published: October 2007

Preconditioning has emerged as a valid strategy against different neurotoxic insults. Although the mechanisms underlying preconditioning are not fully understood, the activation of ATP‐sensitive potassium (KATP) channels has been proposed to play a pivotal role in neuronal preconditioning. In the present work we examine whether minoxidil a KATP channel activator protects against the long‐term toxicity caused by the amphetamine derivative 3,4‐methylenedioxymethamphetamine (MDMA) in rats. Our data show that intrastriatal administration of minoxidil prevents MDMA‐induced long‐term indole depletions in the rat striatum. This effect was not related to an effect on core temperature, as pre‐treatment with minoxidil did not significantly alter MDMA‐induced hyperthermia. Taking into account that minoxidil opens both sarcolemmal and mitochondrial KATP channels, we examined the role of each type of channels in the protective effects of minoxidil using specific inhibitors. The administration of HMR‐1098, a blocker of the sarcolemmal KATP channels, along with minoxidil did not affect the protection afforded by the latter. On the contrary the selective mitochondrial KATP channel blocker 5‐hydroxydecanoic acid completely reversed the protection afforded by minoxidil, thereby implicating the involvement of mitochondrial (but not sarcolemmal) KATP channels. Furthermore our data show the participation of Akt and extracellular signal‐regulated kinases in minoxidil‐afforded protection. Intrastriatal administration of wortmannin or PD98059 (inhibitors of phosphatidylinositol‐3‐kinase and mitogen‐activated protein kinase/extracellular regulated protein kinase, respectively), along with minoxidil abolished the protective effect of minoxidil against the serotonergic toxicity caused by MDMA. These results demonstrate that minoxidil by opening mitochondrial KATP channels completely prevents MDMA toxicity and that Akt and MAP kinases are involved in minoxidil‐afforded neuroprotection.
Atorvastatin attenuates mitochondrial toxin‐induced striatal degeneration, with decreasing iNOS/c‐Jun levels and activating ERK/Akt pathways
Authors: Lee Soon‐Tae , Chu Kon , Park Jung‐Eun , Hong Nan Hyung , Im Woo‐Seok , Kang Lami , Han Zhe , Jung Keun‐Hwa , Kim Min‐Wook , Kim Manho
Published: October 2007

Mitochondrial dysfunction is a major contributor to neurodegeneration, and causes vulnerability to oxidative stress and the activations of downstream cell death pathways. 3‐Hydroxy‐3‐methyl‐glutaryl‐CoA reductase inhibitors, statins, were originally developed as cholesterol lowering agents, and have cholesterol‐independent anti‐excitotoxic and anti‐oxidative properties. We investigated whether atorvastatin can prevent the neurodegeneration induced by a mitochondrial toxin, 3‐nitropropionic acid (3NP), which inhibits succinate dehydrogenase complex II. Male Lewis rats were administered 3NP (63 mg/kg/day) using osmotic pumps for 5 days to induce striatal degeneration, and were also treated with either atorvastatin (1 or 10 mg/kg/day, orally) or vehicle (control) on five consecutive days. Atorvastatin‐treated rats showed fewer neurologic deficits than control animals as measured at day 3–5. Atorvastatin‐treated animals showed reduced striatal lesion volumes by Nissl staining, and decreased numbers of TUNEL‐positive apoptosis and Fluoro‐Jade C‐positive degenerating neurons at 5 days. Atorvastatin reduced the numbers of c‐Jun‐positive and p‐c‐Jun‐positive cells, as well as 3‐nitrotyrosin‐positive cells. In addition, atorvastatin increased p‐extracellular signal‐regulated kinase and p‐Akt levels, and attenuated the up‐regulation of inducible nitric oxide synthase by 3NP. When N(omega)‐nitro‐l‐arginine methyl ester hydrochloride was administered concomitantly with the 3NP infusion, atorvastatin failed to further reduce the striatal lesion volume and c‐Jun levels compared to the vehicle treatment. In summary, atorvastatin decreased striatal neurodegeneration induced by 3NP, with attenuating inducible nitric oxide synthase and c‐Jun levels as well as activating extracellular signal‐regulated kinase and Akt.
Reverse Epidemiology: A Confusing, Confounding, and Inaccurate Term
Authors: Levin Nathan W. , Handelman Garry J. , Coresh Josef , Port Friedrich K. , Kaysen George A.
Published: October 2007

The term “reverse epidemiology” has been proposed to address the apparent different relationship between numerous risk factors and outcomes among dialysis patients: thus, obesity, hypertension, high cholesterol, and elevated creatinine all appear to be associated with decreased risk. Since this is contrary to the general findings in otherwise healthy populations, some kind of “reversal” has been suggested, that would be contrary to classical epidemiology. The authors describe several faults to this conception. The rules of epidemiology have not been reversed in dialysis patients. In fact, the complexity of the population implies a greater need for attention to the distinction between association and causation and the importance of confounding and bias. In particular existing subclinical and clinical disease which is very common among dialysis patients can change associations so drastically that they are dominated by different causal pathways than those seen in the general population. For example, lower cholesterol is a better marker of poor health than of a healthy diet and thus is associated with different outcomes. To the extent the term reverse epidemiology implies either epidemiology or biology is different in dialysis patients it can be misleading and detrimental. The differences between risk factors in end‐stage renal disease (ESRD) and other individuals are surely important, but can themselves be the basis of excellent epidemiology, applied with the classic rules developed for this discipline with the goal of uncovering causal association and hypotheses to be tested in clinical trials.
Volumetric analysis of extraction sockets using cone beam computed tomography: a pilot study on ex vivo jaw bone
Authors: Agbaje Jimoh Olubanwo , Jacobs Reinhilde , Maes Frederik , Michiels Katleen , Van Steenberghe Daniel
Published: October 2007

Aim: The aim of this study was to determine the accuracy of volumetric analysis of extraction sockets using cone beam computed tomography (CBCT).

Material and Methods: The volume of 40 dental alveoli in nine dry skull specimens (four mandibles and five maxillae) was determined by measuring the volume of the tooth socket impression using the water displacement technique. This was considered as the gold standard. Then, the tooth socket was scanned with CBCT and data were uploaded in the semi‐automated Livewire® segmentation software. The software segments the tooth socket in consecutive 1 mm‐thick two‐dimensional slices. After segmentation, the total volume of the delineated socket was computed. The statistical difference between direct volumetric measurements and those obtained with CBCT imaging was assessed using the Student paired t‐test.

Result: The mean socket volume of the skull specimens was 227±91 mm3 when obtained by direct measurement and 225±90 mm3 when obtained by CBCT imaging. Student paired t‐test showed no significant differences between both volume measurements (p>0.1).

Conclusions: CBCT permits imaging of anatomical structures in three planes and allows for reliable volume estimates. The results should be verified in clinical circumstances and might have potential applicability for evaluation of extraction socket healing under different conditions.
Ligation of CD200R by CD200 is not required for normal murine myelopoiesis
Authors: Rijkers Eva S. K. , De Ruiter Talitha , Buitenhuis Miranda , Veninga Henrike , Hoek Robert M. , Meyaard Linde
Published: October 2007

CD200R is an inhibitory receptor involved in the regulation of myeloid cells. It recruits Dok‐1 and Dok‐2, which are potent inhibitors of the Ras signalling pathway used by colony‐stimulating factor (CSF) receptors. Dok‐1/Dok‐2 double knockout (DKO) mice develop leukaemia at 10–12 months of age. We investigated whether disturbed CD200R signalling could be responsible for this phenotype. Therefore, we studied whether CD200−/− mice have altered myelopoiesis and develop leukaemia. We report that CD200R is expressed on haematopoietic progenitor cells. However, CD200−/− mice have normal numbers of myeloid progenitors in the bone marrow and these cells have normal proliferative capacity. These results indicate that the development of leukaemia in Dok‐1/Dok‐2 DKO mice is not solely due to an absence of CD200R signalling. In addition, we show that the previously reported enhanced numbers of myeloid cells do not occur in all CD200−/− mice. We determined whether variations in the numbers of peripheral myeloid cells were due to an enhanced response to granulocyte‐CSF (G‐CSF) or an inflammatory stimulus. Mobilisation of immature neutrophils via G‐CSF and infiltration of mature neutrophils and macrophages upon thioglycolate injection were not altered in CD200−/− mice. We conclude that CD200−/− mice exhibit normal myelopoiesis and that development of leukaemia in Dok‐1/Dok‐2 DKO mice is not caused by a lack of CD200‐mediated CD200R signalling.
The HLA‐A1‐B8 haplotype hitchhiking with the hemochromatosis mutation: does it affect the phenotype?
Authors: Olsson K. Sigvard , Ritter Bernd , Hansson Norbeth
Published: October 2007

Background:  Hemochromatosis is a recessively inherited disorder caused by a point mutation, C282Y of the HFE gene on chromosome 6p21.3 near the human leukocyte antigen (HLA) locus. It is unknown why some homozygotes develop a severe iron loading, while others do not. A recent study suggested that the A1‐B8 haplotype may be associated with higher iron storage.

Methods:  We studied HLA haplotypes of 85 probands, 31 females and 54 males, and their family members from a rural population where A1‐B8 was common. We tested the hypothesis of a modifying effect of the A1‐B8 haplotype.

Results:  Most homozygotes had a mild phenotypic expression, and were often detected accidentally because of a laboratory routine including transferrin saturation. A disease‐related morbidity [serum alanine aminotransferase (S‐ALT) > 43 U] was present in 40%.Three had porphyria cutanea tarda. Two brothers with A1‐B8 died of bronze diabetes, probably caused by co‐inheritance of congenital spherocytosis. In females there were no significant differences in phenotypic expression between groups with regard to the presence or absence of A1‐B8. Two females, <50 yr of age, with this haplotype had iron deficiency. Males with two copies of A1‐B8 had significantly lower serum ferritin (P = 0.02) values than those without. Those with one A1‐B8 haplotype were not different from those without. In men without A1‐B8, those carrying HLA‐A3 were not phenotypically different from those without this ancestral haplotype.

Conclusion:  The A1‐B8 haplotype hitchhiking with the C282Y mutation was not associated with a more efficient iron absorption. On the contrary, males with double copies of this haplotype expressed a milder phenotype, possibly an effect of local (environmental and/or genetic) factors.
Reassessing the Role of Phosphocaveolin‐1 in Cell Adhesion and Migration
Authors: Hill Michelle M. , Scherbakov Nadja , Schiefermeier Natalia , Baran JoAnne , Hancock John F. , Huber Lukas A. , Parton Robert G. , Parat Marie‐Odile
Published: October 2007

Although phosphorylation on tyrosine 14 was identified early in the discovery of caveolin‐1, the functional significance of this modification still remains elusive. Recent evidence points to a role of caveolin‐1 tyrosine 14 phosphorylation in cell adhesion and migration. These results are based on a variety of tools, including a widely used mouse monoclonal anti‐phosphocaveolin‐1 antibody, which labels, in cultured cells, a protein localized at or near focal adhesions. We here report results from three independent laboratories, showing that this antibody recognizes phosphocaveolin‐1 amongst other proteins in immunoblot analyses and that the signal obtained with this antibody in immunostaining experiments is in part due to labeling of paxillin. Published data need to be interpreted keeping in mind that images of phosphocaveolin‐1 cellular localization obtained using this antibody are not valid. We re‐evaluate the current knowledge about the role of caveolin‐1 in cell adhesion and migration in view of this new information.
The Effects of Platelet‐Activating Factor on the Secretion of Interleukin‐8 and Growth‐Regulated Oncogene α in Human Immortalized Granulosa Cell Line (GC1a)
Authors: Kawano Yasushi , Furukawa Yuichi , Fukuda Junichiro , Matsumoto Harunobu , Yuge Akitoshi , Narahara Hisashi
Published: October 2007

Problem

To investigate the role of platelet‐activating factor (PAF) in human ovulation, we studied the regulation of interleukin (IL)‐8 and growth‐regulated oncogene (GRO) α in cultured human immortalized granulosa cell line (GC1a).

Method of study

GC1a was cultured in serum‐free medium, and incubated with carbamyl‐PAF (C‐PAF) and/or PAF receptor antagonist (WEB 2086). The supernatants were collected, and IL‐8 and GRO α were measured by enzyme‐linked immunosorbent assay.

Results

After treatment with C‐PAF, the levels of IL‐8 and GROα increased in a time‐dependent manner. The levels of IL‐8 and GROα were significantly increased after treatment with C‐PAF in a dose‐dependent manner. However, the levels of IL‐8 and GROα were significantly decreased by treatment with C‐PAF and with increasing concentrations of WEB 2086.

Conclusion

Our data indicated that IL‐8 and GROα were regulated by C‐PAF. The results suggested that PAF may play an important role in human pre‐ovulatory processes involving IL‐8 and GROα production.
Relationship Between Human Tumor‐Associated Antigen RCAS1 and Gestational Diabetes Mellitus
Authors: Tskitishvili Ekaterine , Komoto Yoshiko , Kinugasa Yukiko , Kanagawa Takeshi , Song Mihyon , Mimura Kazuya , Tomimatsu Takuji , Kimura Tadashi , Shimoya Koichiro
Published: October 2007

Problem

The human tumor‐associated receptor‐binding cancer antigen expressed on SiSo cells (RCAS1) is considered to play a role in the inhibition of the maternal immune response during pregnancy. The aim of our study was to investigate the expression of RCAS1 protein in the placenta and to compare its concentration in maternal and cord blood sera between normal pregnancies and pregnancies complicated by gestational diabetes mellitus (GDM).

Method of study

Samples were obtained from women with GDM (n = 8), women with type 1 DM (n = 5), and healthy term controls (n = 27) after delivery. Placentas were studied by immunohistochemistry, and real‐time polymerase chain reaction. For assessment of RCAS1 concentrations in maternal and cord blood sera, enzyme‐linked immunosorbent assay was performed.

Results

The RCAS1 protein mRNA expression in the placentas of patients with GDM was significantly lower than that in the controls (P = 0.005). The maternal blood RCAS1 protein concentration of the GDM cases was also significantly lower than that in the controls (P = 0.0411), whereas the cord blood RCAS1 protein concentration was significantly higher in the GDM and type 1 DM groups than in the controls (P = 0.0311 and P = 0.0192, respectively).

Conclusion

The present results suggest that RCAS1 protein might have an important role in the development of GDM.
Microparticles and Exosomes: Impact on Normal and Complicated Pregnancy
Authors: Toth Bettina , Lok Christianne A. R. , Böing Anita , Diamant Michaela , Van Der Post Joris A. M. , Friese Klaus , Nieuwland Rienk
Published: October 2007

Eukaryotic cells release vesicles into their environment by membrane shedding (ectosomes or microparticles) and secretion (exosomes). Microparticles and exosomes occur commonly in vitro and in vivo. The occurrence, composition and function(s) of these vesicles change during disease (progression). During the last decade, the scientific and clinical interest increased tremendously. Evidence is accumulating that microparticles and exosomes may be of pathophysiological relevance in autoimmune, cardiovascular and thromboembolic diseases, as well as inflammatory and infectious disorders. In this review, we will summarize the discovery, biology, structure and function of microparticles and exosomes, and discuss their (patho‐) physiological role during normal and complicated pregnancy.
A highly active adipyl‐cephalosporin acylase obtained via rational randomization
Authors: Otten Linda G. , Sio Charles F. , Reis Carlos R. , Koch Gudrun , Cool Robbert H. , Quax Wim J.
Published: October 2007

There is strong interest in creating an enzyme that can deacylate natural cephalosporins such as cephalosporin C in order to efficiently acquire the starting compound for the industrial production of semisynthetic cephalosporin antibiotics. In this study, the active site of the glutaryl acylase from Pseudomonas SY‐77 was randomized rationally. Several mutations that were found in previous studies to enhance the activity of the enzyme towards adipyl‐7‐aminodesacetoxycephalosporanic acid (ADCA) and cephalosporin C have now been combined, and libraries have been made in which random amino acid substitutions at these positions are joined. The mutants were expressed in a leucine‐deficient Escherichia coli strain and subjected to growth selection with adipyl‐leucine or amino‐adipyl‐leucine as sole leucine source. The mutants growing on these media were selected and purified, and their hydrolysis activities towards adipyl‐7‐ADCA and cephalosporin C were tested. Several mutants with highly improved activities towards the desired substrates were found in these rationally randomized libraries. The best mutant was selected from a library of totally randomized residues: 178, 266, and 375. This mutant comprises two mutations, Y178F + F375H, which synergistically improve the catalytic efficiency towards adipyl‐7‐ADCA 36‐fold. The activity of this mutant towards adipyl‐7‐ADCA is 50% of the activity of the wild‐type enzyme towards the preferred substrate glutaryl‐7‐aminocephalosporanic acid, and therefore the characteristics of this mutant approach those needed for industrial application.
Effect of mutations in the β5–β7 loop on the structure and properties of human small heat shock protein HSP22 (HspB8, H11)
Authors: Kasakov Alexei S. , Bukach Olesya V. , Seit‐Nebi Alim S. , Marston Steven B. , Gusev Nikolai B.
Published: October 2007

The human genome encodes ten different small heat shock proteins, each of which contains the so‐called α‐crystallin domain consisting of 80–100 residues and located in the C‐terminal part of the molecule. The α‐crystallin domain consists of six or seven β‐strands connected by different size loops and combined in two β‐sheets. Mutations in the loop connecting the β5 and β7 strands and conservative residues of β7 in αA‐, αB‐crystallin and HSP27 correlate with the development of different congenital diseases. To understand the role of this part of molecule in the structure and function of small heat shock proteins, we mutated two highly conservative residues (K137 and K141) of human HSP22 and investigated the properties of the K137E and K137,141E mutants. These mutations lead to a decrease in intrinsic Trp fluorescence and the double mutation decreased fluorescence resonance energy transfer from Trp to bis‐ANS bound to HSP22. Mutations K137E and especially K137,141E lead to an increase in unordered structure in HSP22 and increased susceptibility to trypsinolysis. Both mutations decreased the probability of dissociation of small oligomers of HSP22, and mutation K137E increased the probability of HSP22 crosslinking. The wild‐type HSP22 possessed higher chaperone‐like activity than their mutants when insulin or rhodanase were used as the model substrates. Because conservative Lys residues located in the β5–β7 loop and in the β7 strand appear to play an important role in the structure and properties of HSP22, mutations in this part of the small heat shock protein molecule might have a deleterious effect and often correlate with the development of different congenital diseases.
A comparative study of type I and type II tryparedoxin peroxidases in Leishmania major
Authors: König Janine , Fairlamb Alan H.
Published: October 2007

The genome of Leishmania major, the causative agent of cutaneous leishmaniasis, contains three almost identical genes encoding putative glutathione peroxidases, which differ only at their N‐ and C‐termini. Because the gene homologues are essential in trypanosomes, they may also represent potential drug targets in Leishmania. Recombinant protein for the shortest of these showed negligible peroxidase activity with glutathione as the electron donor indicating that it is not a bone fide glutathione peroxidase. By contrast, high peroxidase activity was obtained with tryparedoxin, indicating that these proteins belong to a new class of monomeric tryparedoxin‐dependent peroxidases (TDPX) distinct from the classical decameric 2‐Cys peroxiredoxins (TryP). Mass spectrometry studies revealed that oxidation of TDPX1 with peroxides results in the formation of an intramolecular disulfide bridge between Cys35 and Cys83. Site‐directed mutagenesis and kinetic studies showed that Cys35 is essential for peroxidase activity, whereas Cys83 is essential for reduction by tryparedoxin. Detailed kinetic studies comparing TDPX1 and TryP1 showed that both enzymes obey saturation ping‐pong kinetics with respect to tryparedoxin and peroxide. Both enzymes show high affinity for tryparedoxin and broad substrate specificity for hydroperoxides. TDPX1 shows higher affinity towards hydrogen peroxide and cumene hydroperoxide than towards t‐butyl hydroperoxide, whereas no specific substrate preference could be detected for TryP1. TDPX1 exhibits rate constants up to 8 × 104 m−1·s−1, whereas TryP1 exhibits higher rate constants ∼ 106 m−1·s−1. All three TDPX proteins together constitute ∼ 0.05% of the L. major promastigote protein content, whereas the TryPs are ∼ 40 times more abundant. Possible specific functions of TDPXs are discussed.