ALIMENTARY PHARMACOLOGY & THERAPEUTICS, Issue 11-12 2008
M. CHAPARRO
Summary Background, Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. Unresectable disease patients have median survival of few months. There is a substantial need for novel treatments for patients with advanced HCC. Aim, To provide an update review of mechanism of hepatocarcinigenesis and systemic therapies for HCC and the relevant role of Sorafenib in patients with advanced disease. Methods, A Medline search was performed to identify pertinent original research and review articles. Selected references in these articles were also evaluated. Results, Systemic chemotherapy for HCC has been quite ineffective. Preclinical studies demonstrated that Raf/MAPK-ERK kinase (MEK)/Extracellular signal regulated kinase (ERK) pathway has a role in HCC. HCC tumours are highly vascularized and vascular endothelial growth factor (VEGF) augments HCC development and metastasis. Sorafenib blocks tumour cell proliferation by targeting Raf/MEK/ERK signalling and exerts an antiangiogenic effect by targeting VEGF receptors-2/3 and platelet derived growth factor receptor , tyrosine kinases. Conclusions, Currently available therapies are not effective for patients with advanced HCC. Sorafenib has demonstrated for the first time to prolong survival in patients with advanced HCC, and it is the new reference standard for systemic treatment in these patients. [source]

Mechanisms of glutamate release elicited in rat cerebrocortical nerve endings by ,pathologically' elevated extraterminal K+ concentrations

JOURNAL OF NEUROCHEMISTRY, Issue 3 2007
Luca Raiteri
Abstract Extracellular [K+] can increase during some pathological conditions, resulting into excessive glutamate release through multiple mechanisms. We here investigate the overflow of [3H]d -aspartate ([3H] d -ASP) and of endogenous glutamate elicited by increasing [K+] from purified rat cerebrocortical synaptosomes. Depolarization with [K+] , 15 mmol/L provoked [3H] d -ASP and glutamate overflows almost totally dependent on external Ca2+. Consistent with release by exocytosis, the overflow of [3H] d -ASP evoked by 12 mmol/L K+ was sensitive to clostridial toxins. The overflows evoked by 35/50 mmol/L K+ remained external Ca2+ -dependent by more than 50%. The Ca2+ -independent components of the [3H] d -ASP overflows evoked by [K+] > 15 mmol/L were prevented by the glutamate transporter inhibitors dl - threo -beta-benzyloxyaspartate (dl -TBOA) and dihydrokainate. Differently, the overflows of endogenous glutamate provoked by [K+] > 15 mmol/L were insensitive to both inhibitors; the external Ca2+ -independent glutamate overflow caused by 50 mmol/L KCl was prevented by bafilomycin, by chelating intraterminal Ca2+, by blocking the mitochondrial Na+/Ca2+ exchanger and, for a small portion, by blocking anion channels. In contrast to purified synaptosomes, the 50 mmol/L K+ -evoked release of endogenous glutamate or [3H]D-ASP was inhibited by dl -TBOA in crude synaptosomes; moreover, it was external Ca2+ -insensitive and blocked by dl -TBOA in purified gliosomes, suggesting that carrier-mediated release of endogenous glutamate provoked by excessive [K+] in CNS tissues largely originates from glia. [source]

Ethanol Acutely Inhibits Ionotropic Glutamate Receptor-Mediated Responses and Long-Term Potentiation in the Developing CA1 Hippocampus

ALCOHOLISM, Issue 4 2010
Michael P. Puglia
Background:, Developmental ethanol (EtOH) exposure damages the hippocampus, causing long-lasting alterations in learning and memory. Alterations in glutamatergic synaptic transmission and plasticity may play a role in the mechanism of action of EtOH. This signaling is fundamental for synaptogenesis, which occurs during the third trimester of human pregnancy (first 12 days of life in rats). Methods:, Acute coronal brain slices were prepared from 7- to 9-day-old rats. Extracellular and patch-clamp electrophysiological recording techniques were used to characterize the acute effects of EtOH on ,-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR)- and N -methyl- d -aspartate receptor (NMDAR)-mediated responses and long-term potentiation (LTP) in the CA1 hippocampal region. Results:, Ethanol (40 and 80 mM) inhibited AMPAR- and NMDAR-mediated field excitatory postsynaptic potentials (fEPSPs). EtOH (80 mM) also reduced AMPAR-mediated fEPSPs in the presence of an inhibitor of Ca2+ permeable AMPARs. The effect of 80 mM EtOH on NMDAR-mediated fEPSPs was significantly greater in the presence of Mg2+. EtOH (80 mM) neither affected the paired-pulse ratio of AMPAR-mediated fEPSPs nor the presynaptic volley. The paired-pulse ratio of AMPAR-mediated excitatory postsynaptic currents was not affected either, and the amplitude of these currents was inhibited to a lesser extent than that of fEPSPs. EtOH (80 mM) inhibited LTP of AMPAR-mediated fEPSPs. Conclusions:, Acute EtOH exposure during the third-trimester equivalent of human pregnancy inhibits hippocampal glutamatergic transmission and LTP induction, which could alter synapse refinement and ultimately contribute to the pathophysiology of fetal alcohol spectrum disorder. [source]

Extracellular and intracellular mechanisms that mediate the metastatic activity of exogenous osteopontin

CANCER, Issue 8 2009
Jami Mandelin PhD
Abstract BACKGROUND: Osteopontin affects several steps of the metastatic cascade. Despite direct correlation with metastasis in experimental systems and in patient studies, the extracellular and intracellular basis for these observations remains unsolved. In this study, the authors used human melanoma and sarcoma cell lines to evaluate the effects of soluble osteopontin on metastasis. METHODS: Exogenous osteopontin or negative controls, including a site-directed mutant osteopontin, were used in functional assays in vitro, ex vivo, and in vivo that were designed to test the extracellular and intracellular mechanisms involved in experimental metastasis. RESULTS: In the extracellular environment, the results confirmed that soluble osteopontin is required for its prometastatic effects; this phenomenon is specific, arginine-glycine-aspartic acid (RGD)-dependent, and evident in experimental models of metastasis. In the intracellular environment, osteopontin initially induced rapid tyrosine 418 (Tyr-418) dephosphorylation of the cellular homolog of the Rous sarcoma virus (c-Src), with decreases in actin stress fibers and increased binding to the vascular endothelium. This heretofore undescribed Tyr dephosphorylation was followed by a tandem c-Src phosphorylation after tumor cell attachment to the metastatic site. CONCLUSIONS: The results of this study revealed a complex molecular interaction as well as a dual role for osteopontin in metastasis that depends on whether tumor cells are in circulation or attached. Such context-dependent functional insights may contribute to antimetastasis strategies. Cancer 2009. © 2009 American Cancer Society. [source]

Simultaneous separation of intracellular and extracellular lactate NMR signals of human erythrocytes

MAGNETIC RESONANCE IN MEDICINE, Issue 2 2007
Götz Kohler
Abstract Intracellular/extracellular lactate (Lac) distribution has been determined before in human and animal erythrocytes (red blood cells [RBCs]) with various methods. However, all previous methods determine intra- and extracellular Lac separately or indirectly. Now, 13C-NMR spectroscopy has been used to monitor intra- and extracellular Lac simultaneously in intact RBCs. Isolated human RBCs were incubated with [3- 13C]-Lac, [3- 13C]-pyruvate (Pyr), and [1- 13C]-glucose (Gluc). A distortionless enhancement by polarization transfer (DEPT) sequence was used (TR = 3.3 s, N = 128) to monitor the 13C-NMR resonances in both compartments. The intra- and extracellular methyl group resonances of Lac and Pyr were clearly separated by 9.6 Hz and 7.0 Hz, respectively, under normoxic conditions due to the RBC chemical-shift effect. The results show that the chemical-shift effect of RBCs is convenient to monitor intra- and extracellular Lac simultaneously in intact RBCs under normoxic conditions. Magn Reson Med 58:213,217, 2007. © 2007 Wiley-Liss, Inc. [source]

Intraoperative cytology of clear cell carcinoma of the ovary

CYTOPATHOLOGY, Issue 6 2006
D. Vrdoljak-Mozeti
Objective:, To describe the cytomorphology of clear cell carcinoma (CCC) of the ovary in intraoperative samples of peritoneal fluid, imprint and scraping samples of the tumour tissue. Study design:, Fourteen histologically confirmed cases, stained by standard cytological procedures, were analysed by light microscopy. Results:, In 33.3% of peritoneal fluid samples and 92.9% of imprint and scraping cytological samples, besides variable clear cell cellular morphology, one or both distinct cytological characteristics were observed: eosinophilic, hyaline, extracellular, globular substance with or without formation of a ,raspberry' body and an eosinophilic, intracytoplasmic inclusions. These structures were clearly seen only in samples stained by May-Grünwald,Giemsa. Conclusion:, Using cytological analysis of imprint and scraping samples of ovarian tumours it is possible to make a precise intraoperative cytological diagnosis in most cases of CCC of the ovary. [source]

Immunocytochemical study of activin type IB receptor (XALK4) in Xenopus oocytes

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 2 2003
Akimasa Fukui
Studies have shown that the activin type IB receptor is specific for activin/nodal signaling. Activin is produced by follicle cells in the ovary, and is incorporated into the oocytes. Antisera against three peptides were prepared, encompassing the extracellular, intracellular and serine/threonine kinase domains of the Xenopus type IB activin receptor (XALK4). Immunocytochemistry was done using these antisera to investigate the distribution of XALK4 in the Xenopus ovary. All three antisera stained the mitochondrial cloud of Xenopus previtellogenic oocytes. Purified antibody against the intracellular domain also recognized the mitochondrial cloud. Immunoelectron microscopy localized XALK4 on the endoplasmic reticulum of the mitochondrial cloud, although not on mitochondria. [source]

Improved ,-Glucanase Production by a Recombinant Escherichia coli Strain using Zinc-Ion Supplemented Medium

ENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 3 2007
U. Beshay
Abstract In order to investigate the suitability of different metal chelates for affinity chromatography, an expression vector was constructed. It contained a hybrid ,-glucanase as a model protein fused with a His6 -tag and a secretion cassette providing the ability to secrete ,-glucanase into the culture medium. Supplementation of zinc to the medium led to a rapidly increased expression and release of the target protein into the cultivation medium. Results in respect to the supplementation of the commonly used Terrific Broth "TB-medium" with different metal ions are reported with special emphasis on the influence of zinc ions. A concentration of zinc ions in the order of about 0.175 mM led to optimal results. Batch cultivation under well-controlled conditions showed that the growth behavior did not change significantly by adding zinc ions. Growth in a stirred tank bioreactor was much faster in unsupplemented TB-medium compared to shake flask experiments leading to a much higher biomass concentration (15,g/L instead of 3,g/L). The secretion of ,-glucanase under theses conditions started at the transition into the stationary phase and increased to yield an extracellular activity of 1350,U/mL at the end of the fermentation process. An even higher yield of extracellular ,-glucanase (2800,U/mL) was reached when the fermentation was carried out with TB-medium supplemented with 0.175,mM ZnSO4. [source]

Production of ,-Amylase and Glucoamylase by a New Isolate of Trichoderma sp.

ENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 4 2004
Using Sorghum Starch as a Carbon Source
The performance of a new Trichoderma sp. isolate to produce extracellular ,-amylase and glucoamylase from raw sorghum starch was evalutated. To reduce the costs of starch saccharification and the consumption of amylolytic enzymes, this microorganism has been used for the first time in cultivations using such a carbon source without any prior gelatinization. [source]

Identification of quorum-sensing regulated proteins in the opportunistic pathogen Pseudomonas aeruginosa by proteomics

ENVIRONMENTAL MICROBIOLOGY, Issue 12 2003
Catalina Arevalo-Ferro
Summary The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic human pathogen which is responsible for severe nosocomial infections in immunocompromised patients and is the major pathogen in cystic fibrosis. The bacterium utilizes two interrelated quorum-sensing (QS) systems, which rely on N -acyl-homoserine lactone (AHL) signal molecules, to control the expression of virulence factors and biofilm development. In this study, we compared the protein patterns of the intracellular, extracellular and surface protein fractions of the PAO1 parent strain with those of an isogenic lasI rhlI double mutant by means of two-dimensional gel electrophoresis (2-DE). This analysis showed that the intensities of 23.7% of all detected protein spots differed more than 2.5-fold between the two strains. We only considered those protein spots truly QS regulated that were changed in the mutant in the absence of signal molecules but were rescued to the wild-type situation when the medium was supplemented with AHLs. These protein spots were characterized by MALDI-TOF peptide mapping. Twenty-seven proteins were identified that were previously reported to be AHL controlled, among them several well-characterized virulence factors. For one of the identified proteins, the serine protease PrpL, a biochemical assay was established to verify that expression of this factor is indeed QS regulated. Furthermore, it is shown that the quorum-sensing blocker C-30 specifically interferes with the expression of 67% of the AHL-controlled protein spots of the surface fraction, confirming the high specificity of the compound. Importantly, 20 novel QS-regulated proteins were identified, many of which are involved in iron utilization, suggesting a link between quorum sensing and the iron regulatory system. Two of these proteins, PhuR and HasAp, are components of the two distinct haem-uptake systems present in P. aeruginosa. In agreement with the finding that both proteins are positively regulated by the QS cascade, we show that the lasI rhlI double mutant grows poorly with haemoglobin as the only iron source when compared with the wild type. These results add haemoglobin utilization to the list of phenotypes controlled through QS in P. aeruginosa. The surprisingly high number of AHL-regulated proteins relative to the number of regulated genes suggests that quorum-sensing control also operates via post-transcriptional mechanisms. To strengthen this hypothesis we investigated the role of quorum sensing in the post-translational modification of HasAp, an extracellular protein required for the uptake of free and haemoglobin-bound haem. [source]

Cellular mechanisms of cobalt-induced hippocampal epileptiform discharges

EPILEPSIA, Issue 1 2009
Jiwei He
Summary Purpose:, To explore the cellular mechanisms of cobalt-induced epileptiform discharges in mouse hippocampal slices. Methods:, Hippocampal slices were prepared from adult mice and briefly exposed to a CoCl2 -containing external solution. Population and single cell activities were examined via extracellular and whole-cell patch recordings. Results:, Brief cobalt exposure induced spontaneous, ictal-like discharges originating from the CA3 area. These discharges were suppressed by anticonvulsants, gap junction blockers, or by raising extracellular Ca2+, but their generation was not associated with overall hyperexcitability or impairment in GABAergic inhibition in the CA3 circuit. Electroencephalographic ictal discharges of similar waveforms were observed in behaving rats following intrahippocampal cobalt infusion. Discussion:, Mechanisms involving activity-dependent facilitation of gap junctional communication may play a major role in cobalt-induced epileptiform discharges. [source]

Mechanistic hypotheses for nonsynaptic epileptiform activity induction and its transition from the interictal to ictal state,Computational simulation

EPILEPSIA, Issue 11 2008
Antônio-Carlos G. De Almeida
Summary Purpose:, The aim of this work is to study, by means of computational simulations, the induction and sustaining of nonsynaptic epileptiform activity. Methods:, The computational model consists of a network of cellular bodies of neurons and glial cells connected to a three-dimensional (3D) network of juxtaposed extracellular compartments. The extracellular electrodiffusion calculation was used to simulate the extracellular potential. Each cellular body was represented in terms of the transmembrane ionic transports (Na+/K+ pumps, ionic channels, and cotransport mechanisms), the intercellular electrodiffusion through gap-junctions, and the neuronal interaction by electric field and the variation of cellular volume. Results:, The computational model allows simulating the nonsynaptic epileptiform activity and the extracellular potential captured the main feature of the experimental measurements. The simulations of the concomitant ionic fluxes and concentrations can be used to propose the basic mechanisms involved in the induction and sustaining of the activities. Discussion:, The simulations suggest: The bursting induction is mediated by the Cl, Nernst potential overcoming the transmembrane potential in response to the extracellular [K+] increase. The burst onset is characterized by a critical point defined by the instant when the Na+ influx through its permeable ionic channels overcomes the Na+/K+ pump electrogenic current. The burst finalization is defined by another critical point, when the electrogenic current of the Na+/K+ pump overcomes its influx through the channels. [source]

Decreased hippocampal volume on MRI is associated with increased extracellular glutamate in epilepsy patients

EPILEPSIA, Issue 8 2008
Idil Cavus
Summary Purpose: Temporal lobe epilepsy (TLE) is associated with smaller hippocampal volume and with elevated extracellular (EC) glutamate levels. We investigated the relationship between the hippocampal volume and glutamate in refractory TLE patients. Methods: We used quantitative MRI volumetrics to measure the hippocampal volume and zero-flow microdialysis to measure the interictal glutamate, glutamine, and GABA levels in the epileptogenic hippocampus of 17 patients with medication-resistant epilepsy undergoing intracranial EEG evaluation. The relationships between hippocampal volume, neurochemical levels, and relevant clinical factors were examined. Results: Increased EC glutamate in the epileptogenic hippocampus was significantly related to smaller ipsilateral (R2= 0.75, p < 0.0001), but not contralateral hippocampal volume when controlled for glutamine and GABA levels, and for clinical factors known to influence hippocampal volume. Glutamate in the atrophic hippocampus was significantly higher (p = 0.008, n = 9), with the threshold for hippocampal atrophy estimated as 5 ,M. GABA and glutamine levels in the atrophic and nonatrophic hippocampus were comparable. Decreased hippocampal volume was related to higher seizure frequency (p = 0.008), but not to disease duration or febrile seizure history. None of these clinical factors were related to the neurochemical levels. Conclusions: We provide evidence for a significant association between increased EC glutamate and decreased ipsilateral epileptogenic hippocampal volume in TLE. Future work will be needed to determine whether the increase in glutamate has a causal relationship with hippocampal atrophy, or whether another, yet unknown factor results in both. This work has implications for the understanding and treatment of epilepsy as well as other neurodegenerative disorders associated with hippocampal atrophy. [source]

Comparison of Intrinsic Optical Signals Associated with Low Mg2+, and 4-Aminopyridine,Induced Seizure-Like Events Reveals Characteristic Features in Adult Rat Limbic System

EPILEPSIA, Issue 6 2000
Katharina Buchheim
Summary: Purpose: To analyze the intrinsic optical signal change associated with seizure-like events in two frequently used in vitro models,the low-Mg2+ and the 4-aminopyridine (4-AP) models,and to monitor regions of onset and spread patterns of these discharges by using imaging of intrinsic optical signals (IOS). Methods: Combined hippocampal,entorhinal,cortex slices of adult rats were exposed to two different treatments: lowering extracellular Mg2+ concentrations or application of 100 ,M 4-AP. The electrographic features of the discharges were monitored using extracellular microelectrodes. Optical imaging was achieved by infrared transillumination of the slice and analysis of changes in light transmission using a subtraction approach. The electrographic features were compared with the optical changes. Regions of onset and spread patterns were analyzed in relevant anatomic regions of the slice. Results: Both lowering extracellular Mg2+ concentrations and application of 4-AP induced seizure-like events. The relative duration of the intrinsic optical signal change associated with seizure-like events in the low-Mg2+ model was significantly longer compared with that seen with those occurring in the 4-AP model, although duration of field potentials did not differ significantly in the two models. Seizure-like events of the low-Mg2+ model originated predominantly in the entorhinal cortex, with subsequent propagation toward the subiculum and neocortical structures. In contrast, no consistent region of onset or spread patterns were seen in the 4-AP model, indicating that the seizure initiation is not confined to a particular region in this model. Conclusions: We conclude that different forms of spontaneous epileptiform activity are associated with characteristic optical signal changes and that optical imaging represents an excellent method to assess regions of seizure onset and spread patterns. [source]

Vaccinia virus impairs directional migration and chemokine receptor switch of human dendritic cells

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2007

Abstract A crucial event for the induction of an anti-viral immune response is the coordinated, phenotype-dependent migration of dendritic cells (DC) to sites of infection and secondary lymphoid organs. Here we show that the vaccinia virus (VV) strains Western Reserve (WR) and modified virus Ankara (MVA) inhibit directional migration of mature DC toward the lymphoid chemokines CCL19 and CXCL12 without affecting surface expression of the respective chemokine receptors or impairing undirected cellular locomotion. Instead, infection with VV results in a deficiency of extracellular signal-regulated kinase-1 and a disturbance of intracellular calcium mobilization, indicating a viral interference with signaling events downstream of the surface chemokine receptors. In immature DC, apart from inhibiting chemokine-induced migration of infected DC, infection with both VV strains increases expression of the inflammatory chemokine receptors CCR1 and CXCR1 on non-infected bystander DC, which depends on the activity of IFN-,. Although functional, these chemokine receptors are resistant to lipopolysaccharide-induced down-regulation. In addition, VV-infected and non-infected bystander DC fail to up-regulate the lymphoid chemokine receptor CCR7 upon activation, together pointing to a disability to undergo the chemokine receptor switch. This study shows that VV targets directional migration of professional antigen-presenting cells at multiple functional levels, revealing a potent viral strategy of immune escape. See accompanying commentary: http://dx.doi.org/10.1002/eji.200737215 [source]

Nociceptive spinothalamic tract and postsynaptic dorsal column neurons are modulated by paraventricular hypothalamic activation

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2008
Gerardo Rojas-Piloni
Abstract Previously, we demonstrated that stimulation of the paraventricular hypothalamic nucleus diminishes the nociceptive dorsal horn neuronal responses, and this decrease was mediated by oxytocin in the rat. In addition, we have proposed that oxytocin indirectly inhibits sensory transmission in dorsal horn neurons by exciting spinal inhibitory GABAergic interneurons. The main purpose of the present study was to identify which of the neurons projecting to supraspinal structures to transmit somatic information are modulated by the hypothalamic-spinal descending activation. In anaesthetized rats, single-unit extracellular and juxtacellular recordings were made from dorsal horn lumbar segments, which receive afferent input from the toe and hind-paw regions. The projecting spinothalamic tract and postsynaptic dorsal column system were identified antidromically. Additionally, in order to label the projecting dorsal horn neurons, we injected fluorescent retrograde neuronal tracers into the ipsilateral gracilis nucleus and contralateral ventroposterolateral thalamic nucleus. Hence, juxtacellular recordings were made to iontophoretically label the recorded neurons with a fluorescent dye and identify the recorded projecting cells. We found that only nociceptive evoked responses in spinothalamic tract and postsynaptic dorsal column neurons were significantly inhibited (48.1 ± 4.6 and 47.7 ± 8.2%, respectively) and non-nociceptive responses were not affected by paraventricular hypothalamic nucleus stimulation. We conclude that the hypothalamic-spinal system selectively affects the transmission of nociceptive information of projecting spinal cord cells. [source]

Basolateral amygdala inactivation by muscimol, but not ERK/MAPK inhibition, impairs the use of reward expectancies during working memory

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 12 2007
Lisa M. Savage
Abstract Rats were trained on a delayed matching to position (DMTP) task that embedded either a differential outcomes procedure (DOP) or a non-differential outcomes procedure (NOP). The DOP, via Pavlovian conditioning (stimulus,outcome associations), results in the use of unique reward expectancies that facilitate learning and memory performance above subjects trained with a NOP that requires subjects to retain cue information for accurate choice behavior (stimulus,response associations). This enhancement in learning and/or memory produced by the DOP is called the differential outcomes effect (DOE). After being trained on the DMTP task, rats were implanted with two cannulae aimed at the basolateral amygdala (BLA) nuclei. Rats trained with the DOP, relative to those trained with the NOP, displayed enhanced short-term memory (STM) performance under vehicle conditions (i.e. the DOE). However, injections of the ,-aminobutyric acid (GABA)A agonist muscimol into the BLA dose-dependently (0.0625 and 0.125 µg) impaired STM performance only in DOP-trained rats. These results support the role of the BLA in the use of established reward expectancies during a short-term working memory task. Despite the fact that extracellular signal-regulated kinase/mitogen-activated protein kinases (ERK/MAPK) have been shown to be necessary for amygdala-dependent long-term potentiation and some forms of long-term and STM, inhibition of the ERK/MAPK signaling cascade by U0126 (2.0 or 4.0 µg) in the BLA was not critical for updating the STM of either spatial information or reward expectation. [source]

Proteolytic cleavage of the voltage-gated Ca2+ channel ,2, subunit: structural and functional features

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 6 2007
Arturo Andrade
Abstract By mediating depolarization-induced Ca2+ influx, high-voltage-activated Ca2+ channels control a variety of cellular events. These heteromultimeric proteins are composed of an ion-conducting (,1) and three auxiliary (,2,, , and ,) subunits. The ,2, subunit enhances the trafficking of the channel complex to the cell surface and increases channel open probability. To exert these effects, ,2, must undergo important post-translational modifications, including a proteolytic cleavage that separates the extracellular ,2 from its transmembrane , domain. After this proteolysis both domains remain linked by disulfide bonds. In spite of its central role in determining the final conformation of the fully mature ,2,, almost nothing is known about the physiological implications of this structural modification. In the current report, by using site-directed mutagenesis, the proteolytic site of ,2, was mapped to amino acid residues Arg-941 and Val-946. Substitution of these residues renders the protein insensitive to proteolytic cleavage as evidenced by the lack of molecular weight shift upon treatment with a disulfide-reducing agent. Interestingly, these mutations significantly decreased whole-cell patch-clamp currents without affecting the voltage dependence or kinetics of the channels, suggesting a reduction in the number of channels targeted to the plasma membrane. [source]

Glutamate-induced elevations in intracellular chloride concentration in hippocampal cell cultures derived from EYFP-expressing mice

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2004
Jennifer E. Slemmer
Abstract The homeostasis of intracellular Cl, concentration ([Cl,]i) is critical for neuronal function, including ,-aminobutyric acid (GABA)ergic synaptic transmission. Here, we investigated activity-dependent changes in [Cl,]i using a transgenetically expressed Cl, -sensitive enhanced yellow-fluorescent protein (EYFP) in cultures of mouse hippocampal neurons. Application of glutamate (100 µm for 3 min) in a bath perfusion to cell cultures of various days in vitro (DIV) revealed a decrease in EYFP fluorescence. The EYFP signal increased in amplitude with increasing DIV, reaching a maximal response after 7 DIV. Glutamate application resulted in a slight neuronal acidification. Although EYFP fluorescence is sensitive to pH, EYFP signals were virtually abolished in Cl, -free solution, demonstrating that the EYFP signal represented an increase in [Cl,]i. Similar to glutamate, a rise in [Cl,]i was also induced by specific ionotropic glutamate receptor agonists and by increasing extracellular [K+], indicating that an increase in driving force for Cl, suffices to increase [Cl,]i. To elucidate the membrane mechanisms mediating the Cl, influx, a series of blockers of ion channels and transporters were tested. The glutamate-induced increase in [Cl,]i was resistant to furosemide, bumetanide and 4,4,-diisothiocyanato-stilbene-2,2,-disulphonic acid (DIDS), was reduced by bicuculline to about 80% of control responses, and was antagonized by niflumic acid (NFA) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We conclude that membrane depolarization increases [Cl,]i via several pathways involving NFA- and NPPB-sensitive anion channels and GABAA receptors, but not through furosemide-, bumetanide- or DIDS-sensitive Cl, transporters. The present study highlights the vulnerability of [Cl,]i homeostasis after membrane depolarization in neurons. [source]

High extracellular [Mg2+]-induced increase in intracellular [Mg2+] and decrease in intracellular [Na+] are associated with activation of p38 MAP kinase and ERK2 in guinea-pig heart

EXPERIMENTAL PHYSIOLOGY, Issue 12 2008
Shang-Jin Kim
High extracellular Mg2+ concentrations ([Mg2+]o) caused a remarkable concentration-dependent and reversible increase in intracellular Mg2+ concentrations ([Mg2+]i) in beating and quiescent guinea-pig papillary muscles, accompanied by a definite decrease in intracellular Na+ concentrations ([Na+]i). A change in 1 mm[Mg2+]o evoked a direct change in 0.0161 mm[Mg2+]i and an inverse change in 0.0263 mm[Na+]i. Imipramine completely abolished the high [Mg2+]o -induced decrease in [Na+]i and remarkably diminished the high [Mg2+]o -induced increase in [Mg2+]i in papillary muscles. High [Mg2+]o also produced a significant activation of p38 mitogen-activated protein (MAP) kinase and extracellular signal-related kinase 2 (ERK2) that was inhibited by pretreatment with imipramine. These results suggest that the high [Mg2+]o -induced increase in [Mg2+]i could be coupled with the decrease in [Na+]i, which might involve activation of the reverse mode of Na+,Mg2+ exchange, accompanied by activation of p38 MAP kinase and ERK2 in the guinea-pig heart. [source]

Oxygen binding and its allosteric control in hemoglobin of the primitive branchiopod crustacean Triops cancriformis

FEBS JOURNAL, Issue 13 2007
Ralph Pirow
Branchiopod crustaceans are endowed with extracellular, high-molecular-mass hemoglobins (Hbs), the functional and allosteric properties of which have largely remained obscure. The Hb of the phylogenetically ancient Triops cancriformis (Notostraca) revealed moderate oxygen affinity, cooperativity and pH dependence (Bohr effect) coefficients: P50 = 13.3 mmHg, n50 = 2.3, and , = ,0.18, at 20 °C and pH 7.44 in Tris buffer. The in vivo hemolymph pH was 7.52. Bivalent cations increased oxygen affinity, Mg2+ exerting a greater effect than Ca2+. Analysis of cooperative oxygen binding in terms of the nested Monod,Wyman,Changeux (MWC) model revealed an allosteric unit of four oxygen-binding sites and functional coupling of two to three allosteric units. The predicted 2 × 4 and 3 × 4 nested structures are in accord with stoichiometric models of the quarternary structure. The allosteric control mechanism of protons comprises a left shift of the upper asymptote of extended Hill plots which is ascribable to the displacement of the equilibrium between (at least) two high-affinity (relaxed) states, similar to that found in extracellular annelid and pulmonate molluscan Hbs. Remarkably, Mg2+ ions increased oxygen affinity solely by displacing the equilibrium between the tense and relaxed conformations towards the relaxed states, which accords with the original MWC concept, but appears to be unique among Hbs. This effect is distinctly different from those of ionic effectors (bivalent cations, protons and organic phosphates) on annelid, pulmonate and vertebrate Hbs, which involve changes in the oxygen affinity of the tense and/or relaxed conformations. [source]

Native and subunit molecular mass and quarternary structure of the hemoglobin from the primitive branchiopod crustacean Triops cancriformis

FEBS JOURNAL, Issue 17 2006
Morgane Rousselot
Many branchiopod crustaceans are endowed with extracellular, high-molecular-weight hemoglobins whose exact structural characteristics have remained a matter of conjecture. By using a broad spectrum of techniques, we provide precise and coherent information on the hemoglobin of one of the phylogenetically ,oldest' extant branchiopods, the tadpole shrimp Triops cancriformis. The hemoglobin dissociated under reducing conditions into two subunits, designated TcHbA and TcHbB, with masses of 35 775 ± 4 and 36 055 ± 4 Da, respectively, determined by ESI-MS. Nonreducing conditions showed only two disulfide-bridged dimers, a homodimer of TcHbA, designated D1 (71 548 ± 5 Da), and the heterodimer D2 (71 828 ± 5 Da). Carbamidomethylation of free SH groups revealed the presence of three cysteines per subunit and indicated one intrasubunit and one intersubunit disulfide bridge. Ultracentrifugation and light-scattering experiments under nondenaturating conditions yielded mass estimates that suggested an uneven number of 17 subunits forming the native hemoglobin. This unrealistic number resulted from the presence of two size classes (16-mer and 18-mer), which were recognized by native PAGE and Ferguson plot analysis. ESI-MS revealed three hemoglobin isoforms with masses of 588.1 kDa, 662.0 kDa, and 665.0 kDa. The 16-mer and the smaller 18-mer species are supposed to be composed of TcHbA only, given the dominance of this subunit type in SDS/PAGE. Transmission electron microscopy of negatively stained specimens showed a population of compact molecules with geometrical extensions of 14, 16 and 9 nm. The proposed stoichiometric model of quarternary structure provides the missing link to achieve a mechanistic understanding of the structure,function relationships among the multimeric arthropodan hemoglobins. [source]

Sulfatide with short fatty acid dominates in astrocytes and neurons

FEBS JOURNAL, Issue 8 2006
Giorgis Isaac
Glycosphingolipids are located in cell membranes and the brain is especially enriched. We speculated that the subcellular location of glycosphingolipids depends on their fatty acid chain length because their sugar residues are constant, whereas fatty acid chain length can vary within the same molecule. To test this hypothesis we analysed the glycosphingolipid sulfatide, which is highly abundant in myelin and has mostly long fatty acids. We used a negative ion electrospray tandem mass spectrometry precursor ion scan to analyse the molecular species of sulfatide in cultured astrocytes and a mouse model of the human disease metachromatic leukodystrophy. In these arylsulfatase A (ASA)-deficient mice sulfatide accumulates intracellularly in neurons and astrocytes. Immunocytochemistry was also performed on cultured astrocytes and analysed using confocal laser scanning microscopy. Analyses of the molecular species showed that cultured astrocytes contained sulfatide with a predominance of stearic acid (C18), which was located in large intracellular vesicles throughout the cell body and along the processes. The same was seen in ASA-deficient mice, which accumulated a higher proportion (15 mol% compared with 8 mol% in control mice) of sulfatide with stearic acid. We conclude that the major fatty acid composition of sulfatide differs between white and grey matter, with neurons and astrocytes containing mostly short-chain fatty acids with an emphasis on stearic acid. Based on our results, we speculate that the fatty acid chain length of sulfatide might determine its intracellular (short chain) or extracellular (long chain) location and thereby its functions. [source]

Calcium modulates endopeptidase 24.15 (EC 3.4.24.15) membrane association, secondary structure and substrate specificity

FEBS JOURNAL, Issue 12 2005
Vitor Oliveira
The metalloendopeptidase 24.15 (EP24.15) is ubiquitously present in the extracellular environment as a secreted protein. Outside the cell, this enzyme degrades several neuropeptides containing from 5 to 17 amino acids (e.g. gonadotropin releasing hormone, bradykinin, opioids and neurotensin). The constitutive secretion of EP24.15 from glioma C6 cells was demonstrated to be stimulated linearly by reduced concentrations of extracellular calcium. In the present report we demonstrate that extracellular calcium concentration has no effect on the total amount of the extracellular (cell associated + medium) enzyme. Indeed, immuno-cytochemical analyses by confocal and electron microscopy suggested that the absence of calcium favors the enzyme shedding from the plasma membrane into the medium. Two putative calcium-binding sites on EP24.15 (D93 and D159) were altered by site-directed mutagenesis to investigate their possible contribution to binding of the enzyme at the cell surface. These mutated recombinant proteins behave similarly to the wild-type enzyme regarding enzymatic activity, secondary structure, calcium sensitivity and immunoreactivity. However, immunocytochemical analyses by confocal microscopy consistently show a reduced ability of the D93A mutant to associate with the plasma membrane of glioma C6 cells when compared with the wild-type enzyme. These data and the model of the enzyme's structure as determined by X-ray diffraction suggest that D93 is located at the enzyme surface and is consistent with membrane association of EP24.15. Moreover, calcium was also observed to induce a major change in the EP24.15 cleavage site on distinctive fluorogenic substrates. These data suggest that calcium may be an important modulator of ep24.15 cell function. [source]

Biodiesel fuel production via transesterification of oils using lipase biocatalyst

GCB BIOENERGY, Issue 2 2009
MAN XIAO
Abstract Biodiesel has gained widespread importance in recent years as an alternative, renewable liquid transportation fuel. It is derived from natural triglycerides in the presence of an alcohol and an alkali catalyst via a transesterification reaction. To date, transesterification based on the use of chemical catalysts has been predominant for biodiesel production at the industrial scale due to its high conversion efficiency at reasonable cost. Recently, biocatalytic transesterification has received considerable attention due to its favorable conversion rate and relatively simple downstream processing demands for the recovery of by-products and purification of biodiesel. Biocatalysis of the transesterification reaction using commercially purified lipase represents a major cost constraint. However, more cost-effective techniques based on the immobilization of both extracellular and intracellular lipases on support materials facilitate the reusability of the catalyst. Other variables, including the presence of alcohol, glycerol and the activity of water can profoundly affect lipase activity and stability during the reaction. This review evaluates the current status for lipase biocatalyst-mediated production of biodiesel, and identifies the key parameters affecting lipase activity and stability. Pioneer studies on reactor-based lipase conversion of triglycerides are presented. [source]

One-year longitudinal evaluation of sensorimotor functions in APP751SL transgenic mice

GENES, BRAIN AND BEHAVIOR, Issue 2008
C. Le Cudennec
Intracerebral amyloid-beta (A,) peptide deposition is considered to play a key role in Alzheimer's disease and is designated as a principal therapeutic target. The relationship between brain A, levels and clinical deficits remains, however, unclear, both in human patients and in animal models of the disease. The purpose of the present study was to investigate, in a transgenic mouse model of brain amyloidosis, the consequences of A, deposition on basic neurological functions using a longitudinal approach. Animals were phenotyped at different ages corresponding to graded neuropathological stages (from no extracellular A, deposition to high amyloid loads). Sensory functions were evaluated by assessing visual and olfactory abilities and did not show any effects of the amyloid precursor protein (APP) transgene. Motor functions were assessed using multiple experimental paradigms. Results showed that motor strength was considerably reduced in APP transgenic mice compared with control animals. No deficit was noted in a motor coordination test although APP transgenic mice displayed decreased locomotion on a stationary beam. Hypolocomotion was also observed in the standard open-field test. Measures of anxiety obtained in the elevated plus-maze show some evidence of hyperanxiety in 15-month-old transgenic mice. Some of the neurological impairments showed by APP mice had an early onset and worsened with progressive aging, in parallel to gradual accumulation of A, in brain parenchyma. Relationships between neuropathologically assessed amyloid loads and behavioral deficits were further explored, and it was observed that motor strength deficits were correlated with cortical amyloid burden. [source]

Role of the astrocytic ETB receptor in the regulation of extracellular endothelin-1 during hypoxia

GLIA, Issue 1 2001
Martin Hasselblatt
Abstract Astrocytes are known to possess an effective endothelin (ET) eliminatory system which involves astrocytic ETA and ETB receptors and may become particularly relevant under pathophysiological conditions. The present study has therefore been designed to explore the effect of standardized hypoxia on extracellular concentrations of endothelin-1 (ET-1) and on endothelin-converting enzyme (ECE) activity in primary rat astrocytes genetically (sl/sl) or experimentally (dexamethasone) deficient in ETB receptors. The results revealed (1) a hypoxia-mediated decrease of extracellular ET-1 in wildtype astrocytes (+/+) that was not observed in ETB -deficient (sl/sl) cultures; (2) an ET receptor antagonist-induced increase in ET-1 in the media of both genotypes with further elevation upon hypoxia in +/+ cultures only; (3) augmentation of the dexamethasone-induced increase in extracellular ET-1 by hypoxia in +/+, but not in sl/sl cultures; (4) synergistic reduction of ETB gene transcription by hypoxia and dexamethasone; and (5) significant increases in endothelin-converting enzyme activity in the presence of hypoxia. To conclude, hypoxia stimulates astrocytic release of mature ET-1. This stimulation is (over)compensated for by increased ET-1 binding to functional ETB receptors. ETB deficiency, whether genetic or experimentally induced, impairs elimination of extracellular ET-1. GLIA 34:18,26, 2001. © 2001 Wiley-Liss, Inc. [source]

Immunosuppression using the mTOR inhibition mechanism affects replacement of rat liver with transplanted cells,

HEPATOLOGY, Issue 2 2006
Yao-Ming Wu
Successful grafting of tissues or cells from mismatched donors requires systemic immunosuppression. It is yet to be determined whether immunosuppressive manipulations perturb transplanted cell engraftment or proliferation. We used syngeneic and allogeneic cell transplantation assays based on F344 recipient rats lacking dipeptidyl peptidase IV enzyme activity to identify transplanted hepatocytes. Immunosuppressive drugs used were tacrolimus (a calcineurin inhibitor) and its synergistic partners, rapamycin (a regulator of the mammalian target of rapamycin [mTOR]) and mycophenolate mofetil (an inosine monophosphate dehydrogenase inhibitor). First, suitable drug doses capable of inducing long-term survival of allografted hepatocytes were identified. In pharmacologically effective doses, rapamycin enhanced cell engraftment by downregulating hepatic expression of selected inflammatory cytokines but profoundly impaired proliferation of transplanted cells, which was necessary for liver repopulation. In contrast, tacrolimus and/or mycophenolate mofetil perturbed neither transplanted cell engraftment nor their proliferation. Therefore, mTOR-dependent extracellular and intracellular mechanisms affected liver replacement with transplanted cells. In conclusion, insights into the biological effects of specific drugs on transplanted cells are critical in identifying suitable immunosuppressive strategies for cell therapy. (HEPATOLOGY 2006;44:410,419.) [source]

Functional characterization of human natural killer cells responding to Mycobacterium bovis bacille Calmette-Guérin

IMMUNOLOGY, Issue 1 2004
Semih Esin
Summary The kinetics of activation and induction of several effector functions of human natural killer (NK) cells in response to Mycobacterium bovis bacille Calmette-Guérin (BCG) were investigated. Owing to the central role of monocytes/macrophages (MM) in the initiation and maintenance of the immune response to pathogens, two different experimental culture conditions were analysed. In the first, monocyte-depleted nylon wool non-adherent (NW) cells from healthy donors were stimulated with autologous MM preinfected with BCG (intracellular BCG). In the second, the NW cells were directly incubated with BCG, which was therefore extracellular. In the presence of MM, CD4+ T lymphocytes were the cell subset mainly expressing the activation marker, CD25, and proliferating with a peak after 7 days of culture. In contrast, in response to extracellular BCG, the peak of the proliferative response was observed after 6 days of stimulation, and CD56+ CD3, cells (NK cells) were the cell subset preferentially involved. Such proliferation of NK cells did not require a prior sensitization to mycobacterial antigens, and appeared to be dependent upon contact between cell populations and bacteria. Following stimulation with extracellular BCG, the majority of interferon-, (IFN-,)-producing cells were NK cells, with a peak IFN-, production at 24,30 hr. Interleukin (IL)-2 and IL-4 were not detectable in NK cells or in CD3+ T lymphocytes at any time tested. IL-12 was not detectable in the culture supernatant of NW cells stimulated with extracellular BCG. Compared to the non-stimulated NW cells, the NW cells incubated for 16,20 hr with BCG induced the highest levels of expression of apoptotic/death marker on the NK-sensitive K562 cell line. BCG also induced expression of the activation marker, CD25, and proliferation, IFN-, production and cytotoxic activity, on negatively selected CD56+ CD3, cells. Altogether, the results of this study demonstrate that extracellular mycobacteria activate several NK-cell functions and suggest a possible alternative mechanism of NK-cell activation as the first line of defence against mycobacterial infections. [source]

Ribosomal DNA sequence analysis of mucosa-associated bacteria in Crohn's disease

INFLAMMATORY BOWEL DISEASES, Issue 6 2004
Tom Prindiville MD
Abstract Background: Enteric bacteria are implicated in the pathogenesis of Crohn's disease (CD); however, no specific causative organisms have been identified. Aims: This study was undertaken to correlate disease activity with changes in intestinal biota in patients with CD. Subjects: Ribosomal DNA analysis was used to explore the composition of the intestinal biota in patients with (1) CD undergoing colonoscopy, (2) CD undergoing surgical resection, and (3) no inflammatory bowel disease. Methods: Primers targeting bacterial 16S ribosomal DNA (rDNA) were used to amplify bacterial DNA associated with active CD lesions, comparable normal tissue from patients with CD, and normal control tissue. Each amplicon was cloned. Seven hundred thirty-nine rDNA clones were sequenced from 16 biopsies from CD patients, 15 surgical samples, and 10 biopsies from normal control patients. Results: Known extracellular or intracellular pathogens were not found. No rDNA sequence, phylogenetic group, or subgroup was consistently associated with CD lesions compared with normal tissues from the same patients. Colonic biopsies from CD-afflicted patients compared with biopsies from normal control subjects had an increase in facultative bacteria; in small bowel, CD patients had an increase in the Ruminococcus gnavus subgroup with a decrease in the Clostridium leptum and Prevotella nigrescens subgroups. However, differences in small bowel may have reflected individual variation rather than disease association. Surgical samples showed differences when compared with biopsy-derived samples. Conclusions: These findings suggest that CD is not caused by invasive pathogens associated specifically with the sites of lesions but that dysbiosis exists in this condition. [source]