Targeted deletions of complement lectin pathway genes improve outcome in traumatic brain injury, with MASP-2 playing a major role.

The lectin pathway (LP) of complement activation is believed to contribute to brain inflammation. The study aims to identify the key components of the LP contributing to TBI outcome as possible novel pharmacological targets. We compared the long-term neurological deficits and neuropathology of wild-type mice (WT) to that of mice carrying gene deletions of key LP components after experimental TBI. WT or MASP-2 (Masp2-/-), ficolin-A (Fcna-/-), CL-11 (Colec11-/-), MASP-1/3 (Masp1-/-), MBL-C (Mbl2-/-), MBL-A (Mbl1-/-) or MBL-/- (Mbl1-/-/Mbl2-/-) deficient male C57BL/6J mice were used. Mice underwent sham surgery or TBI by controlled cortical impact. The sensorimotor response was evaluated by neuroscore and beam walk tests weekly for 4 weeks. To obtain a comparative analysis of the functional outcome each transgenic line was rated according to a health score calculated on sensorimotor performance. For selected genotypes, brains were harvested 6 weeks after injury for histopathological analysis. MASP-2-/-, MBL-/- and FCN-A-/- mice had better outcome scores compared to WT. Of these, MASP-2-/- mice had the best recovery after TBI, showing reduced sensorimotor deficits (by 33% at 3 weeks and by 36% at 4 weeks). They also showed higher neuronal density in the lesioned cortex with a 31.5% increase compared to WT. Measurement of LP functional activity in plasma from MASP-2-/- mice revealed the absence of LP functional activity using a C4b deposition assay. The LP critically contributes to the post-traumatic inflammatory pathology following TBI with the highest degree of protection achieved through the absence of the LP key enzyme MASP-2, underlining a therapeutic utility of MASP-2 targeting in TBI.

SAFIIRA: A heavy-ion multi-purpose irradiation facility in Brazil.

This work describes the new facility for applied nuclear physics at the University of Sao Paulo, mainly for irradiation of electronic devices. It is a setup composed of a quadrupole doublet for beam focusing/defocusing plus multiple scattering through gold foils to produce low intensity, large-area, and high-uniformity heavy-ion beams from 1H to 107Ag. Beam intensities can be easily adjusted from 102 particles cm2/s to hundreds of nA for an area as large as 2.0 cm2 and uniformity better than 90%. Its irradiation chamber has a high-precision motorized stage, and the system is controlled by a LabViewTM environment, allowing measurement automation. Design considerations and examples of use are presented.

DOPAL derived alpha-synuclein oligomers impair synaptic vesicles physiological function.

Parkinson's disease is a neurodegenerative disorder characterized by the death of dopaminergic neurons and by accumulation of alpha-synuclein (aS) aggregates in the surviving neurons. The dopamine catabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is a highly reactive and toxic molecule that leads to aS oligomerization by covalent modifications to lysine residues. Here we show that DOPAL-induced aS oligomer formation in neurons is associated with damage of synaptic vesicles, and with alterations in the synaptic vesicles pools. To investigate the molecular mechanism that leads to synaptic impairment, we first aimed to characterize the biochemical and biophysical properties of the aS-DOPAL oligomers; heterogeneous ensembles of macromolecules able to permeabilise cholesterol-containing lipid membranes. aS-DOPAL oligomers can induce dopamine leak in an in vitro model of synaptic vesicles and in cellular models. The dopamine released, after conversion to DOPAL in the cytoplasm, could trigger a noxious cycle that further fuels the formation of aS-DOPAL oligomers, inducing neurodegeneration.

Molecular dynamics simulations of solutions at constant chemical potential.

Molecular dynamics studies of chemical processes in solution are of great value in a wide spectrum of applications, which range from nano-technology to pharmaceutical chemistry. However, these calculations are affected by severe finite-size effects, such as the solution being depleted as the chemical process proceeds, which influence the outcome of the simulations. To overcome these limitations, one must allow the system to exchange molecules with a macroscopic reservoir, thus sampling a grand-canonical ensemble. Despite the fact that different remedies have been proposed, this still represents a key challenge in molecular simulations. In the present work, we propose the Constant Chemical Potential Molecular Dynamics (CµMD) method, which introduces an external force that controls the environment of the chemical process of interest. This external force, drawing molecules from a finite reservoir, maintains the chemical potential constant in the region where the process takes place. We have applied the CµMD method to the paradigmatic case of urea crystallization in aqueous solution. As a result, we have been able to study crystal growth dynamics under constant supersaturation conditions and to extract growth rates and free-energy barriers.

Stress and corticosterone increase the readily releasable pool of glutamate vesicles in synaptic terminals of prefrontal and frontal cortex.

Stress and glucocorticoids alter glutamatergic transmission, and the outcome of stress may range from plasticity enhancing effects to noxious, maladaptive changes. We have previously demonstrated that acute stress rapidly increases glutamate release in prefrontal and frontal cortex via glucocorticoid receptor and accumulation of presynaptic SNARE complex. Here we compared the ex vivo effects of acute stress on glutamate release with those of in vitro application of corticosterone, to analyze whether acute effect of stress on glutamatergic transmission is mediated by local synaptic action of corticosterone. We found that acute stress increases both the readily releasable pool (RRP) of vesicles and depolarization-evoked glutamate release, while application in vitro of corticosterone rapidly increases the RRP, an effect dependent on synaptic receptors for the hormone, but does not induce glutamate release for up to 20 min. These findings indicate that corticosterone mediates the enhancement of glutamate release induced by acute stress, and the rapid non-genomic action of the hormone is necessary but not sufficient for this effect.

Target normal sheath acceleration analytical modeling, comparative study and developments.

Ultra-intense laser interaction with solid targets appears to be an extremely promising technique to accelerate ions up to several MeV, producing beams that exhibit interesting properties for many foreseen applications. Nowadays, most of all the published experimental results can be theoretically explained in the framework of the target normal sheath acceleration (TNSA) mechanism proposed by Wilks et al. [Phys. Plasmas 8(2), 542 (2001)]. As an alternative to numerical simulation various analytical or semi-analytical TNSA models have been published in the latest years, each of them trying to provide predictions for some of the ion beam features, given the initial laser and target parameters. However, the problem of developing a reliable model for the TNSA process is still open, which is why the purpose of this work is to enlighten the present situation of TNSA modeling and experimental results, by means of a quantitative comparison between measurements and theoretical predictions of the maximum ion energy. Moreover, in the light of such an analysis, some indications for the future development of the model proposed by Passoni and Lontano [Phys. Plasmas 13(4), 042102 (2006)] are then presented.

Neuroprotective effect of C1-inhibitor following traumatic brain injury in mice.

BACKGROUND: The goal of the study was to evaluate the effects of Cl-inhibitor (C1-INH), an endogenous glycoprotein endowed with multiple anti-inflammatory actions, on cognitive and histological outcome following controlled cortical impact (CCI) brain injury. METHODS: Male C57B1/6 mice (n=48) were subjected to CCI brain injury. After brain injury, animals randomly received an intravenous infusion of either C1-INH (15 U either at 10 minutes or 1 hour postinjury) or saline (equal volume, 150 microl at 10 min postinjury). Uninjured control mice received identical surgery and saline injection without brain injury. Cognitive function was evaluated at 4 weeks postinjury using the Morris Water Maze. Mice were subsequently sacrificed, the brains were frozen and serial sections were cut. Traumatic brain lesion was assessed by dividing the area of the ipsilateral hemisphere for the area of the contralateral one at the level of the injured area of the brain. FINDINGS: Brain-injured mice receiving C1-INH at 10 min postinjury showed attenuated cognitive dysfunction compared to brain-injured mice receiving saline (p < 0.01). These mice also showed a significantly reduced traumatic brain lesion compared to mice receiving saline (p < 0.01). Mice receiving C1-INH at 1 hour post injury did not show a significant improvement in either cognitive or histological outcome. Conclusions Our results suggest that administration of C1-INH at 10 minutes postinjury attenuates cognitive deficits and histological damage associated with traumatic brain injury.

Effect of traumatic brain injury on cognitive function in mice lacking p55 and p75 tumor necrosis factor receptors.

BACKGROUND: Tumor necrosis factor (TNF)-alpha has been suggested to play both a deleterious and beneficial role in neurobehavioral dysfunction and recovery following traumatic brain injury (TBI). The goal of this study was to evaluate the specific role of tumor necrosis factor (TNF) receptors p55 and p75 in mediating cognitive outcome following controlled cortical impact (CCI) brain injury by comparing post-traumatic cognitive function in mice with genetically engineered deletion of the gene for either p55 (-/-) or p75 (-/-) receptors. METHOD: Male C57B1/6 mice (WT, n=29), and mice genetically engineered to delete p55 TNF (p55 (-/-), n=8) or p75 TNF (p75 (-/-), n=23) receptors were used. They were anesthetized with intraperitoneal (i.p.) administration of sodium pentobarbital (65 mg/kg) and subjected to CCI brain injury of moderate severity. Sham-injured control mice were anesthetized and surgically prepared similarly but they received no impact. Assessment of mRNA expression of inflammatory, proapoptotic and antiapoptotic genes was done by real time-polymerase chain reaction (RT-PCR). Cognitive outcome was evaluated at 4 weeks postinjury using the Morris water maze (MWM). FINDINGS: mRNA expression of inflammatory, proapoptotic and antiapoptotic genes prior to TBI did not reveal any baseline difference between p55 and p75 (-/-) mice. WT mice showed greater baseline expression of inflammatory genes. The learning ability of p55 (-/-) brain-injured mice was significantly better than that observed in p75 (-/-) brain-injured mice (p < 0.05). Cognitive learning in WT control mice fell between the p55 (-/-) and p75 (-/-) mice. CONCLUSIONS: These data suggest that TNF-alpha may both exacerbate cognitive dysfunction via p55 receptor and attenuate it via p75 receptor.

Atomic force microscopy characterization of Xenopus laevis oocyte plasma membrane.

We used atomic force microscopy (AFM) to characterize the plasma membrane of Xenopus laevis oocytes. The samples were prepared according to novel protocols, which allowed the investigation of the extra- and intracellular sides of the membrane, both of which showed sparsely distributed spherical-like protrusions. Regions with comparably sized and densely packed structures arranged in an orderly manner were visualized and dimensionally characterized. In particular, two different arrangements, hexagonal and square packing, were recognizable in ordered regions. The lateral dimension of structures visualized on the external side had a normal distribution centered on 25.5 +/- 0.3 nm (mean value +/- SE), whereas that on the intracellular side showed a normal distribution centered on 30.2 +/- 0.8 nm. The height of the protrusions was 2-5 nm on the external side and 1-3 nm on the intracellular side. The mean number of structures on the external and intracellular sides of the plasma membrane was about 1000 microm(-2) and 850 microm(-2) respectively. Trypsin treatment greatly decreased the size of the membrane protrusions, thus confirming the proteic nature of the structures. These results show that AFM is a useful tool for structural characterization of proteins in a native eukaryotic membrane.

Atomic force microscopy imaging of actin cortical cytoskeleton of Xenopus laevis oocyte.

In this study we report an atomic force microscopy (AFM) investigation of the actin cortical cytoskeleton of Xenopus laevis oocytes. Samples consisted of inside-out orientated plasma membrane patches of X. laevis oocytes with overhanging cytoplasmic material. They were spread on a freshly cleaved mica surface, subsequently treated with Triton X-100 detergent and chemically fixed. The presence of actin fibres in oocyte patches was proved by fluorescence microscopy imaging. Contact mode AFM imaging was performed in air in constant force conditions. Reproducible high-resolution AFM images of a filamentous structure were obtained. The filamentous structure was identified as an actin cortical cytoskeleton, investigating its disaggregation induced by cytochalasin D treatment. The thinnest fibres showed a height of 7 nm in accordance with the diameter of a single actin microfilament. The results suggest that AFM imaging can be used for the high-resolution study of the actin cortical cytoskeleton of the X. laevis oocyte and its modifications mediated by the action of drugs and toxins.

Role of the conserved glutamine 291 in the rat gamma-aminobutyric acid transporter rGAT-1.

We investigated the role of the Q291 glutamine residue in the functioning of the rat gamma-aminobutyric acid (GABA) transporter GAT-1. Q291 mutants cannot transport GABA or give rise to transient, leak and transport-coupled currents even though they are targeted to the plasma membrane. Coexpression experiments of wild-type and Q291 mutants suggest that GAT-1 is a functional monomer though it requires oligomeric assembly for membrane insertion. We determined the accessibility of Q291 by investigating the impact of impermeant sulfhydryl reagents on cysteine residues engineered in close proximity to Q291. The effect of these reagents indicates that Q291 faces the external aqueous milieu. The introduction of a steric hindrance close to Q291 by means of [2-(trimethylammonium)ethyl] methanethiosulfonate bromide modification of C74A/T290C altered the affinity of the mutant for cations. Taken together, these results suggest that this irreplaceable residue is involved in the interaction with sodium or in maintaining the cation accessibility to the transporter.

Adhesion and proliferation of fibroblasts on cluster-assembled nanostructured carbon films: the role of surface morphology.

We have investigated the influence on adhesion and proliferation of NIH 3T3 fibroblasts of the surface morphology of cluster assembled carbon films deposited by Supersonic Cluster Beam Deposition. Nanostructured carbon films exhibit a multi-scale morphology, which resembles the surface structure of the extracellular matrix, and possess a high specific area, while being relatively smooth at all scales. Correlations between measured morphological parameters and adaptive cell response have been brought out. High specific area and smoothness appear to conceivably favour both the early attachment of plated cells and the long-term survival of adherent cells. Moreover, nano-structured carbon films affect the cells morphology as well as the extension and the number of the focal contacts.

Impaired central stress-induced release of noradrenaline in rats with heart failure: a microdialysis study.

Sympathetic hyperactivity in rats with heart failure is associated with increased extracellular noradrenaline in the hypothalamic paraventricular nucleus at rest. However, it is unknown how this nucleus responds to stressful stimuli. In the present study we therefore examined the basal and stress-induced release of noradrenaline in the paraventricular nucleus of conscious Sprague-Dawley rats with heart failure measured by in vivo microdialysis. Basal noradrenaline concentration in the paraventricular nucleus of rats with heart failure was more than double that in sham-operated controls. Immobilization stress decreases noradrenaline levels in the paraventricular nucleus of rats with heart failure to 57% of baseline, while it increased in sham-operated controls to 228%. However, serum corticosterone was similarly elevated at 30 and 90 min post-stress in both experimental groups. We have shown that heart failure causes an impairment of the central noradrenergic system's response to acute sympatho-excitation but does not affect the hypothalamo-pituitary-adrenocortical response.

High glucose causes apoptosis in cultured human pancreatic islets of Langerhans: a potential role for regulation of specific Bcl family genes toward an apoptotic cell death program.

Type 2 diabetes is characterized by insulin resistance and inadequate insulin secretion. In the advanced stages of the disease, beta-cell dysfunction worsens and insulin therapy may be necessary to achieve satisfactory metabolic control. Studies in autopsies found decreased beta-cell mass in pancreas of people with type 2 diabetes. Apoptosis, a constitutive program of cell death modulated by the Bcl family genes, has been implicated in loss of beta-cells in animal models of type 2 diabetes. In this study, we compared the effect of 5 days' culture in high glucose concentration (16.7 mmol/l) versus normal glucose levels (5.5 mmol/l) or hyperosmolar control (mannitol 11 mmol/l plus glucose 5 mmol/l) on the survival of human pancreatic islets. Apoptosis, analyzed by flow cytometry and electron and immunofluorescence microscopy, was increased in islets cultured in high glucose (HG5) as compared with normal glucose (NG5) or hyperosmolar control (NG5+MAN5). We also analyzed by reverse transcriptase-polymerase chain reaction and Western blotting the expression of the Bcl family genes in human islets cultured in normal glucose or high glucose. The antiapoptotic gene Bcl-2 was unaffected by glucose change, whereas Bcl-xl was reduced upon treatment with HG5. On the other hand, proapoptotic genes Bad, Bid, and Bik were overexpressed in the islets maintained in HG5. To define the pancreatic localization of Bcl proteins, we performed confocal immunofluorescence analysis on human pancreas. Bad and Bid were specifically expressed in beta-cells, and Bid was also expressed, although at low levels, in the exocrine pancreas. Bik and Bcl-xl were expressed in other endocrine islet cells as well as in the exocrine pancreas. These data suggest that in human islets, high glucose may modulate the balance of proapoptotic and antiapoptotic Bcl proteins toward apoptosis, thus favoring beta-cell death.

Three kinds of currents in the canine betaine-GABA transporter BGT-1 expressed in Xenopus laevis oocytes.

The cloned canine betaine-GABA cotransporter BGT-1 has been heterologously expressed in Xenopus laevis oocytes in order to characterize its electrophysiological properties. Voltage-clamp experiments on transfected oocytes reveal the presence of three types of membrane current which are absent in non-injected oocytes: (i) an organic substrate-independent current (uncoupled current); (ii) a transport-associated current, seen upon addition of betaine or GABA; (iii) presteady-state currents induced by voltage changes. The three kinds of current are analogous to those reported in structurally similar cotransporters. The transport-associated current is strictly dependent on the presence of Na(+). The good correlation between the amount of charge underlying the presteady-state currents and the transport-associated current indicates that both processes are due to the activity of the transporter.

Inflammatory cytokines and related genes are induced in the rat hippocampus by limbic status epilepticus.

Limbic status epilepticus was induced in rats by unilateral 60-min electrical stimulation of the CA3 region of the ventral hippocampus. As assessed by RT-PCR followed by Southern blot analysis, transcripts of interleukin-1beta, interleukin-6, interleukin-1 receptor antagonist and inducible nitric oxide synthase were significantly increased 2 h after status epilepticus in the stimulated hippocampus. Induction was maximal at 6 h for interleukin-1beta (445%), interleukin-6 (405%) and tumour necrosis factor-alpha (264%) and at 24 h for interleukin-1 receptor antagonist (494%) and inducible nitric oxide synthase (432%). In rats with spontaneous seizures (60 days after status epilepticus), interleukin-1beta mRNA was still higher than controls (241%). Immunocytochemical staining of interleukin-1beta, interleukin-6 and tumour necrosis factor-alpha was enhanced in glia with a time-course similar to that of the respective transcripts. Sixty days after status epilepticus, interleukin-1beta immunoreactivity was increased exclusively in neurons in one third of the animals. Multiple intracerebroventricular injections of interleukin-1 receptor antagonist (0.5 microg/3 microL) significantly decreased the severity of behavioural convulsions during electrical stimulation and selectively reduced tumour necrosis factor-alpha content in the hippocampus measured 18 h after status epilepticus. Thus, the induction of spontaneously recurring seizures in rats involves the activation of inflammatory cytokines and related pro- and anti-inflammatory genes in the hippocampus. These changes may play an active role in hyperexcitability of the epileptic tissue.

The GLT-1 and GLAST glutamate transporters are expressed on morphologically distinct astrocytes and regulated by neuronal activity in primary hippocampal cocultures.

The GLT-1 and GLAST astroglial transporters are the glutamate transporters mainly involved in maintaining physiological extracellular glutamate concentrations. Defects in neurotransmitter glutamate transport may represent an important component of glutamate-induced neurodegenerative disorders (such as amyotrophic lateral sclerosis) and CNS insults (ischemia and epilepsy). We characterized the protein expression of GLT-1 and GLAST in primary astrocyte-neuron cocultures derived from rat hippocampal tissues during neuron differentiation/maturation. GLT-1 and GLAST are expressed by morphologically distinct glial fibrillary acidic protein-positive astrocytes, and their expression correlates with the status of neuron differentiation/maturation and activity. Up-regulation of the transporters paralleled the content of the synaptophysin synaptic vesicle marker p38, and down-regulation was a consequence of glutamate-induced neuronal death or the reduction of synaptic activity. Finally, soluble factors in neuronal-conditioned media prevented the down-regulation of the GLT-1 and GLAST proteins. Although other mechanisms may participate in regulating GLT-1 and GLAST in the CNS, our data indicate that soluble factors dependent on neuronal activity play a major regulating role in hippocampal cocultures.

Mammalian LIN-7 PDZ proteins associate with beta-catenin at the cell-cell junctions of epithelia and neurons.

The heterotrimeric PDZ complex containing LIN-2, LIN-7 and LIN-10 is known to be involved in the organization of epithelial and neuronal junctions in Caenorhabditis elegans and mammals. We report here that mammalian LIN-7 PDZ proteins form a complex with cadherin and beta-catenin in epithelia and neurons. The association of LIN-7 with cadherin and beta-catenin is Ca(2+) dependent and is mediated by the direct binding of LIN-7 to the C-terminal PDZ target sequence of beta-catenin, as demonstrated by means of co-immunoprecipitation experiments and in vitro binding assays with the recombinant glutathione S-transferase:LIN-7A. The presence of beta-catenin at the junction is required in order to relocate LIN-7 from the cytosol to cadherin-mediated adhesions, thus indicating that LIN-7 junctional recruitment is beta-catenin dependent and that one functional role of the binding is to localize LIN-7. Moreover, when LIN-7 is present at the beta-catenin-containing junctions, it determines the accumulation of binding partners, thus suggesting the mechanism by which beta-catenin mediates the organization of the junctional domain.