Negative occurrence between hippocampal rhythmic slow activity and epileptiform spikes: influence of the striatum

The effects of caudate and septal stimulation on hippocampal activity were studied. Sodium penicillin was intravenously injected in order to induce a steady rate of interictal epileptic activity. Penicillin injection caused significant reduction of the rate of occurrence and duration of hippocampal rhythmic slow activity (RSA). Spontaneous RSA as well as RSA-eliciting caudate and septal stimulation induced a marked inhibition on frequency of epileptiform complexes. Lesions of the medial septal nucleus made it impossible to record RSA or to observe any sort of influence on hippocampal epileptiform activity by caudate stimulation. This result suggests that the septum is important for RSA genesis in the striato-hippocampal pathway or in the reciprocal septo-hippocampal connections.     

Feeding behavior of the cat in response to stimulation of the caudate nucleus

In chronic free-moving cats unilateral, low frequency, long duration stimulation of the caudate nucleus causes a significant reduction of food intake. The effects are greatest at the beginning of the stimulation. On cessation of stimulation the animals take enough food to compensate for that lost during the time of stimulation. Results are discussed either considering interrelationships between striatum and lateral hypothalamus, either regarding recent viewpoints distinguishing two striatal compartments: dorsal or "non limbic" striatal region and ventral or "limbic" striatal region.     

Molten globule monomer to condensed dimer: role of disulfide bonds in platelet factor-4 folding and subunit association.

Platelet factor 4 (PF4) exhibits high affinity for heparin and exists as a tetramer in solution under physiologic conditions. Reduction of the two disulfide bridges in PF4 increases the protein's dissociation constant for heparin approximately 20-fold and shifts the highest apparent aggregation state from tetramer to dimer as evidenced by gel filtration, chemical cross-linking, and 1H-NMR studies. 1H-NMR spectra of reduced PF4 monomers generally show narrower, less dispersed, upfield-shifted NH and alpha H resonances, suggesting the presence of an unfolded monomer state. Reduced PF4 monomer folding, however, is evidenced by the presence of about 12 relatively long-lived backbone NHs and by CD spectra that indicate conservation of overall secondary structure. These data suggest the presence of a molten globule-type state. Urea denaturation shifts this apparent molten globule to a fully unfolded state characterized by more random coil-like resonance shifts. The reduced PF4 dimer state yields NMR and CD data consistent with preservation of tertiary structural folds found for the native species. In this regard, the reduced PF4 folding transition is thermodynamically linked with dimer formation which stabilizes tertiary structure. Monomer-dimer association equilibria for reduced PF4 essentially follow the same pH and salt titration trends as reported previously for native PF4 dimers [Mayo, K. H., & Chen, M. J. (1989) Biochemistry 28, 9469-9478], indicating that that dimer interface is generally conserved in the absence of disulfide constraints. Reduced PF4 tetramers are not apparent under any conditions investigated, suggesting that disulfides are necessary for efficient antiparallel beta-sheet alignment between dimer pairs.     

Modulation by glycine on vascular effects of NMDA:in vivo experimental research

Summary The present study has been carried out to determine if glycine, an allosteric modulator of NMDA receptor, is involved in the vascular effect induced by the activation of the CNS NMDA receptors.Icv NMDA (from 0.01 to 1µg/rat in the 3rd ventricle) caused a significant increase in arterial blood pressure in conscious freely moving rats. Moreover, the hypertension was associated with behavioural modifications (jumping, rearing, teething and running). Glycine pretreatment (1 and 10µg/raticv), significantly increased the NMDA hypertension. Glycine alone did not cause any arterial blood pressure modification while it induced a slight sedation. HA-966 (an antagonist of the glycine site on NMDA receptor) administration (1–10µg/raticv 5 min before glycine) significantly antagonized the glycine effects on NMDA hypertension.Alone HA-966 neither modified arterial blood pressure nor antagonized NMDA hypertension. In conclusion, our investigations confirm NMDA receptor involvement in cardiovascular function and they demonstrate thatin vivo glycine positively modulates NMDA receptors.     

Fidelity of mammalian DNA replication and replicative DNA polymerases.

Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon. In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Cannabinoid Receptor Type 2 as Mediator of Mesenchymal Stromal Cell Immunosuppressive Properties

Mesenchymal stromal cells are non-hematopoietic, multipotent progenitor cells producing cytokines, chemokines, and extracellular matrix proteins that support hematopoietic stem cell survival and engraftment, influence immune effector cell development, maturation, and function, and inhibit alloreactive T-cell responses. The immunosuppressive properties of human mesenchymal stromal cells have attracted much attention from immunologists, stem cell biologists and clinicians.Recently, the presence of the endocannabinoid system in hematopoietic and neural stem cells has been demonstrated. Endocannabinoids, mainly acting through the cannabinoid receptor subtype 2, are able to modulate cytokine release and to act as immunosuppressant when added to activated T lymphocytes.In the present study, we have investigated, through a multidisciplinary approach, the involvement of the endocannabinoids in migration, viability and cytokine release of human mesenchymal stromal cells.We show, for the first time, that cultures of human mesenchymal stromal cells express all of the components of the endocannabinoid system, suggesting a potential role for the cannabinoid CB2 receptor as a mediator of anti-inflammatory properties of human mesenchymal stromal cells, as well as of their survival pathways and their capability to home and migrate towards endocannabinoid sources.     

Understanding the Molecular Determinants Driving the Immunological Specificity of the Protective Pilus 2a Backbone Protein of Group B Streptococcus

The pilus 2a backbone protein (BP-2a) is one of the most structurally and functionally characterized components of a potential vaccine formulation against Group B Streptococcus. It is characterized by six main immunologically distinct allelic variants, each inducing variant-specific protection. To investigate the molecular determinants driving the variant immunogenic specificity of BP-2a, in terms of single residue contributions, we generated six monoclonal antibodies against a specific protein variant based on their capability to recognize the polymerized pili structure on the bacterial surface. Three mAbs were also able to induce complement-dependent opsonophagocytosis killing of live GBS and target the same linear epitope present in the structurally defined and immunodominant domain D3 of the protein. Molecular docking between the modelled scFv antibody sequences and the BP-2a crystal structure revealed the potential role at the binding interface of some non-conserved antigen residues. Mutagenesis analysis confirmed the necessity of a perfect balance between charges, size and polarity at the binding interface to obtain specific binding of mAbs to the protein antigen for a neutralizing response.     

Mapping of Alternaria and Pleospora concentrations in Central Italy using meteorological forecast and neural network estimator

Airborne particles (pollens and fungal spores) are recognized as important causes of allergies and many other pathologies whose main symptoms are usually associated with respiratory problems. In addition, these particles seem to be responsible for clinical symptoms of oculorhinitis and bronchial asthma. Many authors showed how pollen and spore concentrations are critically linked to meteorological conditions, while other studies investigated the possibility to estimate these concentrations through meteorological parameters. So, many different approaches have been proposed, and one of the most sophisticated is based on the use of a complex artificial neural network architecture. Once the neural device is calibrated using simultaneous time series of observed meteorological parameters and airborne biological particles, it is straightforward to use the Neural Network to predict spore concentrations using operational Limited Area Meteorological Model. In a previous work, it has been shown that the MM5 meteorological model developed by National Center for Atmospheric Research and Pennsylvania State University can be coupled with the above-cited neural predictor to provide a good prediction of Alternaria and Pleospora spore in the location of L’Aquila (Central Italy). Following the same approach, this work aims to provide the mapping of spore concentration over a wide area covered by high-resolution meteorological prediction in Central Italy. The complex patterns of fungal spore concentrations in selected areas will be described, and the high temporal variability of such fields will be discussed as well. The possibility to infer useful information from the predicted pattern of spore concentrations is discussed, as an example it appears that for people suffering from allergy to fungal spores is more comfortable to spend summertime close to the east coast of Italian Peninsula respect to the west coast. A further step of this work may easily lead to an operational use of the model for supporting the clinical management of allergies and for establishing a preventive strategy in agriculture to avoid unsafe and useless pollution of atmosphere, crops and fields.     

Functional Toll-like receptor 4 expressed in lactotrophs mediates LPS-induced proliferation in experimental pituitary hyperplasia

Toll like receptor 4 (TLR4) has been characterized for its ability to recognize bacterial endotoxin lipopolysaccharide (LPS). Considering that infections or inflammatory processes might contribute to the progression of pituitary tumors, we analyzed the TLR4 functional role by evaluating the LPS effect on lactotroph proliferation in primary cultures from experimental pituitary tumors, and examined the involvement of PI3K-Akt and NF-κB activation in this effect. In addition, the role of 17β-estradiol as a possible modulator of LPS-induced PRL cell proliferation was further investigated. In estrogen-induced hyperplasic pituitaries, LPS triggered lactotroph cell proliferation. However, endotoxin failed to increase the number of lactotrophs taking up BrdU in normal pituitaries. Moreover, incubation with anti-TLR4 antibody significantly reduced LPS-induced lactotroph proliferation, suggesting a functional role of this receptor. As a sign of TLR4 activation, an LPS challenge increased IL-6 release in normal and tumoral cells. By flow cytometry, TLR4 baseline expression was revealed at the plasma membrane of tumoral lactotrophs, without changes noted in the percentage of double PRL/TLR4 positive cells after LPS stimulus. Increases in TLR4 intracellular expression were detected as well as rises in CD14, p-Akt and NF-κB after an LPS challenge, as assessed by western blotting. The TLR4/PRL and PRL/NF-κB co-localization was also corroborated by immunofluorescence and the involvement of PI3K/Akt signaling in lactotroph proliferation and IL-6 release was revealed through the PI3K inhibitor Ly-294002. In addition, 17β-estradiol attenuated the LPS-evoked increase in tumoral lactotroph proliferation and IL-6 release. Collectively these results demonstrate the presence of functional TLR4 in lactotrophs from estrogen-induced hyperplasic pituitaries, which responded to the proliferative stimulation and IL-6 release induced by LPS through TLR4/CD14, with a contribution of the PI3K-Akt and NF-κB signaling pathways.     

Algistatic effect of a quinonamethide triterpene on Microcystis novacekii

Cyanobacteria proliferation represents a problem to water treatment for human consumption supply. Species of the genus Microcystis, commonly found in continental waters, can bloom and produce microcystine and other toxic compounds associated to human and animal poisoning, depending on the nutrient levels present in the water. The use of natural products to control cyanobacteria blooms is promising since they are less harmful to the environment than compounds obtained by organic syntheses. Triterpenes, which are associated with several biological activities, have been isolated from species of the genus Maytenus (Celastraceae), commonly found in Cerrado regions of Brazil. Tingenone is a pentacyclic triterpene that has cytotoxic properties and induces growth inhibition in some microorganisms. In this study, the effects of tingenone isolated from Maytenus gonoclada at distinct concentrations (e.g., 50, 150, 500, 1,500, and 4,500 μg? L^?1) on the growth rates of Microcystis novacekii was investigated. The algicide activity was verified using M. novacekii cultures growing in ASM1 culture medium at 23 °C and continuous illumination for 96 h. Growth inhibition was monitored by light microscopy and optical density (OD_680 nm). The median effective concentration associated to the M. novacekii inhibition growth induced by tingenone was 12.2 μg? L^?1. The result indicates that tingenone has algicide effect and can be potentially applied in water management for public supply, replacing synthetic algicides.