Neuronal assembly detection and cell membership specification by principal component analysis

In 1949, Donald Hebb postulated that assemblies of synchronously activated neurons are the elementary units of information processing in the brain. Despite being one of the most influential theories in neuroscience, Hebb's cell assembly hypothesis only started to become testable in the past two decades due to technological advances. However, while the technology for the simultaneous recording of large neuronal populations undergoes fast development, there is still a paucity of analytical methods that can properly detect and track the activity of cell assemblies. Here we describe a principal component-based method that is able to (1) identify all cell assemblies present in the neuronal population investigated, (2) determine the number of neurons involved in ensemble activity, (3) specify the precise identity of the neurons pertaining to each cell assembly, and (4) unravel the time course of the individual activity of multiple assemblies. Application of the method to multielectrode recordings of awake and behaving rats revealed that assemblies detected in the cerebral cortex and hippocampus typically contain overlapping neurons. The results indicate that the PCA method presented here is able to properly detect, track and specify neuronal assemblies, irrespective of overlapping membership.     

Biochemical and pharmacological characterization of PhTX-I a new myotoxic phospholipase A2 isolated from Porthidium hyoprora snake venom

This paper reports the biochemical and pharmacological characterization of a new myotoxic PLA(2) (EC 3.1.1.4) called PhTX-I, purified from Porthidium hyoprora venom by one step analytical chromatography reverse phase HPLC. The homogeneity of the PhTX-I fraction and its molecular mass were initially evaluated by SDS-PAGE and confirmed by MALDI-TOF spectrometry, indicating a molecular mass of 14.249Da and constituted of a single polipeptidic chain. Amino acid sequence was determined by "de novo sequencing," in tandem mass spectrometry, belonging to D49-PLA(2) enzyme class and exhibiting high identity (44-90%) with other myotoxics PLA(2) from snake venoms. The enzymatic investigation showed maximal activity at pH 8 and 35-45°C. This activity was dependent on Ca(2+), other cations (Mg(2+), Mn(2+), Cd(2+) and Zn(2+)) reduced notably the enzymatic activity, suggesting that the arrangement of the catalytic site presents an exclusive structure for Ca(2+). Ex vivo, whole venom and PhTX-I PLA(2) caused blockade of the neuromuscular transmission in young chick biventer cervicis preparations similar to other isolated snake venom toxins from the Bothrops genus. In vivo, both induced local myotoxicity and systemic interleukin-6 response upon intramuscular injection, additionally, induced moderate footpad edema. In vitro, both induced low cytotoxicity in skeletal muscle myoblasts, however PhTX-I PLA(2) was able to lyse myotubes.     

Placenta growth factor-1 exerts time-dependent stabilization of adherens junctions following VEGF-induced vascular permeability

Increased vascular permeability is an early event characteristic of tissue ischemia and angiogenesis. Although VEGF family members are potent promoters of endothelial permeability the role of placental growth factor (PlGF) is hotly debated. Here we investigated PlGF isoforms 1 and 2 and present in vitro and in vivo evidence that PlGF-1, but not PlGF-2, can inhibit VEGF-induced permeability but only during a critical window post-VEGF exposure. PlGF-1 promotes VE-cadherin expression via the trans-activating Sp1 and Sp3 interaction with the VE-cadherin promoter and subsequently stabilizes transendothelial junctions, but only after activation of endothelial cells by VEGF. PlGF-1 regulates vascular permeability associated with the rapid localization of VE-cadherin to the plasma membrane and dephosphorylation of tyrosine residues that precedes changes observed in claudin 5 tyrosine phosphorylation and membrane localization. The critical window during which PlGF-1 exerts its effect on VEGF-induced permeability highlights the importance of the translational significance of this work in that PLGF-1 likely serves as an endogenous anti-permeability factor whose effectiveness is limited to a precise time point following vascular injury. Clinical approaches that would pattern nature's approach would thus limit treatments to precise intervals following injury and bring attention to use of agents only during therapeutic windows.     

Structural and Biological Characterization of Two Crotamine Isoforms IV-2 and IV-3 Isolated from the <Emphasis Type="Italic">Crotalus durissus cumanensis</Emphasis> Venom

In this work, we isolated the two new crotamine isoforms from the Crotalus durissus cumanensis rattlesnake venom and its “in vitro” neurotoxic, myotoxic and lethality (DL₅₀) intracerebroventricular (i.c.v.) effects were characterized. These proteins were named IV-2 and IV-3 and were purified by combination of two chromatographic steps on molecular exclusion chromatography on Superdex 75 and reverse phase HPLC (μ-Bondapack C18). The molecular mass of the crotamine isoforms was 4905.96 Da for isoform IV-2 and 4956.97 Da for IV-3 and, as determined by mass spectrometry, and both contained six Cys residues. Enzymatic hydrolysis followed by de novo sequencing by tandem mass spectrometry was used to determine the primary structure of both isoforms. The positions of five sequenced tryptic peptides, including the N-terminal of the isoform IV-2 and four from isoform IV-3 were deduced by comparison with a homologous protein from the crotamine family. The isoforms IV-2 and IV-3 had a sequence of amino acids of 42 amino acid residues IV-2: YKRCHIKGGH CFPKEKLICI PPSSDIGKMD CPWKRKCCKK RS and pI value 9.54 and IV-3: YKQCHKKGGH CFPKEVLICI PPSSDFGKMD CRWKRKCCKK RS with a pI value of 9.54. This protein showed high molecular amino acid sequence identity with other crotamine-like proteins from Crotalus durissus terrificus. These new crotamine isoforms induced potent blockade of neuromuscular transmission in young chicken biventer cervicis preparation and potent myotoxic effect. In mice, both isoforms induced myonecrosis, upon intramuscular or subcutaneous injections. These activities were modulated by the presence of positively charged amino acid residues. The LD₅₀ of isoform IV-2 was 0.07 mg/kg and isoform IV-3 was 0.06 mg/kg the animal weight, by i.c.v. route.     

Assembly and architecture of Escherichia coli divisome proteins FtsA and FtsZ

During Escherichia coli cell division, an intracellular complex of cell division proteins known as the Z-ring assembles at midcell during early division and serves as the site of constriction. While the predominant protein in the Z-ring is the widely conserved tubulin homolog FtsZ, the actin homolog FtsA tethers the Z-ring scaffold to the cytoplasmic membrane by binding to FtsZ. While FtsZ is known to function as a dynamic, polymerized GTPase, the assembly state of its partner, FtsA, and the role of ATP are still unclear. We report that a substitution mutation in the FtsA ATP-binding site impairs ATP hydrolysis, phospholipid vesicle remodeling in vitro, and Z-ring assembly in vivo. We demonstrate by transmission electron microscopy and Förster Resonance Energy Transfer that a truncated FtsA variant, FtsA(ΔMTS) lacking a C-terminal membrane targeting sequence, self assembles into ATP-dependent filaments. These filaments coassemble with FtsZ polymers but are destabilized by unassembled FtsZ. These findings suggest a model wherein ATP binding drives FtsA polymerization and membrane remodeling at the lipid surface, and FtsA polymerization is coregulated with FtsZ polymerization. We conclude that the coordinated assembly of FtsZ and FtsA polymers may serve as a key checkpoint in division that triggers cell wall synthesis and division progression.     

Agrobacterium tumefaciens-mediated transformation of the aromatic shrub Lavandula latifolia

Transgenic plants of the aromatic shrub Lavandula latifolia (Lamiaceae) were produced using Agrobacterium tumefaciens-mediated gene transfer. Leaf and hypocotyl explants from 35–40-day old lavender seedlings were inoculated with the EHA105 strain carrying the nptII gene, as selectable marker, and the reporter gusA gene with an intron. Some of the factors influencing T-DNA transfer to L. latifolia explants were assessed. Optimal transformation rates (6.0 ± 1.6% in three different experiments) were obtained when leaf explants precultured for 1 day on regeneration medium were subcultured on selection medium after a 24 h co-cultivation with Agrobacterium. Evidence for stable integration was obtained by GUS assay, PCR and Southern hybridisation. More than 250 transgenic plants were obtained from 37 independent transformation events. Twenty-four transgenic plants from 7 of those events were successfully established in soil. -glucuronidase activity and kanamycin resistance assays in greenhouse-grown plants from two independent transgenic lines confirmed the stable expression of both gusA and nptII genes two years after the initial transformation. Evidence from PCR data, GUS assays and regeneration in the presence of kanamycin demonstrated a 1:15 Mendelian segregation of both transgenes among seedlings of the T1 progeny of two plants from one transgenic L. latifolia line.     

The Role of Lipotoxicity in Smoke Cardiomyopathy

Background/Aims

Experimental and clinical studies have shown the direct toxic effects of cigarette smoke (CS) on the myocardium, independent of vascular effects. However, the underlying mechanisms are not well known.

Methods

Wistar rats were allocated to control (C) and cigarette smoke (CS) groups. CS rats were exposed to cigarette smoke for 2 months.

Results

After that morphometric, functional and biochemical parameters were measured. The echocardiographic study showed enlargement of the left atria, increase in the left ventricular systolic volume and reduced systolic function. Within the cardiac metabolism, exposure to CS decreased beta hydroxy acyl coenzyme A dehydrogenases and citrate synthases and increased lactate dehydrogenases. Peroxisome proliferator-activated receptor alpha (PPARα) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) were expressed similarly in both groups. CS increased serum lipids and myocardial triacylglycerols (TGs). These data suggest that impairment in fatty acid oxidation and the accumulation of cardiac lipids characterize lipotoxicity. CS group exhibited increased oxidative stress and decreased antioxidant defense. Finally, the myocyte cross-sectional area and active Caspase 3 were increased in the CS group.

Conclusion

The cardiac remodeling that was observed in the CS exposure model may be explained by abnormalities in energy metabolism, including lipotoxicity and oxidative stress.     

Evaluation of genotoxic effects induced by exposure to antineoplastic drugs in lymphocytes and exfoliated buccal cells of oncology nurses and pharmacy employees

The continuous introduction of new antineoplastic drugs and their use as complex mixture emphasize the need to carry out correct health risk assessment. The aim of this study was to evaluate genotoxic effects of antineoplastic drugs in nurses (n=25) and pharmacy technicians (n=5) employed in an oncology hospital. The nurses administered antineoplastic drugs in the day-care hospital (n=12) and in the wards (n=13), and pharmacy technicians prepared the drugs in the central pharmacy. We performed the micronucleus (MN) test with lymphocytes and exfoliated buccal cells and conducted traditional analysis of chromosomal aberrations (CA). Thirty healthy subjects were selected as controls. Monitoring of surface contamination with cyclophosphamide, 5-fluorouracil, ifosfamide, cytarabine, and gemcitabine showed the presence of detectable levels only for cyclophosphamide, 5-fluorouracil and ifosfamide. In addition, we measured the 5-fluorouracil metabolite alpha-F-betaalanine in the urine of all subjects and found significant concentrations only in 3 out of 25 nurses. The micronucleus assay with lymphocytes did not show significant differences between exposed and control groups, while the same test with exfoliated buccal cells found higher values in nurses administering antineoplastic drugs than in pharmacy employees. In the CA analysis, we detected in exposed groups a significant increase (about 2.5-fold) of structural CA, particularly breaks (up to 5.0-fold). Our results confirm the genotoxic effect of antineoplastic drugs in circulating blood lymphocytes. Moreover, in exfoliated buccal cells the data show more consistent genetic damage induced during administration of the antineoplastic drugs than during their preparation. The data also stress the use of this non-invasive sampling, to assess occupational exposure to mixture of chemicals at low doses.     

Inward rectifier potassium channels in plants differ from their animal counterparts in response to voltage and channel modulators

We have investigated the electrophysiological basis of potassium inward rectification of the KAT1 gene product from Arabidopsis thaliana expressed in Xenopus oocytes and of functionally related K⁺ channels in the plasma membrane of guard and root cells from Vicia faba and Zea mays. The whole-cell currents passed by these channels activate, following steps to membrane potentials more negative than –100 mV, with half activation times of tens of milliseconds. This voltage dependence was unaffected by the removal of cytoplasmic magnesium. Consequently, unlike inward rectifier channels of animals, inward rectification of plant potassium channels is an intrinsic property of the channel protein itself. We also found that the activation kinetics of KAT1 were modulated by external pH. Decreasing the pH in the range 8.5 to 4.5 hastened activation and shifted the steady state activation curve by 19 mV per pH unit. This indicates that the activity of these K⁺ channels and the activity of the plasma membrane H⁺-ATPase may not only be coordinated by membrane potential but also by pH. The instantaneous current-voltage relationship, on the other hand, did not depend on pH, indicating that H⁺ do not block the channel. In addition to sensitivity towards protons, the channels showed a high affinity voltage dependent block in the presence of cesium, but were less sensitive to barium. Recordings from membrane patches of KAT1 injected oocytes in symmetric, Mg²⁺-free, 100 mM-K⁺, solutions allowed measurements of the current-voltage relation of single open KAT1 channels with a unitary conductance of 5 pS. We conclude that the inward rectification of the currents mediated by the KAT1 gene product, or the related endogenous channels of plant cells, results from voltage-modulated structural changes within the channel proteins. The voltage-sensing or the gating-structures appear to interact with a titratable acidic residue exposed to the extracellular medium. Correspondence to: R. Hedrich     

Structural and functional organization of the Ska complex, a key component of the kinetochore-microtubule interface

The Ska complex is an essential mitotic component required for accurate cell division in human cells. It is composed of three subunits that function together to establish stable kinetochore-microtubule interactions in concert with the Ndc80 network. We show that the structure of the Ska core complex is a W-shaped dimer of coiled coils, formed by intertwined interactions between Ska1, Ska2, and Ska3. The C-terminal domains of Ska1 and Ska3 protrude at each end of the homodimer, bind microtubules in vitro when connected to the central core, and are essential in vivo. Mutations disrupting the central coiled coil or the dimerization interface result in chromosome congression failure followed by cell death. The Ska complex is thus endowed with bipartite and cooperative tubulin-binding properties at the ends of a 350 ?-long molecule. We discuss how this symmetric architecture might complement and stabilize the Ndc80-microtubule attachments with analogies to the yeast Dam1/DASH complex.