La grotte Marcel Clouet à Cognac (Charente)

The Marcel Clouet Cave in Cognac (Charente) is a small cavity, more a shelter than a true cave, in the Cretaceous limestone cliffs along the Antenne, a tributary of the Charente River. The site suffered from a number of clandestine excavations before the work of C. Burnez, who was then followed by one of us (A.D.). The material recovered in stratigraphic context represents both Middle and Upper Paleolithic. The former is an example of Mousterian of Acheulian Tradition (MTA), while the latter includes Aurignacian (probably early) and Perigordian (probably Châtelperronian Gravettian), as well as some Solutrean objects recovered outside of stratigraphic context. It is important to note that this is one of only a few Charentian rockshelter sites, which has yielded an example of MTA.     

Stressful preconditioning and HSP70 overexpression attenuate proteotoxicity of cellular ATP depletion

Rat H9c2 myoblasts were preconditioned by heat or metabolic stress followed by recovery under normal conditions. Cells were then subjected to severe ATP depletion, and stress-associated proteotoxicity was assessed on 1) the increase in a Triton X-100-insoluble component of total cellular protein and 2) the rate of inactivation and insolubilization of transfected luciferase with cytoplasmic or nuclear localization. Both heat and metabolic preconditioning elevated the intracellular heat shock protein 70 (HSP70) level and reduced cell death after sustained ATP depletion without affecting the rate and extent of ATP decrease. Each preconditioning attenuated the stress-induced insolubility among total cellular protein as well as the inactivation and insolubilization of cytoplasmic and nuclear luciferase. Transient overexpression of human HSP70 in cells also attenuated both the cytotoxic and proteotoxic effects of ATP depletion. Quercetin, a blocker of stress-responsive HSP expression, abolished the effects of stressful preconditioning but did not influence the effects of overexpressed HSP70. Analyses of the cellular fractions revealed that both the stress-preconditioned and HSP70-overexpressing cells retain the soluble pool of HSP70 longer during ATP depletion. Larger amounts of other proteins coimmunoprecipitated with excess HSP70 compared with control cells deprived of ATP. This is the first demonstration of positive correlation between chaperone activity within cells and their viability in the context of ischemia-like stress.     

Reactive oxygen species in the elongation zone of maize leaves are necessary for leaf extension

The production and role of reactive oxygen species (ROS) in the expanding zone of maize (Zea mays) leaf blades were investigated. ROS release along the leaf blade was evaluated by embedding intact seedlings in 2',7'-dichlorofluorescein-containing agar and examining the distribution of 2',7'-dichlorofluorescein fluorescence along leaf 4, which was exposed by removing the outer leaves before embedding the seedling. Fluorescence was high in the expanding region, becoming practically non-detectable beyond 65 mm from the ligule, indicating high ROS production in the expansion zone. Segments obtained from the elongation zone of leaf 4 were used to assess the role of ROS in leaf elongation. The distribution of cerium perhydroxide deposits in electron micrographs indicated hydrogen peroxide (H(2)O(2)) presence in the apoplast. 2',7'-Dichlorofluorescein fluorescence and apoplastic H(2)O(2) accumulation were inhibited with diphenyleneiodonium (DPI), which also inhibited O*(2)(-) generation, suggesting a flavin-containing enzyme activity such as NADPH oxidase was involved in ROS production. Segments from the elongation zone incubated in water grew 8% in 2 h. KI treatments, which scavenged H(2)O(2) but did not inhibit O*(2)(-) production, did not modify growth. DPI significantly inhibited segment elongation, and the addition of H(2)O(2) (50 or 500 microM) to the incubation medium partially reverted the inhibition caused by DPI. These results indicate that a certain concentration of H(2)O(2) is necessary for leaf elongation, but it could not be distinguished whether H(2)O(2), or other ROS, are the actual active agents.     

Crystal doping aided by rapid expansion of supercritical solutions

The purpose of this study was to test the utility of rapid expansion of supercritical solution (RESS) based cocrystallizations in inducing polymorph conversion and crystal disruption of chlorpropamide (CPD). CPD crystals were recrystallized by the RESS process utilizing supercritical carbon dioxide as the solvent. The supercritical region investigated for solute extraction ranged from 45 to 100°C and 2000 to 8000 psi. While pure solute recrystallization formed stage I of these studies, stage II involved recrystallization of CPD in the presence of urea (model impurity). The composition, morphology, and crystallinity of the particles thus produced were characterized utilizing techniques such as microscopy, thermal analysis, x-ray powder diffractometry, and high-performance liquid chromatography. Also, comparative evaluation between RESS and evaporative crystallization from liquid solvents was performed. RESS recrystallizations of commercially available CPD (form A) resulted in polymorph conversion to metastable forms C and V, depending on the temperature and pressure of the recrystallizing solvent. Cocrystallization studies revealed the formation of eutectic mixtures and solid solutions of CPD+urea. Formation of the solid solutions resulted in the crystal disruption of CPD and subsequent amorphous conversion at urea levels higher than 40% wt/wt. Consistent with these results were the reductions in melting point (up to 9°C) and in the ΔH_fvalues of CPD (up to 50%). Scanning electron microscopy revealed a particle size reduction of up to an order of magnitude upon RESS processing. Unlike RESS, recrystallizations from liquid organic solvents lacked the ability to affect polymorphic conversions. Also, the incorporation of urea into the lattice of CPD was found to be inadequate. In providing the ability to control both the particle and crystal morphologies of active pharmaceutical ingredients, RESS proved potentially advantageous to crystal engineering. Rapid crystallization kinetics were found vital in making RESS-based doping superior to conventional solvent-based cocrystallizations.     

Heterogeneous amyloid-formed ion channels as a common cytotoxic mechanism: implications for therapeutic strategies against amyloidosis

The amyloidoses consist of human and animal chronic, progressive, and sometimes fatal diseases that are characterized by the deposition of insoluble proteinaceous amyloid fibrils in various tissues. Despite the biochemical diversity of amyloids, they share certain properties. The amphipathic and the charged nature of many amyloid-forming peptides point to their intrinsic ability to form diverse beta-sheet-based aggregates and channel types in negatively charged membranes. We hypothesize that the formation of heterogeneous channels represents a common cytotoxic mechanism that accentuates the changes in the signal transduction that underlie amyloid-induced cell malfunction. One group of amyloid-forming peptides that could mediate their action via the formation of heterogeneous channels includes the extensively examined prions and amyloid beta protein that are associated with conformational neurodegenerative diseases. The aim of this study is to examine heterogeneous channels formed in bilayers with amyloid-forming peptides as a common mechanism of malfunction of signal transduction. The observed amyloid-formed channel types include the following. (1) Natriuretic peptides: (i) 68-pS H2O2- and Ba2+-sensitive channel with fast kinetics. The fast channel had three modes (spike mode, burst mode, and open mode), which differ in their kinetics but not in their conductance properties; (ii) a 273-pS inactivating large conductance channel; and (iii) a 160-pS transiently activated channel. (2) Prions: (i) a 140-pS GSSG- and TEA-sensitive channel with fast kinetics; (ii) a 41-pS dithiothreitol (DTT)-sensitive channel with slow kinetics; (iii) a 900 to 1444-pS large channel. (3) Amyloid beta protein: (i) a 17 to 63-pS AbetaP[1-40]-formed "bursting" fast cation channel, (ii) the AbetaP[1-40]-formed "spiky" fast cation channel with a similar kinetics to the "bursting" fast channel except for the absence of the long intraburst closures, (iii) 275-pS AbetaP[1-40]-formed medium conductance channel, and (iv) 589- to 704-pS AbetaP[1-40]-formed inactivating large conductance channel. This heterogeneity is one of the most common features of these charged cytotoxic amyloid-formed channels, reflecting these channels' ability to modify multiple cellular functions. Although the diversity of these aggregated-peptide-formed channels may indicate that a stochastic mechanism governs their formation, the fact that certain channel types are often observed point to preferential channel protein conformations. In addition, the fact that other amyloids have similar structural properties (e.g. hydrophobicity, charged residues, and beta-structural linkages, suggests that, despite the intrinsic ability to form diverse conformations, certain conformations and, hence, certain channel types could be a common pathologic conformation among these amyloid-forming peptides. It is concluded that conformation-based channel diversity is an important mechanism for enhancing the toxicity of amyloid-forming peptides. The cytotoxic nature of these self-associated beta-based protein channels suggests that under normal physiological conditions cells employ well-evolved protective mechanisms against seeding and/or propagation of channel-forming peptides; for example, (a) compartmentalization of these peptides as membrane bound in internal vesicles and/or (b) degradation of these peptides by enzymes. The pharmacological diversity of the amyloid-forming channels implies that multiple therapeutic interventions may be necessary for blocking and reversing heterogeneous channel formations and preventing their associated diseases.     

Ixodid ticks feeding on humans in South Africa: with notes on preferred hosts,geographic distribution,seasonal occurrence and transmission of pathogens

This paper records the identities of 558 ixodid ticks feeding on 194 humans in South Africa. These ticks belonged to 20 species in six genera and those most frequently encountered were Amblyomma hebraeum, Haemaphysalis leachi, Hyalomma marginatum rufipes, Hyalomma truncatum, Rhipicephalus appendiculatus, Rhipicephalus gertrudae and Rhipicephalus simus. With the exception of the larvae of R. appendiculatus, the incidents of these ticks feeding on humans correlated well with their seasonal occurrences on preferred hosts. Ticks were also collected at monthly intervals, for 14 consecutive months, from the clothing of a game-guard providing protection for field-workers engaged in the collection, by means of flannel strips, of free-living ticks from the vegetation of four localities in the southern region of the Kruger National Park. In addition, with the exception of 3 months when the particular worker was absent, ticks that fed on one of the field-workers were collected over the same 14-month period. A total of 54,429 free-living ticks belonging to 14 species and six genera were collected from the vegetation at the four localities during this time and 3751 ticks belonging to 11 species and six genera from the clothing of the game-guard. The larvae of A. hebraeum and Boophilus decoloratus were the most numerous of the immature ticks, and H. leachi and R. simus of the adults on both the vegetation and the guard’s clothing. Ticks fed on the field-worker on six occasions and 14 were collected, all of which were A. hebraeum larvae. This revised version was published online in July 2006 with corrections to the Cover Date.     

Basolateral Cl- channels in the larval bullfrog skin epithelium

The addition of 150 U/ml nystatin to the mucosal surface of isolated skin from larval bullfrogs increases apical membrane permeability and allows a voltage clamp to be applied to the basolateral membrane. With identical Ringer's solutions bathing either side of the tissue the short-circuit current (I(SC)) averaged 7.60+/-0.78 micro A/cm2, and this current could be increased or decreased by imposing a Cl- concentration gradient. Fluctuation analysis of the I(SC) gave power spectra that could be fit with low- and high-frequency Lorentzian functions having corner frequencies of 1.48+/-0.06 Hz and 48.5+/-11.4 Hz, respectively. The Lorentzian plateau was minimal at the lowest I(SC) and increased as the I(SC) became greater in the positive or negative direction. Current-voltage plots with identical Ringer's on either side of the tissue showed a pattern of outward rectification. Cell attached patches of cells isolated from the skin with collagenase-trypsin treatment showed spontaneous channel activity with a conductance of 20.9 pS at a pipette potential, -Vp=20 mV. Current-voltage plots of single channels showed a similar pattern of rectification to that of the intact skin, and partial replacement of Cl- by gluconate in the pipette solution shifted the reversal potential from zero to about 40 mV, which is close to the expected shift of the reversal potential of the chloride current through a Cl- selective ion channel. These results suggest that the basolateral Cl- conductance of the larval skin is mediated by a channel with properties that resemble a volume-sensing outward-rectifier anion channel that has been described in a variety of cell types     

Penicillin-binding proteins in Streptococcus agalactiae: a novel mechanism for evasion of immune clearance

Group B streptococci (GBS) remain the most significant bacterial pathogen causing neonatal sepsis, pneumonia and meningitis in the USA despite CDC-recommended chemoprophylaxis strategies for preventing infection. To cause infection pathogens such as GBS must evade recognition and clearance by the host's immune system. Strategies for avoidance of opsonization and phagocytic killing include elaboration of antiopsonophagocytic capsules and surface proteins. During screening for mutants of GBS that were attenuated for virulence in a neonatal rat sepsis model, we identified a mutant with a transposon insertion in the ponA gene. ponA encodes an extra-cytoplasmic penicillin-binding protein PBP1a, a newly identified virulence trait for GBS that promotes resistance to phagocytic killing independent of capsular polysaccharide. Complementation analysis in vivo and in vitro confirmed that the altered phenotypes observed in the mutant were due to the transposon insertion in ponA. Deletion of PBP1a does not affect C3 deposition on GBS suggesting that mechanism by which PBP1a protects GBS from phagocytic killing is distinct from the antiopsonic activity of capsular polysaccharide. This is the first report describing expression of an antiphagocytic surface protein by GBS and represents a novel mechanism for evasion of immune recognition and clearance that may explain the decreased virulence observed in Gram-positive bacterial species for penicillin-binding protein mutants.     

Water permeability and mechanical strength of polyunsaturated lipid bilayers

Micropipette aspiration was used to test mechanical strength and water permeability of giant-fluid bilayer vesicles composed of polyunsaturated phosphatidylcholine PC lipids. Eight synthetic-diacyl PCs were chosen with 18 carbon chains and degrees of unsaturation that ranged from one double bond (C18:0/1, C18:1/0) to six double bonds per PC molecule (diC18:3). Produced by increasing pipette pressurization, membrane tensions for lysis of single vesicles at 21 degrees C ranged from approximately 9 to 10 mN/m for mono- and dimono-unsaturated PCs (18:0/1, 18:1/0, and diC18:1) but dropped abruptly to approximately 5 mN/m when one or both PC chains contained two cis-double bonds (C18:0/2 and diC18:2) and even lower approximately 3 mN/m for diC18:3. Driven by osmotic filtration following transfer of individual vesicles to a hypertonic environment, the apparent coefficient for water permeability at 21 degrees C varied modestly in a range from approximately 30 to 40 microm/s for mono- and dimono-unsaturated PCs. However, with two or more cis-double bonds in a chain, the apparent permeability rose to approximately 50 microm/s for C18:0/2, then strikingly to approximately 90 microm/s for diC18:2 and approximately 150 microm/s for diC18:3. The measurements of water permeability were found to scale exponentially with the reduced temperatures reported for these lipids in the literature. The correlation supports the concept that increase in free volume acquired in thermal expansion above the main gel-liquid crystal transition of a bilayer is a major factor in water transport. Taken together, the prominent changes in lysis tension and water permeability indicate that major changes occur in chain packing and cohesive interactions when two or more cis-double bonds alternate with saturated bonds along a chain.