Understanding Bile Micelle Chiral Interactions

Bile salts are known to aggregate into micelles in biological systems; however, the fundamental structure and dynamics of bile molecule micelle formation are poorly understood. Previous studies have established that the bile salt cholate is capable of performing chirally selective micellar electrokinetic capillary chromatography (MEKC) separations of model racemic binaphthyl compounds 1,1¿-binaphthyl-2,2¿-diyl hydrogen phosphate (R,S-BNDHP) and 1,1¿-bi-2-naphthol (R,S-BN). Nuclear magnetic resonance (NMR) has been established as a complementary technique for understanding chiral selectivity and micelle formation events based on changes in proton chemical shifts of the probe molecules BNDHP and BN as well as of cholate. This work investigated the effects of the probe molecule, the alkali cation identity and temperature on cholate micelle aggregation and MEKC separations of R,S-BN and R,S-BNDHP. The probe molecule was found to mediate micelle formation by MEKC and proton NMR. A low (0.1 mM) concentration of probe was found to have minimal effects on micellization events detected by proton NMR while higher probe concentration (2.5 mM) was found to mediate micellization causing micellization events to occur at lower cholate concentrations. This work also investigated the effects of alkali counterion on chiral separation. Generally, counterions with larger crystal cationic radius were found to cause greater chiral separation power. NMR data suggest that protons near the surface of the cholate micelle are most sensitive to the cation identity, suggesting a model of improved separation based on the cation sterically inhibiting binding of one isomer. Finally, the effect of temperature on MEKC separation was investigated. Separation power of R,S-BN and R,S-BNDHP appeared to increase linearly with temperature for 22.0 mM to 50.0 mM pH 12.0 cholate. In total, these results indicate that cholate aggregation is dependent on multiple conditions. Understanding the roles that these factors play in influencing cholate micellization can inform better separation in MEKC.

The strategies of the Theileria parasite: a new twist in host-pathogen interactions

Theileria parasites infect and transform cells of the ruminant immune system. Continuous proliferation and survival of Theileria-transformed cells involves the well-orchestrated activation of several host-cell signalling pathways. Constitutive NF-kappa B (nuclear factor kappa B) activation is accomplished by recruiting the IKK (I kappa B kinase) complex, a central regulator of NF-kappa B pathways, to the surface of the transforming schizont, where it becomes permanently activated. Constitutive activation of the PI-3K-PKB [phosphoinositide 3-kinase-(Akt) protein kinase B] pathway is likely to be indirect and is essential for continuous proliferation. Theileria-transformed T cells express a range of anti-apoptotic proteins that can be expected to provide protection against apoptosis induced by death receptors, as well as cellular control mechanisms that are mobilised to eliminate cells that entered a cycle of uncontrolled proliferation.

An optimized in vitro model of the respiratory tract wall to study particle cell interactions

As a part of the respiratory tissue barrier, lung epithelial cells play an important role against the penetration of the body by inhaled particulate foreign materials. In most cell culture models, which are designed to study particle-cell interactions, the cells are immersed in medium. This does not reflect the physiological condition of lung epithelial cells which are exposed to air, separated from it only by a very thin liquid lining layer with a surfactant film at the air-liquid interface. In this study, A549 epithelial cells were grown on microporous membranes in a two chamber system. After the formation of a confluent monolayer the cells were exposed to air. The morphology of the cells and the expression of tight junction proteins were studied with confocal laser scanning and transmission electron microscopy. Air-exposed cells maintained monolayer structure for 2 days, expressed tight junctions and developed transepithelial electrical resistance. Surfactant was produced and released at the apical side of the air-exposed epithelial cells. In order to study particle-cell interactions fluorescent 1 microm polystyrene particles were sprayed over the epithelial surface. After 4 h, 8.8% of particles were found inside the epithelium. This fraction increased to 38% after 24 h. During all observations, particles were always found in the cells but never between them. In this study, we present an in vitro model of the respiratory tract wall consisting of air-exposed lung epithelial cells covered by a liquid lining layer with a surfactant film to study particle-cell interactions.

Deficiency in parvalbumin, but not in calbindin D-28k upregulates mitochondrial volume and decreases smooth endoplasmic reticulum surface selectively in a peripheral, subplasmalemmal region in the soma of Purkinje cells

The Ca(2+)-binding proteins parvalbumin (PV) and calbindin D-28k (CB) are key players in the intracellular Ca(2+)-buffering in specific cells including neurons and have profound effects on spatiotemporal aspects of Ca(2+) transients. The previously observed increase in mitochondrial volume density in fast-twitch muscle of PV-/- mice is viewed as a specific compensation mechanism to maintain Ca(2+) homeostasis. Since cerebellar Purkinje cells (PC) are characterized by high expression levels of the Ca(2+) buffers PV and CB, the question was raised, whether homeostatic mechanisms are induced in PC lacking these buffers. Mitochondrial volume density, i.e. relative mitochondrial mass was increased by 40% in the soma of PV-/- PC. Upregulation of mitochondrial volume density was not homogenous throughout the soma, but was selectively restricted to a peripheral region of 1.5 microm width underneath the plasma membrane. Accompanied was a decreased surface of subplasmalemmal smooth endoplasmic reticulum (sPL-sER) in a shell of 0.5 microm thickness underneath the plasma membrane. These alterations were specific for the absence of the "slow-onset" buffer PV, since in CB-/- mice neither changes in peripheral mitochondria nor in sPL-sER were observed. This implicates that the morphological alterations are aimed to specifically substitute the function of the slow buffer PV. We propose a novel concept that homeostatic mechanisms of components involved in Ca(2+) homeostasis do not always occur at the level of similar or closely related molecules. Rather the cell attempts to restore spatiotemporal aspects of Ca(2+) signals prevailing in the undisturbed (wildtype) situation by subtly fine tuning existing components involved in the regulation of Ca(2+) fluxes.

Modulation of signal transduction by vitamin E

The ability of vitamin E to modulate signal transduction and gene expression has been observed in numerous studies; however, the detailed molecular mechanisms involved are often not clear. The eight natural vitamin E analogues and synthetic derivatives affect signal transduction with different potency, possibly reflecting their different ability to interact with specific proteins. Vitamin E modulates the activity of several enzymes involved in signal transduction, such as protein kinase C, protein kinase B, protein tyrosine kinases, 5-, 12-, and 15-lipoxygenases, cyclooxygenase-2, phospholipase A2, protein phosphatase 2A, protein tyrosine phosphatase, and diacylglycerol kinase. Activation of some these enzymes after stimulation of cell surface receptors with growth factors or cytokines can be normalized by vitamin E. At the molecular level, the translocation of several of these enzymes to the plasma membrane is affected by vitamin E, suggesting that the modulation of protein-membrane interactions may be a common theme for vitamin E action. In this review the main effects of vitamin E on enzymes involved in signal transduction are summarized and the possible mechanisms leading to enzyme modulation evaluated. The elucidation of the molecular and cellular events affected by vitamin E could reveal novel strategies and molecular targets for developing similarly acting compounds.

High molecular weight kininogen regulates platelet-leukocyte interactions by bridging Mac-1 and glycoprotein Ib

Leukocyte-platelet interaction is important in mediating leukocyte adhesion to a thrombus and leukocyte recruitment to a site of vascular injury. This interaction is mediated at least in part by the beta2-integrin Mac-1 (CD11b/CD18) and its counter-receptor on platelets, glycoprotein Ibalpha (GPIbalpha). High molecular weight kininogen (HK) was previously shown to interact with both GPIbalpha and Mac-1 through its domains 3 and 5, respectively. In this study we investigated the ability of HK to interfere with the leukocyte-platelet interaction. In a purified system, HK binding to GPIbalpha was inhibited by HK domain 3 and the monoclonal antibody (mAb) SZ2, directed against the epitope 269-282 of GPIbalpha, whereas mAb AP1, directed to the region 201-268 of GPIbalpha had no effect. In contrast, mAb AP1 inhibited the Mac-1-GPIbalpha interaction. Binding of GPIbalpha to Mac-1 was enhanced 2-fold by HK. This effect of HK was abrogated in the presence of HK domains 3 or 5 or peptides from the 475-497 region of the carboxyl terminus of domain 5 as well as in the presence of mAb SZ2 but not mAb AP1. Whereas no difference in the affinity of the Mac-1-GPIbalpha interaction was observed in the absence or presence of HK, maximal binding of GPIbalpha to Mac-1 doubled in the presence of HK. Moreover, HK/HKa increased the Mac-1-dependent adhesion of myelomonocytic U937 cells and K562 cells transfected with Mac-1 to immobilized GPIbalpha or to GPIbalpha-transfected Chinese hamster ovary cells. Finally, Mac-1-dependent adhesion of neutrophils to surface-adherent platelets was enhanced by HK. Thus, HK can bridge leukocytes with platelets by interacting via its domain 3 with GPIbalpha and via its domain 5 with Mac-1 thereby augmenting the Mac-1-GPIbalpha interaction. These distinct molecular interactions of HK with leukocytes and platelets contribute to the regulation of the adhesive behavior of vascular cells and provide novel molecular targets for reducing atherothrombotic pathologies.

Characterization of the fetuin-binding fraction of Neospora caninum tachyzoites and its potential involvement in host-parasite interactions

Terminal sialic acid residues on surface-associated glycoconjugates mediate host cell interactions of many pathogens. Addition of sialic acid-rich fetuin enhanced, and the presence of the sialidiase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid reduced, the physical interaction of Neospora caninum tachyzoites and bradyzoites with Vero cell monolayers. Thus, Neospora extracts were subjected to fetuin-agarose affinity chromatography in order to isolate components potentially interacting with sialic acid residues. SDS-PAGE and silver staining of the fetuin binding fraction revealed the presence of a single protein band of approximately 65 kDa, subsequently named NcFBP (Neospora caninum fetuin-binding protein), which was localized at the apical tip of the tachyzoites and was continuously released into the surrounding medium in a temperature-independent manner. NcFBP readily interacted with Vero cells and bound to chondroitin sulfate A and C, and anti-NcFBP antibodies interfered in tachyzoite adhesion to host cell monolayers. In additon, analysis of the fetuin binding fraction by gelatin substrate zymography was performed, and demonstrated the presence of two bands of 96 and 140 kDa exhibiting metalloprotease-activity. The metalloprotease activity readily degraded glycosylated proteins such as fetuin and bovine immunoglobulin G heavy chain, whereas non-glycosylated proteins such as bovine serum albumin and immunoglobulin G light chain were not affected. These findings suggest that the fetuin-binding fraction of Neospora caninum tachyzoites contains components that could be potentially involved in host-parasite interactions.

Meperidine and skin surface warming additively reduce the shivering threshold: a volunteer study

INTRODUCTION: Mild therapeutic hypothermia has been shown to improve outcome for patients after cardiac arrest and may be beneficial for ischaemic stroke and myocardial ischaemia patients. However, in the awake patient, even a small decrease of core temperature provokes vigorous autonomic reactions-vasoconstriction and shivering-which both inhibit efficient core cooling. Meperidine and skin warming each linearly lower vasoconstriction and shivering thresholds. We tested whether a combination of skin warming and a medium dose of meperidine additively would reduce the shivering threshold to below 34 degrees C without producing significant sedation or respiratory depression. METHODS: Eight healthy volunteers participated on four study days: (1) control, (2) skin warming (with forced air and warming mattress), (3) meperidine (target plasma level: 0.9 mug/ml), and (4) skin warming plus meperidine (target plasma level: 0.9 mug/ml). Volunteers were cooled with 4 degrees C cold Ringer lactate infused over a central venous catheter (rate asymptotically equal to 2.4 degrees C/hour core temperature drop). Shivering threshold was identified by an increase of oxygen consumption (+20% of baseline). Sedation was assessed with the Observer's Assessment of Alertness/Sedation scale. RESULTS: Control shivering threshold was 35.5 degrees C +/- 0.2 degrees C. Skin warming reduced the shivering threshold to 34.9 degrees C +/- 0.5 degrees C (p = 0.01). Meperidine reduced the shivering threshold to 34.2 degrees C +/- 0.3 degrees C (p < 0.01). The combination of meperidine and skin warming reduced the shivering threshold to 33.8 degrees C +/- 0.2 degrees C (p < 0.01). There were no synergistic or antagonistic effects of meperidine and skin warming (p = 0.59). Only very mild sedation occurred on meperidine days. CONCLUSION: A combination of meperidine and skin surface warming reduced the shivering threshold to 33.8 degrees C +/- 0.2 degrees C via an additive interaction and produced only very mild sedation and no respiratory toxicity.

CD39 is incorporated into plasma microparticles where it maintains functional properties and impacts endothelial activation

Plasma microparticles (MPs, <1.5 mum) originate from platelet and cell membrane lipid rafts and possibly regulate inflammatory responses and thrombogenesis. These actions are mediated through their phospholipid-rich surfaces and associated cell-derived surface molecules. The ectonucleotidase CD39/ecto-nucleoside triphosphate diphosphohydrolase1 (E-NTPDase1) modulates purinergic signalling through pericellular ATP and ADP phosphohydrolysis and is localized within lipid rafts in the membranes of endothelial- and immune cells. This study aimed to determine whether CD39 associates with circulating MPs and might further impact phenotype and function. Plasma MPs were found to express CD39 and exhibited classic E-NTPDase ecto-enzymatic activity. Entpd1 (Cd39) deletion in mice produced a pro-inflammatory phenotype associated with quantitative and qualitative differences in the MP populations, as determined by two dimensional-gel electrophoresis, western blot and flow cytometry. Entpd1-null MPs were also more abundant, had significantly higher proportions of platelet- and endothelial-derived elements and decreased levels of interleukin-10, tumour necrosis factor receptor 1 and matrix metalloproteinase 2. Consequently, Cd39-null MP augment endothelial activation, as determined by inflammatory cytokine release and upregulation of adhesion molecules in vitro. In conclusion, CD39 associates with circulating MP and may directly or indirectly confer functional properties. Our data also suggest a modulatory role for CD39 within MP in the exchange of regulatory signals between leucocytes and vascular cells.

Biomechanical comparison of different surface modifications for dental implants

Purpose: A satisfactory clinical outcome in dental implant treatment relies on primary stability for immediate load bearing. While the geometric design of an implant contributes to mechanical stability, the nature of the implant surface itself is also critically important. Biomechanical and microcomputerized tomographic evaluation of implant osseointegration was performed to compare alternative structural, chemical and biochemical, and/or pharmaceutical surface treatments applied to an identical established implant design. Materials and Methods: Dental implants with the same geometry but with 6 different surface treatments were tested in vivo in a sheep model (pelvis). Peri-implant bone density and removal torque were compared at 2, 4, and 8 weeks after implantation. Implant surfaces tested were: sandblasted and acid-etched titanium (Ti), sandblasted and etched zirconia, Ti coated with calcium phosphate (CaP), Ti modified via anodic plasma-chemical treatment (APC), bisphosphonate-coated Ti (Ti + Bisphos), and Ti coated with collagen containing chondroitin sulfate (CS). Results: All dental implants were well integrated at the time of sacrifice. There were no significant differences observed in peri-implant bone density between implant groups. After 8 weeks of healing, removal torque values for Ti, Ti + CaP, Ti + Bisphos, and Ti + collagen + CS were significantly higher than those for zirconia and Ti + APC. Conclusions: Whereas the sandblasted/acid-etched Ti implant can still be considered the reference standard surface for dental implants, functional surface modifications such as bisphosphonate or collagen coating seem to enhance early peri-implant bone formation and should be studied further.

Relationship between heart rate variability, interleukin-6, and soluble tissue factor in healthy subjects

Decreased heart rate variability (HRV) has been associated with an increased risk of atherosclerosis. We hypothesized that a decrease in frequency domains of resting HRV would be associated with elevated plasma levels of interleukin (IL)-6 and soluble tissue factor (sTF) both previously shown to prospectively predict atherothrombotic events in healthy subjects. Subjects were 102 healthy and unmedicated black and white middle-aged men and women. We determined IL-6 and sTF antigen in plasma and HRV measures from surface electrocardiogram data using spectral analysis. All statistical analyses controlled for age, gender, ethnicity, smoking status, blood pressure, and body mass index. Low amounts of low frequency (LF) power (beta=-0.31, p=0.007) and high frequency (HF) power (beta=-0.36, p=0.002) were associated with increased amounts of IL-6, explaining 7% and 9% of the variance, respectively. Interactions between LF power and IL-6 (p=0.002) and between HF power and IL-6 (p=0.012) explained 8% and 5%, respectively, of the variance in sTF. Post hoc analyses showed associations between IL-6 and sTF when LF power (beta=0.51, p<0.001) and HF power (beta=0.48, p<0.001) were low but not when LF power and high HF power were high. The findings suggest that systemic low-grade inflammatory activity is associated with a decrease in HRV. Furthermore, there was a positive relationship between plasma levels of IL-6 and sTF antigen when HRV was low. Inflammation and related hypercoagulability might particularly contribute to atherothrombotic events in a setting of decreased HRV.

Interactions between trypanosomes and tsetse flies

African trypanosomes are insect-borne parasites that cause sleeping sickness in humans and nagana in domesticated animals. Successful transmission is the outcome of crosstalk between the trypanosome and its insect vector, the tsetse fly. This enables the parasite to undergo successive rounds of differentiation, proliferation and migration, culminating in the infection of a new mammalian host. Several stage- and species-specific parasite surface molecules have been identified and there are new insights into their regulation in the fly. Tsetse flies are often refractory to infection with trypanosomes. While many environmental and physiological factors are known to influence infection, our detailed understanding of tsetse-trypanosome relationships is still in its infancy. Recent studies have identified a number of tsetse genes that show altered expression patterns in response to microbial infections, some of which have also been implicated in modulating trypanosome transmission.

Effects of decontamination and implant surface characteristics on re-osseointegration following treatment of peri-implantitis

BACKGROUND: Although considerable bone fill may occur following treatment of peri-implantitis, re-osseointegration appears to be limited and unpredictable. Objectives: To evaluate the effects of various decontamination techniques and implant surface configurations on re-osseointegration of contaminated dental implants. MATERIAL AND METHODS: Three months after tooth extraction, implants consisting of a basal part and an exchangeable intraosseous implant cylinder (EIIC) were placed in the mandibles of dogs. The EIIC was machined (M), sandblasted and acid-etched (SLA), or titanium plasma sprayed (TPS). Ligature-induced peri-implantitis was initiated 8 weeks post-implantation and lasted until bone loss reached the junction of the two implant parts. Three treatment modalities were applied: (T1) the EIIC was exchanged for a pristine EIIC; (T2) the EIIC was sprayed in situ with saline; and (T3) the EIIC was removed, cleansed outside the mouth by spraying with saline, steam-sterilized, and remounted. A collagen barrier was placed over each fixture, and 3 months later, samples were processed for histology and histomorphometry. RESULTS: T2 revealed the highest bone-to-implant contact (BIC) level (significantly better than T1 and T3). T2 also yielded the highest bone crest level (significantly better than T1), followed by T3 (significantly better than T1). SLA showed the highest BIC level (significantly better than M), followed by TPS. There were no statistically significant differences in bone crest height between implant types. CONCLUSIONS: Both SLA implants and in situ cleansing resulted in the best re-osseointegration and bone fill of previously contaminated implants.