Unraveling the Na,K-ATPase alpha(4) Subunit Assembling Induced by Large Amounts of C(12)E(8) by Means of Small-Angle X-ray Scattering

In the current work, we studied the effect of the nonionic detergent dodecyloctaethyleneglycol, C(12)E(8), on the structure and oligomeric form of the Na,K-ATPase membrane enzyme (sodium-potassium pump) in aqueous suspension, by means of small-angle X-ray scattering (SAXS). Samples composed of 2 mg/mL of Na,K-ATPase, extracted from rabbit kidney medulla, in the presence of a small amount of C(12)E(8) (0.005 mg/mL) and in larger concentrations ranging from 2.7 to 27 mg/mL did not present catalytic activity. Under this condition, an oligomerization of the alpha subunits is expected. SAXS data were analyzed by means of a global fitting procedure supposing that the scattering is due to two independent contributions: one coming from the enzyme and the other one from C(12)E(8) micelles. In the small detergent content (0.005 mg/mL), the SAXS results evidenced that Na,K-ATPase is associated into aggregates larger than (alpha beta)(2) form. When 2.7 mg/mL of C(12)E(8) is added, the data analysis revealed the presence of alpha(4) aggregates in the solution and some free micelles. Increasing the detergent amount up to 27 mg/mL does not disturb the alpha(4) aggregate: just more micelles of the same size and shape are proportionally formed in solution. We believe that our results shed light on a better understanding of how nonionic detergents induce subunit dissociation and reassembling to minimize the exposure of hydrophobic residues to the aqueous solvent.

Molecular View of the Interaction between iota-Carrageenan and a Phospholipid Film and Its Role in Enzyme Immobilization

Proteins incorporated into phospholipid Langmuir-Blodgett (LB) films are a good model system for biomembranes and enzyme immobilization studies. The specific fluidity of biomembranes, an important requisite for enzymatic activity, is naturally controlled by varying phospholipid compositions. In a model system, instead, LB film fluidity may be varied by covering the top layer with different substances able to interact simultaneously with the phospholipid and the protein to be immobilized. In this study, we immobilized a carbohydrate rich Neurospora crassa alkaline phosphatase (NCAP) in monolayers of the sodium salt of dihexadecylphosphoric acid (DHP), a synthetic phospholipid that provides very condensed Langmuir films. The binding of NCAP to DHP Langmuir-Blodgett (LB) films was mediated by the anionic polysaccharide iota-carrageenan (iota-car). Combining results from surface isotherms and the quartz crystal microbalance technique, we concluded that the polysaccharide was essential to promote the interaction between DHP and NCAP and also to increase the fluidity of the film. An estimate of DHP:iota-car ratio within the film also revealed that the polysaccharide binds to DHP LB film in an extended conformation. Furthermore, the investigation of the polysaccharide conformation at molecular level, using sum-frequency vibrational spectroscopy (SFG), indicated a preferential conformation of the carrageenan molecules with the sulfate groups oriented toward the phospholipid monolayer, and both the hydroxyl and ether groups interacting preferentially with the protein. These results demonstrate how interfacial electric fields can reorient and induce conformational changes in macromolecules, which may significantly affect intermolecular interactions at interfaces. This detailed knowledge of the interaction mechanism between the enzyme and the LB film is relevant to design strategies for enzyme immobilization when orientation and fluidity properties of the film provided by the matrix are important to improve enzymatic activity.

Evaluation of the effects of Candidatus Liberibacter asiaticus on inoculated citrus plants using laser-induced breakdown spectroscopy (LIBS) and chemometrics tools

This study investigated the organic and inorganic constituents of healthy leaves and Candidatus Liberibacter asiaticus (CLas)-inoculated leaves of citrus plants. The bacteria CLas are one of the causal agents of citrus greening (or Huanglongbing) and its effect on citrus leaves was investigated using laser-induced breakdown spectroscopy (LIBS) combined with chemometrics. The information obtained from the LIBS spectra profiles with chemometrics analysis was promising for the construction of predictive models to identify healthy and infected plants. The major, macro- and microconstituents were relevant for differentiation of the sample conditions. The models were then applied to different inoculation times (from 1 to 8 months). The models were effective in the classification of 82-97% of the diseased samples with a 95% significance level. The novelty of this method was in the fingerprinting of healthy and diseased plants based on their organic and inorganic contents. (C) 2010 Elsevier B.V. All rights reserved.

Lipopolysaccharide and lipoteichoic acid induce different innate immune responses in bovine mammary epithelial cells

The objective of the present study was to characterize the innate immune responses induced by in vitro stimulation of bovine primary mammary epithelial cells (bMEC) using gram-negative lipopolysaccharide (LPS) and gram-positive lipoteichoic acid (LTA) bacterial cell wall components. Quantitative real-time PCR (qRT-PCR) was employed to examine the mRNA expression of a panel of 22 cytokines, chemokines, beta-defensins and components of the Toll-Like Receptor signaling pathway. Stimulation of bMEC with LPS for 24 h elicited a marked increase in mRNA expression for IL-1 beta, IL-8, TNF alpha, CXCL6 and beta-defensin while members of the Toll-Like Receptor pathway.. although present, were largely unaffected. Surprisingly, stimulation of these cells with LTA for 24 h did not significantly alter the expression of these genes. A time course of the expression of IL-1 beta, IL-8, TNF alpha, CXCL6 and beta-defensin was subsequently performed. The mRNA levels of all genes increased rapidly after stimulation for 2-4 h with both LPS and LTA but only the former treatment resulted in sustained responses. In contrast, the increased gene expression for LTA stimulated cells returned to resting levels after 8-16 h with the exception of beta-defensin, which remained up-regulated. The limited and unsustained cytokine response to LTA may explain why mastitis caused by gram-positive bacteria has greater potential for chronic intra-mammary infection than gram-negative infection. It was concluded that bovine mammary epithelial cells have a strong but differential capacity to mount innate immune responses to bacterial cell wall components. Crown Copyright (c) 2005 Published by Elsevier Ltd. All rights reserved.

Effect of Diode-Laser and AC Magnetic Field of Bovine Serum Albumin Nanospheres Loaded with Phthalocyanine and Magnetic Particles

This study reports on the development and characterization of bovine serum albumin (BSA) nanospheres containing Silicon(IV) phthalocyanine (NzPc) and/or maghemite nanoparticles (MNP), the latter introduced via ionic magnetic fluid (MF). The nanosized BSA-loaded samples were designed for synergic application while combining Photodynamic Therapy and Hyperthermia. Incorporation of MNP in the albumin-based template, allowing full control of the magnetic content, was accomplished by adding a highly-stable ionic magnetic fluid sample to the albumin suspension, following heat denaturing. The material`s evaluation was performed using Zeta potential measurements and scanning electron microscopy. The samples were characterized by steady-state techniques and time-resolved fluorescence. The in vitro assay, using human fibroblasts, revealed no cytotoxic effect in all samples investigated, demonstrating the potential of the tested system as a synergistic drug delivery system.

Differences in the early biomechanical effects of hyperopic and myopic laser in situ keratomileusis

PURPOSE: To compare changes in corneal hysteresis (CH) and the corneal resistance factor (CRF) in myopic and hyperopic laser in situ keratomileusis (LASIK) and evaluate their relationship to the number of photoablative pulses delivered, a surrogate for ablation volume. SETTING: Cleveland Clinic Cole Eye Institute, Cleveland, Ohio, USA. METHODS: Preoperative and 1-week postoperative Ocular Response Analyzer measurements in eyes that had femtosecond-assisted LASIK were studied retrospectively. Changes in CH and CRF were compared and tested for correlation with the number of excimer laser pulses. RESULTS: Thirteen myopic eyes and 11 hyperopic eyes were evaluated. Preoperative corneal thickness, CH, CRF, programmed correction magnitude, flap thickness, and total number of fixed spotsize photoablative pulses were similar in the 2 groups (P>.1). Decreases in CH and CRF were greater after myopic LASIK than after hyperopic LASIK (P<.005), and changes in CRF were correlated with the number of excimer laser pulses in the myopic group only (r = -0.63, P = .02). Regardless of ablation profile, changes in CH were more strongly correlated with preoperative CH values than with attempted ablation volume. CONCLUSIONS: With comparable flap thickness and attempted ablation volumes, myopic photoablation profiles were associated with greater decreases in CRF and CH than hyperopic profiles. Results indicate that preoperative corneal biomechanical status, ablation volume, and the spatial distribution of ablation are important factors that affect corneal resistance and viscous dissipative properties differently. Preferential tissue removal in the natively thicker paracentral cornea in hyperopia may partially account for the rarity of ectasia after hyperopic LASIK.

The mechanism of inflammation in RelB deficient mice

RelB, NIK and TRAF6-deficient mice die prematurely with multi-organ inflammatory disease and apparent excessive myelopoiesis. While thymic development of CD4+CD25+ regulatory T cells (Treg) is reduced in TRAF6 deficient mice, the impact of this on inflammation is not known. Here we show that while RelB deficient thymic stroma is unable to sustain the development of Treg, surprisingly, FoxP3hi Treg are increased in the periphery. Peripheral expansion of Treg is driven by GITRligand, expressed by immature monocytes maintained by RelBdeficient stroma. RelB-deficient DC fail to activate Treg suppressor function. The data reveal the dual roles of RelB in both hemopoietic and stromal cells to maintain tolerance and contain inflammation through Treg and DC.

Novel Treatment Immediately after Myelomeningocele Repair Applying Low-Level Laser Therapy in Newborns: A Pilot Study

Background/Aims: The use of low-level laser therapy (LLLT) in neurosurgery is still hardly disseminated and there are situations in which the effects of this therapeutic tool would be extremely relevant in this medical field. The aim of the present study is to analyze the effect of LLLT on tissue repair after the corrective surgical incision in neonates with myelomeningocele, in an attempt to diminish the incidence of postoperative dehiscences following surgical repair performed immediately after birth. Materials and Methods: Prospective pilot study with 13 patients submitted to surgery at birth who received adjuvant treatment with LLLT (group A). A diode laser CW, lambda = 685 nm, p = 21 mW, was applied punctually along the surgical incision, with 0.19 J delivered per point, accounting for a total of 4-10 J delivered energy per patient, according to the surgical wound area and then compared with the results obtained in 23 patients who underwent surgery without laser therapy (group B). Results: This pilot study disclosed a significant decline in dehiscences of the surgical wounds in neonates who were submitted to LLLT (7.69 vs. 17.39%). Conclusion: This new adjuvant therapeutic modality with LLLT aided the healing of surgical wounds, preventing morbidities, as well as shortening the period of hospital stay, which implies a reduction of costs for patients and for the institution. Copyright (C) 2010 S. Karger AG, Basel

Prolonged recruitment manoeuvre improves lung function with less ultrastructural damage in experimental mild acute lung injury

The effects of prolonged recruitment manoeuvre (PRM) were compared with sustained inflation (SI) in paraquat-induced mild acute lung injury (ALI) in rats. Twenty-four hours after ALI induction, rats were anesthetized and mechanically ventilated with VT = 6 ml/kg and positive end-expiratory pressure (PEEP) = 5 cmH(2)O for 1 h. SI was performed with an instantaneous pressure increase of 40 cmH(2)O that was sustained for 40 s, while PRM was done by a step-wise increase in positive inspiratory pressure (PIP) of 15-20-25 cmH(2)O above a PEEP of 15 cm H(2)O (maximal PIP = 40 cmH(2)O), with interposed periods of PIP = 10 cmH(2)O above a PEEP = 15 cmH(2)O. Lung static elastance and the amount of alveolar collapse were more reduced with PRM than SI, yielding improved oxygenation. Additionally, tumour necrosis factor-alpha, interleukin-6, interferon-gamma, and type III procollagen mRNA expressions in lung tissue and lung epithelial cell apoptosis decreased more in PRM. In conclusion, PRM improved lung function, with less damage to alveolar epithelium, resulting in reduced pulmonary injury. (C) 2009 Elsevier BLV. All rights reserved.

Attentional modulation of visual cortex activity following right parietal damage in humans

Pathological inattention following parietal damage causes perceptual impairments for visual stimuli in the contralesional hemifield. Here we used functional magnetic resonance imaging (fMRI) to examine visual cortex activity in parietal patients as they performed a spatial attention task. Righthemisphere patients and healthy controls viewed counterphasing checkerboards in which coloured targets appeared briefly within the contralesional and ipsilesional hemifields. In separate fMRI runs participants focused their attention covertiy on the left or right hemifield, or on both hemifields concurrentiy. They were required to detect coloured targets that appeared briefly within the attended hemifield(s), and to withhold responses to distractor stimuli. Neural activit}' was significantly attenuated in early visual areas within the damaged hemisphere. Crucially, although attention significantiy modulated early visual activit}' within the intact (left) hemisphere, there was relatively littie modulation of activity within the affected hemisphere. Our findings suggest that parietal lesions alter early cortical responses to contralesional visual inputs.

Phosphorus overload and PTH induce aortic expression of Runx2 in experimental uraemia

Background. Vascular calcification (VC) is commonly seen in patients with chronic kidney disease (CKD). Elevated levels of phosphate and parathormone (PTH) are considered nontraditional risk factors for VC. It has been shown that, in vitro, phosphate transforms vascular smooth muscle cells (VSMCs) into calcifying cells, evidenced by upregulated expression of runt-related transcription factor 2 (Runx2), whereas PTH is protective against VC. In addition, Runx2 has been detected in calcified arteries of CKD patients. However, the in vivo effect of phosphate and PTH on Runx2 expression remains unknown. Methods. Wistar rats were submitted to parathyroidectomy, 5/6 nephrectomy (Nx) and continuous infusion of 1-34 rat PTH (at physiological or supraphysiological rates) or were sham-operated. Diets varied only in phosphate content, which was low (0.2%) or high (1.2%). Biochemical, histological, immunohistochemistry and immunofluorescence analyses were performed. Results. Nephrectomized animals receiving high-PTH infusion presented VC, regardless of the phosphate intake level. However, phosphate overload and normal PTH infusion induced phenotypic changes in VSMCs, as evidenced by upregulated aortic expression of Runx2. High-PTH infusion promoted histological changes in the expression of osteoprotegerin and type I collagen in calcified arteries. Conclusions. Phosphate, by itself is a potential pathogenic factor for VC. It is of note that phosphate overload, even without VC, was associated with overexpression of Runx2 in VSMCs. The mineral imbalance often seen in patients with CKD should be corrected.

Combined Laser and Intravitreal Triamcinolone for Proliferative Diabetic Retinopathy and Macular Edema: One-year Results of a Randomized Clinical Trial

PURPOSE: To evaluate laser combined with intravitreal triamcinolone acetonide (IVTA) for the management of patients with proliferative diabetic retinopathy (PDR) and clinically significant macular edema (CSME). DESIGN: Randomized clinical trial. METHODS: SETTINGS: Single center. STUDY POPULATION: Twenty-two patients with bilateral treatment,naive moderate PDR and CSME. INTERVENTION: Laser (panretinal and macular) photocoagulation was performed in each eye, followed by IVTA in one randomly assigned eye. Best,corrected visual acuity (BCVA), fundus photography, and optical coherence tomography were performed at baseline and at months 1, 3, 6, 9, and 12. MAIN OUTCOME MEASURES:. Changes in BCVA, central macular thickness (CMT), and total macular volume (TMV). RESULTS: The mean logarithm of the minimal angle of resolution (logMAR) BCVA improved significantly, and mean CMT and TMV were significantly reduced in the IVTA group compared with the laser,only group (controls) at all study follow-up visits (P < .001). The mean logMAR BCVA (Snellen equivalent) was 0.44 (20/50(-2)) for the IVTA group and 0.38 (20/50(+1)) for the controls at baseline, and 0.12 (20/25(-1)) for the IVTA group and 0.32 (20/40(-1)) for the controls at 12 months (P < .001.). The mean CMT and TMV were, respectively, 360 mu m and 8.59 mm(3) for the IVTA group and 331 mu m and 8.44 mm(3) for the controls at baseline, and 236 mu m and 7.32 mm(3) for the IVTA group and 266 mu m and 7.78 mm(3) for the controls at 12 months (P < .001). CONCLUSIONS: The combination of laser photocoagulation with IVTA was associated with improved BCVA and decreased CMT and TMV when compared with laser photocoagulation alone for the treatment of moderate PDR with CSME. (Am J Ophthalmol 2009;147:291-297. (C) 2009 by Elsevier Inc. All rights reserved.)

Trigonal injection of botulinum toxin-A does not cause vesicoureteral reflux in neurogenic patients

Aims: We evaluated the effect of botulinum toxin type A (BTX-A) injections in the trigone on the antireflux mechanism and evaluated its short-term efficacy. Materials and Methods: Between April and December 2006, 21 patients (10 men and 11 women) were prospectively evaluated. All were incontinent due to refractory NDO and underwent detrusor injection of 300 units of BTX-A, including 50 units into the trigone. Baseline and postoperative evaluation after eight weeks included cystogram, urinary tract ultrasound and urodynamics. Results: At baseline, 20 patients had no vesicoureteral (VUR) and one had grade II unilateral VUR. Postoperative evaluation revealed no cases of de novo VUR and the patient with preinjection VUR had complete resolution of the reflux. Ultrasound showed 5 (23.8%) patients with hydronephrosis before BTX-A injection and only one (4.8%) at the followup evaluation (p=0.066). After treatment, 9 (42.8%) patients became dry, 11 (52.4%) were improved and one (4.8%) had no improvement. Improved patients received antimuscarinic treatment and 8 (38.1%) became dry, with a final total continence rate of 80.1%. Cystometric capacity increased from 271 +/- 92 to 390 +/- 1189 ml (p = 0.002), reflex volume varied from 241 +/- 96 to 323 +/- 201 ml (p = 0.020) and maximum detrusor pressure reduced from 66 +/- 39 to 38 +/- 37 cm H2O (p < 0.001). Conclusions: Our results confirm the safety of trigone injections of BTX-A in terms of development of VUR and upper urinary tract damage. Whether they are beneficial for patients with NDO or other causes of voiding dysfunction will need further studies.

Mechanism of ribonuclease inhibition by ribonuclease inhibitor protein based on the crystal structure of its complex with ribonuclease A

We describe the mechanism of ribonuclease inhibition by ribonuclease inhibitor, a protein built of leucine-rich repeats, based on the crystal structure of the complex between the inhibitor and ribonuclease A. The structure was determined by molecular replacement and refined to an R(cryst) of 19.4% at 2.5 Angstrom resolution. Ribonuclease A binds to the concave region of the inhibitor protein comprising its parallel beta-sheet and loops. The inhibitor covers the ribonuclease active site and directly contacts several active-site residues. The inhibitor only partially mimics the RNase-nucleotide interaction and does not utilize the pi phosphate-binding pocket of ribonuclease A, where a sulfate ion remains bound. The 2550 Angstrom(2) of accessible surface area buried upon complex formation may be one of the major contributors to the extremely tight association (K-i = 5.9 x 10(-14) M). The interaction is predominantly electrostatic; there is a high chemical complementarity with 18 putative hydrogen bonds and salt links, but the shape complementarity is lower than in most other protein-protein complexes. Ribonuclease inhibitor changes its conformation upon complex formation; the conformational change is unusual in that it is a plastic reorganization of the entire structure without any obvious hinge and reflects the conformational flexibility of the structure of the inhibitor. There is a good agreement between the crystal structure and other biochemical studies of the interaction. The structure suggests that the conformational flexibility of RI and an unusually large contact area that compensates for a lower degree of complementarity may be the principal reasons for the ability of RI to potently inhibit diverse ribonucleases. However, the inhibition is lost with amphibian ribonucleases that have substituted most residues corresponding to inhibitor-binding residues in RNase A, and with bovine seminal ribonuclease that prevents inhibitor binding by forming a dimer. (C) 1996 Academic Press Limited