Two-minute versus 6-minute walk distances during 6-minute walk test in neuromuscular disease: Is the 2-minute walk test an effective alternative to a 6-minute walk test?

Functional tests such as Motor Function Measure-32 (MFM-32), supine to stand, ascend/descend stairs permit the assessment of task-specific motor function in neuromuscular disease (NMD). The 6-min walk test (6MWT), though functional, is primarily used to assess endurance and disease progression in children with neuromuscular disorders. Barriers to 6MWT administration, in this population, can include reduced attention span due to age and inability to tolerate test length due to weakness. We propose task-specific functional deficits are related to endurance. Additionally, the 2-min walk test (2MWT) could effectively replace the 6MWT in this population. Seventy-seven participants, ages 5-18, with a variety of neuromuscular disorders performed the 6MWT, timed functional tests (TFT), and the MFM-32. Correlation and paired t-test analyses were used to compare the distance walked in the first 2 min (2MWD) to the distance walked in the entire 6 min (6MWD) and to the functional outcome measures above. The 2MWD strongly correlated with 6MWD and the other outcome measures. Paired t-test analysis also showed that the 2MWD did not differ from the distance walked in the last 2 min of the 6MWT. Although equivalence testing could not reject the claim that this difference exceeded the upper practical limit of 9.5 m, it only showed a modest overestimation of the 4-6MWD compared with the 2MWD. Together, our results support the ability of the 2MWD to predict the 6MWD, specifically in the pediatric neuromuscular disease population.

Intervention strategies for preventing excessive gestational weight gain: systematic review and meta-analysis.

BACKGROUND: Interventions relevant to energy intake to prevent excessive gestational weight gain in pregnant overweight and obese women are important but scarce. This review synthesized healthy eating and physical activity strategies and their effects on excessive gestational weight gain prevention. METHODS: Twenty-three randomized controlled trials that included healthy eating and/or physical activity as an intervention in healthy pregnant overweight or obese adult women and gestational weight gain as a primary or secondary outcome were reviewed. FINDINGS: Heathy eating and/or physical activity (21 studies, n = 6,920 subjects) demonstrated 1.81 kg (95% CI: -3.47, -0.16) of gestational weight gain reduction favouring intervention. Healthy eating (-5.77 kg, 95% CI: -9.34, -2.21, p = 0.02) had a larger effect size than combined healthy eating/physical activity (-0.82 kg, 95% CI: -1.28, -0.36, p = 0.0005) in limiting gestational weight gain. Physical activity did not show a significant pooled effect. Healthy eating with prescribed daily calorie and macronutrient goals significantly limited gestational weight gain by 4.28 kg and 4.23 kg, respectively. CONCLUSION: Healthy eating and/or physical activity are effective in gestational weight gain control. Healthy eating with calorie and macronutrient goals are especially effective in limiting excessive gestational weight gain among pregnant overweight and obese women.

Live volumetric (4D) visualization and guidance of in vivo human ophthalmic surgery with intraoperative optical coherence tomography.

Minimally-invasive microsurgery has resulted in improved outcomes for patients. However, operating through a microscope limits depth perception and fixes the visual perspective, which result in a steep learning curve to achieve microsurgical proficiency. We introduce a surgical imaging system employing four-dimensional (live volumetric imaging through time) microscope-integrated optical coherence tomography (4D MIOCT) capable of imaging at up to 10 volumes per second to visualize human microsurgery. A custom stereoscopic heads-up display provides real-time interactive volumetric feedback to the surgeon. We report that 4D MIOCT enhanced suturing accuracy and control of instrument positioning in mock surgical trials involving 17 ophthalmic surgeons. Additionally, 4D MIOCT imaging was performed in 48 human eye surgeries and was demonstrated to successfully visualize the pathology of interest in concordance with preoperative diagnosis in 93% of retinal surgeries and the surgical site of interest in 100% of anterior segment surgeries. In vivo 4D MIOCT imaging revealed sub-surface pathologic structures and instrument-induced lesions that were invisible through the operating microscope during standard surgical maneuvers. In select cases, 4D MIOCT guidance was necessary to resolve such lesions and prevent post-operative complications. Our novel surgical visualization platform achieves surgeon-interactive 4D visualization of live surgery which could expand the surgeon's capabilities.

Cathelicidin suppresses lipid accumulation and hepatic steatosis by inhibition of the CD36 receptor.

BACKGROUND AND OBJECTIVES: Obesity is a global epidemic which increases the risk of the metabolic syndrome. Cathelicidin (LL-37 and mCRAMP) is an antimicrobial peptide with an unknown role in obesity. We hypothesize that cathelicidin expression correlates with obesity and modulates fat mass and hepatic steatosis. MATERIALS AND METHODS: Male C57BL/6 J mice were fed a high-fat diet. Streptozotocin was injected into mice to induce diabetes. Experimental groups were injected with cathelicidin and CD36 overexpressing lentiviruses. Human mesenteric fat adipocytes, mouse 3T3-L1 differentiated adipocytes and human HepG2 hepatocytes were used in the in vitro experiments. Cathelicidin levels in non-diabetic, prediabetic and type II diabetic patients were measured by enzyme-linked immunosorbent assay. RESULTS: Lentiviral cathelicidin overexpression reduced hepatic steatosis and decreased the fat mass of high-fat diet-treated diabetic mice. Cathelicidin overexpression reduced mesenteric fat and hepatic fatty acid translocase (CD36) expression that was reversed by lentiviral CD36 overexpression. Exposure of adipocytes and hepatocytes to cathelicidin significantly inhibited CD36 expression and reduced lipid accumulation. Serum cathelicidin protein levels were significantly increased in non-diabetic and prediabetic patients with obesity, compared with non-diabetic patients with normal body mass index (BMI) values. Prediabetic patients had lower serum cathelicidin protein levels than non-diabetic subjects. CONCLUSIONS: Cathelicidin inhibits the CD36 fat receptor and lipid accumulation in adipocytes and hepatocytes, leading to a reduction of fat mass and hepatic steatosis in vivo. Circulating cathelicidin levels are associated with increased BMI. Our results demonstrate that cathelicidin modulates the development of obesity.

The metabolome profiling and pathway analysis in metabolic healthy and abnormal obesity.

OBJECTIVES: Mechanisms of the development of abnormal metabolic phenotypes among obese population are not yet clear. In this study, we aimed to screen metabolomes of both healthy and subjects with abnormal obesity to identify potential metabolic pathways that may regulate the different metabolic characteristics of obesity. METHODS: We recruited subjects with body mass index (BMI) over 25 from the weight-loss clinic of a central hospital in Taiwan. Metabolic healthy obesity (MHO) is defined as without having any form of hyperglycemia, hypertension and dyslipidemia, while metabolic abnormal obesity (MAO) is defined as having one or more abnormal metabolic indexes. Serum-based metabolomic profiling using both liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry of 34 MHO and MAO individuals with matching age, sex and BMI was performed. Conditional logistic regression and partial least squares discriminant analysis were applied to identify significant metabolites between the two groups. Pathway enrichment and topology analyses were conducted to evaluate the regulated pathways. RESULTS: A differential metabolite panel was identified to be significantly differed in MHO and MAO groups, including L-kynurenine, glycerophosphocholine (GPC), glycerol 1-phosphate, glycolic acid, tagatose, methyl palmitate and uric acid. Moreover, several metabolic pathways were relevant in distinguishing MHO from MAO groups, including fatty acid biosynthesis, phenylalanine metabolism, propanoate metabolism, and valine, leucine and isoleucine degradation. CONCLUSION: Different metabolomic profiles and metabolic pathways are important for distinguishing between MHO and MAO groups. We have identified and discussed the key metabolites and pathways that may prove important in the regulation of metabolic traits among the obese, which could provide useful clues to study the underlying mechanisms of the development of abnormal metabolic phenotypes.

Phosphorylation of glycogen synthase kinase-3ß in metabolically abnormal obesity affects immune stimulation-induced cytokine production.

BACKGROUND: Obesity is a severe health problem worldwide, which leads to multiple comorbidities including type 2 diabetes mellitus and cardiovascular diseases. Inflammation has been found to be an important characteristic of adipose tissue in obese subjects. However, obesity is also associated with compromised immune responses to infections and the impact of obesity on immune function has not been fully understood. SUBJECTS/METHODS: To clarify the role of obesity in the immune responses, we investigated the Toll-like receptor (TLR)-induced cytokine secretion by leukocytes from obese and lean subjects. We also investigated the relationship between insulin-induced intracellular signaling and cytokine production using peripheral blood mononuclear cells (PBMCs) and a monocytic cell line THP-1. RESULTS: We found decreased TLR-induced interferon-γ, interleukin-6 (IL-6) and tumor necrosis factor-α secretions and elevated IL-10 secretion by leukocytes from obese subjects when compared with lean controls. PBMCs from obese subjects showed enhanced basal Akt and glycogen synthase kinase-3ß (GSK-3ß) phosphorylation, which did not further increase with insulin and lipopolysaccharide (LPS) stimulation. We also found that LPS-induced IκBα degradation was inhibited in PBMCs from obese subjects. By using THP-1 cells with GSK-3ß knockdown or cells treated with hyperinsulinemic and high-fatty acid conditions, we found that LPS-induced nuclear factor κB (NF-κB) activation was inhibited and cyclic adenosine monophosphate response element-binding protein (CREB) activation was enhanced. CONCLUSIONS: These findings indicate that GSK-3ß is important in the regulation of NF-κB and CREB activation in leukocytes under the metabolic condition of obesity. Our study revealed a key mechanism through which metabolic abnormalities compromise leukocyte functions in people with obesity.

Dosimetric advantages of generalised equivalent uniform dose-based optimisation on dose-volume objectives in intensity-modulated radiotherapy planning for bilateral breast cancer.

OBJECTIVE: We compared and evaluated the differences between two models for treating bilateral breast cancer (BBC): (i) dose-volume-based intensity-modulated radiation treatment (DV plan), and (ii) dose-volume-based intensity-modulated radiotherapy with generalised equivalent uniform dose-based optimisation (DV-gEUD plan). METHODS: The quality and performance of the DV plan and DV-gEUD plan using the Pinnacle(3) system (Philips, Fitchburg, WI) were evaluated and compared in 10 patients with stage T2-T4 BBC. The plans were delivered on a Varian 21EX linear accelerator (Varian Medical Systems, Milpitas, CA) equipped with a Millennium 120 leaf multileaf collimator (Varian Medical Systems). The parameters analysed included the conformity index, homogeneity index, tumour control probability of the planning target volume (PTV), the volumes V(20 Gy) and V(30 Gy) of the organs at risk (OAR, including the heart and lungs), mean dose and the normal tissue complication probability. RESULTS: Both plans met the requirements for the coverage of PTV with similar conformity and homogeneity indices. However, the DV-gEUD plan had the advantage of dose sparing for OAR: the mean doses of the heart and lungs, lung V(20) (Gy), and heart V(30) (Gy) in the DV-gEUD plan were lower than those in the DV plan (p<0.05). CONCLUSIONS: A better result can be obtained by starting with a DV-generated plan and then improving it by adding gEUD-based improvements to reduce the number of iterations and to improve the optimum dose distribution. Advances to knowledge The DV-gEUD plan provided superior dosimetric results for treating BBC in terms of PTV coverage and OAR sparing than the DV plan, without sacrificing the homogeneity of dose distribution in the PTV.

Studies of optimum conditions for covalent immobilization of Candida rugosa lipase on poly(gamma-glutamic acid) by RSM.

Poly(gamma-glutamic acid) (gamma-PGA) is a material of polymer. Immobilization of Candida rugosa lipase (Lipase AY-30) by covalent binding on gamma-PGA led to a markedly improved performance of the enzyme. Response surface methodology (RSM) and 3-level-3-factor fractional factorial design were employed to evaluate the effects of immobilization parameters, such as immobilization time (2-6h), immobilization temperature (0-26 degrees C), and enzyme/support ratio (0.1-0.5, w/w). Based on the analysis of ridge max, the optimum immobilization conditions were as follows: immobilization time 2.3h, immobilization temperature 13.3 degrees C, and enzyme/support ratio 0.41 (w/w); the highest lipase activity obtained was 1196 U/mg-protein.

The functional insufficiency of human CD4+CD25 high T-regulatory cells in allergic asthma is subjected to TNF-alpha modulation.

BACKGROUND: Natural CD4(+)CD25(high)Foxp3(+) regulatory T (nTreg) cells are important in maintaining immunologic tolerance, but their role in the pathogenesis of allergic asthma is unclear. We studied the function of nTreg cells in allergic asthmatic children and assessed the factors which may relate to the functional insufficiency of nTreg cells. METHODS: The percentage of CD4(+)CD25(high) Treg cells, the expression of Foxp3, and the cell-induced suppressive activity of nTreg cells isolated from nonatopic controls, allergic asthmatics, and allergen-specific immunotherapy (AIT)-treated asthmatic patients were studied. RESULTS: Although the percentage of nTreg in peripheral blood mononuclear cells was increased, the expression of Foxp3 and its cell-induced suppressive activity were significantly lower in Dermatophagoides pteronyssinus (Der p)-sensitive asthmatic children when compared to nonatopic controls. In contrast, the expression of Foxp3 and the functional activity of nTreg cells were reversed in allergic asthmatics who received AIT. The addition of recombinant tumor necrosis factor (TNF)-alpha directly downregulated Foxp3 expression and abrogated the cell-induced suppressive function of Treg cells. The anti-TNF-alpha reagent, etanercept, restored the functional activity and Foxp3 expression of CD4(+)CD25(high) Treg derived from allergic asthmatics. CONCLUSIONS: The functional insufficiency of nTreg cells in patients with allergic asthma may be related to the enhanced production of TNF-alpha and its effect on the Foxp3 expression. These results may explain, in part, the effectiveness of anti-TNF-alpha therapy in the treatment of allergic asthma.

A-272651, a nonpeptidic blocker of large-conductance Ca2+-activated K+ channels, modulates bladder smooth muscle contractility and neuronal action potentials.

BACKGROUND AND PURPOSE: The large-conductance Ca(2+)-activated K(+) channel (BK(Ca), K(Ca)1.1) links membrane excitability with intracellular Ca(2+) signaling and plays important roles in smooth muscle contraction, neuronal firing, and neuroendocrine secretion. This study reports the characterization of a novel BK(Ca) channel blocker, 2,4-dimethoxy-N-naphthalen-2-yl-benzamide (A-272651). EXPERIMENTAL APPROACH: (86)Rb(+) efflux in HEK-293 cells expressing BK(Ca) was measured. Effects of A-272651 on BK(Ca) alpha- and BK(Ca) alphabeta1-mediated currents were evaluated by patch-clamp. Effects on contractility were assessed using low-frequency electrical field stimulated pig detrusor and spontaneously contracting guinea pig detrusor. Effects of A-272651 on neuronal activity were determined in rat small diameter dorsal root ganglia (DRG). KEY RESULTS: A-272651 (10 microM) inhibited (86)Rb(+) efflux evoked by NS-1608 in HEK-293 cells expressing BK(Ca) currents. A-272651 concentration-dependently inhibited BK(Ca) currents with IC(50) values of 4.59 microM (Hill coefficient 1.04, measured at +40 mV), and 2.82 microM (Hill coefficient 0.89), respectively, for BK(Ca) alpha and BK(Ca) alphabeta1-mediated currents. Like iberiotoxin, A-272651 enhanced field stimulated twitch responses in pig detrusor and spontaneous contractions in guinea pig detrusor with EC(50) values of 4.05+/-0.05 and 37.95+/-0.12 microM, respectively. In capsaicin-sensitive DRG neurons, application of A-272651 increased action potential firing and prolonged action potential duration. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that A-272651 modulates smooth muscle contractility and neuronal firing properties. Unlike previously reported peptide BK(Ca) blockers, A-272651 represents one of the first small molecule BK(Ca) channel blockers that could serve as a useful tool for further characterization of BK(Ca) channels in physiological and pathological states.

P2X7-related modulation of pathological nociception in rats.

Growing evidence supports a role for the immune system in the induction and maintenance of chronic pain. ATP is a key neurotransmitter in this process. Recent studies demonstrate that the glial ATP receptor, P2X7, contributes to the modulation of pathological pain. To further delineate the endogenous mechanisms that are involved in P2X7-related antinociception, we utilized a selective P2X7 receptor antagonist, A-438079, in a series of in vivo and in vitro experiments. Injection of A-438079 (10-300 micromol/kg, i.p.) was anti-allodynic in three different rat models of neuropathic pain and it attenuated formalin-induced nocifensive behaviors. Using in vivo electrophysiology, A-438079 (80 micromol/kg, i.v.) reduced noxious and innocuous evoked activity of different classes of spinal neurons (low threshold, nociceptive specific, wide dynamic range) in neuropathic rats. The effects of A-438079 on evoked firing were diminished or absent in sham rats. Spontaneous activity of all classes of spinal neurons was also significantly reduced by A-438079 in neuropathic but not sham rats. In vitro, A-438079 (1 microM) blocked agonist-induced (2,3-O-(4-benzoylbenzoyl)-ATP, 30 microM) current in non-neuronal cells taken from the vicinity of the dorsal root ganglia. Furthermore, A-438079 dose-dependently (0.3-3 microM) decreased the quantity of the cytokine, interleukin-1beta, released from peripheral macrophages. Thus, ATP, acting through the P2X7 receptor, exerts a wide-ranging influence on spinal neuronal activity following a chronic injury. Antagonism of the P2X7 receptor can in turn modulate central sensitization and produce antinociception in animal models of pathological pain. These effects are likely mediated through immuno-neural interactions that affect the release of endogenous cytokines.

Characterization of a novel ATP-sensitive K+ channel opener, A-251179, on urinary bladder relaxation and cystometric parameters.

BACKGROUND AND PURPOSE: ATP-sensitive K(+) channels (K(ATP)) play a pivotal role in contractility of urinary bladder smooth muscle. This study reports the characterization of 4-methyl-N-(2,2,2-trichloro-1-(3-pyridin-3-ylthioureido)ethyl)benzamide (A-251179) as a K(ATP) channel opener. EXPERIMENTAL APPROACH: Glyburide-sensitive membrane potential, patch clamp and tension assays were employed to study the effect of A-251179 in vitro. The in vivo efficacy of A-251179 was characterized by suppression of spontaneous contractions in obstructed rat bladder and by measuring urodynamic function of urethane-anesthetized rat models. KEY RESULTS: A-251179 was about 4-fold more selective in activating SUR2B-Kir6.2 derived K(ATP) channels compared to those derived from SUR2A-Kir6.2. In pig bladder smooth muscle strips, A-251179 suppressed spontaneous contractions, about 27- and 71-fold more potently compared to suppression of contractions evoked by low-frequency electrical stimulation and carbachol, respectively. In vivo, A-251179 suppressed spontaneous non-voiding bladder contractions from partial outlet-obstructed rats. Interestingly, in the neurogenic model where isovolumetric contractions were measured by continuous transvesical cystometry, A-251179 at a dose of 0.3 micromol kg(-1), but not higher, was found to increase bladder capacity without affecting either the voiding efficiency or changes in mean arterial blood pressure. CONCLUSIONS AND IMPLICATIONS: The thioureabenzamide analog, A-251179 is a potent novel K(ATP) channel opener with selectivity for SUR2B/Kir6.2 containing K(ATP) channels relative to pinacidil. The pharmacological profile of A-251179 is to increase bladder capacity and to prolong the time between voids without affecting voiding efficiency and represents an interesting characteristic to be explored for further investigations of K(ATP) channel openers for the treatment of overactive bladder.

Antibody to severe acute respiratory syndrome (SARS)-associated coronavirus spike protein domain 2 cross-reacts with lung epithelial cells and causes cytotoxicity.

Both viral effect and immune-mediated mechanism are involved in the pathogenesis of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infection. In this study, we showed that in SARS patient sera there were autoantibodies (autoAbs) that reacted with A549 cells, the type-2 pneumocytes, and that these autoAbs were mainly IgG. The autoAbs were detectable 20 days after fever onset. Tests of non-SARS-pneumonia patients did not show the same autoAb production as in SARS patients. After sera IgG bound to A549 cells, cytotoxicity was induced. Cell cytotoxicity and the anti-epithelial cell IgG level were positively correlated. Preabsorption and binding assays indicated the existence of cross-reactive epitopes on SARS-CoV spike protein domain 2 (S2). Furthermore, treatment of A549 cells with anti-S2 Abs and IFN-gamma resulted in an increase in the adherence of human peripheral blood mononuclear cells to these epithelial cells. Taken together, we have demonstrated that the anti-S2 Abs in SARS patient sera cause cytotoxic injury as well as enhance immune cell adhesion to epithelial cells. The onset of autoimmune responses in SARS-CoV infection may be implicated in SARS pathogenesis.

Therapeutic effect of surfactant protein D in allergic inflammation of mite-sensitized mice.

BACKGROUND: Surfactant protein D (SP-D) is involved in the innate immunity within the lung and may have important roles in modulating the inflammatory process of asthma. OBJECTIVE: To examine the potential immunomodulating role of SP-D on the allergic response in mice, and its interaction with the alveolar macrophages (AMs) during allergic inflammation. METHODS: A recombinant 60 kDa fragment of human SP-D (rfh SP-D), Survanta, and budesonide were administrated, respectively, to Der p-sensitive BALB/c mice before or after allergen challenge (AC). Total and differential cell counts, levels of cytokines in bronchoalveolar lavage fluids(BALFs), and levels of Der p-specific IgE and IgG1 antibodies in sera, were assayed. The production of nitric oxide (NO), and inducible NO synthase (iNOS) expression, in AMs, were determined by ELISA and RT-PCR, respectively. RESULTS: Instillation of rfh SP-D to sensitized mice 6 h after AC (therapeutic), but not 24 h before AC (preventive), markedly reduced infiltration of eosinophils, and also reduced levels of IL-4, IL-5, eotaxin, and TNF-alpha but elevated levels of IFN-gamma in the BALF. These effects were comparable with those obtained with budesonide treatment, whereas Survanta did not have a suppressive effect, either before or after AC. There was significant inhibition of NO production in the rfh SP-D pre-treated AMs of allergen-sensitized mice, but not in naive mice. CONCLUSIONS: These results indicate that rfh SP-D has a therapeutic effect on allergen-induced bronchial inflammation, and that this might be because of its inhibitory effect on NO and TNF-alpha production by AMs, and it thus prevents the development of T-helper type 2 cytokine response.

Multiple mutagenesis of the Candida rugosa LIP1 gene and optimum production of recombinant LIP1 expressed in Pichia pastoris.

Candida rugosa lipase, a significant catalyst, had been widely employed to catalyze various chemical reactions such as non-specific, stereo-specific hydrolysis and esterification for industrial biocatalytic applications. Several isozymes encoded by the lip gene family, namely lip1 to lip7, possess distinct thermal stability and substrate specificity, among which the recombinant LIP1 showed a distinguished catalytic characterization. In this study, we utilized PCR to remove an unnecessary linker of pGAPZalphaC vector and used overlap extension PCR-based multiple site-directed mutagenesis to convert the 19 non-universal CTG-serine codons into universal TCT-serine codons and successfully express a highly active recombinant C. rugosa LIP1 in the Pichia expression system. Response surface methodology and 4-factor-5-level central composite rotatable design were adopted to evaluate the effects of growth parameters, such as temperature (21.6-38.4 degrees C), glucose concentration (0.3-3.7%), yeast extract (0.16-1.84%), and pH (5.3-8.7) on the lipolytic activity of LIP1 and biomass of P. pastoris. Based on ridge max analysis, the optimum LIP1 production conditions were temperature, 24.1 degrees C; glucose concentration, 2.6%; yeast extract, 1.4%; and pH 7.6. The predicted value of lipolytic activity was 246.9+/-39.7 U/ml, and the actual value was 253.3+/-18.8 U/ml. The lipolytic activity of the recombinant LIP1 resulting from the present work is twofold higher than that achieved by a methanol induction system.

Mite allergen induces nitric oxide production in alveolar macrophage cell lines via CD14/toll-like receptor 4, and is inhibited by surfactant protein D.

BACKGROUND: Previously, we have found that dust mite allergens can directly activate alveolar macrophages (AMs), induce inflammatory cytokines, and enhance T-helper type 2 cytokine production. A molecule of innate immunity in the lung, surfactant protein D (SP-D), is able to bind mite allergens and alleviates allergen-induced airway inflammation. OBJECTIVES: This study was aimed at investigating the activation pathway of mite allergen (Dermatophagoides pteronyassinus, Der p)-induced nitric oxide (NO) production by AMs, and the role of SP-D in the modulation of activated AMs by mite allergens. METHODS: Porcine SP-D was purified from bronchoalveolar lavage fluids of Lan-Yu mini-pigs, by affinity chromatography on maltose-sepharose. NO production, inducible expression of lipopolysaccharides (LPS)-related binding and responding surface receptors complex, CD14 and toll-like receptor 4 (TLR4), as well as inducible NO synthase (iNOs) and nuclear factor-kappaB activation were studied in two AMs cell lines, MH-S (BALB/c strain),and AMJ2-C11 (C57BL/6 strain), and one peritoneal macrophage cell line (RAW264.7), after stimulation with LPS, or Der p. RESULTS: LPS and Der p elicited different responses of NO production in the different cell lines, and the response might depend upon the expression of the cell surface CD14/TLR4 complex in different genetic backgrounds of macrophage cell lines. Pretreatment of macrophages with SP-D could inhibit NO production from Der p or LPS-stimulated alveolar macrophages. CONCLUSION: Mite allergen-induced alveolar macrophage activation is mediated by CD14/TLR4 receptors and can be inhibited by SP-D; it further supports the concept that SP-D may be an important modulator of allergen-induced pulmonary inflammation.

Infradiaphragmatic pulmonary sequestration combined with cystic adenomatoid malformation: unusual postnatal computed tomographic features.

We present a surgically proven case of infradiaphragmatic pulmonary sequestration combined with cystic adenomatoid malformation. Prenatal magnetic resonance imaging revealed a well-defined hyperintense mass with a hypointense septum in the left infradiaphragmatic region. Postdelivery computed tomography (CT) and 3-month follow-up CT showed replacement of intralesional cystic areas by solid content. Such unusual postnatal CT changes, to our knowledge, have not been previously documented.

Modulation of action potential firing by iberiotoxin and NS1619 in rat dorsal root ganglion neurons.

The present study investigated the effects of iberiotoxin (IbTx), a peptide toxin blocker of large-conductance Ca(2+)-activated K(+) (BK(Ca)) channels and NS1619, a BK(Ca) channel opener, on action potential firing of small and medium size afferent neurons from L6 and S1 dorsal root ganglia of adult rats. Application of IbTx (100 nM) reduced whole-cell outward currents in 67% of small and medium size neurons. Analysis of action potential profile revealed that IbTx significantly prolonged the duration of action potential and increased firing frequency of afferent neurons. IbTx did not significantly alter the resting membrane potential, threshold for action potential activation and action potential amplitude. The benzimidazolone NS1619 (10 microM) increased opening activity of a Ca(2+)-dependent channel as assessed by single channel measurements. In contrast to IbTx, NS1619 reversibly suppressed action potential firing, attributable to increases in threshold for evoking action potential, reduction in action potential amplitude and increases in amplitude of afterhyperpolarization. The effect of NS1619 on neuronal firing was sensitive to IbTx, indicating the attenuation of neuronal firing by NS1619 was mediated by opening BK(Ca) channels. NS1619 also reduced neuronal hyperexcitability evoked by 4-aminopyridine (4-AP), a transient-inactivated K(+) channel (A-current) blocker, in an IbTx-sensitive manner. These results indicate that IbTx-sensitive BK(Ca) channels exist in both small and medium diameter dorsal root ganglion (DRG) neurons and play important roles in the repolarization of action potential and firing frequency. NS1619 modulates action potential firing and suppresses 4-AP-evoked hyperexcitability in DRG neurons, in part, by opening BK(Ca) channels. These results suggest that opening BK(Ca) channels might be sufficient to suppress hyperexcitability of afferent neurons as those evoked by stimulants or by disease states.

Optimization of lipase-catalyzed biodiesel by response surface methodology.

Biodiesel prepared by catalyzed mild transesterification has become of much current interest for bioenergy. The ability of a commercial immobilized lipase (Novo Industries--Bagsvaerd, Denmark) from Rhizomucor miehei (Lipozyme IM-77) to catalyze the transesterification of soybean oil and methanol was investigated in this study. Response surface methodology and 5-level-5-factor central composite rotatable design were employed to evaluate the effects on reaction time, temperature, enzyme amount, molar ratio of methanol to soybean oil, and added water content on percentage weight conversion to soybean oil methyl ester by transesterification. Based on ridge max analysis, the optimum synthesis conditions giving 92.2% weight conversion were: reaction time 6.3 h, temperature 36.5 degrees C, enzyme amount 0.9 BAUN (Batch Acidolysis Units NOVO), substrate molar ratio 3.4:1, and added water 5.8%.

Validation of FLIPR membrane potential dye for high throughput screening of potassium channel modulators.

A fluorescence-based assay using the FLIPR Membrane Potential Assay Kit (FMP) was evaluated for functional characterization and high throughput screening (HTS) of potassium channel (ATP-sensitive K+ channel; K(ATP)) modulators. The FMP dye permits a more sensitive evaluation of changes in membrane potential with a more rapid response time relative to DiBAC4(3). The time course of responses is comparable to ligand-evoked activation of the channel measured by patch-clamp studies. The pharmacological profile of the K+ channel evaluated by using reference K(ATP) channel openers is in good agreement with that derived previously by DiBAC4(3)-based FLIPR assays. Improved sensitivity of responses together with the diminished susceptibility to artifacts such as those evoked by fluorescent compounds or quenching agents makes the FMP dye an alternative choice for HTS screening of potassium channel modulators.