The use of dimethylsulfoxide as a vehicle in cell culture experiments using ovarian carcinoma cell lines

We describe here a computational morphology-based approach to the investigation of possible causes of chromatin alterations in sperm. A comprehensive set of state-of-the-art and geometric measures are computationally extracted from toluidine blue stained images and analyzed to infer the possible processes leading to normal and abnormal chromatin formation while seeking a possible taxonomy of chromatin alterations and their influence on sperm head morphology. Using this methodology, we have identified higher chromatin fragility at some specific points of the sperm head. Despite the lack of correlation between morphologies of sperm head and chromatin structure, four main morphological types of chromatin alterations in bull spermatozoa have been identified and their possible causes discussed.     

Intestinal caveolin-1 is important for dietary fatty acid absorption

How dietary fatty acids are absorbed into the enterocyte and transported to the ER is not established. We tested the possibility that caveolin-1 containing lipid rafts and endocytic vesicles were involved. Apical brush border membranes took up 15% of albumin bound ³H-oleate whereas brush border membranes from caveolin-1 KO mice took up only 1%. In brush border membranes, the ³H-oleate was in the detergent resistant fraction of an OptiPrep gradient. On OptiPrep gradients of intestinal cytosol, we also found the ³H-oleate in the detergent resistant fraction, separate from OptiPrep gradients spiked with ³H-oleate or ³H-triacylglycerol. Caveolin-1 immuno-depletion of cytosol removed 91% of absorbed ³H-oleate whereas immuno-depletion using IgG, or anti-caveolin-2 or -3 or anti-clathrin antibodies removed 20%. Electron microscopy showed the presence of caveolin-1 containing vesicles in WT mouse cytosol that were 4 fold increased by feeding intestinal sacs 1 mM oleate. No vesicles were seen in caveolin-1 KO mouse cytosol. Caveolin-1 KO mice gained less weight on a 23% fat diet and had increased fat in their stool compared to WT mice. We conclude that dietary fatty acids are absorbed by caveolae in enterocyte brush border membranes, are endocytosed, and transported in cytosol in caveolin-1 containing endocytic vesicles.     

Inhibition of LPS-induced airway neutrophilic inflammation in healthy volunteers with an oral CXCR2 antagonist

Background

Inhaled lipopolysaccharide (LPS) induces a dose-dependent, acute neutrophilic response in the airways of healthy volunteers that can be quantified in induced sputum. Chemokines, such as CXCL1 and CXCL8, play an important role in neutrophilic inflammation in the lung through the activation of CXCR2 and small molecule antagonists of these receptors have now been developed. We investigated the effect of AZD8309, a CXCR2 antagonist, compared with placebo on LPS-induced inflammation measured in sputum of healthy volunteers.

Methods

Twenty healthy subjects were randomized in a double-blind placebo-controlled, cross-over study. AZD8309 (300 mg) or placebo was dosed twice daily orally for 3 days prior to challenge with inhaled LPS and induced sputum was collected 6 h later.

Results

Treatment with AZD8309 showed a mean 77% reduction in total sputum cells (p < 0.001) and 79% reduction in sputum neutrophils (p < 0.05) compared with placebo after LPS challenge. There was also a reduction in neutrophil elastase activity (p < 0.05) and CXCL1 (p < 0.05) and trends for reductions in sputum macrophages (47%), leukotriene B₄ (39%) and CXCL8 (52%).

Conclusions

AZD8309 inhibited LPS-induced inflammation measured in induced sputum of normal volunteers, indicating that this treatment may be useful in the treatment of neutrophilic diseases of the airways, such as COPD, severe asthma and cystic fibrosis.

Trial registration

{"type":"clinical-trial","attrs":{"text":"NCT00860821","term_id":"NCT00860821"}}NCT00860821.     

Progression,Symptoms and Psychosocial Concerns among Those Severely Affected by Multiple Sclerosis: A Mixed-Methods Cross-Sectional Study of Black Caribbean and White British People

Objective

Multiple sclerosis is now more common among minority ethnic groups in the UK but little is known about their experiences, especially in advanced stages. We examine disease progression, symptoms and psychosocial concerns among Black Caribbean (BC) and White British (WB) people severely affected by MS.

Design

Mixed methods study of 43 BC and 43 WB people with MS (PwMS) with an Expanded Disability Status Scale (EDSS) ≥6 involving data from in clinical records, face-to-face structured interviews and a nested-qualitative component. Progression Index (PI) and Multiple Sclerosis Severity Score (MSSS) were calculated. To control for selection bias, propensity scores were derived for each patient and adjusted for in the comparative statistical analysis; qualitative data were analysed using the framework approach.

Results

Median EDSS for both groups was (6.5; range: 6.0–9.0). Progression Index (PI) and Multiple Sclerosis Severity Score (MSSS) based on neurological assessment of current EDSS scores identified BC PwMS were more likely to have aggressive disease (PI F = 4.04, p = 0.048, MSSS F = 10.30, p<0.001). Patients’ reports of the time required to reach levels of functional decline equivalent to different EDSS levels varied by group; EDSS 4: BC 2.7 years v/s WB 10.2 years (U = 258.50, p = 0.013), EDSS 6∶6.1 years BC v/s WB 12.7 years (U = 535.500, p = 0.011), EDSS 8: BC 8.7 years v/s WB 10.2 years. Both groups reported high symptom burden. BC PwMS were more cognitively impaired than WB PwMS (F = 9.65, p = 0.003). Thematic analysis of qualitative interviews provides correspondence with quantitative findings; more BC than WB PwMS referred to feelings of extreme frustration and unresolved loss/confusion associated with their rapidly advancing disease. The interviews also reveal the centrality, meanings and impact of common MS-related symptoms.

Conclusions

Delays in diagnosis should be avoided and more frequent reviews may be justified by healthcare services. Culturally acceptable interventions to better support people who perceive MS as an assault on identity should be developed to help them achieve normalisation and enhance self-identity.     

Co-administration of Selenium with Inorganic Mercury Alters the Disposition of Mercuric Ions in Rats

Biological Trace Element Research - Mercury (Hg) is a common environmental toxicant to which humans are exposed regularly through occupational and dietary means. Although selenium supplementation...     

Late‐glacial recolonization and phylogeography of European red deer (Cervus elaphus L.)

The Pleistocene was an epoch of extreme climatic and environmental changes. How individual species responded to the repeated cycles of warm and cold stages is a major topic of debate. For the European fauna and flora, an expansion–contraction model has been suggested, whereby temperate species were restricted to southern refugia during glacial times and expanded northwards during interglacials, including the present interglacial (Holocene). Here, we test this model on the red deer (Cervus elaphus) a large and highly mobile herbivore, using both modern and ancient mitochondrial DNA from the entire European range of the species over the last c. 40 000 years. Our results indicate that this species was sensitive to the effects of climate change. Prior to the Last Glacial Maximum (LGM) haplogroups restricted today to South‐East Europe and Western Asia reached as far west as the UK. During the LGM, red deer was mainly restricted to southern refugia, in Iberia, the Balkans and possibly in Italy and South‐Western Asia. At the end of the LGM, red deer expanded from the Iberian refugium, to Central and Northern Europe, including the UK, Belgium, Scandinavia, Germany, Poland and Belarus. Ancient DNA data cannot rule out refugial survival of red deer in North‐West Europe through the LGM. Had such deer survived, though, they were replaced by deer migrating from Iberia at the end of the glacial. The Balkans served as a separate LGM refugium and were probably connected to Western Asia with genetic exchange between the two areas.     

The MIF Antagonist ISO-1 Attenuates Corticosteroid-Insensitive Inflammation and Airways Hyperresponsiveness in an Ozone-Induced Model of COPD

Introduction

Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine associated with acute and chronic inflammatory disorders and corticosteroid insensitivity. Its expression in the airways of patients with chronic obstructive pulmonary disease (COPD), a relatively steroid insensitive inflammatory disease is unclear, however.

Methods

Sputum, bronchoalveolar lavage (BAL) macrophages and serum were obtained from non-smokers, smokers and COPD patients. To mimic oxidative stress-induced COPD, mice were exposed to ozone for six-weeks and treated with ISO-1, a MIF inhibitor, and/or dexamethasone before each exposure. BAL fluid and lung tissue were collected after the final exposure. Airway hyperresponsiveness (AHR) and lung function were measured using whole body plethysmography. HIF-1α binding to the Mif promoter was determined by Chromatin Immunoprecipitation assays.

Results

MIF levels in sputum and BAL macrophages from COPD patients were higher than those from non-smokers, with healthy smokers having intermediate levels. MIF expression correlated with that of HIF-1α in all patients groups and in ozone-exposed mice. BAL cell counts, cytokine mRNA and protein expression in lungs and BAL, including MIF, were elevated in ozone-exposed mice and had increased AHR. Dexamethasone had no effect on these parameters in the mouse but ISO-1 attenuated cell recruitment, cytokine release and AHR.

Conclusion

MIF and HIF-1α levels are elevated in COPD BAL macrophages and inhibition of MIF function blocks corticosteroid-insensitive lung inflammation and AHR. Inhibition of MIF may provide a novel anti-inflammatory approach in COPD.     

Prevention of peroxynitrite-induced apoptosis of motor neurons and PC12 cells by tyrosine-containing peptides

Although peroxynitrite stimulates apoptosis in many cell types, whether peroxynitrite acts directly as an oxidant or the induction of apoptosis is because of the radicals derived from peroxynitrite decomposition remains unknown. Before undergoing apoptosis because of trophic factor deprivation, primary motor neuron cultures become immunoreactive for nitrotyrosine. We show here using tyrosine-containing peptides that free radical processes mediated by peroxynitrite decomposition products were required for triggering apoptosis in primary motor neurons and in PC12 cells cultures. The same concentrations of tyrosine-containing peptides required to prevent the nitration and apoptosis of motor neurons induced by trophic factor deprivation and of PC12 cells induced by peroxynitrite also prevented peroxynitrite-mediated nitration of motor neurons, brain homogenates, and PC12 cells. The heat shock protein 90 chaperone was nitrated in both trophic factor-deprived motor neurons and PC12 cells incubated with peroxynitrite. Tyrosine-containing peptides did not affect the induction of PC12 cell death by hydrogen peroxide. Tyrosine-containing peptides should protect by scavenging peroxynitrite-derived radicals and not by direct reactions with peroxynitrite as they neither increase the rate of peroxynitrite decomposition nor decrease the bimolecular peroxynitrite-mediated oxidation of thiols. These results reveal an important role for free radical-mediated nitration of tyrosine residues, in apoptosis induced by endogenously produced and exogenously added peroxynitrite; moreover, tyrosine-containing peptides may offer a novel strategy to neutralize the toxic effects of peroxynitrite.     

Two-step method to isolate target recombinant protein from co-purified bacterial contaminant SlyD after immobilised metal affinity chromatography

As part of a study to purify the internal domain of HER2 (ICD) from recombinant expression, through metal immobilised affinity chromatography (IMAC), we encountered a contaminant, SlyD, a 29 kDa native E. coli protein. SlyD is a recurrent contaminant, with a histidine rich domain enabling binding to IMAC columns and thus co-elution with the target protein. Research has been carried out on this protein and its purification, however, no work mentions how to treat it as a true contaminant or describe procedures to isolate it from target proteins. In this report, we described a two-step chromatographic method for the purification of ICD, including IMAC as a capture step and size exclusion chromatography (SEC) as a polishing step. IMAC allowed us to purify ICD from bacterial crude with SlyD co-eluting. SEC then allowed us to resolve ICD from SlyD and achieve a purity greater than 95% for ICD. However, this method has been developed to accommodate any protein whose molecular weight is different enough from SlyD to be separated by SEC.