Asymmetric loading of Kar9 onto spindle poles and microtubules ensures proper spindle alignment

Spindle alignment is the process in which the two spindle poles are directed toward preselected and opposite cell ends. In budding yeast, the APC-related molecule Kar9 is required for proper alignment of the spindle with the mother-bud axis. We find that Kar9 localizes to the prospective daughter cell spindle pole. Kar9 is transferred from the pole to cytoplasmic microtubules, which are then guided in a myosin-dependent manner to the bud. Clb4/Cdc28 kinase phosphorylates Kar9 and accumulates on the pole destined to the mother cell. Mutations that block phosphorylation at Cdc28 consensus sites result in localization of Kar9 to both poles and target them both to the bud. Thus, Clb4/Cdc28 prevents Kar9 loading on the mother bound pole. In turn, asymmetric distribution of Kar9 ensures that only one pole orients toward the bud. Our results indicate that Cdk1-dependent spindle asymmetry ensures proper alignment of the mitotic spindle with the cell division axis.     

CFTR directly mediates nucleotide-regulated glutathione flux

Studies have shown that expression of cystic fibrosis transmembrane conductance regulator (CFTR) is associated with enhanced glutathione (GSH) efflux from airway epithelial cells, implicating a role for CFTR in the control of oxidative stress in the airways. To define the mechanism underlying CFTR-associated GSH flux, we studied wild-type and mutant CFTR proteins expressed in Sf9 membranes, as well as purified and reconstituted CFTR. We show that CFTR-expressing membrane vesicles mediate nucleotide-activated GSH flux, which is disrupted in the R347D pore mutant, and in the Walker A K464A and K1250A mutants. Further, we reveal that purified CFTR protein alone directly mediates nucleotide-dependent GSH flux. Interestingly, although ATP supports GSH flux through CFTR, this activity is enhanced in the presence of the non-hydrolyzable ATP analog AMP-PNP. These findings corroborate previous suggestions that CFTR pore properties can vary with the nature of the nucleotide interaction. In conclusion, our data demonstrate that GSH flux is an intrinsic function of CFTR and prompt future examination of the role of this function in airway biology in health and disease.     

Clinical Utility of a Coronary Heart Disease Risk Prediction Gene Score in UK Healthy Middle Aged Men and in the Pakistani Population

BackgroundNumerous risk prediction algorithms based on conventional risk factors for Coronary Heart Disease (CHD) are available but provide only modest discrimination. The inclusion of genetic information may improve clinical utility.MethodsWe tested the use of two gene scores (GS) in the prospective second Northwick Park Heart Study (NPHSII) of 2775 healthy UK men (284 cases), and Pakistani case-control studies from Islamabad/Rawalpindi (321 cases/228 controls) and Lahore (414 cases/219 controls). The 19-SNP GS included SNPs in loci identified by GWAS and candidate gene studies, while the 13-SNP GS only included SNPs in loci identified by the CARDIoGRAMplusC4D consortium.ResultsIn NPHSII, the mean of both gene scores was higher in those who went on to develop CHD over 13.5 years of follow-up (19-SNP p=0.01, 13-SNP p=7x10^-3). In combination with the Framingham algorithm the GSs appeared to show improvement in discrimination (increase in area under the ROC curve, 19-SNP p=0.48, 13-SNP p=0.82) and risk classification (net reclassification improvement (NRI), 19-SNP p=0.28, 13-SNP p=0.42) compared to the Framingham algorithm alone, but these were not statistically significant. When considering only individuals who moved up a risk category with inclusion of the GS, the improvement in risk classification was statistically significant (19-SNP p=0.01, 13-SNP p=0.04). In the Pakistani samples, risk allele frequencies were significantly lower compared to NPHSII for 13/19 SNPs. In the Islamabad study, the mean gene score was higher in cases than controls only for the 13-SNP GS (2.24 v 2.34, p=0.04). There was no association with CHD and either score in the Lahore study.ConclusionThe performance of both GSs showed potential clinical utility in European men but much less utility in subjects from Pakistan, suggesting that a different set of risk loci or SNPs may be required for risk prediction in the South Asian population.     

A plate-based assay system for analyses and screening of the Leishmania major inositol phosphorylceramide synthase

Sphingolipids are key components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is largely conserved. However, in contrast to mammals, which produce sphingomyelin, organisms such as the pathogenic fungi and protozoa synthesize inositol phosphorylceramide (IPC) as the primary phosphosphingolipid. The key step involves the reaction of ceramide and phosphatidylinositol catalysed by IPC synthase, an essential enzyme with no mammalian equivalent encoded by the AUR1 gene in yeast and recently identified functional orthologues in the pathogenic kinetoplastid protozoa. As such this enzyme represents a promising target for novel anti-fungal and anti-protozoal drugs. Given the paucity of effective treatments for kinetoplastid diseases such as leishmaniasis, there is a need to characterize the protozoan enzyme. To this end a fluorescent-based cell-free assay protocol in a 96-well plate format has been established for the Leishmania major IPC synthase. Using this system the kinetic parameters of the enzyme have been determined as obeying the double displacement model with apparent V_max = 2.31 pmol min^?1 U^?1. Furthermore, inhibitory substrate analogues have been identified. Importantly this assay is amenable to development for use in high-throughput screening applications for lead inhibitors and as such may prove to be a pivotal tool in drug discovery.     

A simple cell motility assay demonstrates differential motility of human periodontal ligament fibroblasts, gingival fibroblasts, and pre-osteoblasts

During periodontal regeneration, multiple cell types can invade the wound site, thereby leading to repair. Cell motility requires interactions mediated by integrin receptors for the extracellular matrix (ECM), which might be useful in guiding specific cell populations into the periodontal defect. Our data demonstrate that fibroblasts exhibit differential motility when grown on ECM proteins. Specifically, gingival fibroblasts are twice as motile as periodontal ligament fibroblasts, whereas osteoblasts are essentially non-motile. Collagens promote the greatest motility of gingival fibroblasts in the following order: collagen III>collagen V>collagen I. Differences in motility do not correlate with cell proliferation or integrin expression. Osteoblasts display greater attachment to collagens than does either fibroblast population, but lower motility. Gingival fibroblast motility on collagen I is generally mediated by α2 integrins, whereas motility on collagen III involves α1 integrins. Other integrins (α10 or α11) may also contribute to gingival fibroblast motility. Thus, ECM proteins do indeed differentially promote the cell motility of periodontal cells. Because of their greater motility, gingival fibroblasts have more of a potential to invade periodontal wound sites and to contribute to regeneration. This finding may explain the formation of disorganized connective tissue masses rather than the occurrence of the true regeneration of the periodontium. This research was supported by the Louisiana Board of Regents through the Millennium Trust Health Excellence Fund, HEF-(2000-05)-04.     

Loss of SLC38A5 and FTSJ1 at Xp11.23 in three brothers with non-syndromic mental retardation due to a microdeletion in an unstable genomic region

Using high resolution X chromosome array-CGH we identified an interstitial microdeletion at Xp11.23 in three brothers with moderate to severe mental retardation (MR) without dysmorphic features. The extent of the deletion was subsequently delineated to about 50 kb by regular PCR and included only the SLC38A5 and FTSJ1 genes. The loss of the FTSJ1 MR gene in males is expected to result in the observed phenotype but the contribution of the deletion of the solute carrier SLC38A5 gene is less clear. Their mother also carries the deletion and completely inactivates the aberrant X chromosome. Interestingly, the distal breakpoint is situated within a 200 kb SSX repeat region that appears to stimulate recombination since subtle copy number changes often occur at this location and it is frequently involved in translocations in tumours. Since this apparent SSX unstable structure is flanked proximally by FTSJ1 and PQBP1, subtle deletions or duplications at this location would be expected to cause MR, as in our family. So far, we have screened a cohort of 300 patients but did not find additional aberrations at the FTSJ1 locus indicating that the frequency is likely to be low.     

Synthesis, biological evaluation and molecular modelling of sulfonohydrazides as selective PI3K p110alpha inhibitors

A series of 2-methyl-5-nitrobenzenesulfonohydrazides were prepared and evaluated as inhibitors of PI3K. An isoquinoline derivative shows good selectivity for the p110α isoform over p110β and p110δ, and also demonstrates good in vitro activity in a cell proliferation assay. Molecular modelling provides a rationalisation for the observed SAR.     

Training in the laboratory animal science community: strategies to support adult learning

The essence of learning is change; learning is the process by which learners customize new information to make it personally meaningful and relevant. Training is the process of helping students make those changes. Research indicates that adults learn differently than children or adolescents and that adults consistently use the following six learning strategies: prior experiences; conversations; metacognition; reflection; authentic experiences; and images, pictures, or other types of visuals. Each of these learning strategies can be combined with the other strategies and often build upon each other. A recent study on how health care professionals learn indicated that the learning strategy they used most often was reflection, which supports learning before, during, and after training. Numerous examples are provided in this article describing how to integrate each of the six adult learning strategies into laboratory animal science training. While lectures and other types of direct instruction are appropriate, they are inadequate and ineffective unless they are integrated with and support adult learning strategies. Both the US Department of Agriculture regulations and the Public Health Service Policy mandate that research institutions must ensure that all personnel involved in animal care, treatment, or use are qualified to perform their duties. Applying adult learning strategies to training for the laboratory animal science community will enhance learning and improve both the science and the humane care of the animals, which is a goal our community must continuously strive to achieve.     

Pro-oxidant effect of α-tocopherol in patients with Type 2 Diabetes after an oral glucose tolerance test – a randomised controlled trial

Background

Little information is available on leptin concentrations in individuals with IGT. This study aims to determine and correlate leptin levels to anthropometric measures of obesity in pre-diabetic, (IFG and IGT), type 2 diabetic and normoglycaemic Saudis.

Methods

308 adult Saudis (healthy controls n = 80; pre-diabetes n = 86; Type 2 diabetes n = 142) participated. Anthropometric parameters were measured and fasting blood samples taken. Serum insulin was analysed, using a solid phase enzyme amplified sensitivity immunoassay and also leptin concentrations, using radio-immunoassay. The remaining blood parameters were determined using standard laboratory procedures.

Results

Leptin levels of diabetic and pre-diabetic men were higher than in normoglycaemic men (12.4 [3.2–72] vs 3.9 [0.8–20.0] ng/mL, (median [interquartile range], p = 0.0001). In females, leptin levels were significantly higher in pre-diabetic subjects (14.09 [2.8–44.4] ng/mL) than in normoglycaemic subjects (10.2 [0.25–34.8] ng/mL) (p = 0.046). After adjustment for BMI and gender, hip circumference was associated with log leptin (p = 0.006 with R2 = 0.086) among all subjects.

Conclusion

Leptin is associated with measures of adiposity, hip circumference in particular, in the non-diabetic state among Saudi subjects. The higher leptin level among diabetics and pre-diabetics is not related to differences in anthropometric measures of obesity.     

Home-based exercise rehabilitation in addition to specialist heart failure nurse care: design, rationale and recruitment to the Birmingham Rehabilitation Uptake Maximisation study for patients with congestive heart failure (BRUM-CHF): a randomised controlled trial

Background

We aimed to assess whether we could identify a graded association between increasing number of components of the metabolic syndrome and cardiac structural and functional abnormalities independently of predicted risk of coronary heart disease by the Framingham risk score.

Methods

We conducted a cross-sectional study on a random sample of the urban population of Porto aged 45 years or over. Six hundred and eighty-four participants were included. Data were collected by a structured clinical interview with a physician, ECG and a transthoracic M-mode and 2D echocardiogram. The metabolic syndrome was defined according to ATPIII-NCEP. The association between the number of features of the metabolic syndrome and the cardiac structural and functional abnormalities was assessed by 3 multivariate regression models: adjusting for age and gender, adjusting for the 10-year predicted risk of coronary heart disease by Framingham risk score and adjusting for age, gender and systolic blood pressure.

Results

There was a positive association between the number of features of metabolic syndrome and parameters of cardiac structure and function, with a consistent and statistically significant trend for all cardiac variables considered when adjusting for age and gender. Parameters of left ventricular geometry patterns, left atrial diameter and diastolic dysfunction maintained this trend when taking into account the 10-year predicted risk of coronary heart disease by the Framingham score as an independent variable, while left ventricular systolic dysfunction did not. The prevalence of left ventricular diastolic dysfunction, and the mean left ventricular mass, left ventricular diameter and left atrial diameter increased significantly with the number of features of the metabolic syndrome when additionally adjusting for systolic blood pressure as a continuous variable.

Conclusion

Increasing severity of metabolic syndrome was associated with increasingly compromised structure and function of the heart. This association was independent of Framingham risk score for indirect indices of diastolic dysfunction but not systolic dysfunction, and was not explained by blood pressure level.