P-Glycoprotein is localized in intermediate-density membrane microdomains distinct from classical lipid rafts and caveolar domains

P-glycoprotein (Pgp), a member of the ATP-binding cassette (ABC) superfamily responsible for the ATP-driven extrusion of diverse hydrophobic molecules from cells, is a cause of multidrug resistance in human tumours. Pgp can also operate as a phospholipid and glycosphingolipid flippase, and has been functionally linked to cholesterol, suggesting that it might be associated with sphingolipid-cholesterol microdomains in cell membranes. We have used nonionic detergent extraction and density gradient centrifugation of extracts from the multidrug-resistant Chinese hamster ovary cell line, CH(R)B30, to address this question. Our data indicate that Pgp is localized in intermediate-density membrane microdomains different from classical lipid rafts enriched in Src-family kinases. We demonstrate that Brij-96 can selectively isolate the Pgp domains, separating them from the caveolar and classical lipid rafts. Pgp was found entirely in the Brij-96-insoluble domains, and only partially in the Triton X-100-insoluble membrane microdomains. We studied the sensitivity of these domains to cholesterol removal, as well as their relationship to GM(1) ganglioside- and caveolin-1-enriched caveolar domains. We found that the buoyant density of the Brij-96-based Pgp-containing microdomains was sensitive to cholesterol removal by methyl-beta-cyclodextrin. The Brij-96 domains retained their structural integrity after cholesterol depletion while, in contrast, the Triton X-100-based caveolin-1/GM(1) microdomains did not. Using confocal fluorescence microscopy, we determined that caveolin-1 and GM(1) colocalized, while Pgp and caveolin-1, or Pgp and GM(1), did not. Our results suggest that Pgp does not interact directly with caveolin-1, and is localized in intermediate-density domains, distinct from classical lipid rafts and caveolae, which can be isolated using Brij-96.     

LKB1 as a Gatekeeper of Hepatocyte Proliferation and Genomic Integrity during Liver Regeneration

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Initiation of insulin therapy in elderly patients taking oral antidiabetes drugs

Background

We sought to estimate the rate of initiation of insulin therapy among elderly patients using oral anti-diabetes drugs and to identify the factors associated with this initiation.

Methods

We conducted a population-based cohort study involving people aged 66 or more years who were newly dispensed an oral antidiabetes drug. Individuals who had received acarbose or a thiazolidinedione were excluded. The rate of insulin initiation was calculated by use of the Kaplan–Meier method. Factors associated with insulin initiation were identified by multivariable Cox regression analyses.

Results

In this cohort of 69 674 new users of oral antidiabetes drugs, insulin was initiated at rate of 9.7 cases per 1000 patient-years. Patients who had initially received an insulin secretagogue (rather than metformin), who were prescribed an oral antidiabetes drug by an endocrinologist or an internist, who received higher initial doses of an oral antidiabetes drug, who received oral corticosteroids, used glucometer strips, or were admitted to hospital in the year before initiation of oral antidiabetes therapy, or who received 16 or more medications were more likely than those without these characteristics to have insulin therapy initiated. In contrast, patients who received thiazides or who used up to 12 medications (v. none) were less likely to have insulin therapy initiated.

Interpretation

Several factors related to drugs and health services are associated with the initiation of insulin therapy in elderly patients receiving oral antidiabetes drugs. It is unclear whether these factors predict secondary failure of oral antidiabetes drugs or instead reflect better management of type 2 diabetes.Type 2 diabetes is a progressive disease that requires ongoing increases in doses and complexity of hypo-glycemic pharmacotherapy.¹ Although insulin may be the first agent prescribed to patients with type 2 diabetes who have marked hyperglycemia, oral antidiabetes drugs are usually the first pharmacologic treatment. In general, these drugs are first prescribed as monotherapy; however, combination therapy with 2 oral antidiabetes drugs with different mechanisms may also be a first-line option.^2–4 Unfortunately, oral antidiabetes drugs have limited efficacy for long-term glucose lowering^1,5 and, therefore, many patients may require insulin to achieve better metabolic control.⁶There are several factors that may account for the need to initiate insulin therapy in patients taking oral antidiabetes drugs, including progressive β-cell failure,⁷ deterioration of insulin sensitivity because of glucose toxicity or the development of resistance to the oral antidiabetes drug.^8,9 Disease severity, a younger age at diagnosis^1,10 and poor adherence to treatment may also lead to poor metabolic control in patients with diabetes.¹¹Our study included an outpatient population of elderly patients, all of whom were new users of an oral antidiabetes drug. We sought to estimate the rate of initiation of insulin therapy and to identify factors associated with initiation of insulin therapy.     

Separable roles of UFO during floral development revealed by conditional restoration of gene function

The UNUSUAL FLORAL ORGANS (UFO) gene is required for several aspects of floral development in Arabidopsis including specification of organ identity in the second and third whorls and the proper pattern of primordium initiation in the inner three whorls. UFO is expressed in a dynamic pattern during the early phases of flower development. Here we dissect the role of UFO by ubiquitously expressing it in ufo loss-of-function flowers at different developmental stages and for various durations using an ethanol-inducible expression system. The previously known functions of UFO could be separated and related to its expression at specific stages of development. We show that a 24- to 48-hour period of UFO expression from floral stage 2, before any floral organs are visible, is sufficient to restore normal petal and stamen development. The earliest requirement for UFO is during stage 2, when the endogenous UFO gene is transiently expressed in the centre of the wild-type flower and is required to specify the initiation patterns of petal, stamen and carpel primordia. Petal and stamen identity is determined during stages 2 or 3, when UFO is normally expressed in the presumptive second and third whorl. Although endogenous UFO expression is absent from the stamen whorl from stage 4 onwards, stamen identity can be restored by UFO activation up to stage 6. We also observed floral phenotypes not observed in loss-of-function or constitutive gain-of-function backgrounds, revealing additional roles of UFO in outgrowth of petal primordia.     

Human health risks associated with heavy metals in soil in different areas of San Luis Potosí, México

The aim of this study was to develop a health risk assessment in different areas of San Luis Potosí, México. Four heavy metals (arsenic, mercury, cadmium, and lead) were analyzed in soil from communities assessed. The mean arsenic concentration was significantly higher (p < .05) in the city of San Luis Potosí (51.85 mg/kg) compared to the other assessed areas (5.52–8.43 mg/kg). For cadmium, the mean concentration was significantly higher (p < .05) in Santa Maria Picula (7.46 mg/kg) than in the other areas (3.72–4.15 mg/kg). Regarding mercury levels, a significantly higher (p < .05) mean concentration was found in Mezquitic (1.54 mg/kg) compared to other areas (0.56–0.81 mg/kg). Lastly, when comparing the mean lead concentration in the city of San Luis Potosí (108 mg/kg), it was found to be significantly lower (p < .05) than in other areas (219–227 mg/kg). Subsequently, a probabilistic health risk assessment was performed, ingestion was the major exposure pathway for all four metals. Maximum cumulative hazard index (HI) values showed higher risk in all sampled locations (HIs > 1.0), suggesting that these sites can pose a non-carcinogenic risk to the populations (children) living in those areas. This study highlights the necessity of establishing a biomonitoring program for the surveillance of the child populations living in the assessed locations.     

Comprehensive profiling of 8p11-12 amplification in breast cancer

In human carcinomas, especially breast cancer, chromosome arm 8p is frequently involved in complex chromosomal rearrangements that combine amplification at 8p11-12, break in the 8p12-21 region, and loss of 8p21-ter. Several studies have identified putative oncogenes in the 8p11-12 amplicon. However, discrepancies and the lack of knowledge on the structure of this amplification lead us to think that the actual identity of the oncogenes is not definitively established. We present here a comprehensive study combining genomic, expression, and chromosome break analyses of the 8p11-12 region in breast cell lines and primary breast tumors. We show the existence of four amplicons at 8p11-12 using array comparative genomic hybridization. Gene expression analysis of 123 samples using DNA microarrays identified 14 genes significantly overexpressed in relation to amplification. Using fluorescence in situ hybridization analysis on tissue microarrays, we show the existence of a cluster of breakpoints spanning a region just telomeric to and associated with the amplification. Finally, we show that 8p11-12 amplification has a pejorative effect on survival in breast cancer.     

Does Cognitive Function Increase over Time in the Healthy Elderly?

Background

In dementia screening, most studies have focused on early cognitive impairment by comparing patients suffering from mild dementia or mild cognitive impairment with normal subjects. Few studies have focused on modifications over time of the cognitive function in the healthy elderly. The objective of the present study was to analyze the cognitive function changes of two different samples, born > 15 years apart.

Method

A first sample of 204 cognitively normal participants was recruited in the memory clinic of Broca hospital between 1991 and 1997. A second sample of 177 cognitively normal participants was recruited in 2008–2009 in the same institution. Both samples were from the same districts of Paris and were assessed with the same neuropsychological test battery. Mean cognitive test scores were compared between 1991 and 2008 samples, between < 80 years old and ≥ 80 years old in 1991 and 2008 samples, and finally between subjects < 80 year old of 1991 sample and subjects ≥ 80 years old of the 2008 sample. Means were compared with T-tests stratified on gender, age-groups and educational level.

Results

Cognitive scores were significantly higher in the 2008 sample. Participants < 80 years old outperformed those ≥ 80 in both samples. However, participants < 80 years old in 1991 sample and subjects ≥ 80 in the 2008 sample, born on average in 1923, performed mostly identically.

Conclusion

This study showed a significant increase of cognitive scores over time. Further, contemporary octogenarians in the later sample performed like septuagenarians in the former sample. These findings might be consistent with the increase in life expectancy and life span in good health. The study highlights the necessity to take into account factors which may contaminate and artificially inflate the age-related differences in favor of younger to the older adults.