The asymmetric distribution of the constituents of the Ran system is essential for transport into and out of the nucleus

The GTPase Ran is essential for nuclear import of proteins with a classical nuclear localization signal (NLS). Ran's nucleotide-bound state is determined by the chromatin-bound exchange factor RCC1 generating RanGTP in the nucleus and the cytoplasmic GTPase activating protein RanGAP1 depleting RanGTP from the cytoplasm. This predicts a steep RanGTP concentration gradient across the nuclear envelope. RanGTP binding to importin-beta has previously been shown to release importin-alpha from -beta during NLS import. We show that RanGTP also induces release of the M9 signal from the second identified import receptor, transportin. The role of RanGTP distribution is further studied using three methods to collapse the RanGTP gradient. Nuclear injection of either RanGAP1, the RanGTP binding protein RanBP1 or a Ran mutant that cannot stably bind GTP. These treatments block major export and import pathways across the nuclear envelope. Different export pathways exhibit distinct sensitivities to RanGTP depletion, but all are more readily inhibited than is import of either NLS or M9 proteins, indicating that the block of export is direct rather than a secondary consequence of import inhibition. Surprisingly, nuclear export of several substrates including importin-alpha and -beta, transportin, HIV Rev and tRNA appears to require nuclear RanGTP but may not require GTP hydrolysis by Ran, suggesting that the energy for their nuclear export is supplied by another source.     

Memory and learning abilities in everyday life of the elderly

Self-evaluations by adults (varying in age from 45-92 years) of their memory and learning abilities were investigated and related to performance on laboratory and ecological memory tasks. Hardly any association was found between subjective and objective measures. Self-evaluations were strongly influenced by (systematically varied) frames of reference: optimistic in comparisons with other people, pessimistic in comparisons with their own previous level of functioning. The most frequent problems were 'learning something new' and 'remembering names'. In contrast to external memory aids, cognitive strategies were rarely used spontaneously. Strategy training led to significant improvement of performance, that remained stable at follow-up. A further opportunity for improving performance was realized by ergonomic adaptations of computerized systems (teleshopping). Problems in learning to use such systems were strongly reduced by decreasing the load on working memory and by adapting the system to existing knowledge and skills of the users. A general observation in the different projects was that age-differences could explain only a small percentage of the variance in subjective and objective memory measures.     

Neoplasms of the large intestine in elderly patients

The aim of the study was to compare 2 groups of patients with colorectal cancer treated between 1985-1995. The analysed group consisted of 228 patients aged over 70. Reference group consisted of 330 younger ones. The stage of colorectal cancer (Dukes classification) was similar in both groups. Complications of the colorectal cancer often occurred in the elderly patients (21.9% vs. 10%, respectively). The incidence of associated diseases was also higher (70.6% vs. 59.1%). Operability was similar in both groups (93% vs. 98.8%) but resectability was lower in the elderly group (67.4% vs. 80.1%, respectively). Postoperative complications were observed in the similar percent of cases except those aged over 70 (17.5% vs. 4.9%), especially after emergency operations (31.6% vs. 4.7%, respectively).     

The nuclear cap-binding complex is a novel target of growth factor receptor-coupled signal transduction

In an attempt to further understand how nuclear events (such as gene expression, nuclear import/export, and cell cycle checkpoint control) might be subject to regulation by extracellular stimuli, we sought to identify nuclear activities under growth factor control. Using a sensitive photoaffinity labeling assay that measured [alpha-32P]GTP incorporation into nuclear proteins, we identified the 20-kDa subunit of the nuclear cap-binding complex (CBC) as a protein whose binding activity is greatly enhanced by the extracellular stimulation of serum-arrested cells. The CBC represents a 20- and 80-kDa heterodimer (the subunits independently referred to as CBP20 and CBP80, respectively) that binds the 7-methylguanosine cap on RNAs transcribed by RNA polymerase II. This binding facilitates precursor messenger RNA splicing and export. We have demonstrated that the [alpha-32P]GTP incorporation into CBP20 was correlated with an increased ability of the CBC to bind capped RNA and have used the [alpha-32P]GTP photoaffinity assay to characterize the activation of the CBC in response to growth factors. We show that the CBC is activated by heregulin in HeLa cells and by nerve growth factor in PC12 cells as well as during the G1/S phase of the cell cycle and when cells are stressed with UV irradiation. Additionally, we show that cap-dependent splicing of precursor mRNA, a functional outcome of CBC activation, can be catalyzed by growth factor addition to serum-arrested cells. Taken together, these data identify the CBC as a nuclear target for growth factor-coupled signal transduction and suggest novel mechanisms by which growth factors can influence gene expression and cell growth.     

A second unrelated bone marrow transplant with an unrelated donor marrow: treatment of a patient with relapsed leukemia

To investigate whether statistical parameters used in Down syndrome screening between 15 and 22 weeks of pregnancy can be used at 14 weeks, we assayed alpha-fetoprotein (AFP), unconjugated oestriol (uE3), total human chorionic gonadotrophin (hCG), free alpha-hCG, free beta-hCG, and inhibin-A in 16 pregnancies with Down syndrome in the 14th week of pregnancy and expressed values in multiples of the normal median. The median and standard deviation values for these 16 pregnancies were not materially different from those published for 15-22 weeks. It is reasonable, therefore, to offer Down syndrome screening using these markers starting at 14 completed weeks of pregnancy instead of 15 weeks. It needs to be recognized, however, that serum AFP measurement for neural tube defect screening is less effective at this time than between 16 and 18 weeks of pregnancy.     

Memory training for remembering names: initial results in the healthy elderly

It has been known since the 1940s that nail polishes contain allergenic ingredients. The aim of this study was to clarify whether the nail polishes on the market today contain significant amounts of allergens, and what the solvents are. The following ingredients were determined: toluene, toluene sulfonamide formaldehyde resins, free formaldehyde, acrylates, methacrylates and certain organic solvents. The study comprised 20 brands and 42 samples. All the nail polishes analysed contained allergenic toluene sulfonamide formaldehyde resins (TSFR), in concentrations from 0.08 to 11.0%. The concentration of total formaldehyde varied from 0.02% to 0.5%. The more TSFR a nail polish contained, the higher was its formaldehyde content. Probably not only TSFR-allergic but also formaldehyde-allergic persons may get dermatitis from many of the nail polishes studied. The concentrations of acrylates and methacrylates were so small that they are of practical significance only to those previously sensitized to acrylates. Of the organic solvents, toluene was still widely used, whereas xylene was found in only 1 product. The nail polishes on the market today are not safe for all consumers. However, according to the regulations of the European Union, the packaging labeling of all cosmetic products must be supplied with a list of ingredients from the beginning of 1998. This will help the consumer to avoid allergenic products. A better alternative could, however, be to substitute the most allergenic ingredients with substances possessing minor allergy potency.     

Extended depth of focus in optical microscopy: Assessment of existing methods and a new proposal

Due to depth of focus constraints, the acquisition of a single 2‐D completely in‐focus image of 3‐D objects characterized by a relevant depth dimension is not possible with a standard light microscope. Since the Seventies numerous methods have been proposed to overcome this problem, mainly through different fusion processing techniques to extend the microscope's depth of focus. However, given a specific application, it is very difficult to know which method yields the best results because there are no validated approaches or tested metrics that are suitable for real world cases typically lacking in a reference ground truth. Although the Universal Quality Index (UQI) is widely used to evaluate output quality in image processing, it requires a reference ground truth. Some UQI extensions have been proposed to evaluate the output of fusion methods without a ground truth, but sufficient analyses have not been carried out to confirm their equivalence to the standard UQI in terms of (evaluation) performance. We propose a new method to extend the microscope's depth of focus and, using synthetic stacks of images with ground truth attached, show that it is superior to state‐of‐the‐art methods. We also demonstrate that the output of metrics proposed as UQI extensions is different from that of the UQI. Finally, we validate a new approach to evaluate extended depth of focus methods using real world stacks of slices, as per the UQI, but without the need for a reference ground truth. Microsc. Res. Tech. © 2012 Wiley Periodicals, Inc.     

A validated method for the determination of paracetamol and its glucuronide and sulphate metabolites in the urine of HIV+/AIDS patients using wavelength-switching UV detection

Paracetamol is a safe drug which has been used as an in-vivo probe to determine phase II metabolism in a HIV+/AIDS population. Due to the biohazard nature of HIV-infected samples, a high performance liquid chromatography (HPLC) assay which offers minimal sample manipulation and maximal specificity was developed. This reverse-phase HPLC method uses wavelength-switching UV detection for the simultaneous determination of paracetamol and its glucuronide and sulfate metabolites in HIV-infected urine samples. The solvent systems involves a simple isocratic elution with a composition of 50 mM sodium acetate buffer, pH adjusted to 3.5; acetonitrile (96:4 v/v) modified with 0.35% trifluroacetic acid. The validated method is highly reproducible with an inter-assay variation of < 7%. This method also shows good precision and sensitivity, making it an ideal assay for phenotyping studies to determine the extent of glucurondiation and sulfation activities.     

Copy Number Analysis of 24 Oncogenes: MDM4 Identified as a Putative Marker for Low Recurrence Risk in Non Muscle Invasive Bladder Cancer

Patients with non-muscle invasive bladder cancer (NMIBC) generally have a high risk of relapsing locally after primary tumor resection. The search for new predictive markers of local recurrence thus represents an important goal for the management of this disease. We studied the copy number variations (CNVs) of 24 oncogenes (MDM4, MYCN, ALK, PDGFRA, KIT, KDR, DHFR, EGFR, MET, SMO, FGFR1, MYC, ABL1, RET, CCND1, CCND2, CDK4, MDM2, AURKB, ERBB2, TOP2A, AURKA, AR and BRAF) using multiplex ligation probe amplification technique to verify their role as predictive markers of recurrence. Formalin-fixed paraffin-embedded tissue samples from 43 patients who underwent transurethral resection of the bladder (TURB) were used; 23 patients had relapsed and 20 were disease-free after 5 years. Amplification frequencies were analyzed for all genes and MDM4 was the only gene that showed significantly higher amplification in non recurrent patients than in recurrent ones (0.65 vs. 0.3; Fisher’s test p = 0.023). Recurrence-free survival analysis confirmed the predictive role of MDM4 (log-rank test p = 0.041). Our preliminary results indicate a putative role for the MDM4 gene in predicting local recurrence of bladder cancer. Confirmation of this hypothesis is needed in a larger cohort of NMIBC patients.