Increased risk of leukaemia in children with Down syndrome: a somatic evolutionary view.

Children show a higher incidence of leukaemia compared with young adolescents, yet their cells are less damaged because of their young age. Children with Down syndrome (DS) have an even higher risk of developing leukaemia during the first years of life. The presence of a constitutive trisomy of chromosome 21 (T21) in DS acts as a genetic driver for leukaemia development, however, additional oncogenic mutations are required. Therefore, T21 provides the opportunity to better understand leukaemogenesis in children. Here, we describe the increased risk of leukaemia in DS during childhood from a somatic evolutionary view. According to this idea, cancer is caused by a variation in inheritable phenotypes within cell populations that are subjected to selective forces within the tissue context. We propose a model in which the increased risk of leukaemia in DS children derives from higher rates of mutation accumulation, already present during fetal development, which is further enhanced by changes in selection dynamics within the fetal liver niche. This model could possibly be used to understand the rate-limiting steps of leukaemogenesis early in life.

Systematic generation of in vivo G protein-coupled receptor mutants in the rat.

G-protein-coupled receptors (GPCRs) constitute a large family of cell surface receptors that are involved in a wide range of physiological and pathological processes, and are targets for many therapeutic interventions. However, genetic models in the rat, one of the most widely used model organisms in physiological and pharmacological research, are largely lacking. Here, we applied N-ethyl-N-nitrosourea (ENU)-driven target-selected mutagenesis to generate an in vivo GPCR mutant collection in the rat. A pre-selected panel of 250 human GPCR homologs was screened for mutations in 813 rats, resulting in the identification of 131 non-synonymous mutations. From these, seven novel potential rat gene knockouts were established as well as 45 lines carrying missense mutations in various genes associated with or involved in human diseases. We provide extensive in silico modeling results of the missense mutations and show experimental data, suggesting loss-of-function phenotypes for several models, including Mc4r and Lpar1. Taken together, the approach used resulted not only in a set of novel gene knockouts, but also in allelic series of more subtle amino acid variants, similar as commonly observed in human disease. The mutants presented here may greatly benefit studies to understand specific GPCR function and support the development of novel therapeutic strategies.

Isolation of a deep-sea barophilic bacterium and some of its growth characteristics.

A bacterium, a spirillum, has been isolated from a deep-sea sample and has been found to grow optimally at about 500 bars and 2 degrees to 4 degrees C. These conditions are similar to those prevailing at the 5700-meter depth from which the sample was collected. The organism grows at these pressures and temperatures with a generation time of between 4 and 13 hours; at atmospheric pressure and 2 degrees to 4 degrees C, the generation time is about 3 to 4 days.

The effects of disruption of A kinase anchoring protein-protein kinase A association on protein kinase A signalling in neuroendocrine melanotroph cells of Xenopus laevis.

The secretory activity of melanotroph cells from Xenopus laevis is regulated by multiple neurotransmitters that act through adenylyl cyclase. Cyclic adenosine monophosphate (cAMP), acting on protein kinase A (PKA), stimulates the frequency of intracellular Ca(2+) oscillations and the secretory activity of the melanotroph cell. Anchoring of PKA near target proteins is essential for many PKA-regulated processes, and the family of A kinase anchoring proteins (AKAPs) is involved in the compartmentalisation of PKA type II (PKA II) regulatory subunits. In the present study, we determined to what degree cAMP signalling in Xenopus melanotrophs depends on compartmentalised PKA II. For this purpose, a membrane-permeable stearated form of Ht31 (St-Ht31), which dislodges PKA II from AKAP (thus disrupting PKA II signalling), was used. The effect of St-Ht31 on both secretion of radiolabelled peptides and intracellular Ca(2+) signalling by superfused Xenopus melanotrophs was assessed. St-Ht31 stimulated secretion but had no effect on Ca(2+) signalling. We conclude Xenopus melanotrophs possess a St-Ht31-sensitive PKA II that is associated with the exocytosis machinery and, furthermore, that Ca(2+) signalling is regulated by an AKAP-independent signalling system. Moreover, our results support a recent proposal that AKAP participates in regulating PKA activity independently from cAMP.

Deficiency or inhibition of lysophosphatidic acid receptor 1 protects against hyperoxia-induced lung injury in neonatal rats.

AIM: Blocking of lysophosphatidic acid (LPA) receptor (LPAR) 1 may be a novel therapeutic option for bronchopulmonary dysplasia (BPD) by preventing the LPAR1-mediated adverse effects of its ligand (LPA), consisting of lung inflammation, pulmonary arterial hypertension (PAH) and fibrosis. METHODS: In Wistar rats with experimental BPD, induced by continuous exposure to 100% oxygen for 10 days, we determined the beneficial effects of LPAR1 deficiency in neonatal rats with a missense mutation in cytoplasmic helix 8 of LPAR1 and of LPAR1 and -3 blocking with Ki16425. Parameters investigated included survival, lung and heart histopathology, fibrin and collagen deposition, vascular leakage and differential mRNA expression in the lungs of key genes involved in LPA signalling and BPD pathogenesis. RESULTS: LPAR1-mutant rats were protected against experimental BPD and mortality with reduced alveolar septal thickness, lung inflammation (reduced influx of macrophages and neutrophils, and CINC1 expression) and collagen III deposition. However, LPAR1-mutant rats were not protected against alveolar enlargement, increased medial wall thickness of small arterioles, fibrin deposition and vascular alveolar leakage. Treatment of experimental BPD with Ki16425 confirmed the data observed in LPAR1-mutant rats, but did not reduce the pulmonary influx of neutrophils, CINC1 expression and mortality in rats with experimental BPD. In addition, Ki16425 treatment protected against PAH and right ventricular hypertrophy. CONCLUSION: LPAR1 deficiency attenuates pulmonary injury by reducing pulmonary inflammation and fibrosis, thereby reducing mortality, but does not affect alveolar and vascular development and, unlike Ki16425 treatment, does not prevent PAH in neonatal rats with experimental BPD.

Architecture of the outer membrane of Escherichia coli K12. I. Action of phospholipases A2 and C on wild type strains and outer membrane mutants.

Phospholipids in whole cells of wild type Escherichia coli K12 are not degraded by exogenous phospholipases, whereas those of isolated outer membranes are completely degraded. It is concluded that the resistance of phospholipids in whole cells is due to shielding by one or more other outer membrane components. The nature of the shielding component(s) was investigated by testing the sensitivity of whole cells of a number of outer membrane mutants. Mutants lacking both major outer membrane proteins b and d or the heptose-bound glucose of their lipopolysaccharide, are sensitive to exogenous exogenous phospholipases. Moreover, cells of a mutant which lacks protein d can be sensitized by pretreatment of the cells with EDTA. From these results and from data on the chemical composition of the outer membranes, it is concluded that proteins b and d, the heptose-bound glucose of lipopolysaccharide and divalent cations are responsible for the inaccessibility of phospholipids to to exogenous phospholipases.

Cell surface of the fish pathogenic bacterium Aeromonas salmonicida. I. Relationship between autoagglutination and the presence of a major cell envelope protein.

A comparison was made of membrane protein patterns of various Aeromonas salmonicida strains, initially isolated from different habitats with respect to fish species affected, pathological entity, and geographic location of the outbreak of the disease. A major protein with a molecular weight of 54 000 was found in all autoagglutinating strains, whereas this protein is present in low amounts, or not at all, in non-autoagglutinating strains. Evidence for a causal relationship between the presence of this protein and the phenomenon of autoagglutination came from the observation that a change of the growth medium led simultaneously to an almost complete loss of the additional cell envelope protein and the property of autoagglutination. As it has already been reported that autoagglutination is correlated with the presence of an additional cell surface layer, we hypothesize that the additional cell envelope protein is the (major) subunit of this layer. The application of the gel immuno radio assay, an immunological technique suited to detect antigens in a gel, revealed that the additional cell envelope proteins of all tested strains are immunologically related. The possibility to the use of this protein as a component of a vaccine against A. salmonicida infections is discussed.

Voltage sensing in the PhoE and OmpF outer membrane porins of Escherichia coli: role of charged residues.

The porins PhoE and OmpF form anion and cation-selective pores, respectively, in the outer membrane of Escherichia coli. Each monomer of these trimeric proteins consists of a 16-stranded beta-barrel, which contains a constriction at half the height of the channel. The functional significance of a transverse electrical field that is formed by charged amino acid residues within the constriction zone was investigated. For this purpose, the PhoE residues R37, R75, K18 and E110 were substituted by neutral amino acids. The mutant pores allowed an increased permeation of beta-lactam antibiotics across the outer membrane in vivo, although the single channel conductance, measured in planar lipid bilayers, was not increased or even slightly decreased. Replacement of the positively charged residues resulted in a decreased voltage sensitivity, whereas the substitution of a negatively charged residue resulted in an increased voltage sensitivity. Similar substitutions in OmpF caused the opposite effects, i.e. the substitution of positive and negative charges resulted in increased and decreased voltage sensitivity, respectively. Together, the results suggest that opposite charges, i.e. positive charges in anion-selective and negative charges in cation-selective porins, act as sensors for voltage gating.

Role of the constriction loop in the gating of outer membrane porin PhoE of Escherichia coli.

Porins form voltage-gated channels in the bacterial outer membrane. These proteins are composed of three identical subunits, each forming a 16-stranded beta-barrel. In this study, the role in voltage gating of a loop that forms a constriction within the pore was studied. The channel characteristics of mutant PhoE porins, in which the tip of the constriction loop was connected to the barrel wall, were determined. Whereas the properties of several mutant channels were changed, all of these channels could still be closed at high potential, showing that a gross movement of the constriction loop within the channel is not implicated in voltage gating.

Effect of polyoxyethylene sorbate compounds (Tweens) on colonial morphology, growth, and ultrastructure of Mycobacterium paratuberculosis.

Polyoxyethylene sorbate (Tween) compounds were tested to compare their growth stimulation effects on Mycobacterium paratuberculosis. Three low passage and three high passage clinical isolates and ATCC strain 19698 were used. Tween 20, 40, 60, and 80 were tested at concentrations of 0, 0.001, 0.01, 1.0, and 3.0% (w/v) in radiometric broth culture media and in Middlebrook 7H9 agar plates. Scanning and transmission electron microscopy were used to examine cell wall appearance and ultrastructure, respectively. In broth culture, 0.1% (w/v) Tween 60 most dramatically enhanced growth of M. paratuberculosis ATCC strain 19698. The effects of Tween 40 and 80 on growth took a bimodal form, enhancing growth at concentration ranges of 0-0.01% and 0.1-1.0% (w/v) but suppressing growth at concentrations of 0.01-0.1% (w/v). Two of three high passage clinical isolates grew optimally in the presence of 1.0% (w/v) Tween 80, while the remaining high passage isolate and all three low passage isolates grew best in media containing 0.1% (w/v) Tween 80. Colonial morphology of all strains grown on Middlebrook 7H9 agar without Tween 80 was irregular and granular whereas colonies on plate media containing greater than 0.01% (w/v) Tween 80 were entire, smooth, and domed. Scanning electron microscopy also revealed a transition from rough to smooth cell walls with increasing Tween 80 concentration. Transmission electron microscopy showed the presence of low electron dense intracellular vacuoles in Tween 80 grown M. paratuberculosis cells. Thus, Tweens altered colonial morphology, the cell wall surface, and ultrastructure of M. paratuberculosis and stimulated its growth in vitro in a concentration-dependent, and often bimodal, fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of mutations and deletions on expression of the Enterobacter cloacae ompX gene.

The Enterobacter cloacae outer membrane protein OmpX is involved in resistance to beta-lactams, and possesses virulence characteristics. To gain more insight into the genetic elements that are important for OmpX expression, several mutations were introduced at, and immediately upstream of, the N-terminus of the OmpX coding sequence. These mutations enabled us to delete the 5' untranslated region and the signal peptide coding sequence. The former led to decreased ompX expression, indicating an unexpected and hitherto unexplained role for this region. Deletion of the signal peptide coding sequence blocked transport across the cytoplasmic membrane, indicating that translocation of OmpX across the cytoplasmic membrane is mediated by the general secretory pathway.

Comparison of serum thyroglobulin, 131I and 201Tl scintigraphy in the postoperative follow-up of differentiated thyroid cancer.

To evaluate the significance of 201Tl scintigraphy, 131I scintigraphy and serum thyroglobulin (Tg) levels in the postoperative follow-up of differentiated thyroid carcinoma, 86 patients were examined. Serum Tg was found to have the highest sensitivity (97%) as well as specificity (100%) for the detection of local tumour or metastases. Sensitivity and specificity of 131I scintigraphy after a therapeutic dose of 6100 MBq were 77 and 98%, respectively. For a diagnostic dose of 185 MBq 131I, sensitivity and specificity were 57 and 98%; for 74 MBq 201Tl these figures were 55 and 91%, respectively. The combination of serum Tg and 131I had a sensitivity of 100% and a specificity of 98% to detect the presence, although not always the localization, of persistent or recurrent tumour. 201Tl scintigraphy can provide useful additional information about the localization of local recurrences and metastases, especially in cases where serum Tg becomes positive and 131I uptake is absent. One has to bear in mind the possibility of discrepant results of 131I and 201Tl scintigraphy, in different tumour sites in one patient. There appeared to be no specific tendency for differential uptake of 131I versus 201Tl in relation to tumour type (papillary versus follicular) or localization with the possible exception of a higher preference for 131I uptake in cases of micrometastases of papillary cancer in the lungs.

Problem solving by telephone in palliative care: use of a predetermined assessment tool within a program of home care technology.

In the Rotterdam Cancer Institute, nurses from the palliative care unit take care of accessibility outside office hours for patients transferred home with technical equipment for symptoms control. The nurses use a predetermined assessment tool (PAT) for handling telephone calls. A retrospective evaluation on the registration forms used over the years 1997-1999 was performed to evaluate the telephone service. A total of 124 patients in need of technical support was transferred from the hospital during the study period: 52 in 1997, 33 in 1998, and 39 in 1999. Over the years, 157 calls were registered from 64 (52%) patients. In 1997, the majority of the calls (73%) came from the patient or the family. The frequency of calls from the general practitioner did not change, but calls from the district nurse increased from 12% in 1997 to 35% in 1998, and 48% in 1999. Professionals working in nursing homes have used the telephone service since 1998. The reasons for calling were pain (40%), symptoms other than pain (19%), technical problems (33%), general information and advice (6%), and logistic problems (2%). In 152 of the 157 telephone calls (97%), problems could be solved without admission. The mean time to answer a call was 16 minutes. The telephone service and the use of the PAT made it possible to solve 97% of problems without admission.

The role of SRY-related HMG box transcription factor 4 (SOX4) in tumorigenesis and metastasis: friend or foe?

Development and progression of cancer are mediated by alterations in transcriptional networks, resulting in a disturbed balance between the activity of oncogenes and tumor suppressor genes. Transcription factors have the capacity to regulate global transcriptional profiles, and are consequently often found to be deregulated in their expression and function during tumorigenesis. Sex-determining region Y-related high-mobility-group box transcription factor 4 (SOX4) is a member of the group C subfamily of the SOX transcription factors and has a critical role during embryogenesis, where its expression is widespread and controls the development of numerous tissues. SOX4 expression is elevated in a wide variety of tumors, including leukemia, colorectal cancer, lung cancer and breast cancer, suggesting a fundamental role in the development of these malignancies. In many cancers, deregulated expression of this developmental factor has been correlated with increased cancer cell proliferation, cell survival, inhibition of apoptosis and tumor progression through the induction of an epithelial-to-mesenchymal transition and metastasis. However, in a limited subset of tumors, SOX4 has also been reported to act as a tumor suppressor. These opposing roles suggest that the outcome of SOX4 activation depends on the cellular context and the tumor origin. Indeed, SOX4 expression, transcriptional activity and target gene specificity can be controlled by signaling pathways, including the transforming growth factor-ß and the WNT pathway, as well as at the post-translational level through regulation of protein stability and interaction with specific cofactors, such as TCF, syntenin-1 and p53. Here, we provide an overview of our current knowledge concerning the role of SOX4 in tumor development and progression.

FOXP1 acts through a negative feedback loop to suppress FOXO-induced apoptosis.

Transcriptional activity of Forkhead box transcription factor class O (FOXO) proteins can result in a variety of cellular outcomes depending on cell type and activating stimulus. These transcription factors are negatively regulated by the phosphoinositol 3-kinase (PI3K)-protein kinase B (PKB) signaling pathway, which is thought to have a pivotal role in regulating survival of tumor cells in a variety of cancers. Recently, it has become clear that FOXO proteins can promote resistance to anti-cancer therapeutics, designed to inhibit PI3K-PKB activity, by inducing the expression of proteins that provide feedback at different levels of this pathway. We questioned whether such a feedback mechanism may also exist directly at the level of FOXO-induced transcription. To identify critical modulators of FOXO transcriptional output, we performed gene expression analyses after conditional activation of key components of the PI3K-PKB-FOXO signaling pathway and identified FOXP1 as a direct FOXO transcriptional target. Using chromatin immunoprecipitation followed by next-generation sequencing, we show that FOXP1 binds enhancers that are pre-occupied by FOXO3. By sequencing the transcriptomes of cells in which FOXO is specifically activated in the absence of FOXP1, we demonstrate that FOXP1 can modulate the expression of a specific subset of FOXO target genes, including inhibiting expression of the pro-apoptotic gene BIK. FOXO activation in FOXP1-knockdown cells resulted in increased cell death, demonstrating that FOXP1 prevents FOXO-induced apoptosis. We therefore propose that FOXP1 represents an important modulator of FOXO-induced transcription, promoting cellular survival.

Normal hospital and low-bacterial diet in patients with cytopenia after intensive chemotherapy for hematological malignancy: a study of safety.

BACKGROUND: The purpose of this randomized, controlled pilot study is to address the question whether normal hospital diet (NHD) is safe when compared with low-bacterial diet (LBD) given to prevent infections in cytopenic patients who receive antimicrobial prophylaxis (AP). PATIENTS AND METHODS: The patients were randomized into two groups: one group to receive AP and LBD, the other to receive the same AP and NHD. The primary outcome parameter is colonization of the digestive tract with aerobic gram-negative bacilli and yeasts. Secondary outcome parameters were infections and total societal costs. RESULTS: No statistically significant differences between treatment groups were observed regarding the primary outcome parameter, gut colonization by yeasts or gram-negative bacilli, or infections, use of antimicrobials, days with fever and total societal costs. CONCLUSION: On the basis of the results of this pilot study, NHD appears to be as safe as LBD in patients with chemotherapy-induced cytopenia. Furthermore, the results indicate that LBD may offer no additional benefit as an infection preventive measure to the measures already implemented, such as AP. Thus, a larger randomized study, powered adequately to determine noninferiority of NHD to LBD is warranted and safe to be carried out.

Dependence of reproduction rate on pressure as a hallmark of deep-sea bacteria.

Strains of bacteria in axenic culture were isolated from samples of depths between 1,957 and 10,476 m of the Pacific Ocean. All of the bacteria from this range of depths were barophilic. The pressure at which the rate of reproduction was maximal was found to be correlated with the depth of origin of the isolates.

Reproduction of Bacillus stearothermophilus as a Function of Temperature and Pressure.

The colony-forming ability and the rate of reproduction of Bacillus stearothermophilus were determined as a function of temperature and pressure. Colonies were formed between 39 and 70 degrees C at atmospheric pressure and between 54 and 67 degrees C at 45 MPa. Colonies did not form at 55.9 MPa. The rate of reproduction in broth cultures decreased with increasing pressure at all temperatures. The rate of reproduction diminished rapidly with pressure above 10.4 MPa. Therefore, increased hydrostatic pressure was not sufficient to enable B. stearothermophilus to function beyond the temperature limiting growth and reproduction at atmospheric pressure, and B. stearothermophilus should grow in naturally or artificially warmed regions of the deep sea, where the pressure is less than approximately 50 MPa, although growth rates would be low above 10 MPa.