Activation of the mouse TATA-less and human TATA-containing UDP-glucuronosyltransferase 1A1 promoters by hepatocyte nuclear factor 1.

UDP-glucuronosyltransferase (UGT) 1A1 (UGT1A1) catalyzes the glucuronidation of bilirubin in liver. Among all UGT isoforms identified to date, it is the only relevant bilirubin-glucuronidating enzyme in human. Because glucuronoconjugation is the major route of bilirubin elimination, any genetic alteration that affects bilirubin glucuronosyltransferase activity may result in a more or less severe hyperbilirubinemia. In this study, we report the cloning and characterization of the transcriptional regulation of the mouse UGT1A1 gene. Primary-structure analysis of the mouse Thymidine Adevice promoter revealed marked differences with its human homolog. First, the mouse promoter lacks the highly polymorphic thymidine/adenine repeat occurring in the human promoter, which has been associated with some forms of hyperbilirubinemia. Second, an L1 transposon element, which is absent in the human promoter, is found 480 bp upstream of the transcription start site in mouse. Using the electromobility shift and DNase I footprinting experiments, we have identified a hepatocyte nuclear factor 1-binding site in the mouse UGT1A1 promoter that confers responsiveness to both factors HNF1alpha and HNF1beta in HEK293 cells. Furthermore, we show that this element, which is conserved in the human promoter, also confers strong HNF1 responsiveness to the human UGT1A1 gene. Together, these results provide evidence for a major regulatory function of this liver-enriched transcription factor in UGT1A1 activity in both rodents and human.

Soluble MHC-peptide complexes containing long rigid linkers abolish CTL-mediated cytotoxicity.

Soluble MHC-peptide (pMHC) complexes induce intracellular calcium mobilization, diverse phosphorylation events, and death of CD8+ CTL, given that they are at least dimeric and co-engage CD8. By testing dimeric, tetrameric, and octameric pMHC complexes containing spacers of different lengths, we show that their ability to activate CTL decreases as the distance between their subunit MHC complexes increases. Remarkably, pMHC complexes containing long rigid polyproline spacers (> or =80 A) inhibit target cell killing by cloned S14 CTL in a dose- and valence-dependent manner. Long octameric pMHC complexes abolished target cell lysis, even very strong lysis, at nanomolar concentrations. By contrast, an altered peptide ligand antagonist was only weakly inhibitory and only at high concentrations. Long D(b)-gp33 complexes strongly and specifically inhibited the D(b)-restricted lymphocytic choriomeningitis virus CTL response in vitro and in vivo. We show that complications related to transfer of peptide from soluble to cell-associated MHC molecules can be circumvented by using covalent pMHC complexes. Long pMHC complexes efficiently inhibited CTL target cell conjugate formation by interfering with TCR-mediated activation of LFA-1. Such reagents provide a new and powerful means to inhibit Ag-specific CTL responses and hence should be useful to blunt autoimmune disorders such as diabetes type I.

Experimental cerebral malaria develops independently of caspase recruitment domain-containing protein 9 signaling.

The outcome of infection depends on multiple layers of immune regulation, with innate immunity playing a decisive role in shaping protection or pathogenic sequelae of acquired immunity. The contribution of pattern recognition receptors and adaptor molecules in immunity to malaria remains poorly understood. Here, we interrogate the role of the caspase recruitment domain-containing protein 9 (CARD9) signaling pathway in the development of experimental cerebral malaria (ECM) using the murine Plasmodium berghei ANKA infection model. CARD9 expression was upregulated in the brains of infected wild-type (WT) mice, suggesting a potential role for this pathway in ECM pathogenesis. However, P. berghei ANKA-infected Card9(-/-) mice succumbed to neurological signs and presented with disrupted blood-brain barriers similar to WT mice. Furthermore, consistent with the immunological features associated with ECM in WT mice, Card9(-/-) mice revealed (i) elevated levels of proinflammatory responses, (ii) high frequencies of activated T cells, and (iii) CD8(+) T cell arrest in the cerebral microvasculature. We conclude that ECM develops independently of the CARD9 signaling pathway.

Effect of a whey-predominant starter formula containing LCPUFAs and oligosaccharides (FOS/GOS) on gastrointestinal comfort in infants.

Development of new infant formulas aims to replicate the benefits of breast milk. One benefit of breast milk over infant formulas is greater gastrointestinal comfort. We compared indicators of gastrointestinal comfort in infants fed a whey-predominant formula containing long-chain polyunsaturated fatty acids, galacto-oligo-saccharides and fructo-oligosaccharides, and infants fed a control casein-predominant formula without additional ingredients. The single-centre, prospective, double-blind, controlled trial randomly assigned healthy, full-term infants (n=144) to receive exclusively either experimental or control formula from 30 days to 4 months of age. A group of exclusively breast-fed infants served as reference (n=80). At 1, 2, 3, and 4 months, infants' growth parameters were measured and their health assessed. Parents recorded frequency and physical characteristics of infants' stool, frequency of regurgitation, vomiting, crying and colic. At 2-months, gastric emptying (ultrasound) and intestinal transit time (H2 breath test) were measured, and stool samples collected for bacterial analysis. Compared to the control (n=69), fewer of the experimental group (n=67) had hard stools (0.7 vs 7.5%, p<0.001) and more had soft stools (90.8 vs 82.3%, p<0.05). Also compared to the control, the experimental group's stool microbiota composition (mean % bifidobacteria: 78.1 (experimental, n=17), 63.7 (control, n=16), 74.3 (breast-fed, n=20), gastric transit times (59.6 (experimental, n=53), 61.4 (control, n=62), 55.9 (breast-fed, n=67) minutes) and intestinal transit times (data not shown) were closer to that of the breast-fed group. Growth parameter values were similar for all groups. The data suggest that, in infants, the prebiotic-containing whey-based formula provides superior gastrointestinal comfort than a control formula.

Reversible major histocompatibility complex I-peptide multimers containing Ni(2+)-nitrilotriacetic acid peptides and histidine tags improve analysis and sorting of CD8(+) T cells.

MHC-peptide multimers containing biotinylated MHC-peptide complexes bound to phycoerythrin (PE) streptavidin (SA) are widely used for analyzing and sorting antigen-specific T cells. Here we describe alternative T cell-staining reagents that are superior to conventional reagents. They are built on reversible chelate complexes of Ni(2+)-nitrilotriacetic acid (NTA) with oligohistidines. We synthesized biotinylated linear mono-, di-, and tetra-NTA compounds using conventional solid phase peptide chemistry and studied their interaction with HLA-A*0201-peptide complexes containing a His(6), His(12), or 2×His(6) tag by surface plasmon resonance on SA-coated sensor chips and equilibrium dialysis. The binding avidity increased in the order His(6) < His(12) < 2×His(6) and NTA(1) < NTA(2) < NTA(4), respectively, depending on the configuration of the NTA moieties and increased to picomolar K(D) for the combination of a 2×His(6) tag and a 2×Ni(2+)-NTA(2). We demonstrate that HLA-A2-2×His(6)-peptide multimers containing either Ni(2+)-NTA(4)-biotin and PE-SA- or PE-NTA(4)-stained influenza and Melan A-specific CD8+ T cells equal or better than conventional multimers. Although these complexes were highly stable, they very rapidly dissociated in the presence of imidazole, which allowed sorting of bona fide antigen-specific CD8+ T cells without inducing T cell death as well as assessment of HLA-A2-peptide monomer dissociation kinetics on CD8+ T cells.

Polymers of 3-hydroxyacids in plants

Polyhydroxyalkanoates (PHA) are polyesters of bacterial origin that have properties of biodegradable plastics and elastomers. Synthesis of PHA in crop plants would allow the large-scale production and use of these biodegradable and renewable polymers as substitutes for petroleum-derived plastics. Synthesis of a diversity of PHAs in plants, such as Arabidopsis thaliana, rapeseed, corn and cotton, has been demonstrated through the genetic engineering of metabolic pathways in the cytoplasm, plastid and peroxisome. PHA can also be used as a novel tool to study various aspects of plant metabolism, such as the regulation of carbon flux to the fatty acid biosynthetic and degradation pathways.

Synaptic Plasticity at Intrathalamic Connections via CaV3.3 T-type Ca2+ Channels and GluN2B-Containing NMDA Receptors.

The T-type Ca(2+) channels encoded by the Ca(V)3 genes are well established electrogenic drivers for burst discharge. Here, using Ca(V)3.3(-/-) mice we found that Ca(V)3.3 channels trigger synaptic plasticity in reticular thalamic neurons. Burst discharge via Ca(V)3.3 channels induced long-term potentiation at thalamoreticular inputs when coactivated with GluN2B-containing NMDA receptors, which are the dominant subtype at these synapses. Notably, oscillatory burst discharge of reticular neurons is typical for sleep-related rhythms, suggesting that sleep contributes to strengthening intrathalamic circuits.

Follicular lymphoma at relapse after rituximab containing regimens: comparison of time to event intervals prior to and after (90) Y-ibritumomab-tiuxetan.

Radioimmunotherapies with Zevalin® (RIT-Z) showed encouraging results in patients with relapsed/refractory follicular lymphoma (FL), leading frequently to failure-free intervals longer than those achieved by the last previous therapy. We compared time-to-event variables obtained before and after RIT-Z in patients with relapsed FL, previously exposed to rituximab. All patients with relapsed non-transformed, non-refractory, non-rituximab-naïve FL who have been treated with RIT-Z in two different centres in Europe were included. Staging and response were assessed by contrast-enhanced CT in all patients; PET/CT was performed according to local availability. Event-free survival (EFS) and time to next treatment (TTNT) following the last previous therapy and after RIT-Z were compared. Pre-therapy characteristics were tested in univariate analyses for prediction of outcomes. A description of the patterns of relapse was also provided. Among 70 patients treated, only 16 fulfilled the inclusion criteria. They were treated with a median of 3 prior lines of chemo-immunotherapies, including a median of 2 rituximab-containing regimens; 6 patients had undergone myeloablative chemotherapy with autologous stem cell rescue (ASCT). Overall response rates were 10 (62%) CR/CRu, 3 (19%) PR and 3 (19%) PD; response rates were similar in patients with prior ASCT. After RIT-Z only few patients obtained EFS and TTNT longer than after the last previous therapy. All four patients receiving rituximab maintenance were without progression 12 months after RIT-Z. Relapses occurred in both previously and newly involved sites; a significant association was found between the number of pathologic sites involved prior to RIT-Z and subsequent TTNT. Despite the excellent response rate, the duration of response was shorter than the previous one confirming the known trend of relapses to occur earlier after subsequent treatments. Rituximab maintenance after RIT-Z showed encouraging results in terms of prolonging EFS, warranting further studies. Copyright © 2010 John Wiley &amp; Sons, Ltd.

The emerging importance of the SPX domain-containing proteins in phosphate homeostasis

Plant growth and development are strongly influenced by the availability of nutrients in the soil solution. Among them, phosphorus (P) is one of the most essential and most limiting macro-elements for plants. In the environment, plants are often confronted with P starvation as a result of extremely low concentrations of soluble inorganic phosphate (Pi) in the soil. To cope with these conditions, plants have developed a wide spectrum of mechanisms aimed at increasing P use efficiency. At the molecular level, recent studies have shown that several proteins carrying the SPX domain are essential for maintaining Pi homeostasis in plants. The SPX domain is found in numerous eukaryotic proteins, including several proteins from the yeast PHO regulon, involved in maintaining Pi homeostasis. In plants, proteins harboring the SPX domain are classified into four families based on the presence of additional domains in their structure, namely the SPX, SPX-EXS, SPX-MFS and SPX-RING families. In this review, we highlight the recent findings regarding the key roles of the proteins containing the SPX domain in phosphate signaling, as well as providing further research directions in order to improve our knowledge on P nutrition in plants, thus enabling the generation of plants with better P use efficiency.

Diagnostic performance of Fluorine-18-Fluorodeoxyglucose positron emission tomography in the assessment of pleural abnormalities in cancer patients: A systematic review and a meta-analysis.

OBJECTIVE: To systematically review and meta-analyze published data about the diagnostic performance of Fluorine-18-Fluorodeoxyglucose ((18)F-FDG) positron emission tomography (PET) and PET/computed tomography (PET/CT) in the assessment of pleural abnormalities in cancer patients. METHODS: A comprehensive literature search of studies published through June 2013 regarding the role of (18)F-FDG-PET and PET/CT in evaluating pleural abnormalities in cancer patients was performed. All retrieved studies were reviewed and qualitatively analyzed. Pooled sensitivity, specificity, positive and negative likelihood ratio (LR+ and LR-) and diagnostic odd ratio (DOR) of (18)F-FDG-PET or PET/CT on a per patient-based analysis were calculated. The area under the summary ROC curve (AUC) was calculated to measure the accuracy of these methods in the assessment of pleural abnormalities. Sub-analyses considering (18)F-FDG-PET/CT and patients with lung cancer only were carried out. RESULTS: Eight studies comprising 360 cancer patients (323 with lung cancer) were included. The meta-analysis of these selected studies provided the following results: sensitivity 86% [95% confidence interval (95%CI): 80-91%], specificity 80% [95%CI: 73-85%], LR+ 3.7 [95%CI: 2.8-4.9], LR- 0.18 [95%CI: 0.09-0.34], DOR 27 [95%CI: 13-56]. The AUC was 0.907. No significant improvement considering PET/CT studies only and patients with lung cancer was found. CONCLUSIONS: (18)F-FDG-PET and PET/CT demonstrated to be useful diagnostic imaging methods in the assessment of pleural abnormalities in cancer patients, nevertheless possible sources of false-negative and false-positive results should be kept in mind. The literature focusing on the use of (18)F-FDG-PET and PET/CT in this setting remains still limited and prospective studies are needed.

Characterizing emission and breathing-zone concentrations following exposure cases to fluororesin-based waterproofing spray mists.

Measurements and simulations were performed to assess workers' exposure to solvent vapors and aerosols during the waterproofing of a tiled surface. This investigation followed two recent incidents in the same company where workers experienced acute respiratory illness after spraying a stain-repellent resin containing fluorinated polymers on stone-tiled walls and floors. Because the waterproofing activity had been done for years at the tile company without encountering any exposure problems prior to these cases, it was strongly suspected that the incidents were linked to a recent change in the composition of the coating mixture. Experimental measurements and simulations indicated that the emission rate of particles smaller than 10 microm may be estimated at 0.66 mg/sec (SD 0.10) for the old resin and at 0.37 mg/sec (SD 0.04) for the new one. The measurement of the solvent emission rate from surfaces coated with the two resins indicated that shortly after spraying, the emission was in the range of 18 to 20 mg/sec x m2 and was similar for both products. Solvent and overspray emission rates were introduced in a two-zone compartment model. The results obtained in the near-field indicate significant exposure to overspray mist (7 and 34 mg/m3 for new resin) and solvent vapors (80 to 350 ppm for the new resin). It was also shown that the introduction of the new resin tended to significantly decrease the levels of solvents and particulates in the workers' breathing zone. These results strongly suggest that cases of acute respiratory illness are related to the specific toxicity of the fluorinated polymer itself. The fact that the same polymer is used in various commercial products raises concern regarding other possible occupational and domestic exposures.

Retinal pigment epithelium protein of 65 kDA gene-linked retinal degeneration is not modulated by chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C/ chondroitin sulfate proteoglycan 5.

PURPOSE: To analyze in vivo the function of chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C (gene symbol: Cspg5) during retinal degeneration in the Rpe65⁻/⁻ mouse model of Leber congenital amaurosis. METHODS: We resorted to mice with targeted deletions in the Cspg5 and retinal pigment epithelium protein of 65 kDa (Rpe65) genes (Cspg5⁻/⁻/Rpe65⁻/⁻). Cone degeneration was assessed with cone-specific peanut agglutinin staining. Transcriptional expression of rhodopsin (Rho), S-opsin (Opn1sw), M-opsin (Opn1mw), rod transducin α subunit (Gnat1), and cone transducin α subunit (Gnat2) genes was assessed with quantitative PCR from 2 weeks to 12 months. The retinal pigment epithelium (RPE) was analyzed at P14 with immunodetection of the retinol-binding protein membrane receptor Stra6. RESULTS: No differences in the progression of retinal degeneration were observed between the Rpe65⁻/⁻ and Cspg5⁻/⁻/Rpe65⁻/⁻ mice. No retinal phenotype was detected in the late postnatal and adult Cspg5⁻/⁻ mice, when compared to the wild-type mice. CONCLUSIONS: Despite the previously reported upregulation of Cspg5 during retinal degeneration in Rpe65⁻/⁻ mice, no protective effect or any involvement of Cspg5 in disease progression was identified.

Genetic evidence for a role of adiponutrin in the metabolism of apolipoprotein B-containing lipoproteins.

Adiponutrin (PNPLA3) is a predominantly liver-expressed transmembrane protein with phospholipase activity that is regulated by fasting and feeding. Recent genome-wide association studies identified PNPLA3 to be associated with hepatic fat content and liver function, thus pointing to a possible involvement in the hepatic lipoprotein metabolism. The aim of this study was to examine the association between two common variants in the adiponutrin gene and parameters of lipoprotein metabolism in 23,274 participants from eight independent West-Eurasian study populations including six population-based studies [Bruneck (n = 800), KORA S3/F3 (n = 1644), KORA S4/F4 (n = 1814), CoLaus (n = 5435), SHIP (n = 4012), Rotterdam (n = 5967)], the SAPHIR Study as a healthy working population (n = 1738) and the Utah Obesity Case-Control Study including a group of 1037 severely obese individuals (average BMI 46 kg/m2) and 827 controls from the same geographical region of Utah. We observed a strong additive association of a common non-synonymous variant within adiponutrin (rs738409) with age-, gender-, and alanine-aminotransferase-adjusted lipoprotein concentrations: each copy of the minor allele decreased levels of total cholesterol on average by 2.43 mg/dl (P = 8.87 x 10(-7)), non-HDL cholesterol levels by 2.35 mg/dl (P = 2.27 x 10(-6)) and LDL cholesterol levels by 1.48 mg/dl (P = 7.99 x 10(-4)). These associations remained significant after correction for multiple testing. We did not observe clear evidence for associations with HDL cholesterol or triglyceride concentrations. In conclusion, our study suggests that adiponutrin is involved in the metabolism of apoB-containing lipoproteins.

Immunolocalization of GLUTX1 in the testis and to specific brain areas and vasopressin-containing neurons.

GLUTX1 or GLUT8 is a newly characterized glucose transporter isoform that is expressed at high levels in the testis and brain and at lower levels in several other tissues. Its expression was mapped in the testis and brain by using specific antibodies. In the testis, immunoreactivity was expressed in differentiating spermatocytes of type 1 stage but undetectable in mature spermatozoa. In the brain, GLUTX1 distribution was selective and localized to a variety of structures, mainly archi- and paleocortex. It was found in hippocampal and dentate gyrus neurons as well as amygdala and primary olfactory cortex. In these neurons, its location was close to the plasma membrane of cell bodies and sometimes in proximal dendrites. High GLUTX1 levels were detected in the hypothalamus, supraoptic nucleus, median eminence, and the posterior pituitary. Neurons of these areas synthesize and secrete vasopressin and oxytocin. As shown by double immunofluorescence microscopy and immunogold labeling, GLUTX1 was expressed only in vasopressin neurons. By immunogold labeling of ultrathin cryosections microscopy, GLUTX1 was identified in dense core vesicles of synaptic nerve endings of the supraoptic nucleus and secretory granules of the vasopressin positive neurons. This localization suggests an involvement of GLUTX1 both in specific neuron function and endocrine mechanisms.