Global occurrence of Torque teno virus in water systems.

Bacterial indicator organisms are used globally to assess the microbiological safety of waters. However, waterborne viral outbreaks have occurred in drinking water systems despite negative bacterial results. Using viral markers may therefore provide more accurate health risk assessment data. In this study, fecal, wastewater, stormwater, surface water (fresh and salt), groundwater, and drinking water samples were analyzed for the presence or concentration of traditional indicators, innovative indicators and viral markers. Samples were obtained in the United States, Italy, and Australia and results compared to those reported for studies conducted in Asia and South America as well. Indicators included total coliforms, Escherichia coli, enterococci, male-specific coliphages, somatic coliphages and microviradae. Viral markers included adenovirus, polyomavirus, and a potential new surrogate, Torque teno virus (TTV). TTV was more frequently found in wastewaters (38-100%) and waters influenced by waste discharges (25%) than in surface waters used as drinking water sources (5%). TTV was also specific to human rather than animal feces. While TTV numbers were strongly correlated to other viral markers in wastewaters, suggesting its utility as a fecal contamination marker, data limitations and TTV presence in treated drinking waters demonstrates that additional research is needed on this potential viral indicator.

Docosahexaenoic acid-enriched canola oil increases adiponectin concentrations: a randomized crossover controlled intervention trial.

BACKGROUND AND AIMS: Little is known about the effect of various dietary fatty acids on pro- and anti-inflammatory processes. We investigated the effect of 5 oils containing various amounts of alpha-linolenic acid (ALA), linoleic acid (LA), oleic acid (OA) and docosahexaenoic acid (DHA) on plasma inflammatory biomarkers and expression levels of key inflammatory genes and transcription factors in whole blood cells. METHODS AND RESULTS: In a randomized, crossover controlled nutrition intervention, 114 adult men and women with abdominal obesity and at least one other criterion for the metabolic syndrome consumed 5 experimental isoenergetic diets for 4 weeks each, separated by 4-week washout periods. Each diet provided 60 g/3000 kcal of different oils: 1) control corn/safflower oil blend (CornSaff; LA-rich), 2) flax/safflower oil blend (FlaxSaff; ALA-rich), 3) conventional canola oil (Canola; OA-rich), 4) high oleic canola oil (CanolaOleic; highest OA content), 5) DHA-enriched high oleic canola oil (CanolaDHA; OA- and DHA-rich). Gene expression in whole blood cells was assessed in a subset of 62 subjects. CanolaDHA increased plasma adiponectin concentrations compared with the control CornSaff oil treatment (+4.5%, P = 0.04) and FlaxSaff (+6.9%, P = 0.0008). CanolaDHA also reduced relative expression levels of interleukin (IL)1B compared with CornSaff and Canola (-11% and -13%, respectively, both P = 0.03). High-sensitivity C-reactive protein concentrations were lower after Canola than after FlaxSaff (-17.8%, P = 0.047). CONCLUSION: DHA-enriched canola oil exerts anti-inflammatory effects compared with polyunsaturated fatty acids from plant sources.

Effect of sitagliptin therapy on triglyceride-rich lipoprotein kinetics in patients with type 2 diabetes.

AIM: To investigate the effects of sitagliptin therapy on the kinetics of triglyceride-rich lipoprotein (TRL) apolipoprotein (apo)B-48, VLDL apoB-100, apoE and apoC-III in patients with type 2 diabetes. METHODS: Twenty-two subjects with type 2 diabetes were recruited in this double-blind crossover study, during which the subjects received sitagliptin (100 mg/day) or placebo for a 6-week period each. At the end of each phase of treatment, the in vivo kinetics of the different apolipoproteins were assessed using a primed-constant infusion of l-[5,5,5-D3]leucine for 12 h, with the participants in a constantly fed state. RESULTS: Sitagliptin therapy significantly reduced fasting plasma triglyceride (-15.4%, p = 0.03), apoB-48 (-16.3%, p = 0.03) and free fatty acid concentrations (-9.5%, p = 0.04), as well as plasma HbA1c (placebo: 7.0% ± 0.8 vs. sitagliptin: 6.6% ± 0.7, p < 0.0001) and plasma glucose levels (-13.5%, p = 0.001), without any significant effect on insulin levels. Kinetic results showed that treatment with sitagliptin significantly reduced the pool size of TRL apoB-48 by -20.8% (p = 0.03), paralleled by a reduction in the production rate of these particles (-16.0%, p = 0.03). The VLDL apoB-100 pool size was also significantly decreased by sitagliptin therapy (-9.3%, p = 0.03), mainly because of a reduction in the hepatic secretion of these lipoproteins, although this difference did not reach statistical significance (-9.2%, p = 0.06). CONCLUSIONS: Treatment with sitagliptin for 6 weeks reduced triglyceride-rich apoB-containing lipoprotein levels by reducing the synthesis of these particles.

Surgery combined with controlled-release doxorubicin silk films as a treatment strategy in an orthotopic neuroblastoma mouse model.

BACKGROUND: Neuroblastoma tumour resection goal is maximal tumour removal. We hypothesise that combining surgery with sustained, local doxorubicin application can control tumour growth. METHODS: We injected human neuroblastoma cells into immunocompromised mouse adrenal gland. When KELLY cell-induced tumour volume was >300 mm(3), 80-90% of tumour was resected and treated as follows: instantaneous-release silk film with 100 µg doxorubicin (100IR), controlled-release film with 200 µg (200CR) over residual tumour bed; and 100 and 200 µg intravenous doxorubicin (100IV and 200IV). Tumour volume was measured and histology analysed. RESULTS: Orthotopic tumours formed with KELLY, SK-N-AS, IMR-32, SH-SY5Y cells. Tumours reached 1800±180 mm(3) after 28 days, 2200±290 mm(3) after 35 days, 1280±260 mm(3) after 63 days, and 1700±360 mm(3) after 84 days, respectively. At 3 days post KELLY tumour resection, tumour volumes were similar across all groups (P=0.6210). Tumour growth rate was similar in untreated vs control film, 100IV vs 100IR, and 100IV vs 200IV. There was significant difference in 100IR vs 200CR (P=0.0004) and 200IV vs 200CR (P=0.0003). Tumour growth with all doxorubicin groups was slower than that of control (P: <0.0001-0.0069). At the interface of the 200CR film and tumour, there was cellular necrosis, surrounded by apoptotic cells before reaching viable tumour cells. CONCLUSIONS: Combining surgical resection and sustained local doxorubicin treatment is effective in tumour control. Administering doxorubicin in a local, controlled manner is superior to giving an equivalent intravenous dose in tumour control.

Acquired MET expression confers resistance to EGFR inhibition in a mouse model of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is an aggressive brain tumor for which there is no cure. Overexpression of wild-type epidermal growth factor receptor (EGFR) and loss of the tumor suppressor genes Ink4a/Arf and PTEN are salient features of this deadly cancer. Surprisingly, targeted inhibition of EGFR has been clinically disappointing, demonstrating an innate ability for GBM to develop resistance. Efforts at modeling GBM in mice using wild-type EGFR have proven unsuccessful to date, hampering endeavors at understanding molecular mechanisms of therapeutic resistance. Here, we describe a unique genetically engineered mouse model of EGFR-driven gliomagenesis that uses a somatic conditional overexpression and chronic activation of wild-type EGFR in cooperation with deletions in the Ink4a/Arf and PTEN genes in adult brains. Using this model, we establish that chronic activation of wild-type EGFR with a ligand is necessary for generating tumors with histopathological and molecular characteristics of GBMs. We show that these GBMs are resistant to EGFR kinase inhibition and we define this resistance molecularly. Inhibition of EGFR kinase activity using tyrosine kinase inhibitors in GBM tumor cells generates a cytostatic response characterized by a cell cycle arrest, which is accompanied by a substantial change in global gene expression levels. We demonstrate that an important component of this pattern is the transcriptional activation of the MET receptor tyrosine kinase and that pharmacological inhibition of MET overcomes the resistance to EGFR inhibition in these cells. These findings provide important new insights into mechanisms of resistance to EGFR inhibition and suggest that inhibition of multiple targets will be necessary to provide therapeutic benefit for GBM patients.

Report from the fifth National Cancer Institute Mouse Models of Human Cancers Consortium Nervous System Tumors Workshop.

Cancers of the nervous system are clinically challenging tumors that present with varied histopathologies and genetic etiologies. While the prognosis for the most malignant of these tumors is essentially unchanged despite decades of basic and translational science research, the past few years have witnessed the identification of numerous targetable molecular alterations in these cancers. With the advent of advanced genomic sequencing methodologies and the development of accurate small-animal models of these nervous system cancers, we are now ideally positioned to develop personalized therapies that target the unique cellular and molecular changes that define their formation and drive their continued growth. Recently, the National Cancer Institute convened a workshop to advance our understanding of nervous system cancer mouse models and to inform clinical trials by reconsidering these neoplasms as complex biological systems characterized by heterogeneity at all levels.

Effect of the Mediterranean diet with and without weight loss on cardiovascular risk factors in men with the metabolic syndrome.

BACKGROUND AND AIMS: No study has yet examined how weight loss modifies the impact of the Mediterranean diet (MedDiet) on cardiovascular risk factors in men with the metabolic syndrome (MetS). The objective of the study was to assess the efficacy of MedDiet, with and without weight loss, to modify the cardiometabolic risk profile of male patients with MetS. METHODS AND RESULTS: Twenty-six men aged between 24 and 62 years with the MetS consumed a North American control diet for 5 weeks followed by a 5-week MedDiet, both under weight-maintaining conditions. Participants then underwent a 20-week weight loss period, after which they consumed the MedDiet for five weeks under weight stable conditions. Body weight was reduced by 10.2% ± 2.9% after the weight loss period (p < 0.001). All foods were provided to participants during the weight stable phases of the study. The MedDiet in the absence of weight loss decreased total plasma cholesterol (C) (-7.1%), LDL-C (-9.3%) and the total/HDL-C ratio (-6.5%) compared to the control diet (all p < 0.04). The MedDiet combined with weight loss led to reductions in systolic blood pressure (-4.7%), diastolic blood pressure (-7.7%), triglycerides (-18.2%), ApoB (-10.7%), fasting glucose (-4.2%) and insulin (-29.9%) compared to the control diet (all p < 0.001). CONCLUSION: The MedDiet in the absence of weight loss leads to significant changes in plasma cholesterol concentrations but has little effects on other cardiometabolic risk factors associated with the MetS in men.

Distribution of SPARC during neovascularisation of degenerative aortic stenosis.

OBJECTIVE: To examine the hypothesis that degenerative aortic stenosis (AS) is associated with the development of blood vessels and the expression of the secreted protein, acidic and rich in cysteine/osteonectin (SPARC), a matricellular protein that is involved in ossification, the modulation of angiogenesis and the production of metalloproteinases. METHODS: 30 surgically excised AS valves and 20 normal aortic valves were studied. RESULTS: Blood vessels were detected in the aortic valves from patients with degenerative AS, whereas normal valves were avascular structures. Blood vessels in AS valves expressed endothelial nitric oxide synthase, CD34 and von Willebrand factor (vWF). Blood vessels were located in three distinct regions: near calcified nodules, under the leaflet border and in rich cellular areas forming cell islands. Blood vessels were predominantly present in early and intermediate grades of calcification. Cell islands were densely populated by CD45-positive cells where endothelial cells (CD34+, vWF+) forming cord-like structures were present. Immunoblotting detected SPARC only in AS valves and immunohistological analysis located SPARC in mature blood vessels. The proportion of blood vessels positive for SPARC was higher in valves with a lower grade of calcification. In cell islands, SPARC was distributed to mature blood vessels and to macrophages, where it co-located with matrix metalloproteinase-9, whereas no expression was detected in endothelial cells forming cord-like structures. CONCLUSION: The localisation of SPARC to mature blood vessels and its predominant expression in AS valves with a lower calcification grade suggest that the spatial and temporal distribution of this matricellular protein is tightly controlled to participate in the neovascularisation of AS valves.

Expression of bone-regulatory proteins in human valve allografts.

OBJECTIVE: To test the hypothesis that valve allograft (VA) calcification results from an ossification process in which bone-regulatory proteins are expressed. METHODS: 15 VA that were explanted at the time of surgery for dysfunction were studied. VA were analysed and compared with normal aortic valves (n = 20). RESULTS: All the VA (5 aortic, 10 pulmonary) exhibited heavy calcification and important fibrosis. Immunohistochemistry studies showed that the bone-specific transcription factor Cbfa-1 was expressed by stromal cells. Bone alkaline phosphatase was expressed in calcified regions. Immunostaining for alpha smooth muscle (alpha-SM) actin was increased in VA compared with normal valves and in 6 of the 15 valves formed cellular clusters close to the calcified nodules. In VA osteopontin and osteonectin were expressed by stromal cells, whereas osteocalcin was closely associated with the calcified regions. Furthermore, analysis of the bone-regulatory proteins that control bone resorption showed that receptor activator of nuclear factor kappaB ligand (RANKL), receptor activator of nuclear factor kappaB (RANK) and osteoprotegerin (OPG) were differentially expressed in calcified VA and normal valves. Normal valve leaflets expressed OPG, whereas OPG expression was absent or faint in calcified VA. RANKL and RANK were not detected in normal valves, whereas calcified VA expressed RANKL and RANK. CONCLUSION: These data suggest that calcification of VA results from an ossification process, which relies on tight control of bone-regulatory protein expression. The expression pattern of the RANKL/RANK/OPG system suggests that it may have a regulatory role not only in osteoclastogenesis but also in the calcification of human VA.

Phytosterols in nonfat and low-fat beverages have no impact on the LDL size phenotype.

OBJECTIVE: To examine the impact of nonfat and low-fat phytosterol-enriched beverages on low-density lipoprotein (LDL) electrophoretic characteristics. DESIGN: Double-blind, randomized, crossover, placebo-controlled dietary trial. SETTING: Diets were prepared and consumed at the Mary Emily Clinical Nutrition Research Unit of McGill University. Analyses were performed at the Institute on Nutraceuticals and Functional Foods of Laval University. SUBJECTS AND INTERVENTION: In total, 15 moderately hypercholesterolemic persons consumed each of three experimental diets that each comprised a different beverage: nonfat placebo (NF control), nonfat with phytosterols (NFPS) or low-fat with phytosterols (LFPS). Participants consumed three beverages daily at meal time for a total of 1.8 g of phytosterols per day. Nondenaturing 2-16% polyacrylamide gradient gel electrophoreses were used to characterize LDL size characteristics. RESULTS: The NFPS and LFPS beverage induced no significant changes in several features of the LDL size phenotype compared to the control diet. CONCLUSION: The consumption of phytosterol-supplemented nonfat and low-fat beverages is not associated with clinically meaningful changes in the LDL particle size phenotype.

Are North American nephrologists biased against peritoneal dialysis?

OBJECTIVE: To examine possible bias against peritoneal dialysis (PD) by nephrologists less familiar with it. DESIGN: Secondary analysis of a previously reported survey. PARTICIPANTS: All practicing Canadian nephrologists (n = 290, response rate 66.2%) and a subgroup of American nephrologists who were members of the National Kidney Foundation Council on Dialysis (n = 507, response rate 47.3%). Responses were then subdivided by type of dialysis practice: mainly or only hemodialysis (HD, n = 117), mainly or only PD (n = 16), or both HD and PD (n = 232). INTERVENTION: Self-administered mailed questionnaire. MAIN OUTCOME MEASURES: Opinions and attitudes of nephrologists concerning patient characteristics favoring one dialysis modality over the other, as well as the relative utilization of HD and PD currently and in a hypothetical ideal situation. RESULTS: The main differences were present between physicians practicing mainly HD and physicians practicing mainly PD, with those practicing both giving answers usually intermediate to the others. The maximum weight suitable for PD was 10 kg less according to HD-oriented nephrologists compared with PD-oriented nephrologists (97.8 kg vs 108.5 kg). All nephrologists agreed that, ideally, 40% of prevalent end-stage renal disease patients should be on PD to optimize cost-effectiveness, whereas the proportion should be between 32% and 45% when one optimizes survival, wellness, and quality of life. In general, differences between groups were small. CONCLUSIONS: Most nephrologists favored a proportion of PD higher than the current prevalence seen in either Canada or the U.S.A. If physicians' biases are contributing to the distribution of dialysis modalities, they are not likely to be major factors. Unknown but important factors, external to the physician, may shape modality distribution more than the opinions and attitudes of physicians. If a more balanced and cost-effective dialysis delivery system is desired, more understanding and manipulation of these non physician-related factors will be required.

Association of a novel PDZ domain-containing peripheral Golgi protein with the Q-SNARE (Q-soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor) protein syntaxin 6.

PDZ domains are involved in the scaffolding and assembly of multi-protein complexes at various subcellular sites. We describe here the isolation and characterization of a novel PDZ domain-containing protein that localizes to the Golgi apparatus. Using an in silico cloning approach, we have identified and isolated a cDNA encoding a ubiquitously expressed 59-kDa protein that we call FIG. It is composed of two coiled coil regions, a leucine zipper, and a single PDZ domain. Cytological studies using indirect immunofluorescence microscopy revealed that FIG is a peripheral protein that uses one of its coiled coil domains to localize to the Golgi apparatus. To ascertain the modalities of this Golgi localization, the same coiled coil region was tested for its ability to interact with a panel of coiled coil domain-containing integral membrane Golgi proteins. Using a series of GST fusion protein binding assays, co-immunofluorescence and co-immunoprecipitation experiments, we show that FIG specifically binds to the coiled coil domain-containing Q-SNARE (Q-soluble NSF attachment protein receptor) protein syntaxin 6 both in vitro and in vivo. The structural features of FIG and its interaction with a SNARE protein suggest that FIG may play a role in membrane vesicle trafficking. This is the first example of a PDZ domain-containing peripheral protein that localizes to the Golgi through a coiled coil-mediated interaction with a resident membrane protein. Our results broaden the scope of PDZ domain-mediated functions.

A randomized comparison of intradermal and intramuscular vaccination against hepatitis B virus in incident chronic hemodialysis patients.

Hepatitis B virus (HBV) remains a threat to hemodialysis patients. Nevertheless, the vaccination rate against this virus is low, perhaps because of the low conversion rate. Although intradermal (ID) vaccination has been proven to be effective (even in patients nonresponsive to intramuscular [IM] vaccination), the duration of immunity was short. The impact of vaccination route and a greater peak antibody (Ab) titer on conversion rate and duration of immunity after ID or IM vaccination was compared in incident hemodialysis patients. Forty-nine patients were randomly assigned to treatment with 5 microgram of recombinant hepatitis B vaccine (Engerix B; Smith Kline Beecham Pharma Inc, Oakville, ON, Canada) ID every 2 weeks up to either a peak Ab titer of 1,000 IU/L or greater or 52 doses, whereas 48 patients were administered 40 microgram IM at 0, 1, 2, and 6 months. Group demographics were similar. Conversion was achieved in 97.6% of the ID group and 90.5% of the IM group (P: = 0.16). There was no difference between ID and IM groups in time required to convert, peak Ab titer reached, and proportion of patients with a peak Ab titer of 1,000 IU/L or greater. Overall, the duration of immunity was not different after ID or IM vaccination (P = 0.683), and patients in the IM group with a peak Ab titer of 1,000 IU/L or greater had a longer duration of immunity (P = 0.001). In conclusion, a high conversion rate and long duration of immunity against HBV can be achieved cost-effectively in the end-stage renal disease population using the ID or IM route and aiming for an Ab titer exceeding 1,000 IU/L. Based on these data, we provide recommendations for vaccination and surveillance in this population.

SMAR1, a novel, alternatively spliced gene product, binds the Scaffold/Matrix-associated region at the T cell receptor beta locus.

Rearrangement and expression of the T cell receptor beta gene are critical events for early T lymphocyte development. To characterize cis-regulatory elements and their associated trans-factors that mediate these events, we have previously identified a nuclear matrix/scaffold-associated region, referred to as MARbeta, 400 bp upstream of the Ebeta enhancer. Electrophoretic mobility shift assay showed that two known MAR-binding proteins, SATB1 and Cux, bind MARbeta. In this article, we report the identification of a novel MAR-binding protein, named SMAR1, that also binds MARbeta. SMAR1 shares homology with SATB1 and Cux in the MAR-binding domain/Cut repeat and also with the tetramerization domain of a B cell-specific MAR-binding protein, Bright. The binding of GST-SMAR1 fusion protein to MARbeta is inhibited by the presence of an excess amount of MAR-containing DNA from the immunoglobulin kappa locus. Smar1 transcripts are most abundant in the thymus and are alternatively spliced. The smar1 gene maps to the distal portion of mouse chromosome 8 at a distance of 111.8 cM.

Removal of phenolic compounds from a petrochemical effluent with a methanogenic consortium.

A methanogenic consortium was used to degrade phenol and ortho- (o-) cresol from a specific effluent of a petrochemical refinery. This effluent did not meet the local environmental regulations for phenolic compounds (178 mg/L), oils and greases (61 mg/L), ammoniacal nitrogen (75 mg/L) or sulfides (3.2 mg/L). The consortium, which degrades phenol via its carboxylation to benzoic acid, was progressively adapted to the effluent. Despite the very high effluent toxicity (EC50 of 2% with Microtox), the adapted consortium degraded 97% of 156 mg/L phenol in the supplemented effluent after 13 days in batch cultures (serum bottle). The addition of proteose peptone to the effluent is essential for phenol degradation. o-cresol was also transformed but not meta- or para-cresols. A continuous flow fixed-film anaerobic bioreactor was developed with the consortium. Treating the effluent with the bioreactor reduced phenol and phenolic compounds concentrations by 97 and 83%, respectively, for a hydraulic residence time of 6 h. This treatment also reduced by about half the effluent toxicity. Oils and greases and ammoniacal nitrogen were not affected. Similar microbiological forms were observed in serum bottles and in the bioreactors with or without the petrochemical effluent. These results indicate that this methanogenic consortium can treat efficiently the phenolic compounds in this specific petrochemical effluent.

TATA-driven transcriptional initiation and regulation of the rat serotonin 5-HT1A receptor gene.

The transcriptional initiation and regulation of the rat serotonin 5-HT1A receptor gene were characterized. By three types of analyses, a single brain-specific site of transcriptional initiation was localized to -967 bp upstream of the translation initiation codon that is utilized both in hippocampus and in the rat raphe RN46A cell line. This major site of transcriptional initiation was located 58 bp downstream from a consensus TATA element, suggesting TATA-driven transcription of the rat 5-HT1A receptor. To identify the promoter activity of the receptor gene, progressive 5' deletions of the -2,719/-117-bp fragment of the 5-HT1A promoter linked to luciferase gene were transfected into 5-HT1A-negative (pituitary GH4C1, L6 myoblast, and C6 glioma) and 5-HT1A-positive (septal SN-48 and raphe RN46A) cell lines. Enhancer regions were identified within a fragment between nucleotides -426 and -117 that selectively enhanced transcription in 5-HT1A-positive cells. A nonselective enhancer/promoter that mediated expression in all cell lines was located upstream between -1,519 and -426 bp in a DNA segment containing consensus TATA, CCAAT, SP-1, and AP-1 elements as well as a poly-GT26 dinucleotide repeat. Strong repression of transcription in all cell lines was conferred by the region upstream of -1,519 bp that contains a 152-bp DNA segment with >80% identity to RANTES, tumor necrosis factor-beta, and other immune system genes. Our results indicate that TATA-driven expression of the 5-HT1A receptor is regulated by a novel proximal tissue-specific enhancer region, a nonselective promoter, and an upstream repressor region that is distinct from previously identified neuron-specific repressors.

Protein tyrosine phosphatase-PEST regulates focal adhesion disassembly, migration, and cytokinesis in fibroblasts.

In this article, we show that, in transfected COS-1 cells, protein tyrosine phosphatase (PTP)-PEST translocates to the membrane periphery following stimulation by the extracellular matrix protein fibronectin. When plated on fibronectin, PTP-PEST (-/-) fibroblasts display a strong defect in motility. 3 h after plating on fibronectin, the number and size of vinculin containing focal adhesions were greatly increased in the homozygous PTP-PEST mutant cells as compared with heterozygous cells. This phenomenon appears to be due in part to a constitutive increase in tyrosine phosphorylation of p130(CAS), a known PTP-PEST substrate, paxillin, which associates with PTP-PEST in vitro, and focal adhesion kinase (FAK). Another effect of this constitutive hyperphosphorylation, consistent with the focal adhesion regulation defect, is that (-/-) cells spread faster than the control cell line when plated on fibronectin. In the PTP-PEST (-/-) cells, an increase in affinity for the SH2 domains of Src and Crk towards p130(CAS) was also observed. In (-/-) cells, we found a significant increase in the level of tyrosine phosphorylation of PSTPIP, a cleavage furrow-associated protein that interacts physically with all PEST family members. An effect of PSTPIP hyperphosphorylation appears to be that some cells remain attached at the site of the cleavage furrow for an extended period of time. In conclusion, our data suggest PTP-PEST plays a dual role in cell cytoskeleton organization, by promoting the turnover of focal adhesions required for cell migration, and by directly or indirectly regulating the proline, serine, threonine phosphatase interacting protein (PSTPIP) tyrosine phosphorylation level which may be involved in regulating cleavage furrow formation or disassembly during normal cell division.

Combination of gene targeting and substrate trapping to identify substrates of protein tyrosine phosphatases using PTP-PEST as a model.

Identification of physiological substrates of protein tyrosine phosphatases is a key step in understanding the function of these enzymes. We have generated fibroblast cell lines having a gene-targeted PTP-PEST in order to identify potential substrates with the premise that specific substrates of this enzyme would exist in a hyperphosphorylated state. Analysis of the profile of the phosphotyrosine proteins in the PTP-PEST -/- cells revealed the presence of hyperphosphorylated proteins of 180, 130, and 97 kDa when compared to control cells. The p130 was identified as p130(Cas), and direct immunoprecipitates of p130(Cas) demonstrate that this protein is constitutively hyperphosphorylated in cells lacking PTP-PEST. In addition, p130(Cas) was also isolated by the substrate-trapping mutant of PTP-PEST in the PTP-PEST -/- cell lysates. Interestingly, we have demonstrated for the first time that PTP-PEST, through its first proline-rich sequence 332PPKPPR337, interacts with other members of the p130(Cas) family (Hef1 and Sin) via their SH3 domain in vitro. This result suggests that Hef1 and Sin could also be potential substrates of PTP-PEST. In conclusion, we have combined genetic and biochemical strategies to allow the identification of PTP-PEST substrates. This experimental approach could potentially be used to identify substrates of other PTPases. Furthermore, the Cas-like molecules Hef1 and Sin associate via their SH3 domains with a proline-rich motif found on PTP-PEST, suggesting the possibility that PTP-PEST could be a general modulator of the Cas family of proteins.

Cloning and chromosomal mapping of an orphan chemokine receptor: mouse RDC1.

Degenerate RT-PCR was used to identify a new seven-transmembrane-spanning receptor expressed in astrocytes. A receptor, termed RDC1, displaying the characteristic structural features of a chemokine receptor was cloned. The predicted 362-amino-acid sequence displayed 92% and 91% similarity to the human and dog orphan receptor RDC1, respectively. In addition, RDC1 shares 43% amino acid similarity to rabbit and mouse CXCR2. Transcripts of RDC1 were found in astrocytes, heart, kidney, the mesangial tumor line MES-13, spleen, and neutrophils by means of northern blot. Using linkage analysis of interspecies backcross mice, we localized to chromosome 1 the genes for mouse CXCR2, CXCR4, and RDC1. Mouse RDC1 is linked to and lies between the genes for the mouse CXC chemokine receptors CXCR2 and CXCR4. The combined data of chromosomal location and sequence similarity suggest that RDC1 is an orphan CXC chemokine receptor.

Association of the multisubstrate docking protein Gab1 with the hepatocyte growth factor receptor requires a functional Grb2 binding site involving tyrosine 1356.

Hepatocyte growth factor/scatter factor is a multifunctional factor that induces mitogenesis, motility, invasion, and branching tubulogenesis of several epithelial and endothelial cell lines in culture. The receptor for hepatocyte growth factor has been identified as the Met-tyrosine kinase. Upon stimulation with hepatocyte growth factor, the Met beta subunit becomes highly phosphorylated on tyrosine residues, one of which, tyrosine 1356 within the carboxyl terminus, is crucial for dissociation, motility, and branching tubule formation in Madin-Darby canine kidney epithelial cells. Tyrosine 1356 forms a multisubstrate binding site for the Grb2 and Shc adaptor proteins, the p85 subunit of phosphatidylinositol 3'-kinase, phospholipase Cgamma, and a phosphatase, SHP2. To investigate additional signaling molecules that are activated by the Met receptor, we have identified hepatocyte growth factor-induced phosphoproteins in tubular epithelial cells. We have established that proteins of 100-130 kDa are highly phosphorylated following stimulation of epithelial cells and that one of these is the Grb2-associated binding protein Gab1, a possible insulin receptor substrate-1-like signal transducer. We show that Gab1 is the major substrate for the Met kinase in vitro and in vivo. Association of Gab1 with Met requires a functional Grb2 binding site involving tyrosine 1356 and to a lesser extent tyrosine 1349. Met receptor mutants that fail to induce branching tubulogenesis are impaired in their ability to interact with Gab1, suggesting that Gab1 may play a role in these processes.