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Biomedical subjects

R G Wells

Publications and source records attributed to R G Wells.

At least 19 recordsLinked to original sources

Novel inactivating mutations of transforming growth factor-beta type I receptor gene in head-and-neck cancer metastases.

Carcinoma cell lines are frequently refractory to transforming growth factor-beta (TGF beta)-mediated cell cycle arrest. Whether and how TGF beta signaling is disrupted in the majority of human tumors, however, remains unclear. To investigate whether TGF beta signaling might be disrupted by inactivation of the key signaling molecule, the TGF beta type I (T beta R-I) receptor, and whether or not T beta R-I inactivation is associated with late stage disease, we conducted a comprehensive structural analysis of the T beta R-I gene in fine-needle aspirates of 23 head-&-neck cancer metastases. We encountered 4 different mutations of T beta R-I, 3 of which have not been previously identified. In 1 case, we found a somatic intragenic 4-bp deletion predicting for a truncation of the receptor protein. This is the first example of a true loss-of-function mutation of T beta R-I in a human epithelial neoplasm. In 2 other cases, we identified missense mutations located between the juxtamembrane- and serine-threonine kinase domains. One of these resulted in an alanine-to-threonine substitution (A230T), which disrupts receptor signaling activity by causing rapid protein degradation within the endoplasmatic reticulum. This represents a novel mechanism of inactivation of a TGF beta signaling intermediate. Finally, we identified a serine-to-tyrosine substitution at codon 387 (S387Y) in a metastasis but not in the corresponding primary tumor. We had previously shown this S387Y mutant to be predominantly associated with breast cancer metastases and to have a diminished ability to mediate TGF beta-dependent signaling. In aggregate, these findings provide further support for the hypothesis that inactivation of the TGF beta signaling pathway occurs in a significant subset of human cancers.

Activin Receptors, Type I↗

The effect of changing the excreta moisture of caged laying hens on the excreta and microbial contamination of their egg shells.

1. An experiment that included 1440 caged laying hens in 24 experimental units was conducted to determine the effect of differences in excreta moisture on the proportion of dirty eggs and the microbial contamination of eggs that were ostensibly uncontaminated by excreta. Excreta moisture contents were changed by giving the hens diets that contained 4 different concentrations of sodium. 2. Diets containing 1.6, 5, 10 or 15 g/kg dietary sodium were fed ad libitum to 1140 laying hens for a 12-week feeding period. A sample of excreta was collected from each experimental unit each week and its moisture content determined. All eggs produced were classified as clean or dirty according to the European Community Egg Marketing Regulations. A sample of eggs were collected from each experimental unit on 4 separate occasions in the last 4 weeks of the feeding period and the total bacterial numbers on ostensibly clean egg shells were determined. 3. Increasing dietary sodium concentration gave linear (P<0.01) increases in excreta moisture. Each 100 g/kg increase in excreta moisture increased (P<0.001) dirty egg numbers by 0.52% of the total eggs produced. Increasing excreta moisture gave a linear increase (P<0.001) in the (log-transformed) numbers of microorganisms that contaminated ostensibly clean egg shells.

Animals↗

Effect of excess dietary sodium, potassium, calcium and phosphorus on excreta moisture of laying hens.

1. Four experiments were conducted to investigate the effects of dietary concentrations of sodium, potassium, calcium or phosphate on the water intake and excreta moisture of laying hens. A fifth experiment examined the effect on these variables of increasing amounts of 2 different sodium salts (chloride or bicarbonate) and the interactions with 2 levels of dietary phosphorus. 2. All experiments involved individually caged laying hens fed on diets varying in 1 or 2 minerals in replacement for washed sand. The experimental diets contained mineral concentrations that either met or exceeded the expected requirement of the hens. The diets were given for a 7 or 8 d feeding period and food and water intakes were measured and excreta were collected for the last 48 h of each feeding period. These data were corrected for evaporative water loss to the environment during the collection period. 3. Increasing dietary concentrations of sodium, potassium or phosphorus gave linear increases (P<0.001) in the water intake of the laying hens and linear increases (P<0.01) in the moisture content of their excreta. Each 1 g/kg increase in dietary mineral increased the moisture content of the excreta by 9.04 (+/- 1.57), 11.95 (+/- 2.02) and 5.59 (+/- 0.31) g/kg (+/- standard error) for sodium, potassium and phosphorus, respectively. Increasing concentrations of dietary calcium did not significantly affect the water intakes or excreta moisture levels of the laying hens. 4. The fifth experiment showed that, although there was a sodium x phosphorus interaction (P<0.05), the effects of the 2 mineral additions were approximately additive. There were no significant differences (P>0.05) in water intakes or excreta moisture contents due to the 2 different sodium salts (chloride or bicarbonate).

Animal Feed↗

LROC analysis of detector-response compensation in SPECT.

Localization ROC (LROC) observer studies examined whether detector response compensation (DRC) in ordered-subset, expectation-maximization (OSEM) reconstructions helps in the detection and localization of hot tumors. Simulated gallium (Ga-67) images of the thoracic region were used in the study. The projection data modeled the acquisition of attenuated 93- and 185-keV photons with a medium-energy parallel-hole collimator, but scatter was not modeled. Images were reconstructed with five strategies: 1) OSEM with no DRC; 2) OSEM preceded by restoration filtering; 3) OSEM with iterative DRC; 4) OSEM with an ideal DRC; and 5) filtered backprojection (FBP) with no DRC. All strategies included attenuation correction. There were four LROC studies conducted. In a study using a single tumor activity, the ideal DRC offered the best performance, followed by iterative DRC, restoration filtering, OSEM with no DRC, and FBP. Statistical significance at the 5% level was found between all pairs of strategies except for restoration filtering and OSEM with no DRC. A similar ranking was found for a more realistic study using multiple tumor activities. Additional studies considered the effects of OSEM iteration number and tumor activity on the detection improvement that iterative DRC offered with respect to OSEM with no DRC.

Algorithms↗

Fibrogenesis. V. TGF-beta signaling pathways.

Transforming growth factor (TGF)-beta is a multifunctional peptide growth factor with a wide range of potential effects on growth, differentiation, extracellular matrix deposition, and the immune response. General TGF-beta signaling pathways have been described in detail over the last several years, but factors that determine the nature of the TGF-beta response are poorly understood. In particular, signaling pathways that specifically mediate the matrix effects of TGF-beta have received little attention, although they will be important therapeutic targets in the treatment of pathological fibrosis. This themes article focuses on TGF-beta signaling and highlights potential points for generating matrix-specific responses.

Animals↗

Comparing filtered backprojection and ordered-subsets expectation maximization for small-lesion detection and localization in 67Ga SPECT.

UNLABELLED: Iterative reconstruction of SPECT images has recently become clinically available as an alternative to filtered backprojection (FBP). However, there is conflicting evidence on whether iterative reconstruction, such as with the ordered-subsets expectation maximization (OSEM) algorithm, improves diagnostic performance over FBP. The study objective was to determine if the detection and localization of small lesions in simulated thoracic gallium SPECT images are better with OSEM reconstruction than with FBP, both with and without attenuation correction (AC). METHODS: Images were simulated using an analytic projector acting on the mathematic cardiac torso computer phantom. Perfect scatter rejection was assumed. Lesion detection accuracy was assessed using localization receiver operating characteristic methodology. The images were read by 5 nuclear medicine physicians. For each reconstruction strategy and for each observer, data were collected in 2 viewing sessions of 100 images. Two-way ANOVA and, when indicated, the Scheffé multiple comparisons test were applied to check for significant differences. RESULTS: Little difference in the accuracy of detection or localization was seen between FBP with and without AC. OSEM with AC extended the contrast range for accurate lesion detection and localization over that of the other methods investigated. Without AC, no significant difference between OSEM and FBP reconstruction was detected. CONCLUSION: OSEM with AC may improve the detection and localization of thoracic gallium-labeled lesions over FBP reconstruction.

Algorithms↗

Transforming growth factor-beta induces formation of a dithiothreitol-resistant type I/Type II receptor complex in live cells.

Transforming growth factor-beta (TGF-beta) binds to and signals via two serine-threonine kinase receptors, the type I (TbetaRI) and type II (TbetaRII) receptors. We have used different and complementary techniques to study the physical nature and ligand dependence of the complex formed by TbetaRI and TbetaRII. Velocity centrifugation of endogenous receptors suggests that ligand-bound TbetaRI and TbetaRII form a heteromeric complex that is most likely a heterotetramer. Antibody-mediated immunofluorescence co-patching of epitope-tagged receptors provides the first evidence in live cells that TbetaRI. TbetaRII complex formation occurs at a low but measurable degree in the absence of ligand, increasing significantly after TGF-beta binding. In addition, we demonstrate that pretreatment of cells with dithiothreitol, which inhibits the binding of TGF-beta to TbetaRI, does not prevent formation of the TbetaRI.TbetaRII complex, but increases its sensitivity to detergent and prevents TGF-beta-activated TbetaRI from phosphorylating Smad3 in vitro. This indicates that either a specific conformation of the TbetaRI. TbetaRII complex, disrupted by dithiothreitol, or direct binding of TGF-beta to TbetaRI is required for signaling.

Activin Receptors, Type I↗

Effect of filtering on the detection and localization of small Ga-67 lesions in thoracic single photon emission computed tomography images.

Tumor detection can be significantly affected by filtering so determining an optimal filter is an important aspect of establishing a clinical reconstruction protocol. The purpose of this study was to identify the cut-off frequency of a Butterworth filter used in a filtered backprojection (FBP) reconstruction that maximized the detection and localization accuracy of 1 cm spherical lesions in Ga-67 citrate, thoracic SPECT images. Image quality was evaluated by means of a localization receiver operating characteristic (LROC) study using computer simulated images. Projection data were generated using the mathematical cardiac-torso digital phantom with a clinically realistic background source distribution. The images were reconstructed using FBP with multiplicative Chang attenuation correction and fifth-order Butterworth filtering. The cut-off frequencies considered were 0.25, 0.32, 0.47, and 0.79 cm(-1) for the case of three-dimensional (3D) post-filtering and 0.25, 0.32, and 0.47 cm(-1) for two-dimensional (2D) post-filtering. The images were read by three research scientists and one board certified nuclear medicine clinician. The area under the LROC curve and the localization accuracy for all test conditions were compared using Scheffé's multiple comparisons test. It was found that 3D post-filtering using filters with cut-off frequencies of 0.32 and 0.47 cm(-1) resulted in the highest lesion detectability and localization accuracy. These two test conditions did not differ significantly from each other but were significantly better (p<0.05) than all of the 2D, and the 3D 0.79 cm(-1) cut-off frequency cases.

Analysis of Variance↗

Oligomeric structure of type I and type II transforming growth factor beta receptors: homodimers form in the ER and persist at the plasma membrane.

Transforming growth factor beta (TGF-beta) signaling involves interactions of at least two different receptors, types I (TbetaRI) and II (TbetaRII), which form ligand-mediated heteromeric complexes. Although we have shown in the past that TbetaRII in the absence of ligand is a homodimer on the cell surface, TbetaRI has not been similarly investigated, and the site of complex formation is not known for either receptor. Several studies have indicated that homomeric interactions are involved in TGF-beta signaling and regulation, emphasizing the importance of a detailed understanding of the homooligomerization of TbetaRI or TbetaRII. Here we have combined complementary approaches to study these homomeric interactions in both naturally expressing cell lines and cells cotransfected with various combinations of epitope-tagged type I or type II receptors. We used sedimentation velocity of metabolically labeled receptors on sucrose gradients to show that both TbetaRI and TbetaRII form homodimer-sized complexes in the endoplasmic reticulum, and we used coimmunoprecipitation studies to demonstrate the existence of type I homooligomers. Using a technique based on antibody-mediated immunofluorescence copatching of receptors carrying different epitope tags, we have demonstrated ligand-independent homodimers of TbetaRI on the surface of live cells. Soluble forms of both receptors are secreted as monomers, indicating that the ectodomains are not sufficient to mediate homodimerization, although TGF-beta1 is able to promote dimerization of the type II receptor ectodomain. These findings may have important implications for the regulation of TGF-beta signaling.

Activin Receptors, Type I↗

Biosynthesis of the type I and type II TGF-beta receptors. Implications for complex formation.

The TGF-beta type I and type II receptors (TbetaRI and TbetaRII) are signaling receptors that form heteromeric cell surface complexes with the TGF-betas as one of the earliest events in the cellular response to these multifunctional growth factors. Using TGF-beta-responsive mink lung epithelial cells (Mv1Lu), we have determined the half-lives of the endoplasmic reticulum (ER) and mature forms of these receptors. In metabolically labeled cells, approximately 90% of newly synthesized type II receptor undergoes modification of N-linked sugars in the Golgi, with a half-life of 30-35 min; the Golgi-processed form of the receptor has a relatively short metabolic half-life of 2.5 h. In contrast, only 50% of pulse-labeled type I receptor is converted to the Golgi-processed and therefore endoglycosidase H-resistant form, and the endoglycosidase H-sensitive ER form has a half-life of 2.8-3 h. Addition of 100 pM TGF-beta1 causes the Golgi-processed type II receptor to become less stable, with a half-life of 1.7 h, and also destabilizes the Golgi-processed type I receptor. TGF-beta1 binding and cross-linking experiments on cells treated with tunicamycin for various times confirm different ER to cell surface processing times for TbetaRI and TbetaRII. Our results, which suggest that stable complexes between type I and II TGF-beta receptors do not form until the proteins reach a post-ER compartment (presumably the cell surface), have important implications for our understanding of complex formation and receptor regulation.

Activin Receptors, Type I↗

Wire stent for tracheomalacia in a five-year-old girl.

A wire stent was used successfully to treat life-threatening tracheomalacia in a 5-year-old girl. Wire stents placed bronchoscopically are nonobstructing and have the potential for balloon expansion to accommodate growth.

Bronchoscopy↗

Molecular characteristics of Na(+)-coupled glucose transporters in adult and embryonic rat kidney.

Two distinct Na(+)-coupled glucose transporters (SGLTs) with either a high or a low affinity for glucose were shown to provide reabsorption of filtered glucose in the kidney. We have previously reported the characteristics of the high affinity Na+/glucose cotransporter SGLT1 from rabbit, rat, and human kidney and the low affinity Na+/glucose cotransporter SGLT2 from human kidney. Because the molecular identity of SGLT2 as the kidney cortical low affinity Na+/glucose cotransporter has been recently challenged based on studies of the porcine low affinity Na+/glucoe cotransporter SAAT-pSGLT2 (Mackenzie, B., Panayotova-Heiermann, M., Loo, D. D. F., Lever, J.E., and Wright, E. M. (1994) J. Biol. Chem. 269, 22488-22491), we have reevaluated the properties of SGLT2 in greater detail. We furthermore report new data on the regulation of SGLT1 and SGLT2 during kidney development. To analyze and compare SGLT1 and SGLT2 in adult and embryonic kidney, we have cloned and characterized SGLT2 from rat kidney and determined its tissue distribution based on Northern analysis and in situ hybridization. When expressed in Xenopus oocytes, rat SGLT2 stimulated transport of alpha-methyl-D-glucopyranoside (2 mM) in oocytes up to 4.5-fold over controls with an apparent Km of 3.0 mM. The transport properties (i.e. a Na+ to glucose coupling of 1:1 and lack of galactose transport) generally matched those of the kidney cortical low affinity system. We show that expression of rat SGLT2 mRNA is kidney specific and that it is strongly and exclusively expressed in proximal tubule S1 segments. Hybrid-depletion studies were performed to conclusively determine whether SGLT2 corresponds to the kidney cortical low affinity system. Injection of rat kidney superficial cortex mRNA into oocytes stimulated the uptake of alpha-methyl-D-glucopyranoside (2 mM) 2-3-fold. We show that hybrid depletion of this kidney RNA using an SGLT2 antisense oligonucleotide completely suppresses the uptake. These data strongly indicate that SGLT2 is the major kidney cortical low affinity glucose transporter. We therefore propose that SAAT-pSGLT2 be renamed SGLT3. Experiments addressing the expression of SGLT1 and SGLT2 mRNAs in embryonic rat kidneys reveal that the two Na+/glucose cotransporters are developmentally regulated and that there may be a different splice variant for SGLT2 in embryonic kidney compared to the adult.

Age Factors↗

The soluble exoplasmic domain of the type II transforming growth factor (TGF)-beta receptor. A heterogeneously glycosylated protein with high affinity and selectivity for TGF-beta ligands.

The transforming growth factor (TGF)-beta type II receptor is a transmembrane serine/threonine kinase which is essential for all TGF-beta-induced signals. In several cell types TGF-beta 2 is as potent as TGF-beta or TGF-beta 3 in inducing cellular responses, yet TGF-beta 2 does not bind to the majority of expressed type II receptors. Here we characterized the properties of the soluble extracellular domain of the human TGF-beta type II receptor synthesized in COS-7 cells. Like the membrane-attached type II receptor, the soluble receptor contains complex N-linked oligosaccharides as well as additional sialic acid residues that cause it to migrate heterogenously upon SDS-polyacrylamide gel electrophoresis. 125I-TGF-beta 1 binds to and is chemically cross-linked to this protein. Unlabeled TGF-beta 1 inhibits the binding of 125I-TGF-beta 1 with an apparent dissociation constant (Kd) of approximately 200 pM, similar to the apparent Kd (approximately 50 pM) of the cell-surface type II receptor. TGF-beta 3 inhibits the binding of 125I-TGF-beta 1 to the soluble type II receptor with a similar dissociation constant, approximately 500 pM. In contrast, 125I-TGF-beta 2 cannot bind and be chemically cross-linked to the soluble type II receptor, nor does as much as a 125-fold excess of unlabeled TGF-beta 2 inhibit the binding of 125I-TGF-beta 1 to the soluble receptor. This is the first demonstration of the binding affinities of the type II receptor in the absence of the other cell-surface molecules known to bind TGF-beta. Expressed alone in COS-7 cells the type II receptor also cannot bind TGF-beta 2; co-expression of type III receptor enables the type II receptor to bind TGF-beta 2. Thus, the type III receptor or some other component is required for transmission of TGF-beta 2-induced signals by the type II receptor.

Animals↗