Biomedical subjects

D Yan

Publications and source records attributed to D Yan.

At least 19 recordsLinked to original sources

PubMed2007

Ageing and hearing loss.

Although many adults retain good hearing as they age, hearing loss associated with ageing is common among elderly persons. There are a number of pathophysiolological processes underlying age-related changes to functional components in the inner ear. Genetic factors determine the ageing process but are under the influence of intrinsic and environmental factors. It is difficult to distinguish changes of normal ageing from those of other contributing factors. The effects of age-related deafness can have significant physical, functional and mental health consequences. Although a deficit in hearing can be corrected to some degree by a hearing aid or other appropriate amplification devices, hearing-related rehabilitative needs are much more than simply amplifying external sound. Only by better understanding the process of ageing and its effect on the auditory function can we better accommodate elderly people in our day-to-day interactions. We review here the structure and function of the inner ear, pathophysiology associated with age-related hearing loss (ARHL), heritability, allelism and modifier genes of ARHL, and evaluate the genetic analyses for identification of genetic factors that are involved.

Aging↗

PubMed2006

An isoform of GTPase regulator DOCK4 localizes to the stereocilia in the inner ear and binds to harmonin (USH1C).

The driving forces for the regulation of cell morphology are the Rho family GTPases that coordinate the assembly of the actin cytoskeleton. This dynamic feature is a result of tight coupling between the cytoskeleton and signal transduction and is facilitated by actin-binding proteins (ABPs). Mutations in the actin bundling and PDZ domain-containing protein harmonin are the causes of Usher syndrome type 1C (USH1C), a syndrome of congenital deafness and progressive blindness, as well as certain forms of non-syndromic deafness. Here, we have used the yeast two-hybrid assay to isolate molecular partners of harmonin and identified DOCK4, an unconventional guanine exchange factor for the Rho family of guanosine triphosphatases (Rho GEF GTPases), as a protein interacting with harmonin. Detailed molecular analysis revealed that a novel DOCK4 isoform (DOCK4-Ex49) is expressed in the brain, eye and inner ear tissues. We have further provided evidence that the DOCK4-Ex49 binds to nucleotide free Rac as effectively as DOCK2 and DOCK4 and it is a potent Rac activator. By immunostaining using a peptide antibody specific to DOCK4-Ex49, we showed its localization in the inner ear within the hair bundles along the stereocilia (SC). Together, our data indicate a possible Rac-DOCK4-ABP harmonin-activated signaling pathway in regulating actin cytoskeleton organization in stereocilia.

Actins↗

PubMed2006

The OSBP-related proteins (ORPs): global sterol sensors for co-ordination of cellular lipid metabolism, membrane trafficking and signalling processes?

Protein families related to OSBP (oxysterol-binding protein) are present in eukaryotes from yeast to human. The functions of the ORPs (OSBP-related proteins) have remained largely enigmatic. Even though they have been implicated in the function of ERJs (endoplasmic reticulum junctions), it is evident that any single model for their mechanism of action is insufficient. The existing evidence points in many different directions, such as integration of sterol and sphingomyelin metabolism, regulation of neutral lipid metabolism, control of signalling cascades, regulation of secretory vesicle generation, and function in the microtubule-based motility of endo/lysosomes. Some of these functions could involve ERJ and non-vesicular transport of lipids, but this is unlikely to be the unifying feature. We believe, rather, that the common denominator for ORP function is acting as sterol sensors that relay information to a spectrum of cellular processes.

Animals↗

PubMed2006

A material sensitivity study on the accuracy of deformable organ registration using linear biomechanical models.

Model-based deformable organ registration techniques using the finite element method (FEM) have recently been investigated intensively and applied to image-guided adaptive radiotherapy (IGART). These techniques assume that human organs are linearly elastic material, and their mechanical properties are predetermined. Unfortunately, the accurate measurement of the tissue material properties is challenging and the properties usually vary between patients. A common issue is therefore the achievable accuracy of the calculation due to the limited access to tissue elastic material constants. In this study, we performed a systematic investigation on this subject based on tissue biomechanics and computer simulations to establish the relationships between achievable registration accuracy and tissue mechanical and organ geometrical properties. Primarily we focused on image registration for three organs: rectal wall, bladder wall, and prostate. The tissue anisotropy due to orientation preference in tissue fiber alignment is captured by using an orthotropic or a transversely isotropic elastic model. First we developed biomechanical models for the rectal wall, bladder wall, and prostate using simplified geometries and investigated the effect of varying material parameters on the resulting organ deformation. Then computer models based on patient image data were constructed, and image registrations were performed. The sensitivity of registration errors was studied by perturbating the tissue material properties from their mean values while fixing the boundary conditions. The simulation results demonstrated that registration error for a subvolume increases as its distance from the boundary increases. Also, a variable associated with material stability was found to be a dominant factor in registration accuracy in the context of material uncertainty. For hollow thin organs such as rectal walls and bladder walls, the registration errors are limited. Given 30% in material uncertainty, the registration error is limited to within 1.3 mm. For a solid organ such as the prostate, the registration errors are much larger. Given 30% in material uncertainty, the registration error can reach 4.5 mm. However, the registration error distribution for prostates shows that most of the subvolumes have a much smaller registration error. A deformable organ registration technique that uses FEM is a good candidate in IGART if the mean material parameters are available.

Biomechanical Phenomena↗

PubMed2005

Modelling individual geometric variation based on dominant eigenmodes of organ deformation: implementation and evaluation.

We present a method of modelling inter-fractional organ deformation and correlated motion of adjacent organ structures in terms of so-called eigenmodes. The method is based on a principal component analysis (PCA) of organ shapes and allows for reducing the large dimensionality of geometry information from multiple CT studies to a few-parametric statistical model of organ motion and deformation. Eigenmodes are 3D vectorfields of correlated displacements of the organ surface points and can be seen as fundamental 'modes' of the patient's geometric variability. The amount of variability represented by the eigenmodes is quantified in terms of corresponding eigenvalues. Weighted sums of eigenmodes describe organ displacements/deformations and can be used to generate new organ geometries. We applied the method to four patient datasets of prostate/rectum/bladder with N = 15-18 CTs to assess interfractional geometric variation. The spectrum of eigenvalues was found to be dominated by only few values, indicating that the geometric variability of prostate/bladder/rectum is governed by only few patient specific eigenmodes. We evaluated the capability of this approach to represent the measured organ samples by calculating the residual errors for the organ surface points, using a varying number of eigenmodes. The distribution of residual errors shows fast convergence with the number of eigenmodes. Using 4 dominating modes, the range of residual errors for the four patients was 1.3-2.0 mm (prostate), 1.4-1.9 mm (rectum) and 1.5-1.9 mm (bladder). Thus, individual geometric variation taken from multiple imaging data can be described accurately by few dominating eigenmodes, thereby providing the most important factors to characterize deformable organ motion, which can assist adaptive radiotherapy planning.

Algorithms↗

PubMed2005

Dynamic surface tensions of Athabasca bitumen vacuum residue including the effect of dissolved air.

The pendant drop technique was used to measure the equilibrium and dynamic surface tensions of Athabasca bitumen vacuum residue (500 degrees C+) (AVR) between 150 and 280 degrees C. A significant (16%) slow (over hours) decrease from initial to equilibrium values was found. In addition, the effect of dissolved air on surface tension was studied at 150 degrees C by comparing dynamic surface tensions of air- or nitrogen-saturated AVR in contact with air or nitrogen. It was found that the presence of dissolved air significantly decreases the dynamic change in surface tension (from 16 to 5%). In order to perform the surface tension studies, the density of AVR was required. Archimedes method was used to measure the density of AVR from 98 to 335.8 degrees C.

Journal Article↗

PubMed2005

A novel locus for autosomal dominant non-syndromic deafness, DFNA53, maps to chromosome 14q11.2-q12.

BACKGROUND: Non-syndromic hearing loss is among the most genetically heterogeneous traits known in humans. To date, at least 50 loci for autosomal dominant non-syndromic sensorineural hearing loss (ADNSSHL) have been identified by linkage analysis. OBJECTIVE: To report the mapping of a novel autosomal dominant deafness locus on the long arm of chromosome 14 at 14q11.2-q12, DFNA53, in a large multigenerational Chinese family with post-lingual, high frequency hearing loss that progresses to involve all frequencies. RESULTS: A maximum multipoint LOD score of 5.4 was obtained for marker D14S1280. The analysis of recombinant haplotypes mapped DFNA53 to a 9.6 cM region interval between markers D14S581 and D14S1021. Four deafness loci (DFNA9, DFNA23, DFNB5, and DFNB35) have previously been mapped to the long arm of chromosome 14. The critical region for DFNA53 contains the gene for DFNA9 but does not overlap with the regions for DFNB5, DFNA23, or DFNB35. Screening of the COCH gene (DFNA9), BOCT, EFS, and HSPC156 within the DFNA53 interval did not identify the cause for deafness in this family. CONCLUSIONS: Identifying the DFNA53 locus is the first step in isolating the gene responsible for hearing loss in this large multigeneration Chinese family.

Adolescent↗

PubMed2004

Mutational spectrum in Usher syndrome type II.

Usher syndrome type II is an autosomal recessive disorder characterized by moderate to severe hearing impairment and progressive visual loss due to retinitis pigmentosa (RP). We carried out a mutation screening of the USH2A gene in 88 probands with Usher syndrome type II to determine the frequency of USH2A mutations as a cause for USH2. Six mutations, including 2299delG, 921-922insCAGC, R334W, N346H, R626X, and N357T were identified, with 2299delG mutation being the most frequent (16.5% of alleles), accounting for 77.5% of the pathologic alleles. Thirty-five percent (31/88) of the probands had a USH2A mutation. Nine of them carried two pathogenic mutations: six cases were homozygotes and three were compound heterozygotes. Twenty-two probands (25%) were found to carry only single USH2A mutations. One new missense mutation (N357T) occuring within the laminin N-terminal (type VI) domain of usherin was identified. Eight polymorphisms were found, five of which are novel. Our data support the view that the 2299delG is the most common mutation in USH2A.

Case-Control Studies↗

PubMed2003

Reducing uncertainties in volumetric image based deformable organ registration.

Applying volumetric image feedback in radiotherapy requires image based deformable organ registration. The foundation of this registration is the ability of tracking subvolume displacement in organs of interest. Subvolume displacement can be calculated by applying biomechanics model and the finite element method to human organs manifested on the multiple volumetric images. The calculation accuracy, however, is highly dependent on the determination of the corresponding organ boundary points. Lacking sufficient information for such determination, uncertainties are inevitable-thus diminishing the registration accuracy. In this paper, a method of consuming energy minimization was developed to reduce these uncertainties. Starting from an initial selection of organ boundary point correspondence on volumetric image sets, the subvolume displacement and stress distribution of the whole organ are calculated and the consumed energy due to the subvolume displacements is computed accordingly. The corresponding positions of the initially selected boundary points are then iteratively optimized to minimize the consuming energy under geometry and stress constraints. In this study, a rectal wall delineated from patient CT image was artificially deformed using a computer simulation and utilized to test the optimization. Subvolume displacements calculated based on the optimized boundary point correspondence were compared to the true displacements, and the calculation accuracy was thereby evaluated. Results demonstrate that a significant improvement on the accuracy of the deformable organ registration can be achieved by applying the consuming energy minimization in the organ deformation calculation.

Algorithms↗

PubMed2003

Adapting inverse planning to patient and organ geometrical variation: algorithm and implementation.

Image guided radiotherapy has the potential to improve both tumour control and normal tissue sparing by including temporal patient specific geometry information into the adaptive planning process. In this study we present a practical method of image guided adaptive inverse planning based on computed tomography (CT) and portal image feedback during the treatment course. The method is based on a general description of the radiotherapy optimization problem subject to dynamic geometrical variations of the patient/organs. We will demonstrate the feasibility of off-line image feedback into the inverse planning process with the example of three prostate cancer patients. CT and portal images acquired during the early course of the treatment are used to predict the geometrical variation distribution of a patient and to re-optimize the treatment plan accordingly. We will study the convergence of the optimization problem with respect to the number of image measurements and adaptive feedback loops.

Algorithms↗

PubMed2002

Neural inhibition by c-Jun as a synergizing factor in bone morphogenetic protein 4 signaling.

The transcription factor, activator protein 1 (AP-1) complexes (c-Jun and c-Fos heterodimers) has been shown to interact with transforming growth factor beta signaling in mammalian cells and Drosophila embryo. Here we show that c-Jun alone is involved in the anti-neuralizing activity of bone morphogenetic protein 4, a transforming growth factor beta superfamily member, in Xenopus neurogenesis. Co-injection of mRNAs encoding c-jun and a dominant negative bone morphogenetic protein receptor completely inhibits dominant negative bone morphogenetic protein receptor-induced neuralization and reverses the epidermal fate in the animal cap. Surprisingly, a dominant negative c-Jun does not induce neural tissue in the animal cap, but it synergizes with dominant negative bone morphogenetic protein receptor for neural induction. Temporal analysis using a dexamethasone-inducible c-Jun shows that exogenous c-Jun activity must be turned on before or at stage 11 to fulfill the anti-neuralizing effect. Neural inhibition by c-Jun does not occur until stage 13 suggesting that c-Jun probably acts by suppressing neural maintenance rather than neural initiation. This is also supported by the fact that c-Jun does not inhibit expression of the neural-initializing gene Zic-r1 but the neural cofactor Sox2, and that ectopic expression of Sox2 attenuates the anti-neuralizing effect of c-Jun. Finally, we display that the c-Jun effect is enhanced by an auto-regulatory loop between c-Jun and bone morphogenetic protein. These studies suggest that c-Jun/AP-1 is a converging point in both the fibroblast growth factor and transforming growth factor beta signaling pathways. Based on our findings, we propose that c-Jun synergizes with bone morphogenetic protein 4 signaling to inhibit neural development in Xenopus ectoderm.

Animals↗

PubMed2001

Elevated expression of axin2 and hnkd mRNA provides evidence that Wnt/beta -catenin signaling is activated in human colon tumors.

Genetic studies have identified mutations in key regulators of the Wnt/beta-catenin pathway in a variety of cancers, most frequently in colon cancers. However, whether the pathway is activated in clinical cancer samples is not easily determined, and therefore it is useful to find markers that could be surrogates to show activation of the Wnt/beta-catenin pathway. Gene expression profiles were analyzed in SW620, a colon cancer cell line in which beta-catenin levels are stabilized as a consequence of truncated adenomatous polyposis coli and were compared with profiles of the same cells transfected with antisense oligodeoxynucleotides. Treatment of cells with beta-catenin antisense oligodeoxynucleotides resulted in a decrease in the levels of axin2 and human naked cuticle (hnkd) mRNAs. Interestingly, the proteins encoded by both of these mRNAs are known inhibitors of the beta-catenin pathway. In 30 human cell lines derived from different origins, axin2 and hnkd were expressed only in human colon cancer cell lines that are known to have activating mutations in the Wnt/beta-catenin pathway. Further, levels of both axin2 and hnkd mRNA were also found to be elevated in about 65% of laser microdissected cells from human colon tumors compared with laser microdissected cells of normal morphology from the same patient samples. The increased expression of axin2 and hnkd correlated with truncations in adenomatous polyposis coli in the same patient samples. These results reveal that it is possible to detect activation of a carcinogenic pathway in human cancer samples with specific markers.

Axin Protein↗

PubMed2001

The position and volume of the small bowel during adjuvant radiation therapy for rectal cancer.

PURPOSE: The rate of small bowel toxicity from adjuvant pelvic radiation therapy (RT) for rectal cancer has been reported to be lower for patients treated preoperatively (Preop). This was probably due to a lesser volume of irradiated small bowel; however, studies of postoperative treatment reported that patients with an abdominoperineal resection (APR), who likely have the largest volume of small bowel in the pelvis, had less acute and chronic toxicity than those with a low anterior resection (LAR). In this study, three-dimensional treatment planning techniques were used to characterize the position and volume of small bowel in the pelvis and compare these to repeat studies obtained during the typical 5-week course of treatment to attempt to explain the above observations. METHODS AND MATERIALS: Treatment planning CT scans were obtained in 30 patients with rectal cancer (10 Preop, 10 LAR, 10 APR), including 12 patients with weekly CT scans during RT (65 scans). The position of the small bowel was measured by the distance to the nearest small bowel from the bones of the posterior pelvis and by the volume of small bowel within four anatomically defined regions of the pelvis. The motion of the small bowel was expressed as the standard deviation of the small bowel position measured with both the distance and the volume in the 12 patients with repeat studies. RESULTS: Contrast-containing small bowel was found an average 2.9 cm more anterior than small bowel without contrast below the sacral promontory. The position of the small bowel in Preop patients was significantly more anterior (p < or = 0.01) with less volume (p < or = 0.04) in the pelvis than postoperatively treated patients. The small bowel was also more anterior for patients with an LAR vs. APR (p < or = 0.03) but with similar volume in all pelvic regions. Small bowel motion, expressed as the standard deviation of the distance from the bones of the posterior pelvis to the closest small bowel, was 2.9 cm, 1.4 cm, and 0.2 cm for the Preop, LAR, and APR group, respectively. The LAR group had a considerable degree of motion in the posterior pelvis. Increased bladder volume was associated with reduced small bowel volumes, although this benefit decreased during treatment. CONCLUSION: Because treatment planning CT scans can detect small bowel that does not contain contrast, they may be more accurate than the traditional small bowel series. The Preop patients had significantly less pelvic small bowel supporting the clinical observation of better tolerance to therapy. The higher small bowel toxicity reported for LAR vs. APR patients may be explained by the greater variability of both the position and volume of the small bowel in the posterior pelvis for LAR patients. This finding suggests that a single planning study may not be accurate for the block design used for boost treatment of LAR patients. Bladder-filling techniques were useful for Preop and LAR but not APR patients, and decreased in benefit over time. This study suggested that treatment planning CT scans were more useful than a small bowel series and that more than one treatment planning CT may be obtained in any patient receiving > 45 Gy for rectal cancer. However, further research will be necessary to determine the optimal timing and total number of repeat studies.

Diarrhea↗

PubMed2001

The influence of interpatient and intrapatient rectum variation on external beam treatment of prostate cancer.

PURPOSE: The rectal dose/volume relationship and inherent variations thereof are fundamental parameters to guide dose escalation in prostate cancer treatment. This study evaluates the effect of rectal dose/volume variation on the risk of rectal complication for different planning target volume (PTV) constructions. METHODS AND MATERIALS: Thirty prostate patients with multiple daily CT scans obtained during the treatment course were included in this retrospective study. The dose distribution was calculated based on the pretreatment CT image alone. Treatment plans were generated by applying the four-field-box beam arrangement to each of three different PTVs: PTVs with 0.5-cm and 1.0-cm uniform margins, and a patient-specific PTV constructed using treatment imaging feedback. For each of the 30 patients, the rectal wall as manifested on each of multiple CT images was delineated after image bony registration to the pretreatment CT image, and applied to the corresponding treatment plan to obtain the rectal wall dose-volume histogram (DVH). Interpatient and intrapatient rectal dose/volume variations were quantified accordingly. The corresponding uncertainty and sensitivity of the risk of rectal complication to the variations were evaluated for each of the three PTVs. Finally, the efficacy of using multiple CT images to reduce uncertainty in planning evaluation was examined. RESULTS: Sensitivity of the risk of rectal complication to rectal dose/volume variation strongly depends on the clinical target volume (CTV)-to-PTV margin or prescription dose, or both. Compared to the conventional two-dimensional (2D) prostate cancer treatment, the sensitivity for a conformal treatment can be 3 times higher or more. Due to the interpatient rectal dose/volume variation, the individual normal tissue complication probability (NTCP) was distributed from 10% to 37% when a common prescription dose was applied for all patients. The intrapatient rectal dose/volume variation introduces at least +/- 25% uncertainty in the NTCP calculation for at least 10% or 25% of the patients treated with the PTV of 1.0- or 0.5-cm margin, respectively. These uncertainties were larger for the smaller PTV, with the standard deviation up to 20%. By applying multiple CT image feedback, the NTCP uncertainty could be reduced by a factor of 2. CONCLUSIONS: Shape and position variation of rectum has less influence on treatment planning in the conventional 2D treatment of prostate cancer. However, this influence is quickly growing with high treatment dose or small CTV-to-PTV margins. To reduce the variation and uncertainties in the treatment planning evaluation associated with the inter- and intrapatient rectal dose/volume variation, the iso-NTCP model and treatment image feedback technique can be applied in dose escalation trials of prostate cancer treatment.

Algorithms↗

PubMed2001

Improvement in dose escalation using the process of adaptive radiotherapy combined with three-dimensional conformal or intensity-modulated beams for prostate cancer.

PURPOSE: Advances in technology allow the creation of complex treatment plans with tightly conforming doses. However, variations in positioning of the organ/patient with respect to treatment beams necessitate the use of an appreciable margin, potentially limiting dose escalation in many patients. To (1) reduce this margin and (2) test the hypothesis that the achievable level of dose escalation is patient dependent, a patient-specific, confidence-limited planning target volume (cl-PTV) was constructed using an adaptive radiotherapy (ART) process for prostate cancer treatment developed in-house. The potential dose escalation achievable with this ART process is quantified for both conformal radiotherapy (CRT) delivery and intensity-modulated radiotherapy (IMRT) delivery. MATERIAL AND METHODS: Patients with organ confined prostate cancer were entered prospectively into an ART process developed in-house. This ART process has been designed to improve accuracy and precision of dose delivery, consequently enhancing dose escalation. In this process, a cl-PTV is constructed for each patient in the second week of treatment based upon on-line portal and CT images acquired during the first week of treatment. The treatment prescription dose, defined as the minimum dose to the cl-PTV, is selected based on predefined dose-volume constraints for rectum/bladder and derived from the pretreatment planning CT image. In addition, the treatment modality (CRT or IMRT) is determined based on the level of dose escalation achievable and the risk of inaccurate targeting. The potential for both dose escalation and the application of IMRT was evaluated by comparing the prescription doses delivered using the ART process, with the cl-PTV, to those in the traditional treatment process, with a conventional generic PTV. In addition, the distributions of potential geometric target underdosing and normal tissue overdosing were also calculated to evaluate the quality of the conventional treatment plans. RESULTS: One hundred and fifty patients have been treated with the ART process. When compared to the treatment dose delivered with the conventional treatment process (generic PTV), an average 5% (2.5--10%) more dose could be delivered using the ART process with CRT, and 7.5% (2.5--15%) more dose could be delivered with IMRT. Of the 150 patients, 70% were treated to a minimum cl-PTV dose > or = 77.4 Gy (81.3 Gy ICRU isocenter dose). Dosimetric analysis revealed that 81 Gy to the cl-PTV (or 86.7 Gy ICRU) could be prescribed to at least 50% of patients if IMRT was applied using the ART process. In contrast, IMRT did not yield an obvious dose escalation gain if patients were treated using the generic PTV. Our results also demonstrate that the cl-PTV is significantly smaller than the conventional generic PTV for most patients, with a mean volume reduction of 24% (range, 5--43%). CONCLUSION: These results support our hypothesis that the achievable level of dose escalation using ART is patient dependent. By using the ART process to develop a cl-PTV, one can (1) optimize the dose level, (2) increase the applicability of IMRT, and (3) improve the quality of dose delivery. The ART process provides the foundation to identify a suitable option (CRT or IMRT) for the delivery of a safe treatment and dose escalation. It is now our standard of practice for prostate cancer treatment.

Adenocarcinoma↗

PubMed2001

BeF(3)(-) acts as a phosphate analog in proteins phosphorylated on aspartate: structure of a BeF(3)(-) complex with phosphoserine phosphatase.

Protein phosphoaspartate bonds play a variety of roles. In response regulator proteins of two-component signal transduction systems, phosphorylation of an aspartate residue is coupled to a change from an inactive to an active conformation. In phosphatases and mutases of the haloacid dehalogenase (HAD) superfamily, phosphoaspartate serves as an intermediate in phosphotransfer reactions, and in P-type ATPases, also members of the HAD family, it serves in the conversion of chemical energy to ion gradients. In each case, lability of the phosphoaspartate linkage has hampered a detailed study of the phosphorylated form. For response regulators, this difficulty was recently overcome with a phosphate analog, BeF(3)(-), which yields persistent complexes with the active site aspartate of their receiver domains. We now extend the application of this analog to a HAD superfamily member by solving at 1.5-A resolution the x-ray crystal structure of the complex of BeF(3)(-) with phosphoserine phosphatase (PSP) from Methanococcus jannaschii. The structure is comparable to that of a phosphoenzyme intermediate: BeF(3)(-) is bound to Asp-11 with the tetrahedral geometry of a phosphoryl group, is coordinated to Mg(2+), and is bound to residues surrounding the active site that are conserved in the HAD superfamily. Comparison of the active sites of BeF(3)(-) x PSP and BeF(3)(-) x CeY, a receiver domain/response regulator, reveals striking similarities that provide insights into the function not only of PSP but also of P-type ATPases. Our results indicate that use of BeF(3)(-) for structural studies of proteins that form phosphoaspartate linkages will extend well beyond response regulators.

Aspartic Acid↗

PubMed2001

Cell autonomous regulation of multiple Dishevelled-dependent pathways by mammalian Nkd.

Genetic studies have identified Drosophila Naked Cuticle (Nkd) as an antagonist of the canonical Wnt/beta-catenin signaling pathway, but its mechanism of action remains obscure [Zeng, W., Wharton, K. A., Jr., Mack, J. A., Wang, K., Gadbaw, M., et al. (2000) Nature (London) 403, 789--795]. Here we have cloned a cDNA encoding a mammalian homolog of Drosophila Nkd, mNkd, and demonstrated that mNkd interacts directly with Dishevelled. Dishevelled is an intracellular mediator of both the canonical Wnt pathway and planar cell polarity (PCP) pathway. Activation of the c-Jun-N-terminal kinase has been implicated in the PCP pathway. We showed that mNkd acts in a cell-autonomous manner not only to inhibit the canonical Wnt pathway but also to stimulate c-Jun-N-terminal kinase activity. Expression of mNkd disrupted convergent extension in Xenopus, consistent with a role for mNkd in the PCP pathway. These data suggest that mNkd may act as a switch to direct Dishevelled activity toward the PCP pathway, and away from the canonical Wnt pathway.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Crystal structure of activated CheY. Comparison with other activated receiver domains.

The crystal structure of BeF(3)(-)-activated CheY, with manganese in the magnesium binding site, was determined at 2.4-A resolution. BeF(3)(-) bonds to Asp(57), the normal site of phosphorylation, forming a hydrogen bond and salt bridge with Thr(87) and Lys(109), respectively. The six coordination sites for manganese are satisfied by a fluorine of BeF(3)(-), the side chain oxygens of Asp(13) and Asp(57), the carbonyl oxygen of Asn(59), and two water molecules. All of the active site interactions seen for BeF(3)(-)-CheY are also observed in P-Spo0A(r). Thus, BeF(3)(-) activates CheY as well as other receiver domains by mimicking both the tetrahedral geometry and electrostatic potential of a phosphoryl group. The aromatic ring of Tyr(106) is found buried within a hydrophobic pocket formed by beta-strand beta4 and helix H4. The tyrosine side chain is stabilized in this conformation by a hydrogen bond between the hydroxyl group and the backbone carbonyl oxygen of Glu(89). This hydrogen bond appears to stabilize the active conformation of the beta4/H4 loop. Comparison of the backbone coordinates for the active and inactive states of CheY reveals that only modest changes occur upon activation, except in the loops, with the largest changes occurring in the beta4/H4 loop. This region is known to be conformationally flexible in inactive CheY and is part of the surface used by activated CheY for binding its target, FliM. The pattern of activation-induced backbone coordinate changes is similar to that seen in FixJ(r). A common feature in the active sites of BeF(3)(-)-CheY, P-Spo0A(r), P-FixJ(r), and phosphono-CheY is a salt bridge between Lys(109) Nzeta and the phosphate or its equivalent, beryllofluoride. This suggests that, in addition to the concerted movements of Thr(87) and Tyr(106) (Thr-Tyr coupling), formation of the Lys(109)-PO(3)(-) salt bridge is directly involved in the activation of receiver domains generally.

Bacterial Proteins↗