Biomedical subjects

Xiaohui Wang

Publications and source records attributed to Xiaohui Wang.

At least 19 recordsLinked to original sources

PubMed2026

Structural insights into histone mimicry by the small hepatitis delta antigen.

Hepatitis delta virus (HDV) is a satellite RNA virus that requires hepatitis B virus (HBV) for propagation but replicates its genome independently in the nucleus. The small form of the hepatitis delta antigen (S-HDAg) is essential for replication and is regulated by post-translational modifications. Acetylation at lysine 72 (K72ac) enables S-HDAg to interact with the bromodomain (BRD) of the host chromatin remodeler bromodomain adjacent to zinc finger domain protein 2B (BAZ2B) to promote viral replication. However, the structural basis for this interaction has remained elusive. Here, we provide structural and biophysical insights into this interaction through quantitative binding assays and X-ray crystallography. Isothermal titration calorimetry revealed that BRDs of BAZ2B and its close homolog BAZ2A bind to the viral peptide weakly, with BAZ2A-BRD exhibiting a modestly higher affinity. The crystal structure of BAZ2A-BRD in complex with the S-HDAg-K72ac peptide demonstrates an inverted binding orientation relative to canonical histone ligands, rationalizing the weak interaction. Mutagenesis studies confirmed the critical binding interface both in vitro and in cells. These findings elucidate the molecular mechanism by which HDV co-opts host BAZ2 bromodomains via a unique, weak-affinity interaction, providing a structural framework for understanding viral replication.

Hepatitis delta Antigens↗

PubMed2005

High affinity ATP/ADP analogues as new tools for studying CFTR gating.

Previous studies using non-hydrolysable ATP analogues and hydrolysis-deficient cystic fibrosis transmembrane conductance regulator (CFTR) mutants have indicated that ATP hydrolysis precedes channel closing. Our recent data suggest that ATP binding is also important in modulating the closing rate. This latter hypothesis predicts that ATP analogues with higher binding affinities should stabilize the open state more than ATP. Here we explore the possibility of using N6-modified ATP/ADP analogues as high-affinity ligands for CFTR gating, since these analogues have been shown to be more potent than native ATP/ADP in other ATP-binding proteins. Among the three N6-modified ATP analogues tested, N6-(2-phenylethyl)-ATP (P-ATP) was the most potent, with a K(1/2) of 1.6 +/- 0.4 microm (>50-fold more potent than ATP). The maximal open probability (P(o)) in the presence of P-ATP was approximately 30% higher than that of ATP, indicating that P-ATP also has a higher efficacy than ATP. Single-channel kinetic analysis showed that as [P-ATP] was increased, the opening rate increased, whereas the closing rate decreased. The fact that these two kinetic parameters have different sensitivities to changes of [P-ATP] suggests an involvement of two different ATP-binding sites, a high-affinity site modulating channel closing and a low affinity site controlling channel opening. The effect of P-ATP on the stability of open states was more evident when ATP hydrolysis was abolished, either by mutating the nucleotide-binding domain 2 (NBD2) Walker B glutamate (i.e. E1371) or by using the non-hydrolysable ATP analogue AMP-PNP. Similar strategies to develop nucleotide analogues with a modified adenine ring could be valuable for future studies of CFTR gating.

ATP-Binding Cassette Transporters↗

PubMed2005

[Clinical efficacy of image processing of grid detection and suppression (GDS) in computed radiography].

In projection radiography, stationary grids are indispensable accessories to the improvement of diagnostic imaging. On the other hand, they are becoming one of the issues facing digital image processing. The lead foil that composes the grid can produce moiré on printed films and monitors according to the sampling interval at which the image is read by computed radiography (CR), creating a major obstacle to diagnosing images. The subject of this study on Grid Detection and Suppression (GDS) was the development of comprehensive image-processing software to detect and suppress grid lines automatically. Our results showed that applying GDS parameters 3 approximately 5 could provide a sufficient effect on suppression with little impact on images through the use of a multi-purpose grid (grid ratio 8:1, density 34 lp/cm) for general purposes. In projection radiography, it is expected that soft copy diagnosis will increase because the digital transition is proceeding, and the establishment of high-speed networks is becoming easier. Therefore, the digital environment is expected to improve and the choice of grids and monitors to expand, by using software such as GDS that does not require special skills.

Data Display↗

PubMed2005

Comparison of edge analysis techniques for the determination of the MTF of digital radiographic systems.

The modulation transfer function (MTF) is well established as a metric to characterize the resolution performance of a digital radiographic system. Implemented by various laboratories, the edge technique is currently the most widespread approach to measure the MTF. However, there can be differences in the results attributed to differences in the analysis technique employed. The objective of this study was to determine whether comparable results can be obtained from different algorithms processing identical images representative of those of current digital radiographic systems. Five laboratories participated in a round-robin evaluation of six different algorithms including one prescribed in the International Electrotechnical Commission (IEC) 62220-1 standard. The algorithms were applied to two synthetic and 12 real edge images from different digital radiographic systems including CR, and direct- and indirect-conversion detector systems. The results were analysed in terms of variability as well as accuracy of the resulting presampled MTFs. The results indicated that differences between the individual MTFs and the mean MTF were largely below 0.02. In the case of the two simulated edge images, all algorithms yielded similar results within 0.01 of the expected true MTF. The findings indicated that all algorithms tested in this round-robin evaluation, including the IEC-prescribed algorithm, were suitable for accurate MTF determination from edge images, provided the images are not excessively noisy. The agreement of the MTF results was judged sufficient for the measurement of the MTF necessary for the determination of the DQE.

Algorithms↗

PubMed2005

Androgen receptor mutants detected in recurrent prostate cancer exhibit diverse functional characteristics.

BACKGROUND: Alterations in the function of androgen receptor (AR) and its signaling pathway may be responsible for the progression of prostate cancer. The goal of the present study was to investigate the potential roles of AR structural and functional alterations in the progression of prostate cancer, and the relationship between the structure and function of the AR. METHODS: AR gene in 58 prostate cancer samples was examined for mutations using PCR-single strand conformation polymorphism (SSCP) analysis and DNA sequencing. Effects of mutations on the structure and function of AR were investigated by androgen-binding assays and transactivation assays, respectively. RESULTS: Four novel somatic mutations (G142V, D221H, E872Q, and M886I) were identified from recurrent prostate cancer samples. None of the AR mutants differed from wild-type AR (wtAR) in their abilities to bind the synthetic androgen methyltrienolone. However, these mutated AR exhibited diverse functional characteristics as compared with wtAR. G142V and D221H showed increased responses to DHT. E872Q could be abnormally activated by 17beta-estradiol, progesterone, and cyproterone acetate (CPA). Furthermore, E872Q and M886I presented increased responses to DHT in the presence of coactivators TIF-2 and CBP, but not p300. On the other hand, although overexpression of corepressors N-CoR and SMRT could result in evident inhibition on DHT- or CPA-induced transactivity of wtAR and the AR mutants, N-CoR displayed stronger inhibitory effects on DHT-induced transactivity of the AR mutants (especially for E872Q and M886I) than that of wtAR. To our knowledge, this is the first characterization of enhanced inhibitory effects of corepressors on the transactivity of the AR mutants found in prostate cancer. CONCLUSIONS: The data presented here demonstrate that AR mutants found in prostate cancer had different functional alterations, which might play an important role in the progression of prostate cancer.

Aged↗

PubMed2005

Involvement of death receptor signaling in mechanical stretch-induced cardiomyocyte apoptosis.

Recent evidences suggest that mechanical overload associated with abnormal blood pressure causes apoptosis in cardiovascular system. Still, the intracellular signaling leading to cardiomyocyte apoptosis has not been fully defined. Previous reports ascribed stretch-induced cardiomyocyte apoptosis to rennin-angiotensin-system (RAS) signaling and/or mitochondria-dependent apoptosis pathway. The present study shows the involvement of death receptor signaling in mechanical stretch-induced cardiomyocyte apoptosis. By employing a well-described in vitro stretch model, we studied stretch-induced apoptosis and found that the death receptor-mediated apoptotic signaling was activated in stretch-induced apoptosis in neonatal rat cardiomyocytes. The major finding are as following: (1) The mechanical stretch activated death receptor-mediated apoptotic signaling in cardiomyocytes, including activation of caspases 8, 9 and 3, up-regulation of Fas, FasL expression and cell surface trafficking of death ligands (FasL and TRAIL); (2) That exogenous death ligand (TRAIL) enhanced, while soluble death receptor (sDR5) neutralized, stretch-induced apoptosis; (3) Adenovirus-delivered dominant negative FADD (FADD-DN) significantly reduced apoptosis, caspases 8, 9, and 3 activation, and stretch-induced cyt c release from mitochondria. These data clearly suggested mechanical stretch activated death receptor-mediated apoptotic signaling in cardiomyocytes. In conclusion, our data suggest that the FADD-linked death receptor signaling may contribute to stretch-induced cardiomyocyte apoptosis, probably through activating mitochondria-dependent apoptotic signaling.

Adaptor Proteins, Signal Transducing↗

PubMed2005

CFTR gating I: Characterization of the ATP-dependent gating of a phosphorylation-independent CFTR channel (DeltaR-CFTR).

The CFTR chloride channel is activated by phosphorylation of serine residues in the regulatory (R) domain and then gated by ATP binding and hydrolysis at the nucleotide binding domains (NBDs). Studies of the ATP-dependent gating process in excised inside-out patches are very often hampered by channel rundown partly caused by membrane-associated phosphatases. Since the severed DeltaR-CFTR, whose R domain is completely removed, can bypass the phosphorylation-dependent regulation, this mutant channel might be a useful tool to explore the gating mechanisms of CFTR. To this end, we investigated the regulation and gating of the DeltaR-CFTR expressed in Chinese hamster ovary cells. In the cell-attached mode, basal DeltaR-CFTR currents were always obtained in the absence of cAMP agonists. Application of cAMP agonists or PMA, a PKC activator, failed to affect the activity, indicating that the activity of DeltaR-CFTR channels is indeed phosphorylation independent. Consistent with this conclusion, in excised inside-out patches, application of the catalytic subunit of PKA did not affect ATP-induced currents. Similarities of ATP-dependent gating between wild type and DeltaR-CFTR make this phosphorylation-independent mutant a useful system to explore more extensively the gating mechanisms of CFTR. Using the DeltaR-CFTR construct, we studied the inhibitory effect of ADP on CFTR gating. The Ki for ADP increases as the [ATP] is increased, suggesting a competitive mechanism of inhibition. Single channel kinetic analysis reveals a new closed state in the presence of ADP, consistent with a kinetic mechanism by which ADP binds at the same site as ATP for channel opening. Moreover, we found that the open time of the channel is shortened by as much as 54% in the presence of ADP. This unexpected result suggests another ADP binding site that modulates channel closing.

Adenosine Diphosphate↗

PubMed2005

CFTR gating II: Effects of nucleotide binding on the stability of open states.

Previously, we demonstrated that ADP inhibits cystic fibrosis transmembrane conductance regulator (CFTR) opening by competing with ATP for a binding site presumably in the COOH-terminal nucleotide binding domain (NBD2). We also found that the open time of the channel is shortened in the presence of ADP. To further study this effect of ADP on the open state, we have used two CFTR mutants (D1370N and E1371S); both have longer open times because of impaired ATP hydrolysis at NBD2. Single-channel kinetic analysis of DeltaR/D1370N-CFTR shows unequivocally that the open time of this mutant channel is decreased by ADP. DeltaR/E1371S-CFTR channels can be locked open by millimolar ATP with a time constant of approximately 100 s, estimated from current relaxation upon nucleotide removal. ADP induces a shorter locked-open state, suggesting that binding of ADP at a second site decreases the locked-open time. To test the functional consequence of the occupancy of this second nucleotide binding site, we changed the [ATP] and performed similar relaxation analysis for E1371S-CFTR channels. Two locked-open time constants can be discerned and the relative distribution of each component is altered by changing [ATP] so that increasing [ATP] shifts the relative distribution to the longer locked-open state. Single-channel kinetic analysis for DeltaR/E1371S-CFTR confirms an [ATP]-dependent shift of the distribution of two locked-open time constants. These results support the idea that occupancy of a second ATP binding site stabilizes the locked-open state. This binding site likely resides in the NH2-terminal nucleotide binding domain (NBD1) because introducing the K464A mutation, which decreases ATP binding affinity at NBD1, into E1371S-CFTR shortens the relaxation time constant. These results suggest that the binding energy of nucleotide at NBD1 contributes to the overall energetics of the open channel conformation.

Adenosine Diphosphate↗

PubMed2005

Relative contributions of innate and acquired host responses to bacterial control and arthritis development in Lyme disease.

TLR2(-/-)/scid double-mutant mice were infected with B. burgdorferi to assess the relative importance of acquired and innate host defenses. Although spirochete levels at 4 weeks were lower in TLR2(-/-) mice than in TLR2(-/-)/scid mice, the increased arthritis severity of TLR2 (Toll-like receptor 2)-deficient mice was reduced by the presence of the scid mutation.

Animals↗

PubMed2005

[Adaptive response induced by low concentration of hydroquinone in human embryonic fibroblasts cells].

OBJECTIVE: To study the adaptive response induced by hydroquinone(HQ) on eukaryocyte and its possible mechanism. METHODS: After hydroquinone treatment, AlamarBlue reduce rate, LDH release rate were observed to determine the cell proliferation and death. Annexin V/propidium iodide (PI) staining was performed for each treatment to distinguish viable, early, and late apoptosis or dead cells. The total cellular proteins were separated using two- dimensional gel electrophoresis and visualized by sliver staining. Digital images were analyzed using ImageMaster 2D platinum 5.0 software. The differentially expressed protein spots were picked and digested in gel then identified by tandem mass spectrum. RESULTS: The results of AlamarBlue reduce rate, LDH leakage and Annexin V-FITC/PI staining showed that no effect on cell viability was observed at a concentration of 10 micromol/L HQ while 250 micromol/L HQ significantly decreased cell viability. Cells pretreated with 10 micromol/L HQ for 12h show increasing survival to the following expose to 250 micromol/L HQ. In control MRC-5 cells 1429 +/- 369 protein spots were detected and, 1453 +/- 307 in low HQ group, 1191 +/- 393 in high HQ group, and 1107 +/- 247 in adaptive group by two dimensional gel electrophoresis. Twenty-four protein spots showed significant change after HQ stimulation and 22 protein spots were identified by tandems mass spectra. These identified proteins involved in energy metabolism, translation and RNA processing, protein folding, redox regulation, cell structure and cell signaling. CONCLUSION: Adaptive response can be induced by low concentration of HQ on MRC-5 cells and cellular adaptation is a complex process involving in a modulation of diverse cellular functions.

Apoptosis↗

PubMed2004

Chitosan kills bacteria through cell membrane damage.

The bactericidal activity of chitosan (CS) acetate solution against Escherichia coli and Staphylococcus aureus was evaluated by the enumeration of viable organisms at different incubation times. Morphologies of bacteria treated with CS were observed by transmission electron microscopy (TEM). The integrity of the cell membranes of both species and the permeabilities of the outer membrane (OM) and inner membrane (IM) of E. coli were investigated by determining the release from cells of materials that absorb at 260 nm, changes in the fluorescence of cells treated with the fluorescent probe 1-N-phenylnaphthylamine (NPN) and release of cytoplasmic beta-galactosidase activity. In addition, the interaction of CS with synthetic phospholipid membranes was studied using gel permeation chromatography (GPC), UV-VIS spectrophotometery, Fourier-transform infrared spectroscopy (FT-IR) and thermal analysis. Results showed that CS increased the permeability of the OM and IM and ultimately disrupted bacterial cell membranes, with the release of cellular contents. This damage was likely caused by the electrostatic interaction between NH(3)(+) groups of CS acetate and phosphoryl groups of phospholipid components of cell membranes.

Bacterial Outer Membrane Proteins↗

PubMed2004

High-efficiency, environment-friendly electroluminescent polymers with stable high work function metal as a cathode: green- and yellow-emitting conjugated polyfluorene polyelectrolytes and their neutral precursors.

A series of aminoalkyl-substituted polyfluorene copolymers with benzothiadiazole (BTDZ) of different content were synthesized by Suzuki coupling reaction, and their quaternized ammonium polyelectrolyte derivatives were obtained through a postpolymerization treatment on the terminal amino groups. Copolymers are soluble in environmentally friendlier solvents, such as alcohols. It was found that the efficient energy transfer occurs by exciton trapping on the narrow band gap BTDZ site under UV illumination. Only 1% of BTDZ content is needed to completely quench a fluorene emission for both the neutral and the quaternized copolymers in the neat film. Absolute PL efficiencies of copolymer films were greatly enhanced as a result of the suppression of excimer formation. Light-emitting devices fabricated from these copolymers show high external quantum efficiencies over 3% and 1% for the neutral precursor and the quaternized copolymers, respectively, with high work function metals such as Al as a cathode. To the best of our knowledge, this is the first report on an electroluminescent polymer which bears the high EL efficiency, the electron-injection ability from high work function metals, and the solubility in environment-friendly solvents at the same time. These features make them a promising candidate for the next generation of light-emitting copolymers in PLED flat panel display application.

Journal Article↗

PubMed2004

Influence of a 12-nt insertion present in the 3' untranslated region of classical swine fever virus HCLV strain genome on RNA synthesis.

The function of a 12-nt insertion 'CTTTTTTCTTTT' in the 3' untranslated region (3'UTR) of the HCLV strain, a vaccine strain derived from the Shimen strain of classical swine fever virus (CSFV), was examined in vitro. RNA synthesis increased when the 12-nt insertion was deleted from the 3'UTR of the HCLV strain. RNA synthesis also decreased when the 12-nt insertion was introduced into the 3'UTR of the CSFV Shimen, virulent strain. Therefore, the 12-nt insertion present in the 3'UTR of the HCLV strain may be a marker for avirulence. It was also found that the predicted secondary structure of the 3'UTR of the HCLV strain was more stable when the 12-nt insertion was deleted, and that the 3'UTR of Shimen strain was more unstable when the 12-nt insertion was introduced. Replacement of the 12-nt insertion 'CTTTTTTCTTTT' by another 12-nt fragment 'ATTATTATTTAT' in the 3'UTR of HCLV strain facilitated RNA synthesis and stabilized the predicted secondary structure, which was also observed in the 3'UTR of the Shimen strain.

3' Untranslated Regions↗

PubMed2004

Direct effects of 9-anthracene compounds on cystic fibrosis transmembrane conductance regulator gating.

Anthracene-9-carboxylic acid (9-AC) has been reported to show both potentiation and inhibitory effects on guinea-pig cardiac cAMP-activated chloride channels via two different binding sites, and inhibition of Mg(2+)-sensitive protein phosphatases has been proposed for the mechanism of 9-AC potentiation effect. In this study, we examined the effects of 9-AC on wild-type and mutant human cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels expressed in NIH3T3 or CHO cells. 9-AC inhibits whole-cell CFTR current in a voltage-dependent manner, whereas the potentiation effect is not affected by membrane potentials. Anthracene-9-methanol, an electro-neutral 9-AC analog, fails to block CFTR, but shows a nearly identical potentiation effect, corroborating the idea that two chemically distinct sites are responsible, respectively, for potentiation and inhibitory actions of 9-AC. 9-AC also enhances the activity of deltaR-CFTR, a constitutively active CFTR mutant whose R-domain is removed. In excised inside-out patches, 9-AC increases Po by prolonging the mean burst durations and shortening the interburst durations. We therefore conclude that two different 9-AC binding sites for potentiation and inhibitory effects on CFTR channels are located outside of the R-domain. We also speculate that 9-AC potentiates CFTR activity by directly affecting CFTR gating.

Animals↗

PubMed2004

Capsaicin potentiates wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride-channel currents.

To examine the effects of capsaicin on cystic fibrosis transmembrane conductance regulator (CFTR), we recorded wild-type and mutant CFTR chloride-channel currents using patch-clamp methods. The effects of capsaicin were compared with those of genistein, a well-characterized CFTR activator. In whole-cell experiments, capsaicin potentiates cAMP-stimulated wild-type CFTR currents expressed in NIH 3T3 cells or Chinese hamster ovary cells in a dose-dependent manner with a maximal response approximately 60% of that with genistein and an apparent Kd of 48.4 +/- 6.8 microM. In cell-attached recordings, capsaicin alone fails to activate CFTR in cells that show negligible basal CFTR activity, indicating that capsaicin does not stimulate the cAMP cascade. The magnitude of potentiation with capsaicin depends on the channel activity before drug application; the lower the prestimulated Po, the higher the potentiation. Single-channel kinetic analysis shows that capsaicin potentiates CFTR by increasing the opening rate and decreasing the closing rate of the channel. Capsaicin may act as a partial agonist of genistein because the maximally enhanced wild-type CFTR currents with genistein are partially inhibited by capsaicin. Capsaicin increases DeltaR-CFTR, a protein kinase A (PKA)-independent, constitutively active channel, in cell-attached patches. In excised inside-out patches, capsaicin potentiates the PKA-phosphorylated, ATP-dependent CFTR activity. Both capsaicin and genistein potentiate the cAMP-stimulated G551D-CFTR, DeltaF508-CFTR, and 8SA mutant channel currents. The binding site for capsaicin is probably located at the cytoplasmic domain of CFTR, because pipette application of capsaicin fails to potentiate CFTR activity. In conclusion, capsaicin is a partial agonist of genistein in activation of the CFTR chloride channel. Both compounds affect ATP-dependent gating of CFTR.

Animals↗

PubMed2004

Expression and regulation of alpha-, beta-, and gamma-crystallins in mammalian lens epithelial cells.

PURPOSE: In the mammalian lens, the expression of the beta- and gamma-crystallin families is thought to be limited to fiber cells. However, several studies detected these proteins or their mRNAs in human lens epithelial cells. To resolve this apparent discrepancy, 14 crystallin mRNAs were examined and the expression and subcellular distribution of selected crystallin proteins in lens epithelial cells determined. METHODS: Transcript levels were analyzed by quantitative real-time PCR using mRNA from P3 rat lens epithelia cultured for 0 or 20 hours or 4 or 7 days in basal medium or with added FGF2. Antibodies to betaB1-, gammaS-, alphaA-, and alphaB-crystallins were used for Western blot analysis of proteins extracted from adult mouse, human, bovine, rabbit, and rat lens epithelial and fiber cells. Rat lenses or lens epithelia were rapidly fixed in situ, 30 minutes after death, or after dissection from the lens, and the intracellular distributions of crystallins were examined by immunostaining and confocal microscopy. RESULTS: Four patterns of crystallin gene expression were detected in cultured lens epithelia. Transcripts encoding most beta- and gamma-crystallins were detectable and, in some cases, abundant at the time of explantation. Changes in crystallin protein levels in P3 epithelia cultured in basal or FGF-supplemented medium generally reflected the changes in their mRNAs. betaB1- and gammaS-crystallins were abundant in adult human, mouse, rat, rabbit, and bovine lens epithelial cells. The alpha-, beta- and gamma-crystallins were found in distinct subcellular locations in adult lens epithelial cells. These proteins dramatically relocalized during fiber cell differentiation and after death and/or dissection of the lens epithelium. CONCLUSIONS: BetaB1- and gammaS-crystallins are normally abundant in adult mammalian lens epithelial cells. Complex programs of transcription and degradation regulate the accumulation of crystallin mRNAs in lens epithelial cells after stress, at different ages, and during cell differentiation. Because crystallins selectively localize in distinct subcellular compartments during differentiation or stress, they may function to protect lens cells from injury. After stress, most alphaA- and alphaB-crystallin subunits are not in the same macromolecular complexes.

Animals↗

PubMed2004

Contributions by members of the TGFbeta superfamily to lens development.

Members of the TGFbeta superfamily of growth and differentiation factors, including the TGFbeta, BMP, activin and nodal families, play important signaling roles throughout development. This paper summarizes some of the functions of these ligands in lens development. Targeted deletion of the genes encoding one of the BMP receptors, Alk3 (BMP receptor-1A), showed that signaling through this receptor is essential for normal lens development. Lenses lacking Alk3 were smaller than normal, with thin epithelial layers. The fiber cells of Alk3 null lenses became vacuolated and degenerated within the first week after birth. Lenses lacking Alk3 function were surrounded by abnormal mesenchymal cells, suggesting that the lenses provided inappropriate signals to surrounding tissues. Lens epithelial and fiber cells contained endosomes that were associated with activated (phosphorylated) SMAD1 and SMAD2. Endosomal localization of pSMAD1 was reduced in the absence of Alk3 signaling. The presence of pSMAD2 in lens fiber cell nuclei and the observation that the activin antagonist follistatin inhibited lens cell elongation suggested that an activin-like molecule participates in lens fiber cell differentiation. Lenses deficient in type II TGFbeta receptors were clear and had fiber cells of normal morphology. This suggests that TGFbeta signaling is not essential for the normal differentiation of lens fiber cells. The targeted deletion of single or multiple receptors of the TGFbeta superfamily in the lens should further characterize the role of these signaling molecules in lens development. This approach may also provide a useful way to define the downstream pathways that are activated by these receptors during the development of the lens and other tissues.

Animals↗

PubMed2003

Demonstration of mammalian protein O-mannosyltransferase activity: coexpression of POMT1 and POMT2 required for enzymatic activity.

Defects in O-mannosylation of alpha-dystroglycan are thought to cause certain types of congenital muscular dystrophies with neuronal migration disorders. Among these muscular dystrophies, Walker-Warburg syndrome is caused by mutations in the gene encoding putative protein O-mannosyltransferase 1 (POMT1), which is homologous to yeast protein O-mannosyltransferases. However, there is no evidence that POMT1 has enzymatic activity. In this study, we first developed a method to detect protein O-mannosyltransferase activity in mammalian cells. Then, using this method, we showed that coexpression of both POMT1 and POMT2 (another gene homologous to yeast protein O-mannosyltransferases) was necessary for the enzyme activity, but expression of either POMT1 or POMT2 alone was insufficient. The requirement of an active enzyme complex of POMT1 and POMT2 suggests that the regulation of protein O-mannosylation is complex. Further, protein O-mannosylation appears to be required for normal structure and function of alpha-dystroglycan in muscle and brain. In view of the potential importance of this form of glycosylation for a number of developmental and neurobiological processes, the ability to assay mammalian protein O-mannosyltransferase activity should greatly facilitate progress in the identification and localization of O-mannosylated proteins and the elucidation of their functional roles.

Amino Acid Sequence↗