Biomedical subjects

D Wang

Publications and source records attributed to D Wang.

At least 271 records · Page 15Linked to original sources

PubMed2000

Chemical rescue of a mutant protein-tyrosine kinase.

Protein-tyrosine kinases contain a catalytic loop Arg residue located either two or four positions downstream of a highly conserved Asp residue. In this study, the role of this Arg (Arg-318) in the protein-tyrosine kinase C-terminal Src kinase (Csk) was investigated. The observed k(cat) for phosphorylation of the random copolymer poly(Glu,Tyr) substrate by Csk R318A is approximately 3000-fold smaller compared with that of wild type Csk, whereas the K(m) values for ATP and poly(Glu,Tyr) are only mildly affected. The k(cat) value for poly(Glu,Tyr) phosphorylation by the Csk double mutant A316R,R318A is 100-fold greater than the k(cat) value for the single R318A mutant, suggesting that an Arg positioned at the alternative location fulfills a similar function as in wild type. Csk R318A kinase activity can also be partially recovered by several exogenous small molecules including guanidinium and imidazole. These molecules contain key features whose roles in catalysis can be rationalized from a known x-ray structure of the insulin receptor tyrosine kinase. Imidazole is the best of these activators, enhancing phosphorylation rates by Csk R318A up to 100-fold for poly(Glu,Tyr) and significantly stimulating Csk R318A phosphorylation of the physiologic substrate Src. This chemical rescue of mutant protein kinase activity might find applications in cell signal transduction experiments.

Amino Acid Sequence↗

PubMed2000

Transcriptional regulation of the human osteopontin promoter: functional analysis and DNA-protein interactions.

Synthesis of cell attachment proteins and cytokines, such as osteopontin (OPN), can promote tumor cell remodeling of the extracellular matrix into an environment that promotes tumor cell attachment and migration. We investigated the transcriptional regulation of OPN in the U-251MG and U-87MG human malignant astrocytoma cell lines. Deletion and mutagenesis analyses of the OPN promoter region identified a proximal promoter element (-24 to -94 relative to the transcription initiation site) that is essential for maintaining high levels of OPN expression in the tumor cells. This element, designated RE-1, consists of two cis-acting elements, RE-1a (-55 to -86) and RE-1b (-22 to -45), which act synergistically to regulate the activity of the OPN promoter. Gel shift assays using nuclear extracts of U-251MG cells demonstrated that RE-1a contains binding sites for transcription factors Sp1, the glucocorticoid receptor, and the E-box-binding factors, whereas RE-1b contains a binding site for the octamer motif-binding protein (OCT-1/OCT-2). Inclusion of antibodies directed toward Myc and OCT-1 in the gel shift assays indicated that Myc and OCT-1 participate in forming DNA-protein complexes on the RE-1a and RE-1b elements, respectively. Our results identify two previously unrecognized elements in the OPN promoter that act synergistically to promote upregulation of OPN synthesis by tumor cells but are regulated by different transcription factors.

Animals↗

PubMed2000

Cell-specific changes in expression of mRNAs encoding splice variants of aplysia cell adhesion molecule accompany long-term synaptic plasticity.

Aplysia neurons express several splice variants of apCAM, a member of the Ig superfamily of cell adhesion molecules. The major transmembrane isoform is endocytosed in sensory neurons (SNs) during the early phases of long-term facilitation (LTF) of SN synapses evoked by serotonin (5-HT) or in the motor neuron L7 during the early phases of long-term depression (LTD) of SN synapses evoked by Phe-Met-Arg-Phe-amide (FMRFa). We used single cell RT-PCR to evaluate whether expression of mRNAs encoding for different apCAM isoforms in SNs and L7 is regulated during LTF produced by 5-HT, and LTD produced by FMRFa. Single SNs and L7s express mRNAs encoding for all major isoforms, but the proportion of each isoform expressed differs for the two cells. SN expresses more mRNA encoding for GPI-linked isoforms, while L7 expresses more mRNA encoding for the major transmembrane isoform. The neuromodulators produced significant changes in the proportional levels of mRNAs encoding for specific apCAM isoforms during the first 4 h after treatments without affecting overall levels of apCAM mRNA. 5-HT evoked changes that exaggerated cell-specific differences in isoform expression. FMRFa evoked changes that reduced cell-specific differences in isoform expression. The effects of the neuromodulators on apCAM mRNA expression were not detected when cells were cultured alone or when SNs were cocultured with another motor cell that failed to induce synapse formation (L11). The results suggest that rapid cell-specific regulation of splice variant expression may contribute to different forms of long-term synaptic plasticity.

Alternative Splicing↗

PubMed2000

The alphaBbetaC integrin is expressed on the surface of the sea urchin egg and removed at fertilization.

Integrins are expressed on the surface of some vertebrate eggs where they are thought to have a role in fertilization. The objective of this study is to determine if integrins are expressed on sea urchin eggs. The alphaB and betaC subunits were cloned using the homology polymerase chain reaction. Monoclonal and polyclonal antibodies were developed against bacterially expressed fragments of the extracellular domains of the betaC subunit and the alphaB subunit. As well, a monoclonal antibody was developed against a synthesized peptide corresponding to part of the cytoplasmic domain of betaC. Analysis of biotinylated egg cortex extracts immunoprecipitated with either anti-betaC or anti-alphaB yields bands of 130 and 225 kDa. Immunoblots confirm that betaC is part of the complex immunoprecipitated with anti-alphaB. Confocal immunofluorescence and immunogold electron microscopy show that betaC is present on the surface of the unfertilized egg at the tips of microvilli and in cortical granules. During the cortical reaction, immunoreactivity with antibodies to the extracellular domains of betaC and alphaB disappears from the egg surface, and microvillar casts on the fertilization envelope become immunoreactive. With antibodies to the cytoplasmic domain of betaC, immunoreactivity is lost from the surface of the egg, but the fertilization envelope does not immediately become immunoreactive. In immunoblots of egg cortex there are immunoreactive bands of the predicted sizes for alphaB and betaC. However, in fertilization envelopes, a second band that is slightly lower in molecular weight is also present. Eggs fertilized in the presence of soybean trypsin inhibitor have elongated microvilli that remain bound to the elevating fertilization envelope and immunoreactive to anti-betaC antibodies. Eggs fertilized in the presence of an ovoperoxidase inhibitor, 3-amino-1,2,4-triazole, have a patchy distribution of betaC immunoreactivity in fertilization envelopes. Together, these data suggest that alphaBbetaC integrins are expressed on the surface of unfertilized eggs and, during the cortical reaction, the extracellular domains are cleaved by proteases and cross-linked into the fertilization envelope by ovoperoxidase. The alphaBbetaC integrin receptors may have several potential functions prior to their removal at fertilization, including attachment of the vitelline envelope to the egg surface and anchoring the cortical cytoskeleton.

Amino Acid Sequence↗

PubMed2000

Nuclear factor-kappa B activation by the CXC chemokine melanoma growth-stimulatory activity/growth-regulated protein involves the MEKK1/p38 mitogen-activated protein kinase pathway.

Melanoma growth stimulatory activity/growth-regulated protein (MGSA/GRO), a CXC chemokine, plays an important role in inflammation, wound healing, growth regulation, angiogenesis, and tumorigenesis. Constitutive expression of MGSA/GROalpha in melanoma tumors is associated with constitutive nuclear factor (NF)-kappaB activity. We show here that either exogenous addition or continuous expression of MGSA/GROalpha in immortalized melanocytes enhances NF-kappaB activation, as well as mitogen-activated protein (MAP) kinase kinase kinase (MEKK) 1, MAP kinase kinase (MEK) 3/6, and p38 MAP kinase activation. Expression of dominant negative M-Ras (S27N), dominant negative MEKK1 (K432M), or specific chemical inhibitors for p38 MAP kinase (SB202190 and SB203580) block MGSA/GROalpha-induced NF-kappaB transactivation, demonstrating that Ras, MEKK1, and p38 are involved in the signal pathways of MGSA/GROalpha activation of NF-kappaB. Expression of dominant active Ras or dominant active MEKK1 alone can also stimulate NF-kappaB activation. The expression of dominant negative MEKK1 inhibits the Ras-induced NF-kappaB activation, suggesting that MEKK1 is a downstream target of Ras. Moreover, MGSA/GROalpha induction of NF-kappaB is independent of the MEK1/ERK cascade, because MGSA/GROalpha failed to increase ERK and ELK activation, and specific chemical inhibitors for MEK1 (PD98059) had no effect on MGSA/GROalpha-enhanced NF-kappaB activation. These data demonstrate that NF-kappaB activation is required for MGSA/GROalpha-induced melanocyte transformation through a Ras/MEKK1/p38 cascade in melanocytes.

Animals↗

PubMed2000

Tumor necrosis factor alpha-induced phosphorylation of RelA/p65 on Ser529 is controlled by casein kinase II.

Nuclear factor kappaB (NF-kappaB)/Rel transcription factors are key regulators of a variety of genes involved in immune and inflammatory responses, growth, differentiation, apoptosis, and development. In unstimulated cells, NF-kappaB/Rel proteins are sequestered in the cytoplasm by IkappaB inhibitor proteins. Many extracellular stimuli, such as tumor necrosis factor alpha (TNFalpha), cause rapid phosphorylation of IkappaB at N-terminal serine residues leading to ubiquitination and degradation of the inhibitor. Subsequently, NF-kappaB proteins translocate to the nucleus and activate gene expression through kappaB response elements. TNFalpha, as well as certain other stimuli, also induces the phosphorylation of the NF-kappaB proteins. Previously, we have shown that TNFalpha induces RelA/p65 phosphorylation at serine 529 and that this inducible phosphorylation increases NF-kappaB transcriptional activity on an exogenously supplied reporter (). In this report, we demonstrate that casein kinase II (CKII) interacts with p65 in vivo and can phosphorylate p65 at serine 529 in vitro. A CKII inhibitor (PD144795) inhibited TNFalpha-induced p65 phosphorylation in vivo. Furthermore, our results indicate that the association between IkappaBalpha and p65 inhibits p65 phosphorylation by CKII and that degradation of IkappaBalpha allows CKII to phosphorylate p65 to increase NF-kappaB transactivation potential. These data may explain the ability of CKII to modulate cell growth and demonstrate a mechanism whereby CKII can function in an inducible manner.

Casein Kinase II↗

PubMed2000

MGSA/GRO-mediated melanocyte transformation involves induction of Ras expression.

The MGSA/GRO protein is endogenously expressed in almost 70% of the melanoma cell lines and tumors, but not in normal melanocytes. We have previously demonstrated that over-expression of human MGSA/GROalpha, beta or gamma in immortalized murine melanocytes (melan-a cells) enables these cells to form tumors in SCID and nude mice. To examine the possibility that the MGSA/GRO effect on melanocyte transformation requires expression of other genes, differential display was performed. One of the mRNA's identified in the screen as overexpressed in MGSA/GRO transformed melan-a clones was the newly described M-Ras or R-Ras3 gene, a member of the Ras gene superfamily. Over-expression of MGSA/GRO upregulates M-Ras expression at both the mRNA and protein levels, and this induction requires an intact glutamine-leucine-arginine (ELR)-motif in the MGSA/GRO protein. Western blot examination of Ras expression revealed that K- and N-Ras proteins are also elevated in MGSA/GRO-expressing melan-a clones, leading to an overall increase in the amount of activated Ras. MGSA/GRO-expressing melan-a clones exhibited enhanced AP-1 activity. The effects of MGSA/GRO on AP-1 activation could be mimicked by over-expression of wild-type M-Ras or a constitutively activated M-Ras mutant in control melan-a cells as monitored by an AP-1-luciferase reporter, while expression of a dominant negative M-Ras blocked AP-1-luciferase activity in MGSA/GRO-transformed melan-a clones. In the in vitro transformation assay, over-expression of M-Ras mimicked the effects of MGSA/GRO by inducing cellular transformation in control melan-a cells, while over-expression of dominant negative M-Ras in MGSA/GROalpha-expressing melan-a-6 cells blocked transformation. These data suggest that MGSA/GRO-mediated transformation requires Ras activation in melanocytes.

Animals↗

PubMed2000

Probing the structure of the nicotinic acetylcholine receptor with 4-benzoylbenzoylcholine, a novel photoaffinity competitive antagonist.

[(3)H]4-Benzoylbenzoylcholine (Bz(2)choline) was synthesized as a photoaffinity probe for the Torpedo nicotinic acetylcholine receptor (nAChR). [(3)H]Bz(2)choline acts as an nAChR competitive antagonist and binds at equilibrium with the same affinity (K(D) = 1.4 microm) to both agonist sites. Irradiation at 320 nm of nAChR-rich membranes equilibrated with [(3)H]Bz(2)choline results in the covalent incorporation of [(3)H]Bz(2)choline into the nAChR gamma- and delta-subunits that is inhibitable by agonist, with little specific incorporation in the alpha-subunits. To identify the sites of photoincorporation, gamma- and delta-subunits, isolated from nAChR-rich membranes photolabeled with [(3)H]Bz(2)choline, were digested enzymatically, and the labeled fragments were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high performance liquid chromatography. For the gamma-subunit, Staphylococcus aureus V8 protease produced a specifically labeled peptide beginning at gammaVal-102, whereas for the delta-subunit, endoproteinase Asp-N produced a labeled peptide beginning at deltaAsp-99. Amino-terminal sequence analysis identified the homologous residues gammaLeu-109 and deltaLeu-111 as the primary sites of [(3)H]Bz(2)choline photoincorporation. This is the first identification by affinity labeling of non-reactive amino acids within the acetylcholine-binding sites, and these results establish that when choline esters of benzoic acid are bound to the nAChR agonist sites, the para substituent is selectively oriented toward and in proximity to amino acids gammaLeu-109/deltaLeu-111.

Benzoylcholine↗

PubMed2000

The role of brain-derived neurotrophic factor receptors in the mature hippocampus: modulation of long-term potentiation through a presynaptic mechanism involving TrkB.

The neurotrophin BDNF has been shown to modulate long-term potentiation (LTP) at Schaffer collateral-CA1 hippocampal synapses. Mutants in the BDNF receptor gene trkB and antibodies to its second receptor p75NTR have been used to determine the receptors and cells involved in this response. Inhibition of p75NTR does not detectably reduce LTP or affect presynaptic function, but analyses of newly generated trkB mutants implicate TrkB. One mutant has reduced expression in a normal pattern of TrkB throughout the brain. The second mutant was created by cre-loxP-mediated removal of TrkB in CA1 pyramidal neurons of this mouse. Neither mutant detectably impacts survival or morphology of hippocampal neurons. TrkB reduction, however, affects presynaptic function and reduces the ability of tetanic stimulation to induce LTP. Postsynaptic glutamate receptors are not affected by TrkB reduction, indicating that BDNF does not modulate plasticity through postsynaptic TrkB. Consistent with this, elimination of TrkB in postsynaptic neurons does not affect LTP. Moreover, normal LTP is generated in the mutant with reduced TrkB by a depolarization-low-frequency stimulation pairing protocol that puts minimal demands on presynaptic terminal function. Thus, BDNF appears to act through TrkB presynaptically, but not postsynaptically, to modulate LTP.

Animals↗

PubMed2000

Ethanol regulates calcium channel subunits by protein kinase C delta -dependent and -independent mechanisms.

Chronic exposure to ethanol increases the number of functional L-type voltage-gated calcium channels in neural cells. In PC12 cells, this adaptive response is mediated by protein kinase C delta (PKCdelta), but the mechanisms by which this occurs are not known. Since expression of several different calcium channel subunits can increase the abundance of functional L-type channels, we sought to identify which subunits are regulated by ethanol. Incubation of PC12 cells with 120-150 mm ethanol for 6 days increased levels of alpha(1C), alpha(2), and beta(1b) subunit immunoreactivity in cell membranes and selectively increased the abundance of mRNA encoding the alpha(1C-1) splice variant of alpha(1C). In cells expressing a fragment of PKCdelta (deltaV1) that selectively inhibits PKCdelta, there was no increase in membrane-associated alpha(1C), alpha(2), and beta(1b) immunoreactivity following chronic ethanol exposure. However, ethanol still increased levels of alpha(1C-1) mRNA in these cells. These results indicate that ethanol increases the abundance of L-type channels by at least two mechanisms; one involves increases in mRNA encoding a splice variant of alpha(1C) and the other is post-transcriptional, rate-limiting, and requires PKCdelta.

Alternative Splicing↗

PubMed2000

Analysis of specific gene mutations in the transforming growth factor-beta signal transduction pathway in human ovarian cancer.

Several proteins, including transforming growth factor beta (TGF-beta) receptor type I (RI), TGF-beta receptor type II (RII), Smad2, Smad3, and Smad4/DPC4, have been identified in the transduction pathway of the tumor suppressor TGF-beta. Mutations in TGF-beta RI, TGF-beta RII, Smad2, and Smad4/DPC4 genes are associated with several human cancers. The present study examines these gene mutations in 32 human ovarian cancers and 14 patient-matched normal tissues. For the first time, mutations in the Smad2 and Smad4 genes were analyzed in relation to human ovarian cancer. Gene mutations of TGF-beta RI, TGF-beta RII, Smad2, and Smad4 were analyzed using specific primers by PCR-single-strand conformational polymorphism (SSCP), and the results revealed a frameshift mutation at codons 276-277 (CTCTGG-->CTGCGTGG) in exon 5 of TGF-beta RI in 10 of 32 tumor samples (31.3%). This mutation was associated with reduced or absent expression of TGF-beta RI protein and p53 protein in tumor tissues. We detected SSCP variants of TGF-beta RII in exon 2 in 20 of 32 tumors. Sequence analysis of these variants revealed an A to G transition at the seventh band of intron 2. In this A to G polymorphism in intron 2, 12 samples (37.5%) had A/A alleles, 12 (37.5%) had A/G alleles, and 8 (25%) had G/G alleles. We detected Smad2 SSCP variants in exon 4 in 12 of 32 tumors (37.5%). Sequence analysis revealed a 2-bp deletion in the polypyrimidine tract of intron 3, which is located at position -39 to -56 in the splice acceptor site of the intron 3-exon 4 junction. No SSCP variants were detected in the Smad4 gene. These findings suggest that mutations in the TGF-beta RI and in its signal transduction pathway are likely responsible for human ovarian carcinogenesis.

Activin Receptors, Type I↗

PubMed2000

Cloning and in vitro expression of the cDNA encoding a putative nucleoside transporter from Arabidopsis thaliana.

Nucleoside transporters are integral membrane proteins involved in the uptake or release of nucleosides. Their function constitutes an essential step of the salvage pathway of nucleotide synthesis. In order to study the function of these proteins in higher plants, we cloned the cDNA corresponding to the AtENT1 gene that encodes a putative nucleoside transporter in Arabidopsis thaliana by RT-PCR. The amino acid sequence of the AtENT1 protein deduced from the cloned cDNA shared similarity to those of eukaryotic equilibrative nucleoside transporters. Structure prediction indicated that the deduced AtENT1 protein might possess eleven putative transmembrane domains. Southern hybridization revealed that AtENT1 had one homologue in the Arabidopsis genome. Northern blot analysis showed that AtENT1 might be constitutively expressed in most Arabidopsis organs and in plants at different developmental stages. Two AtENT1 fusion genes, AtENT1-His-tag and GFP-AtENT1-His-tag, were expressed in insect cells. Confocal microscopy demonstrated that the GFP-AtENT1-His-tag fusion protein was targeted specifically to the plasma membrane of insect cells.

Journal Article↗

PubMed2000

Myosin heavy chain gene expression in normal and hyperplastic human prostate tissue.

BACKGROUND: Benign prostatic hyperplasia (BPH) is common among aging men. Over 80% of males 50-60 years and older have various degrees of bladder outlet obstruction secondary to BPH. Despite the tremendous medical impact of BPH, its molecular pathophysiology remains unclear. Current BPH research focuses on steroid hormonal effects, stromal-epithelial cell interaction, and oncogenes and growth factors. But little is known about the potential prostatic smooth muscle (SM) alterations that may occur during stromal hyperplasia. METHODS: To study SM phenotypic modulation in hyperplastic prostatic growth, we isolated and characterized the 3' end of human SM myosin heavy chain (SMMHC) cDNA as a molecular probe. Expression of SMMHC and nonmuscle myosin heavy chain (NMMHC) in human prostates was analyzed using Western blot, Northern blot, and in situ hybridization to determine if BPH tissue expresses significantly less SMMHC and more NMMHC than a normal prostate. In addition, a competitive, reverse transcription (RT) polymerase chain reaction (PCR) method was adapted to quantify SMMHC and NMMHC mRNA expression at the sensitivity level of 10(-21) mole per mg of wet tissue. RESULTS: Western blot, Northern blot, and in situ hybridization results reveal that both SMMHC and NMMHC are expressed in the human prostate, while SMMHC is the predominant form found in normal prostate stroma. Results from competitive RT-PCR analysis indicate that NMMHC mRNA expression is approximately 10(-20) mole/mg of tissue. The SMMHC mRNA expressed is approximately 10(-18) mole/mg. No significant difference was found when NMMHC mRNA expression was compared between normal and BPH periurethral tissues. However, SMMHC expression was reduced almost fivefold in BPH compared to normal prostate, despite an increase in prostatic stromal mass. CONCLUSIONS: Our results suggest the pathogenesis of BPH is associated with a unique type of SM proliferation. Such proliferation is characterized by downregulation of SMMHC mRNA expression but without upregulation of NMMHC mRNA expression, the pattern seen in proliferating SM cells in culture and in other pathologic forms of SM hyperplasia (e.g., atherosclerosis). These findings support a model of BPH typified by active smooth muscle proliferation early in the disease process, and supports clinical observations that suggest ongoing prostate growth of the prostate is minimal in older men. Therapeutic strategies to prevent disease progression should therefore focus on early phases of prostatic growth.

Adult↗

PubMed2000

Recognition of a cognate RNA aptamer by neomycin B: quantitative evaluation of hydrogen bonding and electrostatic interactions.

Aminoglycosides are an important class of antibiotic that selectively target RNA structural motifs. Recently we have demonstrated copper derivatives of amino-glycosides to be efficient cleavage agents for cognate RNA motifs. To fully develop their potential as pharmaceutical agents it is necessary to understand both the structural mechanisms used by aminoglycosides to target RNA, and the relative contributions of hydrogen bonding and electrostatic interactions to recognition selectivity. Herein we report results from a calorimetric analysis of a stem-loop 23mer RNA aptamer complexed to the aminoglycoside neomycin B. Key thermodynamic parameters for complex formation have been determined by isothermal titration calorimetry, and from the metal-ion dependence of these binding parameters the relative contributions of electrostatics and hydrogen bonding toward binding affinity have been assessed. The principal mechanism for recognition and binding of neomycin B to the RNA major groove is mediated by hydrogen bonding.

Base Sequence↗

PubMed2000

Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family.

Two cDNAs encoding NADPH oxidases and constituting the thyroid H(2)O(2) generating system have been cloned. The strategy of cloning was based on the functional similarities between H(2)O(2) generation in leukocytes and the thyroid, according to the hypothesis that one of the components of the thyroid system would belong to the gp91(Phox)/Mox1 gene family and display sequence similarities with gp91(Phox). Screening at low stringency with a gp91(Phox) probe of cDNA libraries from thyroid cells in primary culture yielded two distinct human cDNA clones harboring open reading frames of 1551 (ThOX1) and 1548 amino acids (ThOX2), respectively. The encoded polypeptides display 83% sequence similarity and are clearly related to gp91(Phox) (53 and 47% similarity). The theoretical molecular mass of 177 kDa is close to the apparent molecular mass of 180 kDa of the native corresponding porcine flavoprotein and the protein(s) detected by Western blot in dog and human thyroid. ThOX1 and ThOX2 display sequence similarities of 53% and 61%, respectively, with a predicted protein of Caenorhabditis elegans over their entire length. They show along their first 500 amino acids a similarity of 43% with thyroperoxidase. The corresponding genes of ThOX1 and ThOX2 are closely linked on chromosome 15q15.3. The dog mRNA expression is thyroid-specific and up-regulated by agents activating the cAMP pathway as is the synthesis of the polypeptides they are coding for. In human thyroid the positive regulation by cAMP is less pronounced. The proteins ThOX1 and ThOX2 accumulate at the apical membrane of thyrocytes and are co-localized with thyroperoxidase.

Amino Acid Sequence↗

PubMed2000

gamma-aminobutyric acid- and glycine-immunoreactive neurons postsynaptic to substance P-immunoreactive axon terminals in the superficial layers of the rat medullary dorsal horn.

gamma-Aminobutyric acid (GABA)ergic and glycinergic neurons were examined light- and electron-microscopically in laminae I and II of the medullary dorsal horn (MDH, i.e. spinal trigeminal nucleus caudalis in the rat). The majority of GABA- and glycine (Gly)-immunoreactive (-ir) neurons showed both GABA- and Gly-immunoreactivities (-IRs). Noxious stimulation (subcutaneous injection of formalin into perioral regions) induced Fos-IR in some of GABA- and Gly-ir neurons. GABA- and Gly-ir neuronal profiles were postsynaptic to substance P-ir axon terminals. These results suggest that nociceptive information being carried by primary afferent SP-fibers may be relayed directly to GABAergic and glycinergic neurons in laminae I and II of the MDH.

Animals↗

PubMed2000

A novel smad nuclear interacting protein, SNIP1, suppresses p300-dependent TGF-beta signal transduction.

Members of the transforming growth factor-beta superfamily play critical roles in controlling cell growth and differentiation. Effects of TGF-beta family ligands are mediated by Smad proteins. To understand the mechanism of Smad function, we sought to identify novel interactors of Smads by use of a yeast two-hybrid system. A 396-amino acid nuclear protein termed SNIP1 was cloned and shown to harbor a nuclear localization signal (NLS) and a Forkhead-associated (FHA) domain. The carboxyl terminus of SNIP1 interacts with Smad1 and Smad2 in yeast two-hybrid as well as in mammalian overexpression systems. However, the amino terminus of SNIP1 harbors binding sites for both Smad4 and the coactivator CBP/p300. Interaction between endogenous levels of SNIP1 and Smad4 or CBP/p300 is detected in NMuMg cells as well as in vitro. Overexpression of full-length SNIP1 or its amino terminus is sufficient to inhibit multiple gene responses to TGF-beta and CBP/p300, as well as the formation of a Smad4/p300 complex. Studies in Xenopus laevis further suggest that SNIP1 plays a role in regulating dorsomedial mesoderm formation by the TGF-beta family member nodal. Thus, SNIP1 is a nuclear inhibitor of CBP/p300 and its level of expression in specific cell types has important physiological consequences by setting a threshold for TGF-beta-induced transcriptional activation involving CBP/p300.

Amino Acid Sequence↗

PubMed2000

Posttranslational processing and differential glycosylation of Tractin, an Ig-superfamily member involved in regulation of axonal outgrowth.

Tractin is a novel member of the Ig-superfamily which has a highly unusual structure. It contains six Ig domains, four FNIII-like domains, an acidic domain, 12 repeats of a novel proline- and glycine-rich motif with sequence similarity to collagen, a transmembrane domain, and an intracellular tail with an ankyrin and a PDZ domain binding motif. By generating domain-specific antibodies, we show that Tractin is proteolytically processed at two cleavage sites, one located in the third FNIII domain, and a second located just proximal to the transmembrane domain resulting in the formation of four fragments. The most NH(2)-terminal fragment which is glycosylated with the Lan3-2, Lan4-2, and Laz2-369 glycoepitopes is secreted, and we present evidence which supports a model in which the remaining fragments combine to form a secreted homodimer as well as a transmembrane heterodimer. The extracellular domain of the dimers is mostly made up of the collagen-like PG/YG-repeat domain but also contains 11/2 FNIII domain and the acidic domain. The collagen-like PG/YG-repeat domain could be selectively digested by collagenase and we show by yeast two-hybrid analysis that the intracellular domain of Tractin can interact with ankyrin. Thus, the transmembrane heterodimer of Tractin constitutes a novel protein domain configuration where sequence that has properties similar to that of extracellular matrix molecules is directly linked to the cytoskeleton through interactions with ankyrin.

Animals↗