Search PubMed⌕ Search

Biomedical subjects

X Lin

Publications and source records attributed to X Lin.

At least 145 records · Page 8Linked to original sources
PubMed1999

Inhibition of calcineurin phosphatase activity by a calcineurin B homologous protein.

Calcineurin, a Ca(2+)/calmodulin-stimulated protein phosphatase, plays a key role in T-cell activation by regulating the activity of NFAT (nuclear factor of activated T cells), a family of transcription factors required for the synthesis of several cytokine genes. Calcineurin is the target of the immunosuppressive drugs cyclosporin A and FK506 complexed with their cytoplasmic receptors cyclophilin and FKBP12, respectively. In this study we report that calcineurin is also the target of a recently identified Ca(2+)-binding protein, CHP (for calcineurin homologous protein), which shares a high degree of homology with the regulatory B subunit of calcineurin and with calmodulin. In Jurkat and HeLa cells, overexpression of CHP specifically impaired the nuclear translocation and transcriptional activity of NFAT but had no effect on AP-1 transcriptional activity and only a small (<25%) inhibitory effect on the transcriptional activity of NFkappaB. Further study indicated that CHP inhibits calcineurin activity. In cells overexpressing CHP, the phosphatase activity of immunoprecipitated calcineurin was inhibited by approximately 50%; and in a reconstituted assay, the activity of purified calcineurin was inhibited up to 97% by the addition of purified recombinant CHP in a dose-dependent manner. Moreover, prolonged activation of Jurkat cells was associated with a decreased abundance of CHP, suggesting a possible regulatory mechanism allowing activation of calcineurin. CHP, therefore, is a previously unrecognized endogenous inhibitor of calcineurin activity.

Calcineurin↗
PubMed1999

Sequence and analysis of chromosome 2 of the plant Arabidopsis thaliana.

Arabidopsis thaliana (Arabidopsis) is unique among plant model organisms in having a small genome (130-140 Mb), excellent physical and genetic maps, and little repetitive DNA. Here we report the sequence of chromosome 2 from the Columbia ecotype in two gap-free assemblies (contigs) of 3.6 and 16 megabases (Mb). The latter represents the longest published stretch of uninterrupted DNA sequence assembled from any organism to date. Chromosome 2 represents 15% of the genome and encodes 4,037 genes, 49% of which have no predicted function. Roughly 250 tandem gene duplications were found in addition to large-scale duplications of about 0.5 and 4.5 Mb between chromosomes 2 and 1 and between chromosomes 2 and 4, respectively. Sequencing of nearly 2 Mb within the genetically defined centromere revealed a low density of recognizable genes, and a high density and diverse range of vestigial and presumably inactive mobile elements. More unexpected is what appears to be a recent insertion of a continuous stretch of 75% of the mitochondrial genome into chromosome 2.

Arabidopsis↗
PubMed1999

Postprandial, seasonal and sexual variations in cholecystokinin gene expression in goldfish brain.

Recently, we described the complete nucleotide sequence of cholecystokinin (CCK) mRNA and the deduced amino acid sequence of the precursor on the basis of molecular cloning and sequence analysis of cDNA from goldfish brain. In this study, we investigated the hypothesis that CCK has a role in feeding behavior by examining CCK gene expression in the brain of goldfish using Northern blot. We showed that CCK gene is widely but differentially expressed in broad areas of the goldfish brain, including the olfactory bulbs, telencephalon and preoptic region, hypothalamus, optic tectum-thalamus and posterior brain regions, with highest levels in hypothalamus. We found that CCK mRNA levels in goldfish olfactory bulbs, telencephalon-preoptic region, optic tectum-thalamus, and posterior brain were influenced by sex at least sometime of the seasonal gonadal cycle, with female fish having higher levels than males during at least one of the four seasonal sampling times. We also observed a transient and acute increase in the CCK mRNA levels in the olfactory bulbs, telencephalon-preoptic region, hypothalamus, and posterior brain at 120 min after a meal. These widespread postprandial changes in CCK gene expression in goldfish brain indicate that CCK peptides have multiple roles in regulation of feeding behavior in goldfish. This supports the idea that CCK plays a role as a satiety factor in goldfish.

Animals↗
PubMed1999

Cloning and characterization of cDNA encoding cardosin A, an RGD-containing plant aspartic proteinase.

Cardosin A is an abundant aspartic proteinase from pistils of Cynara cardunculus L. whose milk-clotting activity has been exploited for the manufacture of cheese. Here we report the cloning and characterization of cardosin A cDNA. The deduced amino acid sequence contains the conserved features of plant aspartic proteinases, including the plant-specific insertion (PSI), and revealed the presence of an Arg-Gly-Asp (RGD) motif, which is known to function in cell surface receptor binding by extracellular proteins. Cardosin A mRNA was detected predominantly in young flower buds but not in mature or senescent pistils, suggesting that its expression is likely to be developmentally regulated. Procardosin A, the single chain precursor, was found associated with microsomal membranes of flower buds, whereas the active two-chain enzyme generated upon removal of PSI is soluble. This result implies a role for PSI in promoting the association of plant aspartic proteinase precursors to cell membranes. To get further insights about cardosin A, the functional relevance of the RGD motif was also investigated. A 100-kDa protein that interacts specifically with the RGD sequence was isolated from octyl glucoside pollen extracts by affinity chromatography on cardosin A-Sepharose. This result suggests that the 100-kDa protein is a cardosin A receptor and indicates that the interaction between these two proteins is apparently mediated through RGD recognition. It is possible therefore that cardosin A may have a role in adhesion-mediated proteolytic mechanisms involved in pollen recognition and growth.

Amino Acid Sequence↗
PubMed1999

Dopaminergic regulation of three somatostatin mRNAs in goldfish brain.

Three distinct somatostatin cDNAs characterized previously from goldfish brain encode three preprosomatostatins (PSS), designated as PSS-I, PSS-II and PSS-III. In this study, dopaminergic regulation of PSS gene expression was examined by Northern blot analysis in the forebrain of goldfish. Intraperitoneal injection of the non-selective dopamine (DA) agonist, apomorphine, significantly decreased the levels of all three PSS mRNAs, indicating an inhibitory regulation of PSS gene expression by DA. The involvement of DA receptor subtypes in the regulation of PSS gene expression was examined using the D1 receptor agonist and antagonist drugs SKF 38393 and SCH 23390, and the D2 agonist and antagonist drugs LY 171555 and pimozide, respectively. The results provide evidence for inhibitory and/or stimulatory regulation of PSS gene expression by DA through both D1 and D2 receptors, which are dependent on the temporal pattern of dopamine input and reproductive stage of the fish. Demonstration of involvement of both DA D1 and D2 receptors in the dopaminergic regulation of goldfish brain PSS gene expression is a novel finding, distinct from the observations in mammalian models.

Animals↗
PubMed1999

A new method for the generation and cyclization of iminyl radicals via the Hudson reaction.

[formula: see text] A mild new synthetic procedure has been developed for in situ generation and cyclization of iminyl radicals onto pendant alkenes, followed by functionalization of the resulting carbon radical by one of a variety of trapping reagents. The key process in the method involves production of the iminyl radical via treatment of an aldoxime or ketoxime with readily available 2,6-dimethylbenzenesulfinyl chloride at -50 degrees C to room temperature (Hudson reaction).

Alkenes↗
PubMed1999

Identification, chromosomal assignment, and expression analysis of the human homeodomain-containing gene Orthopedia (OTP).

Homeodomain (HD) genes are helix-turn-helix transcription factors that play key roles in the specification of cell fates. In the central nervous system (CNS), HD genes not only position cells along an axis, but also specify cell migration patterns and may influence axonal connectivity. In an effort to identify novel HD genes involved in the development of the human CNS, we have cloned, characterized, and mapped the human homologue of the murine HD gene Orthopedia (Otp), whose product is found in multiple cell groups within the mouse hypothalamus, amygdala, and brain stem. Human cDNA and genomic libraries were screened with probes derived from mouse Otp sequences to find the human homologue, OTP. The deduced amino acid sequence of the open reading frame of the human cDNA is 99% homologous to mouse Otp and demonstrates a high degree of conservation when compared to sea urchin and Drosophila. OTP was mapped to human chromosome 5q13.3 using radiation hybrid panel mapping and fluorescence in situ hybridization. Flanking markers were identified from YAC clones containing OTP. A single putative OTP gene product was found in 17-week human fetal brain tissue by Western blot analysis using a novel polyclonal antibody raised against a conserved 13-amino-acid sequence at the C-terminus of the OTP protein. Expression in the developing human hypothalamus was confirmed by immunohistochemistry.

Amino Acid Sequence↗
PubMed1999

Abnormal regulation of DNA methyltransferase expression during colorectal carcinogenesis.

Somatic changes in CpG dinucleotide methylation occur quite commonly in human cancer cell DNA. Relative to DNA from normal human colonic cells, DNA from human colorectal cancer cells typically displays regional CpG dinucleotide hypermethylation amid global CpG dinucleotide hypomethylation. The role of the maintenance DNA methyltransferase (DNMT1) in the acquisition of such abnormal CpG dinucleotide methylation changes in colorectal cancer cells remains controversial; in one study, 60-200-fold increases in DNMT1 mRNA expression were detected in colorectal polyps and cancers relative to normal colonic tissue [W. S. El-Deiry et al., Proc. Natl. Acad. Sci. USA, 88: 3470-3474, 1991], whereas in another study, only small increases in DNMT1 mRNA expression, commensurate with differences in cell proliferation accompanying colonic tumorigenesis, were observed [P. J. Lee et al., Proc. Natl. Acad. Sci. USA, 93: 10366-10370, 1996]. To definitively ascertain whether abnormal DNMT1 expression might accompany human colorectal carcinogenesis, we subjected a series of normal and neoplastic colonic tissues to immunohistochemical staining using a polyclonal antiserum raised against a DNMT1 polypeptide. A concordance of DNMT1 expression with the expression of PCNA and other cell proliferation markers, such as Ki-67 and DNA topoisomerase IIalpha, was observed in normal colonic epithelial cells and in cells comprising other normal epithelia and lymphoid tissues. The polypeptide p21, which has been reported to undermine DNMT1 binding to proliferating cell nuclear antigen at DNA replication sites, was not expressed by normal colonic cells containing DNMT1 and other cell proliferation markers. In adenomatous polyps, although DNMT1 expression coincided with the expression of other cell proliferation markers, many DNMT1-expressing cells also expressed p21. The fidelity of DNMT1 expression was further undermined in colorectal carcinomas, in which a striking heterogeneity in DNMT1 expression, with some carcinoma cells containing very high DNMT1 levels and others containing very low DNMT1 levels, was observed. These results indicate that human colorectal carcinogenesis is accompanied by a progressive dysregulation of DNMT1 expression and suggest that abnormalities in DNMT1 expression may contribute to the abnormal CpG dinucleotide methylation changes characteristic of human colorectal carcinoma cell DNA.

Colon↗
PubMed1999

The sleep disorder canine narcolepsy is caused by a mutation in the hypocretin (orexin) receptor 2 gene.

Narcolepsy is a disabling sleep disorder affecting humans and animals. It is characterized by daytime sleepiness, cataplexy, and striking transitions from wakefulness into rapid eye movement (REM) sleep. In this study, we used positional cloning to identify an autosomal recessive mutation responsible for this sleep disorder in a well-established canine model. We have determined that canine narcolepsy is caused by disruption of the hypocretin (orexin) receptor 2 gene (Hcrtr2). This result identifies hypocretins as major sleep-modulating neurotransmitters and opens novel potential therapeutic approaches for narcoleptic patients.

Amino Acid Sequence↗
PubMed1999

Dally cooperates with Drosophila Frizzled 2 to transduce Wingless signalling.

The Drosophila wingless gene (wg) encodes a protein of the Wnt family and is a critical regulator in many developmental processes. Biochemical studies have indicated that heparan sulphate proteoglycans, consisting of a protein core to which heparan sulphate glycosaminoglycans are attached, are important for Wg function. Here we show that, consistent with these findings, the Drosophila gene sulfateless (sfl), which encodes a homologue of vertebrate heparan sulphate N-deacetylase/N-sulphotransferase (an enzyme needed for the modification of heparan sulphate) is essential for Wg signalling. We have identified the product of division abnormally delayed (dally), a glycosyl-phosphatidyl inositol (GPI)-linked glypican, as a heparan sulphate proteoglycan molecule involved in Wg signalling. Our results indicate that Dally may act as a co-receptor for Wg, and that Dally, together with Drosophila Frizzled 2, modulates both short- and long-range activities of Wg.

Amidohydrolases↗
PubMed1999

Construction and characterization of an eightfold redundant dog genomic bacterial artificial chromosome library.

A large insert canine genomic bacterial artificial chromosome (BAC) library was built from a Doberman pinscher. Approximately 166,000 clones were gridded on nine high-density hybridization filters. Insert analysis of randomly selected clones indicated a mean insert size of 155 kb and predicted 8.1 coverage of the canine genome. Two percent of the clones were nonrecombinant. Chromosomal fluorescence in situ hybridization studies of 60 BAC clones indicated no chimerism. The library was hybridized with dog PCR products representing eight genes (ADA, TNFA, GCA, MYB, HOXA, GUSB, THY1, and TOP1). The resulting positive clones were characterized and shown to be compatible with an eightfold redundant library.

Animals↗
PubMed1999

A Theiler's virus alternatively initiated protein inhibits the generation of H-2K-restricted virus-specific cytotoxicity.

In susceptible mouse strains, the wild-type Daniel's (wt-DA) strain of Theiler's murine encephalomyelitis virus induces a persistent central nervous system (CNS) infection with chronic demyelination. The virus is cleared from resistant mice with no resulting demyelination. We characterized the role of the DA L* protein in late demyelination and persistent infection. The DA genome has two alternative reading frames, encoding the virus polyprotein and L*, respectively. The mutant virus DAL*-1 fails to synthesize L* and does not persist in the CNS of wt-DA-susceptible SJL/J or B10.S mice. Since class I-restricted cytotoxicity has been shown to determine resistance to virus persistence and demyelination in this model, virus-specific cytotoxicity in the CNS of DA-resistant (B6 or B10) and -susceptible (SJL/J and B10.S) mice during the acute stage of DA and DAL*-1 infection was characterized. Following intracerebral inoculation with DAL*-1, virus-specific Db- and Kb-restricted CTLs were demonstrated in the CNS of resistant B10 mice, whereas only Db-restricted CTL were found in wt-DA-inoculated mice. CTLs specific to wt-DA or DAL*-1 recognized class I-presented peptides from either of the viruses. Of particular interest, Ks-restricted virus-specific cytotoxicity-restricted CTLs were identified in the CNS of susceptible SJL/J (H-2s) and B10.S (H-2s) mice inoculated with DAL*-1. In contrast, no virus-specific CTLs were identified in the CNS of SJL/J and B10.S mice inoculated with wt-DA. We propose that L* inhibits the generation of H-2K-restricted virus-specific cytotoxicity in the CNS, permitting a persistent infection in susceptible strains, with subsequent inflammatory demyelination in the CNS similar to that in human multiple sclerosis.

Alternative Splicing↗
PubMed1999

Differential expression of corticotropin-releasing factor (CRF) and urotensin I precursor genes, and evidence of CRF gene expression regulated by cortisol in goldfish brain.

Corticotropin-releasing factor (CRF) and urotensin I (UI) precursor cDNAs were cloned and sequenced from a goldfish brain cDNA library in order to investigate the distribution of CRF and UI mRNAs in goldfish brain and the regulation of CRF and UI gene expression. The CRF (966-bp) and UI (769-bp) cDNAs encode 163- and 146-amino acid precursors, respectively, and consist of a signal peptide sequence, a cryptic region, and a 41-amino acid mature peptide at the carboxy terminal. The deduced amino acid sequences of the CRF and UI peptides exhibit a sequence identity of 54%. Northern blot analysis revealed a single size of CRF (1.3 kb) and UI (2.0 kb) mRNAs, which are expressed in the telencephalon-preoptic, hypothalamic, optic tectum-thalamus, and posterior brain regions, but not in the pituitary. In addition, while the CRF gene is strongly expressed in the olfactory bulbs, the UI gene is not. In brain regions in which both genes are expressed, the mRNA levels of CRF were three- to sevenfold higher that those of UI. While the low expression levels of the UI gene prevented further analysis of its regulation, the regulation of CRF gene expression by cortisol was examined. In response to intraperitoneal implants of cortisol (300 microg/g BW) the level of CRF mRNA in the telencephalon-preoptic region decreased to 69% of control values at 6 and 24 h posttreatment. In sham-treated fish, in parallel with a transient injection stress-elicited increase in plasma cortisol, CRF mRNA levels declined to 72% of control value at 6 h postinjection and recovered after 24 h. Injection of the glucocorticoid antagonist, RU-486 (100 microg/g BW), prevented the reduction in CRF gene expression associated with the injection stress at 6 h and increased CRF mRNA levels to 145% of control value after 24 h. In contrast, the various implants had no effect on CRF mRNA levels in either the hypothalamus or the optic tectum-thalamus region. These results provide evidence of differential expression of the CRF and UI genes in hypothalamic and extrahypothalamic regions of goldfish brain. Furthermore, they demonstrate that stress levels of plasma cortisol can lead to a decrease in CRF gene expression that is mediated by glucocorticoid receptors in the telencephalon-preoptic region and give an indication of the regional specificity of the regulation of CRF gene expression by cortisol.

Amino Acid Sequence↗
PubMed1999

The role of DNA mismatch repair in cisplatin mutagenicity.

Cisplatin (DDP) is used with varying success for the treatment of a wide spectrum of human cancers. The most abundant lesions produced in DNA are intrastrand crosslinks, which are believed to account for not only the cytotoxic action but also the mutagenicity of the drug. The molecular basis for the mutagenicity of DDP adducts is believed to be related to bypass replication across the adducts by DNA polymerase. This results in misincorporation of non-complimentary bases by polymerase beta which, if left unpaired, will generate point or frameshift mutations. An important replication-associated correction function is provided by the post-replicative DNA mismatch repair (MMR) system. Loss of MMR activity is well documented to result in increased mutation rates and instability of genomic DNA. Inactivation of the MMR system also augments the intrinsic mutagenicity of DDP and enhances the risk of developing cells resistant to other drugs commonly used in combination with DDP. A future challenge will be to assess the clinical significance of the presence of MMR-deficient cells in tumors, and investigate new approaches to circumvent such multidrug resistance.

Base Pair Mismatch↗
PubMed1999

The role of natural killer cell mediated caspases activation in a graft-versus-host disease model of semiallogeneic small bowel transplantation.

In the clinical setting of solid organ transplantation the event of graft-versus-host disease (GvHD) is rare and not easily predictable. Even intestinal and multivisceral transplants harbour a huge amount of immunocompetent cells and they do not exert a significantly higher risk to trigger serious GvH reactions. A series of our own experimental studies has been conducted to delineate the role of the host's innate immune system in the context of GvHD following parental to F1 hybrid semiallogeneic small bowel transplantation (SBTx). These results clearly demonstrated the immunological significance of the recipient's status of natural killer (NK) cell activity to counteract donor-derived lymphocytes and related cytotoxicity. NK cells and macrophages are both endowed with Ca2+-dependent receptors of the C-type lectin family which interact with a diversity of high-affinity oligosaccharide ligands expressed on potential target cells. One of these proteins of the C-type lectin family, termed NKR-P1, has been cloned and sequenced. Activation of NKR-P1 stimulates activation-induced cell death (AICD) of bound target cells. As intracellular mediators of apoptotic cell death a new family of cysteine proteases, the caspases, have been defined. These proteases appear to be involved in the initiation of apoptosis in response to a number of stimuli. This study was conducted to investigate the impact on the activity level of host NK cells and on target cell lysis of donor-derived lymphocytes after heterotopic semiallogeneic (parental [DA;RT1.aaav1] to F1 [DA x LEW;RT1.(1)]) small bowel transplantation using a rat model. The host's NK activity was either specifically activated (by use of polyinosinic:polycytodilic acid [poly-I:C]) or suppressed (by depletion of host NK cells after intraperitoneal administration of the NKR-P1 monoclonal antibody 3.2.3). The impact of NK-activity on the incidence of GvHD and the recipients' survival was correlated with the frequency of apoptotic cell death and related expression of caspases 1 (ICE) and 3 (CPP-32) from donor and recipient small bowel tissues. Our results confirm that depletion of NK cells in F1 host rats prior to parental small bowel transplantation significantly decreased the mean survival to 11.4 days versus 16.2 days of nondepleted F1 rats (p < 0.01). Conversely, activation of host NK activity with poly-I:C abrogated GvHD in all 12 recipient rats and led to long-term survival in seven of 12 animals. Long-term survival was associated with a substantially higher frequency of apoptotic cell death in donor and recipient small bowel and mesenteric lymph nodes. On day 10 after transplantation, Northern blot analysis of these tissues revealed profound upregulation of mRNA-specific gene expression for caspase 1 and 3 as potential mediators of programmed cell death of activated lymphocytes. Our findings emphasize the importance of NK cell associated innate immunity in the context of GvHD after semiallogeneic small bowel transplantation. Killing of alloreactive donor-derived lymphocytes was mediated by the NKR-P1 protein on NK cells and could be suppressed after pretreatment of F1 hosts with anti-NKR-P1 mAb 3.2.3. Moreover, NK cell-mediated apoptosis induced upregulation of caspases 1 and 3, thus elucidating the involvement of this protein in the context of caspase-mediated target cell killing.

Animals↗