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Biomedical subjects

W Wu

Publications and source records attributed to W Wu.

At least 181 records · Page 10Linked to original sources
PubMed1999

Introduction of protein kinase recognition sites into proteins: a review of their preparation, advantages, and applications.

Labeled proteins are used in a variety of applications. This review focuses on methods that utilize genetic engineering to introduce protein kinase recognition sites into proteins. Many protein kinase recognition sites can be introduced into proteins and serve as useful tags for a variety of purposes. The introduction of protein kinase recognition sites into proteins can be achieved without modifying the essential structure or function of the proteins. Because proteins modified by these procedures retain their activity after phosphorylation, they can be used in many applications. The phosphorylatable proteins can be labeled easily to high specific activity with radioisotopes ((32)P, (33)P, or (35)S), or the nonradioactive (31)P can be used. The use of these radioisotopes provides a convenient and safe method for radiolabeling proteins. Moreover, the use of the nonradioactive (31)P with protein tyrosine kinase recognition sites permits the tagging of proteins and their detection with the many anti-phosphotyrosine antibodies available. Overall, the procedure represents a convenient, safe, and efficient method to label proteins for a variety of applications.

Amino Acid Motifs↗
PubMed1999

The 5' region of the COX4 gene contains a novel overlapping gene, NOC4.

We identified a novel human gene, NOC4 (Neighbor Of COX4), located 5' to COX4, the gene for cytochrome c oxidase subunit IV, on Chr 16q32-ter. Transcripts from this gene were identified among human expressed sequence tags. A full-length, 1.06-kb human retinal NOC4 cDNA encoded a 24-kDa, 210-amino acid hypothetical protein of unknown function. Northern hybridization analysis of human RNAs from various tissues detected NOC4 transcripts of 2.2 and 1.4 kb in all tissues examined, suggesting that NOC4 expression is ubiquitous. Transcription of both the COX4 and NOC4 genes initiates within a 250-bp intergenic promoter and occurs in opposite directions. The bidirectional promoter is G + C-rich, lacks TATA and CCAAT elements, and contains multiple potential binding sites for Sp1 and NRF-2/GABP. Two of the NRF-2/GABP sites are located within 14-bp direct repeats, a conserved feature of mammalian COX4 promoters. The NOC4 and COX4 genes are also linked in the rat, mouse, and bovine genomes. A NOC4-GFP fusion protein is located in both the nucleus and the cytoplasm, including the mitochondria.

Amino Acid Sequence↗
PubMed1999

Dickkopf genes are co-ordinately expressed in mesodermal lineages.

Dickkopf-1 (dkk-1) is member of a novel family of secreted proteins and functions in head induction during Xenopus embryogenesis, acting as a potent inhibitor of Wnt signalling. Here we report: (1) the isolation of two additional murine members of the dkk family, dkk-2 and dkk-3; and (2) analysis of adult and embryonic gene expression of mouse dkk-1,-2, and -3, Xenopus dkk-1 as well as chicken dkk-3. Comparative developmental analyses of the dkk-1, dkk-2 and dkk-3 in mice indicate that these genes are both temporally and spatially regulated. They define overlapping deep domains in mesenchymal lineages suggesting a co-ordinated mode of action. All dkks show distinct and elevated expression patterns in tissues that mediate epithelial- mesenchyme transformations suggesting that they may participate in heart, tooth, hair and whisker follicle, limb and bone induction. In the limb buds expression of these genes are found in regions of programmed cell death. In a given organ, dkk-1 tends to be the earliest member expressed. Comparison with Xenopus dkk-1 and chicken dkk-3 shows evolutionarily conserved expression patterns. Our observations indicate that dkk genes constitute a new family of secreted proteins that may mediate inductive interactions between epithelial and mesenchymal cells.

Adaptor Proteins, Signal Transducing↗
PubMed1999

1,3-Selenazine derivatives induce cytotoxicity and DNA fragmentation in human HT-1080 fibrosarcoma cells.

The inhibitory effects of a series of 5,6-dihydro-4H-1,3-selenazine derivatives, 1,3-selenazole, and 5,6-dihydro-4H-1,3-thiazine derivatives on the proliferation of human HT-1080 fibrosarcoma cells were investigated. The compounds 4-ethyl-4-hydroxy-2-p-tolyl-5, 6-dihydro-4H-1,3-selenazine (TS-2) and 4-hydroxy-4-methyl-6-propyl-2-p-tolyl-5,6-dihydro-4H-1,3-selenazine++ + (TS-6) exhibited the strongest inhibitory effect among 1, 3-selenazine derivatives, and the EC(50) of TS-2 and TS-6 was 7.76 and 8.40 microM, respectively. On the other hand, 1,3-selenazole and 5,6-dihydro-4H-1,3-thiazines had no inhibitory effects. TS-2 and TS-6 inhibited the proliferation of HT-1080 cells time- and dose-dependently. They induced dose-dependent DNA fragmentation in HT-1080 cells, revealing a typical apoptosis characteristics. The present study demonstrated that TS-2 and TS-6 inhibited HT-1080 proliferation through the induction of DNA fragmentation.

Apoptosis↗
PubMed1999

A genetic system yields self-cleaving inteins for bioseparations.

A self-cleaving element for use in bioseparations has been derived from a naturally occurring, 43 kDa protein splicing element (intein) through a combination of protein engineering and random mutagenesis. A mini-intein (18 kDa) previously engineered for reduced size had compromised activity and was therefore subjected to random mutagenesis and genetic selection. In one selection a mini-intein was isolated with restored splicing activity, while in another, a mutant was isolated with enhanced, pH-sensitive C-terminal cleavage activity. The enhanced-cleavage mutant has utility in affinity fusion-based protein purification. These mutants also provide new insights into the structural and functional roles of some conserved residues in protein splicing.

Amino Acid Sequence↗
PubMed1999

An experimental study on estimating human error probability (HEP) parameters for PSA/HRA by using human model simulation.

A framework of Human Error Probability (HEP) parameters, which is needed for Human Reliability Analysis (HRA) within a practice of Probabilistic Safety Assessment (PSA) of Nuclear Power Plant is first proposed. Then a laboratory experiment was conducted in order to construct a computer simulation model (human model) that describes human cognitive behaviour on detecting and diagnosing plant anomaly causes. An inter-comparison between experimental data and human model simulation was performed to estimate Human Cognitive Reliability (HCR) curves, in order to confirm the applicability of a human model for estimating these HEP parameters for PSA/HRA practice.

Cognition↗
PubMed1999

Coiled bodies preferentially associate with U4, U11, and U12 small nuclear RNA genes in interphase HeLa cells but not with U6 and U7 genes.

Coiled bodies (CBs) are nuclear organelles involved in the metabolism of small nuclear RNAs (snRNAs) and histone messages. Their structural morphology and molecular composition have been conserved from plants to animals. CBs preferentially and specifically associate with genes that encode U1, U2, and U3 snRNAs as well as the cell cycle-regulated histone loci. A common link among these previously identified CB-associated genes is that they are either clustered or tandemly repeated in the human genome. In an effort to identify additional loci that associate with CBs, we have isolated and mapped the chromosomal locations of genomic clones corresponding to bona fide U4, U6, U7, U11, and U12 snRNA loci. Unlike the clustered U1 and U2 genes, each of these loci encode a single gene, with the exception of the U4 clone, which contains two genes. We next examined the association of these snRNA genes with CBs and found that they colocalized less frequently than their multicopy counterparts. To differentiate a lower level of preferential association from random colocalization, we developed a theoretical model of random colocalization, which yielded expected values for chi2 tests against the experimental data. Certain single-copy snRNA genes (U4, U11, and U12) but not controls were found to significantly (p < 0.000001) associate with CBs. Recent evidence indicates that the interactions between CBs and genes are mediated by nascent transcripts. Taken together, these new results suggest that CB association may be substantially augmented by the increased transcriptional capacity of clustered genes. Possible functional roles for the observed interactions of CBs with snRNA genes are discussed.

Amino Acid Sequence↗
PubMed1999

Leber congenital amaurosis caused by a homozygous mutation (R90W) in the homeodomain of the retinal transcription factor CRX: direct evidence for the involvement of CRX in the development of photoreceptor function.

The CRX (cone-rod homeobox) gene is specifically expressed in developing and mature photoreceptors and encodes an otd/Otx-like paired homeodomain protein. Mutant alleles of the CRX gene have recently been associated with autosomal dominant cone-rod dystrophy (CORD) as well as dominant Leber congenital amaurosis (LCA). Since LCA is more commonly inherited in an autosomal recessive manner, we examined a cohort of recessive LCA patients for CRX mutations. A homozygous substitution of arginine (R) at codon 90 by tryptophan (W) was identified in the CRX homeodomain of one of the probands who was nearly blind from birth. A group of 48 control individuals and 190 previously characterized CORD probands did not reveal this sequence change. The mutant CRXR90W homeodomain demonstrated decreased binding to the previously identified cis sequence elements in the rhodopsin promoter. In transient transfection experiments, the mutant protein showed significantly reduced ability to transactivate the rhodopsin promoter, as well as lower synergistic activation with the bZIP transcription factor NRL. Heterozygosity of the mutant CRX (R90W) allele was detected in both parents and in an older sibling. Ophthalmologic examination and electro-retinography revealed a subtle abnormality of cone function in both the parents. These data suggest that the R90W mutation results in a CRX protein with reduced DNA binding and transcriptional regulatory activity and that the subsequent changes in photoreceptor gene expression lead to the very early onset severe visual impairment in LCA.

Adolescent↗
PubMed1999

Regulation of rat hepatic hydroxysteroid sulfotransferase (SULT2-40/41) gene expression by glucocorticoids: evidence for a dual mechanism of transcriptional control.

Glucocorticoid-inducible hydroxysteroid sulfotransferase (SULT2-40/41) gene transcription was investigated in primary cultured rat hepatocytes transiently transfected with a series of SULT2-40/41 5'-flanking region-luciferase reporter constructs, with emphasis on examining the functional role of an inverted repeat-0 nuclear receptor motif (IR0). Treatment of transfected cultures with any of four glucocorticoids activated luciferase expression from a construct containing 1938 base pairs (bp) of the SULT2-40/41 gene 5'-flanking sequence, whereas deletion of bp -227 to -158 (containing the IR0 motif) largely abolished the effect. On closer analysis, treatment of hepatocyte cultures with either of the potent glucocorticoids dexamethasone [strong cytochrome P-450 3A (CYP3A) inducer] or triamcinolone acetonide (weak CYP3A inducer) produced dose-dependent increases in luciferase activity when hepatocytes were transiently transfected with a construct containing as little as 158 bp of 5'-flanking sequence or containing a mutated IR0 motif. The dexamethasone dose-dependent increase in luciferase activity continued through a dose of 10(-6) M when the transfected construct contained the IR0 motif, but was maximal at 10(-7) M when the transfected construct lacked the IR0 motif. In contrast, triamcinolone acetonide-induced luciferase activity was maximal at a dose of 10(-7) M, irrespective of the presence or absence of the IR0 motif. Coincubation of transfected hepatocytes with 10(-8) M dexamethasone and the antiglucocorticoid RU486 inhibited luciferase expression. Luciferase induction by the prototypical CYP3A inducer pregnenolone 16alpha-carbonitrile was restricted to constructs containing the IR0 motif. These data suggest that glucocorticoid-inducible SULT2-40/41 gene expression occurs through a dual mechanism, whose components possibly involve the glucocorticoid receptor and the pregnane X receptor.

Animals↗
PubMed1999

Activation of the EGF receptor signaling pathway in human airway epithelial cells exposed to metals.

We have previously shown that exposure to combustion-derived metals rapidly (within 20 min) activated mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK), in the human bronchial epithelial cell line BEAS. To study the mechanisms responsible for metal-induced activation of ERK, we examined the effect of noncytotoxic exposures to As, Cu, V, or Zn on the kinases upstream of ERK in the epidermal growth factor (EGF) receptor signaling pathway. Western blotting using phospho-specific ERK1/2 antibody demonstrated the selective MEK1/2 inhibitor PD-98059 blocked metal-induced phosphorylation of ERK1/2. Meanwhile, Western blotting using a phospho-specific MEK1/2 antibody showed that these metals induce a rapid phosphorylation of MEK1/2. Kinase activity assays confirmed the activation of MEK1/2 by metal treatment. Immunoprecipitation studies demonstrated that As, Cu, V, or Zn induces EGF receptor phosphorylation. Furthermore, the EGF receptor-specific tyrosine kinase inhibitor (PD-153035) significantly blocked the phosphorylation of MEK1/2 initiated by metals. Interestingly, we observed low levels of Raf-1 activity that were not increased by metal exposure in these cells through kinase activity assay. Finally, transfection assays showed that MEK1/2 inhibition could inhibit trans-activation of Elk1, a transcription factor in the ERK pathway, in BEAS cells exposed to metals. Together, these data demonstrate that As, Cu, V, and Zn can activate the EGF receptor signaling pathway in BEAS cells and suggest that this mechanism may be involved in pulmonary responses to metal inhalation.

Air Pollutants↗
PubMed1999

Assignment of a novel bifurcated SET domain gene, SETDB1, to human chromosome band 1q21 by in situ hybridization and radiation hybrids.

We have identified a human gene encoding an unusual bifurcated SET domain protein containing a large "insertion" between the most highly conserved parts of the SET domain. The existence of an evolutionarily related C. elegans gene encoding a similarly bifurcated SET domain suggests that SET domains may generally be composed of two functionally distinct subdomains. We mapped this gene, called SETDB1, to human chromosome 1q21. This region is targeted by a large number of recurrent translocations, suggesting that like the SET domain protein MLL, mutant forms of SETDB1 may be associated with human neoplasias.

Amino Acid Sequence↗
PubMed1999

Tyrosine phosphatases as targets in metal-induced signaling in human airway epithelial cells.

We previously showed that exposure to metal-laden combustion particles disregulates protein tyrosine phosphate homeostasis in human airway epithelial cells (HAEC). More recently, we reported that exposure to certain metal ions activates mitogen-activated protein kinases in HAEC. To study the mechanism responsible, we examined the effects of arsenic (As), vanadium (V), and zinc (Zn) on tyrosine phosphate catabolism in BEAS S6 cells or cultured human bronchial epithelial cells. Western blots and immunocytochemical analyses showed that exposure to noncytotoxic levels of As, V, or Zn resulted in increased levels of protein phosphotyrosines in HAEC. Tyrosine phosphatase activity, measured against [(32)P]-labeled PolyGlu:Tyr, was markedly inhibited in cells treated with V or Zn but was unaffected by exposure to As. Fast performance liquid chromatography fractionation and subsequent in-gel phosphatase activity assay of HAEC protein extracts revealed the presence of numerous tyrosine phosphatases, of varying molecular weights, that were effectively inhibited by exposure to V or Zn ions. As had no discernible effect on these enzymes. The protein tyrosine phosphatase PTP1B, immunoprecipitated from HAEC, was similarly inhibited by V and Zn but not by As ions. These data show that V and Zn may induce tyrosine phosphate accumulation by inhibiting dephosphorylation and implicate kinase activation as the mechanism in HAEC exposed to As. These findings suggest that metal exposure can activate signaling pathways through multiple mechanisms.

Arsenic↗
PubMed1999

"Hot spot" in the PROP1 gene responsible for combined pituitary hormone deficiency.

As pituitary function depends on the integrity of the hypothalamic-pituitary axis, any defect in the development and organogenesis of this gland may account for a form of combined pituitary hormone deficiency (CPHD). Although pit-1 was 1 of the first factors identified as a cause of CPHD in mice, many other homeodomain and transcription factors have been characterized as being involved in different developmental stages of pituitary gland development, such as prophet of pit-1 (prop-1), P-Lim, ETS-1, and Brn 4. The aims of the present study were first to screen families and patients suffering from different forms of CPHD for PROP1 gene alterations, and second to define possible hot spots and the frequency of the different gene alterations found. Of 73 subjects (36 families) analyzed, we found 35 patients, belonging to 18 unrelated families, with CPHD caused by a PROP1 gene defect. The PROP1 gene alterations included 3 missense mutations, 2 frameshift mutations, and 1 splice site mutation. The 2 reported frameshift mutations could be caused by any 2-bp GA or AG deletion at either the 148-GGA-GGG-153 or 295-CGA-GAG-AGT-303 position. As any combination of a GA or AG deletion yields the same sequencing data, the frameshift mutations were called 149delGA and 296delGA, respectively. All but 1 mutation were located in the PROP1 gene encoding the homeodomain. Importantly, 3 tandem repeats of the dinucleotides GA at location 296-302 in the PROP1 gene represent a hot spot for CPHD. In conclusion, the PROP1 gene seems to be a major candidate gene for CPHD; however, further studies are needed to evaluate other genetic defects involved in pituitary development.

Adrenocorticotropic Hormone↗
PubMed1999

Effects of electro-acupuncture on somatostatin and pancreatic polypeptide in ischemic cerebrovascular diseases.

The levels of somatostatin (SS) in CSF and blood and pancreatic polypeptide (PP) in plasma were measured by radioimmunoassay in 64 patients with acute ischemic cerebrovascular diseases (ICVD), randomly divided into two groups: group 1 (n = 31, both electro-acupuncture and routine treatments given) and group 2 (n = 33, routine treatment) and 26 non-ICVD patients were used as controls. The points of electro-acupuncture were Quchi (LI 12), Waiguan (SJ 5) and Huantiao (GB 30) and Zusanli (St 36). After a course of treatment, the SS levels in plasma and CSF were significantly increased in the patients of group 1 with good result and their plasma PP level had no significant change. In the patients with poor result, however, the PP level was significantly decreased. The results suggested that electro-acupuncture might play an active role in alleviating the SS metabolic disturbance in CNS of ICVD patients.

Adult↗
PubMed1999

RWJ 67657, a potent, orally active inhibitor of p38 mitogen-activated protein kinase.

Tumor necrosis factor-alpha (TNF-alpha), a cytokine secreted by activated monocytes/macrophages and T lymphocytes, has been implicated in several disease states, including rheumatoid arthritis, inflammatory bowel disease, septic shock, and osteoporosis. Monocyte/macrophage production of TNF-alpha is dependent on the mitogen-activated protein kinase p38. RWJ 67657 (4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol -2-yl]-3-butyn-1-ol) inhibited the release of TNF-alpha by lipopolysaccharide (a monocyte stimulus)-treated human peripheral blood mononuclear cells with an IC(50) of 3 nM, as well as the release of TNF-alpha from peripheral blood mononuclear cells treated with the superantigen staphylococcal enterotoxin B (a T cell stimulus), with an IC(50) value of 13 nM. This compound was approximately 10-fold more potent than the literature standard p38 kinase inhibitor SB 203580 in all p38 dependent in vitro systems tested. RWJ 67657 inhibited the enzymatic activity of recombinant p38alpha and beta, but not gamma or delta, in vitro and had no significant activity against a variety of other enzymes. In contrast, SB 203580 significantly inhibited the tyrosine kinases p56 lck and c-src (IC(50) = 5 microM). RWJ 67657 did not inhibit T cell production of interleukin-2 or interferon-gamma and did not inhibit T cell proliferation in response to mitogens. RWJ 67657 inhibited TNF-alpha production in lipopolysaccharide-injected mice (87% inhibition at 50 mg/kg) and in rats (91% inhibition at 25 mg/kg) after oral administration. Based on these favorable biological properties, RWJ 67657 may have use as a treatment for inflammatory diseases.

Animals↗
PubMed1999

Nucleation at surfaces: the importance of interfacial energy.

The nucleation and growth of stone-forming minerals on the surfaces of other crystalline phases, cellular material, and immobilized macromolecules must be important in the formation of stones in the urinary tract. The nucleation and growth of calcium oxalate monohydrate (COM) crystals were studied using the constant composition kinetics technique, in solution supersaturated with respect to COM (sigmaCOM = 1.44). The solid phases during the reaction were examined by x-ray diffraction, scanning electron microscopy, and diffuse reflectance Fourier transform infrared spectroscopy. Human serum albumin was found to nucleate COM crystals when immobilized on hydroxyapatite (HAP) surfaces. The induction period for nucleation of COM on HAP surfaces preadsorbed with albumin significantly decreased to about 65 min from about 230 min for pure HAP particles. The initial growth rate of COM on pure HAP particles, Rm approximately/= 0.56 X 10(-7) mol/min per m2, was slower than that for HAP surfaces preadsorbed with albumin, 2.14 x 10(-7) mol/min per m2. The surface properties were characterized using contact angle measurements by sessile drop and thin layer wicking. The thermodynamic results suggested that surfaces with high Lewis base parameter values (gamma-) and low interfacial tension with water (gammaSL) are more effective in the nucleation and growth of crystal phases.

Calcium Oxalate↗
PubMed1999

Cellular DNA, ras p21 and p53 expression in the carcinogenesis of adenomatous colorectal polyps.

OBJECTIVE: To explore the possible roles of cellular DNA, oncogene ras and tumor suppressor gene p53 in the carcinogenesis of colorectal adenomatous polyps (CAP). STUDY DESIGN: Cellular DNA content, oncogene ras and tumor suppressor gene p53 expression at the protein level were quantitatively studied with flow cytometry (FCM) in 16 cases of CAP with mild epithelial dysplasia (CAP-MD), 16 cases of CAP with moderate/severe epithelial dysplasia (CAP-M/SD) and 11 cases of cancer in adenomatous polyps (CIAP). RESULTS: Nuclear DNA contents of CAP-M/SD (DNA [DI] = 1.11 +/- 0.06) and CIAP (DI = 1.29 +/- 0.03) were significantly higher than those of CAP-MD (DI = 1.06 +/- 0.06) and normal controls (DI = 1.00, P < .005) and were in the FCM DNA aneuploidy range. The rates and amount (as determined by the fluoresence index) of mutant p53 protein expression in CAP-M/SD and CIAP were significantly higher than those in the control and CAP-MD groups. Positive rates of ras p21 expression were all high in CAP-MD, CAP-M/SD and CIAP (80%, 75% and 100%, respectively), yet the intensity of expression in the last was significantly stronger than those in the former two groups. DNA aneuploid, ras p21 and p53 coexpression were found in 10 of 11 cases of CIAP. CONCLUSION: The results suggest that cellular DNA, ras p21 and p53 are all involved in the carcinogenesis of CAP. Clinically, the appearance of DNA aneuploidy, ras p21 or p53 overexpression should be considered markers of malignant conversion in CAP.

Adenomatous Polyps↗