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X Yang

Publications and source records attributed to X Yang.

At least 1,045 records · Page 58Linked to original sources
PubMed1993

Identification of JAK2 as a growth hormone receptor-associated tyrosine kinase.

Growth hormone receptor (GHR) forms a complex with a tyrosine kinase, suggesting involvement of a ligand-activated tyrosine kinase in intracellular signaling by growth hormone (GH). Here we identify JAK2, a nonreceptor tyrosine kinase, as a GHR-associated tyrosine kinase. Immunological approaches were used to establish GH-dependent complex formation between JAK2 and GHR, activation of JAK2 tyrosine kinase activity, and tyrosyl phosphorylation of both JAK2 and GHR. The JAK2-GHR and JAK2-erythropoietin receptor interactions described here and in the accompanying paper provide a molecular basis for involvement of tyrosyl phosphorylation in physiological responses to these ligands and suggest a shared signaling mechanism among members of the cytokine/hematopoietin receptor family.

3T3 Cells↗
PubMed1993

Chemically modified antigen preferentially elicits induction of Th1-like cytokine synthesis patterns in vivo.

Differential activation of CD4+ T cell subsets in vivo leads to the development of qualitatively different effector responses. We identify an approach that allows selective activation of strongly Th1-dominated immune responses to protein antigens. Whereas in vivo administration of ovalbumin (OVA) induces cytokine synthesis that is neither Th1 nor Th2 dominated, administration of glutaraldehyde polymerized, high relative molecular weight OVA (OA-POL) leads to 20-fold increase in the ratio of interferon gamma (IFN-gamma)/IL-4 and IFN-gamma/IL-10 synthesis observed after short-term, antigen-mediated restimulation directly ex vivo. In contrast, concurrent in vivo administration of anti-IFN-gamma mAb and OVA or OA-POL results in marked increases in IL-4 and IL-10, and decreased IFN-gamma production, reflecting a polarization of the response towards a Th2-like pattern of cytokine synthesis. These observations may be useful in clinical settings including hypersensitivity, autoimmune diseases, and vaccine development where the ability to actively select specific patterns of cytokine gene expression would be advantageous.

Animals↗
PubMed1993

IFN-gamma, but not IL-4, synthesis by antigen-primed murine T cells is IL-2 dependent. Differential requirements in de novo and established responses.

Ag-induced, CD4 T cell-dependent synthesis of IL-4 and IFN-gamma plays a critical role in the induction, maintenance, and regulation of allergic responses. The requirement for IL-2 and IL-4 in the initial induction of IL-4 T cell responses is well established. However, it remains unclear whether IL-2 is required for priming of IL-4 responses, IL-4 gene expression, or both. In this report, we dissociate the IL-2 dependence of developing and established IL-4 responses. The capacity of spleen cells from naive and allergen-immunized mice to synthesize IL-4 after Ag-specific vs polyclonal restimulation is examined in overnight cultures. Although de novo induction of IL-4 and IFN-gamma production obtained after in vitro activation of cells from naive animals with immobilized anti-CD3 is IL-2 dependent, Ag-driven IL-4 synthesis by CD4 T cells from OVA (alum)- or ragweed extract (alum)-immunized mice is independent of IL-2. The data demonstrate the IL-2 independence of CD4 T cell-derived IL-4 synthesis by allergen-primed cells, distinguishing IL-2-dependent from IL-2-independent stages of IL-4 responses. In contrast to the finding that IL-2 is not required for IL-4 gene expression by primed T cells in allergen-driven responses, both induction and expression of IFN-gamma responses are strictly IL-2 dependent. This differential requirement for IL-2 in IL-4 and IFN-gamma gene expression may be significant in the maintenance of IL-4-dominated responses in allergic individuals.

Animals↗
PubMed1993

Chromosomal localization of glutamate receptor genes: relationship to familial amyotrophic lateral sclerosis and other neurological disorders of mice and humans.

Receptors for the major excitatory neurotransmitter glutamate may play key roles in neurodegeneration. The mouse Glur-5 gene maps to chromosome 16 between App and Sod-1. The homologous human GLUR5 gene maps to the corresponding region of human chromosome 21, which contains the locus for familial amyotrophic lateral sclerosis. This location, and other features, render GLUR5 a possible candidate gene for familial amyotrophic lateral sclerosis. In addition, dosage imbalance of GLUR5 may have a role in the trisomy 21 (Down syndrome). Further characterization of the murine glutamate receptor family includes mapping of Glur-1 to the same region as neurological mutants spasmodic, shaker-2, tipsy, and vibrator on chromosome 11; Glur-2 near spastic on chromosome 3; Glur-6 near waltzer and Jackson circler on chromosome 10; and Glur-7 near clasper on chromosome 4.

Amyotrophic Lateral Sclerosis↗
PubMed1993

Repertoire diversity of antibody response to bacterial antigens in aged mice. III. Phosphorylcholine antibody from young and aged mice differ in structure and protective activity against infection with Streptococcus pneumoniae.

Aging in mice is accompanied by qualitative changes in the antibody repertoire to phosphorylcholine (PC), a natural epitope of certain pneumococci. The PC-specific mAb from young/adult (2-4 mo) BALB/c mice are uniformly encoded by the canonical IgV genes of T15 family, whereas the antibody from aged mice (> or = 20 mo) are molecularly heterogeneous, being encoded by various VH and VL genes of non-T15 families. Interestingly the young/adult and aged BALB/c mice produce comparable amounts of antibodies to PC regardless of the molecular shift in the antibody repertoire. This finding prompted our study on the relative ability of PC antibodies from young and aged donors to protect mice against virulent infection with type 3 pneumococci. Passive administration (i.p.) of pooled, PC-specific mAb generated from young donors (all from the T15 Ig gene family) protected the recipients against subsequent i.p. challenge with a lethal dose of Streptococcus pneumoniae strain WU2, in a dose-dependent manner. In contrast, a mixture of PC mAb from aged donors (all from non-T15 families) failed to protect the mice against the infection, even at the highest amount of administered (100 micrograms of mAb/recipient). Average affinity of the aged mAb for the free hapten (PC-chloride) as well as their binding to the bacteria was lower than that of the young mAb. Similarly, affinity-purified serum PC antibody from S. pneumoniae vaccine-immunized young mice afforded a measurable degree of passive protection against the pneumococcal infection whereas a similar dose of serum PC antibody from aged mice did not. Further experiments showed that PC mAb from young donors were equally protective in either young or aged recipients challenged with the bacteria. These results demonstrate that the aged immune system may, in some instances, produce high levels of antibody that are structurally different and less protective against microbial infection.

Aging↗
PubMed1993

Allergen-specific modulation of cytokine synthesis patterns and IgE responses in vivo with chemically modified allergen.

Hypersensitivity and IgE synthesis are highly dependent on the balance in which production of IL-4 and IFN-gamma is induced. An immunologic approach that alters the dominant pattern of cytokine synthesis and antibody production that is elicited after exposure to native allergen is described. High M(r), glutaraldehyde-polymerized OVA administered (i.p.) before or after immunization with unmodified OVA induces > or = 95% inhibition of specific IgE synthesis concomitant with 300- to 800-fold increases in IgG2a production in C57BL/6 mice. These changes result from a genetically controlled shift in the pattern of cytokine production within the allergen-specific T cell repertoire as demonstrated by i) susceptibility of the changes induced upon administration of modified allergen to in vivo treatment with anti-IFN-gamma mAb and ii) a 5- to 7-fold increase in the ratio of IFN-gamma:IL-4 synthesis after overnight culture directly ex vivo. This system should prove useful in identification of the factors which are influential in the commitment of T cells to Th1- or Th2-like patterns of cytokine synthesis. Moreover, as defective induction of IFN-gamma by allergen-specific T cells appears to play a role in elevated IgE synthesis and human allergy, this approach may have therapeutic potential.

Allergens↗
PubMed1993

Uptake of antisense oligonucleotides and functional block of acetylcholine receptor subunit gene expression in primary embryonic neurons.

Several recent studies have used antisense oligonucleotides in the nervous system to probe the functional role of particular gene products. Since antisense oligonucleotide-mediated block of gene expression typically involves uptake of the oligonucleotides, we have characterized the mechanism of this uptake into developing neurons from embryonic chickens. Antisense oligonucleotides (15 mers) added to the bathing media are taken up into the embryonic chicken sympathetic neurons maintained in vitro. A portion of the oligonucleotide uptake is temperature dependent and saturates at extracellular oligonucleotide concentrations > or = 20 microM. This temperature sensitive, saturable component is effectively completed by single nucleotides of ATP and AMP and is reminiscent of receptor-mediated endocytosis of oligonucleotides described in non-neuronal cells. The efficiency of the oligonucleotide uptake system is dependent on the developmental stage of the animal but independent of the number of days that the neurons are maintained in vitro. Following the uptake of antisense oligonucleotides directed against ion channel subunit genes expressed by these neurons (nicotinic acetylcholine receptor subunit alpha 3; nAChR alpha 3), biophysical assays reveal that the functional expression of the target gene is largely blocked. Thus the number of wild type nAChR channels expressed is decreased by approximately 80%-90%. Furthermore, following antisense deletion of alpha 3, "mutant" nAChRs with distinct functional characteristics are expressed. In sum, these studies characterize the uptake of antisense oligonucleotide and demonstrate the functional block of specific gene expression in primary developing neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Bovine oocyte development following different oocyte maturation and sperm capacitation procedures.

Various procedures have been reported for successful in vitro maturation and in vitro fertilization (IVM/IVF) of bovine follicular oocytes. Direct comparisons of these different recommended procedures have been rare. In this research, involving a total of 5,128 oocytes, a series of experiments were conducted to compare oocyte maturation, fertilization, and development in vitro with 2 maturation systems (with or without added hormones) and 3 types of sperm treatment procedures. Oocytes were collected from ovarian antral follicles (2-7 mm in diameter) within 3 hr after slaughter of cows or heifers. Those with intact or at least 4 layers of cumulus cells were selected for IVM/IVF. Oocytes were incubated for 22 hr in either Medium 199 with 7.5% fetal calf serum (M199 + FCS) alone or M199 + FCS with added hormones (M199 + FCS + H; oFSH 0.5 micrograms/ml, oLH 5.0 micrograms/ml, and E2 1.0 micrograms/ml) at 39 degrees C in 5% CO2 and 95% air. For IVF, frozen-thawed sperm were treated with either 0.1 microM calcium ionophore A23187 (A23187) for 1 min, or 10 or 100 micrograms/ml heparin (H10 or H100) for 15 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Nuclear transfer in cattle: effect of nuclear donor cells, cytoplast age, co-culture, and embryo transfer.

There are many factors affecting the efficiency of nuclear transfer technology. Some are evaluated here using our novel approach by enucleating oocytes at 20-22 hr after in vitro maturation (IVM), culturing the enucleated oocytes (cytoplasts) for 8-10 hr or 18-20 hr to gain activation competence and then conducting nuclear transfer. In the first experiment, we demonstrated that cumulus cell (CC) monolayer can support some cloned embryos to develop into morulae or blastocysts. Co-culture with CC and bovine oviduct epithelial cell (BOEC) monolayers resulted in no differences (P > 0.05) in supporting the development of cloned embryos (Experiment 2). When in vitro matured oocytes were enucleated at 22 hr after IVM followed by nuclear transfer 18-20 hr later, cleavage and morula or blastocyst development of the cloned embryos were similar to those resulting from the enucleated oocytes which had been matured in vivo (Experiment 3). Frozen embryos as nuclear donor cells worked equally well as fresh embryos for cloning in embryo development which was superior to IVF embryos (Experiment 4). However, fresh embryos resulted in a higher proportion (P < 0.05) of blastomere recovery than did frozen of IVF embryos. Finally, embryo transfer of cloned embryos from our procedure produced a viable calf, demonstrating the commercial value of this novel approach of the technology.

Animals↗
PubMed1993

Pluripotency of cultured rabbit inner cell mass cells detected by isozyme analysis and eye pigmentation of fetuses following injection into blastocysts or morulae.

Pluripotency of isolated rabbit inner cell masses (ICMs) and cultured (3 days) inner cell mass (ICM) cells was tested by injecting these donor cells into day 3.5 blastocysts (experiment 1) or day 3 morulae (experiment 2) to produce chimeric embryos. Injected (n = 107) and noninjected (n = 103) embryos were transferred to the opposite uterine horns of the same recipient females. Chimerism was determined by adenosine deaminase (ADA) isozyme analysis on fetal tissue and by eye pigmentation at midgestation. In experiment 1, 53% and 64%, respectively, of blastocysts injected with ICMs or cultured ICM cells developed to midgestation, compared with 52% and 48% for controls. Of these fetuses, four (31%) and one (6%), respectively, had ADA chimerism. In experiment 2, 38% and 62%, respectively, of the morulae injected with ICMs or cultured ICM cells developed to midgestation, compared with 46% and 56% for control morulae. Six (43%) chimeric fetuses from morulae injected with ICMs were detected by ADA analysis, but 12 (86%) chimeric fetuses were detected by eye pigmentation, indicating that eye pigmentation was a more sensitive marker for chimerism than our ADA assay. None of the 14 fetuses recovered after injecting morulae with cultured ICM cells were chimeric with either marker. No chimeras developed from control embryos. These studies demonstrate 1) that pregnancy rates are not compromised by injection of blastocysts or morulae with ICMs or cultured ICM cells, 2) that chimeric rabbit fetuses can be produced by injecting ICMs into either blastocysts or morulae, and 3) that cultured ICM cells can contribute to embryonic development when injected into blastocysts.

Animals↗
PubMed1993

Subzero nonfreezing storage of the mammalian cardiac explant. I. Methanol, ethanol, ethylene glycol, and propylene glycol as colligative cryoprotectants.

We employed hyperosmotic concentrations of penetrating cryoprotective agents (CPA) to store the isolated rat hearts unfrozen at subzero temperatures. The effect of acute exposure to CPA was assessed by flushing the hearts with CP-14, a cardioplegic solution, containing methanol (MeOH), ethanol (EtOH), ethylene glycol (EG), or propylene glycol (PG) for 2 min and reperfusing immediately with Krebs-Henseleit buffer in a working-heart model. The maximal doses that did not cause irreversible suppression of heart function were: MeOH, 1.78 M; EtOH, 1.27 M; EG, 0.84 M; and PG, 0.87 M. For nonfreezing storage, the hearts were flushed with CP-14 containing the highest tolerable concentrations of MeOH, EtOH, EG, or PG, stored for 6 h at -3.7, -2.8, and -1.4 degrees C, respectively, and then reperfused. Control cardiac output (CO) was 76.2 +/- 1.8 ml/min. Post-reperfusional recovery of CO was 86% in MeOH hearts, 82% in EtOH hearts, 76% in EG hearts, and 79% in PG hearts. Thus MeOH offered not only the least cardiac-suppressing effect but the lowest nonfreezing storage temperature. When storage time was extended, recovery and myocardial ATP level decreased with time in hearts flushed with CP-14 + 1.78 M MeOH and stored at -3.7 degrees C. The decay of function was faster than the decay of ATP level, suggesting energy was better preserved than function. The low return of function, however, may be related to CPA toxicity, osmotic stress, and ischemia/reperfusion injury. Nonfreezing storage at subzero temperatures using these CPAs may provide a novel approach to long-term cardiac preservation.

Adenine Nucleotides↗
PubMed1993

High-level production of the mouse epidermal growth factor in a Bacillus brevis expression system.

In order to determine the three-dimensional structure, the folding pathways, and the residues which are critical for biological functions of the mouse epidermal growth factor (mEGF), large amounts of wild-type and site-specific mutants are needed for biological and physiochemical studies. Genes coding for mEGF and its mutants were expressed in a Bacillus brevis system in which the expressed foreign proteins were secreted into the culture medium. However, proteases were also secreted that resulted in partial degradation of the desired foreign proteins. One mutant with much less protease secretion was isolated by applying nitrosoguanidine mutagenesis on a B. brevis strain. A new vector to facilitate DNA mutagenesis and sequencing was also constructed for this system. Methodologies for the transformation of B. brevis and the purification of expressed mEGF were developed. Under the optimized conditions, the production of the mEGF and its mutants was about 50 mg per liter, and yielded up to 10 mg of purified mEGF. Several mEGF mutants have been produced and their receptor binding abilities have been measured.

Amino Acid Sequence↗
PubMed1993

Plasmid diversity in senescent and nonsenescent strains of Neurospora.

A sample of 171 natural isolates of Neurospora crassa and Neurospora intermedia was tested for senescence. Of these, 28 strains senesced within the duration of the experiment. These senescent strains, together with a selection of nonsenescent strains, were examined for the presence of mitochondrial plasmids. This was done by digesting mitochondrial DNA preparations with proteinase K, and running these samples on agarose gels. Most of the strains examined, both senescent and nonsenescent, contained plasmids, many of them new. Some new plasmids were linear, as inferred from their resistance to 5' exonuclease and sensitivity to 3' exonuclease. New circular plasmids were also found. Some strains carry several plasmids, and mixtures of circular and linear elements were common. A cross-homology study was performed on a sample of plasmid-bearing strains, and several cases of apparent relatedness were found, some between strains from distant geographical locations. Linear plasmids homologous to the maranhar linear senescence plasmid were quite common. A new member of the LaBelle circular plasmid homology group was found. In the sample tested for homology, no strains contained elements related to the kalilo linear senescence plasmid. The relationship of the new plasmids to senescence is not known. In addition to plasmid monomers, several different types of derivatives were found. The kalilo linear plasmid was found to occur in linear and circular forms of low mobility, presumed to be giant concatamers, and, in some strains, variant sibling structures and ladders of short derivatives were found. Circular plasmids also gave rise to extensive ladders on electrophoresis, probably representing different relaxation states and head-to-tail concatameric series. Some such forms migrated more slowly than mitochondrial DNA. One unique type of plasmid modification observed was a pair of linear elements that had apparently arisen de novo which showed homology to a circular plasmid.

DNA, Circular↗
PubMed1993

Effect of growth factors on morula and blastocyst development of in vitro matured and in vitro fertilized bovine oocytes.

Growth factors were studied as a means of increasing the development of in vitro matured (IVM) and in vitro fertilized (IVF) oocytes into morulae or blastocysts. Cell numbers of blastocysts were also counted. In Experiment 1, 2- to 8-cell embryos derived from bovine IVM/IVF oocytes were randomly allotted to one of 3 culture groups: a) synthetic oviduct fluid (SOF); b) SOF + 10 ng/ml epidermal growth factor (EGF); or c) SOF + 100 ng/ml EGF; all 3 culture media contained 10% fetal bovine serum. Culture resulted in 12%, 23% and 14% (P>0.05), respectively, developing into morulae and blastocysts. In Experiment 2, 5 ng/ml of transforming growth factor B (1) (TGF B (1)) added to CR(1aa) medium containing BSA increased the percentage of blastocysts to 56% vs 40% for the control (P<0.05). In Experiment 3, EGF and TGF B(1), added singly and in combination to CR(1aa) did not produce a synergistic effect. More embryos developed into morulae and blastocysts (45%) in a bovine oviduct epithelial co-culture than in any other treatment except in CR(1aa) + EGF (34%; P>0.05). In Experiment 4, 0, 1 and 5 ng/ml of platelet derived growth factor (PDGF) added to CR(1aa) yielded 39%, 70% and 52% morulae and blastocysts, respectively (P<0.05). Cell number was not increased, indicating that growth factors can increase the proportion of embryos that develop into morulae and blastocysts without an increase in the cell number.

Journal Article↗
PubMed1993

PCR to identify specific clones of interest for DNA sequencing.

We describe a rapid method for identifying specific clones of interest for the purpose of sequencing. The method essentially is polymerase chain reaction using one internal primer and one vector specific primer. The procedure is particularly useful when relatively large numbers of clones are to be examined either to establish the nucleotide sequence of a full-length cDNA or to find a specific section of a large DNA. The relative orientations of inserts in different clones can also be determined using the same procedure.

Animals↗
PubMed1993

Mechanism of the hypertensive response to central injection of nicotine in conscious rats.

The purpose of this study was to examine the effects of nicotine administered directly into the CNS on mean arterial pressure (MAP) and heart rate to avoid the direct peripheral action of the drug. Also, because nicotine has been reported to enhance the release of endogenous brain acetylcholine, we sought to determine the role of this mechanism in mediating the cardiovascular response. Normotensive Wistar rats were previously implanted with indwelling intracerebroventricular (ICV) cannula guides and an arterial line (iliac artery) for central injection of drugs and measurement of MAP and heart rate, respectively. Rats received a series of increasing doses of nicotine (or saline vehicle) from 2-100 micrograms (in a 10 microliter volume) with each dose separated by at least 1 day. MAP increased immediately following all doses of nicotine; however, the maximal response was obtained following the 50 micrograms dose (higher doses actually produced lower responses). In general, the hypertensive response began immediately after injection, peaked within 2-3 min and returned to baseline within about 20 min. Heart rate changes were often not dramatic and highly variable. In order to examine the dependence of the pressor response to nicotine on brain acetylcholine, rats were pretreated with 20 micrograms (ICV) of hemicholinium-3 (HC-3) 1 h prior to nicotine to deplete endogenous acetylcholine. HC-3 pretreatment resulted in a significant reduction in the magnitude and duration of the pressor response to nicotine. Likewise, pretreatment with atropine inhibited the pressor response to subsequent injection of nicotine. Nicotine enhanced the release of [3H]acetylcholine from brain slices in vitro at concentrations likely achieved in the in vivo studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1993

The effect of bone morphogenetic protein on osseointegration of titanium implants.

This study evaluated the osteointegration of threaded cylindrical uncoated endosseous implants in combination with either bovine bone morphogenetic protein (bBMP) (group 1) or bovine serum albumin (BSA) (group 2) in edentulous dogs. Histologic observations showed that new bone formation was markedly increased by bBMP. The screw-surfaced implants in combination with bBMP exhibited apparent direct bone apposition 4 weeks postimplantation with no evidence of a connective tissue capsule. Thick fibrous connective tissue still encapsulated the control implants, although some new bone was observed slowly growing from the margins of the host bed. Eight weeks after implantation, there was an exact fit between the implant and the bone in group 1, and the surrounding bone was arranged in a regular manner. In group 2, there were still a few areas of thin fibrous tissue surrounding the implants. The results indicate that the rate of osteointegration was increased by bBMP.

Animals↗