Signal perception and transduction in plant defense responses.
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Animals↗
Biomedical subjects
Y Yang
Publications and source records attributed to Y Yang.
Thrombin receptor on rat primary hippocampal neurons: coupled calcium and cAMP responses.
We have tested the hypothesis that hippocampal neurons respond to thrombin via a neuronal thrombin receptor. A human neuroblastoma cell line, SK-N-SH, known to be thrombin responsive morphologically, responded both to thrombin and thrombin receptor agonist peptide (TRAP 42-55) with elevation of intracellular calcium. In Western blots of membranes from SK-N-SH cells and cultured rat hippocampal neurons using an antibody against the N-terminal peptide of the human thrombin receptor, putative receptor proteins of 66 and 47 kDa were detected in both cells. Neurons were treated with thrombin and TRAP 42-55 (TRAP-14) to determine their effects on intracellular levels of calcium and cAMP. Only 10% of the neurons showed a rapid response to thrombin, but most responded rapidly to agonist peptide with a prolonged elevation of intracellular free calcium. Neuronal cAMP levels were decreased by 40% after 24 h thrombin treatment. This decrease in cAMP level could be blocked by both the Gi-protein inhibitor, pertussis toxin, and the thrombin inhibitor, hirudin, suggesting a possible involvement of Gi-protein-coupled receptor activation. Furthermore, rapid calcium and cAMP responses were apparently induced by pre-treatment of neurons with thrombin for 24 h and subsequent washout. In summary, these data indicate that rat primary hippocampal neurons have thrombin receptors whose responses to thrombin apparently are up-regulated by 24 h thrombin pre-treatment. These results may have implications for synaptic remodeling, learning and memory.
Animals↗
Spatial and temporal coherence in perceptual binding.
Component visual features of objects are registered by distributed patterns of activity among neurons comprising multiple pathways and visual areas. How these distributed patterns of activity give rise to unified representations of objects remains unresolved, although one recent, controversial view posits temporal coherence of neural activity as a binding agent. Motivated by the possible role of temporal coherence in feature binding, we devised a novel psychophysical task that requires the detection of temporal coherence among features comprising complex visual images. Results show that human observers can more easily detect synchronized patterns of temporal contrast modulation within hybrid visual images composed of two components when those components are drawn from the same original picture. Evidently, time-varying changes within spatially coherent features produce more salient neural signals.
Contrast Sensitivity↗
Regulation of cytokine-induced iNOS expression by a hairpin oligonucleotide in murine cerebral endothelial cells.
Inducible nitric oxide synthase (iNOS) is expressed in response to cytokines by a number of cell types participating in CNS inflammation, including brain cerebral endothelial cells. NF-kappaB, a transcription factor, mediates effector actions of pro-inflammatory cytokines. A combination of tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) enhanced the expression of iNOS in murine cerebral endothelial cells (MCECs). In an attempt to modulate TNF-alpha+IFN-gamma induced expression of iNOS in MCECs, we designed a double-strand hairpin (hp) oligonucleotide carrying the NF-kappaB motif. This hp oligonucleotide inhibited NF-kappaB binding activity and decreased both iNOS mRNA and protein expression induced by TNF-alpha+IFN-gamma. As a control, a mutant hp oligonucleotide was without effect. The present study confirms the role of transcription factor NF-kappaB in iNOS expression induced by TNF-alpha+IFN-gamma in MCECs. More importantly, it demonstrates that an appropriately designed hp oligonucleotide is an effective tool to modulate iNOS expression and may be of potential pharmacological use.
Animals↗
A case-control study of diet and lung cancer in northeast China.
A case-control study involving interviews with 227 lung-cancer cases and 227 matched hospital controls was conducted in Heilongjiang Province in northeast China to examine the influence of dietary factors on the risk of developing lung cancer. Lung-cancer cases were all incident cases judged to be suitable candidates for tumor removal by surgery. Controls were selected among hospitalized patients with non-neoplastic and non-lung disease. The overall male lung-cancer risks associated with cigarette smoking were similar to those reported in other Chinese studies but quite low compared to risks in Western countries. However, the subjects in this study were relatively young (average age 53.2), had started to smoke on average at a relatively old age (21.3 years), and only smoked an average of 18.7 cigarettes per day. Lung-cancer risk was not strongly associated with any of the nutrients examined, when all cases were compared to all controls. However, the data were suggestive of differences in the relationship of diet to risk among smokers and non-smokers. Cautious interpretation is required because of the wide confidence intervals due to limited sample size. Among the smokers, only higher beta-carotene was associated with estimates suggesting a lowered risk. Among non-smokers, the evidence suggested that increased vegetable consumption might reduce risk, consumption of any fruit might reduce risk but beta-carotene was unrelated to risk. The differences observed in the relationship of diet to lung-cancer risk between Chinese smokers and non-smokers warrant further study.
Case-Control Studies↗
Seizure-induced glutamate release in mature and immature animals: an in vivo microdialysis study.
A glutamate biosensor was used to measure extracellular glutamate concentrations in the hippocampus of mature and immature animals. Significant elevations of extracellular glutamate were observed following seizures induced by either kainic acid or pilocarpine. The degree of glutamate increase following seizures was similar in both mature and immature animals. These results suggest that excitotoxicity may play a role in seizure-induced brain damage in the adult brain. In the immature brain, however, no brain damage is seen after seizures, suggesting that glutamate release may not cause as significant excitotoxic damage early in development.
Aging↗
The beta3-adrenergic receptor in the obesity and diabetes prone rhesus monkey is very similar to human and contains arginine at codon 64.
The beta3-adrenergic receptor (ADRbeta3) is a seven-membrane spanning, G-protein linked receptor expressed in brown adipose tissue in rodents, and visceral adipose tissue in humans. Stimulation of the receptor by norepinephrine leads to lipolysis and thermogenesis. In rodent models of obesity and diabetes, administration of beta3-agonists results in weight loss and improved glucose tolerance. Studies indicate that the pharmacological properties of the ADRbeta3 differ markedly between rodents and humans, making generalizations of rodent studies to humans difficult. We hypothesized that the obesity and diabetes prone rhesus monkey (Macaca mulatta) would provide an excellent animal model to study the role of the ADRbeta3 in the development of obesity and diabetes as well as for assessment of the therapeutic efficacy of beta3-agonists. We sequenced the entire coding region of the rhesus ADRbeta3 gene. Like humans, the rhesus ADRbeta3 has two exons. There is 89% amino acid (aa) identity between human and rhesus compared to 82% aa identity between human and mouse. A single base deletion results in divergence of the intracellular carboxy terminus accounting for 26 of the 45 aa changes and 10 additional aa. Of the 15 rhesus monkeys studied, all were homozygous for Arg64. In humans, Arg64 (rather than Trp) is associated with increased body mass index, insulin resistance, and an earlier onset of type II diabetes mellitus. We conclude that the rhesus ADRbeta3 is more similar to the human ADRbeta3 than to the rodent ADRbeta3 suggesting that this primate model may be more appropriate for physiologic and therapeutic studies of the ADRbeta3 axis, and that Arg64 may influence susceptibility in this species to obesity, insulin resistance, and type II diabetes.
Amino Acid Sequence↗
Downregulation of RAR alpha in mice by antisense transgene leads to a compensatory increase in RAR beta and RAR gamma and development of lymphoma.
Retinoic acid receptors (RARs) alpha, beta, and gamma contain retinoic acid response elements (RAREs) in their promoter regions and respond to their own activation, thus forming an autoregulatory loop. We generated transgenic mice that expressed an antisense construct of the RAR alpha. Homozygous transgenic mice demonstrated 30% to 80% reduction in RAR alpha protein expression in various tissues. Unlike RAR alpha null mice generated by knockout, our antisense mice demonstrated significant compensatory increases in the expression of RAR beta and RAR gamma proteins. Coarse fur, male sterility, and low body weight were other abnormalities observed in these mice. Most importantly, lymphoma developed in 44% of our homozygous transgenic mice at an early stage of life. These data suggest that RAR alpha is necessary for appropriate response of the RAR beta and RAR gamma genes to physiologic changes and deregulation of the RAR alpha in transgenic mice, which resulted in upregulation of RAR beta and RAR gamma, can be associated with lymphomagenesis. Thus, the data support the hypothesis that a balance among the RARs is necessary for appropriate response to various homeostatic needs.
Animals↗
Hypothermia and delayed fever in the male and female rat exposed to chlorpyrifos.
Chlorpyrifos (CHL) is a commonly used organophosphate (OP) pesticide which irreversibly inhibits acetylcholinesterase activity in the CNS. Little is known regarding the thermoregulatory effects of CHL when administered orally and whether the sensitivity to CHL is affected by sex. To address these issues, male and female rats of the Long-Evans strain were administered 0, 10, 50, or 80 mg/kg CHL by gavage while core temperature (T(c)) and motor activity (MA) were monitored continuously by telemetry. Females were generally more sensitive than males to CHL. Significant hypothermic responses to CHL were observed in males administered 80 mg/kg and in females administered 10-80 mg/kg. Following recovery from hypothermia T(c) of both males and females underwent a significant elevation during the light phase 1-2 days after CHL exposure. CHL-induced hyperthermia was blocked in male and female rats by administration of 200 mg/kg sodium salicylate (SS), an antipyretic agent. Male castrated rats were markedly more sensitive to the hypothermic and hyperthermic effects of CHL compared to sham operated controls. On the other hand, ovariectomized female rats responded to CHL in a similar fashion as the sham operated controls. Thus, testicular function may be important in determining greater resistance to CHL in male rats. It appears that exposure to CHL leads to a delayed fever which involves activation of CNS pathways normally involved in fever. This mechanism could be responsible for the febrile response to OP pesticides commonly observed in humans exposed to OPs.
Administration, Oral↗
Thermoregulatory response to chemical toxicants and other insults. Extrapolation from experimental animal to human.
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Animals↗
Molecular cloning and chromosomal localization of a human gene homologous to the murine R-PTP-kappa, a receptor-type protein tyrosine phosphatase.
Tyrosine phosphorylation of proteins plays an important role in cellular signaling and many cellular activities. The levels of cellular phosphorylation are reversibly controlled by protein tyrosine kinases and protein tyrosine phosphatases. The murine R-PTP-kappa, a receptor-type protein tyrosine phosphatase, has recently been cloned (Jiang et al. (1993) Mol. Cell. Biol. 13, 2942-2951). In order to identify the protein tyrosine phosphatases critical to the cellular signal transduction in human keratinocytes, a polymerase chain reaction (PCR)-based strategy was employed, and we have cloned a human homologue of the murine R-PTP-kappa. Here, we report the isolation of a complementary DNA encoding a human R-PTP-kappa. Of the several overlapping cDNA clones, one clone, which we originally termed p55-7, was found to encode a transmembrane protein of 1440 amino acids and was highly conserved with murine R-PTP-kappa with 98% identity at the amino-acid levels. The human R-PTP-kappa gene was localized to chromosome 6 by southern hybridization of DNA from a rodent/human somatic cell mapping panel. Northern blot analysis of RNA from several human tissues revealed, like the murine R-PTP-kappa, the presence of a major mRNA of approx. 7.0 kb and a minor mRNA of approx. 5.3 kb. In contrast to the expression of murine R-PTP-kappa which was highly expressed in liver and kidney, the human R-PTP-kappa was predominantly expressed in spleen, prostate, and ovary. However, the transcripts were detectable at various levels in all examined tissues (thymus, testis, small intestine, and colon) except for PBL (peripheral blood leukocytes). In addition, human R-PTP-kappa displayed a restricted pattern of expression among a series of cell lines, and was apparently expressed in an epidermal cells and cell lines (human normal keratinocytes, HaCaT, and A431), but was not detectable in other cell lines tested after longer exposure.
Amino Acid Sequence↗
Membrane properties and synaptic inputs of suprachiasmatic nucleus neurons in rat brain slices.
1. Whole-cell recordings were made from 390 neurons of the suprachiasmatic nucleus (SCN) in horizontal brain slices during different portions of the circadian day. The locomotor activity of the rats was measured prior to the preparation of brain slices to insure that each rat was entrained to a 12 h-12 h light-dark cycle. 2. The mean input conductance was 42% higher (1.58 nS) in neurons recorded near the subjective dawn than those (1.11 nS) recorded near the subjective dusk. The current required to hold the neurons at -60 mV also showed a circadian variation with a peak in the middle of the subjective day and a nadir in the middle of the subjective night. Analysis of the variations in the input conductance and the holding current at -60 mV suggested that at least two ion conductances are involved in the pacemaking of the circadian rhythms. 3. Voltage-clamped SCN neurons often had both outward and inward spontaneous postsynaptic currents. The outward currents were blocked by bicuculline but not by strychnine, and were identified as IPSCs mediated by GABAA receptors. The inward currents were blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and were identified as EPSCs mediated by glutamate. Most spontaneous synaptic currents were miniature currents but action potential-dependent large events were seen more often in IPSCs than in EPSCs. 4. Stimulation of the optic nerve or chiasm usually evoked a monosynaptic EPSC which was mediated by both NMDA and non-NMDA receptors. In 13% of cells, optic nerve stimulation evoked an outward current or an inward current followed by an outward current; all the evoked currents were blocked by 4-aminophosphonovaleric acid (APV) and CNQX whereas the outward current only was blocked by bicuculline, suggesting involvement of an inhibitory interneuron. 5. SCN neurons sum the excitatory inputs from both optic nerves; on average each SCN cell receives innervation from at least 4.8 retinohypothalamic tract (RHT) axons. 6. Focal stimulation in the vicinity of the recorded neuron revealed that nearly all SCN neurons receive local or extranuclear GABAergic inputs operating via GABAA receptors. The EPSCs activated by such stimulation were not significantly different in amplitude and pharmacological properties from those induced by RHT stimulation. 7. One hundred and one neurons were labelled with neurobiotin during whole-cell recording. Based on the dendritic structures, four types of SCN neurons (monopolar, radial, simple bipolar and curly bipolar) were identified. The curly bipolar cells had a higher membrane conductance, holding current and hyperpolarization-activated current (Ih) amplitude than the other neuronal types. Radial neurons did not respond to optic nerve stimulation, which activated EPSCs in the other cell types.
Animals↗
A picomole-scale method for charge derivatization of peptides for sequence analysis by mass spectrometry.
A highly activated ester containing a fixed positive charge, S-pentafluorophenyl [tris(2,4,6-trimethoxyphenyl)phosphonium]acetate bromide (TMPP-AcSC6F5 bromide), has been synthesized as a reagent for N-terminal modification of peptides. Stable in aqueous acetonitrile solution during extended storage, TMPP-AcSC6F5 bromide reacts with unprotected peptides through p-(dimethylamino)pyridine (DMAP)-promoted amidation in aqueous acetonitrile (15 min, ambient temperature) to form N-TMPP-Ac derivatives of peptides. These peptide derivatives are readily amenable to analysis by fast atom bombardment (FAB) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Greater than 90% conversion has been observed in transforming low-nanomole quantities of analyte using molar ratios of 1:5:10 (peptide/reagent/ DMAP). For reactions at the picomole level a slightly modified stoichiometry, with molar ratios of 1:10:500, is employed. Owing to the high reaction efficiency and the tolerance to moderate excess reagent and base during analysis by FAB- and MALDI-MS, the reaction mixture containing the modified peptides can be analyzed directly in most cases, without sample cleanup. Examples of the preparation and analysis of a variety of N-TMPP-acetyl-peptides (TMPP-Ac-peptides) ranging from hexamers to 15-mers are given. Collisionally activated dissociation tandem mass spectrometry of TMPP-Ac-derivatives showed dominant a-type ions, accompanied by d- and c-type ions in some cases, allowing sequence determination to be made in a straightforward manner.
Amino Acid Sequence↗
A cysteine protease inhibitor prevents activation-induced T-cell apoptosis and death of peripheral blood cells from human immunodeficiency virus-infected individuals by inhibiting upregulation of Fas ligand.
Activation of T-cell hybridomas, preactivated normal T cells, and peripheral blood lymphocytes (PBL) from human immunodeficiency virus (HIV)-infected individuals results in apoptosis. In the first two cases, apoptosis is caused by the upregulation of Fas ligand (FasL) and its subsequent interaction with Fas; the mechanism for the spontaneous and activation-induced death of lymph node cells and PBL from HIV+ blood is not known. A number of protease inhibitors have been shown to prevent T-cell apoptosis under all of these circumstances, but the mechanism of action has not been determined. Here we show that the cysteine protease inhibitor E64d prevent activation-induced T hybridoma cell death by inhibiting the upregulation of FasL. Quantitative polymerase chain reaction (PCR) demonstrated that mRNA for FasL is expressed at low levels in fresh PBL from HIV-infected blood, but increases in cultured PBL from both uninfected and HIV-infected donors. The ex vivo apoptosis of PBL from HIV+ donors was prevented by adding the soluble extracellular domain of Fas, demonstrating a requisite role for Fas/ FasL interactions in this form of cell death. Furthermore, while having no effect on the death of PBL from HIV-infected blood stimulated directly via Fas, E64d inhibited FasL upregulation. Thus, aberrant apoptosis of cultured PBL from HIV-infected individuals is mediated by FasL and Fas, and E64d blocks this apoptosis by inhibiting the upregulation of FasL. These results are consistent with the hypothesis that the abnormal expression of Fas and the inducible expression of FasL, contributes to the immunodeficiency of patients with acquired immune deficiency syndrome and suggest that modulation of FasL expression could be an effective target for therapeutic intervention.
Apoptosis↗
Identification of the glycosylation site and glycan structures of recombinant endopolygalacturonase II by mass spectrometry.
A series of mass spectrometric experiments was performed to characterize the carbohydrate chains attached to endopolygalacturonase II (EPG-II) overexpressed in Aspergillus niger. First, an aliquot of trypsin-digested EPG-II was analyzed by capillary high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) mass spectrometry (HPLC/MS). The ESI mass spectrometer was operated in the tandem mass spectrometric (MS/MS) mode and utilized precursor ion scanning of the HexNAc+ oxonium ion at m/z 204 to selectively detect glycopeptides eluting from the HPLC. Aliquots of the fraction identified by HPLC/MS/MS to contain a glycopeptide were then sequentially digested with Aspergillus saitoi alpha-1-2-mannosidase and peptide N:glycosidase F (N-glycanase), followed by matrix-assisted laser-desorption/ionization mass spectrometric analysis to provide structural information from the carbohydrate chain. The masses of the carbohydrate chains in the glycopeptides were thus determined and subsequently used to search the Complex Carbohydrate Structure Database for the probable structures of the glycans. This analysis determined that recombinant EPG-II has a single N-glycosylation site, the carbohydrate chain at this site is heterogeneous, and the glycan structure is of the high-mannose type. No sites of O-glycosylation were detected in EPG-II.
Amino Acid Sequence↗
Rapamycin inhibits proteasome activator expression and proteasome activity.
Rapamycin (RAPA) is a potent immunosuppressive drug, and certain of its direct or indirect targets might be of vital importance to the regulation of an immune response. In this study, we used differential hybridization to search for human genes whose expression was sensitive to RAPA. Seven RAPA-sensitive genes were found and one of them encoded a protein with high homology to the alpha subunit of a proteasome activator (PA28 alpha). This gene was later found to code for the beta subunit of the proteasome activator (PA28 beta). Activated T and B cells had up-regulated PA28 beta expression at the mRNA level. Such up-regulation could be suppressed by RAPA, FK506, and cyclosporin A. RAPA and FK506 also repressed the up-regulated PA28 alpha messages in phytohemagglutinin (PHA)-stimulated T cells. At the protein level, RAPA inhibited PA28 alpha and PA28 beta in the activated T cells according to immunoblotting and confocal microscopy. Probably as a consequence, there was a fourfold increase of proteasome activities in the peripheral blood mononuclear cell lysate after the PHA activation. RAPA could inhibit the enhanced part of the proteasome activity. Considering the critical role played by the proteasome in degrading regulatory proteins, our data suggest that the proteasome activator is a relevant and important downstream target of rapamycin, and that the immune response could be modulated through the activity of the proteasome.
Cysteine Endopeptidases↗
Biochemical heterogeneity in hysterectomized uterus measured by 31P NMR using SLIM localization.
Ultrasound and magnetic resonance imaging show contrast between the inner and outer myometrium, which is useful in the diagnosis of gynecological disorders. To determine whether the image contrast is associated with biochemical differences between these myometrial regions, phosphorus metabolite concentrations in the inner one third of the myometrium (the junctional zone; JZ) were compared with the outermost one third of the myometrium (OM) in hysterectomized uteri using 31P spectral localization by imaging (SLIM). The technique was validated by comparing the results of SLIM with the results of standard Fourier-encoded spectroscopic imaging (FSI) analysis using phantoms, and by nonlocalized spectroscopy on biopsies taken from the same hysterectomy specimens. As expected theoretically, SLIM yielded better localization than FSI, as judged by spectral intensity and leakage measurements on phantom compartments of known composition. SLIM localization revealed that the JZ has a higher intracellular phosphomonoester (PME) concentration than does the OM, which was confirmed by nonlocalized spectroscopy, and that there is very little NMR-visible phosphorus in the cervix.
Adult↗
Nuclear magnetic resonance assignment and secondary structure of an ankyrin-like repeat-bearing protein: myotrophin.
Multidimensional heteronuclear NMR has been applied to the structural analysis of myotrophin, a novel protein identified from spontaneously hypertensive rat hearts and hypertrophic human hearts. Myotrophin has been shown to stimulate protein synthesis in myocytes and likely plays an important role in the initiation of cardiac hypertrophy, a major cause of mortality in humans. Recent cDNA cloning revealed that myotrophin has 11B amino acids containing 2.5 contiguous ANK repeats, a motif known to be involved in a wide range of macromolecular recognition. A series of two- and three-dimensional heteronuclear bond correlation NMR experiments have been performed on uniformly 15N-labeled or uniformly 15N/13C-labeled protein to obtain the 1H, 15N, and 13C chemical shift assignments. The secondary structure of myotrophin has been determined by a combination of NOEs, NH exchange data, 3JHN alpha coupling constants, and chemical shifts of 1H alpha, 13C alpha, and 13 C beta. The protein has been found to consist of seven helices, all connected by turns or loops. Six of the seven helices (all but the C-terminal helix) form three separate helix-turn-helix motifs. The two full ANK repeats in myotrophin are characteristic of multiple turns followed by a helix-turn-helix motif. A hairpin-like turn involving L32-R36 in ANK repeat #1 exhibits slow conformational averaging on the NMR time scale and appears dynamically different from the corresponding region (D65-169) of ANK repeat #2.
Amino Acid Sequence↗