The role of inducible nitric oxide synthase in cardiac allograft rejection.
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Cell Death↗
Biomedical subjects
X Yang
Publications and source records attributed to X Yang.
Metabolic alteration in neural tissue of rat embryos exposed to beta-hydroxybutyrate during organogenesis.
Hyperketonemia has been identified as an important factor in diabetic pregnancy affecting growth and development of the offspring. In order to assess the immediate metabolic alterations in embryos caused by excess ketone bodies, we studied rat embryonic neural tissue exposed to a high concentration of beta-hydroxybutyrate in vitro. Beta-hydroxybutyrate inhibited oxygen uptake of the neural tissue of day 9 and day 10 embryos by 12.8% and 1 1.2%, but did not affect that of day 11 and day 12 tissue. In contrast, glucose utilization of the neural tissue of day 9 and day 10 embryos was not altered. However, a 30% decrease in glucose utilization was observed in the neural tissue of day 11 and day 12 embryos exposed to beta-hydroxybutyrate. Thus, beta-hydroxybutyrate induced different metabolic alterations in the embryonic neural tissue during early and late organogenesis, which suggests different modes of teratogenic action of ketone bodies in different parts of gestation.
3-Hydroxybutyric Acid↗
Lymphocyte granule-mediated apoptosis: matters of viral mimicry and deadly proteases.
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Animals↗
Tensile strength of lens capsules in eye-bank eyes.
PURPOSE: To measure the tensile strength of the anterior and posterior lens capsules in eye-bank eyes and study the tensile strength of capsules dehydrated with hypertonic glucose solution. SETTING: Tongren Hospital, Beijing, China. METHODS: The in situ anterior and posterior capsules of 35 eye-bank eyes (Group 1) donated by healthy men aged 20 to 40 years (mean 27.1 years) were measured with a biomembrane strength device. Sixteen eyes (Group 2), from donors aged 20 to 37 years (mean 27.0 years) and similar to those in Group 1, were measured in the same way after the anterior and posterior capsules were exposed to glucose 50% solution for 15 minutes. RESULTS: In Group 1, anterior and posterior capsule strength was 6.269 +/- 1.118 and 3.755 +/- 0.835 g, respectively (P < .01), and the correlation between the tensile strength of the anterior and posterior capsules was statistically significant (r = .94; P < .001). In Group 2, the strength of the capsules increased to 9.024 +/- 0.586 and 4.424 +/- 0.761 g, respectively, a finding that was statistically significantly different from that in Group 1 (P < .01 and .05, respectively. CONCLUSION: The tensile strength of the anterior capsule was stronger than that of the posterior capsule under the experimental conditions. Glucose 50% solution has the potential to increase the tensile strength of anterior and posterior capsules.
Adult↗
A cysteine-rich isoform of neuregulin controls the level of expression of neuronal nicotinic receptor channels during synaptogenesis.
We report here that neuregulin (NRG) isoforms with a conserved cysteine-rich domain (CRD) in their N terminus regulate expression of nicotinic acetylcholine receptors (nAChRs) at developing interneuronal synapses and report the isolation of transmembrane NRG isoforms with this CRD within the N-terminal portion. CRD-NRG mRNA and immunoreactive protein are detected early in developing presynaptic (visceral motor) neurons. The levels of expression of CRD-NRG peak prior to the formation of synapses with their postsynaptic partners, the ganglionic sympathetic neurons. Recombinant CRD-NRG mimics the effects of presynaptic input on target neurons. Functional deletion of CRD-NRG from presynaptic neurons abolishes the upregulation of nAChR expression induced by input-derived soluble material. Thus, CRD-NRG appears to be both a necessary and a sufficient signal for the control of neuronal nAChR expression during synaptogenesis.
Acetylcholine↗
Autoproteolytic activation of pro-caspases by oligomerization.
Initiation of apopotosis requires the conversion of procaspases to mature caspases. Here we show that oligomerization of pro-caspases is sufficient to induce proteolytic generation of mature caspase subunits and activation of their cell death activity. Deletion of the protein interaction motif DED from pro-caspase-8 greatly suppresses its apoptotic activity. Cell death activity can be restored by oligomerization of pro-caspase-8 protease domains by two heterologous inducible oligomerization systems. Induced oligomerization also activates the apoptotic activity of pro-caspase-1 but not pro-caspase-3. In vitro, oligomerization leads to pro-caspase processing to from the mature caspase subunits; this processing requires the intrinsic caspase activity of zymogens and proceeds via a novel order of cleavage events.
Apoptosis↗
Expression of a vitamin D-regulated gene (VDUP-1) in untreated- and MNU-treated rat mammary tissue.
Previous studies showed that the expression of an mRNA corresponding to VDUP-1 was decreased within MNU-induced rat mammary tumors. RNA from mammary tissue was DNase treated and reverse transcribed and the resulting cDNA was amplified using primers designed to amplify VDUP-1 (382 bp fragment) and glyceraldehyde-6-phosphate dehydrogenase (GAPDH) (416 bp fragment). Analysis of mammary cDNA derived from untreated or MNU-treated rats indicated that VDUP-1 expression within tumor tissue was significantly decreased, a finding which agrees with previous Northern blotting experiments. The differential expression was confirmed in tissue sections using an antisense VDUP-1 riboprobe for in situ hybridization studies which demonstrated that VDUP-1 staining in all cell types within tumor tissue was greatly decreased. VDUP-1 mRNA was expressed to a greater extent within epithelial cells and to a much lesser extent within stromal cells, including endothelial cells, in untreated mammary tissue. A significant decrease in VDUP-1 expression was detected as early as six weeks after MNU treatment, before tumors had formed. Bilateral ovariectomy did not alter VDUP-1 expression in untreated mammary tissue and ovariectomy prior to MNU treatment prevented tumor formation, as well as the associated decrease in VDUP-1 expression. The relative expression of VDUP-1 was higher in lung tissue than in adrenal, heart, kidney, liver, mammary, muscle, and ovary. Treatment of a cell line derived from an MNU-induced rat mammary tumor (MNU cells) with 1,25-dihydroxyvitamin D3 resulted in a significant increase in VDUP-1 expression and also inhibited cell growth in the absence of serum. The data are consistent with a role for VDUP-1 in mediating the inhibitory effects of 1,25-dihydroxyvitamin D3 on tumor cell growth.
Animals↗
Enzymatic degradation of luteinizing hormone releasing hormone (LHRH)/[D-Ala6]-LHRH in lung pneumocytes.
PURPOSE: To investigate the cellular proteolytic activities of various lung pneumocytes using luteinizing hormone releasing hormone (LHRH) and [D-Ala6]-LHRH as model peptide substrates. METHODS: HPLC analysis was used to investigate the degradation kinetics of LHRH/[D-Ala6]-LHRH and to identify their degradation products in isolated lung pneumocytes. RESULTS: Pulmonary macrophages exhibited the strongest proteolytic activity against LHRH)/[D-Ala6]-LHRH, followed by type II and type I-like pneumocytes. Three major degradation products of LHRH, namely LHRH 4-10, LHRH 6-10, and LHRH 7-10, were identified in macrophages and type II pneumocytes, whereas in type I-like pneumocytes only the LHRH 7-10 was found. Co-incubation of the cells with known enzyme inhibitors including captopril (an ACE inhibitor), thiorphan (an EP24.11 inhibitor), and EDTA (an EP24.15 inhibitor) inhibited the formation of LHRH 4-10, LHRH 7-10, and LHRH 6-10 respectively. In all cell types, the degradation rate of [D-Ala6]-LHRH was about 3-8 times lower than that of LHRH. This peptide analog was resistant to degradation by EP24.15 and EP24.11, but was susceptible to ACE. CONCLUSIONS: ACE, EP24.11, and EP24.15 are the major enzymes responsible for the degradation of LHRH in macrophages and type II pneumocytes. The magnitude of peptidase activities in these cell types are: EP24.15 > EP24.11 approximately ACE. No EP24.15 or ACE activity was observed in type I-like pneumocytes and only a weak EP24.11 activity was detected.
Animals↗
A role for RNA processing in regulating expression from transfected genes.
We have examined the expression of cloned genes following their stable integration into the genome of pluripotent embryonal carcinoma stem cells. Transfected genes integrate into the genome as tandem arrays. Expression of reporter genes from these tandem arrays in embryonal carcinoma cells is inefficient probably because genes are subject to repeat-induced gene silencing. We found that expression of reporter genes was significantly enhanced if co-transfected with cloned fragments derived from the murine Pgk-1 gene. The enhanced expression required (a) that the Pgk-1 fragment carries an active promoter, (b) that the promoter drives transcription through a region of more than 12 kbp, and (c) that this transcribed region contains both introns and exons. Reporter gene activity did not require specific Pgk-1 DNA sequences suggesting that the coupled processes of transcription and RNA processing conferred activity on neighboring genes probably by influencing local chromatin structure. Consistent with this idea, the effect of the Pgk-1 gene could be mimicked by exposing cells to butyrate or trichostatin A, inhibitors of histone deacetylase. Thus, the effect of the co-transfected Pgk-1 gene is to inhibit the process of gene inactivation possibly by functioning like an insulator or boundary element in the chromatin.
Animals↗
Determination of sulfadiazine and sulfamethoxazole by capillary electrophoresis with end-column electrochemical detection.
Capillary electrophoresis (CE) with end-column electrochemical detection (EC) of sulfadiazine (SDZ) and sulfamethoxazole (SMZ) is described. Under the optimum conditions, SDZ and SMZ were separated satisfactorily, and a highly sensitive and stable response was obtained at a potential of 1.1 V versus Ag/AgCl. Optimized end-column detection provides detection limits as low as 0.1 microM for both compounds, which corresponds to 0.024 and 0.021 fmol with peak efficiencies of 394,000 and 335,000 theoretical plates for SDZ and SMZ, respectively. The calibration graph was linear over three order of magnitude. The relative standard deviations (n = 12) of peak currents and migration times were 2.3 and 2.7%, and 0.8 and 1.3%, respectively, for the two compounds. The proposed method was applied to the analysis of tablets and human urine samples with satisfactory results.
Anti-Infective Agents↗
Different roles are played by alpha beta and gamma delta T cells in acquired immunity to Chlamydia trachomatis pulmonary infection.
Using gene knockout and wild-type C57BL/6 mice, we examined the role of alpha beta and gamma delta T cells in the resolution of Chlamydia trachomatis mouse pneumonitis (MoPn) biovar pulmonary infection. The results show that alpha beta T-cell-deficient (alpha-/-) mice, when compared with wild-type control mice, have dramatically increased mortality rate and greater in vivo growth of MoPn. The alpha beta T-cell-deficient mice were as susceptible to MoPn infection as T- and B-lymphocyte-deficient (RAG-1-/-) mice. Moreover, both alpha beta T-cell-deficient and RAG-1 mutant mice failed to mount delayed-type hypersensitivity (DTH) responses to organism-specific challenge and showed undetectable interferon-gamma (IFN-gamma) production by spleen cells upon in vitro organism-specific restimulation. In contrast, gamma delta T-cell-deficient mice exhibited intact DTH responses and their mortality rate and in vivo chlamydial growth were comparable to those in wild-type controls. More interestingly, gamma delta T-cell-deficient mice showed significantly higher levels of IFN-gamma production than did wild-type mice. The data indicate that the alpha beta T cell is the major T-cell population for acquired immunity to chlamydial infection and that gamma delta T cells may play an ancillary role in regulating the magnitude of alpha beta T-cell responses.
Animals↗
Chronic ethanol administration decreases phosphorylation of cyclic AMP response element-binding protein in granule cells of rat cerebellum.
To help define the molecular basis of ethanol's actions on the nervous system, we have in previous studies demonstrated that ethanol administration triggers a robust increase in cyclic AMP-response element-binding protein (CREB) phosphorylation in the cerebellum. The purpose of the present study was to compare the effects of acute and chronic ethanol exposure on the phosphorylation of CREB in rat cerebellum and to determine which cell types in the cerebellum display this response to ethanol. An acute ethanol challenge (3.0 g/kg of body weight) induced a rapid increase in content of the phosphorylated form of CREB, peaking at 30 min and declining to basal levels within 2 h. Immunocytochemical studies revealed prominent ethanol-induced changes in phosphoCREB in the granule cell layer, with little phosphoCREB apparent in Purkinje cells. Following chronic ethanol exposure (5 weeks), induction of CREB phosphorylation by a subsequent acute ethanol challenge was markedly attenuated. The attenuation in CREB phosphorylation was associated with a significant reduction in the levels of the catalytic unit of protein kinase A and calcium/calmodulin-dependent protein kinase IV. In summary, induction of CREB phosphorylation in cerebellum is most prominent in the granule cell layer. Neuroadaptation to chronic ethanol exposure includes a reduction in nuclear protein kinase A and calcium/calmodulin-dependent protein kinase IV levels, an event associated with impaired CREB phosphorylation.
Animals↗
Spatial hearing in children with visual disabilities.
A study is reported of the effect of early visual experience on the development of auditory space perception. The spatial hearing of thirty-five children with visual disabilities (twenty-two with congenital total blindness) was compared with that of eighteen sighted children and seventeen sighted adults. The tests provided a comprehensive assessment of spatial-hearing ability, including psychophysical estimates of spatial resolution in the horizontal, vertical, and distance dimensions, as well as measures of reaching and walking to the locations of sound sources. The spatial hearing of the children with visual disabilities was comparable to or somewhat better than that of the sighted children and adults. This pattern held even when the group with visual disabilities was restricted to those children with congenital total blindness; in fact, some of those children had exceptionally good spatial hearing. These findings imply that the developmental calibration of human spatial hearing is not dependent on a history of visual experience. It seems likely that this calibration arises from the experience of changes in sound-localization cues arising from self-motion, such as turning the head or walking. As a practical matter, orientation and mobility instructors may reasonably assume that individuals with visual disabilities can use their hearing effectively in day-to-day travel situations.
Adolescent↗
Differential inactivation of maturation-promoting factor and mitogen-activated protein kinase following parthenogenetic activation of bovine oocytes.
Bovine oocytes matured for 24 h (young) or 40 h (aged) were treated with calcium ionophore (A23187) alone or followed with 6-dimethylaminopurine (6-DMAP), a protein phosphorylation inhibitor, and were then assayed for histone H1 kinase and mitogen-activated protein kinase (MAPK) activities. Additionally, the changes in chromatin, meiotic spindle, and microfilament were assessed by immunofluoresence microscopy. In both young and aged oocytes, treatment with 6-DMAP following A23187 treatment abolished the activities of both H1 and MAPKs; the decline of H1 kinase preceded the decline in MAPK activity. However, A23187 treatment alone caused a slower decrease in H1 kinase activity and no evident MAPK alteration in young oocytes. In contrast, activities of both kinases decreased in aged oocytes after A23187 treatment, similar to the response in the combined treatments. The inactivation of MAPK was caused by dephosphorylation of MAP42/extracellular signal-regulated kinase 2 (ERK2) as detected by gel mobility shift in the Western blot assay. A23187 treatment of young oocytes led to chromosome separation and second polar body extrusion, but not pronuclear development, with the majority of the oocytes arrested at a transitional stage of metaphase to anaphase known as metaphase III (MIII). However, most of the A23187-treated aged oocytes developed to the pronuclear stage. When oocytes, regardless of age, were treated by A23187 plus 6-DMAP, bivalent chromosomes were clumped into a single mass, the spindle was disassembled, microtubule networks were distributed in the cytoplasm, and a pronucleus appeared. It is suggested that the decrease in H1 kinase activity is involved in the initiation of oocyte activation, i.e., the exit from metaphase II, whereas the decrease in MAPK activity correlates with onset of pronuclear formation. In conclusion, inactivation of maturation-promoting factor and MAPKs probably occurs via two independent processes, and the inactivation of both kinases is required for the metaphase II oocytes to progress through interphase. High MAPK activity might contribute to spindle stabilization, and inactivation of MAPK is associated with microtubular network formation in the cytoplasm.
Adenine↗
CNI-1493 prolongs survival and reduces myocyte loss, apoptosis, and inflammation during rat cardiac allograft rejection.
Cytokines and cytotoxic agents, including nitric oxide (NO) released by macrophages, play important roles during cardiac allograft rejection. In contrast to agents that suppress T-lymphocyte function, CNI-1493 is a multivalent guanylhydrazone compound that inhibits the synthesis and release of proinflammatory cytokines and NO from macrophages. This study investigated the effects of CNI-1493 on rejecting rat cardiac allografts by using Lewis to Wistar-Furth heterotopic cardiac transplants. CNI-1493 (2 mg/kg i.p., b.i.d.) or vehicle (water) was administered beginning the day before surgery. Rat cardiac allograft survival to cessation of heart beat, apoptosis of cardiac myocytes, degree of myocardial inflammation, and inducible nitric oxide synthase (iNOS) messenger RNA (mRNA), protein, and enzyme activity were studied at days 1, 3, 5, and 7 after transplantation. Allograft survival was increased significantly by 26% from 7.5 +/- 0.8 days in vehicle-treated rats (n = 6) to 9.5 +/- 1.2 days in CNI-1493-treated rats (n = 8, p < 0.05). Apoptotic cells per mm2 myocardium decreased from 2.25 +/- 1.25 to 0.84 +/- 0.49 at day 3 and 31.2 +/- 2.9 to 17.6 +/- 5.43 at day 5 after transplantation with CNI-1493 treatment (p < 0.05). The number of apoptotic myocytes and loss of cardiac muscle cells also decreased significantly at day 5 in the treated animals (p < 0.05). The reduction of myocyte loss at day 5 coincided with a significant decrease of the inflammatory response and reduced macrophage influx (p < 0.05). Myocardial iNOS mRNA, protein, and enzyme levels increased during the course of allograft rejection, and CNI-1493 did not significantly reduce iNOS expression in the rejecting rat allograft. CNI-1493 prolongs allograft survival and reduces myocyte loss, apoptosis, and inflammation during rat cardiac allograft rejection. These effects of CNI-1493 appear to be unrelated to altered NO synthesis but may be related to effects of the drug to inhibit macrophage synthesis of cytokines.
Animals↗
The apeE gene of Salmonella typhimurium encodes an outer membrane esterase not present in Escherichia coli.
Salmonella typhimurium apeR mutations lead to overproduction of an outer membrane-associated N-acetyl phenylalanine beta-naphthyl ester-cleaving esterase that is encoded by the apeE gene (P. Collin-Osdoby and C. G. Miller, Mol. Gen. Genet. 243:674-680, 1994). This paper reports the cloning and nucleotide sequencing of the S. typhimurium apeE gene as well as some properties of the esterase that it encodes. The predicted product of apeE is a 69.9-kDa protein which is processed to a 67-kDa species by removal of a signal peptide. The predicted amino acid sequence of ApeE indicates that it is a member of the GDSL family of serine esterases/lipases. It is most similar to a lipase excreted by the entomopathogenic bacterium Photorhabdus luminescens. The Salmonella esterase catalyzes the hydrolysis of a variety of fatty acid naphthyl esters and of C6 to C16 fatty acid p-nitrophenyl esters but will not hydrolyze peptide bonds. A rapid diagnostic test reported to be useful in distinguishing Salmonella spp. from related organisms makes use of the ability of Salmonella to hydrolyze the chromogenic ester substrate methyl umbelliferyl caprylate. We report that the apeE gene product is the enzyme in Salmonella uniquely responsible for the hydrolysis of this substrate. Southern blot analysis indicates that Escherichia coli K-12 does not contain a close analog of apeE, and it appears that the apeE gene is contained in a region of DNA present in Salmonella but not in E. coli.
Amino Acid Sequence↗
Occurrence of homologs of the Escherichia coli lytB gene in gram-negative bacterial species.
The Escherichia coli LytB protein regulates the activity of guanosine 3',5'-bispyrophosphate synthetase I (RelA). A Southern blot analysis of chromosomal DNA with the E. coli lytB gene as a probe revealed the presence of lytB homologs in all of the gram-negative bacterial species examined but not in gram-positive species. The lytB homologs from Enterobacter aerogenes and Pseudomonas fluorescens complemented the E. coli lytB44 mutant allele.
Bacterial Proteins↗
PITALRE, the catalytic subunit of TAK, is required for human immunodeficiency virus Tat transactivation in vivo.
The human cdc2-related kinase PITALRE is the catalytic component of TAK, the Tat-associated kinase. Previously, we have proposed that TAK is a cellular factor that mediates Tat transactivation function. Here we demonstrate that transient overexpression of PITALRE specifically squelches Tat-1 activation of both a transfected and an integrated human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR), suggesting that PITALRE mediates Tat function as a multiprotein complex. A catalytic mutant of PITALRE, D167N, was found to be more efficient than wild-type PITALRE in squelching Tat transactivation. Neither wild-type PITALRE nor D167N was able to squelch transactivation of the human T-cell leukemia type 1 LTR by the Tax protein. Additionally, we show that artificial targeting of PITALRE to a nascent RNA element, in the absence of Tat, activated HIV-1 LTR expression. These results indicate that a PITALRE-containing complex mediates transactivation by Tat and suggest that Tat proteins function by localizing such a PITALRE-containing complex to the site of the transcribing provirus.
Artificial Gene Fusion↗