Localization of tolerogenic epitopes in the alpha 1 helical region of the rat class I major histocompatibility complex molecule.
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Animals↗
Biomedical subjects
X Qu
Publications and source records attributed to X Qu.
Is insulin resistance in the spontaneously hypertensive rat related to changes in protein kinase C in skeletal muscle?
The mechanism of insulin resistance in the spontaneously hypertensive rat (SHR) has not been clearly identified, but protein kinase C (PKC) has been implicated as a mechanism of insulin resistance in obesity and diabetes mellitus and in a diet-induced (fructose-fed) model of insulin resistance and hypertension. This study compared PKC enzyme activity (cytosol and particulate fractions) and expression of the muscle-specific isoform, PKC-theta (Western blotting), in red (soleus) and white (tensor fascia latae) hindlimb muscles from SHR (n = 12) and WKY (n = 12) rats. SHRs were hypertensive and insulin resistant, as shown by higher insulin (188 +/- 34 v 169 +/- 22 pmol/L), triglycerides (1.65 +/- 0.07 v 1.38 +/- 0.06 mmol/L), and nonesterified fatty acids (0.99 +/- 0.05 v 0.78 +/- 0.04 mmol/L) concentrations. PKC activity was significantly greater in the membrane fraction, compared with the cytosol, but there were no significant differences either in PKC activity or subcellular distribution, or expression of PKC-theta, between the two strains. Thus, insulin resistance in the SHR (in contrast to the fructose-fed dietary model of insulin resistance and hypertension) is not related to changes in PKC signaling or expression of PKC-theta in skeletal muscle.
Animals↗
Cytokine mRNA expression in tolerant heart allografts after immunosuppression with cyclosporine, sirolimus or brequinar.
We sought to examine the impact of the preferential activation of Th2 cells on the induction and maintenance of a tolerant state in heart allograft rat recipients treated with a short course of cyclosporine (CsA), sirolimus (SRL) or brequinar (BQR). A quantitative polymerase chain reaction (PCR) method was used to measure the levels of cytokine mRNAs, namely interferon (IFN)-gamma and interleukin (IL)-2 in T helper 1 (Th1) cells and IL-4, IL-5 and IL-10 in Th2 cells. Our main findings were that on day 5 postgrafting allografts from untreated recipients had increased levels of IFN-gamma (216 +/- 119 fg), IL-2 (449 +/- 75 fg), IL-4 (6.2 +/- 1.3 fg), IL-5 (34.8 +/- 9.3 fg) and IL-10 (1554 +/- 184 fg) mRNAs compared with normal hearts. CsA reduced the levels of IFN-gamma, IL-2, IL-5 and IL-10, but not IL-4, mRNAs. SRL did not affect the expression of cytokine mRNAs. BQR decreased the levels of IFN-gamma, IL-2 and IL-10, but not IL-5 or IL-4 mRNAs. Compared with grafts from untreated recipients, those from CsA- or BQR-treated tolerant hosts (day 100) displayed undetectable IL-2 mRNA levels, and reduced levels of IFN-gamma, IL-4 and IL-10 mRNAs. In fact, the patterns of cytokine mRNA expression in grafts from CsA- and BQR-treated tolerant hosts were similar to those of normal hearts. Grafts from SRL-treated tolerant hosts merely showed slightly increased Th2 cell activity. In conclusion the selective activation of Th2 cells is not absolutely required for induction or maintenance of tolerance.
Animals↗
KCNE1 mutations cause jervell and Lange-Nielsen syndrome.
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Female↗
Insecticidal activity of a recombinant baculovirus containing an antisense c-myc fragment.
Attempts to develop baculovirus-based insecticides by insertion of genes encoding enzyme inhibitors, neuropeptides or toxins have met with some success. However, it is often difficult to ensure correct processing or secretion of the encoded peptides. Here we tested a simpler strategy by insertion of an antisense fragment of a host gene to block translation of a protein essential for larval growth and development. We selected the c-myc gene for two main reasons: (i) its protein is known to be well conserved in evolution and to have multiple essential functions during development; and (ii) c-myc family genes have yet to be characterized in insects, thus blockage of essential genes by anti-sense transcripts from a strong virus promoter could provide a sensitive test for the existence of myc-like gene products. An appropriate fragment of the human c-myc gene was inserted downstream from the polyhedrin promoter of Autographa californica nucleopolyhedrovirus and tested in bioassays on Spodoptera frugiperda larvae. Western blot analysis with a human c-myc antibody revealed an endogenous protein band which bound specifically to these antibodies. This band disappeared more rapidly from cells infected with the antisense c-myc recombinant virus than from those infected with c-myc-negative virus. Results of bioassays showed that the antisense construct stopped feeding as soon as the polyhedrin promoter-driven transcripts accumulated, followed shortly by death of the larvae. These results suggest that c-myc-like protein(s) exist in insects and that the antisense strategy is an effective approach to virus insecticide productions.
Animals↗
P-selectin-deficient mice are protected from PAF-induced shock, intestinal injury, and lethality.
In a previous study, we showed that anti-CD11b or anti-CD18 antibody markedly attenuated platelet-activating factor (PAF)-induced shock and intestinal necrosis in rats, whereas anti-P-selectin anti-body was ineffective. Here we used genetically altered mice to study the mechanism of PAF in mice. We found that P-selectin-deficient mice are completely protected from the adverse effects of PAF with no mortality or intestinal injury and only mild hemoconcentration and transient hypotension. In contrast, CD18- or intercellular adhesion molecule 1 (ICAM-1)-deficient mice were not protected from PAF-induced tissue injury and death. However, when ICAM-1-, but not CD18-, deficient mice were pretreated with fucoidin, the adverse effects of PAF were markedly reduced; survival was 100%, although hypotension still developed. Neutrophil-depleted mice were protected from PAF-induced intestinal injury but still developed hypotension and hemoconcentration. PAF increases peripheral blood neutrophil counts, probably by inducing granulopoiesis, since neutrophil-depleted mice still showed granulocytosis 60 min after PAF. Thus P-selectin plays an important role in PAF-induced injury in mice, and the selectins and the integrin-ICAM-1 system work in concert to mediate the inflammatory response to PAF in vivo.
Animals↗
[Study of interaction between cytochrome C and cystine using synchronours fluorescence spectroscopy].
The interaction of cytochrome C with cystine as promoter was studied by synchronous fluorescence spectroscopy. After cystine was added into cytochrome C solution, the synchronous flurorescence spectra of tyrosine and tryptophan residues in cytochrome C were monitered with time. The results indicated that the lysine residues in cytochrome C molecules combine with cystine slowly. It induced the small conformational change of cytochrome C molecules.
Animals↗
Sirolimus in transplantation.
Although sirolimus (SRL) alone was very effective in rodents and pigs, it produced toxic side effects in dogs. Low doses of SRL combined with cyclosporine/brequinar (CsA/BQR) combinations achieved potent immunosuppression in the CsA-resistant mouse model. Similarly, SRL/CsA/BQR therapy protected kidney allografts from rejection in dogs without producing toxic side effects. In the CsA-sensitive rat model SRL/CsA combinations produced a potent synergistic interaction. In addition, recent clinical trials document the beneficial effects of low SRL doses in human kidney transplant recipients. Sirolimus, when combined with standard immunosuppressive therapy, remarkably reduces the incidence of acute rejection and permits individual drug dose reduction.
Animals↗
Isolation, sequencing, and mapping of the human homologue of the yeast transcription factor, SPT5.
We isolated the human homologue, SUPT5H, of the yeast transcription factor, SPT5. The human homologue is 1088 aa long compared to 1063 aa for the yeast gene. SUPT5H maps to 19q13, near the ryanodine receptor. Like its family member, SUPT6H, and like yeast SPT5, SUPT5H has a very acidic 5' domain. Like its family member, SUPT6H, but unlike yeast SPT5 or SPT6, SUPT5H has seven MAP kinase sites at its 5' end. In addition, SUPT5H lacks the novel 6-amino-acid repeat (consensus is S-T/A-W-G-G-A/Q) at the C-terminus of yeast SPT5. This argues that while there is functional similarity between SPT5 and SUPT5H, the molecules differ in the signals to which they respond.
Amino Acid Sequence↗
Effects of the pharmacokinetic interaction between orally administered sirolimus and cyclosporine on the synergistic prolongation of heart allograft survival in rats.
Oral administration, but not continuous intravenous infusion, of sirolimus (SRL) in combination with cyclosporine (CsA) produces a pharmacokinetic interaction, namely increases in the whole blood trough concentrations of SRL ([SRL(WB)]) and CsA ([CsA(WB)]). The effects of this pharmacokinetic interaction on the synergism between SRL and CsA was examined in Wistar Furth (RT1u) recipients of Buffalo (RT1b) heart allografts. A 14-day course of oral SRL produced dose-dependent prolongation of heart allografts: in untreated controls, 0.5 mg/kg SRL per day extended the mean survival time (MST) from 6.4+/-0.5 days to 12.3+/-3.8 days (P<0.05); SRL at 1.0 mg/kg per day prolonged the MST to 18.0+/-5.5 days (P<0.01); at 2.0 mg/kg SRL per day, MST was extended to 52.5+/-13.2 days (P<0.01); and 4.0 mg/kg SRL per day prolonged MST to 90.0+/-41.1 days (P<0.01). Comparison of the in vivo effects after oral versus continuous intravenous SRL administration suggested that the oral bioavailability of SRL is less than 10%. Combinations of oral SRL and CsA synergistically prolonged heart allograft survival, as documented by combination index values of 0.01-0.64 (combination index <1 indicates synergistic interaction). In rats treated with dual drug combinations, CsA increased the bioavailability of SRL by two- to elevenfold, and SRL increased the bioavailability of CsA by two- to threefold, thereby significantly decreasing the oral effective dose (ED) values for each drug. The ED50 for SRL alone is 2.4 mg/kg per day, which produces an average [SRL(WB)] of 13.2 ng/ml. The ED50 for CsA alone is 8.0 mg/kg per day, which produces an average [CsA(WB)] of 1642 ng/ml. However, when the two drugs are combined, the ED50 effect is achieved with only 0.34 mg/kg SRL per day ([SRL(WB)]=1.1 ng/ml) and 2.1 mg/kg CsA per day ([CsA(WB)] =326 ng/ml). Individually, 0.34 mg/kg SRL per day produces an ED9 with an average [SRL(WB)] of 0.6 ng/ml, and 2.1 mg/kg CsA per day produces an ED22 with an average [CsA(WB)] of 174 ng/ml. Thus, the pharmacokinetic interaction between oral SRL and CsA contributes to the in vivo synergism between the two drugs.
Administration, Oral↗
Functional contributions of alpha5 subunit to neuronal acetylcholine receptor channels.
Ligand-gated ion channels are multi-subunit complexes where each subunit-type is encoded by several related genes. Heterologous expression of any one of the neuronal nicotinic acetylcholine receptors (nAChR) alpha-type subunits, either alone or with any beta-type subunit, typically yields functional nAChR channels. A striking exception is the nAChR alpha5 subunit: although apparently complexed with beta2 and beta4 nAChR subunits in neurons, and expressed in a subset of neurons within the central and peripheral nervous systems, heterologous expression of alpha5, either alone or with any beta-type subunit has failed to yield functional channels. We demonstrate here that alpha5 does participate in nAChRs expressed in hetrologous systems and in primary neurons, and further that alpha5 contributes to the lining of functionally unique nAChR channels, but only if coexpressed with both another alpha- and beta-type subunit. Furthermore, channels containing the alpha5 subunit are potently activated and desensitized by nanomolar concentrations of nicotine.
Acetylcholine↗
Induction of specific allograft immunity by soluble class I MHC heavy chain protein produced in a baculovirus expression system.
Spodoptera frugiperda (Sf9) insect cells secreted a class I MHC RT1.Aa heavy chain protein when infected with baculovirus that bore a construct that contained a honeybee melittin secretion (ms) signal attached to RT1.Aa cDNA. The RT1.Aa heavy chain protein in the culture supernatant and cell lysate immunoprecipitated in the presence of 5 individual anti-RT1.Aa-specific mAb. As was revealed by densitometric analysis, the ms signal increased the production (7- to 17-fold) and secretion (20- to 47-fold) of RT1.Aa protein by Sf9 cells (compared with RT1Aa-Sf9 cells without the ms signal). Subcutaneous immunization with secreted RT1.Aa heavy chain protein of Wistar-Furth (WF; RT1u) rats (day -4) accelerated the rejection of ACI (RT1a), but not third-party Brown Norway (BN; RT1n), heart allografts from 5.9 +/- 0.5 days in controls to 4.0 +/- 0.0 days (P < 0.001); cell lysate from RT1.Aa-Sf9 or ms/RT1.Aa-Sf9 cells reduced ACI heart allograft survival to 3.8 +/- 0.4 days or 3.7 +/- 0.5 days, respectively (P < 0.001). Indirect presentation of RT1.Aa heavy chain proteins by syngeneic macrophages shortened the survival of RT1.Aa-disparate PVG.R8 (RT1.AaDuBuCu) heart allografts in PVG.1U (RT1u) hosts from 6.3 +/- 0.5 days in controls to 4.0 +/- 0.0 days (P < 0.01). Finally, RT1.Aa heavy chain proteins injected into the thymus or into the portal vein (day -14) in combination with anti-T cell receptor mAb (days -14 and -13) induced indefinite survival of ACI liver allografts in Lewis (RT1l) recipients ( > 250 days). Thus, indirect presentation of soluble class I MHC heavy chain proteins (produced in a baculovirus/Sf9 cell system) may either sensitize or induce tolerance in the same fashion as native class I MHC alloantigens expressed on donor tissues.
Animals↗
Beneficial effect of graft perfusion with anti-T cell receptor monoclonal antibodies on survival of small bowel allografts in rat recipients treated with brequinar alone or in combination with cyclosporine and sirolimus.
In the present experiments, a multimodality regimen was developed that included an anti-T cell receptor R73 monoclonal antibody and the pharmacologic agents brequinar (BQR), cyclosporine (CsA), and sirolimus (rapamycin; RAPA) to prolong the survival of small bowel (SB) allografts. BQR was the most potent single drug: the 4.0 or 8.0 mg/kg/day BQR doses delivered every second day (q.o.d.) per gavage for 28 days prolonged the survival of Brown Norway (BN; RT1n) SB allografts in Lewis (LEW; RT1l) recipients from a mean survival time of 10.6 +/- 1.9 days in untreated controls to 29.2 +/- 5.8 days, respectively (both P < 0.001). When treatment was extended to 56 days, 8.0 mg/kg/q.o.d BQR produced a mean survival time of 83.8 +/0 33.8 days (P < 0.001), with 2/5 hosts surviving more than 100 days. In a host-versus-graft model, BQR (8.0 mg/kg/q.o.d) delivered for 28 days with CsA (2.0 mg/kg/day) and RAPA (0.04 mg/kg/day) delivered intravenously for 14 days prolonged the survival of BN SB grafts in LEW recipients to 54.4 +/- 21.0 days (P < 0.001). Extending triple-drug therapy to 42 days induced the prolongation of SB allograft survival to greater than 100 days in 5/7 recipients. Although pretransplant perfusion of the grafts with R73 mAb was ineffective alone, the combination of graft perfusion and a 28-day course of BQR (8.0 mg/kg/q.o.d) in the GVH model indefinitely prolonged LEW graft in F1 recipients. Alternatively, indefinite survival of SB allografts ( > 100 days; P < 0.001) was achieved by the combination of a 14-day course of a triple-drug regimen using each agent at subtherapeutic doses, namely BQR (2.0 mg/kg/q.o.d.), CsA (2.0 mg/kg/day), and RAPA (0.04 mg/kg/day). The state of transplantation tolerance is these hosts was documented by the acceptance of donor-type but not third-party heart allografts.
Animals↗
Detailed deletion analysis of sporadic breast tumors defines an interstitial region of allelic loss on 17q25.
Whole genome analyses of breast tumors with polymorphic markers have detected nonrandom loss of heterozygosity on multiple chromosomes, providing clues to the locations of suspected tumor suppressor genes. Tumors are thought to initiate, progress, and metastasize as mutations accumulate in multiple growth-regulatory genes; thus, identification and characterization of these genes are critical to understanding and controlling breast tumorigenesis. To map more precisely a novel breast tumor suppressor gene that has been localized previously to distal 17q, we constructed a detailed deletion map of 17q25 by analyzing eight microsatellite markers on 39 sporadic primary breast tumors. The smallest overlapping region of interstitial loss was narrowed to approximately 3 cM and included D17S937/AFM107ye3, which showed the highest percentage of allelic loss (41%). These results provide a framework from which a genomic contig will be constructed and candidate transcripts will be analyzed.
Alleles↗
A common pathway for p10 and calyx proteins in progressive stages of polyhedron envelope assembly in AcMNPV-infected Spodoptera frugiperda larvae.
The assembly of the polyhedron envelope in baculovirus-infected cells has been the subject of several studies, yet it is still poorly understood. We have used immunogold-labelled antibodies to two baculovirus proteins, p10 and calyx (also referred to as polyhedron envelope protein or PEP), to follow envelope assembly in AcMNPV-infected tissues of Spodoptera frugiperda larvae. We show that, in wild type virus, both proteins colocalize in fibrillar structures and associated electron-dense spacers which progress to encircle the polyhedra, as well as in completed polyhedron envelopes. In cells infected with polyhedrin-negative (PH-) viruses, an unusual proliferation of these spacers was observed suggesting a deregulatory event in the envelope assembly process. Results of Northern and Western blot analysis revealed that synthesis of P10 and calyx mRNA and proteins in PH- AcMNPV is unaffected as compared to wild type virus. Taken together, the observed physical and compositional connection between fibrillar structures, spacers and polyhedron envelopes, as well as the abnormal appearance of the spacers in PH- mutants, provide further evidence in support of a cooperative role of these structures in the assembly of the polyhedron envelope.
Animals↗
Endotoxin induces PAF production in the rat ileum: quantitation of tissue PAF by an improved method.
PAF (platelet-activating factor) is an endogenous mediator of endotoxin (LPS) shock and intestinal injury. In the present study we used an improved method to quantitate intestinal PAF after LPS injection. Both column and thin layer chromatography (TLC) were used to purify PAF. We found that using C18 column eluted sequentially with 10% acetic acid, ethyl acetate and 70% ethanol, yielded consistent results. TLC yielded falsely high PAF values, possibly from an unknown tissue lipid which co-migrated with PAF, or from toxic ingredients in the silica gel. Moreover, addition of optimal amounts of Tween-20 or ethanol in the bioassay samples enhanced PAF solubility and markedly improved PAF recovery. Lastly, dilution and heparinization of platelet-rich plasma greatly improved the sensitivity of the bioassay. The overall PAF recovery under these optimal conditions was 70-80%. We found that LPS (2-10 mg/kg, iv, 90 min) stimulated PAF production in the rat ileum, but not in the jejunum and colon. The difference in PAF production did not correlate to the numbers of sequestered neutrophils (reflected by myeloperoxidase levels) after LPS injection. This selective PAF production may account for the special vulnerability of the ileum to develop injury during endotoxemia.
Animals↗
Activation of acyl-coenzyme A:cholesterol acyltransferase by cholesterol or by oxysterol in a cell-free system.
Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the conjugation of long chain fatty acid and cholesterol to form cholesteryl esters. It is an integral membrane protein located in the endoplasmic reticulum. Experiments performed in intact mammalian cells have shown that the rate of cholesteryl ester synthesis in intact cells, as well as the ACAT activity from cell extracts, are greatly activated by the addition of low density lipoprotein (LDL) or oxygenated sterols such as 25-hydroxycholesterol to the growth medium. However, the molecular mechanism(s) by which sterol(s) stimulate the ACAT activity remains to be elucidated. Recently, our laboratory reported the expression cloning of human ACAT cDNA (Chang, C. C. Y., Huh, H. Y., Cadigan, K. M., and Chang, T. Y. 1993) J. Biol. Chem. 268, 20747-20755). In the current study, we report the expression of human ACAT cDNA in insect Sf9 cells. Uninfected Sf9 cells do not express detectable ACAT-like activity. Infecting these cells with recombinant virus containing ACAT cDNA caused these cells to express high levels of ACAT protein and high levels of ACAT activity when assayed in vitro. The catalytic properties of ACAT expressed in these cells were found to be similar to those found in human tissue culture cells. The combination of high level of ACAT protein expression and the low level of cellular cholesterol content in the infected cells have provided us a novel opportunity to establish a simple cell-free system, whereby stimulation of ACAT by sterols can be readily demonstrated. Using this system, we have shown that cholesterol itself can serve as an ACAT activator in vitro, in addition to its role as an ACAT substrate. The current work provides the experimental basis to hypothesize that, inside mammalian cells, cholesterol itself may serve as a physiological regulator of ACAT.
Amino Acid Sequence↗
Calcification of valved aortic allografts in rats: effects of age, crosslinking, and inhibitors.
Experiments were carried out to investigate rat aortic allograft calcification using valved abdominal aortic allografts. Results indicated that this was a potentially useful model for investigating fresh allograft calcification, as well as mineralization of glutaraldehyde-crosslinked valved allografts. Valve cusp results, however, were not comparable to those noted in large animal or human studies, while aortic wall calcification was more comparable. Calcification inhibitor investigations demonstrated that nearly complete inhibition of the calcification of the aortic wall of glutaraldehyde-crosslinked allografts was achieved using a number of individual inhibitors, including controlled release diphosphonates, and pretreatment with either ferric chloride or aluminum chloride. However, aminopropanehydroxydiphosphonate pretreatment was not efficacious, and sodium dodecyl sulfate pretreatment was only partially effective for inhibiting the aortic wall calcification in the glutaraldehyde-crosslinked allografts. It is concluded that valved aortic allografts in rats provide a useful model for investigating aortic wall (but not valve cusp) calcification and its inhibition.
Aging↗