Biomedical subjects

Y Qiu

Publications and source records attributed to Y Qiu.

At least 91 records · Page 5Linked to original sources

PubMed2000

[Molecular cloning of a novel gene located on chromosome 3p25.3 and an analysis of its expression in nasopharyngeal carcinoma].

OBJECTIVE: To obtain the novel genes associated with human nasopharyngeal carcinoma(NPC) on chromosome 3p24-26. METHODS: Twenty epithelial-derived expressed sequence tags(EST) were selected from chromosome 3p24-26 where loss of heterozygosity(LOH) frequently occurs in NPC tissues. Primers were designed based on the sequences of these ESTs. RT-PCR was used to amply their corresponding cDNA fragments from NPC cell line HNE1 and primary cultures of normal nasopharyngeal epithelial cells. The differential expression of two ESTs, T93093 and R41598, was confirmed by Northern blot. Then, expression of EST T93093 was further detected in 7 normal nasopharyngeal and 19 NPC biopsies. cDNA library screening was used to get its full cDNA sequence and the sequence of this novel gene was analyzed by bioinformatics. RESULTS: Thirteen ESTs(T62511, N39155, N68660, R61275, T95314, R06143, H52697, H66521, AA128685, AA284537, N52379, AA054180, and H98090) showed the similar expression level and 5 ESTs(R00732, R07573, R98052, H91759, H17566) showed no expression in both types of cells. EST T93093 was down-expressed, whereas EST R41598 up-expressed in NPC HNE1 cells. The EST T93093 was also found to be down-expressed in 26.3%(5/19) of NPC biopsies. The full length cDNA of this gene was obtained and named NAG-7, which is located at chromosome 3p25.3. Its 1677 bp full length cDNA has a potential open reading frame(ORF) predicting a 94 amino acid protein with a molecular weight of 11023.87 Dalton. Bioinformatics analysis of the NAG-7 gene shows that it is a transmembrane protein containing a protein kinase C(PKC) phosphorylation site and a myristyl site. It has no significant homology to any reported genes in database of GenBank(AF086709). CONCLUSION: NAG-7 is a novel gene down-expressed in NPC, which may be involved in the development of NPC.

Chromosome Mapping↗

PubMed2000

A cDNA located on chromosome 7q32 shows loss of expression in epithelial cell line of nasopharyngeal carcinoma.

OBJECTIVE: To isolate and clone the tumor suppressor gene on chromosomal region 7q32 correlated with the carcinogenesis of human nasopharyngeal carcinoma (NPC). METHODS: The genotypes of polymorphic microsatellite markers on 7q32 in DNA from 24 biopsies of nasopharyngeal carcinoma and matched normal blood cells were identified. The expression levels of 20 expressed sequence tags (ESTs) on 7q32 between human nasopharyngeal carcinoma epithelial 1 (HNE1) and primary cultures of normal nasopharyngeal epithelial (PNNE) cells were compared using differential RT-PCR and Northern hybridization. The quantity of AA070437 DNA and mRNA was detected by differential PCR and differential RT-PCR, respectively. RESULTS: Loss of heterozygosity (LOH) was found in 25%-46% of NPC biopsies. AA070437 EST expression was down-regulated in HNE1 cell compared to PNNE cells. The down-regulation of AA070437 was found in 30.7% of NPC biopsies and allelic loss of AA070437 was observed in 29.1% of NPC biopsies. CONCLUSION: Our results show that AA070437 EST is negatively related with the occurrence of human NPC and may represent a candidate tumor suppressor gene of NPC on 7q32.

Chromosomes, Human, Pair 7↗

PubMed2000

[Cytokeratin 13 gene expression in human nasopharyngeal carcinoma].

OBJECTIVE: To investigate the possible pathogenetic role of cytokeratin 13 (CK13) gene expression in human nasopharyngeal carcinoma (NPC). METHODS: Tissue samples taken from a total of 40 cases of NPC and 8 cases of chronic inflammation of nasopharyngeal epithelium (CINE) were immunohistochemically studied for CK13 protein expression. Northern blot hybridization was used to detect CK13 gene at mRNA level in 32 of the 40 NPC and in 8 CINE cases. Correlations of CK13 expression with the clinical features of NPC were also investigated. RESULTS: Significantly stronger immunoreactivity for CK13 protein was shown in CINE than in NPC tissues (P < 0.01), so did it at mRNA levels. Significantly higher CK13 expression was demonstrated in patients with cervical lymph node metastasis, as compared to those without (P < 0.05). However, no significant correlation between CK13 expression and clinical staging of the disease. CONCLUSION: The decreased expression of CK13 in NPC suggests that differentiation disturbance of the nasopharyngeal epithelium might play a role in the pathogenesis of NPC.

Adolescent↗

PubMed2000

[Microsurgical treatment of large or giant aneurysms in anterior circle of Willis].

OBJECTIVE: To improve the operative results in large or giant aneurysms of the anterior circle Willis. METHODS: Retrospective analysis was performed on 28 patients with large or giant aneurysms of the anterior circle Willis after operation. Three-D CT angiography was used to detect and delineate aneurysm preoperatively. Improved operative technique included cranial base approach, temporary artery occlusion, intra-aneurysm decompression, obliteration of aneurysm with multiple clips, stepwise coagulation clipping and resection of aneurysms. RESULTS: Good results were noted in 24 patients (85.7%), fair in 2, and poor in 1. One patient died, but no patient with Hunt and Hess 0-III grade died. CONCLUSIONS: Three-D CT angiography is useful in delineating the shape, size, neck of aneurysms, and their relation to near arteries and to cranial base structures. These are helpful to improve operative results of large or giant aneurysms of the anterior circle of Willis.

Adult↗

PubMed2000

[Sagittal profile restoration of thoracolumbar fracture with rod rotation and in situ bending technique].

OBJECTIVE: To restore the sagittal profile at the time of regaining vertebral height during surgery of thoracolumbar fracture by rotation, screw-hook combination and in situ bending technique. METHODS: In 48 patients who have been treated since 1997, 41 were male and 7 female. Their age ranged from 17 to 54 years. Surgical procedures were (L(1) fracture for example): CD or TSRH pedicle screws inserted in T(12) and L(2), offset laminar hooks placed on T(11) and L(2), and two rods contoured according to the normal sagittal profile of the instrumental segment and inserted into the pedicle screws with the rod contouring curve in the frontal plane. Distraction force was used to regain vertebral height. The rods were rotated by 90 degrees and fixed with the set screws. The contouring curve of the rod at this time was transferred from the frontal plane to the sagittal plane where the normal sagittal profile was restored. The offset laminar hooks were compressed toward the screws. If lordosis was not satisfactory, the rods could be bent in situ. Posterolateral bone grafting was finally performed. RESULTS: No death and no neurological worsening were noted except deep infection in one patient. Average vertebral height was restored by 96%. 39 patients had complete restoration of vertebral height. Sagittal kyphotic Cobb angle was corrected from average 24 degrees before operation to average -2 degrees after operation. Follow-up (20 months on average) showed no pseudarthrosis but two screws broken in one patient. CONCLUSIONS: Rod rotation and in situ rod bending technique can satisfactorily restore the normal sagittal profile and regain vertebral height. Offset laminar hooks can effectively protect pedicle screws, and minimise mechanical failure of instrumentation.

Adolescent↗

PubMed2000

[Endoscopy-assisted neurosurgery for intracranial operation].

OBJECTIVE: To investigate the value of endoscope-assisted neurosurgery for intracranial operation. METHODS: 37 patients with intracranial lesions including tumors, aneurysms, cysts, hematomas, hydrocephalus and nerve-vessel compression syndrome were treated with this method. Their results were compared to those of microsurgery and macrosurgery. The items analyzed included exposure and protection of brain tissue and cranial nerves, views of operation field, length of procedure etc. RESULTS: 15 patients with tumors, showed a high rate of complete removal but less complications. The 6th nerves of CPA tumors operation were preserved. Six cases of nerve-vessel compression syndrome showed excellent therapeutic effects. In 8 cases of intraventricle lesions, using endoscope easily found tumor location and the cause of hydrocephalus. As to saccular diseases, endoscope was used to deal with the major location. In 4 cases of hematomas, less blot remained and the procedure shortened. Moreover the operation field was more clear and adequate on the expenses of no more complexity of the procedure. CONCLUSIONS: Endoscope-assisted microsurgery is helpful in intracranial operation. It might become a standard neuro-operation in near future.

Adolescent↗

PubMed2000

[Antigen loading on dendritic cells affects the cell function in stimulating T cells].

OBJECTIVE: To study the effect of antigen loading on dendritic cells (DC). METHODS: DCs collected from peripheral blood monocytes were loaded with a tumor antigen from XG-7 cell line. These DCs were then co-cultured with allogeneic T cells and were compared with those DCs without antigen exposure. RESULTS: Although DCs showed no change in their phenotypes after cultured with the antigen, they secreted more IL-12, and became more powerful in allogenic mixed lymphocyte reaction (MLR). Antigen-loaded DC stimulated more CD(4)(+) cells to proliferate than nonantigen-loaded DCs did. These CD(4)(+) cells did not kill XG-7 cells, but promoted CD(8)(+) cells' ability to inhibit the XG-7 proliferation. CONCLUSION: The proliferation of CD(4)(+) cells after cultured with DC may become an indicator for the function of antigen loaded DC and for the efficiency of DC immunotherapy.

Antigens, Neoplasm↗

PubMed1999

The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO synthase gene.

The goal of this study was to interrogate the role of inducible NO synthase (iNOS) in the late phase of ischemic preconditioning (PC) in vivo. A total of 321 mice were used. Wild-type mice preconditioned 24 h earlier with six cycles of 4-min coronary occlusion/4-min reperfusion exhibited a significant (P < 0.05) increase in myocardial iNOS protein content, iNOS activity (assessed as calcium-independent L-citrulline formation), and nitrite + nitrate tissue levels. In contrast, endothelial NOS protein content and calcium-dependent NOS activity remained unchanged. No immunoreactive neuronal NOS was detected. When wild-type mice were preconditioned 24 h earlier with six 4-min occlusion/4-min reperfusion cycles, the size of the infarcts produced by a 30-min coronary occlusion followed by 24 h of reperfusion was reduced markedly (by 67%; P < 0.05) compared with sham-preconditioned controls, indicating a late PC effect. In contrast, when mice homozygous for a null iNOS allele were preconditioned 24 h earlier with the same protocol, infarct size was not reduced. Disruption of the iNOS gene had no effect on early PC or on infarct size in the absence of PC. These results demonstrate that (i) the late phase of ischemic PC is associated with selective up-regulation of iNOS, and (ii) targeted disruption of the iNOS gene completely abrogates the infarct-sparing effect of late PC (but not of early PC), providing unequivocal molecular genetic evidence for an obligatory role of iNOS in the cardioprotection afforded by the late phase of ischemic PC. Thus, this study identifies a specific protein that mediates late PC in vivo.

Animals↗

PubMed1999

p300 functions as a transcriptional coactivator for the TAL1/SCL oncoprotein.

Activation of the TAL1 (or SCL) gene, originally identified through its involvement by a recurrent chromosomal translocation, is the most frequent gain-of-function mutation recognized in T-cell acute lymphoblastic leukemia (T-ALL). The TAL1 proteins contain a basic helix - loop - helix (bHLH) motif characteristic of a large family of transcription factors that control transcription from an E box target element as heterodimers with the E2A- and HEB-encoded gene products. Gene knockout studies in mice indicate that this transcription factor is required for embryonic and adult hematopoiesis, and considerable evidence suggests it has specific functions in terminal erythroid differentiation. We investigated whether the broadly expressed nuclear protein p300, known to function as a coactivator for other bHLH proteins involved in cellular differentiation, also interacts with TAL1. p300 was found to coimmunoprecipitate with Tal1 in extracts from murine erythroleukemia (MEL) cells induced to differentiate with dimethylsulfoxide (DMSO), and p300 and Tal1 were observed in a common E box DNA-binding complex in extracts from differentiating MEL cells. p300 also interacted with Tal1 in protein pulldown assays, suggesting this was a direct interaction. Finally, p300 augmented transcription by Tal1 from an E box-containing promoter and by a GAL4-Tal1 fusion from a promoter containing the GAL4 DNA-binding element. Deletion analysis identified the bHLH domain of Tal1 and amino-terminal sequences of p300 as necessary for p300-stimulated transactivation and Tal1-p300 interaction in vitro. These results indicate that recruitment of the transcriptional coactivator p300 can positively regulate TAL1-directed gene expression. The dependence of their interaction in MEL cells on addition of a differentiation inducer suggests, further, that this TAL1-p300 complex may have an important role in terminal erythroid differentiation.

3T3 Cells↗

PubMed1999

Methotrexate inhibits the first committed step of purine biosynthesis in mitogen-stimulated human T-lymphocytes: a metabolic basis for efficacy in rheumatoid arthritis?

The immunosuppressive and anti-inflammatory effects of low-dose methotrexate (MTX) have been related directly to inhibition of folate-dependent enzymes by polyglutamated derivatives, or indirectly to adenosine release and/or apoptosis and clonal deletion of activated peripheral blood lymphocytes in S-phase. In this study of phytohaemagglutinin-stimulated primary human T-lymphocytes we show that MTX (20 nM to 20 microM) was cytostatic not cytotoxic, halting proliferation at G(1). This stasis of blastogenesis was associated with an inhibition of purine ribonucleotide synthesis but a stimulation of pyrimidine biosynthesis, the normal mitogen-induced expansion of ATP and GTP pools over 72 h being restricted to concentrations of unstimulated T-cells, whereas the increment in UTP pools exceeded that of controls. Decreased incorporation of H(14)CO(3) or [(14)C]glycine into purine ribonucleotides, with no radiolabel accumulation in any de novo synthetic intermediate but enhanced H(14)CO(3) incorporation into UTP, supported these MTX-related effects. Exaggerated [(14)C]hypoxanthine salvage (which normalized the purine and UTP pools) confirmed the increased availability of 5-phosphoribosyl-1-pyrophosphate (PP-ribose-P) as the molecular mechanism underlying these disparate changes. These results provide the first substantive evidence that the immunosuppressive effects of low-dose MTX in primary blasting human T-lymphocytes relate not to the inhibition of the two folate-dependent enzymes of purine biosynthesis but to inhibition of the first enzyme, amidophosphoribosyltransferase, thereby elevating PP-ribose-P and stimulating UTP synthesis. Varying cell types or incubation conditions employed by other workers, especially malignant/activated cells with high basal metabolic rates, might mask the effects noted in primary human T-lymphocytes. The findings imply the involvement of low-dose MTX in the inhibition of T-lymphocyte proliferation and proliferation-dependent processes in rheumatoid arthritis.

Amidophosphoribosyltransferase↗

PubMed1999

Permeability and absorption of leuprolide from various intestinal regions in rabbits and rats.

The in vitro permeability and in vivo absorption of leuprolide in different intestinal regions were measured to investigate the feasibility for site-specific delivery of leuprolide in the gastrointestinal (GI) tract. In vitro permeability of leuprolide in the rabbit GI tract was performed using a side-by-side diffusion apparatus and the permeability coefficients in the jejunum, ileum and colon were 0.27x10(-7), 2.96x10(-7) and 7.85x10(-7)cm/s, respectively. Varying the donor drug concentrations from 2 to 10 mg/ml, the permeability coefficients were independent of the donor concentration, suggesting the transport mechanism of passive diffusion. Using an intestine loop model in anesthetized rats, bioavailabilities of leuprolide in the jejunum, ileum and colon were 1.28, 5.62 and 9.59%, respectively. Drug recovery from the loop 5 h after dosing was 10.7% in jejunum, 24.5% in ileum and 40.7% in colon. Additional in vivo studies using conscious rats showed that the bioavailability of leuprolide was less than 1% for both ileal and colonic administration. In vivo absorption of leuprolide from ileum was not significantly different from colon in conscious rats. Sodium salicylate, a permeation enhancer, was co-administered with leuprolide to the rat ascending colon, and results showed a 4-fold increase in the bioavailability in conscious rats. Thus, in vivo studies indicate that both absorption and enzymatic degradation of leuprolide in the GI tract is site-dependent and the lower intestine may be an advantageous region for oral delivery of leuprolide.

Animals↗

PubMed1999

[Study on etiology of acute hemorrhagic conjunctivitis in Qingdao during 1997].

OBJECTIVE: To study the etiology of acute hemorrhagic conjunctivitis (AHC) in Qingdao during 1997. METHODS: During the AHC epidemic season viruses from the conjunctival swabs were isolated in Hela and Hep-2 cell cultures and were identified by neutralization test. RESULTS: All isolated virus strains were identified as (CA24) v and adenoviruses (Adv) by neutralization test with antisera of CA24 v, Echo virus 70(EV70), CA24, CA24 v of Beijing local strain, Ad3, Ad7 and Ad11. CONCLUSION: In 1997, a small epidemic of acute hemorrhagic conjunctivitis was caused by adenoviuses and CA24 v.

Conjunctivitis, Acute Hemorrhagic↗

PubMed1999

Etk/Bmx, a PH-domain containing tyrosine kinase, protects prostate cancer cells from apoptosis induced by photodynamic therapy or thapsigargin.

Prostate carcinoma (PCA) is the most frequently diagnosed malignancy in American men. PCA at advanced stages can both proliferate abnormally and resist apoptosis. Among the many known signal transduction pathways, phosphatidylinositide-3'OH kinase (PI3-kinase) has been shown to play an important role in cell survival and resistance to apoptosis. In this study, we investigate the involvement of Etk/Bmx, a newly discovered tyrosine kinase that is a substrate of PI3-kinase, in protection of prostate cancer cells from apoptosis. Parental LNCaP cells and two derivative cell lines, one overexpressing wild type Etk (Etkwt) and the other expressing a dominant negative Etk (EtkDN), were used to study the function of Etk. The cells were treated with photodynamic therapy (PDT), a newly approved cancer treatment which employs a photosensitizer and visible light to produce an oxidative stress in cells, often leading to apoptosis. Our results indicate that PDT induces apoptosis in LNCaP cells, as measured by DNA fragmentation and by cleavage of poly(ADP-ribose) polymerase (PARP), and moreover, the extent of apoptosis was much reduced in Etkwt cells as compared to LNCaP or EtkDN cells. Assay of overall cell viability confirmed that Etkwt cells were considerably less sensitive to PDT than were the parental LNCaP or EtkDN cells. Similar results were found in response to thapsigargin (TG). A specific inhibitor of PI3-kinase, LY294002, abolished Etk activity and markedly increased TG-induced PARP cleavage. The results suggest that Etk/Bmx is an efficient effector of PI3-kinase and that the newly described PI3-kinase/Etk pathway is involved in the protection of prostate carcinoma cells from apoptosis in response to PDT or TG.

Apoptosis↗

PubMed1999

Amplification and overexpression of the hepatocyte growth factor receptor (HGFR/MET) in rat DMBA sarcomas.

In the present study subcutaneous fibrosarcomas were induced by the carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) in rats from F1 generation cross breedings of two different inbred strains. Comparative genomic hybridization (CGH) analysis, which allows detection of DNA sequence copy changes, was applied to one of the tumors and it was found that there were increased copy numbers of sequences at chromosome 4q12-q21 in this tumor. We have previously determined that the loci for the hepatocyte growth factor (Hgf) and hepatocyte growth factor receptor (Hgfr/Met), a protooncogene, are situated in this particular chromosome region. Using probes for the two genes in FISH (fluorescence in situ hybridization) and in Southern blots we found that the Hgfr/Met gene was amplified in five of the 19 sarcomas studied, and that the Hgf gene was coamplified in two of them. Northern and Western blots and tyrosine phosphorylation analysis showed that the HGF receptor was overexpressed and functional in all five tumors, as well as in two additional tumors. In summary, both amplification and overexpression of the Hgfr/Met gene was found in about 25% of DMBA-induced experimental rat sarcomas, and HGF receptor overexpression alone was seen in two additional tumors. Possibly this reflects an involvement in paracrine or autocrine stimulation of growth and invasiveness by HGF. Our finding could provide a rodent model system to increased knowledge about causality and therapy, which may be applicable to the sizeable fraction of human musculoskeletal tumors displaying MET overexpression.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1999

Nuclear factor-kappaB plays an essential role in the late phase of ischemic preconditioning in conscious rabbits.

Although it is recognized that late preconditioning (PC) results from upregulation of cardioprotective genes, the specific transcription factor(s) that govern this genetic adaptation remains unknown. The aim of this study was to test the hypothesis that the development of late PC is mediated by nuclear factor-kappaB (NF-kappaB) and to elucidate the mechanisms that control the activation of NF-kappaB after an ischemic stimulus in vivo. A total of 152 chronically instrumented, conscious rabbits were used. A sequence of six 4-minute coronary occlusion/4-minute reperfusion cycles, which elicits late PC, induced rapid activation of NF-kappaB, as evidenced by a marked increase in p65 content (+164%; Western immunoblotting) and NF-kappaB DNA binding activity (+306%; electrophoretic mobility shift assay) in nuclear extracts isolated 30 minutes after the last reperfusion. These changes were attenuated 2 hours after ischemic PC and resolved by 4 hours. Competition and supershift assays confirmed the specificity of the NF-kappaB DNA complex signals. The mobility of the NF-kappaB DNA complex was shifted by anti-p65 and anti-p50 antibodies but not by anti-c-Rel antibodies, indicating that the subunits of NF-kappaB involved in gene activation after ischemic PC consist of p65-p50 heterodimers. Pretreatment with the NF-kappaB inhibitor diethyldithiocarbamate (DDTC; 150 mg/kg IP 15 minutes before ischemic PC) completely blocked the nuclear translocation and increased DNA binding activity of NF-kappaB. The same dose of DDTC completely blocked the cardioprotective effects of late PC against both myocardial stunning and myocardial infarction, indicating that NF-kappaB activation is essential for the development of this phenomenon in vivo. The ischemic PC-induced activation of NF-kappaB was also blocked by pretreatment with Nomega-nitro-L-arginine (L-NA), a nitric oxide synthase (NOS) inhibitor, N-2-mercaptopropionyl glycine (MPG), a reactive oxygen species (ROS) scavenger, chelerythrine, a protein kinase C (PKC) inhibitor, and lavendustin A, a tyrosine kinase inhibitor (all given at doses previously shown to block late PC), indicating that ischemic PC activates NF-kappaB via formation of NO and ROS and activation of PKC- and tyrosine kinase-dependent signaling pathways. A subcellular redistribution and increased DNA binding activity of NF-kappaB quantitatively similar to those induced by ischemic PC could be reproduced pharmacologically by giving the NO donor diethylenetriamine/NO (DETA/NO) (at a dose previously shown to elicit late PC), demonstrating that NO in itself can activate NF-kappaB in the heart. Taken together, these results provide direct evidence that activation of NF-kappaB is a critical step in the signal transduction pathway that underlies the development of the late phase of ischemic PC in conscious rabbits. The finding that four different pharmacological manipulations (L-NA, MPG, chelerythrine, and lavendustin A) produced similar inhibition of NF-kappaB suggests that this transcription factor is a common downstream pathway through which multiple signals elicited by ischemic stress (NO, ROS, PKC, tyrosine kinases) act to induce gene expression. To our knowledge, this is the first demonstration that NO can promote NF-kappaB activation in the heart, a finding that identifies a new biological function of NO and may have important implications for various pathophysiological conditions in which NO is involved and for nitrate therapy.

Alkaloids↗

PubMed1999

The orphan nuclear receptor COUP-TFII is required for angiogenesis and heart development.

The embryonic expression of COUP-TFII, an orphan nuclear receptor, suggests that it may participate in mesenchymal-epithelial interactions required for organogenesis. Targeted deletion of the COUP-TFII gene results in embryonic lethality with defects in angiogenesis and heart development. COUP-TFII mutants are defective in remodeling the primitive capillary plexus into large and small microcapillaries. In the COUP-TFII mutant heart, the atria and sinus venosus fail to develop past the primitive tube stage. Reciprocal interactions between the endothelium and the mesenchyme in the vascular system and heart are essential for normal development of these systems. In fact, the expression of Angiopoietin-1, a proangiogenic soluble factor thought to mediate the mesenchymal-endothelial interactions during heart development and vascular remodeling, is down-regulated in COUP-TFII mutants. This down-regulation suggests that COUP-TFII may be required for bidirectional signaling between the endothelial and mesenchymal compartments essential for proper angiogenesis and heart development.

Animals↗

PubMed1999

Isoform-selective activation of protein kinase C by nitric oxide in the heart of conscious rabbits: a signaling mechanism for both nitric oxide-induced and ischemia-induced preconditioning.

Although isoform-selective translocation of protein kinase C (PKC) epsilon appears to play an important role in the late phase of ischemic preconditioning (PC), the mechanism(s) responsible for such translocation remains unclear. Furthermore, the signaling pathway that leads to the development of late PC after exogenous administration of NO in the absence of ischemia (NO donor-induced late PC) is unknown. In the present study we tested the hypothesis that NO activates PKC and that this is the mechanism for the development of both ischemia-induced and NO donor-induced late PC. A total of 95 chronically instrumented, conscious rabbits were used. In rabbits subjected to ischemic PC (six 4-minute occlusion/4-minute reperfusion cycles), administration of the NO synthase inhibitor Nomega-nitro-L-arginine (group III), at doses previously shown to block the development of late PC, completely blocked the ischemic PC-induced translocation of PKCepsilon but not of PKCeta, indicating that increased formation of NO is an essential mechanism whereby brief ischemia activates the epsilon isoform of PKC. Conversely, a translocation of PKCepsilon and -eta quantitatively similar to that induced by ischemic PC could be reproduced pharmacologically with the administration of 2 structurally unrelated NO donors, diethylenetriamine/NO (DETA/NO) and S-nitroso-N-acetylpenicillamine (SNAP), at doses previously shown to elicit a late PC effect. The particulate fraction of PKCepsilon increased from 35+/-2% of total in the control group (group I) to 60+/-1% after ischemic PC (group II) (P<0.05), to 54+/-2% after SNAP (group IV) (P<0.05) and to 52+/-2% after DETA/NO (group V) (P<0.05). The particulate fraction of PKCeta rose from 66+/-5% in the control group to 86+/-3% after ischemic PC (P<0.05), to 88+/-2% after SNAP (P<0.05) and to 85+/-1% after DETA/NO (P<0.05). Neither ischemic PC nor NO donors had any appreciable effect on the subcellular distribution of PKCalpha, -beta1, -beta2, -gamma, -delta, - micro, or -iota/lambda; on total PKC activity; or on the subcellular distribution of total PKC activity. Thus, the effects of SNAP and DETA/NO on PKC closely resembled those of ischemic PC. The DETA/NO-induced translocation of PKCepsilon (but not that of PKCeta) was completely prevented by the administration of the PKC inhibitor chelerythrine at a dose of 5 mg/kg (group VI) (particulate fraction of PKCepsilon, 38+/-4% of total, P<0.05 versus group V; particulate fraction of PKCeta, 79+/-2% of total). The same dose of chelerythrine completely prevented the DETA/NO-induced late PC effect against both myocardial stunning (groups VII through X) and myocardial infarction (groups XI through XV), indicating that NO donors induce late PC by activating PKC and that among the 10 isozymes of PKC expressed in the rabbit heart, the epsilon isotype is specifically involved in the development of this form of pharmacological PC. In all groups examined (groups I through VI), the changes in the subcellular distribution of PKCepsilon protein were associated with parallel changes in PKCepsilon isoform-selective activity, whereas total PKC activity was not significantly altered. Taken together, the results provide direct evidence that isoform-selective activation of PKCepsilon is a critical step in the signaling pathway whereby NO initiates the development of a late PC effect both after an ischemic stimulus (endogenous NO) and after treatment with NO-releasing agents (exogenous NO). To our knowledge, this is also the first report that NO can activate PKC in the heart. The finding that NO can promote isoform-specific activation of PKC identifies a new biological function of this radical and a new mechanism in the signaling cascade of ischemic PC and may also have important implications for other pathophysiological conditions in which NO is involved and for nitrate therapy.

Alkaloids↗

PubMed1999

Sublingual absorption of leuprolide: comparison between human and animal models.

Leuprolide is a potent luteinizing hormone releasing hormone agonist used for the treatment of hormone-dependent diseases. It is a decapeptide drug currently administered by subcutaneous and intramuscular injection because it is not orally bioavailable. In the present study, sublingual gel formulations of leuprolide were administered to dogs, monkeys and humans. Plasma samples were analyzed by radioimmunoassay. Absorption and pharmacokinetics of leuprolide following sublingual administration were compared and evaluated. It was found that the extent and rate of absorption were similar between humans and monkeys following sublingual dosing of leuprolide formulations. A prolonged absorption of up to approximately 6 h after dosing was observed in both humans and monkeys. The rate and extent of absorption were significantly higher in dogs than in humans. The estimate of absolute bioavailability of leuprolide was 46.7% in dogs compared with 2.7% in monkeys at an equivalent dose of 0.45 mg/kg. Absolute bioavailabilities in humans were 2.0, 2.3 and 2.4% at doses of 1.125, 2.25 and 4.5 mg, respectively. Based on these results, the dog is not an appropriate animal model for evaluating sublingual absorption of leuprolide. This work suggests that monkey is a preferred model for the development and assessment of sublingual formulations of leuprolide.

Administration, Sublingual↗