Biomedical subjects

X Ye

Publications and source records attributed to X Ye.

At least 37 records · Page 2Linked to original sources

PubMed2001

Evaluation of neurodegeneration in scrapie-infected animals by selective methods that detect cellular degeneration.

Scrapie is a fatal neurodegenerative disease of sheep and goats. The precise details of neuronal and neurite degeneration in scrapie-infected animals remain unknown. Using specific silver staining methods, we compared the neurodegeneration caused by treatment of rats with kainic acid (KA) or ibogaine (IBO) to the neuropathology observed in mice infected with the C602 strain of scrapie. As reported previously, KA resulted in extensive silver labeling of neurons, especially in the cortex, putamen and hippocampus. IBO silver labeling was observed only in small clusters of Purkinje neurons in the paravermal region of the cerebellum. However, in scrapie-infected mice, a few silver stained neurons (differing from the dark degenerating neurons observed following neurotoxic exposure) were found in layer II of cortex, cingulate cortex, zona incerta, thalamus and hypothalamus. Some silver grains were observed in glial-like cells, especially those in the paraventricular region. Degenerating axons were positive for silver staining and were found in the cortex, cingulate cortex, corpus callosum, habenulae, septum, fornix, thalamus, caudate putamen and a few in fasciculus retroflexus and substantia nigra. Our results suggest that the limbic system is one of the important loci for the neurodegenerative effect of at least some scrapie strains.

Animals↗

PubMed2001

Selective loss of sphingosine 1-phosphate signaling with no obvious phenotypic abnormality in mice lacking its G protein-coupled receptor, LP(B3)/EDG-3.

Sphingosine 1-phosphate (S1P) exerts diverse physiological actions by activating its cognate G protein-coupled receptors. Five S1P receptors have been identified in mammals: LP(B1)/EDG-1, LP(B2)/H218/AGR16/EDG-5, LP(B3)/EDG-3, LP(B4)/NRG-1/EDG-8, and LP(C1)/EDG-6. One of these receptors, LP(B1), has recently been shown to be essential for mouse embryonic development. Here we disrupted the lp(B3) gene in mice, resulting in the complete absence of lp(B3) gene, transcript, and LP(B3) protein. LP(B3)-null mice were viable and fertile and developed normally with no obvious phenotypic abnormality. We prepared mouse embryonic fibroblast (MEF) cells to examine effects of LP(B3) deletion on S1P-induced signal transduction pathways. Wild-type MEF cells expressed lp(B1), lp(B2), and lp(B3) but neither lp(B4) nor lp(C1), and they were highly responsive to S1P in phospholipase C (PLC) activation, adenylyl cyclase inhibition, and Rho activation. Identically prepared LP(B3)-null MEF cells showed significant decreases in PLC activation, slight decreases in adenylyl cyclase inhibition, and no change in Rho activation. Retrovirus-mediated rescue of the LP(B3) receptor in LP(B3)-null MEF cells restored S1P-dependent PLC activation and adenylyl cyclase inhibition. These results indicate a nonessential role for LP(B3) in normal development of mouse but show nonredundant cellular signaling mediated by a single type of S1P receptor.

Adenylyl Cyclase Inhibitors↗

PubMed2001

Differences in the human and mouse amino-terminal leader peptides of ornithine transcarbamylase affect mitochondrial import and efficacy of adenoviral vectors.

Mouse models of ornithine transcarbamylase (OTC) deficiency are being used to test the efficacy of viral vectors as possible vehicles for gene therapy. However, it has been demonstrated that virus containing the human OTC cDNA failed to express functional OTC enzyme in the recipient animals. Because functional OTC is assembled as a homotrimer in the mitochondria, there are at least two possible explanations for these results. Either endogenous mutant protein coassembles with the human OTC and has a "dominant-negative effect," or the human version of the protein is not appropriately imported or processed in the mouse mitochondria. To test the importance of processing, which in rodents is thought to depend on the leader peptide, adenoviral vectors containing chimeric OTC cDNAs were prepared. These vectors were evaluated in the OTC-deficient sparse fur mouse models. Although comparable levels of transgene expression were observed in all groups of mice, the only mice that had high levels of OTC activity and mitochondrial OTC immunoreactivity were those mice injected with the vectors containing the mouse leader peptide (mouse OTC and a mouse-human chimera of OTC). To address possible dominant-negative effects, adenoviruses containing mutant human or mouse OTC cDNAs were prepared and evaluated in cell lines or normal C3H mice, respectively. No inhibition of normal OTC activity was observed in either model system. Together, these studies provide no evidence of a dominant-negative effect and suggest that the human and rodent enzymes responsible for transporting of OTC and possibly other mitochondrial proteins have different specificity.

Adenoviridae↗

PubMed2001

Myc represses the p21(WAF1/CIP1) promoter and interacts with Sp1/Sp3.

The cyclin-dependent kinase inhibitor p21((WAF1/CIP1)) inhibits proliferation both in vitro and in vivo, and overexpression of p21 in normal and tumor cell lines results in cell cycle arrest. In contrast, ectopic expression of Myc alleviates G(1) cell cycle arrest. Recent studies showed that Myc can repress p21 transcription, thereby overriding a p21-mediated cell cycle checkpoint. We found that activation of a Myc-estrogen receptor fusion protein by 4-hydroxytamoxifen in mouse cells resulted in suppression of endogenous p21 transcription. This effect was observed in the absence of de novo protein synthesis and was independent of histone deacetylase activity. In transient transfection studies, Myc effectively repressed p21 promoter constructs containing only 119 bp of sequence upstream of the transcription start site. This region contains multiple Sp1-binding sites and a potential initiator element, but no canonical Myc DNA-binding sites. Deletion of the potential initiator element does not affect repression of the p21 promoter by c-Myc. Coimmunoprecipitation and glutathione S-transferase pull-down experiments demonstrate that c-Myc may form complexes with Sp1/Sp3. We found that the central region of c-Myc interacts with the zinc finger domain of Sp1. Because Sp1 is required for p21 transcription, it is possible that Myc may down-regulate p21 transcription, at least in part, by sequestering Sp1. Repression of the p21 promoter may contribute to the ability of c-Myc to promote cell proliferation.

Animals↗

PubMed2001

The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activation.

The expression of DCC (deleted in colorectal cancer) is often markedly reduced in colorectal and other cancers. However, the rarity of point mutations identified in DCC coding sequences and the lack of a tumor predisposition phenotype in DCC hemizygous mice have raised questions about its role as a tumor suppressor. DCC also mediates axon guidance and functions as a dependence receptor; such receptors create cellular states of dependence on their respective ligands by inducing apoptosis when unoccupied by ligand. We now show that DCC drives cell death independently of both the mitochondria-dependent pathway and the death receptor/caspase-8 pathway. Moreover, we demonstrate that DCC interacts with both caspase-3 and caspase-9 and drives the activation of caspase-3 through caspase-9 without a requirement for cytochrome c or Apaf-1. Hence, DCC defines an additional pathway for the apoptosome-independent caspase activation.

Apoptosis↗

PubMed2001

A premature-termination mutation in the Mus musculus cyclin-dependent kinase 3 gene.

Our understanding of the mammalian cell cycle is due in large part to the analysis of cyclin-dependent kinase (CDK) 2 and CDK4/6. These kinases are regulated by E and D type cyclins, respectively, and coordinate the G(1)/S-phase transition. In contrast, little is known about CDK3, a homolog of CDK2 and cell division cycle kinase 2 (CDC2). Previous studies using ectopic expression of human CDK3 suggest a role for this kinase in the G(1)/S-phase transition, but analysis of the endogenous kinase has been stymied by the low levels of protein present in cells and by the absence of an identifiable cyclin partner. Herein we report the presence of a single point mutation in the CDK3 gene from several Mus musculus strains commonly used in the laboratory. This mutation results in the replacement of a conserved tryptophan (Trp-187) within kinase consensus domain IX with a stop codon. The protein predicted to be encoded by this allele is truncated near the T loop, which is involved in activation by CDK-activating kinase. This mutation also deletes motif XI known to be required for kinase function and is, therefore, expected to generate a null allele. In stark contrast, CDK3 from two wild-mice species (Mus spretus and Mus mus castaneus) lack this mutation. These data indicate that CDK3 is not required for M. musculus development and suggest that any functional role played by CDK3 in the G(1)/S-phase transition is likely to be redundant with another CDK.

Amino Acid Sequence↗

PubMed2001

Efficient gene transfer to rat renal glomeruli with recombinant adenoviral vectors.

Recombinant adenoviruses are attractive vectors for renal gene transfer since they can efficiently transduce nondividing cells. However, despite the fact that renal glomeruli are easily accessible via the renal circulation, attempts to deliver foreign genes specifically into renal glomeruli, using adenoviral vectors, have had limited success in rodents. A simple intraarterial injection of adenoviral vectors into the renal circulation or incubation of the virus with the kidney for an extended period of time was found to be insufficient for this purpose. In this study, we have established an efficient gene transfer protocol to express foreign genes in rat renal glomerular cells, using adenoviral vectors. We demonstrated, for the first time, that rat glomerular endothelial cells could be efficiently transduced by slowly infusing a recombinant adenovirus (Ad.CBlacZ) into the right renal artery for a period of 15 min. High levels of lacZ expression were achieved in renal glomeruli without causing significant damage to renal glomeruli or other kidney structures. The virus-mediated expression lasted for at least 21 days. These data demonstrate the feasibility of using recombinant adenoviral vectors as a tool with which to study the effect of foreign gene expression on the structure and function of rat renal glomeruli in vivo.

Adenoviridae↗

PubMed2001

Prospects for prenatal gene therapy in disorders causing mental retardation.

Advances in understanding the genetics and pathogenesis of disease and in prenatal diagnosis have lead to an exploration of ways to intervene earlier and earlier in the disease process. The possibility of prenatal gene therapy for severe genetic and developmental disorders has sparked new research and debate as to its feasibility, reliability, and ethics as a therapeutic option. Recent animal studies have demonstrated the feasibility of introducing a vector into the developing fetus. The optimal timing and best mode of delivery, however, have yet to be defined. Whether or not this research should be pursued also has been the subject of recent bioethical debates. There is additional concern with the possibility of in utero gene transfer inducing mutagenesis and subsequent tumor formation. This review will provide a summary of the current state of knowledge in the field of prenatal gene therapy and possible directions for the future research.

Animals↗

PubMed2001

Monitoring human serum transferrin by capillary zone electrophoresis with end-column amperometric detection.

Capillary zone electrophoresis was employed for the determination of human serum transferrin using end-column amperometric detection with a carbon fiber microelectrode at a constant potential of 1.9 V vs. saturated calomel electrode (SCE). The optimum conditions of separation and detection are 7.5 x 10(-4) mol/L Tris-3.44 x 10(-4) mol/L HCl for the buffer solution, 20 kV for the separation voltage, 5 kV and 10 s for the injection voltage and the injection time, respectively. The limit of detection is 6.7 x 10(-8) mol/L or 440 amol (S/N = 2). The relative standard deviations are 0.67% for the migration time and 1.5% for the electrophoretic peak current. The method was applied to the determination of transferrin in human serum. The recovery is between 93-104%.

Adult↗

PubMed2001

Biosynthesis of beta-carotene (provitamin A) in rice endosperm achieved by genetic engineering.

To obtain a functioning provitamin A (beta-carotene) biosynthetic pathway in rice endosperm, we introduced in a single, combined transformation effort the cDNAs coding for (1) phytoene synthase (psy) and (2) lycopene beta-cyclase (beta-lcy; both from Narcissus pseudonarcissus and both under control of the endosperm-specific glutelin promoter), with (3) a bacterial phytoene desaturase (crtI, from Erwinia uredovora under constitutive 35S promoter control). This combination covers the requirements for beta-carotene synthesis, and yellow, beta-carotene-bearing rice endosperm was obtained in the T0 generation. However, further experiments revealed that the presence of beta-lcy was not necessary, since psy and crtI alone were able to drive beta-carotene synthesis as well as the formation of further downstream xanthophylls. This finding could be explained if these downstream enzymes are either constitutively expressed in rice endosperm or are induced by the transformation, e.g. by products derived therefrom. Based on results in N. pseudonarcissus as a model system, a likely hypothesis can be developed that trans lycopene or a trans lycopene derivative acts as an inductor in a kind of feedback mechanism stimulating endogenous carotenogenic genes.

Alkyl and Aryl Transferases↗

PubMed2001

Long-term and therapeutic-level hepatic gene expression of human factor IX after naked plasmid transfer in vivo.

Naked DNA transfer of a high-expressing human factor IX (hFIX) plasmid yielded long-term (over 1 1/2 years) and therapeutic-level (0.5-2 microg/ml) gene expression of hFIX from mouse livers. The expression cassette contained a hepatic locus control region from the ApoE gene locus, an alpha1-anti-trypsin promoter, hFIX cDNA, a portion of the hFIX first intron, and a bovine growth hormone polyadenylation signal. In contrast, a hFIX plasmid containing the expression cassette without effective regulatory elements produced initially low-level gene expression that rapidly declined to undetectable levels. Southern analyses of the cellular DNA indicated that the majority of the input genome from either vector persisted as episomal forms of the original plasmids. Together with RT-PCR analyses of the transcripts, these data indicated that at least two processes are critical for sustained gene expression: persistence of vector DNA and transcriptional/posttranscriptional activation. Liver regeneration after partial hepatectomy resulted in a significant decline in transgene expression, further suggestive of decreased episomal plasmid maintenance rather than transgene integration. Transaminase levels and liver histology showed that rapid intravenous plasmid injection into mice induced transient focal acute liver damage (< 5% of hepatocytes), which was rapidly repaired within 3 to 10 days and resulted thereafter in histologically normal tissue. No significant differences were observed between rapid injection of plasmid and saline control solutions. Transient, very low level antibodies directed against hFIX did not prevent the circulation of therapeutic levels of the protein. Gene transfer of hFIX plasmid DNA into liver elicited neither transgene-specific cytotoxic effect nor long-term toxicity. These results demonstrate that long-term expression of hFIX can be achieved by nonviral plasmid transfer and suggest that this occurs independent of integration.

Animals↗

PubMed2001

Synthesis and pharmacology of site specific cocaine abuse treatment agents: a new synthetic methodology for methylphenidate analogs based on the Blaise reaction.

In order to make new analogs of the dopamine (DA) uptake inhibitor methylphenidate, a synthetic methodology based on the Blaise reaction was developed. The reaction between alpha-bromophenylacetic acid esters, zinc and alpha-cyano-omega-mesylates gave stable primary enamines. After reduction of the enamines with cyanoborohydride, the amines could be cyclized to methylphenidate analogs in which the amine ring size and aromatic ring were varied. These compounds were tested for inhibitory potency against [(3)H]WIN 35,428 binding to the cocaine recognition site and [(3)H]DA uptake using rat striatal tissue. When the heterocyclic ring size was varied, the six-membered ring of methylphenidate appeared to be the optimum ring size. When the aryl ring was varied the 4-trifluoromethylphenyl analog was less potent than methylphenidate, the beta-naphthyl congener was considerably more potent, whereas the alpha-naphthyl congener was less potent. Most of the compounds tested had ratios of uptake to binding inhibition (discrimination ratio) that were similar to cocaine and were therefore not lead compounds for the development of cocaine antagonists.

Animals↗

PubMed2001

Isolation of lectin and albumin from Pisum sativum var. macrocarpon ser. cv. sugar snap.

A mannose- and glucose-binding lectin bearing considerable sequence similarity to other legume lectins was isolated using a simple procedure, from legumes of the sugar snap Pisum sativum var. macrocarpon. The lectin was unadsorbed on Affi-gel blue gel and Q-Sepharose in 10 mM Tris-HCl buffer (pH 7.2) and adsorbed on SP-Toyopearl in 50 mM NaOAc buffer (pH 5). An albumin could also be purified at the same time. It was unadsorbed on Affi-gel Blue gel, adsorbed on Q-Sepharose and unadsorbed on SP-Toyopearl under the aforementioned chromatographic conditions. The lectin was almost identical in N-terminal sequences of its alpha- and beta-subunit to lectin from P. sativum L. var. Feltham First except for the 19th N-terminal residue of the beta-subunit. The lectin was devoid of antifungal activity. Out of the 15 N-terminal amino acids examined in pea albumin, three were different between the two varieties of P. sativum.

Albumins↗

PubMed2001

Fluoro-Jade and silver methods: application to the neuropathology of scrapie, a transmissible spongiform encephalopathy.

Traditional methods for evaluating neurodegeneration include variations of Nauta's selective silver-staining techniques. The Fluoro-Jade (FJ) method applies a novel fluorescent, anionic stain for localizing degenerating neurons. FJ has produced comparable results to the silver methods, when both have been applied to detect neurodegeneration in animals treated acutely with a variety of neurotoxins, including kainic acid (KA), ibogaine (IBO), 3-nitropropionic acid (3-NPA), domoic acid and others. The potential value of methods selective for neurodegeneration in elucidating the pathophysiology of transmissible spongiform encephalopathies (TSEs), such as the prion disease 'scrapie', has not yet been investigated. Using frozen or paraffin sections stained with FJ or silver, we evaluated the brains of hamsters inoculated with either the 263K or the 139H strains of scrapie, originally passaged from sheep into mice and then into hamsters. As a positive control, we also examined sections from IBO-treated rats, which experience degeneration restricted to small clusters of Purkinje neurons located in the paravermal region of the cerebellum. As expected, both FJ and silver methods delineated this identical pattern of neurodegeneration, characteristic of IBO exposure. Surprisingly, only a small number of FJ or silver-labeled cortical neurons were observed in scrapie-infected hamsters evaluated near the end of their incubation period but before obvious spongiform pathology. Instead, there was intense fluorescent staining of astrocytes in scrapie-infected hamsters, especially in the cortex, corpus callosum, and hypothalamus. Detailed protocols describing the application of the degeneration-selective methods we utilized are presented and compared.

Animals↗

PubMed2001

Protective effect of melatonin on naphthalene-induced oxidative stress and DNA damage in cultured macrophage J774A.1 cells.

Naphthalene is a bicyclic aromatic compound that is widely used in various domestic and commercial applications. Previous studies in our laboratory have demonstrated enhanced production of reactive oxygen species, lipid peroxidation and DNA fragmentation in both in vitro and in vivo models following treatment with naphthalene. Melatonin (N-acetyl-5-methoxytryptamine), an indole hormone, is the chief secretory product of the pineal gland and is an efficient free radical scavenger and antioxidant, both in vitro and in vivo. In this study, we have investigated the ability of 1 mM melatonin to protect against naphthalene-induced oxidative stress and DNA damage in cultured macrophage J774A.1 cells. No significant changes were observed when these macrophage cells were treated with 100 microM naphthalene. Approximately 2.0-, 4.2- and 4.4-fold increases in cytochrome c reduction were observed at 200, 400 and 500 mM concentrations of naphthalene, demonstrating the increased production of superoxide anion. At 24 h, lipid peroxidation increased by approximately 1.4-, 2.1- and 2.2-fold following treatment of these cells with 200, 400 and 500 mM concentrations of naphthalene, respectively, while 1.6-, 2.8- and 2.8-fold increases in DNA fragmentation were observed at these same concentrations. Two hour pretreatment of these cultured cells with 1 mM melatonin provided approximately 26-44% decreases in lipid peroxidation, superoxide anion production and DNA fragmentation in cells treated with 400 and 500 microM naphthalene. Cellular viability decreased significantly when cells were incubated with concentrations of naphthalene greater than 100 microM, while preincubation with melatonin significantly increased the cellular viability. These results demonstrate that naphthalene may induce toxic manifestations by enhanced production of reactive oxygen free radicals, resulting in lipid peroxidation and DNA damage, while preincubation with melatonin significantly suppressed cytoxicity in J774A.1 macrophage cells.

Animals↗

PubMed2001

Pharmacologically regulated gene expression in the retina following transduction with viral vectors.

The availability of inducible expression systems makes regulatable control of therapeutic proteins an attainable goal in gene therapy. We delivered tetracycline-inducible transgenes to the subretinal space using recombinant adenoviruses. Upon administration of doxycycline, we demonstrated reversible expression of green fluorescent protein in the retinal pigment epithelium as well as modulation of human growth hormone produced in the retina and secreted in the blood stream. This mode of delivery and regulation offers a unique way to evaluate gene function in the eye and represents a novel method for introducing therapeutic proteins into the retina.

Adenoviridae↗

PubMed2001

Ischemic mitral valve reconstruction and replacement: comparison of long-term survival and complications.

OBJECTIVE: This study reviews the 223 consecutive mitral valve operations for ischemic mitral insufficiency performed at New York University Medical Center between January 1976 and January 1996. The results for mitral valve reconstruction are compared with those for prosthetic mitral valve replacement. METHODS: From January 1976 to January 1996, 223 patients with ischemic mitral insufficiency underwent mitral valve reconstruction (n = 152) or prosthetic mitral valve replacement (n = 71). Coronary artery bypass grafting was performed in 89% of cases of mitral reconstruction and 80% of cases of prosthetic replacement. In the group undergoing reconstruction, 77% had valvuloplasty with a ring annuloplasty and 23% had valvuloplasty with suture annuloplasty. In the group undergoing prosthetic replacement, 82% of patients received bioprostheses and 18% received mechanical prostheses. RESULTS: Follow-up was 93% complete (median 14.6 mo, range 0-219 mo). Thirty-day mortality was 10% for mitral reconstruction and 20% for prosthetic replacement. The short-term mortality was higher among patients in New York Heart Association functional class IV than among those in classes I to III (odds ratio 5.75, confidence interval 1.25-26.5) and was reduced among patients with angina relative to those without angina (odds ratio 0.26, confidence interval 0.05-1.2). The 30-day death or complication rate was similarly elevated among patients in functional class IV (odds ratio 5.53; confidence interval 1.23-25.04). Patients with mitral valve reconstruction had lower short-term complication or death rates than did patients with prosthetic valve replacement (odds ratio 0.43, confidence interval 0.20-0.90). Eighty-two percent of patients with mitral valve reconstruction had no insufficiency or only trace insufficiency during the long-term follow-up period. Five-year complication-free survivals were 64% (confidence interval 54%-74%) for patients undergoing mitral valve reconstruction and 47% (confidence interval 33%-60%) for patients undergoing prosthetic valve replacement. Results of a series of statistical analyses suggest that outcome was linked primarily to preoperative New York Heart Association functional class. CONCLUSIONS: Initial mortalities were similar among patients undergoing prosthetic replacement and valve reconstruction. Poor outcome was primarily related to preexisting comorbidities. Patients undergoing valve reconstruction had fewer valve-related complications. Valve reconstruction resulted in excellent durability and freedom from complications. These findings suggest that mitral valve reconstruction should be considered for appropriate patients with ischemic mitral insufficiency.

Aged↗

PubMed2001

Identification of new major histocompatibility complex class II restriction fragment length polymorphisms in a closed experimental line of Beltsville Small White turkeys.

Beltsville Small White (BSW) turkeys have been utilized as an experimental model in the study of bacterial, parasitic, and fungal diseases. Given the critical role of MHC antigens in the initial steps of the immune response to specific pathogens, the MHC Class II of BSW turkeys was characterized. Southern blot analysis of PvuII-digested turkey DNA that was hybridized with a chicken Class II beta gene genomic clone revealed two restriction fragment length polymorphism profiles not previously identified in experimental or commercial breeder lines of turkeys. These fingerprint profiles differed in a single 6.0-kb band that was present in approximately 38% of the birds examined. The DNA fragments of 5.0, 4.1, 3.3, and 3.1 were present in both profiles. Furthermore, no mixed lymphocyte reaction was observed between individuals within the BSW turkey line. The present results indicate that BSW turkeys represent a unique source of genetic diversity for MHC Class II haplotypes.

Animals↗