Biomedical subjects

X Ye

Publications and source records attributed to X Ye.

At least 19 recordsLinked to original sources

PubMed2002

Increased c-Fos protein in the brains of scrapie-infected SAMP8, SAMR1, AKR and C57BL mice.

Scrapie is a neurodegenerative disease that occurs naturally in sheep and goats. The histopathological changes include vacuolation, neuronal apoptosis and astrocytosis. The mechanisms involved in neuronal apoptosis are still unknown. Recently, we observed that activated p38 immunohistostaining was increased in scrapie-infected mice. In many neurodegenerative diseases, activation of the p38 pathway and of the immediate-early gene termed c-Fos appears to be required for the initiation of apoptosis. There are similarities in histopathological changes seen in scrapie-infected mice and in an uninfected senescence-accelerated mouse strain (SAMP8). This led us to investigate c-Fos protein levels in the brains of both uninfected and scrapie-infected SAMP8, SAMR1, AKR and C57BL mice using immunohistochemical methods. The SAMR1 strain served as a control in that it is a mouse strain that does not show accelerated ageing, but has a background that is similar to the SAMP8 strain. AKR was used because it is one of the progenitor strains of both SAM strains and, finally, C57BL is a completely unrelated strain. The results showed a low basal c-Fos expression in controls and a marked increase in c-Fos staining in scrapie-infected mice. In scrapie-positive mice, c-Fos immunoreactivity was observed in neurones in the cortex, hippocampus, thalamus, hypothalamus, medulla, midbrain, brainstem, paraterminal body, internal capsule and cerebellar Purkinje cells. Immunoreactivity of c-Fos was also observed in astrocytes in many brain areas of scrapie-infected mice, particularly in the hippocampus and cortex. Our results show that normal mouse brain (NMB)-injected AKR and SAMP8 mice had more c-Fos production than NMB-injected SAMR1 or C57BL mice; scrapie-infection induces significant increases in c-Fos immunoreactivity in all four mouse strains. Our study suggests that the increase in c-Fos levels may play a role in the neuronal apoptosis observed in scrapie-infected mice.

Aging↗

PubMed2001

Lipopolysaccharide regulates constitutive and inducible transcription factor activities differentially in vivo in the rat.

The pathophysiology of septic shock is characterized by the induction of multiple proinflammatory genes and their products. Transcription factors play key roles in the transcription of these genes. Transcription factors may play important roles in the pathophysiology of septic shock. However, little information is available regarding the in vivo transcription factor activities during sepsis. We have studied the in vivo effects of LPS on the DNA binding activities of transcription factors Sp1 (promoter selective transcription factor), AP-2 (activating protein-2), AP-1 (activating protein-1), NF-kappaB (nuclear factor kappa B), and CREB (cAMP response element binding protein). We found that LPS up-regulated the DNA binding activity of inducible transcription factors AP-1, NF-kappaB, and CREB in a time-dependent manner, but down-regulated the DNA binding activity of constitutive transcription factors Sp1 and AP-2. Thus, LPS regulates the inducible and constitutive transcription factors differentially under in vivo conditions.

Animals↗

PubMed2001

Sequence-dependent interactions of two forms of UvrC with DNA helix-stabilizing CC-1065-N3-adenine adducts.

The uvrA, uvrB, and uvrC genes of Escherichia coli control the initial steps of nucleotide excision repair. The uvrC gene product is involved in at least one of the dual incisions produced by the UvrABC complex. Using single-stranded (ss) DNA affinity chromatography, we have separated two forms of UvrC from both wild-type E. coli cells and overproducing cells. UvrCI elutes at 0.4 M KCl, and UvrCII elutes at 0.6 M KCl. In general, both forms, in the presence of UvrA and UvrB, actively incise UV-irradiated and CC-1065-modified DNA in the same fashion; i.e., they incise six to eight nucleotides 5' to and three to five nucleotides 3' to a photoproduct or a CC-1065-N3-adenine adduct. They produce different incisions, however, at a CC-1065-N3-adenine adduct in the sequence 5'-GATTACG- present in the MspI-BstNI 117 bp fragment of M13mp1. UvrABCI incises at both the 5' and 3' sides of the adduct (UvrABCI cut), while UvrABCII incises only at the 5' side (UvrABCII cut). Mixing UvrCI and UvrCII results in both UvrABCI and UvrABCII cuts, and the levels of these two types of cutting are proportional to the amount of UvrCI and UvrCII. DNase I footprints of the MspI-BstNI 117 bp DNA fragment containing a site-directed CC-1065-adenine adduct at the 5'-GATTACG- site show that UvrCII, but not UvrCI, binds to the adduct site. Furthermore, the pattern of DNase I footprints induced by UvrCII binding differs from the pattern of the footprints induced by UvrA, UvrAB, and UvrABCI binding. Interestingly, while the presence of unirradiated DNA enhances the efficiency of UvrABCII in incising UV-irradiated DNA, it does not enhance UvrABCII incision of the CC-1065-N3-adenine adduct formed at 5'-GATTACG-. These results show that two different forms of UvrC differ in DNA binding properties as well as incision modes at some kinds of DNA damage.

Adenine↗

PubMed2001

Phosphorylation-dependent ubiquitination of cyclin E by the SCFFbw7 ubiquitin ligase.

Cyclin E binds and activates the cyclin-dependent kinase Cdk2 and catalyzes the transition from the G1 phase to the S phase of the cell cycle. The amount of cyclin E protein present in the cell is tightly controlled by ubiquitin-mediated proteolysis. Here we identify the ubiquitin ligase responsible for cyclin E ubiquitination as SCFFbw7 and demonstrate that it is functionally conserved in yeast, flies, and mammals. Fbw7 associates specifically with phosphorylated cyclin E, and SCFFbw7 catalyzes cyclin E ubiquitination in vitro. Depletion of Fbw7 leads to accumulation and stabilization of cyclin E in vivo in human and Drosophila melanogaster cells. Multiple F-box proteins contribute to cyclin E stability in yeast, suggesting an overlap in SCF E3 ligase specificity that allows combinatorial control of cyclin E degradation.

Amino Acid Motifs↗

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↗