Biomedical subjects

J Fu

Publications and source records attributed to J Fu.

At least 163 records · Page 9Linked to original sources

PubMed1998

Expression of splice variants of CD44 mRNA and its significance in human primary hepatocellular carcinoma.

OBJECTIVE: To investigate the relationship between the expression of splice variants of CD44 (CD44v) and the metastasis of primary hepatocellular carcinoma (PHCC) in humans. METHODS: The expression of metastasis-associated CD44v mRNA in 54 specimens of human PHCC was detected by reverse transcription and polymerase chain reaction (RT-PCR) method. RESULTS: CD44v mRNA was expressed in 50 (92.59%) of the 54 specimens of PHCC, with moderate or over expression in 42 (77.78%). Among the 42 specimens with moderate or over expression, deficient tumor capsules were found in 25 (59.52%), penetration of tumor capsule in 15 (35.71%), tumor emboli in the portal vein in 34 (80.95%), and daughter tumor nodules in 29 (69.05%). Meanwhile, 12 of the 54 specimens showed negative or weak expression of CD44v mRNA. Of them, all had tumor capsules, 1 (8.33%) had penetrated tumor capsule, 1 (8.33%) had tumor emboli in the portal vein, and 2 (16.67%) had daughter tumor nodules. CONCLUSION: CD44v may contribute to the metastatic potential of PHCC.

Carcinoma, Hepatocellular↗

PubMed1998

Evaluation of left ventricular function with cine magnetic resonance four chamber/multiple slice multiple phase sequence.

OBJECTIVE: To investigate the value of cine magnetic resonance(MR) in determination of cardiac function by using four chamber/multiple slice multiple phase (4CH/MSMP) sequence. METHODS: In 18 healthy subjects, several indices of left ventricular function were measured by using cine MR 4CH/MSMP sequence, and the correlation of the results was compared with ultrasound cardiography by t test. RESULTS: In the measurement of left ventricular short diameter, left ventricular posterior wall thickness, internal ventricular septum thickness, left ventricular end diastolic and end systolic volume, the values of cine MR 4CH/MSMP sequence correlated well with those of echocardiography. The gamma value was between 0.88 and 0.99. CONCLUSION: The cine MR 4CH/MSMP sequence may be considered an important noninvasive modality for accurate assessment of cardiac function.

Adult↗

PubMed1998

[Efficient expression of multidrug resistance gene mdr1 in retroviral vector under control of an internal ribosome entry site].

OBJECTIVE: To test the efficiency of human mdr1 gene expression under the control of an internal ribosome entry site (IRES). METHODS: The expression retroviral vector Halmdr1 was constructed from pSXLC/pHa system which contains an IRES from encephalomyocarditis virus. The vector was introduced into ecotropic packaging cells GP + E86 by liposome-mediated transfection. The retrovirus producing cells were obtained by 50 micrograms/L vincristine selection. The success of transfer and expression of mdr1 gene were determined by polymerase chain reaction (PCR) and flow cytometry (FCM). RESULTS: Virus in the supernatant of the producer cells, in which the integration of mdr1 gene was confirmed by PCR, was titrated to 2.0 x 10(5) cfu/ml. The selected producer cells exhibited a 24-52-fold increase of resistance to vincristine, daunorubicin and taxol in comparison with control cells GP + E86. The expression of mdr1 gene was confirmed by both reverse transcription PCR at RNA level and FCM at protein level, although the HaImdr1 transfectants showed somewhat less expression of P-gp when compared to that with cap-dependent translation of Ha mdr1. CONCLUSION: mdr1 gene can be effectively translated and expressed under the control of IRES in cap-independent manner, it may be used as a dominant selectable marker in bicistronic vectors for gene therapy.

Animals↗

PubMed1998

[The detection of circulating hepatocellular carcinoma cells in peripheral venous blood by reverse transcription-polymerase chain reaction and its clinical significance].

OBJECTIVE: To detect circulating hepatocellular carcinoma by demonstrating hepatocellular carcinoma cells-associated mRNA in the nuclear cell component of peripheral blood (PBL). METHOD: Peripheral blood (5 ml) samples were obtained from 93 patients with hepatocellular carcinoma (HCC) and from 37 controls (15 controls with liver cirrhosis after hepatitis B, 12 chronic hepatitis B, and 10 normal liver function). To identify HCC cells in peripheral blood, liver-specific human alpha-fetoprotein (AFP) mRNA was amplified from total RNA extracted from whole blood by reverse transcription-polymerase chain reaction. RESULT: AFP mRNA was detected in 50 blood samples from the patients with HCC (53.8%). In contrast, there were no clinical control patients whose samples showed detectable AFP mRNA in PBL. The presence of AFP mRNA in blood seemed to be correlated with the stage (by TNM classification) of HCC, the serum AFP value, and the presence of intrahepatic metastasis, portal vein thrombosis, tumor diameter and/or distant metastasis. AFP mRNA was detected in the blood of 21 patients showing metastasis at extrahepatic organs (100%) in contrast to 29 of 72 patients without metastasis (40.3%). CONCLUSION: The presence of AFP mRNA in peripheral blood may be an indicator of malignant hepatocytes, which might predict hematogenous spreading metastasis of tumor cells in patients with HCC.

Adult↗

PubMed1997

Formation and crystallization of yeast RNA polymerase II elongation complexes.

Minimal templates were devised for the efficient generation of yeast RNA polymerase II transcription elongation complexes. A 33-base pair DNA with a 15-residue dC tail at one 3'-end supported the formation of a complex containing the polymerase paused at nucleotide 11 of the duplex region and an RNA of 14-16 residues. The same template could yield an arrested complex with the enzyme at nucleotide 13-15 and RNA of 15-17 residues. These complexes were stable for at least a week under various conditions and could be resolved by gel electrophoresis or purified by ion exchange chromatography. The purified paused complex formed crystals capable of x-ray diffraction to 3.5 A resolution. The complex remained active in the crystal and, in the presence of nucleoside triphosphates, could efficiently extend the transcript in situ.

Base Sequence↗

PubMed1997

Interactions among subunits of the oligosaccharyltransferase complex.

The mammalian oligosaccharyltransferase (OST) is an oligomeric complex composed of three membrane proteins of the endoplasmic reticulum: ribophorin I (RI), ribophorin II (RII), and OST48. In addition, sequence homology between the Ost2 subunit of the yeast OST complex and Dad1 (defender against apoptotic death) suggests that Dad1 may represent a fourth subunit of the mammalian OST complex. In attempts to elucidate the structural organization of this complex, we have studied the interactions among its subunits. Using the yeast two-hybrid system, we have shown that the luminal domains of RI and RII (RIL and RIIL, respectively) interacted with the luminal domain of OST48 (OST48L), but no direct interaction was observed between RIL and RIIL. These results were confirmed by biochemical assays. Deletion analyses using the yeast two-hybrid system showed that subdomain of RIL or RIIL adjacent to the respective transmembrane domains interacted with OST48L. Of the three equal length subdomains of OST48L, the one at the N terminus and the one next to the transmembrane domain interacted with RIL. None of these three subdomains of OST48L interacted with RIIL. The yeast two-hybrid assay also revealed affinity between the cytoplasmically located N-terminal region of Dad1 and the short cytoplasmic tail of OST48, thus placing Dad1 firmly into the OST complex. In addition, we found a homotypic interaction between the cytoplasmic domains of RI, which may play a role in the formation of the oligomeric array formed by components of the translocation machinery.

Binding Sites↗

PubMed1997

An intracellular protein that binds amyloid-beta peptide and mediates neurotoxicity in Alzheimer's disease.

Amyloid-beta is a neurotoxic peptide which is implicated in the pathogenesis of Alzheimer's disease. It binds an intracellular polypeptide known as ERAB, thought to be a hydroxysteroid dehydrogenase enzyme, which is expressed in normal tissues, but is overexpressed in neurons affected in Alzheimer's disease. ERAB immunoprecipitates with amyloid-beta, and when cell cultures are exposed to amyloid-beta, ERAB inside the cell is rapidly redistributed to the plasma membrane. The toxic effect of amyloid-beta on these cells is prevented by blocking ERAB and is enhanced by overexpression of ERAB. By interacting with intracellular amyloid-beta, ERAB may therefore contribute to the neuronal dysfunction associated with Alzheimer's disease.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1997

Amyloid-beta peptide-receptor for advanced glycation endproduct interaction elicits neuronal expression of macrophage-colony stimulating factor: a proinflammatory pathway in Alzheimer disease.

In Alzheimer disease (AD), neurons are thought to be subjected to the deleterious cytotoxic effects of activated microglia. We demonstrate that binding of amyloid-beta peptide (Abeta) to neuronal Receptor for Advanced Glycation Endproduct (RAGE), a cell surface receptor for Abeta, induces macrophage-colony stimulating factor (M-CSF) by an oxidant sensitive, nuclear factor kappaB-dependent pathway. AD brain shows increased neuronal expression of M-CSF in proximity to Abeta deposits, and in cerebrospinal fluid from AD patients there was approximately 5-fold increased M-CSF antigen (P < 0.01), compared with age-matched controls. M-CSF released by Abeta-stimulated neurons interacts with its cognate receptor, c-fms, on microglia, thereby triggering chemotaxis, cell proliferation, increased expression of the macrophage scavenger receptor and apolipoprotein E, and enhanced survival of microglia exposed to Abeta, consistent with pathologic findings in AD. These data delineate an inflammatory pathway triggered by engagement of Abeta on neuronal RAGE. We suggest that M-CSF, thus generated, contributes to the pathogenesis of AD, and that M-CSF in cerebrospinal fluid might provide a means for monitoring neuronal perturbation at an early stage in AD.

Alzheimer Disease↗

PubMed1997

The characterization, molecular cloning, and expression of a novel hematopoietic cell antigen from CD34+ human bone marrow cells.

The adhesion molecule BEN/SC1/DM-GRASP (BEN) is a marker in the developing chicken nervous system that is also expressed on the surface of embryonic and adult hematopoietic cells such as immature thymocytes, myeloid progenitors, and erythroid progenitors. F84.1 and KG-CAM, two monoclonal antibodies to rat neuronal glycoproteins with similarity to BEN, cross-react with an antigen on rat hematopoietic progenitors, but F84.1 only also recognizes human blood cell progenitors. We have defined the antigen recognized by F84.1 as the hematopoietic cell antigen (HCA). HCA expression was detected on 40% to 70% of CD34+ fetal and adult bone marrow cells and mobilized peripheral blood cells. Precursor cell activity for long-term in vitro bone marrow cell culture was confined to the subset of CD34+ cells that coexpress HCA. HCA is expressed by the most primitive subsets of CD34+ cells, including all rhodamine 123(lo), Thy-1+, and CD38(-/lo) CD34+ adult bone marrow cells. HCA was also detected on myeloid progenitors but not on early B-cell progenitors. We also describe here the cloning and characterization of cDNAs encoding two variants of the human HCA antigen (huHCA-1 and huHCA-2) and of a cDNA clone encoding rat HCA (raHCA). The deduced amino acid sequences of huHCA and raHCA are homologous to that of chicken BEN. Recombinant proteins produced from either human or rat HCA cDNAs were recognized by F84.1, whereas rat HCA but not human HCA was recognized by antirat KG-CAM. Expression of either form of huHCA in CHO cells conferred homophilic adhesion that could be competed with soluble recombinant huHCA-Fc. The molecular cloning of HCA and the availability of recombinant HCA should permit further evaluation of its role in human and rodent hematopoiesis.

Adult↗

PubMed1997

Insulin gene region contributes to genetic susceptibility to, but may not to low incidence of, insulin-dependent diabetes mellitus in Japanese.

In the Caucasian population, it has been demonstrated that the insulin gene (INS) region contains the insulin-dependent diabetes mellitus locus (IDDM2). In the Japanese population, however, there has been no report demonstrating the contribution of IDDM2 to the pathogenesis of IDDM. We conducted an association study of IDDM in a large number of Japanese subjects with multiple polymorphisms in INS region. We found a significant association of the INS region with IDDM. Alleles positively associated with IDDM in INS region were the same as those positively-associated with IDDM in Caucasian population, although positively-associated alleles are very common (allele frequencies > 0.9) in the Japanese general population. These data suggest that IDDM2 is involved in the genetic susceptibility to IDDM in Japanese. The high frequencies of disease-associated alleles in the general population suggest that IDDM2 locus is not responsible for the low incidence of IDDM in Japanese.

DNA, Satellite↗

PubMed1997

Expression of the integrin alpha5 subunit and its mediated cell adhesion in hepatocellular carcinoma.

Tumor invasion and metastasis are complex processes, requiring the ability of tumor cells to interact with proteins of the extracellular matrix through cell-adhesion molecules on the cell surface. Integrins are heterodimeric membrane glycoproteins, consisting of alpha and beta subunits, which enable cells to recognize adhesive substrates in the extracellular matrix. The roles of the integrin alpha5beta1 in tumor invasion are highlighted by finding that some tumor cells have lost or reduced alpha5beta1 expression. It therefore functions as a negative signaling regulator. Expression of alpha5beta1 and its mediation of cell adhesion in hepatocellular carcinoma (HCC) have not been elucidated. In surgical specimens of HCC we found, by immunohistochemistry and Northern blot analysis, that the alpha5-positive rates in cancerous tissues were lower than the corresponding rates in non-cancerous tissues. Reduced expression of the integrin alpha5 occurred more frequently in HCC with more malignant phenotypes, such as poor differentiation, large size (more than 10-cm in diameter), absence of capsule and high invasion. Reverse transcription/polymerase chain reaction, a more sensitive assay, was used to detect the alpha5 mRNA level in LCID20, a highly metastatic model of human HCC, and LCID35, a low-metastasis model. The results showed that integrin alpha5 was negative in the former and positive in the latter. Cell adhesion assays showed the maximal percentage inhibition of anti-alpha5 mAb on SMMC 7721 cell adhesion to fibronectin to be 68.9 +/- 4.9% at the saturation concentrations of each antibody (200 microg/ml). If anti-alpha5 mAb was combined with anti-beta1 mAb, the inhibition was 74.1 +/- 11.1%. It is concluded that reduced expression of the integrin alpha5 subunit is correlated with more malignant phenotypes of human HCC. Any change in the adhesion of hepatocellular carcinoma cells to fibronectin is mainly dependent upon the function of the integrin alpha5beta1.

Antigens, CD↗

PubMed1997

The effect of high-dose methylprednisolone on laser-induced retinal injury in primates: an electron microscopic study.

BACKGROUND: Previously we reported an ameliorative effect of high-dose methylprednisolone in laser injury to monkey retinas. The ultrastructural modification by methyl-prednisolone has not been examined. METHODS: Cynomolgus monkeys were given severe (grade III) retinal laser burns and treated with an intravenous megadose of methylprednisolone. Pathologic features of the retinal lesions with or without methylprednisolone treatment were evaluated by light and electron microscopy. RESULTS: Ultrastructurally, the treated lesions showed rapid recanalization of choriocapillaris; proliferation of retinal pigment epithelium to replace the necrotic and damaged cells, resulting in rapid re-establishment of blood retinal barrier; mild macrophagic activity; and rapid reformation of the outer limiting membrane by Mueller cells. CONCLUSION: A high dose of methylprednisolone affected the responses of the choriocapillaris, retinal pigment epithelium, photoreceptor cells and Mueller cells to laser injury, showing an overall beneficial effect. These modifications might be ascribed to methylprednisolone's anti-inflammatory action, protection of the microcirculation and anti-lipid peroxidation effect.

Animals↗

PubMed1997

The production of recombinant dengue virus E protein using Escherichia coli and Pichia pastoris.

The dengue virus envelope protein was expressed as a GST fusion protein using E. coli and P. pastoris as expression hosts. In E. coli the recombinant E protein is expressed initially as a soluble 81 kDa GST fusion protein. Treatment of the fusion protein with thrombin released a 55 kDa protein, which is the expected size for correctly processed, non-glycosylated recombinant E protein. The antiserum from animals immunised with this recombinant E protein was found to specifically recognise the dengue virus E protein in virus-infected cells, thus demonstrating the immunogenic nature of the recombinant E protein. This expression system allowed production of up to 2 mg of purified recombinant E protein from a 1 1 bacterial culture. In contrast, expression of this GST fusion protein in P. pastoris is associated with extensive proteolytic degradation of the recombinant E protein. However, this proteolytic degradation was not observed in the truncated E protein sequences which were expressed. One of these recombinant fusion proteins, GST E401 was secreted into the culture medium at levels of up to 100 microg/l of growth medium.

Animals↗

PubMed1997

Refolding of soluble leukemia inhibitory factor receptor fusion protein (gp 190 sol DAF) from urea.

The insoluble inclusion bodies of soluble leukemia inhibitory factor receptor fusion protein (gp 190 sol DAF) was solubilized in 8 M urea on the unfolding transitions, and several factors on the aggregate formation were indirectly analyzed for the refolding of gp 190 sol DAF. Results indicate that the refolding yield can be considerably increased at lowering concentration of the unfolding protein, a little soluble protein with the slow refolding appears in the process of the aggregate formation and the concentration of the denaturant must be down to a minimum level for its refolding.

Glycerol↗

PubMed1997

Disjunct habitats as islands: genetic variability in the Caucasian rock lizard Lacerta portschinskii.

Genetic diversity at 37 allozyme loci was surveyed in Lacerta portschinskii from contiguous populations and from a disjunct population. Indices of genetic diversity (heterozygosity, number of alleles per locus, and percentage of loci polymorphic) were greater in contiguous populations than in the smaller disjunct population. In this regard, disjunct populations appear to be similar to island populations. Indices of genetic diversity in Caucasian Lacerta are less than those reported from vagile lizard taxa and more similar to those of sit-and-wait predators.

Journal Article↗

PubMed1997

Old age, multiple formations or genetic plasticity? Clonal diversity in the uniparental Caucasian rock lizard, Lacerta dahli.

Allozyme variation at 35 gene loci is investigated in 161 specimens of the uniparental Caucasian lizard Lacerta dahli from several locations in Armenia and Georgia. All individuals are heterozygotic at 12 loci, and homozygotic at 21 loci. Variation at two loci results in five uniparental clones. One clone is widespread whereas four are geographically restricted and are represented by only one or two individuals. Because successful formation of uniparental clones is rare, and because the biparental species forming them are now allopatric, the most probable explanation for the origin of the observed clonal diversity is either mutation or recombination within the common clone. The rare clones have lower levels of enzyme activity at four loci, suggesting that these organisms may be genetically deficient. Although the evidence points to change in a pre-existing clone, the possibility of multiple origins cannot be ruled out.

Journal Article↗

PubMed1997

Expression of the dengue virus structural proteins in Pichia pastoris leads to the generation of virus-like particles.

We have expressed cDNA encoding the dengue virus structural proteins in Pichia pastoris by chromosomal integration of an expression cassette containing the dengue virus structural genes (CprME). The yeast recombinant E protein migrated during SDS-PAGE as a 65 kDa protein when analysed by Western blotting and radioimmunoprecipitation, which is the expected molecular mass for correctly processed and glycosylated E protein. Treatment with endoglycosidases showed that the recombinant E protein was modified by the addition of short mannose chains. The E protein migrated with a buoyant density of 1.13 g/cm3 when analysed using sucrose density gradient centrifugation. Spherical structures with an average diameter of 30 nm, whose morphology resembles dengue virions, were observed in the purified fractions using transmission electron microscopy. Furthermore, the virus-like particles were immunogenic in animals and were able to induce neutralizing antibodies. This is the first report that expression of the structural genes of a flavivirus in yeast is able to generate particulate structures that resemble virions.

Centrifugation, Density Gradient↗

PubMed1997

bFGF induces BCK promoter-driven expression in muscle via increased binding of a nuclear protein.

Changes in gene expression occurring during skeletal muscle differentiation are exemplified by downregulation of brain creatine kinase (BCK) and induction of muscle creatine kinase (MCK). Although both are transcriptionally regulated, there appears to be no transcription factor-element overlap, suggesting that their coordinate expression results from culture medium-related influences. Basic fibroblast growth factor (bFGF) prevents myogenesis and represses MCK expression by inhibiting transcriptional activation. It was hypothesized that bFGF similarly influenced BCK by inducing its expression. Accordingly, BCK promoter constructs were transiently transfected into C2C12 cells and, after a switch to differentiation medium, were treated with bFGF, bFGF plus herbimycin, adenosine 3',5'-cyclic monophosphate (cAMP), or phorbol 12-myristate 13-acetate (PMA). Analyses demonstrated that bFGF responsiveness was contained within a 33-base pair element. Electromobility shift assays showed that bFGF induction increased the abundance of the nuclear factor binding the element. Both effects were prevented by herbimycin. Neither cAMP nor PMA specifically induced the construct containing the bFGF-responsive element. The induced factor required phosphorylation to bind, implying that bFGF-mediated increases in binding may be due to transcription factor phosphorylation.

Animals↗