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Biomedical subjects

Yi Fu

Publications and source records attributed to Yi Fu.

At least 19 recordsLinked to original sources
PubMed2007

Increased expression of integrin beta1 subunit enhances p21WAF1/Cip1 transcription through the Sp1 sites and p300-mediated histone acetylation in human hepatocellular carcinoma cells.

Integrins, a class of membrane receptors, are major players in transmitting the mechanical force across the plasma membrane. We previously reported that overexpression of integrin beta1 subunit imposed a growth inhibitory effect on the hepatocellular carcinoma cell line SMMC-7721 through transcriptional activation of p21(WAF1/CIP1) gene. In this study, we further determined the molecular mechanisms underlying p21(WAF1/CIP1) expression induced by integrin beta1 overexpression. We report herein that overexpression of integrin beta1 subunit upregulates p21(WAF1/Cip1) transcription through a p53-independent pathway. The overexpressed integrin beta1 activates the p21(WAF1/Cip1) promoter through the Sp1/Sp3 sites and makes more transcription factor Sp1 recruited to the proximal p21 promoter region. In addition, it makes the acetylation value of histone proteins increased across some parts of the p21(WAF1/Cip1) gene, especially in the promoter region. The transcriptional co-activator p300, which possesses intrinsic histone acetyltransferase, was found to be involved in the integrin beta1-mediated histone acetylation and p21 transcriptional activation. Therefore, these findings presented the mechanisms by which integrin beta1 induced the elevated p21 expression in hepatic cancer cells.

Acetylation↗
PubMed2006

Enhanced fluorescence of Cy5-labeled oligonucleotides near silver island films: a distance effect study using single molecule spectroscopy.

We investigated fluorescence enhancements and lifetime reductions of Cy5 probe molecules at various distances from the deposited silver island film surface using single molecule spectroscopic methods. The proximity of fluorophore molecules to the surface was controlled by alternating layers of biotinylated bovine serum albumin (BSA-biotin) and avidin, followed by binding of Cy5-labeled oligonucleotides to the top of a BSA-biotin layer structure. We observed dramatically varied brightness of fluorophores with distances from metal structures as well with reduced blinking in the presence of silver island films. In addition, distributions of fluorescence lifetimes and apparent emission intensities from individual molecules indicate an inhomogeneous nature of local matrix surface near metallic nanostructures. These studies illustrate the exclusive information that is otherwise hidden in ensemble measurements.

Binding Sites↗
PubMed2006

Enhanced fluorescence of Cy5-labeled DNA tethered to silver island films: fluorescence images and time-resolved studies using single-molecule spectroscopy.

Methods that increase the total emission per fluorophore would provide increased sensitivity and a wider dynamic range for chemical analysis, medical diagnostics, and in vivo molecular imaging. The use of fluorophore-metal interactions has the potential to dramatically increase the detectability of single fluorophores for bioanalytical monitoring. The fabrication and single-molecule analysis of fluorophore-labeled DNA molecules tethered to silver island films are described in this article. The single-molecule spectroscopic method reveals some insightful information on the behaviors of single molecules, rather than an ensemble of molecules. Analysis of fluorescence images, intensity profiles, total emitted photons, and lifetime distributions reveals some of sample heterogeneities. Investigations of time-dependent emission characteristics of single molecules indicate that the total number of emitted photons on the silvered surface is more than 10 times greater than on free labeled DNA molecules on a glass substrate. In addition, time-correlated single-photon counting results reveal the reduced lifetimes of single molecules tethered to silver island films.

Carbocyanines↗
PubMed2006

Single molecule spectroscopy studies of diffusion in mesoporous silica thin films.

Single molecule spectroscopy is applied in studies of diffusion and surface adsorption in sol-gel-derived mesoporous silica thin films. Mesoporous films are obtained by spin casting surfactant-templated sols onto glass substrates. Small-angle X-ray diffraction results are consistent with hexagonally ordered mesophases in as-synthesized (i.e., surfactant-containing) films. Upon calcination, a 30% contraction and disordering of these structures occurs. Nile Red is used as a fluorescent probe of both the as-synthesized and calcined films. It is loaded into the samples at subnanomolar levels either prior to spin casting or after calcination. Fluorescence imaging and single-point fluorescence time transients show the dye molecules to be relatively mobile in the as-synthesized samples. In contrast, the molecules appear entrapped at fixed locations in dry calcined films. In calcined films rehydrated under high humidity conditions, the Nile Red molecules again become mobile. Time transients obtained from the as-synthesized and rehydrated samples provide clear evidence for frequent reversible adsorption of the dye to the silica surfaces. Autocorrelations of the time transients provide quantitative data on the mean diffusion coefficients (D = 2.4 x 10(-10) and 2.6 x 10(-10) cm2/s) and mean desorption times (1/k = 25 and 40 s) for the as-synthesized and rehydrated films, respectively. The results prove both water and surfactant play important roles in governing matrix interactions and mass transport.

Journal Article↗
PubMed2006

WNK kinases influence TRPV4 channel function and localization.

TRPV4, a renally expressed nonselective cation channel of the transient receptor potential (TRP) family, is gated by hypotonicity. Kinases of the WNK family influence expression and function of the thiazide-sensitive Na+-Cl- cotransporter, and monogenic human hypertension has been linked to mutations in the gene coding for WNK4. Along with TRPV4, WNK isoforms are highly expressed in the distal nephron. We show here that coexpression of WNK4 downregulates TRPV4 function in human embryonic kidney (HEK-293) cells and that this effect is mediated via decreased cell surface expression of TRPV4; total abundance of TRPV4 in whole cell lysates is unaffected. The effect of the related kinase WNK1 on TRPV4 function and surface expression was similar to that of WNK4. Disease-causing point mutations in WNK4 abrogate, but do not eliminate, the inhibitory effect on TRPV4 function. In contrast to wild-type WNK4, a kinase-dead WNK4 point mutant failed to influence TRPV4 trafficking; however, deletion of the entire WNK4 kinase domain did not blunt the effect of WNK4 on localization of TRPV4. Deletion of the extreme COOH-terminal putative coiled-coil domain of WNK4 abolished its effect. In immunoprecipitation experiments, we were unable to detect direct interaction between TRPV4 and either WNK kinase. In aggregate, these data indicate that TRPV4 is functionally regulated by WNK family kinases at the level of cell surface expression. Because TRPV4 and WNK kinases are coexpressed in the distal nephron in vivo and because there is a tendency toward hypercalcemia in TRPV4-/- mice, we speculate that this pathway may impact systemic Ca2+ balance. In addition, because WNK kinases and TRPV4 are activated by anisotonicity, they may comprise elements of an osmosensing or osmotically responsive signal transduction cascade in the distal nephron.

Animals↗
PubMed2006

Integrin beta(1A) upregulates p27 protein amount at the post-translational level in human hepatocellular carcinoma cell line SMMC-7721.

Integrins mediate many fundamental cellular processes by binding to components of the extracellular matrix. We showed previously that integrin beta(1A) could inhibit cell proliferation. Integrin beta(1A) stimulated the promoter activity of p21(cip1) and enhanced its transcription in SMMC-7721 cells. In this study, we demonstrated that integrin beta(1A) upregulated p27(kip1) at the post-translational level in SMMC-7721 cells. Our results showed that integrin beta(1A) increased the p27 protein amount, both in cytoplasm and nucleus, but did not affect the p27 mRNA amount. Cycloheximide treatment experiment revealed that the half-life of p27 protein was prolonged in integrin beta1A overexpressing cells, indicating that integrin beta(1A) inhibited the degradation of p27 protein. Our data also provided evidence that both the proteasome and calpain were involved in the degradation of p27 protein in SMMC-7721 cells. Integrin beta(1A) decreased the Skp2 expression and repressed the activity of calpain during G1 phase in SMMC-7721 cells. Taken together, these results indicated that integrin beta(1A) might upregulate the protein amount of p27 through repressing Skp2-dependent proteasome degradation and calpain-mediated proteolysis in SMMC-7721 cells.

Calpain↗
PubMed2005

Glycosylation of the osmoresponsive transient receptor potential channel TRPV4 on Asn-651 influences membrane trafficking.

We identified a consensus N-linked glycosylation motif within the pore-forming loop between the fifth and sixth transmembrane segments of the osmoresponsive transient receptor potential (TRP) channel TRPV4. Mutation of this residue from Asn to Gln (i.e., TRPV4(N651Q)) resulted in loss of a slower migrating band on anti-TRPV4 immunoblots and a marked reduction in lectin-precipitable TRPV4 immunoreactivity. HEK293 cells transiently transfected with the mutant TRPV4(N651Q) exhibited increased calcium entry in response to hypotonic stress relative to wild-type TRPV4 transfectants. This increase in hypotonicity responsiveness was associated with an increase in plasma membrane targeting of TRPV4(N651Q) relative to wild-type TRPV4 in both HEK293 and COS-7 cells but had no effect on overall channel abundance in whole cell lysates. Residue N651 of TRPV4 is immediately adjacent to the pore-forming loop. Although glycosylation in this vicinity has not been reported for a TRP channel, the structurally related hexahelical hyperpolarization-activated cyclic nucleotide-gated channel, HCN2, and the voltage-gated potassium channel, human ether-a-go-go-related (HERG), share a nearly identically situated and experimentally confirmed N-linked glycosylation site which promotes rather than limits channel insertion into the plasma membrane. These data point to a potentially conserved structural and functional feature influencing membrane trafficking across diverse members of the voltage-gated-like ion channel superfamily.

Amino Acid Sequence↗
PubMed2005

Regulation of TRPV1 by a novel renally expressed rat TRPV1 splice variant.

The capsaicin receptor and transient receptor potential channel TRPV1 senses heat, protons, and vanilloid agonists in peripheral sensory ganglia. Abundant data have suggested the presence of potentially novel splice variants in the kidney. We report a novel rat TRPV1 splice variant, TRPV1(VAR), cloned from kidney papilla. TRPV1(VAR) cDNA was identified in multiple kidney tissues. Its sequence was fully compatible with potential splice donor and acceptor sites in the rat TRPV1 gene. TRPV1(VAR) is predicted to encode a truncated form of TRPV1 consisting of the NH2-terminal 248 residues of TRPV1 (all within the NH2-terminal intracellular domain) followed by five nonconsensus amino acids (Arg-Glu-Ala-Met-Trp) and a stop codon. The variant utilizes the same consensus Kozak sequence as canonical TRPV1. A band of the appropriate molecular mass was identified in rat kidney papillary (but not medullary) lysates immunoblotted with an antibody directed against the NH2 terminus of TRPV1, whereas an antibody recognizing the TRPV1 COOH terminus failed to detect it. Upon heterologous expression in HEK 293 cells, TRPV1(VAR) potentiated the ability of cotransfected TRPV1 to confer calcium influx in response to resiniferatoxin. TRPV1(VAR) did not influence expression or cell surface localization of cotransfected TRPV1. TRPV1(VAR) protein product associated with the NH2 terminus of canonical TRPV1. Interestingly, when expressed in the COS-7 epithelial cell line, TRPV1(VAR) functioned in a dominant-negative acting capacity, partially blocking TRPV1-dependent resiniferatoxin responsiveness. We conclude that TRPV1(VAR) is one of perhaps several TRPV1 splice variants expressed in rat kidney and that it may serve to modulate TRPV1 responsiveness in some tissues.

Alternative Splicing↗
PubMed2005

Cys74 and Cys163 are necessary for IL-18 to elicit IFN-gamma production from peripheral blood lymphoid mononuclear cells.

There are four cysteines (Cys74, Cys104, Cys112 and Cys163) in mature human IL-18 (hIL-18). These cysteines are highly conserved in IL-18s of 11 species cloned so far, suggesting that one or more of the cysteines may be important for hIL-18 function. In this study, each cysteine residue was individually replaced with serine by site-directed mutagenesis. The wild type and mutant IL-18s were expressed in Escherichia coli and renatured by two renaturing methods. The purified wild type and mutant rhIL-18s were assayed for their capacity of inducing IFN-gamma and activating NF-kappaB from ConA-stimulated PBMC. DNA binding activity of NF-kappaB was performed by electrophoretic mobility-shift analysis. Our results showed that the mutant rhIL-18C74S and C163S induced much less amount of IFN-gamma from PBMC and the decrement of NF-kappaB DNA binding activity was also observed from C74S and C163S treated PBMC. These results indicate that functional hIL-18 has an absolute requirement for residues Cys74 and Cys163.

Base Sequence↗
PubMed2005

Single-molecule studies of diffusion by oligomer-bound dyes in organically modified sol-gel-derived silicate films.

Single-molecule fluorescence spectroscopy is used to study dye diffusion within organically modified silicate (ORMOSIL) films. ORMOSIL films are prepared from sols containing tetraethoxysilane and isobutyltrimethoxysilane in 2:1 and 1:9 molar ratios. Nile red and a new silanized form of nile red that can be covalently attached to the silicate matrix are used as fluorescent probe molecules. The number and rate of single molecules diffusing through these films increases dramatically with increasing film organic content. Autocorrelation of the fluorescence images yields a quantitative measure of the relative populations of fixed and diffusing species. Surprisingly, both "free" and silicate-bound nile red exhibit relatively facile translational motions. Single-molecule/single-point fluorescence correlation spectroscopy (FCS) is used to measure the dye diffusion coefficients in submicrometer-scale film regions. The most common diffusion coefficients for "free" and silicate-bound nile red molecules in the 1:9 films are 3.9 x 10(-10) and 1.6 x 10(-10) cm(2)/s, respectively. The unexpectedly rapid diffusion of silicate-bound nile red is attributed to the presence of liquidlike silicate oligomers in the films. A lower bound for the molecular weight of the oligomers is estimated at 2900. Bulk solution-phase FCS experiments performed on "free" and silicate-bound nile red species extracted into chloroform solutions provide valuable support for these conclusions. Comparison of the results derived from experimental and simulated time transients indicates film heterogeneity occurs on sub-100-nm-length scales and likely results from the presence of inorganic- and organic-rich domains.

Journal Article↗
PubMed2005

[The relationship between the five beta-fibrinogen gene polymorphisms and cerebral infarction].

OBJECTIVE: To investigate the relationship among beta-fibrinogen (Fg) concentration, related gene polymorphisms (including -148C/T, -249C/T, -455G/A, 448G/A, 1689T/G) and cerebral infarction. METHODS: Fg level and its five gene polymorphisms were analyzed with by polymerase chain reaction-restriction fragment length polymorphism in 132 patients with cerebral infarction, 79 patients with other neurological diseases and 92 healthy elders. RESULTS: The plasma Fg level in cerebral infarction patients was significantly higher than that in the patients with other neurological diseases or healthy elders (P < 0.05). In the three groups, the plasma Fg levels in individuals with T-148 and A-455 alleles were higher than those in individuals without T-148 and A-455 alleles (P < 0.05). However, there were no statistically significant differences in the genotype and allele frequencies in the five mutation gene polymorphisms among the three groups (P > 0.05). CONCLUSIONS: Cerebral infarction is a multifactorial disease and an increased Fg level is a risk factor for cerebral infarction. T-148 and A-455 allele can lead to elevated Fg concentration.

Aged↗
PubMed2004

Single-molecule spectroscopy studies of microenvironmental acidity in silicate thin films.

Single-molecule (SM) spectroscopic methods were employed to study single site variations in the acidity properties of sol-gel-derived silicate films. The pH-sensitive dye Carboxy SNARF-1 (C.SNARF-1) was used to sense film acidity. Its concentration in the films was maintained at nanomolar levels to allow for SMs to be spectroscopically interrogated. The ratio of C.SNARF-1 fluorescence at 580 nm (protonated form) and 640 nm (deprotonated form) was used to characterize local film pH. SM data were acquired both for "untreated" films and for those treated by immersion for either 1 or 8 h in phosphate solutions of different pH. The SM results prove that the spectral variability observed is dominated by static variations in the local matrix acidity. Shorter immersion times lead to relatively broad histograms and broad "titration" curves, providing clear evidence for kinetic limitations to access of certain film environments by the immersion solutions. Films subjected to longer immersion times generally exhibit narrower histograms. Particularly narrow distributions were obtained for films treated near pH 8-9, while much broader histograms were produced near pH 7. These results are attributed to the buffering effects of surface silanols near pH 9 and enhanced pH sensitivity of the dye near pH 7.

Journal Article↗
PubMed2004

Human parvovirus B19 transgenic mice become susceptible to polyarthritis.

Human parvovirus B19 (B19) often causes acute polyarthritis in adults. In this paper, we analyzed nucleotide sequences of the B19 genome of patients with rheumatoid arthritis (RA), and then introduced the nonstructural protein 1 (NS1) gene of B19 into C57BL/6 mice that had a genetic origin not susceptible to arthritis. The transgenic mice developed no lesions spontaneously, but were susceptible to type II collagen (CII)-induced arthritis. B19 NS1 was expressed in synovial cells on the articular lesions that were histologically characteristic of granulomatous synovitis and pannus formation in cartilage and bone. Serum levels of anti-CII Abs and TNF-alpha increased in NS1 transgenic mice to the same levels as those of DBA/1 mice, which were susceptible to polyarthritis. Stimulation with CII increased secretion of Th1-type- and Th2-type cytokines in NS1 transgenic mice, indicating that a nonpermissive H-2(b) haplotype in the wild type of C57BL/6 mice can be made susceptible to polyarthritis through the expression of NS1. This study is the first to show that a viral agent from the joints in humans can cause CII-induced arthritis resembling RA.

Animals↗
PubMed2004

Integrin beta1 subunit overexpressed in the SMMC-7721 cells regulates the promoter activity of p21(CIP1) and enhances its transcription.

Evidence has been emerging to suggest that integrin could induce growth inhibition in some cell types. Some of the molecular mechanisms underlying growth arrest have been elucidated. We reported here that overexpression of integrin beta1 imposed a growth inhibitory effect on the hepatocellular carcinoma cell line SMMC-7721, and this phenomenon was mainly attributed to the cyclin-dependent kinase inhibitor p21(CIP1). Furthermore, our findings suggested that transcription activity of the p21(CIP1) gene could be upregulated in the integrin beta1-overexpressing cells, and possibly controlled by the cis-elements in the core region of the p21(CIP1) promoter.

Base Sequence↗
PubMed2004

[Assessment and intervention on mental burden of the caregivers of dementia patients].

Along with the aging of population, dementia became a social problem disturbing our life. Western researches focused on caregivers found that, the more severe the dementiais, the more burdens on caregivers. Furthermore, considening the different relationship to the patient, different culture background and different types of dementia, there are different problems in care. Based on comprehensive assessments on the demented and the caregiver, specific health education and other assistant services may improve both the mental and physical health of the caregivers, and reduce the disease burden due to dementia.

Caregivers↗
PubMed2004

[Study on the risk factors of schistosomiasis transmission and control strategy in the Three Gorges Reservoir Areas].

OBJECTIVE: To understand the risk factors of schistosomiasis transmission in the Three Gorges Reservoir Area (TGRA) and to provide evidence for the development of control strategy. METHODS: Approaches including epidemiology, immunology and field survey were applied to investigate the potential risk factors which would involve the importation of infectious resources live mobile and migrant population, and livestock in the reservoir area. Meanwhile, observation on survival and reproductive status of snail under simulation habitats was also carried out, using ecological methods on snails. Strategy in preventing the spread of snail as infectious resources was also provided. RESULTS: 175 mobile people from schistosomaisis endemic area of were tested and one person showed immunology tests positive with indirect hemagglutination test (IHA) and circumoral precipitin test (COPT), with a positive rate of 0.57%. Through the two-year period under observation, data showed that the snails with ribbon/smooth shells could survive and reproduce under habitats of simulation. CONCLUSIONS: Once the infectious resource of schistosomiasis was introduced into the TGRA, the area became a new schistosomiasis epidemic area in TGRA which called for countermeasures to be taken.

Animals↗
PubMed2003

Quartz crystal microbalance sensor for organic vapor detection based on molecularly imprinted polymers.

Molecularly imprinted polymers on quartz crystal microbalances (QCM) are examined for their ability to detect vapors of small organic molecules with greater sensitivity and selectivity than the traditional amorphous polymer coatings. Hydroquinone and phenol serve as noncovalently bound templates that generate shape-selective cavities in a poly(acrylic) or poly(methacrylic) polymer matrix. The imprinted polymers are immobilized on the piezoelectric crystal surface via a precoated poly(isobutylene) layer. The behavior of the imprinted polymer films is characterized by the dynamic and steady-state response of the QCM frequency to pulses of organic vapors in dry air. The apparent partition coefficients are determined for imprinted and nonimprinted polymers prepared by two synthetic methods and for varying mole ratios of template to monomer. The hydroquinone-imprinted polymers and, to a lesser extent, the phenol-imprinted polymers exhibit greater sensitivity and higher selectivity than the nonimprinted polymers toward organic vapors that are structurally related to the templates. These results indicate that molecularly imprinted polymers are promising for the development of selective piezoelectric sensors for organic vapor detection.

Journal Article↗