Biomedical subjects

S Sheng

Publications and source records attributed to S Sheng.

At least 19 recordsLinked to original sources

PubMed2001

Pleiotrophic inhibition of pericellular urokinase-type plasminogen activator system by endogenous tumor suppressive maspin.

Maspin is a novel serine protease inhibitor with tumor suppressive activity, inhibiting tumor invasion and metastasis. To date, the underlying molecular mechanism of maspin remains elusive. Recombinant maspin has been shown to specifically inhibit cell surface-associated urokinase-type plasminogen activator (uPA) and fibrinogen-bound tissue-type plasminogen activator. However, the role of endogenous maspin in plasminogen activation is totally unknown. To address this issue, we generated stable maspin-expressing transfectants using prostate carcinoma cells DU145 as the parental cell line. We report here that endogenous maspin exerts pleiotropic inhibitory effects on the pericellular uPA system. Maspin expression led to a significantly reduced level of cell surface-bound uPA and uPA receptor proteins without altering the steady-state levels of the respective mRNAs. Treatment with receptor-associated protein (RAP), a specific inhibitor of low-density lipoprotein receptor-related protein, lead to a significantly increased level of secreted uPA and cell surface uPAR in maspin transfectants but not in the mock control cells. A combination of enzymatic and molecular analyses revealed that maspin inhibits the cell surface-mediated plasminogen activation by forming an SDS-resistant complex with cell surface-bound uPA. In addition, maspin expression led to a dramatic reduction in the release of active uPA, both high molecular weight and the low molecular weight, into the conditioned culture medium. Consistently, the conditioned medium of maspin transfectant clones had a significantly reduced activity in converting plasminogen to plasmin. The inhibitory effect of maspin on pericellular uPA correlates with significantly decreased cell invasion potential and motility in vitro. The maspin-neutralizing antibody (Abs4A) reversed the subdued invasive potential of maspin transfectant cells in a dose-dependent manner. In summary, this study provides the first evidence that endogenous maspin is a potent inhibitor of pericellular uPA. Furthermore, our results support a current hypothesis that maspin blocks tumor invasion and motility by inhibiting localized pericellular proteolysis.

Cell Membrane↗

PubMed2001

Maspin expression inversely correlates with breast tumor progression in MMTV/TGF-alpha transgenic mouse model.

Maspin is a novel serine protease inhibitor (serpin) with tumor suppressive activity. To date, despite the mounting evidence implicating the potential diagnostic/prognostic and therapeutic value of maspin in breast and prostate carcinoma, the lack of a suitable animal model hampers the in vivo investigation on the role of maspin at different stages of tumor progression. In this study, we used MMTV/TGF-alpha transgenic mouse model to study the expression profile of maspin in mammary tumor progression. Histopathological examinations of MMTV/TGF-alpha transgenic mice revealed TGF-alpha expression leading to hyperproliferation, hyperplasia, and occasional carcinoma in mammary gland. Interestingly, when MMTV/TGF-alpha transgenic mice were breed to homozygocity, they also developed characteristic skin papillomas. Immunohistochemistry analysis of maspin expression in the breast tissues of TGF-alpha transgenic mice showed a direct correlation between down-regulation of maspin expression and tumor progression. The loss of maspin expression was concomitant with the critical transition from carcinoma in situ to invasive carcinoma. Subsequent in-situ hybridization analyses suggest that the down-regulation of maspin expression is primarily a transcriptional event. This data is consistent with the tumor suppressive role of maspin. Furthermore, our data suggests that MMTV/TGF-alpha transgenic mouse model is advantageous for in vivo evaluation of both the expression and the biological function of maspin during the slow multi-stage carcinogenesis of mammary gland.

Animals↗

PubMed2001

Second transmembrane domains of ENaC subunits contribute to ion permeation and selectivity.

Epithelial sodium channels (ENaC) are composed of three structurally related subunits (alpha, beta, and gamma). Each subunit has two transmembrane domains termed M1 and M2, and residues conferring cation selectivity have been shown to reside in a pore region immediately preceding the M2 domains of the three subunits. Negatively charged residues are interspersed within the M2 domains, and substitution of individual acidic residues within human alpha-ENaC with arginine essentially eliminated channel activity in oocytes, suggesting that these residues have a role in ion permeation. We examined the roles of M2 residues in contributing to the permeation pore by individually mutating residues within the M2 domain of mouse alphaENaC to cysteine and systematically characterizing functional properties of mutant channels expressed in Xenopus oocytes by two-electrode voltage clamp. The introduction of cysteine residues at selected sites, including negatively charged residues (alphaGlu(595), alphaGlu(598), and alphaAsp(602)) led to a significant reduction of expressed amiloride-sensitive Na(+) currents. Two mutations (alphaE595C and alphaD602C) resulted in K(+)-permeable channels whereas multiple mutations altered Li(+)/Na(+) current ratios. Channels containing alphaD602K or alphaD602A also conducted K(+) whereas more conservative mutations (alphaD602E and alphaD602N) retained wild type selectivity. Cysteine substitution at the site equivalent to alphaAsp(602) within beta mENaC (betaD544C) did not alter either Li(+)/Na(+) or K(+)/Na(+) current ratios, although mutation of the equivalent site within gamma mENaC (gammaD562C) significantly increased the Li(+)/Na(+) current ratio. Mutants containing introduced cysteine residues at alphaGlu(595), alphaGlu(598), alphaAsp(602), or alphaThr(607) did not respond to externally applied sulfhydryl reagent with significant changes in macroscopic currents. Our results suggest that some residues within the M2 domain of alphaENaC contribute to the channel's conduction pore and that, in addition to the pore region, selected sites within M2 (alphaGlu(595) and alphaAsp(602)) may have a role in conferring ion selectivity.

Amino Acid Sequence↗

PubMed2001

Three-dimensional interaction of Phi29 pRNA dimer probed by chemical modification interference, cryo-AFM, and cross-linking.

Six pRNAs (p for packaging) of bacterial virus phi29 form a hexamer complex that is an essential component of the viral DNA translocating motor. Dimers, the building block of pRNA hexamer, assemble in the order of dimer --> tetramer --> hexamer. The two-dimensional structure of the pRNA monomer has been investigated extensively; however, the three-dimensional structure concerning the distance constraints of the three stems and loops are unknown. In this report, we probed the three-dimensional structure of pRNA monomer and dimer by photo affinity cross-linking with azidophenacyl. Bases 75-81 of the left stem were found to be oriented toward the head loop and proximate to bases 26-31 in a parallel orientation. Chemical modification interference indicates the involvement of bases 45-71 and 82-91 in dimer formation. Dimer was formed via hand-in-hand contact, a novel RNA dimerization that in some aspects is similar to the kissing loops of the human immunodeficiency virus. The covalently linked dimers were found to be biologically active. Both the native dimer and the covalently linked dimer were found by cryo-atomic force microscopy to be similar in global conformation and size.

Bacillus Phages↗

PubMed2001

Epithelial sodium channel pore region. structure and role in gating.

Epithelial sodium channels (ENaC) have a crucial role in the regulation of extracellular fluid volume and blood pressure. To study the structure of the pore region of ENaC, the susceptibility of introduced cysteine residues to sulfhydryl-reactive methanethiosulfonate derivatives ((2-aminoethyl)methanethiosulfonate hydrobromide (MTSEA) and [(2-(trimethylammonium)ethyl]methanethiosulfonate bromide (MTSET)) and to Cd(2+) was determined. Selected mutants within the amino-terminal portion (alphaVal(569)-alphaTrp(582)) of the pore region responded to MTSEA, MTSET, or Cd(2+) with stimulation or inhibition of whole cell Na(+) current. The reactive residues were not contiguous but were separated by 2-3 residues where substituted cysteine residues did not respond to the reagents and line one face of an alpha-helix. The activation of alphaS580Cbetagamma mENaC by MTSET was associated with a large increase in channel open probability. Within the carboxyl-terminal portion (alphaSer(583)-alphaSer(592)) of the pore region, only one mutation (alphaS583C) conferred a rapid, nearly complete block by MTSEA, MTSET, and Cd(2+), whereas several other mutant channels were partially blocked by MTSEA or Cd(2+) but not by MTSET. Our data suggest that the outer pore of ENaC is formed by an alpha-helix, followed by an extended region that forms a selectivity filter. Furthermore, our data suggest that the pore region participates in ENaC gating.

Amiloride↗

PubMed2001

Furanosesquiterpenoids of Commiphora myrrha.

An investigation on the gum exudates of Commiphora myrrha has led to the isolation of six sesquiterpenoids. On the basis of spectroscopic data interpretation, they were determined as two new furanosesquiterpenoids, rel-1S,2S-epoxy-4R-furanogermacr-10(15)-en-6-one (1) and rel-2R-methyl-5S-acetoxy-4R-furanogermacr-1(10)Z-en-6-one (2), and four known furanosesquiterpenoids, rel-3R-methoxy-4S-furanogermacra-1E,10(15)-dien-6-one (3), rel-2R-methoxy-4R-furanogermacr-1(10)E-en-6-one (4), furanogermacra-1(10)Z,4Z-dien-6-one, and curzerenone [6,7-dihydro-5beta-isopropenyl-3,6beta-dimethyl-6-vinylbenzofuran-4(5H)-one]. This is the first report of the relative stereochemistry for the known compounds 3 and 4. Compound 1 exhibited weak cytotoxic activity against a MCF-7 breast tumor cell line in a clonogenic assay, while the other five compounds were inactive in this assay.

Antineoplastic Agents, Phytogenic↗

PubMed2001

Analysis of urinary metabolites of tea catechins by liquid chromatography/electrospray ionization mass spectrometry.

Tea has been proposed to have beneficial health effects which have been attributed to the polyphenolic compounds known as catechins. The bioavailability and biotransformation of these compounds, however, are not clearly understood. In this study, we used liquid chromatography/electrospray ionization-mass spectrometry (LC/ESI-MS) to determine urinary glucuronidated and sulfated tea catechins and their metabolites (including methylated and ring-fission metabolites) based on the detection of deprotonated molecular ions and aglycone fragment ions. The compound resolution was achieved both chromatographically and mass spectroscopically. After green tea administration, the major conjugates appeared in human, mouse, and rat urine samples were identified as monoglucuronides and monosulfates of (-)-epigallocatechin (EGC) and (-)-epicatechin. We also found O-methyl-EGC-O-glucuronides and -O-sulfates and O-methyl-epicatechin-O-sulfates in human urine. (-)-5-(3',4',5'-Trihydroxyphenyl)-gamma-valerolactone (M4) and (-)-5-(3',4'-dihydroxyphenyl)-gamma-valerolactone (M6), the ring-fission metabolites of EGC and (-)-epicatechin, respectively, were also predominantly in monoglucuronide and monosulfate forms in the urine. In comparison to rats, the urinary metabolite profiles of tea catechins in mice resemble more closely to those in humans. This is the first report describing direct simultaneous analysis of multiple tea catechin conjugates in urine samples. This method will allow more thorough investigations of the biotransformation of tea polyphenols.

Adult↗

PubMed2001

The urokinase-type plasminogen activator system in prostate cancer metastasis.

Accumulated clinical and experimental evidence indicates that the urokinase-type plasminogen activator (uPA) and its regulators are causatively involved in the metastatic phenotype of many types of cancers. In the past couple of decades, investigation on the role of the uPA system in human prostate cancer (PC) has been intensified and has yielded valuable insights. This review summarizes recent advances made in several areas regarding the clinical relevance, the function and the molecular mechanisms of the uPA system in PC metastasis. A current consensus suggests that the uPA system promotes PC metastasis by mediating pericellular plasminogen activation. Towards the development of therapeutic strategies that specifically target uPA-mediated PC metastasis, several remaining issues are discussed.

Humans↗

PubMed2001

Epithelial Na(+) channels are regulated by flow.

Na(+) absorption in the renal cortical collecting duct (CCD) is mediated by apical epithelial Na(+) channels (ENaCs). The CCD is subject to continuous variations in intraluminal flow rate that we speculate alters hydrostatic pressure, membrane stretch, and shear stress. Although ENaCs share limited sequence homology with putative mechanosensitive ion channels in Caenorhabditis elegans, controversy exists as to whether ENaCs are regulated by biomechanical forces. We examined the effect of varying the rate of fluid flow on whole cell Na(+) currents (I(Na)) in oocytes expressing mouse alpha,beta,gamma-ENaC (mENaC) and on net Na(+) absorption in microperfused rabbit CCDs. Oocytes injected with mENaC but not water responded to the initiation of superfusate flow (to 4-6 ml/min) with a reversible threefold stimulation of I(Na) without a change in reversal potential. The increase in I(Na) was variable among oocytes. CCDs responded to a threefold increase in rate of luminal flow with a twofold increase in the rate of net Na(+) absorption. An increase in luminal viscosity achieved by addition of 5% dextran to the luminal perfusate did not alter the rate of net Na(+) absorption, suggesting that shear stress does not influence Na(+) transport in the CCD. In sum, our data suggest that flow stimulation of ENaC activity and Na(+) absorption is mediated by an increase in hydrostatic pressure and/or membrane stretch. We propose that intraluminal flow rate may be an important regulator of channel activity in the CCD.

Amiloride↗

PubMed2001

Formation and identification of 4'-O-methyl-(-)-epigallocatechin in humans.

The possible beneficial effects of tea consumption have attracted a great deal of attention. Many of the biological effects have been attributed to tea catechins, but the metabolic fate of these compounds is not clear. In the present study, a major metabolite observed in human blood and urine samples after green tea administration was identified as a O-methylated derivative of (-)-epigallocatechin (EGC) by comparison with products from chemical and enzymatic O-methylation of EGC. The structure of this metabolite was elucidated as 4'-O-methyl-(-)-epigallocatechin (4'-O-MeEGC) by (1)H and (13)C NMR and heteronuclear multiple bond connectivity experiment. The human plasma level of 4'-O-MeEGC reached its peak value within the first 2 h following tea ingestion. Its maximum concentration was 4 to 6 times higher than that of EGC. The half-lives of EGC and 4'-O-MeEGC in the blood were 1.02 +/- 0.07 and 4.39 +/- 1.14 h, respectively. The amount of 4'-O-MeEGC excreted in urine was about 3 times higher than that of EGC, and 88% of 4'-O-MeEGC was excreted in urine within 8 h. The present structural information and concentration-time profile of this metabolite provide the basis for understanding the biotransformation of EGC and for future elucidation of its biological activities.

Catechin↗

PubMed2001

[Effects of seed coating formulation on seedling characters of Atractylodes macrocephala].

The effects of seed coating formulation (SCF) of Atractylodes macrocephala Koidz. on seedling in two experimental plots located in Hangzhou and Jiande were studied, and desinged with L9(3(4)) orthogonal comparison. The results showed that the SCF with paclobutrazol (pp333) had a significant effect on the rate of germination at Hangzhou plot, all factors had no significant effects on length of seedlings in both plots, and both atonik (sodium O-nitrophenolate) and PP333 on the fresh or dry seedling weight had significant effects.

Asteraceae↗

PubMed2000

The surface of prostate carcinoma DU145 cells mediates the inhibition of urokinase-type plasminogen activator by maspin.

Maspin is a novel serine protease inhibitor (serpin) with tumor suppressive potential in breast and prostate cancer, acting at the level of tumor invasion and metastasis. It was subsequently demonstrated that maspin inhibits tumor invasion, at least in part, by inhibiting cell motility. Interestingly, in cell-free solutions, maspin does not inhibit several serine proteases including tissue-type plasminogen activator and urokinase-type plasminogen activator (uPA). Despite the recent biochemical evidence that maspin specifically inhibits tissue-type plasminogen activator that is associated with fibrinogen or poly-L-lysine, the molecular mechanism underlying the tumor-suppressive effect of maspin remains elusive. The goal of this study was to investigate the effect of maspin on cell surface-associated uPA. In our experimental system, we chose prostate carcinoma DU145 cells because these cells mediate plasminogen activation primarily by uPA, as shown by two different colorimetric enzyme activity assays. Purified recombinant maspin produced in baculovirus-infected Spodoptera frugiperda Sf9 insect cells [rMaspin(i)] binds specifically to the surface of DU145 cells, inhibits the DU145 cell surface-bound uPA, and forms a stable complex with the uPA in DU145 cell lysate. The inhibitory effect of rMaspin(i) on cell surface-bound uPA was similar to that of an uPA-neutralizing antibody and was reversed by a polyclonal antibody against the reactive site loop sequence of maspin. The Ki value for rMaspin(i) in cell surface-mediated plasminogen activation was 20 nM, which was comparable to the Ki values for plasminogen activator inhibitor 1 and plasminogen activator inhibitor 2, respectively. Furthermore, the proteolytic inhibitory effect of rMaspin(i) was quantitatively consistent with its inhibitory effect on the motility of DU145 cells in vitro. Our data demonstrate an important role for the prostate carcinoma cell surface in mediating the inhibitory interaction between rMaspin(i) and uPA. Thus, future maspin-based therapeutic strategies may prove useful in blocking the invasion and metastasis of uPA-positive prostate carcinoma.

Antineoplastic Agents↗

PubMed2000

A dimer as a building block in assembling RNA. A hexamer that gears bacterial virus phi29 DNA-translocating machinery.

Six RNA (pRNA) molecules form a hexamer, via hand-in-hand interaction, to gear bacterial virus phi29 DNA translocation machinery. Here we report the pathway and the conditions for the hexamer formation. Stable pRNA dimers and trimers were assembled in solution, isolated from native gels, and separated by sedimentation, providing a model system for the study of RNA dimers and trimers in a protein-free environment. Cryo-atomic force microscopy revealed that monomers displayed a check mark outline, dimers exhibited an elongated shape, and trimers formed a triangle. Dimerization of pRNA was promoted by a variety of cations including spermidine, whereas procapsid binding and DNA packaging required specific divalent cations, including Mg(2+), Ca(2+), and Mn(2+). Both the tandem and fused pRNA dimers with complementary loops designed to form even-numbered rings were active in DNA packaging, whereas those without complementary loops were inactive. We conclude that dimers are the building blocks of the hexamer, and the pathway of building a hexamer is: dimer --> tetramer --> hexamer. The Hill coefficient of 2.5 suggests that there are three binding sites with cooperative binding on the surface of the procapsid. The two interacting loops played a key role in recruiting the incoming dimer, whereas the procapsid served as the foundation for hexamer assembly.

Bacteriophages↗

PubMed2000

Characterization of the selectivity filter of the epithelial sodium channel.

The epithelial sodium channel (ENaC) is composed of three homologous subunits termed alpha, beta, and gamma. Previous studies suggest that selected residues within a hydrophobic region immediately preceding the second membrane-spanning domain of each subunit contribute to the conducting pore of ENaC. We probed the pore of mouse ENaC by systematically mutating all 24 amino acids within this putative pore region of the alpha-subunit to cysteine and co-expressing these mutants with wild type beta- and gamma-subunits of mouse ENaC in Xenopus laevis oocytes. Functional characteristics of these mutants were examined by two-electrode voltage clamp and single channel recording techniques. Two distinct domains were identified based on the functional changes associated with point mutations. An amino-terminal domain (alpha-Val(569)-alpha-Gly(579)) showed minimal changes in cation selectivity or amiloride sensitivity following cysteine substitution. In contrast, cysteine substitutions within the carboxyl-terminal domain (alpha-Ser(580)-alpha-Ser(592)) resulted in significant changes in cation selectivity and moderately altered amiloride sensitivity. The mutant channels containing alphaG587C or alphaS589C were permeable to K(+), and mutation of a GSS tract (positions alpha587-alpha589) to GYG resulted in a moderately K(+)-selective channel. Our results suggest that the C-terminal portion of the pore region within the alpha-subunit contributes to the selectivity filter of ENaC.

Amiloride↗

PubMed2000

Thermal noise reduction of mechanical oscillators by actively controlled external dissipative forces

We show that the thermal fluctuations of very soft mechanical oscillators, such as the cantilever in an atomic force microscope (AFM), can be reduced without changing the stiffness of the spring or having to lower the environment temperature. We derive a theoretical relationship between the thermal fluctuations of an oscillator and an actively controlled external dissipative force. This relationship is verified by experiments with an AFM cantilever where the external active force is coupled through a magnetic field. With simple instrumentation, we have reduced the thermal noise amplitude of the cantilever by a factor of 3.4, achieving an apparent temperature of 25 K with the environment at 295 K. This active noise reduction approach can significantly improve the accuracy of static position or static force measurements in a number of practical applications.

Journal Article↗

PubMed2000

Probing the structure of monomers and dimers of the bacterial virus phi29 hexamer RNA complex by chemical modification.

All dsDNA viruses multiply their genome and assemble a procapsid, a protein shell devoid of DNA. The genome is subsequently inserted into the procapsid. The bacterial virus phi29 DNA translocating motor contains a hexameric RNA complex composed of six pRNAs. Recently, we found that pRNA dimers are building blocks of pRNA hexamers. Here, we report the structural probing of pRNA monomers and dimers by chemical modification under native conditions and in the presence or absence of Mg2+. The chemical-modification pattern of the monomer is compared to that of the dimer. The data strongly support the previous secondary-structure prediction of the pRNA concerning the single-stranded areas, including three loops and seven bulges. However, discrepancies between the modification patterns of two predicted helical regions suggest the presence of more complicated, higher-order structure in these areas. It was found that dimers were formed via hand-in-hand and head-to-head contact, as the interacting sequence of the right and left loops and all bases in the head loop were protected from chemical modification. Cryoatomic force microscopy revealed that the monomer displayed a check-mark shape and the dimer exhibited an elongated shape. The dimer was twice as long as the monomer. Direct observation of the shape and measurement of size and thickness of the images strongly support the conclusion from chemical modification concerning the head-to-head contact in dimer formation. Our results also suggest that the role for Mg2+ in pRNA folding is to generate a proper configuration for the right and head loops, which play key roles in this symmetrical head-to-head organization. This explains why Mg2+ plays a critical role in pRNA dimer formation, procapsid binding, and phi29 DNA packaging.

Bacillus Phages↗

PubMed2000

Structural identification of two metabolites of catechins and their kinetics in human urine and blood after tea ingestion.

Tea is a popular beverage consumed worldwide. The metabolic fate of its major constituents, catechins, however, is not well-known. In this study, two catechin metabolites were detected in the urine and plasma of human volunteers after ingestion of green tea. These metabolites were identified by LC/ESI-MS and NMR as (-)-5-(3',4', 5'-trihydroxyphenyl)-gamma-valerolactone (M4) and (-)-5-(3', 4'-dihydroxyphenyl)-gamma-valerolactone (M6). The renal excretion of M4 and M6 had a 3 h lag time and peaked 7.5-13.5 h after ingestion of a single dose of green tea, while (-)-epigallocatechin (EGC) and (-)-epicatechin peaked at 2 h. M4 and M6 were two major tea metabolites with urinary cumulative excretions as high as 8-25 times the levels of EGC and (-)-epicatechin in some of our subjects, and accounted for 6-39% of the amounts of ingested EGC and (-)-epicatechin. Both the metabolites appeared to be produced by intestinal microorganisms, with EGC and (-)-epicatechin as the precursors of M4 and M6, respectively. Repeated ingestion of green tea produced a slight accumulative effect of the metabolites. They were also detected in the plasma, exhibiting kinetics similar to those of the urinary metabolites, and in the feces. Study on these metabolites may help us further understand the cancer chemopreventive actions and other beneficial effects of tea.

Adult↗

PubMed2000

PNA-dependent gene chemistry: stable coupling of peptides and oligonucleotides to plasmid DNA.

Two approaches are described for stably conjugating peptides, proteins and oligonucleotides onto plasmid DNA. Both methods use a peptide nucleic acid (PNA) clamp, which binds irreversibly and specifically to a binding site cloned into the plasmid. The first approach uses a biotin-conjugated PNA clamp that can be used to introduce functional biotin groups onto the plasmid to which streptavidin can bind. Atomic force microscopy images of linearized plasmid show streptavidin localized at the predicted PNA binding site on the DNA strand. Peptides and oligonucleotides containing free thiol groups were conjugated to maleimide streptavidin, and these streptavidin conjugates were bound to the biotin-PNA-labeled plasmid. In this way, peptides and oligonucleotides could be brought into stable association with the plasmid. A second approach used a maleimide-conjugated PNA clamp. Methods are described for conjugating thiolated peptides and oligonucleotides directly to the maleimide-PNA-DNA hybrid. This straightforward technology offers an easy approach to introduce functional groups onto plasmid DNA without disturbing its transcriptional activity.

Animals↗