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Biomedical subjects

Y Zheng

Publications and source records attributed to Y Zheng.

At least 19 recordsLinked to original sources
PubMed2000

Structure of the BH domain from graf and its implications for Rho GTPase recognition.

Cellular signaling by small G-proteins is down-regulated by GTPase-activating proteins (GAPs), which increase the rate of GTP hydrolysis. The GTPase regulator associated with focal adhesion kinase (Graf) exhibits GAP activity toward the RhoA and Cdc42 GTPases, but is only weakly active toward the closely related Rac1. We determined the crystal structure of a 231-residue fragment of Graf (GrafGAP), a domain containing the GAP activity, at 2.4-A resolution. The structure clarifies the boundaries of the functional domain and yields insight to the mechanism of substrate recognition. Modeling its interaction with substrate suggested that a favorable interaction with Glu-95 of Cdc42 (Glu-97 of RhoA) would be absent with the corresponding Ala-95 of Rac1. Indeed, GrafGAP activity is diminished approximately 40-fold toward a Cdc42 E95A mutant, whereas a approximately 10-fold increase is observed for a Rac1 A95E mutant. The GrafGAP epitope that apparently interacts with Glu-95(Glu-97) contains Asn-225, which was recently found mutated in some myeloid leukemia patients. We conclude that position 95 of the GTPase is an important determinant for GrafGAP specificity in cellular function and tumor suppression.

Amino Acid Sequence↗
PubMed2000

The effects of L-NAME on vestibular compensation and NOS activity in the vestibular nucleus, cerebellum and cortex of the guinea pig.

Nitric oxide (NO) has been implicated in the processes by which animals recover from peripheral vestibular damage ('vestibular compensation'). However, few data exist on the dose-response effects of systemic administration of the nitric oxide synthase (NOS) inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), on the vestibular compensation process. The aim of this study was to investigate the effects on compensation of 5, 10, 50 or 100 mM L-NAME administered by s.c osmotic minipump for 50 h following unilateral vestibular deafferentation (UVD) in guinea pig, either commencing the drug treatment at 4 h pre-UVD or at the time of the UVD (i.e., post-UVD). Post-UVD treatment with L-NAME, at any of the four concentrations used, had no effect on the compensation of spontaneous nystagmus (SN), yaw head tilt (YHT) or roll head tilt (RHT). By contrast, pre-UVD treatment with 100 mM L-NAME resulted in a significant decrease in SN frequency (P<0.05) and a change in the rate of its compensation (P<0.0005). Pre-UVD L-NAME resulted in a significant increase in the overall magnitude of YHT (P<0.005); however, post-hoc comparisons revealed no significant differences between any specific L-NAME and vehicle groups. Pre-UVD L-NAME had no effect on RHT at any concentration. Analysis of NOS activity in the pre-UVD L-NAME treatment groups at 50 h post-UVD showed that only 100 mM L-NAME resulted in a significant decrease in NOS activity in the contralateral medial vestibular nucleus (MVN)/prepositus hypoglossi (PH) (P<0.05) and that NOS activity in the ipsilateral MVN/PH was not significantly affected. However, NOS activity was significantly inhibited in the bilateral cerebellum and cortices for several concentrations of L-NAME. These results suggest that pre-UVD systemic administration of L-NAME can significantly increase the rate of SN compensation in guinea pig and that this effect is correlated with inhibition of NOS activity in several regions of the CNS.

Animals↗
PubMed2000

Dominant negative mutation of the hematopoietic-specific Rho GTPase, Rac2, is associated with a human phagocyte immunodeficiency.

Rho GTPases control a variety of cellular processes, including actin polymerization, integrin complex formation, cell adhesion, gene transcription, cell cycle progression, and cell proliferation. A patient is described who has recurrent infections and defective neutrophil cellular functions similar to those found in Rac2-deficient mice. Molecular methods were used to clone the expressed Rac2 cDNA from this patient, and a single base pair change (G-->A at nucleotide 169) in the coding sequence was identified. This results in an asparagine for aspartic acid mutation at amino acid 57 (D57N), a residue that is involved in nucleotide binding and is conserved in all mammalian Rho GTPases. The cloned cDNA was then introduced into normal bone marrow cells through retrovirus vectors, and neutrophils expressing this mutant exhibited decreased cell movement and production of superoxide in response to fMLP. The expressed recombinant protein was also analyzed biochemically and exhibited defective binding to GTP. Functional studies demonstrated that the D57N mutant behaves in a dominant-negative fashion at the cellular level. The syndrome of Rac2 dysfunction represents a human condition associated with mutation of a Rho GTPase and is another example of human disease associated with abnormalities of small G protein signaling pathways. (Blood. 2000;96:1646-1654)

3T3 Cells↗
PubMed2000

Identification of Rho GTPase-dependent sites in the Dbl homology domain of oncogenic Dbl that are required for transformation.

The Dbl family guanine-nucleotide exchange factors (GEFs) for Rho GTPases share the structural array of a Dbl homology (DH) domain in tandem with a Pleckstrin homology (PH) domain. For oncogenic Dbl, the DH domain is responsible for the GEF activity, and the DH-PH module constitutes the minimum structural unit required for cellular transformation. To understand the structure-function relationship of the DH domain, we have investigated the role of specific residues of the DH domain of Dbl in interaction with Rho GTPases and in Dbl-induced transformation. Alanine substitution mutagenesis identified a panel of DH mutants made in the alpha1, alpha6, and alpha9 regions and the PH junction site that suffer complete or partial loss of GEF activity toward Cdc42 and RhoA. Kinetic and binding analysis of these mutants revealed that although most displayed decreased k(cat) values in the GEF reaction, the substrate binding activities of T506A and R634A were significantly reduced. E502A, Q633A, and N673A/D674A, on the other hand, retained the binding capability to the Rho GTPases but lost the GEF catalytic activity. In general, the in vitro GEF activity of the DH mutants correlated with the in vivo Cdc42- and RhoA-activating potential, and the GEF catalytic efficiency mirrored the transforming activity in NIH 3T3 cells. Moreover, the N673A/D674A mutant exhibited a potent dominant-negative effect on serum-induced cell growth and caused retraction of actin structures. These studies identify important sites of the DH domain involved in binding or catalysis of Rho proteins and demonstrate that maintaining a threshold of GEF catalytic activity, in addition to the Rho GTPase binding activity, is essential for efficient transformation by oncogenic Dbl.

3T3 Cells↗
PubMed2000

The role of Mg2+ cofactor in the guanine nucleotide exchange and GTP hydrolysis reactions of Rho family GTP-binding proteins.

The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cells. Here we have investigated the role of Mg(2+) cofactor in the guanine nucleotide binding and hydrolysis processes of the Rho family members, Cdc42, Rac1, and RhoA. Differing from Ras and Rab proteins, which require Mg(2+) for GDP and GTP binding, the Rho GTPases bind the nucleotides in the presence or absence of Mg(2+) similarly, with dissociation constants in the submicromolar concentration. The presence of Mg(2+), however, resulted in a marked decrease in the intrinsic dissociation rates of the nucleotides. The catalytic activity of the guanine nucleotide exchange factors (GEFs) appeared to be negatively regulated by free Mg(2+), and GEF binding to Rho GTPase resulted in a 10-fold decrease in affinity for Mg(2+), suggesting that one role of GEF is to displace bound Mg(2+) from the Rho proteins. The GDP dissociation rates of the GTPases could be further stimulated by GEF upon removal of bound Mg(2+), indicating that the GEF-catalyzed nucleotide exchange involves a Mg(2+)-independent as well as a Mg(2+)-dependent mechanism. Although Mg(2+) is not absolutely required for GTP hydrolysis by the Rho GTPases, the divalent ion apparently participates in the GTPase reaction, since the intrinsic GTP hydrolysis rates were enhanced 4-10-fold upon binding to Mg(2+), and k(cat) values of the Rho GTPase-activating protein (RhoGAP)-catalyzed reactions were significantly increased when Mg(2+) was present. Furthermore, the p50RhoGAP specificity for Cdc42 was lost in the absence of Mg(2+) cofactor. These studies directly demonstrate a role of Mg(2+) in regulating the kinetics of nucleotide binding and hydrolysis and in the GEF- and GAP-catalyzed reactions of Rho family GTPases. The results suggest that GEF facilitates nucleotide exchange by destabilizing both bound nucleotide and Mg(2+), whereas RhoGAP utilizes the Mg(2+) cofactor to achieve high catalytic efficiency and specificity.

Dose-Response Relationship, Drug↗
PubMed2000

Pseudo-random procedures for rapid presentation rates using optical imaging and spectroscopy.

Optical imaging of rat barrel cortex has provided detailed spatio-temporal maps of functional cortical architecture. We describe an event-related procedure (ERP) for optical imaging based on selective signal averaging as reported by Burock et al., using an anti-correlative pseudo-random event sequence. The sequence used 1 s vibrissal stimulation at 5 Hz, with an interevent interval of 2 s. This rapid presentation rate allows for greater statistical power per unit time, and allows for direct comparison of event-related studies with other imaging modalities. The spatio-temporal characteristics of single wavelength and spectrographic results were found to be comparable with those obtained by standard techniques, although a general lessening of haemodynamic response function (HRF) was noted. We also describe a method of locating barrel activity by spectral analysis of summed event data. Using this technique, the power spectrum of remitted light from the barrel region was found to peak within +/- 0.12 Hz of the inter-event interval frequency.

Algorithms↗
PubMed2000

5-methylcytosine-DNA glycosylase activity is present in a cloned G/T mismatch DNA glycosylase associated with the chicken embryo DNA demethylation complex.

We previously have shown that DNA demethylation by chicken embryo 5-methylcytosine DNA glycosylase (5-MCDG) needs both RNA and proteins. One of these proteins is a RNA helicase. Further peptides were sequenced, and three of them are identical to the mammalian G/T mismatch DNA glycosylase. A 3,233-bp cDNA coding for the chicken homologue of human G/T mismatch DNA glycosylase was isolated and sequenced. The derived amino acid sequence (408 aa) shows 80% identity with the human G/T mismatch DNA glycosylase, and both the C and N-terminal parts have about 50% identity. As for the highly purified chicken embryo DNA demethylation complex the recombinant protein expressed in Escherichia coli has both G/T mismatch and 5-MCDG activities. The recombinant protein has the same substrate specificity as the chicken embryo 5-MCDG where hemimethylated DNA is a better substrate than symmetrically methylated CpGs. The activity ratio of G/T mismatch and 5-MCDG is about 30:1 for the recombinant protein expressed in E. coli and 3:1 for the purified enzyme from chicken embryos. The incubation of a recombinant CpG-rich RNA isolated from the purified DNA demethylation complex with the recombinant enzyme strongly inhibits G/T mismatch glycosylase while slightly stimulating the activity of 5-MCDG. Deletion mutations indicate that G/T mismatch and 5-MCDG activities share the same areas of the N- and C-terminal parts of the protein. In reconstitution experiments RNA helicase in the presence of recombinant RNA and ATP potentiates the activity of 5-MCDG.

Amino Acid Sequence↗
PubMed2000

The effects of cyclic loading on pull-out strength of sacral screw fixation: an in vitro biomechanical study.

STUDY DESIGN: The pull-out strength of sacral screw fixation after cyclic loading was tested using young human cadaveric specimens. OBJECTIVES: To evaluate the effects of fatigue loading on the pull-out strength of medial and lateral unicortical and bicortical sacral screws and to correlate the pull-out strength with sacral bone density and the screw insertion torque. SUMMARY OF BACKGROUND DATA: The immediate biomechanical effects of depth of penetration, screw orientation, and bone density on sacral screw fixation have been studied in aged cadaveric specimens. The effect of cyclic loading on the pull-out strength of sacral screw fixation is unknown, however, and data from young specimens is rare. METHODS: Eleven fresh specimens of human sacrum were used in this study. Bone mineral density at the vertebral body and the ala were determined by peripheral quantitative computed tomography. Seven-millimeter compact Cotrel-Dubousset sacral screws were inserted into the sacrum anteromedially and anterolaterally, both unicortically and bicortically, and the insertion torque for each screw was measured. Cyclic loading from 40 to 400 N was applied to each screw at a frequency of 2 Hz up to 20,000 cycles. Pull-out tests were conducted after completion of the fatigue tests. RESULTS: The average bone density was 0.38 +/- 0.08 g/mL at the S1 body and 0.24 +/- 0.05 g/mL at the S1 ala. The insertion torque and average pull-out force after cyclic loading were significantly higher for bicortical fixation than for unicortical fixation for a particular screw alignment. The pull-out strength and insertion torque of medially oriented fixation was always higher than that for lateral fixation, however, regardless of whether the insertion was unicortical or bicortical. The pull-out force of unicortical and bicortical medial screw fixations after cyclic loading showed significant linear correlations with both the insertion torque and the bone mineral density of the S1 body. CONCLUSIONS: In a young population, screw orientation (anterolateral or anteromedial) was more important in determining pull-out strength than screw depth (unicortical or bicortical) after fatigue loading, anteromedially directed screws being significantly stronger than laterallyplaced screws. Bone mineral density of the S1 body andinsertion torque were good preoperative and intraoperative indicators of screw pull-out strength.

Adult↗
PubMed2000

Genetic deletion of the Pten tumor suppressor gene promotes cell motility by activation of Rac1 and Cdc42 GTPases.

Pten (Phosphatase and tensin homolog deleted on chromosome 10) is a recently identified tumor suppressor gene which is deleted or mutated in a variety of primary human cancers and in three cancer predisposition syndromes [1]. Pten regulates apoptosis and cell cycle progression through its phosphatase activity on phosphatidylinositol (PI) 3,4,5-trisphosphate (PI(3,4,5)P(3)), a product of PI 3-kinase [2-5]. Pten has also been implicated in controlling cell migration [6], but the exact mechanism is not very clear. Using the isogenic Pten(+/+) and Pten(-/-) mouse fibroblast lines, here we show that Pten deficiency led to increased cell motility. Reintroducing the wild-type Pten, but not the catalytically inactive Pten C124S or lipid-phosphatase-deficient Pten G129E mutant, reduced the enhanced cell motility of Pten-deficient cells. Moreover, phosphorylation of the focal adhesion kinase p125(FAK) was not changed in Pten(-/-) cells. Instead, significant increases in the endogenous activities of Rac1 and Cdc42, two small GTPases involved in regulating the actin cytoskeleton [7], were observed in Pten(-/-) cells. Overexpression of dominant-negative mutant forms of Rac1 and Cdc42 reversed the cell migration phenotype of Pten(-/-) cells. Thus, our studies suggest that Pten negatively controls cell motility through its lipid phosphatase activity by down-regulating Rac1 and Cdc42.

Animals↗
PubMed2000

Lead targets GRP78, a molecular chaperone, in C6 rat glioma cells.

Exposure to potentially neurotoxic levels of lead (Pb) occurs in about 9% of American children under 6 years of age. Astroglia in the brain serve as a Pb depot, sequestering Pb and preventing its contact with the more sensitive neurons. Astroglia have the capacity to adapt to Pb exposure, and as such are able to tolerate relatively high intracellular Pb accumulation. This tolerance mechanism has yet to be defined in biochemical terms. In the present study, we present evidence that glucose-regulated protein (GRP78), a molecular chaperone in the ER, participates directly or indirectly in the tolerance mechanism. Exposure of cultured C6 rat glioma cells, an astroglia-like cell line, to 1 microM Pb acetate for 1 week raised the intracellular levels of two proteins, one of which was identified by sequence analysis as GRP78. GRP78 accumulation started within 1 day and progressed with time of exposure. Studies in vitro showed that GRP78 bound tightly to affinity columns with Pb(2+) as the affinity ligand and bound weakly when either Zn(2+) or Ni(2+) replaced the Pb(2+). The reduced form of GSH and BSA did not compete with GRP78 to chelate Pb(2+). However, the heavy metal binding domain (HMB) of Menkes protein competed with GRP78 for chelating Pb(2+). The data provide evidence that GRP78 may be a component of the Pb tolerance mechanism through its direct interaction with Pb(2+). Its increased synthesis could be part of the adaptive response to Pb exposure.

Amino Acid Sequence↗
PubMed2000

C5 cytosine methylation at CpG sites enhances sequence selectivity of mitomycin C-DNA bonding.

We have established that UvrABC nuclease is equally efficient in cutting mitomycin C (MC)-DNA monoadducts formed at different sequences and that the degree of UvrABC cutting represents the extent of drug-DNA bonding. Using this method we determined the effect of C5 cytosine methylation on the DNA monoalkylation by MC and the related analogues N-methyl-7-methoxyaziridinomitosene (MS-NMA) and 10-decarbamoylmitomycin C (DC-MC). We have found that C5 cytosine methylation at CpG sites greatly enhances MC and MS-NMA DNA adduct formation at those sites while reducing adduct formation at non-CpG sequences. In contrast, although DC-MC DNA bonding at CpG sites is greatly enhanced by CpG methylation, its bonding at non-CpG sequences is not appreciably affected. These cumulative results suggest that C5 cytosine methylation at CpG sites enhances sequence selectivity of drug-DNA bonding. We propose that the methylation pattern and status (hypo- or hypermethylation) of genomic DNA may determine the cells' susceptibility to MC and its analogues, and these effects may, in turn, play a crucial role in the antitumor activities of the drugs.

Alkylating Agents↗
PubMed2000

Variation in bone mineral density of the sacrum in young adults and its significance for sacral fixation.

STUDY DESIGN: Bone mineral density variations throughout the sacrum were measured and correlated with sacral screw insertion torque. OBJECTIVE: To quantify bone mineral density variations within the S1 body and ala of young human specimens, especially along the pathways of sacral screws, and to examine the relation between sacral screw fixation and bone mineral density. SUMMARY OF BACKGROUND DATA: Vertebral bone quality is an essential factor in anterior or posterior screw fixation of the spine. Several studies have been conducted regarding bone mineral density variations in the cervical and thoracolumbar spine. However, such variations in bone mineral density in the sacrum have not been well documented. METHODS: The bone mineral density of 13 sacral specimens from young male cadavers (mean age, 31 years) was measured using highly accurate quantitative computed tomography. Variations in bone mineral density were measured in five transverse layers and seven vertical columns within the S1 body, and in four transverse layers and six vertical columns within the ala. The sacral screw insertion torque was measured (unicortical and bicortical), and the correlation with bone mineral density was calculated. RESULTS: The mean bone mineral density of the S1 body was 381.9 +/- 59 mg/cm3, which was 31.9% higher than that of the sacral ala (mean, 296.9 +/- 86 mg/cm3) (P < 0.05). Bone mineral density of the superior sacral endplate was higher than that of any other transverse layer. Columns near the lateral posterior and lateral anterior of the S1 body had the highest bone mineral density. In the ala, bone mineral density values of the internal columns (pedicle) were the highest. Screw insertion torque for bicortical purchase along the S1 pedicle correlated well with the bone mineral density of the S1 body (r = 0.67, P < 0.05). CONCLUSION: This study quantified the volumetric bone mineral density variations within the S1 body and ala, and a significant linear correlation between the screw insertion torque and bone mineral density was found. Optimal sacral screw insertion pathways were also outlined based on bone mineral density values.

Adult↗
PubMed2000

Sphingosine 1-phosphate-induced cell proliferation, survival, and related signaling events mediated by G protein-coupled receptors Edg3 and Edg5.

Sphingosine 1-phosphate (S1P) regulates cell proliferation, apoptosis, motility, and neurite retraction. Contradictory reports propose that S1P acts as either an intracellular second messenger or an extracellular ligand for cell-surface receptors. Hence, the precise signaling mechanisms mediating the diverse cellular effects of S1P remain to be determined. Here, we investigate whether S1P stimulation of cell proliferation, survival, and related signaling events can be mediated by the recently cloned Edg family members of G protein-coupled receptors. We observed that S1P treatment significantly increased proliferation of HTC4 hepatoma cells stably transfected with human S1P receptor Edg3 or Edg5, which was attributable to stimulation of cell growth and inhibition of apoptosis caused by serum starvation. Edg3 and Edg5 transduced S1P-evoked signaling events relevant to cell proliferation and survival, including activation of the ERK/MAP kinases, and immediate-early induction of c-Jun and c-Fos. Trancriptional activation of reporter genes for the c-fos promoter and the serum response element by Edg3 and Edg5 transfected in Jurkat cells was inhibited by pertussis toxin and C3 exoenzyme, implicating G(i/o)- and Rho-dependent pathways. Our data also indicated that Edg3 and Edg5 mediated the serum response element activation through transcriptional factors Elk-1 and serum response factor. Thus, specific G protein-coupled receptors Edg3 and Edg5 account for, at least in part, S1P-induced cell proliferation, survival, and related signaling events.

Cell Division↗
PubMed2000

Ovarian epithelial tumor growth promotion by follicle-stimulating hormone and inhibition of the effect by luteinizing hormone.

OBJECTIVES: The role of gonadotropins in ovarian epithelial cancer development is still controversial. Follicle-stimulating hormone receptor (FSHR) status in ovarian epithelial tumors (OETs) and their presumed precursor lesions has never been studied in detail. The objective of this study was to examine whether FSHR is expressed in OETs and to investigate the possible different roles of the gonadotropins in ovarian cancer development. METHODS: Twenty ovarian epithelial inclusions (entrapments or invaginations of ovarian surface epithelium) from benign ovaries and 60 OETs including 12 cystadenomas, 18 borderline tumors, and 30 carcinomas were examined for FSHR expression by using reverse transcription polymerase chain reaction (RT-PCR), in situ hybridization (ISH), and immunohistochemistry (IHC). We also studied the mitogenic activity of FSH on two FSH and luteinizing hormone (LH) receptor-positive ovarian epithelial carcinoma cell lines (AO and 3AO) and on the modifying effect of LH on this activity. Growth-stimulating effects of the gonadotropins were tested in vitro with measurement of cell numbers, S-phase by flow cytometry, and changes in the cellular proliferative marker Ki-67. RESULTS: Positive FSHR mRNA expression by RT-PCR (the most sensitive method) was found in 100% of epithelial inclusions, 100% of cystadenomas, 94% of borderline tumors, and 60% of carcinomas. There was a nonstatistically significant trend of decreasing positivity with increasing carcinoma grade. ISH and IHC gave similar, but somewhat less sensitive, results. A dose-response effect was seen with FSH, with a 1.6-fold increase in cell numbers with a maximally stimulating FSH concentration of 40 IU/L for a period of 48 h. These proliferative cellular effects were not observed when the cells were stimulated by LH in the range 1 to 100 IU/L. Most significantly, the growth stimulating effects of FSH could be blocked by the simultaneous administration of LH. CONCLUSIONS: FSHR is present in the majority of ovarian epithelial inclusions and OETs. The steady decline of FSHR expression from benign cystadenoma to borderline tumor to carcinoma suggests that FSH may be needed in early ovarian cancer development. Gonadotropins, FSH and LH, may have different roles in ovarian cancer cell proliferation. FSH, not LH, may be an important ovarian epithelial cell growth-promoting factor. The "opposing" effect of LH on FSH stimulation may explain why high FSH levels at postmenopausal ages are not associated with great increases in ovarian cancer risk.

Adult↗
PubMed2000

Algebraic Description of Stretching and Bending Vibrational Spectra of H(2)O and H(2)S.

The vibrational excitations of bent triatomic molecules, including both bending and stretching vibrations, are studied in the framework of the U(4) algebra. For the bent triatomic molecules H(2)O and H(2)S, the highly excited vibrational levels (up to 14) are obtained using the U(4) algebraic approach. We have found that the spectra are made up of clustering structure. The number of levels in one cluster depends on the total quanta of stretching and bending. In addition, some other properties are also discussed. Copyright 2000 Academic Press.

Journal Article↗
PubMed2000

Salmonella infection of a ventricular aneurysm with mural thrombus.

We describe a case of salmonella infection of a left ventricular aneurysm with a mural thrombus and review 12 cases described in the literature. This entity should be looked for in any patient with persistent or relapsing salmonella bacteremia in whom an intracardiac thrombus is demonstrated. Nuclear imaging may help in the diagnosis. A combined medical and surgical approach should be aggressively pursued because patients who do not undergo an aneurysmectomy are unlikely to survive.

Aged↗