Biomedical subjects

Y Zheng

Publications and source records attributed to Y Zheng.

At least 127 records · Page 7Linked to original sources

PubMed2001

Combined intraoperative iliac artery stents and femoro-popliteal bypass for multilevel atherosclerotic occlusive disease.

OBJECTIVE: To review our preliminary experience and evaluate our early results of a combined intraoperative iliac angioplasty and stenting with infrainguinal revascularization in multilevel atherosclerotic occlusive disease. METHODS: From July 1999 to April 2000, intraoperative iliac angioplasty and stenting combined with simultaneous femoro-popliteal bypass were performed on 12 lower extremities of 10 patients suffering from multilevel atherosclerotic occlusive disease. There were 8 men and 2 women, average 72 years. The indications for procedures included disabling claudication in 3 and rest pain in 7 patients. RESULTS: Eleven iliac angioplasty and stent procedures combined with simultaneous 9 femoro-popliteal by-pass and 3 femoro-femoral-popliteal bypass were performed in 12 limbs of 10 patients. Angioplasty and stent placement was technically successful in all patients. One contralateral femoral-popliteal bypass was failure after femoro-femoral-popliteal bypass. There were no additional instances of procedural or postoperative morbidity or mortality. Mean follow-up was 5 months (range 1 approximately 10 months). During the follow-up period, one femoro-infrapopliteal graft became occluded after 7 months and above-knee amputation was required. The cumulative primary patency rate of stented iliac arteries, femoro-femoral bypass grafts and femoro-popliteal bypass grafts were 100% (11/11), 100% (3/3) and 90.9% (10/11) in the follow-up period, respectively. The amputation rate was 8.3% (1/12). CONCLUSIONS: Intraoperative iliac artery PTA and stent placement can be safely and effectively performed simultaneously with infrainguinal revascularization for multilevel atherosclerotic occlusive disease by skilled vascular surgeon, using a portable C arm fluoroscopy inthe operating room. Furthermore, iliac artery PTA and stenting was valuable adjunct to distal bypass either to improve inflow and outflow, or to reduce the extent of traditional surgical intervention, and also, any angioplasty and stenting-related complications can be immediately corrected as well.

Aged↗

PubMed2001

Relationship between human parvovirus B19 infection and aplastic anemia.

OBJECTIVE: To explore the relationship between human parvovirus B19 (HPV B19) infection and aplastic anemia (AA) and to investigate the role of HPV B19 in the occurrence of AA. METHODS: The presence of HPV B19 DNA was detected in the peripheral blood samples of 60 patients with AA (children 38 and adults 22) by nested polymerase chain reaction (PCR) assay, and 30 healthy persons were selected as control. RESULTS: Sixteen (26.7%) of 60 AA cases were HPV B19 DNA positive, while all the samples in the control group were negative for HPV B19 (P = 0.000914). Among the case group, the positive rates of HPV B19 DNA were 21.4% (6/28), 30.0% (3/10), 20.0% (1/5) and 35.3% (6/17) in children acute AA (AAA), children chronic AA (CAA), adults AAA and adults CAA patients respectively, which were significantly higher than that in the control group. Furthermore, there was no remarkable difference between children AA and adults AA in the 16 HPV B19 DNA positive patients; neither was there between AAA and CAA. CONCLUSION: HPV B19 infection is not only correlated with the occurrence of children AAA and CAA, but also with adults AAA and CAA, and might be an important viral cause for AA in humans.

Acute Disease↗

PubMed2000

Oligomerization of Rac1 gtpase mediated by the carboxyl-terminal polybasic domain.

The Rho family GTPase Rac1 mediates a variety of signal transduction processes leading to activation of NADPH oxidase, actin cytoskeleton reorganization, transcription activation, and stimulation of DNA synthesis. In this study, Rac1 was found to form a reversible monomer and oligomer in both the GDP- and GTP-bound states in vitro and in cells. Mutational analysis and peptide competition experiments showed that the unique C-terminal domain of Rac1 consisting of six consecutive basic residues (amino acids 183-188) is required for the homophilic interaction. Oligomerization of Rac1-GTP led to a self-stimulatory GTPase-activating protein (GAP) activity, resulting in a significantly enhanced intrinsic GTP hydrolysis rate of Rac1-GTP. Deletion or mutation of the polybasic residues drastically decreased its intrinsic GTPase activity and resulted in a loss of the self-stimulatory GAP activity. In the oligomeric state, Rac1 became insensitive to the RhoGAP stimulation, albeit maintaining the responsiveness to the guanine nucleotide exchange factor. The ability of the Rac1 C-terminal mutants to activate the effector p21(cdc42/rac)-activated kinase-1 correlated with their oligomerization states, suggesting that oligomer formation potentiates effector activation. Furthermore, the oligomer-to-monomer transition of Rac1-GDP could be driven effectively by interaction with the Rho guanine nucleotide dissociation inhibitor. Building on previous characterizations of Rac1 interaction with regulatory proteins and effectors, these results suggest that Rac1 may employ yet another means of regulation by cycling between the monomeric and oligomeric states to effectively generate a transient and augmented signal.

Amino Acid Sequence↗

PubMed2000

Characterization and reconstitution of Drosophila gamma-tubulin ring complex subunits.

The gamma-tubulin ring complex (gammaTuRC) is important for microtubule nucleation from the centrosome. In addition to gamma-tubulin, the Drosophila gammaTuRC contains at least six subunits, three of which [Drosophila gamma ring proteins (Dgrips) 75/d75p, 84, and 91] have been characterized previously. Dgrips84 and 91 are present in both the small gamma-tubulin complex (gammaTuSC) and the gammaTuRC, while the remaining subunits are found only in the gammaTuRC. To study gammaTuRC assembly and function, we first reconstituted gammaTuSC using the baculovirus expression system. Using the reconstituted gammaTuSC, we showed for the first time that this subcomplex of the gammaTuRC has microtubule binding and capping activities. Next, we characterized two new gammaTuRC subunits, Dgrips128 and 163, and showed that they are centrosomal proteins. Sequence comparisons among all known gammaTuRC subunits revealed two novel sequence motifs, which we named grip motifs 1 and 2. We found that Dgrips128 and 163 can each interact with gammaTuSC. However, this interaction is insufficient for gammaTuRC assembly.

Amino Acid Motifs↗

PubMed2000

The role of Xgrip210 in gamma-tubulin ring complex assembly and centrosome recruitment.

The gamma-tubulin ring complex (gammaTuRC), purified from the cytoplasm of vertebrate and invertebrate cells, is a microtubule nucleator in vitro. Structural studies have shown that gammaTuRC is a structure shaped like a lock-washer and topped with a cap. Microtubules are thought to nucleate from the uncapped side of the gammaTuRC. Consequently, the cap structure of the gammaTuRC is distal to the base of the microtubules, giving the end of the microtubule the shape of a pointed cap. Here, we report the cloning and characterization of a new subunit of Xenopus gammaTuRC, Xgrip210. We show that Xgrip210 is a conserved centrosomal protein that is essential for the formation of gammaTuRC. Using immunogold labeling, we found that Xgrip210 is localized to the ends of microtubules nucleated by the gammaTuRC and that its localization is more distal, toward the tip of the gammaTuRC-cap structure, than that of gamma-tubulin. Immunodepletion of Xgrip210 blocks not only the assembly of the gammaTuRC, but also the recruitment of gamma-tubulin and its interacting protein, Xgrip109, to the centrosome. These results suggest that Xgrip210 is a component of the gammaTuRC cap structure that is required for the assembly of the gammaTuRC.

Amino Acid Sequence↗

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

Drug-resistant Drosophila indicate glutamate-gated chloride channels are targets for the antiparasitics nodulisporic acid and ivermectin.

The fruit fly Drosophila melanogaster was used to examine the mode of action of the novel insecticide and acaricide nodulisporic acid. Flies resistant to nodulisporic acid were selected by stepwise increasing the dose of drug in the culture media. The resistant strain, glc(1), is at least 20-fold resistant to nodulisporic acid and 3-fold cross-resistant to the parasiticide ivermectin, and exhibited decreased brood size, decreased locomotion, and bang sensitivity. Binding assays using glc(1) head membranes showed a marked decrease in the affinity for nodulisporic acid and ivermectin. A combination of genetics and sequencing identified a proline to serine mutation (P299S) in the gene coding for the glutamate-gated chloride channel subunit DmGluClalpha. To examine the effect of this mutation on the biophysical properties of DmGluClalpha channels, it was introduced into a recombinant DmGluClalpha, and RNA encoding wild-type and mutant subunits was injected into Xenopus oocytes. Nodulisporic acid directly activated wild-type and mutant DmGluClalpha channels. However, mutant channels were approximately 10-fold less sensitive to activation by nodulisporic acid, as well as ivermectin and the endogenous ligand glutamate, providing direct evidence that nodulisporic acid and ivermectin act on DmGluClalpha channels.

Animals↗

PubMed2000

5-Methylcytosine DNA glycosylase activity is also present in the human MBD4 (G/T mismatch glycosylase) and in a related avian sequence.

A 1468 bp cDNA coding for the chicken homolog of the human MBD4 G/T mismatch DNA glycosylase was isolated and sequenced. The derived amino acid sequence (416 amino acids) shows 46% identity with the human MBD4 and the conserved catalytic region at the C-terminal end (170 amino acids) has 90% identity. The non-conserved region of the avian protein has no consensus sequence for the methylated DNA binding domain. The recombinant proteins from human and chicken have G/T mismatch as well as 5-methylcytosine (5-MeC) DNA glycosylase activities. When tested by gel shift assays, human recombinant protein with or without the methylated DNA binding domain binds equally well to symmetrically, hemimethylated DNA and non-methylated DNA. However, the enzyme has only 5-MeC DNA glycosylase activity with the hemimethylated DNA. Footprinting of human MBD4 and of an N-terminal deletion mutant with partially depurinated and depyrimidinated substrate reveal a selective binding of the proteins to the modified substrate around the CpG. As for 5-MeC DNA glycosylase purified from chicken embryos, MBD4 does not use oligonucleotides containing mCpA, mCpT or mCpC as substrates. An mCpG within an A+T-rich oligonucleotide is a much better substrate than an A+T-poor sequence. The K:(m) of human MBD4 for hemimethylated DNA is approximately 10(-7) M with a V:(max) of approximately 10(-11) mol/h/microgram protein. Deletion mutations show that G/T mismatch and 5-MeC DNA glycosylase are located in the C-terminal conserved region. In sharp contrast to the 5-MeC DNA glycosylase isolated from the chicken embryo DNA demethylation complex, the two enzymatic activities of MBD4 are strongly inhibited by RNA. In situ hybridization with antisense RNA indicate that MBD4 is only located in dividing cells of differentiating embryonic tissues.

Amino Acid Sequence↗

PubMed2000

Two forms of UvrC protein with different double-stranded DNA binding affinities.

Using phosphocellulose followed by single-stranded DNA-cellulose chromatography for purification of UvrC proteins from overproducing cells, we found that UvrC elutes at two peaks: 0.4 m KCl (UvrCI) and 0.6 m KCl (UvrCII). Both forms of UvrC have a major peptide band (>95%) of the same molecular weight and identical N-terminal amino acid sequences, which are consistent with the initiation codon being at the unusual GTG site. Both forms of UvrC are active in incising UV-irradiated, supercoiled phiX-174 replicative form I DNA in the presence of UvrA and UvrB proteins; however, the specific activity of UvrCII is one-fourth that of UvrCI. The molecular weight of UvrCII is four times that of UvrCI on the basis of results of size exclusion chromatography and glutaraldehyde cross-linking reactions, indicating that UvrCII is a tetramer of UvrCI. Functionally, these two forms of UvrC proteins can be distinguished under reaction conditions in which the protein/nucleotide molar ratio is >0.06 by using UV-irradiated, (32)P-labeled DNA fragments as substrates; under these conditions UvrCII is inactive in incision, but UvrCI remains active. The activity of UvrCII in incising UV-irradiated, (32)P- labeled DNA fragments can be restored by adding unirradiated competitive DNA, and the increased level of incision corresponds to a decreased level of UvrCII binding to the substrate DNA. The sites of incision at the 5' and 3' sides of a UV-induced pyrimidine dimer are the same for UvrCI and UvrCII. Nitrocellulose filter binding and gel retardation assays show that UvrCII binds to both UV-irradiated and unirradiated double-stranded DNA with the same affinity (K(a), 9 x 10(8)/m) and in a concentration-dependent manner, whereas UvrCI does not. These two forms of UvrC were also produced by the endogenous uvrC operon. We propose that UvrCII-DNA binding may interfere with Uvr(A)(2)B-DNA damage complex formation. However, because of its low copy number and low binding affinity to DNA, UvrCII may not interfere with Uvr(A)(2)B-DNA damage complex formation in vivo, but instead through double-stranded DNA binding UvrCII may become concentrated at genomic areas and therefore may facilitate nucleotide excision repair.

Bacterial Proteins↗

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

Gold complexes with potentially tri- and tetradentate phosphinothiolate ligands.

Reactions of [Au(PPh3)Cl], (Bu4N)[AuCl4] and the organometallic gold complex [Au(damp-C1,N)Cl2] (damp- = 2-(N,N-dimethylaminomethyl)phenyl) with the potentially tri- and tetradentate proligands PhP(C6H3-SH-2-R-3)2 (H2L1a, R = SiMe3; H2L1b, R = H) and P(C6H4-SH-2)3 (H3L2) result in the formation of mono- or dinuclear gold complexes depending on the precursor used. Monomeric complexes of the type [AuL1Cl] are formed upon the reaction with [Au(damp-C1,N)Cl2], but small amounts of dinuclear [AuL1]2 complexes with gold in two different oxidation states, +1 and +3, have been isolated as side-products. The dinuclear compounds are obtained in better yields from [AuCl4]-. A dinuclear complex having two Au(III) centers can be isolated from the reaction of [Au(PPh3)Cl] with H3L2, whereas from the reaction with H2L1b the mononuclear [Au(Ph3P)HL1b] is obtained, which contains a three-coordinate gold atom. Comparatively short gold-gold distances have been found in the dinuclear complexes (2.978(2) and 3.434(1) A). They are indicative of weak gold-gold interactions, which is unusual for gold(III).

Journal Article↗

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↗