Biomedical subjects

J Qiu

Publications and source records attributed to J Qiu.

At least 73 records · Page 4Linked to original sources

PubMed2000

Human lactoferrin proteolytic activity: analysis of the cleaved region in the IgA protease of Haemophilus influenzae.

Human lactoferrin proteolytically cleaves and inactivates two colonization factors of non-typable Haemophilus influenzae, the IgA protease precursor protein (Iga), and Hap, the non-pilus adhesin by which microoganisms adhere to host epithelial cells and form microcolonies. Iga and Hap are homologous proteins that are members of the autotransporter family of secreted proteins expressed by gram-negative bacteria. Studies of Iga cleaved by lactoferrin, reported here, show that proteolysis occurred within the helper region of Iga (Iga(beta)) domain which anchors the autotransporter within the Haemophilus outer membrane. The amino-terminus of the extracted Iga protein was not modified. The location of the proteolytic active site in human lactoferrin is under study. Lactoferrin proteolysis may attenuate pathogenicity of H. influenzae, an important cause of otitis media.

Amino Acid Sequence↗

PubMed2000

Cell cycle-dependent and DNA damage-inducible nuclear localization of FEN-1 nuclease is consistent with its dual functions in DNA replication and repair.

Flap endonuclease-1 (FEN-1), a 43-kDa protein, is a structure-specific and multifunctional nuclease. It plays important roles in RNA primer removal of Okazaki fragments during DNA replication, DNA base excision repair, and maintenance of genome stability. Three functional motifs of the enzyme were proposed to be responsible for its nuclease activities, interaction with proliferating cell nuclear antigen, and nuclear localization. In this study, we demonstrate in HeLa cells that a signal located at the C terminus (the nuclear localization signal (NLS) motif) facilitates nuclear localization of the enzyme during S phase of the cell cycle and in response to DNA damage. Truncation of the NLS motif prevents migration of the protein from the cytoplasm to the nucleus, while having no effect on the nuclease activities and its proliferating cell nuclear antigen interaction capability. Site-directed mutagenesis further revealed that a mutation of the KRK cluster to three alanine residues completely blocked the localization of FEN-1 into the nucleus, whereas mutagenesis of the KKK cluster led to a partial defect of nuclear localization in HeLa cells without observable phenotype in yeast. Therefore, the KRKXXXXXXXXKKK motif may be a bipartite NLS driving the protein into nuclei. Yeast RAD27Delta cells transformed with human mutant M(krk) survived poorly upon methyl methanesulfonate treatment or when they were incubated at an elevated temperature.

Cell Cycle↗

PubMed2000

Mutational spectrum analysis of RNase H(35) deficient Saccharomyces cerevisiae using fluorescence-based directed termination PCR.

Mutational spectrum analysis has become an informative genetic tool to understand those protein functions involved in mutation avoidance pathways since specific types of mutations are often associated with particular protein defects involved in DNA replication and repair. In this study, we describe a novel, fluorescence-based procedure for direct determination of deletions and insertions with 100% accuracy. We performed two complementary directed termination PCR with near infrared dye-labeled primers, followed by visualization of termination fragments using an automated Li-cor DNA sequencer. This method is used for rapid analysis of mutational spectra generated in nuclease-defective strains of Saccharomyces cerevisiae to elucidate the role of RNase H(35) in RNA primer removal during DNA replication and in mutation avoidance. Strains deficient in RNH35 displayed a distinct spontaneous mutation spectrum of deletions characterized by a unique 4 bp deletion in a lys2-Bgl allele. This was in sharp contrast to strains deficient in rad27 that displayed duplication mutations. Further analysis of mutations in a rnh35/rad27 double mutant revealed a mixed spectrum. These results indicate that RNase H(35) may participate in a redundant pathway in Okazaki fragment processing and that mutational spectra caused by protein deficiencies may be more intermediate-specific than pathway-specific.

DNA Mutational Analysis↗

PubMed2000

Fas receptor and neuronal cell death after spinal cord ischemia.

Cell death from spinal cord injury is mediated in part by apoptotic mechanisms involving downstream caspases (e.g., caspase-3). Upstream mechanisms may involve other caspases such as procaspase-8, a 55 kDa apical caspase, which we found constitutively expressed within spinal cord neurons along with Fas. As early as 1.5 hr after transient ischemia, activated caspase-8 (p18) and caspase-8 mRNA appeared within neurons in intermediate gray matter and in medial ventral horn. We also detected evidence for an increase in death receptor complex by co-immunoprecipitation using Fas and anti-procaspase-8 after ischemia. At early time points, Fas and p18 were co-expressed within individual neurons, as were activated caspase-8 and caspase-3. Moreover, we detected p18 in cells before procaspase-3 cleavage product (p20), suggesting sequential activation. The appearance of cytosolic cytochrome c and gelsolin cleavage after ischemia was consistent with mitochondrial release and caspase-3 activation, respectively. Numerous terminal deoxynucleotidyl transferase-mediated DNA nick end-labeling-positive neurons contained p18 or p20 (65 and 80%, respectively), thereby supporting the idea that cells undergoing cell death contain both processed caspases. Our data are consistent with the idea that transient spinal cord ischemia induces the formation of a death-inducing signaling complex, which may participate in caspase-8 activation and sequential caspase-3 cleavage. Death receptors as well as downstream caspases may be useful therapeutic targets for limiting the death of cells in spinal cord.

Animals↗

PubMed2000

Universality in the nuclear dependence coefficient alpha(q(T))

We derive the nuclear dependence coefficient alpha(q(T)) for Drell-Yan and J/psi production. We show that at small q(T), alpha(q(T)) is given by a universal functional form: alpha(q(T)) = a+bq(2)(T), and the parameters a and b are completely determined by either perturbatively calculable or independently measurable quantities. This universal functional form alpha(q(T)) is insensitive to A, and is consistent with existing data.

Journal Article↗

PubMed2000

Unique structural and stabilizing roles for the individual pseudouridine residues in the 1920 region of Escherichia coli 23S rRNA.

The synthesis of a 5'-O-BzH-2'- O -ACE-protected pseudouridine phosphoramidite is reported [BzH, benzhydryloxy-bis(trimethylsilyloxy)silyl; ACE, bis(2-acetoxyethoxy)methyl]. The availability of the phosphoramidite allows for reliable and efficient syntheses of hairpin RNAs containing single or multiple pseudouridine modifications in the stem or loop regions. Five 19-nt hairpin RNAs representing the 1920-loop region (G(1906)-C(1924)) of Escherichia coli 23S rRNA were synthesized with pseudouridine residues located at positions 1911, 1915 and 1917. Thermodynamic parameters, circular dichroism spectra and NMR data are presented for all five RNAs. Overall, three different structural contexts for the pseudouridine residues were examined and compared with the unmodified RNA. Our main findings are that pseudouridine modifications exhibit a range of effects on RNA stability and structure, depending on their locations. More specifically, pseudouridines in the single-stranded loop regions of the model RNAs are slightly destabilizing, whereas a pseudo-uridine at the stem-loop junction is stabilizing. Furthermore, the observed effects on stability are approximately additive when multiple pseudouridine residues are present. The possible relationship of these results to RNA function is discussed.

Base Sequence↗

PubMed2000

The interaction of heparin sulfate and adeno-associated virus 2.

Recently heparan sulfate was proposed as the host cell receptor for the dependovirus, adeno-associated virus type 2 (AAV2). We show that although heparan sulfate on the cell surface may contribute to the binding of AAV2 to permissive cells, the amount of heparan sulfate on the cell surface as determined by flow cytometry using four different monoclonal antibodies does not correlate with AAV2 binding to cells or recombinant AAV2 transduction efficiency. Experiments with either mutant CHO cells or cells treated with chlorate to remove sulfate groups showed that sulfation was not absolutely required for infection or binding: in the absence of cell surface sulfation, recombinant AAV2 was still able to be transduced in previously permissive cells. Heparin is commonly used as a substitute in studies of the interaction between heparan sulfate and ligand, and we demonstrate that the binding affinity of AAV2/heparin is low, with a K(d) value of approximately 2.0 nM. A study of the direct interaction between AAV2 and artificial glycosaminoglycans showed that a high degree of sulfation on heparin was critical for the ability to bind AAV2 and compete rAAV2 transduction and that both O- and N-sulfate groups are required. Overall, our data suggest that, as has been shown for other viruses, the presence of a high-affinity AAV2 receptor mediates AAV2 infection in addition to the low-affinity heparan sulfate binding.

Animals↗

PubMed2000

A new class of conformationally rigid analogues of 4-amino-5-halopentanoic acids, potent inactivators of gamma-aminobutyric acid aminotransferase.

Recently, we found (Qiu, J.; Pingsterhaus, J. M.; Silverman, R. B. J. Med. Chem. 1999, 42, 4725-4728) that conformationally rigid analogues of the GABA aminotransferase (GABA-AT) inactivator vigabatrin were not inactivators of GABA-AT. To determine if this is a general phenomenon of GABA-AT inactivators, several mono- and di-halogen-substituted conformationally rigid analogues (7-15) of other GABA-AT inactivators, 4-amino-5-halopentanoic acids, were synthesized as potential inactivators of GABA-AT. Four of them, (+)-7, (-)-9, (+)-10, and (+)-15, were inactivators, although not as potent as the corresponding open-chain analogues. The maximal inactivation rate constants, k(inact), for the fluoro- and bromo-substituted analogues were comparable, indicating that cleavage of the C-X bond is not rate determining. Consistent with that observation is the finding that [3-(2)H]-10 exhibits a deuterium isotope effect on inactivation of 3.3, suggesting that C-H bond cleavage is the rate-determining step. The rate of inactivation of GABA-AT by the fluorinated analogue 7 is 1/15 that of inactivation by the corresponding open-chain analogue, 4-amino-5-fluoropentanoic acid (3a). Whereas inactivation by 3a releases only one fluoride ion, inactivation by 7 releases 148 fluoride ions, accounting for the less efficient inactivation rate. Inactivation leads to covalent attachment of 2 equiv of inactivator after gel filtration; upon urea denaturation, 1 equiv of radioactivity remains bound to the enzyme. This suggests that, unlike the open-chain anlogue, the conformationally rigid analogue becomes, at least partially, attached to an active-site residue. It appears that the conformational constraint has a larger effect on inactivators that inactivate by a Michael addition mechanism than by an enamine mechanism.

4-Aminobutyrate Transaminase↗

PubMed2000

Glial inhibition of nerve regeneration in the mature mammalian CNS.

The lack of axonal regeneration in the adult mammalian CNS is due to both unfavorable environmental glial factors and the intrinsic neuronal state. Inhibitors associated with myelin and the glial scar have been extensively studies and it has been shown that neutralizing at least some of the inhibitors can lead to improved growth. Meanwhile, important advances have also been made towards our understanding of the neuronal intrinsic state, particularly the intracellular levels of cyclic nucleotide, that influence the capacity of mature CNS neurons to initiate and maintain a regrowth response. It is well recognized that successful regeneration may only be achieved by application of a combination of strategies that both block glial inhibitors and enhance the intrinsic neuronal growth capacity.

Animals↗

PubMed2000

Spin-locking mechanism of spin I = 3/2 quadrupolar nuclei undergo magic angle spinning.

The spin-locking mechanism of the spin I = 3/2 quadrupolar nuclei under magic angle spinning (MAS) has been theoretically and experimentally investigated, and the criterion of adiabatic passage around zero-crossings of the quadrupole splitting was inferred from the time-dependent Shrödinger equation in this article. The theory, numerical simulations, and experiments conducted in this work all indicated that second-order quadrupole interaction and off-resonance play important roles in the spin-locking of the quadrupolar nuclei, and they were responsible for the great loss of the spin-locking signals. The spin-locking for a spin I = 3/2 nucleus might be achieved by minimizing the effect of the second-order quadrupole interaction by using a radio frequency (RF) offset. This offset was realized by setting the RF to the opposite position of the isotropic second-order quadrupolar shift of single quantum coherences.

Magnetic Resonance Spectroscopy↗

PubMed2000

Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts.

Osteoblasts subjected to fluid shear increase the expression of the early response gene, c-fos, and the inducible isoform of cyclooxygenase, COX-2, two proteins linked to the anabolic response of bone to mechanical stimulation, in vivo. These increases in gene expression are dependent on shear-induced actin stress fiber formation. Here, we demonstrate that MC3T3-E1 osteoblast-like cells respond to shear with a rapid increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) that we postulate is important to subsequent cellular responses to shear. To test this hypothesis, MC3T3-E1 cells were grown on glass slides coated with fibronectin and subjected to laminar fluid flow (12 dyn/cm(2)). Before application of shear, cells were treated with two Ca(2+) channel inhibitors or various blockers of intracellular Ca(2+) release for 0. 5-1 h. Although gadolinium, a mechanosensitive channel blocker, significantly reduced the [Ca(2+)](i) response, neither gadolinium nor nifedipine, an L-type channel Ca(2+) channel blocker, were able to block shear-induced stress fiber formation and increase in c-fos and COX-2 in MC3T3-E1 cells. However, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, an intracellular Ca(2+) chelator, or thapsigargin, which empties intracellular Ca(2+) stores, completely inhibited stress fiber formation and c-fos/COX-2 production in sheared osteoblasts. Neomycin or U-73122 inhibition of phospholipase C, which mediates D-myo-inositol 1,4,5-trisphosphate (IP(3))-induced intracellular Ca(2+) release, also completely suppressed actin reorganization and c-fos/COX-2 production. Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform of U-73122, did not inhibit these shear-induced responses. These results suggest that IP(3)-mediated intracellular Ca(2+) release is required for modulating flow-induced responses in MC3T3-E1 cells.

3T3 Cells↗

PubMed2000

[Molecular cytogenetics of bone tumors].

OBJECTIVE: To study alterations of molecular cytogenetics of bone tumor serially. METHODS: Chromosomal analysis, immunohistochemistry, molecular hybridization, nested-PCR/SSCP, DNA sequence analysis, and gene transfection techniques were used to investigate the common bone tumors. RESULTS: Chromosomal aberrations were found in 50 cases of aggressive bone tumors, with a chromosome number aberration rate of 86.96% and a structure aberration rate of 93.94%. However, there were differences among different kinds of bone tumors. The incidence of chromosomal instability in 53 patients with bone tumors was higher than that in normal controls. Meanwhile, the fragile sites of chromosomes of the patients were closely correlated with the high nonrandom chromosome breakpoints of tumor cells and related oncogene loci. The data on p53 and Rb from 305 cases and 89 cases, respectively, indicated that abnormalities of p53 and Rb were common events in bone tumors. The accumulation of p53 protein and loss of Rb protein were more common in aggressive bone tumors. Structures and their products of related proto-oncogenes (ras, myc, fos, bcl-2, met) were studied in 124 cases. Bone tumors presented multi-oncogenes abnormalities with differences in different pathological kinds. Exogenous wild-type p53 gene transfected into osteosarcoma cell line induced growth inhibition and apoptosis of tumor cells. CONCLUSIONS: Abnormalities of chromosomes, proto-oncogenes, and tumor suppressor genes frequently exist in bone tumor cells and are correlated with oncogenesis.

Bone Neoplasms↗

PubMed2000

[The influence of external stimulation on content and quality of volatile oil in Lignun Santali albi].

The authors analyzed the quality of Ligmum Santali Albi formed by the external stimulation of hormone and windburn by GC-MS-DS. The results showed that the content of volatile oil is 2.34% in the heart wood formed in 10 years tree age of Santalum album (SA) after 2 years stimulation continuously with a definite concentration of hormone, which is near to the 25 years tree age of SA in the same place. The GC-MS analysis showed that the content of santalol and other chemical components in volatile oil are similar to the 25 years tree age of SA. It is indicated that a definite concentration of hormone stimulated the SA may shorten the formation of the heart wood. The heart wood can be also formed by the broken branches after 2 years windburn, but its content of volatile oil is only 1/2 of the heart wood formed by hormone stimulation.

Age Factors↗

PubMed1999

Adenines at -11, -9 and -8 play a key role in the binding of Bacillus subtilis Esigma(A) RNA polymerase to -10 region single-stranded DNA.

The sigma subunit of RNA polymerase interacts with the promoter DNA in at least two regions: the -35 and the -10 consensus elements. The latter contacts are involved both in recognition and in melting of the promoter DNA to form the transcriptionally-competent open complex. RNA polymerase holoenzyme, but neither core nor sigma alone, binds with high selectivity to single-stranded DNA (ssDNA) containing the non-template -10 consensus sequence. We have used equilibrium competition to assess the specificity of holoenzyme binding to a 19 base oligonucleotide containing a -10 consensus element, TATAAT. Analysis of all 18 possible single point mutations in the -10 consensus sequence reveals that binding by Bacillus subtilis Esigma(A)holoenzyme depends critically upon adenine at position -11 and, unexpectedly, is strongly affected by substitutions of the poorly conserved adenines at -9 and -8. Similarly, ssDNA binding by Escherichia coli Esigma(70)holoenzyme is most strongly affected by substitutions of adenines within the -10 region consensus. The critical role of -11A in binding ssDNA supports a key role for this base in the nucleation of DNA melting. A novel role for -9A and -8A is proposed in the context of recent models of promoter melting.

Adenine↗

PubMed1999

Integrin alphaVbeta5 is not involved in adeno-associated virus type 2 (AAV2) infection.

alphaVbeta5 integrin was recently proposed as a coreceptor for adeno-associated virus type 2 (AAV2) infection (Summerford et al., 1999, Nat. Med. 5, 78-82), based mainly on the direct binding of AAV2 to denatured beta5 by virus overlay assay. In studies using purified natural or recombinant human integrin alphaVbeta5 we were unable to demonstrate AAV2 binding, either by virus overlay or by liquid binding assay. Furthermore, neither purified integrin alphaVbeta5, nor RGD peptides, nor functional blocking monoclonal antibody blocked rAAV2 transduction. These data strongly suggest that integrin alphaVbeta5 is not involved in AAV2 infection.

Animals↗

PubMed1999

Inhibition and substrate activity of conformationally rigid vigabatrin analogues with gamma-aminobutyric acid aminotransferase.

Several cyclopentene GABA analogues were synthesized as conformationally rigid analogues of the epilepsy drug vigabatrin and tested as inhibitors and substrates of gamma-aminobutyric acid aminotransferase (GABA-AT). None of these compounds produced time-dependent inhibition. (1R, 4S)-(+)-4-Amino-2-cyclopentene-1-carboxylic acid ((+)-3), (4R)-(-)-4-amino-1-cyclopentene-1-carboxylic acid ((-)-4), and d, l-3-amino-1-cyclopentene-1-carboxylic acid (6) are good substrates. The K(m) and k(cat) values for the latter two compounds are very similar to those of GABA, suggesting that they bind in an orientation similar to that of GABA. The K(m) value for (+)-3 is 24 times lower than that for GABA, although its k(cat) value is only one-fourth that for GABA; nonetheless, it is a better substrate for GABA-AT than is GABA. All of these compounds, as well as the enantiomers of 3 and 4 and d, l-trans-4-amino-2-cyclopentene-1-carboxylic acid (5), are competitive inhibitors of GABA-AT. These results demonstrate the effects of the carboxylate group orientation and the stereochemistry of the amino and carboxylate groups on the substrate activity and inhibitor activity, and this should be important to the future design of inhibitors of GABA-AT.

4-Aminobutyrate Transaminase↗

PubMed1999

Aminomethyl-2,6-difluorophenols as a novel class of increased lipophilicity GABA(C) receptor antagonists.

3- and 4-(Aminomethyl)-2,6-difuorophenols were tested for activity against the three major classes of GABA receptors. 4-(Amninomethyl)-2,6difluorophenol was shown to be a competitive and somewhat selective antagonist at p1 GABA(C) receptors expressed in Xenopus oocytes (K(B) = 75.5 microM with a 95% Confidence Interval range of 75.2 microM to 75.8 microM). This is the first in a novel class of increased lipophilicity GABA(C) receptor antagonists with little activity at alpha1beta2gamma2 GABA(A) and GABA(B) receptors.

Animals↗

PubMed1999

Statistics aided optimization for high-performance liquid chromatographic analysis of organic acids in tobacco.

HPLC analysis for organic acids in tobacco was optimized with the aid of statistical experimental design, a central composite face-centered design. In the design, only thirteen HPLC analyses were needed for identifying two optimal separation parameters. A Bio-Rad Aminex HPX-87H column was used for the analyses. An optimal separation for seven acids in tobacco was found at a temperature of 57 degrees C and a mobile phase of 0.032 N sulfuric acid solution, or at a temperature of 70 degrees C and a mobile phase of 0.024 N sulfuric acid solution, with a flow rate of 0.6 ml min(-1).

Chromatography, High Pressure Liquid↗