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Biomedical subjects

J Zhang

Publications and source records attributed to J Zhang.

At least 19 recordsLinked to original sources
PubMed2001

Efficacy of 3'-azido 3'deoxythymidine (AZT) in preventing HTLV-1 transmission to human cord blood mononuclear cells.

The present study investigated the effect of 3'-azido 3'deoxythymidine (AZT) treatment on in vitro infection of human cord blood mononuclear cells (CBMCs) exposed to HTLV-1 by cocultivation with the MT-2 cell line. Cultures of CBMCs were grown in IL-2 and were either left untreated or were treated with concentrations of AZT ranging from 0.0078 to 32 microM. HTLV-1-infected cultures were monitored at different times of culture by evaluating proliferation activity, cell growth and the presence and expression of HTLV-1 genes. Results showed that untreated cultures infected with HTLV-1 were able to grow for several weeks, while those treated with AZT at 0.03 microM or higher concentrations were limited in their growth capacity. Moreover, the addition of AZT at the moment of infection significantly inhibited cell proliferation in a dose-dependent fashion. In the presence of AZT, detection of proviral DNA and, more remarkably, viral RNA expression were clearly reduced. In addition, treatment with AZT resulted in a noticeable decrease in Tax protein expression. Using treatment with relatively low doses of AZT, effective in exerting an antiviral action, cytotoxicity on CBMCs was not observed, whereas higher doses induced apoptosis in uninfected CBMCs. These data show that CBMCs are protected by AZT against HTLV-1 transmission even at low, non-toxic doses.

Apoptosis↗
PubMed2001

Neuroblastoma tumor cell-binding peptides identified through random peptide phage display.

Random peptide phage display libraries have been employed widely to identify protein-protein interactions, using as targets either purified proteins, intact cells, or organs. To isolate peptides that bind to human neuroblastoma cells, we have used a phage display approach with the neuroblastoma cell line WAC 2 as the target. In particular, two bacteriophages, t147 and t160, displaying peptides p147 and p160, respectively, were isolated by repeated display cycles. Binding of t147 and t160 to WAC 2 cells was abrogated by pretreatment with the peptides p147 and p160, respectively, which strongly support that cellular binding of both phages is dictated by their displayed peptides. Immunofluorescence analysis by confocal light microscopy revealed that the major proportion of t147 remains on the surface of WAC 2 cells and that only a fraction is taken up into the cells. In contrast, the vast majority of t160 is internalized. K(+) depletion reduced the number of the phages internalized by the cells to approximately 20% for t160 and to 10% for t147, indicating that the phage internalization was through receptor-mediated endocytosis. Phage t147 appears to bind to a range of tumor cell lines, including neuroblastoma, breast cancer, glioblastoma and C-cell carcinoma, but less so to non-tumor lines, such as erythrocytes, lymphocytes, monocytes and epithelial cells. Phage t160 bound to a range of neuroblastoma cell lines and a breast cancer cell line, but not to other tested cell lines. While neither of the displayed peptides conferred a narrow tissue specific binding ability, they do provide a basis for targeted drug delivery in selected experimental or natural tumor systems.

Amino Acid Sequence↗
PubMed2001

Differential osteopontin expression in lung cancer.

Osteopontin (OPN) is a phosphorylated glycoprotein with diverse functions including cancer development, progression and metastasis. Its expression is induced by a variety of stimuli such as TNF-alpha and Ras proto-oncogene. However, differential OPN expression and its regulation in each histologic type of lung cancer are not well established. In this study, we assessed expression of OPN in lung cancer tissues with immunohistochemical analysis. OPN was predominantly expressed in tumor cells of non-small cell lung cancer (NSCLC) tissues: 11 of 16 cases (68.8%) of squamous cell carcinoma (SCC), five of 24 cases (20.8%) of adenocarcinoma (AD), but only two of 18 cases (11%) of small cell lung cancer (SCLC). Expectedly, OPN was principally expressed in NSCLC cell lines (H322 cells and HL460 cells) but not in SCLC cell line (H69 cells) by Western blotting and Northern blotting. Interestingly, Ras-p21 was specifically co-expressed with OPN staining in eight of eight cases with SCC (100%), whereas it was demonstrated in three of ten cases (30%) with AD and only one of 18 cases (5%) with SCLC. Collectively, these results suggest that OPN is mainly expressed in NSCLC, especially among SCC. OPN expression may be tightly regulated by Ras oncogene, and its concomitant induction with Ras activation may play a crucial role in the development of SCC.

Adenocarcinoma↗
PubMed2001

Secretin facilitates GABA transmission in the cerebellum.

Secretin was the first hormone discovered in human history, and yet, its function as a neuropeptide has been overlooked in the past. The recent discovery of the potential use of secretin in treating autistic patients, together with the conflicting reports on its effectiveness, urges an in-depth investigation of this issue. We show here that in the rat cerebellar cortex, mRNAs encoding secretin are localized in the Purkinje cells, whereas those of its receptor are found in both Purkinje cells and GABAergic interneurons. Immunoreactivity for secretin is localized in the soma and dendrites of Purkinje cells. In addition, secretin facilitates evoked, spontaneous, and miniature IPSCs recorded from Purkinje cells. We propose that secretin is released from the somatodendritic region of Purkinje cells and serves as a retrograde messenger modulating GABAergic afferent activity.

Adenylyl Cyclases↗
PubMed2001

Adenovirus-mediated tissue-targeted expression of a caspase-9-based artificial death switch for the treatment of prostate cancer.

Clinical experience with suicide gene therapy for prostate cancer using first-generation approaches has provided a basis for developing improved strategies. Given the low proliferation rate exhibited by prostate cancer, one improvement would be to develop suicide genes that effectively kill both dividing and nondividing cells. A second improvement would be to restrict cytotoxicity to prostate cancer cells, limiting injury of nondiseased tissue. Here we describe a novel approach to achieving both goals based on: (a) the use of a small, but potent, prostate-specific composite promoter, ARR(2)PB, based on the rat probasin gene; and (b) the use of a powerful artificial death switch, called inducible caspase-9 (iCaspase-9). ARR(2)PB includes two copies of the androgen response region (ARR), each containing two androgen receptor (AR)-binding sites, placed upstream of the probasin promoter elements necessary for basal transcription. Because iCaspase-9 contains two binding sites for the dimeric ligand, AP20187, administration of chemical inducers of dimerization leads to aggregation and caspase activation, followed by rapid apoptosis in both dividing and nondividing cells. Using both reagents, we constructed two novel adenoviruses (ADVs), ADV.ARR(2)PB-iCasp9 expressing iCaspase-9 and control ADV.ARR(2)PB-EGFP expressing enhanced green fluorescent protein (EGFP). We demonstrate that tissue specificity is not sacrificed in an ADV backbone because the marker protein, EGFP, is expressed in R1881-stimulated ADV.ARR(2)PB-EGFP-transduced LNCaP cells but not in AR(-) PC-3, 293, HuH-7, U-87, and MCF-7 cells. Similarly, Pro-iCaspase-9 is expressed in ADV.ARR(2)PB-iCasp9-infected LNCaP cells after R1881 administration and is activated after AP20187 administration. In vitro experiments revealed rapid and efficient iCaspase-9-induced apoptosis of LNCaP cells in both an R1881- and AP20187-dependent manner. Only 28, 8, and 0.5% survival of LNCaP cells was seen at multiplicities of infection of 2, 10, and 25, respectively. Furthermore, at a multiplicity of infection of 10, extraordinary sensitivity to AP20187 was seen (IC(50), approximately 3 pM). In vivo experiments showed that ADV.ARR(2)PB-iCasp9 induced apoptosis in LNCaP but not in HuH-7 xenograft tumors in an AP20187-dependent manner. Furthermore, a simple i.p. injection of AP20187 dramatically suppressed LNCaP tumor growth in nude mice and led to a significantly increased host survival. This study demonstrates the feasibility of using tissue-specific expression of cell cycle-independent iCaspases as a nonmutagenic alternative modality for prostate cancer suicide gene therapy.

Adenocarcinoma↗
PubMed2001

Stereoselective synthesis of methyl 4,6-O-benzylidene-2-C-methoxycarbonylmethyl-alpha-D-ribo-hexopyranosid-3-ulose, and its X-ray crystallographic analysis.

Methyl 4,6-O-benzylidene-2-C-methoxycarbonylmethyl-alpha-D-ribo-hexopyranosid-3-ulose has been stereoselectively synthesized in 65% yield by reaction of methyl 4,6-O-benzylidene-alpha-D-arabino-hexopyranosid-2-ulose with diethyl malonate. X-ray crystallographic structure analysis reveals that the chain-branch and the OH group are bonded to C-2 in axial and equatorial positions, respectively. The molecules in the crystal lattice are stacked along a one-dimensional chain, with intermolecular hydrogen bonds between O-8 of one molecule and 2-OH of the next as well as intramolecular hydrogen bonds between O-3 and 2-OH. All phenyl groups are parallel as well as the planes of sugar rings in the molecular columnar stacking.

Carbohydrate Conformation↗
PubMed2001

The C terminus of the human nicotinic alpha4beta2 receptor forms a binding site required for potentiation by an estrogenic steroid.

In addition to actions mediated by changes in gene expression, steroids can directly modulate several transmitter-gated and voltage-gated ion channels. Despite numerous studies showing that steroids enhance or reduce ion channel activity, the site(s) that mediates steroid recognition is not known. To identify the regions in which steroids bind and affect ion channel activity, we have taken advantage of the observation that human alpha4beta2 neuronal nicotinic receptors are potentiated by an estrogen steroid, 17beta-estradiol, whereas a rat alpha4beta2 receptor is not. Mutations indicate that a sequence (AGMI) at the end of the C terminus of the human alpha4 subunit forms a binding site required for 17beta-estradiol potentiation. In contrast, ethynyl beta-estradiol (an oral contraceptive) potentiates both human and rat alpha4beta2 receptors. A single tryptophan in the C terminus of both the rat and the human alpha4 subunit is sufficient for potentiation by ethynyl beta-estradiol, probably through a pi-pi interaction. Mutation of this tryptophan eliminates the ability of ethynyl beta-estradiol to potentiate rat receptors. However, in human receptors it was necessary to mutate both the AGMI sequence and the tryptophan to eliminate potentiation by ethynyl beta-estradiol. The findings that beta-estradiol requires the AGMI sequence but that a single C-terminal tryptophan is sufficient for potentiation by ethynyl beta-estradiol indicate that the C terminus forms a binding site for these steroids. The binding site(s) for block appears to differ from those involved in potentiation because the C-terminal sequence does not affect block by steroids such as progesterone, and progesterone does not competitively inhibit potentiation.

Acetylcholine↗
PubMed2001

Requirement of interleukin 17 receptor signaling for lung CXC chemokine and granulocyte colony-stimulating factor expression, neutrophil recruitment, and host defense.

Bacterial pneumonia is an increasing complication of HIV infection and inversely correlates with the CD4(+) lymphocyte count. Interleukin (IL)-17 is a cytokine produced principally by CD4(+) T cells, which induces granulopoiesis via granulocyte colony-stimulating factor (G-CSF) production and induces CXC chemokines. We hypothesized that IL-17 receptor (IL-17R) signaling is critical for G-CSF and CXC chemokine production and lung host defenses. To test this, we used a model of Klebsiella pneumoniae lung infection in mice genetically deficient in IL-17R or in mice overexpressing a soluble IL-17R. IL-17R-deficient mice were exquisitely sensitive to intranasal K. pneumoniae with 100% mortality after 48 h compared with only 40% mortality in controls. IL-17R knockout (KO) mice displayed a significant delay in neutrophil recruitment into the alveolar space, and had greater dissemination of K. pneumoniae compared with control mice. This defect was associated with a significant reduction in steady-state levels of G-CSF and macrophage inflammatory protein (MIP)-2 mRNA and protein in the lung in response to the K. pneumoniae challenge in IL-17R KO mice. Thus, IL-17R signaling is critical for optimal production of G-CSF and MIP-2 and local control of pulmonary K. pneumoniae infection. These data support impaired IL-17R signaling as a potential mechanism by which deficiency of CD4 lymphocytes predisposes to bacterial pneumonia.

Animals↗
PubMed2001

Cyclin A-dependent phosphorylation of the ETS-related protein, MEF, restricts its activity to the G1 phase of the cell cycle.

MEF, a recently identified member of the E74 family of ETS-related transcription factors, is a strong transcriptional activator of cytokine gene expression. Using a green fluorescent protein gene reporter plasmid regulated by an MEF-responsive promoter, we determined that the transcriptional activity of MEF is largely restricted to the G1 phase of the cell cycle. MEF-dependent transcription was suppressed by the expression of cyclin A but not by cyclin D or cyclin E. This effect was due to the kinase activity generated by cyclin A expression, as co-expression of the cyclin-dependent kinase inhibitors p21 or p27, or a dominant negative form of CDK2 (DNK2), abrogated the reduction of MEF transcriptional activity by cyclin A. Cyclin A-CDK2 phosphorylated MEF protein in vitro more efficiently than cyclin D-CDK4 or cyclin E-CDK2, and phosphorylation of MEF by cyclin A-CDK2 reduced its ability to bind DNA. We determined one site of phosphorylation by cyclin A-CDK2 at the C terminus of MEF, using mass-spectrometry; mutation of three serine or threonine residues in this region significantly reduced phosphorylation of MEF by cyclin A and reduced cyclin A-mediated suppression of its transactivating activity. These amino acid substitutions also reduced the restriction of MEF activity to G1. Phosphorylation of MEF by the cyclin A-CDK2 complex controls its transcriptional activity during the cell cycle, establishing a novel link between the ETS family of proteins and the cell cycle machinery.

Amino Acid Sequence↗
PubMed2001

Dna fragmentation factor 45 mutant mice exhibit resistance to kainic acid-induced neuronal cell death.

Excitotoxicity is a process where glutamate or other excitatory amino acids induce neuronal cell death. Emerging evidence suggests that apoptosis plays a key part in excitotoxic neurodegeneration. The DNA fragmentation factor 45 (DFF45 or ICAD) is a subunit of a heterodimeric DNase complex crucial for DNA fragmentation during apoptosis. Using a DFF45 mutant mouse model, we previously found that DFF45 deficient cells are more resistant to apoptosis than normal control cells. To investigate whether the lack of DFF45 may attenuate neuronal cell death induced by excitotoxicity, we compared kainic acid-induced seizure behavior and neuronal cell death in DFF45 mutant and wild-type control mice. We found that the mutant mice exhibit similar kainic acid-induced seizure severity compared to control mice. However, DFF45 mutant mice are more resistant than control mice to kainic acid-induced CA3 neuronal cell death. Interestingly, residual DNA degradation can be detected in the hippocampus of DFF45 mutant mice that exhibit KA-induced lesions. Our results suggest that a lack of DFF45 can lead to neuronal resistance to excessive activity-induced toxicity.

Animals↗
PubMed2001

Monitoring for adsorption of human serum albumin and bovine serum albumin onto bare and polystyrene-modified silver electrodes by quartz crystal impedance analysis.

The adsorption of human serum albumin (HSA) and bovine serum albumin (BSA) from PBS (pH 7.4) onto bare and polystyrene (PS)-modified silver electrodes was in situ monitored using quartz crystal impedance analysis. The adsorption characteristics of HSA and BSA were discussed by analyzing piezoelectric parameter simultaneous responses. Experimental results indicated that for both HSA and BSA, the amount adsorbed on bare silver was more than that on PS-modified surface. The BSA amount adsorbed on the two surfaces was more than that of adsorbed HSA. A kinetic model was developed to describe the adsorption process and fitted to the experimental data of frequency shift. It was shown that HSA adsorption could be described by a kinetic equation involving two consecutive reactions. At lower concentration, BSA adsorption only involved the first reaction. At higher concentration, BSA adsorption on PS-modified surface involved two consecutive reactions. All fitted results were well in agreement with the corresponding experimental results. The regression values of reaction rate constants for the HSA and BSA adsorption were obtained. These data exhibited difference in adsorption kinetics under different conditions.

Adsorption↗
PubMed2001

Crystal structure of the FAD/NADPH-binding domain of rat neuronal nitric-oxide synthase. Comparisons with NADPH-cytochrome P450 oxidoreductase.

Nitric-oxide synthase (NOS) is composed of a C-terminal, flavin-containing reductase domain and an N-terminal, heme-containing oxidase domain. The reductase domain, similar to NADPH-cytochrome P450 reductase, can be further divided into two different flavin-containing domains: (a) the N terminus, FMN-containing portion, and (b) the C terminus FAD- and NADPH-binding portion. The crystal structure of the FAD/NADPH-containing domain of rat neuronal nitric-oxide synthase, complexed with NADP(+), has been determined at 1.9 A resolution. The protein is fully capable of reducing ferricyanide, using NADPH as the electron donor. The overall polypeptide fold of the domain is very similar to that of the corresponding module of NADPH-cytochrome P450 oxidoreductase (CYPOR) and consists of three structural subdomains (from N to C termini): (a) the connecting domain, (b) the FAD-binding domain, and (c) the NADPH-binding domain. A comparison of the structure of the neuronal NOS FAD/NADPH domain and CYPOR reveals the strict conservation of the flavin-binding site, including the tightly bound water molecules, the mode of NADP(+) binding, and the aromatic residue that lies at the re-face of the flavin ring, strongly suggesting that the hydride transfer mechanisms in the two enzymes are very similar. In contrast, the putative FMN domain-binding surface of the NOS protein is less positively charged than that of its CYPOR counterpart, indicating a different nature of interactions between the two flavin domains and a different mode of regulation in electron transfer between the two flavins involving the autoinhibitory element and the C-terminal 33 residues, both of which are absent in CYPOR.

Amino Acid Sequence↗
PubMed2001

Site-directed mutation of the highly conserved region near the Q-loop of the cytochrome bd quinol oxidase from Escherichia coli specifically perturbs heme b595.

Cytochrome bd is one of the two quinol oxidases in the respiratory chain of Escherichia coli. The enzyme contains three heme prosthetic groups. The dioxygen binding site is heme d, which is thought to be part of the heme-heme binuclear center along with heme b(595), which is a high-spin heme whose function is not known. Protein sequence alignments [Osborne, J. P., and Gennis, R. B. (1999) Biochim. Biophys Acta 1410, 32--50] of cytochrome bd quinol oxidase sequences from different microorganisms have revealed a highly conserved sequence (GWXXXEXGRQPW; bold letters indicate strictly conserved residues) predicted to be on the periplasmic side of the membrane between transmembrane helices 8 and 9 in subunit I. The functional importance of this region is investigated in the current work by site-directed mutagenesis. Several mutations in this region (W441A, E445A/Q, R448A, Q449A, and W451A) resulted in a catalytically inactive enzyme with abnormal UV--vis spectra. E445A was selected for detailed analysis because of the absence of the absorption bands from heme b(595). Detailed spectroscopic and chemical analyses, indeed, show that one of the three heme prosthetic groups in the enzyme, heme b(595), is specifically perturbed and mostly missing from this mutant. Surprisingly, heme d, while known to interact with heme b(595), appears relatively unperturbed, whereas the low-spin heme b(558) shows some modification. This is the first report of a mutation that specifically affects the binding site of heme b(595).

Alanine↗
PubMed2001

Absorption of femtosecond laser pulses in interaction with solid targets.

We have studied the effects of the plasma density scale length on the absorption mechanism of the femtosecond (fs) laser pulses interacting with solid targets. Experiments and particle-in-cell (PIC) simulations demonstrate that the vacuum heating is the main absorption in the plasma in the interaction of fs laser pulses with solid targets when no prepulses are applied. The energy spectrum of hot electrons ejected out of or injected into the plasma show a bitemperature distribution. While the first temperature of the two groups of hot electrons can be attributed to the "pull-and-push" exertion of the laser field, the second temperature refers to the electrons accelerated by the static part (in front of the target) and the oscillating part (in the plasma layer) of the laser-induced electric field, respectively. PIC simulations also show that with an appropriate density scale length, the femtosecond laser energy can be absorbed locally through different mechanisms.

Journal Article↗
PubMed2001

Determination of puerarin, daidzein and rutin in Pueraria lobata (Wild.) Ohwi by capillary electrophoresis with electrochemical detection.

A method based on capillary electrophoresis with electrochemical detection was developed for the determination of puerarin, daidzein and rutin. Effects of several important factors such as the acidity and concentration of running buffer, separation voltage, injection time, and detection potential were investigated to acquire the optimum conditions. The working electrode was a 300-microm diameter carbon disc electrode positioned opposite the outlet of capillary. The three analytes could be well separated within 12 min in a 40 cm length capillary at a separation voltage of 9 kV in a 50 mmol/l borate buffer (pH 9.0). The relationship between peak currents and analyte concentrations was linear over about three orders of magnitude with detection limits (SIN=3) ranging from 0.241 x 10(-6) to 0.511 x 10(-6) mol/l for all compounds. This proposed method demonstrated long-term stability and reproducibility with relative standard deviations of less than 5% for both migration time and peak current (n=7). It has been successfully applied for the determination of puerarin, daidzein and rutin in Chinese traditional drugs, the vines of Pueraria lobata (Wild.) Ohwi and Puerariae Radix.

Buffers↗