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Biomedical subjects

K Zhang

Publications and source records attributed to K Zhang.

At least 109 records · Page 6Linked to original sources
PubMed2000

Differential mRNA fingerprinting by preferential amplification of coding sequences.

The need for rapid identification of differentially expressed genes will persist even after the complete human genomic sequence becomes available. The most popular method for identifying differentially expressed genes acquires expressed sequence tags (ESTs) from the extreme 3' non-coding end of mRNAs. Such ESTs have limitations for downstream applications. We have developed a method, termed preferential amplification of coding sequences (PACS), that was applied to identify differentially expressed coding sequence tags (dCSTs) between osteoblasts and osteosarcoma cells. PACS was achieved by PCR with a set of primers to anchor at sequences complementary to AUG sequences in mRNAs and another set of primers to anchor at a PCR-amplifiable distance from AUG sequences. An initial screen identified 103 candidate dCSTs after screening approximately 15% of the expressed genes between the two cell types. Of these sequences, 27 represent CSTs of known genes and two are from 3'-ESTs of known mRNAs. Thus, PACS identified CSTs approximately 13.5 times more often than it identified 3' ESTs, attesting to the objective of the method. Since many of the dCSTs represent known genes, their identity and potential relevance to osteosarcoma could be immediately hypothesized. Differential expression of many of the dCSTs was further demonstrated by northern blotting or RT-PCR. Since PACS is not dependent on the existence of a poly A tail on an mRNA, it should have application to identify dCSTs for both prokaryotic and eukaryotic organisms. Additionally, PACS should aid in the identification of cell-specific or tissue-specific genes and bidirectional acquisition of cDNA sequence enabling rapid retrieval of full-length cDNA sequence of novel genes.

Adenosine Triphosphatases↗
PubMed2000

Identification of differentially expressed genes by mutually subtracted RNA fingerprinting.

A mutually subtracted RNA fingerprinting (SuRF) method has been developed that allows efficient identification of differentially expressed sequence tags between two samples. Mutual subtractions of two RNA samples are achieved by first synthesizing cDNAs using oligo(dT) coupled with magnetic beads which are then reciprocally hybridized to starting RNA samples to remove common mRNAs between them. The second step involves differential fingerprinting of the subtracted RNA samples by polymerase chain reaction with specially designed degenerate primers. SuRF was applied to identify alteration in gene expression pertinent to osteogenic sarcoma which was achieved by employing the method between FOB (an immortalized fetal osteoblast) and MG63 (an osteosarcoma) cell lines. An estimated 10% of the total expressed genes in these two cell types were screened by the method. This analysis identified 96 differentially expressed sequences, none of which was identified repeatedly. A subset of these sequences was subsequently confirmed to have differential expression between the two cell types. Removal of common mRNAs prior to differential display should diminish redundant identification of abundant genes and increase the chance of identifying rare differentially expressed genes.

DNA, Complementary↗
PubMed2000

Effects of nigrostriatal dopamine denervation on ionotropic glutamate receptors in rat caudate-putamen.

Changes in ionotropic glutamate NMDA, AMPA and KA receptor binding in rat caudate-putamen were examined by quantitative in vitro receptor autoradiography 5 weeks after lesioning nigrostriatal dopaminergic projections. In this animal model of Parkinson's disease, density of binding in caudate-putamen increased at KA, but not NMDA or AMPA receptors. The findings indicate that nigrostriatal dopamine denervation can selectively enhance KA receptor levels in rat basal ganglia, suggest that KA receptors contribute to the pathophysiology of Parkinson's disease, and may suggest innovative treatments.

Adrenergic Agents↗
PubMed2000

Alkylation of rat dopamine transporters and blockade of dopamine uptake by EEDQ.

Effects of the alkylating agent EEDQ (N-ethoxycarbonyl-2-ethoxy-1, 2-dihydroquinoline) on levels of dopamine transporter (DA(T)) and function were examined in caudate-putamen (CPu) tissue from rat brain. EEDQ produced profound, dose-dependent decreases in DA(T) binding in homogenates (IC(50)=78 microM) and frozen sections (IC(75)=200 microM) that were not reversed by washing. EEDQ also blocked uptake of [(3)H]DA in CPu synaptosomes (IC(50)=17 microM). However, single (10 mg/kg) or repeated administration of EEDQ in vivo (15 mg/kg/day x 3) did not alter DA(T) levels or DA uptake in CPu. Pretreatment of rats with alpha-methyl-p-tyrosine and reserpine to deplete endogenous dopamine also failed to lower DA(T) levels in CPu after injections of EEDQ. EEDQ is an effective alkylating agent for DA(T) in vitro, but not to evaluate metabolic turnover or function of DA(T) in vivo. The results encourage development of selective and in vivo-active DA(T)-alkylating agents.

Alkylating Agents↗
PubMed2000

Production, properties and application to nonaqueous enzymatic catalysis of lipase from a newly isolated Pseudomonas strain.

A potent bacterium for lipase production was isolated from soil and identified as Pseudomonas species. It produced lipase constitutively. A mutant of this strain with a lipase productivity 3.25-fold higher was obtained by treatment with ultraviolet (UV) and nitrosoguanidine (NTG). Its fermentation condition was optimized to a lipase yield of 87.5 U/ml. The lipase had maximum activity at pH 9.0 and 45 degrees C. It was stable at pHs from 7.0 to 11.0 and below 60 degrees C. The effects of metal ions, surfactants and bile salts were also studied. The lipase was 1,3-specific. In organic solvents, the thermal stability of the lipase was significantly enhanced. Its optimum temperature was also slightly increased. The optimum water activity was found between 0.5 and 0.6. The lipase was successfully applied in organic phase to catalyze the glycerolysis of palm oil for monoglyceride (MG) production, and the enantioselective esterification of (R,S)-2-octanol. The enantioselectivity of the lipase could be enhanced substantially by treatment with an amphipathic.

Journal Article↗
PubMed2000

Proliferation-associated SNF2-like gene (PASG): a SNF2 family member altered in leukemia.

To identify genes involved in cell growth and/or apoptosis in leukemia, differential display was used to identify mRNAs that showed altered expression levels after cytokine withdrawal from the cytokine-dependent MO7e cell line. Sequence analysis of one transcript that showed a profound decrease in expression after cytokine withdrawal revealed it to be a member of the SNF2 family of chromatin remodeling ATPases. This cDNA had a 2514-nucleotide (838-amino acid) open reading frame and encoded an additional 230 amino acids at the NH2 terminus compared with the murine homologue, lsh, and the human counterpart, Hells. This gene locus has been designated SMARCA6 (SWI/SNF2-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 6). The highest levels of mRNA expression in humans are observed in proliferative tissues such as the thymus, testis, and bone marrow. Whereas cytokine withdrawal in MO7e cells leads to apoptosis and decreased mRNA expression, growth arrest without the induction of apoptosis of MO7e cells also leads to down-regulation of mRNA expression, suggesting an association with cell proliferation and not suppression of apoptosis. Nuclear localization of this SNF2-like putative helicase is dependent on a nuclear localization sequence located in the NH2-terminal region. Based on sequence homology to other SNF2-like helicases, the pattern of tissue expression, and the association of expression with cell proliferation, we refer to the protein product as proliferation-associated SNF2-like gene product [PASG (D. W. Lee et al., Blood, 94: 594a, 1999)]. Examination of acute myelogenous leukemia and acute lymphoblastic leukemia samples revealed a high frequency of a PASG transcript containing an in-frame 75-nucleotide deletion, which codes for a conserved motif known to be critical for the transactivation activity of a related yeast SWI/SNF polypeptide. These results extend our knowledge of this SNF2-like family member and suggest a role for PASG in leukemogenesis.

Alternative Splicing↗
PubMed2000

Olanzapine, quetiapine, and risperidone: long-term effects on monoamine transporters in rat forebrain.

Long-term effects of novel atypical antipsychotic drugs on monoamine transporters are unknown. We compared labeling of dopamine (DAT) and serotonin (SERT) transporter proteins in subregions of rat corpus striatum by quantitative autoradiography with [(3)H]2-beta-carbomethoxy-3-beta-[4'-iodophenyl]tropane ([(3)H]beta-CIT) and [(3)H]paroxetine after 28 days of continuous subcutaneous infusion of olanzapine, quetiapine, risperidone, or vehicle controls. Drug treatment did not significantly alter the abundance of either transporter type in caudate-putamen or nucleus accumbens, indicating that transporter proteins required to inactivate synaptically released dopamine and serotonin resist adaptations to long-term treatment with novel antipsychotics that affect neurotransmission by these amines.

Animals↗
PubMed2000

Noradrenergic lesions differentially alter the expression of two subtypes of low Km cAMP-sensitive phosphodiesterase type 4 (PDE4A and PDE4B) in rat brain.

This study examined the effects of selective, central noradrenergic dennervation with 6-hydroxydopamine (6-OHDA) on the expression of type 4 phosphodiesterases (PDE4). Twenty-one days following i.c.v. injection of 6-OHDA (200 microg) hypothalamus, neostriatum, and cerebellum were dissected. Infusion of 6-OHDA reduced norepinephrine (NE) content in all the brain areas examined (to 17%, 76% and 16% of sham-operated controls in hypothalamus, striatum, and cerebellum, respectively). 6-OHDA injections also reduced dopamine levels in hypothalamus (53%) and neostriatum (68%). Administration of desipramine (20 mg/kg, i.p.) 30 min prior to 6-OHDA injection protected neostriatal and cerebellar noradrenergic neurons NE levels (110-122% of the control levels). Desipramine partially attenuated the 6-OHDA-mediated decrease in NE content of hypothalamus, but had little or no effect on either striatal or hypothalamic dopamine (DA) levels. Western blot analysis using a PDE4A-selective antibody revealed three major bands (109 kDa PDE4A5, 102 kDa PDE4AX and 76 kDa PDE4A1) in hypothalamus and striatum. Infusion of 6-OHDA decreased the expression of PDE4A5 and PDE4AX but not of PDE4A1 in hypothalamus, as determined by quantitative Western blotting. Pretreatment of rats with desipramine attenuated the 6-OHDA-induced down-regulation of PDE4A5 and PDE4AX bands in hypothalamus. The PDE4B selective antibody K118 labels 5 major bands in all the brain regions studied. One hundred kDa PDE4B3, 86 kDa PDE4B2 and a 78 kDa PDE4B band was identified using recombinant proteins. Treatment of rats with 6-OHDA resulted in a 52% decrease in the PDE4B3 and 58% decrease in 78 kDa PDE4B variant in hypothalamus; administration of desipramine attenuated the 6-OHDA-induced down-regulation of both PDE4B variants. Neither 6-OHDA nor desipramine altered striatal PDE4A or PDE4B isozymes. In contrast, cerebellar PDE4B3 variant is up-regulated by 6-OHDA treatment and were partially normalized to control values by desipramine pretreatment. These data demonstrate that PDE4 subtypes are differentially regulated by presynaptic noradrenergic activity and may play an important role in the maintaining homeostasis of noradrenergic signal transduction in rat brain.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed2000

Neuronal expression of Fos protein in the hypothalamus of rats with heart failure.

We sought to identify the areas that have altered neuronal activity within the hypothalamus of rats with heart failure (HF) by mapping neuronal staining of c-Fos protein (Fos) 6-8 weeks following coronary artery ligation (HF group; n=17) or sham surgery (sham-operated control group, n=15). Fos-like immunoreactivity was observed in the paraventricular nucleus (PVN), supraoptic nucleus (SON), median preoptic nucleus (MnPO), anterior hypothalamus (AH) and posterior hypothalamus (PH) using a standard ABC immunocytochemical protocol. The rats in the HF group displayed infarcts averaging 34+/-2% of the outer circumference and 41+/-1% of the inner circumference of the left ventricular wall. Sham-operated control rats had no observable damage to the myocardium. Rats with chronic heart failure (n=5) but no manipulation (no surgery) had a similar number of Fos-staining cells in PVN SON, MnPO, AH and PH compared to sham-operated rats. Acute surgery for isolation of vagus nerves and anesthesia for 90 min increased the number of Fos positive cells in PVN, SON and MnPO of both sham-operated rats and rats with HF. Furthermore, rats with heart failure (n=5) had significantly higher number of Fos-staining cells in PVN (four times), SON (4.5 times) and MnPO (1.5 times) compared to sham-operated rats after acute surgery for isolation of the vagus. The number of Fos-staining cells remained unaltered in AH and PH in both groups of rats. However, in a third series of experiments vagotomy reduced the number of Fos-staining cells in the PVN, SON or MnPO of rats with HF (n=5) to those observed in sham-operated vagotomized rats. This study shows that: (1) there is augmented neuronal activity as indicated by increased number of Fos staining neurons in the PVN, SON and MnPO due to acute surgical stress in rats with HF, and (2) vagal afferents are responsible for the increased neuronal activity in PVN, SON and MnPO of rats with HF during acute surgical stress. These data support the conclusion that vasopressin producing neurons and autonomic areas within the hypothalamus influenced by vagal afferents are activated during HF and are sensitive to 'acute surgical stress' and may contribute to the elevated levels of vasopressin and sympatho-excitation commonly observed in heart failure.

Animals↗
PubMed2000

Bruton's tyrosine kinase links the B cell receptor to nuclear factor kappaB activation.

The recognition of antigen by membrane immunoglobulin M (mIgM) results in a complex series of signaling events in the cytoplasm leading to gene activation. Bruton's tyrosine kinase (BTK), a member of the Tec family of tyrosine kinases, is essential for the full repertoire of IgM signals to be transduced. We examined the ability of BTK to regulate the nuclear factor (NF)-kappaB/Rel family of transcription factors, as the activation of these factors is required for a B cell response to mIgM. We found greatly diminished IgM- but not CD40-mediated NF-kappaB/Rel nuclear translocation and DNA binding in B cells from X-linked immunodeficient (xid) mice that harbor an R28C mutation in btk, a mutation that produces a functionally inactive kinase. The defect was due, in part, to a failure to fully degrade the inhibitory protein of NF-kappaB, IkappaBalpha. Using a BTK-deficient variant of DT40 chicken B cells, we found that expression of wild-type or gain-of-function mutant BTK, but not the R28C mutant, reconstituted NF-kappaB activity. Thus, BTK is essential for activation of NF-kappaB via the B cell receptor.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed2000

A universal scaling law between gray matter and white matter of cerebral cortex.

Neocortex, a new and rapidly evolving brain structure in mammals, has a similar layered architecture in species over a wide range of brain sizes. Larger brains require longer fibers to communicate between distant cortical areas; the volume of the white matter that contains long axons increases disproportionally faster than the volume of the gray matter that contains cell bodies, dendrites, and axons for local information processing, according to a power law. The theoretical analysis presented here shows how this remarkable anatomical regularity might arise naturally as a consequence of the local uniformity of the cortex and the requirement for compact arrangement of long axonal fibers. The predicted power law with an exponent of 4/3 minus a small correction for the thickness of the cortex accurately accounts for empirical data spanning several orders of magnitude in brain sizes for various mammalian species, including human and nonhuman primates.

Animals↗
PubMed2000

Spherical Oscillatory alpha2 Dynamo Induced by Magnetic Coupling between a Fluid Shell and an Inner Electrically Conducting Core: Relevance to the Solar Dynamo.

A two-layer spherical alpha2 dynamo model consisting of an inner electrically conducting core (magnetic diffusivity lambdai and radius ri) with alpha=0 surrounded by an electrically conducting spherical shell (magnetic diffusivity lambdao and radius ro) with a constant alpha is shown to exhibit oscillatory behavior for values of beta=lambdai&solm0;lambdao and ri&solm0;ro relevant to the solar dynamo. Time-dependent dynamo solutions require ri&solm0;ro>/=0.55 and beta</=O&parl0;1&parr0;. For the Sun, ri&solm0;ro is about 0.8 and beta approximately 10-3. The timescale of the oscillations matches the 22 yr period of the sunspot cycle for lambda0=O(102 km2 s-1). It is unnecessary to hypothesize an alpha-omega dynamo to obtain oscillatory dynamo solutions; an alpha2 dynamo suffices provided the spherical shell region of dynamo action lies above a large, less magnetically diffusive core, as is the case for the solar dynamo.

Journal Article↗
PubMed2000

In vivo antitumor activity of ONYX-015 is influenced by p53 status and is augmented by radiotherapy.

The E1B-deleted, replication-competent ONYX-015 (dl1520) adenovirus was originally described as being able to selectively kill p53-deficient cells due to a requirement of p53 inactivation for efficient viral replication. This hypothesis has become controversial because subsequent in vitro studies have demonstrated that the host range specificity of ONYX-015 is independent of p53 gene status. Using a pair of isogenic cell lines that differ only in their p53 status, we demonstrate here that although ONYX-015 can replicate in both p53 wild-type and mutant cells in vitro, the virus demonstrates significantly greater antitumor activity against mutant p53 tumors in vivo. Moreover, ONYX-015 viral therapy can be combined with radiation to improve tumor control beyond that of either monotherapy. The results demonstrate that ONYX-015 can discern in vivo between tumors having a different p53 status and that it may be an effective neoadjuvant to radiation therapy.

Adenoviridae↗
PubMed2000

Glutathione-related factors are not correlated with sensitivity of human tumour cells to actinomycin D.

Glutathione (GSH) contents and activities of glutathione S-transferases (GST), glutathione reductase (GSH-RD), glutathione peroxidase (GSHpx) and glutathione conjugate export pump (GS-X pump) were determined in eight human tumour cell lines with different sensitivities to melphalan, a substrate of glutathione conjugation, and actinomycin D which has not been shown to be detoxified by glutathione-related mechanisms. Chang liver cells with highest GSH content and highest activities of GST, GSH-RD, GSHpx and GS-X pump were found to be most resistant to melphalan. Statistical analysis showed significant correlations between sensitivities of the human tumour cells to melphalan and the glutathione-related factors (r = 0.72-0.79; except for GST, r = 0.65, P = 0.08), while there were no significant correlations observed between sensitivities of the human tumour cells to actinomycin D and all the glutathione-related factors tested (r = -0.25-0.14). Significant correlations of the glutathione-related factors to resistance of human tumour cells to melphalan, a substrate of glutathione conjugation, but not to resistance of the human tumour cells to actinomycin D which has not been shown to be detoxified by glutathione-related mechanisms suggested that glutathione-related mechanisms contribute to drug resistance by increased detoxification of the drugs involved.

Antibiotics, Antineoplastic↗
PubMed2000

Synthesis and evaluation of furo[3,4-d]pyrimidinones as selective alpha1a-adrenergic receptor antagonists.

Furo[3,4-d]pyrimidinones were found to be metabolites of dihydropyrimidinones such as 1a-b that are subtype-selective antagonists of the alpha1a-adrenergic receptor. A versatile synthesis that provides access to furo[3,4-d]pyrimidinones in high yield and in enantiomerically pure forms is described along with structure-activity relationships in the series.

Adrenergic alpha-Antagonists↗
PubMed2000

Cell-free recombination of immunoglobulin switch-region DNA with nuclear extracts.

We have developed an in vitro recombination system employing cell-free nuclear extracts from human B lymphocytes capable of detecting the recombination between human mu switch (Smu) and Sepsilon sequences in a model plasmid. Nuclear extracts from CD40-stimulated B lymphocytes gave a higher frequency of recombination in the assay than the unstimulated B cells. Recombination between Smu and Sepsilon was mediated by the nuclear extracts as the recombinational products could be amplified prior to bacterial transformation. Characterization of the recombination products demonstrated that the recombination process had the characteristics of immunoglobulin (Ig) isotype switching, as it was (i) switch-region-sequence specific, (ii) nonhomologous recombination, and (iii) enhanced by CD40 stimulation. Transcription through the S region DNA was not required for recombination in the system. These results demonstrate that Ig switch-region DNA recombination can be accomplished in vitro by cell-free nuclear extracts. This in vitro system for Ig switch-region DNA recombination using cell-free nuclear extracts will permit the dissection of the events involved in IgE class switch recombination, a critical event in the development of allergic diseases.

B-Lymphocytes↗
PubMed2000

Assessing reliability of gene clusters from gene expression data.

The rapid development of microarray technologies has raised many challenging problems in experiment design and data analysis. Although many numerical algorithms have been successfully applied to analyze gene expression data, the effects of variations and uncertainties in measured gene expression levels across samples and experiments have been largely ignored in the literature. In this article, in the context of hierarchical clustering algorithms, we introduce a statistical resampling method to assess the reliability of gene clusters identified from any hierarchical clustering method. Using the clustering trees constructed from the resampled data, we can evaluate the confidence value for each node in the observed clustering tree. A majority-rule consensus tree can be obtained, showing clusters that only occur in a majority of the resampled trees. We illustrate our proposed methods with applications to two published data sets. Although the methods are discussed in the context of hierarchical clustering methods, they can be applied with other cluster-identification methods for gene expression data to assess the reliability of any gene cluster of interest.

Algorithms↗