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Biomedical subjects

K Chen

Publications and source records attributed to K Chen.

At least 235 records · Page 13Linked to original sources
PubMed1998

Mathematical model of the fission yeast cell cycle with checkpoint controls at the G1/S, G2/M and metaphase/anaphase transitions.

All events of the fission yeast cell cycle can be orchestrated by fluctuations of a single cyclin-dependent protein kinase, the Cdc13/Cdc2 heterodimer. The G1/S transition is controlled by interactions of Cdc13/Cdc2 and its stoichiometric inhibitor, Rum1. The G2/M transition is regulated by a kinase-phosphatase pair, Wee1 and Cdc25, which determine the phosphorylation state of the Tyr-15 residue of Cdc2. The meta/anaphase transition is controlled by interactions between Cdc13/Cdc2 and the anaphase promoting complex, which labels Cdc13 subunits for proteolysis. We construct a mathematical model of fission yeast growth and division that encompasses all three crucial checkpoint controls. By numerical simulations we show that the model is consistent with a broad selection of cell cycle mutants, and we predict the phenotypes of several multiple-mutant strains that have not yet been constructed.

Anaphase↗
PubMed1998

Predictors of cessation of marijuana use: an event history analysis.

Event history analysis was applied to monthly life and drug histories of a representative community sample of 706 marijuana users, followed from ages 15-16 to 34-35, to investigate factors associated with cessation of marijuana use from adolescence to adulthood. In addition to age and gender, the most important determinants of cessation are the phenomenology of marijuana use, social role participation, depressive symptoms and deviance. Frequent users, those who started using early and those who use illicit drugs other than marijuana are more likely to continue their marijuana use. Using marijuana for social reasons accelerates cessation, using to change one's mood reduces cessation. Becoming pregnant and a parent is the most important social role leading to marijuana cessation for women. There is also a very important experimental effect of the interview itself on the reported timing of a cessation. The effect of a social context favorable to marijuana use appears to reflect selection rather than social influence.

Adolescent↗
PubMed1998

A multicenter investigation with interphase fluorescence in situ hybridization using X- and Y-chromosome probes.

Twenty-six laboratories used X and Y chromosome probes and the same procedures to process and examine 15,600 metaphases and 49,400 interphases from Phaseolus vulgaris-leucoagglutinin (PHA)-stimulated lymphocytes. In Part I, each laboratory scored 50 metaphases and 200 interphases from a normal male and a normal female from its own practice. In Part II, each laboratory scored 50 metaphases and 200 interphases on slides prepared by a central laboratory from a normal male and a normal female and three mixtures of cells from the male and female. In Part III, each laboratory scored 50 metaphases (in samples of 5, 10, 15, and 20) and 100 interphases (in samples of 5, 10, 15, 20, and 50) on new, coded slides of the same specimens used in Part II. Metaphases from male specimens were scored as 98-99% XY with no XX cells, and 97-98% of interphases were scored as XY with 0.04% XX cells. Metaphases from female specimens were scored as 96-97% XX with 0.03% XY cells, and 94-96% of interphases were scored as XX with 0.05% XY cells. Considering the data as a model for any probe used with fluorescence in situ hybridization (FISH), a statistical approach assessing the impact of analytical sensitivity on the numbers of observations required to assay for potential mosaicisms and chimerisms is discussed. The workload associated with processing slides and scoring 50 metaphases and 200 interphases using FISH averaged 27.1 and 28.6 minutes, respectively. This study indicates that multiple laboratories can test/develop guidelines for the rapid, efficacious, and cost-effective integration of FISH into clinical service.

Cytogenetics↗
PubMed1998

The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53.

The INK4a gene encodes two distinct growth inhibitors--the cyclin-dependent kinase inhibitor p16Ink4a, which is a component of the Rb pathway, and the tumor suppressor p19Arf, which has been functionally linked to p53. Here we show that p19Arf potently suppresses oncogenic transformation in primary cells and that this function is abrogated when p53 is neutralized by viral oncoproteins and dominant-negative mutants but not by the p53 antagonist MDM2. This finding, coupled with the observations that p19Arf and MDM2 physically interact and that p19Rrf blocks MDM2-induced p53 degradation and transactivational silencing, suggests that p19Arf functions mechanistically to prevent MDM2's neutralization of p53. Together, our findings ascribe INK4a's potent tumor suppressor activity to the cooperative actions of its two protein products and their relation to the two central growth control pathways, Rb and p53.

Animals↗
PubMed1998

Effects of endogenous acetylcholine on spontaneous activity in rat dorsal cochlear nucleus slices.

We have examined the contribution of endogenous acetylcholine (ACh) release to the spontaneous firing of both regular (probably fusiform cells) and bursting neurons (probably cartwheel cells) in the dorsal cochlear nucleus (DCN) in rat brainstem slices. The muscarinic antagonists atropine, scopolamine, and tropicamide (1-2 microM) caused substantial decreases of firing rates in a majority of the neurons. Reversible acetylcholinesterase (AChE) inhibitors typically caused large transient increases in firing that decayed more slowly than responses to carbachol. The irreversible AChE inhibitor diisopropyl fluorophosphate (DFP) usually caused a sustained increase, with an initial peak followed by a gradual change to a final level higher than before DFP. Tropicamide caused large decreases in firing after DFP, confirming sustained ACh release. Both neostigmine and DFP applied after AChE inhibition by DFP sometimes elicited a transient response. We conclude that the level of sustained response to DFP is determined by the rate of endogenous ACh release, and that DFP and reversible AChE inhibitors exert an initial transient agonist effect that overlaps the initial effect of acetylcholinesterase inhibition. The slice experiments provide a model for cholinergic mechanisms in vivo, confirm that the release of endogenous ACh increases the firing rates of regular and bursting neurons in superficial DCN, and support the hypothesis that spontaneous firing of DCN neurons is sustained in part by cholinergic inputs.

Acetylcholine↗
PubMed1998

The kinetics of cytokine gene expression in the thyroids of mice developing granulomatous experimental autoimmune thyroiditis.

To study the potential roles of cytokines in development and resolution of granulomatous experimental autoimmune thyroiditis (EAT), the kinetics of in vivo expression of cytokine genes in thyroid infiltrates was analysed using reverse transcriptase-PCR (RT-PCR). Both Th1 (IL-2 and IFN-gamma) and Th2 (IL-4 and IL-10) cytokines as well as TGF-betaTNF-alphaIL-12 and IL-1beta were detected in thyroids during both the initial phase and peak of granulomatous EAT. Maximal expression of cytokine genes generally occurred 11-14 days after cell transfer, prior to maximal EAT severity, which occurred 19-21 days after cell transfer. The relative ratios of Th1:Th2 cytokines and mouse thyroglobulin-(MTg)-specific IgG1 and IgG2a autoantibody levels were similar during both the initial phase and peak of EAT. Depletion of CD8(+) T cells did not decrease the severity of EAT but delayed resolution of lesions. Cytokine gene expression in thyroids was not decreased by anti-CD8 treatment. Together, these data indicate that both Th1 and Th2 cytokines produced by CD4(+) T cells are involved in induction and development of granulomatous EAT, and CD8-dependent resolution of granulomatous EAT is apparently not mediated by these cytokines.

Animals↗
PubMed1998

Determination of regions important for monoamine oxidase (MAO) A and B substrate and inhibitor selectivities.

MAO-A and -B are defined by their substrate and inhibitor preferences. To determine which regions of the isoenzymes confer these preferences, we have constructed six chimeric MAO enzymes by reciprocally exchanging corresponding N-terminal, C-terminal, and internal segments of MAO-A and -B then determined the catalytic properties of these chimeric enzymes. N-terminal chimerics A45B and B36A were made by exchanging amino acid segments 1-45 and 1-36 of MAO-A and -B respectively. C-terminal chimerics A402B and B393A were made by exchanging amino acid segments 403-527 and 394-520 of MAO-A and -B respectively, and internal chimerics AB161-375A and BA152-366B were made by exchanging amino acid segments 161-375 and 152-366 of MAO-A and -B respectively. The enzymatic properties observed for the chimerics suggest that the exchanged internal regions but not the N- or C-terminal regions confer substrate and inhibitor preferences.

Amino Acid Sequence↗
PubMed1998

New method for the analysis of multiple positron emission tomography dynamic datasets: an example applied to the estimation of the cerebral metabolic rate of oxygen.

Positron emission tomography (PET) provides the ability to extract useful quantitative information not available through other radiological techniques. In certain studies, the physiological parameters of interest cannot be determined from the data obtained from a single PET experiment alone. In this case, multiple experiments are required. At present, the methods used to analyse measurements acquired from multiple experiments often involve considering them separately during the modelling procedures. These methods of analysis may cause errors to be propagated through successive modelling procedures and do not fully utilise the information content provided by the PET measurements. A new method is presented, based on linear least squares for the analysis of PET dynamic data acquired from multiple experiments. This method simultaneously considers the complete set of measurements obtained and provides reliable parameter estimates. The efficient use of the information content provided by multiple experiments is considered and the propagation of errors is discussed. To facilitate our discussion, we apply this new method to the estimation of the cerebral metabolic rate of oxygen and the parameters of the oxygen utilisation model as a practical example. The results demonstrate a significant improvement in the reliability and estimation accuracy of the estimates for this new method. Furthermore, this method reduced the likelihood of errors being propagated. Therefore, the proposed method is suitable for the analysis of multiple PET dynamic datasets.

Brain↗
PubMed1998

Differential effects of two protein tyrosine kinase inhibitors, tyrphostin and genistein, on human bone cell proliferation as compared with differentiation.

Protein tyrosyl phosphorylation is a key determinant of cell proliferation and differentiation. The aim of this study was to test the hypothesis that the signal transduction pathway(s) responsible for human bone cell proliferation may involve different groups of protein tyrosine kinase (PTKs) as compared with that for differentiation. To achieve this, we investigated the effects of two structurally different PTK inhibitors, viz, tyrphostin A51 and genistein, on the proliferation ([3H]thymidine incorporation) and differentiation [alkaline phosphatase (ALP) specific activity and collagen synthesis] of two normal human bone cell types: mandible-derived and vertebra-derived bone cells. Tyrphostin A51 and genistein each markedly reduced cellular tyrosyl phosphorylation level (assessed by Western analysis using a commercial anti-phosphotyrosine antibody and the enhanced chemiluminescence detection assay), confirming that these two effectors are potent PTK inhibitors in human bone cells. Regarding bone cell proliferation, tyrphostin A51 (5-30 microM) caused, a dose-dependent inhibition of basal [3H]thymidine incorporation of both human bone cell types. In contrast, genistein (5-20 microM), not only did not inhibit, but significantly stimulated [3H]thymidine incorporation of these same cell types in a dose-dependent, biphasic manner, with the optimal stimulatory dose between 10 and 20 microM. These effects on cell proliferation were confirmed by cell number counting. In addition, whereas the mitogenic activity of 10 ng/ml epidermal growth factor (EGF) on human mandible-derived bone cells was completely abolished by 5-30 microM tyrphostin A51, genistein at 5-30 microM enhanced the EGF-induced bone cell proliferation in an additive manner. With respect to bone cell differentiation, tyrphostin A51 and genistein each significantly increased basal ALP specific activity and collagen synthesis in human bone cells. In summary, (1) PTKs are involved in human bone cell proliferation and differentiation; (2) tyrphostin A51 inhibited both basal and EGF-induced cell proliferation, thus tyrphostin-sensitive PTKs are involved in basal and EGF-induced human bone cell proliferation; (3) genistein stimulated basal proliferation and enhanced EGF-mediated cell proliferation, suggesting that genistein-sensitive PTKs may play an inhibitory role in human bone cell proliferation; and (4) these differential effects of PTK inhibitors on human bone cell proliferation and differentiation are independent of basal differentiation status of the cells.

Alkaline Phosphatase↗
PubMed1998

Tissue-specific effects of estrogen on monoamine oxidase A and B in the rat.

Estrogen replacement therapy is widely used in postmenopausal women. The current study examines the effect of varying concentrations of estrogen on the levels of activity of monoamine oxidase A and -B in brain and in other tissues. Adult female rats were ovariectomized and randomized to receive a subcutaneous, slow-release preparation of either placebo or one of three doses of 17-beta-estradiol (0.05, 0.5, or 5.0 mg/pellet, estimated serum levels of 20-25 pg/ml, 100-600 pg/ml, and 1-2 ng/ml, respectively). Animals were sacrificed at 3 weeks and MAO-A and -B activity was assessed in homogenates of heart, liver, lung, uterus, kidney, adrenal and small intestine using 5-hydroxytryptamine and phenylethylamine as substrates. Cortex, amygdala and hypothalamus were microdissected from frozen sections of the brain and were also assayed for MAO-A and -B activity. High dose estrogen (5 mg/pellet) significantly decreased MAO-B activity and resulted in lesser or insignificant changes in MAO-A activity, respectively in liver (-30%, +1%), kidney (-22%, -11%), and uterus (-57%, -35%) (p < 0.05). No significant changes in enzyme activity were observed in heart, adrenal, lung and small intestine. In brain, estrogen (5 mg/pellet) decreased MAO-A activity in the hypothalamus (-28%) and amygdala (-21%), with no significant change seen in MAO-B. Our results suggest that estrogen exerts a tissue-specific, differential regulation of MAO-A and -B activity.

Animals↗
PubMed1998

Is there a "non-MAO" macromolecular target for L-deprenyl?: Studies on MAOB mutant mice.

Several irreversible inhibitors of monoamine oxidase B, can promote survival of damaged neurons in several animal models of cerebral injury. Today it is evident that this effect is not a consequence of monoamine inhibition. Instead it has been proposed that L-deprenyl may act via an unrelated non-monoamine oxidase type- of binding site. In the present study we have investigated if brain tissue from MAOB knockout mice contain such "non-MAO" binding sites for radiolabelled deprenyl. Interestingly, no binding of L-deprenyl was observed indicating that the mother compound does not act directly via a macromolecular target. This is enticing since several alternative mechanisms of action have been proposed in the literature.

Animals↗
PubMed1998

The pro-convulsant actions of corticotropin-releasing hormone in the hippocampus of infant rats.

Whole-cell patch-clamp and extracellular field recordings were obtained from 450-microns-thick brain slices of infant rats (10-13 days postnatal) to determine the actions of corticotropin-releasing hormone on glutamate- and GABA-mediated synaptic transmission in the hippocampus. Synthetic corticotropin-releasing hormone (0.15 microM) reversibly increased the excitability of hippocampal pyramidal cells, as determined by the increase in the amplitude of the CA1 population spikes evoked by stimulation of the Schaffer collateral pathway. This increase in population spike amplitude could be prevented by the corticotropin-releasing hormone receptor antagonist alpha-helical (9-41)-corticotropin-releasing hormone (10 microM). Whole-cell patch-clamp recordings revealed that, in the presence of blockers of fast excitatory and inhibitory synaptic transmission, corticotropin-releasing hormone caused only a small (1-2 mV) depolarization of the resting membrane potential in CA3 pyramidal cells, and it did not significantly alter the input resistance. However, corticotropin-releasing hormone, in addition to decreasing the slow afterhyperpolarization, caused an increase in the number of action potentials per burst evoked by depolarizing current pulses. Corticotropin-releasing hormone did not significantly change the frequency, amplitude or kinetics of miniature excitatory postsynaptic currents. However, it increased the frequency of the spontaneous excitatory postsynaptic currents in CA3 pyramidal cells, without altering their amplitude and single exponential rise and decay time constants. Corticotropin-releasing hormone did not change the amplitude of the pharmacologically isolated (i.e. recorded in the presence of GABAA receptor antagonist bicuculline) excitatory postsynaptic currents in CA3 and CA1 pyramidal cells evoked by stimulation of the mossy fibers and the Schaffer collaterals, respectively. Current-clamp recordings in bicuculline-containing medium showed that, in the presence of corticotropin-releasing hormone, mossy fiber stimulation leads to large, synchronized, polysynaptically-evoked bursts of action potentials in CA3 pyramidal cells. In addition, the peptide caused a small, reversible decrease in the amplitude of the pharmacologically isolated (i.e. recorded in the presence of glutamate receptor antagonists) evoked inhibitory postsynaptic currents in CA3 pyramidal cells, but it did not significantly alter the frequency, amplitude, rise and decay time constants of spontaneous or miniature inhibitory postsynaptic currents. These data demonstrate that corticotropin-releasing hormone, an endogenous neuropeptide whose intracerebroventricular infusion results in seizure activity in immature rats, has diverse effects in the hippocampus which may contribute to epileptogenesis. It is proposed that the net effect of corticotropin-releasing hormone is a preferential amplification of those incoming excitatory signals which are strong enough to reach firing threshold in at least a subpopulation of CA3 cells. These findings suggest that the actions of corticotropin-releasing hormone on neuronal excitability in the immature hippocampus may play a role in human developmental epilepsies.

Action Potentials↗
PubMed1998

Androgen receptor YAC transgenic mice carrying CAG 45 alleles show trinucleotide repeat instability.

X-linked spinal and bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene. Disease-associated alleles (37-66 CAGs) change in length when transmitted from parents to offspring, with a significantly greater tendency to shift size when inherited paternally. As transgenic mice carrying human AR cDNAs with 45 and 66 CAG repeats do not display repeat instability, we attempted to model trinucleotide repeat instability by generating transgenic mice with yeast artificial chromosomes (YACs) carrying AR CAG repeat expansions in their genomic context. Studies of independent lines of AR YAC transgenic mice with CAG 45 alleles reveal intergenerational instability at an overall rate of approximately 10%. We also find that the 45 CAG repeat tracts are significantly more unstable with maternal transmission and as the transmitting mother ages. Of all the CAG/CTG repeat transgenic mice produced to date the AR YAC CAG 45 mice are unstable with the smallest trinucleotide repeat mutations, suggesting that the length threshold for repeat instability in the mouse may be lowered by including the appropriate flanking human DNA sequences. By sequence-tagged site content analysis and long range mapping we determined that one unstable transgenic line has integrated an approximately 70 kb segment of the AR locus due to fragmentation of the AR YAC. Identification of the cis -acting elements that permit CAG tract instability and the trans -acting factors that modulate repeat instability in the AR YAC CAG 45 mice may provide insights into the molecular basis of trinucleotide repeat instability in humans.

Age Factors↗
PubMed1998

Noninvasive quantification of the cerebral metabolic rate for glucose using positron emission tomography, 18F-fluoro-2-deoxyglucose, the Patlak method, and an image-derived input function.

The authors developed and tested a method for the noninvasive quantification of the cerebral metabolic rate for glucose (CMRglc) using positron emission tomography (PET), 18F-fluoro-2-deoxyglucose, the Patlak method, and an image-derived input function. Dynamic PET data acquired 12 to 48 seconds after rapid tracer injection were summed to identify carotid artery regions of interest (ROIs). The input function then was generated from the carotid artery ROIs. To correct spillover, the early summed image was superimposed over the last PET frame, a tissue ROI was drawn around the carotid arteries, and a tissue time activity curve (TAC) was generated. Three venous samples were drawn from the tracer injection site at a later time and used for the spillover and partial volume correction by non-negative least squares method. Twenty-six patient data sets were studied. It was found that the image-derived input function was comparable in shape and magnitude to the one obtained by arterial blood sampling. Moreover, no significant difference was found between CMRglc estimated by the Patlak method using either the arterial blood sampling data or the image-derived input function.

Brain↗
PubMed1998

Generalized linear least squares method for fast generation of myocardial blood flow parametric images with N-13 ammonia PET.

In this paper, we developed and tested strategies for estimating myocardial blood flow (MBF) and generating MBF parametric images using positron emission tomography (PET), N-13 ammonia, and the generalized linear least square (GLLS) method. GLLS was generalized to the general linear compartment model, modified for the correction of spillover, validated using simulated N-13 ammonia data, and examined using PET data from several patient studies. In comparison to the standard model-fitting procedure, the GLLS method provided similar accuracy and superior computational speed.

Aged↗
PubMed1998

Development and characterization of monoclonal antibodies specific to the serotonin 5-HT2A receptor.

Serotonin (5-hydroxytryptamine, 5-HT) mediates many functions of the central and peripheral nervous systems by its interaction with specific neuronal and glial receptors. Fourteen serotonin receptors belonging to seven families have been identified through physiological, pharmacological, and molecular cloning studies. Monoclonal antibodies (MAbs) specific for each of these receptor subtypes are needed to characterize their expression, distribution, and function in embryonic, adult, and pathological tissues. In this article we report the development and characterization of MAbs specific to the serotonin 5-HT2A receptor. To generate MAbs against 5-HT2AR, mice were immunized with the N-terminal domain of the receptor. The antigens were produced as glutathionine S-transferase (GST) fusion proteins in insect cells using a Baculovirus expression system. The hybridomas were initially screened by ELISA against the GST-5-HT2AR recombinant proteins and subsequently against GST control proteins to eliminate clones with unwanted reactivity. They were further tested by Western blotting against recombinant GST-5-HT2AR, rat and human brain lysate, and lysate from cell lines transfected with 5-HT2AR cDNA. One of the MAbs G186-1117, which recognizes a portion of the 5-HT2AR N-terminus, was selected for further characterization. G186-1117 reacted with a band of molecular size 55 kD corresponding to the predicted size of 5-HT2AR in lysates from rat brain and a 5-HT2AR-transfected cell line. Its specificity was further confirmed by adsorption of immunoreactivity with recombinant 5-HT2AR but not with recombinant 5-HT2BR and 5-HT2CR. Rat brain sections and Schwann cell cultures were immunohistochemically labeled with this MAb. G186-1117 showed differential staining in various regions of the rat brain, varying from regions with no staining to regions of intense reactivity. In particular, staining of cell bodies and dendrites of the pyramidal neurons in the cortex was observed, which is in agreement with observations of electrophysiological studies.

Aged↗