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Biomedical subjects

K Chen

Publications and source records attributed to K Chen.

At least 55 records · Page 3Linked to original sources
PubMed2001

Role of TGFbeta in development of spontaneous autoimmune thyroiditis in NOD.H-2h4 mice.

Nearly 100% of NOD.H-2h4 mice develop spontaneous autoimmune thyroiditis (SAT) and produce anti-mouse thyroglobulin autoantibodies when they receive 0.05% NaI in their drinking water beginning at 8 wk of age. Our previous studies showed that TGFbeta1 mRNA was constitutively expressed in thyroids and spleens of normal NOD.H-2h4 mice but not other strains of mice. To determine whether TGFbeta might have a role in SAT, mice were given anti-TGFbeta mAb at various times during development of SAT. Anti-TGFbeta markedly inhibited development of SAT and production of anti-mouse thyroglobulin IgG1 autoantibodies. Anti-TGFbeta was most effective in inhibiting SAT when given during the time thyroid lesions were developing, i.e., starting 4 wk after administration of NaI water. The active form of the TGFbeta1 protein was present in thyroids of mice with SAT but not in normal NOD.H-2h4 thyroids. However, thyrocytes of normal NOD.H-2h4 thyroids did express latent TGFbeta1. TGFbeta1 protein expression in the thyroid correlated with SAT severity scores, and administration of anti-TGFbeta inhibited TGFbeta1 protein expression in both the thyroid and spleen. TGFbeta1 was produced primarily by inflammatory cells and was primarily localized in areas of the thyroid containing clusters of CD4(+) T and B cells. Depletion of CD8(+) T cells had no effect on TGFbeta1 protein expression. Activation of splenic T cells was apparently not inhibited by anti-TGFbeta, because up-regulation of mRNA for cytokines and other T cell activation markers was similar for control and anti-TGFbeta-treated mice. TGFbeta1 may function by promoting migration to, or retention of, inflammatory cells in the thyroid.

Animals↗
PubMed2001

Expression and regulation of Fas and Fas ligand on thyrocytes and infiltrating cells during induction and resolution of granulomatous experimental autoimmune thyroiditis.

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by mouse thyroglobulin-sensitized spleen cells activated in vitro with mouse thyroglobulin, anti-IL-2R, and IL-12. G-EAT lesions reach maximal severity 19-21 days after cell transfer, and lesions almost completely resolve by day 35. Depletion of CD8+ cells delays resolution and reduces Fas ligand (FasL) mRNA expression in thyroids. This study was undertaken to analyze Fas and FasL protein expression in the thyroid during induction and resolution of G-EAT and to determine whether CD8+ cells might regulate Fas or FasL expression in the thyroid. Fas and FasL expression was analyzed by immunohistochemical staining or in situ hybridization in thyroids of mice with or without depletion of CD8+ cells. Fas and FasL proteins were not detectable in normal thyroids, but expression of both proteins increased during development of G-EAT. Fas was expressed primarily by inflammatory cells; some enlarged thyrocytes were also Fas+. Thyrocytes had intense FasL immunoreactvity, and many CD8+ cells were also FasL positive. Depletion of CD8+ cells resulted in decreased FasL expression by thyrocytes and inflammatory cells, but had no effect on Fas expression. TUNEL assay detected many apoptotic inflammatory cells in proximity to thyrocytes. CD8-depleted thyroids had ongoing inflammation with fewer apoptotic infiltrating cells at day 35. Administration of a neutralizing anti-FasL mAb had no apparent effects on development of G-EAT, but anti-FasL was as effective as anti-CD8 in preventing G-EAT resolution. These results suggested that CD8+ T cells and thyrocytes may kill inflammatory cells through the Fas pathway, contributing to G-EAT resolution.

Animals↗
PubMed2001

Modulation of [3H]quinpirole binding at striatal D2 dopamine receptors by a monoamine oxidaseA-like site: evidence from radioligand binding studies and D2 receptor- and MAO(A)-deficient mice.

[3H]Quinpirole is a dopamine agonist with high affinity for the D2 and D3 dopamine receptors. A variety of monoamine oxidase inhibitors (MAOIs) inhibit equilibrium binding of [3H]quinpirole binding in rat striatal membranes suggesting that MAOIs interact with a novel binding site that is labeled by [3H]quinpirole or that allosterically modulates [3H]quinpirole binding. To determine whether the D2 receptor is essential for [3H]quinpirole binding and/or modulation of [3H]quinpirole binding by MAOIs, D2 receptor-deficient mice were studied. [3H]Quinpirole binding was decreased in D2 receptor-deficient mice to 3% of that observed in wild-type controls indicating that [3H]quinpirole binding is associated with the D2 dopamine receptors. Then, in an attempt to label the site mediating the modulation of [3H]quinpirole binding, binding of the MAOI [3H]Ro 41-1049 was characterized in rat striatal membranes. [3H]Ro-41-1049 labeled a single binding site with a pharmacological profile with respect to MAOIs that was similar to both [3H]quinpirole binding (Spearman r=0.976) and MAO(A) activity. To determine whether MAO(A) plays a role in the modulation of [3H]quinpirole binding by MAOIs, MAO(A)-deficient mice were examined. In these mice, [3H]Ro-41-1049 binding was decreased to 7% of wild-type control. [3H]Spiperone binding was unaltered. Spiperone-displaceable [3H]quinpirole binding was decreased to 43% of wild-type control; however, the remaining [3H]quinpirole binding in MAO(A)-deficient animals was inhibited by Ro 41-1049 similar to wild-type. [3H]Ro-41-1049 binding was not decreased in D2 receptor-deficient mice. These data suggest that [3H]Ro-41-1049 labels multiple sites and that MAOIs modulate [3H]quinpirole binding to the D2 receptor via interactions at a novel, non-MAO binding site with MAO(A)-like pharmacology.

Animals↗
PubMed2001

Biochemical, behavioral, physiologic, and neurodevelopmental changes in mice deficient in monoamine oxidase A or B.

The availability of mutant mice that lack either MAO A or MAO B has created unique profiles in the central and peripheral availability of serotonin, norepinephrine, dopamine, and phenylethylamine. This paper summarizes some of the current known phenotypic findings in MAO A knock-out mice and contrast these with those of MAO B knock-out mice. Differences are discussed in relation to the biochemical, behavioral, and physiologic changes investigated to date, as well as the role played by redundancy mechanisms, adaptational responses, and alterations in neurodevelopment.

Animals↗
PubMed2001

Negative feedback on the effects of stem cell factor on hematopoiesis is partly mediated through neutral endopeptidase activity on substance P: a combined functional and proteomic study.

Hematopoietic regulation is a complex but dynamic process regulated by intercellular and intracellular interactions within the bone marrow (BM) microenvironment. Through neurokinin-1 (NK-1) and NK-2 receptors, peptides (eg, substance P [SP]) encoded by the preprotachykinin-I gene mediate distinct hematopoietic effects. Cytokines, associated with hematopoietic stimulation, and SP regulate the expression of each other in BM mesenchymal and immune cells. Neutral endopeptidase (NEP) uses SP as a substrate to produce SP(1-4), which inhibits the proliferation of matured myeloid progenitor. This study determines whether the degradation of SP to SP(1-4) by endogenous NEP in BM stroma could be a feedback on hematopoietic stimulation by stem cell factor (SCF). SP(1-4) induced the production of transforming growth factor (TGF)-beta and tumor necrosis factor-alpha in BM stroma. TGF-beta production accounted for part of the inhibitory effects by SP(1-4) on the proliferation of early (granulocyte-macrophage colony-forming units) and late (long-term culture-initiating cells) hematopoietic progenitors. Enzyme-linked immunosorbent assays and/or protein-chip arrays indicated a timeline change of SP to SP(1-4) in BM stroma stimulated with SCF, which correlated with increase in NEP messenger RNA. Since SP and its fragment, SP(1-4), interact with the same receptor to mediate opposing hematopoietic effects, 2 interactive studies were done to understand the dual responses of NK-1: (1) a 3-dimensional molecular model of NK-1 and SP and (2) screening of a random dodecapeptide library for SP(1-4) interacting sites. The effects of SP(1-4) on hematopoietic progenitors and the timeline change of SP to SP(1-4), together with the 3-dimensional model, provide a partial explanation for the feedback on the stimulatory effects of SCF and SP on hematopoiesis.

Binding Sites↗
PubMed2001

Induction of experimental autoimmune thyroiditis in IL-12-/- mice.

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by transfer of mouse thyroglobulin (MTg)-sensitized spleen cells activated in vitro with MTg and anti-IL-2R or MTg and IL-12. Previous work suggested that IL-12 was required in vitro for development of G-EAT. To determine whether IL-12 was also required during the induction and/or effector phases, DBA/1 mice with a disrupted IL-12-P40 gene (IL-12(-/-)) were used for EAT induction. Cells from MTg-sensitized IL12(-/-) donors activated in vitro by MTg or MTg and anti-IL2R induced severe EAT in recipient mice. Compared with effector cells from IL-12(+/+) donors, effector cells from IL-12(-/-) donors induced thyroid lesions dominated by lymphocytes with minimal granulomatous changes. Thyroids of recipients of IL-12(-/-) cells expressed less IFN-gamma mRNA and more TGF-beta, IL-4, and IL-10 compared with recipients of IL-12(+/+) cells. When IL-12 was added during in vitro activation, cells from both IL-12(-/-) and IL-12(+/+) donors induced severe G-EAT, and expression of all cytokines except IL-12 was comparable in thyroids of both IL-12(+/+) and IL-12(-/-) recipients. Transfer of cells from IL-12(+/+) or IL-12(-/-) donors into IL-12(+/+) or IL-12(-/-) recipients indicated that IL-12 expressed in thyroids was derived from recipients. Thus, endogenous IL-12 is not absolutely essential for the sensitization and activation of EAT effector cells to induce severe EAT, although it is required in vitro to promote activation of cells to induce severe granulomatous histopathology.

Adoptive Transfer↗
PubMed2001

Stereospecific alkane hydroxylation by non-heme iron catalysts: mechanistic evidence for an Fe(V)=O active species.

High-valent iron-oxo species have frequently been invoked in the oxidation of hydrocarbons by both heme and non-heme enzymes. Although a formally Fe(V)=O species, that is, [(Por(*))Fe(IV)=O](+), has been widely accepted as the key oxidant in stereospecific alkane hydroxylation by heme systems, it is not established that such a high-valent state can be accessed by a non-heme ligand environment. Herein we report a systematic study on alkane oxidations with H(2)O(2) catalyzed by a group of non-heme iron complexes, that is, [Fe(II)(TPA)(CH(3)CN)(2)](2+) (1, TPA = tris(2-pyridylmethyl)amine) and its alpha- and beta-substituted analogues. The reactivity patterns of this family of Fe(II)(TPA) catalysts can be modulated by the electronic and steric properties of the ligand environment, which affects the spin states of a common Fe(III)-OOH intermediate. Such an Fe(III)-peroxo species is high-spin when the TPA ligand has two or three alpha-substituents and is proposed to be directly responsible for the selective C-H bond cleavage of the alkane substrate. The thus-generated alkyl radicals, however, have relatively long lifetimes and are susceptible to radical epimerization and trapping by O(2). On the other hand, 1 and the beta-substituted Fe(II)(TPA) complexes catalyze stereospecific alkane hydroxylation by a mechanism involving both a low-spin Fe(III)-OOH intermediate and an Fe(V)=O species derived from O-O bond heterolysis. We propose that the heterolysis pathway is promoted by two factors: (a) the low-spin iron(III) center which weakens the O-O bond and (b) the binding of an adjacent water ligand that can hydrogen bond to the terminal oxygen of the hydroperoxo group and facilitate the departure of the hydroxide. Evidence for the Fe(V)=O species comes from isotope-labeling studies showing incorporation of (18)O from H(2)(18)O into the alcohol products. (18)O-incorporation occurs by H(2)(18)O binding to the low-spin Fe(III)-OOH intermediate, its conversion to a cis-H(18)O-Fe(V)=O species, and then oxo-hydroxo tautomerization. The relative contributions of the two pathways of this dual-oxidant mechanism are affected by both the electron donating ability of the TPA ligand and the strength of the C-H bond to be broken. These studies thus serve as a synthetic precedent for an Fe(V)=O species in the oxygen activation mechanisms postulated for non-heme iron enzymes such as methane monooxygenase and Rieske dioxygenases.

Alkanes↗
PubMed2001

Permeability of lipophilic compounds in drug discovery using in-vitro human absorption model, Caco-2.

Highly lipophilic compounds are often encountered in the early stages of drug discovery. The apparent permeability (Papp) of these compounds in Caco-2 cell could be underestimated because of considerable retention by the Caco-2 monolayer and non-specific binding to transwell surface. We have utilized a general approach for the determination of permeability of these compounds, which includes the addition of 1-5% DMSO in the apical (AP) and 4% bovine serum albumin (BSA) in the basolateral (BA) side. Two highly lipophilic and highly protein bound Schering compounds, SCH-A and SCH-B, exhibited poor recovery and low Papp in the conventional Caco-2 system that included 1% DMSO in the AP and BA sides. In contrast, both compounds were well absorbed in cynomolgus monkeys. Inclusion of BSA (up to 4%) in the BA side provided necessary absorptive driving force similar to in vivo sink conditions improving both recovery and Papp of these compounds as well as progesterone, a model highly lipophilic and highly protein bound compound. Whereas, the recovery and Papp of mannitol (high recovery, low permeability) and propranolol (high recovery, high permeability) remained unaffected. The presence of 4% BSA increased Papp of SCH-A, SCH-B, and progesterone by five-, four-, and three-fold, respectively. We also compared this approach with a second, based on the disappearance of the compound from the AP side, which resulted in a reasonable estimate of the permeability (23.3x10(-6) cm/s) for SCH-A. The results demonstrated that the reliable estimates of permeability of highly lipophilic compounds that are subjected to considerable retention by the cell monolayer and exhibit non-specific binding are obtained by the addition of BSA to the BA side.

Caco-2 Cells↗
PubMed2001

"Intermolecular" trapping of a nonheme Fe(IV)=O intermediate.

The reactions of Fe(III)(TPA) (TPA = tris(2-pyridylmethyl)amine) complexes with 2-methyl-1-phenyl-2-propyl-hydroperoxide (MPPH) delivered by syringe pump under anaerobic conditions afford nearly quantitative conversion of MPPH to products derived from the benzyl radical. These results unequivocally show that MPPH breaks down by O-O bond homolysis, leading to the formation of the benzyl radical and a high valent Fe(IV)=O species. Without added substrates, the benzyl radical reacts with the high valent species to form benzyl alcohol or benzyl halides. The Fe(IV)=O species can also effect the two-electron oxidation of added substrates such as thioanisole, cyclohexanol, and cyclooctene under appropriate conditions. The oxidation of thioanisole and cyclohexanol is likely facilitated by pre-equilibrium binding of the substrate to the metal center, allowing these substrates to intercept the high valent iron-oxo species as it forms. These results suggest the importance of close proximity to direct the high valent metal center down a desired pathway.

Chemical Phenomena↗
PubMed2001

3D-QSAR model of flavonoids binding at benzodiazepine site in GABAA receptors.

With flavone as a structural template, three-dimensional quantitative structure-activity relationship (3D-QSAR) studies and ab initio calculations were performed on a series of flavonoids. A reasonable pharmacophore model was built through CoMFA, CoMSIA, and HQSAR analyses and electrostatic potential calculations. A plausible binding mode for flavonoids with GABA(A) receptors was rationalized. On the basis of the commonly recognized binding site, the specific S1 and S2 subsites relating to substituent positions were proposed. The different binding affinities could be explained according to the frontier orbitals and electrostatic potential (ESP) maps. The ESP could be used as a novel starting point for designing more selective BZ-binding-site ligands.

Benzodiazepines↗
PubMed2001

Effects of carboxyl-terminal truncations on the activity and solubility of human monoamine oxidase B.

Monoamine oxidase is an outer mitochondrial membrane protein that catalyzes the deamination of a number of neurotransmitters and dietary amines. To determine the roles of the carboxyl-terminal amino acids on the activity and solubility of human monoamine oxidase (MAO B), 10 sequential mutants were made with stop codons at amino acid positions 511, 504, 498, 492, 486, 481, 476, 467, 417, and 397, respectively. All truncated mutants were expressed in Sf21 insect cells using baculovirus, and the enzyme kinetic parameters were determined. Truncations at amino acid positions 511, 504, and 498 slightly decreased MAO B catalytic activity and had no significant changes on deprenyl inhibition. Further deletions up to amino acid 417 decreased the specific activity 10--100-fold without significant changes of the K(m) for phenylethylamine or dopamine or the IC(50) for deprenyl and clorgyline. The truncation mutant C397, which lacks covalently attached FAD, was inactive. Progressive carboxyl-terminal truncations up to position 481 were correlated with increased solubility of MAO B mutants. 47% of the activity of the truncated C481 was found in the 105,000 x g supernatant in the absence of detergent. However, further truncated mutants, C476, C467, and C417, remained associated with the membrane fraction. In contrast to crude homogenate, the water-soluble C481 mutant was rapidly inactivated at 4 degrees C and 37 degrees C, which indicates that the membrane environment is required for the stability of MAO B. Expression of the green fluorescent protein-MAO B C481 fusion protein revealed that this mutant was located in the cytoplasm, whereas its counterpart in MAO A, truncated mutant C490, was located on the mitochondria. These results suggest that the carboxyl-terminal amino acid residues 417--520 of MAO B are not directly involved in the active site but are required for maintaining the appropriate conformation and interaction with the outer mitochondrial membrane. The different solubilities of the various carboxyl-terminal truncation mutants indicate that the interaction of MAO B with mitochondrial membrane is not simply anchoring through the carboxyl-terminal hydrophobic tail. Further, our results suggest that the carboxyl-terminal of MAO A and B plays different roles in mitochondrial attachment.

Amino Acid Sequence↗
PubMed2001

Scale dependent dimension of luminous matter in the universe.

We suggest a geometrical model for the distribution of luminous matter in the Universe, where the apparent dimension, D(l), increases linearly with the logarithm of the scale l. Beyond the correlation length, xi, the Universe is homogeneous, and D = 3. Comparison with data from the SARS redshift catalog, and the LEDA database provides a good fit with a correlation length xi approximately 300 Mpc. This type of scaling structure was recently discovered in a simple reaction-diffusion "forest-fire" model, indicating a broad class of scaling phenomena.

Journal Article↗
PubMed2001

OSP/claudin-11 forms a complex with a novel member of the tetraspanin super family and beta1 integrin and regulates proliferation and migration of oligodendrocytes.

Oligodendrocyte-specific protein (OSP)/claudin-11 is a major component of central nervous system myelin and forms tight junctions (TJs) within myelin sheaths. TJs are essential for forming a paracellular barrier and have been implicated in the regulation of growth and differentiation via signal transduction pathways. We have identified an OSP/claudin-11-associated protein (OAP)1, using a yeast two-hybrid screen. OAP-1 is a novel member of the tetraspanin superfamily, and it is widely expressed in several cell types, including oligodendrocytes. OAP-1, OSP/claudin-11, and beta1 integrin form a complex as indicated by coimmunoprecipitation and confocal immunocytochemistry. Overexpression of OSP/claudin-11 or OAP-1 induced proliferation in an oligodendrocyte cell line. Anti-OAP-1, anti-OSP/claudin-11, and anti-beta1 integrin antibodies inhibited migration of primary oligodendrocytes, and migration was impaired in OSP/claudin-11-deficient primary oligodendrocytes. These data suggest a role for OSP/claudin-11, OAP-1, and beta1 integrin complex in regulating proliferation and migration of oligodendrocytes, a process essential for normal myelination and repair.

Amino Acid Sequence↗
PubMed2001

Declining brain activity in cognitively normal apolipoprotein E epsilon 4 heterozygotes: A foundation for using positron emission tomography to efficiently test treatments to prevent Alzheimer's disease.

Cross-sectional positron emission tomography (PET) studies find that cognitively normal carriers of the apolipoprotein E (APOE) epsilon4 allele, a common Alzheimer's susceptibility gene, have abnormally low measurements of the cerebral metabolic rate for glucose (CMRgl) in the same regions as patients with Alzheimer's dementia. In this article, we characterize longitudinal CMRgl declines in cognitively normal epsilon4 heterozygotes, estimate the power of PET to test the efficacy of treatments to attenuate these declines in 2 years, and consider how this paradigm could be used to efficiently test the potential of candidate therapies for the prevention of Alzheimer's disease. We studied 10 cognitively normal epsilon4 heterozygotes and 15 epsilon4 noncarriers 50-63 years of age with a reported family history of Alzheimer's dementia before and after an interval of approximately 2 years. The epsilon4 heterozygotes had significant CMRgl declines in the vicinity of temporal, posterior cingulate, and prefrontal cortex, basal forebrain, parahippocampal gyrus, and thalamus, and these declines were significantly greater than those in the epsilon4 noncarriers. In testing candidate primary prevention therapies, we estimate that between 50 and 115 cognitively normal epsilon4 heterozygotes are needed per active and placebo treatment group to detect a 25% attenuation in these CMRgl declines with 80% power and P = 0.005 in 2 years. Assuming these CMRgl declines are related to the predisposition to Alzheimer's dementia, this study provides a paradigm for testing the potential of treatments to prevent the disorder without having to study thousands of research subjects or wait many years to determine whether or when treated individuals develop symptoms.

Aged↗