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Biomedical subjects

Y J Kim

Publications and source records attributed to Y J Kim.

At least 19 recordsLinked to original sources
PubMed2001

Xenobiotic response in humanized double transgenic mice expressing tetracycline-controlled transactivator and human CYP1B1.

The cytochrome P450 enzymes (P450s or CYPs) are a superfamily of hemeproteins that catalyze the monooxygenation of a wide range of endobiotic and xenobiotic substrates. A typical strategy in toxicological research and testing involves applying a toxicant at high doses for a short period to homogeneous animals under controlled conditions. However, the conditions of this approach have very little in common with actual human exposure. Transgenic (Tg) mice carrying human genes encoding a drug-metabolizing enzyme (CYP) offer a solution to many of the difficulties in the evaluation of chemical toxicity. It has been demonstrated that the expression of human CYP transgenes under the control of mammalian-inducible promoters exhibits relatively poor fold increases after induction. In this study, we used the tetracycline-regulated (tet) promoter system to increase the expression of the human CYP1B1 (hCYP1B1) gene in the tissues of transgenic mice. By mating two lineages of transgenic mice, double transgenic (dTg) mice expressing both tTA and hCYP1B1 genes under the control of the tet promoter were successfully produced, into which the two transgenes were introduced in an embryo. The expression pattern of tTA-driven hCYP1B1 transgene featured a fold induction of more than 3 to 12 in the brain, heart, and lung and 2- to 4-fold induction in the liver, kidney, and intestine upon doxycycline removal. Immunohistochemical staining with hCYP1B1 antibody was also increased by the removal of doxycycline. In addition, the activities of CYP liver microsomes in the dTg mice without doxycycline showed an increase compared to that in the dTg mice treated with doxycycline. The level of activities correspond to the levels of human CYP1B1 protein expression in the Tg mice (-dox) that was increased by 2-fold induction as compared to that of the dTg mice with doxycycline. Thus, overproduction in Tg can be purified and the activity of purified human CYP1B1 can be characterized by alterations to the coding sequence in order to solve the physiological function of this enzyme in a humanized in vivo system. It is also possible to examine the activity of purified human CYP1B1 using several environmental toxicants such as procarcinogens.

Animals↗
PubMed2001

Caspase 3-cleaved N-terminal fragments of wild-type and mutant huntingtin are present in normal and Huntington's disease brains, associate with membranes, and undergo calpain-dependent proteolysis.

The Huntington's disease (HD) mutation is a polyglutamine expansion in the N-terminal region of huntingtin (N-htt). How neurons die in HD is unclear. Mutant N-htt aggregates in neurons in the HD brain; expression of mutant N-htt in vitro causes cell death. Other in vitro studies show that proteolysis by caspase 3 could be important in regulating mutant N-htt function, but there has been no direct evidence for caspase 3-cleaved N-htt fragments in brain. Here, we show that N-htt fragments consistent with the size produced by caspase 3 cleavage in vitro are resident in the cortex, striatum, and cerebellum of normal and adult onset HD brain and are similar in size to the fragments seen after exogenous expression of human huntingtin in mouse clonal striatal neurons. HD brain extracts treated with active caspase 3 had increased levels of N-htt fragments. Compared with the full-length huntingtin, the caspase 3-cleaved N-htt fragments, especially the mutant fragment, preferentially segregated with the membrane fraction. Partial proteolysis of the human caspase 3-cleaved N-htt fragment by calpain occurred in vitro and resulted in smaller N-terminal products; products of similar size appeared when mouse brain protein extracts were treated with calpain. Results support the idea that sequential proteolysis by caspase 3 and calpain may regulate huntingtin function at membranes and produce N-terminal mutant fragments that aggregate and cause cellular dysfunction in HD.

Adult↗
PubMed2001

Cerebral activation and distribution of inducible hsp110 and hsp70 mRNAs following focal ischemia in rat.

A potential function for inducible heat shock protein 70 (hsp70i) expression in the pathophysiology of ischemic brain has been well documented. The recently cloned hsp70 superfamily member, hsp110, was shown to be highly expressed in the brain and suggested to have a similar functional property as members of the hsp70 family. In this study, as an initial step to probe for its physiological significance in the ischemic brain, cerebral activation and distribution of hsp110 mRNA was comparatively evaluated with that of hsp70i mRNA by in situ hybridization. A rat focal cerebral ischemia model was employed to examine the distribution and localization of hsp110 and hsp70i mRNAs in both affected (ipsilateral) and unaffected (contralateral) hemispheres of the same animal. Our results demonstrated a significant accumulation of hsp110 as well as hsp70i mRNAs following ischemia; although the magnitude and kinetics of induction differ slightly, spatial expression profiles of hsp110 and hsp70i mRNAs were highly correlated in the affected region. In control brain, limited hybridization signal was observed with 3'-untranslated region (UTR) containing hsp110 probe, suggesting a possible existence of inducible hsp110 and a selective recognition of our 3'-UTR containing probe for the inducible hsp110 mRNA species. Subsequent 2D western analysis with Hsp110 specific Ab was consistent with our view, which resolved constitutive and inducible immunostained spots in rat ischemic brain. Considering a regulatory similarity as well as previously documented structural and functional similarities between hsp110 and hsp70i, we propose that coordinated cerebral activation of hsp110 and hsp70i is likely to be of significant relevance in the context of pathophysiology of ischemic brain. Further study is required to characterize the genetic and biochemical nature of rat inducible hsp110 identified in the current study.

Animals↗
PubMed2001

Surgical treatment of congenital kyphosis.

STUDY DESIGN: In this study, 26 cases of congenital kyphosis and kyphoscoliosis treated surgically were retrospectively reviewed. OBJECTIVE: To assess the clinical outcomes and surgical indications for posterior only versus anteroposterior surgery in the child. SUMMARY OF BACKGROUND DATA: Congenital kyphosis usually is progressive without surgical intervention. Current recommended treatment includes posterior arthrodesis for deformities of less than 50 degrees to 60 degrees, and anterior release or decompression, anterior fusion, and posterior instrumented arthrodesis for large deformities and cord compression. METHODS: Cases involving myelodysplasia, spinal dysgenesis, and skeletal dysplasia were excluded from the study. Kyphoscoliosis was included if the kyphotic deformity was greater than the scoliotic deformity. Patients were grouped by age and surgical technique. The patients in group P1 underwent posterior arthrodesis at an age younger than 3 years, and those in group P2 underwent the procedure at an age older than 3 years. The patients in group AP1 underwent anterior and posterior procedures at an age younger than 3 years, and those in group AP2 underwent the procedures at an age older than 3 years. The preoperative deformity, complications, and postoperative deformity correction were analyzed. There were nine Type 1 (failure of formation), nine Type 2 (failure of segmentation), and eight Type 3 (mixed) deformities. Four patients had associated spinal dysraphism. Three patients with Type 1 deformities had clinical or radiographic evidence of cord compression. RESULTS: In Group P1, five patients at an average age of 16 months underwent posterior arthrodesis alone for an average kyphotic deformity of 49 degrees. The immediate postoperative correction improved over a period of 6 years and 9 months by an additional 10 degrees, resulting in a final deformity of 26 degrees. Pseudarthrosis developed in two patients, requiring fusion mass augmentation or anterior arthrodesis. Neither patient was instrumented. In Group P2, five patients at an average age of 13 years and 7 months underwent posterior arthrodesis with instrumentation for kyphotic deformity of 59 degrees. Approximately 30 degrees of intraoperative correction was achieved safely using compression instrumentation and positioning. No further correction occurred with growth. The final residual kyphotic deformity was 29 degrees after a follow-up period of 4 years and 5 months. In Group AP1, seven patients underwent anterior release or vertebra resection for deformity correction and posterior arthrodesis for an average kyphotic deformity of 48 degrees at the age of 16 months. There were no iatrogenic neurologic injuries. The final residual kyphotic deformity was 22 degrees after a follow-up period of 6 years and 3 months. In Group AP2, nine patients underwent anterior release or decompression with posterior arthrodesis for kyphotic deformity of 77 degrees at the age of 11 years and 6 months. The deformity was corrected to 37 degrees, with no significant loss over a follow-up period of 5 years and 2 months. There were two postoperative neurologic complications. CONCLUSIONS: After reviewing their experience, the authors made the following observations: 1) The pseudarthrosis rate was low even without routine augmentation of fusion mass if instrumentation was used; 2) gradual correction of kyphosis may occur with growth in patients younger than 3 years with Types 2 and 3 deformities after posterior fusion, but appears to be unpredictable; 3) the risk of neurologic injury with anterior and posterior fusion for kyphotic deformity was associated with greater age, more severe deformity, and preexisting spinal cord compromise.

Adolescent↗
PubMed2001

Mechanistic insight into the inactivation of carboxypeptidase A by alpha-benzyl-2-oxo-1,3-oxazolidine-4-acetic acid, a novel type of irreversible inhibitor for carboxypeptidase A with no stereospecificity.

On the basis of the active site topology and enzymic catalytic mechanism of carboxypeptidase A (CPA), a prototypical zinc-containing proteolytic enzyme, alpha-benzyl-2-oxo-1,3-oxazolidine-4-acetic acid (1), was designed as a novel type of mechanism-based inactivator of the enzyme. All four possible stereoisomers of the inhibitor were synthesized in an enantiomerically pure form starting with optically active aspartic acid, and their CPA inhibitory activities were evaluated to find that surprisingly all of the four stereoisomers inhibit CPA in a time dependent manner. The inhibited enzyme did not regain its enzymic activity upon dialysis. The inactivations were prevented by 2-benzylsuccinic acid, a competitive inhibitor that is known to bind the active site of the enzyme. These kinetic results strongly support that the inactivators attach covalently to the enzyme at the active site. The analysis of ESI mass spectral data of the inactivated CPA ascertained the conclusion from the kinetic results. The values of second-order inhibitory rate constants (k(obs)/[I](o)) fall in the range of 1.7-3.6 M(-1) min(-1). The lack of stereospecificity shown in the inactivation led us to propose that the ring cleavage occurs by the nucleophilic attack at the 2-position rather than at the 5-position and the ring opening takes place in an addition-elimination mechanism. The tetrahedral transition state that would be generated in this pathway is thought to be stabilized by the active site zinc ion, which was supported by the PM3 semiemprical calculations. In addition, alpha-benzyl-2-oxo-1,3-oxazolidine-5-acetic acid (18), a structural isomer of 1 was also found to inactivate CPA in an irreversible manner, reinforcing the nucleophilic addition-elimination mechanism. The present study demonstrates that the transition state for the inactivation pathway plays a critical role in determining stereochemistry of the inactivation.

Carboxypeptidases↗
PubMed2001

The structural and functional organization of the yeast mediator complex.

The Mediator complex of Saccharomyces cerevisiae is required for diverse aspects of transcription by RNA polymerase II (pol II). Mediator is composed of two functionally distinct subcomplexes, Rgr1 and Srb4. To identify the structures and functions of each subcomplex, we expressed recombinant proteins for each subunit and assayed their interactions with each other and with basal transcription proteins. The Rgr1 subcomplex is composed of the Gal11 module, which binds activators, and the Med9/10 module. The Med9/10 module is required for both transcriptional activation and repression, and these activities appear to be carried out by two submodules. Proteins in the Med9 submodule interact physically and genetically with Srb10/11, suggesting that the Med9 submodule mediates the repression of pol II. Purified recombinant Srb4 subcomplex stimulated basal transcription of pol II but had little effect on activated transcription and phosphorylation of the C-terminal domain of the Rpb1 subunit of pol II. Both subcomplexes of Mediator interacted with a distinct set of basal transcription factors and pol II. The modular organization of Mediator and the associated functions suggest that the Mediator complex may recruit and/or stabilize the preinitiation complex through several points of contact with transcriptional regulators and basal transcription factors.

Fungal Proteins↗
PubMed2001

Blocking of c-FLIP(L)--independent cycloheximide-induced apoptosis or Fas-mediated apoptosis by the CC chemokine receptor 9/TECK interaction.

Chemokines play a pivotal role in regulating leukocyte migration as well as other biological functions. CC chemokine receptor 9 (CCR9) is a specific receptor for thymus-expressed CC chemokine (TECK). It is shown here that engagement of CCR9 with TECK leads to phosphorylation of Akt (protein kinase B), mitogen-activated protein kinases (MAPKs), glycogen synthase kinase--3 beta (GSK-3 beta), and a forkhead transcription factor, FKHR, in a human T-cell line, MOLT4, that naturally expresses CCR9. By means of chemical inhibitors, it is shown that phosphoinositide-3 kinase (PI-3 kinase), but not MAPK, is required for CCR9-mediated chemotaxis. Akt, GSK-3 beta, FKHR, and MAPK have been previously implicated in cell survival signals in response to an array of death stimuli. When MOLT4 cells, which expressed Fas as well as CXCR4, were stimulated with cycloheximide (CHX), an agonistic anti-Fas antibody, or a combination of these, the cells rapidly underwent apoptosis. However, costimulation of MOLT4 cells with TECK or stromal derived factor--1 significantly blocked CHX-mediated apoptosis, whereas stimulation only with TECK partially blocked Fas-mediated apoptosis. Concomitant with this blocking, cleavage of poly (adenosine 5'-diphosphate--ribose) polymerase and activation of caspase 3 were significantly attenuated, but the expression level of FLICE inhibitory protein c-FLIP(L), which had been shown to be regulated by CHX, was unchanged. This demonstrates that activation of CCR9 leads to phosphorylation of GSK-3 beta and FKHR and provides a cell survival signal to the receptor expressing cells against CHX. It also suggests the existence of a novel pathway leading to CHX-induced apoptosis independently of c-FLIP(L). (Blood. 2001;98:925-933)

Apoptosis↗
PubMed2001

Why is alkylation of an enolate accompanied by so much polyalkylation?

[reaction: see text] The lithium enolate 1-Li of 6-phenyl-alpha-tetralone forms a monomer-tetramer equilibrium in THF at 25 degrees C with K(1,4) = 4.7E+10 M(-3). The lithium enolate 2-Li, however, forms a monomer-dimer equilibrium with K(1,2) = 3800 M(-1). In both cases reaction with benzyl bromide is dominantly with the monomer. The results support an earlier conjecture of House that alkylation of an enolate is frequently accompanied by extensive polyalkylation because the less substituted enolates are more aggregated.

Journal Article↗
PubMed2001

Caffeine inhibits exercise-induced increase in tryptophan hydroxylase expression in dorsal and median raphe of Sprague-Dawley rats.

Effect of caffeine on the expression of tryptophan hydroxylase (TPH), rate limiting enzyme of serotonin synthesis, in dorsal and median raphe was investigated via immunohistochemistry. In exercise groups, Sprague-Dawley rats were put on treadmill running for 30 min per day for 6 consecutive days. On the seventh day, animals of control-with-caffeine group were injected subcutaneously with 4 mg/kg caffeine, while control-without-caffeine group were injected with 0.9% NaCl, sacrificed 2 h later. Exercise-with-caffeine group and exercise-without-caffeine group were injected with caffeine and NaCl, respectively; all-out time was determined 1 h after injection, and then sacrificed. Caffeine increased all-out time in exercised rats, and inhibited the exercise-induced elevation in TPH expression. The suppressive effect of caffeine on TPH expression in exercised rats can be suggested as one possible ergogenic mechanism of caffeine.

Animals↗
PubMed2001

Med9/Cse2 and Gal11 modules are required for transcriptional repression of distinct group of genes.

The yeast Mediator is composed of two subcomplexes, Rgr1 and Srb4, known to be required for diverse aspects of transcriptional regulation; however, their structural and functional organizations have not yet been deciphered in detail. Biochemical analyses designed to determine the subunit composition of the Rgr1 subcomplex revealed that the regulator-interacting subcomplex has a modular structure and is composed of the Gal11, Med9/Cse2, and Med10/Nut2 modules. Genome-wide gene expression and Northern analyses performed in the presence or absence of the various Mediator modules revealed a distinct requirement for the Gal11, Med9/Cse2, and Med10/Nut2 modules in transcriptional repression as well as activation. GST pull-down analysis revealed that the transcriptional repressor Tup1 binds to distinct but overlapping regions of the Gal11 module that were shown previously to be transcriptional activator binding sites. These data suggest that competition between transcriptional activators and repressors for a common binding site in the Mediator and distinct conformational changes in the Mediator induced by repressor binding may underlie the mechanism of transcriptional repression in eukaryotes.

Animals↗
PubMed2001

Auricular acupuncture decreases neuropeptide Y expression in the hypothalamus of food-deprived Sprague-Dawley rats.

The aim of the present study was to investigate whether stimulation of auricular acupuncture point has any effects on the expression of neuropeptide Y (NPY), appetite-inducing factor particularly abundant in the mammalian hypothalamus. In food-deprived condition, enhanced NPY expression was detected in both the arcuate nucleus (ARN) and the paraventricular nucleus (PVN) of the hypothalamus via immunohistochemistry in Sprague-Dawley rats. Needling the unfed rats on the auricular point resulted in decreased NPY levels in both the ARN and PVN, while it increased NPY levels in the ARN and PVN of fed rats. The present findings indicate that auricular acupuncture may affect NPY expression in the ARN and PVN of the hypothalamus.

Acupuncture, Ear↗
PubMed2001

Synthesis of the trisaccharide portion of the immunologic adjuvant QS-21A via sulfonium-mediated oxidative and dehydrative glycosylation.

[see structure]. The first synthesis of the trisaccharide fragment of the potent immunologic adjuvant QS-21A is reported. The key steps involve the application of sulfonium-mediated oxidative and dehydrative glycosidic couplings to construct the anomeric linkages in a short and convergent assembly of the branched trisaccharide.

Adjuvants, Immunologic↗
PubMed2001

Cyclophilin a binds to peroxiredoxins and activates its peroxidase activity.

Six distinct peroxiredoxin (Prx) proteins (Prx I-VI) from distinct genes have been identified in mammalian tissues. Prxs are members of a group of peroxidases that have conserved reactive cysteine residue(s) in the active site(s). An immediate physiological electron donor for the peroxidase catalysis for five Prx proteins (Prx I-V) has been identified as thioredoxin (Trx), but that for Prx VI (1-Cys Prx) is still unclear. To identify an immediate electron donor and a binding protein for Prx VI, we performed a Prx VI protein overlay assay. A 20-kDa binding protein was identified by the Prx VI protein overlay assay with flow-through fractions from a High-Q column with rat lung crude extracts. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) and MS-Fit, we identified the 20-kDa Prx VI-binding protein as a cyclophilin A (CyP-A). The binding of recombinant human CyP-A (hCyP-A) to Prx VI was confirmed by using the hCyP-A protein overlay assay and Western immunoblot analysis with hCyP-A-specific antibodies. hCyP-A enhanced the antioxidant activity of Prx VI, as well as the other known mammalian Prx isotypes. hCyP-A supported antioxidant activity of Prx II and Prx VI both against thiol (dithiothreitol)-containing metal-catalyzed oxidation (MCO) systems and ascorbate-containing MCO systems. Prx II was reduced by hCyP-A without help from any other reductant, and the reduction was cyclosporin A-independent. These results strongly suggest that CyP-A not only binds to Prx proteins but also supports its peroxidase activity as an immediate electron donor. In addition, Cys(115) and Cys(161) of hCyP-A were found to be involved in the activation and the reduction of Prx.

Animals↗
PubMed2001

Structure and expression of the Arabidopsis thaliana homeobox gene Athb-12.

We have isolated the Arabidopsis thaliana homeobox gene Athb-12, determined its structure and activation domain, demonstrated that its promoter is inducible in response to abscisic acid (ABA) treatment, and characterized the cellular distribution of its transcripts. The single intron of the gene interrupted the leucine-zipper domain region. The 5' regulatory region of Athb-12 can drive beta-glucuronidase (GUS) expression in tobacco transgenic plants. Athb-12 gene expression was further examined using in situ hybridization to determine the cellular distribution of Athb-12 transcripts during ABA induction. A complex pattern of Athb-12 expression was observed, often associated with regions of developing vascular tissues. Analysis of chimeras constructed from Athb-12 and the DNA-binding domain of the Saccharomyces cerevisiae transcription factor GAL4 revealed that the activation domain of Athb-12 lies in the C-terminal region (amino acids 180 to 235). Taken together, our data suggest that Athb-12 is a transcriptional activator important in regulating certain developmental processes as well as in the plant's response to water stress involving ABA-mediated gene expression.

5' Untranslated Regions↗
PubMed2001

Mutation in PMR1, a Ca(2+)-ATPase in Golgi, confers salt tolerance in Saccharomyces cerevisiae by inducing expression of PMR2, an Na(+)-ATPase in plasma membrane.

Sodium tolerance in yeast is enhanced by continuous activation of calcineurin, a Ca(2+)/calmodulin-dependent protein phosphatase that is required for modulation of the Na(+) efflux mechanism. We isolated several salt-tolerant mutations with the treatment of ethylmethane sulfonate under high salt stress. One of the mutations was mapped in the PMR1 gene. Pmr1p, the P-type Ca(2+)-ATPase in the Golgi apparatus, regulates a cytosolic Ca(2+) level in various responses. Cytosolic Ca(2+) concentration in the pmr1 mutant is highly maintained, and thus calcineurin is activated continuously. The treatment of FK506, a specific inhibitor of calcineurin, abolishes the salt-tolerant phenotype of the pmr1 mutant. Activated calcineurin induces the expression of PMR2, encoding the P-type Na(+)-ATPase, through the specific transcription factor, Tcn1p/Crz1p. Also, expression of the PMR2::lacZ reporter gene in the pmr1 mutant was higher than that in wild type. We propose that the pmr1 mutation confers salt tolerance through continuous activation of calcineurin and that Pmr1p might act as a major Ca(2+)-ATPase under high salt stress.

Adenosine Triphosphatases↗