Biomedical subjects
Publications and source records attributed to K Kim.
The [2 + 2] photoreaction of (E)-diaminostilbene dihydrochloride proceeds with large rate acceleration and high stereoselectivity via formation of a stable 1:2 host-guest complex with curcurbit[8]uril in solution.
Gold-silver alloy nanoparticles can be produced by pulsed laser irradiation of bulk alloy metals in water, preserving the stoichiometry of the target metals.
Proteolytic processing plays a significant role in the process of invasion by the obligate intracellular parasite Toxoplasma gondii. We have cloned a gene, TgSUB1, encoding for a subtilisin-type serine protease found in T. gondii tachyzoites. TgSUB1 protein is homologous to other Apicomplexan and bacterial subtilisins and is processed within the secretory pathway of the parasite. Initial cleavage occurs in the endoplasmic reticulum, after which the protein is transported to micronemes, vesicles that secrete early during host cell invasion. Upon stimulation of microneme secretion, TgSUB1 is cleaved into smaller products that are secreted from the parasite. This secondary processing is inhibited by brefeldin A and serine protease inhibitors. TgSUB1 is a candidate processing enzyme for several microneme proteins cleaved within the secretory pathway or during invasion.
A series of umbelliferone derivatives was prepared and their 5alpha-reductase type 1 inhibitory activities were evaluated in cell culture systems. Our studies have identified a new series of potent 5alpha-reductase type 1 inhibitors and provided the basis for further development for the treatment of human endocrine disorders associated with overproduction of DHT by 5alpha-reductase type 1. The preliminary structure-activity relationship was described to elucidate the essential structural requirements.
Peptidyl-prolyl isomerases (PPIases) are ubiquitous cellular enzymes that play roles in cellular signaling and protein folding. In addition, these proteins are the receptors for the widely used immunosuppressants cyclosporin A and FK506. We report the first structure-activity studies of de novo designed inhibitors of cyclophilin, the cellular target of cyclosporin A. Our mechanism-based inhibitors were modeled on the ground- and transition-state structures of proline-containing peptides, the natural substrates of the enzyme. Both ground-state analogues 1 and transition-state analogues 2 were prepared as single enantiomers from L-proline following a "self-reproduction of chirality" procedure. The binding affinities of the analogues for the active site of cyclophilin were measured by a fluorescence perturbation assay. While the transition-state analogues 2 did not display significant avidity for the active site (K(d) = 77 microM for 2b), several ground-state analogues bound to the enzyme with low micromolar affinity (K(d) = 1.5 microM for 1e). These results proclaim that properly designed small molecular weight molecules can form strong complexes with cyclophilin and may find use as probes in cell biology and as therapeutic agents.
Upon host cell invasion the apicomplexan parasite Toxoplasma gondii resides in a specialized compartment termed the parasitophorous vacuole that is derived from the host cell membrane but modified by the parasite. Despite the segregation of the parasitophorous vacuole from the host endocytic network, the intravacuolar parasite has been shown to acquire cholesterol from the host cell. In order to characterize further the role of sterol metabolism in T. gondii biology, we focused our studies on the activity of acyl-CoA:cholesterol acyltransferase (ACAT), a key enzyme for maintaining the intracellular homeostasis of cholesterol through the formation of cholesterol esters. In this study, we demonstrate that ACAT and cholesterol esters play a crucial role in the optimal replication of T. gondii. Moreover, we identified ACAT activity in T. gondii that can be modulated by pharmacological ACAT inhibitors with a consequent detrimental effect on parasite replication.
There have been many studies for tumor therapy mediated by cytotoxic T lymphocytes (CTL) that recognize tumor-associated antigen. It is generally accepted that CTL responses are induced when antigen is delivered into the cytosol. The pH-sensitive liposomes as vehicles are well known for their capacity to deliver the antigen into the cytosol. In this work, immunization of mice with CTL epitope peptides from Hantaan nucleocapsid protein (M6) or human papilloma virus E7 encapsulated in pH-sensitive liposomes induced effective antigen-specific CTL responses. The CTL responses induced by M6 peptide encapsulated in pH-sensitive liposomes blocked the formation of tumor mass from Hantaan NP transfected B16 melanoma cells in C57BL/6 mice and delayed the growth of preinoculated melanoma cells. During the blockade of the tumor growth, the CTL response was maintained for at least approximately 6 weeks, and the mice secreted Th1 type cytokines such as IL-2 and IFN-gamma. These results suggested that the pH-sensitive liposomes might provide an effective peptide delivery system for CTL-mediated tumor therapy.
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.
The size distribution of biodegradable polymer microspheres critically impacts the allowable routes of administration, biodistribution, and release rate of encapsulated compounds. We have developed a method for producing microspheres of precisely controlled and/or monodisperse size distributions. Our apparatus comprises spraying a polymer-containing solution through a nozzle with (i) acoustic excitation to produce uniform droplets, and (ii) an annular, non-solvent carrier stream allowing further control of the droplet size. We used this apparatus to fabricate poly(D,L-lactide-co-glycolide) (PLG) spheres. The acoustic excitation method, by itself, produced uniform microspheres as small as 30 microm in diameter in which > or =95% of the spheres were within 1.0-1.5 microm of the average. The carrier stream method alone allowed production of spheres as small as approximately 1-2 microm in diameter from a 100-microm diameter nozzle, but generated broader size distributions. By combining the two devices, we fabricated very uniform spheres with average diameters from approximately 5 to >500 microm. Furthermore, by discretely or continuously varying the experimental parameters, we fabricated microsphere populations with predefined size distributions. Finally, we demonstrate encapsulation and in vitro release of a model drug compound, rhodamine B. In summary, our apparatus provides unprecedented control of microsphere size and may allow development of advanced controlled-release delivery systems.
Early T progenitors in the thymus have been reported to have the capacity to develop into B cells, thymic dendritic cells, and NK cells. Here we describe conditions that induce early T progenitors to develop into macrophages. Initially, we observed that early T progenitors could be induced to develop into macrophages by cytokines produced from a thymic stromal cell line, TFGD, and later we found that the cytokine mixture of M-CSF plus IL-6 plus IL-7 also induced macrophage differentiation from pro-T cells. M-CSF by itself was unable to induce macrophage differentiation from early T progenitors. To correlate this observation with the developmental potential of early T progenitors, mouse embryonic thymocytes were sorted into four populations, pro-T1 to pro-T4, based on the expression of CD44 and CD25, and then cultured with TFGD culture supernatant. We found that pro-T1 and pro-T2 cells, but not pro-T3 and pro-T4 cells, generate macrophages. Limiting dilution analysis of the differentiation capability of sorted pro-T2 cells also confirmed that pro-T2 cells could generate macrophages. These results suggest that T cells and thymic macrophages could originate from a common intrathymic precursor.
STUDY OBJECTIVE: To examine the relationship between lifestyle, health status factors and sleep loss. DESIGN: A cross-sectional questionnaire survey conducted by the Ministry of Health and Welfare, Japan. SETTING: N/A. PATIENTS OR PARTICIPANTS: Approximately 30,000 subjects selected from the general population in Japan. INTERVENTIONS: N/A. MEASUREMENTS AND RESULTS: This study indicated that approximately 28% of the general population sleep less than 6 hours nightly and approximately 65% sleep less than 7 hours. However, approximately 80% of the population reported getting sufficient sleep. Multiple logistic regression analysis showed that being females, being of younger age, living in an urban environment, being unemployed, and having an unhealthy lifestyle (i.e., lack of exercise, poor health status, and irregular eating habits) were associated with sleep loss. CONCLUSION: In this study, sleep loss was found to be associated with having an unhealthy lifestyle and being in poor general health. These findings suggest that health education and promotion of a healthy lifestyle should be advocated.
The effects of clozapine on the activities of nicotinic acetylcholine receptors (nAChRs) and voltage-sensitive calcium channels (VSCCs) were investigated and compared with those of chlorpromazine (CPZ) in bovine adrenal chromaffin cells. [(3)H]Norepinephrine ([(3)H]NE) secretion induced by activation of nAChRs was inhibited by clozapine and CPZ with half-maximal inhibitory concentrations (IC(50)) of 10.4 +/- 1.1 and 3.9 +/- 0.2 microM, respectively. Both cytosolic calcium increase and inward current in the absence of extracellular calcium induced by nicotinic stimulation were also inhibited by clozapine and CPZ, but the greater inhibition was achieved by CPZ. In addition, [(3)H]nicotine binding to chromaffin cells was inhibited by clozapine and CPZ with IC(50) values of approximately 19 and 2 microM, respectively. On the other hand, [(3)H]NE secretion induced by high K(+) was inhibited by clozapine and CPZ with similar IC(50) values of 15.5 +/- 3.8 and 17.1 +/- 3.9 microM, respectively. Our results suggest that clozapine, as well as CPZ, inhibits nAChRs and VSCCs, thereby causing inhibition of catecholamine secretion, and that clozapine is much less potent than CPZ in inhibiting nAChRs.
A phase I dose-escalation clinical trial of peritumoral injections of interleukin 12 (IL-12)-transduced autologous fibroblasts was performed in patients with disseminated cancer for whom effective treatment does not exist. The goals of this study were to assess the safety and toxicities as well as the efficacy, and ancillarily the immunomodulatory effects, of peritumoral IL-12 gene transfer. Primary dermal fibroblasts cultured from the patients were transduced with retroviral vector carrying human IL-12 genes (p35 and p40) as well as the neomycin phosphotransferase gene (TFG-hIL-12-Neo). Patients received four injections at intervals of 7 days. Nine patients were enrolled in this dose-escalation study, with secreted IL-12 doses ranging from 300 ng/24 hr for the first three patients to 1000, 3000, and 5000 ng/24 hr for two patients in each subsequent dosage level. Although a definite statement cannot be made, there appears to be perturbation of systemic immunity. Also, the locoregional effects mediated by tumor necrosis factor alpha (TNF-alpha) and CD8+ T cells were observed with tumor regression. Treatment-related adverse events were limited to mild to moderate pain at the injection site; clinically significant toxicities were not encountered. Transient but clear reductions of tumor sizes were observed at the injected sites in four of nine cases, and at noninjected distant sites in one melanoma patient. Hemorrhagic necrosis of tumors was observed in two melanoma patients. These data indicate that gene therapy by peritumoral injection of IL-12-producing autologous fibroblasts is feasible, and promising in patients with advanced cancer.
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BACKGROUND: Left liver graft from a small donor will not meet the metabolic demands of a larger adult recipient. One solution to this problem is to use a right liver graft without a middle hepatic vein (MHV). However, the need for drainage from the MHV tributaries has not yet been described. METHODS: Five right liver grafts without a MHV were transplanted in patients including two hepatitis B virus-cirrhosis, two fulminant hepatic failure and one secondary biliary cirrhosis. The graft weight ranged from 650 to 1,000 g, corresponding to 48 to 83% of the standard liver volume of the recipients. RESULTS: Two of five recipients were complicated with severe congestion of the right median sector immediately after reperfusion, followed by prolonged massive ascites and severe liver dysfunction. One of the patients died of sepsis with progressive hepatic dysfunction 20 days after the operation. CONCLUSIONS: Preservation and reconstruction of the MHV tributaries is recommended to prevent congestion of the right liver graft without MHV.
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In contrast to pseudobinary alloys, the relative number of bonds in quaternary alloys cannot be determined uniquely from the composition. Indeed, we do not know if the Ga0.5In0.5As0.5N0.5 alloy should be thought of as InAs+GaN or as InN+GaAs. We study the distribution of bonds using Monte Carlo simulation and find that the number of In-N and Ga-As bonds increases relative to random alloys. This quaternary-unique short range order affects the band structure: we calculate a blueshift of the band gap and predict the emergence of a broadband tail of localized states around the conduction band minimum.
We previously reported an enhancing effect of all-trans-retinoic acid (all-trans-RA) on gonadotropin-releasing hormone (GnRH) gene transcription via distal promoter elements of the rat GnRH gene. The present study examined the effects of another biologically active retinoid, 9-cis-retinoic acid (9-cis-RA), on GnRH transcription in GT1-1 cells. Similar to the action of all-trans-RA, 9-cis-RA significantly induced the luciferase activity of the strong retinoic acid response element (RARE) reporter construct, 3X beta RARE-Luc, by about 60-fold, indicating that GT1-1 cells are also responsive to 9-cis-RA. In contrast to the stimulatory effect of all-trans-RA on GnRH transcription, 9-cis-RA inhibited the GnRH promoter activity in a dose- and time-dependent manner. Significant inhibition by 9-cis-RA required at least an 18 h treatment and a further decrease of GnRH promoter-driven luciferase activity was observed up to 48 h of incubation. Accordingly, GnRH mRNA levels were decreased by 9-cis-RA treatment in a similar dose- and time-related manner, indicating that mouse GnRH expression is also negatively regulated by 9-cis-RA. Transient transfections of serial deletion constructs of the rat GnRH promoter revealed that the --230/--110 sequence of the rat GnRH promoter is responsible for 9-cis-RA-induced inhibition of GnRH transcription. Within this region, however, no consensus retinoid X receptor response element was found. To gain insights into the role of retinoid X receptors (RXRs) in GnRH expression, we examined the effects of RXR overexpression on GnRH transcriptional activity. Interestingly, co-transfection of RXR overexpression vectors significantly increased the GnRH promoter-driven luciferase activity, while treatment with 9-cis-RA not only nullified the enhancing effect of RXR overexpression but also decreased the basal GnRH promoter-driven luciferase activity by 50% compared to vehicle-treated controls. This implies that RXRs in the absence of its cognate ligand 9-cis-RA contribute to the maintenance of basal GnRH gene transcription. Northern blot analysis revealed that 9-cis-RA, but not all-trans-RA, down-regulated RXR beta expression in GT1-1 cells, suggesting that one possible mechanism of 9-cis-RA-induced repression involves down-regulation of RXR expression. In conclusion, the present study clearly demonstrates that 9-cis-RA is a negative regulator of GnRH gene expression in immortalized GnRH neurons.