Crystal structure of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from Thermus caldophilus.
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Bacterial Proteins↗
Biomedical subjects
Sung Hyun Kim
Publications and source records attributed to Sung Hyun Kim.
CT angiography demonstration of the development of intraventricular hemorrhage during aneurysm rupture.
The authors present the 3D reconstructed CT angiography image of extravasation from a rebleeding aneurysm. Direct hemorrhage of a bleeding aneurysm into the lateral ventricle was detected by CT angiography. This image clearly visualized that extravasated contrast medium spurting from a rupturing anterior communicating artery aneurysm, penetrated the ventricle wall and passed through the lateral ventricle to reach the temporal horn. This case provides a real-time demonstration of the way how an intraventricular hemorrhage develops during rupture of an anterior communicating artery aneurysm.
Aged↗
The parthenogenetic activation of canine oocytes with Ca-EDTA by various culture periods and concentrations.
In the present study, canine oocytes were exposed to various concentrations of and durations of exposure to EDTA saturated with Ca(2+) (Ca-EDTA), a cell membrane-impermeable metal ion chelator, to determine if parthenogenetic activation could be induced. When oocytes were cultured for 48 or 72 h in parthenogenetic activation medium (PAM) without Ca-EDTA (control) or PAM supplemented with 1 or 5mM Ca-EDTA, the highest rate of pronuclear formation (PN) was obtained in oocytes cultured in 1mM Ca-EDTA for 48 h (8.0%; P<0.05). There was no pronuclear formation in the control group (PAM without Ca-EDTA). Oocytes treated with 5mM Ca-EDTA for 48 h or 1mM Ca-EDTA for 72 h formed a parthenogenetic pronucleus (3.1 and 4.5, respectively). However, there was no pronuclear formation in oocytes treated with 5mM Ca-EDTA for 72 h. In summary, exposure to Ca-EDTA can induce pronuclear formation in canine oocytes.
Animals↗
Ectopic expression of tethered human follicle-stimulating hormone (hFSH) gene in transgenic mice.
To determine whether the mammary gland can be used to secrete large quantities of a bioactive heterodimeric protein into milk, we used a bovine beta-casein promoter to target and express human follicle-stimulating hormone (hFSH) in the mammary gland into the milk of transgenic mice. We also identified the effects of hFSH leaked into the bloodstream. Transgenic mice produced a high level (up to 300 mIU/ml) of recombinant hFSH in the mammary gland. Human FSH was expressed in the mammary gland and brain, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemistry. In vitro bioactivity was also identified by cyclic AMP (cAMP) assay. The highest activity was showed in the transgenic mice line 11. However, hFSH leaked into the bloodstream was a powerful factor in the generation of breast and ovarian tumors from the transgenic mice line 11. These results suggest that change of endogenous hormones (FSH and progesterone) may affect the morphology and blood cell counts of peripheral blood and, especially, provoke breast and ovarian tumors.
Animals↗
Down-regulation of lactate dehydrogenase-A by siRNAs for reduced lactic acid formation of Chinese hamster ovary cells producing thrombopoietin.
Lactate, one of the major waste products in mammalian cell culture, can inhibit cell growth and affect cellular metabolism at high concentrations. To reduce lactate formation, lactate dehydrogenase-A (LDH-A), an enzyme catalyzing the conversion of glucose-derived pyruvate to lactate, was down-regulated by an expression vector of small interfering RNAs (siRNA) in recombinant Chinese hamster ovary (rCHO) cells producing human thrombopoietin (hTPO). Three clones expressing low levels of LDH-A, determined by reverse transcription-PCR and an enzyme activity test, were established in addition to a negative control cell line. LDH-A activities in the three clones were decreased by 75-89%, compared with that of the control CHO cell line, demonstrating that the effect of siRNA is more significant than that of other traditional methods such as homologous recombination (30%) and antisense mRNA (29%). The specific glucose consumption rates of the three clones were reduced to 54-87% when compared to the control cell line. Similarly, the specific lactate production rates were reduced to 45-79% of the control cell line level. In addition, reduction of LDH-A did not impair either cell proliferation or hTPO productivity. Taken together, these results show that the lactate formation rate in rCHO cell culture can be efficiently reduced through the down-regulation of LDH via siRNA.
Animals↗
SPIN90/WISH interacts with PSD-95 and regulates dendritic spinogenesis via an N-WASP-independent mechanism.
SPIN90/WISH (SH3 protein interacting with Nck, 90 kDa/Wiskott-Aldrich syndrome protein (WASP) interacting SH3 protein) regulates actin polymerization through its interaction with various actin-regulating proteins. It is highly expressed in the brain, but its role in the nervous system is largely unknown. We report that it is expressed in dendritic spines where it associates with PSD-95. Its overexpression increased the number and length of dendritic filopodia/spines via an N-WASP-independent mechanism, and knock down of its expression with small interfering RNA reduced dendritic spine density. The increase in spinogenesis is accompanied by an increase in synaptogenesis in contacting presynaptic neurons. Interestingly, PSD-95-induced dendritic spinogenesis was completely abolished by knock down of SPIN90/WISH. Finally, in response to chemically induced long-term potentiation, SPIN90/WISH associated with PSD-95 and was redistributed to dendritic spines. Our results suggest that SPIN90/WISH associates with PSD-95, and so becomes localized to dendritic spines where it modulates actin dynamics to control dendritic spinogenesis. They also raise the possibility that SPIN90/WISH is a downstream effector of PSD-95-dependent synaptic remodeling.
Actins↗
Neoplastic transformation and tumorigenesis associated with overexpression of IMUP-1 and IMUP-2 genes in cultured NIH/3T3 mouse fibroblasts.
Immortalization-upregulated protein 1 (IMUP-1) and immortalization-upregulated protein 2 (IMUP-2) genes have been recently cloned and are known to be involved in SV40-mediated immortalization. IMUP-1 and IMUP-2 genes were strongly expressed in various cancer cell lines and tumors, suggesting the possibility that they might be involved in tumorigenicity. To directly elucidate the functional role of IMUP-1 and IMUP-2 on neoplastic transformation and tumorigenicity, we stably transfected IMUP-1 and IMUP-2 into NIH/3T3 mouse fibroblast cells. Cellular characteristics of the neoplastic transformation were assessed by transformation foci, growth in soft agar, and tumor development in nude mice. We found that IMUP-1 and IMUP-2 overexpressing cells showed altered growth properties, anchorage-independent growth in soft agar and inducing tumor in nude mice. Furthermore, IMUP-1 and IMUP-2 transformants proliferated in reduced serum and shortened cell cycle. These results suggest that ectopic overexpression of IMUP-1 and IMUP-2 may play an important role in acquiring a transformed phenotype, tumorigenicity in vivo, and be related to cellular proliferation.
3T3 Cells↗
Crystal structure of visfatin/pre-B cell colony-enhancing factor 1/nicotinamide phosphoribosyltransferase, free and in complex with the anti-cancer agent FK-866.
Visfatin/pre-B cell colony-enhancing factor 1 (PBEF)/nicotinamide phosphoribosyltransferase (NAmPRTase) is a multifunctional protein having phosphoribosyltransferase, cytokine and adipokine activities. Originally isolated as a cytokine promoting the differentiation of B cell precursors, it was recently suggested to act as an insulin analog via the insulin receptor. Here, we describe the first crystal structure of visfatin in three different forms: apo and in complex with either nicotinamide mononucleotide (NMN) or the NAmPRTase inhibitor FK-866 which was developed as an anti-cancer agent, interferes with NAD biosynthesis, showing a particularly high specificity for NAmPRTase. The crystal structures of the complexes with either NMN or FK-866 show that the enzymatic active site of visfatin is optimized for nicotinamide binding and that the nicotinamide-binding site is important for inhibition by FK-866. Interestingly, visfatin mimics insulin signaling by binding to the insulin receptor with an affinity similar to that of insulin and does not share the binding site with insulin on the insulin receptor. To predict binding sites, the potential interaction patches of visfatin and the L1-CR-L2 domain of insulin receptor were generated and analyzed. Although the relationship between the insulin-mimetic property and the enzymatic function of visfatin has not been clearly established, our structures raise the intriguing possibility that the glucose metabolism and the NAD biosynthesis are linked by visfatin.
Acrylamides↗
Effect of conditioned medium of mouse embryonic fibroblasts produced from EC-SOD transgenic mice in nuclear maturation of canine oocytes in vitro.
The rate of in vitro maturation (IVM) of canine oocytes has not improved in comparison to that of other mammalian species. This study aims to improve the efficiency of canine oocytes IVM using the antioxidant, extracellular superoxide dismutase (EC-SOD). Thus, the effect of conditioned medium of EC-SOD transgenic mouse embryonic fibroblasts cultured with MEF culture medium (DMEM + 5% FBS) for in vitro nuclear maturation in canine oocytes was investigated. In experiment I, oocytes were collected from the ovaries of domestic bitches, which were allotted to one of two groups: (1) TCM199 + 1% FBS (n = 108) or (2) DMEM + 5% FBS (n = 112), cultured for 48 h and investigated for in vitro nuclear maturation of canine oocytes using Hoechst staining. Meiotic progression to metaphase II in group 1 was 1.8% compared to 1.8% in group 2. In experiment II, EC-SOD levels were examined in NTg-CMEF and Tg-CMEF at 0, 2 and 4 days obtained from EC-SOD transgenic mice generated in our laboratory. The concentration of EC-SOD in Tg-CMEF at day 2 (371.7 +/- 3.1 ng/ml) was the highest for all groups (P < 0.05). EC-SOD levels in Tg-CMEF were higher than in NTg-CMEF; therefore, the efficiency of Tg-CMEF for IVM was investigated. In experiment III, oocytes were allotted to one of three groups: (1) Tg-CMEF at day 0 (n = 84), (2) Tg-CMEF at day 2 (n = 92) or (3) Tg-CMEF at day 4 (n = 98), cultured for 48 h and the IVM of canine oocytes investigated. The mean percentage of MII oocytes in IVM was 2.4, 4.4 and 2.0% for groups 1, 2 and 3, respectively. In experiment IV, the effects of conditioned medium of EC-SOD transgenic mouse embryonic fibroblasts (Tg-CMEF) cultured in MEF culture medium were compared with conditioned medium acquired from non-transgenic mouse embryonic fibroblasts (NTg-CMEF) on IVM of canine oocytes. In this experiment, meiotic progression to metaphase II was 7.1% in Tg-CMEF versus 0% in NTg-CMEF (P < 0.05). Tg-CMEF was more effective than NTg-CMEF. In conclusion, it was verified that canine oocytes were able to effectively progress to metaphase II in IVM when cultured in Tg-CMEF.
Animals↗
Characterization of a brain tumor cell line established from transgenic mice expressing the vasopressin SV-40 T antigen.
We previously reported that transgenic mice produced with a transgene consisting of the SV40 T antigen and vasopressin without the 3'-flanking region exhibit brain tumors and lymphoma. In this study, transgenic mice were produced with the fusion gene containing the SV40 T antigen and the whole vasopressin gene with the 3'-flanking region. Six transgenic mice were generated, five which died after 2-6 weeks. The remaining founder mouse was investigated for fusion gene expression and tumor progression at the age of 6 weeks. Brain tumor cells were characterized for phenotypes and transgene expression. During in vitro cell cultures, the phenotypic appearances at 10, 20, and 30 passages were as a uniform monolayer with similar growth rates. The site of SV40 T antigen integration was in the A2 region of chromosome 11, and SV40 T antigen was expressed at the same level in cells of both earlier and later passages. Thirty passages were probably insufficient to reach crisis and immortalization. These cells enriched brain tumor cell compositions with astrocytes and neuronal cells.
Animals↗
Interaction of SPIN90 with syndapin is implicated in clathrin-mediated endocytic pathway in fibroblasts.
SPIN90, a 90-kDa Nck-interacting protein with a SH3 domain, plays a role in sarcomere formation and myofibril assembly, and its phosphorylation is modulated by cell adhesion and Erk activation. Here we demonstrate that SPIN90 participates in receptor-mediated endocytic pathway in fibroblasts. We identified syndapin (synaptic dynamin-binding protein) as a SPIN90 interacting protein using yeast two-hybrid screening. SPIN90 directly binds the SH3 domain of syndapin via its proline rich domain in vitro and in vivo and also associates with clathrin. Over-expression of SPIN90-full length in COS-7 cells inhibited transferrin uptake, a marker of endocytosis. Interestingly, SPIN90-PRD, a syndapin-binding domain, significantly inhibited endocytosis, and the inhibition was reversed by co-expression of syndapin. Depleting SPIN90 through antibody microinjection or Knocking it down using siRNAs also significantly inhibited transferrin internalization. Moreover, early endosomal marker proteins (EEA1 and Rab5) appeared to closely associate or partially co-localize with SPIN90 in endosomes and an internalized FITC-dextran and Texas Red-EGF were found on the endosomes in association with SPIN90. Time-lapse video showed that GFP-SPIN90 travels with moving vesicles within living cells. Taken together, these findings suggest that SPIN90 is implicated in receptor-mediated endocytic pathway in fibroblasts.
Animals↗
Repeat endovascular treatment in post-embolization recurrent intracranial aneurysms.
OBJECTIVE: The purpose of this study was to describe clinical situations requiring repeat embolization in patients previously treated by endovascular coil embolization for intracranial aneurysms, and to report on our experiences of repeat embolization (RE). METHODS: A total of 466 patients harboring 522 intracranial aneurysms were treated by endovascular coil embolization at our institution during the period between December 1992 and August 2004. We studied 32 patients who underwent repeat coil embolization (RE) owing to recanalization or aneurysm recurrence. Radiological and clinical data were reviewed to determine the reasons, results, and technical problems of RE. RESULTS: Thirty-nine sessions of RE were performed in 32 patients; four patients underwent RE twice and another patient three times. The major reason for RE was asymptomatic aneurysmal recanalization owing to coil compaction and/or loosening. The time interval between RE and the previous embolization was 12 months or less in 27 sessions. Complete or near complete occlusion of the aneurysm was achieved in all cases without procedure-related morbidity or mortality. Radiolucent gaps between the coil masses were observed in 17 cases. CONCLUSION: RE is a safe and effective treatment option in cases of recanalized or recurrent aneurysms. Close follow-up evaluation is essential in patients with intracranial aneurysms after coil embolization.
Adult↗
Therapeutic effect and outcome predictors of sciatica treated using transforaminal epidural steroid injection.
OBJECTIVE: The objectives of this retrospective study were to assess the therapeutic effect of transforaminal epidural steroid for sciatica and to identify outcome predictors. MATERIALS AND METHODS: Transforaminal epidural steroid injections were performed in 248 patients from June 2003 to May 2004. Fifty-six patients (33 women, 23 men; mean age, 53.3 years; age range, 30-83 years) were included. Therapeutic effects were evaluated 2 weeks after injection. The possible outcome predictors were as follows: intraepineural or extraepineural injection, saddle-type distribution pattern (contrast material distributed rostrally to the epidural portion of the preganglionic nerve root) or not saddle type, cause of sciatica (spinal stenosis vs herniated disk), patient age, patient sex, and duration of sciatica (acute or subacute [< 6 months] vs chronic [> 6 months]). The relationships between possible outcome predictors and therapeutic effects were analyzed. Statistical analysis was performed using Fisher's exact test, the chi-square test, and multiple logistic regression analysis. RESULTS: Forty-three (76.8%) of the 56 patients achieved a satisfactory result 2 weeks after transforaminal epidural steroid injection. Nineteen (65.5%) of the 29 patients treated by intraepineural injection and 24 (88.9%) of the 27 patients treated by extraepineural injection achieved a satisfactory result, and this difference was significantly different (p < 0.05). Other possible predictors of a better outcome were identified--that is, saddle-type pattern of contrast distribution, a herniated disk, and sciatica of less than 6 months' duration. Multiple regression analysis showed that the only factor significantly associated with outcome was the type of injection (p = 0.04, odds ratio: 5.01). CONCLUSION: Transforaminal epidural steroid is an effective tool for managing sciatica, and an extraepineural injection may be a predictor of a better outcome for sciatica treated using transforaminal epidural steroid.
Adult↗
A case of benign metastasizing leiomyoma with multiple metastasis to the soft tissue, skeletal muscle, lung and breast.
Benign metastasizing leiomyoma (BML) is composed of well-differentiated smooth muscle cells and dense connective tissue. BML affects middle-aged women who have had previous hysterectomies due to a histologically benign-appearing uterine leiomyoma. We report here on a case of BML from the uterine leiomyoma in a 39-year-old woman that involved the soft tissues, skeletal muscles, lungs and breasts. She underwent a hysterectomy for the uterine leiomyoma, double oophorectomy for hormonal ablation and lung wedge resection to confirm the diagnosis. The microscopic findings of the breast and lung tumor were similar to those of the benign uterine leiomyoma. Therefore, we consider that these lesions were breast and pulmonary metastases of the uterine leiomyoma. We report here on a rare case of benign metastasizing uterine leiomyoma that involved the soft tissue, skeletal muscles, lungs and breasts, and we include a review of the relevant literature.
Adult↗
Interaction of SPIN90 with the Arp2/3 complex mediates lamellipodia and actin comet tail formation.
The appropriate regulation of the actin cytoskeleton is essential for cell movement, changes in cell shape, and formation of membrane protrusions like lamellipodia and filopodia. Moreover, several regulatory proteins affecting actin dynamics have been identified in the motile regions of cells. Here, we provide evidence for the involvement of SPIN90 in the regulation of actin cytoskeleton and actin comet tail formation. SPIN90 was distributed throughout the cytoplasm in COS-7 cells, but exposing the cells to platelet-derived growth factor (PDGF) caused a redistribution of SPIN90 to the cell cortex and the formation of lamellipodia (or membrane ruffles), both of which were dramatically inhibited in SPIN90-knockdown cells. In addition, the binding of the C terminus of SPIN90 with both the Arp2/3 complex (actin-related proteins Arp 2 and Arp 3) and G-actin activates the former, leading to actin polymerization in vitro. And when coexpressed with phosphatidylinositol 4-phosphate 5 kinase, SPIN90 was observed within actin comet tails. Taken these findings suggest that SPIN90 participates in reorganization of the actin cytoskeleton and in actin-based cell motility.
Actin Cytoskeleton↗
Interaction of SPIN90 with dynamin I and its participation in synaptic vesicle endocytosis.
SH3 protein interacting with Nck, 90 kDa (SPIN90) is an Nck-binding protein that contains one Src homology 3 (SH3) domain, three proline-rich domains (PRDs), a serine/threonine-rich region, and a hydrophobic C-terminal region. Previously, we have shown that SPIN90 plays roles in the sarcomere assembly in cardiac muscles and in the formation of focal contacts in HeLa cells. Besides in heart, SPIN90 is also highly expressed in the brain, but its role in the neuronal system is completely unknown. Here, we found that SPIN90 is expressed in the presynaptic compartment in which it binds dynamin I, a key component of the endocytic machinery, and that it participates in synaptic vesicle endocytosis. Pull-down analysis and coimmunoprecipitation proved the associations of SPIN90 with dynamin I through SH3-PRD interaction. Overexpression of SPIN90 or knocking down SPIN90 by small interfering RNA impaired synaptic vesicle endocytosis. We further confirmed by the rescue experiments that the endocytic defects by SPIN90 expression come from its interaction with dynamin I. Exocytosis kinetics was not affected by SPIN90 expression. Together, our findings suggest that SPIN90 could modulate the interactions of dynamin I with other endocytic proteins that cooperate in the coated vesicle formation, thus regulating synaptic vesicle endocytosis.
Adaptor Proteins, Signal Transducing↗
Transgene expression of biological active recombinant human granulocyte-colony stimulating factor (hG-CSF) into mouse urine.
We have generated transgenic mice that expressed human granulocyte-colony stimulating factor (hG-CSF) in their urine. In particular, the expression plasmid DNA containing mouse uroplakin II promoter was used to direct the uroepithelium-specific transcription of the transgene. In this study, the hG-CSF transcript was detected only in bladder, as was determined by RT-PCR analysis. Furthermore, hG-CSF protein was detected in the suprabasal layer of the uroepithelium and ureter, as was demonstrated by immunohistochemistry. The hG-CSF was secreted into urine at a high level (approx. 500 pg/ml), and it was able to enhance the proliferation of DMSO treated HL-60 cells, suggesting that the transgenic urine-derived hG-CSF was bioactive. However, the recombinant hG-CSF was leaked to peripheral circulation system. To examine the relationship between hG-CSF in the blood stream and the proliferation of hematopoietic cells, we tested the transgenic mouse blood with hematocrit analysis. An increase of the total number of neutrophils in the transgenic mice peripheral blood was not observed; therefore, the leakage of human G-CSF can probably be expected to do no harm to the transgenic mouse. Our results demonstrate that bladder can be safely used as a bioreactor to produce biologically important substances such as recombinant G-CSF.
Animals↗
Fiber tracking by diffusion tensor imaging in corticospinal tract stroke: Topographical correlation with clinical symptoms.
Fiber tracking of the white matter using diffusion tensor imaging is a new imaging technique to visualize the integrity of the white matter. This study investigated the capability of this technique to localize the lacunar infarctions, particularly with respect to the body parts affected, by correlating the location of the lesion with the clinical symptoms topographically. Twenty-seven patients with capsular and pericapsular small acute infarctions underwent diffusion tensor imaging and subsequent fiber tracking of the corticospinal tract (CST). According to the lesion topography with regard to the CST, the infarctions were classified into four types: (1) the anterior type (n = 9) involving the anterior part of the CST, (2) the central type (n = 9) involving the middle or whole part of the CST, (3) the posterior type (n = 5) involving the posterior part of the CST and (4) the intact type (n = 4) not involving the CST. Motor weakness of the face, upper extremities and lower extremities was found at 100%, 67% and 44%, respectively in the anterior type, at 89%, 100% and 89%, respectively in the central type and at 20%, 80% and 100%, respectively in the posterior type. The intact type was not associated with motor weakness. In conclusion, the fiber tracking technique of the CST enables the specific localization of capsular and pericapsular infarctions with regard to the body parts affected. These results also confirm the topographical accuracy of the fiber tracking of the CST.
Aged↗