Determination of the disulfide bond and its possible role in tobacco acetolactate synthase.
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Acetolactate Synthase↗
Biomedical subjects
H J Shin
Publications and source records attributed to H J Shin.
Metal-catalyzed oxidation of alpha-synuclein in the presence of Copper(II) and hydrogen peroxide.
alpha-Synuclein is a component of abnormal protein depositions of Lewy bodies and senile plaques found in Parkinson's and Alzheimer's diseases, respectively. By using chemical coupling reagents such as dicyclohexylcarbodiimide or N-(ethoxycarbonyl)-2-ethoxy-1, 2-dihydroquinoline, the protein was shown to experience self-oligomerization in the presence of either copper(II) or Abeta25-35. The oligomers which appeared as a ladder on a 10-20% Tricine/SDS-PAGE have been suggested to participate in the formation of protein aggregations by possibly providing a nucleation center. Since oxidatively modified protein could increase its own tendency toward protein aggregation, metal-catalyzed oxidation of alpha-synuclein has been examined with copper(II) and hydrogen peroxide in the absence of the coupling reagent. Intriguingly, the protein was also self-oligomerized into an SDS-resistant ladder on the gel. This biochemically specific copper-mediated oxidative oligomerization was shown to be dependent upon the acidic C-terminus of alpha-synuclein because the C-terminally truncated proteins such as alpha-syn114 and alpha-syn97 were not affected by the metal and hydrogen peroxide. More importantly, the oxidative oligomerization was synergistically enhanced by the presence of Abeta25-35, indicating that the peptide interaction with alpha-synuclein facilitated the copper(II) binding to the acidic C-terminus and subsequent oxidative crosslinking. It has been, therefore, suggested that abnormalities in copper and H(2)O(2) homeostasis and certain pathological factors functionally similar to the Abeta25-35 could play critical roles in the metal-catalyzed oxidative oligomerization of alpha-synuclein, which may lead to possible protein aggregation and neurodegenerations.
Alkylating Agents↗
Involvement of SPARC in in vitro differentiation of skeletal myoblasts.
SPARC (secreted protein acidic and rich in cysteine) is an extracellular Ca(2+)-binding glycoprotein associated with the morphogenesis and remodeling of various tissues. Here, involvement of SPARC in the myogenesis of skeletal myoblasts was investigated in vitro. First, the differential expression of SPARC mRNA during the myogenesis was initially identified by a differential display reverse transcription (DDRT)-PCR method. The expression of the SPARC gene was significantly up-regulated during the differentiation of C2C12 mouse myoblasts. Second, the treatment with anti-SPARC antibody almost completely prevented the differentiation of myoblasts. Third, the treatment with EGTA, a Ca(2+) chelator that is known to inhibit the fusion of C2C12 myoblasts, reversibly inhibited the up-regulation of SPARC gene expression. On the other hand, the treatment with A23187, a Ca(2+) ionophore, rapidly and dramatically increased the level of SPARC transcript. Taken together, these results suggest that SPARC may play a critical role(s) in the morphological change of myoblasts, and that the expression of SPARC gene may be controlled by Ca(2+)-dependent pathway in myogenesis.
Animals↗
Structural and functional role of cysteinyl residues in tobacco acetolactate synthase.
Acetolactate synthase (ALS) is the common enzyme in the biosynthesis of valine, leucine, and isoleucine. The role of four cysteinyl residues in tobacco ALS was determined using site-directed mutagenesis and cysteine-specific cleavage. The C411A mutation abolished the enzymatic activity, as well as the binding affinity for the cofactor FAD. The activation constant of C411S for FAD is approximately 50-fold higher than that of wALS. The C607S mutation did not significantly affect the kinetic parameters. The IC(50) values of C411S and C607S for ALS-inhibiting herbicides are not much different from those of wALS. Two mutants, C163S and C309S, are labile and readily degraded to peptide fragments. The treatment of wALS with 2-nitro-5-thiocyanobenzoic acid, specific for cleavage of the N-terminal side of cysteine, yielded three peptides of 37.0, 22. 0, and 7.0 kDa. This fragmentation pattern is consistent with that deduced from the amino acid sequence of tobacco ALS, assuming the disulfide bond between Cys163 and Cys309. These results suggest that Cys411 is involved in the binding of FAD and that the intrachain disulfide bond between Cys163 and Cys309 plays a key role in maintaining the correct conformation of tobacco ALS.
Acetolactate Synthase↗
Vascular endothelial growth factor expression under ischemic stress in human meningiomas.
Vascular endothelial growth factor (VEGF) is an endothelial cell-specific antigen and angiogenic factor that plays a role in angiogenesis. We analyzed the expression of four VEGF mRNA isoforms in meningiomas. Among 35 meningiomas, 11 came from patients who underwent complete (n=4) or partial (n=7206=189 in all samples. However, the VEGF121 and 165 isoforms were significantly upregulated in samples from patients who underwent partial preoperative embolization. The diffusible VEGF121 isoform may be important for vascularity and edema formation in meningiomas.
Adolescent↗
Epithelial cells and other cytologic features of pseudomyxoma peritonei in patients with ovarian and/or appendiceal mucinous neoplasms: a study of 12 patients including 5 men.
BACKGROUND: Pseudomyxoma peritonei (PP) is a rare condition. Cytologic evaluation of peritoneal fluid often is an initial diagnostic test for possible ovarian and/or appendiceal primary tumors. Previous studies suggest that patients with PP who have epithelial cells (ECs) in their peritoneal fluid usually have a less favorable prognosis than patients with acellular PP. To the authors' knowledge, few reports of PP in the cytologic literature cite the presence of ECs. METHODS: Twelve cases of PP diagnosed by cytologic examination at the University of Texas M. D. Anderson Cancer Center over 15 years were identified. In all cases, primary tumors were confirmed histologically. All available cytologic smears and cell block sections were reviewed for cytomorphology, with particular attention given to the presence of ECs. A correlation between the presence of ECs and patient outcome also was sought (median follow-up, 26 months). RESULTS: Two patients had ovarian neoplasms, six patients (one female and five males) had appendiceal neoplasms, and four patients had synchronous ovarian and appendiceal tumors. Cytologic features included mucin pools (12 of 12 patients), ECs (11 of 12 patients), mesothelial or mesothelial-like cells (10 of 12 patients), histiocytes (11 of 12 patients), and fibroblast-like or spindle cells (6 of 12 patients). ECs were columnar with mucinous features in the majority of cases, and the number of ECs in each case was variable, ranging from 1+ (rare) to 3+ (many). Of the 11 patients with available follow-up data, 6 had recurrent disease, 4 had persistent disease, and 1 patient with acellular PP was alive without clinical evidence of disease after 24 months of follow-up. CONCLUSIONS: Unlike previous PP cytology reports, the current study frequently identified ECs (92%). Because of the potential prognostic implication of ECs in patients with PP, a diligent search for ECs is warranted. Indication of the presence or absence of ECs in the cytology report may be useful when PP is diagnosed.
Adenocarcinoma, Mucinous↗
Biochemopreventive therapy for patients with premalignant lesions of the head and neck and p53 gene expression.
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Anticarcinogenic Agents↗
Expression of stable hepatitis B viral polymerase associated with GRP94 in E. coli.
We here presented evidence that a 94-kDa glucose-regulated protein (GRP94) was associated with hepatitis B viral (HBV) polymerase in the human liver cell HepG2 and this association could be applied even in Escherichia coli. We investigated the role of GRP94 in the expression and stabilization of HBV polymerase in Escherichia coli by coexpression of the two proteins. The affinity column-purified glutathione S-transferase-tagged HBV polymerase (GST-P, 130 kDa) showed a proper molecular size and reverse transcriptase activity on several exogenous templates and was sensitive to specific inhibitors. The GST-P was associated with the maltose-binding protein-tagged GRP94 (MBP-GRP94, 130 kDa) using analyses by an affinity chromatography, native gel electrophoresis and glycerol gradient centrifugation. However, nondenaturing and partially denaturing activity gel analyses showed two active bands of approximately 260 kDa and approximately 130 kDa, respectively. Furthermore, in the presence of the encapsidation signal RNA template (HBV epsilon RNA), the approximately 260-kDa active band was gradually converted to approximately 130 kDa, which implies that HBV polymerase was dissociated from the chaperone GRP94 and bound preferentially to the HBV epsilon RNA. These results suggested that the chaperone GRP94 was necessary for the stabilization and production of HBV polymerase as an active form.
Cell Line↗
Specific detection of avian pneumovirus (APV) US isolates by RT-PCR.
This report details the development of an RT-PCR assay for the specific detection of US isolates of avian pneumovirus (APV). Of the several primer pairs tested, two sets of primers derived from the matrix gene of APV were able to specifically detect the viral RNA of APV. The nucleotide sequence comparison of the PCR products of APV isolates from Minnesota suggested that these viruses were closely related to the Colorado strain of APV, but were distinct from subtypes A and B European isolates of turkey APV (turkey rhinotracheitis: TRT). This M gene-based PCR was found to be very specific and sensitive. APV as low as 8 x 10(-5) TCID50 (0.0323 microg/ml) could be detected using this assay. In addition, the two primers were able to differentiate isolates from turkeys in Minnesota.
Animals↗
Brain tumor imaging with 99mTc-tetrofosmin: comparison with 201Tl, 99mTc-MIBI, and 18F-fluorodeoxyglucose.
The purpose of the present study was to assess the ability of technetium-99m-tetrofosmin (99mTc-TF) to predict tumor malignancy and to compare its uptake with that of thallium-201 (201Tl), technetium-99m-hexakis-2-methoxyisobutyl isonitrile (99mTc-MIBI) and fluorine-18-fluorodeoxyglucose (18F-FDG) in brain tumors. 99mTc-TF single-photon emission computed tomography (SPECT) imaging was performed in 22 patients with brain tumors and 3 healthy controls. Some of the patients underwent 201Tl (n = 12) and 99mTc-MIBI SPECT (n = 14) and 18F-FDG positron emission tomography (PET) (n = 12). The radioactivity ratio of tumor to contralateral normal tissue (T/N) and the ratio of tumor to contralateral white matter (T/WM) were calculated in SPECT and PET images, respectively. In healthy controls, 99mTc-TF uptake was seen only in scalp, in the choroid plexus and pituitary gland, but not in normal cerebral parenchyma. TF T/N in low grade gliomas (2.8+/-0.4) was significantly lower than that in high grade gliomas (22.5+/-29.8) and malignant non-gliomas (8.3+/-2.8) without overlap of values (p = 0.003 and p = 0.014, respectively). TF T/N was significantly correlated with MIBI T/N (p = 0.92, p = 0.001), Tl T/N (p = 0.72, p = 0.017), and FDG T/WM (p = 0.65. p = 0.031). There was an excellent agreement between TF T/N and MIBI T/N values on linear regression analysis (MIBI T/N = -0.63+/-0.97 x TF T/N). These preliminary results indicate that SPECT imaging with 99mTc-TF may be useful for the non-invasive grading of brain tumors. They also suggest that 99mTc-TF and 99mTc-MIBI may accumulate in brain tumors by a similar mechanism or in relation to a similar process of tumor cell proliferation.
Adolescent↗
Characterization and gene cloning of monoclonal antibody specific for the hepatitis B virus X protein.
The hepatis B virus X protein (HBx) has been thought to be implicated in the development of hepatocellular carcinoma. Although many functions of HBx have been reported, it is not clear which of HBx functions is important in hepatocellular carcinogenesis. To study HBx function, we produced a monoclonal anti-HBx Ab secreted by hybridoma cell clone H7 and mapped its epitope to a region of HBx between amino acids 29 and 48 by Western blot with truncated forms of HBx and by enzyme-linked immunoadsorbent assay (ELISA) with synthetic HBx peptides. The variable regions of H7 anti-HBx Ab were cloned by polymerase chain reaction using the degenerate-primers and by the 5' rapid amplification-cDNA end method. The sequence analyses revealed that the variable gene segments of the heavy and light chains are the members of mouse heavy chain variable gene 1 family and kappa light chain variable gene 2 family, respectively. In addition, J(H)2 or Jkappa4 gene segment at the end of the heavy-chain or light-chain variable region and DSP2.x gene segment in the CDR 3 of heavy chain were identified.
Amino Acid Sequence↗
Apoptosis of primary-culture rat microglial cells induced by pathogenic Acanthamoeba spp.
To determine whether trophozoites and lysates of pathogenic Acanthamoeba spp. induce apoptosis in primary-culture microglial cells, transmission electron microscopic (TEM) examinations, assessment of DNA fragmentation by agarose gel electrophoresis, and the TdT-mediated dUTP nick-end labeling assay were performed. When a trophozoite of pathogenic Acanthamoeba culbertsoni came in contact with a microglial cell, the digipodium was observed by TEM. Nuclear chromatin condensation was observed in 10% of microglial cells, while it was not revealed when they were cocultured with weakly pathogenic Acanthamoeba royreba trophozoites. DNA fragmentation in microglial cells cocultured with the A. culbertsoni lysate was detected by electrophoresis, showing DNA ladder formation, whereas it was hardly observed in microglial cells cocultured with A. royreba. DNA fragmentation of microglial cells was also confirmed by flow cytometry analysis. The fluorescence of TdT-stained apoptotic bodies became intensely visible with microglial cells cocultured with the A. culbertsoni lysate. In contrast, with microglial cells cocultured with the A. royreba lysate, only a background level of fluorescence of TdT-stained apoptotic bodies was detected. These results suggest that some rat microglial cells cocultured with pathogenic A. culbertsoni undergo cytopathic changes which show the characteristics of the apoptotic process, such as nuclear condensation and DNA fragmentation.
Acanthamoeba↗
Avian pneumovirus (APV) RNA from wild and sentinel birds in the United States has genetic homology with RNA from APV isolates from domestic turkeys.
Nasal turbinates or swabs were collected from wild ducks, geese, owls, sparrows, swallows, and starlings and from sentinel ducks placed next to turkey farms experiencing avian pneumovirus (APV) infections and were analyzed for APV genome and infectious particles. APV RNA was detected in samples examined from geese, sparrows, and starlings. APV RNA and antibodies were also detected in two different groups of sentinel ducks. Infectious APV was recovered from sentinel duck samples. The APV M gene isolated from the wild birds had over 96% predicted amino acid identity with APV/Minnesota 2A, which was isolated earlier from domestic turkeys showing respiratory illness, suggesting that wild birds may be involved in spreading APV infection.
Amino Acid Sequence↗
MR angiography of moyamoya disease before and after encephaloduroarteriosynangiosis.
OBJECTIVE: Our purpose was to evaluate the usefulness of MR angiography in revealing moyamoya disease before and after encephaloduroarteriosynangiosis. SUBJECTS AND METHODS: Twenty-six patients (51 hemispheres) with angiographically confirmed moyamoya disease who underwent encephaloduroarteriosynangiosis were included in the study. Findings on preoperative MR angiography were compared with those on conventional angiography. Postoperative neurologic status was categorized as poor, fair, good, or excellent. Postoperative MR angiography was examined for the appearance of the superficial temporal artery, changes in moyamoya vessels, and transdural collateral vessels into the middle cerebral artery territory. RESULTS: Preoperative MR angiography revealed moyamoya disease in all patients (diagnostic accuracy, 100%). MR angiography correctly depicted the degree of internal carotid artery stenosis in 37 arteries (73%), moyamoya vessels in 33 hemispheres (65%), and the degree of stenosis in the middle, anterior, and posterior cerebral arteries in 125 (82%) of 153 arteries. After surgery, 39 hemispheres showed an excellent outcome, eight showed a good outcome, two a fair outcome, and two a poor outcome. On postoperative MR angiography, vascular supply to the middle cerebral artery territory via transdural collateral vessels increased in 28 hemispheres (55%) and decreased in four (8%). The size of the superficial temporal artery increased in 41 (80%) of 51 hemispheres. The extent of moyamoya vessels decreased in 27 hemispheres (53%) after surgery. CONCLUSION: MR angiography can show the changes in the superficial temporal artery and development of transdural collateral vessels after encephaloduroarteriosynangiosis. Because MR angiography is noninvasive, it is valuable for evaluating postoperative changes.
Adolescent↗
Intracranial oculomotor nerve rhabdomyoma.
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Adolescent↗
Surgical treatment of intractable epilepsy accompanying cortical dysplasia.
OBJECT: Surgical treatment of cortical dysplasia (CD) together with intractable seizures is challenging because both visualization and localization of the lesion are difficult, correlation with seizure foci requires comprehensive study, and the surgical outcomes reported thus far are unsatisfactory. The authors report their experience in the surgical treatment of CD classified according to a surgical point of view. METHODS: The definition of CD used in this study was a dysplastic lesion visible on magnetic resonance (MR) images or a lesion that, although not visible on MR images, was diagnosed as moderate-to-severe dysplasia by using pathological analysis. During the last 4.5 years, the authors treated 36 patients with intractable epilepsy accompanied by CD. They divided the 36 cases of CD into four characteristic groups: Group A, diffuse bilateral hemispheric dysplasia; Group B, diffuse lobar dysplasia; Group C, focal dysplasia; and Group D, a moderate to severe degree of CD with a normal appearance on MR images. All but one patient in Group C were monitored in the epilepsy monitoring unit by using subdural electrodes for seizure localization and functional mapping. The incidence of CD among a cohort of 291 patients who had undergone epilepsy surgery at the authors' center during the study period was 12.4%. The mean age of the 36 patients was 21.3 years and the mean age at seizure onset was 8.5 years. The mean follow-up period was 26 months. Twenty-six patients (72.2%) belonged to Engel Class I or II (20 and six, respectively). There were five cases in Group A, nine in Group B, nine in Group C, and 13 in Group D. Patients in Groups A and B were significantly younger at seizure onset and had significantly poorer surgical outcomes compared with patients in Groups C and D (p < 0.05). If outcome is compared on the basis of the extent of removal of CD, patients in whom CD was completely removed had significantly better outcomes than those in whom CD was only partially removed (p < 0.001). CONCLUSIONS: The authors conclude that intractable epilepsy accompanied by CD can be treated surgically using comprehensive preoperative approaches. Deliberate resective procedures aimed at complete removal of dysplastic tissue ensure excellent seizure control without permanent neurological deficit.
Adolescent↗
In vitro cytotoxicity of Acanthamoeba spp. isolated from contact lens containers in Korea by crystal violet staining and LDH release assay.
In order to observe the cytotoxicity of Acanthamoeba spp., which were isolated from contact lens containers as ethiological agents for the probable amoebic keratitis in Korea, the crystal violet staining method and LDH release assay were carried out. In the crystal violet staining method, among eight contact lens container isolates, isolate 3 (Acanthamoeba KA/LS5) showed 83.6% and 81.8% of cytotoxicity, and isolate 7 (Acanthamoeba KA/LS37) showed 28.2% and 25.1% of cytotoxicity, in 1 mg/ml and 0.5 mg/ml lysate treatments, respectively. Acanthamoeba culbertsoni and A. healyi showed 84.0% and 82.8% of cytotoxicity. Similar results were observed in A. castellanii and A. hatchetti which showed 83.6% and 75.5% of cytotoxicity. Acanthamoeba royreba and A. polyphaga showed 9.0% and 1.7% of cytotoxicity. In the LDH release assay, isolate 3 (20.4%) showed higher cytotoxicity than other isolates in 1 mg/ml lysate treatment. The results provide that at least isolate 3 has the cytotoxic effect against CHO cells and seems to be the pathogenic strain.
Acanthamoeba↗
Utility of punch biopsy for lesions that are hard to aspirate by conventional fine-needle aspiration.
BACKGROUND: Fine-needle aspiration (FNA) is a fast, reliable, and cost-efficient technique for diagnosing palpable masses. However, when the lesion is small, dermal in location, shallow in depth, or fibrotic, the cellular yield by FNA may be limited and thus hinder an accurate diagnosis. The authors examined the value of punch biopsy (PB) in diagnosing such hard-to-aspirate lesions. METHODS: The authors reviewed 49 PB specimens from 47 patients who presented in their FNA clinic from June 1994 to July 1997. RESULTS: The lesions were typically described as ill-defined erythematous skin lesions or as papules or small, firm, subcutaneous nodules (average size, 0.7 cm). Patients' previous history included breast carcinoma (in 36 cases), nonmammary carcinoma (in 3 cases), melanoma (in 2 cases), squamous carcinoma of the skin (in 2 cases), cutaneous T-cell lymphoma (in 2 cases), small lymphocytic lymphoma (in 1 case), and no history of malignancy (in 1 case). PB sites included chest wall, breast, extremities, abdominal wall, neck, back, scalp, and forehead. Of the 37 cases in which FNA was performed before PB, 21 aspirates (57%) were nondiagnostic because of scant cellularity, 11 aspirates (31%) were positive (9) or suspicious/atypical (2) for malignancy, and 5 aspirates (14%) were negative for malignancy. Seventeen (81%) of the 21 nondiagnostic aspirates and 10 of the 11 suspicious/atypical aspirates were positive for malignancy on PB specimens. Twelve PBs were done without prior FNA, 8 (67%) were positive for malignancy, and 4 (33%) were negative. In 7 patients, the findings from the PB specimens (new diagnosis of malignancy in 5 cases and recurrence of disease in 2 cases) led to surgical excision of the lesion. CONCLUSIONS: PB is a valuable adjunct to FNA for diagnosing hard-to-aspirate lesions.
Adult↗