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2.  Characterization of ASKP1240, a Fully Human Antibody Targeting Human CD40 With Potent Immunosuppressive Effects 
American Journal of Transplantation  2014;14(6):1290-1299.
Blocking the CD40–CD154 interaction is reported to be effective for transplantation management and autoimmune disease models in rodents and nonhuman primates. However, clinical trials with anti-CD154 mAbs were halted because of high incidence of thromboembolic complications. Thus, we generated and characterized a fully human anti-CD40 mAb ASKP1240, as an alternative to anti-CD154 mAb. In vitro ASKP1240 concentration-dependently inhibited human peripheral blood mononuclear cell proliferation induced by soluble CD154. In addition, ASKP1240 did not destabilize platelet thrombi under physiological high shear conditions while mouse anti-human CD154 mAb (mu5C8) did. And ASKP1240 itself did not activate platelet and endothelial cells. In vivo administration of ASKP1240 (1 or 10 mg/kg, intravenously) to cynomolgus monkeys, weekly for 3 weeks, significantly attenuated both delayed-type hypersensitivity and specific antibody formation evoked by tetanus toxoid. The immunosuppressive effect was well correlated with the CD40 receptor saturation. Thus, these results suggest that ASKP1240 is immunosuppressive but not prothromboembolic, and as such appears to be a promising therapeutic candidate for the management of solid organ transplant rejection and autoimmune diseases therapy.
PMCID: PMC4225473  PMID: 24731050
CD40; costimulation; immunosuppressive therapy; mAbs
3.  A novel approach to biomarker discovery in head and neck cancer using an autoantibody signature 
Oncogene  2012;32(42):5026-5037.
Despite the dismal prognosis for patients with squamous cell carcinoma of the head and neck (SCCHN), there have been no novel treatments in over 40 years. Identification of novel tumor antigens in SCCHN will facilitate the identification of potential novel treatment targets. Tumor antigens are proteins selectively expressed by tumor cells and recognized by the host immune system. Phage-displayed tumor antigens were enriched by biopanning with normal and then SCCHN-specific serum. Ninety-six phage clones were sequenced for identification, and 21 clones were validated using Luminex. One of these proteins, L23, a novel tumor antigen in SCCHN, was validated as an oncogene. L23 is upregulated in SCCHN compared with normal keratinocytes. Knockdown of L23 inhibited proliferation, invasion and cell survival. Overexpression of L23 had the reverse effect. Overexpression of L23 in non malignant cells led to transformation. Injection of SCCHN cells with knockdown of L23 in mice, induced tumors that were significantly smaller than control tumors. In conclusion, the immunomic screen yielded a panel of antigens specific to SCCHN; one of these proteins, L23, is a novel oncogene in SCCHN.
PMCID: PMC4083474  PMID: 23160375
L23; proliferation; tumor antigen
4.  Arginine vasopressin neuronal loss results from autophagy-associated cell death in a mouse model for familial neurohypophysial diabetes insipidus 
Cell Death & Disease  2014;5(3):e1148-.
Familial neurohypophysial diabetes insipidus (FNDI) characterized by progressive polyuria is mostly caused by mutations in the gene encoding neurophysin II (NPII), which is the carrier protein of the antidiuretic hormone, arginine vasopressin (AVP). Although accumulation of mutant NPII in the endoplasmic reticulum (ER) could be toxic for AVP neurons, the precise mechanisms of cell death of AVP neurons, reported in autopsy studies, remain unclear. Here, we subjected FNDI model mice to intermittent water deprivation (WD) in order to promote the phenotypes. Electron microscopic analyses demonstrated that, while aggregates are confined to a certain compartment of the ER in the AVP neurons of FNDI mice with water access ad libitum, they were scattered throughout the dilated ER lumen in the FNDI mice subjected to WD for 4 weeks. It is also demonstrated that phagophores, the autophagosome precursors, emerged in the vicinity of aggregates and engulfed the ER containing scattered aggregates. Immunohistochemical analyses revealed that expression of p62, an adapter protein between ubiquitin and autophagosome, was elicited on autophagosomal membranes in the AVP neurons, suggesting selective autophagy induction at this time point. Treatment of hypothalamic explants of green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) transgenic mice with an ER stressor thapsigargin increased the number of GFP-LC3 puncta, suggesting that ER stress could induce autophagosome formation in the hypothalamus of wild-type mice as well. The cytoplasm of AVP neurons in FNDI mice was occupied with vacuoles in the mice subjected to WD for 12 weeks, when 30–40% of AVP neurons are lost. Our data thus demonstrated that autophagy was induced in the AVP neurons subjected to ER stress in FNDI mice. Although autophagy should primarily be protective for neurons, it is suggested that the organelles including ER were lost over time through autophagy, leading to autophagy-associated cell death of AVP neurons.
PMCID: PMC3973212  PMID: 24675466
autophagy; autophagy-associated cell death; ER stress; ER-associated compartment; familial neurohypophysial diabetes insipidus
5.  Phase I first-in-human study of TAK-285, a novel investigational dual HER2/EGFR inhibitor, in cancer patients 
British Journal of Cancer  2012;106(4):666-672.
This phase I first-in-human study was conducted in Japanese patients to investigate the safety, pharmacokinetics (PKs), and determine the maximum tolerated dose (MTD) of oral TAK-285, a novel dual erbB protein kinase inhibitor that specifically targets human epidermal growth factor receptor (EGFR) and HER2.
The TAK-285 dose was escalated until MTD was determined. A second patient cohort received TAK-285 at the MTD for at least 4 weeks.
In all, 26 patients received TAK-285 at doses ranging from 50 to 400 mg once daily (q.d.) or twice daily (b.i.d.); 20 patients made up the dose escalation cohort and the remaining 6 patients were the repeated administration cohort. TAK-285 was well tolerated. Dose-limiting toxicities noted in two patients who received 400 mg b.i.d. were grade 3 increases in aminotransferases and grade 3 decreased appetite. Consequently, the MTD was determined to be 300 mg b.i.d. Absorption of TAK-285 was rapid after oral dosing, and plasma exposure at steady-state increased in a dose-proportional fashion for doses ranging from 50 to 300 mg b.i.d. A partial response was observed for one patient with parotid cancer who received 300 mg b.i.d.
The toxicity profile and PK properties of oral TAK-285 warrant further evaluation.
PMCID: PMC3322948  PMID: 22240796
first-in-human; phase I TAK-285; epidermal growth factor receptor; dual erbB protein kinase inhibitor family; receptor tyrosine kinase inhibitor
6.  Post-traumatic rapidly enlarging mucinous carcinoma of the breast with intratumoural haemorrhage: MRI appearances with pathological correlation 
The British Journal of Radiology  2011;84(1002):e118-e120.
Pure mucinous carcinoma of the breast is a histological type of invasive carcinoma and generally shows a slow growth pattern. Rapid growth and intratumoural haemorrhage are rare and there have been no reports presenting such a clinical course and associated radiographic findings. We report a case with atypical rapidly enlarging mucinous carcinoma of the breast after trauma, in which MRI closely reflected the histopathological background and was thought to be useful for differential diagnosis from other highly malignant breast tumours.
PMCID: PMC3473633  PMID: 21606064
7.  Chronic Atherosclerotic Mesenteric Ischemia That Started to Develop Symptoms Just after Anaphylaxis 
Case Reports in Gastroenterology  2012;6(2):300-308.
An 83-year-old woman was referred to our emergency department with acute urticaria and sudden shortness of breath approximately 30 min after taking rectal diclofenac potassium for lumbago. After treatment with adrenaline and corticosteroids, the patient became hemodynamically stable and left the hospital on the next day. She attended our hospital 1 week after the onset of anaphylaxis because of repeated postprandial epigastric pain. No abnormal lesions were found in endoscopy. Radiographic selective catheter angiography revealed chronic mesenteric ischemia caused by atherosclerosis and abundant collateral arteries between the celiac trunk, the superior mesenteric artery and the inferior mesenteric artery. Patients with chronic mesenteric ischemia usually present with a clinical syndrome characterized by painful abdominal cramps and colic occurring typically during the postprandial phase. Fear of eating resulted in malnutrition. She was prescribed proton pump inhibitor, digestants, anticholinergic agents, serine protease inhibitors, prokinetics, antiplatelet agents and transdermal nitroglycerin intermittently, but these had no beneficial effects. It was most probable that this patient with chronic atherosclerotic mesenteric ischemia was suffering from functional abdominal pain syndrome induced by anaphylaxis. Since psychiatric disorders were associated with alterations in the processing of visceral sensation, we facilitated the patient's understanding of functional abdominal pain syndrome with the psychologist. Postprandial abdominal pain gradually faded after administration of these drugs and the patient left the hospital. Developing a satisfactory patient-physician relationship was considered more effective for the management of persistent abdominal pain caused by complicated mechanisms.
PMCID: PMC3376342  PMID: 22754490
Atherosclerosis; Mesenteric ischemia; Anaphylaxis; Functional abdominal pain syndrome; Patient-physician relationship
8.  Imaging features contributing to the diagnosis of ameloblastomas and keratocystic odontogenic tumours: logistic regression analysis 
Dentomaxillofacial Radiology  2011;40(3):133-140.
The aim of this study was to clarify the characteristic imaging features that can be used to differentiate ameloblastomas from keratocystic odontogenic tumours and to examine the significant imaging features contributing to a correct diagnosis.
60 observers (39 specialists in oral and maxillofacial radiology and 21 non-specialists) examined CT and/or panoramic images of 10 ameloblastomas and 10 keratocystic odontogenic tumours shown on a webpage and made diagnoses. Their correct answer ratios were then calculated. The imaging features of the tumours were evaluated and expressed as binary numbers or quantitative values. The imaging features that contributed to a correct diagnosis were elucidated using logistic regression analysis.
The mean correct answer ratio was 61.3% ± 17.2% for the diagnosis of ameloblastomas and keratocystic odontogenic tumours. CT images produced higher correct answer ratios for diagnosis of keratocystic odontogenic tumours by specialists. The significantly different imaging features between ameloblastomas and keratocystic odontogenic tumours were the degree of bone expansion and the presence of high-density areas. The significant imaging features contributing to a correct imaging diagnosis were the number of locules, the presence of high-density areas and the inclusion of impacted teeth.
The presence of high-density areas is the most useful feature in the differential diagnosis of ameloblastomas and keratocystic odontogenic tumours based on comparison of the imaging features of both tumours and examination of the diagnostic contributions of these features.
PMCID: PMC3611454  PMID: 21346078
ameloblastomas; keratocystic odontogenic tumours; logistic regression analysis; computed tomography
9.  Speriolin is a novel human and mouse sperm centrosome protein 
Human Reproduction (Oxford, England)  2010;25(8):1884-1894.
Oocytes in humans, mice and other mammals lack identifiable centrioles. The proximal centriole brought in by the fertilizing sperm in humans and most other mammals appears to gives rise to the centrioles at the spindle poles in the zygote, and is believed to indicate that centrioles are inherited through the paternal lineage. However, both the proximal and distal sperm centrioles degenerate in mice and other rodents. A bipolar mitotic spindle nucleates from multiple centrosome-like structures in the mouse zygote and centrioles are not seen until the blastocyst stage, suggesting that centrioles are inherited through the maternal lineage in mice. We previously identified speriolin as a spermatogenic cell-specific binding partner of Cdc20 that co-localizes with pericentrin in mouse spermatocytes and is present in the centrosome in round spermatids.
The nature and localization of speriolin in mouse and human sperm and the fate of speriolin following fertilization in the mouse were determined using immunofluorescence microscopy, immunoelectron microscopy and western blotting.
Speriolin surrounds the intact proximal centriole in human sperm, but is localized at the periphery of the disordered distal centriole in mouse sperm. Human speriolin contains an internal 163-amino acid region not present in mouse that may contribute to localization differences. Speriolin is carried into the mouse oocyte during fertilization and remains associated with the decondensing sperm head in zygotes. The speriolin spot appears to undergo duplication or splitting during the first interphase and is detectable in 2-cell embryos.
Speriolin is a novel centrosomal protein present in the connecting piece region of mouse and human sperm that is transmitted to the mouse zygote and can be detected throughout the first mitotic division.
PMCID: PMC2907228  PMID: 20542897
centriole; flagellum; fertilization; paternal inheritance; zygote
10.  Promoter CpG methylation in cancer cells contributes to the regulation of MUC4 
British Journal of Cancer  2009;100(2):344-351.
Mucin 4 (MUC4) is a high molecular weight transmembrane mucin that is overexpressed in many carcinomas and is a risk factor associated with a poor prognosis. In this study, we show that the DNA methylation pattern is intimately correlated with MUC4 expression in breast, lung, pancreas and colon cancer cell lines. We mapped the DNA methylation status of 94 CpG sites from −3622 to +29 using MassARRAY analysis that utilises base-specific cleavage of nucleic acids. MUC4-negative cancer cell lines and those with low MUC4 expression (eg, A427) were highly methylated near the transcriptional start site, whereas MUC4-positive cell lines (eg, NCI-H292) had low methylation levels. Moreover, 5-aza-2′-deoxycytidine and trichostatin A treatment of MUC4-negative cells or those with low MUC4 expression caused elevation of MUC4 mRNA. Our results suggest that DNA methylation in the 5′ flanking region play an important role in MUC4 gene expression in carcinomas of various organs. An understanding of epigenetic changes in MUC4 may contribute to the diagnosis of carcinogenic risk and prediction of outcome in patients with cancer.
PMCID: PMC2634723  PMID: 19127263
MUC4; mucin; epigenetics; DNA methylation; massARRAY
11.  MUC4 expression is a novel prognostic factor in patients with invasive ductal carcinoma of the pancreas 
Journal of Clinical Pathology  2005;58(8):845-852.
Background: Many patients with invasive ductal carcinoma of the pancreas (IDC) have a poor outcome. MUC4 expression has been implicated as a marker for diagnosis and progression of IDC, but there are no studies of the relation between MUC4 expression and patient prognosis in IDC.
Aims: To investigate the prognostic significance of MUC4 expression in IDC.
Methods: The expression profiles of MUC4, ErbB2, p27, and MUC1 were investigated in IDC tissues from 135 patients by means of immunohistochemistry.
Results: MUC4 was expressed in 43 of the 135 patients with IDC (31.9%). The survival of 21 patients with high MUC4 expression (>20% of neoplastic cells stained) was significantly worse than that of the 114 patients with low MUC4 expression (<20% of neoplastic cells stained) (p  =  0.0043). Univariate analysis showed that high MUC4 expression (p  =  0.0061), large primary tumour status (>T2) (p  =  0.0436), distant metastasis (p  =  0.0383), lymphatic invasion (p  =  0.0243), and surgical margins (p  =  0.0333) were significant risk factors affecting the outcome of patients with IDC. Backward stepwise multivariate analysis showed that MUC4 expression (p  =  0.0121), lymph node metastasis (p  =  0.0245), and lymphatic invasion (p  =  0.0239) were significant independent risk factors. ErbB2, p27, and MUC1 were not independent risk factors.
Conclusions: This study shows that MUC4 expression in IDC is a new independent factor for poor prognosis and predicts the outcome of patients with IDC.
PMCID: PMC1770880  PMID: 16049287
pancreatic cancer; mucin; immunohistochemistry; cumulative survival rate; multivariate analysis
12.  Mucin expression in pleomorphic adenoma of salivary gland: a potential role for MUC1 as a marker to predict recurrence 
Journal of Clinical Pathology  2004;57(8):813-821.
Background: Pleomorphic adenoma of the salivary gland (PA) is essentially a benign neoplasm. However, patients with recurrent PA are difficult to manage. There are rare reports on useful immunohistochemical markers to detect a high risk of recurrence when the primary lesions are resected.
Aims: To find a new marker to predict the recurrence of PA.
Methods: Primary lesions of PA were collected from nine patients showing subsequent recurrence and from 40 patients without recurrence during at least 10 years of follow up of the disease. Paraffin wax embedded tumour samples of the two groups were examined for the expression profiles of MUC1 (differentially glycosylated forms), MUC2, MUC4, MUC5AC, and MUC6 using immunohistochemistry. Several clinicopathological factors were also examined.
Results: In univariate analysis of the factors examined, MUC1/DF3 high expression (more than 30% of the neoplastic cells stained) in the primary lesions was seen more frequently in patients with recurrence (four of nine) than in those without recurrence (three of 40; p  =  0.011). Larger tumour size (more than 3.0 cm) of the primary PA was also a significant (p  =  0.035) risk factor for the recurrence of PA. In multivariate analysis, only high expression of MUC1/DF3 was found to be a significant independent risk factor for the recurrence of PA (p  =  0.021).
Conclusions: Expression of MUC1/DF3 in PA is a useful marker to predict its recurrence. Those patients with PA showing positive MUC1/DF3 expression should be followed up carefully.
PMCID: PMC1770389  PMID: 15280401
salivary gland neoplasms; local recurrence; mucins; MUC1 mucin; DF3 breast carcinoma associated antigen; immunohistochemistry
15.  Some problems found in HIV-1 RNA quantification 
Journal of Clinical Pathology  2000;53(8):645-646.
PMCID: PMC1762923  PMID: 11002778
16.  Insulin-like growth factor-1 content and pattern of expression correlates with histopathologic grade in diffusely infiltrating astrocytomas. 
Neuro-Oncology  1999;1(2):109-119.
Studies of experimental tumorigenesis have strongly implicated signaling of the insulin-like growth factor 1 (IGF-1) as a key component in astrocytic neoplasia; however, its role in the growth of low-grade and malignant human tumors is not well understood. Correlative analyses of IGF-1, p53, and Ki-67 (MIB-1) immunohistochemistry and IGF-1 receptor (IGF-1R) mRNA expression were performed to examine the cellular pattern of IGF-1 signaling in 39 cases of astrocytoma (World Health Organization grades II-IV). Tumor cells expressing IGF-1 and IGF-1R were present in all tumor grades. The proportion of tumor cells that expressed IGF-1 correlated with both histopathologic grade and Ki-67 labeling indices, while expression of IGF-1R mRNA correlated with Ki-67 indices. In cases where stereotactic tissue sampling could be identified with a specific tumor area by neuroimaging features, the numbers of IGF-1 immunoreactive cells correlated with the tumor zones of highest cellularity and Ki-67 labeling. In glioblastomas, the localization of IGF-1 immunoreactivity was notable for several features: frequent accentuation in the perivascular tumor cells surrounding microvascular hyperplasia; increased levels in reactive astrocytes at the margins of tumor infiltration; and selective expression in microvascular cells exhibiting endothelial/pericytic hyperplasia. IGF-1R expression was particularly prominent in tumor cells adjacent to both microvascular hyperplasia and palisading necrosis. These data suggest that IGF-1 signaling occurs early in astroglial tumorigenesis in the setting of cell proliferation. The distinctive correlative patterns of IGF-1 and IGF-1R expression in glioblastomas also suggest that IGF-1 signaling has an association with the development of malignant phenotypes related to aberrant angiogenesis and invasive tumor interactions with reactive brain.
PMCID: PMC1920755  PMID: 11550306
17.  Werner syndrome helicase contains a 5'-->3' exonuclease activity that digests DNA and RNA strands in DNA/DNA and RNA/DNA duplexes dependent on unwinding. 
Nucleic Acids Research  1999;27(11):2361-2368.
We show that WRN helicase contains a unique 5'-->3' exonuclease activity in the N-terminal region. Adeletion mutant lacking 231 N-terminal amino acid residues, made in a baculovirus system, did nothave this activity, while it showed ATPase and DNA helicase activities. This exonuclease activity was co-precipitated with the helicase activity using monoclonal antibodies specific to WRN helicase, indicating that it is an integral component with WRN helicase. The exonuclease in WRN helicase does not digest free single-stranded DNA or RNA, but it digests a strand in the duplex DNA or an RNA strand in a RNA/DNA heteroduplex in a 5'-->3' direction dependent on duplex unwinding. The digestion products were identified as 5'-mononucleotides. Our data show that WRN helicase needs a single-stranded 3' overhang region for efficient binding and unwinding of duplex molecules, while blunt-ended or 5' overhang duplex molecules were hardly unwound. These findings suggest that the WRN helicase and integral 5'-->3' exonuclease activities are involved in preventing a hyper-recombination by resolving entangled structures of DNA and RNA/DNA heteroduplexes that may be generated during rep-lication, repair and/or transcription.
PMCID: PMC148803  PMID: 10325426
18.  Hydrocephalus and Hirschsprung's disease in a patient with a mutation of L1CAM. 
Journal of Medical Genetics  1997;34(8):670-671.
Abnormalities of the L1CAM gene, a member of the immunoglobulin gene superfamily of neural cell adhesion molecules, are associated with X linked hydrocephalus and some allelic disorders. We describe a patient with X linked hydrocephalus and Hirschsprung's disease (HSCR) with a novel mutation in the L1CAM gene. This is the first report of HSCR with a mutant neural cell adhesion molecule. Although the disease phenotypes of this patient may well be independent, the alternative explanation that L1CAM mutations may contribute to both phenotypes cannot be excluded in view of an earlier report on another patient with both X linked hydrocephalus and HSCR.
PMCID: PMC1051030  PMID: 9279760
19.  DNA helicase activity in Werner's syndrome gene product synthesized in a baculovirus system. 
Nucleic Acids Research  1997;25(15):2973-2978.
The gene responsible for Werner's syndrome (WRN) contains a region homologous to the Escherichia coli RecQ type DNA helicase and was thought to code for a DNA helicase belonging to this helicase family. However, no evidence has been shown before to substantiate this prediction. Here, we show data that the product of the WRN gene is indeed a DNA helicase. The gene product, a polypeptide with a relative molecular mass of 170 kDa, expressed in the insect Spodoptera frugiperda (Sf21) cell and purified by affinity column chromatography contained both the ATPase and DNA unwinding activities characteristic of DNA helicase. Expressions in Sf21, as well as in HeLa cells, showed that the WRN DNA helicase is exclusively transported to the nucleoplasm, which is consistent with its function in DNA metabolism. Our studies on strand displacement suggest that WRN helicase can unwind not only a duplex DNA, but also an RNA-DNA heteroduplex, while the latter reaction seems less efficient. Enzymological features learned from the purified WRN helicase are discussed with respect to the biological function, which remains to be clarified.
PMCID: PMC146849  PMID: 9224595
20.  Overexpression of apolipoprotein AII in transgenic mice converts high density lipoproteins to proinflammatory particles. 
Journal of Clinical Investigation  1997;100(2):464-474.
Previous studies showed that transgenic mice overexpressing either apolipoprotein AI (apoAI) or apolipoprotein AII (apoAII), the major proteins of HDL, exhibited elevated levels of HDL cholesterol, but, whereas the apoAI-transgenic mice were protected against atherosclerosis, the apoAII-transgenic mice had increased lesion development. We now examine the basis for this striking functional heterogeneity. HDL from apoAI transgenics exhibited an enhanced ability to promote cholesterol efflux from macrophages, but HDL from apoAII transgenics and nontransgenics were not discernibly different in efflux studies. In contrast with HDL from nontransgenics and apoAI transgenics, HDL from the apoAII transgenics were unable to protect against LDL oxidation in a coculture model of the artery wall. Furthermore, HDL taken from apoAII-transgenic mice, but not HDL taken from either the apoAI transgenics or nontransgenic littermate controls, by itself stimulated lipid hydroperoxide formation in artery wall cells and induced monocyte transmigration, indicating that the apoAII-transgenic HDL were in fact proinflammatory. This loss in the ability of the apoAII-transgenic HDL to function as an antioxidant/antiinflammatory agent was associated with a decreased content of paraoxonase, an enzyme that protects against LDL oxidation. Reconstitution of the apoAII transgenic HDL with purified paraoxonase restored both paraoxonase activity and the ability to protect against LDL oxidation. We conclude that overexpression of apoAII converts HDL from an anti- to a proinflammatory particle and that paraoxonase plays a role in this transformation.
PMCID: PMC508211  PMID: 9218525
21.  Expression and functional analysis of a hyperglycosylated glucoamylase in a parental host, Aspergillus awamori var. kawachi. 
A modified glucoamylase gene (glaA) with an extra Thr- and Ser-rich Gp-I domain (T. Semimaru, M. Goto, K. Furukawa, and S. Hayashida, Appl. Environ. Microbiol. 61:2885-2890, 1995) was introduced into a mutant parental host, Aspergillus awamori var. kawachi, in which the original glaA gene had been completely deleted and replaced with the hygromycin phosphotransferase gene. The modified glaA was successfully expressed and secreted. The modified glucoamylase possessed higher digestibility of raw corn starch and higher stabilities in response to heat and extreme pH.
PMCID: PMC168589  PMID: 9212440
22.  Functional analyses of a variety of chimeric dioxygenases constructed from two biphenyl dioxygenases that are similar structurally but different functionally. 
Journal of Bacteriology  1997;179(12):3936-3943.
The biphenyl dioxygenases (BP Dox) of strains Pseudomonas pseudoalcaligenes KF707 and Pseudomonas cepacia LB400 exhibit a distinct difference in substrate ranges of polychlorinated biphenyls (PCB) despite nearly identical amino acid sequences. The range of congeners oxidized by LB400 BP Dox is much wider than that oxidized by KF707 BP Dox. The PCB degradation abilities of these BP Dox were highly dependent on the recognition of the chlorinated rings and the sites of oxygen activation. The KF707 BP Dox recognized primarily the 4'-chlorinated ring (97%) of 2,5,4'-trichlorobiphenyl and introduced molecular oxygen at the 2',3' position. The LB400 BP Dox recognized primarily the 2,5-dichlorinated ring (95%) of the same compound and introduced O2 at the 3,4 position. It was confirmed that the BphA1 subunit (iron-sulfur protein of terminal dioxygenase encoded by bphA1) plays a crucial role in determining the substrate selectivity. We constructed a variety of chimeric bphA1 genes by exchanging four common restriction fragments between the KF707 bphA1 and the LB400 bphA1. Observation of Escherichia coli cells expressing various chimeric BP Dox revealed that a relatively small number of amino acids in the carboxy-terminal half (among 20 different amino acids in total) are involved in the recognition of the chlorinated ring and the sites of dioxygenation and thereby are responsible for the degradation of PCB. The site-directed mutagenesis of Thr-376 (KF707) to Asn-376 (LB400) in KF707 BP Dox resulted in the expansion of the range of biodegradable PCB congeners.
PMCID: PMC179202  PMID: 9190809
23.  Functional domains of transcription factor hGABP beta1/E4TF1-53 required for nuclear localization and transcription activation. 
Nucleic Acids Research  1996;24(24):4954-4961.
Transcription factor E4TF1 is the human homolog of GABP and has been renamed hGABP (human GABP). hGABP is composed of two types of subunits; hGABP beta1/E4TF1-53 and the ets-related protein hGABP alpha/E4TF1-60. Both bind together to form an (alpha)2(beta1)2 heterotetrameric complex on DNA and activate transcription at specific promoters in vitro. Tetramer formation depends on two regions of hGABP beta1; the N-terminal region containing the Notch/ankyrin-type repeats is necessary for binding to hGABP alpha and the C-terminal region is necessary for homodimerization. In this report, we constructed various deletion mutants of hGABP beta1 in order to delimit the functional regions required for nuclear localization and transcription activity. We found that hGABP beta1 localization in the nucleus is dependent on a region located between amino acids 243 and 330 and that the presence of hGABP beta1 influences the efficiency of hGABP alpha transport into the nucleus. Next, we demonstrated that the hGABP complex composed of alpha and beta1 subunits activates transcription from the adenovirus early 4 promoter in vivo. This transcription activation needs the C-terminal region of hGABP beta1 and is consistent with results obtained with the in vitro assay. Furthermore, site-directed mutagenesis analysis of the C-terminal region reveals that the alpha-helix structure and the leucine residues are important for formation of a heterotetrameric complex with hGABP alpha in vitro and for transcription activation in vivo. These results suggest that hGABP beta1 stimulates transcription as part of a heterotetrameric complex with hGABP alpha in vivo.
PMCID: PMC146336  PMID: 9016666
24.  Functional analysis of the threonine- and serine-rich Gp-I domain of glucoamylase I from Aspergillus awamori var. kawachi. 
Glucoamylase I (GAI) from Aspergillus awamori var. kawachi hydrolyzes raw starch efficiently and is composed of three functional domains: the amino-terminal catalytic GAI' domain (A-1 to V-469), the threonine- and serine-rich O-glycosylated Gp-I domain (A-470 to V-514), and the carboxy-terminal raw starch-binding Cp domain (A-515 to R-615). In order to investigate the role of the Gp-I domain, an additional repeat of Gp-I and internal deletions of the entire Gp-I sequence or parts of the Gp-I sequence were introduced within Gp-I. All mutant genes as well as the wild-type gene were inserted into a yeast-secretion vector, YEUp3H alpha, and expressed in Saccharomyces cerevisiae. Wild-type GAI expressed in yeast cells (GAY), GAGpI, having an extra Gp-I, and GA delta 470-493, lacking the A-470-to-T-493 sequences of Gp-I, were successfully secreted into the culture medium. On the other hand, GA delta 470-507, lacking A-470 to S-507, and GA delta GpI, lacking the entire Gp-I (A-470-to-V-514) sequence, failed to be secreted and remained in the yeast cells. The carbohydrate content of GAGpI was 1.2 times higher than that of GAY and 2.4 times higher than that of the original GAI. The raw starch digestibility of GAGpI was almost the same as that of GAY but was 1.5 times faster than that of GAI.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC167565  PMID: 7487021
25.  Constitutive production of angiotensin converting enzyme from rheumatoid nodule cells under serum free conditions. 
Annals of the Rheumatic Diseases  1992;51(6):741-742.
Angiotensin converting enzyme was assayed in serum free culture supernatants from unstimulated rheumatoid nodule cells. Angiotensin converting enzyme was released spontaneously and the angiotensin converting enzyme derived from rheumatoid nodule cells was suppressed in a dose and time dependent manner by the protein synthesis inhibitor cycloheximide. These data suggest the constitutive de novo synthesis of angiotensin converting enzyme by rheumatoid nodule cells.
PMCID: PMC1004737  PMID: 1319698

Results 1-25 (45)