Site-directed mutagenesis of the amino acid residues in β-strand III [Val30-Val36] of d-amino acid aminotransferase of Bacillus sp. YM-1

AbstractThe β-strand III formed by amino acid residues Val30-Val36 is located across the active site of the thermostable d-amino acid aminotransferase (d-AAT) from thermophilic Bacillus sp. YM-1, and the odd-numbered amino acids (Tyr31, Val33, Lys35) in the strand are revealed to be directed toward the active site. Interestingly, Glu32 is also directed toward the active site. We first investigated the involvement of these amino acid residues in catalysis by alanine scanning mutagenesis. The Y31A and E32A mutant enzymes showed a marked decrease in kcat value, retaining less than 1% of the wild-type enzyme activity. The kcat values of V33A and K35A were changed slightly, but the Km of K35A for α-ketoglutarate was increased to 35.6 mM, compared to the Km value of 2.5 mM for the wild-type enzyme. These results suggested that the positive charge at Lys35 interacted electrostatically with the negative charge at the side chain of α-ketoglutarate. Site-directed mutagenesis of the Glu32 residue was conducted to demonstrate the role of this residue in detail. From the kinetic and spectral characteristics of the Glu32-substituted enzymes, the Glu32 residue seemed to interact with the positive charge at the Schiff base formed between the aldehyde group of pyridoxal 5′-phosphate (PLP) and the ε-amino group of the Lys145 residue

Molecular mechanisms of heptaplatin effective against cisplatin-resistant cancer cell lines: less involvement of metallothionein

BACKGROUND: Heptaplatin is a new platinum derivative with anticancer activity against various cancer cell lines, including cisplatin-resistant cancer cell lines (Cancer Chemother Pharmacol 1995; 35: 441). METHODS: Molecular mechanisms of heptaplatin effective against cisplatin-resistant cancer cell lines has been investigated in connection with metallothionein (MT). Cytotoxicity was determined by an MTT assay. MT mRNA, was determined by RT-PCR assay. Transfection study was carried out to examine the function of MT. RESULTS: Of various gastric cancer cell lines, SNU-638 and SNU-601 showed the highest and lowest levels of MT mRNA, respectively, showing 80-fold difference. The IC(50 )values of SNU-638 to cisplatin, carboplatin and heptaplatin were 11.2-fold, 5.1-fold and 2.0-fold greater than those of SNU-601, respectively. Heptaplatin was more effective against cisplatin-resistant and MT-transfected gastric cancer sublines than cisplatin or carboplatin was. In addition, heptaplatin attenuated cadmium, but not zinc, induction of MT. CONCLUSION: These results indicate that molecular mechanisms of heptaplatin effective against cisplatin-resistant gastric cancer sublines is at least in part due to the less involvement of MT in heptaplatin resistance as well as its attenuation of MT induction

Association of Oncogenic Human Papillomaviruses(HPV 16, 18) with Cervical Intraepithelial Neoplasia and Cervical Cancer

The prevalence of oncogenic human papillomavirus (HPV) type 16 and 18 was investigated by polymerase chain reaction (PCR) method in cervical scrapes omitting prior DNA extraction. Samples were obtained from 70 gynecologic inpatients with normal cervix and 160 women with cervical neoplastic lesion ( N = 50 in cervical intraepithelial neoplasia (CIN) I, N = 50 in CIN II, N = 30 in CIN III, N = 30 in invasive cervical cancer). Eight members were excluded from the data due to failure of ,f3-g10bin amplification during the PCR procedure. The HPV 16 prevalence rate was 19.1 % (13/68) in the normal group, 38.8 % (19/49) in CIN 1,57.1 % (28/49) in CIN 11,75.9 % (22/29) in CIN III, 88.9% (24/27) in invasive cancer. For HPV type 18, DNA positivity was 4.4 % (3/68), 8.2 % (4/49), 12.2 % (6/49), 13.8 % (4/29), 18.5 % (5/27), respectively. In the whole series a consistent correlation was found between HPV positivity and severity of cervical lesion. HPV 16 was the more prevalent type and about five times more common than HPV 18. These results suggest that HPV 16 and 18 may be strongly associated with carcinogenesis of cervical cancer. The high risk HPV typing by direct PCR from cervical scrapes can be used as a useful marker for the presence of neoplastic cells and also served as a simple tool in identifying women who are at risk of developing dysplasia and cervical cancer

Tide-related changes in mRNA abundance of aromatases and estrogen receptors in the ovary and brain of the threespot wrasse Halichoeres trimaculatus

Author Posting. © The Author(s), 2018. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in Ocean Science Journal 53 (2018): 239-249, doi:10.1007/s12601-018-0016-0.The threespot wrasse (Halichoeres trimaculatus; Family Labridae) is a common coral reef species of the Indo-Pacific Ocean. Given that this species spawns daily at high tide (HT), we hypothesized that endocrine changes in relation to gonadal development are synchronized with the tidal cycle. To test this, we examined the transcript abundance of two cytochrome P450 aromatases (cyp19a and cyp19b) and two estrogen receptors (er and er) in the ovary and brain of this species in response to tidal change. When fish were collected around four tidal points [low tide (LT), flood tide (FT), high tide (HT), and ebb tide (ET)], gonadosomatic index and oocyte diameter increased around HT and FT, respectively. Ovulatory follicles were observed in ovaries around HT. Real-time quantitative polymerase-chain reaction revealed that mRNA abundance of cyp19a and er, but not er, in the ovary increased around ET and HT, respectively. On the other hand, mRNA levels of cyp19b in the forebrain were significantly higher around FT. Increases of er and er mRNA abundance around FT were observed in all areas of the brain and the midbrain, respectively. The changes in mRNA abundance of key genes involved in reproduction at specific tidal cycles, along with the development of the vitellogenic oocytes in the ovary, support our hypothesis that synchronization of endocrine changes to the tidal periodicity plays a role in the gonadal development of this species. We hypothesize that conversion of testosterone to E2 in the brain may be associated with the spawning behavior given that the wrasse exhibits group spawning with a territory-holding male around HT.This study was supported partially by the 21st Century COE program “The Comprehensive Analyses on Biodiversity in Coral Reef and Island Ecosystems in Asian and Pacific Regions” from the Ministry of Education, Culture, Sports, Science and Technology, Japan, JSPS KAKENHI Grant number 16H05796, and the Natural Sciences and Engineering Research Council (NSERC, Canada) Discovery Grant program

UKIRT Widefield Infrared Survey for Fe+

The United Kingdom Infrared Telescope (UKIRT)Widefield Infrared Survey for Fe+ (UWIFE) is a 180 deg2 imaging survey of the first Galactic quadrant (7° < l < 62° |b| <1°.5) that uses a narrow-band filter centred on the [Fe II] 1.644-μm emission line. The [Fe II] 1.644-μm emission is a good tracer of dense, shock-excited gas, and the survey will probe violent environments around stars: star-forming regions, evolved stars, and supernova remnants, among others. The UWIFE survey is designed to complement the existing UKIRTW idefield Infrared Survey for H2 (UWISH2). The survey will also complement existing broad-band surveys. The observed images have a nominal 5Ï? detection limit of 18.7 mag for point sources, with a median seeing of 0.83 arcsec. For extended sources, we estimate a surface brightness limit of 8.1 Ã? 10-20 W m-2 arcsec-2. In this paper, we present an overview and some preliminary results of this survey. © 2014 The Authors Published by Oxford University Press on behalf of the Royal Astronomical Society

Evaluation of the Efficacy and Cross-Protectivity of Recent Human and Swine Vaccines against the Pandemic (H1N1) 2009 Virus Infection

The current pandemic (H1N1) 2009 virus remains transmissible among humans worldwide with cases of reverse zoonosis, providing opportunities to produce more pathogenic variants which could pose greater human health concerns. To investigate whether recent seasonal human or swine H1N1 vaccines could induce cross-reactive immune responses against infection with the pandemic (H1N1) 2009 virus, mice, ferrets or mini-pigs were administered with various regimens (once or twice) and antigen content (1.77, 3.5 or 7.5 µg HA) of a-Brsibane/59/07, a-CAN01/04 or RgCA/04/09xPR8 vaccine. Receipt of a-CAN01/04 (2-doses) but not a-Brisbane/59/07 induced detectable but modest (20–40 units) cross-reactive serum antibody against CA/04/09 by hemagglutinin inhibition (HI) assays in mice. Only double administration (7.5 µg HA) of both vaccine in ferrets could elicit cross-reactivity (30–60 HI titers). Similar antigen content of a-CAN01/04 in mini-pigs also caused a modest ∼30 HI titers (twice vaccinated). However, vaccine-induced antibody titers could not suppress active virus replication in the lungs (mice) or virus shedding (ferrets and pigs) of immunized hosts intranasally challenged with CA/04/09. Furthermore, neither ferrets nor swine could abrogate aerosol transmission of the virus into naïve contact animals. Altogether, these results suggest that neither recent human nor animal H1N1 vaccine could provide complete protectivity in all animal models. Thus, this study warrants the need for strain-specific vaccines that could yield the optimal protection desired for humans and/or animals

Differential Diagnosis of Benign and Malignant Intraductal Papillary Mucinous Tumors of the Pancreas: MR Cholangiopancreatography and MR Angiography

Objective: To compare the usefulness of magnetic resonance cholangiopancreatography (MRCP) and MR angiography (MRA) in differentiating malignant from benign intraductal papillary mucinous tumors of the pancreas (IPMTs), and to determine the findings which suggest malignancy. Materials and Methods: During a 6-year period, 46 patients with IPMT underwent MRCP. Morphologically, tumor type was classified as main duct, branch duct, or combined. The diameter of the main pancreatic duct (MPD), the extent of the dilated MPD, and the location and size of the cystic lesion, septum, and communicating channel were assessed. For all types of IPMTs, enhanced mural nodules and portal vein narrowing were evaluated at MRA. Results: Combined-type IPMTs were more frequently malignant (78%) than benign (42%) (p < 0.05). Compared with benign lesions, malignant lesions were larger, and the caliber of the communicating channel was also larger (p < 0.05). Their dilated MPD was more extensive and of greater diameter (p < 0.05), and the presence of mural nodules was more frequent (p < 0.001). Conclusion: Combined MRCP and MRA might be useful for the differential diagnosis of malignant and benign IPMTs of the pancreas. Index terms: Pancreas, neoplasms Pancreas, M

A Nationwide Survey of Lymphangioleiomyomatosis in Korea: Recent Increase in Newly Diagnosed Patients

In 2007, the Korean Interstitial Lung Disease Society had collected clinical data of patients who have diagnosed as Lymphangioleiomyomatosis (LAM) since 1990 through nationwide survey, which showed that LAM patients had increased sharply after 2004. The present study was performed to show the clinical features of Korean patients with LAM, and to establish the reason for the recent increase in the diagnosis. All 63 patients were women and the mean age at diagnosis was 36 yr. The most common presenting symptom was dyspnea and 8 patients had tuberous sclerosis complex. The survival rate at 5 yr after diagnosis was 84%. Compared with patients diagnosed after 2004 (n=34), the patients diagnosed before 2004 (n=29) complained with dyspnea more (P=0.016) and had lower FEV1% predicted (P=0.003), and DLco% predicted (P=0.042). The higher proportion of patients diagnosed after 2004 showed the normal chest radiography, and they were detected by routine chest CT screening (P=0.016). This study showed that clinical features of Korean patients with LAM were not different from those reported elsewhere. It is concluded that the reason for the increase of newly diagnosed patients is the result of increase in detection of the early stage LAM by the widespread use of chest CT screening