Continuous assay of proteases using a microtiter plate fluorescence reader

The present study was designed to determine if spinal calcium channels, calmodulin, and calcium/calmodulin-dependent protein kinase II were involved in the production of antinociception induced by cold water swimming stress (CWSS). The effects of intrathecal (i.t.) injection of nimodipine, omega-conotoxin GVIA, calmidazolium, or (S)-5-isoquinolinesulfonic acid, 4-[2-[(5-isoquinolinyl-sulfonyl)methylamino]-3-oxo-3-(4-phenyl-1-piperaz inyl)-propyl]phenyl ester (KN-62) on CWSS-induced antinociception were studied in ICR mice. The antinociception was assessed by the tail-flick test. CWSS produced inhibition of the tail-flick response. Various doses of nimodipine (10-40 ng), omega-conotoxin GVIA (5-40 ng), calmidazolium (10-40 ng), or KN-62 (5-40 ng) injected i.t. alone did not show any antinociceptive effect in the tail-flick test. I.t. pretreatment with omega-conotoxin GVIA, calmidazolium, or KN-62 dose dependently attenuated the CWSS-induced inhibition of the tail-flick response. However, i.t. pretreatment with nimodipine did not affect the inhibition of the tail-flick response induced by CWSS. Our results suggest that spinal N-type calcium channel, calmodulin and calcium/calmodulin-dependent protein kinase II may be involved in the production of antinociception induced by CWSS. On the other hand, CWSS-induced antinociception appears not to be mediated via the spinal L-type calcium channel.     

EFFECT OF PROCESSING ON NUTRITIONAL AND ANTINUTRITIONAL FACTORS OF MOONGBEAN CULTIVARS

Ordinary cooking of presoaked and unsoaked seeds of six cultivars of moongbean (Vigna radiata L.) lowered phytic acid, polyphenols, trypsin inhibitors and improved in vitro digestibility of protein and starch, and availability of Ca, Fe and Zn when compared to unprocessed seeds. However, the effect was more pronounced on ordinary cooking of presoaked seeds followed by cooking of unsoaked seeds and presoaked seeds alone. A significant increase in vitro digestibility of protein (34–48%), and starch (31–62%) may be ascribed to the reduced content of phytic acid (45–65%) polyphenols (34–70%) and trypsin inhibitor activity (42–55%) after ordinary cooking of presoaked seeds of moongbean cultivars. Therefore, ordinary cooking of soaked seeds was the most effective treatment in improving the nutritional quality of moongbean.     

Cloning, tissue expression, and chromosomal localization of the mouse IRS-3 gene

Insulin receptor substrate (IRS) proteins are key regulators of basic functions such as cellular growth and metabolism. They provide an interface between multiple receptors and a complex network of intracellular signaling molecules. Two members of this family (IRS-1 and IRS-2) have been identified previously. In this investigation, we analyzed a mouse expressed sequence tag clone that proved to be a new member of the IRS family. Sequence analysis of this clone and comparison with the sequences deposited in GenBank demonstrates this protein may be the murine homolog of rat IRS-3, recently purified and cloned from rat adipocytes. Accordingly, we have named our protein mouse IRS-3. The expressed sequence tag clone contains the complete coding sequence of 1485 bp, encoding a protein of 495 amino acids. Sequence alignment with the other members of the IRS family shows that this protein contains pleckstrin homology and phosphotyrosine-binding domains that are highly conserved. In addition, there is conservation of many tyrosine phosphorylation motifs responsible for interactions with downstream signaling molecules containing SH2 domains. The murine IRS-3 messenger RNA (2.4 kilobases in length) is expressed in many tissues, with highest levels in liver and lung. Mouse IRS-3 is highly expressed in the first part of the embryonic life, when IRS-1 messenger RNA is barely detectable. Unlike the genes encoding IRS-1 and IRS-2, the IRS-3 gene contains an intron (344 bp in length) in the region between the pleckstrin homology and the phosphotyrosine-binding domains. Fluorescent in situ hybridization localized the mouse IRS-3 gene on the telomeric region of chromosome 5G2. Cloning of the murine IRS-3 gene will make it possible to apply genetic approaches to elucidate the physiological role of this new member of the IRS family of proteins.     

Effects of the neem tree compounds azadirachtin, salannin, nimbin, and 6-desacetylnimbin on ecdysone 20-monooxygenase activity

The effects of azadirachtin, salannin, nimbin, and 6-desacetylnimbin on ecdysone 20-monooxygenase (E-20-M) activity were examined in three insect species. Homogenates of wandering stage third instar larvae of Drosophila melanogaster, or abdomens from adult female Aedes aegypti, or fat body or midgut from fifth instar larvae of Manduca sexta were incubated with radiolabeled ecdysone and increasing concentrations (from 1 x 10(-8) to 1 x 10(-3) M) of the four compounds isolated from seed kernels of the neem tree, Azadirachta indica. All four neem tree compounds were found to inhibit, in a dose-dependent fashion, the E-20-M activity in three insect species. The concentration of these compounds required to elicit a 50% inhibition of this steroid hydroxylase activity in the three insect species examined ranged from approximately 2 x 10(-5) to 1 x 10(-3).     

Single-dose versus multiple instillations of epirubicin as prophylaxis for recurrence after transurethral resection of pTa and pT1 transitional-cell bladder tumours: a prospective, randomized controlled study

OBJECTIVE: To compare single-dose and multiple instillations of epirubicin in the chemoprophylaxis of superficial bladder tumours. PATIENTS AND METHODS: In a prospective randomized and controlled study, 168 evaluable patients were assigned to three groups after transurethral resection of bladder tumour (TURBT) and histological confirmation of its superficial nature (pTa and pT1). The groups were comparable for tumour stage, grade and other tumour characteristics. In group 1, patients received a single dose of 50 mg epirubicin in 50 mL normal saline immediately after TURBT; group 2 received 50 mg epirubicin in 50 mL normal saline 1-2 weeks after TURBT and the instillations were repeated for 8 weeks and thereafter monthly to complete one year of treatment: group 3 (control group) received no adjuvant therapy after TURBT. The patients were assessed by cysto-urethroscopy, urine cytology and DNA flow cytometry 8 weeks after resection and then every 3 months during the first 2 years and 6 monthly thereafter during the next 2 years. Intravenous urography was performed annually and when otherwise indicated. RESULTS: The recurrence rate was significantly lower in the patients treated with epirubicin than in the control group (24, 25 and 52%, respectively; P < 0.001). In those receiving epirubicin, the rates of recurrence were statistically comparable (P = 0.9). Patients who had a large tumour burden showed slightly lower recurrence rates with single-dose epirubicin than with delayed maintenance therapy but the difference was statistically insignificant. Patients with a history of bladder tumours before treatment had lower recurrence rates with maintenance treatment than with a single dose (34.6 and 22.6% in groups 1 and 2, respectively); again this difference was statistically insignificant. Patients with grade 3 tumours showed a marginal difference in favour of maintenance therapy. The rates of progression amongst the three groups were 5.5, 3.4 and 9.3%, respectively, with no significant differences. The overall toxicity rates were comparable in the two treated groups (22 and 25%). CONCLUSION: With the possible exception of grade 3 tumours, single-dose immediate epirubicin is as effective as delayed maintenance therapy, with the advantage of being more cost-effective.     

Partial cricotracheal resection with primary anastomosis in the pediatric age group

The traditional approach to severe subglottic stenosis (SGS) in the pediatric age group is laryngotracheal reconstruction (LTR). This approach may be complex and multistaged, with variable and unpredictable success rates in the individual patient. Excellent results have been reported in adults who had severe SGS and underwent partial resection of the cricoid and primary thyrotracheal anastomosis. This procedure has not been widely reported in infants and children. We report our experience with this procedure in 16 pediatric patients with grade III or IV SGS. Eleven patients had multiple previous LTR operations. The preoperative evaluation, surgical techniques, postoperative care, complications, and final results are described and discussed. Fourteen patients were decannulated after the procedure, 1 patient needed a second open procedure prior to decannulation, and 1 patient with concomitant bronchopulmonary dysplasia remains cannulated, for an overall 94% decannulation rate. Fourteen patients have no limitation of respiration, and 1 patient has moderate exercise intolerance. The results of this series suggest that partial cricotracheal resection with primary anastomosis is a relatively safe and effective procedure for pediatric patients with severe SGS.     

Task Planning For A Mobile Robot In An Indoor Environment Using Object-oriented Domain Information

For a mobile robot to be practical, it needs to navigate in dynamically changing environments and manipulate objects in the environment with operating ease. The main challenges to satisfying these requirements in mobile robot research include the collection of robot environment information, storage and organization of this information, and fast task planning based on available information. Conventional approaches to these problems are far from satisfactory due to their requirement of high computation time. In this paper, we specifically address the problems of storage and organization of the environment information and fast task planning in the area of robotic research. We propose an special object-oriented data model (OODM) for information storage and management in order to solve the first problem. This model explicitly represents domain knowledge and abstracts a global perspective about the robot's dynamically changing environment. To solve the second problem, we introduce a fast task planning algorithm that fully uses domain knowledge related to robot applications and to the given environment. Our OODM based task planning method presents a general frame work and representation, into which domain specific information, domain decomposition methods and specific path planners can be tailored for different task planning problems. This method unifies and integrates the salient features from various areas such as database, artificial intelligence, and robot path planning, thus increasing the planning speed significantly     

Protein expression profiles in human breast ductal carcinoma and histologically normal tissue

Reference two-dimensional (2-D) gels are presented for human breast ductal carcinoma and histologically normal tissue. Whole biopsy fragments were analyzed, including epithelial and nonepithelial components. Thirty-five spots have been assigned by gel matching to the human liver SWISS-2DPAGE reference map and/or to the human primary keratinocyte IPG map from the Danish Center for Human Genome. N-terminal microsequencing was applied to confirm randomly chosen matching assignments and to identify six new spots. Protein expression profiles in ductal carcinoma and in normal breast tissue appeared to be similar, except for a pattern consisting of 32 spots, which were highly expressed in all carcinoma specimens, and less intense and occasionally undetectable in normal tissue. This difference was statistically significant. Assignment has been obtained for several spots, namely GRP94, GRP78, GRP75, mitochondrial HSP60, calreticulin, protein disulfide isomerase, peptidyl-prolyl cis-trans isomerase, collagen-binding protein 2, fructose bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, thioredoxin, cytochrome c oxidase VA subunit, tubulin beta isoform and macrophage migration inhibitory factor (MIF). The cancer- and tissue-specificity of the described pattern was assessed by matching to the Swiss-2DPAGE human liver, hepatoma, lymphoma, erythroleukemia reference maps. The pattern of 32 spots was found to be indicative of epithelial neoplasia.