Fast MR abdominal imaging using the HASTE sequence.
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Abdomen↗
Biomedical subjects
B Nguyen
Publications and source records attributed to B Nguyen.
Combined cisplatin and gemcitabine for non-small cell lung cancer: influence of scheduling on toxicity and drug delivery.
The scheduling of cytotoxic chemotherapy has important bearing on the toxicity and ability to deliver chemotherapy at or close to full dose. We report new data on the influence of different schedules of cisplatin and gemcitabine on toxicity and drug delivery in phase II studies of non-small cell lung cancer. Patients in six phase II studies had standard entry criteria for advanced non-small cell lung cancer. Whereas gemcitabine was given on days 1, 8, and 15 of a 28-day cycle in all these studies, the scheduling of cisplatin varied and was given either on day 1, day 2, day 15 (in two studies), or days 1, 8, and 15 (in two studies). The protocol dose per infusion for gemcitabine was 1,000 mg/m2 (five studies) and 1,500 mg/m2 (one study); for cisplatin, it was 100 mg/m2 when given once per cycle and 30 mg/m2 when given on days 1, 8, and 15. Similar dose reduction schedules were implemented in the event of grade 3 or higher drug toxicity for all studies except for the day 1 cisplatin study, in which the dose was omitted for grade 2 thrombocytopenia. Nonhematologic toxicity was very low. Hematologic toxicity was moderate, and in patients who developed grade 3 or 4 toxicity, there was no hemorrhage from thrombocytopenia and neutropenic sepsis was rare. The incidence of grade 3 or 4 thrombocytopenia with the day 1, day 2, day 15 (two studies combined), and days 1, 8, and 15 (two studies combined) cisplatin regimens was 50%, 52%, 26%, and 38%. The incidence of grade 3 or 4 neutropenia with these four regimens was 51%, 37%, 56%, and 49%, respectively. Although the hematologic toxicity might appear relatively similar, it represents the toxicity at the administered rather than the intended (protocol) dose, because drug delivery was reduced or omitted in the event of grade 3 or 4 toxicity. Differences between the schedules are revealed by analysis of the actual dosages delivered. The median dosage of gemcitabine per scheduled infusion was statistically higher with the day 15 cisplatin regimens (combined) compared with any of the other regimens treating at 1,000 mg/m2 (P < .003, z-score). The dose with the day 1, day 2, day 15, and days 1, 8, and 15 cisplatin regimens was 664, 829, 889, and 774 mg/m2, respectively. Both the percentages of cycles in which gemcitabine infusions were given at full dose and in which there were no omissions of gemcitabine infusions (including infusions with dose reductions) were statistically higher in the day 15 cisplatin regimen than with any of the other regimens (P < .0001, chi-square test). The percentage of cycles containing full-dose gemcitabine with the day 1, day 2, day 15, and days 1, 8, and 15 cisplatin regimens was 24%, 44%, 75%, and 46%, respectively. The percentage of cycles in which there were no omissions of gemcitabine infusions for the four regimens above was 32%, 55%, 83%, and 72%, respectively. Apart from the once-weekly regimen (days 1, 8, and 15) in which the protocol gemcitabine dose was 1,250 mg/m2, the day 15 cisplatin schedule allowed for the highest median concentration of gemcitabine. More importantly, the day 15 cisplatin schedule provided the longest duration of gemcitabine exposure, which is particularly important for its activity as gemcitabine is a phase-specific agent. The day 15 cisplatin schedule is associated with the best dose intensity and the longest median duration of exposure to gemcitabine, and best meets the goal of administering both agents at full doses in combination.
Adult↗
Delivering labeled teaching images over the Web.
The Web provides educators with the best opportunity to date for distributing teaching images across the educational enterprise and within the clinical environment. Experience in the pre-Web era showed that labels and information linked to parts of the image are crucial to student learning. Standard Web technology does not enable the delivery of labeled images. We have developed an environment called OverLayer that succeeds in the authoring and delivering of such images in a variety of formats. OverLayer has a number of functional specifications, based on the literature and on our experience, among them, the following: Users should be able to find components by name or by image; to receive feedback about their choice to test themselves. The image should be of arbitrary size; should be reusable; should be linked to further information; should be stand-alone files. The labels should not obscure the image; should be linked to further information. Images should be stand-alone files that can be transferred among faculty members. Implemented in Java, OverLayer (http:/(/)omie.med.jhmi.edu/overlayer) has at its heart a set of object classes that have been reused in a number of applets for different teaching purposes and a file format for creating OverLayer images. We have created a 350-image histology library and a 500-image pathology library, and are working on a 400-image GI endoscopy library. We hope that the OverLayer suite of classes and implementations will help to further the gains made by previous image-based hyperlinked technologies.
Anatomy↗
Pyrrolidine-3-carboxylic acids as endothelin antagonists. 2. Sulfonamide-based ETA/ETB mixed antagonists.
When the N,N-dialkylacetamide side chain of the highly ETA-selective endothelin antagonist ABT-627 (1; [2R,3R,4S]-2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)-1-[[N, N-dibutylamino)-carboxyl]methyl]pyrrolidine-3-carboxylic acid; A-147627) is replaced by N,S-dialkylsulfonamidoethyl, the resultant analogs retain ETA affinity, but exhibit substantial ETB affinity as well. Structure-activity studies reveal that modifications in the length of the two alkyl groups, and in the substitution on the anisyl ring, are important in optimizing this "balanced" antagonist profile. In particular the combination of an N-n-propyl group, an S-alkyl chain between four and six carbons in length, and a fluorine atom ortho to the aromatic OCH3 provides compounds with sub-nanomolar affinities for both receptor subtypes, and with ETA/ETB ratios close to 1. A number of these compounds also exhibit oral bioavailabilities (in rats) in the 30-50% range and have substantial plasma half-lives. The balanced receptor-binding profile of these potent and orally bioavailable compounds complements the ETA selectivity observed with 1.
Administration, Oral↗
Regulation of inducible nitric oxide synthase expression by macrophage purinoreceptors and calcium.
Macrophage activation is central to the progression of multiple diseases via the release of inflammatory mediators such as cytokines and nitric oxide. Despite the recognized overlap in the regulatory mechanisms involved in mediator production, little formation exists regarding receptor-initiated signaling pathways that coordinately control multiple end points, such as tumor necrosis factor-alpha (TNF-alpha) and nitric oxide production. In this study, the expression of inducible nitric oxide synthase (iNOS) in macrophages is shown to be regulated by calcium and by a purinoreceptor signaling system. The P2Y purinoreceptor partial agonist, 2-methylthio-ATP (2-MeS-ATP), inhibits the expression of iNOS induced by lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma) in primary macrophages. Additionally, 2-MeS-ATP attenuates the expression of iNOS in macrophages isolated from CD-1 mice challenged with LPS, and it inhibits LPS-induced TNF-alpha and interleukin-1 alpha (IL-1 alpha) release, thereby preventing endotoxic death. Thus, purinoreceptors and calcium are likely to be critical for macrophage activation and the production of inflammatory mediators stimulated by LPS.
Adenosine Triphosphate↗
Expression of human dopamine beta-hydroxylase in Drosophila Schneider 2 cells.
Human dopamine beta-hydroxylase (DBH) has been expressed in transformed Drosophila Schneider 2 (S2) cells with yields of > 16 mg/l. Most of the activity was found in the culture fluid. Similarly, human neuroblastoma cells also secrete native DBH into the medium, but at a much lower level than recombinant Drosophila cells. We have purified native and recombinant human DBH by a modified purification procedure using SP-Sepharose, lentil lectin-Sepharose and gel-filtration chromatography and carried out studies to compare the two enzymes. Two variants of human DBH that differ by a single amino acid (either serine or alanine) at position 304 were expressed in Drosophila cells, purified, and found to have no significant difference in enzyme activity. The molecular mass of human DBH monomer has been determined from SDS/PAGE to be 73 kDa, but the recombinant DBH from Drosophila is smaller at 66 kDa. The difference may be due to glycosylation as deglycosylated enzymes from both sources are identical in size (61 kDa). The Km of tyramine for native and recombinant human enzymes are virtually the same but higher than bovine DBH by about 3-fold. Likewise, the inhibition of native and recombinant human DBH by fusaric acid and SKF102698 is not significantly different but IC50 values are 2-3-fold higher than that for the bovine enzyme. These results strongly support the conclusion that recombinant human DBH from Drosophila S2 cells can be used in place of human neuroblastoma-derived DBH for drug screening, characterization of the enzyme's physicochemical properties, and determination of structure-function relationships. The Drosophila expression system has thus provided a convenient source for large quantities of human DBH enzyme.
Amino Acid Sequence↗
Predictors of success in a smoking cessation clinic.
We identified factors associated with success among the first 531 patients referred to a Veterans Affairs Medical Center smoking cessation clinic. Both patients and providers completed questionnaires covering smoking history, other health habits, and comorbidity. The 3-month success rate was 23% of all referrals or 42% of clinic attendees. Among those referred, factors associated with their success were being at least 50 years of age, having hyperlipidemia, and not currently drinking alcohol. Among those who attended the clinic at least once, factors associated with success were being at least 50 years of age, being less physically active, and not currently drinking alcohol. Neither patients nor providers could predict who would successfully stop smoking.
Activities of Daily Living↗
Effects of GABA uptake inhibitors on posthypoxic myoclonus in rats.
Male Sprague-Dawley rats developed posthypoxic myoclonus following 10-min cardiac arrest and resuscitation. Previous results showed that dysfunction of central GABAergic neurotransmission may contribute to the disease. In current studies, effects of GABA uptake inhibitors, guvacine hydrochloride (1,2,5,6-tetrahydro-3-pyridine carboxylic acid hydrochloride) and (+/-)-cis-4-hydroxynipecotic acid ([+/-]-cis-4-hydroxy-3-piperidine carboxylic acid), in the pathophysiology of posthypoxic myoclonus were investigated. Administration of guvacine (1 or 10 mg/kg, IP) or nipecotic acid (0.5 or 5 mg/kg, IP) significantly attenuated myoclonus scores of the animals. Tolerance to antimyoclonus effects of these two compounds did not develop after chronic administration (twice a day for 14 days) of guvacine (10 mg/kg, IP) or nipecotic acid (5 mg/kg, IP). On the other hand, tolerance was noticed with clonazepam (2.5 mg/kg, IP twice a day for 7 days). The results indicate that guvacine or nipecotic acid may be used in combination with (at reduced doses) or as alternatives to clonazepam to treat patients with the disease so as to reduce tolerance phenomenon usually associated with clonazepam.
Animals↗
High-level production from a baculovirus expression system and biochemical characterization of human GMP synthetase.
GMP synthetase, a key enzyme in the de novo synthesis of guanine nucleotides, is a potential target for immunosuppression and anticancer chemotherapy. In order to closely examine the catalytic mechanism and active-site topography of this enzyme, large amounts of pure protein are needed. Catalytically active human GMP synthetase was expressed in a baculovirus system. A high-level production system has been established from which the yield of pure protein is routinely more than 50 mg/10 liters of cell culture. The recombinant enzyme was purified to homogeneity and characterized. Like native GMP synthetase, the recombinant enzyme was resolved into two forms by ion-exchange chromatography. The two forms are both monomers and they differ in their isoelectric points. There is no evidence that these forms are in equilibrium or interconvertible. Protein sequence analysis reveals that both forms are blocked at the amino-terminus and they are essentially identical in sequence. Since they can be produced by a cDNA with a single open reading frame, we believe that they represent post-translational modification variants. The recombinant GMP synthetase is not distinguishable from the native enzyme in terms of chromatographic profiles, subunit composition, molecular weight, and kinetic properties. The inhibition constants and the modes of inhibition toward decoyinine, a selective inhibitor of GMP synthetase, are also the same as the native enzyme. The high-level production of active enzyme is invaluable to the determination of the three-dimensional structure and the discovery of potent and selective drug candidates.
Animals↗
The biology of growth regulation in normal and malignant breast epithelium: from bench to clinic.
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Breast↗
Purification, characterization and selective inhibition of human prostaglandin G/H synthase 1 and 2 expressed in the baculovirus system.
Human prostaglandin G/H synthase 1 and 2 were expressed in the baculovirus expression system and purified to high levels. Both enzymes were glycosylated. PGHS-1 appeared to be homogeneous by SDS-PAGE analysis but two closely migrating bands were detected in PGHS-2 preparation which were evidently due to heterogeneity in glycosylation. The amino-acid sequence of the N-termini of both isoforms indicated that the signal sequences were efficiently cleaved by the insect cells. The recombinant human PGHS-1 and PGHS-2 possessed both cyclooxygenase and peroxidase activities. Both had high affinities for arachidonate as substrate and underwent self-inactivation during catalysis. The recombinant isoforms were not pharmacologically identical, since some NSAIDs were selective inhibitors of either PGHS-1 or PGHS-2. This is the first report of high levels of expression and purification of human PGHS isoforms. The recombinant enzymes are invaluable in developing potent PGHS-2 selective inhibitors that may be efficacious anti-inflammatory drugs with no or low levels of toxicity.
Amino Acid Sequence↗
Production, purification, and characterization of human matrilysin (PUMP) from recombinant Chinese hamster ovary cells.
A process for semicontinuous production of matrilysin zymogen secreted from recombinant Chinese hamster ovary (CHO) cells was developed. The zymogen was purified to apparent homogeneity by sequential ion-exchange and metal chelation chromatography. These processes were scaled-up to purify gram quantities of the zymogen. The N-terminus of the secreted zymogen from the recombinant cells was the same as the observed sequence of the zymogen from natural sources. Furthermore, activation and autocatalysis of the recombinant zymogen resulted in a form with an N-terminus identical to that of the corresponding native enzyme. The three C-terminal amino acids of both the recombinant zymogen and the corresponding smaller activated form were missing. Activated matrilysin was shown to have activity against a synthetic peptide substrate. The large quantities of matrilysin that can be produced and purified from the recombinant CHO cells will be useful in determination of the structure of matrilysin.
Amino Acid Sequence↗
[Tussive form of Horton disease].
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Aged↗
Overexpression, purification and characterization of human recombinant 15-lipoxygenase.
Human 15-lipoxygenase was expressed to high levels (approx. 20% of cellular protein) in a baculovirus/insect cell expression system. Catalytically active enzyme was readily purified (90-95% pure) from cytosolic fractions by anion-exchange chromatography on a Mono Q column with approx. 95% recovery of enzymatic activity. Routinely, a yield of 25-50 mg of pure enzyme per L of culture and a specific activity of 7.1-21 mumol 13-hydroxyoctadecadienoic acid (13-HODE)/mg.min (turnover rate of 8.4-25 s-1) were obtained. Both the specific activity and the enzyme's iron content was significantly increased by the addition of ferrous ions to either the purified enzyme or to the insect cell culture medium during production. An isoelectric point of 5.85 was determined and the N-terminal amino acid sequence was found to be identical to that predicted from the cDNA. The purified recombinant enzyme exhibits a dual positional specificity with arachidonic acid (formation of 15S- and 12S-hydroxyeicosatetraenoic acid (12S-HETE) in a ratio of 12:1). Double oxygenation products 14R,15S- and various 8,15-DiHETE isomers were also identified. With linoleic acid as substrate, a pH-optimum of 7.0 and a KM of 3 microM were determined. The enzyme undergoes suicidal inactivation during fatty acid oxygenation, is sensitive to standard lipoxygenase inhibitors, and oxygenates phospholipids, cholesterol esters, biomembranes and human low-density lipoprotein. Contrary to prior studies on the rabbit enzyme, no glycosylation was detected.
Animals↗
[Rehabilitation of fresh, surgically managed anteromedial knee instabilities].
In between 1983 and 1988, 33 patients suffering from an acute antero-medial instability of the knee were operated on for the anterior cruciate ligament. The reconstruction of the ligament was performed by transcondylar fixation. 25 of these patients took part in a retrospective examination. The postoperative treatment had been carried out according to various concepts of rehabilitation. Our investigations were supposed to measure the restoration of the muscular efficiency of the knee-extensor and -flexor by isokinetic training arrangements. The results revealed that by the operation a sufficient stabilization of the knee had not been achieved. 18 of 25 patients once more showed an antero-medial instability of the knee. Twelve of these 25 patients had an extended anterior instability. Only in seven cases the muscular system was completely restored with average isokinetic muscle efficiency. The high rate of postoperative knee-instability proves the persistent sports-incapacity. We must draw the conclusion that the after-treatment of ligamentous injuries about the knee must focus on the muscular rehabilitation.
Adult↗
Fed-batch culture of insect cells: a method to increase the yield of recombinant human nerve growth factor (rhNGF) in the baculovirus expression system.
A fed-batch method was developed which increased the density of insect cells (Spodoptera frugiperda, Sf-9 cells) in suspension culture and the feeding of nutrients improved the yield of a recombinant protein produced by a baculovirus expression system. Analysis of spent medium samples indicated that depletions of glucose and glutamine correlated with the retardation of cell growth. Feeding of a mixture of nutrients consisting of glucose, glutamine, yeastolate and lipids solution restored the growth rate. In fed-batch culture, cell density was increased from 3 x 10(6) cells per ml to 1.2 x 10(7) cells per ml and the increased cell density enhanced the yield of the desired recombinant product, in this case, human nerve growth factor (rhNGF). The optimal conditions for the production of rhNGF were also defined by selecting the appropriate viral multiplicity of infection (MOI). At a cell density of 5 x 10(6) ml-1, a MOI of 0.05 (plaque forming units per cell) gave the highest yield of rhNGF in culture fluid 3 d post-infection. The yield of rhNGF was 20 mg l-1. The fed-batch method was scaled up to 12 l stirred bioreactor.
Animals↗
Use of polyethylene glycol for purification of DNA from leaf tissue of woody plants.
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Biotechnology↗
Physicochemical characterization of recombinant human nerve growth factor produced in insect cells with a baculovirus vector.
Recombinant human nerve growth factor (rhNGF) secreted by insect cells was purified by ion-exchange and reversed-phase chromatography to near homogeneity. The N-terminus of the secreted molecule was analogous to that of mouse salivary gland NGF. In its native conformation, the insect cell produced rhNGF molecules were homodimers consisting of 120 amino acid polypeptide chains. Mature rhNGF was found not to be significantly glycosylated (less than 0.08 mol of N-acetylglucosamine/mol of protein). The rhNGF was homogeneous with regard to molecular weight and amino acid sequence. Isoelectric focusing resolved the rhNGF into one major and one minor component. Because rhNGF from insect cells can be obtained in large quantities, purified to near homogeneity, and is similar to natural NGF with regard to physicochemical properties and biological activity, it is suitable for further evaluation in animal models as a therapeutic molecule for neurodegenerative diseases such as Alzheimer's disease.
Amino Acid Sequence↗