Home About us Contact
|
Home About us Contact | ||
|
|
||
Nmol/min/mg Protein (nmol/min/mg + protein)
Selected AbstractscDNA cloning, functional expression and characterization of kynurenine 3-hydroxylase of Anopheles stephensi (Diptera: Culicidae)INSECT MOLECULAR BIOLOGY, Issue 5 2002M. Hirai Abstract Kynurenine 3-hydroxylase (K3H) is a NADPH-dependent flavin monooxygenase involved in the tryptophan pathway. Xanthurenic acid (XA) is a metabolite of this pathway and has recently been identified as a gamete activating factor (GAF) of the malarial parasite. We cloned K3H cDNA from Anopheles stephensi (AsK3H), because anopheline mosquitoes are a vector of the human malaria parasite, Plasmodium falciparum and the catalytic function of AsK3H in XA production. Recombinant AsK3H protein was expressed in Sf-9 cells using the baculovirus system and its enzymatic properties were characterized. The specific activities of crude cell lysate and affinity purified protein were 94.9 ± 6.2 and 865.6 ± 10.5 nmol/min/mg protein, respectively. The optimum pH of AsK3H was 7.0. Analysis of AsK3H gene expression using RT-PCR revealed that AsK3H was constitutively expressed in egg, larva, pupa and adult. [source] Expression of GFAT1 and OGT in podocytes: Transport of glucosamine and the implications for glucose uptake into these cellsJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2010Dorota Rogacka Glutamine:fructose-6-phosphate amidotransferase (GFAT) and N -acetylglucosaminyltransferase (OGT) participate in glucosamine (GlcN) production and its utilization in O -glycosylation, one of key post-translational modifications of nuclear and cytoplasmic proteins. For this purpose, cells require a high rate of intracellular production of GlcN and/or significant GlcN delivery. We studied the expression of GFAT1 and OGT and measured uptake of glucose and GlcN in cultured rat podocytes, the main cellular component of glomerular filtration barrier. RT-PCR revealed the presence of both GFAT1 and OGT mRNA. Immunofluorescence of GFAT1 has shown staining signal diffused within the cytoplasm of the cell body and processes. However, OGT was distinctly visible around the nucleus and, in diffuse form, within the cytoplasm of cell bodies and processes. Glucose was transported (1.3,±,0.2,nmol/min/mg protein) mainly by facilitative transporter systems whilst GlcN uptake (1.1,±,0.2,nmol/min/mg protein) in a significant part, involved a sodium-dependent transporter. There was interplay between glucose and GlcN uptake. In the presence of GlcN (50,µM), the rate of glucose uptake decreased by about 50%. The rate of GlcN uptake decreased by 28% in the presence of 5.6,mM glucose. Our results suggest that cultured podocytes possess limited ability to synthesize GlcN internally and therefore may need to receive GlcN from the extracellular environment. J. Cell. Physiol. 225: 577,584, 2010. © 2010 Wiley-Liss, Inc. [source] Mechanisms of cholinergic dysfunction in rabbits following recurrent aspiration of cow's milk,PEDIATRIC PULMONOLOGY, Issue 6 2001Gary L. Larsen MD Abstract Recurrent aspiration of cow's milk has been shown to alter neural control of airways in young rabbits (Gelfand et al., 1997). The purpose of this study was to define the mechanisms responsible for in vitro cholinergic hyperresponsiveness in this model. Beginning at 1 week of age, rabbits received either 0.5 mL/kg whole cow's milk or sterile saline intranasally while under light anesthesia. This was repeated each weekday for 2 weeks. At 8 weeks of age, rabbits were sacrificed. Portions of lungs underwent lavage with sterile saline. Tracheal smooth muscle (TSM) segments were also removed. Segments were assessed for acetylcholine (ACh) release by high-performance liquid chromatography ( HPLC) with electrochemical detection or acetylcholinesterase (AChE) kinetic activity by spectrophotometry. Substance P (SP), a neuropeptide that can increase ACh release from nerves, was also assessed using an enzyme immunoassay to define the content in lavage and TSM segments. Immunohistochemistry for SP within airways was also assessed. We found that recurrent aspiration of milk led to statistically significant alterations in many parameters. Acetylcholine release was significantly greater in segments of airways from rabbits that had aspirated cow's milk (27.5,±,1.7 vs. 20.1,±,1.6 pmol/min/g tissue) than saline. At the same time, AChE activity was less in the group that aspirated milk (8.7,±,0.4 vs. 10.2,±,0.5 nmol/min/mg protein) compared to saline. The amount of SP within both lavage as well as tissue homogenates was greater in the group that had aspirated the foreign protein (159.1,±,28.9 vs. 41.9,±,5.2 pmol/mg protein in lavage; 158.7,±,31.9 vs. 80.5,±,7.8 pmol/mg protein in tissues) than saline controls. While total cholinergic nerve density as assessed by choline acetyltransferase was not significantly different between groups, SP-positive immunoreactive nerves were easily identified in the group that aspirated cow's milk. This study suggests that cholinergic hyperresponsiveness caused by repeated aspiration of milk is due to several abnormalities, including prejunctional (increase in ACh release) as well as junctional (decrease in AChE) mechanisms within the airways. In addition, an upregulation of SP within airways is part of this process. Pediatr Pulmonol. 2001; 32:409,417. © 2001 Wiley-Liss, Inc. [source] Esterase-based resistance in the tobacco-adapted form of the green peach aphid, Myzus persicae (Sulzer) (Hemiptera: Aphididae) in the eastern United StatesARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 2 2009Lakshmipathi Srigiriraju Abstract Organophosphates and carbamates represent alternative insecticides in managing the tobacco-adapted form of the green peach aphid (TGPA), Myzus persicae (Sulzer), a major pest of tobacco in the United States and around the world. General esterases that detoxify these insecticides were assessed in green, red, and orange morphs of field-collected M. persicae. A total of 136 aphid colonies were collected from 2004 though 2007 and screened for total esterase activity. The green morphs had lower esterase levels, with a mean of 77±6.6,nmol/min/mg protein, as compared to red (84±2.9,nmol/min/mg protein) and orange morphs (172±16.5,nmol/min/mg protein). Overall esterase activities, and those for the red and green morphs, were positively correlated with LC50 values for acephate (organophosphate) and methomyl (carbamate) assessed in leaf-dip bioassays. Esterase genes responsible for higher esterase activities were diagnosed by gene amplification studies. Twenty-three of 24 colonies tested had either the E4 or FE4 gene amplified, both known to confer esterase-based resistance. Fifteen out of the 24 colonies tested had amplified E4 gene and four colonies had FE4 gene amplification. All orange morphs and one green morph had both E4 and FE4 genes amplified. This unique phenotype, where two esterase genes were amplified had an 865-bp band characteristic of the FE4 gene and an additional 381-bp band characteristic of a deleted upstream region of the E4 gene. Changes that occurred in esterase-based resistance in the TGPA over the past two decades and their implications on insecticide resistance management are discussed. © 2009 Wiley Periodicals, Inc. [source] A case of multiple angiomas without any angiokeratomas in a female heterozygote with Fabry diseaseAUSTRALASIAN JOURNAL OF DERMATOLOGY, Issue 1 2010Vesna Mirceva ABSTRACT Fabry disease is a rare, X-chromosome-linked lysosomal storage disease caused by a deficient ,-galactosidase A enzyme. The disease manifests primarily in affected hemizygous males and to some extent in heterozygous females (,carrier'). A 45-year-old female Fabry disease patient without angiokeratomas but with numerous angiomas is presented. Her leukocyte ,-galactosidase A activity was reduced (0.35 nmol/min/mg protein; normal range: 0.4,1). The analysis of her ,-galactosidase A gene (exon 1,7) showed the transition c.427 G>A. An intrafamilial follow-up search detected a reduced leukocyte ,-galactosidase A activity in her father, who suffered exclusively from coronary heart disease. Our case report underlines the possible wide range of clinical signs in Fabry disease patients, sometimes complicated by missing typical lesions (e.g. angiokeratomas). In oligosymptomatic Fabry disease cases, genetic analysis is recommended. [source] A rapid assay method for catechol- O -methyltransferase activity by flow injection analysisBIOMEDICAL CHROMATOGRAPHY, Issue 4 2002Nozomi Aoyama A rapid assay employing flow injection analysis (FIA) to determine the activity of purified catechol- O -methyltransferase (COMT) from porcine liver is described. The method was based on the determination of normetanephrine, the 3- O -methyl metabolite of the substrate norepinephrine. Excess norepinephrine was removed from the incubation mixture by alumina extraction twice to allow normetanephrine to be subjected to flow injection analysis, coulometrical oxidation, fluorogenic reaction with ethylenediamine and fluorescence detection. Km and Vmax values for COMT obtained with the system were 503,µM and 4.51 nmol/min/mg protein, respectively. The method is suitable for screening of COMT inhibitors or activators, as a large number of samples, up to 200, can be processed in one working day. Copyright © 2002 John Wiley & Sons, Ltd. [source] Enzymatic stability of 2,-ethylcarbonate-linked paclitaxel in serum and conversion to paclitaxel by rabbit liver carboxylesterase for use in prodrug/enzyme therapyBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 5 2008Tadatoshi Tanino Abstract In prodrug/enzyme therapy for cancer, information on the sensitivity of hydrolytic enzymes to prodrug is required to reduce adverse effects of the parental drug and to find the activating enzyme. The aim of this study was to characterize the enzymatic stability of 2,-ethylcarbonate-linked paclitaxel (TAX-2,-Et) in the sera of several different species including humans. TAX-2,-Et disposition in serum was kinetically analysed using models with hydrolytic and/or degradation processes. To further evaluate the capability of liver carboxylesterases (CESs) in TAX-2,-Et hydrolysis, a CES isolated from rabbit liver (Ra-CES) was utilized as a model enzyme. Rat serum provided rapid enzymatic hydrolysis of TAX-2,-Et with a half-life of 4 min. The degradation of paclitaxel (TAX) (degradation rate constant, 0.16,h,1) was accompanied by the formation of an unknown compound. The conversion to TAX was almost completely inhibited by phenylmethyl sulfonylfluoride (PMSF) and bis(p-nitrophenyl) phosphate (BNPP). In human and rabbit sera, the degradation rate constant of TAX-2,-Et was 5.1,×,10,2 and 0.15,h,1, respectively, when excepting hydrolysis. The degradation products had the same molecular weight as TAX-2,-Et. The amount of TAX produced accounted for only 8,11% of the decrease in TAX-2,-Et after a 9 h exposure to rabbit or human serum. PMSF, but not BNPP, inhibited more than 90% of the TAX production in a 1.5,h incubation with human or rabbit serum. Ra-CES enzyme converted TAX-2,-Et to TAX with Vmax and Km of 74.7±13.8 nmol/min/mg protein and 8.8±2.8 µM, respectively. These results indicate that TAX-2,-Et is sensitive to serum CESs, but not cholinesterases. However, serum CESs show species-dependent hydrolysis of TAX-2,-Et. Although human serum allows the slow release of TAX, TAX-2,-Et is expected to reduce the side-effects of TAX. The Ra-CES enzyme is capable of hydrolysing TAX-2,-Et, which may be beneficial for the development of a TAX-2,-Et/enzyme therapy strategy for ovarian cancer. Copyright © 2008 John Wiley & Sons, Ltd. [source] In vitro activation of the corticosteroid ciclesonide in animal nasal mucosal homogenatesBIOPHARMACEUTICS AND DRUG DISPOSITION, Issue 2 2007H. Sato Abstract Ciclesonide, a new corticosteroid for allergic rhinitis, is administered as an inactive parent compound that is converted by esterases to the pharmacologically active metabolite, desisobutyryl-ciclesonide (des-CIC). This study investigated the in vitro activation of ciclesonide in nasal mucosa of multiple animal species. Nasal mucosal homogenates from rats, guinea-pigs, rabbits and dogs were incubated with ciclesonide 0.5 µmol/l (0.271 µg/ml) or 5 µmol/l (2.71 µg/ml) for up to 120 min. Concentrations of ciclesonide and des-CIC were measured by high-performance liquid chromatography with tandem mass spectrometry. Ciclesonide was metabolized to des-CIC in nasal mucosal homogenates of each species. The initial velocities of des-CIC formation ranged from 0.0038 to 0.0150 nmol/min/mg protein and 0.0319 to 0.0983 nmol/min/mg protein in nasal mucosal homogenates incubated with ciclesonide 0.5 µmol/l and 5 µmol/l, respectively. Furthermore, the initial velocities of ciclesonide metabolism ranged from 0.0032 to 0.0142 nmol/min/mg protein and 0.0445 to 0.1316 nmol/min/mg protein in nasal mucosal homogenates incubated with ciclesonide 0.5 µmol/l and 5 µmol/l, respectively. This study confirms that ciclesonide is converted to des-CIC in nasal mucosal homogenates without any marked differences among animal species. Copyright © 2007 John Wiley & Sons, Ltd. [source] | ||||||||||||||||