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1.
Appl Biochem Biotechnol ; 175(4): 1789-804, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25424286

ABSTRACT

Twenty bacterial strains, which are capable of degrading monocrotophos, were isolated from five soil samples collected from agriculture soils in India. The ability of the strains to mineralize monocrotophos was investigated under different culture conditions. A potential strain degrading monocrotophos was selected and named KPA-1. The strain was identified as a Bacillus subtilis on the basis of the results of its cellular morphology, physiological and chemotaxonomic characteristics, and phylogenetic conclusion of 16S ribosomal DNA (rDNA) gene sequences. Organophosphate hydrolase (opdA gene) involved in the initial biodegradation of monocrotophos in KPA-1 was quantitatively expressed, which was a constitutively expressed cytosolic enzyme. RT-qPCR data revealed that KPA-1 harboring opdA gene in an early stage was significantly downregulated from opdA gene in a degradation stage (1.5 fold more) with a p value of 0.0375 (p < 0.05). We have optimized culture conditions for the efficient degradation (94.2 %) of monocrotophos under aerobic conditions. Growth and degradation kinetic studies proved that KPA-1 was able to grow in minimal salt medium containing 1000 ppm monocrotophos as the only carbon source. Hence, KPA-1 culture has a great potential utility for the bioremediation of agriculture soils contaminated with organophosphorus pesticides, particularly monocrotophos.


Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/metabolism , Gene Expression Regulation, Bacterial , Insecticides/metabolism , Monocrotophos/metabolism , Phosphoric Monoester Hydrolases/metabolism , Soil Microbiology , Bacillus subtilis/classification , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Biodegradation, Environmental , Culture Media/chemistry , Genes, rRNA , Kinetics , Phosphoric Monoester Hydrolases/genetics , Phylogeny , RNA, Ribosomal, 16S/genetics
2.
J Muscle Res Cell Motil ; 21(4): 345-55, 2000 May.
Article in English | MEDLINE | ID: mdl-11032345

ABSTRACT

Myosin heavy chain (MyHC) is the major contractile protein of muscle. We report the first complete cosmid cloning and definitive physical map of the tandemly linked human skeletal MyHC genes at 17p13.1. The map provides new information on the order, size, and relative spacing of the genes. and it resolves uncertainties about the two fastest twitch isoforms. The physical order of the genes is demonstrated to contrast with the temporal order of their developmental expression. Furthermore, nucleotide sequence comparisons allow an approximation of the relative timing of five ancestral duplications that created distinct genes for the six isoforms. A firm foundation is provided for molecular analysis in patients with suspected primary skeletal myosinopathies and for detailed modelling of the hypervariable surface loops which dictate myosin's kinetic properties.


Subject(s)
Muscle, Skeletal/embryology , Myosin Heavy Chains/genetics , 3' Untranslated Regions , Amino Acid Sequence , Base Sequence , Chromosome Mapping , Cloning, Molecular , Cosmids , Exons , Humans , Molecular Sequence Data , Muscle Development , Muscle, Skeletal/growth & development , Muscular Diseases/genetics , Oculomotor Muscles/embryology , Oculomotor Muscles/growth & development , Protein Isoforms/genetics , Sarcomeres/chemistry , Sequence Alignment
3.
Mol Pharmacol ; 50(2): 306-13, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8700138

ABSTRACT

The mechanism by which agonists induce 5-hydroxytryptamine2A (5-HT2A) receptor internalization was investigated in a clonal cell line stably transfected with the 5-HT2A receptor cDNA. Confocal laser microscopy of immunolabeled 5-HT2A receptors in control (untreated) cells demonstrated that most of the immunoreactivity was associated with the cell surface. After quipazine administration, a significant increase in intracellular immunofluorescence was measured. Time course studies demonstrated rapid agonist-dependent internalization of 5-HT2A receptors, with significant internalization occurring as early as 5 min after agonist administration at 37 degrees. In GF-62 cells, agonist-induced internalization was blocked by preincubation with the 5-HT2A receptor antagonist ketanserin. Internalization was also temperature sensitive because agonist-induced internalization did not occur at 4 degrees. Dual-label experiments disclosed that 5-HT2A and transferrin receptors were internalized via the same endocytotic vesicles. These results suggest that 5-HT2A receptors and transferrin receptors are internalized via the endosomal pathway in GF-62 cells. Although 5-HT2A receptors were internalized, down-regulation, or loss of radioligand binding sites, did not occur. Our results demonstrate that agonists rapidly induce 5-HT2A receptor internalization via the endosomal pathway and that internalization can be dissociated from down-regulation.


Subject(s)
Endosomes/metabolism , Receptors, Serotonin/metabolism , Serotonin Receptor Agonists/pharmacology , 3T3 Cells , Animals , Down-Regulation , Ketanserin/pharmacology , Mice , Receptors, Serotonin/analysis , Receptors, Transferrin/analysis , Temperature
4.
J Pharm Sci ; 83(11): 1537-42, 1994 Nov.
Article in English | MEDLINE | ID: mdl-7891270

ABSTRACT

Supercritical fluid extraction (SFE) was shown to be an accurate and precise alternative to liquid extraction for sample preparation of sustained-release felodipine tablets (5 mg potency) while realizing an 80% reduction in solvent consumption. Extractions of felodipine spiked on an inert support were used to evaluate the solubility of felodipine in CO2 as well as analyte trapping after SFE. Even though the pure drug was found to be soluble in pure CO2, extractions of felodipine from the tablet matrix required moderate modifier concentrations [8.7% (v/v) methanol in CO2] in order to overcome strong matrix-drug interactions. Sequential static/dynamic extraction steps were also required to quantitatively recover the drug from the tablet matrix, indicating that the drug extraction was diffusion-limited. Average recoveries (n = 5) for the optimized SFE method were determined to be 4.93 mg felodipine tablet (98.6% claim) with an RSD of 1.2% versus those for the liquid extraction procedure (n = 5, 4.98 mg/tablet, 99.6% claim, 2.4% RSD). Similar levels of drug degradation (0.12% expressed as felodipine) were also obtained with both the traditional liquid extraction and with the SFE method.


Subject(s)
Felodipine/analysis , Chromatography, High Pressure Liquid , Delayed-Action Preparations , Felodipine/administration & dosage , Spectrophotometry, Ultraviolet
6.
JPEN J Parenter Enteral Nutr ; 13(3): 324-5, 1989.
Article in English | MEDLINE | ID: mdl-2503646

ABSTRACT

The use of small amounts of a dilute solution of heparin (less than or equal to 100 IU) to keep indwelling intravenous needles or catheters patent for intermittent venous access either for intravenous therapy or timed blood sampling is a common clinical practice. It is considered safe since the amount of heparin required is much less than that required for heparinization. Herein, we describe a 13-yr-old patient with malabsorption who developed clinically significant bleeding shortly after a diagnostic test which required multiple small injections of heparin for intermittent venous access (total amount of heparin administered was 600 units over 5 hr). The coagulopathy was corrected by a single dose (10 mg) of parenteral vitamin K. As our patient had multiple risk factors for the development of vitamin K deficiency including malabsorption, decreased food intake, and antibiotic use, we postulate that the small amount of heparin precipitated the coagulopathy by increasing the antiprotease activity of antithrombin III on abnormal factors X and II formed in the vitamin K deficient state. We would therefore recommend administration of vitamin K to patients who are at risk of developing vitamin K deficiency before using even small amounts of heparin.


Subject(s)
Blood Coagulation Disorders/etiology , Heparin/adverse effects , Vitamin K Deficiency/complications , Vitamin K/administration & dosage , Adolescent , Blood Coagulation Disorders/therapy , Humans , Malabsorption Syndromes/complications , Male , Parenteral Nutrition , Vitamin K/therapeutic use
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