Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0040822 (tremor)
 18,428 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Immunocytochemical and biochemical techniques have been used to localize and characterize a novel plasma membrane-associated, neutral-pH-optimum alpha-L-fucosidase from rat spermatozoa. Light and electron microscopy specifically localized the fucosidase on the plasma membrane of the convex region of the principal segment of testicular and cauda epididymal sperm heads. Immunoreactivity for alpha-L-fucosidase was also detected in the Golgi apparatus of spermatocytes and spermatids but no immunoreactivity was observed in the acrosome. 2. Fractionation of epididymal sperm homogenates indicated that over 90% of the alpha-L-fucosidase activity was associated with the 48,000 g pellet. This pellet-associated activity could be solubilized with 0.5 M NaCl but not with 0.5% Triton X-100, suggesting that fucosidase is peripherally associated with membranes. Sucrose-density-gradient centrifugation of sperm homogenates indicated that fucosidase was enriched in the plasma membrane-enriched fraction. Analysis of alpha-L-fucosidase on intact epididymal sperm indicated that the enzyme was active, displayed linear kinetics and had a pH-activity curve (with an optimum near 7) which was comparable to that of fucosidase from epididymal sperm extracts. These results further suggest that fucosidase is associated with plasma membranes, and that its active site is accessible to fucoconjugates. Evidence that most of the fucosidase is associated with the exterior of the plasma membrane came from studies in which intact sperm had fucosidase activity comparable to that of sperm sonicates, and from studies in which approx. 90% of the fucosidase activity on intact sperm could be released from the sperm by gentle shaking with 0.5 M NaCl. Isoelectric focusing indicated that the NaCl-solubilized epididymal sperm fucosidase appears to have one major and one minor isoform with pIs near 7.2 and 5.2, respectively. SDS/PAGE and Western blotting indicated that the NaCl-solubilized extract of epididymal sperm contains two protein bands of 54 and 50 kDa which were highly immunoreactive with the IgG fraction of anti-fucosidase antibodies. Although the function of the novel sperm fucosidase is not known, its specific localization to the plasma membrane of the region of the rat sperm head involved in sperm-egg binding and its high enzymic activity at neutral pH on intact sperm suggest that this enzyme may have a role in sperm-egg interactions.
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PMID:Immunocytochemical localization and biochemical characterization of a novel plasma membrane-associated, neutral pH optimum alpha-L-fucosidase from rat testis and epididymal spermatozoa. 883 25

Ashbya gossypii can grow on triacyglycerol as carbon source. A degradation rate of 0.05 g x g-1 mycelial dry mass x h-1 was detected for soybean oil. Although this rate was within the sensitivity range of lipase assays no activity was detectable. On the other hand, extracellular lipase activity could be visualized by clearance halos round the growing mycelium when trioleoylglycerol was emulsified as the sole carbon source in agar plates. Variation of the culture conditions revealed that reduced shaking speed and decreased fat content in the medium led to detectable amounts of lipase in the supernatant of flask cultures. A maximal activity of 800 U x l-1 was obtained after 32 h of cultivation in flasks containing 1% yeast extract and incubated at 60 rpm. Because of its pI of 9.0, the enzyme could be purified in a single step by preparative isoelectric focusing. It appeared as a homogeneous protein in analytical isoelectric focusing and SDS/PAGE (M 35,000). The lipase was inactivated within minutes in stirred gas/water, trioleoylglycerol/water or oleic acid/water mixtures. These effects suggested an interface inactivation. This idea was supported by a stability modulation observed with the surfactant Pluronic F-68. Inactivation by oleic acid led to an aggregation of the lipase shown by gel filtration. Growth experiments performed under lipase-stabilizing conditions revealed a negative influence of glucose, glycerol or oleic acid on detectable lipase activity, probably due to a regulation of lipase formation. Inactivation and regulation thus explained the lack of detectable lipase activity in cultures of A. gossypii growing on triacylglycerol.
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PMID:Regulation and properties of a fungal lipase showing interfacial inactivation by gas bubbles, or droplets of lipid or fatty acid. 906 67

The present study reports on a 17-year-old male who ingested approximately 70 ml trichloroethene (TRI) in a suicide attempt. The patient developed fever, tremor, general motor restlessness, and sinus tachycardia and lost consciousness 5 h after poisoning. After 5 days of intubation under narcosis with forced hyperventilation and diuresis he regained consciousness. During this period blood and urine were collected and TRI and its metabolites were quantified. The highest concentration of TRI in blood was detected 13 h after ingestion. Trichloroethanol and trichloroacetic acid, metabolites of the cytochrome P450-mediated pathway, and N-acetyl-S-(1, 2-dichlorovinyl)-l-cysteine and N-acetyl-S-(2, 2-dichlorovinyl)-l-cysteine from the glutathione-dependent pathway of TRI were quantified in urine samples. Besides these known metabolites in humans, chloroacetic acid and dichloroacetic acid were identified for the first time in urine of a human exposed to TRI. Although the patient exhibited normal levels of glucose and total protein in urine, excretion of alpha1- and beta2-microglobulin as well as beta-NAG was significantly increased. In addition to these typical markers of selective tubule damage, analysis of the urinary protein pattern by SDS-PAGE revealed increased excretion of several low-molecular-mass proteins between 10,000 and 50,000 Da, clearly indicating tubular damage. Based on the elucidated glutathione-dependent mechanism for the nephrotoxicity of TRI, activation of the formed S-conjugates by beta-lyases to reactive intermediates may account for the observed renal effects after a single, high dose of TRI.
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PMID:Acute intoxication with trichloroethene: clinical symptoms, toxicokinetics, metabolism, and development of biochemical parameters for renal damage. 952 Mar 51

The aim of the present work was to study the release of a model protein, bovine serum albumin (BSA) encapsulated within biodegradable poly (D,L-lactide-co-glycolide) (PLGA) microspheres prepared by a modified solvent evaporation method using a double emulsion. These microspheres were characterized for size, morphology, surface adsorbed protein, encapsulation efficiency and release kinetics. Two types of in vitro assays were developed to evaluate the influence of shaking and the addition of surfactants on the release profile of encapsulated protein. Scanning electron microscopy (SEM) observation showed spherical and smooth surface particles, with a mean particle size of 20 microm and an encapsulation efficiency of 81%. Surface associated protein was about 25%. The in vitro release profile showed a biphasic pattern described by means of a biexponential equation. There was an initial burst effect due to the release of the protein adsorbed on the microsphere surface and a sustained release phase due to protein diffusion through the channels or pores formed in the polymer coat. The release obtained profiles in static and dynamic assays showed statistically significant differences in the amount of the released protein, whereas the release rate was not affected. The burst effect was 28.30+/-1.63% and 35.20+/-1.50% of the total encapsulated protein for the static and dynamic assays respectively. The addition of surfactants (SDS) to the release medium increased the rate and the amount of drug released. In both assays the value of the slow release rate constant, beta, was 0.029+/-0.002 days(-1) when the surfactant was added, and 0.017+/-0.0014 days(-1) in the samples without surfactant. It is believed that the surfactant leads to an increase in the microsphere surface polarity which allows channel and pore formation inside the polymer through which the protein diffuses easily.
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PMID:Influence of shaking and surfactants on the release of bsa from plga microspheres. 972 63

Hair pigmentation is a critical factor in the interpretation of the concentration of certain compounds and their metabolites incorporated into hair. Melanin is responsible for the pigmentation. The color and the melanin content of human hair samples differs over a wide range. Once deposited into hair, drug may remain detectable for a period of months to years. However, if drug disposition into hair is influenced by those properties attributed to hair color, then certain persons may test positive more frequently than other persons. Removal of the melanin from hair digests prior to drug analysis may reduce the effect of melanin on the total drug concentration by excluding the drug bound to the pigment. In this study, the effect of melanin removal by centrifugation of hair digests on cocaine concentrations was investigated. Two sets of hair samples from five cocaine users were analyzed for cocaine and metabolites. A solution consisting of 10 mL of 0.5M Tris buffer (pH 6.4) to which is added 60 mg D,L-dithiothreitol, 200 mg SDS, and 200 U Proteinase K, was used to digest the hair. Two milliliters of this solution was added to 20 mg of hair and incubated at 37 degrees in a shaking water bath (90 oscillations/min) overnight. The samples were removed from the water bath and mixed. One set was centrifuged at 2000 rpm and divided into supernatant and melanin pellet. The other set was not centrifuged. Internal standards were added to all tubes. The samples were further extracted, derivatized, and analyzed by gas chromatography-mass spectrometry. A mean of 8.8% (standard deviation [SD] 7.0%) of the total cocaine concentration (supernatant and pellet) was left behind in the pellet. The same experiment was repeated except that the melanin pellet was redigested with 0.1 N HCl. After redigestion of the melanin pellet, the mean cocaine concentration in the pellet was 3.8% +/- 4.0% (mean +/- SD) of the total cocaine concentration in hair. These data demonstrate that removal of melanin from hair digests by centrifugation does not eliminate hair color bias when interpreting cocaine concentrations.
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PMID:Quantitation of cocaine in human hair: the effect of centrifugation of hair digests. 978 14

A portable device was developed and assembled from a stationary differential continuous flow centrifuge usually employed for blood cell separation, for the purpose of concentrating Cryptosporidium and Giardia from large volumes of water. Following compaction onto the wall of the disposable plastic centrifuge bowl and aspiration of residual water, the oocysts and cysts were dislodged by injection of a 20 ml solution containing 0.01% Tween-80 and 1% SDS and vigorous shaking. Following aspiration, the oocysts were pelleted, reacted with specific FITC-conjugated monoclonal antibodies, and enumerated via fluorescence microscopy. The entire procedure required about 2 h. Initially, 55% and 87% of Cryptosporidium oocysts and Giardia cysts, respectively, were recovered from 45 litres of tap water, and 27% and 57%, respectively, from river water. Adjustments in centrifuge speed and flow rates improved recovery to about 90% for Cryptosporidium oocysts and hence, this method compared favourably with the recently developed calcium carbonate flocculation method. It was superior in time requirement and volume flexibility, and showed a distinct advantage over the standard cartridge filtration method in all respects. The continuous flow centrifugation equipment is compact, mobile, flexible, and yields reproducibly high recovery rates. The ease of handling, speed of performance and minimal requirements for post-concentration equipment, reagents and labour make the system highly cost-effective. It appears to offer an improved method, well suited for use by water utilities for monitoring the burden of water-borne protozoan pathogens.
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PMID:Evaluation of a portable differential continuous flow centrifuge for concentration of Cryptosporidium oocysts and Giardia cysts from water. 1038 44

Conditions for the optimal expression of the human CYP1B1 hemoprotein in Escherichia coli have been investigated. CYP1B1 cDNA was prepared from a retinal cDNA template and used to generate cDNA fragments with modified 5'-sequences reported to allow enhanced expression in E. coli DH5alpha. Plasmids were constructed, using the pCWori+ expression vector and were used to examine necessity for thiamine, delta-aminolevulinic acid (ALA), and IPTG. The optimal shaking speed in an orbital incubator was 150 rpm at 30 degrees C. Higher speeds resulted in increased cell death and lower speeds resulted in lower expression of cytochrome P450. IPTG was necessary for this expression system, which makes use of the lac repressor, but levels above 0.5 mM were without additional benefit. We were able to show thiamine to be unnecessary in this expression system, although included by others expressing CYP1B1. ALA has been reported to enhance expression of several different forms of cytochrome P450. We examined the dependence of CYP1B1 expression on ALA. The expression proved to be highly dependent upon this heme precursor, with levels of CYP1B1 increasing approximately 20-fold, to 920 nmol/l in the presence of up to 2.5 mM ALA. The question of whether heme synthesis and apoprotein synthesis were coupled was then investigated. It could be shown that although heme synthesis was not limiting (CYP101 holoenzyme expression in the absence of ALA was four times higher than the ALA-supported CYP1B1 holoenzyme expression), it was necessary for optimal expression of CYP1B1. CYP1B1 protein synthesis appears to be coupled to heme precursor availability, as seen by SDS-PAGE, because in the absence of heme precursor apocytochrome P450 1B1 does not accumulate.
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PMID:Enhanced expression of CYP1B1 in Escherichia coli. 1078 90

A lipase-producing bacterium, Acinetobacter calcoacetius LP009, was isolated from raw milk. The optimum conditions for growth and lipase production by A. calcoaceticus LP009 were 15 degrees C with shaking at 200 rpm in LB supplemented with 1.0% (v/v) Tween 80. The crude lipase was purified to homogeneous state by ultrafiltration and gel filtration chromatography on Sephadex G-100. Its molecular weight determined by SDS-PAGE was 23 kDa and it exhibited maximum activity at pH 7.0 and 50 degrees C. It was stable over the pH range of 4.0 to 8.0 and at temperatures lower than 45 degrees C. It was a metalloenzyme that is positionally non-specific and had the ability to improve fat hydrolysis in soybean meal and in premixed animals feed.
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PMID:Purification and characterization of lipase from psychrophilic Acinetobacter calcoaceticus LP009. 1095 Jan 91

Glycoxidative modification of various body proteins, including fibronectin (FN), has been shown to change their structural and functional properties, and be implicated in pathogenesis of diabetic complications. Little is known about the role of secondary structure of glycoxidative FN (gFN) in its domain functions. gFN was prepared by incubation with 25 and 200 mM glucose in 0.2 M sodium phosphate buffer at 37 degrees C on a shaking plate under aerobic and sterile conditions for various time intervals up to 49 days, being defined as gFN25 and gFN200, respectively. Unmodified FN (uFN) was prepared by incubation in 0.2 M sodium phosphate buffer without any glucose at 4 degrees C for 49 days. The extent of glycoxidative modification was examined using a noncompetitive enzyme-linked immunosorbent assay with an antibody against N(epsilon) -(carboxymethyl)lysine (CML), one of the major glycoxidation products. The binding activities of uFN and gFN to collagen, gelatin and heparin were determined by a solid phase enzyme immunoassay or heparin-affinity HPLC. Cell attachment was estimated by the extent of adhesion of FITC-labeled smooth muscle cells to uFN or gFN. Conformational change in gFN was detected by SDS-polyacrylamide gel electrophoresis and spectroscopy (circular dichroism). CML was detected in gFN25 and gFN200 after 49 and 21 days of incubation, respectively. Levels of CML were about six-fold higher in gFN200 than in gFN25 after 49 days. Both gFN25 and gFN200 showed a significant decrease in the ability of binding to collagen and gelatin after 7 days of incubation. The binding activity for heparin was significantly decreased in both gFN25 and gFN200 after one day. Cell attachment activity was reduced to 89% and 76% of the unmodified form in both gFN25 and gFN200 after 49 days, respectively. High molecular weight materials were found in gFN25 and gFN200 after 21 and 7 days, respectively. CD spectrum showed that gFN25 had lost its native conformation after 3 days of incubation, depending upon the concentration and incubation interval of the applied glucose. These in vitro results suggest that the loss of native conformation may reduce the domain functions of gFN, including binding activity to macromolecular ligands and cell attachment, and may play a major role in the pathogenesis of diabetic complications.
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PMID:Causal relationship between conformational change and inhibition of domain functions of glycoxidative fibronectin. 1099 58

Eighty-three isolates from different soil samples exhibited the potential for producing active extracellular phytase. The most active fungal isolate with phytase activity was identified as Penicillium simplicissimum. In shaking culture with enrichment medium, the highest extracellular phytase activity of the producing strain was 3.8 U/mL. The crude enzyme filtrate was purified to homogeneity using ultrafiltration. IEC and gel filtration chromatography. The molar mass of the purified enzyme was estimated to be 65 kDa on SDS-PAGE. The saccharide identification with periodic acid-Schiff reagent (PAS) and activity recognition by 1-naphthyl phosphate was all positive. The isoelectric point of the enzyme, as deduced by isoelectric focusing, was pH 5.8, the optimum pH and temperature being pH 4.0 and 55 degrees C, respectively. The purified enzyme revealed broad substrate specificity and was strongly inhibited by Fe2+, Fe3+ and Zn2+; however, no inhibition was found by EDTA and PMSF. Phytase activity was inhibited when 2 mmol/L of dodecasodium phytate was added and the Km for it was determined to be 813 mmol/L.
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PMID:Isolation and characterization of a novel phytase from Penicillium simplicissimum. 1127 18


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