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PubMed2004

Comparing dexmedetomidine prescribing patterns and safety in the naturalistic setting versus published data.

BACKGROUND: In clinical practice, new drugs may be used differently than the product labeling recommends. Furthermore, it often takes several years of use before adverse drug reactions (ADRs) are reported. OBJECTIVE: To compare prescribing patterns and safety of the newly released drug dexmedetomidine as observed in clinical practice with published data on the drug. METHODS: Information from a convenience sample of adults receiving dexmedetomidine as part of routine patient care at 10 institutions was retrospectively collected from June 27, 2001, to May 31, 2002. Investigators reviewed medical records daily and entered dosing information, patient demographics, and predefined categories of ADR severity and probability anonymously via the Internet on a secure server. RESULTS: Only 33% of the total sample (n = 136) of patients received a loading infusion of dexmedetomidine; however, maintenance dosing was usually within product labeling guidelines. Of note, 27.2% of patients received dexmedetomidine above the maximum dose and 33.8% received the drug beyond 24 hours. Some patients (15.4%) were never mechanically ventilated, while 59.5% received dexmedetomidine following extubation for an average of 11.3 hours. ADRs were reported in 30% of patients: 20% of the reactions required treatment or increased length of stay. Hypotension was the most common ADR, occurring in 22.7% of patients. Bradycardia was reported in 4.4% of patients. The rate and type of ADRs were similar in patients receiving dexmedetomidine >24 hours compared with the total population. CONCLUSIONS: Dexmedetomidine is prescribed within product labeling guidelines except for low use of a loading dose, some patients received the drug at doses above the maximum, and others received it for longer than 24 hours. Since ADR rates are similar to those of other published reports, dexmedetomidine maintained its expected safety profile in our patients.

Dexmedetomidine↗
PubMedDate not supplied

Extension of AOAC official method 999.14 (choline in infant formula and milk) to the determination of choline in dietary supplements.

AOAC Official Method 999.14 is applicable for the determination of choline in milk and infant formulas. To date, its use has not been extended beyond these matrixes. We modified Official Method 999.14 and applied it to the determination of choline in a range of choline-containing dietary supplements. Dietary supplement tablets, capsules, wafers, softgels, liquid products, and drink powders were included. We found that the standard curve could be extended to cover a wider range of choline concentrations and defined a procedure for the use of Norit for samples in which the vitamin C content was high enough to interfere with the analysis. Recoveries of choline added to infant formula powders and to representative dietary supplement tablets, capsules, powdered drink mix, and wafer products were 85-114%. The use of Norit during the procedure did not affect the recovery of choline added to infant formula powders or to dietary supplements. An alkaline digestion was included for use with a product containing lecithin as the sole source of choline. Ten of 11 dietary supplement products analyzed by the modified method contained amounts of choline at or above declarations found on the product labels. The remaining product contained about 40% of the label-declared amount of choline.

Animals↗
PubMed1997

Role of pyruvate carboxylase in facilitation of synthesis of glutamate and glutamine in cultured astrocytes.

CO2 fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with 14CO2 and the incorporation of 14C into medium or cell extract products was determined. After chromatographic separation of 14C-labelled products, the fractions of 14C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 mM increased pyruvate carboxylase flux as observed by increases in the 14C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 mM almost doubled CO2 fixation. When 25 mM bicarbonate plus 0.5 mM pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 mM bicarbonate plus 5 mM pyruvate, the rate of appearance of [14C] glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate --> alpha-ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 mM) had no effect on net 14CO2 fixation, but did alter the distribution of 14C-labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or 14C incorporation). Low rates of conversion of alpha-[14C]ketoglutarate to [14C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of alpha-ketoglutarate is inactive under conditions studied in these cultured astrocytes. We conclude that pyruvate carboxylase is required for de novo synthesis of glutamate plus glutamine, but that conversion of alpha-ketoglutarate to glutamate may frequently be the rate-limiting step in this process of glutamate synthesis.

Ammonia↗
PubMed1989

Transcription-terminating lesions induced by bifunctional alkylating agents in vitro.

The present study was initiated to determine if DNA damage induced by the bifunctional anti-tumor alkylating agents melphalan, nitrogen mustard, a spontaneously activated derivative of cyclophosphamide or chlorambucil inhibits transcription in vitro, and to determine if the potential sites of transcription termination correlate with the sites of N7 guanine adducts predominantly formed by these agents. To assess drug effects on in vitro transcription, linearized plasmid DNA containing the 420-bp PstI fragment of exon two of the human c-myc oncogene was incubated with various concentrations of the drugs. After drug removal and further drug-free incubation, the sense strand of the c-myc insert was transcribed with either of two bacteriophage RNA polymerases in the presence of [32P]UTP. The labeled products of the reaction were electrophoresed next to the labeled products of RNA sequencing reactions, and the location of transcription termination along the DNA template was determined. The sites of transcription termination were then compared with the sites of drug-induced guanine N7 alkylation in the template, as determined by modified Maxam-Gilbert sequencing. At the drug exposures examined, all the drugs were shown to alkylate any guanine in the template. Transcription of this alkylated DNA, however, resulted in RNA molecules truncated not at every alkylated guanine, but at various discrete sites throughout the template. Transcription was terminated at every adenine pair examined in the melphalan-treated template, at selected guanine pairs in the nitrogen-mustard-treated template, and at selected adenine-guanine and guanine-adenine pairs in the chlorambucil-treated template. Transcription of cyclophosphamide-derivative-treated DNA was unaffected. These results suggest that only some bifunctional alkylating agents induce DNA damage capable of terminating transcription in vitro, and that these agents do so in a sequence-specific, drug-specific manner inconsistent with patterns of guanine N7 alkylation.

Alkylating Agents↗
PubMed1984

In vitro synthesis of low molecular weight proteins in human platelets: absence of labelled release products.

Isolated human blood platelets incubated at 37 degrees C in vitro incorporated labelled amino acids into compounds which included some low molecular weight (less than 80KDa) proteins, as determined by autoradiography after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Non-dialysable plasma factor(s) inhibited both uptake and incorporation, which although unaffected by Actinomycin D, was inhibited partly by Chloramphenicol and almost completely by Puromycin and Cycloheximide, results which confirm that synthesis is directed by pre-existing mRNAs, some of which is mitochondrial. Assuming that the mRNA coding for proteins which are truly "platelet specific" must be present in megakaryocyte cytoplasm, we investigated the possibility that such RNA may be sufficiently stable for its translation to continue in platelets. Although leakage from platelet alpha-granules and cytoplasm during incubation was negligible and platelets retained their secretory potential we were unable to detect radiolabelled proteins in thrombin-released material after incubation. We conclude that either alpha-granule proteins are not synthesised in platelets or their megakaryocyte progenitors, or that their mRNAs become degraded by the time platelets reach the peripheral circulation. Alternatively, the mechanism which concentrates these proteins in granules does not function in circulating platelets.

Amino Acids↗
PubMed1972

Initiation of picornavirus protein synthesis in ascites cell extracts.

The current model of picornavirus protein formation implies that initiation of protein synthesis occurs at a single site on the viral RNA, and that the large polypeptide formed is later cleaved. A direct test of this model was made in vitro by studying the incorporation of [(35)S]methionine from rabbit liver Met-tRNA(M) (Met) and fMet-tRNA(F) (Met) into encephalomyocarditis virus RNA-coded proteins in extracts of Ehrlich ascites cells. The incorporation of N-formylmethionine was complete within 5 min, while utilization of Met-tRNA(M) (Met) continued for 20 min. Tryptic digests of [(35)S]methionine-labeled products from Met-tRNA(M) (Met) analyzed by anion-exchange chromatography yielded more than 30 peptides, as compared to about 15 [(35)S]methionine-labeled peptides from purified encephalomyocarditis virus. In contrast, products labeled with fMet-tRNA(F) (Met) yielded one major (26)S-labeled tryptic peptide. The N-terminal location of methionine in this peptide was verified by Edman degradation. One predominant N-terminal tryptic peptide was also obtained with fMet-tRNA(F) (Met) when mouse Elberfeld and mengo-virus RNAs were used as messengers. On the basis of N-terminal compared with internal labeling of the products, no evidence for in vitro post-translational cleavage was found. The results are consistent with a single initiation site for synthesis of picornavirus proteins.

Animals↗
PubMed2006

Unidentified inert ingredients in pesticides: implications for human and environmental health.

BACKGROUND: By statute or regulation in the United States and elsewhere, pesticide ingredients are divided into two categories: active and inert (sometimes referred to as other ingredients, adjuvants, or coformulants). Despite their name, inert ingredients may be biologically or chemically active and are labeled inert only because of their function in the formulated product. Most of the tests required to register a pesticide are performed with the active ingredient alone, not the full pesticide formulation. Inert ingredients are generally not identified on product labels and are often claimed to be confidential business information. OBJECTIVES: In this commentary, we describe the shortcomings of the current procedures for assessing the hazards of pesticide formulations and demonstrate that inert ingredients can increase the toxicity of and potential exposure to pesticide formulations. DISCUSSION: Inert ingredients can increase the ability of pesticide formulations to affect significant toxicologic end points, including developmental neurotoxicity, genotoxicity, and disruption of hormone function. They can also increase exposure by increasing dermal absorption, decreasing the efficacy of protective clothing, and increasing environmental mobility and persistence. Inert ingredients can increase the phytotoxicity of pesticide formulations as well as the toxicity to fish, amphibians, and microorganisms. CONCLUSIONS: Pesticide registration should require full assessment of formulations. Evaluations of pesticides under the National Environmental Policy Act, the Endangered Species Act, and similar statutes should include impact assessment of formulations. Environmental monitoring for pesticides should include inert ingredients. To enable independent research and risk assessment, inert ingredients should be identified on product labels.

Environment↗
PubMed2003

The rheology of liquids: a comparison of clinicians' subjective impressions and objective measurement.

Texture-modified diets are commonly prescribed for patients with dysphagia; it is therefore important to demonstrate that clinicians form accurate impressions of the rheological (flow) properties of the items that they recommend for their clients. We explored the correlation between objective rheological measurement and clinicians' subjective impressions of liquid consistency, rated on the bases of product labeling and sampling. Ten liquids, ranging from thin through nectar-thick and honey-thick to spoon-thick consistencies, were selected for study. Rheological analysis was conducted using a CarriMed CSL Controlled Stress Rheometer. Fifty speech-language pathologists ranked the liquids in order of perceived viscosity, based on their interpretation of the product packaging and label. Product nomenclature proved insufficient to accurately represent the consistency class to which each liquid belonged. A second group of 16 speech-language pathologists rated the perceived relative viscosity and density of nectar-thick and honey-thick juice items in blinded two-point discrimination tests of stirring-resistance, oral manipulation, and vessel weight. Physical sampling of these two products enabled clinicians to reliably perceive relative viscosity and density differences between the nectar- and honey-thick items.

Deglutition Disorders↗
PubMed1975

Regulation of oxaloacetate, aspartate, and malate formation in mesophyll protoplast extracts of three types of c(4) plants.

The use of mesophyll protoplast extracts from various C(4) species has provided an effective method for studying light-and substrate-dependent formation of oxaloacetate, malate, and asparate at rates equivalent to whole leaf C(4) photosynthesis. Conditions regulating the formation of the C(4) acids were studied with protoplast extracts from Digitaria sanguinalis, an NADP-malic enzyme C(4) species, Eleusineindica, an NAD-malic enzyme C(4) species, and Urochloa panicoides, a phosphoenolpyruvate (PEP) carboxykinase C(4) species. Light-dependent induction of CO(2) fixation by the mesophyll extracts of all three species was relatively low without addition of exogenous substrates. Pyruvate, alanine and alpha-ketoglutarate, or 3-phosphoglycerate induced high rates of CO(2) fixation in the mesophyll extracts with oxaloacetate, malate, and aspartate being the primary products. In all three species, it appears that pyruvate, alanine, or 3-phosphoglycerate may serve as effective precursors to the formation of PEP for carboxylation through PEP-carboxylase in C(4) mesophyll cells. Induction by pyruvate or alanine and alpha-ketoglutarate was light-dependent, whereas 3-phosphoglycerate-induced CO(2) fixation was not.Several differences between these species representing the three C(4) groups were observed. Substrate induction of CO(2) fixation in mesophyll protoplast extracts of D. sanguinalis gave malate as a major product; only by an apparent exchange reaction with cold aspartate did substantial label appear in aspartate (up to 53% of labeled products). In contrast, aspartate was a major product when alanine and alpha-ketoglutarate served as inducing substrates with E. indica (up to 57%) and U. panicoides (up to 86%). With induction by pyruvate or 3-phosphoglycerate, mesophyll preparations of U. panicoides and E. indica were less effective in forming malate (up to 31% of products) than D. sanguinalis (up to 87% of products). After 2 seconds of whole leaf (14)CO(2) fixation, malate was the major labeled product (57%) with D. sanguinalis, whereas with E. indica and U. panicoides aspartate was the predominant product (73% and 76%, respectively).With mesophyll protoplast extracts of D. sanguinalis, aspartate inhibited CO(2) fixation (about 50% at 0.6 mm), while malate was relatively uninhibitory at comparable concentrations. CO(2) fixation by mesophyll protoplast extracts of E. indica was inhibited by malate (about 50% at 0.6 mm), while aspartate was relatively uninhibitory. With mesophyll preparations of U. panicoides, malate or aspartate (2 mm) caused only slight inhibition of CO(2) fixation. The regulation of aspartate and malate synthesis in C(4) mesophyll cells is discussed relative to initial products of photosynthesis in C(4) species in vivo and species differences in the mechanisms of C(4) photosynthesis.

Journal Article↗
PubMed1988

[New aspects of nuclear medicine diagnosis of kidney function: improved potential by pharmacologic intervention and quantitative analytic procedures].

In nuclear medicine new trends in the diagnosis of renal function are based on the introduction of new radiopharmaceuticals, improvements in the methodological part of the procedure and precise pharmacological intervention in response to given indications. Tc99m mercaptoacetyltriglycine (Tc99m MAG3) was tested as replacement for I123 orthoiodohippuric acid (I123 oIH) both in the form of a HPLC purified substance and as an impure kit preparation. HPLC purified Tc99m MAG3 clearance determinations in anuric patients showed a low extrarenal excretion amounting to only about 5% of the total clearance in normal patients. Kit preparations yielded about 90% of the labelled product; impurities were pertechnetate, reduced hydrolyzed Tc99m and chemically unidentified labelled products which showed a significantly lower renal, but increased hepatobiliary excretion in comparison with Tc99m MAG3. The renal clearance with kit preparations of Tc99m MAG3 was 55% of the clearance with oIH at a comparable urinary excretion. Significantly higher protein binding and therefore, a decrease in the distribution volume of Tc99m was found in comparison with I123 oIH. No difference was recorded between the two substances with respect to the renogram curves in normal subjects, apart from a modest delay in the elimination of Tc99m MAG3. For clinical purposes kit preparations of Tc99m MAG3 proved equal to I123 oIH. The influence of angiotensin converting enzyme (ACE) inhibitors (captopril) leads to characteristic changes in the renograms of patients with Goldblatt hypertension. Quantitative criteria for the evidence of haemodynamically significant renal artery stenosis were derived from investigations without and with captopril (25 mg) (I123 oIH and Tc99m DTPA) in 21 patients with essential hypertension. The criteria were defined as follows: a delay in peak activity (Tmax) in the I123 oIH captopril renogram exceeding 2 minutes as compared with the baseline value and/or a lower uptake of Tc99m DTPA in comparison with the uptake of I123 oIH (uptake quotient I123 oIH/Tc99m DTPA greater than 1.2). The diagnostic and prognostic potential of the captopril renogram was compared with that of the captopril test by investigating 34 patients with renal artery stenosis (23 uni-, 11 bilateral) (atherosclerosis: 23, fibromuscular hyperplasia: 11). The captopril renogram was positive more often (n = 12) than the captopril test (n = 4) in patients without renal functional impairment of the stenosed kidney. Similar results were obtained with both methods in patients with atrophic kidneys: captopril renography was positive in all cases with a positive captopril test.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed1996

Use of terfenadine and contraindicated drugs.

OBJECTIVE: To assess changes in concurrent use of products containing terfenadine and contraindicated macrolide antibiotics (erythromycin, clarithromycin, troleandomycin) and imidazole antifungals (ketoconazole, itraconazole) following reports of serious drug-drug interactions and changes in product labeling. DESIGN: Retrospective review of computerized pharmacy claims. SETTING: A large health insurer in New England. PATIENTS: Health plan members with 1 or more paid pharmacy claims for products containing terfenadine between January 1990 and June 1994. MAIN OUTCOME MEASURES: Among persons with paid claims for terfenadine in any given month, percentage with a prescription for any contraindicated drug that alternatively was dispensed on the same day as ("same-day dispensing") or had therapy days that overlapped those of ("overlapping use") a prescription for terfenadine. RESULTS: Concurrent use of terfenadine and contraindicated drugs declined over the study period. The rate of same-day dispensing declined by 84% from an average of 2.5 per 100 persons receiving terfenadine in 1990 to 0.4 per 100 persons during the first 6 months of 1994, while the rate of overlapping use declined by 57% (from 5.4 to 2.3 per 100 persons). Most cases involved erythromycin. CONCLUSIONS: Despite substantial declines following reports of serious drug-drug interactions and changes in product labeling, concurrent use of terfenadine and contraindicated macrolide antibiotics and imidazole antifungals continues to occur.

Anti-Bacterial Agents↗
PubMed1998

[Immunohistochemical study on the progression of colorectal cancer--with respect to apoptotic index, expression of apoptosis-related gene products, and labeling index of proliferating cell nuclear antigen (PCNA)].

To clarify the biological changes in the development and progression of colorectal cancer, immunohistochemical examination was performed with a particular focus on the activity of apoptosis, the expression of apoptosis-related gene products and cell proliferation activity, assessed by the labeling index of proliferating cell nuclear antigen (PCNA). Seventy-six resected specimens of colorectal cancer were used to investigate the expression of apoptosis-related gene products, Bcl-2 protein and PCNA labeling index, as well as the apoptotic index using the TUNEL method. Seventy-five percent of 60 advanced cancer specimens was negative for Bcl-2 protein, and the proportion was higher than that in early cancer specimens. The apoptotic index (AI) in the advanced cancers was significantly higher than in the early stage of cancers. Meanwhile, the percentage of PCNA-positive cells for the advanced cancers was significantly higher than for early cancer. This study demonstrated a decrease in Bcl-2 protein expression, an increase in tumor cell apoptosis, and opposite an increase of cellular proliferation activity in the progression of colon cancer from early to the advanced stage of the disease.

Apoptosis↗
PubMed1987

Newly synthesized proinsulin/insulin and stored insulin are released from pancreatic B cells predominantly via a regulated, rather than a constitutive, pathway.

The pancreatic B cell has been used as a model to compare the release of newly synthesized prohormone/hormone with that of stored hormone. Secretion of newly synthesized proinsulin/insulin (labeled with [3H]leucine during a 5-min pulse) and stored total immunoreactive insulin was monitored from isolated rat pancreatic islets at basal and stimulatory glucose concentrations over 180 min. By 180 min, 15% of the islet content of stored insulin was released at 16.7 mM glucose compared with 2% at 2.8 mM glucose. After a 30-min lag period, release of newly synthesized (labeled) proinsulin and insulin was detected; from 60 min onwards this release was stimulated up to 11-fold by 16.7 mM glucose. At 180 min, 60% of the initial islet content of labeled proinsulin was released at 16.7 mM glucose and 6% at 2.8 mM glucose. Specific radioactivity of the released newly synthesized hormone relative to that of material in islets indicated its preferential release. A similar degree of isotopic enrichment of released, labeled products was observed at both glucose concentrations. Quantitative HPLC analysis of labeled products indicated that glucose had no effect on intracellular proinsulin to insulin conversion; release of both newly synthesized proinsulin and insulin was sensitive to glucose stimulation; 90% of the newly synthesized hormone was released as insulin; and only 0.5% of proinsulin was rapidly released (between 30 and 60 min) in a glucose-independent fashion. It is thus concluded that the major portion of released hormone, whether old or new, processed or unprocessed, is directed through the regulated pathway, and therefore the small (less than 1%) amount released via a constitutive pathway cannot explain the preferential release of newly formed products from the B cell.

Animals↗
PubMed1995

Dosimetry of a D2/D3 dopamine receptor antagonist that can be used with PET or SPECT.

UNLABELLED: FIDA-2 (R-(+)-2,3-dimethoxy-5-iodo-N-[(1-4'-fluorobenzyl)-2-pyrrolidinyl) methyl] benzamide) is a simultaneously fluorinated and iodinated D2/D3 dopamine receptor antagonist. The purpose of this study was to measure its biodistribution and radiation dosimetry in humans. METHODS: Whole-body emission scans were sequentially acquired in eight healthy volunteers 24-43 hr after the intravenous administration of 101-150 MBq 123I FIDA-2. Regions of interest (ROIs) were placed on the initial set of conjugate emission images and transposed as a single set onto all the other scans without manipulating any of the regions for solid organs independently. The counts in each ROI were corrected for attenuation with transmission scans and compared to the net counts in images of the injection syringe containing the administered dose. The radiation doses were estimated with the MIRD formalism from the residence times for both the 18F- and 123I-labeled ligands. RESULTS: There were no subjective or objective pharmacological effects of the tracer on any of the subjects. The findings showed that the dose-limiting organ for the 123I-labeled product was the thyroid gland in this sample. If the 18F-labeled product had been used, then the urinary bladder would have received 0.086 mGy/MBq (0.32 rads/mCi) and become the dose-limiting organ. The effective dose equivalents were 0.025 mSv/MBq (0.092 rem/mCi) for both the 123I- and the 18F-labeled versions of the tracer. CONCLUSION: The data suggest that FIDA-2 can be used to produce relatively high contrast images of the D2/D3 dopaminergic system with substantially less than the maximum allowable radiation dose for research volunteers.

Adult↗
PubMed1994

Labelling of proteins with 11C in high specific radioactivity: [11C]albumin and [11C]transferrin.

A method for the 11C-labelling of proteins for use in positron emission tomography is presented. Human iron-free transferrin and human serum albumin were treated with [11C]cyanogen bromide of high specific radioactivity under physiological conditions to give the labelled proteins in high radiochemical yields using a simple and rapid procedure. In a typical experiment, 2.0 GBq of [11C]albumin was obtained in 28-32 min synthesis time counted from the end of bombardment. The radiochemical purity was in all cases > 98% and the specific radioactivity varied with the amount of protein between 10.7-141 GBq mumol-1 (0.3-3.8 Ci mumol-1). The radiochemical yield of labelled product as a function of pH and protein concentration was studied. The labelled products were characterized using both high performance liquid chromatography and high performance capillary electrophoresis with u.v.-and radiodetection in series.

Carbon Dioxide↗
PubMed1997

Serine protein kinase activity associated with rotavirus phosphoprotein NSP5.

The rotavirus nonstructural protein NSP5, a product of the smallest genomic RNA segment, is a phosphoprotein containing O-linked N-acetylglucosamine. We investigated the phosphorylation of NSP5 in monkey MA104 cells infected with simian rotavirus SA11. Immunoprecipitated NSP5 was analyzed with respect to phosphorylation and protein kinase activity. After metabolic labeling of NSP5 with 32Pi, only serine residues were phosphorylated. Separation of tryptic peptides revealed four to six strongly labeled products and several weakly labeled products. Phosphorylation at multiple sites was also shown by two-dimensional polyacrylamide gel electrophoresis (PAGE), where several isoforms of NSP5 with different pIs were identified. Analysis by PAGE of protein reacting with an NSP5-specific antiserum showed major forms at 26 to 28 and 35 kDa. Moreover, there were polypeptides migrating between 28 and 35 kDa. Treatment of the immunoprecipitated material with protein phosphatase 2A shifted the mobilities of the 28- to 35-kDa polypeptides to the 26-kDa position, suggesting that the slower electrophoretic mobility was caused by phosphorylation. Radioactive labeling showed that the 26-kDa form contained additional phosphate groups that were not removed by protein phosphatase 2A. The immunoprecipitated NSP5 possessed protein kinase activity. Incubation with [gamma-32P]ATP resulted in 32P labeling of 28- to 35-kDa NSP5. The distribution of 32P radioactivity between the components of the complex was similar to the phosphorylation in vivo. Assays of the protein kinase activity of a glutathione S-transferase-NSP5 fusion polypeptide expressed in Escherichia coli demonstrated autophosphorylation, suggesting that NSP5 was the active component in the material isolated from infected cells.

Animals↗
PubMed1983

Mechanism of synthesis of D-glucans by D-glucosyltransferases from Streptococcus mutans 6715.

Two glucosyltransferases from Streptococcus mutans 6715 were purified and separated. One of the glucosyltransferases synthesized an insoluble glucan, and the other, a soluble glucan. The enzymes were immobilized on Bio-Gel P-2 beads, and the mechanism of glucan synthesis was studied by pulse and chase techniques with 14C-sucrose. Label was associated with the immobilized enzymes. The label could be quantitatively released by heating at pH 2. Analysis of the labeled products from the pulse experiment showed labeled glucose and labeled glucan; the chase experiment showed labeled glucan and a significant decrease in labeled glucose. The glucans from the pulse and the chase experiments were separated from glucose by chromatography on Bio-Gel P-6. They were reduced with sodium borohydride, and the products hydrolyzed with acid. Analysis of the labeled products from the reduced and hydrolyzed, pulsed glucans showed labeled glucose and labeled glucitol; label in the glucitol was greatly decreased in the chase experiment. These experiments showed that glucose and glucan were covalently attached to the active site of the enzymes during synthesis, and that the glucose was being transferred to the reducing end of the glucan chain. A mechanism for the synthesis of the glucans is proposed in which there are two catalytic groups on each enzyme that holds glucosyl and glucanosyl units. During synthesis, the glucosyl and glucanosyl units alternate between the two sites, giving elongation of the glucans from the reducing end. The addition of increasing amounts of B-512F dextran to the insoluble-glucan-forming glucosyltransferase produced a decrease in the proportion of insoluble glucan formed and a concomitant increase in a soluble glucan. The total amount of glucan synthesized (soluble plus insoluble) was increased 1.6 times over the amount of insoluble glucan formed when no exogenous dextran was added. It is shown that the addition of B-512F dextran affects the solubility of the synthesized alpha-(1 to 3)-glucan by accepting alpha-(1-3)-glucan chains at various positions along the dextran chain, to give a soluble, graft polymer.

Enzymes, Immobilized↗
PubMed1985

Radioiodination and preliminary in vivo investigation of the alkaloid cryptolepine.

In this paper we report the successful labelling of cryptolepine with 131I using the chloramine T method. Quality control by ITLC of the labelled product showed 80% labelling but when the mixture was equilibrated with some amberlite (Cl-) anion resin to exchange the excess free iodide, the radiochemical purity was raised to greater than 90%. Preliminary biodistribution in a rat did not show specific localisation of the tracer; rather, rapid clearance from the blood was indicated. It appears that its main excretory pathway is the hepatobiliary tract despite its relatively small molecular weight of 365. Also, despite i.v. administration, an appreciable 8% uptake was found in the stomach at 1 h post injection, indicating the process of enterohepatic reflux. These findings may have some bearings on the antimicrobial property of the plant.

Adrenergic alpha-Antagonists↗