Monitoring biotransformations in polyamide fibres

The enzymatic hydrolysis of polyamide fibres yields amino and carboxylic groups. These groups can be found in solution treatments as polyamide monomers and soluble oligomers. The amino groups can also be found at the surface of the fibres as end group chains. In this paper we report two methods to quantify the formation of these groups as a result of the enzymatic action. Soluble amino groups can be quantified with 2,4,6-trinitrobenzenesulfonic acid (TNBS), which yields a coloured complex which can be determined spectrophotometrically. The amino groups on the fibre surface can be quantified by reaction with a wool reactive dye and determination of colour intensities after a dyeing procedure below the glass transition temperature of polyamide.     

Direct enzymatic procedure for the determination of liver glycogen

A method is proposed to measure glycogen content in liver homogenates without extraction and acid hydrolysis of tissue glycogen. Homogenates were treated with amyloglucosidase, which degrades glycogen to glucose, and the glucose was the determined enzymatically by the use of glucose oxidase and peroxidase. The method was shown to yield nearly complete (99%) recoveries of standard glycogen, while 5 hr of acid hydrolysis of standard glycogen were required to obtain comparable recoveries. When compared to an acid hydrolysis method for liver, amyloglucosidase degradation of rat liver glycogen and subsequent determination of glucose resulted in higher values for glycogen content. The amyloglucosidase, glucose oxidase: peroxidase method has the advantage of rapidity, whereas the traditional method consisting of extraction, precipitation, and acid hydrolysis is not only time consuming, but may also be subject to losses of glycogen in each step.     

Determination of the glycogen content in single neutrophil leukocytes using a micromodel of leukocyte glycogen.

The kinetics of the periodic acid oxidation as part of the periodic acid-Schiff reaction was studied by combined microinterferometry and microspectrophotometry in micromodel systems of liver glycogen and leukocyte glycogen as well as in neutrophil leukocytes. The initial formation of Schiff-positive chromogens was more rapid in neutrophil leukocytes than in liver or leukocyte glycogen. The chromogen formation was, however, practically complete within 60 min in both neutrophil leukocytes and leukocyte glycogen, but this did not appear to be the case in liver glycogen. Differences in the rate of chromogen formation may depend on various factors such as differences in the source and treatment of the glycogen. The complete periodic acid-Schiff reaction appears to be a measure of the glycogen amount in neutrophil leukocytes and the microdroplet system of leukocyte glycogen is considered to be an appropriate model for the estimation of the glycogen amount in single neutrophil leukocytes. A mean value of 13.3 10-12 g glycogen per normal human neutrophil was found.     

On the activities of glycogen phosphorylase and glycogen synthase in the liver of the rat.

A procedure was developed for determination of glycogen synthase and phosphorylase activities in liver after various in vivo physiological treatments. Liver samples were obtained from anaesthetised rats by freeze-clamping in situ. Other procedures were shown to stimulate the activity of phosphorylase and depress the activity of glycogen in the liver. The direction of glycogen metabolism appears to be regulated by the relative proportions of the two enzymes, as shown by a strong positive correlation between total activities and active forms of phosphorylase and synthase. The enzyme activities responded as expected to stimuli such as insulin and glucose, which depressed phosphorylase and increased synthase activity, and glucagon, which increased phosphorylase and decreased synthase activity. In fasted animals approximately 50% of each enzyme was in the active form, which suggests the existence of a potential futile cycle for glycogen metabolism. The role for such a cycle in the regulation of glycogen synthesis and degradation is discussed.     

On the activities of glycogen phosphorylase and glycogen synthase in the liver of the rat

A procedure was developed for determination of glycogen synthase and phosphorylase activities in liver after various in vivo physiological treatments. Liver samples were obtained from anaesthetised rats by freeze-clamping in situ. Other procedures were shown to stimulate the activity of phosphorylase and depress the activity of glycogen in the liver. The direction of glycogen metabolism appears to be regulated by the relative proportions of the two enzymes, as shown by a strong positive correlation between total activities and active forms of phosphorylase and synthase. The enzyme activities responded as expected to stimuli such as insulin and glucose, which depressed phosphorylase and increased synthase activity, and glucagon, which increased phosphorylase and decreased synthase activity. In fasted animals approximately 50% of each enzyme was in the active form, which suggests the existence of a potential futile cycle for glycogen metabolism. The role for such a cycle in the regulation of glycogen synthesis and degradation is discussed.     

Continuous cultivation in a chemostat of the phototrophic procaryote,anacystis nidulans,under nitrogen-limiting conditions

Anacystis nidulans was grown photoautotrophically in a chemostat in the presence of light, air and CO2 as the sole carbon source. Either the amount of the nitrogen source in the medium or light intensity were used as growth-limiting parameters. 1. Cells of high glycogen content obtained by pre-incubation under nitrogen starvation conditions maintained their glycogen content during continuous cultivation. Both growth rate and the amount of cell-mass and of glycogen depended on the nitrate content of the medium and the light intensity. The values for the growth rate, the maximal rates of glycogen synthesis and of cell mass formation were 0.1 h-1, 6 mg/l.h and 17 mg/l.h, respectively. 2. Cells without glycogen which had been transferred from an exponentially growing batch culture to chemostat conditions showed increasing rates of growth and of cell mass formation when the light intensity was increased. A determination of specific values resulted in 0.15 h-1 for growth rate and 23 mg/1.h for cell mass formation. 3. The chemostat apparatus is described in detail.     

Enzymatic microdetermination of glycogen.

A method for the determination of isolated glycogen was developed. Glucose was released from glycogen with an amyloglucosidase from Rhizopus. The released glucose was determined with glucose oxidase and peroxidase utilizing diammonium 2,2′-azino-di-[3-ethyl-benzthiazoline sulfonate (6)] (ABTS) as a chromogenic substrate. The ABTS method was found to be three times as sensitive as the older o-dianisidine method. For rabbit liver glycogen, the results obtained with amyloglucosidase correlated highly with those obtained by acid hydrolysis.     

The effect of fixation on the chemical extraction of glycogen from rat liver

Synopsis Small samples of rat liver, weighing 15 mg or less, were either (a) frozen in liquid nitrogen or (b) fixed at 4°C for 5 min to 2 hr in absolute alcohol, alcoholic picric acid (Rossman's fluid), or aqueous picric acid (Bouin's fluid). The tissue samples were analysed for total glycogen content by a modification of the procedure described by Goodet al. (1933).Comparable yields of glycogen were extracted from freshly frozen and fixed tissue samples. The time of fixation had no apparent effect on the amount of glycogen that could be extracted chemically. Dissolved glycogen was not detectable in the fixatives.It is concluded that (a) the fixatives used in this study do not significantly affect the yield of chemically extractable glycogen from liver; (b) fixation is extremely rapid; and (c) alcoholic fixatives are not significantly superior to aqueous picric acid fixatives for preservation of chemically extractable glycogen in very small samples of tissue.     

A micromethod for the enzymatic estimation of the degree of glycogen ramification

A comparison of methods for the evaluation of glycogen content in liver tissue of rats has been carried out by determining the recoveries in the differential ethanol precipitation of glycogen from alkaline tissue digests as well as the actual quantitative equivalence between glycogen content and actual glucose measured. Hydrolytic/enzymatic methods gave lower results than non-specific chemical methods such as anthrone. These lower values, combined with the losses in the purification process resulted in much lower glycogen estimations than the actual estimated tissue content. A method has been devised for the measurement of glycogen ramification in small liver tissue samples, using neutral periodate oxidation of the molecule, followed by determination of the formic acid evolved from the branch ends with formic acid dehydrogenase. The method gave results very similar to the classical methods in which the acid formed is measured titrimetrically. Rat liver tissue contained a mean 323 +/- 69 mmol of glucose equivalents of glycogen per gram of tissue; this glycogen had a mean chain length of 11.4 +/- 0.8 units.     

Structural and functional studies on rabbit liver glycogenin

Glycogenin, the protein primer required for the biogenesis of muscle glycogen, has been isolated from rabbit liver glycogen. The protein comprised 0.0025% of liver glycogen by mass, 200-fold lower than the glycogenin content of muscle glycogen. Structural analyses, including determination of the amino acid sequence surrounding the glucosylated-tyrosine residue, showed identity with muscle glycogenin. Catalytically active liver glycogenin was partially purified and, like the skeletal muscle protein, catalysed an intramolecular, Mn2+- and UDP-Glc-dependent autoglucosylation reaction, forming a primer on which glycogen synthase could act. The results demonstrate that hepatic and muscle glycogenins are almost certainly identical proteins and that liver and skeletal muscle share a common mechanism for the biogenesis of glycogen molecules. The results also indicate that there is about one glycogenin molecule/liver glycogen alpha particle.     

Use of the penicillanic acid formation reaction for the analysis of penicillins. The quantitative determination of oxacillin]

A procedure for quantitative determination of oxacillin by means of spectrophotometry is described. The procedure is based on the capacity of penicillins to transform on heating in acid buffer solutions containing copper ions into respective penicillenic acids having specific absorption characteristics in the UV-spectrum region. The amount of oxacillin was estimated by the optical density of the solutions at a wave length of 335 nm corresponding to the absorption maximum of the penicillenic acid of oxacillin. The optimal conditions for the reaction of the penicillenic acid formation, i.e. pH of the buffer solution and the level of the copper ions in it, time and temperature of heating were determined. The data on the comparison of the specificity and reproducibility of the new method with the currently used ones for determination of oxacillin are presented.     

High-performance liquid chromatographic determination of d-amino acid oxidase activity

A new procedure for the assay of d-amino acid oxidase activity has been developed. α-Ketoisovaleric acid, derived from d-valine, was estimated by high-performance liquid chromatography after reaction with o-phenylenediamine to give the corresponding quinoxalinol derivative. α-Ketovaleric acid was used as an internal standard to ensure the reproducibility of the method. As an example of application, kidney cortex homogenates were analyzed for their d-amino acid oxidase activity. The advantages of the presented procedure for the determination of the enzymatic activity in biological samples compared with previously reported procedures are discussed.     

Directed screening of bacteria--destructive agents of surface-active substances

A procedure for detection of bacteria destructing cationic surface active substances (CSAS) was developed. The procedure is based on changing colour of pyrocatechin violet, an organic dye from geen-blue in the presence of a CSAS to red-brown in its absence. After application of pyrocatechin violet to bacterial macrocolonies grown on a synthetic medium with a CSAS as the only source of C red-brown zones developed around the colonies which was probably indicative of the CSAS biodestruction. 55 strains of bacteria destructing undecylpyridinium bromide, a CSAS were isolated from water of sewage purification plants. The study of their spectrum with respect to CSASs of different chemical structure revealed 6 bacterial strains capable of destructing all the nine test preparations. It was shown that biodestruction of the CSASs was associated with the long carbon chain and depended on the presence of the substitute in the aromatic ring and branching in the side chain. Therefore, the procedure provides screening of microbes destructing CSASs and may be used for rapid determination of biodestruction of CSASs with different chemical structures.     

Protein Synthesis in Relation to Sporulation and Meiosis in Yeast

The dependence upon protein synthesis of physiological and biochemical events occurring during yeast sporulation was investigated. Protein synthesis was inhibited by cycloheximide. There was an early, irreversible sensitivity to inhibition with respect to cell viability and ascus formation; inhibition was reversible only if the cells were inhibited after, but not prior to, 2 to 3 h in sporulation medium. Interruption of protein synthesis of any time during sporulation inhibited all measurable metabolic and sporulation-specific processes except protein breakdown and, to some extent, ribonucleic acid synthesis. The time interval between the occurrence of an event and the protein synthesis necessary for that event was determined to be 2 to 3 h for ascus formation, 相似文献   

Ultrastructural localization of glycogen in the granulocytes of normal rabbit bone marrow.

The glycogen of rabbit granulocytes has been studied in glutaraldehyde and osmium tetroxide fixed bone marrow by the periodic acid-thiocarbohydrazide-silver proteinate procedure (PA-TCH-SP). The PA-TCH-SP procedure involved the staining of intracytoplasmic glycogen more densely than the routine lead citrate staining. The PA-TCH-SP procedure demonstrated the intracytoplasmic glycogen in all three kinds of granulocytes. Though a sequence of intensity was observed in each stage of cell maturation, intracytoplasmic glycogen increased generally in accordance with cell maturation in the granulocytes. Functional significance of the glycogen in the granulocytes was discussed in relation to its staining. A very weak reaction in the granules of the granulocytes was described in relation to their contents.     

Ultrastructural localization of glycogen in the granulocytes of normal rabbit bone marrow

Summary The glycogen of rabbit granulocytes has been studied in glutaraldehyde and osmium tetroxide fixed bone marrow by the periodic acid-thiocarbohydrazide-silver proteinate procedure (PA-TCH-SP). The PA-TCH-SP procedure involved the staining of intracytoplasmic glycogen more densely than the routine lead citrate staining. The PA-TCH-SP procedure demonstrated the intracytoplasmic glycogen in all three kinds of granulocytes. Though a sequence of intensity was observed in each stage of cell maturation, intracytoplasmic glycogen increased generally in accordance with cell maturation in the granulocytes. Functional significance of the glycogen in the granulocytes was discussed in relation to its staining. A very weak reaction in the granules of the granulocytes was described in relation to their contents.     

Characteristic properties of the complex of glycogen synthase with glycogen

The glycogen synthase I--glycogen complex isolated from rabbit skeletal muscles is stable during precipitation with trichloroacetic acid and Sepharose 2B chromatography. The complex catalyzes the synthesis (lengthening) of the alpha-1.4-glucosyl chains when endogenous or exogenous enzyme-free glycogen is used, the initial rates of this synthesis being identical. Preincubation with glycogen does not cause activation of the complex or formation of additional glycogen synthase I--polysaccharide bonds. The complex is characterized by saturation with respect to glycogen; the molar concentration ratios of the non-reducible chain and protein monomer within the complex does not exceed 100. An increase in the length of the synthesized alpha-1.4-glycosyl chains of glycogen results in a decrease of the rate of the glycogen synthase reaction in time.     

Spectrofluorimetric determination of certain biologically active phenothiazines in commercial dosage forms and human plasma

A validated simple and sensitive spectrofluorimetric method was developed for the determination of chlorpromazine hydrochloride, promethazine hydrochloride, trifluperazine hydrochloride, thioridazine hydrochloride, perazine maleate and oxomemazine. The method was based on condensation of malonic acid/acetic anhydride (MAA) under the catalytic effect of the tertiary amine moiety of the studied phenothiazines to provide a deep yellow to brown colour with green florescence. Relative fluorescence intensity of the products was measured at λ_exc 398 nm and λ_em 432 nm. Different variables affecting the reaction were studied and optimized. The method was successfully applied for the determination of the studied drugs in commercial dosage forms. The lower detection limits allowed the application of this method for the determination of the compounds in plasma as an example of a biological fluid. In addition, the method was considered specific for the determination of tertiary amines in the presence of primary and secondary amines; as a result, it was deemed suitable for the determination of the cited drugs in the presence of their degradation products resulting from N‐dealkylation or oxidation of the corresponding sulphoxides or sulphones. Copyright © 2012 John Wiley & Sons, Ltd.     

The heterogeneity of the protein content of liver and muscle glycogens

The portions of both liver and muscle glycogen that have a high protein content have been investigated. In liver the high molecular weight protions of glycogen may be rendered insoluble by treatment with trichloroacetic acid. This shows that reported desmo- (or insoluble) glycogen is an artefact of the extraction process and therefore of no physiological significance. In contrast, muscle glycogen isolubility is not associated with any specific molecular size range. Insolubility of muscle glycogen is shown to be related to partial degradation of the polysaccharide and to the high protein content remaining after the gentle extraction procedure. Since the molecular weight profile is unaltered by the removal of the insoluble glycogen it does not interfere with the interpretation of metabolic studies.     

Bromophenol blue protein assay: Improvement in buffer tolerance and adaptation for the measurement of proteolytic activity

A modification of the bromophenol blue dye binding procedure of protein estimation is described. Substritution of glycine/phosphoric acid, pH 2.6, for dilute acetic acid in the colour reagent extended the applicability of the procedure to protein solutions containing buffers of various pH values. This was, however, accompained by approximately 25% loss in the sensitivity of the procedure. The mofified reagent exhibited very marked tolerance to detergents and could be successfuly adapted for the measurement of proteolytic activity in acidic, neutral or alkaline pH ranges.