A postcoupling method for the demonstration of N-acetyl-beta-D-glucosaminidase in unfixed frozen tissue sections.

The localization and activity of the lysosomal enzyme, N-acetyl-beta-glucosaminidase, has been studied in unfixed frozen sections of tissues from normal and hemorrhaged rats with the use of a modified postcoupling technique. Discrete localization of the end product of the reaction was achieved in this method by the incorporation of 20% (w/v) polyvinyl alcohol (molecular weight 14,000) in the incubation medium. Advantages of the present method include the ability to overcome the inhibitory effects on enzyme activity of both tissue fixation and the presence of diazonium salts in the incubation medium. The staining reaction obtained with this technique demonstrates the enzyme activity at specific cytoplasmic sites within cells and has a wider applicability to the comparative study of N-acetyl-beta-glucosaminidase activity in normal and injured tissues.     

A histochemical study of the oxidation of 17β-estradiol in human term placenta at different incubation conditions

Summary The present study deals with the histochemical demonstration of 17-estradiol dehydrogenase in human term placenta using the polyvinyl alcohol method to reduce diffusion artefacts. Incubations took place with both NAD⁺ and NADP⁺ as coenzymes and at different pH values of the incubation medium. The NAD⁺ linked enzyme reaction showed a greater activity than the NADP⁺ linked, both in the trophoblast as well as in connective tissue. There were differences in staining intensity at the different pH values, and strongest reaction was observed using glycine-NaOH buffer pH 10 in the incubation medium. Owing to a non-enzymatically reduction of nitro blue tetrazolium by reduced NAD⁺, the demonstration of 17-estradiol dehydrogenase is independent of diaphorase at this high pH. The findings are discussed in relation to data about nothing dehydrogenase and biochemically determined pH optima for the enzymatic reactions dealt with in this work.The following Abbreviations are used in this Article NAD⁺ -nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - NBT nitro blue tetrazolium - PVA polyvinyl alcohol - tris tris (hydroxymethyl)-aminomethane - 17-OH-SDH 17-OH-steroid-dehydrogenase Supported by The Norwegian Research Council for Science and the Humanities. Skilful assistance of Mrs. E. Alvestad, Mrs. Aa. Flatnes and Mrs. F. Sørensen is greatfully acknowledged.     

The histochemical localization of lactic dehydrogenase isoenzymes in the rat nephron by means of an improved polyvinyl alcohol method

Summary The histochemical localization of lactic dehydrogenase (LDH) activity and the prevailing type of isoenzyme in different segments of the nephron in male and female rats are described. Polyvinyl alcohol was added to the incubation medium in order to reduce enzyme diffusion. Localization of the reaction product was further improved by the use of a high concentration of Nitro BT (and of PMS and NAD).The three segments of the proximal tubules exhibited clearly different staining patterns. In glomeruli, terminal parts of the third proximal segments, thin limbs of Henle, and collecting ducts M subunits of LDH predominated, whereas in the remaining tubular segments H subunits prevailed. Sex differences were observed in respect to the LDH reaction in the proximal tubules, but not in the rest of the nephron. The localization of -hydroxy acid oxidase was also investigated, as this enzyme oxidizes lactate and therefore contributes to the reaction. Under certain conditions, i.e. high substrate concentration, the activity of -hydroxy acid oxidase was negligible in comparison with that of LDH.The Abbreviations used are PVA polyvinyl alcohol - PMS phenazine methosulfate - NAD nicotinamide adenine dinucleotide - Nitro BT nitroblue tetrazolium - Tris Tris (hydroxymethyl) aminomethane - PCMB p-chloromercuribenzoate Supported by grants from Fonden til Lægevidenskabens Fremme, and the Danish Medical Research Council.The terms H isoenzyme and M isoenzyme denote isoenzymes of LDH containing predominantly H subunits and M subunits, respectively.     

A direct colorimetric assay for Ca2+ -stimulated ATPase activity

A simple and rapid colorimetric assay for measuring the high affinity Ca2+-ATPase activity in subcellular fractions is presented. With this method a one-step addition of a malachite green/molybdate/polyvinyl alcohol reagent to the assay mixture at the end of the incubation period is all that is required for the spectrophotometric quantification of the phosphomolybdate-malachite green complex. The presence of polyvinyl alcohol allows the quantification of released phosphate without having to separate it from protein. We have validated this assay by characterizing the high affinity Ca2+-ATPase activity in isolated rat liver microsomes. Comparable Ca2+-ATPase activities in rat liver microsomes and adipocyte plasma membranes were found when measured with this colorimetric assay and an isotopic assay. This method is applicable to the measurement of other types of ATPase activities.     

The clinical, histologic, and ultrastructural presentation of polyvinyl sponge (Ivalon) breast prostheses removed for massive fluid accumulation.

The current study describes what we believe is the first report of bilateral massive seromas associated with open-cell Ivalon sponges. Additionally, the gross, histologic, and ultrastructural features consistent with previous reports of polyvinyl alcohol prostheses are presented. Despite the reported chemical inertness of polyvinyl alcohol, this material may incite a biologic response in some patients, leading to dense fibrosis and occasional foreign-body giant-cell reaction. It is postulated that the molecular breakdown products of the polyvinyl alcohol polymer may create an osmotic gradient across the periprosthetic capsule, which may lead to intracapsular fluid accumulation, as presented in this case.     

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase has found many applications in biomedical research. However, up to several years ago, the methods used often appeared to be unreliable because many artefacts occurred during processing and staining of tissue sections or cells. The development of histochemical methods preventing loss or redistribution of the enzyme by using either polyvinyl alcohol as a stabilizer or a semipermeable membrane interposed between tissue section and incubation medium, has lead to progress in the topochemical localization of glucose-6-phosphate dehydrogenase. Optimization of incubation conditions has further increased the precision of histochemical methods. Precise cytochemical methods have been developed either by the use of a polyacrylamide carrier in which individual cells have been incorporated before staining or by including polyvinyl alcohol in the incubation medium. In the present text, these methods for the histochemical and cytochemical localization of glucose-6-phosphate dehydrogenase for light microscopical and electron microscopical purposes are extensively discussed along with immunocytochemical techniques. Moreover, the validity of the staining methods is considered both for the localization of glucose-6-phosphate dehydrogenase activity in cells and tissues and for cytophotometric analysis. Finally, many applications of the methods are reviewed in the fields of functional heterogeneity of tissues, early diagnosis of carcinoma, effects of xenobiotics on cellular metabolism, diagnosis of inherited glucose-6-phosphate dehydrogenase deficiency, analysis of steroid-production in reproductive organs, and quality control of oocytes of mammals. It is concluded that the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase is of highly significant value in the study of diseased tissues. In many cases, the first pathological change is an increase in glucose-6-phosphate dehydrogenase activity and detection of these early changes in a few cells by histochemical means only, enables prediction of other subsequent abnormal metabolic events. Analysis of glucose-6-phosphate dehydrogenase deficiency in erythrocytes has been improved as well by the development of cytochemical tools. Heterozygous deficiency can now be detected in a reliable way. Cell biological studies of development or maturation of various tissues or cells have profited from the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and properties of two distinct groups of ADH isozymes from Chinese hamster liver

Two major classes of alcohol dehydrogenase isozymes have been purified from Chinese hamster liver. The two isozyme classes have the same subunit molecular weights but different electrophoretic mobilities. They have a similar range of substrates but different KROM values and sensitivities to the inhibitor pyrazole. The ADHs are immunologically related as determined by Ouchterlony double diffusion experiments. These results suggest that the isozymes are encoded by different structural gene loci derived from a common ancestral gene.Financial support was provided by the National Cancer Institute of Canada and the Medical Research Council of Canada, Grant MT4860. J.-P. Thirion is a Research Scholar of the Science Research Council of Quebec.     

In vitro study of the interaction of polyalkilimide and polyvinyl alcohol hydrogels with cells

Hydrogels are a class of polymers that in the last decade have had a great development and application for soft tissue augmentation, due to their similarity to this tissue for their high water content. The in vitro effects of polyalkylmide hydrogel (pAI) and polyvinyl alcohol hydrogel (pVOH) on human lymphocytes and U937 cells viability, apoptosis and cell shape were investigated. Cell viability was always higher than 70%, thus showing the hydrogels were not cytotoxic for both cell lines. Some differences were, however, found. At short time, lymphocytes were very sensitive to the hydrogels incubation, while at long time, U937 cells were the most sensitive cells. Other differences on cell viability were related to the time of incubation, to the type of hydrogel and to the polymers concentration. Cell viability decreased only at the longest time of incubation and with the highest hydrogel concentration. Accordingly, cell death by apoptosis increased; necrosis was never observed in the cultures. Concentration- and hydrogel-dependent modifications of cell shape (bigger cell volume, elongations of cells) were observed in a few percentage of viable cells. In conclusion, the very high in vitro degree of biocompatibility shown by both hydrogels encourages their use as dermal fillers.     

Improvement of the method of Karnovsky and Roots for the histochemical demonstration of acetylcholinesterase

The aqueous reaction medium of Karnovsky and Roots for the demonstration of acetylcholinesterase (AChE) activity was modified to obtain a gel-incubation medium that prevents diffusion of the reaction product and the soluble part of AChE into the incubation medium and along the plane of the section. The incubation medium contained 27% polyvinyl alcohol and suitable concentrations of the components of the medium of Karnovsky and Roots. The application of this incubation medium to sections of rat hippocampus resulted in intense staining, with the reaction products being mainly localized in fibrous structures.     

Preparation of DNA-polyintercalator conjugate and its functional property

Psoralen immobilized polyvinyl alcohol (PVA-P) was synthesized from chloromethylmethoxypsoralen and polyvinyl alcohol. The psoralen part of PVA-P intercalated into the double-stranded DNA and formed covalent bonding between the psoralen and nucleic acid base after 365 nm UV irradiation. As a result, DNA and PVA-P produced a water-insoluble conjugate. This DNA-PVA-P conjugate maintained the double-stranded structure of DNA and possessed the DNA's property, such as intercalation. Therefore, the DNA-PVA-P conjugate selectively accumulated the planar-structure containing chemical compounds, such as biphenyl and dibenzofuran, from an aqueous multi-component solution. These DNA-PVA-P conjugates may have the potential to be utilized as a separation material for the selective removal of harmful compounds.     

Diffusion during dehydrogenase reactions: the effects of intermediate electron acceptors

Summary A hamster cheek pouch model has been used to study the diffusion of reactants from the epithelium into adjacent muscle and connective tissue during the histochemical demonstration of glucose-6-phosphate dehydrogenase activity. The effects of the addition of intermediate electron acceptors to the incubation medium varied considerably from one acceptor to another, but were independent of the grade of polyvinyl alcohol incorporated into the medium. Menadione was the least effective intermediate both in transferring reducing equivalents from the primary dehydrogenase to Neotetrazolium chloride and in preventing diffusion. Phenazine methosulphate, Methylene Blue and Thionin were more efficient intermediates. Nevertheless, considerable diffusion occurred in the presence of Phenazine methosulphate, although very little diffusion was detectable with either of the thiazine dyes. It is suggested that these differences are related to different modes and sites of action of the carriers.     

The hormonal control of wheat leaf unrolling

Summary The unrolling of etiolated wheat leaf sections in the dark is stimulated by the application of gibberellic acid (GA₃). GA₃ is most effective if applied for a short time at the beginning of incubation. Kinetin also stimulated leaf unrolling in the dark. AMO1618 and CCC inhibit red light and kinetin-stimulated unrolling. Gibberellin-like substances extracted from red light-treated leaf tissue are effective in stimulating leaf unrolling.Ethylene production in leaf sections is stimulated by IAA, GA₃ and kinetin and inhibited by ABA. A brief exposure to red light decreases the ability of the tissue to produce ethylene. It is concluded that ethylene plays no important role in the control of leaf unrolling by red light or by the application of hormones.Holder of a Science Research Council Studentship.     

The ultrastructure of the sinus gland of Carcinus maenas (Crustacea: Decapoda)

Summary The sinus gland of Carcinus maenas consists of the swollen axonal endings of the neurosecretory cells of the major ganglia and acts as a storage release centre for the membrane bound neurosecretory material. These neurosecretory granules fall into five different types based on size and electron density. Their contents are released by exocytosis of the primary granules or smaller units budded from the primary granules.I thank Professor E. Naylor for his constant advice and Professor E. W. Knight-Jones, Department of Zoology, University College, Swansea, for the provision of laboratory facilities. I am grateful to the Science Research Council for the financial support. Finally, I thank the Electron Microscope Unit, Southampton General Hospital, where the work was completed.     

Improvement of the method of Karnovsky and Roots for the histochemical demonstration of acetylcholinesterase

Summary The aqueous reaction medium of Karnovsky and Roots for the demonstration of acetylcholinesterase (AChE) activity was modified to obtain, a gel-incubation medium that prevents diffusion of the reaction product and the soluble part of AChE into the incubation medium and along the plane of the section. The incubation medium contained 27% polyvinyl alcohol and suitable concentrations of the components of the medium of Karnovsky and Roots. The application of this incubation medium to sections of rat hippocampus resulted in intense staining, with the reaction products being mainly localized in fibrous structures.Supported by the Deutsche Forschungsgemeinschaft (Ku 541/2-1)     

On the role of oxygen in dehydrogenase reactions using tetrazolium salts

Summary Fundamental aspects of the reduction fo tetrazolium salts were investigated and, in particular, the role of oxygen in the reduction. It was found that oxygen had a competitive inhibitory effect on the reduction of (Tetra)Nitro BT mediated by NADH and phenazine methosulphate. This competitive effect, under aerobic conditions, could be reversed by using tetrazolium concentrations of 5mm. Oxygen did not have a signIficant effect on BPST reduction, whereas the inhibitory effect of oxygen on the reduction of Neotetrazolium was not reversed by increasing the tetrazolium concentration. The oxygen effect on Nitro BT reduction was considerably less when macromolecular substances such as albumin or polyvinyl alcohol were added to the medium. This may be due to increased Nitro BT concentrations being built up at the surface of macromolecules due to the nonpolar components of the Nitro BT molecule. When demonstrating glucose-6-phosphate dehydrogenase activityin vitro or in tissue sections with the use of Nitro BT, oxygen also had a direct inhibitory effect, even when azide was added to the medium for the inhibition of flavoprotein-mediated electron transfer to oxygen. Again, this direct inhibition of Nitro BT reduction by oxygen could be excluded by using a high Nitro BT concentration. Macromolecules present in the incubation medium or in tissue sections counteracted the oxygen effect. It is concluded that the maximum reaction rate and optimum localization of dehydrogenases is obtained when histochemical media are used containing 5mm (Tetra)Nitro BT and 20% polyvinyl alcohol.     

Linearity in dehydrogenase reaction rate studies in tissue sections is affected by loss of endogenous substrates during the reaction

We studied the effect of section thickness on the reaction rate of glucose-6-phosphate dehydrogenase (G6PD) activity in unfixed sections of rat liver by use of continuous monitoring by microdensitometry of the reaction product as it formed in the section during incubation. Tetranitro BT or nitro BT was used as final electron acceptor and polyvinyl alcohol as tissue stabilizer. Each test minus control reaction curve deviated from linearity during the first 2 min of incubation. This was mainly due to loss of low molecular weight endogenous dehydrogenase substrates from the surface of the section. For any given reaction, the same absolute amount of endogenous substrate was lost from each section, and hence a much greater proportion was lost from the thinner sections. Such losses lead to a deficit in (nonspecific) formazan production. There was a greater loss from, and hence a greater deficit in, formazan production in sections incubated at 30 degrees C than at 37 degrees C and when nitro BT was used instead of tetranitro BT, but the greatest loss of endogenous substrates occurred in sections incubated in control media. Therefore, greater losses seemed to occur when the reactions were slower because of failure to overcome the critical supersaturation level of the formazan. A consequence of this was a non-linear test minus control response during the first minutes of the incubation.     

The appearance of monoamines in the adult and developing neurohypophysis of Gallus gallus

Summary Most of the specific monoamine fluorescence of the fowl neurohypophysis is found in the eminentia mediana and the infundibular stem. The densest accumulation of fluorescent structures is located to the zona externa and the subependymal layer, whereas generally only scattered fluorescence is demonstrable in the fiber layer. The neural lobe tissue is provided with very fine smooth fibers often difficult to distinguish. Spectrofluorimetric determinations have shown that noradrenaline is the major catecholamine in the chick neurohypophysis. From the embryological studies it is evident that the monoamine fluorescence first appears in the subependymal layer, the fiber layer and the neural lobe (after about 15 days of incubation). The zona externa fluorescence is not visible until just before hatching. 10 days after hatching the fluorescence intensity of the chick neurohypophysis is similar to that of the adult. Some comparisons are also made with the appearance of monoamines in the mouse.The authors take great pleasure in expressing their warmest thanks for laboratory facilities and good advice provided by Dr. Bengt Falck at the Institute of Histology, Lund, Sweden.This work was supported by grants from the Swedish Natural Science Research Council (project no. 99-35 and 2180-16), from the United States Public Health Service (NB-06701-02) and from the Swedish Medical Research Council (B-69-14 x -56-05 C).     

Über den Einfluß der Gelinkubation mit Polyvinylalkohol auf die Löslichkeit der Fructose-1,6-diphosphat-Aldolase

Zusammenfassung Die Verminderung der Löslichkeit der Fructose-1,6-diphosphat-Aldolase (Fructose-1,6-diphosphat-D-Glycerinaldehyd-3-phosphat-Lyase, E.C. 4.1.2.13) durch Zusatz von Polyvinylalkohol zum Inkubationsmedium wurde unter Einhaltung histochemischer Bedingungen überprüft. Bei der Inkubation in wäßrigem Medium sind nach 100 min in einem Schnitt des Herzmuskels und der Niere der Ratte weniger als 10% der ursprünglichen Enzymaktivität nachweisbar. Nach Zusatz von Polyvinylalkohol zum Inkubationsmedium beträgt die im Schnitt verbleibende Enzymaktivität nach gleicher Inkubationsdauer für den Rattenherzmuskel etwa 80%, für die Niere 30–40%. Die Bedeutung dieser Befunde für den histochemischen Nachweis der Aldolase wird am Beispiel des Rattenherzmuskels gezeigt. Als Ursache der organbedingten Unterschiede im Löslichkeitsverhalten wird die Anwesenheit verschiedener Isoenzyme in beiden Organen in Betracht gezogen.

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The effect of gel incubation using polyvinyl alcohol on the solubility of fructose-1,6-diphosphate-aldolase

Summary The decrease in the solubility of fructose- 1,6-diphosphate-aldolase (fructose-1, 6-diphosphate-D-glyceraldehyde-3-phosphate lyase, E.C.4.1.2.13) brought about by the addition of polyvinyl alcohol to the incubation medium was tested observing histochemical conditions. After 100 minutes of incubation in an aqueous medium less than 10% of the original enzymic activity was demostrated in sections of rat cardiac muscle and kidney. After polyvinyl alcohol was added to the incubation medium the enzymic activity in the sections (measured over the same period) was 80% in rat cardiac muscle and 30–40% in kidney sections. The importance of these findings for the histochemical demonstration of aldolase is shown in the rat cardiac muscle. The organ dependend differences in the solubility may find an explanation in the presence of various iso-enzymes in the two organs under investigation.

     

Effects of allatotropin and allatostatin on in vitro production of juvenile hormones by the corpora allata of virgin females of the moths of Heliothis virescens and Manduca sexta

Retrocerebral complexes (RCs) were isolated from adult females of the moths Heliothis virescens and Manduca sexta. Different homologs of juvenile hormone (JH) produced by the isolated RCs were identified and amounts measured by capillary gas chromatography-chemical ionization (isobutane)-mass spectroscopy. Only JH I, II and III were identified. Incubation of RCs from both species in media containing acetate, but no propionate, induced production of approximately equal amounts of JH II and JH III, but the amount of JH I present was very low in all samples. Incubation of RCs with synthetic Manduca sexta allatotropin stimulated significant increases in production of all three homologs but increases in JH I and JH II were greater than those for JH III. The effect of allatotropin was mimicked by addition of propionate to the medium, which indicated that allatotropin increased supply of acetyl- and propionyl-CoA precursors. Incubation of tissue from H. virescens females during the first 24 h after eclosion with synthetic Manduca sexta allatostatin did not reduce production of JH. However, incubation of tissue from 3-day-old females with allatostatin significantly reduced production of JH. Similarly, incubation of tissue from H. virescens females during the first 24 h after eclosion with both allatotropin and allatostatin did not increase JH over the amount present in extracts from tissue incubated without the neuropeptides, indicating that allatostatin negated the action of allatotropin. Incubation of tissue from H. virescens females with allatostatin plus farnesol or JH III acid resulted in significant production of JH III, but neither JH I nor JH II was detected. These findings indicated that allatostatin acts prior to formation of the sesquiterpene alcohol precursors of JH.     

Entrapment of Sorghum root oxalate oxidase into polyvinyl alcohol membrane

A Cl- and NO3- insensitive oxalate oxidase, purified from the roots of 10-day old seedlings of grain Sorghum has been immobilized on polyvinyl alcohol (PVA) membrane through entrapment with 96.07% retention of initial activity. The membrane bound enzyme showed an increase in optimum pH (from 5.0 to 6.5), time of incubation (from 5 to 10 min) and Km for oxalate (from 0.38 to 6.23 mM), but decrease in incubation temperature for maximum activity (from 37 to 30 degrees C) and Vmax (from 70 nmol/min/ml to 9.7 nmol H2O2/min) and was unaffected by Cl- and NO3. The membrane bound enzyme lost 50% of its initial activity after 30 days of storage at room temperature. The use of membrane bound oxalate oxidase in determination of serum oxalate of urinary stone patients is demonstrated.