Quantitative microspectrophotometrical determination of protein thiols and disulfides with 2,2′-dihydroxy-6,6′-dinaphthyldisulfide (DDD)

Summary 2,2-dihydroxy-6,6-dinaphthyldisulfide (DDD) reacts with both protein thiol groups and with protein disulfides (Nöhammer 1977). By varying the pH of the DDD-reaction, as well as the reaction times, the complex reaction became specific with respect to the histochemical demonstration of protein-SH groups. Furthermore, the application of the histochemical DDD-reaction following quantitative blockade of the protein-SH groups enabled the demonstration of distinctive DDD-reactive disulfides. The specifity and the extent of the different histochemical DDD-staining methods were investigated by comparing macroscopically determined values of the protein-SH-contents, and the contents of the different kinds of disulfides in Ehrlich-ascites-tumor cells (EATC) (Modig 1968; Hofer 1975), with microspectrometrical values determined with the MCN-method of Nöhammer et al. (1981), and with microspectrometrical values measured on EATC after staining with the modified DDD-methods. Also, the method for the histochemical demonstration of protein-SH with DDD after the reduction of the disulfides with thioglycolate was investigated and conditions were found by which the protein-SH content could be determined quantitatively with DDD and Fast blue B after the reduction of the disulfides. With the aid of the MCN-method (Nöhammer et al. 1981), the intracellular disulfide interchange reaction was investigated, leading to pH-dependent changes of the SH-SS-ratio of fixed cells during their incubation in aqueous media. In addition the possibility of protein loss during the long incubation times of the fixed cells in the DDD-solutions was investigated. For the quantitative microscpecrometrical determination of the protein content of EATC the so-called tetrazonium-coupling method, optimized by Nöhmmer (1978) and calibrated by Nöhammer et al. (1981), was used.Dedicated to Prof. Dr. E. Ziegler on the occasion of his 70^th birthday     

Multiple localizations of adenylate cyclase in rat hippocampus

Summary A new method for the histochemical demonstration of adenylate cyclase activity, introduced and biochemically tested by Poeggel et al. (1981a), was employed in nervous tissue. Using this method a multiple pattern of activity was detectable. Activity occurs in nervous as well as glial elements. Biochemical results and physiological conclusions could be confirmed by ultrahistochemical visualization of adenylate cyclase activity in nervous tissue. The specificity of the reaction is controlled by a number of variations of the incubation methods.     

Reversible inhibition of adenylate cyclase activity of rat brain caudate nucleus by oxidized glutathione

Basal and dopamine-stimulated adenylate cyclase (EC 4.6.1 1.) activities were strongly inhibited by GSSG, but not by GSH. Adenylate cyclase that had been inactivated by GSSG was reactivated by incubation with various sulfhydryl compounds including GSH. Formation of mixed disulfides by reaction between GSSG and protein-SH groups increased on incubation with GSSG and returned to the normal level on subsequent incubation with DTT.     

Mercurochrom can be used for the histochemical demonstration and microphotometric quantitation of both protein thiols and protein (mixed) disulfides

 Mercurochrom [2,7-dibromo-4-(hydroxymercuri)-fluorescein disodium salt] used for staining of protein thiols in addition binds to other groups of proteins. Experimental evidence is provided that mercurochrom bound to non-thiol groups forms a 1:1 adduct with protein (mixed) disulfides. The disulfide contents of three different types of cells determined biochemically correlated with the corresponding mean integrated optical densities determined microphotometrically after mercurochrom staining of groups other than thiols. Intracellular disulfide exchange has been studied, leading to a transformation of protein mixed disulfides to protein disulfides and an equimolar loss of protein thiols. Protein mixed disulfides were generated from protein thiols using both methyl methanethiosulfonate (MMTS) and 2,2′-dihydroxy-6,6′-dinaphthyldisulfide (DDD). Loss of thiols as well as the equimolar increase of protein mixed disulfides were followed using both mercurochrom staining for thiols and for disulfides. Generation of protein mixed disulfides due to the DDD reaction was also followed by azocoupling with Fast blue B. On the basis of the observed stoichiometry between the loss of protein thiols and the quantity, increase or conversion of protein disulfides determined microphotometrically using both mercurochrom staining and DDD Fast blue B staining, we conclude that: (1) 1 mol of mercurochrom is bound per mol of protein (mixed) disulfide; and (2) the molar absorptivity of mercurochrom bound to disulfides is ɛ₅₂₀=34940. This study demonstrates that mercurochrom can be used for the quantitative determination of the oxidative status of protein thiols in cells. Accepted: 17 December 1996     

Quantitative Bestimmung von Sulfhydrylgruppen mit Mercurochrom

Zusammenfassung Native Ehrlich-Ascites Tumorzellen (EATZ) werden in einer 1×10^–3 M Lösung von Dibrommercurifluoreszein (DBMF) in Sörensen-Phosphatpuffer pH 6.7+0,9% NaCl 30 min inkubiert und sodann viermal mit dem gleichen Puffer, der 0,01 M in bezug auf NaCN ist, bis zur Farblosigkeit des Überstandes gewaschen. Die Zellen zeigen nun ein Absorptionsmaximum zwischen 520 und 525 nm, das mit dem von Komplexen reiner Korpuskularproteine mit DBMF identisch ist. Die Zellen werden im Scanning bei 520 nm photometriert und daraus ihre Gesamtextinktionen ermittelt. Unter Zugrundelegung des in früheren Arbeiten bei den Protein-DBMF-Komplexen bestimmten Extinktions-koeffizienten =33 000 ergibt sich ein Gehalt von rund 1,1×10^–14 Molen Proteinthiolen (Prot-SH) pro Zelle. Dieser Wert entspricht sowohl dem von Nöhammer 1982 mit Dihydroxydinaphthyldisulfid gefundenen Gehalt an reaktiven, d.h. schnell reagierenden Prot-SH, als auch dem von Rindler et al. 1970 bestimmten SH-Gehalt der primär löslichen Zellproteine. Da aus nativ mit DBMF behandelten Zellen jedoch keine im Homogenat-Puffer löslichen Proteine gefunden wurden, ist es wahrscheinlich, daß DBMF mit den schnellen SH-Gruppen der Cytosol-Proteine reagiert, die dabei strukturelle Veränderungen erfahren, die zum Löslichkeitsverlust führen. DBMF erfaßt jedoch die gesamten Prot-SH, reaktive und maskierte, wenn es auf vorher fixierte Zellen einwirkt (Nöhammer et al. 1981). Ist der zum Waschen verwendete Puffer Cyanid-frei, so wird die identische Absorptionsbande gemessen; die für E _tot,520 ermittelten Werte sind jedoch um 60% höher. Die Differenz wird nicht-kovalent, reversibel an die Zellen gebundenem DBMF zugeschrieben.

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Quantitative determination of sulfhydryl groups with MercurochromeII. Detection of fast reacting protein thiols in native cells

Summary Native Ehrlich ascites tumor cells (EATC) are incubated during 30 min in a 1×10^–3 M solution of dibrommercurifluoresceine (DBMF) in Sörensen phosphate buffer pH 6.7+0.9% NaCl. Subsequently the cells are washed four times in the same buffer containing additionally 0.01 M NaCN until the supernate appears to be colourless. They show an absorption maximum between 520 and 525 nm which is identical with that of complexes between pure corpuscular proteins and DBMF, investigated previously. Scanning at 520 nm yields the total extinction of the stained cells E _tot which is calculated into moles of protein bound thiol groups (prot-SH), with an extinction coefficient =33,000 previously determined with the protein-DBMF-complexes. A mean protein-SH content of 1.1×10^–14 moles per single cell is found which corresponds with both the content of fast reacting prot-SH previously determined by Nöhammer with dihydroxydinaphthyldisulfide, and the SH-content of the soluble cellular proteins determined by Rindler et al. with DTNB. As no soluble proteins could be obtained from DBMF-treated cells, it can be assumed that in native cells DBMF reacts preferentially with the fast reacting SH-groups of the soluble proteins which, in the course of structural changes, became insoluble. According to Nöhammer et al. (1981), however, DBMF also reacts with the total of protein-SH content of 1.1×10^–14 moles per single cell is found which fixed with ethanol/ether. When the buffer used for washing is free from CN^–, the identical absorption band is measured; however, the values determined for E _tot,520 are approximately 60% higher. The extinction difference is ascribed to DBMF, noncovalently and reversibly bound to the cells.

     

Histochemical demonstration of asparagine-linked oligosaccharides in glycoproteins of human placenta and umbilical cord tissues by means of almond glycopeptidase digestion

Almond glycopeptidase is an enzyme which cleaves specifically beta-aspartylglucosylamine linkages in glycoproteins with asialo-carbohydrate moieties. With this enzyme, it was possible to demonstrate the localization of asparagine-linked oligosaccharides in glycoproteins of human placenta and umbilical cord tissues. In these tissues, the oligosaccharides were shown to react positively for a series of histochemical procedures for neutral complex carbohydrates such as periodic acid-Schiff (PAS), peroxidase-labelled Ricinus communis agglutinin-I-diaminobenzidine (PO-RCA-DAB) and concanavalin A-peroxidase-diaminobenzidine (Con A-PO-DAB). The asparagine-linked carbohydrates were localized in the placental villi, blood vessels and perivascular tissues and the umbilical cord blood vessels and matrix. The results of previous biochemical analyses performed upon the same tissues (Takahashi et al., 1981) have corroborated the results of the histochemical studies. The present results appear to substantiate the usefulness of almond glycopeptidase for the histochemical demonstration of the particular oligosaccharides of glycoproteins in tissues in general.     

PROTEIN-BOUND SULFHYDRYL GROUPS IN COLEUS WOUND MERISTEMS

Roberts , Lorin W. (U. Idaho, Moscow.) Protein-bound sulfhydryl groups in Coleus wound meristems. Amer. Jour. Bot. 47(2): 110—114. Illus. 1960.–A hisiochemical examination was conducted of the development of the stem wound-meristem of Coleus blumei Benth. with 2,2‘-dihydroxy-6,6‘-dinaphthyl disulfide (DDD) and 1-(4-chloromercuriphenylazo)-naphthol-2 (Mercury Orange) to test for protein-bound sulfhydryl groups. Specificity for the histochemical reactions was indicated by complete blocking of staining in sections pretreated with 0.001 M iodine in npropanol and 0.1 M aqueous solutions of iodoaceiamide. Both histochemical methods yielded comparable results in protein-SH localization (zone of cell division and differentiating xylem elemments) and staining intensity. The zone of cell division did not exhibit staining at the time of initial cell division (3 days). However, the cytoplasm of the dividing cells of the wound-meristem demonstrated protein-SH 4—5 days following wounding. A high concentration of protein-SH was observed in the cytoplasm of the dividing cells 7 days after wounding. Redifferentiating parenchyma cells, destined to become wound-xylem elements, first exhibited wall staining at the time of protoplast contraction. The subsequent walled xylem stage exhibited strong staining in the reticulated striations of the secondary walls.     

Quantitative cytospectrophotometrical determination of the total protein thiols with “Mercurochrom”

Summary Mercurochrom (2,7-dibromo-4-(hydroxymercuri)-fluoresceine-disodium salt) reacts histochemically not only with protein-SH-groups, but is also bound unspecifically to cellular proteins. The amount of the unspecifical staining approximately equals the specific SH-staining. Two methods are described to remove the unspecifically bound Mercurochrom, without influencing the specific reaction with the protein thiols. The first applies 0,1 m thioglycolate pH 4.0 (MT4-method), the other a special tris-cyanidebuffer pH 7.4 (MCN)-method). Aliquots from preparations of rat hepatocytes, Yoshida ascites tumor cells, Ehrlich ascites tumor cells, isolated nuclei of Ehrlich-cells and chicken thymocytes were investigated as well for the protein thiol content of the cells macroscopically with DTNB as microspectrophotometrically for the extinctions of the cells after staining with MT4-or MCN-method. A strong correlation was found between the macroscopically determined total-protein-SH-contents and the microphotometrically determined mean-total-extinctions of the cells. Additionally the molar absorptivities determined macroscopically by Schauenstein and Scheuringer (1980) coincide excellently with the values found microspectrometrically on MT4- and MCN-stained cells.     

A modification of the amidoblack-TCA-staining for quantitative microspectrometrical determination of proteins in tissue sections

Summary Conditions are described by which cells and fresh frozen tissues, following fixation with ethanol-ether, and after staining with the amidoblack (AB)-TCA-staining method, show a modified dye/protein ratio of 0.9 moles AB/10⁵ g protein compared to 8.5 moles AB/10⁵ g protein (Schauenstein et al. 1980) as in a previously used method. In contrast to the AB-TCA-method, which leads to extremly high and unmeasurable extinctions in tissue sections, staining with the modified AB-TCA-23st-method with 10 m tissue sections produces easily measurable extinction values.A correlation of the microspectrometrically determined mean total extinction values of different cell types and nuclei after staining with the tetrazonium method (Nöhammer 1978; Nöhammer and Desoye 1981) and on the other hand with the AB-TCA 23st-method has been found. The microspectrometrically determined extinctions after AB-TCA 23st-staining can be calculated; an extinction of 0.04248 corresponds to 1 pgm protein.Dedicated to Prof. Dr. E. Schauenstein on the occasion of his 65th birthday     

Membrane thiol-disulfide status in glucose-6-phosphate dehydrogenase deficient red cells. Relationship to cellular glutathione

The behavior of glucose-6-phosphate dehydrogenase (G6PD)-deficient red cell membrane proteins upon treatment with diamide, the thiol-oxidizing agent (Kosower, N.S. et al. (1969) Biochem. Biophys. Res. Commun. 37, 593–596), was studied with the aid of monobromobimane, a fluorescent labeling agent (Kosower, N.S., Kosower, E.M., Newton, G.L. and Ranney, H.M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3382–3386) convenient for following membrane thiol group status. In diamide-treated G6PD-deficient red cells (and in glucose deprived normal cells), glutathione (GSH) is oxidized to glutathione disulfide (GSSG). When cellular GSH is absent, membrane protein thiols are oxidized with the formation of intrachain and interchain disulfides. Differences in sensitivity to oxidation are found among membrane thiols. In diamidetreated normal red cells, GSH is regenerated in the presence of glucose and membrane disulfides reduced. In G6PD-deficient cells, GSSG is not reduced, and the oxidative damage (disulfide formation) in the membrane not repaired. Reduction of membrane disulfides does occur after the addition of GSH to these membranes. A direct link between the thiol status of the cell membrane and cellular GSH is thereby established. GSH serves as a reductant of membrane protein disulfides, in addition to averting membrane thiol oxidation.     

Microphotometric determination of enzymes in brain sections. V. Glycerophosphate dehydrogenases

An incubation medium was adapted for the microphotometric determination (kinetic and end-point measurements) of the activities of mitochondrial alpha-glycerophosphate dehydrogenase (GPDH) in the rat hippocampus. For comparison, the activities of the cytoplasmic NAD-linked alpha-glycerophosphate dehydrogenase were also measured. The study showed that in the demonstration of both enzymes the use of an exogenous electron carrier is necessary. Both enzymes react to phenazine methosulfate (PMS) which transfers reduction equivalents to the electron acceptor nitroblue tetrazolium chloride (NBT), thus causing a coreaction of GPDH in the demonstration of NAD-GPDH. Therefore, only the NAD-independent GPDH which is stimulated by menadione, can be selectively demonstrated in the histochemical procedure applied. The final incubation medium of GPDH consisted of 15 mM L-glycerol 3-phosphate, 5 mM NBT, 0.4 mM menadione, 7.5% polyvinyl alcohol in 0.5 M Hepes buffer, pH 8; the final pH of the incubation medium was 7.5. A linear response of the reaction lasted about 5 min. There was a linear relationship between section thickness and the formation of reaction product up to a section thickness of 14 microns. The apparent Km value at 25 degrees C was 0.6 mM. It is concluded that using menadione histochemical methods are suited to determine the mitochondrial GPDH activities in brain sections whereas using PMS a coreaction of GPDH takes place in the demonstration of NAD-GPDH, so that a histochemical quantification of NAD-GPDH cannot be recommended.     

Antipain microinjection prevents progesterone to inhibit adenyl cyclase in Xenopus oocytes

Microinjection of antipain, an inhibitor of thiol and Ca2+-dependent proteases, in immature Xenopus oocytes inhibited meiotic maturation induced by progesterone, but not by transfer of cytoplasm taken from maturing oocytes. Oocytes could be released from antipain inhibition by increasing progesterone concentration. alpha-32P-ATP was microinjected to study adenylcyclase in ovo. As already reported, neosynthesis of cAMP was decreased following progesterone application. This decrease was not observed, or it was considerably reduced, in oocytes previously injected with antipain. In amphibian, full-grown ovarian oocytes are arrested at first meiotic prophase, and have a large nucleus known as the germinal vesicle. Progesterone induces the production of a cytoplasmic maturation-promoting factor (MPF), which itself triggers germinal vesicle breakdown (GVBD), and subsequent events of meiotic maturation (Masui and Markert, 1971; Gerhart et al., 1984). A considerable body of evidences support the view that release from prophase block is due to inactivation of a cAMP-dependent protein kinase (reviewed by Maller, 1983). On the other hand, progesterone has been shown to induce a transient decrease in cAMP level (Speaker and Butcher, 1977; Schorderet-Slatkine et al., 1982; Cicirelli et al., 1985), and this initial drop of cAMP, along with a number of studies indicating a decrease in adenylate cyclase activity (Mulner et al., 1979; Baltus et al., 1981; Sadler and Maller, 1981; Finidori-Lepicard et al., 1981; Jordana et al., 1981), provided key support to the theory that an early drop in cAMP led to the dephosphorylation of a hypothetical protein which initiates maturation.(ABSTRACT TRUNCATED AT 250 WORDS)     

A critical examination of some histochemical methods for demonstrating tetrahydrofolate dehydrogenase

Synopsis Two different methods, one proposed by us (1969, 1970) and the other by Onicescuet al. (1970), for the histochemical demonstration of tetrahydrofolate dehydrogenase have been compared using as test materials frozen sections of mouse liver and blood smears of laboratory animals that had been infected experimentally with malaria.Our method appears to be of practical value, especially for blood smears. It is based on the following principles: (a) the enzymic reaction proceeds via the reduction of the pyridine coenzyme NADP by tetrahydrofolic acid (dissolved in 2-mercaptoethanol to prevent its spontaneous oxidation); (b) an intermediate electron carrier, phenazine methosulphate, is used to increase the reaction sensitivity; and (c) antifolate metabolic inhibitors are added in specificity controls.The differences between other methods, which have failed in our hands, and our method are discussed critically.Centre for the Study of Histochemistry of the C.N.R.     

Methylations in human hemoglobin

Levels of N-Methylvaline (MeVal) and N tau-methylhistidine (MeHis) were measured in male smokers and non-smokers in a program aimed at mapping background alkylations of hemoglobin (Hb) as potential indicators of doses of exogenous and endogenous genotoxic agents. MeVal was also determined in Hb from rats, Syrian golden hamsters, mice and chickens. MeVal was found to occur at levels around 0.5 nmole/g Hb, with relatively little variation between individuals and species. MeVal was not significantly affected by smoking. This result contrasts with elevated levels of N-hydroxyethylvaline (HOEtVal) measured in the same persons (T?rnqvist et al., 1986b). Levels of S-methylcysteine (MeCys) (Bailey et al., 1981) and MeHis were much higher than those of MeVal. The high levels of MeCys and MeHis may be due partly to misincorporation during protein synthesis and to artifacts. S-Adenosylmethionine and formaldehyde are possible endogenous sources of MeVal. One individual (smoker) out of 21 selected for measurement of MeVal was an outlier, with raised levels of both MeVal and HOEtVal, as would be expected in case of a defective detoxification system.     

Azoindoxyl methods for the histochemical investigation of hydrolases. I. Lactase (lactase-beta-glucosidase complex) (author's transl)]

An azoindozyl method for the histochemical demonstration of lactase (lactase-beta-glucosidase complex) is described. The incubation medium consists of 5 mg 5-Br-4-Cl-3-indolyl-beta-D-fucoside (dissolved in 0.5 ml N,N-dimethylformamide) and 0.02 ml hexazotized prosaniline in 10 ml 0.1 M citric acid phosphate buffer, pH 6-6.5. By means of this method lactase can be exactly localized in the brush border of the enterozytes in the jejunum of suckling rats. Compared to the corresponding indigogenic method the azoindoxyl reaction proceeds faster and the reaction product is often precipitated more precisely.     

Disulfide Bond Formation in the Mammalian Endoplasmic Reticulum

The formation of disulfide bonds between cysteine residues occurs during the folding of many proteins that enter the secretory pathway. As the polypeptide chain collapses, cysteines brought into proximity can form covalent linkages during a process catalyzed by members of the protein disulfide isomerase family. There are multiple pathways in mammalian cells to ensure disulfides are introduced into proteins. Common requirements for this process include a disulfide exchange protein and a protein oxidase capable of forming disulfides de novo. In addition, any incorrect disulfides formed during the normal folding pathway are removed in a process involving disulfide exchange. The pathway for the reduction of disulfides remains poorly characterized. This work will cover the current knowledge in the field and discuss areas for future investigation.One of the characteristics of proteins that enter the secretory pathway is that they frequently contain covalent linkages called disulfide bonds within and between constituent polypeptide chains. The presence of these linkages is thought to confer stability when secreted proteins are exposed to the extracellular milieu or when membrane proteins are recycled through acidic endocytic compartments. In addition to structural disulfides it is now clear that a number of proteins use the formation and breaking of disulfides as a mechanism for regulation of activity (Schwertassek et al. 2007). Hence, it is important that we have a clear understanding of how correct disulfides are formed within proteins both during the protein folding process and to regulate protein function. The focus of this article will be on how correct disulfides are introduced into proteins within the secretory pathway, specifically within the endoplasmic reticulum (ER) during folding and assembly.The formation of disulfides within polypeptides begins as the protein is being cotranslationally translocated into the ER (Chen et al. 1995). The initial collapse of the polypeptide and formation of secondary structure brings cysteine residues into close enough proximity for them to form disulfides. Correct disulfide formation requires enzymes to both introduce disulfides between proximal cysteines and to reduce disulfides that form during folding but that are not present in the final native structure (Jansens et al. 2002). In addition, proteins that do not fold correctly are targeted for degradation and may require their disulfides to be broken before dislocation across the ER membrane into the cytosol (Ushioda et al. 2008). Hence, there must be a reduction and oxidation pathway present in the ER to ensure that native disulfides form and nonnative disulfides are broken during protein folding.Central to both reduction and oxidation pathways is the protein disulfide isomerase (PDI) family of enzymes (Ellgaard and Ruddock 2005) that are capable of exchanging disulfides with their substrate proteins (Fig. 1). Whether disulfide exchange results in the formation or breaking of a disulfide depends on the relative stability of the disulfides in the enzyme and substrate. To drive the formation of disulfides, the PDI family member must itself be oxidized. It is now clear that there are a number of ways for the disulfide exchange proteins to be oxidized by specific oxidases. Importantly, these oxidases do not introduce disulfides into nascent polypeptide chains; rather, they specifically oxidize members of the PDI family. The exception to this rule is the enzyme quiescin sulfydryl oxidase (QSOX; see below). The pathway for disulfide reduction is not as well characterized. It is known that the PDI family members can be reduced by the low molecular mass thiol glutathione (GSH) (Chakravarthi and Bulleid 2004; Jessop and Bulleid 2004; Molteni et al. 2004) but no enzymatic process for reduction has been identified. The glutathione redox balance within the ER is significantly more oxidized than in the cytosol (Hwang et al. 1992; Dixon et al. 2008), indicating that GSH is actively oxidized to glutathione disulfide either during the reduction of PDI family members or by reducing disulfides in nascent polypeptides directly. However, there is currently no clear indication as to how glutathione disulfide is itself reduced.Open in a separate windowFigure 1.PDI family of enzymes catalyzes disulfide exchange reactions in the endoplasmic reticulum. Nascent polypeptide chains are cotranslationally translocated across the ER membrane whereupon cysteines in close proximity can form disulfides. The reaction is catalyzed by members of the PDI family (depicted as PDI) by a disulfide exchange reaction resulting in the reduction of the PDI active site. If nonnative disulfides are formed these can be reduced by the reverse disulfide exchange reaction, resulting in the oxidation of the PDI active site.Both the formation and breaking of disulfides can be thought of as electron transport pathways that require suitable electron acceptors or donors to drive the flow of electrons. For the purposes of this article the two pathways will be discussed separately, but it should be appreciated that each pathway occurs within the same organelle so the possibility of crossover between them is real. Whether futile redox reactions occur between the pathways is unclear but any kinetic segregation of the pathways will be highlighted where it is known to occur.     

On the method of Glenner for the histochemical demonstration of the monoamine oxidase (MAO).

The method of Glenner et al. for the histochemical demonstration of MAO activity was studied by means of scanning microdensitometry and discrete measurement of optical density (lambda=590 nm) of the liver and brain tissues sections. The experiments indicated that: (1) The optimal time of incubation (the thickness of sections is 15 mum) is 60-90 min. (2) The histochemical reaction proceeds with the following substrates: dopamine, noradrenalin, serotonin, and tryptamine. (3) Iproniazid is the best inhibitor for preincubation whereas for simultaneous inhibition the substrate semicarbazide is better. (4) The incubation under the anaerobic conditions caused a considerable decrease of the stain intensity of the sections. We consider these data to indicate that both the aldehydes and acids formed under oxidation may take part in direct reduction of NBT to diformazan. (5) The histochemical reaction for MAO without substrates testifies to the presence of endogenous amines or other redox reactions leading to the reduction of NBT.     

Freezing injury in spinach leaf tissue: Effects on water-soluble proteins, protein-sulfhydryl and water-soluble non-protein-sulfhydryl groups

Freezing of spinach leaf discs ( Spinacia aleracea L. cv. Estivato) resulted in an irreversible and parallel loss of protein-sulfhydryl (SH) and water-soluble protein. This decrease was inversely related to the increase in freezing injury as determined by the loss of electrolytes from the tissue after thawing. Loss of proteins and protein-SH occurred during freezing of the tissue and was not enhanced by thawing. The parallel decreases in content of soluble proteins and SH groups make it impossible to determine whether oxidation of protein-SH groups is the primary step in decline of protein content. During freezing the content of non-protein-SH compounds, mainly glutathione (GSH), was decreased to a lesser extent than that of protein-SH. Contrary to protein-SH, the levels of non-protein-SH declined substantially after thawing. The data indicate that GSH is not directly involved in protection of soluble proteins against freezing-induced denaturation.     

Microphotometric determination of enzymes in brain sections

Summary An incubation medium was adapted for the microphotometric determination (kinetic and end-point measurements) of the activities of mitochondrial alpha-glycerophosphate dehydrogenase (GPDH) in the rat hippocampus. For comparison, the activities of the cytoplasmic NAD-linked alpha-glycerophosphate dehydrogenase were also measured. The study showed that in the demonstration of both enzymes the use of an exogenous electron carrier is necessary. Both enzymes react to phenazine methosulfate (PMS) which transfers reduction equivalents to the electron acceptor nitroblue tetrazolium chloride (NBT), thus causing a coreaction of GPDH in the demonstration of NAD-GPDH. Therefore, only the NAD-independent GPDH which is stimulated by menadione, can be selectively demonstrated in the histochemical procedure applied. The final incubation medium of GPDH consisted of 15 mM l-glycerol 3-phosphate, 5 mM NBT, 0.4 mM menadione, 7.5% polyvinyl alcohol in 0.05 M Hepes buffer, pH 8; the final pH of the incubation medium was 7.5. A linear response of the reaction lasted about 5 min. There was a linear relationship between section thickness and the formation of reaction product up to a section thickness of 14 microns. The apparent K _m value at 25°C was 0.6 mM. It is concluded that using menadione histochemical methods are suited to determine the mitochondrial GPDH activities in brain sections whereas using PMS a coreaction of GPDH takes place in the demonstration of NAD-GPDH, so that a histochemical quantification of NAD-GPDH cannot be recommended.     

Histochemistry and functional significance of putative neocortical transmitter substances: a review

1. Intrinsic neuronal chains of the neocortex communicate most probably with amino acid transmitters. These involve both excitatory (glutamate, aspartate--Nadler et al. 1976) both inhibitory (GABA--Ribak 1978) amino acids, and ensure fast, ionotropic postsynaptic actions (Eccles, McGeer 1979). 2. Some interneurons of the neocortex seemingly operate with the peptide transmitter VIP (Lorén et al. 1979). Presumably, this is a metabotropic, slowly acting substance (Dodd, Kelly and Said 1979). 3. The existence of intrinsic cholinergic neurons in the neocortex is a matter of question (Krnjevic and Silver 1965). It is worth to mention that in the periphery, cholinergic terminals also contain and release VIP (H?kfelt et al. 1980). It is not known, whether this transmitter dualism can be found in neocortex, too. An ascending cholinergic system projecting from the basal forebrain to the neocortex exists and exerts profound influence on cortical function (Shute and Lewis 1967). 4. Diffusely terminating, ascending monoamine axons innervate the neocortex and modulate interneuronal transmission (Thiery et al. 1977; Morrison et al. 1981, Lidov et al. 1981). 5. The neuropeptide SP excites cortical neurons (Phillis and Limacher 1974), and its presence in thin axons can be demonstrated immunohistochemically (H?kfelt et al. 1976). 6. Neocortical efferents to the thalamus and striatum seemingly use glutamate or aspartate (Fonnum et al. 1981). The transmitters of other corticofugal projections are not known. 7. The transmitters of specific thalamic afferents and those of callosal and association projections are unknown, too. 8. The main task of future histochemistry is to explore the synaptology of neocortical neurons and afferent systems with identified or evidenced transmitters, viz. to explore the neurochemical subsystems of cortical organization. The tool for it could be the immunohistochemistry, and future development depends mainly on the synthesis and purification of suitable antigens. The knowledge on the synaptology of identified neurochemical units of the cortex would be the basis of the understanding at least partly of the pharmacological effects exerted by the putative neocortical transmitters.