Diphosphopyridine Nucleotide Diaphorase Research Articles

Alcoholic hyalin-containing hepatocytes--a characteristic morphologic appearance.

We studied the morphologic appearance of alcoholic hyalin (AH)-containing hepatocytes in liver biopsies from 14 patients with alcoholic liver disease. Most hepatocytes had a characteristic appearance. The cells were swollen and hydropic with an intact cell membrane. The mitochondria had variable-sized cristae which were both shortened and elongated. The smooth endoplasmic reticulum was markedly decreased. The rough endoplasmic reticulum was bizarre, with detachment of the ribosomes that surrounded the AH. The hepatocytes that contained AH bodies had lost almost all the glucose-6-phosphate activity but had variable amounts of succinic dehydrogenase and diphosphopyridine nucleotide diaphorase activities. The neutrophils admixed with mononuclear cells attached themselves to the hepatocytes and then invaginated into the hepatocytic cytoplasm with focal lysis of the cell membrane mediated via the release of neutrophilic lysosomes. The distortion of protein-synthesizing organelles and decrease in glucose-6-phosphatase activity suggest that the AH-containing hepatocyte is metabolically decompensated. The final cell death may be related to the neutrophilic attack, rather than the metabolic derangement.

Acute viral hepatitis: Morphologic and functional correlations in human livers

The usual histologic pattern in acute viral hepatitis (AVH) includes cellular abnormalities predominantly in the perivenular (zone 3) hepatocytes and changes interpreted as representing regenerative activity in the periportal (zone 1) hepatocytes. Enzyme histochemical and ultrastructural studies of livers of 12 patients with AVH were undertaken to see whether these features support the concept of regeneration of hepatocytes in zone 1. The swollen hepatocytes in the perivenular areas were hydropic, with dilated or eccentric rough endoplasmic reticulum and decreased or vesicular smooth endoplasmic reticulum; correspondingly, the glucose-6-phosphatase activity (reflecting, when present, intact and functional endoplasmic reticulum) was markedly decreased. Succinic dehydrogenase and diphosphopyridine nucleotide diaphorase activities, representing mitochondrial enzymes, were limited to the perinuclear or pericanalicular cytoplasm of swollen hepatocytes. gamma-Glutamyl transpeptidase activity was increased. The periportal hydropic hepatocytes were small and arranged in clusters displacing sinusoids. Ultrastructurally, these hepatocytes had nearly normal organelles but scanty smooth endoplasmic reticulum. Activities of the enzymes glucose-6-phosphatase, succinic dehydrogenase, and diphosphopyridine nucleotide diaphorase were weak, although glycogen was abundant. gamma-Glutamyl transpeptidase activity was scanty in these hepatocytes. These findings from enzyme histochemical and electron microscopic studies could be interpreted as evidence of functional deterioration of perivenular swollen hepatocytes and relative functional immaturity of periportal hydropic clustered hepatocytes, suggesting regeneration of zone 1 hepatocytes.

Histochemistry and ultrastructure of the heart in experimental cobalt cardiomyopathy in the dog

Experimental cobalt cardiomyopathy was studied in dogs given cobalt twice weekly through a chronic indwelling iv catheter. The dogs became dyspneic and lethargic and had exercise intolerance. Cardiac failure developed in some animals. The hearts in these dogs were pale and flabby with dilatation of the atria. All had scattered myocardial degeneration characterized by diffuse cytoplasmic vacuolation, loss of cross striations, and interstitial edema. No inflammation was seen. Histochemical reactions for succinic dehydrogenase and reduced diphosphopyridine nucleotide (DPN) diaphorase were done on frozen heart muscle. There was no differences in activity of reduced DPN diaphorase when muscle from control dogs was compared to muscle from treated dogs. The activity of succinic dehydrogenase was decreased in cardiac muscle taken from dogs in the two cobalt-treated groups. Ultrastructurally, the mitochondria were extremely swollen with loss of cristae. Electron-dense particles were seen in some damaged mitochondria. The sarcoplasmic reticulum and the T system contained numerous dilated vesicles in cells with mitochondrial and myofibrillar damage. Loss of cross striations and Z-band thickening were present in degenerating myofibers. Lipid droplets of varying sizes, some containing dense osmiophilic particles at the periphery, were observed in the myocytes.

Laminar organization of the extraocular muscles of the rabbit

The functional organization of the inferior rectus muscle of the rabbit was studied with electrophysiological, mechanical, and anatomical techniques. The activity of single inferior rectus muscle fibers was recorded with a microelectrode while the isometric force of the inferior rectus muscle was modulated by vestibuloocular reflex activation. The active isometric force of the inferior rectus muscle at which a muscle fiber was recruited into activity was defined as the recruitment threshold for that particular muscle fiber. Recruitment thresholds were measured for 100 muscle fibers. Fibers with low recruitment thresholds were scattered throughout the inferior rectus, but concentrated most densely in the orbital rim. Multiple-unit recordings at various depths within the inferior rectus muscle demonstrated an almost exclusive activity of the orbital rim fibers at active isometric forces of less than 1 g. The development of active tension in the orbital layer of a surgically divided inferior rectus muscle precedes the development of tension in the nonorbital layer. Histochemically, the orbital layer of the inferior rectus muscle has a high oxidative metabolism as evinced by succinic dehydrogenase, diphosphopyridine nucleotide diaphorase, and acid-preincubated ATPase stains. The entire inferior rectus muscle stained intensely for ATPase with alkali preincubation. An analysis of serial cross sections of the inferior rectus muscle near its insertion revealed that the fibers of the orbital rim have the most lateral and the most anterior insertions. These data suggest that muscle fibers of the orbital rim of the inferior rectus muscle maintain the stability of the eye during horizontal eye movements by moving the “effective insertion” of the muscle in the opposite direction to the movement of the “anatomic insertion.”

Cytoenzymology of benign and malignant tumours of the corpus uteri: I. Respiratory enzymes

The activity of a group of respiratory enzymes was studied in normal menopausal as well as benign and malignant tumours. A decrease in the activity of succinic dehydrogenase, diphosphopyridine nucleotide diaphorase and cytochrome oxidase in malignant tumours especially in spindle cell sarcoma and undifferentiated adenocarcinoma was observed. Benign tumours manifested variable results, thus cellular fibromyoma showed no changes in the activity of succinic dehydrogenase, diphosphopyridine nucleotide diaphorase and cytochrome oxidase whereas fibromyomal cells exhibited less enzymatic activity. In addition, no marked difference was observed in the activity of glucose-6-phosphate dehydrogenase in the aforementioned tumours as compared with normal menopausal uterine tissues.

Histochemical properties of skeletal muscle fibers in streptozotocin-diabetic rats.

The response of rat gastrocnemius muscle fibers to chronic streptozotocindiabetes was studied. Transverse sections of this muscle from normal and diabetic rats were histochemically assayed for reduced diphosphopyridine nucleotide-diaphorase, myofibrillar adenosine triphosphatase, mitochondrial alpha-glycerophosphate dehydrogenase, beta-hydroxybutyrate dehydrogenase, and alkaline phosphatase activities. Cross-sectional areas of the fiber types were measured, and fiber capillarization and populations estimated. Chemically-induced diabetes appeared to have little effect on the metabolic or morphological properties of slow-twitch fibers. However, a general dedifferentiation occurred in the 2 fast-twitch fiber populations. There was a loss of oxidative potential in the fast-twitch-oxidative-glycolytic fibers, and a significant decrease in size in the fast-twitch-glycolytic fibers. No change in the proportions of slow- and fast-twitch fibers in the muscles of diabetic rats occurred. It is concluded that hypoinsulinism has differential effects on the 3 fiber types in heterogeneous rat skeletal muscle, and that slow-twitch fibers are least affected by the diabetic condition.

Histochemical demonstration of mitochondrial dehydrogenases in developing normal and nervous mutant mouse Purkinje cells.

The mouse mutation nervous (nr), expressed as a recessive, causes a morphologic change in cerebellar Purkinje cell perikaryonal mitochondria during the 2nd postnatal week which is followed by the selective degeneration of most Purkinje cells. In the present study, mitochondrial dehydrogenase activities (succinic acid dehydrogenase, lactic acid dehydrogenase, malic acid dehydrogenase, reduced diphosphopyridine nucleotide diaphorase and succinic semialdehyde dehyrogenase) are found by light microscopy to be similar in the perikarya of normal and nervous Purkinje cells until after mitochondrial rounding has begun to occur, but then staining increases markedly in nervous. Although only a small fraction of mitochondrial functions were examined, those enzymes assayed were present and competent. In a sense, the only mitochondrial abnormalities demonstrated thus far in nervous Purkinje cells are their unusual shape and probable loss of motility.

Ototoxic Drug Effects on Cochlear Histochemistry

Cochlear histochemical changes after administration of kanamycin sulfate and ethacrynic acid were studied in normal guinea pigs. The activities of cochlear succinic dehydrogenase and reduced diphosphopyridine nucleotide diaphorase were more suppressed by a large amount of kanamycin administered over a shorter duration than by a moderate amount given over a longer duration and were suppressed by ethacrynic acid given intravenously in one large dose or by middle ear injection. Both ethacrynic acid and kanamycin had a greater effect on the respiratory enzyme activities of the basal turn than the other turns. Kanamycin had a greater effect on cochlear respiratory enzyme activities than on adenosine triphosphatase (ATPase) activity. Ethacrynic acid, in the doses given, had no effect on cochlear ATPase activity.

Enzyme cytochemistry of the abraded gastric surface epithelium. An aid in the diagnosis of early atrophic gastritis and focal intestinal metaplasia.

Enzyme cytochemistry of the abraded gastric mucosa was here developed as a new procedure and applied to the study of atrophic gastritis. This new technic consists of determining 5 enzymes in the superficial epithelium of the gastric mucosa, abraded by conventional cytologic brush technic—namely, alkaline phosphatase, acid phosphatase, leucine amino peptidase, lactic acid dehydrogenase and diphosphopyridine nucleotide diaphorase. Enzyme cytochemistry of the abraded surface epithelial cells was correlated, in 49 individuals, with the histology and cytology of the gastric mucosa, as obtained by gastric suction biopsy and conventional cytologic technic. In addition, in 31 of these patients, enzyme histochemistry of the gastric surface epithelium, obtained by gastric suction biopsy, was studied and correlated with enzyme cytochemistry of the smears of the abraded surface epithelium of the stomach. The results obtained indicate similar yields of cytochemical and histochemical methods in individuals with normal gastric mucosa and chronic superficial gastritis. However, enzyme cytochemical study of the abraded mucosa appears to be superior to histologic and enzyme histochemical examination of the gastric suction biopsy specimens in recognizing the early atrophic lesions of the gastric mucous membrane. These manifest themselves by positive reactions of the abraded surface epithelium for acid phosphatase, lactic acid dehydrogenase and diphosphopyridine nucleotide diaphorase. Enzyme cytochemistry is especially important in diagnosing the focally distributed intestinal metaplasia, which is often missed in gastric suction biopsy. This can be easily recognized by strongly positive reactions of the abraded surface epithelium for alkaline phosphatase and leucine amino polypeptidase. In view of the relation existing between the foci of intestinal metaplasia and development of gastric cancer in this area such a recognition may be significant in the followup of patients presenting this type of lesion.

Histochemical localization of brain succinic semialdehyde dehydrogenase--a -aminobutyric acid degradative enzyme.

Succinic semialdehyde dehydrogenase is the final enzyme in the degradative pathway of γ-aminobutyric acid, a postulated inhibitory synaptic transmitter. This study reports a specific, sensitive technique for the localization of succinic semialdehyde dehydrogenase in rat brain. The technique utilizes the direct reduction of nitroblue tetrazolium by reduced pyridine nucleotide at an alkaline pH, a mechanism of formazan production substantiated by biochemical studies. The succinic semialdehyde dehydrogenase histochemical reaction is completely inhibited by p-hydroxybenzaldehyde and m-hydroxybenzaldehyde, compounds which have no effect on succinic acid dehydrogenase and reduced diphosphopyridine nucleotide diaphorase. Conversely, succinic acid dehydrogenase and reduced diphosphopyridine nucleotide diaphorase are inhibited by malonate and nordihydroguaiaretic acid, respectively, substances which have no effect on succinic semialdehyde dehydrogenase. The distribution and intensity of staining for succinic semialdehyde dehydrogenase are different from that seen following the succinic acid dehydrogenase and reduced diphosphopyridine nucleotide diaphorase reactions and can be localized to groups of neurons and their cell processes, e.g., the Purkinje cell dendritic tree, and in specific regions of neuropil.

Histochemically demonstrable fibre abnormalities in normal skeletal muscle and in muscle from carriers of Duchenne muscular dystrophy.

Deltoid muscle was removed at motor point biopsy from 10 female relatives of patients with Duchenne muscular dystrophy and from seven others, with no evidence of neuromuscular disease. Transverse cryostat sections of the muscle from each case were stained for reduced diphosphopyridine nucleotide diaphorase and it was found that all contained varying numbers of degenerating type 1 fibres. The percentage of abnormal fibres in the type 1 fibre population was then calculated for each case and it was found that the muscles from the patients with dystrophic relatives contained considerably higher percentages of abnormal fibres, which also showed more severe degeneration, than did the muscles from the normal cases. There was no absolute correlation between serum creatine kinase levels and degree of pathological change, though patients with the most severe changes in their muscles had abnormally high serum creatine kinase levels. It is suggested that histochemical studies could be a useful addition to the present diagnostic tests for the carrier state in Duchenne muscular dystrophy.

Neuronal dynamics and axonal flow. IV. Blockage of intra-axonal enzyme transport by colchicine.

The fact that the nucleated center of the nerve cell is the major source of the macromolecular materials required in the maintenance and function of the whole neuron requires the operation of a steady cellulifugal convection of these supplies into and down the nerve fiber. This proximo-distal traffic has been firmly established, but the mechanisms involved in it are still poorly understood. Besides the slow (ca. 1 mm per day) advance of the axonal column as a whole ("axonal flow" in the strict sense), the demonstration of additional, much faster, traffic rates (up to several cm per day) calls for special conduits within the axon ("intra-axonal flow"). To test the possible role of neurotubules (average width:220 A) in this traffic, the drug colchicine, known for its immobilizing effect on microtubules in other types of cells, was locally injected into peripheral nerves. This resulted in a major blockage of the proximo-distal movement of a test enzyme, acetylcholinesterase, into and through the injected zone, the extent of blockage varying with the applied dosage. By analogy, the neurotubules thus seem to be definitely implicated in the motile mechanism of intra-axonal transport. By contrast, the movement of a mitochondrion-associated marker enzyme, diphosphopyridine nucleotide diaphorase, was not perceptibly affected (in the submaximal dosage range), which seems to signify that the proximo-distal shift of mitochondria, for which the slow axonal flow acts as carrier, has gone on uninterruptedly. The experiments thus indicate the possibility of uncoupling the axonal and intra-axonal transport mechanisms.

Protein and enzyme changes in cold injury edema.

Protein and enzyme changes in the edematous white matter and the CSF were studied during the course of experimental cold injury edema. The edematous white matter showed an increase in total content of albumin which on disc gel electrophoresis separated as four distinct patterns and appeared even more complex in the two-dimensional fractionation patterns. No increase was noted in γ-globulins, although they revealed a greater variety, as compared with the normal white matter. In the CSF there was a persistent increase in percentage of γ-globulin. It is speculated that although there is an increased entry into the CSF of both albumin and globulin, the levels of the former are reduced to normal values by a selective resorption by the choroid plexus. Enzymatic assays on the edematous white matter revealed an increase in the levels of α-glycero-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase. There was a marked decrease in lactic dehydrogenase and diphosphopyridine nucleotide diaphorase. In the CSF enzyme determinations showed up to 10-fold increase in lactic dehydrogenase levels with the highest values recorded in the fluid from the lateral ventricle underlying the edematous white matter.

A morphologic and histochemical study of three oxidative enzymes in the hamster submandibular gland

Several oxidative enzymes have been examined histochemically in the normal adult hamster submandibular gland. Patterns and levels of activity of succinic dehydrogenase, diphosphopyridine nucleotide diaphorase and triphosphopyridine nucleotide diaphorase were recorded. Strikingly different patterns of activity were found with each of the three enzymes. In general, ductal cells exhibited more intense enzyme activity than acinar cells. Characteristic staining of different intralobular segments was observed with each enzyme. These findings indicate a probable functional specificity of the various cell types.

Generalized eruptive histiocytoma. Enzyme histochemistry and electron microscopy.

Histochemical study of generalized eruptive histiocytoma (GEH) showed large amounts of hydrolytic enzymes, including nonspecific esterase, aminopeptidase, nucleoside triphosphatase, and acid phosphatase, as well as the oxidative enzymes, succinic dehydrogenase and dephosphopyridine nucleotide diaphorase, in moderate amounts. Succinic dehydrogenase was evenly distributed throughout the tumor cells with diphosphopyridine nucleotide diaphorase only in localized foci. The enzymatic signature of GEH is similar to that in histiocytes of normal skin as well as that in other histiocytic disorders. The enzymes seem characteristic of the histiocytic cells themselves and may not be dependent on cellular change occurring in inflammation and active phagocytosis. Ultrastructural observations supported the light microscopic impression of a cellular proliferative lesion in which significant phagocytosis was not evident.

Enzyme-histochemical Studies on the Brain Stem of Spontaneously Hypertensive Rats

1. Enzyme-histochemical study was per-formed on the brain stem of spontaneously hypertensive rats (OKAMOTO and AOKI). Four kinds of enzyme activity (acid phosphatase, diphosphopyridine nucleotide diaphorase, monoamine oxidase and acetylcholine esterase) were examined on 11 nuclei of brain stem of a total of 11 spontaneously hypertensive rats and 11 normotensive control rats of Wistar strain which were subdivided into two groups according to survival duration (40-60 days after birth (Stage I) and 3.5-4 months after birth (Stage II)). Each positive finding in spontaneously hypertensive rats was obtained by comparing it to that of normotensive controls. 2. Mean systolic blood pressure of spontaneously hypertensive rats was significantly higher than that of controls, despite that, it was still in normotensive range in Stage I. Mean body weight did not show any significant difference between spontaneously hypertensive rats and control rats. 3. As to the normal distribution of enzyme activities in the brain stem, an outline of the reactions observed in the brain stem nuclei of normotensive control rats was described. 4. Comparing the results from spontaneously hypertensive rats with those of controls the following findings were obtained. Increased enzyme activities were found in Nucl. originalis dorsalis nervi vagi (acid phosphatase, diphosphopyridine nucleotide diaphorase, monoamine oxidase and acetylcholine esterase in Stage I ; acid phosphatase and diphosphopyridine nucleotide diaphorase in Stage II), Nucl. reticularis lateralis (acid phosphatase in Stage II), Nucl. reticularis pontis oralis (diphosphopyridine nucleotide diaphorase in Stage I), Nucl. reticularis gigantocellularis (diphosphopyridine nucleotide diaphorase in Stage I), Edinger-Westphal's nucleus (monoamine oxidase in Stage I and diphosphopyridine nucleotide diaphorase in Stage II). Decreased activity was observed only in Nucl. reticularis tegmenti (acid phosphatase and diphosphopyridine nucleotide diaphorase in Stage I). 5. From the above-mentioned findings it was assumed that the autonomic nervous sys-tem in the brain stem of the spontaneously hypertensive rats may be in overactive state, being particularly distinct in the parasympathetic zones centering around the dorsal vagal nucleus in this study, though detailed etiological participation of this area could not be clarified.

Pitfalls in the histochemistry of the inner ear.

THE CORRELATION of structure and function in the inner ear is a real challenge to the morphologist. Because of its complex anatomy, the inner ear lends itself to histochemical investigations. It is important, however, to emphasize the dangers inherent in the application of histochemical methods to the inner ear. Results of histochemical staining may depend on techniques used.¹Variations in the distribution pattern of diformazan may result from the use of fixatives. The purpose of this paper is to demonstrate these differences in the staining pattern of reduced diphosphopyridine nucleotide (DPNH) diaphorase activity. <h3>Material and Methods</h3> The cochleas of 25 adult albino guinea pigs were used in this study. The substrate solution was prepared by dissolving 1.5 mg of DPNH and 1.5 mg of potassium cyanide in 5 ml of 0.1 M veronal buffer at pH 7.4. Nitro blue tetrazolium was added to the substrate solution to form

Enzyme histochemical studies on the hypothalamus of spontaneously hypertensive rats with special reference to that of rats subjected to various endocrine interferences.

Enzyme histochemical studies were performed on the hyhothalamus of spontaneously hypertensive rats (OKAMOTO and AOKI) in the pre-hypertensive stage (40 to 60 days after birth). Each positive finding in spontaneously hypertensive rats was obtained by comparing it to the reaction of normotensive Wistar strain rats with almost the same survival duration. On the other hand, in order to clarify the meaning of the findings thus obtained in spontaneously hypertensive rats, the hypothalamus of normotensive Wistar strain adult rats subjected to various endocrine interferences were similarly examined histochemically and compared with the reaction of the control adult rats. 1) As far as the normal distribution of enzyme activities in the hypothalamus is concerned, acid phosphatase activity was clearly demonstrated in the cytoplasma of nerve cells in contrast with glial substances. Most prominent staining was found in nerve cells of Nucl. paraventricularis and Nucl. supraopticus. Nucl. perifornicalis and Nucl. mamillaris prelateralis were stained relatively deeply. Most of the others were stained moderately. By diphosphopyridine nucleotide diaphorase activity determination, all hypothalamic nuclei showed more or less the activity in the perikaryon of nerve cells. Monoamine oxidase activity was mainly detected diffusely in neuropils and hardly demonstrated in the perikaryon. 2) By enzyme histochemical studies on the hypothalamus of spontaneously hypertensive rats in the pre-hypertensive stage, increase of acid phophatase activity was observed in Nucl. preopticus pars lateralis, Nucl. supraopticus, Nucl. ventromedialis pars anterior, medialis, centralis et lateralis, Nucl. dorsomedialis, Nucl. perifornicalis, Area lateralis hypothalami pars tuberalis, Nucl. arcuatus pars tuberalis et mamillaris, Nucl. posterior hypothalami, Nucl. mamillaris prelateralis, Nucl. supramamillaris and Nucl. mamillaris medialis. Increase of diphosphopyridine nucleotide diaphorase activity was detected in Nucl. premamillaris dorsalis, Nucl. premamillaris ventralis and decrease of the activity was noted in Nucl. arcuatus pars mamillaris and Nucl. mamillaris lateralis. Enhancement of monoamine oxidase activity was observed in Nucl. arcuatus pars tuberalis, Nucl. ventromedialis pars medialis, centralis et lateralis and the activity was reduced in Nucl. suprachiasmaticus and Nucl. mamillaris prelateralis.

Metabolic activities of the endolymphatic sac. An enzyme histochemical and autoradiographic study.

The distribution of diphosphopyridine nucleotide diaphorase, triphos-phopyridine nucleotide diaphorase, lactic dehydrogenase, malic dehydrogenase, succinic dehydrogenase, isocitric dehydrogenase, glucose-6-phosphate dehydrogenase, and β-hydroxybutyric dehydrogenase, cytochrome oxidase, alkaline and acid phosphatase, leucine- and alanine-aminopeptidase activity was demonstrated histochemically in the endolymphatic sac and in the perisaccular connective tissue. Enzyme activity was stronger in the epithelial cells lining the intermediate portion and in the cells floating free in the endolymph than in other parts of the sac, except for aminopeptidase activity which was marked in the perisaccular connective tissue, too.Foreign protein (peroxidase) was injected into the cochlear duct and after two days was found phagocytized in cells floating free in the endolymphatic sac but not in the lining cells themselves or elsewhere in the membranous labyrinth. Peroxidase was also observed in fibroblasts of the perisaccu...

Development of the Lactic Dehydrogenase — Enzyme System in the Testes and Epididymis of Young Dairy Bulls ,

Development of the lactic dehydrogenase diphosphopyridine nucleotide diaphorase system (LDH) in the testis and epididymis of sexually immature dairy bulls has been investigated. The study included tissues from seven animals varying in age from one to 367 days. The degree of staining, indicating LDH activity, for the cells of the seminiferous tubules and interstitual tissue is described. Staining of spermatozoa, when found, was confined to the midpiece.The cytological distribution of the LDH system in the epididymis at various ages is also presented. In the cells of the body portion of the epididymal canal high levels of activity were observed in the apex of the epithelial cells, with low levels of activity prevailing in the general cytoplasm. In the head and tail portions of the canal the system showed low levels of activity homogeneously distributed throughout the cytoplasm. It appears from these observations that the physiological properties of various portions of the epididymal canal are highly specialized.