Reduced Nicotinamide Adenine Dinucleotide Diaphorase Research Articles

Enzyme Histochemistry of Hemal Nodes of Indian Buffalo (Bubalus bubalis)

The present study was conducted on hemal nodes of buffalo to study the histoenzymic distribution of phosphatases and oxidoreductases. The fresh unfixed buffalo hemal nodes were immediately collected after sacrifice and were subjected to cryostat sectioning at -200 C with cryostat microtome. The sections were incubated with substrates for demonstration of various enzymes. The study revealed that in both the groups, weak alkaline phosphatase (AKPase) activity was observed in capsule of the hemal node. The AKPase activity was moderate in subcapsular sinus, periphery of lymphoid follicles and diffused lymphocytes. Uniform weak Glucose 6-phosphate activity was observed in entire hemal node. Succinic dehydrogenase (SDH) activity was moderate in capsule and lymphoid follicles and weak in subcapsular sinuses. Lactic dehydrogenase activity was moderate to strong in capsule, sub-capsular sinus and peripheral area of lymphoid follicles. Nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-D) and reduced Nicotinamide adenine dinucleotide diaphorase (NADH-D) was weak to moderate in the capsule and moderate to strong in lymphoid follicles. The presence of variable activity of different enzymes in hemal nodes was correlated with maturation of lymphocytes and development of different metabolic pathways.

Different fibre populations distinguished by their calcium transient characteristics in enzymatically dissociated murine flexor digitorum brevis and soleus muscles

Enzymatically dissociated flexor digitorum brevis (FDB) and soleus fibres from mouse were used to compare the kinetics of electrically elicited Ca2+ transients of slow and fast skeletal muscle fibres, using the fast Ca2+ dye MagFluo4-AM, at 20-22 degrees C. For FDB two Ca2+ transient morphologies, types I (MT-I, 11 fibres, 19%) and II (MT-II, 47 fibres, 81%), were found, the kinetic parameters (amplitude, rise time, half width, decay time, and time constants of decay) being statistically different. For soleus (n = 20) only MT-I was found, with characteristics similar to MT-I from FDB. Correlations with histochemically determined mATPase, reduced nicotinamide adenine dinucleotide diaphorase and alpha-glycerophosphate dehydrogenase activities, as well as immunostaining and myosin heavy chain electrophoretic analysis of both muscles suggest that signals classified as MT-I may correspond to slow type I and fast IIA fibres while those classified as MT-II may correspond to fast IIX/D fibres. The results point to the importance of Ca2+ signaling for characterization of muscle fibres, but also to its possible role in determining fibre function.

Lesion progression with time and the effect of vascular occlusion following radiofrequency ablation of the liver.

The effectiveness of radiofrequency ablation (RFA) under selective vascular occlusion and its effects on architecture and viability of normal liver parenchyma was studied in a porcine model. RFA was applied in the liver under general anaesthesia in 18 pigs. Six animals were killed immediately after the procedure and 12 at 24 h. RFA was performed sequentially under four conditions: (1) without vascular occlusion, (2) during occlusion of the hepatic artery, (3) during occlusion of the portal vein and (4) during occlusion of the hepatic artery and portal vein. Liver biopsies from the treated area were stained for conventional histological examination, reduced nicotinamide adenine dinucleotide diaphorase and 5'-nucleotidase activity. Vascular occlusion significantly increased the size of the coagulation centre after RFA. Combined portal venous and arterial occlusion had no additional effect on lesion size compared with venous or arterial occlusion alone. After 24 h, deterioration of viability was observed in the parenchyma up to 3 cm from the coagulated area. The efficacy of RFA in liver increases with occlusion of the portal vein or hepatic artery. The extent of secondary heat-induced necrosis in liver parenchyma should be considered for determination of the final size of the ablated area.

Manganese superoxide dismutase and reduced nicotinamide adenine dinucleotide diaphorase colocalize in the rat gut

Background & Aims Superoxide and other free radicals participate in inflammatory bowel disease and ischemia-reperfusion injury. Manganese superoxide dismutase (SOD) scavenges superoxide. Mn SOD is colocalized with reduced nicotinamide adenine dinucleotide (NADH) diaphorase in some tissues. NADH diaphorase histochemistry selectively stains enteric nerves. The aim of this study was to seek colocalization of Mn SOD with NADH diaphorase in the gut, especially in enteric nerves. Methods Indirect immunofluorescent staining and histochemistry were used to localize Mn SOD and NADH diaphorase in rat gut. Results Strong Mn SOD immunoreactivity was found in parietal cells, most intramural nerve cell bodies, the colonic interstitial cells of Cajal (at the submucosa-circular muscle layer interface), and intestinal epithelium cells. Weak to moderate Mn SOD immunoreactivity characterized smooth muscle cells, small submucosal arteries, esophageal striated muscle, esophageal epithelium, gastric epithelium, and intestinal glands. NADH diaphorase histochemistry (with Triton X-100) resulted in identical staining. Conclusions Mn SOD and NADH diaphorase are colocalized throughout rat gut with strong activity in enteric nerves and colonic interstitial cells of Cajal.

The pretectal complex of the rabbit: distribution of acetylcholinesterase and reduced nicotinamide adenine dinucleotide diaphorase activities.

The chemoarchitecture of the pretectal complex of the rabbit was examined in sections stained by acetylcholinesterase (AChE) and reduced nicotinamide adenine dinucleotide (NADH) diaphorase in the coronal, horizontal and sagittal plane. Twelve different subdivisions can be identified in the rabbit pretectum on the basis of the distribution of both histochemical markers. According to the standard terminology, the pretectal complex of the rabbit consists of: the nucleus of the optic tract; the anterior, posterior, olivary and medial pretectal nuclei; the nucleus of the posterior commissure; the periventricular subcommissural gray; the suprageniculate and internal suprageniculate nuclei, and the dorsal, lateral and medial terminal nuclei of the accessory optic system. The combined use of several sectioning planes and the histochemical mapping of AChE and NADH diaphorase have been of value in resolving the structural limits within transitional regions of the pretectum.

Comparative Mapping of Acetylcholinesterase and Reduced Nicotinamide Adenine Dinucleotide Diaphorase in the Rabbit Dorsal Thalamus

The distribution of acetylcholinesterase and reduced nicotinamide adenine dinucleotide (NADH) diaphorase enzymatic activities was mapped histochemically in the dorsal thalamus of the rabbit. A comparison of the resulting patterns helped in the histochemical delimitation of a number of nuclei, as well as in the detection of some subdivisions, that showed differential expression of these enzymes. It was observed that AChE and NADH diaphorase tend to appear in a complementary fashion in many dorsal thalamic neuropiles, so that intense activity of the one was accompanied by low activity of the other. However, coincident expression of both enzymes was also obtained in a small number of areas. The correlation of these patterns with other chemo-architectonic and hodologic data does not yet disclose an explanation of these regularities, which however suggest some functional significance.

Neuromuscular organization of the pectoralis (pars thoracicus) of the pigeon (Columba livia): implications for motor control.

The pectoralis (pars thoracicus) of the domestic pigeon (Columba livia) is divisible into two anatomical parts, the pars sternobrachialis (SB) and the pars thoracobrachialis (TB). Innervation to this complex is from rostral and caudal branches of the brachial ventral cord. In four anesthetized pigeons, the distribution of muscle units associated with each nerve branch was mapped after prolonged stimulation of each nerve and subsequent analysis for muscle fiber glycogen. An additional three animals were used to analyze the morphology, distribution, and histochemical profiles of the muscle fibers in the SB and TB subregions. Fibers were characterized on the basis of their reactions for myofibrillar adenosine triphosphates (alkaline and acid preincubation) and reduced nicotinamide adenine dinucleotide diaphorase (NADH-D). The SB is primarily innervated by the rostral nerve branch and the TB by the caudal nerve branch. For two-thirds of the muscle's length, the SB is separated from the TB by an aponeurosis, the membrana intermuscularis (MI). SB and TB fibers located posteroventral to the caudal margin of the MI are innervated variously by both nerves. Two populations of fibers were recognized, distinguishable primarily by 1) fiber diameter and 2) density of the NADH-D reaction product. Compared to the TB, the SB possesses a higher average percentage of large fibers. Within the SB but not the TB the percentage of large fibers increases from deep to superficial. These data support our previous findings that the pars thoracicus of the pigeon is partitioned into at least two functional subunits, each with a potential for independent action on the wing during flight.

Effect of endurance exercise on fibre type composition and muscle weight of reinnervating rat plantaris muscle.

The purpose of this study was to evaluate the effect of endurance exercise on the histochemistry and wet weight of the reinnervating rat plantaris muscle. Two groups of young female Wistar rats (6 weeks old), 1 sedentary denervated control (n = 13) and 1 exercised denervated experimental (n = 17), were denervated unilaterally by cutting and resecting the sciatic nerve. To effect reinnervation a skin grafting operation was carried out on the nerve so that the gap caused by resection was bridged. The third group was the sedentary non-denervated normal control (n = 10). A progressive training program of 18 weeks of treadmill running was carried out by the experimental group. Approximately 5 months after denervation, the plantaris muscles were studied histochemically for reduced nicotinamide adenine dinucleotide diaphorase (NADH-D) and mitochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD) activities. Fibres were classified as "red", "white", or "intermediate" with NADH-D. Alpha-GPD differentiates "intermediate" from "red" fibre types in case of difficulty in differentiating these fibre types from each other with NADH-D. The weight of the reinnervated plantaris muscle increased significantly after exercise. The exercise did not change the fibre type proportions--including "red" fibre type--in the deep region of the reinnervating plantaris. There were significant differences between normal control and denervated control or experimental groups in histochemical fibre populations in the deep region of the plantaris. The findings of this study suggest that: treadmill running did not increase the oxidative capacity of the deep region of the reinnervating rat plantaris muscle; treadmill training did not damage the reinnervating plantaris.(ABSTRACT TRUNCATED AT 250 WORDS)

Histochemical and physiological properties of cat motor units after self-and cross-reinnervation.

1. This report describes selected histochemical and physiological properties of the motor units of adult cat soleus muscle approximately one year after self- and cross-reinnervation with the nerve of the heterogenous flexor hallucis longus (f.h.l.). Self-reinnervated f.h.l. motor units are also considered. Whole muscles were tested for fibre reaction to alkaline pre-incubated ATPase, alpha-glycerophosphate dehydrogenase (alpha-GPD) and reduced nicotinamide adenine dinucleotide diaphorase (NADH-D). Motor units were isolated and studied by splitting the ventral root in acute preparations.2. The histochemical fibre type profile in the self-reinnervated muscle was comparable to normal muscle as was mean twitch contraction time, twitch-tetanus ratio and fatigue index. The mean tetanic tension of the soleus self- and cross-reinnervated motor units appeared close to a normal soleus whereas the mean tetanic tension of the f.h.l. self-reinnervated units was significantly less than a normal f.h.l.3. An average of 14% of the fibres of the soleus cross-reinnervated muscles had high ATPase and a alpha-GPD staining intensity in contrast to normal and self-reinnervated soleus in which such fibres are absent. Thus alkaline lability of myofibrillar ATPase increased in some fibres of what was originally a homogeneous population. The small increase in the number of densely staining fibres for ATPase at an alkaline pH (14%) was associated with a 73% decrease in (mean) contraction time (41 +/- 11 ms) of the thirty-three cross-reinnervated muscle units studied, with no unit's contraction time greater than 60 ms. Mean contraction times for the self-reinnervated soleus and f.h.l. muscles were 78 +/- 31 ms and 27 +/- 8 ms respectively.4. All fibres of the soleus cross-reinnervated muscles showed intense reaction to NADH-D, as was true of self-reinnervated soleus. This staining pattern is typical of normal soleus. In concordance, these motor units consistently demonstrated a high resistance to fatigue when stimulated for a four-minute period.5. These results suggest that in the adult self-and cross-reinnervated soleus muscle, there is some active mechanism which regulates the eventual size of motor units as reflected by tetanic tension.6. Change in contraction time from that typical for a soleus unit to that similar to an f.h.l. unit remains incomplete one year after cross-reinnervation. Within this time this partial change in single motor units reflects incomplete neural control of this property rather than a mixture of self- and foreign-innervation.7. A greater degree of independence from neural control to conversion of the histochemically demonstrated myofibrillar ATPase activity exists than is the case for contraction time.

Comparison of the membrane composition of Spiroplasma citri and the corn stunt Spiroplasma

Components of membranes isolated from Spiroplasma citri and corn stunt spiroplasma grown at 28 degrees C were analyzed. On a protein basis, lipid phosphorus was lower and cholesterol was higher in S. citri. Only minor differences between the two species were found in fatty acid composition, reduced nicotinamide adenine dinucleotide diaphorase, and adenosine triphosphatase.

Properties of immobilized hind-limb muscles of the Galago senegalensis

Morphological, biochemical, and physiological properties of longterm (6 mo) immobilized skeletal muscles of a nonhuman primate were studied in seven Galago senegalensis which were immobilized at the ankle and knee of one hind limb with an external brace. Electromyographic activity of the ipsilateral and contralateral quadriceps and muscles of the calf were assessed after 5 and 6 mo of immobilization. The EMG was markedly reduced in the immobilized muscles compared to the contralateral control when the brace was intact and the animal moving freely. Without exception, extensor muscles atrophied more than flexors. The soleus, a slow-twitch muscle atrophied more than any other muscle of the lower leg but the same was not true of the vastus intermedius the analogous muscle of the thigh. Slow-twitch oxidative fibers (SO) were atrophied more than fast-twitch oxidative glycolytic fibers (FOG), and FOG tended to atrophy more than fast-twitch glycolytic fibers (FG). The immobilized soleus and vastus intermedius had a smaller percentage of SO fibers than their controls, suggesting that they had faster contraction times. With respect to alterations in reduced nicotinamide adenine dinucleotide diaphorase activity, no consistent pattern was observed except for a greater coarseness of staining granules and more homogeneous dispersion of the granules throughout the cross-section of the fibers. No changes were found in phosphorylase, lactate dehydrogenase, or succinate dehydrogenase specific activity or in myoglobin concentration in homogenates of ankle flexors or the vastus lateralis. Myosin ATPase, but not actomyosin ATPase activity was significantly less in the immobilized gastrocnemius-plantaris muscles. No change in contractile properties related to speed were observed in the plantaris. This muscle did exert more twitch and tetanic tension per gram of muscle in the immobilized leg.

Hindlimb muscle fiber populations of five mammals.

The fiber type profiles of hindlimb muscles in the Hartley guinea pig, Sprague-Dawley rat, cat, Galago senegalensis (lesser bushbaby) and Nycticebus coucang (slow loris) were estimated histochemically. Fibers were classified as fast oxidative glycolytic, fast glycolytic or slow oxidative according to their myosin adenosine triphosphatase, α-glycerophosphate dehydrogenase and reduced nicotinamide adenine dinucleotide diaphorase activities. It was found that the soleus and vastus intermedius muscles had the highest proportion of slow oxidative fibers in all five species, demonstrating the constancy of muscle fiber profiles dependent upon anatomical position and functional utilization. The tensor fascia latae and white vastus lateralis of the guinea pig were mostly fast glycolytic, while the red vastus lateralis of the guinea pig consisted of predominantly fast oxidative glycolytic fibers. The majority of muscles investigated in these five mammals were heterogeneous, having a wide range of percentages of the three fiber types.

Types of muscle fibers in the extraocular muscles of birds

Eye muscles of the quail, pigeon, sparrow and canary were examined histologically to see if their fibers are differentiated into two or more types, as has been described for mammals. In each oblique and rectus muscle examined, an area along the orbital border was found to be occupied by fibers of small cross-sectional area, and a more extensive central portion predominantly by large-caliber fibers. These fiber populations were separable on the basis of levels of α-glycerophosphate dehydrogenase and reduced nicotinamide adenine dinucleotide diaphorase activity, and to a lesser degree by glycogen content. All fibers stained strongly with the myosin adenosine triphosphatase reaction, the level of the activity being comparable to that in fast-twitch, mammalian limb muscles. The differences in features of location, size and histochemical reaction indicate that the rectus and oblique muscles in birds contain at least two types of fibers. The quadratus and pyramidalis muscles, which serve to advance the nictitating membrane, did not show segregation into zones of fibers, although in the pyramidalis two intermixed fibers were generally of larger size in the pyramidalis. Both fiber types stained darkly with myosin adenosine triphosphatase, suggesting that they were fast-twitch fibers.

The intermediate muscle fiber of rats and guinea pigs.

The soleus, plantaris and gastrocnemius muscles of 60 Sprague-Dawley rats and 70 Hartley guinea pigs were studied histochemically. In the plantaris and gastrocnemius muscles, myosin adenosine triphosphatase activity differentiated intermediate fibers from red and white fibers as determined by malate dehydrogenase and succinate dehydrogenase and reduced nicotinamide adenine dinucleotide diaphorase activities. Contrary to what is commonly reported, red fibers could not be distinguished from white fibers on the basis of myosin adenosine triphosphatase activity as is commonly reported. The intermediate fiber was characterized by minimal menadione-mediated α-glycerophosphate dehydrogenase, phosphorylase and myosin adenosine triphosphatase activity and moderate malate dehydrogenase and succinate dehydrogenase and reduced nicotinamide adenine dinucleotide diaphorase activities. It is suggested that fibers with intermediate oxidative enzyme activity are physiologically slow, white fibers are fast and red fibers are moderately slow or even fast contracting fibers.

Alpha-glycerophosphate oxidase in Streptococcus faecium F 24.

alpha-Glycerophosphate oxidase, in a strain of Streptococcus faecium, was found to be adaptive to aerated conditions of growth. The enzyme was purified and found to mediate electron transfer from alpha-glycerophosphate to O(2), with the production of stoichiometric concentrations of H(2)O(2) and dihydroxyacetone phosphate. The enzyme is an anionic flavoprotein, with flavine adenine dinucleotide as the apparent prosthetic group. By manometric methods, a K(m) of 6 x 10(-3)m, with reference to substrate concentration, was obtained. An active reduced nicotinamide adenine dinucleotide diaphorase was closely associated with this enzyme in chromatographic mobility on ECTEOLA-cellulose. The purified alpha-glycerophosphate oxidase was not inhibited by KCN, azide, or sulfhydryl reagents, nor was it stimulated by alpha-lipoate, yeast extract, or other supplements.

Histochemical changes in rat skeletal muscle after exercise

Previous studies led us to suspect that the proportion of fibers within a given muscle which show relatively high activity of the enzymes associated with mitochondria should be related to the nature of the work load placed on that muscle. Following a 52-day swimming program for white rats (one and two 30-min exercise bouts each day) the proportion of fibers having intermediate and high malate dehydrogenase, succinate dehydrogenase, and reduced nicotinamide adenine dinucleotide diaphorase was not altered in the soleus. Neither was the proportion of fibers having low and high mitochondrial α-glycerophosphate dehydrogenase (mito α-GPD) and myosin adenosine triphosphatase altered in the soleus. Myosin adenosine triphosphatase showed no changes in the plantaris. However, the plantaris of moderately and heavily exercised rats did show a greater proportion of fibers having high malate and succinate dehydrogenase, and nicotinamide adenine dinucleotide diaphorase than did the plantaris of sedentary rats. This increase in the proportion of fibers having these high enzyme activities appears to be one mechanism through which a muscle can adapt to a chronic repetitive overload. If one wishes to think in terms of fiber types as determined by these enzymes, this study demonstrates that muscle fiber types can be changed in a nonpathologic condition.