NADH-tetrazolium Reductase Activity Research Articles

POLA DISTRIBUSI MITOKONDRIA SEL-SEL TROFOBLAS BLASTOSIS MENCIT (Mus Muculus Albinus) DAN PENGARUHNYA TERHADAP KEGAGALAN HATCHING DAN IMPLANTASI

Implantation is the most critical stage in the establishment of pregnancy. In mammals, it has been estimated that between 25% and 60% of conceptuses are lost before or at the time of implantation. The objectives of this study were to investigate the ability of blastocyst hatching and attachment of trophoblast cells in in vitro , the outgrowth and differentiation of trophoblast cells in in vitro culture of hatched and non hatched blastocyst, the activity of mitochondria NADH-CoQ reductase and the pattern of mitochondrial distribution. Blastocysts were collected from mice cornua utery at day-4 of pregnancy and were divided into 3 groups: blastocysts undergo hatching within 24 hours, 48 hours and non hatching. Embryos were cultured in DMEM medium in 5% CO 2 incubator at 37°C for 10 days. The trophoblasts monolayer were processed for Giemsa staining and histochemistry analysis of NADH-tetrazolium reductase activity. The outgrowth of trophoblast cells were measured using eyespiece micrometer. The results showed the ability of blastocyst hatching in in vitro were different. The trophoblast outgrowth diameter of 24 h hatched blastocyst was significantly different with the 48 h hatched and non hatched blastocyst. The activity of NADH-CoQ reductase of 24 h and 48 h hatched blastocysts showed higher intensity than the non hatched blastocysts (P<0,05) and the distribution of mitochondrial in trophoblast cell cytoplasma of 24 h and 48 h hatched blastocyst were homogen around nucleus whereas thoes of non hatched blastocyst were cluster and heterogen. In conclusion, in in vitro study the failure of blastocysts hatching and implantation was due to the failure of outgrowth and differentiation of the trofoblast cells and the impairment of NADH-CoQ reductase activity in complex I mitochondria to produce energy and the pattern of mitochondrial distribution. Key words : mitochondrial distribution, NADH- tetrazolium reductase, trophoblast, implantation

Muscle biopsy in Pompe disease

Pompe disease (PD) can be diagnosed by measuring alpha-glucosidase levels or by identifying mutations in the gene enzyme. Muscle biopsies can aid diagnosis in doubtful cases. A review of muscle biopsy from 19 cases of PD (infantile, 6 cases; childhood, 4 cases; and juvenile/adult, 9 cases). Vacuoles with or without glycogen storage were found in 18 cases. All cases had increased acid phosphatase activity. The vacuole frequency varied (almost all fibers in the infantile form to only a few in the juvenile/adult form). Atrophy of type 1 and 2 fibers was frequent in all forms. Atrophic angular fibers in the NADH-tetrazolium reductase and nonspecific esterase activity were observed in 4/9 of the juvenile/adult cases. Increased acid phosphatase activity and vacuoles were the primary findings. Most vacuoles were filled with glycogen, and the adult form of the disease had fewer fibers with vacuoles than the infantile or childhood forms.

Increased Expression of Wild-Type or a Centronuclear Myopathy Mutant of Dynamin 2 in Skeletal Muscle of Adult Mice Leads to Structural Defects and Muscle Weakness

Dynamin 2 (DNM2) is a large GTPase implicated in many cellular functions, including cytoskeleton regulation and endocytosis. Although ubiquitously expressed, DNM2 was found mutated in two genetic disorders affecting different tissues: autosomal dominant centronuclear myopathy (ADCNM; skeletal muscle) and peripheral Charcot-Marie-Tooth neuropathy (peripheral nerve). To gain insight into the function of DNM2 in skeletal muscle and the pathological mechanisms leading to ADCNM, we introduced wild-type DNM2 (WT-DNM2) or R465W DNM2 (RW-DNM2), the most common ADCNM mutation, into adult wild-type mouse skeletal muscle by intramuscular adeno-associated virus injections. We detected altered localization of RW-DNM2 in mouse muscle. Several ADCNM features were present in RW-DNM2 mice: fiber atrophy, nuclear mislocalization, and altered mitochondrial staining, with a corresponding reduction in specific maximal muscle force. The sarcomere and triad structures were also altered. We report similar findings in muscle biopsy specimens from an ADCNM patient with the R465W mutation. In addition, expression of wild-type DNM2 induced some muscle defects, albeit to a lesser extent than RW-DNM2, suggesting that the R465W mutation has enhanced activity in vivo. In conclusion, we show the RW-DNM2 mutation acts in a dominant manner to cause ADCNM in adult muscle, and the disease arises from a primary defect in skeletal muscle rather than secondary to peripheral nerve involvement. Therefore, DNM2 plays important roles in the maintenance of adult muscle fibers.

ИЗСЛЕДВАНЕ НА НАДН-ТР В СПЕРМАТОЗОИДИ ОТ КОЧ ПРИ КРАТКРОТРАЙНО СЪХРАНЕНИЕ И КРИОКОНСЕРВАЦИЯ

The investigations for the determination of NADH-tetrazolium reductase enzyme system activity in ram semen during liquid and cryopreservation in relation with their viability, were carried out. The semen samples with lower motility of spermatozoa after collection, possess lower values of mean cytochemical coefficient for NADH-tetrazolium reductase activity ( р<0.5, р<0.5) after liquid and cryopreservation.

Neuromuscular compartments and fiber-type regionalization in the human inferior pharyngeal constrictor muscle.

The inferior pharyngeal constrictor (IPC) muscle functions during swallowing, respiration, and vocalization. The most-caudal portion of the IPC is believed to be part of the functional upper esophageal sphincter (UES). We hypothesized that the caudal fibers of the human IPC may have enzyme-histochemical characteristics similar to those of the cricopharyngeus muscle, a major component of the UES. In this study, human IPC muscles obtained from autopsy were studied using Sihler's stain to examine innervation patterns, and using myofibrillar ATPase, NADH tetrazolium reductase (NADH-TR), and succinic dehydrogenase (SDH) techniques to investigate the distribution and oxidative capacity of the slow- (type I) and fast- (type II) twitch fibers in the muscle. The results showed that the human IPC consists of at least two neuromuscular compartments (NMCs): rostral and caudal. Each of the NMCs was innervated by a separate nerve branch derived from the pharyngeal branch of the vagus nerve. The rostral NMC is faster (39% type I, 61% type II) than the caudal NMC (70% type I, 30% type II). In addition, two histochemically-delineated fiber layers were identified in the human IPC: a slow inner layer (SIL) with predominantly type I fibers (66%), and a fast outer layer (FOL) with predominantly type II fibers (62%) (P < 0.01). However, the dimensions of both fiber layers and proportions of the muscle fiber types varied with the NMCs. Specifically, the ratio of the thickness of the SIL to FOL was approximately 2:1 for the caudal NMC and approximately 1:2 for the rostral NMC, respectively. In the SIL the type I fibers accounted for 84% for the caudal NMC and 69% and 44% for the lower and upper portions of the rostral NMC. In contrast, the type II fibers in the FOL accounted for 46% for the caudal NMC and 67% and 74% for the lower and upper portions of the rostral NMC, respectively (P < 0.01). The caudal NMC of the IPC shared histochemical characteristics with the cricopharyngeus muscle, in that it contained predominantly slow oxidative fibers. Overall, the caudal NMC and the SIL in the IPC had high NADH-TR and SDH activities. However, different patterns of oxidative enzyme activity were identified in both type I and type II fibers. This study provided histochemical evidence for the concept that the caudal NMC within the IPC contributes to the functional UES. In addition, the two histochemically-defined fiber layers in the IPC may be a specialized adaptation in humans to enable different upper-airway functions during respiration, swallowing, and speech.

Microphotometric analysis of NADH-tetrazolium reductase deficiency in fibroblasts of patients with Leber hereditary optic neuropathy.

We employed a microphotometric approach to examine whether a defect in the mitochondrial respiratory complex I expected in Leber hereditary optic neuropathy (LHON) as the consequence of a mtDNA (11778G>A) mutation in the ND4 gene coding for a subunit of the respiratory complex I can be detected at the single-cell level. Genetically stable fibroblast cell lines were established from skin biopsies of two members of a Chinese Indonesian family with LHON. The fibroblasts were homoplasmic for the 11778G>A mutation. The activity of the respiratory complex I was examined histochemically by staining for NADH-tetrazolium reductase. The histochemical staining showed a typical pattern with an apparent concentration of the activity around the nucleus, suggested as the reflection of the gradient in the thickness of the unsectioned fibroblast cells. Microphotometric quantification of the staining intensity showed that the activity is linear for at least 60 min. The activity shows a discontinuity in its Arrhenius kinetics with a break point at 13.0-13.5 degrees C (activation energy at 50-58 J/mol and 209-238 J/mol above and below the break temperature, respectively), indicating the membrane association of the NADH-tetrazolium reductase activity. Both patients showed lower fibroblast NADH-tetrazolium reductase activity, with a reduction of degrees 30%. Our results demonstrate the utility of microphotometric analysis in the study of biochemical defects associated with mutations in the mtDNA.

Histomorphology of rabbit thigh muscles: establishment of standard control values.

The thigh muscles of New Zealand White (NZW) rabbits are frequently used in experimental surgery, particularly for evaluation after reinnervation or ischaemia. Although histomorphometric analyses are regularly performed, morphological data for untreated thigh muscles in previously unoperated animals are not available. Specimens from the rectus femoris (RF), vastus medialis (VM) and adductor magnus (AM) muscles from both thighs were harvested in 7 untreated rabbits and were processed for histomorphometric evaluation. The right RF and VM were harvested in a further 5 rabbit hindlimbs after experimental denervation and reinnervation of the contralateral RF and subsequently processed for histomorphometric analysis. Muscle fibre type distribution, diameter and connective tissue content were evaluated on serial transverse cryosections reacted for ATPase and NADH tetrazolium reductase activity and statistical analysis was performed. In all untreated animals RF revealed the highest proportion of type I muscle fibres (right: 8.4+/-4%, left: 11.4+/-4.9%), whereas VM showed the highest percentage of IIa fibres (right: 31.9+/-5.5%, left: 28.3+/-7.8%) and AM the highest proportion of IIb/d fibres (right: 80.5+/-8.6%, left: 84.4+/-6.3%). Fibre type distribution and diameter in rabbits after contralateral experimental operations revealed a statistically significant difference from the data obtained in bilaterally untreated animals. Knowledge of the morphology of untreated muscles is fundamental to the understanding of changes induced by intervention to the ipsi and/or contralateral thigh muscles.

Abnormal mitochondria and sarcoplasmic changes in rabbit skeletal muscle induced by immobilization.

Immobilization of the rabbit knee in extended position results in damage to the vastus intermedius profundus (VIP) muscle. To examine the mechanisms involved in initiation of the injury, we studied the light and electron microscopic morphology of the VIP muscle, as well as the activity and distribution of NADH tetrazolium reductase (NADH-TR) in the affected muscle, and determined serum total creatine kinase (CK) activity in immobilized rabbits. The VIP muscle of the immobilized right hindlimb was removed at various time points (10 h, 24 h, 36 h and 48-72 h, n=5 for each time point). The nonimmobilized left hindlimb and five nonimmobilized animals served as controls. No morphological changes were observed by light microscopy within 48-72 h in routine stainings. Transient ultrastructural abnormalities, including abnormal cristae, matrix lucencies and mild swelling of mitochondria, were observed between 10 h and 36 h of immobilization, subsiding by 48-72 h. On the other hand, progressive disorganization of myofibrils with breaking-up of Z-bands and an increase in the number and size of sarcoplasmic lipid vacuoles was seen with increasing duration of immobilization. NADH-TR activity at subsarcolemmal locations had decreased by 10 h and disappeared by 24 h of immobilization, while the intermyofibrillar mitochondria remained unaltered. Serum total CK activity began to increase by 2 h of immobilization and reached a peak by 24 h. The results indicate that already a few hours of immobilization of the rabbit knee in extension leads to signs of metabolic disturbance of the VIP muscle and sarcolemmal leakage. The simultaneous occurrence of transient mitochondrial abnormalities, transient CK efflux and progressive myofibrillar damage suggests the operation of multiple adverse mechanisms already at the onset of disuse muscle atrophy.

Long term effects of spinal cord transection in Zebrafish: swimming performances, and metabolic properties of the neuromuscular system

This study concerns functional recovery of zebrafish following spinal cord transection. Spinal cords were transected at the level of the 14th vertebra, just rostral to the dorsal fin. Recovery was tested at one month after transection when descending fibers start to regrow across the transection site and at three months after transection when fish perform kick and glide swimming. To estimate the rate of regrowth across the lesion site we analysed the tyrosine hydroxylase (TH) and dorsal 5-hydroxytryptamine (5-HT) systems in distal parts of lesioned cords. Both systems have cell bodies in the brainstem and in control fish TH- and dorsal 5-HT-containing fibers descend to all spinal segments. Swimming performance was studied by subjecting lesioned fish to endurance tests in a swimming tunnel with water flowing at a constant rate of 2 or 4.5 body lengths per second (BL/s). At 2 BL/s slow myotomal muscles are active whereas at 4.5 BL/s fast myotomal muscles are recruited. Control fish endured sustained swimming at both speeds for at least 3 hours. As a measure for the condition of the neuromuscular system in trunk and tail, we analysed aerobic metabolic capacities, assessed by NADH-tetrazolium reductase (NADH-TR) histochemistry of myotomal muscle fibers and spinal lateral neuropil. We found that TH- and dorsal 5-HT-immunoreactive fibers were absent in the entire distal part of lesioned cords at one month but at two months after transection they were present at approximately 6000 microns caudally to the site of the lesion. Thus the rate of outgrowth of these fibers is at least 200 microns per day. Sustained swimming at the slow speed (2 BL/s) could be endured for about 14.4 min at one month and for 23.5 min at two months after transection; there was no further improvement in the period that followed. In contrast, in the 10 weeks following transection, fast swimming (4.5 BL/s) could be endured for about 5 to 6 minutes. A significant improvement was gained in the period of 10 to 12 weeks after transection when fish could endure the high speed for almost 15 min. The aerobic capacity of muscle fibers in distal parts of the body was not strongly affected by the lesion. The only important change in aerobic capacity was observed in the neuropil of distal parts of the cords where, at three months after transection, NADH-TR activity was increased to approximately 150% of control values. On the basis of our findings, we assume that it is not the condition of the neuromuscular system, but rather a deficient co-ordination between proximal and distal body parts of lesioned fish that accounts for the relatively poor performances in endurance tests. Furthermore, differences in timing of improvements in swimming at 2 and 4.5 BL/s indicate that the spinal circuitries serving the slow parts of the neuromuscular system recover at an earlier stage than those serving the fast parts.

Stability of enzymatic indicators of metabolic and neuronal activity in post-ganglionic neurons supplying the urinary tract of aged rats

Aged post-ganglionic neurons supplying the urinary tract exhibit stability in their metabolic and functional status, as demonstrated by quantification of enzyme histochemical reaction products. The major pelvic and thirteenth thoracic (T13) ganglia from 4- and 24-month-old male Wistar rats were incubated under standardized conditions to demonstrate succinate dehydrogenase, NADH-tetrazolium reductase and cytochrome oxidase activities. The amount of reaction product for each enzyme, localized in neuronal perikarya throughout the ganglia, was quantified by microdensitometry. Comparison of mean totals demonstrated stability of all three enzymes with age in both the major pelvic and T13 ganglia and in target organs. Also, the distribution and range of reaction product densities were similar. In both ganglia, the number of NADH-tetrazolium reductase-stained neurons per unit area was reduced in the aged animals. Additionally, nicotinic (DMPP) and muscarinic (McN-A-343) agonists were used to mimic the effects of preganglionic stimulation in the major pelvic ganglia before performing enzyme histochemical reaction. Quantification of these activities demonstrated that the response of aged neurons to preganglionic stimulation was unchanged. These results indicate an overall metabolic stability in post-ganglionic autonomic neurons innervating the urinary tract and in their targets in aged rats. © 1998 Chapman & Hall

Vagal nuclei in the medulla oblongata: structure and activity are maintained in aged rats

The neurones and microvessels of the dorsal motor nucleus of the vagus (DMN), the nucleus ambiguus (NA) and the nucleus tractus solitarius (NTS) of 4, 24 and 30 month male Wistar rats have been examined morphometrically and by quantitative enzyme histochemical methods (4 and 24 months only) to assess the affects of old age on the structure and activity of their neurones. DMN and NTS neuronal soma area increased whilst NA neuronal area was reduced in the aged groups; the changes in neuronal size were reflected in the density of neurones per unit area. The mean diameter and percentage area occupied by microvessels was unchanged with increased age in all three nuclei. Quantitative assessment of cytochrome oxidase and NADH-tetrazolium reductase activities in the nuclei revealed no changes in old age, indicating that old age does not affect neuronal or metabolic activity of central vagal neurones. These results compliment previous age-related studies on the vagus nerve and nodose ganglion in which little change has been reported, suggesting that the vagal system is well preserved in aged rats.

Changes in enzyme histochemical profiles of identified spinal motoneurons of the European eel, Anguilla anguilla, following cordotomy.

The enzyme histochemical profiles of glucose-6-phosphate dehydrogenase (a marker of synthetic performance), succinate dehydrogenase (an indicator of oxidative metabolism), and NADH tetrazolium reductase (a marker of overall neuronal activity) were determined for identified white muscle motoneurons in six control and six cordotomized cels. Images were digitized and mean integrated absorbances obtained using appropriate hardware and software. For motoneurons caudal to the transection site there was a significant decrease in the mean absorbance value for NADH tetrazolium reductases which declines from 0.28 a.u. (arbitrary units) in control animals to 0.23 a.u. in cordotomized animals. However, no significant changes were detected in the activities of glucose-6-phosphate and succinate dehydrogenases. The cross-sectional area of the motoneuronal cell body was not affected by cordotomy. The decrease by around 20% in overall neuronal activity, as expressed by NADH tetrazolium reductase activity, might be expected from the decline in body motility that follows cordotomy. Changes in SDH and G6PDH activities would also be expected to follow this surgery, but none were seen, perhaps because they are compensated for by changes in neuronal metabolism that result from deafferentation.

Hereditary myopathy of the diaphragmatic muscles in Holstein-Friesian cattle.

We describe a family line with an autosomal recessive disease of muscular dystrophy of the diaphragmatic muscles in Holstein-Friesian cattle. Histopathological examination in the present cases revealed various degenerative changes in the diaphragmatic and other thoracic muscles as follows: variation in muscle fiber diameter, fiber splitting, sarcoplasmic masses, ring fiber, vacuolar and hyalinized degeneration of muscle fibers. In addition, central core-like structures were the prominent features in the diaphragmatic muscles, occupying the center of the fiber or scattered within the fiber. These pathological alterations are consistent with the diaphragmatic myopathy previously reported in Meuse-Rhine-Yssel cattle in the Netherlands. The fibers containing core-like structures consisted of three distinct zones which could be well distinguished by NADH-tetrazolium reductase activity. This activity was absent in the innermost zone, decreased in the intermediate zone, and normal or increased in the periphery. Electron microscopically, this structure appeared to be composed of focal myofibrillar degeneration beginning with streaming or disintegration of the Z disk. We discuss here the similarity between this core-like structure and the other alternative organelles that have been reported previously, and a possible defect or storage in the cytoskeleton from the findings of the Z disk abnormalities.

Effect of thawing regimens on the morphofunctional state of canine spermatozoa

The effect of 2 thawing regimens (37 °C for 8 sec and 55 °C for 5 sec) was followed up on semen parameters related to the viability of canine spermatozoa. The ejaculates were frozen in the form of pellets on dry ice in the following cryoprotective extenders: TRIS-fructose (TF), TRIS-glucose (TG), and sucrose-lactose (SL). For the 3 extenders, significant differences were found in the percentage of motile spermatozoa and their survival rate up to 300 min in favor of the 55 °C vs the 37 °C thawing regimens. Structural changes such as swelling, breakage and absence of acrosomes were observed in the samples frozen in the 3 cryoprotective extenders. A considerably lower percentage of spermatozoa with damaged acrosomes was recorded at 55 °C in comparison with that found at 37 °C (P < 0.05 for TG, TF and SL).Enzymocytochemical analysis was made of NADH-tetrazolium reductase activity in thawed spermatozoa. Cells showing moderate and strong intensity of the cytochemical reaction were found after both regimens of thawing. The percentage of spermatozoa manifesting strong intensity of the reaction was comparatively higher after thawing at 55 °C (31.8 ± 2.06) than at 37 °C (23.7 ± 1.41; P < 0.01). The thawing regimens were the factors that exerted influence on the morphofunctional state of frozen canine spermatozoa, irrespective of the cryoprotective extenders used, in the present study. Thus the optimal preservation of sperm viability was achieved by thawing at 55 °C for 5 sec.

Muscle fiber type composition and fiber size in successfully and unsuccessfully endurance-raced horses

Triplicate biopsies from three different depths of the gluteus medius muscle were obtained in 36 endurance-raced horses, aged 8.42 +/- 2.85 yr. Twenty of the horses were considered excellent endurance performers according to the mean speed of their three fastest records in endurance events for the past 2 or 3 years, whereas 16 were moderate performers, with a mean racing speed < 12.5 km/h (in 120- to 180-km endurance rides), < 14 km/h (in 80- to 120-km endurance rides), or < 13.5 km/h (in 40- to 60-km endurance rides). Significant differences in muscle fiber type composition and fiber size were recorded; excellent performers had a higher percentage and a larger size of type I and type IIa fibers (high and low myosin adenosinetriphosphatase activity at pH 4.5, respectively) and a lower percentage of type IIb fibers (moderate myosin adenosinetriphosphatase activity at pH 4.5), including both type IIb oxidative (moderate to high NADH-tetrazolium reductase activity) and IIb nonoxidative (low NADH-tetrazolium reductase activity). The differences in distribution of myofiber types and in fiber sizes were more marked in the deeper parts compared with the superficial regions of muscle. Our results also imply a greater homogeneity among the fiber type sizes across the muscle in horses with a superior endurance performance than in horses that had been poorly or moderately endurance raced. Thus the results show that fiber type proportions and fiber size in equine skeletal muscle are directly related to the athletic ability of the horse for endurance events.

Composition of myofiber types in the pectoral girdle musculature of sheep

The muscles of the pectoral girdle in domestic animals attach the forelimbs to the trunk and function as the suspensory apparatus. In the present study the composition of the pectoral girdle musculature of sheep by myofiber types was examined. Myofibers showing a strong reaction for alkali-stable myosin ATPase were classified into fast-twitch/glycolytic (FG) myofibers with a weak activity for NADH tetrazolium reductase (NADH-TR) and fast-twitch/oxidative/glycolytic (FOG) myofibers with a moderate and strong NADH-TR activity. Myofibers showing a weak reaction for alkali-stable myosin ATPase and a strong activity for NADH-TR were classified as slow-twitch/oxidative (SO) myofibers. The SO myofibers that showed a granular and striped pattern of diformazan deposits in NADH-TR activity were classified as SO-1 myofibers, whereas the SO myofibers characterized by a reticular pattern of diformazan deposits were classified as SO-2 myofibers. The trapezius, rhomboideus cervicis, and pectoralis descendens muscles situated superficially in the cranial regions of the back and chest had about 50% SO (SO-1 plus SO-2) myofibers. The deeply situated serratus ventralis cervicis and thoracis muscles had 37.5% SO myofibers. These five muscles included more SO-2 myofibers with large diameters than did all other muscles, and had about 50% and more cross-sectional area of SO myofibers. The other muscles had less than 32% SO myofibers and fewer SO-2 myofibers. The FOG and FG myofibers accounted for 50% or less in the muscles examined. Many muscles of the pectoral girdle had many fast-twitch (FOG plus FG) myofibers; they seem to meet locomotory requirements. In the pectoral girdle musculature, the SO myofibers were not necessarily distributed more in the deep regions than in the superficial regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical and structural adaptation of autologous skeletal muscle used for counterpulsation

We have studied in a normal animal model (sheep), the biochemical and morphological adaptation of electrically stimulated skeletal muscle used for extra aortic counterpulsation. Immunocytochemical analysis of latissimus dorsi, using monoclonal antibodies to slow and fast myosin heavy chains, indicated an increase in the population of mixed fibres after stimulation for one week. By one month, up to 70% of fibres expressed both slow and fast myosin heavy chains in addition to the 15% of fibres expressing only slow myosin heavy chains. After 4 months, the population of mixed fibres was further transformed towards purely slow fibres to give values of 40 and 67% of fibres expressing only slow myosin heavy chain at 4 and 6 months, respectively. Increased staining, both in intensity and area, for NADH tetrazolium reductase activity (an enzyme of the oxidative metabolic pathway) was detected by 28 days. An increase in mitochondrial number was observed also by 28 days, further indicating a shift towards an oxidative metabolism. The molecular adaptation of latissimus dorsi was achieved by stimulation every fourth cardiac cycle at 35 Hz, 3 V, initiated 48 hours after the operation; this being a marked reduction in the delay from operation prior to stimulation. Evaluation of other regimes indicated that more frequent modes, or an increase in voltage or frequency, caused damage to the muscle during the early phase of molecular adaptation. A thorough understanding of the time sequence of the different adaptive processes is required to determine the ideal regime of stimulation initiated promptly after mobilisation of the muscle; aimed at harvesting the maximum amount of energy from the autologous muscle.

Histochemical classification of myofiber types in the triceps surae and flexor digitorum superficialis muscle of Japanese macaques.

Leg muscles of Japanese macaques (Macaca fuscata) were examined to characterize histochemical properties of myofiber types. Myofiber types were classified by differences in reactivity for myosin ATPase, NADH tetrazolium reductase (NADH-TR), and menadione-linked glycerol-3-phosphate dehydrogenase (3-GPD). The myofibers that reacted strongly for acid-stable myosin ATPase and were weakly reactive or unreactive for alkali-stable myosin ATPase were classified as slow-twitch/oxidative (SO) myofibers, which reacted strongly for NADH-TR and weakly to moderately for 3-GPD. The myofibers that were unreactive to moderately reactive for acid-stable myosin ATPase and strongly reactive for alkali-stable myosin ATPase were classified into fast-twitch/oxidative/glycolytic (FOG) myofibers with a moderate to strong activity for NADH-TR and 3-GPD and into fast-twitch/glycolytic (FG) myofibers with a weak NADH-TR activity and a strong 3-GPD activity. Slow-twitch myofibers with a strong activity for NADH-TR and 3-GPD were characterized as slow-twitch/oxidative/glycolytic (SOG) myofibers. The presence or absence of the SOG myofibers depended on the individuals. The soleus muscle had larger percentages of SO myofibers than of FOG and FG myofibers. The gastrocnemius and flexor digitorum superficialis muscles generally had larger percentages of FG myofibers than of FOG and SO myofibers including SOG myofibers.

The effects of high intensity training upon respiratory gas exchanges during fixed term maximal incremental exercise in man.

The response of respiratory gas exchanges to a 6 week high intensity training program was examined in 5 healthy males during fixed term maximal incremental treadmill exercise. Training was performed 3 d.wk-1 and consisted of a progressive series of repeated 15 sec and 30 sec maximal runs, and weight training exercises for the leg extensor muscles. Respiratory gases during the tests were continuously monitored using an on-line system. Muscle biopsy samples were obtained from the m. vastus lateralis before and after training for histochemical determination of fibre distribution based on myosin ATP-ase activity, and fibre cross-sectional area based on NADH-Tetrazolium Reductase activity. Training significantly increased the proportion of type IIa fibres (+5.9 +/- 2.0%, p less than 0.001) and decreased type I fibres (-6.3 +/- 2.0%, p less than 0.001), the distribution of type IIb fibres remained unchanged (+0.4 +/- 0.9%). Muscle cross-sectional area also showed a significant increase after training in type I (+318 +/- 215 microns 2, p less than 0.05), IIa (+652 +/- 207 microns 2, p less than 0.001) and IIb (+773 +/- 196 microns 2, p less than 0.001) fibres. During fixed term maximal incremental exercise the mean carbon dioxide output (VCO2) and mean respiratory exchange ratio (R = VCO2/VO2) were significantly increased (p less than 0.01) after training. The R-time relationship was at all times shifted to the left after training, being significantly (p less than 0.01) so over the final five min of exercise. No changes in mean exercise oxygen uptake (VO2), maximum oxygen uptake (VO2max) and maximum heart rate (FHRmax) were observed between tests.(ABSTRACT TRUNCATED AT 250 WORDS)

Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle

Combined histochemical and biochemical analyses were performed on rat skeletal muscles in order to determine the myosin heavy chain patterns in specific fiber types. Four myosin heavy chain isoforms were separated by gradient polyacrylamide gel electrophoresis of extracts from single fibers and whole muscle homogenates. Their electrophoretic mobility increased in the order HCIIa, HCIIb, and HCI. HCIIa, HCIIb and HCI were present as unique isoforms in histochemically defined fiber types IIA, IIB and I, respectively. The isoforms HCI and HCIIa coexisted at variable ratios in type IC and IIC fibers. An additional fast myosin heavy chain isoform with an electrophoretic mobility between HCIIa and HCIIb was designated as HCIId because of its abundance in fast fibers of large diameter in the diaphragm. With the exception of slight differences in mATPase staining intensity after acid preincubation, these fibers were almost indistinguishable from type IIB fibers. In view of their specific myosin heavy chain composition (HCIId), these fibers were named type IID. In the extensor digitorum longus muscle, type IID fibers were of smaller size than type IIB and differed from the latter by higher NADH tetrazolium reductase activities. Circumstantial evidence suggests that type IID fibers are identical with the 2X fibers, previously described by Schiaffino et al. (1986).