Tetrazolium Reductase Activity Research Articles

Discriminating Susceptibility of Xanthine Oxidoreductase Family to Metals.

The xanthine oxidoreductase (XOR) family are metal-containing enzymes that use the molybdenum cofactor (Moco), 2Fe-2S clusters, and flavin adenine dinucleotide (FAD) for their catalytic activity. This large molybdoenzyme family includes xanthine, aldehyde, and CO dehydrogenases. XORs are widely distributed from bacteria to humans due to their key roles in the catabolism of purines, aldehydes, drugs, and xenobiotics, as well as interconversions between CO and CO2. Assessing the effect of excess metals on the Rubrivivax gelatinosus bacterium, we found that exposure to copper (Cu) or cadmium (Cd) caused a dramatic decrease in the activity of a high-molecular-weight soluble complex exhibiting nitroblue tetrazolium reductase activity. Mass spectrometry and genetic analyses showed that the complex corresponds to a putative CO dehydrogenase (pCOD). Using mutants that accumulate either Cu+ or Cd2+ in the cytoplasm, we show that Cu+ or Cd2+ is a potent inhibitor of XORs (pCOD and the xanthine dehydrogenase [XDH]) in vivo. This is the first in vivo demonstration that Cu+ affects Moco-containing enzymes. The specific inhibitory effect of these compounds on the XOR activity is further supported in vitro by direct addition of competing metals to protein extracts. Moreover, emphasis is given on the inhibitory effect of Cu on bovine XOR, showing that the XOR family could be a common target of Cu. Given the conservation of XOR structure and function across the tree of life, we anticipate that our findings could be transferable to other XORs and organisms. IMPORTANCE The high toxicity of Cu, Cd, Pb, As, and other metals arises from their ability to cross membranes and target metalloenzymes in the cytoplasm. Identifying these targets provides insights into the toxicity mechanisms. The vulnerability of metalloenzymes arises from the accessibility of their cofactors to ions. Accordingly, many enzymes whose cofactors are solvent exposed are likely to be targets of competing metals. Here, we describe for the first time, with in vivo and in vitro experiments, a direct effect of excess Cu on the xanthine oxidoreductase family (XOR/XDH/pCOD). We show that toxic metal affects these Moco enzymes, and we suggest that access to the Moco center by Cu ions could explain the Cu inhibition of XORs in living organisms. Human XOR activity is associated with hyperuricemia, xanthinuria, gout arthritis, and other diseases. Our findings in vivo highlight XOR as a Cu target and thus support the potential use of Cu in metal-based therapeutics against these diseases.

Interaction of Soluble and Immobilized Manganese-Stabilizing PsbO Protein with Manganese Ions and Isolated D1/D2/cyt b559 Complex of the PSII Reaction Center

Interaction of water-soluble and immobilized (on BrCN-activated agarose) manganese-stabilizing protein PsbO with Mn2+ and Mn3+ cations and with preparations of D1/D2/cyt b559 reaction center (RC) of photosystem II was studied. By native electrophoresis, the formation of dimeric and aggregated forms of PsbO protein were found in the presence of Mn2+, Mg2+, or Fe2+ ions. The dimerization of PsbO occurred after ultraviolet irradiation of the protein preparation. The presence of protein-bound Mn3+ cations increased the electrostatic interaction of the immobilized PsbO with the RC. This was evidenced to by higher amounts of CaCl2 that were required for dissociation of the PsbORC complex. It was first demonstrated that the protein exhibited superoxide dismutase (SOD) activity after an electrophoresis in PAAG upon incubation of the gel in an Mn2+-containing solution. Tetrazolium-reductase activity was also ascertained in the protein after its electrophoresis in a mixture with preparations of the oxygen-evolving complex (OEC). It is suggested that the protein interaction with Mn ions and superoxide radicals, as well as short-term UV irradiation, reduces tyrosine and a disulfide bond in the PsbO protein. This yields tyrosil radical and SH-groups participating in redox reactions with ETC components. The interactions of PsbO with Mn cations and UV light, taking place in the chloroplast thylakoids, may regulate the protein binding to RC, modify structural organization of the protein, and promote its participation in alternative pathways of electron transport under the influence of stress factors. The hypothetical scheme of interaction of the immobilized PsbO protein with Mn ions and RC is discussed.

The Pharmacological Blockade of Phosphodiesterase IV Potentiates the Anti-Tumor Effects of Arsenic Trioxide (ATO) in Acute Myelogenous Leukemia Cells through Multiple Signaling Pathways

Preclinical in vitro and in vivo studies showed that Arsenic Trioxide (ATO) has antileukemic effects both as a single agent and in combination with conventional therapies or with other molecularly targeted agents . Furthermore, analogs of cAMP have been shown to enhance the therapeutic effects of ATRA and ATO in APL cells. Because cAMP pathway is modulated by various Inhibitiors of phosphodiesterase IV (PDEIV), some of which are already in the clinics for the treatment of asthma, the aim of this study was to investigate whether the pharmacological inhibition of PDEIV potentiates the cytodifferentiating and/or proapoptotic activities of ATO in acute myelogenous leukemia (AML) cells.We found that PDEIV inhibitors induced the upregulation of the myeloid markers (CD38 and CD11b), and potentiated the cytodifferentiating action of ATO in APL cells via induction of cAMP; the cytodifferentiating effects of the PDEIV inhibitors /ATO combination in leukemic cells was also confirmed by nitro blue tetrazolium reductase activity assay. Accordingly, we also found that the inhibition of PDEIV accelerated the ATO-induced degradation of PML-retinoic acid receptor α (PML-RARα) oncoprotein in NB4 cells, thus removing the PML-RARα-induced maturation blocks.Furthermore we also demonstrated that PDEIV inhibitors synergized with ATO to induce apoptosis in both APL NB4 and non-APL HL-60, MOLM-13 and OCI-AML-3 AML cell lines and significantly (P<.01) potentiated the ATO-induced cell death of fresh purified leukemic blasts in 8 out of 10 AML patients analyzed with different genetic abnormalities; in contrast, no significant cytotoxicity in peripheral blood mononuclear cells from 3 healthy volunteers was observed after PDEIV inhibitors/ATO treatment. We consistently found that the inhibition of PDEIV strongly potentiated the ATO-induced activation of caspase-3 and PARP fragmentation in all the tested cell lines as well as in primary AML blasts from patients, and, by using a peptide inhibitor approach, we demonstrated that caspase activity was indispensable for PDEIV inhibitors / ATO-induced apoptosis. Furthermore, siRNA knockdown of endogenous PDE4A and PDE4B gene expression recapitulated the ability of PDEIV inhibitors to sensitize leukemic cells to ATO, thereby confirming the significant role of these enzymes in mediating ATO sensitization of leukemic cells to PDEIV inhibitors /ATO-induced apoptosis. Interestingly, by using cAMP analogs and cAMP competitors we demonstrated that PDEIV inhibitors /ATO -induced apoptosis was cAMP independent.In order to investigate the molecular effectors involved in PDEIV inhibitors /ATO-induced apoptosis we first evaluated the effects of the combined treatment on Bcl-2 and p53 family proteins and their biologic relevance in the synergism observed between PDEIV inhibitors and ATO. By western blot analysis we demonstrated that co-treatment with PDEIV inhibitors and ATO increased protein expression of pro-apoptotic of Bax, Bak and/or Puma and decrease anti-apoptotic Mcl-1, A1/Bfl1 and/or Bcl-2; in agreement with the biochemical findings indicating the activation of the mitochondrial apoptotic pathway, we found that combined treatment with PDEIV inhibitors and ATO strongly potentiated mitochondrial depolarization induced by ATO alone in all the tested cell lines. Preliminary data indicate the involvement of p73 pathway which is consistent with the observed upregulation of Bax, Bak and/or Puma in AML cells; ongoing experiment are in progress to evaluate whether p53 pathway is activated by the combined treatment in MOLM-13 and OCI-AML-3 AML cell lines as well as in fresh purified leukemic blasts.Additionally, we found that co-treatment with PDEIV inhibitors and ATO also induced prosurvival signals by increasing ERK and Bad phosphorylation and the expression of anti-apoptotic Bcl-xL, and the pharmacological or genetic disruption of ERK or Bcl-xL consistently and significantly potentiated the cytotoxic effects of the combination PDEIV inhibitors /ATO. Altogether these findings suggest to extend the study to more patients and strongly support the rationale for testing these promising combinations in clinically relevant in vivo mouse models of drug-resistant leukemia. DisclosuresNo relevant conflicts of interest to declare.

Effect of synbiotic dietary supplementation on histological and histopathological parameters of Pectoralis major muscle of broiler chickens

Bioactive substances may constitute an alternative to antibiotics. Probiotics are mixtures of microorganisms that enhance the effectiveness and yield of nutrient absorption by competing for the substrate against pathogens that may cause intestinal infections. Prebiotics are organic substances which include complex carbohydrates and have an influence on the growth and activity of desirable intestinal microflora. Synbiotics are a combination of both of them. The aim of this study was to analyze the impact of synbiotics added to commercial feed on Pectoralis major muscle microstructure in broiler chickens. Research material consisted of 240 one-day-old Ross 308 female chicks. Birds were divided into 2 groups, 12 broilers each (replicated 10 times). The control group (C) was fed a commercial diet, and the SYN group was fed the same diet with added synbiotic: 0.8% of prebiotic RFO (raffinose family oligosaccharides) extracted from lupin seeds and 1% of probiotic Lavipan&lt;sup&gt;®&lt;/sup&gt;. Birds were slaughtered at day 42. Immediately after slaughter, samples of the Pectoralis major muscle for histological analysis were taken and frozen in liquid nitrogen. The staining procedures performed were: hematoxylin and eosin staining to evaluate fibre diameter and histopathological changes, oil red staining to determine intramuscular fat content, NADH-TR (tetrazolium reductase) activity to distinguish muscle fibre types and alkaline phosphatase reaction for counting the number of capillaries. The tendency to reduced muscle fibre diameter in chickens supplemented with synbiotics indicates an increase in muscle fibre density, which gives meat a more delicate structure. When assessing the occurrence of histopathological changes, significantly more fibre splitting was observed in the control group. Moreover, the positive effect of feed supplementation with synbiotics on muscle microvascularization could have an important practical meaning, because low physical activity of chickens during rearing may lead to ischemic muscles, increased changes in the structure of muscle fibres, and reduction of meat quality.

Evaluation of a human generic formulation of ciclosporin in the treatment of canine atopic dermatitis with in vitro assessment of the functional capacity of phagocytic cells

To compare the efficacy, tolerability and safety of a generic formulation of ciclosporin for human beings with prednisone in the treatment of canine atopic dermatitis), human generic ciclosporin A (hgCsA)...

Comparing the use of glycerol preserved and cryopreserved allogenic skin for the treatment of severe burns: differences in clinical outcomes and in vitro tissue viability

Cryopreserved (CryoPA) and Glycerol-preserved (GPA) skin allografts are commonly used in the treatment of severe burn injuries. However, comparable data on their differences in clinical outcome is scarce. This retrospective review aims to study the effect of allograft viability on clinical outcomes. The records of 48 severe burn patients who either received CryoPA or GPA were reviewed. Key burn mortality determinants were used to match the 2 groups. Clinical outcomes such as mortality rate (MR) and the length of hospital stay (LOS) were obtained. A separate in vitro comparison included histological assessments and the use of tetrazolium reductase activity to compare tissue viability. Both groups showed a comparable profile in burn mortality determinants. Patients who received CryoPA had a lower MR of 25% compared to 34.8% (P=0.250) in the GPA group and a lower LOS of 39.2-45.9 days (P=0.730), respectively. The histological structural integrity was found to be well preserved with both methods although CryoPA was confirmed to be the more viable product (P<0.05). The lower MR associated with CryoPA cannot be totally ignored. However, the mechanism through which viable skin allografts improves MR of severe burns patients remains to be elucidated.

Human Extraocular Muscles in ALS

PURPOSE. To investigate the general morphology, fiber type content, and myosin heavy chain (MyHC) composition of extraocular muscles (EOMs) from postmortem donors with amyotrophic lateral sclerosis (ALS) and to evaluate whether EOMs are affected or truly spared in this disease. METHODS. EOM and limb muscle samples obtained at autopsy from ALS donors and EOM samples from four control donors were processed for immunohistochemistry with monoclonal antibodies against distinct MyHC isoforms and analyzed by SDS-PAGE. In addition, hematoxylin and eosin staining and nicotinamide tetrazolium reductase (NADH-TR) activity were studied. RESULTS. Wide heterogeneity was observed in the appearance of the different EOMs from each single donor and between donors, irrespective of ALS type or onset. Pathologic morphologic findings in ALS EOMs included presence of atrophic and hypertrophic fibers, either clustered in groups or scattered; increased amounts of connective tissue; and areas of fatty replacement. The population of fibers stained with anti-MyHCslow tonic was smaller than that of MyHCIpositive fibers and was mostly located in the orbital layer in most of the ALS EOM samples, whereas an identical staining pattern for both fiber populations was observed in the control specimens. MyHCembryonic was notably absent from the ALS EOMs. CONCLUSIONS. The EOMs showed signs of involvement with altered fiber type composition, contractile protein content, and cellular architecture. However, when compared to the limb muscles, the EOMs were remarkably preserved. EOMs are a useful model for the study of the pathophysiology of ALS.

Nitric Oxide Synthase-3 Overexpression Causes Apoptosis and Impairs Neuronal Mitochondrial Function: Relevance to Alzheimer's-Type Neurodegeneration

Dementia in Alzheimer's disease (AD) is correlated with cell loss that is mediated by apoptosis, mitochondrial (Mt) dysfunction, and possibly necrosis. Previous studies demonstrated increased expression of the nitric oxide synthase 3 (NOS3) gene in degenerating neurons of AD brains. For investigating the role of NOS3 overexpression as a mediator of neuronal loss, human PNET2 central nervous system–derived neuronal cells were infected with recombinant adenovirus vectors that expressed either human NOS3 or green fluorescent protein cDNA under the control of a CMV promoter. NOS3 overexpression resulted in apoptosis accompanied by increased levels of p53, p21/Waf1, Bax, and CD95. In addition, NOS3 overexpression impaired neuronal Mt function as demonstrated by the reduced levels of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide and nicotinamide adenine dinucleotide (reduced form)-tetrazolium reductase activities and MitoTracker Red fluorescence. These adverse effects of NOS3 were associated with increased cellular levels of reactive oxygen species and impaired membrane integrity and were not produced in cells that were transfected with a cDNA encoding catalytically inactive NOS3. Importantly, modest elevations in NOS3 expression, achieved by infection with low multiplicities of adenovirus-NOS3 infection, did not cause apoptosis but rendered the cells more sensitive to oxidative injury by H2O2 or diethyldithiocarbamate. In contrast, treatment with NO donors did not enhance neuronal sensitivity to oxidative injury. These results suggest that NOS3-induced neuronal death is mediated by Mt dysfunction, oxidative injury, and impaired membrane integrity, rather than by NO production, and that neuroprotection from these adverse effects of NOS3 may be achieved by modulating intracellular levels of oxidative stress.

Oxidases and reductases are involved in metronidazole sensitivity in Helicobacterpylori

Helicobacter pylori is a contributing factor to the development of gastric and duodenal ulcers and some gastric cancers. Some therapeutic regimes comprise of a number of components, one of which is the antimicrobial metronidazole. A problem with these therapies is the increasing prevalence of metronidazole-resistant (MtrR) H. pylori strains. Several resistance mechanisms have been proposed, and this study addresses the ‘scavenging of oxygen’ hypothesis. Spectrophotometric assays of cytosolic fractions indicated that metronidazole-sensitive (MtrS) H. pylori isolates had 2.6-fold greater nicotinamide adenine dinucleotide (NADH) oxidase activity, 34-fold greater NADH nitroreductase activity, and eightfold greater nicotinamide adenine dinucleotide phosphate (NADPH) nitroreductase activity than cytosolic fractions from matched MtrR strains. Electrophoresis of cytosolic fractions in non-denaturing gels showed up to 10 protein bands when stained with Coomassie blue. Activity staining of non-denaturing, non-reducing polyacrylamide gels detected NAD(P)H oxidase, disulphide reductase, tetrazolium reductase and nitroreductase activities in the protein bands. Oxidase and reductase activities observed in a band from MtrS strains were absent in the corresponding band from MtrR strains. This band comprised at least 13 proteins, and the major constituent was identified as an alkyl hydroperoxide reductase AhpC subunit. The absence of oxidase and reductase activities in the band from MtrR strains indicated a correlation between the activity of the proteins in this band and the metronidazole-sensitive phenotype.

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.

Metabolic Correction of Cerebral Radiation Syndrome

DNA strand breaks that occur after irradiation activate the repair enzyme adenosine diphosphoribosyl transferase, which consumes NAD as a substrate and causes depletion first of neuronal NAD and then of the ATP pool. This is considered to be the crucial link in the mechanism underlying the cerebral radiation syndrome (CRS). In this study, two ways to correct the CRS metabolically were examined: (a) prevention of depletion of NAD after irradiation by administration of the enzyme inhibitor nicotinamide and (b) shunting the NAD-dependent oxidative phosphorylation pathway of ATP resynthesis by administration of a substrate of NAD-independent oxidation, succinate. Cerebral lesions induced by radiation were modeled by irradiation of rats or rat brain homogenates with 150 Gy of X rays. The manifestations of CRS in rats (excitement, convulsions, etc.) closely resembled those seen after acute hypoxia. In brain homogenates, pyruvate tetrazolium-reductase activity decreased after irradiation and could be corrected by addition of NAD after irradiation. Succinate tetrazolium-reductase activity was not affected by irradiation. Oxygen consumption by brain homogenates after irradiation in vitro and in situ decreased, as did oxygen consumption by rats in vivo after cranio-caudal irradiation. Administration of nicotinamide or succinate prevented both the postirradiation decrease in respiration (in both rats in vivo and brain homogenates in vitro) and the development of cerebral radiation syndrome. These results help to clarify the mechanisms underlying CRS and its metabolic correction.

Structural and functional characteristics of muscle fibres in pigs with different malignant hyperthermia susceptibility (MHS) and different meat quality

In pigs, intensive growth of the musculature is often accompanied by malignant hyperthermia susceptibility (MHS; n gene) and poorer meat quality. Using histological and histochemical methods, different fibre characteristics in the Longissimus muscle were found in Pietrain×German Landrace pigs with this gene defect. Compared to MHS homozygous negative pigs, groups with the n gene had increased diameters of the mean fibre types and increased glycolytic metabolic potential, as shown by a higher frequency of the fast twitch glycolytic type and a lower frequency of the slow twitch oxidative fibre type. Differences between the groups were also found in the number of angular and giant fibre types. Furthermore, a positive correlation was found between the frequency of oxidative fibres and the relative enzyme activity of NADH tetrazolium reductase. The changes correlated with lower pH and higher drip loss in meat from the MHS homozygous positive group. In conclusion, the different muscle fibre characteristics can be interpreted as endogenous factors which influence the physiological condition in the muscle of the live animal and meat quality post mortem.

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.

Morphologic and Histochemical Evaluation of Muscle in Patients with Chronic Pulmonary Emphysema Manifesting Generalized Emaciation

Twelve severely emaciated patients with emphysema and 4 control patients with mild emaciation were studied. Arterial blood gas analysis, pulmonary function tests, and muscle biopsy were performed. According to the percentage of ideal body weight (%IBW), patients were divided into two groups of 6 patients each; one with %IBW values greater than 70.0 (group 1) and the other with %IBW values less than 70.0 (group 2), and 4 control patients with emphysema whose %IBW values were greater than 85.5. %FEV1.0, PaO2 and Hugh-Jones scores in group 2 patients were consistent with significantly greater deterioration as compared with those in group 1 patients and controls. In the muscle fibers of 11 patients and 4 controls, nicotinamide and adenosine dinucleotide tetrazolium reductase activity was studded with spots. Nemalin rods were detected in 3 specimens in group 2 and in 1 specimen each in both group 1 and controls. These changes probably result from chronic hypoxemia. Fiber type grouping accompanied by type II dominant fiber atrophy was demonstrated in 5 patients of group 2, whereas type II fiber atrophy was shown in 2 specimens from patients of group 1. Diameters of both types I and II fibers in group 2 were smaller than those in group 1 and controls. Significant correlations were observed between fiber diameters and %IBW, %FEV1.0 and %IBW, and PaO2 and %IBW. Neurogenic and disuse muscular atrophy due to both hypoxic axonal disorder and disuse is likely to be the cause of the emaciation, and a 'vicious circle' between muscular atrophy, respiratory function and hypoxemia probably exists in group 2. Since mild or moderate emaciation was observed in controls and group 1 in spite of the lack of fiber atrophy, involvement of fat and connective tissue should also be taken into consideration to determine the cause of emaciation.

Preservation of the cervical vagus nerve in aged rats: morphometric and enzyme histochemical evidence

The vagus nerve trunk, sampled at a mid-cervical level, has been analysed quantitatively by light and electron microscopy principally with respect to the numbers and sizes of the myelinated and unmyelinated axon populations in Wistar rats of 4, 24 and 30 months. No significant differences in total myelinated axon numbers were seen over the age range in counts made on light microscope montages of the entire cross-section of the nerve. The overall histological organisation and appearance of the nerve trunk did not change with age but age-associated alterations in the ultrastructure of some myelinated fibres and their Schwann cells was seen. Unmyelinated axons and their associated Schwann cells rarely showed age-associated changes. The numbers of myelinated and unmyelinated axons per mm 2 determined from electron micrographs were, however, slightly decreased but the ratio of myelinated to unmyelinated axons was approximately 1:4 at all ages. Measurements of myelinated fibres showed a small but significant increase in size between young and old animals. There was an increase in the thickness of the myelin sheath, a decrease in myelinated axon diameter and in the the g ratio. The diameter of unmyelinated axons decreased with age and the number of unmyelinated axons per Schwann cell unit increased. The numbers, diameters and thickness of the walls of the vagal microvessels remained unchanged. Quantitation by microdensitometry of the activity of NADH tetrazolium reductase and succinate dehydrogenase in longitudinal sections of the vagus indicated an increase in the activity of these two metabolic markers whilst an increase in the activity of cytochrome oxidase indicated that neuronal activity in the vagus was unimpaired in old age. It is concluded that the structure of the rat vagus nerve, and in particular of its predominantly unmyelinated axon population, is not significantly affected in old age.

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.

The Effect of Amphotericin B on the Viability of Cryopreserved Human Skin

To evaluate whether amphotericin B has a detrimental effect on the viability of cryopreserved skin, human skin samples were incubated during 7 and 24 h in three different media: 0.9% phosphate-buffered saline and Medium 199 with and without 10 μg/ml amphotericin B. The viability of the tissue was assessed by measuring the tetrazolium reductase activity before and after cryopreservation. A significant decrease in cryopreserved human skin viability was observed after 7 and 24 h incubation with amphotericin B. These results indicate that in the processing of skin preservation, the beneficial effect of using amphotericin B as an antifungal agent may be reduced by its negative effect on tissue viability.

Aldehyde fixation differentially affects distribution of diaphorase activity but not of nitric oxide synthase immunoreactivity in rat brain

The effect of aldehyde fixation on NADPH- and NADH-dependent diaphorase (d) histochemistry and nitric oxide synthase (NOS) immunocytochemistry in the brain was investigated by comparing the distribution of these enzymes in in situ nitrocellulose blots of unfixed brain sections with that in aldehyde-fixed brain sections. Substitution of NADPH by NADH yielded no gross differences in cellular distribution in the native blot, whereas in fixed sections NADH produced nonspecific staining of the entire section. In the in situ blot NADPHd histochemistry therefore visualized general nitroblue tetrazolium reductase (NBTr) activity, which was particularly strong in hippocampal pyramidal neurons and cerebellar Purkinje cells. Aldehyde fixation abolished the anatomical pattem of general NBTr activity and changed the histochemical distribution in that of the NADPHd activity associated with the distribution of NOS-I immunoreactivity (ir). Fixation intensified NADPHd histochemical staining in specific neurons, resulting in outstanding, Golgi-like staining of these neurons in several brain regions, whereas the general NBTr activity in pyramidal and Purkinje cells disappeared. In contrast to the histochemical diaphorase distribution, the distribution of NOS-I it on blots and in aldehyde-fixed brain sections was similar. No NOS was observed in hippocampal pyramidal and cerebellar Purkinje neurons. In regions like cerebral and cerebellar cortex and striatum the applied anti NOS-I serum had a higher affinity for the native protein. It is concluded that aldehydes, rather than to progressively suppress NOS-unrelated enzymes, differentially elicit NADPHd activity in some groups of neurons while leaving NOS-ir unaffected.

The Effects of Variation of Cryopreservation Protocols on the Immunogenicity of AIIogeneic Skin Grafts

The use of allografts to effect wound closure on full thickness skin injuries is limited by unpredictable graft rejection times. If the period of graft take could be extended, the use of allografts would reduce the morbidity and mortality associated with these wounds. This study evaluated the effects of variation in the cryopreservation protocol on the viability and immunogenicity of skin using a murine model system. lmmunogenicity was assessed by the stimulatory activity of C3H (H-2K) skin-derived epidermal cells (EC) in primary one-way EC/lymphocyte reactions with BALB/c (H-2d) and CBA (H-2K) responder lymphocytes. Viability was determined by measuring tetrazolium reductase activity. The following cryopreservation protocols were assessed: freezing at 1, 30, 64, and > 100°C/min in 10 and 15% (v/v) Me2SO and freezing at 30°C/min in 5 to 20% (v/v) Me2SO or glycerol. A cryopreservation protocol of 30°C/min in 15% (v/v) Me2SO proved optimal for murine skin allograft storage and immunomodulation. The viability of skin treated by this protocol was maintained (78% of fresh skin viability, no significant difference analysis of variance). The stimulatory capacity of treated EC for H-2K and H-2d lymphocytes was 5 ± 4 and 5 ± 9% (±95% confidence limits) of fresh EC (100%) activity. Langerhans cell numbers in epidermal sheets and EC suspensions did not correlate with the stimulatory capacity of fresh and treated EC for allogeneic Iymphocytes. A functional impairment of Langerhans cell immunostimulatory capacity was implied. Thus it may be feasible to provide a single stage storage and immunomodulation treatment for allogeneic skin.

Quantitative Reduction of MTT by Hearts Biopsies In Vitro is an Index of Viability

The aim of this study is to evaluate the use of tetrazolium reductase (TR) activity as an indicator of myocardial viability in an isolated arrested pig heart biopsy model. Methyl Tetrazolium (MTT) is cleaved by an enzyme in the presence of coenzymes NAD, NADP. Cleavage yields a highly colored formazan product which is DMSO soluble. Efficient bioreduction of MTT has been investigated with heart biopsies. The relationship between MTT reduction and (1) oxygen consumption (r = 0.96, P < 0.001), (2) the sum of the adenine nucleotide levels (r = 0.87, P < 0.001) and (3) localization of coloration, has been established. The use of MTT in colorimetric assays offers high sensitivity. MTT reduction is a valid method. It is rapid and reproducible, and can be used as an indicator of myocardial viability. The MTT test has been used to rapidly compare the effect of different cardioplegic solutions (St Thomas and improved St Thomas) on hypothermic cardiac preservation. Significant differences have been the two solution (P < 0.01).