NADH-diaphorase Activity Research Articles

Fumonisin-containing diets decrease the metabolic activity of myenteric neurons in rats

ABSTRACT Fumonisins are naturally occurring mycotoxins that contaminate food for human and animal consumption. They have neurotoxic effects, but the mechanisms by which these toxins affect the nervous system are not fully known. In the present study, male Wistar rats were fed between 21 and 63 days of age with diets that contained fumonisins B1+B2 at 0, 1, and 4 mg/kg. The following variables were assessed: food consumption, growth, body weight gain, and blood parameters. Morphoquantitave analyses of the most metabolically active myenteric neurons were performed, detected by NADH-diaphorase activity. Nitrergic neurons were detected by NADPH-diaphorase activity. The fumonisin-containing diets did not significantly alter food consumption or the body or plasma parameters. These diets decreased the metabolic activity of jejunal myenteric neurons, reducing neuronal density of the most metabolic active neurons by 30.8% and the cell body area by 4.3%. The diets also decreased the cell body area of nitrergic neurons by 22.1%. The effects of fumonisin B1 on the respiratory metabolism of isolated mitochondria in the brain and liver were also assessed. A decrease in oxygen consumption up to a 29% in the brain and 38% in the liver was observed in mitochondrial isolates to which 50 µM fumonisin B1 was added. The decrease in respiratory activity that was triggered by exposure to fumonisins was related to the lower metabolic activity of myenteric neurons, which had a negative impact on neuroplasticity of the enteric nervous system.

Evidence for a hexaheteromeric methylenetetrahydrofolate reductase in Moorella thermoacetica.

Moorella thermoacetica can grow with H₂ and CO₂, forming acetic acid from 2 CO₂ via the Wood-Ljungdahl pathway. All enzymes involved in this pathway have been characterized to date, except for methylenetetrahydrofolate reductase (MetF). We report here that the M. thermoacetica gene that putatively encodes this enzyme, metF, is part of a transcription unit also containing the genes hdrCBA, mvhD, and metV. MetF copurified with the other five proteins encoded in the unit in a hexaheteromeric complex with an apparent molecular mass in the 320-kDa range. The 40-fold-enriched preparation contained per mg protein 3.1 nmol flavin adenine dinucleotide (FAD), 3.4 nmol flavin mononucleotide (FMN), and 110 nmol iron, almost as predicted from the primary structure of the six subunits. It catalyzed the reduction of methylenetetrahydrofolate with reduced benzyl viologen but not with NAD(P)H in either the absence or presence of oxidized ferredoxin. It also catalyzed the reversible reduction of benzyl viologen with NADH (diaphorase activity). Heterologous expression of the metF gene in Escherichia coli revealed that the subunit MetF contains one FMN rather than FAD. MetF exhibited 70-fold-higher methylenetetrahydrofolate reductase activity with benzyl viologen when produced together with MetV, which in part shows sequence similarity to MetF. Heterologously produced HdrA contained 2 FADs and had NAD-specific diaphorase activity. Our results suggested that the physiological electron donor for methylenetetrahydrofolate reduction in M. thermoacetica is NADH and that the exergonic reduction of methylenetetrahydrofolate with NADH is coupled via flavin-based electron bifurcation with the endergonic reduction of an electron acceptor, whose identity remains unknown.

Quercetin up-regulates mitochondrial complex-I activity to protect against programmed cell death in rotenone model of Parkinson’s disease in rats

We tested quercetin, a dietary bioflavonoid with potent free radical scavenging action and antioxidant activity, for its neuroprotective effects in rotenone-induced hemi-parkinsonian rats. Rats were infused unilaterally with rotenone into the substantia nigra, and quercetin (25–75mg/kg, i.p.) was administered at 12-h intervals for 4days, and analyzed on the 5th day. Amphetamine- or apomorphine-induced unilateral rotations were significantly reduced in quercetin-treated rats, when analyzed on 14th or 16th day post-surgery, respectively. Quercetin possessed potent hydroxyl radical scavenging action in a cells-free, Fenton-like reaction in test tubes, and in isolated mitochondria when measured by salicylate hydroxylation method. We observed dose-dependent attenuation of the rotenone-induced loss in striatal dopamine, and nigral oxidized and reduced glutathione, as well as the increases in endogenous antioxidant enzymes (catalase and superoxide dismutase) activities supporting the notion that quercetin-effect is mediated via its powerful hydroxyl radicals-scavenging and antioxidant actions. Quercetin’s dose-dependent ability to up-regulate mitochondrial complex-I activity, as evidenced by NADH-oxidation, and as seen in blue native-polyacrylamide gel electrophoresis (PAGE) staining in both the contra- and ipsi-lateral nigra suggests the containment of reactive oxygen production at the mitochondrial level. Rotenone-induced induction of NADH-diaphorase activity in the nigral neurons, and its attenuation by quercetin pointed to the possible involvement of nitric oxide too. Reversal of neuronal death induced by rotenone as observed by increased tyrosine hydroxylase-positive cells and decreased TdT-mediated dUTP nick end-labeling (TUNEL) staining in the substantia nigra confirmed the potential of quercetin to revamp dopaminergic cells following oxidative stress mediated programmed cell death and neuronal demise. The present study strongly implicates quercetin’s potential ability to repair mitochondrial electron transport defects and to up-regulate its function as the basis of neuroprotection observed in a mitochondrial neurotoxin-induced Parkinsonism.

Muscle fiber types, growth and development in the whole myotome of cultured Pacific bluefin tuna Thunnus orientalis

Following the successful development of Pacific bluefin tuna (PBT) aquaculture, it is of considerable importance to determine the muscle fiber types and their growth patterns for future development. Muscle fiber profiles of dorsal ordinary, lateral ordinary (LO) and dark muscles and their growth patterns in PBT from 3.0 to 54.3 kg body weight were studied. Muscle fibers were histochemically stained for NADH-diaphorase and myosin adenosine triphosphatase activity (mATPase), and immunohistochemically stained with S-58 slow-muscle myosin antibody. All muscle fibers in dorsal and LO muscles showed low NADH-diaphorase activity, and acid-labile (pH 4.0 or 4.3) and alkali-stable mATPase activity. In LO muscle adjacent to dark muscle, three intensities of mATPase activity were observed after acid pre-incubation at pH 4.5 or 5.0, and the activity was related to the muscle fiber diameter. In dark muscle, all small and some large fibers stained intensely for NADH-diaphorase activity, related to their high aerobic metabolism. The high-active fibers with NADH-diaphorase in dark muscle were positive for S-58 antibody. Some large fibers in dark muscle showed intermediate NADH-diaphorase activity and high mATPase activity after alkali pre-incubations. These are fast-twitch oxido-glycolytic fibers in dark muscle and transformed to red muscle fibers with increasing body weight.

Expression of the P2X2receptor in different classes of ileum myenteric neurons in the female obese ob/ob mouse

To examine whether the ob/ob mouse model of obesity is accompanied by enteric nervous system abnormalities such as altered motility. The study examined the distribution of the P2X₂ receptor (P2X₂R) in myenteric neurons of female ob/ob mice. Specifically, we used immunohistochemistry to analyze the co-expression of the P2X₂R with neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and calretinin (CalR) in neurons of the small intestine myenteric plexus in ob/ob and control female mice. In these sections, we used scanning confocal microscopy to analyze the co-localization of these markers as well as the neuronal density (cm²) and area profile (μm²) of P2X₂R-positive neurons. In addition, enteric neurons were labeled using the nicotinamide adenine dinucleotide (NADH) diaphorase method and analyzed with light microscopy as an alternate means by which to analyze neuronal density and area. In the present study, we observed a 29.6% increase in the body weight of the ob/ob animals (OG) compared to the control group (CG). In addition, the average small intestine area was increased by approximately 29.6% in the OG compared to the CG. Immunoreactivity (IR) for the P2X₂R, nNOS, ChAT and CalR was detectable in the myenteric plexus, as well as in the smooth muscle, in both groups. This IR appeared to be mainly cytoplasmic and was also associated with the cell membrane of the myenteric plexus neurons, where it outlined the neuronal cell bodies and their processes. P2X₂R-IR was observed to co-localize 100% with that for nNOS, ChAT and CalR in neurons of both groups. In the ob/ob group, however, we observed that the neuronal density (neuron/cm²) of P2X₂R-IR cells was increased by 62% compared to CG, while that of NOS-IR and ChAT-IR neurons was reduced by 49% and 57%, respectively, compared to control mice. The neuronal density of CalR-IR neurons was not different between the groups. Morphometric studies further demonstrated that the cell body profile area (μm²) of nNOS-IR, ChAT-IR and CalR-IR neurons was increased by 34%, 20% and 55%, respectively, in the OG compared to controls. Staining for NADH diaphorase activity is widely used to detect alterations in the enteric nervous system; however, our qualitative examination of NADH-diaphorase positive neurons in the myenteric ganglia revealed an overall similarity between the two groups. We demonstrate increases in P2X₂R expression and alterations in nNOS, ChAT and CalR IR in ileal myenteric neurons of female ob/ob mice compared to wild-type controls.

Relationship of sperm superoxide dismutase-like activity with other sperm-specific enzymes and experimentally induced lipid peroxidation in infertile men.

Superoxide dismutase-like activity (SOD-like), isoenzyme lactate dehydrogenase-C4 (LDH-C4) and NADH-diaphorase activities in spermatozoa have been investigated from 58 normozoospermic and 27 oligozoospermic men. Significantly higher SOD-like, LDH-C4 and diaphorase activities (P < 0.01, P < 0.005 and P < 0.0001, respectively) were detected in spermatozoa from oligozoospermic men, compared to the activities found in normozoospermic samples. SOD-like activity (mean +/- SE) in oligozoospermic samples amounted to 8.3 +/- 1.6 U 10(-8) spermatozoa, while in spermatozoa in normozoospermic men with a sperm concentration above 20 million of spermatozoa per ml amounted to 4.2 +/- 0.5 U 10(-8). There was a close correlation between the SOD-like activity and biochemical indicators of the presence of residual cytoplasm i.e. isoenzyme LDH-C4 and NADH-diaphorase (r = 0.53 and r = 0.66 in normozoospermic and r = 0.63 and r = 0.54 in oligozoospermic men, respectively). A positive relationship between SOD-like activity and experimentally-induced lipid peroxidation was detected in 54 infertile men (r = 0.30; P < 0.05). These findings suggest that a higher level of superoxide dismutase-like activity may reflect a defect in the development or maturation of spermatozoa and, thereby, a decreased fertility potential. Hence, determination of SOD-like activity may give information on the state of maturity of human spermatozoa, while its role in the antioxidative protection remains to be determined.

Growth and viability of mycorrhizal extraradical mycelia associated with three temperate orchid species

Growth and enzymatic activities of extraradical mycelia (ERM) of native mycorrhizal symbionts associated with three orchid species, Dactylorhiza fuchsii, D. majalis and Platanthera bifolia, were studied. ERM extracted from the mycorrhizosphere of these species showed features typical for fungi that form orchid mycorrhiza. In the first pot experiment, three different treatments were applied on tubers of D. fuchsii transplanted from a natural site: control (no specific treatment), reinoculated (surface-sterilized tubers reinoculated with mycorrhizal fungi-colonised roots), and benomyl (nonsterilized tubers treated with fungicide). However, no significant differences in ERM growth and intensity of root mycorrhizal colonisation at harvest were observed among these treatments. ERM associated with reinoculated D. fuchsii plants showed significantly higher alkaline phosphatase (ALP) enzymatic activity at week 36 than at week 24, but no differences were observed for NADH diaphorase activity. Benomyl application significantly reduced ALP activity in comparison with reinoculated plants at week 36. In the second experiment, plants of all three species were either untreated (control), or repeatedly treated with benomyl. Similarly to the results of the first experiment, benomyl application did not reduce the ERM growth of mycorrhizal symbionts associated with D. majalis and D. fuchsii. The low ERM growth associated with benomyl-treated P. bifolia was probably caused by poor root system development in this treatment. Significantly higher mycorrhizal colonisation was found for D. fuchsii compared to P. bifolia in control treatments at the end of cultivation. The ERM of native symbionts of the three orchid species studied seemed to have a different growth pattern over time and responded differently to fungicide application.

Novel tumor‐ablation device for liver tumors utilizing heat energy generated under an alternating magnetic field

We have developed a novel tumor-ablation device for liver tumors utilizing heat energy induced by magnesium ferrite (MgFe(2)O(4)) particles under an alternating magnetic field (AMF) produced by electric currents. This novel device can repeatedly heat liver tumors at lower temperature than usual heating devices, such as radiofrequency ablation therapy, with slight infliction of pain. This study assesses its heating effect on rat liver tumors as local therapy. The small needle was manufactured from MgFe(2)O(4) particles by sintering at 1100 degrees C. After a MgFe(2)O(4) needle was inserted into liver tumors comprising of dRLh-84 cells, the tumors were heated for 30 min under an AMF. We examined cellular activity by using nicotinamide adenine dinucleotide (NADH) diaphorase staining and terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling (TUNEL) staining, and evaluated the effect of suppressing tumor growth by sequentially comparing the tumor diameter with that of the control group. The mean temperature of the heated tumors was 60.2 +/- 1.8 degrees C. The tumor cells were constricted, and chromatin of nuclei had shrunk immediately after heating. The heat-injury area that contained the tumors was negative for NADH diaphorase activity. After 3 days, the tumor cells in the heat-injury area became positive for TUNEL staining, which detects cell death. At 7 days, the mean tumor diameters were significantly smaller in the heating group than in the control group (6.15 +/- 0.47 mm vs 16.89 +/- 2.69 mm; P < 0.05). This device, utilizing heat energy induced by ferromagnetic metal under an AMF, appears useful as local thermotherapy for human liver cancer.

Long-term consequences of prenatal exposure to lead on brain development in rats

Administration of singe doses of lead citrate (200 mg/kg) to pregnant rats (on day 18 of pregnancy) was followed by the appearance of destructive changes in brains at age 40 days, with cysts, foci of gliocyte proliferation, pyknotic neurons, and decreases in NADH and NADPH diaphorase activities in neocortical and hippocampal neurons. Decreases in the density of neurons in the cortex and decreases in cortical thickness were also observed. The intensity of free-radical oxidation in the cortex increased three-fold, along with a 3.9-fold increase in the concentration of lipid peroxides, providing evidence of oxidative stress. The possible mechanisms by which these alterations develop are analyzed.

MORPHO-FUNCTIONAL CHANGES OF THE ESOPHAGEAL EPITHELIUM AFTER CYTOSTATIC TREATMENT

Using histological, morphometric, histochemical and immunocytochemical methods, the effect of cytotoxic treatment on structural and functional characteristics of the epithelium of esophageal mucosa was studied in mice together with the reversibility of the changes induced by cytotoxic drug. Fourfold intraperitoneal injection of cyclophosphamide (400 mg/kg of body mass) resulted in such morpho-functional changes, as thickening of epithelial layer, increase in proportion of its stratum corneum and its loosening, disturbances in cornification process, hyperkeratosis, vacuolization of cell cytoplasm in stratum basale and stratum spinosum, interstitial edema, nuclear hypertrophy and parakeratosis. Mitotic activity and the activity of NADH-diaphorase were significantly reduced, while the number of PCNA+ cells was increased. Cyclophosphamide had no significant affect on the concentration of total proteins. 15 days after the discontinuation of cytostatic treatment, most of the indexes did not return to normal values, indicating profound disturbances in the esophageal epithelium.

Nitric oxide in blastocyst implantation in the rhesus monkey

Successful blastocyst implantation depends on the interaction between cells of maternal endometrium and conceptus, as well as adequate blood supply to the site of blastocyst implantation. Nitric oxide (NO) generally plays a significant role in the local regulation of vascular physiology in a variety of mammalian tissue systems, however, its role in blastocyst implantation and placentation in the primate is not known. The aim of the present study was to examine: (i) NADH-diaphorase activity and expression of three isoforms of nitric oxide synthase (NOS), namely endothelial NOS (eNOS), inducible NOS (iNOS) and neuronal NOS (nNOS) in pre-implantation stage monkey embryos, morula (n = 4) and blastocyst (n = 10), as well as, in different compartments of conceptus and maternal endometrium at primary implantation sites during lacunar (n = 6) and villous (n = 9) stages of placentation in the rhesus monkey, and (ii) the potential anti-nidatory effect of vaginal administration of NOS inhibitor during the peri-implantation period of conception cycles in rhesus monkeys. Pre-implantation stage blastocysts exhibited marked NADPH-diaphorase activity along with immunopositive iNOS mainly in the inner cell mass. During the lacunar stage, marked eNOS expression was observed in cytotrophoblast cells lining the embryonic cavity. However, cytotrophoblast cells lining villi, forming columns, and constituting anchoring villi expressed all the three isoforms of NOS in villous placenta stage tissue. During the lacunar stage, eNOS and iNOS protein expressions were observed in epithelial and decidual cells of endometrium. As gestation advanced, mRNAs for all three isoforms of NOS were observed to increase in epithelial and decidual cells, however, with no marked change in protein expression. Vaginal administration of a NOS inhibitor (N(G)-nitro-l-arginine methyl ester, L-NAME, 4, 6, and 8 mg/kg body weight or aminoguanidine, AG, 4 mg/kg body weight) during days 6 to 12 after ovulation resulted in pregnancy failure in a higher number of animals (L-NAME: 8 confirmed pregnancies in 25 animals; AG: 2 confirmed pregnancies in 8 animals) compared with control animals (5 pregnancies in 7 animals). It appears that NO may play an important role in the establishment of pregnancy in the rhesus monkey.

Synergistic effect of Glomus intraradices and Frankia spp. on the growth and stress recovery of Alnus glutinosa in an alkaline anthropogenic sediment

The presence of actinorhizas and arbuscular mycorrhizas may reduce plant stresses caused by adverse soil conditions. A greenhouse experiment was conducted using a sediment with a high pH, resulting from the disposal of waste originated at an acetylene and polyvinylchloride factory, in which Black alder ( Alnus glutinosa) seedlings were inoculated either with Glomus intraradices BEG163 (originally isolated from the same sediment), Frankia spp. or both symbionts. After a 6-month growth period, plants inoculated with both symbionts had significantly greater leaf area, shoot height and total biomass when compared with the uninoculated control, the Frankia spp. and the G. intraradices treatments alone. In dual inoculated plants the N and P leaf content was significantly increased. A defoliation experiment was performed to evaluate the stress recovery of A. glutinosa and plants inoculated with both symbionts had a faster leaf regrowth and produced greater numbers of leaves. The dual inoculation resulted in greater numbers of and larger root nodules than when inoculated with Frankia spp. alone. The length and NADH diaphorase activity of the extraradical mycelium of G. intraradices was also significantly greater when dual inoculation was performed. The inoculation with Frankia spp. alone was shown to improve A. glutinosa growth, whereas G. intraradices alone had no positive effect under these environmental conditions. However, when the two symbionts were inoculated together a synergistic effect was observed resulting in a greater benefit for the plants and for both symbionts. The relevance of these findings for the phytorestoration of anthropogenic stressed sediments with high pH is discussed.

Effects of inoculation with Glomus intraradices on lead uptake by Zea mays L. and Agrostis capillaris L.

An inhibitory effect of lead (Pb) on hyphal growth of Glomus intraradices was observed after the incubation of colonised root segments in the media containing Pb concentrations higher than 0.01 mM. The comparison of two G. intraradices isolates that originated either in Pb-contaminated or non-contaminated soil suggests higher Pb tolerance of the native isolate from Pb-contaminated soil in comparison to the reference isolate from non-contaminated soil. The role of both isolates in Pb accumulation by two plant species with different tolerance to heavy metals—common bent grass ( Agrostis capillaris) from a Pb-contaminated site and maize ( Zea mays)—was investigated in a sand-based hydroponic experiment with simulated Pb stress. Increased Pb concentrations influenced neither the growth of host plants nor the development of AM fungi (root colonisation, ERM length and NADH-diaphorase activity). In spite of low Pb toxicity, mycorrhizal inoculation substantially influenced Pb accumulation by the plants and the translocation into their shoots, but its effect differed considerably between plant species. When maize plants were treated with 0.01 mM Pb, mycorrhizal inoculation with both G. intraradices isolates resulted in decreased Pb concentrations in both shoots and roots in comparison with non-inoculated plants. At a higher Pb level (0.1 mM), the inoculation decreased Pb concentrations in maize roots, but not shoots, when compared to non-inoculated plants. For the common bent, shoot Pb concentrations were not influenced by the inoculation in either Pb treatment. At a lower Pb level, the G. intraradices isolate from contaminated soil increased Pb concentrations in the roots of Agrostis plants in comparison with control plants or plants inoculated with the isolate from non-contaminated soil. No significant differences in Pb concentrations in Agrostis roots were found between the inoculation treatments at the higher Pb level.

Trypanosoma brucei: in vitro slender-to-stumpy differentiation of culture-adapted, monomorphic bloodstream forms

Pleomorphic Trypanosoma brucei strains are characterized by their ability to differentiate from replicating long slender forms into non-dividing short stumpy forms in the mammalian host. The differentiation process can be efficiently induced in vitro by treatment with the membrane-permeable cAMP derivative 8-(4-chlorophenylthio)-cAMP (pCPTcAMP). In contrast, monomorphic T. brucei strains do not differentiate to stumpy forms in the host. Here, we show that exposure of monomorphic, culture-adapted T. brucei bloodstream forms to pCPTcAMP allowed their subsequent differentiation into short stumpy forms. The stumpy nature of pCPTcAMP-treated parasites was confirmed by (1) morphological change, (2) inhibition of growth and DNA synthesis, (3) cell cycle arrest in the G 1/G 0 phase, (4) expression of NADH diaphorase activity and dihydrolipoamide dehydrogenase, (5) disappearance of the small subunit of ribonucleotide reductase, (6) up-regulation of the major lysosomal membrane protein, and (7) efficient transformation into replicating procyclic insect forms after induction with citrate/ cis-aconitate. Our results indicate that the inability of monomorphic T. brucei bloodstream forms to differentiate into short stumpy forms in the host may be due to a failure in the signalling pathway rather than in the differentiation process itself. Treatment of monomorphic bloodstream trypanosomes with pCPTcAMP could be a useful method for identifying the genes involved in the slender-to-stumpy differentiation process. Index Descriptors and Abbreviations: Trypanosoma brucei; culture-adapted bloodstream forms; differentiation; cell cycle arrest; cAMP, adenosine 3 ′,5 ′-cyclic monophosphate; pCPTcAMP, 8-(4-chlorophenylthio)-cAMP; pCPTcGMP, 8-(4-chlorophenylthio)-guanosine 3 ′,5 ′-cyclic monophosphate; 4 ′,6-diamidino-2-phenylindole, DAPI; PBS, phosphate-buffered saline; HBSS, Hank’s balanced salt solution.

Purification and characterization of ferredoxin–NAD(P) + reductase from the green sulfur bacterium Chlorobium tepidum

Ferredoxin–NAD(P) + reductase [EC 1.18.1.3, 1.18.1.2] was isolated from the green sulfur bacterium Chlorobium tepidum and purified to homogeneity. The molecular mass of the subunit is 42 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular mass of the native enzyme is approximately 90 kDa, estimated by gel-permeation chromatography, and is thus a homodimer. The enzyme contains one FAD per subunit and has absorption maxima at about 272, 385, and 466 nm. In the presence of ferredoxin (Fd) and reaction center (RC) complex from C. tepidum, it efficiently catalyzes photoreduction of both NADP + and NAD +. When concentrations of NADP + exceeded 10 μM, NADP + photoreduction rates decreased with increased concentration. The inhibition by high concentrations of substrate was not observed with NAD +. It also reduces 2,6-dichlorophenol-indophenol (DPIP) and molecular oxygen with either NADPH or NADH as efficient electron donors. It showed NADPH diaphorase activity about two times higher than NADH diaphorase activity in DPIP reduction assays at NAD(P)H concentrations less than 0.1 mM. At 0.5 mM NAD(P)H, the two activities were about the same, and at 1 mM, the former activity was slightly lower than the latter.

Cardiac denervation in mice infected with Trypanosoma cruzi.

The neuronal features of the hearts of mice that were acutely or chronically infected with the Y strain of Trypanosoma cruzi were compared with those of control hearts from uninfected mice. Whole-mount preparations of the murine atria, isolated by microdissection, were stained to reveal neurons with NADH-diaphorase activity. Counts, by a microscopist who was blind to the infection status of the donor mouse, revealed that there were significantly (38%) fewer such neurons in the atria from the acutely infected mice than in the atria from the control hearts. The ganglia of the infected mice were also irregularly distributed, severely damaged ganglia being found beside slightly degenerated or morphologically normal ones. Although the ganglia contained small, medium and large neurons, the apparent destruction caused by T. cruzi was confined to the large ones. As neuron counts in preparations of hearts from mice with chronic infections were 32% lower than those in the control hearts, there appears to be no additional loss of cardiac neurons as the acute infection in mice progresses to the chronic phase.

Rhodococcus sp. RB1 grows in the presence of high nitrate and nitrite concentrations and assimilates nitrate in moderately saline environments.

Rhodococcus sp. RB1 was able to thrive in media with up to 0.9 M NaCl or KCl and in the presence of high concentrations of nitrate (up to 0.9 M) and nitrite (up to 60 mM), but only under oxic conditions. An adaptation period was not required for salt tolerance, but a rapid extrusion of K+ and intake of Na+ was observed after addition of 0.5 M NaCl. Nitrate assimilation was limited by the carbon supply, but nitrite was not accumulated in the culture medium, even at nitrate concentrations as high as 0.8 M, thus suggesting that nitrite reduction does not limit nitrate assimilation. The presence of NaCl or KCl did not affect nitrate or nitrite uptake, which were completely inhibited by ammonium or glutamine. Rhodococcus sp. RB1 nitrate reductase had an apparent molecular mass of 142 kDa and used NADH and reduced bromophenol blue or viologens as electron donors, independently of the presence of salt. The enzyme was associated with an NADH-diaphorase activity and was induced by nitrate and repressed by ammonium or glutamine, thus showing typical biochemical and regulatory properties of bacterial assimilatory NADH-nitrate reductases. The enzyme was active in vitro in the presence of 3 M NaCl or KCI, but the maximal activity was observed at 0.5 M salt. Addition of 2 M NaCl increased the optimal temperature of the enzyme from 12 to 32 degrees C, but the optimal pH (10.3) was unaffected.

TNT nitroreductase from a Pseudomonas aeruginosa strain isolated from TNT-contaminated soil

Microbial degradation and detoxification of TNT (2,4,6-trinitrotoluene) in soils ultimately depends on the presence of effective intracellular or extracellular enzymes. TNT was rapidly degraded by an enzyme obtained from a Pseudomonas aeruginosa strain isolated from TNT-contaminated soil. The nitroreductase enzyme was purified to apparent electrophoretic homogeneity by sequential chromatography on phenyl-sepharose, hydroxyapatite, and ion exchange resins. The TNT-nitroreductase catalyzed reduction of TNT to 4-hydroxylamino-4,6-dinitrotoluene (4HADNT) and 4-amino-2,6-dinitrotoluene (4ADNT). TNT reduction to 4ADNT in cell-free enzyme preparations corresponded to 2ADNT production by intact cells. The enzyme required NADH as a cosubstrate and the optimal pH for the reaction was approximately 6.5. The purified enzyme also exhibited NADH diaphorase activity and its denatured molecular weight was approximately 54kDa.

Metabolic activity and DNA integrity in human hepatic metastases after interstitial laser coagulation (ILC)

Background and Objective For investigations into interstitial laser coagulation (ILC) of solid tumors, tissue whitening is used as a parameter for the extent of coagulation. This obvious demarcation is associated with global thermal denaturation, but it is not clear whether this finding is a good indicator of the exact outer boundary of the lethal tissue effect. Study Design/Materials and Methods ILC with portal inflow occlusion was performed in human hepatic metastases of colorectal carcinoma directly after surgical resection (n = 5) or before surgical resection (n = 5) with laser parameters adapted to tumor diameter. Mitochondrial NADH-diaphorase activity and DNA integrity were assessed by histoenzymatic staining. Results In 7 of 10 tumors (mean diameter, 3.7 cm), an area of macroscopic coagulation (mean diameter, 4.2 cm) encircled the tumor in all three axes. Macroscopic coagulation corresponded to absent metabolism and disintegrated DNA. Furthermore, the macroscopic volumes of coagulation produced in tumor were comparable to the dimensions in normal porcine liver with the same laser parameters. Conclusion ILC with portal inflow occlusion results in areas with complete cell avitality in the zone of tissue whitening in human hepatic liver metastases. Lasers Surg. Med. 28:80–86, 2001. © 2001 Wiley-Liss, Inc.

POST VITRO MYCORRHIZATION AND BACTERIZATION OF MICROPROPAGATED STRAWBERRY, POTATO AND AZALEA

A greenhouse experiment showed that inoculations with certain combinations of arbuscular mycorrhizal fungi (AMF) and rhizosphere bacteria enhanced mini-tuber production of micropropagated potato after transplanting from in vitro to a non-sterile peat-based substrate. In the second experiment inoculation using the AMF isolate Glomus fistulosum BEG23 and a mix of three rhizosphere bacteria was found to increase shoot dry weight of micropropagated strawberry transplanted to one of the three non-sterile soil used. The majority of combinations of AMF / bacteria inoculants promoted root mycorrhizal colonisation, growth of extraradical hyphae and NADH-diaphorase activity of mycelium associated with strawberry roots in all three non-sterile soils. In a third experiment it was found that the inoculation with ericoid mycorrhizal fungi requires thorough selection since a mixture of two mycorrhizal fungal strains tested did not show stimulatory effects on growth of micropropagated azaleas after transplantation to Lignocel cultivation substrate. The separate inoculation of substrate with the biocontrol agents T. harzianum showed a stimulatory effect on azalea growth, however, when inoculated together with ericoid mycorrhizal fungi no significant effects were found compared to the non-inoculated control. To acquire the beneficial effect of microbial biotization of plants, a careful selection of the most effective combinations of fungal and bacterial inoculants should be carried out for the particular type of growth substrate.