Titrating the effects of mitochondrial complex I impairment in the cell physiology.

The mitochondrial oxidative phosphorylation system consists of five multimeric enzymes (complexes I-V). NADH dehydrogenase or complex I (CI) is affected in most of the mitochondrial diseases and in some neurodegenerative disorders. We have studied the physiological consequences of a partial CI inhibition at the cellular level. We used a genetic model (40% CI-inhibited human-ape xenomitochondrial cybrids) and a drug-induced model (0-100% CI-inhibited cells using different concentrations of rotenone). We observed a quantitative correlation between the level of CI impairment and cell respiration, cell growth, free radical production, lipid peroxidation, mitochondrial membrane potential, and apoptosis. We showed that cell death was quantitatively associated with free radical production rather than with a decrease in respiratory chain function. The results obtained with human xenomitochondrial cybrid cells were compatible with those observed in rotenone-induced 40% CI-inhibited cells. At high concentrations (5-6-fold higher than the concentration necessary for 100% CI inhibition), rotenone showed a second toxic effect at the level of microtubule assembly, which also led to apoptosis. The correlation found among all the parameters studied helped clarify the physiological consequences of partial CI inhibitions at the cellular level.     

Phylogeography of the cactophilic species Drosophila gouveai: demographic events and divergence timing in dry vegetation enclaves in eastern Brazil

Aim  The aim of this study was to assess the causal mechanisms underlying populational subdivision in Drosophila gouveai , a cactophilic species associated with xeric vegetation enclaves in eastern Brazil. A secondary aim was to investigate the genetic effects of Pleistocene climatic fluctuations on these environments.
Location  Dry vegetation enclaves within the limits of the Cerrado domain in eastern Brazil.
Methods  We determined the mitochondrial DNA haplotypes of 55 individuals (representing 12 populations) based on sequence data of a 483-bp fragment from the cytochrome c oxidase subunit II (COII) gene. Phylogenetic and coalescent analyses were used to test for the occurrence of demographic events and to infer the time of divergence amongst genetically independent groups.
Results  Our analyses revealed the existence of two divergent subclades (G1 and G2) plus an introgressed clade restricted to the southernmost range of D. gouveai . Subclades G1 and G2 displayed genetic footprints of range expansion and segregated geographical distributions in south-eastern and some central highland regions, east and west of the Paraná River valley. Molecular dating indicated that the main demographic and diversification events occurred in the late to middle Pleistocene.
Main conclusions  The phylogeographical and genetic patterns observed for D. gouveai in this study are consistent with changes in the distribution of dry vegetation in eastern Brazil. All of the estimates obtained by molecular dating indicate that range expansion and isolation pre-dated the Last Glacial Maximum, occurring during the late to middle Pleistocene, and were probably triggered by climatic changes during the Pleistocene. The current patchy geographical distribution and population subdivision in D. gouveai is apparently closely linked to these past events.     

Haematological and biochemical reference intervals for farmed channel catfish

The reference intervals for biochemical variables and red blood cell indices of healthy intensively bred channel catfish Ictalurus punctatus were determined. The blood variables were determined using standardized clinical methods. The reference intervals (25th and 75th percentiles) were established using a non-parametric method. Reference intervals for plasma glucose, serum total protein, sodium, potassium, calcium, magnesium, chloride concentration, primary and secondary red blood cell indices were established. The haematological and biochemical reference intervals established may allow important clinical decisions about channel catfish.     

Effect of temperature and sperm supply on the reproductive potential ofTetranychus evansi (Acari: Tetranychidae)

The biology ofTetranychus evansi Baker and Pritchard was studied at five different temperatures on excised leaves ofSolanum douglasii Dunal. The theoretical lower threshold temperature of development was ca. 13°C. The combined immature stages required ca. 148 day-degrees for completion. Progressively later initiation of mating resulted in higher peak proportions of females produced, up to an 8-day period between emergence and mating at 30°C. The proportion of male offspring always increased towards the end of the oviposition period, apparently because of sperm depletion. The calculated intrinsic rate of increase was highest (0.432) at 35°C, the highest temperature tested. At that same temperature, daily oviposition rate and net reproductive rate were maximum (9.5 and 111.77, respectively), and mean generation time and doubling time were minimum (10.92 and 1.6 days, respectively).     

Induction of an immune response through the idiotypic network with monoclonal anti-idiotype antibodies in the carcinoembryonic antigen system.

Anti-idiotype antibodies can mimic the conformational epitopes of the original antigen and act as antigen substitutes for vaccination and/or serological purposes. To investigate this possibility concerning the tumor marker carcinoembryonic antigen (CEA), BALB/c mice were immunized with the previously described anti-CEA monoclonal antibody (MAb) 5.D11 (AB1). After cell fusion, 15 stable cloned cell lines secreting anti-Ids (AB2) were obtained. Selected MAbs gave various degrees of inhibition (up to 100%) of the binding of 125I-labeled CEA to MAb 5.D11. Absence of reactivity of anti-Id MAbs with normal mouse IgG was first demonstrated by the fact that anti-Id MAbs were not absorbed by passage through a mouse IgG column, and second because they bound specifically to non-reduced MAb 5.D11 on Western blots. Anti-5.D11 MAbs did not inhibit binding to CEA of MAb 10.B9, another anti-CEA antibody obtained in the same fusion as 5.D11, or that of several anti-CEA MAbs reported in an international workshop, with the exception of two other anti-CEA MAbs, both directed against the GOLD IV epitope. When applied to an Id-anti-Id competitive radioimmunoassay, a sensitivity of 2 ng/ml of CEA was obtained, which is sufficient for monitoring circulating CEA in carcinoma patients. To verify that the anti-Id MAbs have the potential to be used as CEA vaccines, syngeneic BALB/c mice were immunized with these MAbs (AB2). Sera from immunized mice were demonstrated to contain AB3 antibodies recognizing the original antigen, CEA, both in enzyme immunoassay and by immunoperoxidase staining of human colon carcinoma. These results open the perspective of vaccination against colorectal carcinoma through the use of anti-idiotype antibodies as antigen substitutes.     

Isolation and characterization of a heparin with high anticoagulant activity from Anomalocardia brasiliana

The isolation, some structural features, physicochemical properties and pharmacological activities of a heparin from Anomalocardia brasiliana are reported. It is shown that the mollusc heparin is very similar to those present in mammalian tissues with regard to chemical composition, physicochemical properties, pharmacological activities and susceptibility to heparinase and heparitinase II from Flavobacterium heparinum, as well as to the types of products formed by the action of these enzymes. Three significant quantitative differences were observed for the mollusc heparin when compared with the ones from mammalian origin, namely, a higher degree of binding with antithrombin III (45%), higher molecular weight (27-43 kDa) and higher anticoagulant activity (320 I.U./mg). The possible biological role of heparin is discussed in view of the present findings.     

Glutamate synthase in greening callus of Bouvardia ternifolia Schlecht

The distribution of the two glutamate-synthase (GOGAT) activities known to exist in higher plants (NADH dependent, EC 2.6.1.53; and ferredoxin dependent, EC 1.4.7.1) was studied in non-chlorophyllous and chlorophyllous cultured tissue as well as in young leaves of Bouvardia ternifolia. The NADH-GOGAT was present in all three tissues. Using a sucrose gradient we found it in both the soluble and the plastid fraction of non-chlorophyllous and chlorophyllous tissue, but exclusively in the chloroplast fraction of the leaves. Ferredoxin-GOGAT was found only in green tissues and was confined to the chloroplasts. Ferredoxin-GOGAT activity increased in parallel with the chlorophyll content of the callus during the greening process in Murashige-Skoog medium (nitrate and ammonium as the nitrogen sources), while NADH-GOGAT was not affected by the greening process in this medium. Furthermore, both activities were differentially affected by either nitrate or ammonium as the sole nitrogen source in the medium during this process. It is suggested that each GOGAT activity is a different entity or is differently regulated.Abbreviations GOGAT glutamate synthase - MS Murashige-Skoog (1962) medium - PMSF phenylmethylsulfonyl fluoride     

Lack of sensitivity to ouabain in natural killer activity

Natural killer (NK) activity against K562 target cells is not an ouabain-sensitive process. Inhibition of 40% of cytotoxicity was achieved only with an ouabain concentration much higher than that required to inhibit cell activation in other systems such as leukocyte chemotaxis and B lymphocyte plaque formation. Pretreatment of effector cells with biological agents such as phorbol-ester 12-O-tetradecanoylphorbol-13-acetate or interferon increased the cytotoxicity. This activation was not counteracted by ouabain. The effect of ouabain on NK activity was compared with a well-known ouabain-sensitive process, for example, phytohemagglutinin-induced peripheral blood lymphocyte proliferation. Ouabain completely blocked [3H]thymidine incorporation, independent of the stage of the culture when the drug was added, with exception of the last 6 h. This inhibition could be partially reversed by addition of KCl. Ouabain was equally effective when whole blood cultures were used. These results suggest that NK activity is ouabain resistant, unlike other systems of cell activation that lead or do not lead to proliferation.     

Isolation of the replication DNA region from a Rhizobium plasmid and examination of its potential as a replicon for Rhizobiaceae cloning vectors

The DNA region essential for replication and stability of a native plasmid (pTM5) from Rhizobium sp. (Hedysarum) has been identified and isolated within a 5.4-kb PstI restriction fragment. The isolation of this region was accomplished by cloning endonuclease-restricted pTM5 DNA into a ColE1-type replicon and selecting the recombinant plasmids containing the pTM5 replicator (pTM5 derivative plasmids) by their ability to replicate in Rhizobium. DNA homology studies revealed that pTM5-like replicons are present in cryptic plasmids from some Rhizobium sp. (Hedysarum) strains but not in plasmids from strains of other Rhizobium species or Agrobacterium tumefaciens. The pTM5 derivative plasmids were able to replicate in Escherichia coli and A. tumefaciens and in a wide range of Rhizobium species. On the basis of stability assays in the absence of antibiotic selective pressure, the pTM5 derivative plasmids were shown to be highly stable in both free-living and symbiotic cells of Rhizobium sp. (Hedysarum). The stability of these plasmids in other species of Rhizobium and in A. tumefaciens varied depending on the host and on the plasmid. Most pTM5 derivative plasmids tested showed significantly higher symbiotic stability than RK2 derivative plasmids pRK290 and pAL618 in Rhizobium sp. (Hedysarum), R. meliloti, and R. leguminosarum bv. phaseoli. Consequently, we consider that the constructed pTM5 derivative plasmids are potentially useful as cloning vectors for Rhizobiaceae.