Phenol oxidases from Rhodnius prolixus: Temporal and tissue expression pattern and regulation by ecdysone

Rhodnius prolixus 5th instar nymphs have significant PO enzymatic activity in the anterior midgut, fat body and hemolymph. The tissue with the major amount of PO activity is the anterior midgut while those with higher specific activities are the fat body and hemolymph. In this work the temporal pattern of PO enzymatic activity in different tissues was investigated. In fat body, PO peaks occur at 7, 12 and 16 days after a blood meal. In hemolymph, PO diminishes until day 7, and then recovers by day 14. In the anterior midgut tissue, PO peaks on day 9 and just before ecdysis; a similar pattern was observed in the anterior midgut contents. Some of these activities are dependent on the release of ecdysone, as feeding blood meal containing azadirachtin suppresses them and ecdysone treatment counteracts this effect. These results suggest that during the development of the 5th instar, the insect has natural regulating cycles of basal PO expression and activation, which could be related to the occurrence of natural infections. The differences in temporal patterns of activity and the effects of azadirachtin and ecdysone in each organ suggest that, at least in R. prolixus, different tissues are expressing different PO genes.     

Water and ions in a high resolution structure of B-DNA.

A detailed picture of hydration and counterion location in the B-DNA duplex d(GCGAATTCG) is presented. Detailed data have been obtained by single crystal x-ray diffraction at atomic resolution (0.89 A) in the presence of Mg(2+). The latter is the highest resolution ever obtained for a B-DNA oligonucleotide. Minor groove hydration is compared with that found in the Na(+) and Ca(2+) crystal forms of the related dodecamer d(CGCGAATTCGCG). High resolution data (1.45 A) of the Ca(2+) form obtained in our laboratory are used for that purpose. The central GAATTC has a very stable hydration spine identical in all cases, independent of duplex length and crystallization conditions (counterions, space group). However, the organization of the water molecules (tertiary and quaternary layers) associated with the central spine vary in each case.     

Protein binding and stability of norepinephrine in human blood plasma. Involvement of prealbumin, alpha 1-acid glycoprotein and albumin

The binding of norepinephrine (NE) to plasma proteins of fresh human blood obtained from healthy volunteers was studied by ultrafiltration at different NE concentrations and incubation times at 37 degrees C. At 1.7 nM L-[3H]-NE binding was approximately 25%. The binding was rapid and was not influenced by the incubation time. [3H]-NE could be dissociated from its binding sites by acid precipitation and, after HPLC, showed to be unchanged NE. No difference in NE binding was found between plasma collected in EGTA-GSH or heparin solution. There was no degradation of NE when incubated in plasma at 37 degrees C for 10 h, even without the addition of antioxidants. Therefore, in the present study, binding represented interaction of unchanged NE with plasma proteins. The whole plasma binding was saturable over the range of 0.66 nM to 0.59 mM of NE. Scatchard plot of specific binding revealed high-affinity sites with a Kd of 5.4 nM and a Bmax of 3.9 fmoles.mg-1 protein, and low-affinity sites with a Kd of 2.7 microM and a Bmax of 3.3 pmoles.mg-1 protein. Electrophoretic characterization of NE-binding proteins showed that about 60% of bound NE was associated to albumin, and 20% to prealbumin. NE binding to pure human plasma proteins was also studied using ultrafiltration. Scatchard analyses revealed a single class of very high-affinity binding sites for prealbumin (Kd 4.9 nM), a single class of binding sites for alpha 1-acid glycoprotein (Kd 54 microM) and two classes of binding sites for albumin with high (Kd 1.7 microM) and low (Kd 0.8 mM) affinities respectively. The main results obtained in this study - a) reversibility of NE binding, b) stability of free and bound NE in plasma, c) involvement of the prealbumin as a specific binding protein - point out to a specific transport for NE in human blood plasma.     

Effect of mixing on biogas production during mesophilic anaerobic digestion of screened dairy manure in a pilot plant

The effect of mixing on biogas production of a 1.5‐m³ pilot continuous stirred tank reactor (CSTR) processing screened dairy manure was evaluated. Mixing was carried out by recirculation of reactor content with a mono pump. The experiment was conducted at a controlled temperature of 37±1°C and hydraulic retention times (HRTs) of 20 and 10 days. The effect of continuous and intermittent operation of the recirculation pump on biogas production was studied. At 10 days of HRT, the results showed a minimal influence of recirculation rate on biogas production and that continuous recirculation did not improve reactor performance. At 20 days of HRT, the recirculation rate did not affect reactor performance. Combination of low solid content in feed animal slurry and long HRTs results in minimal mixing requirements for anaerobic digestion.     

A phylogenetic analysis of species in the Bufo crucifer group (Anura: Bufonidae), based on indolealkylamines and proteins from skin secretions

This research tested the utility of two classes of skin secretion compounds to the phylogeny of the Bufo crucifer group. Skin secretions from specimens of nine populations of B. crucifer group were obtained and submitted to qualitative analysis. We observed a clear difference in the composition of the skin secretion molecules obtained from the species of Bufo studied. Fifty-nine molecules, 16 indolealkylamines and 43 proteins, were used as characters, and 39 of these were parsimonious informative. The tree topology of the skin secretion combined data showed areas of congruence and conflict when compared to an mtDNA phylogeny of the B. crucifer group. We used the Templeton test to evaluate the heterogeneity between the skin secretion and mtDNA data. Although not recommended, we performed a combined analysis with the two partitions. The skin secretion characters from the species of Bufo studied have phylogenetic signal. These data are indicative, at least as a preliminary study, of the phylogenetic relationships among the B. crucifer group taxa.     

Renal cortical intermediary metabolism in the recovery phase of postischemic acute renal failure in the dog.

Renal metabolism has been studied in eight dogs before and 48 hr after a 60-min period of renal ischemia induced by clamping the left renal artery with the simultaneous removal of the right kidney, and in 12 sham-operated animals. The study involved the measurement of renal uptake and production of lactate, glutamine, glutamate, alanine, ammonium, and oxygen, and the measurement of the tissue concentrations of ATP, glutamine, lactate, alpha-ketoglutarate, aspartate, and alanine in the renal cortex. Two days after a temporary renal ischemia, the remaining kidney showed a 22% decrease in glomerular filtration rate (GFR) and a 25% decrease in renal plasma flow. Fractional sodium and potassium excretions were similar to those of control dogs. Renal production or extraction of glutamine, glutamate, alanine, ammonium, and oxygen (all expressed by 100 ml of GFR) was not significantly different in basal conditions or 2 days after ischemia, but lactate extraction was reduced in postischemic kidneys (-101 +/- 29 vs -204 +/- 38 mumol/100 ml GFR in control dogs). The cortical concentrations of glutamine and glutamate were lower in postischemic than in control kidneys. No differences were found in cortical concentration of alpha-ketoglutarate, aspartate, lactate, pyruvate, or ATP, but total nucleotides and inorganic phosphate were decreased in postischemic kidneys. It is concluded that in the recovery phase of the ischemia, a decreased lactate uptake is the main metabolic change, and total ATP production is adapted to the decrease of GFR and sodium reabsorption.     

Electron microscopic determination of phenoloxidase (EC 1.14.18.1) in eosinophilic granulocytes of the small intestine of white rats

It is shown that the light microscopic permanent detectable phenoloxidase activity, using dihydroxyphenylalanine as substrate, of the small intestine of white rats is localized in the most cases in eosinophilic granulocytes. The enzyme has been found as well in the cytoplasma as in the granules. The enzyme proof triggered in the granules the so called reverse effect. The results allow to conclude new aspects for the cell mediated immunological defense reaction.     

Cortinarius sarcoflammeus sp. nov, a new species of subgenus Dermocybe (Agaricales) growing in Sphagnum bogs

 Cortinarius sarcoflammeus is proposed as a new species belonging to subgenus Dermocybe, on the basis of its morphological, chemical and ecological characters. The strong red-orange colour of the context and stipe base, large spores, sphagnicolous habitat and high dermorubin content are characteristic for the new species. Holotypes of C. huronensis and C. huronensis var. olivaceus have been examined for comparison, and their differences discussed. Photographs and line drawings of C. sarcoflammeus are added. Received February 13, 2001 Accepted March 23, 2001     

Genetic analysis of hypertropic mutants of Phycomyces

A new class of Phycomyces behavioral mutants with enhanced tropic responses has been analyzed genetically to determine the number of genes involved and the nature of their expression. These hypertropic mutants carry pleiotropic nuclear mutations. Besides their effects on sensory behavior, they also affect morphology and meiotic processes. Behavioral analyses of heterokaryons containing hypertropic and wild-type nuclei in varying proportions show that the hypertropic mutations in strains L82, L84, L86, and L88 are strongly dominant. Conversely, the hypertropic mutations carried by the strains L83, L85, and L87 are strongly recessive. We performed recombination analyses between hypertropic mutants and mutants with diminished phototropism, affected in the seven genes madA to madG. We found no evidence of linkage between the hypertropic mutations and any of these mad mutations. From crosses, we isolated double mutants carrying hypertropic mutations together with madC (night blind) and madG (stiff) mutations. The behavioral phenotypes of the double mutants are intermediate between those of the parentals. Complementation analyses show that the three recessive hypertropic mutations affect the same gene, which we call madH. The expression of the recessive hypertropic allele becomes dominant in heterokaryons carrying madC and madH nuclei; the madC gene has been implicated separately with the photoreceptor at the input to the sensory pathway, while the madH gene is associated with the growth control output. This result suggests the physical interaction of both gene products, madH and madC, in a molecular complex for the photosensory transduction chain.