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.     

Product of extracellular-superoxide dismutase catalysis

Extracellular-superoxide dismutase is a tetrameric enzyme containing four copper atoms. It has previously been shown to catalyse the decay of the superoxide radical, but the resulting product was not determined. In a xanthine oxidase-xanthine system in which about 30% of the electron flux resulted in superoxide radical formation, accumulation of hydrogen peroxide was determined. Catalysis of superoxide radical decay by extracellular-superoxide dismutase was found to result in hydrogen peroxide formation. The catalysed reaction is thus identical to those of previously investigated superoxide dismutases. Human manganese superoxide dismutase was also found to dismute the superoxide radical to hydrogen peroxide and water.     

Acyltransferase-catalyzed cleavage of arachidonic acid from phospholipids and transfer to lysophosphatides in lymphocytes and macrophages

The cleavage of fatty acyl moieties from phospholipids was compared in intact cells and homogenates of mouse lymphocytes (thymocytes, spleen cells) and macrophages. Liberation of free arachidonic acid during incubations of intact cells was only detectable in the presence of albumin. Homogenization of prelabeled thymocytes and further incubation of these homogenates at 37 degrees C resulted in a pronounced decrease of phospholipid degradation and cleavage of arachidonoyl residues, while further incubation of homogenates from prelabeled macrophages produced a greatly increased phospholipid degradation. Homogenates of macrophages but not those of thymocytes contain substantial activities of phospholipase A2 detectable using exogenous radiolabeled substrates. These findings indicate that in thymocytes cleavage of arachidonic acid from phosphatidylcholine is an active process that is not catalyzed by phospholipase A2. Addition of CoA and lysophosphatidylethanolamine to prelabeled thymocyte homogenates induced a fast breakdown of phosphatidylcholine and transfer of arachidonic acid to phosphatidylethanolamine, as in seen during incubations of intact thymocytes or macrophages. The transfer is restricted to arachidonic acid and does not require addition of ATP. Sodium cholate, a known inhibitor of the acyl-CoA:lysophosphatide acyltransferase, completely inhibited this transfer reaction. These results suggest that the CoA-mediated, ATP-independent breakdown of phosphatidylcholine and transfer of arachidonic acid is catalyzed by the acyl-CoA:lysophosphatide acyltransferase operating in reverse.     

Perfusion method for preparing pig brain cortex for Golgi-Cox impregnation

The perfusion procedure described in this paper produces high quality impregnation of pig visual and somatosensory cortical neurons with a Golgi-Cox solution. Starting within 30 min after death, pig heads were perfused with a fixative solution composed of a mixture (v/v) of liquid phenol, 5%; formalin, 14%; ethylene glycol, 25%; methanol, 28%; and water, 28% for two periods of 4 hr each. After perfusion, the heads were chilled for at least 18 hr. The entire brain was removed from the skull and then placed in 10% buffered formalin, where it remained for at least 10 days before taking the blocks that were to be immersed in the Golgi-Cox solution. Three weeks spent in the Golgi-Cox solution typically produced uniform neuron impregnation. The tissue blocks were then embedded in celloidin and sectioned at 120 micron. This procedure avoids the following difficulties: Golgi-Cox methods that produced excellent results with rodent or primate tissue were unsuccessful with pig tissue, placing fresh tissue in Golgi-Cox solution resulted in incomplete neuron impregnation, and immersion fixation in 10% buffered formalin without perfusion resulted in excessive staining of glia.     

Studies of the seminal receptacle of the crab Portunus sanguinolentus (Portunidae: Brachyura)

The seminal receptacle or spermatheca of Portunus sanguinolentus consists of two parts--an anterior glandular and a posterior chitinous part. The chitinous part continues as the oviduct, which opens on the sternite of the sixth thoracic segment. Significant morphological and histological differences were observed between the spermatheca, as well as the oviduct, of mated and unmated crabs. In mated crabs the spermatheca is much more bulging, owing to receipt of a copious supply of seminal products, and its cells are hyperactive. Further stages of ovarian development were observed as indicators of sequential changes in the spermatheca. The secretory cells gradually disintegrate by way of holocrine secretion; this results in cellular stratification and the formation of distinct furrows in the chitinous posterior part.