Effect of stocking density on the behavior of newly transformed juvenile Macrobrachium rosenbergii

This study characterized the behavioral activity of Macrobrachium rosenbergii in the early stages of development, under different stocking densities (25 and 40 animals/m²), and during the light and dark phases of a 24-h cycle. Observations of individuals were made in 8 aquariums. Behavioral recording lasted 15 min/aquarium, 4 times/day and 4 days/week, 4 weeks in total. Food was offered twice daily. Observational methods included a combination of behavioral sampling and scan sampling. During the light phase, inactivity, cleaning and remaining in a shelter were the most frequent behaviors. During the dark phase the subjects displayed a higher frequency of feeding, exploration, swimming, and digging. At low density, the animals gained more weight and exhibited greater growth overall. These results indicate a behavioral pattern that is more favorable to animals in the lower density cultivation environment that can also create better living conditions for these shrimp, favor survival rates and therefore improve management success.     

Effect of metabolic or respiratory acidosis on rabbit renal medullary proton-ATPase

Distal urinary acidification is thought to be mediated by an H+-ATPase sensitive to N-ethylmaleimide and dicyclohexyl-carbodiimide. We have studied the effect of chronic metabolic acidosis (NH4Cl for 3 days) or respiratory acidosis (inhalation of 10% CO2 for 2 days) on the H+-ATPase of plasma membranes prepared from the medulla. The enzymatic assay for the H+-ATPase was performed in the presence of ouabain and oligomycin and in the absence of Ca. H+-transport activity was assessed by the quenching of acridine orange in the presence of ATP. The 15-25% sucrose gradient fraction was enriched 40-fold in enzymatic activity over the homogenate, and 8-fold in enzymatic activity and 4-fold in H+-transport activity over the fluffy fraction (38,000 X g). Metabolic acidosis (pH less than 7.31) or chronic hypercapnia (PCO2 greater than 66 mmHg; 1 mmHg = 133.3 Pa) was induced for 2-3 days. Both groups showed the same enrichment factor in enzymatic and H+-transport assays as the control rabbits. Enzymatic and H+-transport activities, however, were not different between animals with respiratory acidosis and controls. Kinetic studies failed to disclose an increase in Vmax (673 vs. 702 mumol/(mg protein.min] or a decrease in Km (0.43 vs. 0.48 mM) in chronic hypercapnia as compared with controls. Metabolic acidosis also failed to increase H+-ATPase activity. These data demonstrate that the H+-ATPase of renal medulla does not display the expected increase in activity during acidosis. The role of this H+-ATPase in the adaptation to acidosis remains to be determined.     

Dual effect of cyclic GMP on renal brush border Na-H antiporter.

The Na-H antiporter of renal-brush border membranes is inhibited by cyclic AMP and stimulated by protein kinase C. The proximal tubule contains guanylate cyclase and is capable of cyclic GMP production. The effect of cGMP on renal Na-H antiporter activity was analyzed in phosphorylated brush border membranes by 22Na uptake in the presence or absence of 1 mM amiloride. 8-Bromo cyclic GMP (1 microM) increased the amiloride-sensitive 22Na uptake in control from 1.26 +/- 0.13 to 1.54 +/- 0.12 nmol/mg/protein/10 sec, P less than 0.01, without altering the amiloride-insensitive component. In the absence of exogenous ATP, cGMP also stimulated the amiloride-sensitive 22Na uptake, which can be explained by the presence of endogenous ATP in concentrations of up to 50 microM in the membranes. In ATP-depleted membrane vesicles, however, cGMP inhibited the amiloride-sensitive 22Na uptake. These data indicate that cGMP acts on the Na-H antiporter by at least two different mechanisms, one of which is ATP dependent. It is likely that cGMP-dependent protein kinase mediates the stimulatory effects seen in the presence of ATP, and the inhibition seen in ATP-depleted membranes results from cGMP direct action on the Na-H antiporter.     

Plasma membrane proton-ATPase of a turtle bladder epithelial cell line

Urinary acidification by the turtle bladder is mediated by a proton ATPase located in the apical membrane. The present study describes a proton ATPase in the plasma membrane of a cell line of turtle bladder epithelial cells. In the presence of ouabain to inhibit Na+,K+-ATPase and in the absence of Ca2+ to inhibit Ca2+-ATPase, we measured ATPase activity of the plasma membranes of the cultured cells. This ATPase was resistant to oligomycin but sensitive to dicyclohexylcarbodiimide, N-ethylmaleimide, and vanadate. In the presence of ATP, the ATPase was capable of acidification as assessed by quenching of acridine orange. Acidification could not be elicited by other nucleotides (GTP, UTP). Acidification was inhibited by dicyclohexylcarbodiimide, N-ethylmaleimide, and vanadate but was not affected by replacement of Na+ by K+. The acidification response was dependent on the presence of chloride, abolished in the presence of gluconate, and inhibited partially by nitrate. Experiments utilizing the voltage-sensitive dye 3,3'-dipropylthiodicarbocyanine iodide showed that the proton ATPase was electrogenic and capable of responding to a favorable electric gradient. In summary, the turtle bladder epithelial cell line has a plasma membrane proton ATPase which is similar to the proton ATPase of turtle bladder epithelium and thus should allow purification and characterization of this enzyme.     

Effect of vanadate on water transport by the toad bladder

Vanadate increases renal Na and water excretion. The mechanism whereby vanadate impairs water transport was examined in the toad bladder. Vanadate did not alter baseline water transport but caused a significant inhibition of water transport elicited by high doses of AVP. The inhibition of AVP stimulated water flow by vanadate was dose dependent with inhibition present with concentration as low as 10(-7) and maximal inhibition occurring at 10(-5) M. Vanadate also inhibited water transport stimulated by cyclic AMP or by phosphodiesterase inhibition indicating that vanadate has an effect beyond cyclic AMP step, in addition to whatever effect it might have on adenylate cyclase. The inhibitory effect of vanadate on AVP stimulated water flow was not altered by prior Na-K-ATPase or prostaglandin inhibition. Since vanadate has been shown to stimulate adenylate cyclase in other tissues we examined whether addition of vanadate 10 minutes after addition of AVP would enhance water transport. Vanadate caused a transient enhancement of AVP stimulated water flow. These data demonstrate that vanadate can inhibit or stimulate water flow in the toad bladder.     

Renal Cortical Basolateral Na+/HCO− 3 Cotransporter: IV Characterization and Localization with Polyclonal Antibodies

We have previously partially purified the basolateral Na⁺/HCO⁻ ₃ cotransporter from rabbit renal cortex and this resulted in a 400-fold purification, and an SDS-PAGE analysis showed an enhancement of a protein band with a MW of approximately 56 kDa. We developed polyclonal antibodies against the Na⁺/HCO⁻ ₃ cotransporter by immunizing Dutch-belted rabbits with a partially purified protein fraction enriched in cotransporter activity. Western blot analysis of renal cortical basolateral membranes and of solubilized basolateral membrane proteins showed that the antibodies recognized a protein with a MW of approximately 56 kDa. The specificity of the purified antibodies against the Na⁺/HCO⁻ ₃ cotransporter was tested by immunoprecipitation. Solubilized basolateral membrane proteins enriched in Na⁺/HCO⁻ ₃ cotransporter activity were incubated with the purified antibody or with the preimmune IgG and then reconstituted in proteoliposomes. The purified antibody fraction caused a concentration-dependent inhibition of the Na⁺/HCO⁻ ₃ cotransporter activity, while the preimmune IgG failed to elicit any change. The inhibitory effect of the antibody was of the same magnitude whether it was added prior to (inside) or after (outside) reconstitution in proteoliposomes. In the presence of the substrates (NaHCO₃ or Na₂CO₃) for the cotransporter, the inhibitory effect of the antibody on cotransporter activity was significantly blunted as compared with the inhibition observed in the absence of substrates. Western blot analysis of rabbit kidneys showed that the antibodies recognized strongly a 56 kDa protein band in microsomes of the inner stripe of outer medulla and inner medulla, but not in the outer stripe of outer medulla. A 56 kDa protein band was recognized in microsomes of the stomach, liver, esophagus, and small intestine but was not detected in red blood cell membranes. Localization of the Na⁺/HCO⁻ ₃ cotransporter protein by immunogold technique revealed specific labeling of the cotransporter on the basolateral membranes of the proximal tubules, but not in the brush border membranes. These results demonstrate that the polyclonal antibodies against the 56 kDa basolateral protein inhibit the activity of the Na⁺/HCO⁻ ₃ cotransporter suggesting that the 56 kDa protein represents the cotransporter or a component thereof. These antibodies interact at or near the substrate binding sites. The Na⁺/HCO cotransporter protein is expressed in different regions of the kidneys and in other tissues. Received: 27 January 1996/Revised: 23 July 1996     

Experimental paracoccidioidomycosis of hamster inoculated in the cheek pouch

We compared the granuloma morphology and immune response of hamsters inoculated withParacoccidioides brasiliensis (Pb) into the cheek pouch, which lacks lymphatic drainage, and into the footpad, which is rich in lymphatics. Our objective was to better understand the modulation ofPb granuloma in an immunocompetent animal inoculated in an immunologically privileged site. The humoral immune response (ELISA) and cell mediated immunity (footpad test) became positive on days 7 and 14, respectively in animals inoculated into footpad and on days 35 and 60 in animals inoculated into the pouch. Typical epithelioid granulomas were observed at both sites on day 14. The number of fungi gradually decreased from the beginning of the experiment in footpad lesions, but only after day 35 in pouch granulomas, when cell mediated immunity was detectable. The results indicate that typical epithelioid paracoccidioidomycotic granulomas may develop in the absence of a detectable immune response; however, they are incapable of controlling fungal reproduction. Lack of lymphatic drainage delays the appearance of a detectable immune response, but with time fungi escape from the pouch, elicit an immune response and reach other organs. Our results further indicate the importance of the lymphatics in the pathogenesis of paracoccidioidomycosis.Abbreviations HCP hamster cheek pouch - Pb Paracoccidioides brasiliensis - Pbmycosis Paracoccidioidomycosis     

Structural characterization and promoter activity analysis of the γ-kafirin gene from sorghum

A genomic clone encoding the γ-kafirin gene from sorghum was isolated and sequenced. A 2938 bp sequenced fragment includes an intronless open reading frame of 636 nucleotides encoding a putative polypeptide of 212 amino acids. Comparison of the deduced amino acid sequence of γ-kafirin with the published sequences of γ-prolamins of maize, and Coix revealed highly conserved domains. The N-terminal region of these proteins contains the conserved hexapeptide PPPVHL, which is repeated eight times in γ-zein, four times in γ-kafirin and three times in γ-coixin. The number of PPPVHL repeats accounts predominantly for the differences in the molecular weights of γ-prolamins. Several putative regulatory sequences common to the γ-kafirin and γ-zein genes were identified in both the 5′ and the 3′ flanking regions. Putative GCN4-like regulatory sequences were found at positions ?192 and ?476 in the 5′ flanking region of γ-kafirin. In the 3′ noncoding region, three putative polyadenylation signals, two AATAAT and one AATGAA, were found at positions + 658, + 716, and + 785, respectively. In order to investigate the role of the putative GCN4-like motifs and other possible cis-acting element(s) of the γ-kafirin promoter, a series of deleted and chimeric promoter constructs were introduced into maize, Coix and sorghum tissues by particle bombardment. Histochemical analysis of β-glucuronidase (GUS) activity in different tissues indicated that the element(s) responsible for tissue specificity is probably located in the 285-bp proximal region of the promoter, while the remaining promoter sequence seems to carry the element(s) responsible for the quantitative response.     

Diversity and evolution of leaflet anatomical characters in the Pterocarpus clade (Fabaceae: Papilionoideae)

Journal of Plant Research - The Pterocarpus clade includes 23 genera previously attributed to different Fabaceae tribes. The recent rearrangements of many genera in the clade do not recognize...