Lecithotrophic development and metamorphosis in the Indo-West Pacific brittle star Ophiomastix venosa (Echinodermata: Ophiuroidea)

Summary

The larval development of the ophiocomid ophiuroid Ophiomastix venosais described using SEM. The gastrula transforms into a uniformly ciliated early larva which progressively changes into a lecithotrophic late premetamorphic larva with a continuous bilateral ciliated band. This stage is short-lived and equivalent to a highly reduced ophiopluteus. Comparisons between O. venosa and other ophiuroid species whose development has been investigated suggest that, whatever the developmental mode (lecithotrophic or planktotrophic), a pluteus stage always occurs in ophiuroids with planktonic development. Two metamorphic stages were identified, the late metamorphic larva differing from the early one by the closure of the larval mouth. The appearance of the permanent mouth marks the end of the metamorphosis. The postlarva still possesses remnants of larval features. The transformation of the reduced ophiopluteus into a barrel-shaped metamorphic larva with transverse ciliated bands, a vitellaria larva, is followed. The possible occurrence of a unique type of metamorphic larva in non-brooding ophiuroids is discussed. Verification of this, however, needs further SEM investigations on metamorphic larva from species having “regular” planktotrophic development.     

A cleavage enzyme-cytometric bead array provides biochemical profiling of resistance mutations in HIV-1 Gag and protease

Most protease-substrate assays rely on short, synthetic peptide substrates consisting of native or modified cleavage sequences. These assays are inadequate for interrogating the contribution of native substrate structure distal to a cleavage site that influences enzymatic cleavage or for inhibitor screening of native substrates. Recent evidence from HIV-1 isolates obtained from individuals resistant to protease inhibitors has demonstrated that mutations distal to or surrounding the protease cleavage sites in the Gag substrate contribute to inhibitor resistance. We have developed a protease-substrate cleavage assay, termed the cleavage enzyme- cytometric bead array (CE-CBA), which relies on native domains of the Gag substrate containing embedded cleavage sites. The Gag substrate is expressed as a fluorescent reporter fusion protein, and substrate cleavage can be followed through the loss of fluorescence utilizing flow cytometry. The CE-CBA allows precise determination of alterations in protease catalytic efficiency (k(cat)/K(M)) imparted by protease inhibitor resistance mutations in protease and/or gag in cleavage or noncleavage site locations in the Gag substrate. We show that the CE-CBA platform can identify HIV-1 protease present in cellular extractions and facilitates the identification of small molecule inhibitors of protease or its substrate Gag. Moreover, the CE-CBA can be readily adapted to any enzyme-substrate pair and can be utilized to rapidly provide assessment of catalytic efficiency as well as systematically screen for inhibitors of enzymatic processing of substrate.     

The role of tyrosine 207 in the reaction catalyzed by Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

The functional significance of tyrosine 207 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase was explored by examining the kinetic properties of the Tyr207Leu mutant. The variant enzyme retained the structural characteristics of the wild-type protein as indicated by circular dichroism, intrinsic fluorescence spectroscopy, and gel-exclusion chromatography. Kinetic analyses of the mutated variant showed a 15-fold increase in K(m)CO?, a 32-fold decrease in V(max), and a 6-fold decrease in K(m) for phosphoenolpyruvate. These results suggest that the hydroxyl group of Tyr 207 may polarize CO? and oxaloacetate, thus facilitating the carboxylation/decarboxylation steps.     

Synthesis and activity of quinolinyl-methylene-thiazolinones as potent and selective cyclin-dependent kinase 1 inhibitors

A novel series of quinolinyl-methylene-thiazolinones has been identified as potent and selective cyclin-dependent kinase 1 (CDK1) inhibitors. Their synthesis and structure activity relationships (SAR) are described. Representative compounds from this class reversibly inhibit CDK1 activity in vitro, and block cell cycle progression in human tumor cell lines, suggesting a potential use as antitumor agents.     

Acceptor specificity of the human leukocyte alpha3 fucosyltransferase: role of FucT-VII in the generation of selectin ligands

The alpha3 fucosyltransferase, FucT-VII, is one of the key glycosyltransferases involved in the biosynthesis of the sialyl Lewis X (sLex) antigen on human leukocytes. The sialyl Lewis X antigen (NeuAcalpha(2-3)Galbeta(1-4)[Fucalpha(1-3)]GlcNAc-R) is an essential component of the recruitment of leukocytes to sites of inflammation, mediating the primary interaction between circulating leukocytes and activated endothelium. In order to characterize the enzymatic properties of the leukocyte alpha3 fucosyltransferase FucT-VII, the enzyme has been expressed in Trichoplusia ni insect cells. The enzyme is capable of synthesizing both sLexand sialyl-dimeric-Lexstructures in vitro , from 3'-sialyl-lacNAc and VIM-2 structures, respectively, with only low levels of fucose transfer observed to neutral or 3'-sulfated acceptors. Studies using fucosylated NeuAcalpha(2-3)-(Galbeta(1- 4)GlcNAc)3-Me acceptors demonstrate that FucT-VII is able to synthesize both di-fucosylated and tri-fucosylated structures from mono- fucosylated precursors, but preferentially fucosylates the distal GlcNAc within a polylactosamine chain. Furthermore, the rate of fucosylation of the internal GlcNAc residues is reduced once fucose has been added to the distal GlcNAc. These results indicate that FucT-VII is capable of generating complex selectin ligands, in vitro , however the order of fucose addition to the lactosamine chain affects the rate of selectin ligand synthesis.      

Dopamine control of aldosterone secretion in end-stage renal failure

The role of the tonic inhibitory effect of dopamine on aldosterone secretion has been investigated in 10 patients with chronic renal failure (CRF) on hemodialysis, in 8 normotensive renal transplant recipients (Tx) with normal renal function and in 8 normotensive volunteers (NV). The following tests were performed: the response of plasma aldosterone (PA) to metoclopramide administration; the response of plasma prolactin (PRL) to TRH administration, and the changes induced by Lisuride (a dopaminergic agonist, on the values of PA and PRL). The basal values of PA and PRL were higher in CRF than in NV and Tx. The inverse was true for plasma renin activity (PRA) values. The response of PA and PRL to metoclopramide showed blunted increases in CRF when compared to NV, in the absence of changes of PRA, cortisol and potassium. After TRH administration, PRL increase in CRF was also inferior. Lisuride induced a decrease of both PA and PRL both in CRF and NV. In Tx, basal values of PA and PRL were similar to NV. Nevertheless, the response to metoclopramide and TRH were partially blunted when compared to that of NV. These results point to the existence of a deranged dopaminergic regulation of aldosterone secretion in end-stage renal failure patients. The alterations are partially corrected by a well-functioning kidney graft.     

Histological development of the digestive system of Mayan cichlid Cichlasoma urophthalmus (Günther 1862)

The ontogeny of the digestive tract in Cichlasoma urophthalmus was studied by means of optical microscopy from hatching to 30 days post‐hatching (dph; 855 degree days, dd). The development of the digestive system in this precocial species was a very intense and asynchronous process, which proceeded from both distal ends interiorly. At hatching, the digestive tract consisted of a straight tube with a smooth lumen dorsally attached to the yolk‐sac. The digestive accessory glands were already differentiated and eosinophilic zymogen granules were visible in the exocrine pancreas. At the onset of exogenous feeding between 5 and 6 dph (142.5–171.0 cumulative thermal units, CTU), the buccopharynx, oesophagus, intestine, liver and pancreas were almost completely differentiated, with the exception of the gastric stomach that completed its differentiation between 11 and 14 dph (313.5–399.0 CTU). The development of gastric glands at 14 dph and the differentiation of the stomach in the fundic, cardiac and pyloric regions at 19 dph (541.5 CTU) were the last major events in digestive tract development and designated the onset of the juvenile period. Remnants of yolk were still detected until 16 dph (456.0 CTU), indicating a long period of mixed nutrition that lasted between 10 and 11 days (285.0–313.5 CTU). The results of the organogenesis of larvae complement previous data on the functionality of the digestive system and represent a useful tool for establishing the functional systemic capabilities and physiological requirements of larvae to ensure optimal welfare and growth under aquaculture conditions, which might be useful for improving current larval rearing practices for this cichlid species.