The expression of Visinin-like 1 during mouse embryonic development

Visinin like 1 (Vsnl1) encodes a calcium binding protein which is well conserved between species. It was originally found in the brain and its biological functions in central nervous system have been addressed in several studies. Low expression levels have also been found in some peripheral organs, but very little information is available regarding its physiological roles in non-neuronal tissues. Except for the kidney, the expression pattern of Vsnl1 mRNA and protein has not yet been addressed during embryogenesis. By in situ hybridization and immunolabeling we have extensively analyzed the expression pattern of Vsnl1 during murine development. Vsnl1 specifies the cardiac primordia and its expression becomes restricted to the atrial myocardium after heart looping. However, in the adult heart, Vsnl1 is expressed by all four cardiac chambers. It also serves as a specific marker for the cardiomyocyte-derived structures in the systemic and pulmonary circulation. Vsnl1 is dynamically expressed also by many other organs during development e.g. taste buds, cochlea, thyroid, tooth, salivary and adrenal gland. The stage specific expression pattern of Vsnl1 makes it a potentially useful marker particularly in studies of cardiac and vascular morphogenesis.     

Effect of GABA A receptor activation on UT-coupled signaling pathways in rat cortical astrocytes

Cultured rat cortical astrocytes express two types of urotensin II (UII) binding sites: a high affinity site corresponding to the UT (GPR14) receptor and a low affinity site that has not been fully characterized. Activation of the high affinity site in astroglial cells stimulates polyphosphoinositide (PIP) turnover and provokes an increase in intracellular calcium concentration. We have hypothesized that the existence of distinct affinity sites for UII in rat cortical astrocytes could be accounted for by a possible cross-talk between UT and the ligand-gated ion channel GABA(A) receptor (GABA A R). Exposure of cultured astrocytes to UII provoked a bell-shaped increase in cAMP production, with an EC50 stimulating value of 0.83+/-0.04 pM, that was totally blocked in the presence of the adenylyl cyclase inhibitor SQ 22,536. In contrast, UII was found to inhibit forskolin-induced cAMP formation. In the presence of the specific PKA inhibitor H89, UII provoked a sustained stimulation of cAMP formation. Inhibition of PKA by H89 strongly reduced the stimulatory effect of UII on PIP metabolism. GABA and the GABA A R agonist isoguvacine provoked a marked inhibition of UII-induced cAMP synthesis and a significant reduction of UII-evoked PIP turnover. These data suggest that functional interaction between UT and GABA(A)R negatively regulates coupling of UT to the classical PLC/IP(3) signaling cascade as well as to the adenylyl cyclase/PKA pathway.     

Mechanism of hydrolysis of dicholine esters with long polymethylene chain by human butyrylcholinesterase

Human butyrylcholinesterase hydrolyzes long chain dicholine esters more rapidly than short chain dicholine esters. The active site of butyrylcholinesterase is deeply buried within the enzyme molecule and there is limited space for binding of large compounds. Our goal was to understand how butyrylcholinesterase accommodates long chain dicholine esters to make them better substrates than short chain dicholine esters. For this purpose we studied the rate of hydrolysis of adipyldicholine (n=4) and sebacyldicholine (n=8) with mass spectrometry, a method that allowed monitoring the dicholine substrates, the monocholine intermediates, the dicarboxylic acid and choline products. It was shown that hydrolysis of adipyldicholine involves two consecutive steps, dicholine ester hydrolysis followed by relatively slow monocholine ester hydrolysis. However, sebacyldicholine was hydrolyzed at both choline ester sites, though hydrolysis of dicholine was faster than hydrolysis of monocholine. Sebacyldicholine was completely converted to sebacic acid and choline within 90 min, whereas only 15% of the adipyldicholine was converted to adipic acid in this time. Molecular modeling indicated that these dicholine esters can bind to butyrylcholinesterase in two energetically equivalent alternative conformations that may theoretically lead to hydrolysis. The long chain dicholine ester makes closer contact than the short chain ester between one of its carbonyl carbons and the catalytic Ser198, thus explaining why long-chain dicholine esters are hydrolyzed more rapidly by butyrylcholinesterase.     

Learning Differences between Feral Pigeons and Zenaida Doves: The Role of Neophobia and Human Proximity

Learning differences predicted from ecological variables can be confounded with differences in wariness of novel stimuli (neophobia). Previous work on feral pigeons ( Columba livia ), as well as on group-feeding and territorial zenaida doves ( Zenaida aurita ), reported individual and social learning differences predicted from social foraging mode. In the present study, we show that speed of learning a foraging task covaries with neophobia and latency to feed from a familiar dish in the three types of columbids. Pigeons were much faster than either territorial or group-feeding zenaida doves on all tests conducted in captivity, but showed unexpectedly strong neophobia in some urban flocks during field tests. Human proximity strongly affected performance in group-feeding doves both in the field and in captivity. They were slightly faster at learning than their territorial conspecifics in cage tests. In multiple regressions, species identity, but not social foraging mode, significantly predicted individual variation in learning, as did individual variation in neophobia. Wariness of novel stimuli and species differences associated with artificial selection appear to be more important than foraging mode and wariness of humans in accounting for learning differences between these columbids.     

Cloning and characterization of the rat homologues of the Inhibitor of Apoptosis protein 1, 2, and 3 genes.

Background  

Inhibitor of Apoptosis (IAP) proteins are key intrinsic regulators of apoptosis induced by a variety of triggers. We isolated the rat Inhibitor of Apoptosis genes 1, 2 and 3 and characterized their tissue distribution and expression.     

Correlation of androgen-responsiveness of Shionogi mouse mammary carcinoma cell lines with binding of dihydrotestosterone to nuclear envelopes

Purified nuclear envelopes have been isolated from an androgen-responsive and two androgen-unresponsive cell lines of the Shionogi mouse mammary carcinoma. The binding of dihydrotestosterone to nuclear envelope fractions isolated from the three variant cell lines is correlated with the androgen-responsiveness of the cell line. Nuclear envelopes prepared from the two androgen-unresponsive cell lines did not bind dihydrotestosterone specifically following incubation with radioactive dihydrotestosterone from 2.5 to 45.0 nM at 20 degrees C for 18 h. Under the same binding conditions, nuclear envelopes prepared from the androgen-responsive cell line demonstrated saturable, specific binding of dihydrotestosterone. Scatchard analysis revealed a class of binding sites with an apparent Kd of 14.2 nM and a maximum binding capacity of 28.7 fmol/mg protein. Proteinase and heat treatments resulted in the complete loss of androgen-binding activity, whereas DNAase treatment resulted in the loss of 38% of the binding activity. The binding sites were specific for dihydrotestosterone. Testosterone was only a weak competitor and estradiol did not compete. Extraction with concentrations of KCl up to 1.0 M did not result in loss of androgen binding.     

Interaction of steroids with the nuclear envelope

Three approaches have been taken to determine the molecular mechanism by which steroid hormones traverse the nuclear envelope on their way to the genome. The first approach involved characterization of steroid binding to nuclear envelope preparations. We have characterized androgen binding to nuclear envelopes isolated from the rat ventral prostate, the rat liver, and androgen-responsive and androgen-unresponsive cell lines of the Shionogi mouse mammary carcinoma and glucocorticoid binding to rat liver. Relatively high affinity binding sites for steroids have been identified on nuclear envelopes. Importantly, the number and specificity of the sites correlates with the responsiveness of the tissue to the steroid. In the second approach, we have undertaken to identify the steroid binding site directly. As the characteristics of the rat ventral prostate site resembled those of the nuclear androgen receptor, we have begun purifying that receptor and have found fast protein liquid chromatography to be very effective. By affinity labelling studies, the dexamethasone binding site on the rat liver nuclear envelope has been identified as a peptide of molecular weight of approximately 90,000. The third approach we have used is to identify androgen-dependent peptides in nuclear envelope preparations. In both the rat ventral prostate and an androgen-responsive cell line of the Shionogi mouse mammary carcinoma, we have identified abundant androgen-dependent peptides. The relationship of these peptides to the binding sites identified by the first two approaches and their role in steroid transport is being investigated.