Biophysical and molecular properties of annexin-formed channels.

The annexins are water soluble proteins possessing a hydrophilic surface, which belong to a family of proteins which (a) bind ('annex') both calcium and phospholipids, and (b) form voltage-dependent calcium channels within planar lipid bilayers. Annexins types are diverse (94 annexins in 45 species) and they belong to an enormous multigene family that ranges throughout all eukaryotic kingdoms. Although the structure of these proteins is now well known their functional and physiological roles remain largely unknown and circumstantial. Various experimental approaches provided evidence that annexins function as Ca(2+) channels that could act as regulators of membrane fusion. The identity of annexins is derived from the conserved 34 kDa C-terminal domain which comprises four repeats - except for annexin VI, with eight repeats - of a sequence of approximately seventy amino acids, which holds the area known as the 'endonexin fold', with its identifying GXGTDE. Annexins have been placed into three subgroups of (1) tetrad core and short amino terminal, (2) tetrad core and long amino terminal, and (3) octad core and short amino terminal. The repeats are highly conserved, each forming a compact alpha-helical domain comprising five alpha-helices wound in a right-handed superhelix. Four domains are formed, arranged in a nearly flat and cyclical array, with domains I and IV, and II and III respectively forming two tightly organised modules with almost twofold symmetry. A hydrophilic pore lies at the centre of the molecule, forming a prominent ion channel coated with charged and highly conserved residues. The annexin molecule is slightly curved, with both a convex and a concave face. The cation/anion permeability ratios and the selectivity sequence of the ion channels formed by several annexins confirm the selectivity of the annexins for Ca(2+) over other divalent cations, and reveals the importance of structural sites, e.g. amino acid positions 17, 78, 95 and 112 for the identification of the ion channel's position, function and regulation. Some are sensitive to low doses of the phenothiazine drugs, trifluoperazine (an anti-schizophrenia drug) and promethazine (anti nausea drug) La(3+) and Cd(2+), (blockers of voltage-gated Ca(2+) channels) nifedipine (an inhibitor of non-activating Ca(2+) channels). There are two main competing models used to explain in vitro ion channel activity of annexins: one involves changes in the conductance of ion via electrostatic disturbance of the membrane surface; the other involves a much more extensive alteration in protein structure and a correspondingly deeper penetration into the membrane.

Comparative expression of the mouse Sox1, Sox2 and Sox3 genes from pre-gastrulation to early somite stages.

Whole mount in situ hybridisation was used to study the embryonic expression of the mouse HMG box-containing genes Sox1, Sox2 and Sox3 between 6.5 and 9.0 days post coitum (dpc). Sox2 and Sox3 are expressed in the epiblast and extraembryonic ectoderm of the egg cylinder, becoming restricted to the prospective neural plate and chorion at the onset of gastrulation. Sox3 is upregulated in the posterior ectoderm during late streak and neural plate stages and is concomitantly downregulated in the chorion. Sox1 transcripts are first detected in the neural fold ectoderm at the headfold stage. During early somitogenesis, all three genes are expressed in the neuroectoderm, and Sox2 and Sox3 are also expressed in the primitive streak ectoderm, gut endoderm and prospective sensory placodes.

Prorhombomeric subdivision of the mammalian embryonic hindbrain: is it functionally meaningful?

The technique of whole embryo culture has made significant contributions to understanding the mechanisms of morphogenesis in mammalian embryos, especially with respect to cranial neurulation and neural crest cell migration. This study traces the fate of two specifically mammalian structures, the preotic and otic sulci. Their formation at the 1/2- and 3-somite stages respectively, divides the hindbrain neuroepithelium into prorhombomeres A, B and C. The preotic sulcus is a deeply recessed structure that forms the rostral boundary of expression of both Hoxb-2 and the first domain of Krox-20. The otic sulcus is a shallow concavity in which the second Krox-20 domain is expressed. DiI labeling followed by whole embryo culture confirmed that the later fate of the preotic sulcus is the rhombomere 2/3 boundary, and the fate of the otic sulcus is the cranial part of rhombomere 5. Structurally, the preotic and otic sulci show no specialization with respect to actin, tubulin or proteoglycans, but their maintenance depends on contact with the subjacent mesenchyme. Their formation is inhibited by exposure of embryos to retinoic acid prior to the onset of somitic segmentation, indicating that the molecular events governing prorhombomeric subdivision of the hindbrain are retinoic acid-sensitive. The preotic sulcus may be essential for neuroepithelial cell movement towards and into the rapidly enlarging forebrain; the otic sulcus may simply delineate the caudal boundary of prorhombomere B, an area with a discrete neural crest cell population discontinuous with those rostral and caudal to it. Understanding the positional relationships of the preotic and otic sulci to later rhombomeric segments makes them useful landmarks for experimental purposes, but there is no evidence that prorhombomeres are functionally significant as the precursors of rhombomeric segments.

A theoretical study of the chorismate synthase reaction.

Density functional calculations (B3LYP/6-31+G(d,p)) were carried out to investigate the mechanism of the anti-1,4-elimination of phosphate from 5-enolpyruvylshikimate-3-phosphate 1 that is catalyzed by chorismate synthase. Of particular interest was the functional role of the reduced flavin cofactor. [reaction: see text]

Absence of mutagenicity of coralyne and related antileukemic agents: structural comparison with the potent carcinogen 7,12-dimethylbenz[a]anthracene.

The structural similarity between antileukemic alkaloid coralyne and the carcinogenic and antineoplastic hydrocarbon 7, 12-dimethylbenz[a]anthracene, as well as the similarity between the antileukemic alkaloid nitidine and the carcinogenic hydrocarbon 5-methylchrysene, prompted a mutagenicity evaluation of coralyne sulfoacetate, nitidine chloride, the 8-ethyl homolog of coralyne, nitidine methosulfate, and the tetramethoxy analog of nitidine by the Ames method against the histidine-auxotroph strains of Salmonella typhimurium TA-1537, TA-1538, TA-98, and TA-100; 7,12-dimethylbenz[a]anthracene was used as a reference standard. The mutagenicity of these antileukemic compounds was either completely eliminated or drastically reduced, but the mutagenic response was generally high for 7,12-dimethylbenz[a]anthracene. The results suggest that the presence of a quaternary nitrogen atom and alkoxy groups could be important in alleviating the mutagenicity of the parent mutagenic and carcinogenic hydrocarbons.

Effects of all-trans-retinoic acid on skeletal pattern, 5'HoxD gene expression, and RAR beta 2/beta 4 promoter activity in embryonic mouse limbs.

Mouse embryos were exposed to all-trans-retinoic acid on day 11 or day 12 of development and the resulting skeletal pattern alterations compared with early effects on Hoxd-11 and Hoxd-13 expression domains and RAR-beta 2/beta 4 promoter activity. The effects on skeletal pattern showed a clear correlation between the timing of retinoic acid exposure and the sequence of mesenchymal condensation. Ectopic RAR-beta 2/beta 4 promoter activity was detected within 2 hr of exposure to retinoic acid, and was present throughout the limb bud after 5 hr; it remained high in the apical ectodermal ridge and proximal mesenchyme after 12 hr, by which time the abnormal digital pattern could be seen. HoxD gene expression domains in the distal handplate were narrowed by 5 hr after maternal retinoic acid administration on day 11. Following retinoic acid treatment on both day 11 and day 12, the normal downregulation of Hoxd-11 and Hoxd-13 in the digital mesenchymal condensations was retarded. There was no evidence to suggest that RAR-beta 2/beta 4 promoter activity mediates the effects of RA on HoxD gene expression, but ectopic promoter activity is a useful indicator of at least some of the sites in which RA levels are raised. We suggest (1) that the apical ectodermal ridge is the most functionally significant of these sites, (2) that raised retinoic acid levels in the ridge result in altered gene expression and/or altered cell proliferation within this epithelium, (3) that both altered HoxD gene expression domains and altered skeletal pattern formation are secondary to this effect. There was a good correlation between the effects of retinoic acid on Hoxd-11 and Hoxd-13 expression and delay of skeletal differentiation, suggesting that this may be a direct effect.

CD34 expression patterns during early mouse development are related to modes of blood vessel formation and reveal additional sites of hematopoiesis.

CD34 is a cell surface glycoprotein that is selectively expressed within the human hematopoietic system on stem and progenitor cells, and in early blood vessels. To elucidate its functions during early blood vessel formation and hematopoiesis, we analyzed the expression patterns, in day 8 to day 10 mouse embryos, of CD34 RNA by in situ hybridization and protein by immunohistochemistry using the monclonal antibody RAM 34. Levels of expression in embryonic blood vessels were correlated with the mode of vessel formation, being high in pre-endothelial cells and in vessels forming by vasculogenesis (particularly the dorsal aortae) or angiogenesis, but low in vessels forming by coalescence (the cardinal veins). CD34+ erythroid cells, presumably of yolk sac origin, were present in the liver of day 10 embryos; at the same stage, putative definitive hematopoietic cells, strongly CD34+, were present in the para-aortic mesenchyme. Possible sites of hemangioblastic differentiation were detected in the form of CD34+ endothelium-attached hematopoietic cells in the dorsal aorta and in two previously unreported locations, the proximal umbilical and vitelline arteries. These observations suggest functions for CD34 in relation to specific modes of blood vessel formation, and a hemangioblastic role in both embryonic and extraembryonic sites.

The basal SAR of a novel insulin receptor activator.

The synthesis and SAR of analogues prepared from novel insulin receptor activator 1 are described. Changes to the dihydroxyquinone core were not tolerated while functionalization of the two indoles contained in 1 resulted in little effect upon activation of the insulin receptor.