Suppression of programmed neuronal death by a thapsigargin-induced Ca2+ influx

Rat sympathetic neurons undergo programmed cell death (PCD) in vitro and in vivo when they are deprived of nerve growth factor (NGF). Chronic depolarization of these neurons in cell culture with elevated concentrations of extracellular potassium ([K⁺]_o) prevents this death. The effect of prolonged depolarization on neuronal survival is thought to be mediated by a rise of intracellular calcium concentration ([Ca²⁺]_i) caused by Ca²⁺ influx through voltage-gated channels. In this report we investigate the effects of chronic treatment of rat sympathetic neurons with thapsigargin, an inhibitor of intracellular Ca²⁺ sequestration. In medium containing a normal concentration of extracellular Ca²⁺ ([Ca²⁺]_o), thapsigargin caused a sustained rise of intracellular Ca²⁺ concentration and partially blocked death of NGF-deprived cells. Elevating [Ca²⁺]_o in the presence of thapsigargin further increased [Ca²⁺]_i, suggesting that the sustained rise of [Ca²⁺]_i was caused by a thapsigargin-induced Ca²⁺ influx. This treatment potentiated the effect of thapsigargin on survival. The dihydropyridine Ca²⁺ channel antagonist, nifedipine, blocked both a sustained elevation of [Ca²⁺]_i and enhanced survival caused by depolarization with elevated [K⁺]_o, suggesting that these effects are mediated by Ca²⁺ influx through L-type channels. Nifedipine did not block the sustained rise of [Ca²⁺]_i or enhanced survival caused by thapsigargin treatment, indicating that these effects were not mediated by influx of Ca²⁺ through L-type channels. These results provide additional evidence that increased [Ca²⁺]_i can suppress neuronal PCD and identify a novel method for chronically raising neuronal [Ca²⁺]_i for investigation of this and other Ca²⁺-dependent phenomena. © 1995 John Wiley & Sons, Inc.     

Cadherin 23-like polypeptide in hair bundle mechanoreceptors of sea anemones

We investigated hair bundle mechanoreceptors in sea anemones for a homolog of cadherin 23. A candidate sequence was identified from the database for Nematostella vectensis that has a shared lineage with vertebrate cadherin 23s. This cadherin 23-like protein comprises 6,074 residues. It is an integral protein that features three transmembrane alpha-helices and a large extracellular loop with 44 contiguous, cadherin (CAD) domains. In the second half of the polypeptide, the CAD domains occur in a quadruple repeat pattern. Members of the same repeat group (i.e., CAD 18, 22, 26, and so on) share nearly identical amino acid sequences. An affinity-purified antibody was generated to a peptide from the C-terminus of the cadherin 23-like polypeptide. The peptide is expected to lie on the exoplasmic side of the plasma membrane. In LM, the immunolabel produced punctate fluorescence in hair bundles. In TEM, immunogold particles were observed medially and distally on stereocilia of hair bundles. Dilute solutions of the antibody disrupted vibration sensitivity in anemones. We conclude that the cadherin 23-like polypeptide likely contributes to the mechanotransduction apparatus of hair bundle mechanoreceptors of anemones.     

Relationships between lake macrophyte cover and lake and landscape features

We examined the ability of lake and landscape features to predict a variety of macrophyte cover metrics using 54 north temperate lakes. We quantified submersed cover, emergent cover, floating leaf cover, Eurasian watermilfoil cover and total macrophyte cover. Measured lake features included lake physio-chemical and morphometric variables and landscape features included hydrologic, catchment and land use/cover variables. Univariate regression analyses demonstrated that these macrophyte cover metrics are predicted by a wide range of predictor variables, most commonly by: Secchi disk depth, maximum or mean depth, catchment morphometry, road density and the proportion of urban or agricultural land use/cover in the riparian zone or catchment (r² = 0.06–0.46). Using a combination of lake and landscape features in multiple regressions, we were able to explain 29–55% of the variation in macrophyte cover metrics. Total macrophyte cover and submersed cover were related to Secchi disk depth and mean depth, whereas the remaining metrics were best predicted by including at least one land use/cover variable (road density, proportion local catchment agriculture land use/cover, proportion cumulative catchment urban land use/cover, or proportion riparian agriculture land use/cover). The two main conclusions from our research are: (1) that different macrophyte growth forms and species are predicted by a different suite of variables and thus should be examined separately, and (2) that anthropogenic landscape features may override patterns in natural landscape or local features and are important in predicting present-day macrophytes in lakes.     

Mutations affecting a regulated, membrane-associated esterase in Salmonella typhimurium LT2

Mutations at the apeA locus in Salmonella typhimurium lead to loss of a soluble enzyme (protease I) that hydrolyzes the chromogenic endoprotease substrate N-acetyl phenylalanine -naphthyl ester. We have isolated pseudorevertants of S. typhimurium apeA mutations that have regained the ability to hydrolyze this compound. These pseudorevertants contain mutations (apeR) that lead to overproduction of a membrane-bound esterase different from protease I. The apeR locus is phage P1 cotransducible with ilvC (83 map units) and is unlinked to apeA. Mutations at still another locus, apeE, lead to loss of the membrane-associated esterase. The apeE locus is P1 cotransducible with purE (12 map units). In an apeE-lacZ operon fusion strain, an apeR mutation increases the level of -galactosidase approximately 60-fold. We propose that apeR encodes a repressor of apeE. The evidence available suggests that the ApeE protein is not a protease.     

Imidazo[1,2-a]pyrazine diaryl ureas: Inhibitors of the receptor tyrosine kinase EphB4

Inhibition of receptor tyrosine kinases (RTKs) such as vascular endothelial growth factor receptors (VEGFRs) and platelet-derived growth factor receptors (PDGFRs) has been validated by recently launched small molecules Sutent^® and Nexavar^®, both of which display activities against several angiogenesis-related RTKs. EphB4, a receptor tyrosine kinase (RTK) involved in the processes of embryogenesis and angiogenesis, has been shown to be aberrantly up regulated in many cancer types such as breast, lung, bladder and prostate. We propose that inhibition of EphB4 in addition to other validated RTKs would enhance the anti-angiogenic effect and ultimately result in more pronounced anti-cancer efficacy. Herein we report the discovery and SAR of a novel series of imidazo[1,2-a]pyrazine diarylureas that show nanomolar potency for the EphB4 receptor, in addition to potent activity against several other RTKs.     

Gastro-intestinal handling of water and solutes in three species of elasmobranch fish,the white-spotted bamboo shark,Chiloscyllium plagiosum,little skate,Leucoraja erinacea and the clear nose skate Raja eglanteria

The present study reports aspects of GI tract physiology in the white-spotted bamboo shark, Chiloscyllium plagiosum, little skate, Leucoraja erinacea and the clear nose skate, Raja eglanteria. Plasma and stomach fluid osmolality and solute values were comparable between species, and stomach pH was low in all species (2.2 to 3.4) suggesting these elasmobranchs may maintain a consistently low stomach pH. Intestinal osmolality, pH and ion values were comparable between species, however, some differences in ion values were observed. In particular Ca²⁺ (19.67 ± 3.65 mM) and Mg²⁺ (43.99 ± 5.11 mM) were high in L. erinacea and Mg²⁺ was high (130.0 ± 39.8 mM) in C. palgiosum which may be an indication of drinking. Furthermore, intestinal fluid HCO₃^? values were low (8.19 ± 2.42 and 8.63 ± 1.48 mM) in both skates but very high in C. plagiosum (73.3 ± 16.3 mM) suggesting ingested seawater may be processed by species-specific mechanisms. Urea values from the intestine to the colon dropped precipitously in all species, with the greatest decrease seen in C. plagiosum (426.0 ± 8.1 to 0 mM). This led to the examination of the molecular expression of both a urea transporter and a Rhesus like ammonia transporter in the intestine, rectal gland and kidney in L. erinacea. Both these transporters were expressed in all tissues; however, expression levels of the Rhesus like ammonia transporter were orders of magnitude higher than the urea transporter in the same tissue. Intestinal flux rates of solutes in L. erinacea were, for the most part, in an inward direction with the notable exception of urea. Colon flux rates of solutes in L. erinacea were all in an outward direction, although absolute rates were considerably lower than the intestine, suggestive of a much tighter epithelia. Results are discussed in the context of the potential role of the GI tract in salt and water, and nitrogen, homeostasis in elasmobranchs.     

Conformation of a synthetic antigenic peptide from HIV-1 p24 protein induced by ionic micelles

We studied the interaction of the peptide AAMQMLKETINEEAAEWDRVHPVHAGPIA from the HIV-1 p24 protein in the presence of SDS (anionic) and CTABr (cationic) micelles at pH 7.0 by circular dichroism, fluorescence, and electron spin resonance (ESR). The micelles induced secondary structure as well as a blue shift in the tryptophan fluorescence emission, indicating an interaction between the peptide and the micelles. However, different contents of secondary structure elements were found when the peptide interacts with SDS or CTABr micelles. Steady-state anisotropy indicates a constraint on the rotational mobility of the tryptophan residue of the peptide upon interaction with micelles. ESR studies pointed to different locations for the peptide in either micelle. Our results suggested that at least part of the peptide might be located at the hydrophobic core of the CTABr micelles, probably at the C-terminal region, while it is more inserted into the SDS micelles.     

Ultrastructure of the ovarian follicles in the placentotrophic Andean lizard of the genus Mabuya (Squamata: Scincidae)

We studied the ultrastructural organization of the ovarian follicles in a placentotrophic Andean lizard of the genus Mabuya. The oocyte of the primary follicle is surrounded by a single layer of follicle cells. During the previtellogenic stages, these cells become stratified and differentiated in three cell types: small, intermediate, and large globoid, non pyriform cells. Fluid‐filled spaces arise among follicular cells in late previtellogenic follicles and provide evidence of cell lysis. In vitellogenic follicles, the follicular cells constitute a monolayered granulosa with large lacunar spaces; the content of their cytoplasm is released to the perivitelline space where the zona pellucida is formed. The oolemma of younger oocytes presents incipient short projections; as the oocyte grows, these projections become organized in a microvillar surface. During vitellogenesis, cannaliculi develop from the base of the microvilli and internalize materials by endocytosis. In the juxtanuclear ooplasm of early previtellogenic follicles, the Balbiani's vitelline body is found as an aggregate of organelles and lipid droplets; this complex of organelles disperses in the ooplasm during oocyte growth. In late previtellogenesis, membranous organelles are especially abundant in the peripheral ooplasm, whereas abundant vesicles and granular material occur in the medullar ooplasm. The ooplasm of vitellogenic follicles shows a peripheral band constituted by abundant membranous organelles and numerous vesicular bodies, some of them with a small lipoprotein core. No organized yolk platelets, like in lecithotrophic reptiles, were observed. Toward the medullary ooplasm, electron‐lucent vesicles become larger in size containing remains of cytoplasmic material in dissolution. The results of this study demonstrate structural similarities between the follicles of this species and other Squamata; however, the ooplasm of the mature oocyte of Mabuya is morphologically similar to the ooplasm of mature oocytes of marsupials, suggesting an interesting evolutionary convergence related to the evolution of placentotrophy and of microlecithal eggs. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Hematological,biochemical, respiratory,cardiovascular and electroneurophysiological parameters in African green monkeys (Cercopithecus aethiops sabaeus). Its use in non‐clinical toxicological studies

Background The purpose of this study is to better characterize the hematological, biochemical, respiratory, cardiovascular and electroneurophysiological parameters in young adult Cercopithecus aethiops sabaeus of both sexes. The rhesus and cynomolgus monkeys are widely used as experimental primate models. However, only few articles have been published testing toxicological effects of pharmaceuticals on African green monkey. Methods The present study was carried out with the recompilation of all parameters recorded before the first drug administration in five sub‐chronic or chronic toxicological studies performed on 66 Cercopithecus aethiops sabaeus, born in Cuba. Results This study provides hematological, biochemical, respiratory, cardiovascular and electroneurophysiological data for both choosing animals to be included into experiments and monitoring these parameters during the study. Conclusions We conclude that this study provides valuable integrated data for determining the health status, including electroneurophysiological parameters, data not previously reported for this species, of the African green monkey.