N-cadherin detected in the membrane fraction of lens fiber cells

Summary N-cadherin was identified as a glycoprotein present in the fiber cell membranes of frog, chick, bovine, rabbit and human lenses. The molecular size of N-cadherin varies with the species. Homogenization of the chick lens in the presence of Ca²⁺ resulted in a decrease in the concentration of N-cadherin. This suggests that the lens contains a Ca²⁺-activated protease which can act on N-cadherin.     

The effect of chloroquine upon the developing lens

Applied to the developing lens of the 14-day-old chick embryo, in organ culture conditions, chloroquine prevented the elongation of the primary lens fibres, destroyed the equatorial ones and provoked vacuolisation and/or destruction in the epithelial cells.     

Polypeptides of the lens fibre cell intracellular matrix

Summary The cytoskeletal proteins of the vertebrate lens fibre cell comprise polypeptides ranging in mol.wt from 43,000 to 250,000 daltons. The main intermediate filament polypeptide of the pickerel, Northern frog, chick, bovine, and human lens has a mol.wt of 54,000 daltons. Peptide analysis revealed that the chick 54,000 dalton protein was more similar to the bovine protein than to the pickerel protein.Supported by grants EY 01417 (H. Maisel) and HD-06390 (J.C. Brown, University of Virginia, Charlottesville) from the National Institutes of Health, U.S.P.H.S.     

The effect of chloroquine upon the developing lens

Summary Applied to the developing lens of the 14-day-old chick embryo, in organ culture conditions, chloquine prevented the elongation of the primary lens fibres, destroyed the equatorial ones and provoked vacuolisation and/or destruction in the epithelial cells.This work was supported by a grant from the office of the Chief Scientist, Ministry of Health, Israel     

Surface molecules regulating rolling and adhesion to endothelium of neutrophil granulocytes and MDA-MB-468 breast carcinoma cells and their interaction

The extravasation of leukocytes and tumor cells is a multi-step process with the involvement of various adhesion molecules depending on the three steps rolling, adhesion, and diapedesis. We have developed an in vitro model, by which we investigated the rolling and adhesion of neutrophil granulocytes and MDA-MB-468 human breast carcinoma cells to lung endothelial cells under physiological flow-conditions. We found that norepinephrine had an inhibitory function on the fMLP-promoted adhesion of neutrophil granulocytes due to a down-regulation of β2-integrin. Furthermore, neutrophil granulocytes serve as linking cells for the interaction of the MDA-MB-468 cells with the endothelium, which are both β2-integrin negative, but express the β2-integrin ligand ICAM-1. In addition, we show here that N-cadherin is up-regulated on the endothelial cells and on neutrophil granulocytes in response to fMLP. This up-regulation resulted in a significant increase of adherent MDA-MB-468 cells, which are also N-cadherin positive. Received 3 September 2007; received after revision 17 October 2007; accepted 22 October 2007     

Development of rhythmic melatonin synthesis in cultured pineal glands and pineal cells isolated from chick embryo

The chick pineal gland exhibits circadian rhythms in melatonin synthesis under in vivo and in vitro conditions. A daily rhythm of melatonin production was first detectable in pineal glands isolated from chick embryos at embryonic day 16 and incubated under a LD cycle. All pineal glands isolated from 17-day-old and older embryos were rhythmic while no gland isolated at embryonic day 14 and 15 exhibited a daily rhythm in melatonin synthesis. Melatonin production in static cultures of embryonic pineal cells was rhythmic over 48 h if the cells were kept under a LD cycle. When embryonic pineal cells were incubated in constant darkness the rhythm in melatonin production was damped within 48 h. These results suggest that chick pineal cells from embryonic day 16 onwards are photosensitive but that the endogenous component of the melatonin rhythm is not completely developed at that age. A soluble analogue of cAMP stimulated and norepinephrine inhibited melatonin synthesis in cultured embryonic pineal cells. These findings indicate that the stimulatory and inhibitory pathways controlling melatonin synthesis in the mature pineal gland are effective in pineal cells isolated from chick embryos at least 2 days before hatching.     

Signaling mechanisms of neurite outgrowth induced by the cell adhesion molecules NCAM and N-Cadherin

Formation of appropriate neural circuits depends on a complex interplay between extracellular guiding cues and intracellular signaling events that result in alterations of cytoskeletal dynamics and a neurite growth response. Surface-expressed cell adhesion molecules (CAMs) interact with the surroundings via the extracellular domain and bind to the cytoskeleton via their intracellular domain. In addition, several CAMs induce signaling events via direct interactions with intracellular proteins or via interactions with cell surface receptors. Thus, CAMs are obvious candidates for transmitting extracellular guidance cues to intracellular events and thereby regulating neurite outgrowth. In this review, we focus on two CAMs, the neural cell adhesion molecule (NCAM) and N-cadherin, and their ability to mediate signaling associated with a neurite outgrowth response. In particular, we will focus on direct interaction between NCAM and N-cadherin with a number of intracellular partners, as well as on their interaction with the fibroblast growth factor receptor (FGFR). Received 23 May 2008; received after revision 14 July 2008; accepted 21 July 2008     

Cytosolic free Ca2+ in insulin secreting cells and its regulation by isolated organelles

The role of Ca2+ in secretagogue-induced insulin release is documented not only by the measurements of 45Ca fluxes in pancreatic islets, but also, by direct monitoring of cytosolic free Ca2+, [Ca2+]i. As demonstrated, using the fluorescent indicator quin 2, glyceraldehyde, carbamylcholine and alanine raise [Ca2+]i in the insulin secreting cell line RINm5F, whereas glucose has a similar effect in pancreatic islet cells. The regulation of cellular Ca2+ homeostasis by organelles from a rat insulinoma, was investigated with a Ca2+ selective electrode. The results suggest that both the endoplasmic reticulum and the mitochondria participate in this regulation, albeit at different Ca2+ concentrations. By contrast, the secretory granules do not appear to be involved in the short-term regulation of [Ca2+]i. Evidence is presented that inositol 1,4,5-trisphosphate, which is shown to mobilize Ca2+ from the endoplasmic reticulum, is acting as an intracellular mediator in the stimulation of insulin release.     

Calcium signalling: a historical account, recent developments and future perspectives

Ca2+ is a uniquely important messenger that penetrates into cells through gated channels to transmit signals to a large number of enzymes. The evolutionary choice of Ca2+ was dictated by its unusual chemical properties, which permit its reversible complexation by specific proteins in the presence of much larger amounts of other potentially competing cations. The decoding of the Ca2+ signal consists in two conformational changes of the complexing proteins, of which calmodulin is the most important. The first occurs when Ca2+ is bound, the second (a collapse of the elongated protein) when interaction with the targeted enzymes occurs. Soluble proteins such as calmodulin contribute to the buffering of cell Ca2+, but membrane intrinsic transporting proteins are more important. Ca2+ is transported across the plasma membrane (channel, a pump, a Na+/Ca2+ exchanger) and across the membrane of the organelles. The endoplasmic reticulum is the most dynamic store: it accumulates Ca2+ by a pump, and releases it via channels gated by either inositol 1,4,5-trisphosphate (IP3) and cyclic adenosine diphosphate ribose (cADPr). The mitochondrion is more sluggish, but it is closed-connected with the reticulum, and senses microdomains of high Ca2+ close to IP3 or cADPr release channels. The regulation of Ca2+ in the nucleus, where important Ca(2+)-sensitive processes reside, is a debated issue. Finally, if the control of cellular Ca2+ homeostasis somehow fails (excess penetration), mitochondria 'buy time' by precipitating inside Ca2+ and phosphate. If injury persists, Ca2(+)-death eventually ensues.     

Nuclear calcium signalling

The topic of nuclear Ca2+ signalling is beset by discrepant observations of substantial nuclear/cytoplasmic gradients. The reasons why some labs have recorded such gradients, whilst other workers see equilibration of Ca2+(cyt) and Ca2+(nuc) using the same cells and techniques, is unexplained. Furthermore, how such gradients could arise across the NE that possesses many highly-conductive NPCs is a mystery. Although nuclei may have the capacity to be autonomous signalling entities, with functional Ca2+ release channels and an inositide cycle, the balance of evidence suggests that Ca2+ release on the inner NE does not occur during physiological stimulation. Our work suggests that elementary Ca2+ release events originating in the cytoplasm can give rise to Ca2+ signals without causing elevation of the bulk cytoplasm. Clearly, the many Ca2+ signalling mechanisms that may impinge on Ca2+(nuc) will remain a topic of controversy and debate for some time.     

Latent differentiation in the endoderm of the young chick embryo]

Initiation and localization of the capacity of self-differentiation in the 1-2 day old chick embryo were studied by means of culturing endoderm fragments, wrapped in a piece of viteline membrane and completely deprived of mesenchyme.     

Molecular and Cellular Basis of Regeneration and Tissue Repair

Although early after birth the central nervous system is more plastic than in the adult, it already displays limited regenerative capability. This becomes severely impaired at specific stages of embryonic development; however, the precise cellular and molecular basis of this loss is not fully understood. The chick embryo provides an ideal model for direct comparisons of regenerating and non-regenerating spinal cord within the same species because of its accessibility in ovo, the extensive knowledge of chick neural development and the molecular tools now available. Regenerative ability in the chick is lost at around E13, a relatively advanced stage of spinal cord development. This is most likely due to a complex series of events: there is evidence to suggest that developmentally regulated changes in the early response to injury, expression of inhibitory molecules and neurogenesis may contribute to loss of regenerative capacity in the chick spinal cord.     

Signaling pathways between the plasma membrane and endoplasmic reticulum calcium stores

This review discusses multiple ways in which the endoplasmic reticulum participates in and is influenced by signal transduction pathways. The endoplasmic reticulum provides a Ca2+ store that can be mobilized either by calcium-induced calcium release or by the diffusible messenger inositol 1,4,5-trisphosphate. Depletion of endoplasmic reticulum Ca2+ stores provides a signal that activates surface membrane Ca2+ channels, a process known as capacitative calcium entry. Depletion of endoplasmic reticulum stores can also signal long-term cellular responses such as gene expression and programmed cell death or apoptosis. In addition to serving as a source of cellular signals, the endoplasmic reticulum is also functionally and structurally modified by the Ca2+ and protein kinase C pathways. Elevated cytoplasmic Ca2+ causes a rearrangement and fragmentation of endoplasmic reticulum membranes. Protein kinase C activation reduces the storage capacity of the endoplasmic reticulum Ca2+ pool. In some cell types, protein kinase C inhibits capacitative calcium entry. Protein kinase C activation also protects the endoplasmic reticulum from the structural effects of high cytoplasmic Ca2+. The emerging view is one of a complex network of pathways through which the endoplasmic reticulum and the Ca2+ and protein kinase C signaling pathways interact at various levels regulating cellular structure and function.     

Intrinsic forces alone are sufficient to cause closure of the neural tube in the chick

An isolated neural plate or a postnodal piece of early chick embryos, when cultured under appropriate experimental conditions, can undergo morphogenetic movements and form tubular structures closely resembling neural tubes of early chick embryos.     

Cytosolic calcium in platelet activation

Experiments with permeabilised platelets, and with intact platelets loaded with fluorescent Ca2+-indicators, over the past several years have greatly extended our knowledge and understanding of cytosolic Ca2+ as a platelet activator and its interactions with other cytosolic regulators. This article outlines insights, gained from the use of the fluorescent dyes, into maintenance and restoration of basal [Ca2+]i, mechanisms of receptor-mediated Ca2+-mobilisation and quantitation of [Ca2+]i/response relations in intact human platelets.     

Development of melatonin rhythm in the pineal gland and eyes of chick embryo.

A melatonin rhythm was observed in the pineals of 18-day-old chick embryos incubated under a light-dark regime of 18: 6 h. A low pineal melatonin content was found during the light phase of the day. Concentrations started to increase 2 h after dark onset and reached maximum levels after 4 h of darkness. The amplitude of the pineal melatonin rhythm increased considerably after 2 days and night-time concentrations in 20-day-old embryos were more than 5 times higher than in 18-day-old ones. Significant day/night differences in melatonin production were found both in pineals and eyes. Exposure of eggs to 1 h of light during the dark period decreased the high melatonin concentrations in the eyes but not in the pineals of the 20-day-old chick embryo. The results suggest that in this precocial bird at least part of the circadian system may already operate during embryonic life.     

Effects of N2, O2'-dibutyril cyclic GMP on the nucleoside phosphotransferase activity of the retina of the chick embryos.

In the retina of the chick embryo, 2 different forms of nucleoside phosphotransferase take part in the phosphorylation of thymidine. One is an unstable form with higher molecular weight. The other with lower m. wt is a stable form. This paper shows that N2,O2'-dibutyril cyclic GMP causes a marked decrement of the activity of the unstable nucleoside phosphotransferase.     

Biochemical events associated with rapid cellular damage during the oxygen- and calcium-paradoxes of the mammalian heart

The O2- and Ca2(+)-paradoxes have a number of features in common and it is suggested that release of cytosolic proteins in both paradoxes is initiated by the activation of a sarcolemma NAD(P)H dehydrogenase which can generate a transmembrane flow of H+ and e- and also oxygen radicals or redox cycling which damage ion channels and membrane proteins (phase I). Entry of Ca2+ through the damaged ion channels then exacerbates the damage by further activating this system, either directly or indirectly, and the redox cycling and/or oxygen radicals cause further damage to integral and cytoskeletal proteins of the sarcolemma resulting in microdamage to the integrity of the membrane (phase II) and the consequent release or exocytosis of cytoplasmic proteins and, under specialised conditions, the blebbing of the sarcolemma. The system may be primed either by removal of extracellular Ca2+ or by raising [Ca2+]i by a variety of measures, these two actions being synergistic. The system is initially activated in the Ca2(+)-paradox by the membrane perturbation associated with removal of extracellular Ca2+; prolonged anoxia in the metabolically active cardiac muscle causes a depletion of the ATP supply, particularly in the absence of glucose, and hence a rise in [Ca2+]i in phase I of the oxygen paradox with the consequent activation of the NAD(P)H oxidase at the sarcolemma. Oxygen radicals are probably generated in both paradoxes and may have a partial role in the genesis of damage, but are not essential in the Ca2(+)-paradox which continues under anoxia. Massive entry of Ca2+ also activates an intracellularly localised dehydrogenase (probably at the SR) which produces myofilament damage by redox cycling.     

Mast cells are present during angiogenesis in the chick extraembryonic vascular system

The extraembryonic vascular membranes of 3-day-18-day chick embryos were examined for the presence of mast cells. As early as 3.5 days mast cells were found on the area vasculosa. It is suggested that these cells have a role in angiogenesis of the chick extraembryonic vascular system.     

Regulation of the type III InsP3 receptor and its role in beta cell function

The type III inositol 1,4,5-trisphosphate receptor (InsP3R) is an important intracellular calcium (Ca2+) release channel in the pancreatic beta cell. Pancreatic beta cells secrete insulin following a characteristic change in membrane potential that leads to an increase in cytoplasmic Ca2+. Both extracellular Ca2+ and Ca2+ mobilized from InsP3-sensitive stores contribute to this increase. RIN-m5F cells, an insulin-secreting beta cell line, preferentially express the type III InsP3R. These cells have been useful in determining the regulatory properties of the type III InsP3R and the role of this isoform in an intact cell. The type III InsP3R is ideal for signal initiation because high cytoplasmic Ca2+ does not inhibit its activity. Altered insulin secretion, the result of changes in Ca2+ handling by the beta cell, has significant clinical consequences.