Electrodeposition of ordered Bi2Te3 nanowire arrays.
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Letter↗
Biomedical subjects
A L Prieto
Publications and source records attributed to A L Prieto.
Outer segment phagocytosis by cultured retinal pigment epithelial cells requires Gas6.
The function and viability of vertebrate photoreceptors requires the daily phagocytosis of photoreceptor outer segments (OS) by the adjacent retinal pigment epithelium (RPE). We demonstrate here a critical role in this process for Gas6 and by implication one of its receptor protein tyrosine kinases (RTKs), Mertk (Mer). Gas6 specifically and selectively stimulates the phagocytosis of OS by normal cultured rat RPE cells. The magnitude of the response is dose-dependent and shows an absolute requirement for calcium. By contrast the Royal College of Surgeons (RCS) rat RPE cells, in which a mutation in the gene Mertk results in the expression of a truncated, non-functional receptor, does not respond to Gas6. These data strongly suggest that activation of Mertk by its ligand, Gas6, is the specific signaling pathway responsible for initiating the ingestion of shed OS. Moreover, photoreceptor degeneration in the RCS rat retina, which lacks Mertk, and in humans with a mutation in Mertk, strongly suggests that the Gas6/Mertk signaling pathway is essential for photoreceptor viability. We believe that this is the first demonstration of a specific function for Gas6 in the eye.
Animals↗
Expression of the receptor protein-tyrosine kinases Tyro-3, Axl, and mer in the developing rat central nervous system.
Tyro-3, Axl, and Mer are three related receptor protein-tyrosine kinases (RPTKs) characterized by an extracellular domain exhibiting significant amino acid sequence similarity to neural cell adhesion molecules. The molecule Gas6 (for growth arrest-specific gene-6) has been shown to activate each of these receptors. Gas6 is expressed extensively in the central nervous system (CNS), suggesting that interactions between Gas6 and its receptors are likely to have physiologically relevant functions. To identify and localize the relevant Gas6/RPTK pairs, we have characterized the developmental expression of Tyro-3, Axl, and Mer in rat CNS using blotting and mRNA in situ hybridization analyses. Throughout development, Tyro-3 was the most widely expressed of the three receptors in the CNS, with Axl and Mer detected in only a limited number of sites in the adult. Tyro-3 expression was low in the embryo and increased markedly during early postnatal stages, with a time course paralleling that of synaptogenesis. Axl and Mer were expressed at low but relatively constant levels throughout development. In the cerebellum, all three receptors were found in Purkinje cells, and Tyro-3 was also detected in both granule neurons and Bergmann glia. Insofar as Gas6 has been previously shown to also be expressed by Purkinje cells, it may be engaged in both autocrine and paracrine signaling. The three receptors were also detected in cerebellar white matter, primarily during myelination. In the cortex, Tyro-3 was expressed at high levels during postnatal development and in the adult. Beginning at P6 in the hippocampus, Tyro-3 was expressed at high levels in CA1 pyramidal neurons and at lower levels in CA3 and was not detected in dentate granule neurons. Axl and Mer were found in the molecular layer of the dentate gyrus and were absent from the pyramidal and dentate granule neurons. In that Gas6 is expressed throughout the pyramidal cell layer, it may activate these cells in both an autocrine and a paracrine manner. These studies provide initial clues for elucidating the cellular functions of the Axl subfamily members and suggest potential complex Gas6/RPTK as well as RPTK/RPTK signaling interactions in the mature and developing CNS.
Age Factors↗
Inequalities in health care use and expenditures: empirical data from eight developing countries and countries in transition.
This paper summarizes eight country studies of inequality in the health sector. The analyses use household data to examine the distribution of service use and health expenditures. Each study divides the population into "income" quintiles, estimated using consumption expenditures. The studies measure inequality in the use of and spending on health services. Richer groups are found to have a higher probability of obtaining care when sick, to be more likely to be seen by a doctor, and to have a higher probability of receiving medicines when they are ill, than the poorer groups. The richer also spend more in absolute terms on care. In several instances there are unexpected findings. There is no consistent pattern in the use of private providers. Richer households do not devote a consistently higher percentage of their consumption expenditures to health care. The analyses indicate that intuition concerning inequalities could result in misguided decisions. It would thus be worthwhile to measure inequality to inform policy-making. Additional research could be performed using a common methodology for the collection of data and applying more sophisticated analytical techniques. These analyses could be used to measure the impact of health policy changes on inequality.
Data Collection↗
Gas6, a ligand for the receptor protein-tyrosine kinase Tyro-3, is widely expressed in the central nervous system.
Gas6 (growth arrest specific gene-6) is a ligand for members of the Axl subfamily of receptor protein-tyrosine kinases. One of these receptors, Tyro-3, is widely expressed in the central nervous system. We have used biochemical and histological techniques, including in situ hybridization, to determine the expression patterns of Gas6 mRNA and protein during development. Gas6 is widely expressed in the rat central nervous system (CNS) beginning at late embryonic stages and its levels remain high in the adult. Gas6 is detected as a single 85 kDa protein, which is encoded by a single 2.5 kb mRNA species. At embryonic day 14 it is detected in the heart, blood vessels, testes, choroid plexus, and in the ventral spinal cord. In the adult, Gas6 is expressed in the cerebral cortex, (predominantly in layer V), the piriform cortex, and the hippocampus (areas CA1, CA3 and the dentate gyrus). It is also expressed in thalamic and hypothalamic structures, the midbrain, and in a subset of motor and trigeminal nuclei. In the cerebellum, it is expressed in Purkinje neurons and deep cerebellar nuclei. Protein S, a protein related to Gas6, is only detected at low levels in the CNS. The spatial and temporal profiles of Gas6 expression suggest that it could potentially serve as the physiologically relevant ligand for Tyro-3 in the postnatal rat nervous system.
Animals↗
Cell-cell adhesion molecules in epithelial-mesenchymal transformations.
The functional units in most inductive and morphogenetic processes in the embryo are not single cells, but rather collectives of interacting cells that give rise to the tissues and organs. Cell adhesion molecules (CAMs) are involved in defining cell collectives and their borders as they interact during inductive events in morphogenesis. The expression patterns of CAMs are highly dynamic and changes are known to occur during epithelial-mesenchymal transformations. Alterations in CAM expression are correlated with changes in morphology. Conversely, experimentally induced changes in morphology result in changes in CAM expression. The structure, function, distribution, and control of CAM gene expression are presented in this review, and discussed with regard to their importance to normal developmental processes, particularly epithelial-mesenchymal transformations.
Animals↗
The integrin alpha 9 beta 1 mediates cell attachment to a non-RGD site in the third fibronectin type III repeat of tenascin.
We have previously reported the sequence of the integrin alpha 9 subunit, a partner of the beta 1 subunit that is expressed in basal keratinocytes, hepatocytes, airway epithelial cells, and smooth and skeletal muscle. In the present study, we have stably expressed alpha 9 beta 1 on the surface of the human embryonic kidney cell line 293 and the human colon carcinoma cell line SW480 and used these transfected cells lines to identify ligand(s) for this integrin. Transfected cells did not appear to utilize alpha 9 beta 1 for attachment to the extracellular matrix proteins fibronectin, laminin, vitronectin, fibrinogen, thrombospondin, or type I or IV collagen. However, in contrast to mock transfectants, both 293 cells and SW480 cells expressing alpha 9 beta 1 adhered to intact chicken tenascin. By utilizing a variety of recombinant fragments of tenascin, we were able to localize the binding site for alpha 9 beta 1 to the third type III repeat. This repeat contains the arginine-glycine-aspartic acid (RGD) tripeptide that has been shown to serve as a binding site in tenascin for alpha v-integrins. However, the RGD site does not appear to be the binding site for alpha 9 beta 1, as the attachment of alpha 9 transfectants to this fragment was not inhibited by RGD peptide, nor by changing the RGD site to RAD or RAA.
Amino Acid Sequence↗
Differential effects of cytotactin/tenascin fusion proteins on intracellular pH and cell morphology.
Cytotactin/tenascin is a multidomain extracellular matrix protein that inhibits both cell spreading and intracellular alkalinization. The protein has multiple different domains which are homologous to regions in epidermal growth factor, fibronectin, and fibrinogen. In previous studies, we produced nonoverlapping fusion proteins corresponding to these domains and examined their effects on cell attachment and spreading. Based on their ability either to promote or to inhibit cell attachment, two of these fusion proteins were shown to be adhesive and two were shown to be counteradhesive. To determine how the adhesive and counteradhesive activities of different cytotactin/tenascin domains alter intracellular pH (designated pHi), we have measured pHi, in NIH3T3 and U251MG cells in the presence of the cytotactin/tenascin fusion proteins and intact cytotactin/tenascin, as well as fibronectin. Cells incubated in the presence of intact cytotactin/tenascin or of the counteradhesive fusion proteins had a pHi lower than control cells. In contrast, the presence of the adhesive fusion proteins or of fibronectin caused cells to have higher pHi values than control cells. When two fragments were simultaneously presented, one of which alone increased pHi and the other of which alone decreased pHi, the predominant effect was that of lowered pHi. Incubation with an RGD-containing peptide derived from the cytotactin/tenascin sequence inhibited alkalinization promoted by the adhesive fragment containing the second through sixth fibronectin type III repeats that was known to bind to integrins. Incubation of the cells with heparinase I or III inhibited the intracellular alkalinization of cells plated in the presence of the other adhesive fusion protein containing the fibrinogen domain, suggesting that heparan sulfate proteoglycans were involved in these pHi changes. The activity of protein kinase C appeared to be important for the changes in pHi mediated by all of the proteins. The protein kinase C inhibitor Calphostin C blocked the rise in pHi elicited by the adhesive fusion proteins and by fibronectin. Moreover, activation of protein kinase C by the addition of phorbol esters increased the pHi in cells plated on cytotactin/tenascin or counteradhesive fusion proteins and reversed their effects. The results of this study support the hypothesis that cytotactin/tenascin can bind to multiple cell surface receptors and thereby elicit different physiological responses. Decreases in pHi are correlated with the phenomenon of counteradhesion whereas the ability to increase pHi is associated with cell attachment via at least two different types of cell surface receptors. The data raise the possibility that binding of cytotactin/tenascin may influence primary cellular processes such as migration and proliferation through the differential regulation of pHi.
3T3 Cells↗
Multiple integrins mediate cell attachment to cytotactin/tenascin.
To identify potential cell surface receptors for chicken cytotactin (CT), we have characterized the ability of recombinant fusion proteins spanning the proximal fibronectin (FN) type III repeats of the molecule to support attachment of glioma and carcinoma cell lines. The third FN type III repeat, which contains the RGD tripeptide, supported cell attachment and cell spreading; however, mutation of RGD to RAD did not result in significant loss of either activity. In addition, the same repeat of mouse CT, which contains a natural mutant, RVD, also supported cell attachment and spreading, although at a lower level; both activities were increased by mutation of the RVD sequence to RGD. Studies utilizing RGD-containing peptides and well-characterized antibodies to integrins indicated that cell attachment to the third FN type III repeat was mediated by at least two different integrin receptors of the alpha v subtype. Additional cellular receptors may also be involved in cell attachment to CT. For example, an antibody to the beta 1 subfamily of integrins partially inhibited binding of cells to intact CT but did not inhibit cell binding to the third FN type III repeat. These findings suggest that the RGD site in CT is able to mediate cell attachment to integrins and thus is not a cryptic adhesion site. They also open the possibility that the functions of CT in processes such as counteradhesion, cell migration, cell proliferation, and cell differentiation may be mediated in part by interaction with multiple integrins.
Amino Acid Sequence↗
Expression patterns of the cell adhesion molecule Nr-CAM during histogenesis of the chick nervous system.
Neuron-glia-related cell adhesion molecule (Nr-CAM) is a recently characterized cell adhesion molecule in the family of immunoglobulin-related molecules of which the neural cell adhesion molecule, N-CAM, is the prototype. Nr-CAM shares structural properties with another member of this family (neuron-glia CAM, Ng-CAM) and both molecules exhibit homophilic and heterophilic binding properties. To understand better the role of such molecules in development, we have examined the sites of synthesis and expression of Nr-CAM by means of in situ hybridization and immunohistochemistry. Both methods indicated that Nr-CAM is expressed only in the nervous system. The molecule was observed on neurons in both the peripheral and central nervous systems and on epithelial floor plate cells in the spinal cord, but it was absent in the germinal zones. The protein was present on perikarya, but was found preferentially on axonal tracts. As observed for messenger RNAs specifying other cell adhesion molecules, messenger RNA for Nr-CAM was localized in the perikarya. The temporal expression of Nr-CAM was correlated with various neural morphoregulatory events, including cell proliferation and migration, axonal outgrowth and myelination. The molecule was expressed during the onset of neurogenesis at embryonic day 3 in the floor plate epithelium, and then on postmitotic ventral horn motor neurons of the spinal cord. At later stages, it was expressed throughout the spinal cord but disappeared from the floor plate. In the cerebellum, Nr-CAM was found on granule and Purkinje neurons and afferent fibers. Both local and projection neurons in the optic tectum, as well as axonal pathways throughout the telencephalon, expressed Nr-CAM. In the peripheral nervous system, Nr-CAM was expressed strongly in sensory and autonomic ganglia and in the enteric nervous system. At the onset of myelination, there was a general decrease in staining for Nr-CAM protein in the central nervous system but not in the periphery. Comparison of the expression of Nr-CAM to that of the structurally related Ng-CAM showed considerable overlap in their distributions, although there were differences in the levels at which each CAM was observed in particular structures. For example, sympathetic ganglia stained more intensely for Nr-CAM protein than for Ng-CAM. This differential but co-distributed pattern is consistent with the idea that although similar cell adhesion molecules have independent binding specificities, they may have related functions that act synergistically in the development of the nervous system.
Animals↗
Characterization of multiple adhesive and counteradhesive domains in the extracellular matrix protein cytotactin.
The extracellular matrix molecule cytotactin is a multidomain protein that plays a role in cell migration, proliferation, and differentiation during development. To analyze the structure-function relationships of the different domains of this glycoprotein, we have prepared a series of fusion constructs in bacterial expression vectors. Results obtained using a number of adhesion assays suggest that at least four independent cell binding regions are distributed among the various cytotactin domains. Two of these are adhesive; two others appear to be counteradhesive in that they inhibit cell attachment to otherwise favorable substrates. The adhesive regions were mapped to the fibronectin type III repeats II-VI and the fibrinogen domain. The morphology of the cells plated onto these adhesive fragments differed; the cells spread on the fibronectin type III repeats as they do on fibronectin, but remained round on the fibrinogen domain. The counteradhesive properties of the molecule were mapped to the EGF-like repeats and the last two fibronectin type III repeats, VII-VIII. The latter region also contained a cell attachment activity that was observed only after proteolysis of the cells. Several cell types were used in these analyses, including fibroblasts, neurons, and glia, all of which are known to bind to cytotactin. The different domains exert their effects in a concentration-dependent manner and can be inhibited by an excess of the soluble molecule, consistent with the hypothesis that the observed properties are mediated by specific receptors. Moreover, it appears that some of these receptors are restricted to particular cell types. For example, glial cells bound better than neurons to the fibrinogen domain and fibroblasts bound better than glia and neurons to the EGF fragment. These results provide a basis for understanding the multiple activities of cytotactin and a framework for isolating different receptors that mediate the various cellular responses to this molecule.
Animals↗
Cytotactin expression in somites after dorsal neural tube and neural crest ablation in chicken embryos.
The spatiotemporal expression of the extracellular matrix protein cytotactin/tenascin during somitogenesis suggests that it plays a role in the morphogenetic events that give rise to the pattern of neural crest (NC) development. In the present study, the spatial distribution and molecular forms of cytotactin in somites were examined using in situ hybridization, Western blotting, and immunohistochemistry during normal development and after injury. In situ hybridization showed that prior to NC cell invasion cytotactin mRNA was restricted to the caudal half of the newly formed epithelial somites. As each epithelial somite matured, giving rise to a sclerotome and dermamyotome, the mRNA was first restricted to the dermamyotome and later restricted to the rostral protion of the sclerotome, consistent with the previously reported protein distribution. Immunocytochemical analysis of the distribution of cytotactin and NC cells in embryos with ablations that removed NC cells, or with simple wounds that left NC cells in place, demonstrated that the presence of NC cells is neither necessary nor sufficient for the correct positioning of cytotactin. Immunoblotting analysis showed that cytotactin synthesized by sclerotomes in the absence of NC cells was of similar molecular mass to that produced in their presence. These findings are in accord with the notion that the abnormalities of cytotactin distribution are related to the wounding process. We conclude that, contrary to the suggestion of Stern et al. [Stern, C. D., Norris, W. E., Bronner-Fraser, M., Carlson, G. J., Faissner, A., Keynes, R. J. & Schachner, M. (1989) Development 107, 309-319], there is no causal link between the presence of NC cells and the distribution and molecular mass of sclerotomal cytotactin.
Animals↗
Expression of adhesion molecules and the establishment of boundaries during embryonic and neural development.
Evidence is accumulating that molecules involved in cell-cell and cell-substratum interactions are important in the establishment and maintenance of borders between cell groups during development. In this report, we review evidence supporting this conclusion, particularly in regard to the role of adhesion molecules in the formation of cell collectives and in the modulation of cell and neurite movements.
Animals↗
Localization during development of alternatively spliced forms of cytotactin mRNA by in situ hybridization.
Cytotactin, an extracellular glycoprotein found in neural and nonneural tissues, influences a variety of cellular phenomena, particularly cell adhesion and cell migration. Northern and Western blot analysis and in situ hybridization were used to determine localization of alternatively spliced forms of cytotactin in neural and nonneural tissues using a probe (CT) that detected all forms of cytotactin mRNA, and one (VbVc) that detected two of the differentially spliced repeats homologous to the type III repeats of fibronectin. In the brain, the levels of mRNA and protein increased from E8 through E15 and then gradually decreased until they were barely detectable by P3. Among the three cytotactin mRNAs (7.2, 6.6, and 6.4 kb) detected in the brain, the VbVc probe hybridized only to the 7.2-kb message. In isolated cerebella, the 220-kD polypeptide and 7.2-kb mRNA were the only cytotactin species present at hatching, indicating that the 220-kD polypeptide is encoded by the 7.2-kb message that contains the VbVc alternatively spliced insert. In situ hybridization showed cytotactin mRNA in glia and glial precursors in the ventricular zone throughout the central nervous system. In all regions of the nervous system, cytotactin mRNAs were more transient and more localized than the polypeptides. For example, in the radial glia, cytotactin mRNA was observed in the soma whereas the protein was present externally along the glial fibers. In the telencephalon, cytotactin mRNAs were found in a narrow band at the edge of a larger region in which the protein was wide-spread. Hybridization with the VbVc probe generally overlapped that of the CT probe in the spinal cord and cerebellum, consistent with the results of Northern blot analysis. In contrast, in the outermost tectal layers, differential hybridization was observed with the two probes. In nonneural tissues, hybridization with the CT probe, but not the VbVc probe, was detected in chondroblasts, tendinous tissues, and certain mesenchymal cells in the lung. In contrast, hybridization with both probes was observed in smooth muscle and lung epithelium. Both epithelium and mesenchyme expressed cytotactin mRNA in varying combinations: in the choroid plexus, only epithelial cells expressed cytotactin mRNA; in kidney, only mesenchymal cells; and in the lung, both of these cell types contained cytotactin mRNA. These spatiotemporal changes during development suggest that the synthesis of the various alternatively spliced cytotactin mRNAs is responsive to tissue-specific local signals and prompt a search for functional differences in the various molecular forms of the protein.
Animals↗
Phosphatidylinositol-specific phospholipase C solubilized G2 acetylcholinesterase from plasma membranes of chromaffin cells.
Using whole homogenates and defined subcellular fractions of bovine adrenal medulla, we investigated the properties of the dimeric G2 molecular form of acetylcholinesterase (AChE), its distribution, and the mode of attachment to chromaffin cells. Our studies indicate that a substantial fraction of the G2 form is specifically susceptible to solubilization by phosphatidylinositol-specific phospholipase C (PIPLC) from subcellular fractions enriched with plasma membrane fragments. The results suggest that the G2 form of AChE is anchored in the plasma membrane to a glycolipid domain that contains phosphatidylinositol. Since a Ca+2-dependent PIPLC has been previously described in chromaffin granules, it is possible that the adrenal AChE could be released by a system reminiscent of that involved in the case of the surface glycoprotein of Trypanosoma brucei.
Acetylcholinesterase↗
Localization of mRNA for neural cell adhesion molecule (N-CAM) polypeptides in neural and nonneural tissues by in situ hybridization.
The differential expression of the mRNA for the neural cell adhesion molecule (N-CAM) has been studied by in situ hybridization and compared with protein localization analyzed by immunohistochemical staining. The distribution of mRNA during chicken embryonic development was analyzed in neural and nonneural tissues by using an RNA probe that detects all N-CAM mRNAs and a probe specific for the mRNA of the large cytoplasmic domain (ld) of N-CAM. The results provide a detailed description of the mRNA distribution for N-CAM. The distribution of mRNA for total N-CAM generally corresponded to that of protein but differed at a more detailed level of analysis. For example, the mRNA was localized only within the cell bodies of neurons, whereas the protein was also in neuronal processes; this differential localization was most clearly seen in the alternating layers of cell bodies and fibers in the optic tectum and cerebellum. N-CAM ld mRNA, which arises from alternative RNA splicing, was expressed only in neural tissues, confirming previous biochemical and histological studies. Differential expression of the ld mRNA was detected in specific neural cell types: N-CAM mRNA was present in the ependymal cells of the spinal cord and optic tectum, but mRNA for the ld form was absent. In contrast, the ld mRNA was among the N-CAM mRNAs found in the Purkinje cells and internal granule cells in the cerebellum. The differential expression of mRNAs for the N-CAM forms emphasizes the potential importance of alternative mRNA splicing in modulating adhesive events during embryonic development, particularly in the nervous system.
Animals↗