Primitive streak-forming cells of the chick invaginate through a basement membrane

Summary Recently fibronectin was shown to appear in the development of the chick for the first time as a thin band on the epiblastic side facing the hypoblast just prior to primitive streak formation. It was thus suggested that fibronectin might be instrumental in the migration of cells that lead to axis formation during primitive streak formation. In the present work we have examined simultaneously for the presence of fibronectin and the specific basement membrane glycoprotein laminin during primitive streak formation using immunofluorescence methods. Laminin was found to be expressed between the epiblast and the hypoblast of stage XIII¹ chick blastoderms. During the immediately following process of streak formation the laminin was found to be continuously detectable throughout the area covered by the hypoblast, but disrupted on the streak area. Fibronectin was found to co-distribute with laminin in stage XIII and in the early primitive streak chick blastoderms. It is concluded that at stage XIII laminin and fibronectin form part of a basement membrane that is partially disrupted during the immediately following process of primitive streak formation in order to allow the migration of the streak-forming epiblastic cells during this morphogenetic process.     

Expression pattern and secretion of human and chicken heparanase are determined by their signal peptide sequence

Cleavage of heparan sulfate (HS) proteoglycans affects the integrity and function of tissues and thereby fundamental phenomena, involving cell migration and response to changes in the extracellular microenvironment. The role of HS-degrading enzymes, commonly referred to as heparanases, in normal development has not been identified. The present study focuses on cloning, expression, and properties of a chicken heparanase and its distribution in the developing chicken embryo. We have identified a chicken EST, homologous to the recently cloned human heparanase, to clone and express a functional chicken heparanase, 60% homologous to the human enzyme. The full-length chicken heparanase cDNA encodes a 60-kDa proenzyme that is processed at the N terminus into a 45-kDa highly active enzyme. The most prominent difference between the chicken and human enzymes resides in the predicted signal peptide sequence, apparently accounting for the chicken heparanase being readily secreted and localized in close proximity to the cell surface. In contrast, the human enzyme is mostly intracellular, localized in perinuclear granules. Cells transfected with a chimeric construct composed of the chicken signal peptide preceding the human heparanase exhibited cell surface localization and secretion of heparanase, similar to cells transfected with the full-length chicken enzyme. We examined the distribution pattern of the heparanase enzyme in the developing chicken embryo. Both the chicken heparanase mRNA and protein were expressed, as early as 12 h post fertilization, in cells migrating from the epiblast and forming the hypoblast layer. Later on (72 h), the enzyme is preferentially expressed in cells of the developing vascular and nervous systems. Cloning and characterization of heparanase, the first and single functional vertebrate HS-degrading enzyme, may lead to identification of other glycosaminoglycan degrading enzymes, toward elucidation of their significance in normal and pathological processes.     

Kidney derived micro-scaffolds enable HK-2 cells to develop more in-vivo like properties

Many cell lines, despite the fact that they are easy to culture, tend to lose some of their in vivo characteristics in vitro, we therefore decided to investigate whether culturing HK-2 cells on kidney derived micro-scaffolds (KMS) could improve proximal tubule functionality to these cells. Kidney derived micro-scaffolds (KMS) have been prepared that, due to the fact that they are only 300 µm in depth, allow for transfer of gasses and nutrients via diffusion whilst maintaining the kidney's intricate microstructure. Culturing HK-2 on KMS shows significant increase in expression of AQP-1, ATP1B1, SLC23A1 and SLC5A2 after 1, 2 and 3 weeks compared with HK-2 grown under standard tissue culture conditions. Additionally, very high levels of expression of CCL-2 (15–30 fold increase) and LRP-2 (25–200 fold increase) were observed when the HK-2 were grown on KMS compared with HK-2 grown under standard tissue culture conditions. Furthermore, HK-2 cells grown under standard conditions released higher levels of Il-6 and Il-8 compared with primary tubule cells (Asterand AS-9-2) and secreted no MCP-1 or RANTES as opposed to primary cells that released MCP-1 and RANTES following stimulation. However, HK-2 grown on KMS showed both a marked decrease in Il-6/Il-8 secretion in line with the primary cells and secreted MCP-1 as well. These results show that the micro-environment of the KMS assists in restoring in vivo like properties to the HK-2 cells.     

Autonomic involvement in the permanent metabolic programming of hyperinsulinemia in the high-carbohydrate rat model

Exposure to a high-carbohydrate (HC) milk formula during the suckling period results in permanent metabolic programming of hyperinsulinemia in HC rats. Previous studies have shown that hyperinsulinemia in HC rats involves a programmed hyperresponsiveness to glucose. However, the immediate onset and persistence of enhanced insulin secretion throughout life suggests a role for numerous factors that control insulin secretion. Present in vivo and in vitro studies have shown a role for altered autonomic activity, including increased parasympathetic and decreased sympathetic activities, in the maintenance of hyperinsulinemia in 100-day-old HC rats. HC rats were shown to be more sensitive to cholinergic-induced potentiation of glucose-stimulated insulin secretion (GSIS) in response to acetylcholine and showed increased sensitivity to blockade of cholinergic-induced insulin secretion by the muscarinic-type 3 receptor-specific antagonist 4-diphenylacetoxy-N-methylpiperidine. In addition, HC rats were less sensitive to adrenergic-induced inhibition of insulin secretion by oxymetazoline, whereas treatment with yohimbine resulted in increased GSIS. Furthermore, HC rats showed greater reductions in plasma insulin levels after vagotomy, as well as an attenuation of yohimbine-induced potentiation of GSIS, suggesting that yohimbine-mediated changes are mediated by parasympathetic activity. Changes in autonomic regulation of GSIS are supported by increased mRNA levels of the parasympathetic signaling molecules muscarinic-type 3 receptor, phospholipase Cbeta1, and protein kinase C-alpha and decreased levels of alpha(2a)-adrenergic receptors in islets from adult HC rats. In conclusion, metabolic programming of hyperinsulinemia throughout adulthood of HC rats involves changes in autonomic activity in response to the HC dietary intervention in the suckling period.     

Differentiation of dissociated-reconstituted epiblasts of the chick under the influence of a normal hypoblast

Abstract. A cell suspension of chick epiblast cells cultured under defined conditions to form a flat disk, can differentiate and generate axial embryonic structures when covered with a primary hypoblast. Macroscopically identifiable axes developed in 26 out of 33 cases. In all cases axes developed in a direction consistent with the posteroanterior polarity of the normal hypoblast. Almost invariably the epiblast cells differentiated into ectoderm, neural plates or tubes, and endoderm. In some cases typical primitive streaks were found, sometimes accompanied by signs of axial mesoderm, whereas in other cases the primitive streaks seemed to regress. In the absence of a hypoblast no differentiation of neural tissue or any signs of axial development were observed.     

A subacute treatment of L-methionine induces an increase in the number of [3H]spiperone binding sites in the striatum of the rat

A subacute treatment, 500 mg/kg I.P. twice daily during 5 days, by L-methionine provoked an increase in the Bmax of [3H]-spiperone binding in the striatum of the rat. This increase was associated to a decrease in membrane microviscosity. However in these conditions no changes were found in the [3H]-DHA, [3H]QNB bindings or in the brain dopamine sensitive adenylate cyclase activity. L-methionine treatment reduced the accumulation of Dopa after NSD 1015 and antagonized the decrease in striatal acetylcholine provoked by haloperidol. Thus L-methionine might be a new potential drug for Parkinson's disease treatment.     

Retinoic acid inhibits growth in agarose of early chick embryonic cells and may be involved in regulation of axis formation

The mechanisms involved in the generation of axial structures in the chick are well documented, yet, little is known about the actual factors that generate such a complex pattern. The recent demonstrations that all-trans-retinoic acid (RA) acts as a morphogen during limb development (Thaller and Eichele, 1987) lead us to examine whether during axis formation in the developing chick, RA could be one of the factors involved. We now show that retinoic acid can block a very unusual property of normal early chick embryonic cells, mainly their capacity to grow in semisolid medium. We also present experiments that suggest that RA may play a direct role during axis formation in the developing chick.     

Effects on striatal and mesolimbic dopamine systems of a new potential antipsychotic drug -mezilamine- with weak cataleptogenic properties.

Mezilamine (2-methylamino-4-N-methylpiperazino-5-methylthio-6-chloropyrimidine) inhibits dopamine-sensitive adenylate cyclase in rat nucleus accumbens and striatum both in vitro and in vivo. After parenteral administration, mezilamine produces a dose dependent increase in homovanillic acid in rat and rabbit brain. As with clozapine, this increase is more marked in the limbic system than in the striatum of rabbit brain whereas the reverse holds true in the rat. However, in rats pretreated with probenecid, mezilamine and clozapine produce a greater activity in the limbic system while that of chlorpromazine is more pronounced in the striatum. In both regions mezilamine is more active than chlorpromazine. After chronic treatment (15 days), the activity of mezilamine decreases in the striatum but not in the limbic system. The low cataleptogenic activity of mezilamine cannot be explained by anticholinergic properties, neither can it be related to GABA-mimetic properties. Among the hypotheses discussed that attributing α-adrenergic properties to mezilamine is supported by the catalepsy-inducing effect observed in the rat when associating mezilamine and phenoxybenzamine.     

Maternal hyperinsulinemia predisposes rat fetuses for hyperinsulinemia, and adult-onset obesity and maternal mild food restriction reverses this phenotype

We have previously shown that artificial rearing of newborn female rat pups on a high-carbohydrate (HC) milk formula resulted in chronic hyperinsulinemia and adult-onset obesity (HC phenotype) and that the maternal HC phenotype was transmitted to their progeny (2-HC rats) because of fetal development in the HC female rat. The aims of this study were to investigate 1) the fetal adaptations that predisposed the progeny for the expression of the HC phenotype in adulthood and 2) whether the transfer of the HC phenotype to the progeny could be reversed by maternal food restriction. Fetal parameters such as plasma insulin and glucose levels, mRNA level of preproinsulin gene, pancreatic insulin content, and islet insulin secretory response in vitro were determined. On gestational day 21, 2-HC fetuses were hyperinsulinemic, had increased insulin content and mRNA level of the preproinsulin gene in their pancreata and demonstrated an altered glucose-stimulated insulin secretory response by isolated islets. Modification of the intrauterine environment in HC female rats was achieved by pair feeding them to the amount of diet consumed by age-matched control rats from the time of their weaning. This mild dietary restriction reversed their HC phenotype and also prevented the development of the HC phenotype in their progeny. These findings show that mal-programming of the progeny of the hyperinsulinemic-obese HC female for the expression of the HC phenotype is initiated in utero and that normalization of the maternal environment in HC female rats by mild food restriction resulted in the normal phenotype in their progeny.