Morphological and functional effects of extracellular matrix on pancreatic islet cell cultures

Extracellular matrix (ECM) has been reported to enhance epithelial cell attachment and proliferation as well as to induce differentiation in vitro. In an attempt to determine the benefits of culturing pancreatic islet cells on ECM, we studied the morphological and functional patterns of rat islet cells and an insulin-secreting tumor cell line. ECM enhanced islet cell attachment and proliferation when compared to plastic, as suggested by a higher specific activity of DNA synthesis and a higher mitotic index. Cells on ECM were heterogeneous in size and insulin content. They showed extended areas of confluence. Cultures on plastic demonstrated an organisation in clusters and low mitotic activity. However, ECM did not allow for reconstitution of an islet-like structure. When compared to plastic, an initial decrease in basal and stimulated insulin secretion per million cells was observed on ECM, but B-cell activity was restored after 6 days of culture. Glucagon and somatostatin secretion were similar on both substrates. These data suggest that ECM enhances markedly islet cells attachment and proliferation, as well as long-term culture maintenance.     

Ciona intestinalis ParaHox genes: evolution of Hox/ParaHox cluster integrity,developmental mode,and temporal colinearity

The Hox gene cluster, and its evolutionary sister the ParaHox gene cluster, pattern the anterior-posterior axis of animals. The spatial and temporal regulation of the genes seems to be intimately linked to the gene order within the clusters. In some animals the tight organisation of the clusters has disintegrated. We note that these animals develop in a derived fashion relative to the norm of their respective lineages. Here we present the genomic organisation of the ParaHox genes of Ciona intestinalis, and note that tight clustering has been lost in evolution. We present a hypothesis that the Hox and ParaHox clusters are constrained as ordered clusters by the mechanisms producing temporal colinearity; when temporal colinearity is no longer needed or used during development, the clusters can fall apart. This disintegration may be mediated by the invasion of transposable elements into the clusters, and subsequent genomic rearrangements.     

Differential regulation of ParaHox genes by retinoic acid in the invertebrate chordate amphioxus (Branchiostoma floridae)

The ParaHox cluster is the evolutionary sister to the Hox cluster. Like the Hox cluster, the ParaHox cluster displays spatial and temporal regulation of the component genes along the anterior/posterior axis in a manner that correlates with the gene positions within the cluster (a feature called collinearity). The ParaHox cluster is however a simpler system to study because it is composed of only three genes. We provide a detailed analysis of the amphioxus ParaHox cluster and, for the first time in a single species, examine the regulation of the cluster in response to a single developmental signalling molecule, retinoic acid (RA). Embryos treated with either RA or RA antagonist display altered ParaHox gene expression: AmphiGsx expression shifts in the neural tube, and the endodermal boundary between AmphiXlox and AmphiCdx shifts its anterior/posterior position. We identified several putative retinoic acid response elements and in vitro assays suggest some may participate in RA regulation of the ParaHox genes. By comparison to vertebrate ParaHox gene regulation we explore the evolutionary implications. This work highlights how insights into the regulation and evolution of more complex vertebrate arrangements can be obtained through studies of a simpler, unduplicated amphioxus gene cluster.     

Combined-method phylogenetic analysis of Hox and ParaHox genes of the metazoa

The clustered Hox genes show a conserved role in patterning the body axis of bilaterian metazoans. Increasingly, a broader phylogenetic sampling of non-model system organisms is being examined to detect a correlation, if any, between Hox gene evolution, and body plan innovations. To assess how Hox gene expression and function evolve with changing cluster arrangements, we must be able to reliably assign gene orthologies between Hox genes. Recent evidence suggests that a four-gene proto-Hox cluster duplicated to form the precursor of the present cluster and an additional sister-cluster, the ParaHox group. Here, phylogenetic methods are used to determine Hox-gene orthologies and to infer probable clustering events leading to the current bilaterian Hox complement. This analysis supports the ParaHox hypothesis and gives first confirmation that ind (intermediate neuroblasts defective) is an anterior ParaHox ortholog from protostomes. This analysis supports a proto-Hox cluster of four genes in which the central-class member of the ParaHox cluster may have been lost. It is also proposed here that ancestral diploblasts had central-class members of both Hox and ParaHox clusters. Primitive Hox gene ancestors are estimated by phylogenetic methods and found to have no strong affinity to any particular class of extant Hox members.     

Hox and ParaHox genes in Nemertodermatida, a basal bilaterian clade

Molecular evidence suggests that Acoelomorpha, a proposed phylum composed of acoel and Nemertodermatida flatworms, are the most basal bilaterian animals. Hox and ParaHox gene complements characterised so far in acoels consist of a small set of genes, comprising representatives of anterior, central and posterior genes, altogether Hox and ParaHox, but no PG3-Xlox representatives have been reported. It has been proposed that this might be the ancestral Hox repertoire in basal bilaterians. However, no studies of the other members of the group, the Nemertodermatida, have been done. In order to get a more complete picture of the basal bilaterian Hox and ParaHox complement, we have analysed the Hox/ParaHox complement of the nemertodermatid Nemertoderma westbladi. We have found representatives of two central and one posterior Hox genes, as well as an Xlox and a Caudal ParaHox gene. From our data we conclude that a PG3-Xlox gene was present in the ancestor of bilaterians. These findings support the speculation that basal bilaterians already had the beginnings of the extended central Hox set, driving back gene duplications in the central part of the Hox cluster deeper in phylogeny than previously suggested.     

Hox and ParaHox genes in evolution, development and genomics

The discovery of the homeobox motif and its presence in each gene of the Hox clusters revolutionized the fields of developmental biology and evolutionary developmental biology (1, 2),providing a rapid entrance into investigating the mechanisms of development of almost any animal taxon as well as dramatically altering conceptions on the extent of genetic conservation across the animal kingdom.     

Stimulation of density-inhibited cell cultures by insulin

Cell proliferation in density-inhibited chick embryo cell cultures was induced by microgram quantities of insulin, neuraminidase, trypsin or papain. Other proteins tested, including albumin, fetuin, ribonuclease and hyaluronidase were inactive except in very high concentrations (> 100 μg/ml). The insulin chick embryo model was selected for detailed analysis of the initiation of proliferation. Insulin insolubilized by conjugation with Sepharose particles was also active, but only in so far as it was released in soluble form from the particles. This was measured by a radioimmunoassay. Under the conditions giving maximal cell proliferation less than 0.002-0.2% of insulin was taken up by the cells. This suggests that an interaction of insulin with the cell surface only is sufficient to stimulate the cells. Insulin released the density-inhibited cells from G₁ phase to produce an almost synchronous wave of proliferation. The following sequence of events was characteristic of the cells after stimulation by insulin: an early increase in sugar uptake and decrease in leucine uptake, increase in cell volume, stimulation of RNA and protein synthesis, increase in thymidine uptake, DNA synthesis, mitosis and cell division.     

Toxic effects of resazurin on cell cultures

Resazurin, introduced as a cell viability indicator under the trade name alamarBlue^®, is generally regarded as nontoxic when used according to manufacturer’s suggested shorter-term incubation time specifications. However, problems arise when exposure times are extended to longer-term cultures on the order of days. To assess the effect of resazurin over longer incubation times, MCF7 (HTB-22), MCF10A (CRL-10317), 3T3-L1 (CL-173), and D1 (CRL-12424) cultures were tested with varying amounts of resazurin over 4- and 8-day periods. MCF7, 3T3-L1, and D1 cells cultured for 8 days with 20 % alamarBlue^® had significantly less cell survivability. Specifically, levels of metabolic activity, deoxyribonucleic acid (DNA) concentration, and glucose consumption of the cell lines cultured for 8 days in medium with 20 % alamarBlue^® were significantly lower (p < 0.05) than metabolic activity, DNA concentration, and glucose consumption of MCF7 cells cultured for 8 days in medium with no alamarBlue^®. MCF7, 3T3-L1, and D1 cells used less glucose at concentrations as low as 5 %. Data also suggests the toxic effects are more pronounced in the cancerous cell line as compared to the noncancerous cells.     

Independent regulation of nonallelic pancreatic amylase genes in diabetic mice

Administration of streptozotocin produces a diabetic condition in mice characterized by a specific decrease in amylase synthesis in the pancreas as well as a substantial reduction in amylase mRNA concentration. We have studied this effect in mice of the congenic strains C3H.AmyYBR and C3H.AmyCE with multiple active copies of the pancreatic amylase structural gene. When mice of these strains are treated with streptozotocin, the magnitude of reduction in the synthesis of each amylase isozyme is different. These differences are reflected in the relative activities of isozyme-specific mRNAs in an in vitro translation assay. Administration of insulin results in partial restoration of normal phenotypes. The results provide genetic evidence that individual copies of the amylase structural gene are associated with divergent cis-acting insulin-responsive sequences.     

All the three ParaHox genes are present in Nuttallochiton mirandus (Mollusca: polyplacophora): evolutionary considerations

The ParaHox gene cluster contains three homeobox genes, Gsx, Xlox and Cdx and has been demonstrated to be an evolutionary sister of the Hox gene cluster. Among deuterostomes the three genes are found in the majority of taxa, whereas among protostomes they have so far been isolated only in the phylum Sipuncula.We report the partial sequences of all three ParaHox genes in the polyplacophoran Nuttallochiton mirandus, the first species of the phylum Mollusca where all ParaHox genes have been isolated. This finding has phylogenetic implications for the phylum Mollusca and for its relationships with the other lophotrochozoan taxa.     

Environmental and nutritional effects on the epigenetic regulation of genes

Major efforts have been directed towards the identification of genetic mutations, their use as biomarkers, and the understanding of their consequences on human health and well-being. There is an emerging interest, however, in the possibility that environmentally-induced changes at levels other than the genetic information could have long-lasting consequences as well. This review summarises our current knowledge of how the environment, nutrition, and ageing affect the way mammalian genes are organised and transcribed, without changes in the underlying DNA sequence. Admittedly, the link between environment and epigenetics remains largely to be explored. However, recent studies indicate that environmental factors and diet can perturb the way genes are controlled by DNA methylation and covalent histone modifications. Unexpectedly, and not unlike genetic mutations, aberrant epigenetic alterations and their phenotypic effects can sometimes be passed on to the next generation.