Observations that amelogenins, in the form of enamel matrix derivative (EMD), have a stimulatory effect on mesenchymal cells and tissues, and on the regeneration of alveolar bone, justified investigations into the effect of EMD on bone-forming cells. The binding and uptake of EMD in primary osteoblastic cells was characterized, and the effect of EMD on osteoblast gene expression, protein secretion, and mineralization was compared with the effect of parathyroid hormone (PTH). Although no specific receptor(s) has yet been identified, EMD appeared to be taken up by osteoblasts through clathrin-coated pits via the interaction with clathrin adaptor protein complex AP-2, the major mechanism of cargo sorting into coated pits in mammalian cells. EMD had a positive effect on factors involved in mineralization in vitro, causing an increased alkaline phosphatase (ALP) activity in the medium as well an as increased expression of osteocalcin and collagen type 1. Several hundred genes are regulated by EMD in primary human osteoblasts. There appear to be similarities between the effects of EMD and PTH on human osteoblasts. The expression pattern of several mRNAs and proteins upon EMD stimulation also indicates a secondary osteoclast stimulatory effect, suggesting that the osteogenic effect of EMD in vivo, at least partly, involves stimulation of bone remodelling.
The aims of this study were firstly, to investigate the expression of E-cadherin complex proteins in ovarian carcinoma cells in serous effusions and in primary and metastatic lesions; and secondly to study the value of these four proteins and calretinin, a mesothelial marker, in the differential diagnosis of ovarian carcinoma cells from reactive mesothelial cells in effusions. Sixty-seven malignant effusions and 97 corresponding primary (n=36) and metastatic (n=61) lesions were immunohistochemically stained for E-cadherin and alpha-, beta-, and gamma-catenin. Staining extent and intensity were scored. Effusion specimens were additionally analysed for calretinin immunoreactivity. Membrane immunoreactivity for E-cadherin and alpha-, beta-, and gamma-catenin was detected on carcinoma cells in the majority of the effusions, but rarely on reactive mesothelial cells (p<0.001 for all markers). Calretinin immunoreactivity was confined to mesothelial cells (p<0.001). An association was seen between E-cadherin and alpha-catenin expression, in both effusions and solid tumours, and for beta-catenin in solid tumours (range p<0. 001 to p=0.014). Up-regulation of all four cadherin complex proteins was seen in carcinoma cells in effusions, when compared with corresponding primary tumours (range p<0.001 to p=0.028). As with effusions, metastatic lesions showed up-regulation of alpha-, beta-, and gamma-catenin when compared with primary carcinomas (p=0.002-0. 015). Carcinoma cells in effusions showed in addition elevated levels of E-cadherin when compared with metastatic lesions (p<0.001). Staining results in effusions showed no association with effusion site, tumour type or histological grade. Immunoblotting on 29 malignant effusions confirmed the presence of all four proteins in the majority of samples and co-precipitation of E-cadherin and beta-catenin was seen in ten specimens examined. E-cadherin complex proteins are widely expressed in ovarian carcinoma cells. Together with calretinin, they form a powerful battery of markers for the cytological diagnosis of carcinoma cells in effusions. The up-regulation of E-cadherin complex proteins in serous effusions and metastatic lesions may mark an early metastatic phenotype and possibly mediates survival of tumour cells at these sites through the inhibition of apoptosis.
Previously, B12D and B22E have been characterized as Barley aleurone and embryo (Balem) transcripts, expressed during seed maturation and embryo germination. The open reading frame of B12D cDNAs encodes a protein of unknown function highly conserved in mono‐ and dicotyledonous species, while B22E encodes a metallothionein‐like protein. Several slightly different B22E transcripts have earlier been identified. Our objective was to investigate the number of B12D genes, and B12D and B22E expression patterns in mature aleurone. Genomic Southern hybridization and primer extension experiments suggest the presence of a B12D gene family in barley with at least 8 or 9 members. B12D transcripts can also be identified in the starchy endosperm, and a primer extension analysis indicates that some of these genes are expressed in the starchy endosperm only. A number of genes appear to be transcribed in all tissues investigated; starchy endosperm, pericarp, immature and mature embryos and aleurone, and mature aleurone incubated with GA3. One B12D gene, HvB12Dg1, was isolated and shown by particle bombardment with a promoter‐GUS construct to be transcriptionally active. The HvB12Dg1 promoter contains elements similar to those of the gibberellic acid response complex (GARC). B12D transcripts are found in the aleurone of imbibed embryoless grains, while B22E transcripts are barely detectable. However, both transcripts are up‐regulated by the presence of the germinating embryo. For B22E this effect is not mimicked by applying GA3 exogenously to imbibed embryoless grains, while the B12D transcript level increases 2‐ to 3‐fold, at most. On the other hand, ABA can suppress B12D expression. Our investigations indicate that gibberellic acid may not be directly involved in the up‐regulation of all transcripts induced in the aleurone during germination.
The presence of estrogen receptors (ERs) in breast carcinomas is important for clinical response to endocrine therapy. However, the cellular mechanisms following ER activation are not fully understood. It has been indicated that expression of the ER is associated with the expression of c-erbB-4. To address this question, 103 breast carcinoma samples were studied using reverse transcriptase polymerase chain reaction (RT-PCR) analysis after application of a microselection method for RNA isolation. Total RNA for RT-PCR was isolated from 20-microm-thick frozen sections, which were made from microselected areas. Paraffin blocks from 98 of these 103 tumors were also immunohistochemically examined. Significant associations between ER-alpha and c-erbB-4 mRNA and protein expressions were found in the present study with both methods. One-fourth of the tumors did not express ER-alpha (22%, 24%, and 26% with chemical binding, immunohistochemistry, and RT-PCR, respectively). About one-half of the ER-alpha negative tumors did not express c-erbB-4 on both mRNA and protein levels (48% with RT-PCR and 46% with immunohistochemistry, P=0.001 for both methods). The endocrine therapy responsive breast cancer cell lines MCF-7 and T47-D were positive for both ER-alpha and c-erbB-4 expression, while the endocrine therapy nonresponsive breast cancer cell lines MDA-MD-231 and SK-BR-3 were not. Thus, we confirm the association between the expression of ER-alpha and c-erbB-4 mRNA and protein in breast carcinomas, indicating a role for c-erbB-4 in estrogen signal transduction.
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