Basophils are not the key antigen-presenting cells in allergic patients.

BACKGROUND: Recent data obtained in mouse models have initiated a controversy whether basophils are the key antigen-presenting cells (APCs) in allergy. Here, we investigate whether basophils are of importance for the presentation of allergen and the induction of T cell proliferation in allergic patients. METHODS: T cells, basophils, and APCs depleted of basophils were purified from allergic patients. Co-culture systems based on purified major allergens were established to study allergen-specific T cell responses using proliferation assays. RESULTS: Only co-cultures of T cells with APCs depleted of basophils but not with basophils proliferated in response to allergen. Even addition of IL-3 to T cell-basophil co-cultures failed to induce allergen-specific T cell proliferation. CONCLUSIONS: Our data demonstrate by classical in vitro proliferation assays that basophils are not key antigen-presenting cells that promote T cell proliferation in secondary immune responses to allergen in allergic patients.

Photooxidation technology for correlated light and electron microscopy.

The combination of the capabilities of light microscopical techniques with the power of resolution of electron microscopy along with technical advances has led to a gradual decline of the gap between classical light and electron microscopy. Among the correlative techniques using the synergistic opportunities, photooxidation methods have been established as valuable tools for visualizing cell structures at both light and electron microscopic level. Fluorescent dyes are used to oxidize the substrate diaminobenzidine, which in its oxidized state forms fine granular precipitates. Stained with osmium, the diaminobenzidine precipitates are well discernible in the electron microscope, thus labelling and defining the cellular structures, which at light microscopy level are recorded by fluorescent probes. The underlying photooxidation reaction is based on the excitation of free oxygen radicals that form upon illumination of fluorochromes; this is a central step in the procedure, which mainly influences the success of the method. This article summarizes basic steps of the technology and progresses, shows efforts and elaborated pathways, and focuses on methodical solutions as to the applicability of different fluorochromes, as well as conditions for fine structural localizations of the reaction products.

Effect of comprehensive validation of the template isolation procedure on the reliability of bacteraemia detection by a 16S rRNA gene PCR.

The influence of the DNA extraction method on the sensitivity and specificity of bacteraemia detection by a 16S rRNA gene PCR assay was investigated. The detection limit of the assay was 5 fg with purified DNA from Escherichia coli or Staphylococcus aureus, corresponding to one bacterial cell. However, with spiked blood samples, the detection limits were 10(4) and 10(6) CFU/mL, respectively. The sensitivity of the S. aureus assay was improved to the level of the E. coli test with the addition of proteinase K to the commercial DNA extraction kit protocol. Ten (16.6%) of 60 amplification reactions were positive with templates isolated from sterile blood, while PCR reagent controls were negative, thereby indicating contamination during the DNA extraction process. Blood samples were spiked with serial dilutions of E. coli and S. aureus cells, and six PCR results were obtained from three extractions for each blood sample. A classification threshold system was devised, based on the number of positive reactions for each sample. Samples were deemed positive if at least four positive reactions were recorded, making it possible to avoid false-positive results caused by contamination. These results indicate that a comprehensive validation procedure covering all aspects of the assay, including DNA extraction, can improve considerably the validity of PCR assays for bacteraemia, and is a prerequisite for the meaningful detection of bacteraemia by PCR in the clinical setting.

Proteome analysis of nuclear matrix proteins during apoptotic chromatin condensation.

The nuclear matrix (NM) is considered a proteinaceous scaffold spatially organizing the interphase nucleus, the integrity of which is affected during apoptosis. Caspase-mediated degradation of NM proteins, such as nuclear lamins, precedes apoptotic chromatin condensation (ACC). Nevertheless, other NM proteins remain unaffected, which most likely maintain a remaining nuclear structure devoid of chromatin. We, therefore, screened various types of apoptotic cells for changes of the nuclear matrix proteome during the process of apoptotic ACC. Expectedly, we observed fundamental alterations of known chromatin-associated proteins, comprising both degradation and translocation to the cytosol. Importantly, a consistent set of abundant NM proteins, some (e.g. hNMP 200) of which displaying structural features, remained unaffected during apoptosis and might therefore represent constituents of an elementary scaffold. In addition, proteins involved in DNA replication and DNA repair were found accumulated in the NM fraction before cells became irreversibly committed to ACC, a time point characterized in detail by inhibitor studies with orthovanadate. In general, protein alterations of a consistent set of NM proteins (67 of which were identified), were reproducibly detectable in Fas-induced Jurkat cells, in UV-light treated U937 cells and also in staurosporine-treated HeLa cells. Our data indicate that substantial alterations of proteins linking chromatin to an elementary nuclear protein scaffold might play an intriguing role for the process of ACC.

Placental alkaline phosphatase expression at the apical and basal plasma membrane in term villous trophoblasts.

Human placental alkaline phosphatase (PLAP) was localized at the apical and basal plasma membrane of syncytiotrophoblasts and at the surface of cytotrophoblasts in term chorionic villi using immunoelectron microscopy. Similarly, apical and basolateral PLAP expression was found in polarized trophoblast-derived BeWo cells. Trophoblasts isolated from term placentas exhibited mainly vesicular PLAP immunofluorescence staining immediately after isolation. After in vitro differentiation into syncytia, PLAP plasma membrane expression was upregulated and exceeded that observed in mononuclear trophoblasts. These data call for caution in using PLAP as a morphological marker to differentiate syncytiotrophoblasts from cytotrophoblasts or as a marker enzyme for placental brush-border membranes. (J Histochem Cytochem 49:1155-1164, 2001)

Aged mother cells of Saccharomyces cerevisiae show markers of oxidative stress and apoptosis.

Recently, we and others have shown that genetic and environmental changes that increase the load of yeast cells with reactive oxygen species (ROS) lead to a shortening of the life span of yeast mother cells. Deletions of yeast genes coding for the superoxide dismutases or the catalases, as well as changes in atmospheric oxygen concentration, considerably shortened the life span. The presence of the physiological antioxidant glutathione, on the other hand, increased the life span of yeast cells. Taken together, these results pointed to a role for oxygen in the yeast ageing process. Here, we show by staining with dihydrorhodamine that old yeast mother cells isolated by elutriation, but not young cells, contain ROS that are localized in the mitochondria. A relatively large proportion of the old mother cells shows phenotypic markers of yeast apoptosis, i.e. TUNEL (TdT-mediated dUTP nick end labelling) and annexin V staining. Although it has been shown previously that apoptosis in yeast can be induced by a cdc48 allele, by expressing pro-apoptotic human cDNAs or by stressing the cells with hydrogen peroxide, we are now showing a physiological role for apoptosis in unstressed but aged wild-type yeast mother cells.

Autophagic and apoptotic types of programmed cell death exhibit different fates of cytoskeletal filaments.

Programmed cell death comprises several subtypes, as revealed by electron microscopy. Apoptosis or type I programmed cell death is characterized by condensation of cytoplasm and preservation of organelles, essentially without autophagic degradation. Autophagic cell death or type II programmed cell death exhibits extensive autophagic degradation of Golgi apparatus, polyribosomes and endoplasmatic reticulum, which precedes nuclear destruction. In the present study, we analysed the fate of cytokeratin and F-actin during autophagic cell death in the human mammary carcinoma cell line MCF-7 because recent studies suggest that an intact cytoskeleton is necessary for autophagocytosis. Programmed cell death was induced by 10(-)(6) M tamoxifen. For quantitative light microscopic analysis, autophagic vacuoles were visualized by monodansyl cadaverin, which stains autophagic vacuoles as distinct dot-like structures. In control cultures, the number of monodansylcadaverin-positive cells did not exceed 2%. Tamoxifen induced a dramatic increase 2-4 days after treatment to a maximum of 60% monodansylcadaverin-positive cells between days 5 and 7. Cell death, as indicated by nuclear condensation, increased more gradually to about 18% of all cells on day 7. In cells with pyknotic nuclei cytokeratin appeared disassembled but retained its immunoreactivity; actin was still polymerized to filaments, as demonstrated by its reaction with phalloidin. Western blot analysis showed no significant cleavage of the monomeric cytokeratin fraction. For comparison, apoptotic or type I cell death was studied using the human colon cancer cell HT29/HI1 treated with the tyrosine kinase inhibitor tyrphostin A25 as a model. Cleavage of cytokeratin was already detectable in early morphological stages of apoptosis. F-actin was found to depolymerize; its globular form could be detected by antibodies; western blot analysis revealed no products of proteolytic cleavage. In conclusion, in our model of apoptosis, early stages are associated with depolymerization of actin and degradation of intermediate filaments. In contrast, during autophagic cell death intermediate and microfilaments are redistributed, but largely preserved, even beyond the stage of nuclear collapse. The present data support the concept that autophagic cell death is a separate entity of programmed cell death that is distinctly different from apoptosis.

Advanced glycosylation end-products and NO-dependent vasodilation in renal afferent arterioles from diabetic rats.

Systemic pressor responses to acetylcholine (ACh) are reduced in DM, an effect thought to be related to quenching of nitric oxide (NO) by advanced glycosylation end-products (AGE). We studied the effects of AGE in juxtamedullary (JM) afferent arterioles (AA) from rats with 40-50 days diabetes mellitus (DM) induced via streptozotocin. JM AA were perfused in vitro with solutions containing fresh RBCs suspended in either 6% bovine albumin or 6% AGE-albumin in euglycaemic Krebs-Ringer. Autoregulatory responses were evident in the DM vessels: AA constricted 31 +/- 2% (n=9) when perfusion pressure (PP) was raised from 60 to 140 mmHg. ACh (10 microM) caused a 43 +/- 15% dilation and Ca2+-channel blockade elicited a 95 +/- 14% dilation at 100 mmHg PP, indicating substantial basal vascular tone in DM AA. L-NAME (0.1 mM) constricted DM AA by 21 +/- 2% (n=9) at 100 mmHg PP, indicating significant basal NO production in DM vessels. Segments of renal resistance arteries from DM rats perfused in vitro responded to muscarinic stimulation and elevated glucose levels with significant increments in NO production, as measured with an NO-sensitive electrode. This observation shows that the renal endothelial NO system is intact in DM. While AGE in the perfusate dilated control AA, they had no effect on DM AA at all PP levels, although they blunted ACh-induced dilation. Hence, although AGE do appear to have vasoactive properties in the absence of hyperglycaemia, the results of this study are inconsistent with substantial NO quenching by AGE.

Programmed cell death (PCD). Apoptosis, autophagic PCD, or others?

The occurrence of cell death as a physiological event in multicellular organisms has been known for more than 150 years; in 1972 the term apoptosis was introduced on morphological grounds. However, accumulating evidence suggests that programmed cell death (PCD) is not confined to apoptosis, but that cells use different pathways for active self-destruction as reflected by different morphology: condensation prominent, type I or apoptosis; autophagy prominent, type II; etc. Autophagic PCD appears to be a phylogenetically old phenomenon; it may occur in physiological and disease states. We have studied the relation between morphological and biochemical events during autophagic and apoptotic PCD in human mammary, lymphoblast, and colon cancer cells using electron microscopy and proteom analysis. We find that autophagic cell death (type II) PCD includes degradation of Golgi apparatus, polyribosomes, and endoplasmic reticulum, which precedes nuclear destruction. Intermediate and microfilaments are largely preserved; presumably the cytoskeleton is required for autophagocytosis. Apoptosis (type I) PCD is characterized by condensation of cytoplasm and preservation of organelles; cytoskeletal elements disintegrate in early stages. Either type of PCD involves synthesis of distinct proteins. Finally, both types of PCD share features some of a cell's stress response (e.g., translocation of hsp90). In conclusion our findings support the concept that autophagic cell death is a separate pathway of PCD distinctly different from "classical" apoptosis. However, autophagic and apoptotic PCD should not be considered as mutually exclusive phenomena. Rather, they appear to reflect a high degree of flexibility in a cell's response to changes of environmental conditions, both physiological or pathological.

Yeast ascospore wall assembly requires two chitin deacetylase isozymes.

Chitin deacetylases are required for spore wall rigidity in Saccharomyces cerevisiae. Two chitin deacetylase genes (CDA1 and CDA2) have been identified in yeast. In this report we studied the biochemical properties of the chitin deacetylases encoded by CDA1 and CDA2 and we show how their elimination directly affects the ascospore wall assembly.

Mechanism of antimitogenic action of vitamin D in human colon carcinoma cells: relevance for suppression of epidermal growth factor-stimulated cell growth.

Because the efficacy of 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] in treatment of colon cancer might critically depend on its ability to specifically counteract epidermal growth factor (EGF)-stimulated tumor cell growth, we utilized human colon adenocarcinoma-derived cells in primary culture as well as the Caco-2 cell line to elucidate possible sites of interaction of 1alpha,25-(OH)2D3 with signaling from EGF receptor activation. In both types of colon cancer cells investigated, 10(-8) M 1alpha,25-(OH)2D3 reduced basal cell proliferation by about 50%, and prevented any rise in proliferation when colon cancer cells were treated with 25 ng/ml EGF: this can be explained by a marked inhibitory effect of 1alpha,25-(OH)2D3 on EGFR mRNA and protein expression. The steroid hormone also seemingly promotes EGF-induced internalization of apical and basolateral membrane EGFR. In addition, 1alpha,25-(OH)2D3 significantly reduced basal and EGF-stimulated expression of cyclin D1 at the mRNA and protein level in primary cultures as well as in the Caco-2 cell line. The ability of 1alpha,25-(OH)2D3 to interfere with a key event in cell cycle control and thereby to block mitogenic signaling from EGF could be seen as advantageous for the potential use of vitamin D compounds in colon cancer therapy.

The effects of partner insistence of condom usage on perceptions of the partner, the relationship, and the experience.

The present study examined the effects of an individual insisting on condom usage on how he or she is viewed by their partner. Participants were led through a realistic role-play scenario in which, after dating a new partner for "a while", they engaged in sexual intercourse. Results were such that after first time sex, participants indicated feeling more responsible, less at-risk, and less worried when a condom was used than when no condom was used. Participants evaluated their partner as more responsible, more caring, and less likely to have a sexually transmitted disease when a condom was used. Further, when a condom was used, the relationship was evaluated as enhanced, closer, more intimate, and more likely to be long lasting. The insistence of condom use by the participant's partner also resulted in less guilt and regret regarding the sexual experience itself. These results generally held true for both male and female participants.

Tri-iodothyronine inhibits multilayer formation of the osteoblastic cell line, MC3T3-E1, by promoting apoptosis.

Cell death through apoptosis is a well-known mechanism for maintaining homoeostasis in many developmental and pathological processes. We have recently presented evidence for the occurrence of apoptosis during the formation of bone-like tissue in vitro. MC3T3-E1 osteoblast-like cells in culture develop features of the osteoblastic phenotype and form many cell layers embedded in extracellular matrix which can mineralise. Tri-iodothyronine (T3), even though it enhances the expression of many osteoblastic features, attenuates the multilayer formation to about two layers. The aim of this study was to investigate how T3 prevents multilayer formation. MC3T3-E1 cells were seeded at different densities and cultured for up to 2 weeks. Thereafter we analysed proliferation rate and the distribution of the phases of the cell cycle and studied apoptosis. We found that T3 did not inhibit DNA synthesis. Analysis of the cell cycle phases showed an increase in the number of cells in G0/G1 with increasing cell density, but no significant effect of T3 treatment was found. Morphological investigations showed apoptotic features in both cell layers and culture supernatants. The cells exhibited typical plasma membrane blebbings, chromatin condensation, DNA fragmentation and phagocytosed apoptotic bodies. T3 treatment significantly increased the number of apoptotic cells. We conclude from our data that T3 inhibits multilayer formation of MC3T3-E1 cells by increasing the rate of apoptosis and not by inhibition of proliferation. Because apoptosis is a fundamental regulatory event during bone tissue differentiation, our findings emphasise the importance of thyroid hormones in bone maintenance and development.

Matrix mineralization in MC3T3-E1 cell cultures initiated by beta-glycerophosphate pulse.

MC3T3-E1 cells, grown in the presence of serum and ascorbate, express alkaline phosphatase and produce an extensive collagenous extracellular matrix that can be mineralized by the addition of beta-glycerophosphate (beta-GP). In the present work, we study the influence of concentration and duration of beta-GP treatment on the mineralization pattern in 4-week-old cell cultures. Amount and structure of mineral deposition were monitored by von Kossa staining, light, and electron microscopy, as well as small-angle X-ray scattering (SAXS) of unstained specimens. SAXS measures the total surface of the mineral phase and is therefore preferentially sensitive to very small crystals (typically <50 nm). It was used to determine the ratio (M) of small crystals to collagen matrix. A variety of mineralization patterns was observed to occur simultaneously, some associated with collagen within nodules or in deeper layers of the cultures and some independent of it. At a beta-GP concentration of 10 mmol, mineralization was initiated after about 24 h and continued to increase, irrespective of whether the high level of beta-GP was maintained or reduced to 2 mmol. With shorter pulses (<24 h), no significant mineralization was observed in the week following beta-GP pulse. With continuous treatment at 5 mmol beta-GP, the first signs of mineralization were detected 14 days after the beginning of treatment in the 4-week-old cultures, but no mineralization at all occurred at lower beta-GP concentrations. When cells were grown without ascorbic acid for 4 weeks, only two cell layers without collagen matrix were found. In these cultures, no mineralization detectable by SAXS could be induced with beta-GP. These data indicate that, in viable cells, high doses of beta-GP are essential for the nucleation of mineral crystals, but not for the progression of mineralization once crystals had been nucleated. In contrast, when 4-week-old cell cultures were devitalized, M was found to increase immediately, even at 2 mmol beta-GP. These results suggest that, in MC3T3-E1 cell cultures, cell viability is essential for prevention of spontaneous mineralization of the extracellular matrix.

Survival of normal colonic epithelial cells from both rats and humans is prolonged by coculture with rat embryo colonic fibroblasts.

Primary cultures of normal colonic epithelial cells from both humans (HCEC) and rats (RCEC) have been established using coculture with colon fibroblasts isolated from rat term embryos. While no other factors we have analyzed had any effect on the survival of epithelial cells, which is normally 3-4 days, coculture with viable fibroblasts extended this period to at least 2 weeks. The effects depended on early passages and low seeding densities of the fibroblasts and on direct cell-cell contact. We have obtained cultures of epithelial cells expressing keratin, laminin, and uvomorulin, displaying a polygonal, epithelial morphology and forming microvilli. DNA synthesis as measured by BrdU uptake into DNA varied widely between colonies of the same culture depending on cell morphology: flat colonies of RCECs contained 5.7% +/- 0.56% BrdU-positive cells, while the proportion in dense three-dimensional colonies reached 50.3% +/- 2.6%. In HCECs the growth fraction was lower, but showed the same distribution between classes of colonies. In the presence of rat embryonic colon fibroblasts, growth factors exerted survival activity on colonic epithelial cells. Consecutive addition of insulin and epidermal growth factor/fibroblast growth factor (EGF/FGF) increased colony number (15.0 +/- 1.0 and 23.0 +/- 2.0 colonies/well respectively; p < or = 0.05 increased above control) and size (1022 +/- 155 and 1207 +/- 158 cells/colony respectively; p < or = 0.05 increased above control) compared to serum-free control medium and basic MEM without growth factors. BrdU labeling index was not increased, however: EGF/FGF actually decreased BrdU labeling from 33.2% +/- 3.9% in controls to 21.3% +/- 3.8% in the EGF/FGF group (p < or = 0.05) owing to the high proportion of flat colonies consisting of resting cells. The newly established culture model can now be used to investigate growth control mechanisms in colonic mucosa and the effects of toxic and/or tumor-promoting substances on these mechanisms.

[The role of nitric oxide in reproduction]. / Die Rolle des Stickstoffmonoxids bei der Reproduktion.

Nitrix oxide (NO) is a highly reactive and short-lived radical (half-life time: 10-12 s), which is derived from L-arginine by the NO synthases (NOS) in several organ systems. The release of NO by endothelial cells leads to rapid relaxation of vascular smooth muscle cells, whereas release by several neuronal cells causes neurotransmission. When NOS is actively induced in immune cells or certain epithelia it causes cytotoxicity and/or apoptosis of these cells. In the reproductive organs NO is now considered to be an important trigger molecule for several physiological mechanisms. Follicular synthesized NO is involved in rupture of the follicle during ovulation. Moreover, NO participates in the acrosome reaction of spermatozoa during capacitation. Apoptosis and collagenolysis of the functional endometrium may be involved in endometrial shedding during menstruation. Since NO induces both apoptosis and collagenolysis, the newly discovered production of NO in late secretory endometrium could act as a key mechanism in the process of menstrual disintegration of the endometrium. Additionally, NO is necessary to support and maintain the decidualization process and plays a pivotal role in implantation.

Epidermal growth factor receptor expression in primary cultured human colorectal carcinoma cells.

In situ hybridization on human colon tissue demonstrates that epidermal growth factor receptor (EGFR) mRNA expression is strongly increased during tumour progression. To obtain test systems to evaluate the relevance of growth factor action during carcinogenesis, primary cultures from human colorectal carcinomas were established. EGFR distribution was determined in 2 of the 27 primary cultures and was compared with that in well-defined subclones derived from the Caco-2 cell line, which has the unique property to differentiate spontaneously in vitro in a manner similar to normal enterocytes. The primary carcinoma-derived cells had up to three-fold higher total EGFR levels than the Caco-2 subclones and a basal mitotic rate at least fourfold higher. The EGFR affinity constant is 0.26 nmol l(-1), which is similar to that reported in Caco-2 cells. The proliferation rate of Caco-2 cells is mainly induced by EGF from the basolateral cell surface where the majority of receptors are located, whereas primary cultures are strongly stimulated from the apical side also. This corresponds to a three- to fivefold higher level of EGFR at the apical cell surface. This redistribution of EGFR to apical plasma membranes in advanced colon carcinoma cells suggests that autocrine growth factors in the colon lumen may play a significant role during tumour progression.

Endocytic routes to the Golgi apparatus.

The endocytic routes of labelled lectins as well as cationic ferritin were studied in cells with a regulated secretion, i.e. pancreatic beta cells, and in constitutively secreting cells, i.e. fibroblasts and HepG2 hepatoma cells, paying particular attention to routes into the Golgi apparatus. Considerable amounts of internalised molecules were taken up into the trans Golgi network (TGN) and into Golgi subcompartments in all three cell types as well as in secretory granules of the pancreatic beta cells. The internalised materials did not pass rapidly the TGN and Golgi stacks, but were still present hours after internalisation, being then particularly concentrated in TGN-elements and in the transmost Golgi cisterna. Endocytosed materials reached forming secretory granules present in the TGN. Further, direct fusion between endocytotic vesicles and mature secretory granules was observed. Golgi subcompartments as well as endocytic TGN containing endocytosed materials were in close apposition to specialised regions of the endoplasmic reticulum. The Golgi apparatus including its parts containing endocytosed materials were transformed into a tubular reticulum upon treatment with the fungal metabolite Brefeldin A. Rarely, internalised material was observed in the lumen of the endoplasmic reticulum, thus providing evidence for an endocytic plasma membrane to endoplasmic reticulum route.

Apoptosis in human colorectal tumours: ultrastructure and quantitative studies on tissue localization and association with bak expression.

Apoptotic cell death in human tumours has been demonstrated by electron and light microscopy. In adenomas, fragmented and apoptotic nuclei and signs of phagocytosis have been observed close to the basement membrane. In carcinomas the characteristic structures were apoptotic bodies with small fragments of chromatin. DNA fragmentation was shown by in situ end-labelling. Quantitative assessment of apoptosis and proliferation revealed a high apoptotic index (AI) in all types of adenoma (tubular: 1.77+/-0.35%, tubulovillous: 2.38+/-0.41%; villous: 3.3+/-0.39%) as well as loss of compartmentalization of proliferating and dying cells. In carcinomas a shift towards proliferation was evident, as shown by lower AIs than in adenomas (0.9+/-0.68% and 1.1+/-0.12% for moderately and poorly differentiated tumours), higher Ki67 indices (38.32+/-2.23% and 57+/-3.89%, respectively) and higher mitosis (0.9+/-0.56% and 1.21+/-0.17%, respectively). However, apoptosis was observed in all tumours and is available as a target for therapeutic intervention. Expression of the apoptosis related proteins bcl-2 and bak also reflected loss of compartmentalization. While bcl-2 did not show a consistent relationship to AI in tumour specimens, bak was positively correlated with apoptosis in 4 of 8 adenomas and 4 of 7 carcinomas, suggesting a role for this protein in the induction of apoptosis in a subset of tumours.

1,25-Dihydroxy vitamin D3 and tri-iodothyronine stimulate the expression of a protein immunologically related to osteocalcin.

Osteocalcin (OC), a bone-specific protein, is a marker of late osteoblastic differentiation. Its expression is influenced by various growth factors and hormones. We investigated the effect of 1, 25-dihydroxy vitamin D3 (D3) and tri-iodothyronine (T3) on OC expression in osteoblast-like MC3T3-E1 cells. A heterologous OC green fluorescence protein (GFP) fusion vector was established and expressed to study possible effects on protein transport. Immunostaining of endogenous OC revealed a significant increase in the percentage of positive cells after D3 and T3 treatment. This was consistent for MC3T3-E1 cells as well as nonosteogenic NIH-3T3 and mammary carcinoma cells, but not for neuroblastoma cells. The perinuclear immunostaining corresponded to the NBD C6 ceramide Golgi staining. Conversely, we found a strong induction of OC in MC3T3-E1 cells at the mRNA and protein levels only with T3 and not with D3. OC mRNA and protein expression was not detected in NIH fibroblasts. OC GFP transfection experiments indicate rapid transport and secretion of OC, because OC GFP was not found to be accumulated at intracellular compartments after hormone treatment. We conclude that the strong perinuclear immunostaining does not represent OC but a protein immunologically related to OC, as indicated by preabsorption experiments. The expression of this OC epitope-sharing protein is regulated by both D3 and T3 in the osteoblastic MC3T3-E1 and in nonosteogenic cells.