Tissue-dependent subcellular localization of Drosophila arginine methyl-transferase 4 (DART4), a coactivator whose overexpression affects neither viability nor differentiation

Drosophila arginine methyl-transferase 4 (DART4) belongs to the type I class of arginine methyltransferases. It catalyzes the methylation of arginine residues to monomethylarginines and asymmetrical dimethylarginines. The DART4 sequence is highly similar to mammalian PRMT4/CARM1, and DART4 substrate specificity has been conserved, too. Recently it was suggested that DART4/Carmer functions in ecdysone receptor mediated apoptosis of the polytene larval salivary glands and an apparent up-regulation of DART4/Carmer mRNA levels before tissue histolysis was reported. Here we show that in Drosophila larvae, DART4 is mainly expressed in the imaginal disks and in larval brains, and to a much lesser degree in the polytene larval tissue such as salivary glands. In glands, DART4 protein is present in the cytoplasm and the nucleus. The nuclear signal emanates from the extrachromosomal domain and gets progressively restricted to the region of the nuclear lamina upon pupariation. Surprisingly, DART4 levels do not increase in salivary glands during pupariation, and overexpression of DART4 does not cause precautious cell death in the glands. Furthermore, over- and misexpression of DART4 under the control of the alpha tubulin promoter do not lead to any major problem in the life of a fly. This suggests that DART4 activity is regulated at the posttranslational level and/or that it acts as a true cofactor in vivo. We present evidence that nuclear localization of DART4 may contribute to its function because DART4 accumulation changes from a distribution with a strong cytoplasmic component during the transcriptional quiescence of the young embryo to a predominantly nuclear one at the onset of zygotic transcription.

Sex determination and temperature-induced sex differentiation in three natural populations of Nile tilapia (Oreochromis niloticus) adapted to extreme temperature conditions

As a species of major interest for aquaculture, the sex determination system (SDS) of Nile tilapia, Oreochromis niloticus, has been widely investigated. In this species, sex determination is considered to be governed by the interactions between a complex system of genetic sex determination factors (GSD) and the influence of temperature (TSD) during a critical period. Previous studies were exclusively carried out on domestic stocks with the genetic and maintenance limitations associated. Given the wide distribution and adaptation potential of the Nile tilapia, we investigated under controlled conditions the sex determination system of natural populations adapted to three extreme thermal regimes: stable extreme environments in Ethiopia, either cold temperatures in a highland lake (Lake Koka), or warm temperatures in hydrothermal springs (Lake Metahara), and an environment with large seasonal variations in Ghana (Kpandu, Lake Volta). The sex ratio analysis was conducted on progenies reared under constant basal (27 degrees C) or high (36 degrees C) temperatures during the 30 days following yolk-sac resorption. Sex ratios of the progenies reared at standard temperature suggest that the three populations share a similar complex GSD system based on a predominant male heterogametic factor with additional influences of polymorphism at this locus and/or action of minor factors. The three populations presented a clear thermosensitivity of sex differentiation, with large variations in the intensity of response depending on the parents. This confirms the presence of genotype-environment interactions in TSD of Nile tilapia. Furthermore the existence of naturally sex-reversed individuals is strongly suggested in two populations (Kpandu and Koka). However, it was not possible here to infer if the sex-inversion resulted from minor genetic factors and/or environmental influences. The present study demonstrated for the first time the conservation of a complex SDS combining polymorphic GSD and TSD components in natural populations of Nile tilapia. We discuss the evolutionary implications of our findings and highlight the importance of field investigations of sex determination. (c) 2007 Elsevier B.V. All rights reserved.

HIC1 tumour suppressor gene is suppressed in acute myeloid leukaemia and induced during granulocytic differentiation

A hallmark of acute myeloid leukaemia (AML) is a block in differentiation caused by deregulated gene expression. The tumour suppressor Hypermethylated In Cancer 1 (HIC1) is a transcriptional repressor, which is epigenetically silenced in solid cancers. HIC1 mRNA expression was found to be low in 128 patient samples of AML and CD34+ progenitor cells when compared with terminally differentiated granulocytes. HIC1 mRNA was induced in a patient with t(15;17)-positive acute promyelocytic leukaemia receiving all-trans retinoic acid (ATRA) therapy. We therefore investigated whether HIC1 plays a role in granulocytic differentiation and whether loss of function of this gene might contribute to the differentiation block in AML. We evaluated HIC1 mRNA levels in HL-60 and U-937 cells upon ATRA-induced differentiation and in CD34+ progenitor cells after granulocyte colony-stimulating factor-induced differentiation. In both models of granulocytic differentiation, we observed significant HIC1 induction. When HIC1 mRNA was suppressed in HL-60 cells using stably expressed short hairpin RNA targeting HIC1, granulocytic differentiation was altered as assessed by CD11b expression. Bisulphite sequencing of GC-rich regions (CpG islands) in the HIC1 promoter provided evidence that the observed suppression in HL-60 cells was not because of promoter hypermethylation. Our findings indicate a role for the tumour suppressor gene HIC1 in granulocytic differentiation. Low expression of HIC1 may very well contribute to pathogenic events in leukaemogenesis.

Chondrogenic differentiation of bovine synovium: bone morphogenetic proteins 2 and 7 and transforming growth factor beta1 induce the formation of different types of cartilaginous tissue

OBJECTIVE: To compare the potential of bone morphogenetic proteins 2 and 7 (BMP-2 and BMP-7) and transforming growth factor beta1 (TGFbeta1) to effect the chondrogenic differentiation of synovial explants by analyzing the histologic, biochemical, and gene expression characteristics of the cartilaginous tissues formed. METHODS: Synovial explants derived from the metacarpal joints of calves were cultured in agarose. Initially, BMP-2 was used to evaluate the chondrogenic potential of the synovial explants under different culturing conditions. Under appropriate conditions, the chondrogenic effects of BMP-2, BMP-7, and TGFbeta1 were then compared. The differentiated tissue was characterized histologically, histomorphometrically, immunohistochemically, biochemically, and at the gene expression level. RESULTS: BMP-2 induced the chondrogenic differentiation of synovial explants in a dose- and time-dependent manner under serum- and dexamethasone-free conditions. The expression levels of cartilage-related genes increased in a time-dependent manner. BMP-7 was more potent than BMP-2 in inducing chondrogenesis, but the properties of the differentiated tissue were similar in each case. The type of cartilaginous tissue formed under the influence of TGFbeta1 differed in terms of both cell phenotype and gene expression profiles. CONCLUSION: The 3 tested members of the TGFbeta superfamily have different chondrogenic potentials and induce the formation of different types of cartilaginous tissue. To effect the full differentiation of synovial explants into a typically hyaline type of articular cartilage, further refinement of the stimulation conditions is required. This might be achieved by the simultaneous application of several growth factors.

A multiplex real-time PCR assay for rapid detection and differentiation of 25 bacterial and fungal pathogens from whole blood samples

Early detection of bloodstream infections (BSI) is crucial in the clinical setting. Blood culture remains the gold standard for diagnosing BSI. Molecular diagnostic tools can contribute to a more rapid diagnosis in septic patients. Here, a multiplex real-time PCR-based assay for rapid detection of 25 clinically important pathogens directly from whole blood in <6 h is presented. Minimal analytical sensitivity was determined by hit rate analysis from 20 independent experiments. At a concentration of 3 CFU/ml a hit rate of 50% was obtained for E. aerogenes and 100% for S. marcescens, E. coli, P. mirabilis, P. aeruginosa, and A. fumigatus. The hit rate for C. glabrata was 75% at 30 CFU/ml. Comparing PCR identification results with conventional microbiology for 1,548 clinical isolates yielded an overall specificity of 98.8%. The analytical specificity in 102 healthy blood donors was 100%. Although further evaluation is warranted, our assay holds promise for more rapid pathogen identification in clinical sepsis.

Steady-state diffusion imaging for MR in-vivo evaluation of reparative cartilage after matrix-associated autologous chondrocyte transplantation at 3 tesla--preliminary results

OBJECTIVES: To demonstrate the feasibility of time-reversed fast imaging with steady-state precession (FISP) called PSIF for diffusion-weighted imaging of cartilage and cartilage transplants in a clinical study. MATERIAL AND METHODS: In a cross-sectional study 15 patients underwent MRI using a 3D partially balanced steady-state gradient echo pulse sequence with and without diffusion weighting at two different time points after matrix-associated autologous cartilage transplantation (MACT). Mean diffusion quotients (signal intensity without diffusion-weighting divided by signal intensity with diffusion weighting) within the cartilage transplants were compared to diffusion quotients found in normal cartilage. RESULTS: The global diffusion quotient found in repair cartilage was significantly higher than diffusion values in normal cartilage (p<0.05). There was a decrease between the earlier and the later time point after surgery. CONCLUSIONS: In-vivo diffusion-weighted imaging based on the PSIF technique is possible. Our preliminary results show follow-up of cartilage transplant maturation in patients may provide additional information to morphological assessment.

Longitudinal evaluation of cartilage composition of matrix-associated autologous chondrocyte transplants with 3-T delayed gadolinium-enhanced MRI of cartilage

OBJECTIVE: The purposes of this study were to use delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) to evaluate the zonal distribution of glycosaminoglycans (GAGs) in normal cartilage and repair tissue and to use 3-T MRI to monitor the GAG content in matrix-associated autologous chondrocyte transplants. SUBJECTS AND METHODS: Fifteen patients who underwent matrix-associated autologous chondrocyte transplantation in the knee joint underwent MRI at baseline and 3-T follow-up MRI 1 year later. Total and zonal changes in longitudinal relaxivity (deltaR1) and relative deltaR1 were calculated for repair tissue and normal hyaline cartilage and compared by use of analysis of variance. RESULTS: There was a significant difference between the mean deltaR1 of repair tissue and that of reference cartilage at baseline and follow-up (p < 0.001). There was a significant increase in deltaR1 value and a decrease in GAG content from the deep layer to the superficial layer in the reference cartilage and almost no variation and significantly higher values for the repair tissue at both examinations. At 1-year follow-up imaging, there was a 22.7% decrease in deltaR1 value in the deep zone of the transplant. CONCLUSION: T1 mapping with dGEMRIC at 3 T shows the zonal structure of normal hyaline cartilage, highly reduced zonal variations in repair tissue, and a tendency toward an increase in global and zonal GAG content 1 year after transplantation.

In vivo biochemical 7.0 Tesla magnetic resonance: preliminary results of dGEMRIC, zonal T2, and T2* mapping of articular cartilage

INTRODUCTION: Ultra-high-field whole-body systems (7.0 T) have a high potential for future human in vivo magnetic resonance imaging (MRI). In musculoskeletal MRI, biochemical imaging of articular cartilage may benefit, in particular. Delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) and T2 mapping have shown potential at 3.0 T. Although dGEMRIC, allows the determination of the glycosaminoglycan content of articular cartilage, T2 mapping is a promising tool for the evaluation of water and collagen content. In addition, the evaluation of zonal variation, based on tissue anisotropy, provides an indicator of the nature of cartilage ie, hyaline or hyaline-like articular cartilage.Thus, the aim of our study was to show the feasibility of in vivo dGEMRIC, and T2 and T2* relaxation measurements, at 7.0 T MRI; and to evaluate the potential of T2 and T2* measurements in an initial patient study after matrix-associated autologous chondrocyte transplantation (MACT) in the knee. MATERIALS AND METHODS: MRI was performed on a whole-body 7.0 T MR scanner using a dedicated circular polarization knee coil. The protocol consisted of an inversion recovery sequence for dGEMRIC, a multiecho spin-echo sequence for standard T2 mapping, a gradient-echo sequence for T2* mapping and a morphologic PD SPACE sequence. Twelve healthy volunteers (mean age, 26.7 +/- 3.4 years) and 4 patients (mean age, 38.0 +/- 14.0 years) were enrolled 29.5 +/- 15.1 months after MACT. For dGEMRIC, 5 healthy volunteers (mean age, 32.4 +/- 11.2 years) were included. T1 maps were calculated using a nonlinear, 2-parameter, least squares fit analysis. Using a region-of-interest analysis, mean cartilage relaxation rate was determined as T1 (0) for precontrast measurements and T1 (Gd) for postcontrast gadopentate dimeglumine [Gd-DTPA(2-)] measurements. T2 and T2* maps were obtained using a pixelwise, monoexponential, non-negative least squares fit analysis; region-of-interest analysis was carried out for deep and superficial cartilage aspects. Statistical evaluation was performed by analyses of variance. RESULTS: Mean T1 (dGEMRIC) values for healthy volunteers showed slightly different results for femoral [T1 (0): 1259 +/- 277 ms; T1 (Gd): 683 +/- 141 ms] compared with tibial cartilage [T1 (0): 1093 +/- 281 ms; T1 (Gd): 769 +/- 150 ms]. Global mean T2 relaxation for healthy volunteers showed comparable results for femoral (T2: 56.3 +/- 15.2 ms; T2*: 19.7 +/- 6.4 ms) and patellar (T2: 54.6 +/- 13.0 ms; T2*: 19.6 +/- 5.2 ms) cartilage, but lower values for tibial cartilage (T2: 43.6 +/- 8.5 ms; T2*: 16.6 +/- 5.6 ms). All healthy cartilage sites showed a significant increase from deep to superficial cartilage (P < 0.001). Within healthy cartilage sites in MACT patients, adequate values could be found for T2 (56.6 +/- 13.2 ms) and T2* (18.6 +/- 5.3 ms), which also showed a significant stratification. Within cartilage repair tissue, global mean values showed no difference, with 55.9 +/- 4.9 ms for T2 and 16.2 +/- 6.3 ms for T2*. However, zonal assessment showed only a slight and not significant increase from deep to superficial cartilage (T2: P = 0.174; T2*: P = 0.150). CONCLUSION: In vivo T1 dGEMRIC assessment in healthy cartilage, and T2 and T2* mapping in healthy and reparative articular cartilage, seems to be possible at 7.0 T MRI. For T2 and T2*, zonal variation of articular cartilage could also be evaluated at 7.0 T. This zonal assessment of deep and superficial cartilage aspects shows promising results for the differentiation of healthy and affected articular cartilage. In future studies, optimized protocol selection, and sophisticated coil technology, together with increased signal at ultra-high-field MRI, may lead to advanced biochemical cartilage imaging.

Pineal parenchymal tumor of intermediate differentiation: diagnostic pitfalls and discussion of treatment options of a rare tumor entity

Tumors of the pineal region are uncommon, comprising approximately 0.4-1% of all intracranial tumors in adults in European and American series. Histopathologically, they are a very heterogeneous group of tumors. Of genuine pineal tumors, pineal parenchymal tumors of intermediate differentiation (PPTIDs) are the least frequently found type. In this paper, we report on the case of a patient with an unexpected and difficult-to-diagnose PPTID. A 2.2 x 2.2-cm midline mass within the posterior part of the third ventricle with consecutive obstructive hydrocephalus was found in a 44-year-old man presenting with diplopia and gait disturbances. There was no clear connection of the tumor to the pineal gland. Differential diagnosis included all intraventricular and midline tumors, therefore a biopsy was taken. Preliminary histopathological diagnosis was germinoma or primitive neuroectodermal tumor, and the tissue sample was reexamined by a referential neuropathological institute. Final diagnosis was PPTID. The tumor was then resected through a transventricular/transchoroidal approach. Histopathological examination of tumor specimen confirmed the diagnosis of a PPTID. Postoperatively, the patient received gamma-knife radiosurgery. At 1-year follow-up, there are no signs of tumor regrowth. Diagnosis of pineal parenchymal tumors in general and PPTIDs in particular can be troublesome. Their histopathological features are still being defined, as is the biological behavior of the different tumor entities. Thus, treatment options including surgery, radiation therapy, and chemotherapy remain controversial. We recommend surgical removal of PPTID, preferably in toto whenever the size of the tumor permits that kind of excision.

SFRP-4 abrogates Wnt-3a-induced beta-catenin and Akt/PKB signalling and reverses a Wnt-3a-imposed inhibition of in vitro mammary differentiation

ABSTRACT: BACKGROUND: Conserved Wnt ligands are critical for signalling during development; however, various factors modulate their activity. Among these factors are the Secreted Frizzled-Related Proteins (SFRP). We previously isolated the SFRP-4 gene from an involuting rat mammary gland and later showed that transgenic mice inappropriately expressing SFRP-4 during lactation exhibited a high level of apoptosis with reduced survival of progeny. RESULTS: In order to address the questions related to the mechanism of Wnt signalling and its inhibition by SFRP-4 which we report here, we employed partially-purified Wnt-3a in a co-culture model system. Ectopic expression of SFRP-4 was accomplished by infection with a pBabepuro construct. The co-cultures comprised Line 31E mouse mammary secretory epithelial cells and Line 30F, undifferentiated, fibroblast-like mouse mammary cells. In vitro differentiation of such co-cultures can be demonstrated by induction of the beta-casein gene in response to lactogenic hormones.We show here that treatment of cells with partially-purified Wnt-3a initiates Dvl-3, Akt/PKB and GSK-3beta hyperphosphorylation and beta-catenin activation. Furthermore, while up-regulating the cyclin D1 and connexin-43 genes and elevating transepithelial resistance of Line 31E cell monolayers, Wnt-3a treatment abrogates differentiation of co-cultures in response to the lactogenic hormones prolactin, insulin and glucocorticoid. Cells which express SFRP-4, however, are largely unaffected by Wnt-3a stimulation. Since a physical association between Wnt-3a and SFRP-4 could be demonstrated with immunoprecipitation/Western blotting experiments, this interaction, presumably owing to the Frizzled homology region typical of all SFRPs, explains the refractory response to Wnt-3a which was observed. CONCLUSION: This study demonstrates that Wnt-3a treatment activates the Wnt signalling pathway and interferes with in vitro differentiation of mammary co-cultures to beta-casein production in response to lactogenic hormones. Similarly, in another measure of differentiation, following Wnt-3a treatment mammary epithelial cells could be shown to up-regulate the cyclin D1 and connexin-43 genes while phenotypically they show increased transepithelial resistance across the cell monolayer. All these behavioural changes can be blocked in mammary epithelial cells expressing SFRP-4. Thus, our data illustrate in an in vitro model a mechanism by which SFRP-4 can modulate a differentiation response to Wnt-3a.

Insulin-like growth factor (IGF)-binding protein-3 (IGFBP-3) is closely associated with the chondrocyte nucleus in human articular cartilage

OBJECTIVE: Insulin-like growth factor-I (IGF-I) is critically involved in the control of cartilage matrix metabolism. It is well known that IGF-binding protein-3 (IGFBP-3) is increased during osteoarthritis (OA), but its function(s) is not known. In other cells, IGFBP-3 can regulate IGF-I action in the extracellular environment and can also act independently inside the cell; this includes transcriptional gene control in the nucleus. These studies were undertaken to localize IGFBP-3 in human articular cartilage, particularly within cells. DESIGN: Cartilage was dissected from human femoral heads derived from arthroplasty for OA, and OA grade assessed by histology. Tissue slices were further characterized by extraction and assay of IGFBPs by IGF ligand blot (LB) and by enzyme-linked immunosorbent assay (ELISA). Immunohistochemistry (IHC) for IGF-I and IGFBP-3 was performed on cartilage from donors with mild, moderate and severe OA. Indirect fluorescence and immunogold-labeling IHC studies were included. RESULTS: LBs of chondrocyte lysates showed a strong signal for IGFBP-3. IHC of femoral cartilage sections at all OA stages showed IGF-I and IGFBP-3 matrix stain particularly in the top zones, and closely associated with most cells. A prominent perinuclear/nuclear IGFBP-3 signal was seen. Controls using non-immune sera or antigen-blocked antibody showed negative or strongly reduced stain. In frozen sections of human ankle cartilage, immunofluorescent IGFBP-3 stain co-localized with the nuclear 4',6-diamidino-2-phenyl indole (DAPI) stain in greater than 90% of the cells. Immunogold IHC of thin sections and transmission electron immunogold microscopy of ultra-thin sections showed distinct intra-nuclear staining. CONCLUSIONS: IGFBP-3 in human cartilage is located in the matrix and within chondrocytes in the cytoplasm and nuclei. This new finding indicates that the range of IGFBP-3 actions in articular cartilage is likely to include IGF-independent roles and opens the door to studies of its nuclear actions, including the possible regulation of hormone receptors or transcriptional complexes to control gene action.

Effect of reduced oxygen tension and long-term mechanical stimulation on chondrocyte-polymer constructs

We have investigated the influence of long-term confined dynamic compression and surface motion under low oxygen tension on tissue-engineered cell-scaffold constructs. Porous polyurethane scaffolds (8 mm x 4 mm) were seeded with bovine articular chondrocytes and cultured under normoxic (21% O(2)) or hypoxic (5% O(2)) conditions for up to 4 weeks. By means of our joint-simulating bioreactor, cyclic axial compression (10-20%; 0.5 Hz) was applied for 1 h daily with a ceramic ball, which simultaneously oscillated over the construct surface (+/-25 degrees; 0.5 Hz). Culture under reduced oxygen tension resulted in an increase in mRNA levels of type II collagen and aggrecan, whereas the expression of type I collagen was down-regulated at early time points. A higher glycosaminoglycan content was found in hypoxic than in normoxic constructs. Immunohistochemical analysis showed more intense type II and weaker type I collagen staining in hypoxic than in normoxic cultures. Type II collagen gene expression was slightly elevated after short-term loading, whereas aggrecan mRNA levels were not influenced by the applied mechanical stimuli. Of importance, the combination of loading and low oxygen tension resulted in a further down-regulation of collagen type I mRNA expression, contributing to the stabilization of the chondrocytic phenotype. Histological results confirmed the beneficial effect of mechanical loading on chondrocyte matrix synthesis. Thus, mechanical stimulation combined with low oxygen tension is an effective tool for modulating the chondrocytic phenotype and should be considered when chondrocytes or mesenchymal stem cells are cultured and differentiated with the aim of generating cartilage-like tissue in vitro.