Distinct regions control transcriptional activation of the alpha1(VI) collagen promoter in different tissues of transgenic mice.

To identify regions involved in tissue specific regulation of transcription of the alpha1(VI) collagen chain, transgenic mice were generated carrying various portions of the gene's 5'-flanking sequence fused to the E. coli beta-galactosidase gene. Analysis of the transgene expression pattern by X-gal staining of embryos revealed that: (a) The proximal 0.6 kb of promoter sequence activated transcription in mesenchymal cells at sites of insertion of superficial muscular aponeurosis into the skin; tendons were also faintly positive. (b) The region between -4.0 and -5.4 kb from the transcription start site was required for activation of the transgene in nerves. It also drove expression in joints, in intervertebral disks, and in subepidermal and vibrissae mesenchyme. (c) The fragment comprised within -6.2 and -7.5 kb was necessary for high level transcription in skeletal muscle and meninges. Positive cells in muscle were mostly mononuclear and probably included connective tissue elements, although staining of myoblasts was not ruled out. This fragment also activated expression in joints, in intervertebral disks, and in subepidermal and vibrissae mesenchyme. (d) beta-Galactosidase staining in vibrissae induced by the sequences -4.0 to -5.4 and -6.2 to -7.5 was not coincident: with the latter sequence labeled nuclei were found mainly in the ventral and posterior quadrant, and, histologically, in the outer layers of mesenchyme surrounding and between the follicles, whereas with the former the remaining quadrants were positive and expressing cells were mostly in the inner layers of the dermal sheath. (e) Other tissues, notably lung, adrenal gland, digestive tract, which produce high amounts of collagen type VI, did not stain for beta-galactosidase. (f) Central nervous system and retina, in which the endogenous gene is inactive, expressed the lacZ transgene in most lines. The data suggest that transcription of alpha1(VI) in different tissues is regulated by distinct sequence elements in a modular arrangement, a mechanism which confers high flexibility in the temporal and spatial pattern of expression during development.

Report of a case of discoid lupus erythematosus localised to the oral cavity: immunofluorescence findings.

Discoid Lupus Erythematosus (DLE) is a chronic disease with a typical cutaneous involvement. This pathology rarely involves mucosa: oral cavity is interested in 20 percent of DLE patients. We describe a case of oral DLE in a 50-year-old woman with an anamnesis for autoimmune disorders. This study shows the helpful role of immunofluorescence in the diagnosis of autoimmune diseases. The first diagnostic step was the clinical observation of the oral mucosa: the lesion area was erythematous, atrophic and hyperkeratotic. The patient then underwent laboratory examination. We utilized human epithelial cells (Hep-2010) for Indirect Immuno-Fluorescence (IIF). Moreover, the biopsy site for Direct Immuno-Fluorescence (DIF) and histopathological analysis was the untreated oral lesion. IIF detected an increase of Anti-Nuclear Antibody (ANA) and positivity for SSA-RO. By DIF, we observed IgG/IgA/fibrinogen along basal layer. Multiple biopsies reported signs of chronic basal damage. Steroid systemic therapy induced a considerable lesion regression. We suggest the use of immunofluorescence with the integration of further data to improve diagnosis of rare diseases and to establish a suitable therapy.

A microRNA code for prostate cancer metastasis.

Although the development of bone metastasis is a major detrimental event in prostate cancer, the molecular mechanisms responsible for bone homing and destruction remain largely unknown. Here we show that loss of miR-15 and miR-16 in cooperation with increased miR-21 expression promote prostate cancer spreading and bone lesions. This combination of microRNA endows bone-metastatic potential to prostate cancer cells. Concomitant loss of miR-15/miR-16 and gain of miR-21 aberrantly activate TGF-ß and Hedgehog signaling, that mediate local invasion, distant bone marrow colonization and osteolysis by prostate cancer cells. These findings establish a new molecular circuitry for prostate cancer metastasis that was validated in patients' cohorts. Our data indicate a network of biomarkers and druggable pathways to improve patient treatment.

Identification of a recognition element for CAAT-enhancer binding proteins (C/EBPs) in the elastin promoter.

DNase I footprinting experiments with a DNA fragment of the human elastin promoter have revealed a protected segment comprised between -156 and -172 nucleotides from the translation start site. Various types of gel retardation experiments indicate that the protected element binds different members of the C/EBP family of transcription factors. CAT (chloramphenicol acetyltransferase) fusion constructs carrying the wild type or a mutated promoter sequence were transfected into NIH3T3 and chick embryo aorta cells. The mutation significantly lowered CAT expression in NIH3T3 cells, but was ineffective in aorta cells. Cotransfection of the CAT promoter constructs with eucaryotic vectors expressing C/EBPs, did not affect the production of the reporter gene in NIH3T3 cells; on the contrary a several-fold increase of CAT activity was observed in aortic cells. This increase, however, was identical for the wild type and the mutated constructs. Taken together the data indicate that the elastin promoter contains a recognition site for proteins of the C/EBP family and that the function of this cis-acting element on basal elastin transcription varies with the cell type.

Lulwoana sp., a dark septate endophyte in roots of Posidonia oceanica (L.) Delile seagrass.

Posidonia oceanica is the most common, widespread and important monocotyledon seagrass in the Mediterranean Basin, and hosts a large biodiversity of species, including microorganisms with key roles in the marine environment. In this study, we ascertain the presence of a fungal endophyte in the roots of P. oceanica growing on different substrata (rock, sand and matte) in two Sicilian marine meadows. Staining techniques on root fragments and sections, in combination with microscope observations, were used to visualise the fungal presence and determine the percentage of fungal colonisation (FC) in this tissue. In root fragments, statistical analysis of the FC showed a higher mean in roots anchored on rock than on matte and sand. In root sections, an inter- and intracellular septate mycelium, producing intracellular microsclerotia, was detected from the rhizodermis to the vascular cylinder. Using isolation techniques, we obtained, from both sampling sites, sterile, slow-growing fungal colonies, dark in colour, with septate mycelium, belonging to the dark septate endophytes (DSEs). DNA sequencing of the internal transcribed spacer (ITS) region identified these colonies as Lulwoana sp. To our knowledge, this is the first report of Lulwoana sp. as DSE in roots of P. oceanica. Moreover, the highest fungal colonisation, detected in P. oceanica roots growing on rock, suggests that the presence of the DSE may help the host in several ways, particularly in capturing mineral nutrients through lytic activity.

TAZ is required for metastatic activity and chemoresistance of breast cancer stem cells.

Metastatic growth in breast cancer (BC) has been proposed as an exclusive property of cancer stem cells (CSCs). However, formal proof of their identity as cells of origin of recurrences at distant sites and the molecular events that may contribute to tumor cell dissemination and metastasis development are yet to be elucidated. In this study, we analyzed a set of patient-derived breast cancer stem cell (BCSC) lines. We found that in vitro BCSCs exhibit a higher chemoresistance and migratory potential when compared with differentiated, nontumorigenic, breast cancer cells (dBCCs). By developing an in vivo metastatic model simulating the disease of patients with early BC, we observed that BCSCs is the only cell population endowed with metastatic potential. Gene-expression profile studies comparing metastagenic and non-metastagenic cells identified TAZ, a transducer of the Hippo pathway and biomechanical cues, as a central mediator of BCSCs metastatic ability involved in their chemoresistance and tumorigenic potential. Overexpression of TAZ in low-expressing dBCCs induced cell transformation and conferred tumorigenicity and migratory activity. Conversely, loss of TAZ in BCSCs severely impaired metastatic colonization and chemoresistance. In clinical data from 99 BC patients, high expression levels of TAZ were associated with shorter disease-free survival in multivariate analysis, thus indicating that TAZ may represent a novel independent negative prognostic factor. Overall, this study designates TAZ as a novel biomarker and a possible therapeutic target for BC.

Technetium-99m-methylene diphosphonate scintimammography to image primary breast cancer.

UNLABELLED: Technetium-99m-methylene diphosphonate (MDP) uptake within breast lesions was investigated during routine presurgical bone scintigraphy in a cohort of women at high risk for cancer who were candidates for surgery or excisional biopsy. The aim was twofold: (a) to demonstrate positive 99mTc-MDP uptake in primary breast cancer and (b) to differentiate malignant from benign lesions. METHODS: Anterior and oblique lateral views of the breasts were acquired 0-4 min, 10-20 min and 2 hr after intravenous injection of 740 MBq of 99mTc-MDP in 200 women with elevated suspicion or proven diagnosis of breast cancer (Group 1) and in 80 women with other solid tumor types (Group 2). RESULTS: Physical examination and mammography revealed breast abnormalities in all Group 1 subjects. The mammographic findings were definitely positive for carcinoma in 120 patients, highly suspicious in 27 and indeterminate in 53. Breast cancer was later histologically diagnosed in 172 women (86%) and benign disease found in 28 women (14%). Of these patients, 158 (92%) showed focal uptake of 99mTc-MDP in the images collected 10-20 min after injection. This was found to be the best timing for imaging, with tumor-to-background ratios as high as 4.3 (mean +/- s.d. = 3.8 +/- 0.4). Two hr after injection, only 61 of the 158 (38%) malignant lesions were clearly detectable. CONCLUSION: Technetium-99m-MDP is concentrated by primary breast carcinoma 10-20 min after injection, enabling successful external gamma imaging. Scintimammography with 99mTc-MDP is an accurate test that differentiates malignant from benign breast lesions, particularly in patients with indeterminate mammograms.

Scintimammography with 99mTc-MDP: experience of the National Cancer Institute of Naples.

The role of scintimammography with 99mTc-MDP was investigated in patients with mammographic or clinical evidence of breast lesions, suspicious for malignancy, in our Department at the National Cancer Institute of Naples. The end-point of the study was to assess the uselfulness of this test in diagnosing or ruling out breast cancer in more than 2000 women. Scintimammography results were compared with those of mammography and ultrasound and categorized according to histological findings. Overall sensitivity was 92%, specificity was 90%, and accuracy 91%. Sensitivity was affected by the lesions exceeding 12 mm and specificity by sclerotic and/or hyaline or myxoid fibroadenomas, which may be positive. The major advantages of scintimammography appeared in the study of calcifications without a mass and of the indirect mammographic signs of breast cancer, such as distortion and asymmetry. Scintimammography with 99mTc-MDP is a reliable, safe and highly cost-effective procedure to diagnose or to rule out breast cancer, after mammography and ultrasound have yielded questionable results.

Ocular larva migrans and histopathological lesions in mice experimentally infected with Baylisascaris transfuga embryonated eggs.

The ability of Baylisascaris transfuga larvae to cause ocular larva migrans (OLM) in mice was examined. Mice were given approximately 3500 infective eggs of B. transfuga per os. Their eyes were removed and examined either microscopically or histologically at various intervals post-infection. Larvae were recovered beginning 7 days after infection. Histologically, free larvae were observed in the posterior chamber and within the ocular membranes. Larval granulomas were present in the choroid with involvement of retinal layers. It was concluded that B. transfuga larvae have the ability to produce OLM in mice following oral infection and should be considered as possible agents of OLM in other animals and in human beings.

BTG2 loss and miR-21 upregulation contribute to prostate cell transformation by inducing luminal markers expression and epithelial-mesenchymal transition.

Prostate cancer is one of the leading causes of cancer-related death in men. Despite significant advances in prostate cancer diagnosis and management, the molecular events involved in the transformation of normal prostate cells into cancer cells have not been fully understood. It is generally accepted that prostate cancer derives from the basal compartment while expressing luminal markers. We investigated whether downregulation of the basal protein B-cell translocation gene 2 (BTG2) is implicated in prostate cancer transformation and progression. Here we show that BTG2 loss can shift normal prostate basal cells towards luminal markers expression, a phenotype also accompanied by the appearance of epithelial-mesenchymal transition (EMT) traits. We also show that the overexpression of microRNA (miR)-21 suppresses BTG2 levels and promotes the acquisition of luminal markers and EMT in prostate cells. Furthermore, by using an innovative lentiviral vector able to compete with endogenous mRNA through the overexpression of the 3'-untranslated region of BTG2, we demonstrate that in prostate tumor cells, the levels of luminal and EMT markers can be reduced by derepression of BTG2 from microRNA-mediated control. Finally, we show that the loss of BTG2 expression confers to non-tumorigenic prostate cells ability to grow in an orthotopic murine model, thus demonstrating the central role of BTG2 downregulaton in prostate cancer biology.

Signaling crosstalk between TGFß and Dishevelled/Par1b.

Crosstalk of signaling pathways is critical during metazoan development and adult tissue homeostasis. Even though the transforming growth factor-beta (TGFß) transduction cascade is rather simple, in vivo responsiveness to TGFß ligands is tightly regulated at several steps. As such, TGFß represents a paradigm for how the activity of one signaling system is modulated by others. Here, we report an unsuspected regulatory step involving Dishevelled (Dvl) and Par1b (also known as MARK2). Dvl and Par1b cooperate to enable TGFß/bone morphogenetic protein (BMP) signaling in Xenopus mesoderm development and TGFß responsiveness in mammalian cells. Mechanistically, the assembly of the Par1b/Dvl3/Smad4 complex is fostered by Wnt5a. The association of Smad4 to Dvl/Par1 prevents its inhibitory ubiquitination by ectodermin (also known as transcriptional intermediary factor 1 gamma or tripartite motif protein 33). We propose that this crosstalk is relevant to coordinate TGFß responses with Wnt-noncanonical and polarity pathways.

beta-Catenin initiates tooth neogenesis in adult rodent incisors.

beta-Catenin signaling is required for embryonic tooth morphogenesis and promotes continuous tooth development when activated in embryos. To determine whether activation of this pathway in the adult oral cavity could promote tooth development, we induced mutation of epithelial beta-catenin to a stabilized form in adult mice. This caused increased proliferation of the incisor tooth cervical loop, outpouching of incisor epithelium, abnormal morphology of the epithelial-mesenchymal junction, and enhanced expression of genes associated with embryonic tooth development. Ectopic dental-like structures were formed from the incisor region following implantation into immunodeficient mice. Thus, forced activation of beta-catenin signaling can initiate an embryonic-like program of tooth development in adult rodent incisor teeth.

Is quantitative histologic examination useful to predict nonorgan-confined prostate cancer when saturation biopsy is performed?

OBJECTIVES: To evaluate the role of quantitative histologic findings in predicting nonorgan-confined (non-OC) prostate cancer (PCa) in patients undergoing saturation prostate biopsy (SPBx). METHODS: A total of 69 patients who had undergone SPBx underwent radical retropubic prostatectomy. Their prostate-specific antigen level was <10 ng/mL, and 49 and 20 patients had T1c and T2 PCa, respectively. The following biopsy variables from the quantitative histologic examination were evaluated as predictive of OC vs non-OC PCa: Gleason score (6), total percentage of PCa (20%), greatest percentage of PCa (50%), number of PCa-positive cores (2), presence of PCa-positive cores in both lateral margins (yes vs no), and PCa localization (unilateral vs bilateral). The results obtained from patients who had undergone SPBx were compared with those of 183 patients who had undergone 12-core prostate biopsy before radical retropubic prostatectomy. RESULTS: Overall, 32 patients had non-OC PCa. Among the men with Stage T1c PCa, the quantitative histologic findings were predictive of non-OC PCa in 12 of 17 cases. The area under the receiver operating characteristic curve was 0.935 +/- 0.29, supporting the high accuracy of quantitative histologic examination in predicting for non-OC PCa. The sensitivity in patients who underwent SPBx vs the 12-core biopsy was 78.1% and 89.4%, respectively. Also, although the specificity of each histologic parameter was significantly lower in the SPBx group, it was equivalent using quantitative histologic examination (85.6% vs 86.5%). CONCLUSIONS: In the preoperative staging of patients with clinical Stage T1c-T2 PCa and a prostate-specific antigen level <10 ng/mL who had undergone SPBx, quantitative histologic examination demonstrated good accuracy in predicting for non-OC PCa only when all pathological variables were considered.

A mutually stimulating loop involving emx2 and canonical wnt signalling specifically promotes expansion of occipital cortex and hippocampus.

The correct size of the different areas composing the mature cerebral cortex depends on the proper early allocation of cortical progenitors to their distinctive areal fates, as well as on appropriate subsequent tuning of their area-specific proliferation-differentiation profiles. Whereas much is known about the genetics of the former process, the molecular mechanisms regulating proliferation and differentiation rates within distinctive cortical proto-areas are still largely obscure. Here we show that a mutual stimulating loop, involving Emx2 and canonical Wnt signalling, specifically promotes expansion of the occipito-hippocampal anlage. Collapse of this loop occurring in Emx2-/- mutants leads progenitors within this region to slow down DNA synthesis and exit prematurely from the cell cycle, due to misregulation of cell cycle-, proneural- and lateral inhibition-molecular machineries, and eventually results in dramatic and selective size-reduction of occipital cortex and hippocampus. Reactivation of canonical Wnt signalling in the same mutants rescues a subset of molecular abnormalities and corrects differentiation rates of occipito-hippocampal progenitors.

[The head inducer Cerberus in a multivalent extracellular inhibitor]. / L'inducteur céphalique Cerberus est un inhibiteur multivalent extracellulaire.

Cerberus encodes for a secreted protein which when overexpressed ventrally in a Xenopus embryo induces head differentiation without trunk (Bouwmeester et al., 1996). We have recently shown that Cerberus can bind BMP-4 (Bone Morphogenetic Protein-4), Xnr-1 (Xenopus Nodal-related 1) and Xwnt-8 in the extracellular space (Piccolo et al., 1999). We present here studies showing that Cerberus does not have a receptor nor a dedicated transduction pathway but rather acts as an extracellular inhibitor. Our results suggest that the action of Cerberus in head induction can be explained by an inhibitory activity upstream of the Nodal-related and BMP-4 receptors. In addition, using dominant negative receptor mutants which block both the Xnr-1 and BMP-4 transduction pathways, we show that this double inhibition is sufficient for head induction in ventral mesoderm explants.

Cleavage of Chordin by Xolloid metalloprotease suggests a role for proteolytic processing in the regulation of Spemann organizer activity.

The Xolloid secreted metalloprotease, a tolloid-related protein, was found to cleave Chordin and Chordin/BMP-4 complexes at two specific sites in biochemical experiments Xolloid mRNA blocks secondary axes caused by chordin, but not by noggin, follistatin, or dominant-negative BMP receptor, mRNA injection. Xolloid-treated Chordin protein was unable to antagonize BMP activity. Furthermore, Xolloid digestion released biologically active BMPs from Chordin/BMP inactive complexes. Injection of dominant-negative Xolloid mRNA indicated that the in vivo function of Xolloid is to limit the extent of Spemann's organizer field. We propose that Xolloid regulates organizer function by a novel proteolytic mechanism involving a double inhibition pathway required to pattern the dorsoventral axis: [formula in text].

Frzb-1 is a secreted antagonist of Wnt signaling expressed in the Spemann organizer.

Frzb-1 is a secreted protein containing a domain similar to the putative Wnt-binding region of the frizzled family of transmembrane receptors. Frzb-1 is widely expressed in adult mammalian tissues. In the Xenopus gastrula, it is expressed and regulated as a typical Spemann organizer component. Injection of frzb-1 mRNA blocks expression of XMyoD mRNA and leads to embryos with enlarged heads and shortened trunks. Frzb-1 antagonizes the effects of Xwnt-8 ectopic expression in a non-cell-autonomous manner. Cultured cells transfected with a membrane-tethered form of Wnt-1 bind epitope-tagged Frzb-1 in the 10(-10) M range. The results strengthen the view that the Spemann organizer is a source of secreted inhibitory factors.

Dorsoventral patterning in Xenopus: inhibition of ventral signals by direct binding of chordin to BMP-4.

Chordin (Chd) is an abundant protein secreted by Spemann organizer tissue during gastrulation. Chd antagonizes signaling by mature bone morphogenetic proteins (BMPs) by blocking binding to their receptors. Recombinant Xenopus Chd binds to BMP-4 with high affinity (KD, 3 x 10(-10) M), binding specifically to BMPs but not to activin or TGF-beta1. Chd protein is able to dorsalize mesoderm and to neuralize ectoderm in Xenopus gastrula explants at 1 nM. We propose that the noncell-autonomous effects of Spemann's organizer on dorsoventral patterning are executed in part by diffusible signals that directly bind to and neutralize ventral BMPs during gastrulation.