Human MATER localization in specific cell domains of oocytes and follicular cells.

MATER (Maternal Antigen That Embryos Require) is an oocyte-specific protein dependent on the maternal genome and required for early embryonic development. The gene products expressed in oocytes play important roles in folliculogenesis, fertilization and pre-implantation development. The aim of this study was to characterize the localization and distribution pattern of the human MATER protein during follicular development and after ovulation, to determine its functional role. Immunocytochemistry experiments coupled with confocal and electron microscopy analysis were carried out to determine the ultrastructural localization of MATER in human ovarian tissue and in isolated oocytes, obtained during IVF procedures. Human cumulus cells were cultured, with or without cycloheximide, to confirm endogenous biosynthesis of the protein. Human MATER is detectable at the onset of the follicular maturation process, suggesting this protein has a role at earlier stages in the human compared with other mammalian species. The presence of MATER is specific to the oocyte and follicular cells that, during maturation, are spatially and functionally associated with the oocyte. The nuclear, nucleolar and mitochondrial localization hints at a possible role in RNA processing and the metabolic activity of the cell.

Shockwave lithotripsy and protective role of inosine: early and late evaluation in an experimental model.

PURPOSE: Extracorporeal shockwave lithotripsy (SWL) is one of the most common treatments for urinary stones. Despite technological improvements, it may cause side effects varying from minor reversible microscopic damage to severe large renal hematomas. The aim of our experimental study is to assess the efficacy of inosine in avoidance of acute renal damage after SWL. MATERIALS AND METHODS: We used 25 Wistar rats that had previously had left nephrectomy. The rats were divided into three groups: group A consisted of 10 rats undergoing renal SWL; group B consisted of 10 rats that received adjunctive treatment with IP injection of inosine 40 minutes before SWL; and group C consisted of 5 rats that served as controls. N-acetylglucosaminidase (NAG) and lactate dehydrogenase (LDH) concentrations were evaluated 24 hours before and 24 hours after SWL. All the rats were subsequently sacrificed (4 rats in group A and 4 in group B at 48 hours post-SWL, and the remaining rats were sacrificed 30 days post-SWL). Renal tissue was submitted to histologic and electron microscopic examination to assess early and late alterations. RESULTS: NAG and LDH values were significantly increased after SWL in group A (P<0.001), while no significant NAG and LDH differences were detected in group B (P<0.16). Early histologic examination revealed a considerable amount of cellular degeneration in group A with ultrastructural vacuolization and disruption of lysosomal membranes; the tubular features and cellular structures appeared to be well preserved in group B. No late histologic alterations were evident in any of the specimens. CONCLUSIONS: Inosine is helpful and protective in the prevention of early microscopic damage to renal parenchyma due to SWL.

Subcellular localization of beta-catenin and APC proteins in colorectal preneoplastic and neoplastic lesions.

Adenomatous polyposis coli (APC) is a tumor suppressor gene whose main function is the destabilization of beta-catenin, a key effector of the Wnt signaling pathway. This gene is defective in familial adenomatous polyposis (FAP), a dominantly inherited disease, but inactivation of APC has been reported also in most sporadic colorectal tumors and it is considered an early event in colorectal tumorigenesis. The aim of the present study was to evaluate the intracellular ultrastructural distribution of beta-catenin and APC proteins in epithelial cells of normal colorectal mucosa, aberrant crypt foci (ACF, an early premalignant lesion) and cancer. We used the immunogold electron microscopic method to identify both proteins. Normal colonic epithelial cells showed a strong membranous expression of beta-catenin and lacked cytoplasmic and nuclear expression. Normal cells showed APC localization pattern characterized by diffuse nuclear expression and along the plasma membrane. In ACF and in carcinoma an absent or reduced membranous expression of beta-catenin was associated with an increased nuclear and cytoplasmatic expression. In aberrant crypt foci and carcinoma, APC was evident inside the nucleus and at the level of cell-cell junctions, but it was decreased in the cytoplasm. This method allowed the accurate localization of proteins of the Wnt signaling pathway in the early steps of colorectal carcinogenesis. The similar pattern of subcellular distribution of APC and beta-catenin in dysplastic ACF and colorectal cancer suggests that ACF are precursor lesions of sporadic and FAP-associated colorectal carcinoma.

Matrix metalloproteinases 15 and 19 are stromal regulators of colorectal cancer development from the early stages.

Matrix metalloproteinases (MMPs) have been well characterized for their ability to degrade extracellular matrix proteins and, thus, they have been studied to elucidate their involvement in both tumor development and progression. In the present study, attention was focused on MMP-15 and MMP-19, two less known members of the MMP family. The expression profile of MMP-15 and -19 was assayed in samples of normal colorectal mucosa, microadenomas and cancer using confocal analysis, western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Both qRT-PCR and western blotting showed that MMP-15 and MMP-19 appeared to be upregulated during colorectal tumorigenesis, with different expression patterns: MMP-15 expression level increases from normal mucosa to microadenomas, with a reduced level in cancer with respect to microadenomas; the semiquantitative immunofluorescence analysis showed a stromal localization of this protein in the early phases of neoplastic transformation. Increasing amount of MMP-19 mRNA and protein levels were observed in the progression of colonic lesions; MMP-19 staining increased in the normal mucosa-microadenoma-carcinoma sequence. Such different expression patterns, are probably due to the different roles played in colorectal tumorigenesis by these two molecules. Conflicting data on the role of these proteins in tumor progression have been reported, thus, an improved understanding of the biological roles of MMPs, in particular the lesser known members such as MMP-15 and 19, in colorectal cancer may lead to a re-evaluation of the use of MMP inhibitors and suggests the need of integrated translational studies on MMP expression patterns.

Human amniotic fluid stem cells seeded in fibroin scaffold produce in vivo mineralized matrix.

This study investigated the potential of amniotic fluid stem cells (AFSCs) to synthesize mineralized extracellular matrix (ECM) within different porous scaffolds of collagen, poly-D,L-lactic acid (PDLLA), and silk fibroin. The AFSCs were initially differentiated by using an osteogenic medium in two-dimensional culture, and expression of specific bone proteins and the physiologic mineral production by the AFSCs were analyzed. In particular, during differentiation process, AFSCs expressed proteins like Runt-related transcription factor 2 (Runx2), Osterix, Osteopontin, and Osteocalcin with a sequential expression, analogous to those occurring during osteoblast differentiation, and produced extracellular calcium stores. AFSCs were then cultured on three-dimensional (3D) scaffolds and evaluated for their ability to differentiate into osteoblastic cells in vivo. Stem cells were cultured in vitro for 1 week in collagen, fibroin, and PDLLA scaffolds. The effect of predifferentiation of the stem cells in scaffolds on the subsequent bone formation in vivo was determined in a rat subcutaneous model. With the addition of a third dimension, osteogenic differentiation and mineralized ECM production by AFSCs were significantly higher. This study demonstrated the strong potential of AFSCs to produce 3D mineralized bioengineered constructs in vivo and suggests that fibroin may be an effective scaffold material for functional repair of critical size bone defects.

Altered expression of apoptosis biomarkers in human colorectal microadenomas.

Human colorectal microadenomas are considered the earliest detectable premalignant lesions in the colon. They can be identified as aggregates of enlarged crypts with thicker epithelial linings and elongated luminal openings on the colonic mucosal surface after methylene blue staining and observation under a dissecting microscope. Multiple lines of evidence suggest that a central role in neoplastic development is played by the inhibition of apoptosis, followed by disruption of DNA repair. Understanding the early mechanisms of colorectal carcinogenesis may help develop new approaches of colorectal cancer prevention and treatment. The aim of the present study was to quantify poly-ADP ribose polymerase 1 (PARP-1)-positive cells and to evaluate apoptotic control mechanisms through Caspase-3 active and Bcl-2 protein expression in human microadenomas and in normal colorectal mucosa using immunofluorescence techniques coupled with confocal microscopy and immunoblot experiments. The mean percentage of PARP-1-positive epithelial cells was 3.0 +/- 0.37% (SD) and 15.67 +/- 0.40% in microadenoma and in normal mucosa, respectively. Proteins involved in programmed cell death were differently expressed in microadenoma and in normal mucosa. Indeed, by semiquantitative immunofluorescence analysis, confirmed by Western blot, microadenoma showed low levels of Caspase-3 active and high levels of Bcl-2 expression, whereas the opposite was true for normal colorectal mucosa [corrected]. In the stroma of normal colorectal mucosa, fibroblast-like cells and neutrophils were the cells that underwent apoptosis to a greater extent. In conclusion, malfunction of the control mechanisms of programmed cell death seems present in the early stages of colorectal cancer development.

Cyclooxygenase-2 and Hypoxia-Inducible Factor-1alpha protein expression is related to inflammation, and up-regulated since the early steps of colorectal carcinogenesis.

Chronic mucosal inflammation is considered a risk factor for colorectal cancer. Neutrophils are a major source of oxidants, whereas cyclooxygenase 2 (COX-2) and Hypoxia Inducible Factor-1alpha (HIF-1alpha) protein expression levels are increased in inflammatory and malignant lesions. The main purpose of the present study was to evaluate myeloperoxidase (MPO) positive cell infiltration, COX-2 and HIF-1alpha protein expression in colorectal carcinogenesis, especially in its early phases, using immunohistochemistry and immunofluorescence confocal microscopy techniques. MPO, COX-2 and HIF-1alpha proteins were expressed at higher rates in the normal colorectal mucosa of patients with inflammatory bowel diseases and colorectal tumours than in patients with normal colonoscopy. A gradual increase in COX-2 and HIF-1alpha protein expression was observed in dysplastic aberrant crypt foci, adenomas and carcinomas, showing a strong relation to dysplasia. In conclusion, the present study supports the hypothesis of a key role of inflammation in malignant transformation of colorectal mucosa. The evaluation of some early markers related to inflammation in the mucosa of the large bowel may serve as potential tool for prognosis and therapeutic strategies.

Human ovarian tissue cryopreservation: effect of sucrose concentration on morphological features after thawing.

Recent improvements in techniques in clinical assisted reproduction have led to an increased interest in the cryopreservation of human ovarian tissue as a way of preserving fertility and ovarian steroidogenic activity in young cancer patients. Acceptable follicular survival in frozen-thawed human ovarian tissue has generally been reported. Since a 0.3 mol/l sucrose concentration in cryopreservation solutions evidently increases human oocyte survival after cryopreservation, the aim of this study was to observe the effect of sucrose concentrations of 0.2 mol/l and 0.3 mol/l on human ovarian tissue survival after thawing. Ovarian cortical slices from 10 patients, 22-36 years of age, were cryopreserved slowly using 0.2 mol/l or 0.3 mol/l sucrose with 1,2-propanediol (1.5 mol/l) as the cryoprotectants. Light and electron microscopy were used for the histological analyses. Results showed that both treatments produced an increase in damaged cells; however, the use of 0.3 mol/l sucrose showed a smaller percentage of damaged germ cells than 0.2 mol/l sucrose, and therefore was less detrimental to the thawed ovarian tissue. However as the damage occurred principally in the stroma and follicular cells rather than in the oocytes, the suitability of these cryopreservation protocols must be further evaluated prior to considering the use of stored ovarian cortex for autografting after thawing.