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1.
Reproduction ; 152(6): 695-704, 2016 12.
Article in English | MEDLINE | ID: mdl-27651518

ABSTRACT

The multifactorial pathological condition, that is, severe low sperm motility is a frequent cause of infertility. However, mechanisms underlying the development of this condition are not completely understood. Single abnormalities have been reported in sperm of patients with asthenozoospermia. In this study, we characterized, in 22 normozoospermic men and in 37 patients with asthenozoospermia, biochemical, molecular and genomic abnormalities that frequently occur in sperm of patients with asthenozoospermia. We evaluated a panel of sperm biomarkers that may affect the motility and fertilizing ability of sperm of patients with severe asthenozoospermia. Since reactive oxygen species (ROS) production is involved in the pathogenesis of such sperm abnormalities, we determined the association between ROS production and sperm abnormalities. High percentage of patients with severe asthenozoospermia showed increased basal and stimulated ROS production. Moreover, these patients showed increased mitochondrial DNA (mtDNA) copy number but decreased mtDNA integrity and they were associated with elevated ROS levels. Furthermore, mitochondrial membrane potential was also significantly decreased and again associated with high ROS production in these patients. However, the rate of nuclear DNA fragmentation was increased only in less than one-fifth of these patients. An important cohort of these patients showed multiple identical biochemical, molecular and genomic abnormalities, which are typical manifestations of oxidative stress. The most frequent association was found in patients with high ROS levels, increased mtDNA copy number and decreased integrity, and low MMP. A smaller cohort of the aforementioned patients also showed nDNA fragmentation. Therefore, patients with asthezoospermia likely present reduced fertilizing potential because of such composed abnormalities.


Subject(s)
Asthenozoospermia/genetics , Asthenozoospermia/pathology , DNA, Mitochondrial/genetics , Spermatozoa/chemistry , Adult , Case-Control Studies , Genomics , Humans , Male , Membrane Potential, Mitochondrial , Reactive Oxygen Species/metabolism , Spermatozoa/metabolism , Young Adult
2.
Int J Oncol ; 45(2): 516-24, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24899250

ABSTRACT

Cutaneous melanoma is an aggressive cancer with a poor prognosis for patients with advanced disease. The identification of several key molecular pathways implicated in the pathogenesis of melanoma has led to the development of novel therapies for this devastating disease. In melanoma, both the Ras/Raf/MEK/ERK (MAPK) and the PI3K/AKT (AKT) signalling pathways are constitutively activated through multiple mechanisms. Targeting various effectors of these pathways with pharmacologic inhibitors may inhibit melanoma cell growth and angiogenesis. Ongoing clinical trials provide hope to improve progression-free survival of patients with advanced melanoma. This review summarizes the most relevant studies focused on the specific action of these new molecular targeted agents. Mechanisms of resistance to therapy are also discussed.


Subject(s)
Antineoplastic Agents/therapeutic use , Melanoma/drug therapy , Molecular Targeted Therapy/methods , Skin Neoplasms/drug therapy , Humans
3.
Int J Oncol ; 40(3): 639-44, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22200790

ABSTRACT

The most common therapeutic approach for many cancers is chemotherapy. However, many patients relapse after treatment due to the development of chemoresistance. Recently, targeted therapies represent novel approaches to destroy cancer cells. The PI3K/PTEN/AKT pathway is a key signaling pathway involved in the regulation of cell growth. Dysregulated signaling of this pathway may be associated with activating mutations of PI3K-related genes. Analyses of these mutations reveal that they increase the PI3K signal, stimulate downstream Akt signaling, promote growth factor-independent growth and increase cell invasion and metastasis. In this review, we summarize the PI3K/PTEN/AKT pathway genetic alterations in cancer and their potential clinical applications.


Subject(s)
Neoplasms/genetics , PTEN Phosphohydrolase/genetics , Phosphatidylinositol 3-Kinases/genetics , Proto-Oncogene Proteins c-akt/genetics , Animals , Drug Resistance , Genetic Variation , Humans , Neoplasms/drug therapy , Neoplasms/metabolism , PTEN Phosphohydrolase/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction
4.
Int J Mol Med ; 17(3): 523-8, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16465402

ABSTRACT

In this study, we evaluated the expression of genes probably involved in spermatogenesis in the mouse. We examined cytosolic chaperonin theta subunit (CCTtheta), Ngg1 interacting factor 3 like 1 binding protein 1 (NIF3L1 BP1) and apolipoprotein H (ApoH) expression during mouse onto-geny using RT-PCR. Testicular tissue was obtained from mice 3, 6, 8, 10, 12, 14, 18, 20 and 40 (adult) days after birth. For each mouse, one testis was used for histological examination, whereas RNA was extracted from the controlateral testis for expression analysis. RT-PCR analysis showed that CCTtheta gene expression was low until day 10, but increased drastically afterwards. At this age, spermatocytes started to be present in the mouse testis. Therefore, CCT protein could be involved in chromatin packaging and remodeling during spermiogenesis, as also suggested by other studies. NIF3L1 BP1 expression increased steadily during ontogenesis reaching maximum levels in the adult mouse when all germ cell stages are present. This finding suggests that NIF3L1 BP1 is a gene not expressed by a specific germ cell type. ApoH expression was very low or absent during prepuberal stages, whereas it was detectable in the adult testis when spermatogenesis was completed. This suggests that ApoH may be involved in clearing apoptotic bodies during spermatogenesis since apoptotic events increase during spermatogenesis. This study contributes to understanding the role played by genes important for spermatogenesis.


Subject(s)
Gene Expression Regulation, Developmental , Spermatogenesis/genetics , Animals , Chaperonin Containing TCP-1 , Chaperonins/genetics , Co-Repressor Proteins , Glycoproteins/genetics , Male , Mice , Proteins/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Testis/cytology , Transcription Factors , beta 2-Glycoprotein I
5.
Int J Oncol ; 27(4): 1071-7, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16142325

ABSTRACT

Bovine seminal-ribonuclease (BS-RNase) is a member of the 'ribonucleases with special biological actions' family since it possesses specific anti-tumour, anti-spermatogenic and embryotoxic activities and exerts an immunosuppressive effect on T lymphocytes. In previous studies it was demonstrated that BS-RNase induced apoptosis in proliferating, malignant and normal cells and that telomerase activity loss also caused apoptotic death in neoplastic cells. Since an obvious relationship between cell proliferation and telomerase activity exists, the aim of this work was to study if the pro-apoptotic cytotoxic action exerted by BS-RNase on proliferating malignant cells (HT29) and proliferating normal cells (PHA-stimulated lymphocytes) could be linked to a possible BS-RNase effect on telomerase activity. In BS-RNase-treated HT29 cells (Na-butyrate-differentiated or not) and human lymphocytes (proliferating or not), we investigated cell vitality (MTT method) and morphology (SEM), BS-RNase localization (immunofluorescence), telomerase activity (TRAP-ELISA method), hTR mRNA expression (RT-PCR), and hTERT levels (western blot). While no BS-RNase effect was detectable on not proliferating cells, a clear relationship was noticed between the diminished number of vital elements of both proliferating cell populations after treatment (48 h and 72 h for HT29 and PHA-stimulated lymphocytes, respectively) with 50 microg/ml BS-RNase and the decrease of their telomerase activity. At the same time, we found that hTR levels, the RNA subunit of telomerase, in proliferation-inhibited BS-RNase-treated cells were diminished. Moreover, by immunofluorescence technique, we detected BS-RNase in the HT29 cell nucleolus after 3-h treatment. Therefore, as hTR has been recently proven to co-fractionate with nucleoli, we hypothesize that a BS-RNase direct action on the telomerase hTR subunit could be a possible mechanism of action by which BS-RNase exerts its pro-apoptotic effects only on proliferating cells.


Subject(s)
Endoribonucleases/chemistry , Endoribonucleases/pharmacology , Telomerase/biosynthesis , Animals , Antineoplastic Agents/pharmacology , Apoptosis , Blotting, Western , Cattle , Cell Differentiation , Cell Line, Tumor , Cell Nucleolus/metabolism , Cell Proliferation , Down-Regulation , Enzyme-Linked Immunosorbent Assay , Humans , Immunosuppressive Agents/pharmacology , Leukocytes, Mononuclear/cytology , Lymphocytes/metabolism , Microscopy, Electron, Scanning , Microscopy, Fluorescence , RNA/metabolism , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , T-Lymphocytes/drug effects , Telomerase/metabolism , Tetrazolium Salts/pharmacology , Thiazoles/pharmacology , Time Factors
6.
Mol Diagn ; 9(4): 201-4, 2005.
Article in English | MEDLINE | ID: mdl-16392899

ABSTRACT

INTRODUCTION: Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive disorder of cholesterol biosynthesis, resulting from deficient 7-dehydrocholesterol reductase (3beta-hydroxysterol Delta7-reductase) activity, the enzyme responsible for conversion of 7-dehydrocholesterol to cholesterol. SLOS is most common among people of European descent, with a reported incidence of 1 per 20,000-60,000 newborns, depending on the diagnostic criteria and the reference population. More than 80 different mutations have been identified in several hundred patients. In Italy, SLOS appears to be a rare condition, probably because of underdiagnosis. METHOD: We analyzed by direct sequencing the 7-dehydrocholesterol reductase gene (DHCR7) in a Sicilian patient with Smith-Lemli-Opitz syndrome and his parents in order to characterize the molecular defect. RESULTS: The molecular analysis of the coding exons and the intron-exon boundaries of the DHCR7 gene demonstrated the presence of two missense mutations: a novel mutation (I251N) and a known mutation (E288K) responsible in a compound heterozygous state for a severe form of SLOS. CONCLUSION: The present study describes a Sicilian patient, a carrier of a novel mutation of the DHCR7 gene (I251N), which is responsible in a compound heterozygous state for a severe form of SLOS.


Subject(s)
Oxidoreductases Acting on CH-CH Group Donors/genetics , Smith-Lemli-Opitz Syndrome/genetics , Heterozygote , Humans , Infant , Male , Mutation, Missense , Pedigree , Sicily
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