Staining of human sperm with fluorochrome-labeled inhibitor of caspases to detect activated caspases: correlation with apoptosis and sperm parameters

BACKGROUND: Detection of apoptosis in sperm samples may help evaluate sperm quality. Recently, it has been suggested that in some ejaculated sperm populations, apoptosis is caspase dependent. The aim of this study was to investigate the presence of activated caspases and examine possible correlations with apoptosis and sperm parameters in semen samples prepared for IVF. METHODS: To detect activated caspases, neat semen from infertile patients and sperm prepared by PureSperm gradient were stained with the fluorescein isothyocyanate-Val-Ala-Asp-fluoromethylketone (FITC-VAD-fmk) and analysed by flow cytometry. Cell death was determined by DNA fragmentation (TUNEL) and mitochondrial membrane potential. Sperm parameters were studied by conventional microscopy. RESULTS: FITC-VAD-fmk stained sperm cells in situ and the subcellular labeling pattern was compatible with the known localization of caspases. A significant correlation was found between the frequency of FITC-VAD-fmk stained cells and cell death markers. In both prepared sperm and neat semen a negative correlation was found between the percentage of FITC-VAD-fmk positive cells and standard parameters (concentration/motility). FITC-VAD-fmk positive cells negatively correlated with high fertilization rates after IVF. CONCLUSIONS: Labelling of sperm cells with the activated caspases-reacting fluorochrome provides a sensitive assay for detection of sperm apoptosis. This cytometric assay can be helpful to test sperm before IVF.     

NF1 gene analysis based on DHPLC

The high mutation rate at the NF1 locus results in a wide range of molecular abnormalities. The majority of these mutations are private and rare, generating elevated allelic diversity with a restricted number of recurrent mutations. In this study, we have assessed the efficacy of denaturing high-performance liquid chromatography (DHPLC), for detecting mutation in the NF1 gene. DHPLC is a fast and highly sensitive technique based on the detection of heteroduplexes in PCR products by ion pair reverse-phase HPLC under partially denaturing conditions. We established theoretical conditions for DHPLC analysis of all coding exons and splice junctions of the NF1 gene using the WAVEmaker software version 4.1.40 and screened for mutations a panel of 40 unrelated NF1 patients (25 sporadic and 15 familial), genetically uncharacterized. Disruptive mutations were identified in 29 individuals with an overall mutation detection rate of 72.5%. The mutations included eight deletions (exons 4b, 7, 10a, 14, 26, and 31), one insertion (exon 8), nine nonsense mutation (exons 10a, 13, 23.1, 27a, 29, 31, and 36), six missense mutations (exons 15, 16, 17, 24, and 31), four splice errors (exons 11, 14, 36, and 40) and a complex rearrangement within exon 16. Eighteen (62%) of the identified disruptive mutations are novel. Seven unclassified and three previously reported polymorphisms were also detected. None of the missense mutations identified in this study were found after screening of 150 controls. Our results suggest that DHPLC provides an accurate method for the rapid identification of NF1 mutations.     

CDK9/CYCLIN T1 expression during normal lymphoid differentiation and malignant transformation

CDK9 is a member of the CDC2-like family of kinases. Its cyclin partners are members of the CYCLIN T family (T1, T2a, and T2b) and CYCLIN K. The CDK9/CYCLIN T1 complex is very important in the differentiation programme of several cell types, controlling specific differentiation pathways. Limited data are available regarding the expression of CDK9/CYCLIN T1 in haematopoietic and lymphoid tissues. The aim of this study was to analyse the expression of the CDK9/CYCLIN T1 complex in lymphoid tissue, in order to assess its role in B- and T-cell differentiation and lymphomagenesis. CDK9/CYCLIN T1 expression was found by immunohistochemistry in precursor B and T cells. In peripheral lymphoid tissues, germinal centre cells and scattered B- and T-cell blasts in interfollicular areas expressed CDK9/CYCLIN T1, while mantle cells, plasma cells, and small resting T-lymphocytes displayed no expression of either molecule. CDK9/CYCLIN T1 expression therefore appears to be related to particular stages of lymphoid differentiation/activation. CDK9 and CYCLIN T1 were highly expressed in lymphomas derived from precursor B and T cells, from germinal centre cells, such as follicular lymphomas, and from activated T cells (ie anaplastic large cell lymphomas). Hodgkin and Reed-Sternberg cells of classical Hodgkin's lymphoma also showed strong nuclear staining. Diffuse large B-cell, Burkitt's lymphomas, and peripheral T-cell lymphomas, among T-cell lymphoproliferative disorders, showed a wide range of values. No expression of CDK9 or CYCLIN T1 was detected in mantle cell and marginal zone lymphomas. However, at the mRNA level, an imbalance in the CDK9/CYCLIN T1 ratio was found in follicular lymphoma and diffuse large B-cell lymphomas with germinal centre phenotype, and in the cell lines of classical Hodgkin's lymphomas, Burkitt's lymphomas, and anaplastic large cell lymphoma, in comparison with reactive lymph nodes. These results suggest that the CDK9/CYCLIN T1 complex may affect the activation and differentiation programme of lymphoid cells. The molecular mechanism through which the CDK9/CYCLIN T1 complex is altered in malignant transformation needs to be elucidated.     

Spontaneous and MNNG‐induced reversion of an EGFP construct in HeLa cells: An assay for observing mutations in living cells by fluorescent microscopy

A HeLa cell line stably expressing the enhanced green fluorescence protein (EGFP) gene, interrupted by the HBB IVS2‐654 intron, was studied without treatment and after treatment with a single standard dose of 15 μM of N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG). This assay was done in order to prove that such a construct can revert by a variety of mechanisms and that it produces a visible phenotype, i.e., green fluorescence. The system permits visual detection of living mutant cells among a background of non‐mutant cells and does not require a selective medium. The results show that the construct reverts by large deletions (–62, –100, and –162 bp), small insertions (+4 bp), small rearrangements (19 bp duplication), base substitutions at purines (G₆₅₂, G₆₅₃, A₆₅₅, G₅₇₉), and a pyrimidine (T₆₅₄) between nucleotide positions 579 and 837. Splice‐site mutations were recovered, and some of the mechanisms underlying these mutations are discussed. Because of the ease of detection of revertant cells under fluorescent light and the wide variety of mutations that can be recovered, further development of this system could make it a useful new mammalian cell mutagenicity assay. Hum Mutat 18:526–534, 2001. © 2001 Wiley‐Liss, Inc.     

Effects of extracellular fluid volume changes on renal response to low-dose dopamine infusion in normal women

Dopamine (DA) i.v. infused in a low dose (0.1 microgram/kg/min) in healthy women during sustained hypotonic polyuria, produced different renal functional effects as variations in extracellular fluid volume occurred. (1) In hydro-saline retention (n = 23), induced by desoxycorticosterone acetate treatment, DA produced typical vasodilator and hydro-natriuretic effects (Goldberg, 1972). (2) In hydro-saline depletion (n = 19), induced by diuretic treatment and low dietary sodium intake, DA lost its vasodilator and natriuretic efficacy, manifesting, on the other hand, renal sodium conserving effects mainly dependent on the increase in distal sodium reabsorption and a trend towards afferent arteriolar vasoconstriction. (3) Treatment with prazosin (n = 9) or propranolol (n = 9) in hydro-saline depletion, was efficacious in partly restoring the typical vasodilator and natriuretic effects of DA. Thus, in hydro-saline depletion, DA produced sympathomimetic effects which were sufficiently intense to outweigh those due to activation of specific DA renal receptors.     

Hepatic lymphoid aggregates in chronic hepatitis C and mixed cryoglobulinemia

Summary We have examined the clinical (virological and immunological), histological and immunohistochemical features of liver lymphoid nodules in hepatitis C virus-positive (HCV⁺)/mixed cryoglobulinemia (type II and III) and chronic hepatitis C. The clinical features of liver disease were found to be similar in all patients. In all these groups, liver lymphoid nodules were observed to a similar extent, being more frequent in earlier phases of liver disease and less in more advanced stages. These data were confirmed by studies in serial biopsy samples taken from individual patients with type II mixed cryoglobulinemia; the loss of lymphoid nodules with progression to more advanced histological stages of disease in these patients was accompanied by a decrease of the serum levels of cryoglobulins (although not statistically significant). By immunohistochemical analysis, the liver lymphoid nodules contained predominantly B cells with a CD5⁺/bcl2⁺/Ki67^– phenotype, which were always polyclonal in type III mixed cryoglobulinemia and chronic hepatitis C, and monoclonal in type II mixed cryoglobulinemia. These immunological features were consistent with an active role of the immune system in HCV-associated liver necro-inflammation. Only in type II mixed cryoglobulinemia was there a clonal restriction of B cells. The immunological profile (autoantibodies) and viral genotypes were examined in some patients, but no significant correlation with clinical and immunohistochemical findings was found; however, the prevalence of genotype 2a was significantly higher in type II mixed cryoglobulinemia than in type III and chronic hepatitis without cryoglobulinemia.