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Query: UNIPROT:P06889 (Mol)
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Transition metal complexes containing vanadium IV have been shown to modulate the cellular redox potential and catalyse the generation of reactive oxygen intermediates (ROI). Since sperm function is exquisitely susceptible to ROI, we examined the effects of stable chelate complexes of vanadocenes on human sperm motility. We synthesized seven structurally distinct chelate complexes of bis(cyclopentadienyl)vanadium(IV) with bidentate ligands [i.e. vanadocene acetylacetonato monotriflate (VDacac), vanadocene hexafluoro acetylacetonato monotriflate (VDHfacac), vanadocene N-phenyl benzohydroxamato monotriflate (VDPH), vanadocene acethydroxamato monotriflate (VDH), vanadocene catecholate (VDCAT), vanadocene bipyridino ditriflate (VDBPY), and vanadocene dithiocarbamate monotriflate (VDDTC)], and evaluated their spermicidal activity using computer-assisted sperm analysis (CASA; Hamilton-Thorne). All seven chelate complexes of vanadocene elicited potent spermicidal activity at micromolar concentrations (EC50 values: 3.9-106 microM) without affecting the sperm acrosome integrity. The catecholate and acetylacetonate complexes of vanadocene were the most active and the bipyridyl complex the least active with an order of efficacy VDCAT > VDacac > VDDTC > VDPH > VDH > VDHfacac > VDBPY. The spermicidal activity of chelate complexes of vanadocenes was rapid and irreversible since the treated spermatozoa underwent apoptosis, as determined by the flow cytometric analysis of mitochondrial membrane potential, surface annexin V binding assay, in-situ nick-end labelling of sperm nuclei, and confocal laser scanning microscopy. These results provide unprecedented evidence that chelate complexes of vanadocene with bidentate ligands have spermicidal and apoptosis inducing properties. These vanadocene complexes, especially VDacac, may be useful as contraceptive agents.
Mol Hum Reprod 1998 Jul
PMID:Spermicidal activity of chelated complexes of bis(cyclopentadienyl)vanadium(IV). 970 91

Nonionic and anionic water-soluble amphiphiles were shown to increase strongly the binding of fluorescein isothiocyanate-conjugated annexin V (FITC-annexin V) in human erythrocytes pretreated with the aminophospholipid translocase (APLT) inhibitor n-ethylmaleimide (NEM). At high sublytic amphiphile-concentrations the binding of FITC-annexin V, monitored in a flow cytometer, was time- and temperature-dependent and occurred heterogeneously in the cell population, with 43-81% of cells being stained above background following incubation for 60 minutes at 37 degrees C. The increased FITC-annexin V binding apparently indicates an increased flop rate of phosphatidylserine (PS) to the outer membrane leaflet. When the NEM-pretreatment was omitted, the FITC-annexin V binding was markedly, but not completely, reduced. In erythrocytes incubated with a zwitter-ionic amphiphile, a small increase in FITC-annexin V binding was detected, while cationic amphiphiles did not induce an increased FITC-annexin V binding. The potency of amphiphiles to induce PS exposure was not related to the type of shape alteration or vesiculation induced. Our results indicate a significant role of the charge status of a membrane intercalated amphiphile for its capability to induce PS exposure.
Mol Membr Biol
PMID:Amphiphile-induced phosphatidylserine exposure in human erythrocytes. 972 27

Confocal laser scanning microscopy was used to observe human arrested and fragmented preimplantation embryos obtained by in-vitro fertilization. Observation of the cellular actin cortex and chromatin showed a high frequency of embryos with blastomeres exhibiting two or more nuclei, while others had nuclei displaying chromatin condensation and fragmentation patterns. Many of the abnormal chromatin images could be due to the process of programmed cell death (apoptosis). The possible link between abnormalities of the blastomeres and apoptosis was investigated using two detection methods for cells undergoing apoptosis. Detection of phosphatidylserine exposure was performed using annexin V; the chromosomal breakdown preceding the nuclear collapse of apoptotic nuclei was tested using the terminal transferase-mediated DNA end labelling (TUNEL) assay. Annexin V staining was observed in all arrested and/or fragmented human embryos, but not in cryopreserved embryos which continued to develop normally after thawing. The TUNEL assay was positive in 30% (15/50) of arrested embryos, all of which had cytoplasmic fragments. In contrast, embryos showing regular size blastomeres without fragments were TUNEL negative.
Mol Hum Reprod 1998 Aug
PMID:Annexin V labelling and terminal transferase-mediated DNA end labelling (TUNEL) assay in human arrested embryos. 973 35

C2-ceramide, a cell-permeable analogue of ceramide, induced significant, dose- and time-dependent death in human retinoblastoma Y79 cells. Dying cells strongly displayed the morphology of apoptosis as characterized by microscopic evidence of cell shrinkage, membrane blebbing, nuclear and chromatin condensation and degeneration of the nucleus into membrane-bound apoptotic bodies. Upon induction of apoptosis Y79 cells evidence early phosphatidylserine externalization, as shown by annexin V-FITC. Apoptosis was also assessed by monitoring changes in cell granularity by staining with the combined fluorescent dyes acridine orange and ethidium bromide. C2-ceramide induced these morphological changes without a concomitant production of oligonucleosomal fragments responsible for the DNA ladder and without changes in p53 protein level. Apoptosis was accompanied by accumulation of a modified Bcl-2 protein with a slower-mobility form, and by proteolytic cleavage of PARP. The effect seemed to be specific for C2-ceramide, as C2-dihydroceramide, or other amphiphilic lipid analogues, or products of ceramide hydrolysis were ineffective. The effect also depended on mRNA and protein synthesis as it was markedly inhibited by actinomycin D and cycloheximide. Sphingomyelinase and interleukin-1beta, which are known to activate the sphingomyelin turnover leading to ceramide generation, also induced apoptosis mimicking the effects of ceramide. These findings propose ceramide as an activator of the suicidal program in Y79 cells.
Mol Cell Biochem 1998 Aug
PMID:Induction of programmed cell death in human retinoblastoma Y79 cells by C2-ceramide. 974 6

The tetraacetate ester of 2-deoxy-D-glucose was found to cause a concentration-related (10 microM to 1.0 mM) inhibition of activated T cell proliferation and decrease in viability of both unstimulated and anti-CD3 monoclonal antibody-stimulated peripheral blood mononuclear cells. Comparable findings were made in primary fibroblast cultures initiated from human skin explants. Likewise, 2-deoxy-D-glucose tetraacetate exerted a time-related (4-72 h) and concentration-related (0.1-1.0 mM) cytotoxic action upon human melanoma cells of the Colo 38 line. In this cell line, multiparameter flow cytometric analysis of cells stained with annexin V and propidium iodide revealed a necrotic, rather than apoptotic, form of cell death after exposure to 2-deoxy-D-glucose tetraacetate.
Int J Mol Med 1998 Feb
PMID:Cytotoxic action of 2-deoxy-D-glucose tetraacetate upon human lymphocytes, fibroblasts and melanoma cells. 985 46

The mechanism by which radiation induces human peripheral T cell apoptosis is not known. We examined sequential changes in post-irradiated peripheral blood mononuclear cells (PBMC(S)) taken from normal volunteers, by using flow-cytometer and an anti-CD3 monoclonal antibody, annexin V, propidium iodide, anti-Fas antibody, and anti-Fas ligand antibody. After 5 or 10 Gy of irradiation with a 60Co radiation therapy unit, most of the human peripheral T cells showed positivity against annexin V in 15 h, and positivity against propidium iodide in 23 h after irradiation. On a microscopy-video system, approximately 80% of mononuclear cells revealed apoptotic changes in 24 h after irradiation. Because of its proposed role in activation-induced cytotoxicity, we also examined the Fas (CD95/Apo-1) pathway in killing T cells by irradiation. Irradiated PBMC, displayed no increase in surface Fas expression and caspase-3 activity relative to non-irradiated cells. In addition, the anti-Fas ligand failed to eliminate the apoptotic death of PBMC, after irradiation. These results suggest that irradiation induces direct apoptosis of T cells by a Fas-independent mechanism.
Int J Mol Med 1998 Oct
PMID:Radiation kills human peripheral T cells by a Fas-independent mechanism. 985 24

When the cell membrane is disturbed, phospholipid phosphatidylserine (PS) is translocated from the inner to the outer leaflet of the plasma membrane. This is one of the earliest signs of apoptosis and can be monitored by the calcium-dependent binding of annexin V. Therefore, annexin V-binding, in conjunction with flow cytometry, was used to evaluate the integrity of the sperm plasma membrane after different cryostorage protocols: i.e. 10% (v/v) glycerol; sperm maintenance medium (MM); freezing medium TEST yolk buffer (TYB); or cryostorage without protection (cryoshock). Using a combination of two fluorescent dyes, annexin V and propidium iodide (PI), led to three groups of spermatozoa being identified: (i) viable spermatozoa (annexin V-negative and PI-negative); (ii) dead spermatozoa (annexin V-positive and PI-positive); and (iii) cells with impaired but integer plasma membrane (annexin V-positive and PI-negative). The percentage of vital annexin V-negative spermatozoa increased significantly (P < 0.05) from spermatozoa treated by cryoshock (15.0+/-1.2%) to spermatozoa cryopreserved by TYB (26.6+/-2.2%) via cryopreservation by 10% (v/v) glycerol (19.9+/-1.6%) and by MM (22.2 1.8%) and was associated with the percentage of motile spermatozoa (17.6+/-3.4% by glycerol; 19.6+/-3.7% by MM and 22.6+/-3.9% by TYB; P = 0.0001). Of the spermatozoa, 12-22% were annexin V-positive even though they did not bind to PI, indicating viability before as well as after cryostorage. The percentage of vital annexin V-positive spermatozoa was significantly correlated with different sperm motility parameters (velocity straight linear, r = 0.601, P = 0.018; percentage of linearly motile spermatozoa: r = 0.549, P = 0.034). We, therefore, concluded that annexin V-binding is more sensitive in detecting a deterioration of membrane functions than PI staining, and that a considerable percentage of spermatozoa might have dysfunctional plasma membranes besides dead or moribund cells. Of the cryopreservation protocols tested, TYB yielded the most viable spermatozoa. Therefore, we advocate the use of the annexin V-binding assay for the evaluation of the quality and integrity of spermatozoa.
Mol Hum Reprod 1999 Feb
PMID:Binding of annexin V to plasma membranes of human spermatozoa: a rapid assay for detection of membrane changes after cryostorage. 1006 65

The annexins comprise a family of soluble Ca2+- and phospholipid-binding proteins. Although highly similar in three-dimensional structure, different annexins are likely to exhibit different biochemical and functional properties and to play different roles in various membrane related events. Since it must be expected that these functional differences arise from differences in the characteristic thermodynamic parameters of these proteins, we performed high-sensitivity differential scanning microcalorimetry (DSC) and isothermal guanidinium hydrochloride (GdnHCl)-induced unfolding studies on annexin I and compared its thermodynamic parameters with those of annexin V published previously. The DSC data were analyzed using a model that permits quantitative treatment of the irreversible reaction. It turned out, however, that provided a heating rate of 2 K min-1 is used, unfolding of annexin I can be described satisfactorily in terms of a simple two-state reaction. At pH 6.0 annexin I is characterized by the following thermodynamic parameters: t1/2=61.8 degrees C, DeltaHcal=824 kJ mol-1 and DeltaCp=19 kJ mol-1 K-1. These parameters result in a stability value of DeltaG0D (20 degrees C)=51 kJ mol-1. The GdnHCl induced isothermal unfolding of annexin I in Mes buffer (pH 6.0), yielded DeltaG0D (buffer) values of 48, 60 and 36 kJ mol-1 at 20, 12 and 5 degrees C, respectively. These DeltaG0D values are in reasonable agreement with the values obtained from the DSC studies. The comparison of annexin I and annexin V under identical conditions (pH 8.0 or pH 6.0) shows that despite the pronounced structural homology of these two members of the annexin familiy, the stability parameters are remarkably different. This difference in stability is consistent with and provides a thermodynamic basis for the potential different in vivo functions proposed for these two annexins.
J Mol Biol 1999 May 21
PMID:A comparison of the energetics of annexin I and annexin V. 1032 95

Recent work from this laboratory demonstrated potent inhibition of apoptosis in human alveolar epithelial cells (AECs) by the angiotensin-converting enzyme inhibitor captopril [B. D. Uhal, C. Gidea, R. Bargout, A. Bifero, O. Ibarra-Sunga, M. Papp, K. Flynn, and G. Filippatos. Am. J. Physiol. 275 (Lung Cell. Mol. Physiol. 19): L1013-L1017, 1998]. On this basis, we hypothesized that apoptosis in this cell type might be induced by angiotensin II (ANG II) through its interaction with the ANG II receptor. Purified ANG II induced dose-dependent apoptosis in both the human AEC-derived A549 cell line and in primary type II pneumocytes isolated from adult Wistar rats as detected by nuclear and chromatin morphology, caspase-3 activity, and increased binding of annexin V. Apoptosis also was induced in primary rat AECs by purified angiotensinogen. The nonselective ANG II-receptor antagonist saralasin completely abrogated both ANG II- and angiotensinogen-induced apoptosis at a concentration of 50 microgram/ml. With RT-PCR, both cell types expressed the ANG II-receptor subtypes 1 and 2 and angiotensin-converting enzyme (ACE). The nonthiol ACE inhibitor lisinopril blocked apoptosis induced by angiotensinogen, but not apoptosis induced by purified ANG II. These data demonstrate the presence of a functional ANG II-dependent pathway for apoptosis in human and rat AECs and suggest a role for the ANG II receptor and ACE in the induction of AEC apoptosis in vivo.
 ...
PMID:Angiotensin II induces apoptosis in human and rat alveolar epithelial cells. 1033 45

Accumulating evidence indicates that fragmentation of ovulated murine oocytes, resulting spontaneously or following exposure to lethal stimuli such as anticancer drugs during in-vitro culture, occurs with several hallmark features of apoptosis. However, recent work has failed to demonstrate a correlation between DNA cleavage, as assessed by DNA 3'-end-labelling, or of phosphatidylserine exposure on the outer leaflet of the plasma membrane, as measured by annexin V-staining, with fragmentation of ovulated mouse or human oocytes maintained in vitro. Consequently, these authors stated that it is 'premature to conclude that apoptosis occurs in ovulated oocytes or that such a mechanism is involved in the elimination or prevention of fertilization of oocytes with cytoplasmic or chromosomal defects'. Here, we have re-assessed DNA cleavage in normal and fragmented murine oocytes, have provided new evidence of an additional biochemical marker of apoptosis in fragmented oocytes (i.e. caspase activity), and have re-evaluated published reports regarding oocyte fragmentation, in an effort to clarify these discrepant findings. The results and discussions presented herein fully support previous conclusions reached by ourselves and others that fragmentation of ovulated oocytes is in fact an unequivocal example of apoptotic cell death.
Mol Hum Reprod 1999 May
PMID:Fragmentation and death (a.k.a. apoptosis) of ovulated oocytes. 1033 64


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