Fractal organization of feline oocyte cytoplasm.

The present study aimed at verifying whether immature cat oocytes with morphologic irregular cytoplasm display self-similar features which can be analytically described by fractal analysis. Original images of oocytes collected by ovariectomy were acquired at a final magnification of 400x with a CCD video camera connected to an optic microscope. After greyscale thresholding segmentation of cytoplasm, image profiles were submitted to fractal analysis using FANAL++, a program which provided an analytical standard procedure for determining the fractal dimension (FD). The presentation of the oocyte influenced the magnitude of the fractal dimension with the highest FD of 1.91 measured on grey-dark cytoplasm characterized by a highly connected network of lipid droplets and intracellular membranes. Fractal analysis provides an effective quantitative descriptor of the real cytoplasm morphology, which can influence the acquirement of in vitro developmental competence, without introducing any bias or shape approximation and thus contributes to an objective and reliable classification of feline oocytes.

Resveratrol modulates apoptosis and oxidation in human blood mononuclear cells.

BACKGROUND: We examined the effect of resveratrol (RS), a nonflavonoid polyphenolic phytoalexin found in grapes and red wine, and RS coincubated with the oxidant 2-deoxy-D-ribose (dR), on apoptosis and on the oxidative metabolic status of normal human peripheral blood mononuclear cells (PBMNCs) isolated ex vivo from healthy donors. MATERIAL AND METHODS: Apoptosis was measured by changes of membrane permeability to propidium iodide (PI), plasma membrane exposure of phosphatidylserine (PS) and intracellular caspase activity. Oxidative status was assessed by recording the intracellular glutathione concentration (GSH), the activities of the enzymes y-glutamyltransferase (y-GT) and glutathione-S-transferase (GST), and intracellular lipid peroxidation (MDA). RESULTS: Neither apoptotic nor oxidative parameters were affected by culturing PBMNCs in medium containing RS up to 20 micro M for 5 days, while the frequency of cells with intermediate permeability to PI (17% +/- 5) increased at 50 micro M of RS. Thus resveratrol was slightly toxic, but there was little apoptosis in these cells. Peripheral blood mononuclear cells were also grown first in medium plus RS for 24 h and then for 96 h in medium containing RS plus 10 mM of dR, an oxidant sugar that is apoptogenic for human lymphocytes. The apoptotic changes triggered by dR were counteracted by the phytoalexin in a dose-dependent manner, but RS activity was absent at the lowest concentration (5 micro M) and significantly reduced at the highest concentration used (50 micro M). In PBMNCs coincubated with 20 micro M of RS and 10 mM of dR the antioxidant effect of RS manifested with a significant reduction of caspases-3, -8, y-GT, GST activities and MDA content. CONCLUSIONS: Peripheral blood mononuclear cells acquire antioxidant capacity when treated with RS. Grape resveratrol may make a useful dietary supplement for minimizing oxidative injury in immune-perturbed states and human chronic degenerative diseases.

Ultrastructural complexity of nuclear components during early apoptotic phases in breast cancer cells.

Fractal morphometry was used to investigate the ultrastructural features of the plasma membrane, perinuclear membrane and nuclear chromatin in SK-BR-3 human breast cancer cells undergoing apoptosis. Cells were incubated with 1 microM calcimycin (A23187) for 24 h. Cells in the early stage of apoptosis had fractal dimension (FD) values indicating that their plasma membranes were less rough (lower FD) than those of control cells, while their perinuclear membranes were unaffected. Changes of the chromatin texture within the entire nucleus and in selected nuclear domains were more pronounced in treated cells. This confirms that the morphological reorganization imputable to a loss of structural complexity (reduced FD) occurs in the early stage of apoptosis, is accompanied by the inhibition of distinct enzymatic events and precedes the onset of conventional cellular markers, which can only be detected during the active phases of the apoptotic process.

Spontaneous apoptosis, oxidative status and immunophenotype markers in blood lymphocytes of AIDS patients.

Peripheral blood mononuclear cells (PBMC) from 251 HIV-positive drug abusers of known clinical stage and from 40 healthy donors were tested for conventional immunologic markers (CD3, CD4, CD8, CD19, CD14, CD16/CD56, CD45 and HLA-DR). Additional cell parameters and the occurrence of spontaneous apoptosis (programmed cell death) were investigated on freshly isolated PBMC by flow cytometric measurement of either annexin-V bound to plasma membrane phosphatidylserine or propidium iodide uptake. The activity of gamma-glutamyltransferase (gamma-GT), an ectoenzyme contributing to the synthesis of the intracellular antioxidant glutathione (GSH) and involved in early apoptosis, was also determined in these cells. Immunocompetent T-cell counts were lower in HIV+ patients, with the exception of CD8+ and HLA-DR+ lymphocytes. The external binding of annexin-V was significantly higher in HIV+ PBMC and occurred in both CD8+ and CD4+ T-lymphocyte subsets. The activity of gamma-GT, was significantly lower in the PBMC from HIV+ patients, indicating that the redox status of PBMC may be affected in HIV+ individuals. Finally, the most dominant features characterising patients receiving antiretroviral therapy were greater long-term stability in the distribution of various cell parameters excepted the level of apoptosis.

Effects of steroid hormones on nuclear membrane and membrane-bound heterochromatin from breast cancer cells evaluated by fractal morphometry.

OBJECTIVE: To evaluate the effect of steroid hormones on the ultrastructure of nuclear heterochromatin and perinuclear membranes in human MCF-7 breast cancer cells. STUDY DESIGN: MCF-7 cells were cultured briefly (five minutes) in the presence of 10(-9) M estrogen 17 beta-estradiol, a stimulator of cell proliferation and/or 10(-9) M glucocorticoid dexamethasone. Changes in the morphologic complexity of nuclear membrane-bound heterochromatin (NMBHC) and nuclear membranes (ENM) were assessed by means of the fractal capacity dimension, D, a noneuclidean geometric descriptor of complex, irregular bodies. RESULTS: 17 beta-estradiol (10(-9) M) enhanced the ultrastructural irregularity of NMBHC, as documented by the increased value of D, whereas dexamethasone (10(-9) M) reduced it when compared to NMBHC from untreated MCF-7 control cells. In contrast, neither steroid modified ENM ultrastructure. Changes in the nuclear heterochromatin complexity induced by estrogen 17 beta-estradiol occurred concomitantly with functional changes at the cell periphery, such as activation of the phospholipase C, a cell membrane-associated enzyme involved in signal transduction. Dexamethasone reduced the ultrastructural complexity of NMBHC without affecting functional processes. CONCLUSION: Fractal morphometry proved its usefulness in quantifying early ultrastructural changes in nuclear components induced in MCF-7 cells by steroid hormones, 17 beta-estradiol and dexamethasone.

Apoptosis and oxidative status in peripheral blood mononuclear cells of diabetic patients.

We have compared the concentrations of intracellular glutathione (GSH), glutathione-dependent antioxidative enzymes, the cell death rate and immunophenotype profile of peripheral blood mononuclear cells (PBMC) from healthy donors and from patients with insulin-dependent type II (NIDDM) diabetes mellitus. The IDDM and NIDDM patients had above-normal absolute lymphocyte counts, whereas the percentages of CD3, CD4 adn CD8 T lymphocytes were significantly reduced. In contrast, the absolute number and percentage of B lymphocytes was higher in diabetic patients than in healthy donors. The low intracellular reduced glutathione(GSH) and the unbalanced profile of key enzymes involved in GSH metabolism, gamma-glutamyltransferase (gamma-GT) and glutathione-S-transferase (GST), account for the increased oxidative status of PBMC from diabetic patients. The plasma membranes of PBMC for diabetic patients were less permeable to propidium iodide than those of PBMC from healthy donors, indicating that the apoptotic cell death rate was lower in the cells from diabetic patients. These differences are potentially useful markers of pathogenic metabolic changes which occur during clinical diabetes and if they are confirmed could be use dot identify the onset of diabetes.

Steroid hormones modify nuclear heterochromatin structure and plasma membrane enzyme of MCF-7 cells. A combined fractal, electron microscopical and enzymatic analysis.

Ultrastructural features of the nuclear membrane envelope (ENM) and the nuclear membrane-bound heterochromatin (NMBHC) were investigated in MCF-7 human breast cancer cells by fractal morphometry. The fractal dimension D established by the box counting method proved to be effective for quantifying nuclear changes in MCF-7 cells treated with steroid hormones, namely the estrogen 17 beta-estradiol, which stimulates cell proliferation, and the glucocorticoid dexamethasone. When MCF-7 cells were briefly (5 min) cultured in the presence of 17 beta-estradiol (10(-9) M), the irregularity of the NMBHC outline was increased as documented by the increased fractal dimension D. Changes in the ultrastructural complexity of the nuclear heterochromatin were observed in concomitance with functional changes at the cell periphery, namely the modulation of the estrogen-induced activity of phospholipase C, a cell membrane-associated enzyme involved in the signal transduction pathway via phosphoinositides metabolism. Dexamethasone did not affect the in vitro proliferation, the phospholipase C activity nor the shape of the ENM of MCF-7 cells, but reduced the structural complexity of the nuclear membrane-bound heterochromatin.

Apoptotic cell death and the proliferative capacity of human breast cancers.

The proliferative capacity (%S-phase fraction), DNA ploidy, apoptosis frequency (DNA fragmentation) and steroid hormone receptor status (estrogen receptor, ER; progesterone receptor, PR) of 110 samples of human breast tissues with ductal invasive carcinoma were measured using biochemical and cytofluorimetric procedures. The DNA fragmentation had a left-skewed frequency distribution and an overall median value of 1.64%, whilst the median %S-phase fraction was 8%. The median %DNA fragmentation and %S-phase fraction were 1.96% and 16% in hyperdiploid tumours (n = 29; DNA index >1.1) higher than in hypodiploid tumors (n = 10; DNA index <0.96), 0.38% and 7.5%. DNA diploid tumours (n = 71) had median %DNA fragmentation and %S-phase values of 1.68% and 6%, consistently lower than the median values of DNA hyperdiploid tumours. The ER content of hypodiploid tumours was about one half (median: 5.9 fmol/mg) the median values in hyperdiploid (10.6 fmol/mg) and diploid tumours (14.6 fmol/mg). This may correlate with the lowest frequency of apoptosis in hypodiploid tumours, at least when measured by biochemical methods which only detect cells in the late phases of apoptosis. In contrast, the median PR was lowest in hyperdiploid tumours than in hypo and/or diploid tumours. The %S-phase/%fragmented DNA ratio for the hypodiploid tumours was 19.7, significantly higher than the ratios for hyperdiploid (8.2) and diploid tumours (3.6). These findings indicated that there is an imbalance between proliferative capacity and cell death or growth arrest in human breast tumours. This imbalance may well be linked to a loss of steroid hormone control.

Calcium Dobesilate protects human peripheral blood mononuclear cells from oxidation and apoptosis.

The antioxidant effects of Calcium Dobesilate (CD, Doxium) were investigated in relation to the oxidative status, apoptosis and in vitro proliferation of human peripheral blood mononuclear cells (PBMC) isolated from healthy donors. CD alone did not modify cell growth in vitrountil 10 microM. This molecule counteracted oxidative damages generated by the high reducing sugar dR and was shown to reduce apoptosis by delaying both membrane permeability changes and DNA fragmentation. CD 10 microM affected in a time-dependent dynamics several parameters representative of the cellular oxidative status. In particular, CD significantly increased the activity of glutathione S-transferase (GST) after three days of treatment and also, but to a lower extent, the activity of gamma-glutamyltransferase (gamma-GT). Both enzymes are known to be involved in the glutathione (GSH) metabolic cycle. This enzymatic behaviour was reversed at seven days of treatment, with a significant GST decrease and a gamma-GT activation. After seven days of CD exposure, the intracellular GSH content was enhanced and this resulted in a dramatic decrease in lipid peroxidation, underlining the powerful antioxidant properties of CD in human PBMC.

Self-similarity and fractal irregularity in pathologic tissues.

The irregularity and self-similarity under scale changes are the main attributes of the morphologic complexity of cells and tissues, either normal or pathologic. In other words, the shape of a self-similar object does not change when scales of measure change because any part of it might be similar to the original object. Size and geometric parameters of an irregular object, however, differ when inspected at increasing resolution, which reveals more details. Significant progress has been made over the past three decades in understanding how to analyze irregular shapes and structures in the physical and biologic sciences. Dominant influences have been the discovery by B.B. Mandelbrot of a new, practical geometry of nature, now called fractal geometry, and the continuous improvements in computational capabilities. The application of the principles of fractal geometry, unlike the conventional Euclidean geometry developed for describing regular and ideal geometric shapes practically unknown in nature, enables one to measure the fractal dimension, contour length, surface area, and other dimensional parameters of almost all irregular and complex biologic tissues. During the past decade, a large amount of experimental evidence has accumulated showing that even in biomedical sciences fractal patterns could be observed. Through several examples borrowed from the recent literature, we focus on the application of the fractal approach to measuring irregular and complex features of pathologic cells and tissues and also on its potential role in the understanding of tumor biology.

Changes in membrane enzymes and glycosphingolipids in lymphocytes from HIV-1--infected and noninfected intravenous drug users.

The amounts of cell-surface glycosphingolipids and plasma membrane enzymes produced on the peripheral blood mononuclear cells (PBMNCs) isolated from 101 intravenous drug users (IDUs), of whom 91 were HIV-1 seropositive, were examined. Seronegative IDUs and age-matched healthy donors served as controls. The numbers of circulating CD3+, CD4+, and CD8+ T lymphocytes decreased during the advanced stages of the infection. There were also fewer CD4+ T-helper cells in HIV-1--seronegative IDU drug addicts. PBMNCs from HIV-1--seropositive subjects had abnormal surface enzyme kinetics. The phospholipase C had two pH optima, whereas the enzyme on normal cells has only one. The specific activity in cells from AIDS subjects was 4 times lower than that in normal PBMNCs. 5'-Nucleotidase showed a similar trend, whereas neutral endopeptidase activity did not correlate with the amounts of surface common acute lymphoblastic leukemia antigen (CALLA). These enzyme activities were decreased in HIV-seronegative IDUs. The subcellular distribution of enzymes and the profile of surface glycosphingolipids were also markedly changed, indicating the profound alterations in the membranes of PBMNCs from HIV-1--seropositive IDUs. These data suggest that intravenous drug use compromises the biochemical and structural integrity of the membrane surface of PBMNCs even before the onset of HIV.

Enzymatic, biophysical and ultrastructural changes of plasma membranes in chemical-induced rat hepatoma.

Plasma membranes from liver of control rats or from chemical-induced hepatoma were prepared. The basal activity of adenylate cyclase was increased significantly in the rat plasma membranes of DEN-induced hepatoma compared to normal tissue. The glucagon-induced response on the cellular effector systems via guanine nucleotide-binding regulatory proteins (G proteins) was inhibited in hepatoma plasma membranes. These findings suggest that in hepatoma membranes, unlike normal hepatic membranes, the response to hormonal stimuli through regulatory G proteins results in a loss of response to glucagon, as well as to GTP plus glucagon or to GTP gamma S. However, the activating effects of forskolin, which catalyses the formation of cyclic AMP from ATP acting on the catalytic subunit, were to some extent retained. The methyltransferase-I behaved in the opposite direction to the adenylate cyclase, showing a decreased activity in hepatoma plasma membranes compared to control membranes. In contrast, the activity of the ecto-5'-nucleotidase was significantly increased in hepatoma. These enzymatic changes have been found to influence the membrane fluidity and to be responsible for the ultrastructural modifications of hepatoma plasma membranes which are induced by chemical carcinogens.

Apoptosis in human lymphoblastoid cells induced by acivicin, a specific gamma-glutamyltransferase inhibitor.

We examined the effects of acivicin, a specific inhibitor of the ectoenzyme gamma-glutamyltransferase (gamma-GT), on gamma-GT activity and apoptosis in 2 human T-lymphoblastoid CEM cell lines, CCRF and VBL-100. In both cell lines, acivicin was found to cause morphological and biochemical changes of apoptosis in a dose-dependent manner. There was a close correlation between inhibition of gamma-GT activity and the emergence of apoptotic cells. However, VBL-100 cells had a 50% higher gamma-GT basal activity than CCRF-CEM cells, their enzyme activity was more inhibited, and, they had a greater apoptotic response to acivicin. The gamma-GT-specific activity in apoptotic/dead cells was also almost totally inhibited, while that of cells that remained alive after 5 days of acivicin treatment was not. These findings confirm that gamma-GT is implicated in the process of apoptosis of CEM cells.

Fractals in pathology: are they really useful?

Through several examples selected from functional and pathological cells and tissues, including surface, cytoplasmic and nuclear membranes one will focus on the application of the fractal approach to measuring irregular and complex structures, on the importance of evaluating real morphology and on its potential role in understanding of tumor biology.

Changes in the activities of signal transduction and transport membrane enzymes in CEM lymphoblastoid cells by glucocorticoid-induced apoptosis.

The behaviour of the membrane enzymes PIP2-phospholipase C (PLC), that modulates the extracellular signals, and gamma-glutamyltransferase (gamma-GT), involved in metabolite transport, was followed during the early and active phases of apoptosis induced in CCRF-CEM cells by glucocorticoid (10(-6) M dexamethasone, DEX). The activities of gamma-GT and PLC increased significantly at 15 and 30 s after dexamethasone addition. Both activities decreased to the control level after 2 min but increased again at 5 min. The enzyme activities were high in apoptotic cells. Apoptosis was seen after incubation with 10(-6)M dexamethasone for 48 h, with decreased cell number, cellular activation (MTT) and S-phase percentage; enhanced DNA fragmentation and propidium iodide uptake; and ultrastructural changes in the chromatin and cell membranes. The changes in enzyme activity are indicators which occur much earlier than the cellular events related to the apoptotic death in CD4(+)-T CEM lymphoblastoid cells.

Digital image analysis of self-similar cell profiles.

Many biological objects appear to have self-similar structures which can be characterized by their fractal dimension D. However, applications of the concept of fractal geometry are rather scarce in cell and tissue biology. Here we adapt and analyse critically 3 methods of digital image analysis to measure D of cellular profiles. As prototype examples we investigate in detail 2 samples of cells: (i) human T-lymphocytes from normal donors, and (ii) hairy leukemic cells. It is shown that D correlates to the structural complexity of the individual cell contour. The calculated D values for cells out of the same cell line scatter around a mean value D = 1.15 for T-lymphocytes (S.D. = 0.03) and D = 1.34 for hairy leukemic cells (S.D. = 0.04). Consequently, we interprete D as a statistical measure for the sample's fractal dimension.

Effect of cocaine and morphine on neutral endopeptidase activity of human peripheral blood mononuclear cells cultured with lectins.

We have tested the effect of alkaloids (cocaine, morphine) and enkephalins on neutral endopeptidase of peripheral blood mononuclear cells activated by lectins. When treated with concanavalin A and cocaine, peripheral blood mononuclear cells showed an enhanced activity (+110 per cent) of the membrane neutral endopeptidase, which was not related to the expression of the common acute lymphoblastic leukemia antigen at the cell surface, although both molecules have the identical amino acid sequence. Phytohemagglutinin-P, morphine and synthetic enkephalins did not induce the activity of neutral endopeptidase nor the expression of common acute lymphoblastic leukemia antigen. Our findings suggested that the drugs of abuse, cocaine and morphine, affected specific membrane constituents without altering proliferation, subcellular localization of membrane enzymes or the surface immune phenotype of peripheral blood mononuclear cells.

Sulfated proteoglycans in the extracellular matrix of human breast tissues with infiltrating carcinoma.

The size, content and distribution of sulfated proteoglycans, constituents of the extracellular matrix, were investigated in non-neoplastic human breast tissues and in tissues with infiltrating carcinoma. Sulfated proteoglycans were identified as cuprolinic-blue-positive elements on electron microscopy preparations by the method of the critical electrolyte concentration. Morphometric data indicated that the numerical density of PG per surface area (NA) in normal tissues was about 2 to 7 times higher respectively in dense or loose connective zone then that found in the corresponding zones of malignant tissues. By referring to the unit volume of tissue, the overall PG content (Nv) was found to be increased about 5 fold in nonneoplastic tissues. However, in tissues with carcinoma the average length of sulfated proteoglycans was about twice as long in dense and loose connective layers than in the corresponding connective zones of non-neoplastic breast tissues. Our findings provide evidence of profound structural alterations of non-collagenous constituents in the extracellular matrix of human mammary tissues concerned with infiltrating carcinoma.

Lectins and anti-T monoclonal antibodies-induced changes of second messengers generating enzymes in human peripheral blood mononuclear cells.

A five min. incubation of peripheral blood mononuclear cells (PBMN) with either phytohaemagglutinin (PHA) or concanavalin A (ConA) resulted in distinct subcellular redistribution patterns of phosphatidylinositol 4,5-bisphosphate phospholipase C [PtdIns(4,5)P2-PLC] and myo-inositol 1,4,5-trisphosphate monophosphatase [Ins(1,4,5)P3-monophosphatase] activities. When compared to control cells, PHA-treated PBMN cells displayed a significant increase of PtdIns(4,5)P2-PLC and Ins(1,4,5)P3-monophosphatase relative specific activities in the nuclear fraction along with an increment (D) in enzyme amount of 6.5% and 7.3%, respectively. Incubation with B66.6, an anti-CD4 monoclonal antibody (Mab) which specifically activates CD4(+)-T cells in the absence of any other stimuli, also induced changes of these activities in the nuclear fraction, thus mimicking the effect of PHA observed in helper T cell subpopulation. No changes were detected after incubation of PBMN cells with the non mitogenic anti-CD4 MAb 101-69, or with an anti-CD3 MAb which activates T cells only in the presence of a second stimulus. On the other hand, after incubation with ConA, PtdIns(4,5)P2-PLC relative specific activity was enhanced in the microsomal fraction while the Ins(1,4,5)P3-monophosphatase activity increased in both nuclear and microsomal fractions and decreased in cytosol. An increment D of 4.6% and 10.9% for PtdIns(4,5)P2-PLC and Ins(1,4,5)P3-monophosphatase, respectively, was measured in the microsomal fraction. Only after three days of incubation with a mitogenic anti-CD2 MAb Lau-2.1.2, the PtdIns(4,5)P2-PLC activity increased in the particulate fraction of PBMN similar to ConA treatment.

Subcellular localization of inositide enzymes in established T-cell lines and activated lymphocytes.

The specific activity of phosphatidylinositol 4,5-bisphosphate phospholipase C (PIP2-PLC) and inositol 1,4,5-trisphosphate monophosphatase (IP3-MP) involved in phosphoinositide catabolism was found to be significantly lower in the total homogenate of four human lymphoblastoid cell lines, HSB-2, MOLT-4, CEM and JURKAT, than in resting and activated peripheral blood mononuclear cells, ranging from 0.8 to 3.2 and from 1.3 to 3.7 nmol/min/mg for PIP2-PLC and IP3-MP, respectively. In PHA-stimulated cells, the specific activities were enhanced 25 and 35% respectively over the values (8.02 and 7.83 nmol/min/mg, respectively) measured in resting peripheral blood mononuclear cells. After centrifugation on discontinuous sucrose gradient of cell homogenate, PIP2-PLC and IP3-MP activities were found to be predominantly associated with the cytosol fraction (> 69%) in HSB-2 and MOLT-4 cells, with a distribution similar to that found in PHA-stimulated and in resting lymphocytes. In CEM cells, about half of the total activity remained in this fraction, while in JURKAT lymphoblastic cells more than 45% of the total activity was recovered in the high-density membrane fraction (d = 1.20-1.25), the soluble PIP2-PLC and IP3-MP activity accounting for only 13 and 25%, respectively. Conversely, in less differentiated leukemic cells HSB-2 and MOLT-4, conspicuous activity of the ectoenzymes 5'-nucleotidase (5'-NT) and gamma-glutamyltransferase (gamma-GT) was recovered in the soluble fraction. Growing leukemic cells at a distinct level of differentiation have a general reduction in activity but a characteristic distribution of enzymes involved in the transmission of signals usually targeting the cell surface.