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G Carruba

Publications and source records attributed to G Carruba.

At least 55 records · Page 3Linked to original sources
PubMed1991

Simple approach to measure metabolic pathways of steroids in living cells.

A simple, rapid approach to the study of conversion rates and metabolic patterns of the steroids testosterone and estradiol is presented. It includes an optimized isocratic high-performance liquid chromatographic procedure in the reversed-phase mode and radioactive on-line detection. The purpose was to estimate the activity of key enzymes of steroid pathways, such as 17 beta-hydroxysteroid dehydrogenase and 5 alpha-reductase, in in vivo conditions. Using this system, we obtained good efficiency and linearity of radio detection, under continuous flow conditions. Sensitivity limits were of the order of 50 and 70 cpm for [3H]estradiol and [14C]estrone, respectively, even though the efficiency was quite dissimilar (17.3% versus 56.2%). The applicability of this approach to studies of steroid metabolic pathways in growing cancer cells in culture is illustrated with examples of the conversion rates of both testosterone and estradiol. The high reproducibility (coefficients of variation of 2.7 and 5.1% for 3H and 14C, respectively) and good extraction efficiency (ranging from 86 to 94%) indicate the feasibility and reliability of this approach.

17-Hydroxysteroid Dehydrogenases↗
PubMed1991

Androgen receptor assays in specimens of prostatic tissue obtained by transurethral resection and transvesical adenomectomy.

The main goal of this study was to ascertain whether routine transurethral resection (TUR) of prostate may provide useful material for the evaluation of androgen receptor (AR) status. At the same time, either intracellular distribution of binding affinity and capacity of receptor molecules were particularly taken into account. Based on our previous findings in breast and endometrial cancer, we suggest that a "functional" receptor status may correspond to the presence of type I (high affinity, low capacity) AR in both soluble and nuclear fractions. However, the precise significance of type II (lower affinity, higher capacity) binding sites remains to be clarified. Ten samples of large prostatic adenomas, obtained by transvesical adenomectomy (TVA), were compared with ten parallel specimens obtained by an in vitro TUR, whereby a pure cutting current was used. The AR assay was carried out with a standard competition method using tritiated mibolerone as the radioligand and Scatchard analysis for data processing. No significant difference between the TUR and TVA groups emerged concerning type I AR content of soluble, nuclear or soluble together with nuclear fractions; this was also true when the results were expressed either as fmol/ml homogenate or as fmol/mg DNA. Similarly, concentrations of type II AR in TVA and TUR samples did not differ significantly in either cell compartment, although they were widely scattered, especially in the soluble fraction. In the light of our findings, it is suggested that TUR specimens represent suitable material for receptor studies, provided that only cutting current is employed and that the use of coagulation current, to control bleeding from the prostatic bed, is confined to the final step of the TUR procedure.

Electrocoagulation↗
PubMed1991

DNA fingerprinting and electrophoretic karyotype of environmental and clinical isolates of Candida parapsilosis.

The endonuclease restriction pattern (DNA fingerprinting) and the electrophoretic karyotype of 16 Candida parapsilosis isolates from environmental and clinical sources were investigated. DNA from both whole cells and separated mitochondria was digested with enzymes, including EcoRI, BamHI, KpnI, BglII, HpaII, PvuII, and HindIII. Regardless of their source and pathogenic properties, all isolates showed a uniform, reproducible, and overlapping whole-cell DNA fingerprinting with each endonuclease digest. Mitochondrial DNA fragments were, in all cases, major contributors to the total cellular DNA restriction pattern. In contrast, the electrophoretic karyotype generated by rotating field gel electrophoresis (RFGE) or contour clamped homogeneous field electrophoresis (CHEF) showed a remarkable polymorphism among the isolates. This polymorphism concerned the smaller molecular size section of the karyotype (range, 1.8 to 0.7 Mb), where at least two to five chromosomal bands could be consistently detected by both RFGE and CHEF. Larger (greater than or equal to 3.0 to 1.9 Mb) chromosome-sized DNA bands (four in CHEF and three in RFGE) were quite distinct and common to all isolates. Thus, seven karyotype classes could be defined, on the basis of both the number and size of putative chromosomes. The three categories of isolates (soil, vaginal, and hematological) were not randomly distributed among the seven classes. In particular, the four hematological isolates had a karyotype pattern which was clearly distinct from that shown by the three environmental isolates, and of the nine vaginal isolates only one shared a class with isolates from another source (soil). Although tentative, the classification was totally consistent with the independent and reproducible results obtained by the two pulse-field electrophoretic techniques employed. It is suggested that the electrophoretic analysis of the karyotype might be particularly useful for epidemiological and pathogenicity studies on biotypes of C. parapsilosis.

Candida↗
PubMed1990

Prostate long-term epithelial cell lines. Biological and biochemical features.

This review reports studies on long-term prostate cell lines using multiple experimental approaches. The main goal was to investigate the metabolism of testosterone (T) through in vitro conversion rates. Extensive studies were also carried out on growth curves, tritiated thymidine incorporation, and morphometry by either hormone-responsive or hormone-unresponsive, normal and neoplastic human (PC3 and DU-145) and canine (CAPE and CPA) cell lines. All of them were characterized for their content of both soluble and nuclear androgen receptors. Receptor studies at site I binding in both soluble and nuclear fractions were carried out to establish the hormone sensitivity status of cells. In two prostate epithelial cells, steroid metabolic conversions in vitro show predominantly an oxidative metabolism of T, forming mainly androstenedione. Conversion rates were greater than 50% in the first 24 hours and still higher after 72 hours. At the same time and under exactly the same experimental conditions, the other cells showed metabolic pathways in which reductive metabolism prevails, dihydrotestosterone (DHT) being the prevalent metabolite. Different metabolic patterns of steroids of several cell lines relate to the hormone sensitivity status of the cells; steroid receptor-endowed cells are maintaining higher levels of unconverted precursor than are receptor-empty cells. In fact, hormone-sensitive cells, such as cancer canine CPA and human DU-145, produced DHT early through slowly converting T. On the contrary, unresponsive cells such as human cancer cells PC3 and normal canine CAPE quickly metabolize T, but DHT formation was not observed. These significant differences between cells are highly reproducible provided the proportion between cell number and molar concentration of precursors is constant. Differences we observe cannot be attributed to different experimental conditions. Cell viability, extraction efficiency, and all other parameters used for monitoring cell growth kinetics do not substantiate these reported significant differences in metabolic abilities of cells. The divergent steroid metabolic pathway we observe in different prostate long-term cells appears to be an intrinsic, consistent, highly reproducible property of each cell line.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1989

Morphometry of in vitro systems. An image analysis of two human prostate cancer cell lines (PC3 and DU-145).

Morphometric analysis on two human prostate cancer cell lines (PC3 and DU-145) was carried out to better characterize these cells and to approach some problems concerning their polymorphism. In our hands, these cells appear to differ either in their ability to oxidize testosterone or in their androgen receptor content. Morphometric evaluation was performed by means of an Interactive Image Analysis System (IBAS 1), which computes several parameters related to the whole cell and to the nucleus. Our results clearly indicate that nuclear parameters alone may discriminate PC3 from DU-145 cells. Particularly, the Nuclear Roundness Factor appeared important, since it quantifies changes in nuclear shape. Nevertheless, the value of this parameter for "in vitro" morphometry may be overshadowed by the polymorphism common to the cultured cells. However, morphometric evaluation of PC3 and DU-145 cells is relevant to a better understanding and definition of the biologic nature of these cells, even if polymorphism, heterogeneity and genetic instability, specially of PC3, require further investigations.

Cell Nucleus↗
PubMed1989

Do multiple oestrogen receptor assays give significant additional information for the management of breast cancer?

In 101 breast cancer patients, measurement of oestrogen receptor status in multiple biopsies across a tumour reveals a highly significant difference in the proportion of patients remaining either disease-free (P less than 0.04) or alive (P less than 0.005), when those with uniformly receptor positive (++) primary tumours are matched with clinically comparable patients whose tumours were homogeneously receptor negative (--). Mean follow-up time was 85 months. The prognostic value of this discriminant is particularly striking in the 53 patients with involved nodes at presentation. Of these, 13 were (++) and seven remain alive of whom six are disease-free, whereas 24 of the 29 (--) patients are dead. These results further suggest that receptor assay on a single homogenate gives less clinical information than do assays on multiple biopsies across the tumour. For patients with involved nodes, clinical management may best be decided after determination of 'macroheterogeneity'.

Breast Neoplasms↗
PubMed1987

Heterogeneity of soluble and nuclear oestrogen receptor status of involved nodes in relation to primary breast cancer.

Both soluble and nuclear oestrogen receptors were measured in at least two different portions of primary breast cancer and in concurrent metastatic tissue from axillary nodes. Oestrogen receptor (ER) status of involved nodes was found highly consistent with that of primary tumours. Of the 67 patients studied, 30 had metastatic nodes which contained both soluble and nuclear ER. Of these, 27 were associated with a primary cancer which also had both soluble and nuclear ER, determined in at least two separate parts of the primary cancer. Conversely, none of the completely negative primaries gave rise to fully receptor positive metastatic tissue. Surprisingly, 17 out of 20 heterogeneous primary tumours, i.e. those containing both receptor positive and negative components, generated receptor negative metastatic nodes. Moreover, in 7 of the 8 patients with N-2 stage nodal involvement, the metastatic disease had arisen from primaries which were either completely receptor negative or with a heterogeneous ER status. It is suggested that macroscopic heterogeneity of ER status in primary breast cancer is associated with poor prognosis.

Adult↗
PubMed1986

Estrone conversion rates by human endometrial cancer cell lines.

Studies on estrone metabolism by long term human endometrial cancer HEC-1A and Ishikawa cell lines are reported. These cell lines grow well in epithelial mono or plurilayer form, as previously reported. Ishikawa cells appear to be estrogen responsive whereas HEC-1A appear non responsive. In our experience Ishikawa cells show high affinity--low capacity estrogen binding sites in both soluble and nuclear fractions of the same range of ZR 75-1 and of MCF7, but HEC-1A cytosols gave Kd values in the order of 10(-8)-10(-9). These values are probably more representative of estrogen receptors of low affinity-high capacity (site II) and this is in agreement with previous results regarding their poor response to estrogens. These two different endometrial cancer cell lines exhibit at the same time, common and quite dissimilar metabolic patterns of estrogens. In fact, metabolic conversion studies carried out after 24th incubation at not far from physiological concentrations by using high pressure liquid chromatography in reverse phase mode plus "on line" radioactive detection showed: Both these well established cell lines are fast growing in culture with sufficient morphological or biochemical stability, at least during a limited number of passages and appear a useful material for studies on steroid metabolism. In both, estradiol (E2) and estrone (E1) were most part of converted products (more than 95%); negligible amounts of other radio-metabolites were observed. Quite different conversion rates of E1 to E2 have been shown by HEC-1A cells (6 times or more), with respect to Ishikawa.

Cell Line↗
PubMed1985

Modulation of oestrogen excretion profiles by adjuvant chemotherapy in pre- and postmenopausal breast cancer.

Modulation of steroid status by conventional chemotherapy was studied in 31 breast cancer patients receiving CMF and in 31 age-matched breast cancer patients without any therapy, taken as controls. This was achieved through the study of oestrogen excretion profiles using previously identified parameters and referring not only to classical but also to the "other", namely catechol and unusual, oestrogen metabolites. After CMF treatment the premenopausal patients exhibit a modified excretion pattern, mainly concerning a marked and significant reduction of classical oestrogens, as shown by pattern indices. Because there is evidence that oestriol metabolism is not markedly affected by CMF treatment, such a significant decrease in classical oestrogens must be attributed to the secretory function, presumably ovarian ab origine. To the contrary, after treatment, pattern indices show significantly higher median values in postmenopausal patients. Mean oestriol ratio values also display a significant increase, thus supporting the hypothesis that conventional cytotoxic drugs may act by enhancing oestrogen metabolic rates. In fact, the postmenopausal treated subgroup proved to have significantly higher excretion levels of most of the oestrogens considered to date. Surprisingly, E1 + E1-S fractions were strongly reduced in this subgroup and this leads to the suggestion of an increased steroid metabolic rate by CMF treatment. However, comparing 9 breast cancer patients, when having had both short-term and non-short-term CMF treatment, the effects on steroid excretion patterns appear to arise at an early stage.

Adrenal Cortex↗
PubMed1985

Differential expression of surface proteins, virus receptors and histocompatibility antigens in SV40-transformed human choroid cells and their clones.

Human choroid (HC) cells transformed by Simian virus 40 (HC/SV40) and some of their soft agar-derived clones were utilized to study the influence of SV40 transformation on the expression of some differentiation parameters. The presence of receptors for DNA and RNA viruses, known to induce cytopathic effects in target cells, always occurred in normal HF and HC/SV40 cells as well as in their derived clones, whereas differences were observed in the expression of influenza A and Sindbis virus receptors. Immunofluorescence assays showed an increase in the expression of class I (HLA-A/B/C) and class II (DR) histocompatibility antigens in hyperdiploid clones compared with both the hypodiploid clones and the normal human cell counterpart. The two surface glycoproteins, fibronectin and laminin, appear only partially correlated with the transformed phenotype of the cells both at optical (immunofluorescence) and supraoptical (immunoelectron microscopy) level. This study confirms the SV40 transformation induces in human choroid cells a modulation of the considered differentiation parameters and that this may be due to a different insertion mode of the SV40 genome in HC cells.

Amnion↗
PubMed1984

Transformation of human choroid cells in vitro by SV40. Ultrastructural and cytogenetic analysis of cloned cell lines.

Several clones were isolated from a Simian Virus 40-transformed human choroid cell line (HC/SV40) by the soft agar plating technique. Five of them which showed different morphology, growth capacity and cell density, were plated in monolayer and subjected to ultrastructural, immunological and cytogenetic analyses. The morphological features at both TEM and SEM were distinct for every examined clone but uniform in the cells of the same clone. In particular clone 3 (HC/SV40-C13) showed a greater increase of intracytoplasmic granules, a marker of all cells of choroid origin, while clone 2 (HC/SV40-C12) was totally devoid of them. No virus production was observed at the electron microscope. Immunofluorescence experiments revealed 100% positivity for nuclear T-antigen in all the clones, thus demonstrating the persistence of SV40 genome in these cells. Cytogenetic analysis showed a hyperdiploid number of chromosomes in clones 1 and 3, while clones 2 and 4 were clearly hypodiploid. All clones showed a general tendency to cytogenetic stabilization as compared to the karyotypes found in HC/SV40 parental cell line.

Cell Division↗
PubMed1983

Ultrastructural and biological characterization of human choroid cell cultures transformed by Simian Virus 40.

A human diploid cell line of choroid origin was isolated from the retrouveal portion of an enucleated eye and designated HC. After 10 passages, when the proliferative capacity of HC cells decreased, they were infected and transformed by Simian Virus 40 (SV40). A proliferating long-term cultured cell line designated HC/SV40 was established and it has been maintained as monolayer for more than 100 passages so far. The two cell lines, HC and HC/SV40, were compared for growth characteristics, capacity to form colonies in soft agar, presence of nuclear T-antigen, and ultrastructure. Cytogenetic analysis was also performed to determine the presence of chromosomal aberrations due to the permanent viral transformation of the cell line. The results indicate that HC/SV40 should be considered the transformed counterparts of HC cells because they are morphologically similar to the latter but can grow in soft agar, possess T-antigen, and show a pattern of karyotypic changes similar to that induced by SV40 in human fibroblasts. The choroid origin of HC and HC/SV40 cell lines was confirmed by the presence, in their cytoplasm, of typical electron dense granules. Their neural origin will make these cell lines very useful for neuropharmacological and differentiation studies.

Antigens, Viral↗
PubMed1983

Can SV40 transformation reverse commitment to terminal division in human adult cells in vitro?

Adult Human Eye diploid Fibroblasts (HEF) had an extremely low growth potential in vitro (12 duplication cycles), that was identical in all the isolated clones. HEF cells were transformed with SV40 virus at the 10th duplication cycle, at which stage they were already approaching the crisis phase as demonstrated by the slowing down in the proliferation rate and the inability to produce clones. After SV40-transformation the proliferation rate rapidly increased, reaching a maximal level in a few days, and the transformed cells recovered the ability to produce clones. All the SV40-transformed isolated clones underwent 37 duplication cycles and did not show any slowing down in the proliferation rate or any sign of aging to date. We suggest that SV40 virus was able to "rescue" the cells from an irreversible commitment to cease dividing.

Adolescent↗
PubMed1982

Induction of germ tube formation by N-acetyl-D-glucosamine in Candida albicans: uptake of inducer and germinative response.

A number of strains of Candida albicans were tested for germ tube formation after induction by N-acetyl-D-glucosamine (GlcNAc) and other simple (proline, glucose plus glutamine) or complex (serum) compounds. A proportion of strains (high responders) were induced to form germ tubes evolving to true hyphae by GlcNAc alone or by proline or glucose plus glutamine mixture. The majority of strains were low responders because they could be induced only by serum or GlcNAc-serum medium. Two strains were found to be nonresponders: they grew as pseudohyphae in serum. Despite minor quantitative differences, all strains efficiently utilized GlcNAc for growth under the yeast form at 28 degrees C. They also had comparable active, inducible, and constitutive uptake systems for GlcNAc. During germ tube formation in GlcNAc, the inducible uptake system was modulated, as expected from induction and decay of GlcNAc kinase. Uranyl acetate, at a concentration of 0.01 mM, inhibited both GlcNAc uptake and germ tube formation and was reversed by phosphates. Germinating and nongerminating cells differed in the rapidity and extent of GlcNAc incorporation into acid-insoluble and alkali-acid-insoluble cell fractions. During germ tube formation induced by proline, GlcNAc was almost totally incorporated into the acid-insoluble fraction after 60 min. Moreover, hyphal development on induction by either GlcNAc or proline was characterized by an apparent "uncoupling" between protein and polysaccharide metabolism, the ratio between the two main cellular constituents falling from more than 1 to less than 0.5 after 270 min of development. The data suggest that utilization of the inducer for wall synthesis is a determinant of germ tube formation C. albicans but that the nature and extent of inducer uptake is not a key event for this phenomenon to occur.

Acetylglucosamine↗