Giancarlo Gazzanelli

Ultrastructural analysis of pancreatic acinar cells from mice fed on genetically modified soybean

No direct evidence that genetically modified (GM) food may represent a possible danger for health has been reported so far; however, the scientific literature in this field is quite poor. Therefore, we investigated the possible effects of a diet containing GM soybean on mouse exocrine pancreas by means of ultrastructural, morphometrical and immunocytochemical analyses. Our observations demonstrate that, although no structural modification occurs in pancreatic acinar cells of mice fed on GM soybean, quantitative changes of some cellular constituents take place in comparison to control animals. In particular, a diet containing significant amount of GM food seems to influence the zymogen synthesis and processing.

Promotion of tumour metastases and induction of angiogenesis by native HIV-1 Tat protein from BK virus/tat transgenic mice

ObjectiveTo characterize the T53 cell line and its clones derived from an adenocarcinoma of BK virus (BKV)/tat transgenic mice and to establish the role of native Tat in tumorigenicity, induction of metastases and angiogenesis. Design and methodsTat was quantified by flow cytometry and chloramphenicol acetyltransferase (CAT) assays. Tumorigenicity and metastatic ability of cell lines were assayed in nude mice. Production of proteases was evaluated by a plasmin chromogenic assay and gelatinase zymography. The angiogenic effect was studied in vivo with conditioned medium from tumour cell lines. ResultsTat protein was detected in tumour cell lines in amounts from 600–7000 molecules/cell. Conditioned medium from tumour cell lines was able to transactivate an LTR-CAT in HL3T1 cells, indicating release of extracellular Tat. Tumour cell lines, inoculated into nude mice, induced angiogenic tumours with remarkable recruitment of host endothelial cells. Metastases were detected in lymph nodes, lungs, kidneys, and heart. Cell lines produced relevant amounts of proteases. Conditioned medium implanted in mice with matrigel induced an angiogenic response, enhanced by addition of heparin. Preincubation with an anti-Tat antibody abolished the angiogenic effect. ConclusionsTat from cells from BKV/tat transgenic mice promotes tumorigenesis and formation of metastases and induces angiogenic activity. Angiogenesis occurs at physiological concentrations of Tat lower than 20 ng/ml. The effects of Tat on induction of metastases and angiogenesis appear to be mediated by activation of proteases.

Biochemical and Ultrastructural Features of Human Milk and Nipple Aspirate Fluids

Breast duct epithelium produces, secretes, and metabolises several biologically important compounds, which are found in breast secretions obtained in physiologic and pathologic conditions (milk and nipple aspirate fluids, respectively). In order to preliminarily evaluate the ultrastructural morphology of the cells found in Type II nipple aspirate fluids (NAF) and correlate it with the biochemical profile of the extracellular fluid present in these breast secretions and in human milk, we analyzed 72 NAFs from nonlactating premenopausal women affected by various breast diseases and 10 normal milk samples. Although several constitutive proteins were detected in all samples examined, the preliminary biochemical analyses and electrophoretic profiles revealed characteristic behaviours for several biologic constituents, suggesting a possible basic mechanism of production by breast epithelial cells during both physiologic and pathologic conditions. The ultrastructural analysis of milk cellular components give preliminary evidence of the apocrine secretion mechanism peculiar of breast gland, whereas Type II NAF cells appeared as biosynthetically active cells, showing a possible modified secretion mechanism. Our multidisciplinary approach seems to support the hypothesis that cellular and biochemical behaviour of Type II NAF may be an useful tool to identify aberrated breast epithelial cells in nonlactating women that might be prone to premalignant transformation. J. Clin. Lab. Anal. 14:330–335, 2000.

Prostate-specific antigen (PSA/hK3): a further player in the field of breast cancer diagnostics?

Since its identification, much information has been obtained about prostate-specific antigen (PSA, or human glandular kallikrein 3 [hK3]), a kallikrein-like serine protease that is the most valuable tumour marker for the screening, diagnosis and management of human prostate carcinoma. Recently, it has become widely accepted that PSA is also present in many nonprostatic sources, casting doubts about the specificity of its tissue expression. Here we summarize the findings on the biomolecular expression of PSA in breast secretions, cells and tissues of healthy and diseased females. Although several studies have strongly suggested that the molecular forms of PSA seem to represent a potential tool for the risk assessment of breast cancer, recent reports have yielded conflicting results. Although several studies have suggested new biological function(s) for PSA in breast physiopathology, more studies are needed to enlist PSA unequivocally as an additional weapon in the anticancer armoury in breast cancer diagnostics.

Nuclear bodies are usual constituents in tissues of hibernating dormice

In previous studies we demonstrated in several tissues of the hazel dormouse Muscardinus avellanarius that during hibernation cell nuclei contain particular structural constituents absent in euthermia. In the present study we examine the same tissues in euthermic and hibernating individuals of the edible dormouse Glis glis in order to investigate possible modifications of nuclear structural constituents occurring during hibernation in this species.

Quantification of prostate-specific antigen immunoreactivity in human breast cyst fluids

SummaryThe frequency of gross cystic breast disease in premenopausal women and its possible association with in-creased breast cancer risk emphasises the importance of investigations relating to breast cyst fluid composition. In order to contribute to a better analysis of this medium, we have measured the presence of prostate-specific antigen immuno-reactivity in sixty-four human breast cyst fluids. Data analyses show that 35% of samples presented a level of this antigen < 0.05 µg/L, whereas 42 out of 64 cysts show a significant increase in the mean value of metabolically active apocrine cysts when compared to flattened cysts (p < 0.01). We report the first evidence that breast epithelium of gross cysts produces, secretes, and accumulates large amounts of prostate-specific antigen, a glycoprotein produced by prostatic tissue but recently detected in breast tumours, normal tissues, and during pregnancy. The production and intracystic accumulation of this serine protease in biosynthetically active apocrine type cyst can play a feasible role in the natural history of gross cystic breast disease as well as in the mechanism of cyst formation, enlargement, and transformation.

FINE STRUCTURAL MODIFICATIONS OF LIVER, PANCREAS AND BROWN ADIPOSE TISSUE MITOCHONDRIA FROM HIBERNATING, AROUSING AND EUTHERMIC DORMICE

An ultrastructural and morphometric study was performed on mitochondria of euthermic, hibernating and arousing hazel dormice (Muscardinus avellanarius), in order to investigate possible modifications during the seasonal cycle. Hepatocytes, pancreatic acinar cells and brown adipocytes were considered. Our results demonstrated that: (1) the general morphology of mitochondria of all cell types shows slight modifications during the seasonal cycle; (2) mitochondrial size and inner membrane length significantly increase from euthermia to hibernation and decrease upon arousal in all cell types; (3) mitochondrial matrix granules drastically increase in number during hibernation and decrease upon arousal in hepatocytes and pancreatic acinar cells, whereas they do not change in brown adipocytes. These structural modifications are probably related to the changes in cellular energy needs during the euthermia—hibernation—arousal cycle.

Altered RNA structural constituents in aging and vitamin E deficiency.

Ribonucleoprotein (RNP) containing structural constituents in hepatocyte nuclei of adult, old and adult, vitamin E-deficient rats were investigated to assess the effect of aging and increased oxidative stress on nuclear functions. Fibrillar centres (FCs), dense fibrillar (DFC) and granular (GC) components of nucleoli as well as perichromatin granules (PGs) in the nucleoplasm were preferentially evidenced by the ethylenediaminetetracetic acid (EDTA) method and measured by computer-assisted morphometric procedures. FCs size and the percentage of nucleolar surface occupied by FCs significantly decreased during aging and vitamin E-deficiency. The percentage of nucleolar surface occupied by GC and DFC remained unchanged in adult and old rats, but in vitamin E-deficient animals GC increased and DFC decreased significantly. PG density significantly changed in aging and vitamin E-deficiency. Functionally, FCs, DFC and GC constitute sites of transcription and processing of ribosomal RNA while PGs are involved in intranuclear storage and transport of messenger RNA. Thus, the present structural changes during aging and vitamin E-deficiency correlate with a decay of nuclear responsiveness to cellular metabolic needs. Considering the antioxidant action of alpha-tocopherol, our data lend further support to the importance of free radical production and control in the aging process.

Nucleoli undergo structural and molecular modifications during hibernation

Abstract. The nucleolus is a very dynamic structure able rapidly to adapt its activity to the cellular metabolic state. An interesting physiological model characterized by drastic modifications of cellular metabolism is represented by hibernating animals. In the present study we investigated the hepatocyte nuclei of euthermic and hibernating edible dormice (Glis glis) with the aim of revealing, by means of ultrastructural and immunocytochemical analyses, possible modifications of nucleolar components during hibernation. Our observations demonstrate that, in deep hibernation, nucleoli undergo structural and molecular modifications: (a) they show numerous nucleoplasmic invaginations and clumps of dense fibrillar component extend from the nucleolar surface; (b) they are frequently in contact with coiled bodies and fibro-granular material, two nuclear bodies usually occurring in the nucleoplasm; (c) the dense fibrillar component contains significant amounts of small nuclear ribonucleoproteins, splicing factors usually distributed in the nucleoplasm. Taken together, these results suggest that during hibernation complex relationships are established between the nucleolus and nucleoplasm, probably related to functional activities peculiar to this physiological phase. However, since no evident nucleolar modification was found in early hibernating dormice, it seems likely that the particular structural and molecular arrangement of nucleoli establishes progressively during hibernation, becoming evident only in the deepest phase, and then disappears upon arousal.

Development and characterization of hydrogen peroxide-resistant Chinese hamster ovary cell variants. I: relationship between catalase activity and the induction/stability of the oxidant-resistant phenotype

Hydrogen peroxide (H2O2)-resistant sublines of Chinese hamster ovary (CHO) cells were isolated by in vitro exposure to the oxidant (treatment for 1 hr followed by 3 days of growth in peroxide-free medium). Stepwise increase in low level H2O2 concentrations produced variants which were progressively more resistant to the growth inhibitory effect elicited by the oxidant. Removal from H2O2 decreased resistance and the curve describing this process was biphasic in nature. In addition, the rate of loss of the H2O2-resistant phenotype was more rapid for the toxicity elicited by low concentrations of hydrogen peroxide, compared to that produced by high concentrations. Changes in total cell proteins were found to parallel the variations in sensitivity to the oxidant, since the protein content constantly increased during the adaptation process and decreases upon removal from H2O2. Catalase activity did not show large variations in resistant sublines with respect to the parental cell line, and these changes were at least partially related to differences in cell size/amount of total cell proteins of the sublines. In addition, the minor changes observed for catalase activity did not correlate with the degree of resistance to growth inhibition elicited by the oxidant. It may therefore be suggested that the H2O2-resistant phenotype of mammalian cells, initially adapted to low--then gradually increased--concentrations of the oxidant, is the result of a complex phenomenon which only partially involves over-expression of catalase.