An analysis of DNA loss and synthesis in the rat adrenal medulla nuclei upon cold stimulation.

The peculiar changes previously observed in DNA content of rat adrenal medulla cell nuclei upon intermittent cold exposure (15 hr at +4 degrees C followed by 9 hr at room temperature) have been further studied with the aid of Feulgen histophotometry and H(3)-thymidine radioautography. The amount of DNA decreases progressively with increasing length of cold exposure until 300 hr (-32%). Later a rapid change takes place, whereby DNA content per nucleus returns to values which are slightly, but consistently lower than normal. At termination of a period of cumulative exposure to cold, an analysis of a whole-day experimental cycle shows that the DNA decrease is due to loss of DNA during cold exposure and that DNA synthesis occurs upon return to room temperature. The balance between these two processes can be divided into three stages: (a) loss of DNA up to 300 hr of cumulative cold exposure; (b) marked increase in DNA by 350 hr; (c) oscillation around zero or slightly negative at 400 hr and beyond. These variations are due to: (1) the extension of DNA synthesis into the period of cold exposure as clearly demonstrated by radioautography (stage b), and (2) a later still greater DNA loss (stage c) which partly offsets the increased synthesis. A complex pattern of adaptation of the adrenal medulla cells, as regards DNA content, to the repetitive cold stimulus is thus demonstrated.

A radioautographic study with H3-thymidine on adrenal medulla nuclei of rats intermittently exposed to cold.

A considerable decrease (24 to 40%) of DNA content per nucleus previously observed in the adrenal medulla of rats exposed intermittently to cold is followed by restoration to normal and supranormal values. This phenomenon has now been studied by use of H(3)-thymidine, which was given to normal rats, to rats exposed to cold, and to animals brought to room temperature after cold exposure. In the first two conditions, no significant labeling of nuclei was observed. In the third, labeling took place clearly in the 1st 3 days. The grain counts showed that the early labeled nuclei had more grains than those labeled later, indicating differences in the rate of DNA synthesis. A statistically significant correlation was found, on the same nuclei, between amount of Feulgen dye and number of grains. It is concluded that net synthesis of DNA takes place in the phase of recovery from cold. This fact is not related to cell division, as no mitoses could ever be detected, but rather to the cold-induced loss of DNA. Clear demonstration is thus given of a marked variation in the amount of DNA per nucleus in relation to the functional conditions of adrenal medulla cells.

I. Quantitative determination of the amount of DNA per nucleus by interference microscopy.

A method for the determination of the DNA content of isolated nuclei of different ploidy has been developed. It is based on measurement of the nuclear dry mass, with an integrating microinterferometer, before and after DNase treatment. The values found are slightly low, because, as indicated by biochemical determinations, consistently 5% to 8% of DNA is not extracted by DNase under these conditions. The average DNA values thus obtained for diploid and tetraploid nuclei of adult rat liver are 7.7 and 15.6 pg (10(-12) g), respectively. Definite advantages of this procedure are: i) comparisons with biochemical determinations to give DNA values for each class of ploidy, ii) comparisons with histophotometry of the Feulgen dye-DNA complex to give absolute values instead of arbitrary units.

II. Differences in adrenal medulla nuclear DNA content among rats of different strains following intermittent exposure to cold.

The amount of DNA per nucleus in the adrenal medulla cells of four different strains of rats (Wistar, Sprague-Dawley, Long-Evans, and Italico) is determined both under control conditions and after 300 hr of intermittent exposure to cold. The adrenal medulla nuclei of the four strains of rats contain the same amount of DNA; however, the loss of DNA observed after the same experimental treatment differs markedly in the different strains. The loss is small in Wistar and Sprague-Dawley rats (8-13%), larger in Long-Evans rats (20%) and still larger in Italico rats (45%). The DNA loss in Wistar rats increases if the animals are fed the same diet as the Italico rats, and the DNA loss in Italico rats is reduced if the animals are fed the same diet as the Wistar rats. The different behavior of the four strains is discussed in terms of turnover of DNA.

3. Decrease of labeled DNA in cells of the adrenal medulla after intermittent exposure to cold.

Italico rats were injected with thymidine-(3)H 6 hr after the end of 300 hr of intermittent cold treatment. This plan of experiment ensured replacement in the adrenal medulla of lost DNA which is specifically sensitive to cold treatment and has a labeling index sufficiently high for statistical evaluation. The labeling index in the adrenal medulla decreases to one-half of the initial value within 10 days in animals subjected to further intermittent cold treatment and within 32 days in animals kept at room temperature. The very low mitotic index and the absence of doubling of the labeling index show that the observed labeling cannot be ascribed to pre-mitotic DNA synthesis. The concept of metabolic DNA adequately explains the findings.

The nuclear ceramide/diacylglycerol balance depends on the physiological state of thyroid cells and changes during UV-C radiation-induced apoptosis.

Intranuclear lipid metabolism modifications in relation to cell proliferation and/or apoptosis were demonstrated in hepatocytes. The aim of this study was to establish whether nuclear lipid metabolites influence cell function in different experimental models using a rat thyroid cell line (FRTL-5) treated with UV-C radiation. After UV-C irradiation cells proliferate and undergo apoptosis in the presence of thyrotropin, are quiescent and resistant to radiation-induced apoptosis in its absence and finally are proapoptotic for nutrition withdrawal. In nuclei purified from proliferating cells, irradiation stimulates neutral-sphingomyelinase activity and inhibits sphingomyelin-synthase, phosphatidylcholine-specific phospholipase C and phosphatidylinositol-specific phospholipase C activity with a consequent increase in the ceramide/diacylglycerol ratio. This effect is marked in proapoptotic cell nuclei and low in quiescent cell nuclei. In conclusion, UV-C radiation induces apoptosis, modifying nuclear lipid metabolism in relation to the physiological state of cells.

Low levels of serum cholesterol/phospholipids are associated with the antiphospholipid antibodies in monoclonal gammopathy.

A decrease in cholesterol blood level, not due to a decrease synthesis by the liver, has been observed in patients suffering from tumors. In this work cholesterol blood was evaluated in patients affected by monoclonal gammopathy who were not subjected to any treatment. The blood of 25 patients were analyzed for protein and lipid content. Patients were divided according to the gamma protein content into three groups, and it was demonstrated that the group with high levels of gamma proteins presented a strong decrease in blood cholesterol and phospholipids. In these patients the presence of antibodies against phospholipids by using cardiolipin and phosphatidylinositol as antigens has also been demonstrated. The antibodies were rare in patients with a low content of gamma proteins and normal level of lipids, but the frequency was more than 80% in patients with low blood lipid levels.

Antiphospholipid antibodies in patients with cancer.

Antiphospholipid antibodies are generally associated with Antiphospholipid Syndrome, which can occur as a primary disorder or may be secondary to connective tissue disease or tumour. The presence of antiphospholipid antibodies in patients with tumour disease is responsible for thrombotic complications. In a population of 53 tumor patients with positive carcinoembryonic antigen CEA, carbohydrate antigen CA19.9, CA125 and CA15.3 markers, IgM and IgG anticardiolipin and antiphosphatidylinositol were detected by solid-phase immunoassays. Our results show that moderate or high levels of antiphospholipid antibodies are present in a great number of patients with CEA and CA19.9 markers, suggesting a specific association with gastroenteric tumors. By testing for antiphosphatidylinositol antibodies, many patients not evidenced by the standard anticardiolipin assay were found to be antiphospholipid-positive. The analysis of antiphosphatidylinositol antibodies as a diagnostic tool in gastroenteric cancer to highlight patients with the risk of thromboembolic complications is discussed.

Base composition changes in hepatocyte nuclei DNA of rats at different ages.

DNA extracted from isolated hepatic nuclei of rats at different aged (1 h, 6 and 30 days of life) has been characterized by (i) melting temperature, (ii) buoyant density, (iii) thermal denaturation on hydroxyapatite and (iv) nucleoside composition. The melting midpoint (Tm) determined spectrophotometrically in 0.1 X SSC (0.15 M NaCl/0.0015 M sodium citrate) is 71.9 +/- 0.4 for 1-h-old rats and decreases to 70.7 +/- 0.3 in 6-day-old animals. The buoyant densities of DNAs determined by CsCl on both native and alkaline-denaturated and reneutralized DNA were also found to decrease with age. Hydroxyapatite thermal denaturation of sonicated DNA confirmed the significant difference between the Tm values of 1-h-old and 6-day-old rats (86.5 +/- 0.5 and 85.2 +/- 0.1, respectively). The possibility that these differences in Tm values could be due to an increase in methyl bases, has been ruled out by the finding that the amount of [3H]methyl incorporated in relation to the DNA synthesis is constant at these two ages. The alternative possibility of a change in base composition has been tested by the chromatographic analysis of nucleosides. The dG + dC content is 0.433 +/- 0.003 in 1-h-old rats and decreases to 0.411 +/- 0.002 and to 0.403 +/- 0.005 in 6-day- and 30-day-old rats, respectively. The physiological significance of the different base composition is discussed in relation to the possibility that specific DNA sequences are synthesized during the non-premitotic synthesis which has been found to take place during the first 6 days of life.

Age-related changes in the cell proliferation of ATPC+ mouse ascites tumour.

The growth of ATPC+, an ascites tumour derived from a spontaneous mammary carcinoma in BALB/c+ mice, was studied at different ages. It was observed that the number of cells increases rapidly during the first 5 days after implantation. Thereafter, the cell number increases more slowly, reaching a plateau after 8 days. This slowing-down is not due to a reduction in the growth fraction but to a lengthening of the cell cycle. Between 5 and 8 days the duration of all phases increases, including the S phase, which increases from 5.2 h in 5-day tumours to 8.2 h in 8-day tumours. In 12-day tumours both the cell cycle and S phase are only slightly longer than in 8-day tumours whereas the growth fraction is reduced. The slowing-down of cell growth can be attributed to growth fraction reduction rather than cell loss, which is maximal in the 5-day tumour. At this age the time course of the percent labelled cells and of the number of grains/nucleus suggests reutilization of [3H]-thymidine. Incorporation of [3H]-thymidine/cell decreases sharply in 12-day tumours due to a reduced availability of thymidine, which is degraded to thymine in the in vivo ascitic fluid faster than in 8-day tumours. This indicates an age-related change in the ascitic fluid composition.

Changes in oncogene expression in ascite tumour cells during ageing.

The expression of two oncogenes, c-myc and c-fos, was studied in an ascitic tumour (ATPC+) at different times after implantation. The specific mRNA synthesis was analysed by Northern blot analysis. The presence of the oncogene proteins was shown by immunofluorescence using flow cytometry and referred to the distribution of the cells in the different cell phases. The results show that both oncogenes are expressed by ATPC+ tumour cells. c-myc is expressed 5, 8 and 12 days after implantation, although with a different intensity, and the protein is mainly present in S or S+G2 phase cells. The c-fos oncogene is expressed only 12 days after tumour implantation and the cells labelled with the specific antibody are mainly in G1 phase. We conclude that c-myc is principally correlated with proliferative activity, whereas c-fos is expressed by non-cycling cells.

Antiphosphatidylinositol antibody in deep venous thrombosis patients.

Antiphospholipid antibodies are a heterogeneous group of immunoglobulins with specificity for a number of phospholipids, phospholipid-binding proteins and phospholipid-protein complexes. The association between antiphospholipid antibodies and a variety of pathologic disorders, such as arterial and venous thrombosis and recurrent pregnancy loss is recognized as Antiphospholipid Syndrome. The immunoassay currently used to detect antiphospholipid antibodies is the anticardiolipin test. Anticardiolipin antibodies are believed to be polyspecific antibodies that cross-react with all the anionic phospholipids. Therefore, testing only for anticardiolipin antibodies does not always permit detection of all antiphospholipid antibodies, specially when only IgG are evaluated. In a selected population of 74 idiopathic and secondary deep venous thrombosis patients, IgG anticardiolipin, antiphosphatidylinositol and antiphosphatidylserine antibodies were detected by solid-phase immunoassays. Our results show that by testing for each antiphospholipid family, many patients, not evidenced by the standard anticardiolipin assay, were found to be antiphospholipid-positive. The anticardiolipin positive patients have always low, moderate or high levels of antiphospholipid antibodies, suggesting that the antiphospholipid positivity is predictive of anticardiolipin positivity. It should be noted that the patients with only antiphosphatidylinositol positive antibody have a story of nervous system pathology. The meaning of these results is at present under discussion.

Rat liver chromatin phospholipids.

To shed light on the question whether the phospholipids present in chromatin are native or are due to contamination from nuclear membranes, we labeled the phospholipids of isolated nuclei and determined the amount of phospholipids (PL) and PL fatty acid composition in nuclei and chromatin. The hepatocyte nuclei were isolated and radioiodinated by the lactoperoxidase method under saturating and nonsaturating conditions, and the radioactivity associated with chromatin extracted from these nuclei was monitored. Whereas 97% the label was recovered in the nuclear membranes, only 0.08-0.6% was found in chromatin. The PL present in chromatin were relative to the amounts present in the entire nuclei and calculated as percentage of total, phosphatidylethanolamine (10%), phosphatidylserine (22%), phosphatidylinositol (19%) phosphatidylcholine (14%), and sphingomyelin (35%). In sphingomyelin of chromatin-associated PL an enrichment in polyunsaturated fatty acids was seen. The data indicated that the PL found in isolated chromatin do not seem to be due to contamination from the nuclear membrane.

Tyrosine aminotransferase gene regulation during aging.

Tyrosine aminotransferase activity (TAT) and its expression were measured in rats of different ages (3, 12 and 24 months). The enzyme activity showed a circadian rhythm with a peak at midnight due to different levels of transcription during the day. The circadian rhythm was present at all ages studied but showed some differences: an age-related shift of the peak was more evident in the actual levels of transcript. Both specific mRNA and enzyme activity analyses provide better insight into the complex modifications in gene-expression of aging animals.

[Hepatic resection in the fetal rabbit. Histologic comparison of tissue regeneration in the fetus versus the adult]. / Resezione epatica fetale nel coniglio. Confronto istologico della rigenerazione tissutale del feto e dell'adulto.

Fetal tissues present peculiar features of repair after injury. Although the development of fetal hepatocytes have already been studied in vitro and in transplant models, an in vivo study of fetal liver regeneration is still missed in the literature, to the best of our knowledge. Eight time-dated pregnant California rabbits (23, 24, 25, 30 days of gestational age) and 2 adult male California rabbits were anesthetized following a standardized i.v. protocol (ketamine 50 mg/kg; xilazine 5 mg/kg; propiopromazine 0.75 mg/kg; spontaneous breathing; no anesthetic gas). All the pregnant does underwent a midline laparotomy and a minimal hysterotomy to approach a fetus per each animal. In 2 cases, 1 fetus was delivered and prior to sacrifice the fetal liver was sampled in toto (30 days of gestational age). These pregnancies were allowed to continue to term and were uneventful with a full-term spontaneous delivery of the remaining fetuses. In the other 6 pregnancies, after the hysterotomy, the fetal abdomen was entered through a right-sided longitudinal incision and the liver was partially resected by thermocauterization. Fetal abdomen was closed in 1 layer (non absorbable suture 7-0). The fetus was then returned in the uterus and, after amniotic fluid restoration with warmed saline, the hysterotomy was sutured in double layer (polyglycolic 5-0). Maternal abdomen was closed in 1 layer (polyglycolic 4-0) and the skin in a continuous overlying fashion (silk 3-0). The abdominal cavity of the 2 adult male rabbits was entered through a right subcostal incision. Partial liver resection was performed, and abdominal and skin closure followed the same techniques used for the pregnant does. The treated livers were then sampled in toto at 24, 48, 72 hrs and 4 days after surgery from the fetuses, and at 7 days from the adult rabbits. Histological stains were: H & E; Van Gieson; Masson; Alcian Bleu; PAS. Fetal histology showed a low inflammatory reaction poor in PMN cells with minimal deposition of collagen and a high amount of glycogen in the hepatocytes. The inflammatory response to resection was much more evident in the adult samples as much as the abundant intra and extra-cellular deposition of collagen associated to a minor amount of intracellular glycogen. The peculiar features of liver regeneration in the fetus, deserve further experimental studies.

Progress towards the 'Golden Age' of biotechnology.

Biotechnology uses substances, materials or extracts derived from living cells, employing 22 million Europeans in a € 1.5 Tn endeavour, being the premier global economic growth opportunity this century. Significant advances have been made in red biotechnology using pharmaceutically and medically relevant applications, green biotechnology developing agricultural and environmental tools and white biotechnology serving industrial scale uses, frequently as process feedstocks. Red biotechnology has delivered dramatic improvements in controlling human disease, from antibiotics to overcome bacterial infections to anti-HIV/AIDS pharmaceuticals such as azidothymidine (AZT), anti-malarial compounds and novel vaccines saving millions of lives. Green biotechnology has dramatically increased food production through Agrobacterium and biolistic genetic modifications for the development of 'Golden Rice', pathogen resistant crops expressing crystal toxin genes, drought resistance and cold tolerance to extend growth range. The burgeoning area of white biotechnology has delivered bio-plastics, low temperature enzyme detergents and a host of feedstock materials for industrial processes such as modified starches, without which our everyday lives would be much more complex. Biotechnological applications can bridge these categories, by modifying energy crops properties, or analysing circulating nucleic acid elements, bringing benefits for all, through increased food production, supporting climate change adaptation and the low carbon economy, or novel diagnostics impacting on personalized medicine and genetic disease. Cross-cutting technologies such as PCR, novel sequencing tools, bioinformatics, transcriptomics and epigenetics are in the vanguard of biotechnological progress leading to an ever-increasing breadth of applications. Biotechnology will deliver solutions to unimagined problems, providing food security, health and well-being to mankind for centuries to come.

Biotechnology worldwide and the 'European Biotechnology Thematic Network' Association (EBTNA).

The European Biotechnology Congress 2011 held under the auspices of the European Biotechnology Thematic Network Association (EBTNA) in conjunction with the Turkish Medical Genetics Association brings together a broad spectrum of biotechnologists from around the world. The subsequent abstracts indicate the manner in which biotechnology has permeated all aspects of research from the basic sciences through to small and medium enterprises and major industries. The brief statements before the presentation of the abstracts aim to introduce not only Biotechnology in general and its importance around the world, but also the European Biotechnology Thematic Network Association and its aims especially within the framework of education and ethics in biotechnology.