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.

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.

The role of intranuclear lipids.

The presence of phospholipids as a component of chromatin is now well documented and many enzymes such as sphingomyelinase, sphingomyelin-synthase, reverse sphingomyelin-synthase and phosphatidylcholine-dependent phospholipase C have been described and characterised. Other lipids were demonstrated inside the nucleus especially plasmalogens and cholesterol. The chromatin phospholipids, comprising 10% of that present in the nucleus, show a different metabolism with respect to those present in either microsomes or in nuclear membranes; they increase also during the DNA duplication as shown during both liver regeneration and cell maturation. They appear localised near newly synthesized RNA in decondensed chromatin. Digestion of chromatin with RNase, but not with DNase, causes a loss of phospholipids. The composition of the chromatin phospholipid fraction shows an enrichment in sphingomyelin and phosphatidylserine. In this review the behaviour of single lipids in relation to cell proliferation, cell differentiation and apoptosis is described. Sphingomyelin, the lipid most represented in chromatin with respect to microsomes and nuclear membranes, is localised near to newly synthesized RNA, its presence appearing to protect RNA from RNase digestion. This effect is reversed by sphingomyelinase which digests sphingomyelin and, as a consequence, RNA may be hydrolysed. The amount of sphingomyelin is restored by sphingomyelin-synthase. Sphingomyelin increases during the differentiation process and apoptosis. An increase of sphingomyelinase with consequent decrease in sphingomyelin is observed at the beginning of S-phase of the cell cycle. A possible role in stabilising the DNA double helix is indicated. Phosphatidylserine behaves similarly during differentiation and appears to stimulate both RNA and DNA polymerases. Phosphatidylcholine is implicated in cell proliferation through the activation of intranuclear phosphatidylcholine-dependent phospholipase C and diacylglycerol production. The increase in diacylglycerol stimulates phosphatidylcholine synthesis through the major pathway from cytidyltriphosphate. An inhibition of phosphatidylcholine synthesis is responsible for the initiation of apoptosis. The presence of reverse sphingomyelin-synthase favours the formation of phosphatidylcholine, the donor of phosphorylcholine, from sphingomyelin. Little information has been reported for phospatidylethanolamine, but phosphtidylinositol appears to influence cell differentiation and proliferation. This last effect is due to the action of two enzymes: PI-PLCss1 having a role in the onset of DNA synthesis and PC-PLCgamma1 acting in G2 transit. Phosphoinositides also may have an important role: in membrane-stripped nuclei isolated from mitogen stimulated cells a decrease in PIP and PIP2 followed by an increase in diacylglycerol and a translocation of protein kinase C inside the nucleus is observed. On the other hand, overexpression of the enzyme inositol polysphosphate-1-phosphatase reduced DNA synthesis by 50%. Nevertheless, an enhanced rate of phosphorylation has been demonstrated in cells induced to differentiate. These molecules probably favour RNA transcription, counteracting the inhibition of H1 on RNA polymerase II. Plasmalogens were demonstrated in the nucleus and their increase favours the increased activity of phosphatidylcholine-dependent phospholipase C when DNA synthesis starts. Moreover, two forms of cholesterol has been described in chromatin: one, a less soluble sphingomyelin-linked form and a free fraction. Cholesterol increases during liver regeneration, first as a linked fraction and then, when DNA synthesis starts, as a free fraction. The changes of these components have been summarised in relation to cell function in order to give an overview of their possible roles in the different phases of cell duplication and their influence on cell differentiation and during apoptosis. Finally, the relevance of these molecules as intranuclear signals is discussed and future directions are indicated in clarifying pathological process such as tumour cell transformation and the possibility in finding new therapeutic tools.

Chromatin-associated sphingomyelin: metabolism in relation to cell function.

After the first histochemical demonstration by Chayen and Gahan of the presence of phospholipids and especially of sphingomyelin in chromatin, this became the object of long debate and of contradictory results. The general conclusion was that the presence of phospholipids may due to contamination during the isolation of chromatin. More recently the existence of a phospholipid chromatin fraction was confirmed by demonstrating that isolated hepatocyte nuclei, labelled by saturated and unsaturated radioiodination method, showed the presence of radioactivity only in the membrane and not in the isolated chromatin. The phospholipid composition showed an enrichment in sphingomyelin which increased during hepatocyte maturation or erythroleukemic cell differentiation induced by DMSO. A decrease in sphingomyelin was observed at the beginning of the S-phase in regenerating liver or in cultured proliferating cells. These changes were due to the presence of sphingomyelinase and sphingomyelin synthase in the chromatin, the activity of which paralleled the variation in sphingomyelin content. The sphingomyelin was co-localized with RNA as shown by biochemical and electron microscopy methods. Using bromo-uridine it was demonstrated that labelled RNA and sphingomyelin were present in actively transcribing nuclear regions. Isolated nuclear complexes after DNase and RNase digestion contained not only protein, but also RNA and sphingomyelin. After hydrolysis of sphingomyelin the RNAse-resistant RNA becomes RNAse sensitive. It can therefore be concluded that sphingomyelin and the related enzymes are present in the chromatin; sphingomyelin may have a role in RNA transcription protecting RNA by RNAse digestion before its transfer to the cytoplasm.

Chromatin sphingomyelin changes in cell proliferation and/or apoptosis induced by ciprofibrate.

It has been shown that neutral-sphingomyelinase and sphingomyelin-synthase activities are present in chromatin and they modify the sphingomyelin (SM) content. The activity of the first enzyme is stimulated and the second inhibited, when the hepatocytes enter into the S-phase after partial hepatectomy, thus suggesting that ceramide may have a pivotal role in cell proliferation. An opposite function was attributed to ceramide in hepatocytes which undergo apoptosis after lobular ligature. In order to clarify this point, a model was developed in which the same liver cells undergo proliferation followed by induced apoptosis. To this purpose, the rats were treated for 7 days with ciprofibrate and then left without treatment for 4 days. During the treatment, the peroxisome enzyme markers increase their activity and the number of proliferating cells increases, reaching a maximum after 3 days of treatment, as shown by the number of cells positive for the proliferating cell nuclear antigen. At the same time, the chromatin sphingomyelinase activity reaches the maximum, while a similar increase is not found in the cytoplasm or in the isolated nuclei. On the contrary, SM-synthase activity is depressed in chromatin, but not in the nuclei in which a peak is shown after 3 days of ciprofibrate treatment. After drug withdrawal, the hepatocytes undergo apoptosis as confirmed by the increase of Bax and tissue transglutaminase (tTGase) expression; the chromatin SM increases as a consequence of an increase of SM-synthase activity. It can be hypothesised that chromatin SM may have a role in cell duplication by influencing the chromatin structure stability.

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.

In utero partial liver resection in the rabbit model: a study on fetal tissue regeneration.

In this study we developed a model of in vivo intrauterine partial liver resection in the fetal rabbit to analyze fetal liver regeneration. After intravenous anesthesia, 12 time-dated pregnant, California rabbits underwent a midline laparotomy and minimal hysterotomy at 24-25 days of gestational age. One fetus was exposed from each pregnant doe and the fetal liver was partially resected. Cesarean sections were performed 24, 48 and 72 h and 4 days after surgery. Three fetuses operated at 24 days of gestational age and 3 fetuses operated at 25 days were alive at retrieval. The fetuses and the sampled livers were weighed at retrieval and fetal liver weight showed a well-maintained value in all cases. Fetal livers were processed for the common histologic stains. Lymphocytes, polymorphonuclear leukocytes and phagocytes were counted from sections obtained in areas close to the edge of resection. Inflammatory cells showed a peculiar pattern of infiltration at different stages of repair, with a constantly increased number of phagocytes peaking 48 h after resection. Fetal liver seems to present a specific pattern of repair that differs from both the adult liver and other fetal tissues healing after injury.

Choline base exchange activity in rat hepatocyte nuclei and nuclear membranes.

Previous investigations have demonstrated the presence of phospholipids as a component of chromatin; however the mechanism of their synthesis, namely if they are synthesized in the nuclei or in the cytoplasm (microsomal fraction), from where they may eventually be transported to the nucleus, has not yet been clarified. The phosphatidylcholine, for example, can be formed, albeit in a limited amount, by an interconversion reaction between bases. The aim of the present research was to ascertain the presence of the enzyme complex responsible for this reaction in hepatocyte nuclei and in isolated nuclear membrane. The incorporation of [14C]-choline in phosphatidylcholine was assayed in microsomes, hepatocyte nuclei, liver nuclei and nuclear membranes of rat liver. The reaction was Ca(2+)-dependent and the specific activity was higher in microsomes but was present, albeit at a low level, also in nuclei and in nuclear membranes. Possible contaminations were excluded by specific microsomal markers and by the reaction time course. In fact, the nuclear reaction reached the maximum level slowly with respect to microsomes. Since the phosphatidylcholine extracted from the nuclei show an enrichment in unsaturated fatty acids of monoenoic fraction, such as oleic acid, the difference in reaction kinetics has been tentatively explained as due to the phosphatidylcholine fatty acid content. The presence of this base exchange enzyme complex may allow a fast change in chromatin phospholipid composition.

[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.

Phospholipids and nuclear RNA.

It has been demonstrated that in hepatocyte nuclei the chromatin phospholipid fraction is localized near the RNA in decondensed chromatin. The aim of the present study was to see if there is any linkage between phospholipids and other nuclear components. Isolated hepatocyte nuclei and nuclear membranes were treated with deoxyribonuclease and ribonuclease. No loss of phospholipids was observed after DNA digestion, whereas 48% was lost following enzymatic RNA removal. This loss of phospholipids, localized either near the membrane or inside the nucleus, was not homogeneous for all phospholipids: phosphatidylserine and sphingomyelin being the most affected. It can be concluded that 48% of nuclear phospholipids, in particular sphingomyelin, is lost with RNA removal. This result is discussed in view of a possible role of phospholipids in DNA synthesis and RNA transcription.

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.

GM2 activator protein expression in mouse tissues.

The GM2 activator protein is an essential cofactor of hexosaminidase A in the degradation of GM2 ganglioside and it is responsible for the variant AB of GM2 gangliosidosis in man. In this study GM2 activator protein and its mRNA were determined in different mouse tissues. It was found that this protein is expressed mostly in the spleen and testis followed by brain and kidney which represent the main source in man. It is also interesting that in mouse testis there is a higher expression of the alpha subunit of hexosaminidase, thus suggesting a relationship between alpha subunit and GM2 activator protein. Furthermore the results indicate that the expression of GM2 activator protein is regulated, at least in part, at the transcriptional level.

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.

Different expression of beta-N-acetylhexosaminidase in mouse tissues.

The expression of genes encoding for the alpha and beta-subunits of the lysosomal enzyme beta-N-acetylhexosaminidase was investigated in different mouse tissues. It was found, using fluorogenic substrates, that the amounts of alpha and beta subunits were not the same in different tissues: alpha-subunit was more abundant in the brain, beta-subunit in epididymis and brain. The different isoenzyme patterns and specific activities in mouse tissues are due to the differences in the amount of hexosaminidase subunits. The mRNA, evaluated by Northern blotting analyses, revealed a greater expression of alpha-subunit in the testis and of beta-subunit in the brain and epididymis. The results indicate, therefore, that gene expression and the amount of subunits are in good relationship for beta-subunit, whereas there is no correlation for alpha-subunit.

TEM cytochemical study of the localization of phospholipids in interphase chromatin in rat hepatocytes.

The electron microscopy cytochemical detection of phospholipids in well-defined areas in the interphase nuclei of hepatocytes has been obtained by the acid haematein test, modified for electron microscopy and by the phospholipase A2-colloidal gold method. The specificity of both methods were controlled by enzymatic digestion with phospholipase. The main intra-nuclear localization of phospholipids is at the border between the condensed and dispersed chromatin, where non-ribosomal RNA is also revealed by RNase-gold labelling. Phospholipids are detected, too, over the clusters of interchromatin granules and in the fibrillar component of the nucleolus.

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.