The mid-Holocene extinction of silver fir (Abies alba) in the Southern Alps: a consequence of forest fires? Palaeobotanical records and forest simulations

Pollen records suggest that Abies alba played a dominating role in both the montane and lowland forests at the border of the Southern Alps between ca. 8500 and 5700 years ago. Two major declines in fir, at about 7300–7000 cal b.p. and at ca. 6000 cal b.p., followed by the local extinction of the species are characteristic of the area below ca. 1000 m a.s.l. In order to test the impact of fire on the population dynamics of silver fir, a dynamic model (DisCForm) with a fire module was applied to simulate the early- and mid-Holocene forest development. Simulation outputs based on different fire scenarios were compared with the pollen record from Lago di Annone (226 m a.s.l.). The marked Abies decreases shown in the pollen record can be simulated with very intensive fire scenarios, but they do not result in an extinction of silver fir in the model. Low charcoal influx values related to the Abies declines in the palaeobotanical record suggest that fire was not the only reason for the extinction of silver fir. Human impact, as well as Holocene climatic changes leading to temporary moisture deficits and reduced adaptability due to low genetic variation may have had a significant impact on the Abies forests.     

Poly(ADP-ribosyl)ation of proteins and germ cell development in hyperthyroid rat testes

The effect of increased serum levels of thyroid hormone (triiodothyronine, T3) on young rat testis spermatogenesis was studied by analysing molecular and morphological parameters. Hyperthyroidism was induced by either T3-treatment or 2- and 10-day cold exposure. The poly(ADP-ribosyl)ation of proteins catalysed by poly(ADP-ribose) polymerase, which is particularly active at specific stages of rat spermatogenesis, was analysed as molecular index of DNA damage and cell stress. Poly(ADP-ribose) polymerase activity rose after both T3-treatment and 2- and 10-day cold exposure, with a trend of 10-day cold-exposed rats towards control values. In all hyperthyroid rats poly(ADP-ribose) turnover, as a contribution of both poly(ADP-ribose) polymerase and poly(ADP-ribose) glycohydrolase), was enhanced with respect to euthyroid animals. Poly(ADP-ribosyl)ation of proteins occurred with long and branched polymers suggesting an increased involvement of the modification system in DNA repair. Morphological changes of germ tissue were observed in hyperthyroid rats, mainly a high reduction of mature cells in the seminiferous tubule, and evidence of germ cell apoptosis was obtained by TUNEL method. In control animals germ cell apoptosis was within physiological levels. Conversely, in hyperthyroid rats a dramatic increase in the number of TUNEL-positive cells (some spermatogonia and numerous primary spermatocytes) was found, even though the increase was lower in 10-day than in 2-day cold-exposed animals.     

Soluble polyphenols: Synthesis and bioavailability of 3,4′,5-tri(α-d-glucose-3-O-succinyl) resveratrol

We report the development of a chemical modification method of general applicability to polyphenols, which increases solubility to influence absorption. Glucosyl groups were added to the resveratrol kernel via a succinate linker, yielding 3,4′,5-tri-(α-d-glucose-3-O-succinyl) resveratrol. The construct was only slowly hydrolyzed in acid and at pH 6.8, but it was destroyed by blood esterases in less than 1 h. In rats its administration resulted in a blood concentration versus time curve shifted to longer times in comparison to resveratrol, a useful modulation of pharmacokinetics. The area-under-curve parameter and the metabolite mix were similar to those of resveratrol. The method may be advantageously employed to solubilize other polyphenols and to make them more palatable.     

BRCA1 Interaction of Centrosomal Protein Nlp Is Required for Successful Mitotic Progression

Breast cancer susceptibility gene BRCA1 is implicated in the control of mitotic progression, although the underlying mechanism(s) remains to be further defined. Deficiency of BRCA1 function leads to disrupted mitotic machinery and genomic instability. Here, we show that BRCA1 physically interacts and colocalizes with Nlp, an important molecule involved in centrosome maturation and spindle formation. Interestingly, Nlp centrosomal localization and its protein stability are regulated by normal cellular BRCA1 function because cells containing BRCA1 mutations or silenced for endogenous BRCA1 exhibit disrupted Nlp colocalization to centrosomes and enhanced Nlp degradation. Its is likely that the BRCA1 regulation of Nlp stability involves Plk1 suppression. Inhibition of endogenous Nlp via the small interfering RNA approach results in aberrant spindle formation, aborted chromosomal segregation, and aneuploidy, which mimic the phenotypes of disrupted BRCA1. Thus, BRCA1 interaction of Nlp might be required for the successful mitotic progression, and abnormalities of Nlp lead to genomic instability.The successful mitosis requires the assembly of a strictly bipolar mitotic apparatus that will ensure that chromosomes equally distribute to the daughter cells. This process is controlled by the centrosomes that are required for spindle formation and function (1). Abnormalities of centrosome have been demonstrated to cause chromosomal missegregation and generation of aneuploidy, consequently leading to cell malignant transformation and tumorigenesis (2, 3). The machinery that controls centrosome stability involves multiple important cellular proteins, including p53 (4), BRCA1 (5), Gadd45 (6, 7), p21 (8), and Cdk2/cyclin E (9). The precise coordination among those regulators maintains centrosome duplication and stability. Prior to mitosis, centrosomes undergo maturation (10), which is characterized by centrosome enlargement, recruitment of γ-tubulin, and an increased microtubule nucleation activity (11, 12). Centrosome maturation is regulated by several mitotic kinases (13), such as Plk1 (Polo-like kinase 1) (14), Aurora-A (15), and Nek2, a member of NIMA (never in mitosis gene A)-related kinase (16). Recently, a Plk1-regulated ninein-like protein, termed Nlp, has been characterized as an important molecule involved in centrosome maturation (17). Nlp interacts with γ-tubulin ring complex and stimulates microtubule nucleation in the interphase. Upon the G₂/M transition, Nlp is subjected to phosphorylation by Plk1 and Nek2 (17, 18) and departs from the centrosome. It is thus suggested that the delicate association of Nlp with the centrosome is required for proper centrosome maturation and spindle assembly (17).BRCA1, a breast cancer susceptibility gene that accounts for more than 70% of hereditary breast cancer cases, is a critical regulator in the control of cell cycle progression (19, 20). BRCA1 interacts with multiple important cellular proteins, including RAD51 (21), BRCA2 (22), p53 (23), c-Myc (24), and p300 proteins (25). It is speculated that the BRCA1 protein may exert its control over cellular functions by acting as a platform for these proteins to converge and interact and may, therefore, create interactive modes for regulating their respective functions. BRCA1 is linked to the control of centrosome stability (26). Mouse embryonic fibroblasts (MEFs)³ carrying targeted deletion of exon 11 of the Brca1 gene exhibit centrosome amplification and abnormalities of spindle formation (5). BRCA1 may regulate centrosome duplication, probably through its interacting proteins such as p53 (23), BRCA2 (27), Cdk2 (28), and γ-tubulin (29–31), or its downstream genes such as p21 (32) and Gadd45a (33, 34). Most recently, BRCA1 was reported to be required for mitotic spindle assembly through its interaction with three spindle pole proteins, TPX2, NuMA, nuclear mitotic apparatus protein; and XRHAMM, Xenopus homolog to human RHAXX (35). These findings strongly suggest that BRCA1 is involved in the mitotic machinery. However, the importance of BRCA1 in the control of mitotic progression still remains to be further defined.In this report, we demonstrate that BRCA1 physically interacts and colocalizes with Nlp. Nlp centrosomal localization and its protein stability are likely dependent on normal cellular BRCA1 function. Suppression of Nlp using the siRNA approach disturbs the process of chromosomal segregation and results in aberrant spindle formation, failure of chromosomal segregation, and aneuploidy.     

GSH and analogs in antiviral therapy

Reduced glutathione (GSH) is the most prevalent non-protein thiol in animal cells. Its de novo and salvage synthesis serves to maintain a reduced cellular environment. GSH is the most powerful intracellular antioxidant and plays a role in the detoxification of a variety of electrophilic compounds and peroxides via catalysis by glutathione-S-transferases (GST) and glutathione peroxidases (GPx). As a consequence, the ratio of reduced and oxidized glutathione (GSH:GSSG) serves as a representative marker of the antioxidative capacity of the cell. A deficiency in GSH puts the cell at risk for oxidative damage. An imbalance in GSH is observed in a wide range of pathologies, such as cancer, neurodegenerative diseases, cystic fibrosis (CF), several viral infections including HIV-1, as well as in aging. Several reports have provided evidence for the use of GSH and molecules able to replenish intracellular GSH levels in antiviral therapy. This non-conventional role of GSH and its analogs as antiviral drugs is discussed in this chapter.     

Differential SELEX in Human Glioma Cell Lines

The hope of success of therapeutic interventions largely relies on the possibility to distinguish between even close tumor types with high accuracy. Indeed, in the last ten years a major challenge to predict the responsiveness to a given therapeutic plan has been the identification of tumor specific signatures, with the aim to reduce the frequency of unwanted side effects on oncologic patients not responding to therapy. Here, we developed an in vitro evolution-based approach, named differential whole cell SELEX, to generate a panel of high affinity nucleic acid ligands for cell surface epitopes. The ligands, named aptamers, were obtained through the iterative evolution of a random pool of sequences using as target human U87MG glioma cells. The selection was designed so as to distinguish U87MG from the less malignant cell line T98G. We isolated molecules that generate unique binding patterns sufficient to unequivocally identify any of the tested human glioma cell lines analyzed and to distinguish high from low or non-tumorigenic cell lines. Five of such aptamers act as inhibitors of specific intracellular pathways thus indicating that the putative target might be important surface signaling molecules. Differential whole cell SELEX reveals an exciting strategy widely applicable to cancer cells that permits generation of highly specific ligands for cancer biomarkers.     

New pyrrole-based histone deacetylase inhibitors: binding mode, enzyme- and cell-based investigations

Aroyl-pyrrolyl-hydroxy-amides (APHAs) are a class of synthetic HDAC inhibitors described by us since 2001. Through structure-based drug design, two isomers of the APHA lead compound 1, the 3-(2-benzoyl-1-methyl-1H-pyrrol-4-yl)-N-hydroxy-2-propenamide 2 and the 3-(2-benzoyl-1-methyl-1H-pyrrol-5-yl)-N-hydroxy-2-propenamide 3 (iso-APHAs) were designed, synthesized and tested in murine leukemia cells as antiproliferative and cytodifferentiating agents. To improve their HDAC activity and selectivity, chemical modifications at the benzoyl moieties were investigated and evaluated using three maize histone deacetylases: HD2, HD1-B (class I human HDAC homologue), and HD1-A (class II human HDAC homologue). Docking experiments on HD1-A and HD1-B homology models revealed that the different compounds selectivity profiles could be addressed to different binding modes as observed for the reference compound SAHA. Smaller hydrophobic cap groups improved class II HDAC selectivity through the interaction with HD1-A Asn89-Ser90-Ile91, while bulkier aromatic substituents increased class I HDAC selectivity. Taking into account the whole enzyme data and the functional test results, the described iso-APHAs showed a behaviour of class I/IIb HDACi, with 4b and 4i preferentially inhibiting class IIb and class I HDACs, respectively. When tested in the human leukaemia U937 cell line, 4i showed altered cell cycle (S phase arrest), joined to high (51%) apoptosis induction and significant (21%) differentiation activity.     

Microaerophilic–aerobic sequential decolourization/biodegradation of textile azo dyes by a facultative Klebsiella sp. strain VN-31

Four different azo dyes were decolourized and biodegraded in a sequential microaerophilic–aerobic treatment by a facultative Klebsiella sp. strain VN-31, a bacterium isolated from activated sludge process of the textile industry. Dye decolourization was performed under microaerophilic conditions until no colour was observed (decolourization percentage >94%). The medium was then aerated to promote the biodegradation of the amines produced. The presence of aromatic amine in the microaerophilic stage and its absence in the aerobic stage demonstrate azo bond reduction and an oxidative biodegradation process, respectively. Total Organic Carbon (TOC) reduction for the growth medium plus dyes was ～50% in the microaerophilic stage and ～80% in the aerobic stage. The degradation products were also characterized by FT-IR and UV–vis techniques and their toxicity measured using Daphnia magna. The results provide evidence that the successive microaerophilic/aerobic stages, using a single Klebsiella sp. strain VN-31 in the same bioreactor, were able to form aromatic amines by the reductive break down of the azo bond and to oxidize them into non-toxic metabolites.     

A Missense Mutation in the Arabidopsis COPII Coat Protein Sec24A Induces the Formation of Clusters of the Endoplasmic Reticulum and Golgi Apparatus

How the endoplasmic reticulum (ER) and the Golgi apparatus maintain their morphological and functional identity while working in concert to ensure the production of biomolecules necessary for the cell''s survival is a fundamental question in plant biology. Here, we isolated and characterized an Arabidopsis thaliana mutant that partially accumulates Golgi membrane markers and a soluble secretory marker in globular structures composed of a mass of convoluted ER tubules that maintain a connection with the bulk ER. We established that the aberrant phenotype was due to a missense recessive mutation in sec24A, one of the three Arabidopsis isoforms encoding the coat protomer complex II (COPII) protein Sec24, and that the mutation affects the distribution of this critical component at ER export sites. By contrast, total loss of sec24A function was lethal, suggesting that Arabidopsis sec24A is an essential gene. These results produce important insights into the functional diversification of plant COPII coat components and the role of these proteins in maintaining the dynamic identity of organelles of the early plant secretory pathway.