Stabilization of the Escherichia coli DNA polymerase III ε subunit by the θ subunit favors in vivo assembly of the Pol III catalytic core

Escherichia coli DNA polymerase III holoenzyme (HE) contains a core polymerase consisting of three subunits: α (polymerase), ε (3'-5' exonuclease), and θ. Genetic experiments suggested that θ subunit stabilizes the intrinsically labile ε subunit and, furthermore, that θ might affect the cellular amounts of Pol III core and HE. Here, we provide biochemical evidence supporting this model by analyzing the amounts of the relevant proteins. First, we show that a ΔholE strain (lacking θ subunit) displays reduced amounts of free ε. We also demonstrate the existence of a dimer of ε, which may be involved in the stabilization of the protein. Second, θ, when overexpressed, dissociates the ε dimer and significantly increases the amount of Pol III core. The stability of ε also depends on cellular chaperones, including DnaK. Here, we report that: (i) temperature shift-up of ΔdnaK strains leads to rapid depletion of ε, and (ii) overproduction of θ overcomes both the depletion of ε and the temperature sensitivity of the strain. Overall, our data suggest that ε is a critical factor in the assembly of Pol III core, and that this is role is strongly influenced by the θ subunit through its prevention of ε degradation.     

Distribution, ecology and morphology of Lytocarpia myriophyllum (Cnidaria: Hydrozoa), a Mediterranean Sea habitat former to protect

In the Mediterranean Sea, as well as in other parts of the word, intense bottom trawling threatens deep and mesophotic assemblages, compromising mainly the survivorship of erect organisms and of the habitat complexity they shape. Protection of species able to affect their habitats, by increasing spatial complexity and enhancing interspecific interactions, is crucial for biodiversity conservation. It is urgent to highlight the occurrence of those species which act as ecosystem engineers and/or habitat former to enhance awareness on their ecological role and to develop focused conservation strategies. Lytocarpia myriophyllum is the largest Leptomedusan hydroid of the Mediterranean Sea, with colonies up to 1 m high, and the most abundant Aglaopheniid in the eastern part of the North Atlantic Ocean. This species creates wide forests on soft bottoms stabilizing sediments, providing refuge and food for several other associated organisms and could be defined both a habitat former and an ecosystem engineer. Thanks to trimix diving here we report on new insights on the morphological, biological and ecological features of L. myriophyllum meadows from the Mediterranean Sea furnishing a baseline for protection plans focused on these facies. This work demonstrates that direct studies of mesophotic habitats allow to collect far more detailed information than grabs, ROVs, or towed camera arrays and highlights the urgent need to redefine the vertical extension of several marine protected areas.     

The chronological life span of Saccharomyces cerevisiae

Simple model systems have played an important role in the discovery of fundamental mechanisms of aging. Studies in yeast, worms and fruit flies have resulted in the identification of proteins and signalling pathways that regulate stress resistance and longevity. New findings indicate that these pathways may have evolved to prevent damage and postpone aging during periods of starvation and may be conserved from yeast to mammals. We will review the yeast S. cerevisiae model system with emphasis on the chronological life span as a model system to study aging and the regulation of stress resistance in eukaryotes.     

Centrally acting leptin induces a resuscitating effect in haemorrhagic shock in rats

Centrally acting leptin induces the activation of the sympathetic nervous system with a pressor effect in normotensive rats. The purpose of the study was to examine central leptin-evoked action in critical haemorrhagic hypotension. In anaesthetized male Wistar rats subjected for irreversible haemorrhagic shock with mean arterial pressure (MAP) 20-25 mmHg haemodynamic parameters and plasma concentrations of adrenaline and noradrenaline were measured. Leptin given intracerebroventricularly (20 μg) evoked long-lasting rises in MAP and heart rate (HR), with a subsequent increase in renal, mesenteric and hindquarters blood flows and a 100% survival at 2 h. MAP and peripheral blood flow changes were inhibited by a pre-treatment with α(1)- and α(2)-adrenoceptor antagonists prazosin (0.5 mg/kg) and yohimbine (1 mg/kg), while β-adrenoceptor antagonist propranolol (1 mg/kg) blocked leptin-induced HR changes, without influence on MAP, peripheral blood flows and survival. Twenty min after leptin treatment, there were higher plasma concentrations of noradrenaline, but not adrenaline, in comparison with the saline-treated control group. In conclusion, centrally acting leptin induces a long-lasting pressor effect with an improvement in the survival rate in haemorrhage-shocked rats. The effect may be associated with the activation of the sympathetic nervous system.     

Identification and seasonal distribution of airborne fungi in three horse stables in Italy

Fungal agents are responsible for a variety of respiratory diseases both in humans and animals. The nature and seasonal variations of fungi have been investigated in many environments with wide ranging results. The aims of the present report were (i) to evaluate the quality and magnitude of exposure to airborne fungi in three differently structured equine stalls (open air, partially and completely enclosed buildings) during a one-year period, using an air sampling technique and (ii) to compare the distribution and frequency of fungal species, with regards to these different environments. Air samples were collected monthly from December 2001 to November 2002 by means of a surface air sampler (SAS) Super-90, (PBI International, Milan, Italy). Penicillium and Aspergillus spp. were cultured from all the stables in all seasons. Mucoraceae were also recovered in all seasons in stalls 1 and 2, while they were not isolated in spring and fall in stall 3. These fungi were detected in 28.4%, 72.9% and 60.5% of the total number of samples, respectively. Other fungal genera such as Alternaria, Cladosporium, Fusarium, Beauveria and Drechslera were also occasionally recovered.Viable fungal concentrations varied greatly, ranging from below the limit of detection to more than 3000 CFU/m³ for stables 1 and 2, and 1750 CFU/m³ for stable 3. The median fungal concentration was approximately 178 CFU/m³. Total fungal concentration appeared to be highest in summer, winter and spring, and lowest in the fall.     

Induction of metal binding compounds and antioxidative defence in callus cultures of two black poplar (<Emphasis Type=Italic>P. nigra</Emphasis>) clones with different tolerance to cadmium

Callus cultures of two parental clones of Populus nigra L., Poli and 58-861, originating from contrasting environments, were exposed to different cadmium concentrations (0, 150 and 250 μM CdSO₄). Clones showed different growth responses to cadmium, evaluated by the tolerance index (Ti), with Poli being more tolerant to the metal at both concentrations. The cadmium concentration at the end of the treatment was very similar between clones at 150 μM CdSO₄, while a higher value in 58-861 compared to Poli was detected at 250 μM CdSO₄. The bioconcentration factor evidenced the lowest value in Poli at 250 μM CdSO₄. Unlike 58-861, cadmium provoked a strong induction of thiols and phytochelatins in clone Poli. In both clones, organic acid concentration differed notably in untreated calli and cadmium treatment induced a general lowering of these compounds. A notably higher antioxidant enzyme activity (ascorbate peroxidase, APX; catalase, CAT; guaiacol peroxidase, GPX) was measured in control calli of clone Poli compared to 58-861. Cadmium induced a remarkable enhancement of APX and CAT, but not GPX, activity at 150 μM CdSO₄ in Poli. Conversely, in 58-861 at 150 μM CdSO₄, and in both clones at 250 μM CdSO₄, a decrease in the antioxidant activity occurred. This investigation provided evidence that these two contrasting genotypes of P. nigra are characterised by a different response to cadmium in callus cultures. In particular, in Poli, the higher tolerance to cadmium is associated with a higher activity of antioxidative enzymes and the ability to strongly increase thiol and PC concentration in response to metal exposure.     

Growth at low temperature suppresses readthrough of the UGA stop codon during the expression of Bacillus subtilis flgM gene in Escherichia coli

The efficient production of recombinant proteins in Escherichia coli requires a proper termination of translation to ensure the synthesis of only the desired product. During the recombinant production of Bacillus subtilis flgM in E. coli, we detected an additional polypeptide of molecular mass higher than the expected, corresponding to a product of a translational readthrough of the UGA stop codon. In this paper we show that the readthrough was abolished when the synthesis of the recombinant protein was carried out at 25 degrees C. The possible causes that contribute to reduce the proportion of readthrough protein species against the correct terminated product are discussed.     

Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin,beta-catenin,and the phosphatase DEP-1/CD148

Confluent endothelial cells respond poorly to the proliferative signals of VEGF. Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation. VE-cadherin truncated in beta-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. beta-Catenin-null endothelial cells are not contact inhibited by VE-cadherin and are still responsive to VEGF, indicating that this protein is required to restrain growth factor signaling. A dominant-negative mutant of high cell density-enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation. Overall the data indicate that VE-cadherin-beta-catenin complex participates in contact inhibition of VEGF signaling. Upon stimulation with VEGF, VEGFR-2 associates with the complex and concentrates at cell-cell contacts, where it may be inactivated by junctional phosphatases such as DEP-1. In sparse cells or in VE-cadherin-null cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.     

Over-Expression of hNGF in Adult Human Olfactory Bulb Neural Stem Cells Promotes Cell Growth and Oligodendrocytic Differentiation

The adult human olfactory bulb neural stem/progenitor cells (OBNC/PC) are promising candidate for cell-based therapy for traumatic and neurodegenerative insults. Exogenous application of NGF was suggested as a promising therapeutic strategy for traumatic and neurodegenerative diseases, however effective delivery of NGF into the CNS parenchyma is still challenging due mainly to its limited ability to cross the blood–brain barrier, and intolerable side effects if administered into the brain ventricular system. An effective method to ensure delivery of NGF into the parenchyma of CNS is the genetic modification of NSC to overexpress NGF gene. Overexpression of NGF in adult human OBNSC is expected to alter their proliferation and differentiation nature, and thus might enhance their therapeutic potential. In this study, we genetically modified adult human OBNS/PC to overexpress human NGF (hNGF) and green fluorescent protein (GFP) genes to provide insight about the effects of hNGF and GFP genes overexpression in adult human OBNS/PC on their in vitro multipotentiality using DNA microarray, immunophenotyping, and Western blot (WB) protocols. Our analysis revealed that OBNS/PC-GFP and OBNS/PC-GFP-hNGF differentiation is a multifaceted process involving changes in major biological processes as reflected in alteration of the gene expression levels of crucial markers such as cell cycle and survival markers, stemness markers, and differentiation markers. The differentiation of both cell classes was also associated with modulations of key signaling pathways such MAPK signaling pathway, ErbB signaling pathway, and neuroactive ligand-receptor interaction pathway for OBNS/PC-GFP, and axon guidance, calcium channel, voltage-dependent, gamma subunit 7 for OBNS/PC-GFP-hNGF as revealed by GO and KEGG. Differentiated OBNS/PC-GFP-hNGF displayed extensively branched cytoplasmic processes, a significant faster growth rate and up modulated the expression of oligodendroglia precursor cells markers (PDGFRα, NG2 and CNPase) respect to OBNS/PC-GFP counterparts. These findings suggest an enhanced proliferation and oligodendrocytic differentiation potential for OBNS/PC-GFP-hNGF as compared to OBNS/PC-GFP.     

Chromatin dynamics of the developmentally regulated P. lividus neural alpha tubulin gene

Over 40 years ago, Allfrey and colleagues (1964) suggested that two histone modifications, namely acetylation and methylation, might regulate RNA synthesis. Nowadays it is universally accepted that activation of gene expression strictly depends on enzymatic mechanisms able to dynamically modify chromatin structure. Here, using techniques including DNaseI hypersensitive site analysis, chomatin immunoprecipitation and quantitative PCR analysis, we have analyzed the dynamics of histone post-translation modifications involved in developmentally/spatially controlled activation of the sea urchin PlTalpha2 tubulin gene. We have demonstrated that only when the PlTalpha2 core promoter chromatin is acetylated on H3K9, tri-methylated on H3K4 and not di-methylated on H3K27, RNA pol II can be enrolled. In contrast, we have shown that when chromatin is methylated both on H3K9 (me2/3) and H3K27 (me2) and mono methylated on H3K4 the promoter is not accessible to RNA pol II. Our results suggest that, during P. lividus embryogenesis, both HAT/HDAC and HMT/HDM activities, which are able to regulate accessibility of the PlTalpha2 basal promoter to RNA polymerase II, are coordinately switched-on.