Carbohydrate synthesis by two Navicula strains isolated from benthic and pelagic mucilages in the Tyrrhenian Sea (Tuscan Archipelago)

Two exopolysaccharide (EPS)-producing strains of the diatom Navicula, were isolated from benthic and pelagic mucilaginous aggregates sampled in the Tyrrhenian Sea and cultured under laboratory conditions. The amount of carbohydrate formed over the growth period and on a per cell basis was quite similar. However, the benthic strain showed a preferential synthesis of the bound (i.e., cellular and capsular) carbohydrate fraction, whereas the pelagic strain preferentially synthesised soluble, polymeric carbohydrates. The polysaccharides released into the medium by the two strains showed the same qualitative monosaccharidic composition, being constituted by two acidic and six neutral sugars. It is suggested that the difference between the benthic and the pelagic strain in the synthesis of bound or soluble carbohydrates may be related to the different role of these compounds in the particular habitats of the strains.     

Amplification and excision of integrated polyoma DNA sequences require a functional origin of replication

Cells transformed by Polyoma virus (Py) can undergo a high rate of excision or amplification of integrated viral DNA sequences, and these phenomena require the presence of homology (i.e., repeats) within the viral insertion as well as a functional viral large T antigen (T-Ag). To determine whether the main role of large T-Ag in excision and amplification was replicative or recombination-promoting, we studied transformed rat cell lines containing tandem insertions of a ts-a Py molecule (encoding a thermolabile large T-Ag) with a deletion of the origin of viral DNA replication. Culturing of these cells at the temperature permissive for large T-Ag function did not result in any detectable excision or amplification of integrated Py sequences. We then introduced into origin-defective lines a recombinant plasmid containing the viral origin of replication and the gene coding for resistance to the antibiotic G418. All G418-resistant clones analyzed readily amplified the integrated plasmid molecules when grown under conditions permissive for large T-Ag function, showing that these cells produced viral large T-Ag capable of promoting amplification in trans of DNA sequences containing the Py origin. These observations strongly suggest that Polyoma large T antigen promotes excision or amplification of viral DNA by initiating replication at the integrated origin, providing a favorable substrate for subsequent recombination.     

Biophysical properties and cellular toxicity of covalent crosslinked oligomers of α-synuclein formed by photoinduced side-chain tyrosyl radicals

Alpha-synuclein (αS), a 140 amino acid presynaptic protein, is the major component of the fibrillar aggregates (Lewy bodies) observed in dopaminergic neurons of patients affected by Parkinson's disease. It is currently believed that noncovalent oligomeric forms of αS, arising as intermediates in its aggregation, may constitute the major neurotoxic species. However, attempts to isolate and characterize such oligomers in vitro, and even more so in living cells, have been hampered by their transient nature, low concentration, polymorphism, and inherent instability. In this work, we describe the preparation and characterization of low molecular weight covalently bound oligomeric species of αS obtained by crosslinking via tyrosyl radicals generated by blue-light photosensitization of the metal coordination complex ruthenium (II) tris-bipyridine in the presence of ammonium persulfate. Numerous analytical techniques were used to characterize the αS oligomers: biochemical (anion-exchange chromatography, SDS-PAGE, and Western blotting); spectroscopic (optical: UV/Vis absorption, steady state, dynamic fluorescence, and dynamic light scattering); mass spectrometry; and electrochemical. Light-controlled protein oligomerization was mediated by formation of Tyr-Tyr (dityrosine) dimers through -C-C- bonds acting as covalent bridges, with a predominant involvement of residue Y39. The diverse oligomeric species exhibited a direct effect on the in vitro aggregation behavior of wild-type monomeric αS, decreasing the total yield of amyloid fibrils in aggregation assays monitored by thioflavin T (ThioT) fluorescence and light scattering, and by atomic force microscopy (AFM). Compared to the unmodified monomer, the photoinduced covalent oligomeric species demonstrated increased toxic effects on differentiated neuronal-like SH-SY5Y cells. The results highlight the importance of protein modification induced by oxidative stress in the initial molecular events leading to Parkinson's disease.     

AN ELECTRON MICROSCOPE STUDY OF DENERVATION ATROPHY IN RED AND WHITE SKELETAL MUSCLE FIBERS

A study, mainly by electron microscopy, has been made on two leg muscles of rat, in the course of atrophy experimentally induced by total denervation. As a preliminary the chief distinctive features of the soleus, chosen as a representative of pure red muscle, and of the gastrocnemius, representative of pure white muscle, are described. Two major phases of atrophy, somewhat overlapping in time, were observed. In the first, a degenerative autolytic process takes place in areas of the fiber, with loss of striation. It can be detected as early as the 7th day, but the maximum is observed at the 14th day, and accounts for a gross weight loss of 50 per cent. The first alteration appears in the Z lines; disorder in the disposition of filaments then follows. The process occurs very rapidly, leaving large areas in the cell in which one can detect only ground substance, glycogen, rare randomly disposed vesicular elements, and some mitochondria. Several lysosomes and masses of lipoproteins, which assume the configuration of concentric lamellae, show up in these fibers. Subsequently large parts of the waste sarcoplasm are discarded into the intercellular spaces. In the second major phase the so called "simple" atrophy takes place. The process starts early, but its effects are more detectable after 1 month. In this period, single myofibrils undergo different degrees of reduction in diameter, while the spatial disposition of primary and secondary filaments inside the fibrils remains normal. The appearance of the fibrils in longitudinal sections suggests that the process takes place by the detachment of filaments from the periphery of the fibrils and by their subsequent breakdown in the interfibrillary spaces. The sarcoplasmic reticulum is still well preserved, and relatively overdeveloped. Mitochondria disappear in parallel with the contractile material.     

Mechanical analysis of the ring opening test applied to human ascending aortas

This work presents experiments, modelling and numerical simulation aimed at describing the mechanical response of human ascending aortas in the ring opening test. The objective is to quantify, from the opening angles measured in the test, the residual stress distribution along the artery wall and, afterwards, how this stress pattern changes when the artery is subjected to standard physiological pressures. The cases studied correspond to four groups including both healthy and pathological arteries. The tissues are characterized via tensile test measurements that enable to derive the material parameters of two constitutive models adopted in the present analysis. Overall, the numerical results obtained for all groups were found to be a useful data that allow to estimate the residual stress and their influence on the vessels under normal and hypertension physiological conditions.     

Prothymosin alpha protects cardiomyocytes against ischemia-induced apoptosis via preservation of Akt activation

The human prothymosin alpha (PTα) gene encodes a 12.5 kDa highly acidic nuclear protein that is widely expressed in mammalian tissues including the heart and importantly, is detectable also in blood serum. During apoptosis or necrosis, PTα changes its nuclear localization and is able to exert an important cytoprotective effect. Since the role of PTα in the heart has never been evaluated, the aim of the present study was to investigate the effects of PTα on cardiomyocytes during ischemic injury. Our data show that seven after myocardial infarction (MI), PTα expression levels are significantly increased both in blood serum and in cardiac tissue, and notably we observe that PTα translocates from the nuclei to cytoplasm and plasma membrane of cardiomyocytes following MI. Furthermore, in vitro experiments in cardiomyocytes, confirm that after 6 h of simulated ischemia (SI), PTα protein levels are upregulated compared to normoxic cells. Importantly, treatment of cardiomyocytes with a recombinant PTα (rPTα), during SI results in a significant decrease in the apoptotic response and in a robust increase in cell survival. Moreover, these effects are accompanied to a significant preservation of the activated levels of the anti-apoptotic serine-threonine kinase Akt. Consistent with our in vitro observation, rPTα-treated MI mice exhibit a strong reduction in infarct size at 24 h, compared to the MI control group and at the molecular level, PTα treatment induces activation of Akt. The present study provides for the first time the demonstration that PTα offers cardioprotection against ischemic injury by an Akt-dependent mechanism.     

RNA metabolism: putting the brake on the UPR

The unfolded protein response (UPR) is a major signaling cascade that determines cell fate under conditions of endoplasmic reticulum (ER) stress. The kinetics and amplitude of UPR responses are tightly controlled by several feedback loops and the expression of positive and negative regulators. In this issue of EMBO Reports, the Wilkinson lab uncovers a novel function of nonsense‐mediated RNA decay (NMD) in fine‐tuning the UPR 1 . NMD is an mRNA quality control mechanism known to destabilize aberrant mRNAs that contain premature termination codons. In this work, NMD was shown to determine the threshold of stress necessary to activate the UPR, in addition to adjusting the amplitude of downstream responses and the termination phase. These effects were mapped to the control of the mRNA stability of IRE1, a major ER stress transducer. This study highlights the dynamic crosstalk between mRNA metabolism and the proteostasis network demonstrating the physiological relevance of normal mRNA regulation by the NMD pathway.     

Quantitative image analysis identifies pVHL as a key regulator of microtubule dynamic instability

Von Hippel-Lindau (VHL) tumor suppressor gene mutations predispose carriers to kidney cancer. The protein pVHL has been shown to interact with microtubules (MTs), which is critical to cilia maintenance and mitotic spindle orientation. However, the function for pVHL in the regulation of MT dynamics is unknown. We tracked MT growth via the plus end marker EB3 (end-binding protein 3)-GFP and inferred additional parameters of MT dynamics indirectly by spatiotemporal grouping of growth tracks from live cell imaging. Our data establish pVHL as a near-optimal MT-stabilizing protein: it attenuates tubulin turnover, both during MT growth and shrinkage, inhibits catastrophe, and enhances rescue frequencies. These functions are mediated, in part, by inhibition of tubulin guanosine triphosphatase activity in vitro and at MT plus ends and along the MT lattice in vivo. Mutants connected to the VHL cancer syndrome are differentially compromised in these activities. Thus, single cell–level analysis of pVHL MT regulatory function allows new predictions for genotype to phenotype associations that deviate from the coarser clinically defined mutant classifications.