Lamin A and the LINC complex act as potential tumor suppressors in Ewing Sarcoma

Lamin A, a main constituent of the nuclear lamina, is involved in mechanosignaling and cell migration through dynamic interactions with the LINC complex, formed by the nuclear envelope proteins SUN1, SUN2 and the nesprins. Here, we investigated lamin A role in Ewing Sarcoma (EWS), an aggressive bone tumor affecting children and young adults. In patients affected by EWS, we found a significant inverse correlation between LMNA gene expression and tumor aggressiveness. Accordingly, in experimental in vitro models, low lamin A expression correlated with enhanced cell migration and invasiveness and, in vivo, with an increased metastatic load. At the molecular level, this condition was linked to altered expression and anchorage of nuclear envelope proteins and increased nuclear retention of YAP/TAZ, a mechanosignaling effector. Conversely, overexpression of lamin A rescued LINC complex organization, thus reducing YAP/TAZ nuclear recruitment and preventing cell invasiveness. These effects were also obtained through modulation of lamin A maturation by a statin-based pharmacological treatment that further elicited a more differentiated phenotype in EWS cells. These results demonstrate that drugs inducing nuclear envelope remodeling could be exploited to improve therapeutic strategies for EWS.Subject terms: Nuclear organization, Cancer     

Sialic Acid Derivatives and their Distribution in Rat Sublingual Gland Acini during Pre- and Post-natal Development

Sialoglycoconjugates in rat sublingual gland acinar cells, at different stages of pre- and post-natal development, were investigated in situ with specific lectins and by the selective removal of terminal sialic acids. Cleavage of acetyl substituents sited in the pyranose ring and/or polyhydroxyl side chain was used as an additional means of characterising the glycoconjugates. The first expression of terminal sialic acid linked to -galactose was found at gestational day 17 and progressive different derivatives were observed. The terminal disaccharide sialic acid-N-acetylgalactosamine was constantly visualized in the sublingual gland from gestational day 18. In both terminal disaccharides, sialic acids were characterized by variable degrees of acetylation and were found to be highly packaged and responsible for the hydration coat. The complex data obtained indicated that the sublingual gland is characterized by a marked fluctuation of complex sialoglycoconjugates that differ from those in the submandibular gland of the same species.     

The metal function in the reactions of bovine serum amine oxidase with substrates and hydrazine inhibitors

 Bovine serum amine oxidase (BSAO) reacts with 2-hydrazinopyridine, which binds the organic cofactor 2,4,5-trihydroxyphenylalanine quinone, forming a band at 435 nm. The band shifts to 526 nm around 60  °C, to 415 nm upon denaturation, but only shifts to 429 nm upon Cu²⁺ depletion. Its wavelength and intensity suggest that the adduct has the azo conformation, whilst the same adduct of crystallineEscherichia coli amine oxidase (ECAO) shows the hydrazone conformation in the X-ray structure. The steady state kinetics of aminomethyl- and aminoethylpyridines confirm that the formation of the product Schiff base, analogous to the azo form of the 2-hydrazinopyridine adduct, is not hindered in solution. The structural stability of the adduct in the absence of Cu²⁺ is taken to imply hydrogen bonding of the pyridyl nitrogen to a conserved aspartate, as in the ECAO adduct. Thus the ECAO adduct provides a good model for a transient intermediate leading to formation of the BSAO azo adduct. On the basis of this model and of the catalytic competence of Co²⁺-substituted BSAO, confirmed by the present data, a catalytic reaction scheme is proposed. Received: 2 December 1998 / Accepted: 22 March 1999     

Biased T-cell receptor usage is associated with allelic variation in the MHC class II peptide binding groove

 A comprehensive analysis was carried out of the tri-molecular complex of peptide, major histocompatibility class II molecule, and T-cell receptor (TcR) involved in the recognition of the promiscuous HA (306–318) peptide, restricted by one of two closely related HLA-DR alleles, HLA-DRB1*0101 and HLA-DRB1*0103. These two DR molecules differ by only three amino acids at positions 67, 70, and 71, in the third variable region of the DRB1 chain. None of the HA (306–318)-specific T-cell clones restricted by these two DR molecules tolerated amino acid substitution at the peptide-binding position 71, despite the fact that the substitution did not interfere with peptide binding. The majority of the DRB1*0103-restricted clones tolerated substitution of the amino acid at the TcR-contacting position 70, while the DRB1*0101-restricted T cells did not. Biased usage of TRVA and TRVB segments was observed for the DRB1*0103-restricted clones; in contrast, apparently random usage was seen in the DRB1*0101-restricted T cells. Finally, limiting dilution analysis revealed a lower frequency of T cells reactive with the HA peptide in a DRB1*0103 compared with a DRB1*0101 individual. Taken together these data suggest that biased TcR gene usage may reflect a relatively low precursor frequency of T cells, and the need for clonal expansion of a limited set of high avidity T cells. Received: 7 August 1998 / Revised: 19 November 1998     

PTX3 plays a key role in the organization of the cumulus oophorus extracellular matrix and in in vivo fertilization

PTX3 is a prototypic long pentraxin that plays a non-redundant role in innate immunity against selected pathogens and in female fertility. Here, we report that the infertility of Ptx3(-/-) mice is associated with severe abnormalities of the cumulus oophorus and failure of in vivo, but not in vitro, oocyte fertilization. PTX3 is produced by mouse cumulus cells during cumulus expansion and localizes in the matrix. PTX3 is expressed in the human cumulus oophorus as well. Cumuli from Ptx3(-/-) mice synthesize normal amounts of hyaluronan (HA), but are unable to organize it in a stable matrix. Exogenous PTX3 restores a normal cumulus phenotype. Incorporation in the matrix of inter-alpha-trypsin inhibitor is normal in Ptx3(-/-) cumuli. PTX3 does not interact directly with HA, but it binds the cumulus matrix hyaladherin tumor necrosis factor alpha-induced protein 6 (TNFAIP6, also known as TSG6) and thereby may form multimolecular complexes that can cross-link HA chains. Thus, PTX3 is a structural constituent of the cumulus oophorus extracellular matrix essential for female fertility.     

Beneficial effects of GH/IGF-1 on skeletal muscle atrophy and function in experimental heart failure

Muscle atrophy is a determinant of exercise capacity in heart failure (CHF). Myocyte apoptosis, triggered by tumor necrosis factor- (TNF-) or its second messenger sphingosine (SPH), is one of the causes of atrophy. Growth hormone (GH) improves hemodynamic and cardiac trophism in several experimental models of CHF, but its effect on skeletal muscle in CHF is not yet clear. We tested the hypothesis that GH can prevent skeletal muscle apoptosis in rats with CHF. CHF was induced by injecting monocrotaline. After 2 wk, 2 groups of rats were treated with GH (0.2 mg·kg^–1·day^–1 and 1.0 mg·kg^–1·day^–1) subcutaneously. A third group of controls had saline. After 2 additional weeks, rats were killed. Tibialis anterior cross-sectional area, myosin heavy chain (MHC) composition, and a study on myocyte apoptosis and serum levels of TNF- and SPH were carried out. The number of apoptotic nuclei, muscle atrophy, and serum levels of TNF- and SPH were decreased with GH at high but not at low doses compared with CHF rats. Bcl-2 was increased, whereas activated caspases and bax were decreased. The MHC pattern in GH-treated animals was similar to that of controls. Monocrotaline slowed down both contraction and relaxation but did not affect specific tetanic force, whereas absolute force was decreased. GH treatment restored contraction and relaxation to control values and brought muscle mass and absolute twitch and tetanic tension to normal levels. These findings may provide an insight into the therapeutic strategy of GH given to patients with CHF to improve exercise capacity. apoptosis; cytokines; myosin heavy chains     

Enhancement of vitamin E production in sunflower cell cultures

The most biologically active component of vitamin E, -tocopherol, is synthesized in its most effective stereoisomeric form only by photosynthetic organisms. Using sunflower cell cultures, a suitable in vitro production system of natural -tocopherol was established. The most efficient medium was found to be MS basal medium with naphthaleneacetic acid and 6-benzylaminopurine with the addition of casaminoacids and myo-inositol. Culture feeding experiments using biosynthetic precursors showed that -tocopherol production improved by 30% when homogentisic acid was used. Interestingly, time-course experiments with sunflower suspension cultures showed a possible increase of 78% in -tocopherol production when using cultures of longer subculture intervals. Compared to the starting plant tissue, an overall 100% increase of -tocopherol was reached by these sunflower cell cultures.     

Construction of replicative and integrative plasmids for setting up the in vivo expression technology in Helicobacter pylori

Some pathogenic factors of Helicobacter pylori, a bacterium involved in peptic ulcer and gastric cancer, have already been identified using either global or particular approach, but there are still some orphan genes and unidentified pathogenic factors. One of the methods used successfully for the identification of virulence genes of many pathogens is the in vivo expression technology. We describe here the construction and sequences of three different plasmids, one integrative and two replicatives, for the identification of virulence genes by using in vivo expression technology in H. pylori, and of potential use in other bacteria such as Campylobacter spp. Moreover, the use of the green fluorescent protein could allow to classify the genes according to the strength of their expression and to identify those which are repressed upon interaction with gastric mucosa.