Estrogen binding proteins of calf uterus. Inhibition of aggregation and dissociation of receptor by chemical perturbation with NaSCN.

Sodium thiocyanate up to 0.5 M is compatible with a stable estradiol-t-receptor complex during sucrose gradient centrifugation; however, the maximum permissible concentration in 0.1 M during Sephadex G-100 and G-200 chromatography. When NaSCN 0.1 M is added to low-salt cytosol (approximately 7 mg of protein/ml); (1) age-dependent aggregation of receptor is inhibited; (2) peaks of estrogen-binding activity in sucrose gradients and on Sephadex chromatography are sharp; (3) instead of the usual larger molecular states ("8S") found in low salt, most of estrogen receptor is under the following form: 4.1S; Stokes radius, 36 A; mol wt 61 000; flfo, 1.25; homogeneous at electrofocusing, with isoelectric point at 6.0. When cytosol containing NaSCN 0.1 M is diluted down to 2-3 mg of protein/ml or, only for sucrose gradients, NaSCN concentration is increased to 0.4-0.5 M, the 61000 dalton species decreases, being substituted, without loss of bound estradiol-t, by the following estrogen-binding entity: 28S; Stokes radius, 28 A; mol wt 32 000; flfo, 1.44. In the presence of NaSCN, KCl up to 0.4 M does not affect in a significant manner the molecular properties of the above forms. When NaSCN is dialyzed out, most receptor reverts to a 8-9S state. When cytosol is preincubated with Ca2+ (4 mM) and KCl (0.4 M) before addition of NaSCN, the above picture is modified only in the following aspects: (1) Sephadex chromatography peaks are broader and slightly but reproducibly shifted toward higher elution volumes; (2) the electrofocusing pattern consists of a two-peak heterogeneous band shifted toward higher pH (isoelectric points 6.4 and 6.6); (3) upon dialysis of NaSCN there is little or no reversion to faster sedimenting states. These modifications appear to depend on limited proteolytic attack of receptor by Ca2+ -activated receptor transforming factor (RTF), not on binding of Ca2+ to receptor. Present data suggest that the 4.1S entity is a dimer resulting from side-by-side pairing of 2.8S subunits. Molecular dimension of larger receptor forms purified from cytosol are consistent with the hypothesis that under native conditions in vivo dimers are coupled end-by-end into tetrameric structures with two stronger (between subunits) and two weaker (between dimers) bonding regions, and that tetramers may further self-associate. While NaSCN reversibly releases native dimers and subunits by direct impairment of intersubunit bonds, Ca2+ activated RTF irreversibly and specifically releases slightly modified, about 60000 mol wt dimers, by preferential proteolytic attack of the weaker bonding regions and indirect destruction of involved bonds. In vivo, this effect of RTF may be instrumental in mobilization and nuclear penetration of receptor-estradiol complex. Heteroassociation of receptor with other proteins of cytosol is not excluded by the above hypothesis.     

A standardized laboratory and surgical method for in vitro culture isolation and expansion of primary human Tenon’s fibroblasts

Good manufacturing practices guidelines require safer and standardized cell substrates especially for those cell therapy products to treat ocular diseases where fibroblasts are used as feeder layers. However, if these are unavailable for stem cells culturing, murine fibroblasts are regularly used, raising critical issues as accidentally transplanting xenogenous graft and adversely affecting stem cell clinical trials. Moreover, human fibroblasts play a significant role in testing novel ophthalmologic drugs. Accordingly, we developed a standardized laboratory and surgical approach to isolate normal and undamaged Tenon’s fibroblasts to implement the setting up of banks for both stem cells-based ocular cell therapy and in vitro drug testing. A 2–3 cm² undamaged Tenon’s biopsy was surgically obtained from 28 patients without mutually correlated ocular diseases. Nineteen dermal biopsies were used as control. Fibroblasts were isolated with or without collagenase, cultured in autologous, fetal bovine or AB serum, tested for viability by trypan blue, vimentin expression and standardized until passage 6. Successful Tenon’s fibroblasts isolation was age dependent (P = 0.001) but not sex, pathology or surgery related. A significant rate of successful cultures were obtained when biopsies were not digested by collagenase (P = 0.013). Moreover, cultures in autologous or fetal bovine serum had comparable proliferative properties (P = 0.77; P = 0.82). Through our surgical and laboratory standardized procedure, we elucidated for the first time key points of this human primary culture system, the role of the autologous serum, comparing Tenon’s and dermal fibroblasts. Our protocol may be clinically useful to reduce the risk above mentioned and may be potentially more effective for ophthalmological clinical purposes.     

Characterization of caveolae from rat heart: localization of postreceptor signal transduction molecules and their rearrangement after norepinephrine stimulation

Caveolae are plasma membrane subcompartments that have been implicated in signal transduction. In many cellular systems, caveolae are rich in signal transduction molecules such as G proteins and receptor-associated tyrosine kinases. An important structural component of the caveolae is caveolin. Recent evidence show that among the caveolin gene family, caveolin-3 is expressed in skeletal and cardiac muscle and caveolae are present in cardiac myocyte cells. Both the ANP receptor as well as the muscarinic receptor have been localized to the caveolae of cardiac myocytes in culture. These findings prompted us to conduct a further analysis of cardiac caveolae. In order to improve our understanding of the mechanisms of signal transduction regulation in cardiac myocytes, we isolated cardiac caveolae by discontinuous sucrose density gradient centrifugation from rat ventricles and rat neonatal cardiocytes. Our analysis of caveolar content demonstrates that heterotrimeric G proteins, p21ras and receptor-associated tyrosine kinases are concentrated within these structures. We also show that adrenergic stimulation induces an increase in the amount of diverse alpha- and beta-subunits of G proteins, as well as p21ras, in both in vivo and in vitro experimental settings. Our data show that cardiac caveolae are an important site of signal transduction regulation. This finding suggests a potential role for these structures in physiological and pathological states.     

Reinitiation of protein synthesis in Escherichia coli can be induced by mRNA cis-elements unrelated to canonical translation initiation signals

In Eubacteria, de novo translation of some internal cistrons may be inefficient or impossible unless the 5' neighboring cistron is also translated (translational coupling). Translation reinitiation is an extreme case of translational coupling in which translation of a message depends entirely on the presence of a nearby terminating ribosome. In this work, the characteristics of mRNA cis-elements inducing the reinitiation process in Escherichia coli have been investigated using a combinatorial approach. A number of novel translational reinitiation sequences (TRSs) were thus identified, which show a wide range of reinitiation activities fully dependent on a translational coupling event and unrelated to the presence/absence of secondary structure or mRNA stability. Moreover, some of the isolated TRSs are similar to intercistronic sequences present in the E. coli genome.     

Interaction of p53 with Mdm2 and azurin as studied by atomic force spectroscopy

Azurin, a bacterial protein, can be internalized in cancer cells and induce apoptosis. Such anticancer effect is coupled to the formation of a complex with the tumour‐suppressor p53. The mechanism by which azurin stabilizes p53 and the binding sites of their complex are still under investigation. It is also known that the predominant mechanism for p53 down‐regulation implies its association to Mdm2, the main ubiquitin ligase affecting its stability. However, the p53/Mdm2 interaction, occurring at the level of both their N‐terminal domains, has been characterized so far by experiments involving only partial domains of these proteins. The relevance of the p53/Mdm2 complex as a possible target of the anticancer therapies requires a deeper study of this complex as made up of the two entire proteins. Moreover, the apparent antagonist action of azurin against Mdm2, with respect of p53 regulation, might suggest the possibility that azurin binds p53 at the same site of Mdm2, preventing in such a way p53 and Mdm2 from association and thus p53 from degradation. By following the interaction of the two entire proteins by atomic force spectroscopy, we have assessed the formation of a specific complex between p53 and Mdm2. We found for it a binding strength and a dissociation rate constant typical of dynamical protein–protein interactions and we observed that azurin, even if capable to bind p53, does not compete with Mdm2 for the same binding site on p53. The formation of the p53/Mdm2/azurin ternary complex might suggest an alternative anti‐cancer mechanism adopted by azurin. Copyright © 2009 John Wiley & Sons, Ltd.     

Hemorrhagic Fever with Renal Syndrome Complicated by Orchitis

Hemorrhagic fever with renal syndrome (HFRS) is a disease caused by viruses of the family Bunyaviridae,genus Hantavirus.HFRS from Dobrava virus (DOBV) is a seldom reported disease in Albania.Clinically HFRS is manifested as mild,moderate,or severe.Therefore,the number of cases of Hantavirus' infection may be underestimated,and should be included in the differential diagnosis of many acute infections,hematologic diseases,acute abdominal diseases and renal diseases complicated by acute renal failure.We report...     

Mesenchymal stromal cells are present in the heart and promote growth of adult stem cells in vitro

Background aimsFor many years the human heart has been considered a terminally differentiated organ with no regenerative potential after injury. Recent studies, however, have cast doubt on this long-standing dogma. The objective of this study was to investigate the presence of and characterize mesenchymal stromal cells (MSC) in the adult mouse heart. The impact of MSC on growth and differentiation of adult cardiac stem cells (CSC) was also analyzed.MethodsA combination of lineage-negative/c-kit-negative (Lin^?/c-kit^?) immunoselection with a plastic-adhesion technique was used to isolate cardiac-derived MSC. The differentiation capacity and expression of surface markers were analyzed. To investigate the impact of MSC on growth and differentiation of adult CSC, Green Fluorescent Protein (GFP⁺) adult CSC were co-cultured with GFP^? cardiac-derived MSCResultsMSC were present in the adult mouse heart and they met the criteria established to define mouse MSC. They expressed surface markers and were able to differentiate, in a controlled manner, into multiple lineages. In addition, cardiac-derived MSC promoted the survival and expansion of adult CSC in vitroConclusionsMSC can be isolated from the mouse heart and they promote growth and differentiation of adult CSC. The findings from this study could have a significant beneficial impact on future heart failure treatment. Co-culture and co-implantation of cardiac-derived MSC with adult CSC could provide extensive cardiac regeneration and maintenance of the CSC population after implanted into the heart.