Characterization of plasma factors that alter the permeability to albumin within isolated glomeruli

Focal segmental glomerulosclerosis (FSGS) is responsible for intractable proteinuria and has become the leading cause of renal insufficiency in children. Protenuria in FSGS is probably due to the effect of one or more permeability plasma factors which increase the glomerular permeability to proteins. We fractioned serum from children with FSGS using two mixed chromatographic-electrophoretic approaches and have purified ten proteins among several hundreds which maintained the original permeability activity after renaturation, utilizing an isolated rat glomeruli assay. Six proteins were successfully characterized by mass spectometry as fibulin, apolipoprotein J, vitronectin, albumin isoforms, gamma chain fibrinogen and mannan-binding lectin-associated serine protease. Both procedures utilized for purification were based on affinity chromatography with Protein A-Sepharose and ended with two-dimensional electrophoresis, whereas the intermediate steps were different. Cross inhibition with zinc and aprotinin of purified factors and whole FSGS serum indicate strong homology. These are the first data demonstrating permeability activity for serum proteins, an observation with important implications in pathogenesis of proteinuria. Determination of the serum levels of each protein and a careful differentiation of FSGS from normal serum could provide the basis for clarifying the mechanism of proteinuria.     

Cytochrome c551 as a model system for protein folding

Considerable progress was made over the last few years in understanding the mechanism of folding of cytochrome c₅₅₁, a small acidic hemeprotein from Pseudomonas aeruginosa. Comparison of our results with those obtained by others on horse heart cytochrome c allows to draw some general conclusions on the structural features that are common determinants in the folding of members of the cytochrome c family.     

Diet and retarded growth.

The diets of 36 children below the third centile for height but with no organic disease were compared with the diets of a control group. In most cases retarded growth was associated with a long-continued deficiency in calorie intake. When the diets were reassessed about a year later the shortfall in calorie intake was significantly reduced. This improvement, which tended to be followed by an increase in the rate of growth in height, might have been due to alteration in the child''s circumstances or improvement in the family attitudes and feeding habits or both. Advice given at the clinic is thought to have played a part in bringing these changes about.     

Frequency of the ΔF508 mutation in a sample of 175 Italian cystic fibrosis patients

Summary A sample of 175 Italian cystic fibrosis patients has been analysed for the presence of the ΔF508 mutation. The frequency of this mutation among 137 patients with pancreatic insufficiency is equal to 57%; in 23 patients with pancreatic sufficiency it is 26%. A high proportion of the unknown mutations is associated with the same rare haplotype found in association with ΔF508, suggesting that at least another mutation occurred on a chromosome characterized by the same haplotype.     

Reactivity of ferric Aplysia myoglobin towards anionic ligands in the acidic region: Proposal for a structural model

The equilibrium binding properties of ferric Aplysia myoglobin have been studied for a number of anionic ligands in the pH region from neutrality to ~4. For all the ligands studied, the intrinsic affinity of Aplysia metmyoglobin increases by more than one order of magnitude as the pH is lowered well below neutrality.The spectroscopic properties of the ligand-free and the ligand-bound molecules show a pH dependence with apparent pK values of 4.7 and 6.1, respectively.On the basis of temperature-jump experiments, a kinetic scheme has been proposed and rate constants have been measured for the binding of azide at pH 6 and pH 4.Kinetic and thermodynamic features match each other, suggesting that a single ionizing group is responsible for all the observed effects.By inspection of the three-dimensional structure, this group has been tentatively identified as the proximal imidazole. Protonation of the N_ε of proximal histidine would be associated to the rupture of the proximal bond, giving rise to the formation of a tetra-co-ordinate, ligand-free and penta-co-ordinate, ligand-bound molecule.     

Conservation genetics of an endangered rainforest tree (Fontainea oraria – Euphorbiaceae) and implications for closely related species

Four new eastern Australian Fontainea species have beenrecently described and all have a limited distribution. F.oraria is the rarest, being restricted to 10 adult individualswithin a single site in regrowth littoral rainforest. In order todevelop adequate management strategies, this study was aimed atsurveying the genetic variability remaining within the species by usingRAPD analysis. To assist with the correct interpretation of the results,a matching study was conducted on four populations of the closelyrelated F. australis. Similar amounts of within-populationgenetic diversity were recorded for both species. The RAPD-based studysuggested that adult plants are contributing unevenly to successivegenerations. RAPD analysis also recognised a close evolutionaryrelationship between F. oraria and F. australis.Sequencing of cpDNA (trnL-F) and nrDNA (ITS2) regions,confirmed recent divergence and possibly some historical reticulationbetween these two species and two other members of the genus. Ofparticular interest was the recognition that one of the F.australis populations (Limpinwood) represented a novel genotypiccombination in need of conservation attention. The implications of theRAPD and sequencing results are discussed in reference to theirinfluence upon the development of adequate conservation strategies forall important conservation units.     

Cardiac disease modeling using induced pluripotent stem cell-derived human cardiomyocytes

Causative mutations and variants associated with cardiac diseases have been found in genes encoding cardiac ion channels, accessory proteins, cytoskeletal components, junctional proteins, and signaling molecules. In most cases the functional evaluation of the genetic alterationhas been carried out by expressing the mutated proteins in in-vitro heterologous systems. While these studies have provided a wealth of functional details that have greatly enhanced the understanding of the pathological mechanisms, it has always been clear that heterologous expression of the mutant protein bears the intrinsic limitation of the lack of a proper intracellular environment and the lack of pathological remodeling. The results obtained from the application of the next generation sequencing technique to patients suffering from cardiac diseases have identified several loci, mostly in non-coding DNA regions, which still await functional analysis. The isolation and culture of human embryonic stem cells has initially provided a constant source of cells from which cardiomyocytes(CMs) can be obtained by differentiation. Furthermore, the possibility to reprogram cellular fate to a pluripotent state, has opened this process to the study of genetic diseases. Thus induced pluripotent stem cells(i PSCs) represent a completely new cellular model that overcomes the limitations of heterologous studies. Importantly, due to the possibility to keep spontaneously beating CMs in culture for several months, during which they show a certain degree of maturation/aging, this approach will also provide a system in which to address the effect of long-term expression of the mutated proteins or any other DNA mutation, in terms of electrophysiological remodeling. Moreover, since i PSC preserve the entire patients’ genetic context, the system will help the physicians in identifying the most appropriate pharmacological intervention to correct the functional alteration. This article summarizes the current knowledge of cardiac genetic diseases modelled with i PSC.