Extracellular Matrix Molecular Remodeling in Human Liver Fibrosis Evolution

Chronic liver damage leads to pathological accumulation of ECM proteins (liver fibrosis). Comprehensive characterization of the human ECM molecular composition is essential for gaining insights into the mechanisms of liver disease. To date, studies of ECM remodeling in human liver diseases have been hampered by the unavailability of purified ECM. Here, we developed a decellularization method to purify ECM scaffolds from human liver tissues. Histological and electron microscopy analyses demonstrated that the ECM scaffolds, devoid of plasma and cellular components, preserved the three-dimensional ECM structure and zonal distribution of ECM components. This method has been then applied on 57 liver biopsies of HCV-infected patients at different stages of liver fibrosis according to METAVIR classification. Label-free nLC-MS/MS proteomics and computation biology were performed to analyze the ECM molecular composition in liver fibrosis progression, thus unveiling protein expression signatures specific for the HCV-related liver fibrotic stages. In particular, the ECM molecular composition of liver fibrosis was found to involve dynamic changes in matrix stiffness, flexibility and density related to the dysregulation of predominant collagen, elastic fibers and minor components with both structural and signaling properties. This study contributes to the understanding of the molecular bases underlying ECM remodeling in liver fibrosis and suggests new molecular targets for fibrolytic strategies.     

Towards boron neutron capture therapy: The formulation and preliminary in vitro evaluation of liposomal vehicles for the therapeutic delivery of the dequalinium salt of bis-nido-carborane

Liposomes of phosphatidylcholine or of dimyristoylphosphatidylcholine that incorporate bis-nido-carborane dequalinium salt are stable in physiologically relevant media and have in vitro toxicity profiles that appear to be compatible with potential therapeutic applications. These features render the structures suitable candidate boron-delivery vehicles for evaluation in the boron neutron capture therapy of cancer.     

The essential cytosolic iron-sulfur protein Nbp35 acts without Cfd1 partner in the green lineage

In photosynthetic eukaryotes assembly components of iron-sulfur (Fe-S) cofactors have been studied in plastids and mitochondria, but how cytosolic and nuclear Fe-S cluster proteins are assembled is not known. We have characterized a plant P loop NTPase with sequence similarity to Nbp35 of yeast and mammals, a protein of the cytosolic Cfd1-Nbp35 complex mediating Fe-S cluster assembly. Genome analysis revealed that NBP35 is conserved in the green lineage but that CFD1 is absent. Moreover, plant and algal NBP35 proteins lack the characteristic CXXC motif in the C terminus, thought to be required for Fe-S cluster binding. Nevertheless, chemical reconstitution and spectroscopy showed that Arabidopsis (At) NBP35 bound a [4Fe-4S] cluster in the C terminus as well as a stable [4Fe-4S] cluster in the N terminus. Holo-AtNBP35 was able to transfer an Fe-S cluster to an apoprotein in vitro. When expressed in yeast, AtNBP35 bound 55Fe dependent on the cysteine desulfurase Nfs1 and was able to partially rescue the growth of a cfd1 mutant but not of an nbp35 mutant. The AtNBP35 gene is constitutively expressed in planta, and its disruption was associated with an arrest of embryo development. These results show that despite considerable divergence from the yeast Cfd1-Nbp35 Fe-S scaffold complex, AtNBP35 has retained similar Fe-S cluster binding and transfer properties and performs an essential function.     

Morphology and biogeography of Apiaceae subfamily Saniculoideae as inferred by phylogenetic analysis of molecular data

The phylogenetic placements of several African endemic genera at the base of Apiaceae subfamilies Saniculoideae and Apioideae have revolutionized ideas of relationships that affect hypotheses of character evolution and biogeography. Using an explicit phylogeny of subfamily Saniculoideae, we reconstructed the evolutionary history of phenotypic characters traditionally important in classification, identified those characters most useful in supporting relationships, and inferred historical biogeography. The 23 characters examined include those of life history, vegetative morphology, inflorescences, and fruit morphology and anatomy. These characters were optimized over trees derived from maximum parsimony analysis of chloroplast DNA trnQ-trnK sequences from 94 accessions of Apiaceae. The results revealed that many of these characters have undergone considerable modification and that traditional assumptions regarding character-state polarity are often incorrect. Infrasubfamilial relationships inferred by molecular data are supported by one to five morphological characters. However, none of these morphological characters support the monophyly of subfamilies Saniculoideae or Apioideae, the clade of Petagnaea, Eryngium and Sanicula, or the sister-group relationship between Eryngium and Sanicula . Southern African origins of Saniculoideae and of its tribes Steganotaenieae and Saniculeae are supported based on dispersal-vicariance analysis.     

Cleavage of the iron-methionine bond in c-type cytochromes: Crystal structure of oxidized and reduced cytochrome c2 from Rhodopseudomonas palustris and its ammonia complex

The three-dimensional structures of the native cytochrome c(2) from Rhodopseudomonas palustris and of its ammonia complex have been obtained at pH 4.4 and pH 8.5, respectively. The structure of the native form has been refined in the oxidized state at 1.70 A and in the reduced state at 1.95 A resolution. These are the first high-resolution crystal structures in both oxidation states of a cytochrome c(2) with relatively high redox potential (+350 mV). The differences between the two oxidation states of the native form, including the position of internal water molecules, are small. The unusual six-residue insertion Gly82-Ala87, which precedes the heme binding Met93, forms an isolated 3(10)-helix secondary structural element not previously observed in other c-type cytochromes. Furthermore, this cytochrome shows an external methionine residue involved in a strained folding near the exposed edge of the heme. The structural comparison of the present cytochrome c(2) with other c-type cytochromes has revealed that the presence of such a residue, with torsion angles phi and psi of approximately -140 and -130 degrees, respectively, is a typical feature of this family of proteins. The refined crystal structure of the ammonia complex, obtained at 1.15 A resolution, shows that the sulphur atom of the Met93 axial ligand does not coordinate the heme iron atom, but is replaced by an exogenous ammonia molecule. This is the only example so far reported of an X-ray structure with the heme iron coordinated by an ammonia molecule. The detachment of Met93 is accompanied by a very localized change in backbone conformation, involving mainly the residues Lys92, Met93, and Thr94. Previous studies under typical denaturing conditions, including high-pH values and the presence of exogenous ligands, have shown that the detachment of the Met axial ligand is a basic step in the folding/unfolding process of c-type cytochromes. The ammonia adduct represents a structural model for this important step of the unfolding pathway. Factors proposed to be important for the methionine dissociation are the strength of the H-bond between the Met93 and Tyr66 residues that stabilizes the native form, and the presence in this bacterial cytochrome c(2) of the rare six-residue insertion in the helix 3(10) conformation that increases Met loop flexibility.     

Effect of joint rotation correction when measuring elongation of the gastrocnemius medialis tendon and aponeurosis

It is well known that during maximal plantar flexion contractions the ankle joint rotation overestimates the actual elongation of the tendon and aponeurosis. The aim of this study was to examine the influence of the curve length changes of the Achilles tendon on the joint rotation corrected elongation and strain of the gastrocnemius medialis (GM) tendon and aponeurosis. Nine subjects (age: 29.4 ± 5.7 years, body mass: 78.8 ± 6.8 kg, body height: 178 ± 4 cm) participated in the study. The subjects performed maximal voluntary isometric plantarflexion contractions in the prone position on a Biodex-dynamometer. Ultrasonography (Aloka SSD 4000) was used to visualize the muscle belly of the GM muscle-tendon unit. To calculate the curve length changes of the Achilles tendon its surface contour was reconstructed using a series of small reflective skin markers having a diameter of 2.5 mm. The elongation of the GM tendon and aponeurosis was calculated (a) as the difference of the measured and the passive (due to joint rotation) displacement of the tendon and aponeurosis and (b) as the difference of the measured displacement and the length changes of the reconstructed Achilles tendon surface contour. The absolute difference between the elongation obtained by both methods were 1.2 ± 0.4 mm. These differences were due to the higher changes in length obtained by the reconstruction of the tendon curved surface contour as compared to the changes observed in the passive displacement of the digitised point at the aponeurosis. Without correcting for angle joint rotation, the measured elongation clearly overestimates the actual elongation of the GM tendon and aponeurosis. After the passive displacement correction the calculated elongation still overestimates the actual elongation of the GM tendon and aponeurosis. However, this overestimation has a negligible effect on the examined in vivo strain (0.3%) of the tendon and aponeurosis.     

Drosophila double-parked is sufficient to induce re-replication during development and is regulated by cyclin E/CDK2

It is important that chromosomes are duplicated only once per cell cycle. Over-replication is prevented by multiple mechanisms that block the reformation of a pre-replicative complex (pre-RC) onto origins in S and G2 phase. We have investigated the developmental regulation of Double-parked (Dup) protein, the Drosophila ortholog of Cdt1, a conserved and essential pre-RC component found in human and other organisms. We find that phosphorylation and degradation of Dup protein at G1/S requires cyclin E/CDK2. The N terminus of Dup, which contains ten potential CDK phosphorylation sites, is necessary and sufficient for Dup degradation during S phase of mitotic cycles and endocycles. Mutation of these ten phosphorylation sites, however, only partially stabilizes the protein, suggesting that multiple mechanisms ensure Dup degradation. This regulation is important because increased Dup protein is sufficient to induce profound rereplication and death of developing cells. Mis-expression has different effects on genomic replication than on developmental amplification from chorion origins. The C terminus alone has no effect on genomic replication, but it is better than full-length protein at stimulating amplification. Mutation of the Dup CDK sites increases genomic re-replication, but is dominant negative for amplification. These two results suggest that phosphorylation regulates Dup activity differently during these developmentally specific types of DNA replication. Moreover, the ability of the CDK site mutant to rapidly inhibit BrdU incorporation suggests that Dup is required for fork elongation during amplification. In the context of findings from human and other cells, our results indicate that stringent regulation of Dup protein is critical to protect genome integrity.     

Constitutively active PTH/PTHrP receptor in odontoblasts alters odontoblast and ameloblast function and maturation

Parathyroid hormone (PTH)-related protein (PTH-rP) is an important autocrine/paracrine attenuator of programmed cell differentiation whose expression is restricted to the epithelial layer in tooth development. The PTH/PTHrP receptor (PPR) mRNA in contrast is detected in the dental papilla, suggesting that PTHrP and the PPR may modulate epithelial-mesenchymal interactions. To explore the possible interactions, we studied the previously described transgenic mice in which a constitutively active PPR is targeted to osteoblastic cells. These transgenic mice have a vivid postnatal bone and tooth phenotype, with normal tooth eruption but abnormal, widened crowns. Transgene mRNA expression was first detected at birth in the dental papilla and, at 1 week postnatally, in odontoblasts. There was no transgene expression in ameloblasts or in other epithelial structures. Prenatally, transgenic molars and incisors revealed no remarkable change. By the age of 1 week, the dental papilla was widened, with disorganization of the odontoblastic layer and decreased dentin matrix. In addition, the number of cusps was abnormally increased, the ameloblastic layer disorganized, and enamel matrix decreased. Odontoblastic and, surprisingly, ameloblastic cytodifferentiation was impaired, as shown by in situ hybridization and electron microscopy. Interestingly, ameloblastic expression of Sonic Hedgehog, a major determinant of ameloblastic cytodifferentiation, was dramatically altered in the transgenic molars. These data suggest that odontoblastic activation of the PPR may play an important role in terminal odontoblastic and, indirectly, ameloblastic cytodifferentiation, and describe a useful model to study how this novel action of the PPR may modulate mesenchymal/epithelial interactions at later stages of tooth morphogenesis and development.