Molecular simulation of the binding of nerve growth factor peptide mimics to the receptor tyrosine kinase A

Nerve growth factor (NGF) mimics play an important role for therapies that target the receptor tyrosine kinase A (trkA). The N-terminal fragment of the NGF (N-term@NGF) was previously demonstrated to be an important determinant for affinity and specificity in the binding to trkA. Here we use a variety of computational tools (contact surface analysis and free energy predictions) to identify residues playing a key role for the binding to the receptor. Molecular dynamics simulations are then used to investigate the stability of complexes between trkA and peptides mimicking N-term@NGF. Steered molecular dynamics calculations are finally performed to investigate the process of detaching the peptide from the receptor. Three disruptive events are observed, the first involving the breaking of all intermolecular interactions except two salt bridges, which break subsequently.     

Intramolecular aldol cyclization of L-lyxo-hexos-5-ulose derivatives: a new diastereoselective synthesis of D-chiro-inositol

The DBU-promoted intramolecular aldol condensation of two partially protected L-lyxo-hexos-5-ulose derivatives (8 and 9), in turn obtained starting from methyl beta-D-galactopyranoside, takes place with fairly good yield and complete diastereoselectivity to give 2L-(2,3,6/4,5)-pentahydroxycyclohexanone derivatives, 10 and 11. The stereoselective reduction of inosose 10 with sodium triacetoxyborohydride leads, after catalytic debenzylation, to D-chiro-inositol (1), while the sodium borohydride reduction furnishes, with opposite stereoselectivity, a derivative of allo-inositol.     

Insulin and insulin-like growth factor II differentially regulate endocytic sorting and stability of insulin receptor isoform A

The insulin receptor isoform A (IR-A) binds both insulin and insulin-like growth factor (IGF)-II, although the affinity for IGF-II is 3-10-fold lower than insulin depending on a cell and tissue context. Notably, in mouse embryonic fibroblasts lacking the IGF-IR and expressing solely the IR-A (R-/IR-A), IGF-II is a more potent mitogen than insulin. As receptor endocytosis and degradation provide spatial and temporal regulation of signaling events, we hypothesized that insulin and IGF-II could affect IR-A biological responses by differentially regulating IR-A trafficking. Using R-/IR-A cells, we discovered that insulin evoked significant IR-A internalization, a process modestly affected by IGF-II. However, the differential internalization was not due to IR-A ubiquitination. Notably, prolonged stimulation of R-/IR-A cells with insulin, but not with IGF-II, targeted the receptor to a degradative pathway. Similarly, the docking protein insulin receptor substrate 1 (IRS-1) was down-regulated after prolonged insulin but not IGF-II exposure. Similar results were also obtained in experiments using [NMeTyr(B26)]-insulin, an insulin analog with IR-A binding affinity similar to IGF-II. Finally, we discovered that IR-A was internalized through clathrin-dependent and -independent pathways, which differentially regulated the activation of downstream effectors. Collectively, our results suggest that a lower affinity of IGF-II for the IR-A promotes lower IR-A phosphorylation and activation of early downstream effectors vis à vis insulin but may protect IR-A and IRS-1 from down-regulation thereby evoking sustained and robust mitogenic stimuli.     

Do the phenology and functional stem attributes of woody species allow for the identification of functional groups in the semiarid region of Brazil?

The phenology of tree species in environments that are subject to strong climatic seasonality is mainly determined by water availability, which may vary as a function of wood density. The relationship among phenology, water potential, wood density and the capacity of water storage in the stem were determined for woody species of caatinga vegetation (dry forest) in the semiarid region of NE Brazil. Leaf flush and fall, flowering and fruiting events were recorded over a 31-month period, and the water potential was measured over a two-year period. These data were related to precipitation, water availability in the soil and photoperiod. Seven deciduous species exhibited low wood density (DLWD,?<0.5?g?cm^?3), high capacity of water storage in the stem (until 250?% of the dry weight) and high water potential during the year, as opposed to 15 deciduous species that showed high wood density (DHWD,?≥0.5?g?cm^?3). Leaf flush, flowering and the fruiting of DHWD species were related to precipitation, whereas these phenological events occurred at the end of the dry season and/or the beginning of the rainy season for DLWD species and were related to the photoperiod. The two evergreen species showed variations of water potential that were intermediate between those of DHWD and DLWD deciduous species, leaf flush during the dry season and flowering at the end of dry season. These results suggest the existence of three functional groups: evergreen species, DHWD deciduous species and DLWD deciduous species.     

RACK1 is a ribosome scaffold protein for β-actin mRNA/ZBP1 complex

In neurons, specific mRNAs are transported in a translationally repressed manner along dendrites or axons by transport ribonucleic-protein complexes called RNA granules. ZBP1 is one RNA binding protein present in transport RNPs, where it transports and represses the translation of cotransported mRNAs, including β-actin mRNA. The release of β-actin mRNA from ZBP1 and its subsequent translation depends on the phosphorylation of ZBP1 by Src kinase, but little is known about how this process is regulated. Here we demonstrate that the ribosomal-associated protein RACK1, another substrate of Src, binds the β-actin mRNA/ZBP1 complex on ribosomes and contributes to the release of β-actin mRNA from ZBP1 and to its translation. We identify the Src binding and phosphorylation site Y246 on RACK1 as the critical site for the binding to the β-actin mRNA/ZBP1 complex. Based on these results we propose RACK1 as a ribosomal scaffold protein for specific mRNA-RBP complexes to tightly regulate the translation of specific mRNAs.     

Analysis of endothelial protein C receptor gene and metabolic profile in Prader-Willi syndrome and obese subjects

The endothelial protein C receptor (EPCR) has a critical role in the regulation of anticoagulant and anti-inflammatory functions of activated protein C (APC). Abnormalities in EPCR might be associated with an increased risk of thrombosis. In this respect, a 23 bp insertion in the exon 3 of the EPCR gene predicts a truncated protein which cannot bind APC. High levels of C-reactive protein (CRP), a strong predictor of cardiovascular events, are found both in the obese and in subjects with Prader-Willi syndrome (PWS). Several cardiovascular risk factors are already present in prepubertal PWS children, but it is uncertain which mechanism contributes to the increased risk of cardiovascular disease in PWS. We analyzed the distribution of 23 bp insertion in the EPCR gene in 81 overweight and obese PWS subjects, 52 adults and 29 children, and in 58 overweight and obese children and adolescents (controls). We found that 1/58 (1.7%) of the controls was heterozygous for the 23 bp insertion, while this mutation was never found in PWS subjects. Furthermore, we evaluated CRP levels, glucose, insulin, and lipid profile, and we found higher CRP values in PWS adults with respect to children with PWS and controls, and a better insulin sensitivity in all PWS subjects than in the controls. This study suggests that in PWS subjects there is no predisposition to develop thrombotic events in association with EPCR gene alteration and demonstrates substantial differences regarding metabolic and inflammatory profile between PWS and non-PWS obese children, with further impairment in adults with PWS.     

Golgi complex fragmentation in G2/M transition: An organelle-based cell-cycle checkpoint

In mammalian cells, the Golgi complex is organized into a continuous membranous system known as the Golgi ribbon, which is formed by individual Golgi stacks that are laterally connected by tubular bridges. During mitosis, the Golgi ribbon undergoes extensive fragmentation through a multistage process that is required for its correct partitioning into the daughter cells. Importantly, inhibition of this Golgi disassembly results in cell-cycle arrest at the G2 stage, suggesting that accurate inheritance of the Golgi complex is monitored by a "Golgi mitotic checkpoint." Here, we discuss the mechanisms and regulation of the Golgi ribbon breakdown and briefly comment on how Golgi partitioning may inhibit G2/M transition.     

First report of Melittobia australica Girault in Europe and new record of M. acasta (Walker) for Italy

Melittobia acasta and Melittobia australica are newly recorded from Sicily, Italy, and the second species is reported in Europe for the first time. A short historical background about Melittobia parasitoid wasps, their hosts, and distribution, with emphasis in those two species is presented together with illustrations to facilitate their identification. Brief discussion about the presence and possible distribution of the species in Sicily is also included.     

Biophysical Characterization of Met-G-CSF: Effects of Different Site-Specific Mono-Pegylations on Protein Stability and Aggregation

The limited stability of proteins in vitro and in vivo reduces their conversion into effective biopharmaceuticals. To overcome this problem several strategies can be exploited, as the conjugation of the protein of interest with polyethylene glycol, in most cases, improves its stability and pharmacokinetics. In this work, we report a biophysical characterization of the non-pegylated and of two different site-specific mono-pegylated forms of recombinant human methionyl-granulocyte colony stimulating factor (Met-G-CSF), a protein used in chemotherapy and bone marrow transplantation. In particular, we found that the two mono-pegylations of Met-G-CSF at the N-terminal methionine and at glutamine 135 increase the protein thermal stability, reduce the aggregation propensity, preventing also protein precipitation, as revealed by circular dichroism (CD), Fourier transform infrared (FTIR), intrinsic fluorescence spectroscopies and dynamic light scattering (DLS). Interestingly, the two pegylation strategies were found to drastically reduce the polydispersity of Met-G-CSF, when incubated under conditions favouring protein aggregation, as indicated by DLS measurements. Our in vitro results are in agreement with preclinical studies, underlining that preliminary biophysical analyses, performed in the early stages of the development of new biopharmaceutical variants, might offer a useful tool for the identification of protein variants with improved therapeutic values.