Biomechanics of actin filaments: a computational multi-level study

The actin microfilament (F-actin) is a structural and functional component of the cell cytoskeleton. Notwithstanding the primary role it plays for the mechanics of the cell, the mechanical behaviour of F-actin is still not totally explored. In particular, the relationship between the mechanics of F-actin and its molecular architecture is not completely understood. In this study, the mechanical properties of F-actin were related to the molecular topology of its building monomers (G-actin) by employing a computational multi-level approach. F-actins with lengths up to 500 nm were modelled and characterized, using a combination of equilibrium molecular dynamics (MD) simulations and normal mode analysis (NMA). MD simulations were performed to analyze the molecular rearrangements of G-actin in physiological conditions; NMA was applied to compute the macroscopic properties of F-actin from its vibrational modes of motion. Results from this multi-level approach showed that bending stiffness, bending modulus and persistence length are independent from the length of F-actin. On the contrary, the orientations and motions of selected groups of residues of G-actin play a primary role in determining the filament flexibility. In conclusion, this study (i) demonstrated that a combined computational approach of MD and NMA allows to investigate the biomechanics of F-actin taking into account the molecular topology of the filament (i.e., the molecular conformations of G-actin) and (ii) that this can be done using only crystallographic G-actin, without the need of introducing experimental parameters nor of reducing the number of residues.     

Direct chromatographic enantioresolution of fully constrained β-amino acids: exploring the use of high-molecular weight chiral selectors

To the best of our knowledge enantioselective chromatographic protocols on β-amino acids with polysaccharide-based chiral stationary phases (CSPs) have not yet appeared in the literature. Therefore, the primary objective of this work was the development of chromatographic methods based on the use of an amylose derivative CSP (Lux Amylose-2), enabling the direct normal-phase (NP) enantioresolution of four fully constrained β-amino acids. Also, the results obtained with the glycopeptide-type Chirobiotic T column employed in the usual polar-ionic (PI) mode of elution are compared with those achieved with the polysaccharide-based phase. The Lux Amylose-2 column, in combination with alkyl sulfonic acid containing NP eluent systems, prevailed over the Chirobiotic T one, when used under the PI mode of elution, and hence can be considered as the elective choice for the enantioseparation of this class of rigid β-amino acids. Moreover, the extraordinarily high α (up to 4.60) and R _S (up to 10.60) values provided by the polysaccharidic polymer, especially when used with camphor sulfonic acid containing eluent systems, make it also suitable for preparative-scale enantioisolations.     

Tryptophanyl-tRNA synthetase is a major soluble protein species in bovine pancreas

Besides their central role in protein synthesis, aminoacyl-tRNA synthetases have been found or thought to be involved in other processes. We present here a study showing that tryptophanyl-tRNA synthetase has a surprising tissular distribution. Indeed, immunochemical determinations showed that in several bovine organs such as liver, kidney and heart, tryptophanyl-tRNA synthetase constitutes, as expected, about 0.02% of soluble proteins. In spleen, brain cortex, stomach, cerebellum or duodenum, this amount is about 10-times higher, and in pancreas it is 100-fold. There is no correlation between these amounts and the RNA content of the organs. Moreover, the concentration of another aminoacyl-tRNA synthetase (methionyl-tRNA synthetase) is higher in liver than in pancreas, while the amount of tRNATrp is not higher in pancreas than in liver as compared to other tRNAs. Among several interpretations, it is possible that tryptophanyl-tRNA synthetase is involved in a function other than tRNA aminoacylation. This unknown function would be specific to the differentiated organs, since fetal cerebellum and fetal pancreas contain the same amount of tryptophanyl-tRNA synthetase as adult liver.     

Asymmetric synthesis of the four diastereoisomers of a novel non-steroidal farnesoid X receptor (FXR) agonist: Role of the chirality on the biological activity

An asymmetric synthetic strategy was designed for the preparation of the four possible diastereoisomers of 3,6-dimethyl-1-(2-methylphenyl)-4-(4-phenoxyphenyl)-4,8-dihydro-1H-pyrazolo[3,4-e][1,4]thiazepin-7-one, a non-steroidal FXR agonist, we recently discovered following a virtual screening approach. The results obtained from an AlphaScreen assay clearly demonstrated that only the isomer endowed with 4R,6S absolute configuration is responsible for the biological activity. A deep investigation of the different putative binding modes adopted by these enantiomerically pure ligands using computational modeling studies confirmed the enantioselectivity of FXR towards this class of molecules.     

Grossly Defective nef Gene Sequences in a Human Immunodeficiency Virus Type 1-Seropositive Long-Term Nonprogressor

We have been investigating a long-term nonprogressor who was found to be human immunodeficiency virus type 1 (HIV-1) seropositive in 1985 and has survived with stable CD4⁺ T-cell counts (>1,000 CD4 cells/μl) without any AIDS-related illness. We have previously reported that repeated attempts to measure HIV-1 RNA in the peripheral mononuclear cells obtained from this subject have invariably failed. In the present study, we have analyzed the molecular nature of the HIV-1 quasispecies infecting this patient by PCR amplification of two proviral regions, the 5′ long terminal repeat (5′LTR)/gag leader and the nef gene, directly from fresh uncultured peripheral mononuclear cells, followed by length polymorphism analysis (with 1994, 1995, and 1996 samples) and sequencing (with a 1996 sample). Only proviral forms with nef deletions were revealed by length polymorphism analysis in samples from all three time points. Sequence analysis of the nef gene from the 1996 sample confirmed the presence of similar proviral quasispecies characterized by the presence of several deletions located in the nef-alone and the nef/U3 overlapping regions. Length polymorphism analysis of the 5′LTR/gag leader region suggested the existence of two major quasispecies populations, one characterized by the presence of forms carrying deletions in the U3 region and the other showing a completely intact, full-length 5′LTR. Evidence of the role of nef gene defects in long-term survival of HIV-1-infected patients has been provided so far in two independent investigations involving patients infected with HIV through blood transfusion. Here we show the existence of a similar condition in a subject who acquired HIV-1 seropositivity through the sexual route.     

Structural chemistry of sulfonated fluorobenzylphosphonate salts. I

A study of the salts of 4-fluoro-3-sulfobenzylphosphonic acid has resulted in the synthesis of two new compounds with unusual layered structures. The crystal structures of these salts and the parent acid have been determined by single crystal X-ray methods. Crystal data: 4-F-3-SO₃H-C₆H₃CH₂PO₃H₂·H₂O: triclinic, space group

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for 1519 observations (l>3σ(l)) and 182 variables; [Ni(NH₃)₂(H₂O)₄]₃(4-F-3-SO₃-C₆H₃-CH₂PO₃)₂·4H₂O: triclinic, space group

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for 2689 observations (l>3σ(I)) and 511 variables: Na₃(4-F-3-SO₃-C₆H₃CH₂PO₃)- 8.5H₂O: monoclinic, C2/c, Z = 8, A = 25.636(4), B = 6.218(4), C = 24.136(2) Å, β = 98.33(1)°, V = 3807(3) ų, R(F) = 0.047 for 2262 observations (I>2;3σ(I)) and 254 variables. The parent acid monohydrate crystallizes in layers with the acidic groups directed to the faces of the layer. The water molecules are in between the layers hydrogen-bonded to the sulfonate oxygen atoms. The nickel salt contains three independent cations, each of which is hexacoordinated to a mixture of water and ammonia molecules. Thus, there is no direct coordination of the nickel by either the sulfonate or phosphonate oxygen atoms. The structure has alternating layers, the first formed by the anions and one of the nickel complexes, and the second by the other two complexes and the free water molecules. The sodium salt also has the anions arranged in layers but with an interpenetrating three-dimensional network of ionic and hydrogen bonds involving the cations and water molecules. The sodium ions are coordinated to a mixture of sulfonate oxygen atoms and bridging water molecules in irregular six-fold environments. These structures are discussed in terms of the coordination behavior of the difunctional anions in the context of known monofunctional phosphonate and sulfonate compounds.