Residual dipolar couplings: are multiple independent alignments always possible?

RDCs for the 14 kDa protein hen egg-white lysozyme (HEWL) have been measured in eight different alignment media. The elongated shape and strongly positively charged surface of HEWL appear to limit the protein to four main alignment orientations. Furthermore, low levels of alignment and the protein’s interaction with some alignment media increases the experimental error. Together with heterogeneity across the alignment media arising from constraints on temperature, pH and ionic strength for some alignment media, these data are suitable for structure refinement, but not the extraction of dynamic parameters. For an analysis of protein dynamics the data must be obtained with very low errors in at least three or five independent alignment media (depending on the method used) and so far, such data have only been reported for three small 6–8 kDa proteins with identical folds: ubiquitin, GB1 and GB3. Our results suggest that HEWL is likely to be representative of many other medium to large sized proteins commonly studied by solution NMR. Comparisons with over 60 high-resolution crystal structures of HEWL reveal that the highest resolution structures are not necessarily always the best models for the protein structure in solution.     

Phylogenetic relationships and character evolution in Primula L.: The usefulness of ITS sequence data

ABSTRACT

The main goals of this research were to reconstruct the infrageneric phylogeny of the genus Primula based on both nuclear and chloroplast DNA sequences, and to use the resulting phylogenies to elucidate the evolution of breeding systems, morphological characters, chromosome number, and biogeographic distribution in the genus. In this paper, the results of a pilot study based on the nuclear ribosomal Internal Transcribed Spacer (ITS) region are described. ITS sequences from 21 taxa produced a number of variable characters sufficient to resolve relationships among sections. The resulting phylogeny confirmed the monophyly of sections Auricula and Aleuritia. Sections Armerina, Proliferae, Crystallophlomis, Parryi, and Auricula, with a base chromosome number of x = 11, and sect. Aleuritia, with a base chromosome number of x = 9, formed two well supported clades. The ITS topology also suggested that leaves with revolute vernation, previously believed to be a derived state, might represent the ancestral condition in Primula, with later reversals to the involute condition. Finally, this initial ITS tree provides preliminary support to the proposed role of the widespread, diploid and heterostylous P. mistassinica as having given origin to the polyploid, homostylous P. incana and P. laurentiana.     

Acylguanidine inhibitors of beta-secretase: optimization of the pyrrole ring substituents extending into the S1' substrate binding pocket

The proteolytic enzyme beta-secretase (BACE-1) produces amyloid beta (Abeta) peptide, the primary constituent of neurofibrillary plaques, implicated in Alzheimer's disease, by cleavage of the amyloid precursor protein. A small molecule inhibitor of BACE-1, (diaminomethylene)-2,5-diphenyl-1H-pyrrole-1-acetamide (1, BACE-1 IC(50)=3.7 microM), was recently described, representing a new small molecule lead. Initial SAR investigation demonstrated the potential of accessing the nearby S(3) and S(1)(') substrate binding pockets of the BACE-1 enzyme by building substituents off one of the phenyl substituents and guanidinyl functional group. We report here the optimization of guanidinyl functional group substituents on 1, leading to potent submicromolar BACE-1 inhibitors.     

Novel 1-aminoethyl-3-arylsulfonyl-1H-pyrrolo[2,3-b]pyridines are potent 5-HT6 agonists

A series of 1-aminoethyl-3-arylsulfonyl-1H-pyrrolo[2,3-b]pyridines 10a–z was prepared as novel 5-HT₆ ligands. The best compounds were high affinity, full agonists at 5-HT₆ receptors. Several agonists demonstrated good selectivity over other serotonergic and dopaminergic receptors. Acute administration of selective agonist 10e significantly increased extracellular GABA concentrations in rat frontal cortex. This compound also reduced adjunctive drinking behavior in the rat schedule-induced polydipsia assay, possibly predictive of efficacy in obsessive compulsive disorder and other anxiety related disorders.     

Human Plasmacytoid Dendritic Cells Sense Lymphocytic Choriomeningitis Virus-Infected Cells In Vitro

We previously reported that exosomal transfer of hepatitis C virus (HCV) positive-strand RNA from human Huh-7 hepatoma cells to human plasmacytoid dendritic cells (pDCs) triggers pDC alpha/beta interferon (IFN-α/β) production in a Toll-like receptor 7 (TLR7)-dependent, virus-independent manner. Here we show that human pDCs are also activated by a TLR7-dependent, virus-independent, exosomal RNA transfer mechanism by human and mouse hepatoma and nonhepatoma cells that replicate the negative-strand lymphocytic choriomeningitis virus (LCMV).     

Predicting the permeability of trabecular bone by micro-computed tomography and finite element modeling

The intrinsic permeability of bone plays an important role in the transport of nutrients and minerals within the tissue, and affects the mechanical stimuli that are related to the fate of the stem cells. The objective of this study was to establish a method to assess trabecular bone permeability using experimental and finite element (FE) modeling approaches based on micro computed tomography (µCT) images. Human cadaveric tibia cube specimens (N=23) were scanned with µCT. The permeability was measured experimentally using a custom-developed constant-head permeameter, and computationally by a poroelastic formulation to simulate the fluid flow within the discretized bone matrix and pore phase. The average of the experimentally measured permeability was 4.84×10⁻¹⁰ m² with a standard deviation of 3.70×10⁻¹⁰ m². A regression model of the µCT determined that the maximum bone area to total area ratio (maxBA/TA) for all slices that are perpendicular to the direction of fluid flow explained 84% of the variability of the natural logarithm of the experimentally measured permeability. The 2D measure of maxBA/TA performed better than 3D measures in general, although some parameters were reasonably well associated with permeability such as bone volume ratio (BV/TV, r=−0.71), the bone surface/bone volume (BS/BV, r=0.73), and the trabecular thickness (TbTh, r=−0.71). The correlation between the permeability predicted with FE models and experimentally measured permeability was reasonable (r=0.69), but the FE approach did not accurately represent the wide variability of permeability measured experimentally. The results of this study suggest that the changes in the trabecular bone microarchitecture have an exponential relationship with permeability, and the use of µCT-based 2D measurement of maxBA/TA performs well at predicting permeability, thus providing a convenient approach to measure this important aspect affecting biomechanical functions in the tissue.     

Selman A. Waksman,Winner of the 1952 Nobel Prize for Physiology or Medicine

The history of the discovery and development of streptomycin is reviewed here from the personal standpoint of a member of Dr. Selman Waksman''s antibiotic screening research team. The team approach of eight individuals illustrates how the gradual enhancement of the screening methodology was developed. I illustrate three study periods with key aspects in the development of streptomycin which led to a Nobel Prize being granted to Professor Waksman. One item not previously emphasized is the employment of a submerged culture technique for large-scale production of streptomycin, thus enabling rapid animal testing and human clinical trials with Mycobacterium tuberculosis. Another is that purified streptomycin was shown by Dr. Waksman to be distinctly different from the substances called natural products, which are no longer patentable in the United States; therefore, streptomycin was found to be patentable. A third item not previously emphasized is his emphasis on the screening of actinomycetes, including the newly named Streptomyces genus. All of these factors contributed to the success of streptomycin in the treatment of tuberculosis. In combination, their successes led to Dr. Waksman''s department becoming a new pharmacological research area, specializing in drug discovery. These unique accomplishments all burnish the prior rationales used by the Karolinska Institute in granting Dr. Waksman alone the 1952 Nobel Prize for Physiology or Medicine.