Squaric acid derivatives as VLA-4 integrin antagonists

SAR studies aimed at improving the rate of clearance by the incorporation of a 3,4-diamino-3-cyclobutene-1,2-dione group as an amino acid isostere in a series of VLA-4 integrin antagonists are described.     

In vitro articular cartilage growth with sequential application of IGF-1 and TGF-β1 enhances volumetric growth and maintains compressive properties

In vitro cultures with insulin-like growth factor-1 (IGF-1) and transforming growth factor-β1 (TGF-β1) have previously been shown to differentially modulate the growth of immature bovine articular cartilage. IGF-1 stimulates expansive growth yet decreases compressive moduli and increases compressive Poisson's ratios, whereas TGF-β1 maintains tissue size, increases compressive moduli, and decreases compressive Poisson's ratios. The current study's hypothesis was that sequential application of IGF-1 and TGF-β1 during in vitro culture produces geometric and compressive mechanical properties that lie between extreme values produced when using either growth factor alone. Immature bovine articular cartilage specimens were harvested and either untreated (D0, i.e., day zero) or cultured in vitro for either 6 days with IGF-1 (D6 IGF), 12 days with IGF-1 (D12 IGF), or 6 days with IGF-1 followed by 6 days with TGF-β1 (D12 SEQ, i.e., sequential). Following treatment, all specimens were tested for geometric, biochemical, and compressive mechanical properties. Relative to D0, D12 SEQ treatment enhanced volumetric growth, but to a lower value than that for D12 IGF. Furthermore, D12 SEQ treatment maintained compressive moduli and Poisson's ratios at values higher and lower, respectively, than those for D12 IGF. Considering the previously described effects of 12 days of treatment with TGF-β1 alone, D12 SEQ induced both growth and mechanical property changes between those produced with either IGF-1 or TGF-β1 alone. The results suggest that it may be possible to vary the durations of select growth factors, including IGF-1 and TGF-β1, to more precisely modulate the geometric, biochemical, and mechanical properties of immature cartilage graft tissue in clinical repair strategies.     

Acquisition of Hox codes during gastrulation and axial elongation in the mouse embryo

Early sequential expression of mouse Hox genes is essential for their later function. Analysis of the relationship between early Hox gene expression and the laying down of anterior to posterior structures during and after gastrulation is therefore crucial for understanding the ontogenesis of Hox-mediated axial patterning. Using explants from gastrulation stage embryos, we show that the ability to express 3' and 5' Hox genes develops sequentially in the primitive streak region, from posterior to anterior as the streak extends, about 12 hours earlier than overt Hox expression. The ability to express autonomously the earliest Hox gene, Hoxb1, is present in the posterior streak region at the onset of gastrulation, but not in the anterior region at this stage. However, the posterior region can induce Hoxb1 expression in these anterior region cells. We conclude that tissues are primed to express Hox genes early in gastrulation, concomitant with primitive streak formation and extension, and that Hox gene inducibility is transferred by cell to cell signalling. Axial structures that will later express Hox genes are generated in the node region in the period that Hox expression domains arrive there and continue to spread rostrally. However, lineage analysis showed that definitive Hox codes are not fixed at the node, but must be acquired later and anterior to the node in the neurectoderm, and independently in the mesoderm. We conclude that the rostral progression of Hox gene expression must be modulated by gene regulatory influences from early on in the posterior streak, until the time cells have acquired their stable positions along the axis well anterior to the node.     

The R403Q myosin mutation implicated in familial hypertrophic cardiomyopathy causes disorder at the actomyosin interface

Background

Mutations in virtually all of the proteins comprising the cardiac muscle sarcomere have been implicated in causing Familial Hypertrophic Cardiomyopathy (FHC). Mutations in the β-myosin heavy chain (MHC) remain among the most common causes of FHC, with the widely studied R403Q mutation resulting in an especially severe clinical prognosis. In vitro functional studies of cardiac myosin containing the R403Q mutation have revealed significant changes in enzymatic and mechanical properties compared to wild-type myosin. It has been proposed that these molecular changes must trigger events that ultimately lead to the clinical phenotype.

Principal Findings

Here we examine the structural consequences of the R403Q mutation in a recombinant smooth muscle myosin subfragment (S1), whose kinetic features have much in common with slow β-MHC. We obtained three-dimensional reconstructions of wild-type and R403Q smooth muscle S1 bound to actin filaments in the presence (ADP) and absence (apo) of nucleotide by electron cryomicroscopy and image analysis. We observed that the mutant S1 was attached to actin at highly variable angles compared to wild-type reconstructions, suggesting a severe disruption of the actin-myosin interaction at the interface.

Significance

These results provide structural evidence that disarray at the molecular level may be linked to the histopathological myocyte disarray characteristic of the diseased state.     

Use of equine chorionic gonadotrophin in female mink

This study was comprised of three trials to determine the effects of equine chrionic gonadotrophin (eCG) on induction of sexual receptivity in female mink that had failed to mate by late in the breeding season. In the first trial one ovary was removed from unmated mink, which were then injected with 100 IU eCG. This treatment induced ovarian activity, including ovulation in the remaining ovary. In the second experiment, mink that had not been observed to mate were treated with 100 IU eCG or saline, resulting in mating of 10 11 of the eCG-treated animals, compared to 5 11 controls. Litter sizes were larger in mink in the control group, suggesting that eCG interfered with some phase of the reproductive process. In the third trial, 226 mink that had failed to mate until late in the breeding season were treated with 100 IU eCG. Of the 191 that subsequently mated, 99 produced litters, but litter sizes were reduced slightly from those observed in the remainder of the herd that bred without hormone treatment prior to March 20. Neonatal kit loss per female whelping was greater in mink treated with eCG. It is concluded that eCG treatment will induce mating in mink that refuse to mate, but this treatment results in reduced whelping success and greater neonatal kit loss. Its utility may be restricted to salvage situations where large numbers of mink fail to mate.     

The structure of the C-terminal actin-binding domain of talin

Talin is a large dimeric protein that couples integrins to cytoskeletal actin. Here, we report the structure of the C-terminal actin-binding domain of talin, the core of which is a five-helix bundle linked to a C-terminal helix responsible for dimerisation. The NMR structure of the bundle reveals a conserved surface-exposed hydrophobic patch surrounded by positively charged groups. We have mapped the actin-binding site to this surface and shown that helix 1 on the opposite side of the bundle negatively regulates actin binding. The crystal structure of the dimerisation helix reveals an antiparallel coiled-coil with conserved residues clustered on the solvent-exposed face. Mutagenesis shows that dimerisation is essential for filamentous actin (F-actin) binding and indicates that the dimerisation helix itself contributes to binding. We have used these structures together with small angle X-ray scattering to derive a model of the entire domain. Electron microscopy provides direct evidence for binding of the dimer to F-actin and indicates that it binds to three monomers along the long-pitch helix of the actin filament.