Characterization of the full‐length human Grb7 protein and a phosphorylation representative mutant

It is well known the dimerization state of receptor tyrosine kinases (RTKs), in conjunction with binding partners such as the growth factor receptor bound protein 7 (Grb7) protein, plays an important role in cell signaling regulation. Previously, we proposed, downstream of RTKs, that the phosphorylation state of Grb7SH2 domain tyrosine residues could control Grb7 dimerization, and dimerization may be an important regulatory step in Grb7 binding to RTKs. In this manner, additional dimerization‐dependent regulation could occur downstream of the membrane‐bound kinase in RTK‐mediated signaling pathways. Extrapolation to the full‐length (FL) Grb7 protein, and the ability to test this hypothesis further, has been hampered by the availability of large quantities of pure and stable FL protein. Here, we report the biophysical characterization of the FL Grb7 protein and also a mutant representing a tyrosine‐phosphorylated Grb7 protein form. Through size exclusion chromatography and analytical ultracentrifugation, we show the phosphorylated‐tyrosine‐mimic Y492E‐FL‐Grb7 protein (Y492E‐FL‐Grb7) is essentially monomeric at expected physiological concentrations. It has been shown previously the wild‐type FL Grb7(WT‐FLGrb7) protein is dimeric with a dissociation constant (Kd) of approximately 11μM. Our studies here measure a FL protein dimerization K_d of WT‐FL‐Grb7 within one order of magnitude at approximately 1μM. The approximate size and shape of the WT‐FL‐Grb7 in comparison the tyrosine‐phosphorylation mimic Y492E‐FL‐Grb7 protein was determined by dynamic light scattering methods. In vitro phosphorylation of the Grb7SH2 domain indicates only one of the available tyrosine residues is phosphorylated, suggesting the same phosphorylation pattern could be relevant in the FL protein. The biophysical characterization studies in total are interpreted with a view towards understanding the functionally active Grb7 protein conformation.     

Substituted 3-amino biaryl propionic acids as potent VLA-4 antagonists

A series of substituted N-(3,5-dichlorobenzenesulfonyl)-(L)-prolyl- and (L)-azetidyl-beta-biaryl beta-alanine derivatives was prepared as selective and potent VLA-4 antagonists. The 2,6-dioxygenated biaryl substitution pattern is important for optimizing potency. Oral bioavailability was variable and may be a result of binding to circulating plasma proteins.     

Distinct self-interaction domains promote Multi Sex Combs accumulation in and formation of the Drosophila histone locus body

Nuclear bodies (NBs) are structures that concentrate proteins, RNAs, and ribonucleoproteins that perform functions essential to gene expression. How NBs assemble is not well understood. We studied the Drosophila histone locus body (HLB), a NB that concentrates factors required for histone mRNA biosynthesis at the replication-dependent histone gene locus. We coupled biochemical analysis with confocal imaging of both fixed and live tissues to demonstrate that the Drosophila Multi Sex Combs (Mxc) protein contains multiple domains necessary for HLB assembly. An important feature of this assembly process is the self-interaction of Mxc via two conserved N-terminal domains: a LisH domain and a novel self-interaction facilitator (SIF) domain immediately downstream of the LisH domain. Molecular modeling suggests that the LisH and SIF domains directly interact, and mutation of either the LisH or the SIF domain severely impairs Mxc function in vivo, resulting in reduced histone mRNA accumulation. A region of Mxc between amino acids 721 and 1481 is also necessary for HLB assembly independent of the LisH and SIF domains. Finally, the C-terminal 195 amino acids of Mxc are required for recruiting FLASH, an essential histone mRNA-processing factor, to the HLB. We conclude that multiple domains of the Mxc protein promote HLB assembly in order to concentrate factors required for histone mRNA biosynthesis.     

Determination of Fc function with frozen red blood cells.

The Fc function of immunoglobulins is commonly determined by an assay based on monitoring immunoglobulin induced, complement mediated red cell lysis. This assay requires a continuous source of fresh red cells. We have shown that the assay can be successfully performed with frozen red cells. The possibility of access to a stored standard stock of red cells will improve the convenience of performing the assay and could contribute to improved assay reproducibility.     

Habitat use and population characteristics of potentially spawning shovelnose sturgeon Scaphirhynchus platorynchus (Rafinesque, 1820), blue sucker (Cycleptus elongatus (Lesueur, 1817), and associated species in the lower Wisconsin River, USA

The goal of this study was to compare the possible locations, timing, and characteristics of potentially spawning shovelnose sturgeon (Scaphirhynchus platorynchus), blue sucker (Cycleptus elongatus), and associated species during the spring of 2007–2015 in the 149‐km‐long lower Wisconsin River, Wisconsin, USA, a large, shallow, sand‐dominated Mississippi River tributary. A 5‐km index station of two pairs of rocky shoals surrounded by sandy areas was electrofished for shovelnose sturgeon and blue sucker in a standardized fashion a total of 40 times from late March through mid‐June, the presumed spawning period. On one date in 2008 and two dates in 2012, all rocky shoals and adjacent sandy areas in the lowermost 149 km of the river were also electrofished for both species. Shovelnose sturgeon and blue sucker appeared to spawn in the limited rocky areas of the river along with at least four other species: mooneye (Hiodon tergisus), quillback (Carpiodes cyprinus), smallmouth buffalo (Ictiobus bubalus), and shorthead redhorse (Moxostoma macrolepidotum), usually at depths of 0.8–2.0 m and surface velocities of 0.4–1.0 m/s. However, apparently spawning shovelnose sturgeon were found only on mid‐channel cobble and coarse gravel shoals within a single 7‐km segment that included the 5‐km index station, whereas apparently spawning blue suckers were encountered on these same shoals but also more widely throughout the river on eroding bluff shorelines of bedrock and boulder and on artificial boulder wing dams and shoreline rip‐rap. Both species showed evidence of homing to the same mid‐channel shoal complexes across years. Blue sucker tended to concentrate on the shoals earlier in the spring than shovelnose sturgeon, usually from late April through mid‐May at water temperatures of 8.0–15.5°C along with quillback and shorthead redhorse. In comparison, shovelnose sturgeon usually concentrated on the shoals from mid‐May through early June at 13.5–21.8°C along with mooneye and smallmouth buffalo. Based on recaptures of tagged fish, at least some shovelnose sturgeon and blue sucker returned to the shoals at one‐year intervals, although there was evidence that female blue sucker may have been more likely to return at two‐year intervals. Most shovelnose sturgeon could not be reliably sexed based on external characteristics. Spawning shovelnose sturgeon ranged from 487 to 788 mm fork length, 500–2400 g weight, and 5–20 years of age, whereas spawning blue sucker ranged from 495 to 822 mm total length, 900–5100 g weight, and 5–34 years of age, although age estimates were uncertain. Females were significantly larger than males for both species although there was overlap. Growth in length was negligible for tagged and recaptured presumably spawning shovelnose sturgeon and low (3.5 mm/y) for blue sucker, suggesting that nearly all growth may have occurred prior to maturity and that fish may have matured at a wide range of sizes.     

The contribution of genetic variation and infection to the pathogenesis of ANCA-associated systemic vasculitis

The aetiology of anti-neutrophil cytoplasmic antibody (ANCA)-associated systemic vasculitis has not been well defined. Here we review two factors which may play a role in the pathogenesis of the disease: genetics and infection. In particular, we discuss the role of autoantibodies to LAMP-2, which may arise following infection with Gram-negative bacteria, and may contribute to the development of ANCA-associated systemic vasculitis in genetically susceptible individuals.     

Delayed transition to new cell fates during cellular reprogramming

In many embryos specification toward one cell fate can be diverted to a different cell fate through a reprogramming process. Understanding how that process works will reveal insights into the developmental regulatory logic that emerged from evolution. In the sea urchin embryo, cells at gastrulation were found to reprogram and replace missing cell types after surgical dissections of the embryo. Non-skeletogenic mesoderm (NSM) cells reprogrammed to replace missing skeletogenic mesoderm cells and animal caps reprogrammed to replace all endomesoderm. In both cases evidence of reprogramming onset was first observed at the early gastrula stage, even if the cells to be replaced were removed earlier in development. Once started however, the reprogramming occurred with compressed gene expression dynamics. The NSM did not require early contact with the skeletogenic cells to reprogram, but the animal cap cells gained the ability to reprogram early in gastrulation only after extended contact with the vegetal halves prior to that time. If the entire vegetal half was removed at early gastrula, the animal caps reprogrammed and replaced the vegetal half endomesoderm. If the animal caps carried morpholinos to either hox11/13b or foxA (endomesoderm specification genes), the isolated animal caps failed to reprogram. Together these data reveal that the emergence of a reprogramming capability occurs at early gastrulation in the sea urchin embryo and requires activation of early specification components of the target tissues.     

Endogenous fluorescence signatures in living pluripotent stem cells change with loss of potency

The therapeutic potential of stem cells is limited by the non-uniformity of their phenotypic state. Thus it would be advantageous to noninvasively monitor stem cell status. Driven by this challenge, we employed multidimensional multiphoton microscopy to quantify changes in endogenous fluorescence occurring with pluripotent stem cell differentiation. We found that global and cellular-scale fluorescence lifetime of human embryonic stem cells (hESC) and murine embryonic stem cells (mESC) consistently decreased with differentiation. Less consistent were trends in endogenous fluorescence intensity with differentiation, suggesting intensity is more readily impacted by nuances of species and scale of analysis. What emerges is a practical and accessible approach to evaluate, and ultimately enrich, living stem cell populations based on changes in metabolism that could be exploited for both research and clinical applications.     

Innervation of the synovial membrane of the cats joint capsule

Summary Results of investigations on the occurrence of nerve fibres and endings in the synovial membrane of the knee and elbow joint in the cat are reported. The stratum synoviale contains only autonomic fibres, running in the adventitia of arteries.Free nerve endings are lacking in the stratum synoviale. Simple Pacinian corpuscles with an inner core are occasionally observed in the border zone between the stratum synoviale and fibrosum. The ultrastructure of these endorgans resembles that of Pacinian corpuscles in the hairless and hairy skin of the cat.