Endoderm-derived Fgf3 is necessary and sufficient for inducing neurogenesis in the epibranchial placodes in zebrafish

In vertebrates, epibranchial placodes are transient ectodermal thickenings that contribute sensory neurons to the epibranchial ganglia. These ganglia innervate internal organs and transmit information on heart rate, blood pressure and visceral distension from the periphery to the central nervous system. Despite their importance, the molecular mechanisms that govern the induction and neurogenesis of the epibranchial placodes are only now being elucidated. In this study, we demonstrate that endoderm is required for neurogenesis of the zebrafish epibranchial placodes. Mosaic analyses confirm that endoderm is the source of the neurogenic signal. Using a morpholino knockdown approach, we find that fgf3 is required for the majority of placode cells to undergo neurogenesis. Tissue transplants demonstrate that fgf3 activity is specifically required in the endodermal pouches. Furthermore, ectopic fgf3 expression is sufficient for inducing phox2a-positive neurons in wild-type and endoderm-deficient embryos. Surprisingly, ectodermal foxi1 expression, a marker for the epibranchial placode precursors, is present in both endoderm-deficient embryos and fgf3 morphants, indicating that neither endoderm nor Fgf3 is required for initial placode induction. Based on these findings, we propose a model for epibranchial placode development in which Fgf3 is a major endodermal determinant required for epibranchial placode neurogenesis.     

(Ca2+ + Mg2+)-ATPase in enriched sarcolemma from dog heart

An enriched fraction of plasma membranes was prepared from canine ventricle by a process which involved thorough disruption of membranes by vigorous homogenization in dilute suspension, sedimentation of contractile proteins and mitochondria at 3000 X g followed by sedimentation of a microsomal fraction at 200 000 X g. The microsomal suspension was then fractionated on a discontinuous sucrose gradient. Particles migrating in the density range 1.0591--1.1083 were characterized by (Na+ + K+)-ATPase activity and [3H]ouabain binding as being enriched in sarcolemma and were comprised of nonaggregated vesicles of diameter approx. 0.1 micron. These fractions contained (Ca2+ + Mg2+)-ATPase which appreared endogenous to the sarcolemma. The enzyme was solubilized using Triton X-100 and 1 M KCl and partially purified. Optimal Ca2+ concentration for enzyme activity was 5--10 microM. Both Na+ and K+ stimulated enzyme activity. It is suggested that the enzyme may be involved in the outward pumping of Ca2+ from the cardiac cell.     

Endoplasmic reticulum calcium tunnels integrate signalling in polarised cells

One of the most crucial aspects of Ca(2+) signalling is the ability to generate highly localised transient elevations of the cytosolic Ca(2+) concentration at specific strategically important target sites. Inevitably this necessitates a relatively high Ca(2+) buffering power of the cytoplasm, which in turn makes movement of Ca(2+) from one part of a cell to another difficult. Nature has evolved an elegant solution to this problem by creating operational Ca(2+) tunnels through the endoplasmic reticulum. Very recently direct evidence that such tunnelling also occurs in neurons has been provided.     

A mechanical model of cell segregation driven by differential adhesion

From simulations that begin with a random mix of two cell types, we monitor progress towards segregation driven by contact-mediated linkage of model cells, which is equivalent to the cell-cell adhesion of real cells. In comparison with real cell experiments, we show that this mechanical model can account for the observed extent of segregation obtained by differential adhesion in a 2D cell culture assay of cells with differentially expressed cadherin molecules. Calibration of virtual to real time allowed us to estimate a time course for these experiments that was within 50% agreement for the simulations compared to differential adhesion of cells. In contrast, simulations of differential adhesion do not account for the rate of segregation driven by interactions between EphB2 receptor and ephrinB1 expressing cells which occurs an order of magnitude faster. The latter result suggests that mechanisms additional or alternative to differential adhesion contribute to Eph-ephrin mediated cell segregation.     

Activation loop targeting strategy for design of receptor-interacting protein kinase 2 (RIPK2) inhibitors

Development of selective kinase inhibitors remains a challenge due to considerable amino acid sequence similarity among family members particularly in the ATP binding site. Targeting the activation loop might offer improved inhibitor selectivity since this region of kinases is less conserved. However, the strategy presents difficulties due to activation loop flexibility. Herein, we report the design of receptor-interacting protein kinase 2 (RIPK2) inhibitors based on pan-kinase inhibitor regorafenib that aim to engage basic activation loop residues Lys169 or Arg171. We report development of CSR35 that displayed >10-fold selective inhibition of RIPK2 versus VEGFR2, the target of regorafenib. A co-crystal structure of CSR35 with RIPK2 revealed a resolved activation loop with an ionic interaction between the carboxylic acid installed in the inhibitor and the side-chain of Lys169. Our data provides principle feasibility of developing activation loop targeting type II inhibitors as a complementary strategy for achieving improved selectivity.     

Coherent Neutron Scattering and Collective Dynamics in the Protein,GFP

Collective dynamics are considered to be one of the major properties of soft materials, including biological macromolecules. We present coherent neutron scattering studies of the low-frequency vibrations, the so-called boson peak, in fully deuterated green fluorescent protein (GFP). Our analysis revealed unexpectedly low coherence of the atomic motions in GFP. This result implies a low amount of in-phase collective motion of the secondary structural units contributing to the boson peak vibrations and fast conformational fluctuations on the picosecond timescale. These observations are in contrast to earlier studies of polymers and glass-forming systems, and suggest that random or out-of-phase motions of the β-strands contribute greater than two-thirds of the intensity to the low-frequency vibrational spectra of GFP.     

Egg production and early development of Thysanoessa inermis and Euphausia pacifica (Crustacea: Euphausiacea) in the northern Gulf of Alaska

Early life history patterns were studied in the dominant euphausiids from the northern Gulf of Alaska (GOA) in 2001-2004. Gravid females of Thysanoessa inermis were observed in April and May. Brood size varied from 10 to 1021 eggs with an average of 138 ± 19 (95% CI) eggs female^− 1. Most gravid females started to release eggs within the first 2 days of incubation. The average number of eggs released per female was similar in incubation Day 1 and 2, but significantly smaller on Day 3 and 4. About 25% of the females were continuously releasing eggs over 3 days rather than producing a single distinctive brood. In contrast, gravid females of Euphausia pacifica were observed from early July through October. Most gravid females released eggs on the first day of observation, while only 2% of females produced eggs repeatedly. Brood size varied from 20 to 246 eggs with an average of 102 ± 12 (95% CI) eggs female^− 1. The relationship between E. pacifica brood size and ambient chlorophyll-a concentration was sigmoidal (r² = 0.73), with food saturated brood size of 144 ± 14(SE, P < 0.001) eggs, and half-saturation occurring at 0.46 ± 0.02(SE, P < 0.001) mg chlorophyll-a m^− 3. The average interbrood interval of E. pacifica reared at 12 °C and satiated food conditions in the laboratory was ∼ 8 days, suggesting their potential individual fecundity in the GOA was 1148-1530 eggs per spawning season. Hatching and early development (from egg to furcilia stage) was studied under 5 °C, 8 °C and 12 °C. Hatching was nearly synchronous and lasted 3-6 h, depending on incubation temperature. Development times from egg to the first furcilia stage ranged between 20 and 33 days for T. inermis, and 15 and 45 days for E. pacifica at 12 °C and 5 °C, respectively.     

Bacterial expression of functional, biotinylated peripheral cannabinoid receptor CB2

A biotin-protein ligase recognition site (BRS) was inserted into a polypeptide comprised of the maltose-binding protein, the peripheral cannabinoid receptor (CB2), thioredoxin A, and a polyhistidine tag at the carboxy terminus. Expression levels of the recombinant receptor in Escherichia coli BL21(DE3) cells were approximately 1mg per liter of bacterial culture. The biotinylated CB2-fusion fully retained its ligand-binding capacity. Introduction of the BRS at the C-terminus of the CB2 fusion protein (construct CB2-109) resulted in its complete in vivo biotinylation; the biotinylated protein was streptavidin-binding competent. Positioning of the BRS near the N-terminus of CB2 (CB2-112) resulted in a very low level of biotinylation in vivo. However, the detergent solubilized and purified CB2-112 fusion protein were successfully biotinylated in vitro by action of a BirA biotin-protein ligase. The biotinylated CB2-112 fusion protein was cleaved by the tobacco etch virus protease at specifically inserted sites, and deposited onto monomeric avidin agarose beads. Biotinylation of the recombinant CB2 receptor enabled not only purification but also immobilization of the GPCR on a solid support in homogeneous orientation which is beneficial for subsequent structural characterization.     

Structure and activity of the NAD(P)+‐dependent succinate semialdehyde dehydrogenase YneI from Salmonella typhimurium

Aldehyde dehydrogenases are found in all organisms and play an important role in the metabolic conversion and detoxification of endogenous and exogenous aldehydes. Genomes of many organisms including Escherichia coli and Salmonella typhimurium encode two succinate semialdehyde dehydrogenases with low sequence similarity and different cofactor preference (YneI and GabD). Here, we present the crystal structure and biochemical characterization of the NAD(P)⁺‐dependent succinate semialdehyde dehydrogenase YneI from S. typhimurium. This enzyme shows high activity and affinity toward succinate semialdehyde and exhibits substrate inhibition at concentrations of SSA higher than 0.1 mM. YneI can use both NAD⁺ and NADP⁺ as cofactors, although affinity to NAD⁺ is 10 times higher. High resolution crystal structures of YneI were solved in a free state (1.85 Å) and in complex with NAD⁺ (1.90 Å) revealing a two domain protein with the active site located in the interdomain interface. The NAD⁺ molecule is bound in the long channel with its nicotinamide ring positioned close to the side chain of the catalytic Cys268. Site‐directed mutagenesis demonstrated that this residue, as well as the conserved Trp136, Glu365, and Asp426 are important for activity of YneI, and that the conserved Lys160 contributes to the enzyme preference to NAD⁺. Our work has provided further insight into the molecular mechanisms of substrate selectivity and activity of succinate semialdehyde dehydrogenases. © 2012 Wiley Periodicals, Inc.