Dual requirement of ectodermal Smad4 during AER formation and termination of feedback signaling in mouse limb buds

BMP signaling is pivotal for normal limb bud development in vertebrate embryos and genetic analysis of receptors and ligands in the mouse revealed their requirement in both mesenchymal and ectodermal limb bud compartments. In this study, we genetically assessed the potential essential functions of SMAD4, a mediator of canonical BMP/TGFß signal transduction, in the mouse limb bud ectoderm. Msx2‐Cre was used to conditionally inactivate Smad4 in the ectoderm of fore‐ and hindlimb buds. In hindlimb buds, the Smad4 inactivation disrupts the establishment and signaling by the apical ectodermal ridge (AER) from early limb bud stages onwards, which results in severe hypoplasia and/or aplasia of zeugo‐ and autopodal skeletal elements. In contrast, the developmentally later inactivation of Smad4 in forelimb buds does not alter AER formation and signaling, but prolongs epithelial‐mesenchymal feedback signaling in advanced limb buds. The late termination of SHH and AER‐FGF signaling delays distal progression of digit ray formation and inhibits interdigit apoptosis. In summary, our genetic analysis reveals the temporally and functionally distinct dual requirement of ectodermal Smad4 during initiation and termination of AER signaling. genesis 51:660–666. © 2013 Wiley Periodicals, Inc.     

Oxidative modification of citrate synthase by peroxyl radicals and protection with novel antioxidants

In mammals, aging is linked to a decline in the activity of citrate synthase (CS; E.C. 2.3.3.1), the first enzyme of the citric acid cycle. We used 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH), a water-soluble generator of peroxyl and alkoxyl radicals, to investigate the susceptibility of CS to oxidative damage. Treatment of isolated mitochondria with AAPH for 8–24?h led to CS inactivation; however, the activity of aconitase, a mitochondrial enzyme routinely used as an oxidative stress marker, was unaffected. In addition to enzyme inactivation, AAPH treatment of purified CS resulted in dityrosine formation, increased protein surface hydrophobicity, and loss of tryptophan fluorescence. Propyl gallate, 1,8-naphthalenediol, 2,3-naphthalenediol, ascorbic acid, glutathione, and oxaloacetate protected CS from AAPH-mediated inactivation, with IC₅₀ values of 9, 14, 34, 37, 150, and 160?μM, respectively. Surprisingly, the antioxidant epigallocatechin gallate offered no protection against AAPH, but instead caused CS inactivation. Our results suggest that the current practice of using the enzymatic activity of CS as an index of mitochondrial abundance and the use of aconitase activity as an oxidative stress marker may be inappropriate, especially in oxidative stress-related studies, during which alkyl peroxyl and alkoxyl radicals can be generated.     

Synthesis and Biochemical Evaluation of the Novel Steroid Androsta-4,6,8(9)-Triene-3,17-Dione

Abstract

According to a proposed aromatisation mechanism by which estrogens are biosynthesized from androgens, the novel steroid androsta-4,6,8(9)-triene-3,17-dione (FCE 24918) should behave as a suicide substrate for aromatase. The synthesis of this triene steroid has been accomplished starting from androsta-4,7-diene-3,17-dione (4) by the acid-catalysed cleavage of the corresponding 7,8α-epoxide, 5, and it was obtained together with androsta-4,6,8(14)-triene-3,17-dione (FCE 24917) as a side product. The time-dependent inactivation of placental aromatase by the two isomers was studied comparatively and showed that the 4,6,8(9)-triene moiety acts as a latent alkylating group.     

Visualization of inactive X chromosome in preimplantation embryos utilizing MacroH2A–EGFP transgenic mouse

One of the two X chromosomes is inactivated in female eutherian mammals. MacroH2A, an unusual histone variant, is known to accumulate on the inactive X chromosome (Xi) during early embryo development, and can thus be used as a marker of the Xi. In this study, we produced a transgenic mouse line expressing the mouse MacroH2A1.2–enhanced green fluorescent protein (EGFP) fusion protein (MacroH2A–EGFP) under the control of a CAG promoter and verified whether MacroH2A–EGFP would be useful for tracing the process of X chromosome inactivation by visualizing Xi noninvasively in preimplantation embryos. In transgenic female mice, MacroH2A–EGFP formed a fluorescent focus in nuclei throughout the body. In female blastocysts, the MacroH2A–EGFP focus colocalized with Xist RNA, well known as a marker of Xi. Fluorescence marking of Xi was first observed in some embryonic cells between the 4‐ and 8‐cell stages. These results demonstrate that MacroH2A can bind to the Xi by around the 8‐cell stage in female mouse embryos. These MacroH2A–EGFP transgenic mice might be useful to elucidate the process of X chromosome inactivation during the mouse life cycle. genesis 51:259–267. © 2013 Wiley Periodicals, Inc.     

Surface modifications of influenza proteins upon virus inactivation by β‐propiolactone

Inactivation of intact influenza viruses using formaldehyde or β‐propiolactone (BPL) is essential for vaccine production and safety. The extent of chemical modifications of such reagents on viral proteins needs to be extensively investigated to better control the reactions and quality of vaccines. We have evaluated the effect of BPL inactivation on two candidate re‐assortant vaccines (NIBRG‐121xp and NYMC‐X181A) derived from A/California/07/2009 pandemic influenza viruses using high‐resolution FT‐ICR MS‐based proteomic approaches. We report here an ultra performance LC MS/MS method for determining full‐length protein sequences of hemagglutinin and neuraminidase through protein delipidation, various enzymatic digestions, and subsequent mass spectrometric analyses of the proteolytic peptides. We also demonstrate the ability to reliably identify hundreds of unique sites modified by propiolactone on the surface of glycoprotein antigens. The location of these modifications correlated with changes to protein folding, conformation, and stability, but demonstrated no effect on protein disulfide linkages. In some cases, these modifications resulted in suppression of protein function, an effect that correlated with the degree of change of the modified amino acids’ side chain length and polarity.     

Bacterial cheating drives the population dynamics of cooperative antibiotic resistance plasmids

Inactivation of β ‐lactam antibiotics by resistant bacteria is a ‘cooperative’ behavior that may allow sensitive bacteria to survive antibiotic treatment. However, the factors that determine the fraction of resistant cells in the bacterial population remain unclear, indicating a fundamental gap in our understanding of how antibiotic resistance evolves. Here, we experimentally track the spread of a plasmid that encodes a β ‐lactamase enzyme through the bacterial population. We find that independent of the initial fraction of resistant cells, the population settles to an equilibrium fraction proportional to the antibiotic concentration divided by the cell density. A simple model explains this behavior, successfully predicting a data collapse over two orders of magnitude in antibiotic concentration. This model also successfully predicts that adding a commonly used β ‐lactamase inhibitor will lead to the spread of resistance, highlighting the need to incorporate social dynamics into the study of antibiotic resistance.     

Pharmacological blockade of gap junctions induces repetitive surging of extracellular potassium within the locust CNS

The maintenance of cellular ion homeostasis is crucial for optimal neural function and thus it is of great importance to understand its regulation. Glial cells are extensively coupled by gap junctions forming a network that is suggested to serve as a spatial buffer for potassium (K⁺) ions. We have investigated the role of glial spatial buffering in the regulation of extracellular K⁺ concentration ([K⁺]_o) within the locust metathoracic ganglion by pharmacologically inhibiting gap junctions. Using K⁺-sensitive microelectrodes, we measured [K⁺]_o near the ventilatory neuropile while simultaneously recording the ventilatory rhythm as a model of neural circuit function. We found that blockade of gap junctions with either carbenoxolone (CBX), 18β-glycyrrhetinic acid (18β-GA) or meclofenamic acid (MFA) reliably induced repetitive [K⁺]_o surges and caused a progressive impairment in the ability to maintain baseline [K⁺]_o levels throughout the treatment period. We also show that a low dose of CBX that did not induce surging activity increased the vulnerability of locust neural tissue to spreading depression (SD) induced by Na⁺/K⁺-ATPase inhibition with ouabain. CBX pre-treatment increased the number of SD events induced by ouabain and hindered the recovery of [K⁺]_o back to baseline levels between events. Our results suggest that glial spatial buffering through gap junctions plays an essential role in the regulation of [K⁺]_o under normal conditions and also contributes to a component of [K⁺]_o clearance following physiologically elevated levels of [K⁺]_o.     

XLMR protein related to neurite extension (Xpn/KIAA2022) regulates cell–cell and cell–matrix adhesion and migration

X-linked mental retardation (XLMR) is a common cause of moderate to severe intellectual disability in males. XLMR protein related to neurite extension (Xpn, also known as KIAA2022) has been implicated as a gene responsible for XLMR in humans. Although Xpn is highly expressed in the developing brain and is involved in neurite outgrowth in PC12 cells and neurons, little is known about the functional role of Xpn. Here, we show that Xpn regulates cell–cell and cell–matrix adhesion and migration in PC12 cells. Xpn knockdown enhanced cell–cell and cell–matrix adhesion mediated by N-cadherin and β1-integrin, respectively. N-Cadherin and β1-integrin expression at the mRNA and protein levels was significantly increased in Xpn knockdown PC12 cells. Furthermore, overexpressed Xpn protein was strongly expressed in the nuclei of PC12 and 293T cells. Finally, depletion of Xpn perturbed cellular migration by enhancing N-cadherin and β1-integrin expression in a PC12 cell wound healing assay. We conclude that Xpn regulates cell–cell and cell–matrix adhesion and cellular migration by regulating the expression of adhesion molecules.     

Cortical control of appropriate tongue protrusion during licking in cats—Increase in regional cerebral blood flow (rCBF) of the contralateral area P and in tongue protrusion after the unilateral area P lesion

Adequate tongue protrusion may be regulated by cat bilateral area P (the motor cortex for jaw and tongue movements) (Hiraba and Sato, Somatosens Mot Res 2005b;22:183–192). The ICMS (intracortical microstimulation) in the unilateral area P evoked motor effects of tongue protrusion without deviation (Hiraba and Sato, Somatosens Mot Res 2004;23:1–12), and cats with the unilateral lesion of area P showed abnormal tongue protrusion without deviation during licking (Hiraba and Sato, Somatosens Mot Res 2005b;22:183–192). Further, the measurements of the regional cerebral blood flow (rCBF) in the bilateral jaw and tongue motor cortical areas were shown to have the same activity rate during the lateral licking (Hiraba and Sato, Somatosens Mot Res 2005c;22:307–317). We assumed from these results that cortical control for tongue protrusion was executed by networks between the bilateral area P including inhibitory interneurons. We prepared the measurable cats of the rCBF in the contralateral side after the unilateral area P lesion. Changes in the rates of rCBF and tongue protrusion during licking were examined over a long time course of about 1–2 months after the unilateral area P lesion. All cats after the unilateral area P lesion showed increased rate (double or triple in comparison with the normal ones) of rCBF of the contralateral area P in the early (0–20 days) phase. On the other hand, increased rates of tongue protrusion were about 120% in the early phase, and about 180% in the middle (21–35 days) and late (36–last days) phases. The results support the organization of networks between bilateral area P including the inhibitory interneurons.     

Cortical control of tongue protrusion and lateral movements in the cat

Based on area P lesion experiments, we hypothesized that tongue protrusion adapted for licking might be regulated by the lateral wall of the presylvian sulcus (bilateral areas P) of the cerebral cortex (Hiraba H, Sato T, Nakakawa K, Ueda K. . Cortical control of appropriate tongue protrusion during licking in cats—Increase in regional cerebral blood flow (rCBF) of the contralateral area P and in tongue protrusion after the unilateral area P lesion. Somatosens Mot Res 26:82–89). We propose that the right and left lingual muscles are dominated by the right and left hypoglossal nucleus, respectively, and that right and left pyramidal cells projecting to the right and left hypoglossal nucleus, respectively, exist in unilateral area P. These cells project via an inhibitory interneuron relay to the lateral branches toward the left or right pyramidal cells in contralateral area P. In this study, we aimed to demonstrate the existence of inhibitory interneurons using injections of a gamma-aminobutyric acid (GABA) agonist (muscimol), a GABA antagonist (bicuculline), and kainic acid into unilateral area P, followed by examination of tongue protrusion and lateral movements during trained licking and changes in regional cerebral blood flow (rCBF) values in the contralateral area P. We found disordered protrusion toward both sides and a marked decrease in rCBF values in the contralateral area P after bicuculline injection. We also found abnormal tongue protrusion toward the front and a marked increase in rCBF values after muscimol and kainic acid injections. These results suggest that cortical networks between the bilateral areas P are relayed by inhibitory interneurons.