IkappaB kinase beta phosphorylates the K63 deubiquitinase A20 to cause feedback inhibition of the NF-kappaB pathway

Misregulation of NF-kappaB signaling leads to infectious, inflammatory, or autoimmune disorders. IkappaB kinase beta (IKKbeta) is an essential activator of NF-kappaB and is known to phosphorylate the NF-kappaB inhibitor, IkappaBalpha, allowing it to undergo ubiquitin-mediated proteasomal degradation. However, beyond IkappaBalpha, few additional IKKbeta substrates have been identified. Here we utilize a peptide library and bioinformatic approach to predict likely substrates of IKKbeta. This approach predicted Ser381 of the K63 deubiquitinase A20 as a likely site of IKKbeta phosphorylation. While A20 is a known negative regulator of innate immune signaling pathways, the mechanisms regulating the activity of A20 are poorly understood. We show that IKKbeta phosphorylates A20 in vitro and in vivo at serine 381, and we further show that this phosphorylation event increases the ability of A20 to inhibit the NF-kappaB signaling pathway. Phosphorylation of A20 by IKKbeta thus represents part of a novel feedback loop that regulates the duration of NF-kappaB signaling following activation of innate immune signaling pathways.     

A Nonlinear Cable Framework for Bidirectional Synaptic Plasticity

Finding the rules underlying how axons of cortical neurons form neural circuits and modify their corresponding synaptic strength is the still subject of intense research. Experiments have shown that internal calcium concentration, and both the precise timing and temporal order of pre and postsynaptic action potentials, are important constituents governing whether the strength of a synapse located on the dendrite is increased or decreased. In particular, previous investigations focusing on spike timing-dependent plasticity (STDP) have typically observed an asymmetric temporal window governing changes in synaptic efficacy. Such a temporal window emphasizes that if a presynaptic spike, arriving at the synaptic terminal, precedes the generation of a postsynaptic action potential, then the synapse is potentiated; however if the temporal order is reversed, then depression occurs. Furthermore, recent experimental studies have now demonstrated that the temporal window also depends on the dendritic location of the synapse. Specifically, it was shown that in distal regions of the apical dendrite, the magnitude of potentiation was smaller and the window for depression was broader, when compared to observations from the proximal region of the dendrite. To date, the underlying mechanism(s) for such a distance-dependent effect is (are) currently unknown. Here, using the ionic cable theory framework in conjunction with the standard calcium based plasticity model, we show for the first time that such distance-dependent inhomogeneities in the temporal learning window for STDP can be largely explained by both the spatial and active properties of the dendrite.     

Inhibition of the inositol trisphosphate receptor of mouse eggs and A7r5 cells by KN-93 via a mechanism unrelated to Ca2+/calmodulin-dependent protein kinase II antagonism

KN-93, a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) inhibitor, concentration-dependently and reversibly inhibited inositol 1,4,5-trisphosphate receptor (IP(3)R)-mediated [Ca(2+)](i) signaling in mouse eggs and permeabilized A7r5 smooth muscle cells, two cell types predominantly expressing type-1 IP(3)R (IP(3)R-1). KN-92, an inactive analog, was ineffective. The inhibitory action of KN-93 on Ca(2+) signaling depended neither on effects on IP(3) metabolism nor on the filling grade of Ca(2+) stores, suggesting a direct action on the IP(3)R. Inhibition was independent of CaMKII, since in identical conditions other CaMKII inhibitors (KN-62, peptide 281-309, and autocamtide-related inhibitory peptide) were ineffective and since CaMKII activation was precluded in permeabilized cells. Moreover, KN-93 was most effective in the absence of Ca(2+). Analysis of Ca(2+) release in A7r5 cells at varying [IP(3)], of IP(3)R-1 degradation in eggs, and of [(3)H]IP(3) binding in Sf9 microsomes all indicated that KN-93 did not affect IP(3) binding. Comparison of the inhibition of Ca(2+) release and of [(3)H]IP(3) binding by KN-93 and calmodulin (CaM), either separately or combined, was compatible with a specific interaction of KN-93 with a CaM-binding site on IP(3)R-1. This was also consistent with the much smaller effect of KN-93 in permeabilized 16HBE14o(-) cells that predominantly express type 3 IP(3)R, which lacks the high affinity CaM-binding site. These findings indicate that KN-93 inhibits IP(3)R-1 directly and may therefore be a useful tool in the study of IP(3)R functional regulation.     

A bacterial artificial chromosome (BAC) library for sunflower, and identification of clones containing genes for putative transmembrane receptors

Sunflower (Helianthus annuus L.) is an economically important oil seed crop with an estimated genome size of 3000 Mb. We have constructed a bacterial artificial chromosome (BAC) library for sunflower, which represents an estimated 4- to 5-fold coverage of the genome. Nuclei isolated from young leaves were used as a source of high-molecular-weight DNA and a partial restriction endonuclease digestion protocol was used to cleave the DNA. A random sample of 60 clones indicated an average insert size of 80 kb, implying a 95% probability of recovering any specific sequence of interest. The library was screened with chloroplast DNA probes. Only 0.1% of the clones were identified to be of chloroplast origin, indicating that contamination with organellar DNAs is very low. The utility of the library was evaluated by screening for the presence of genes for putative transmembrane receptors sharing epidermal growth factor (EGF) and integrin-like domains. First, a homologous sunflower EST (HaELP1) was obtained by degenerate RT-PCR cloning, using Arabidopsis thaliana genes (AtELP) as a source of consensus sequences. Three different BACs yielded positive hybridization signals when HaELP1 was used as a probe. BAC subcloning and sequencing demonstrated the presence of two different loci putatively homologous to genes for transmembrane proteins with EGF- and integrin-like domains from sunflower. This work demonstrates the suitability of the library for homology map-based cloning of sunflower genes and physical mapping of the sunflower genome.     

Reproduction in captive common marmosets (Callithrix jacchus)

Though sexual maturation may begin at around one year of age, first successful reproduction of the common marmoset (Callithrix jacchus) is likely to be later, and it is generally recommended that animals not be mated before 1.5 years of age. The average gestation period is estimated to be 143 to 144 days. A crown-rump length measurement taken by use of ultrasonography during the linear, rapid, prenatal growth phase (between approx. days 60 and 95) can be compared against standard growth curves to estimate delivery date to within 3 to 4 days, on average. Marmosets produce more young per delivery than does any other anthropoid primate, and have more variation in litter size. Many long-established colonies report that triplets are the most common litter size, and there is documented association between higher maternal body weight and higher ovulation numbers. Higher litter sizes generally do not generate higher numbers of viable young. Marmosets are unusual among primates in having a postpartum ovulation that typically results in conception and successful delivery; reported median inter-birth intervals range from 154 to 162 days. However, pregnancy losses are quite common; one study of a large breeding colony indicated 50 percent loss between conception and term delivery. The average life span for breeding females is around six years; the range of reported average lifetime number of litters for a breeding pair is 3.45 to 4.0. Our purpose is to provide an overview of reproduction in the common marmoset, including basic reproductive life history, lactation and weaning, social housing requirements, and common problems encountered in the captive breeding of this species. A brief comparison between marmoset and tamarin reproduction also will be provided.     

Modulation of cardiac troponin C-cardiac troponin I regulatory interactions by the amino-terminus of cardiac troponin I

Multidimensional heteronuclear magnetic resonance studies of the cardiac troponin C/troponin I(1-80)/troponin I(129-166) complex demonstrated that cardiac troponin I(129-166), corresponding to the adjacent inhibitory and regulatory regions, interacts with and induces an opening of the cardiac troponin C regulatory domain. Chemical shift perturbation mapping and (15)N transverse relaxation rates for intact cardiac troponin C bound to either cardiac troponin I(1-80)/troponin I(129-166) or troponin I(1-167) suggested that troponin I residues 81-128 do not interact strongly with troponin C but likely serve to modulate the interaction of troponin I(129-166) with the cardiac troponin C regulatory domain. Chemical shift perturbations due to troponin I(129-166) binding the cardiac troponin C/troponin I(1-80) complex correlate with partial opening of the cardiac troponin C regulatory domain previously demonstrated by distance measurements using fluorescence methodologies. Fluorescence emission from cardiac troponin C(F20W/N51C)(AEDANS) complexed to cardiac troponin I(1-80) was used to monitor binding of cardiac troponin I(129-166) to the regulatory domain of cardiac troponin C. The apparent K(d) for cardiac troponin I(129-166) binding to cardiac troponin C/troponin I(1-80) was 43.3 +/- 3.2 microM. After bisphosphorylation of cardiac troponin I(1-80) the apparent K(d) increased to 59.1 +/- 1.3 microM. Thus, phosphorylation of the cardiac-specific N-terminus of troponin I reduces the apparent binding affinity of the regulatory domain of cardiac troponin C for cardiac troponin I(129-166) and provides further evidence for beta-adrenergic modulation of troponin Ca(2+) sensitivity through a direct interaction between the cardiac-specific amino-terminus of troponin I and the cardiac troponin C regulatory domain.     

A linkage map for the hybridising toads Bombina bombina and B. variegata (Anura: Discoglossidae)

Stable hybrid zones in which ecologically divergent taxa give rise to a range of recombinants are natural laboratories in which the genetic basis of adaptation and reproductive isolation can be unraveled. One such hybrid zone is formed by the fire-bellied toads Bombina bombina and B. variegata (Anura: Discoglossidae). Adaptations to permanent and ephemeral breeding habitats, respectively, have shaped numerous phenotypic differences between the taxa. All of these are, in principle, candidates for a genetic dissection via QTL mapping. We present here a linkage map of 28 codominant and 10 dominant markers in the Bombina genome. In an F2 cross, markers that were mainly microsatellites, SSCPs or allozymes were mapped to 20 linkage groups. Among the 40 isolated CA microsatellites, we noted a preponderance of compound and frequently interleaved CA-TA repeats as well as a striking polarity at the 5' end of the repeats.     

Proteomic identification of glycosylphosphatidylinositol anchor-dependent membrane proteins elevated in breast carcinoma

The glycosylphosphatidylinositol (GPI) anchor is a lipid and glycan modification added to the C terminus of certain proteins in the endoplasmic reticulum by the activity of a multiple subunit enzyme complex known as the GPI transamidase (GPIT). Several subunits of GPIT have increased expression levels in breast carcinoma. In an effort to identify GPI-anchored proteins and understand the possible role of these proteins in breast cancer progression, we employed a combination of strategies. First, alpha toxin from Clostridium septicum was used to capture GPI-anchored proteins from human breast cancer tissues, cells, and serum for proteomic analysis. We also expressed short interfering RNAs targeting the expression of the GPAA1 and PIGT subunits of GPIT in breast cancer cell lines to identify proteins in which membrane localization is dependent on GPI anchor addition. Comparative membrane proteomics using nano-ESI-RPLC-MS/MS led to the discovery of several new potential diagnostic and therapeutic targets for breast cancer. Furthermore, we provide evidence that increased levels of GPI anchor addition in malignant breast epithelial cells promotes the dedifferentiation of malignant breast epithelial cells in part by increasing the levels of cell surface markers associated with mesenchymal stem cells.