Bulk Segregant Analysis of an Induced Floral Mutant Identifies a MIXTA-Like R2R3 MYB Controlling Nectar Guide Formation in Mimulus lewisii

The genetic and developmental basis of many ecologically important floral traits (e.g., carotenoid pigmentation, corolla tube structure, nectar volume, pistil and stamen length) remains poorly understood. Here we analyze a chemically induced floral mutant of Mimulus lewisii through bulk segregant analysis and transgenic experiments and identify a MIXTA-like R2R3 MYB gene that controls nectar guide formation in M. lewisii flowers, which involves epidermal cell development and carotenoid pigmentation.     

Pretreatment with the probiotic VSL#3 delays transition from inflammation to dysplasia in a rat model of colitis-associated cancer

Evidence supports involvement of microflora in the transition of chronic inflammation to neoplasia. We investigated the protective efficacy of the probiotic VSL#3 in a model of colitis-associated colorectal cancer. Chronic colitis was induced in Sprague-Dawley rats by administration of trinitrobenzene sulfonic acid (TNBS), followed 6 wk later by systemic reactivation. To induce colitis-associated dysplasia and cancer, the animals received TNBS (intravenously) twice a week for 10 wk. One group received VSL#3 in drinking water from 1 wk before colitis induction until death. The colons were examined for damage and presence of dysplasia or cancer. Samples were analyzed for cell proliferation and apoptosis, vitamin D receptor (VDR) expression, angiogenic factors, and presence of alkaline sphingomyelinase or phosphatase. Microbial community composition was evaluated by terminal restriction fragment-length polymorphism analysis of the bacterial 16S rRNA gene. None of the probiotic-treated animals developed carcinoma, and no high-grade dysplasia was found in either the proximal or mid colon. In contrast, 29% of the animals in the control group developed carcinoma in one or more regions of the colon. VSL#3-treated animals had significantly less damage than the vehicle treated-controls in all areas of the colon, and this correlated with decreased richness and diversity of the mucosally adherent microbiota. Treatment with the probiotic increased the antiangiogenic factor angiostatin, VDR expression, and alkaline sphingomyelinase. We concluded that pretreatment with the probiotic VSL#3 can attenuate various inflammatory-associated parameters, delaying transition to dysplasia and cancer, thus offering its potential therapeutic use in patients with long-standing colitis.     

Revisioning Women, Health, and Healing: Feminist, Cultural, and Technoscience Perspectives

Revisioning Women, Health, and Healing: Feminist, Cultural, and Technoscience Perspectives. Adele E. Clarke and Virginia L. Olesen. eds. New York and London: Routledge, 1999. + 374 pp.     

Monarch-1 suppresses non-canonical NF-kappaB activation and p52-dependent chemokine expression in monocytes

CATERPILLER (NOD, NBD-LRR) proteins are rapidly emerging as important mediators of innate and adaptive immunity. Among these, Monarch-1 operates as a novel attenuating factor of inflammation by suppressing inflammatory responses in activated monocytes. However, the molecular mechanisms by which Monarch-1 performs this important function are not well understood. In this report, we show that Monarch-1 inhibits CD40-mediated activation of NF-kappaB via the non-canonical pathway in human monocytes. This inhibition stems from the ability of Monarch-1 to associate with and induce proteasome-mediated degradation of NF-kappaB inducing kinase. Congruently, silencing Monarch-1 with shRNA enhances the expression of p52-dependent chemokines.     

Regulation of beta-adrenergic receptor signaling by S-nitrosylation of G-protein-coupled receptor kinase 2

beta-adrenergic receptors (beta-ARs), prototypic G-protein-coupled receptors (GPCRs), play a critical role in regulating numerous physiological processes. The GPCR kinases (GRKs) curtail G-protein signaling and target receptors for internalization. Nitric oxide (NO) and/or S-nitrosothiols (SNOs) can prevent the loss of beta-AR signaling in vivo, but the molecular details are unknown. Here we show in mice that SNOs increase beta-AR expression and prevent agonist-stimulated receptor downregulation; and in cells, SNOs decrease GRK2-mediated beta-AR phosphorylation and subsequent recruitment of beta-arrestin to the receptor, resulting in the attenuation of receptor desensitization and internalization. In both cells and tissues, GRK2 is S-nitrosylated by SNOs as well as by NO synthases, and GRK2 S-nitrosylation increases following stimulation of multiple GPCRs with agonists. Cys340 of GRK2 is identified as a principal locus of inhibition by S-nitrosylation. Our studies thus reveal a central molecular mechanism through which GPCR signaling is regulated.     

Z-disc-associated,Alternatively Spliced,PDZ Motif-containing Protein (ZASP) Mutations in the Actin-binding Domain Cause Disruption of Skeletal Muscle Actin Filaments in Myofibrillar Myopathy

The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP)/Cypher interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We show that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8–11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle.     

Interactome of the Amyloid Precursor Protein APP in Brain Reveals a Protein Network Involved in Synaptic Vesicle Turnover and a Close Association with Synaptotagmin-1

Knowledge of the protein networks interacting with the amyloid precursor protein (APP) in vivo can shed light on the physiological function of APP. To date, most proteins interacting with the APP intracellular domain (AICD) have been identified by Yeast Two Hybrid screens which only detect direct interaction partners. We used a proteomics-based approach by biochemically isolating tagged APP from the brains of transgenic mice and subjecting the affinity-purified complex to mass spectrometric (MS) analysis. Using two different quantitative MS approaches, we compared the protein composition of affinity-purified samples isolated from wild-type mice versus transgenic mice expressing tagged APP. This enabled us to assess truly enriched proteins in the transgenic sample and yielded an overlapping set of proteins containing the major proteins involved in synaptic vesicle endo- and exocytosis. Confocal microscopy analyses of cotransfected primary neurons showed colocalization of APP with synaptic vesicle proteins in vesicular structures throughout the neurites. We analyzed the interaction of APP with these proteins using pulldown experiments from transgenic mice or cotransfected cells followed by Western blotting. Synaptotagmin-1 (Stg1), a resident synaptic vesicle protein, was found to directly bind to APP. We fused Citrine and Cerulean to APP and the candidate proteins and measured fluorescence resonance energy transfer (FRET) in differentiated SH-SY5Y cells. Differentially tagged APPs showed clear sensitized FRET emission, in line with the described dimerization of APP. Among the candidate APP-interacting proteins, again only Stg1 was in close proximity to APP. Our results strongly argue for a function of APP in synaptic vesicle turnover in vivo. Thus, in addition to the APP cleavage product Aβ, which influences synaptic transmission at the postsynapse, APP interacts with the calcium sensor of synaptic vesicles and might thus play a role in the regulation of synaptic vesicle exocytosis.