Analysis of Dysferlin Direct Interactions with Putative Repair Proteins Links Apoptotic Signaling to Ca Elevation via PDCD6 and FKBP8.

Quantitative surface plasmon resonance (SPR) was utilized to determine binding strength and calcium dependence of direct interactions between dysferlin and proteins likely to mediate skeletal muscle repair, interrupted in limb girdle muscular dystrophy type 2B/R2. Dysferlin canonical C2A (cC2A) and C2F/G domains directly interacted with annexin A1, calpain-3, caveolin-3, affixin, AHNAK1, syntaxin-4, and mitsugumin-53, with cC2A the primary target and C2F lesser involved, overall demonstrating positive calcium dependence. Dysferlin C2 pairings alone showed negative calcium dependence in almost all cases.

Development of high-affinity nanobodies specific for Na1.4 and Na1.5 voltage-gated sodium channel isoforms.

Voltage-gated sodium channels, Nas, are responsible for the rapid rise of action potentials in excitable tissues. Na channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-Na nanobodies (Nbs), Nb17 and Nb82, that recognize the Na1.

Strategy for Tumor-Selective Disruption of Androgen Receptor Function in the Spectrum of Prostate Cancer.

Purpose: Testosterone suppression in prostate cancer is limited by serious side effects and resistance via restoration of androgen receptor (AR) functionality. ELK1 is required for AR-dependent growth in various hormone-dependent and castration-resistant prostate cancer models. The amino-terminal domain of AR docks at two sites on ELK1 to coactivate essential growth genes.

Imaging EGFR and HER3 through Zr-labeled MEHD7945A (Duligotuzumab).

Tumor resistance to treatment paved the way toward the development of single agent drugs that target multiple molecular signatures amplified within the malignancy. The discovered crosstalk between EGFR and HER3 as well as the role of HER3 in mediating EGFR resistance made these two receptor tyrosine kinases attractive targets. MEHD7945A or duligotuzumab is a single immunotherapy agent that dually targets both molecular signatures.

Analysis of Protein Interactions by Surface Plasmon Resonance.

Surface plasmon resonance is an optical technique that is utilized for detecting molecular interactions, such as interactions that occur between proteins or other classes of molecules. Binding of a mobile molecule (analyte) to a molecule immobilized on a thin metal film (ligand) changes the refractive index of the film. The angle of extinction of light that is completely reflected after polarized light impinges upon the film, is altered and monitored as a change in detector position for a dip in reflected intensity (the surface plasmon resonance phenomenon).

Dopamine D1A directly interacts with otoferlin synaptic pathway proteins: Ca2+ and phosphorylation underlie an NSF-to-AP2mu1 molecular switch.

Dopamine receptors regulate exocytosis via protein-protein interactions (PPIs) as well as via adenylyl cyclase transduction pathways. Evidence has been obtained for PPIs in inner ear hair cells coupling D1A to soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE)-related proteins snapin, otoferlin, N-ethylmaleimide-sensitive factor (NSF), and adaptor-related protein complex 2, mu 1 (AP2mu1), dependent on [Ca] and phosphorylation. Specifically, the carboxy terminus of dopamine D1A was found to directly bind t-SNARE-associated protein snapin in teleost and mammalian hair cell models by yeast two-hybrid (Y2H) and pull-down assays, and snapin directly interacts with hair cell calcium-sensor otoferlin.

The Amino-terminal Domain of the Androgen Receptor Co-opts Extracellular Signal-regulated Kinase (ERK) Docking Sites in ELK1 Protein to Induce Sustained Gene Activation That Supports Prostate Cancer Cell Growth.

The ETS domain transcription factor ELK1 is in a repressive association with growth genes and is transiently activated through phosphorylation by ERK1/2. In prostate cancer (PCa) cells the androgen receptor (AR) is recruited by ELK1, via its amino-terminal domain (A/B), as a transcriptional co-activator, without ELK1 hyper-phosphorylation. Here we elucidate the structural basis of the interaction of AR with ELK1.

Surface Plasmon Resonance (SPR) Analysis of Binding Interactions of Inner-Ear Proteins.

Surface plasmon resonance is an optical technique that is utilized for detecting molecular interactions. Binding of a mobile molecule (analyte) to a molecule immobilized on a thin metal film (ligand) changes the refractive index of the film. The angle of extinction of light that is completely reflected after polarized light impinges upon the film, is altered, and monitored as a change in detector position for a dip in reflected intensity (the surface plasmon resonance phenomenon).

Antigen itself antibody: an alternative one-step immunoassay for measuring the anti-idiotypic antibody titer.

Immunoassay designs rely on the great specificity of antibodies and a suitable marker that facilitates generation of a quantitative signal. Currently, there is no reliable method for measuring the titers of an anti-idiotypic antibody. Our initial attempt to measure titers of mouse anti-idiotypic antibody after idiotypic vaccination with HM-1 killer toxin neutralizing monoclonal antibody (nmAb-KT) failed.

Cyclic nucleotide-gated channel α-3 (CNGA3) interacts with stereocilia tip-link cadherin 23 + exon 68 or alternatively with myosin VIIa, two proteins required for hair cell mechanotransduction.

Previously, we obtained evidence for a photoreceptor/olfactory type of CNGA3 transcript in a purified teleost vestibular hair cell preparation with immunolocalization of CNGA3 protein to stereocilia of teleost vestibular and mammalian cochlear hair cells. The carboxyl terminus of highly Ca(2+)-permeable CNGA3 expressed in the mammalian organ of Corti and saccular hair cells was found to interact with an intracellular domain of microfibril interface-located protein 1 (EMILIN 1), a member of the elastin superfamily, also immunolocalizd to hair cell stereocilia (Selvakumar, D., Drescher, M.

CNGA3 is expressed in inner ear hair cells and binds to an intracellular C-terminus domain of EMILIN1.

The molecular characteristics of CNG (cyclic nucleotide-gated) channels in auditory/vestibular hair cells are largely unknown, unlike those of CNG mediating sensory transduction in vision and olfaction. In the present study we report the full-length sequence for three CNGA3 variants in a hair cell preparation from the trout saccule with high identity to CNGA3 in olfactory receptor neurons/cone photoreceptors. A custom antibody targeting the N-terminal sequence immunolocalized CNGA3 to the stereocilia and subcuticular plate region of saccular hair cells.

The role of the histidine-35 residue in the cytocidal action of HM-1 killer toxin.

Diethylpyrocarbonate modification and site-directed mutagenesis studies of histidine-35 in HM-1 killer toxin (HM-1) have shown that a specific feature, the imidazole side chain of histidine-35, is essential for the expression of the killing activity. In subcellular localization experiments, wild-type HM-1 was in the membrane fraction of Saccharomyces cerevisiae BJ1824, but not the HM-1 analogue in which histidine-35 was replaced by alanine (H35A HM-1). Neither wild-type nor H35A HM-1 was detected in cellular fractions of HM-1-resistant yeast S.

Recombinant single-chain anti-idiotypic antibody: an effective fungal beta-1,3-glucan synthase inhibitor.

Recombinant single-chain fragment variable anti-idiotypic antibodies were produced to represent the internal image of HM-1 killer toxin and were used as novel and effective antifungal agents to inhibit in vitro beta-1,3-glucan synthase and cell growth. The mechanism of cytocidal activity of anti-idiotypic antibodies was investigated and was compared with the actions of aculeacin A and papulacandin B, the most common antibiotics acting as beta-1,3-glucan synthase inhibitors. The degree of inhibition of beta-1,3-glucan synthase by both antibodies and antibiotics were examined for yeasts Saccharomyces cerevisiae A451, Cryptococcus albidus NBRC 0612 and Candida albicans IFM 40215.

Inhibition of fungal beta-1,3-glucan synthase and cell growth by HM-1 killer toxin single-chain anti-idiotypic antibodies.

Single-chain variable-fragment (scFv) anti-idiotypic antibodies of an HM-1 killer toxin (HM-1) from the yeast Williopsis saturnus var. mrakii IFO 0895 have been produced by recombinant DNA technology from the splenic lymphocytes of mice immunized by idiotypic vaccination with a neutralizing monoclonal antibody (nMAb-KT). The fungicidal activity of scFv anti-idiotypic antibodies against the isolates of four Candida species was assessed by MIC analysis.

Inhibition of beta-1,3-glucan synthase and cell growth of Cryptococcus species by recombinant single-chain anti-idiotypic antibodies.

Recombinant single-chain fragment variable (scFv) anti-idiotypic antibodies were produced to represent the internal image of a HM-1 killer toxin, which is characterized by a wide spectrum of anti-fungal activity through inhibiting beta-1,3-glucan synthase (GS). We examined if scFv antibodies are active against Cryptococcus species, a human pathogen of increasing medical importance. The anti-cryptococcal activity of scFv antibodies and HM-1 were assessed by MIC analysis for C.

Identification and characterization of a neutralizing monoclonal antibody for the epitope on HM-1 killer toxin.

Killer toxin-neutralizing monoclonal antibody (nmAb-KT) against HM-1 killer toxin (HM-1) produced by yeast Williopsis saturnus var. mrakii IFO 0895 reduces both the killing and glucan synthase inhibitory activity of HM-1. nmAb-KT is classified as IgG1kappa and has been shown to be ineffective against HYI killer toxin produced by the related yeast W.

Monoclonal antibodies and sandwich ELISA for quantitation of HM-1 killer toxin.

To establish a method for quantitative analysis of HM-1 killer toxin (HM-1), two purified mouse monoclonal antibodies, 1F1 and 4A2, and rabbit polyclonal antiserum against HM-1 were prepared. Both monoclonal antibodies were classified as IgG1(kappa) subtype, and did not neutralize the killing activity of HM-1. By SPOTs analysis, the epitope of 1F1 was found in the sequence of CDPNTG with a corresponding sequence of 11-16 from N-terminal amino acid residues of HM-1, but the epitope of 4A2 was not determined.