Ivermectin and nodulisporic acid receptors in Drosophila melanogaster contain both gamma-aminobutyric acid-gated Rdl and glutamate-gated GluCl alpha chloride channel subunits

35S-labeled derivatives of the insecticides nodulisporic acid and ivermectin were synthesized and demonstrated to bind with high affinity to a population of receptors in Drosophila head membranes that were previously shown to be associated with a glutamate-gated chloride channel. Nodulisporic acid binding was modeled as binding to a single population of receptors. Ivermectin binding was composed of at least two kinetically distinct receptor populations, only one of which was associated with nodulisporic acid binding. The binding of these two ligands was modulated by glutamate, ivermectin, and antagonists of invertebrate gamma-aminobutyric acid (GABA)ergic receptors. Because solubilized nodulisporic acid and ivermectin receptors comigrated as 230-kDa complexes by gel filtration, antisera specific for both the Drosophila glutamate-gated chloride channel subunit GluCl alpha (DmGluCl alpha) and the GABA-gated chloride channel subunit Rdl (DmRdl) proteins were generated and used to examine the possible coassembly of these two subunits within a single receptor complex. DmGluCl alpha antibodies immunoprecipitated all of the ivermectin and nodulisporic acid receptors solubilized by detergent from Drosophila head membranes. DmRdl antibodies also immunoprecipitated all solubilized nodulisporic receptors, but only approximately 70% of the ivermectin receptors. These data suggest that both DmGluCl alpha and DmRdl are components of nodulisporic acid and ivermectin receptors, and that there also exists a distinct class of ivermectin receptors that contains the DmGluCl alpha subunit but not the DmRdl subunit. This co-association of DmGluCl alpha and DmRdl represents the first biochemical and immunological evidence of coassembly of subunits from two different subclasses of ligand-gated ion channel subunits.     

Mitochondrial protein import: recognition of internal import signals of BCS1 by the TOM complex

BCS1, a component of the inner membrane of mitochondria, belongs to the group of proteins with internal, noncleavable import signals. Import and intramitochondrial sorting of BCS1 are encoded in the N-terminal 126 amino acid residues. Three sequence elements were identified in this region, namely, the transmembrane domain (amino acid residues 51 to 68), a presequence type helix (residues 69 to 83), and an import auxiliary region (residues 84 to 126). The transmembrane domain is not required for stable binding to the TOM complex. The Tom receptors (Tom70, Tom22 and Tom20), as determined by peptide scan analysis, interact with the presequence-like helix, yet the highest binding was to the third sequence element. We propose that the initial recognition of BCS1 precursor at the surface of the organelle mainly depends on the auxiliary region and does not require the transmembrane domain. This essential region represents a novel type of signal with targeting and sorting functions. It is recognized by all three known mitochondrial import receptors, demonstrating their capacity to decode various targeting signals. We suggest that the BCS1 precursor crosses the TOM complex as a loop structure and that once the precursor emerges from the TOM complex, all three structural elements are essential for the intramitochondrial sorting to the inner membrane.     

The Product of the Herpes Simplex Virus Type 1 UL25 Gene Is Required for Encapsidation but Not for Cleavage of Replicated Viral DNA

The herpes simplex virus type 1 (HSV-1) UL25 gene contains a 580-amino-acid open reading frame that codes for an essential protein. Previous studies have shown that the UL25 gene product is a virion component (M. A. Ali et al., Virology 216:278–283, 1996) involved in virus penetration and capsid assembly (C. Addison et al., Virology 138:246–259, 1984). In this study, we describe the isolation of a UL25 mutant (KUL25NS) that was constructed by insertion of an in-frame stop codon in the UL25 open reading frame and propagated on a complementing cell line. Although the mutant was capable of synthesis of viral DNA, it did not form plaques or produce infectious virus in noncomplementing cells. Antibodies specific for the UL25 protein were used to demonstrate that KUL25NS-infected Vero cells did not express the UL25 protein. Western immunoblotting showed that the UL25 protein was associated with purified, wild-type HSV A, B, and C capsids. Transmission electron microscopy indicated that the nucleus of Vero cells infected with KUL25NS contained large numbers of both A and B capsids but no C capsids. Analysis of infected cells by sucrose gradient sedimentation analysis confirmed that the ratio of A to B capsids was elevated in KUL25NS-infected Vero cells. Following restriction enzyme digestion, specific terminal fragments were observed in DNA isolated from KUL25NS-infected Vero cells, indicating that the UL25 gene was not required for cleavage of replicated viral DNA. The latter result was confirmed by pulsed-field gel electrophoresis (PFGE), which showed the presence of genome-size viral DNA in KUL25NS-infected Vero cells. DNase I treatment prior to PFGE demonstrated that monomeric HSV DNA was not packaged in the absence of the UL25 protein. Our results indicate that the product of the UL25 gene is required for packaging but not cleavage of replicated viral DNA.     

3D Modeling for TM Receptors: Algorithms and Validations

This paper outlines the basic strategy to build 3D models of transmembrane G-protein coupled receptors (GPCRs) starting from their amino acid sequences in a block-by-block manner: (i) locate possible TM helical fragments in the GPCR sequence; (ii) build 3D structures for these helices; (iii) arrange isolated helices across the membrane; (iv) calculate all pairwise helix-helix interactions; (v) assemble helical bundle(s); (vi) restore interhelical loops and N- and C-termini; and (vii) refine the entire 3D structure(s). Computer algorithms and preliminary results for most of the steps are discussed.     

SBOL Visual: A Graphical Language for Genetic Designs

Synthetic Biology Open Language (SBOL) Visual is a graphical standard for genetic engineering. It consists of symbols representing DNA subsequences, including regulatory elements and DNA assembly features. These symbols can be used to draw illustrations for communication and instruction, and as image assets for computer-aided design. SBOL Visual is a community standard, freely available for personal, academic, and commercial use (Creative Commons CC0 license). We provide prototypical symbol images that have been used in scientific publications and software tools. We encourage users to use and modify them freely, and to join the SBOL Visual community: http://www.sbolstandard.org/visual.     

Effect of Calcifications on Breast Ultrasound Shear Wave Elastography: An Investigational Study

PurposeTo investigate the effects of macrocalcifications and clustered microcalcifications associated with benign breast masses on shear wave elastography (SWE).MethodsSuperSonic Imagine (SSI) and comb-push ultrasound shear elastography (CUSE) were performed on three sets of phantoms to investigate how calcifications of different sizes and distributions influence measured elasticity. To demonstrate the effect in vivo, three female patients with benign breast masses associated with mammographically-identified calcifications were evaluated by CUSE.ResultsApparent maximum elasticity (E_max) estimates resulting from individual macrocalcifications (with diameters of 2mm, 3mm, 5mm, 6mm, 9mm, 11mm, and 15mm) showed values over 50 kPa for all cases, which represents more than 100% increase over background (~21kPa). We considered a 2cm-diameter circular region of interest for all phantom experiments. Mean elasticity (E_mean) values varied from 26 kPa to 73 kPa, depending on the macrocalcification size. Highly dense clusters of microcalcifications showed higher E_max values than clusters of microcalcification with low concentrations, but the difference in E_mean values was not significant.ConclusionsOur results demonstrate that the presence of large isolated macrocalcifications and highly concentrated clusters of microcalcifications can introduce areas with apparent high elasticity in SWE. Considering that benign breast masses normally have significantly lower elasticity values than malignant tumors, such areas with high elasticity appearing due to presence of calcification in benign breast masses may lead to misdiagnosis.     

Operant Procedures for Assessing Behavioral Flexibility in Rats

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these processes is the ability to adjust behavior in response to changes in one’s environment (i.e., behavioral flexibility). These processes are essential to normal human behavior, and may be disrupted in diverse neuropsychiatric conditions, including schizophrenia, alcoholism, depression, stroke, and Alzheimer’s disease. Understanding of the neurobiology of executive functions has been greatly advanced by the availability of animal tasks for assessing discrete components of behavioral flexibility, particularly strategy shifting and reversal learning. While several types of tasks have been developed, most are non-automated, labor intensive, and allow testing of only one animal at a time. The recent development of automated, operant-based tasks for assessing behavioral flexibility streamlines testing, standardizes stimulus presentation and data recording, and dramatically improves throughput. Here, we describe automated strategy shifting and reversal tasks, using operant chambers controlled by custom written software programs. Using these tasks, we have shown that the medial prefrontal cortex governs strategy shifting but not reversal learning in the rat, similar to the dissociation observed in humans. Moreover, animals with a neonatal hippocampal lesion, a neurodevelopmental model of schizophrenia, are selectively impaired on the strategy shifting task but not the reversal task. The strategy shifting task also allows the identification of separate types of performance errors, each of which is attributable to distinct neural substrates. The availability of these automated tasks, and the evidence supporting the dissociable contributions of separate prefrontal areas, makes them particularly well-suited assays for the investigation of basic neurobiological processes as well as drug discovery and screening in disease models.