Regulators of Autophagosome Formation in Drosophila Muscles

Given the diversity of autophagy targets and regulation, it is important to characterize autophagy in various cell types and conditions. We used a primary myocyte cell culture system to assay the role of putative autophagy regulators in the specific context of skeletal muscle. By treating the cultures with rapamycin (Rap) and chloroquine (CQ) we induced an autophagic response, fully suppressible by knockdown of core ATG genes. We screened D. melanogaster orthologs of a previously reported mammalian autophagy protein-protein interaction network, identifying several proteins required for autophagosome formation in muscle cells, including orthologs of the Rab regulators RabGap1 and Rab3Gap1. The screen also highlighted the critical roles of the proteasome and glycogen metabolism in regulating autophagy. Specifically, sustained proteasome inhibition inhibited autophagosome formation both in primary culture and larval skeletal muscle, even though autophagy normally acts to suppress ubiquitin aggregate formation in these tissues. In addition, analyses of glycogen metabolic genes in both primary cultured and larval muscles indicated that glycogen storage enhances the autophagic response to starvation, an important insight given the link between glycogen storage disorders, autophagy, and muscle function.     

Novel lynx spider toxin shares common molecular architecture with defense peptides from frog skin

A unique 30-residue cationic peptide oxyopinin 4a (Oxt 4a) was identified in the venom of the lynx spider Oxyopes takobius (Oxyopidae). Oxt 4a contains a single N-terminally located disulfide bond, Cys4-Cys10, and is structurally different from any spider toxin studied so far. According to NMR findings, the peptide is disordered in water, but assumes a peculiar torpedo-like structure in detergent micelles. It features a C-terminal amphipathic α-helical segment (body; residues 12-25) and an N-terminal disulfide-stabilized loop (head; residues 1-11), and has an unusually high density of positive charge in the head region. Synthetic Oxt 4a was produced and shown to possess strong and broad-spectrum cytolytic and antimicrobial activity. cDNA cloning showed that the peptide is synthesized in the form of a conventional prepropeptide with an acidic prosequence. Unlike other arachnid toxins, Oxt 4a exhibits striking similarity with defense peptides from the skin of ranid frogs that contain the so-called Rana-box motif (a C-terminal disulfide-enclosed loop). Parallelism or convergence is apparent on several levels: the structure, function and biosynthesis of a lynx spider toxin are mirrored by those of Rana-box peptides from frogs.     

A method for two-dimensional registration and construction of the two-dimensional atlas of gene expression patterns in situ

We apply the fast redundant dyadic wavelet transform to the spatial registration of two-dimensional gene expression patterns of 736 Drosophila melanogaster embryos. This method is superior to the Fourier transform or windowed Fourier transform because of its ability to reduce noise and is of high resolution. In registration of the dataset we use two cost functions based on computing the Euclidean or Mahalanobis distance. The algorithm shows a high level of accuracy. For early temporal classes the cost function based on Mahalanobis distance gives better results. We have reported a method for construction of an integrated dataset elsewhere. In this paper the method is extended to the two-dimensional case. The procedure for data assembly provides for the preservation of some aspects of the nuclear structure of a two-dimensional gene expression pattern. It is based on creating an averaged model that reproduces the spatial distribution of nuclei over the embryo image. The average concentrations of each protein in each averaged nucleus are computed from the series of embryos of the same age.     

Spatial registration of data on gene expression in situ]

For registering data on the in situ expression of segmentation genes, a method of image registration was developed basing on the spline approximation. The reference points for the registration were the coordinates of extrema in one-dimensional patterns of gene expression. This registration method is characterized by a very high accuracy. A method of creating a generalized pattern of gene expression in single cells is proposed. Such patterns were constructed for nine segmentation genes belonging to the gap and pair-rule classes of genes.     

Aminotransferases in early development of salmonid fish

The activities of alanine and aspartate aminotransferases were assessed in the embryos and juveniles of the rainbow trout Parasalmo mukiss L. and chum salmon Oncorchyncus keta W. Changes in subcellular localization of these enzymes and their activities were found at different pH optima in each subcellular fraction of the rainbow trout during ontogenesis.     

Enzyme immunoassay of ampicillin in milk

An indirect immunoassay for quantitative determination of ampicillin (range, 10-1000 ng/ml) in buffer or milk has been developed. Polyclonal antibodies were obtained against ampicillin conjugated with bovine serum albumin; the conjugate was synthesized by direct condensation using carbodiimide. The antibodies were specific for ampicillin and exhibited low cross-reactivity to other penicillins (azlocillin, 17%; penicillin G, 10%; piperacillin, 5%; and carbenicillin, 4%). Matrix effects were minimized by combining the use of a casein-supplemented buffer (content of casein, 1%) with sample dilution. The threshold of ampicillin detection in milk (diluted tenfold) was equal to 5.0 ng/ml (which corresponded to 50 ng/ml of the original sample).     

A database for management of gene expression data in situ

MOTIVATION: To create a spatiotemporal atlas of Drosophila segmentation gene expression at cellular resolution. RESULTS: The expression of segmentation genes plays a crucial role in the establishment of the Drosophila body plan. Using the IBM DB2 Relational Database Management System we have designed and implemented the FlyEx database. FlyEx contains 2832 images of 14 segmentation gene expression patterns obtained from 954 embryos and 2,073,662 quantitative data records. The averaged data is available for most of segmentation genes at eight time points. FlyEx supports operations on images of gene expression patterns. The database can be used to examine the quality of data, analyze the dynamics of formation of segmentation gene expression domains, as well as estimate the variability of gene expression patterns. We also provide the capability to download data of interest. AVAILABILITY: http://urchin.spbcas.ru/flyex, http://flyex.ams.sunysb.edu/flyex