A new quinoline-based chemical probe inhibits the autophagy-related cysteine protease ATG4B.

The cysteine protease ATG4B is a key component of the autophagy machinery, acting to proteolytically prime and recycle its substrate MAP1LC3B. The roles of ATG4B in cancer and other diseases appear to be context dependent but are still not well understood. To help further explore ATG4B functions and potential therapeutic applications, we employed a chemical biology approach to identify ATG4B inhibitors. Here, we describe the discovery of 4-28, a styrylquinoline identified by a combined computational modeling, in silico screening, high content cell-based screening and biochemical assay approach. A structure-activity relationship study led to the development of a more stable and potent compound LV-320. We demonstrated that LV-320 inhibits ATG4B enzymatic activity, blocks autophagic flux in cells, and is stable, non-toxic and active in vivo. These findings suggest that LV-320 will serve as a relevant chemical tool to study the various roles of ATG4B in cancer and other contexts.

Germline transformation of the western corn rootworm, Diabrotica virgifera virgifera.

The western corn rootworm (WCR), a major pest of maize, is notorious for rapidly adapting biochemically, behaviourally and developmentally to a variety of control methods. Despite much effort, the genetic basis of WCR adaptation remains a mystery. Since transformation-based applications such as transposon tagging and enhancer trapping have facilitated genetic dissection of model species such as Drosophila melanogaster, we developed a germline-transformation system for WCR in an effort to gain a greater understanding of the basic biology of this economically important insect. Here we report the use of a fluorescent-marked Minos element to create transgenic WCR. We demonstrate that the transgenic strains express both an eye-specific fluorescent marker and piggyBac transposase. We identified insertion-site junction sequences via inverse PCR and assessed insertion copy number using digital droplet PCR (ddPCR). Interestingly, most WCR identified as transgenic via visual screening for DsRed fluorescence proved to carry multiple Minos insertions when tested via ddPCR. A total of eight unique insertion strains were created by outcrossing the initial transgenic strains to nontransgenic WCR mates. Establishing transgenic technologies for this beetle is the first step towards bringing a wide range of transformation-based tools to bear on understanding WCR biology.

Programmed cell death takes flight: genetic and genomic approaches to gene discovery in Drosophila.

Programmed cell death (PCD) is an essential and wide-spread physiological process that results in the elimination of cells. Genes required to carry out this process have been identified, and many of these remain the subjects of intense investigation. Here, we describe PCD, its functions, and some of the consequences when it goes awry. We review PCD in the model system, the fruit fly, Drosophila melanogaster, with a particular emphasis on cell death gene discovery resulting from both genetics and genomics-based approaches.

Acidic conditions stabilise intermediates populated during the folding of Im7 and Im9.

The helical bacterial immunity proteins Im7 and Im9 have been shown to fold via kinetic mechanisms of differing complexity, despite having 60 % sequence identity. At pH 7.0 and 10 degrees C, Im7 folds in a three-state mechanism involving an on-pathway intermediate, while Im9 folds in an apparent two-state transition. In order to examine the folding mechanisms of these proteins in more detail, the folding kinetics of both Im7 and Im9 (at 10 degrees C in 0.4 M sodium sulphate) have been examined as a function of pH. Kinetic modelling of the folding and unfolding data for Im7 between pH 5.0 and 8.0 shows that the on-pathway intermediate is stabilised by more acidic conditions, whilst the native state is destabilised. The opposing effect of pH on the stability of these states results in a significant population of the intermediate at equilibrium at pH 6.0 and below. At pH 7.0, the folding and unfolding kinetics for Im9 can be fitted adequately by a two-state model, in accord with previous results. However, under acidic conditions there is a clear change of slope in the plot of the logarithm of the folding rate constant versus denaturant concentration, consistent with the population of one or more intermediate(s) early during folding. The kinetic data for Im9 at these pH values can be fitted to a three-state model, where the intermediate ensemble is stabilised and the native state destabilised as the pH is reduced, rationalising previous results that showed that an intermediate is not observed experimentally at pH 7.0. The data suggest that intermediate formation is a general step in immunity protein folding and demonstrate that it is necessary to explore a wide range of refolding conditions in order to show that intermediates do not form in the folding of other small, single-domain proteins.

A screen for dominant modifiers of the irreC-rst cell death phenotype in the developing Drosophila retina.

Programmed cell death (PCD) in the Drosophila retina requires activity of the irregular chiasmC-roughest (irreC-rst) gene. Loss-of-function mutations in irreC-rst block PCD during retinal development and lead to a rough eye phenotype in the adult. To identify genes that interact with irreC-rst and may be involved in PCD, we conducted a genetic screen for dominant enhancers and suppressors of the adult rough eye phenotype. We screened 150,000 mutagenized flies and recovered 170 dominant modifiers that localized primarily to the second and third chromosomes. At least two allelic groups correspond to previously identified death regulators, Delta and dRas1. Examination of retinae from homozygous viable mutants indicated two major phenotypic classes. One class exhibited pleiotropic defects while the other class exhibited defects specific to the cell population that normally undergoes PCD.

Posttranslational modification and plasma membrane localization of the Drosophila melanogaster presenilin.

Mutations in two genes, presenilin 1 (PS1) and presenilin 2, are linked to early onset cases of familial Alzheimer's disease. The presenilins are thought to contribute to the pathogenesis of Alzheimer's disease by directly or indirectly affecting the proteolytic processing of the amyloid precursor protein. They have also been implicated in the proteolytic processing of Notch. In PS1-deficient mammalian cells, the proteolytic release of the Notch intracellular domain is reduced. Likewise, loss-of-function mutations in Drosophila presenilin (Psn) prevent the production of the intracellular Notch signaling fragment and lead to phenotypes resembling Notch mutants. Here we characterize the Drosophila Psn protein and demonstrate that it undergoes a proteolytic cleavage. We describe Psn expression at different developmental stages of the fly and show Psn localization near both apical and basal plasma membranes. Furthermore, we demonstrate that portions of the Psn protein span the plasma membrane in S2 cells.

Expression of protein tyrosine phosphatase genes during oogenesis in Drosophila melanogaster.

The spatial and temporal expression of seven Drosophila protein tyrosine phosphatase genes during oogenesis was examined by whole mount in-situ hybridization of antisense RNA probes to ovaries. Our observations indicate diverse expression patterns consistent with multiple roles for protein tyrosine phosphatases in the ovary. DPTP99A and corkscrew transcripts are expressed in follicle cells, consistent with possible roles in the EGF receptor signaling pathway. Transcripts from corkscrew and DPTP10D are detected in the germline during oogenesis and localized to the oocyte during egg chamber development. Localization of the two transcripts is disrupted by mutations in egalitarian and Bicaudal D. DLAR and DPTP4E transcripts are found in the germline during the same developmental stages as DPTP10D transcripts, but their transcripts are not localized to the oocyte. DPTP61F transcription is detected only after stage 6 of oogenesis. After stage 10B these transcripts are transported to the oocyte; thus ovarian transcription of DPTP61F may reflect a maternal contribution of the mRNA for later use during embryogenesis. DPTP69D transcripts are sequestered in the nucleus from stage 7 to stage 10, and then released to the cytoplasm. Our observations suggest that the export of DPTP69D mRNA from the nucleus is temporally regulated during oogenesis.

Linkage analysis of X-linked cleft palate and ankyloglossia in Manitoba Mennonite and British Columbia Native kindreds.

A locus (CPX) responsible for X-linked cleft palate and ankyloglossia was previously mapped to the proximal long arm of the X chromosome through DNA marker linkage studies in two large kindred: an Icelandic family and a British Columbia (B.C.) Native family. In this study, additional linkage analyses have been performed in the B.C. family and in a newly identified Manitoba Mennonite family with X-linked cleft palate and ankyloglossia. The Manitoba CPX locus maps to the same region as Icelandic and B.C. CPX. Two-point disease-to-marker linkage analyses in the Manitoba family indicate a maximum lod score (Zmax) between CPX and DXS349 (Zmax = 3.33 at theta = 0.0). In multipoint linkage analysis, combined data from the B.C. and Manitoba families suggest that the most likely location for CPX is at DXS447 in Xq21.1 (multipoint Z = 13.5). The support interval for CPX at DXS447 extends approximately from PGK1 to DXYS1 and includes a newly isolated polymorphic locus DXS1109.

The phylogeny of echinoderm classes based on mitochondrial gene arrangements.

Previous analyses have demonstrated that, among the echinoderms, the sea star (class: Asteroidea) mitochondrial genome contains a large inversion in comparison to the mitochondrial DNA of sea urchins (class: Echinoidea). Polymerase chain reaction amplification, DNA cloning, and sequencing have been used to examine the relationships of the brittle stars (class: Ophiuroidea) and sea cucumbers (class: Holothuroidea) to the sea stars and sea urchins. The DNA sequence of the regions spanning potential inversion junctions in both brittle stars and sea cucumbers has been determined. This study has also revealed a highly modified tRNA cluster in the ophiuroid mitochondrial genome. Our data indicate mitochondrial gene arrangement patterns that group the sea cucumbers with sea urchins and sea stars with brittle stars. This use of molecular characters clarifies the relationships among these classes.

The gene responsible for X-linked cleft palate (CPX) in a British Columbia native kindred is localized between PGK1 and DXYS1.

Human craniofacial malformations are a class of common congenital anomalies in which the etiology is heterogeneous and often poorly understood. To better delineate the molecular basis of craniofacial development, we have undertaken a series of experiments directed toward the isolation of a gene involved in human secondary palate formation. DNA marker linkage studies have been performed in a large British Columbia (B.C.) Native family in which cleft palate segregates as an X-linked trait. We have examined 62 family members, including 15 affected males and 8 obligate carrier females. A previous clinical description of the clefting defect in this kindred included submucous cleft palate and bifid or absent uvula. Our recent reevaluation of the family has indicated that ankyloglossia (tongue-tie) is also a feature of X-linked cleft palate in some of the affected males and carrier females. Ankyloglossia has previously been associated with X-linked cleft palate in an Icelandic kindred in which a gene responsible for cleft palate (CPX) was assigned to the Xq21.3-q22 region between DXYS12 and DXS17. For the B.C. kindred reported here, we have mapped the gene responsible for cleft palate and/or ankyloglossia to a more proximal position on the X chromosome. No recombination was observed between B.C. CPX and the DNA marker DXS72 (peak lod score [Zmax] = 7.44 at recombination fraction [theta] = .0) localized to Xq21.1. Recombination was observed between CPX and PGK1 (Zmax = 7.35 at theta = .03) and between CPX and DXYS1 (Zmax = 5.59 at theta = .04). These recombination events localize B.C. CPX between PGK1 and DXYS1 in the Xq13-q21.31 region.

Double pattern of relationship between skin temperature, thermoregulation and sensory nerve conduction.

Electroneurographic and thermographic investigations were done in 32 persons. Sensory nerve conduction velocity, amplitude of sensory nerve potential, subjective and objective sensory thresholds were determined during stimulation of each finger. The maximal and minimal skin temperatures for each finger were evaluated from thermograms taken from the dorsal and palmar surface of the hand before and after standard cooling test. The measurements were done in a thermostabilised room at 19-21 degrees C effective ambient temperature. The second degree correlations between the electroneurographic and thermographic parameters were calculated. The statistical analysis revealed the presence of a double correlation pattern depending on the homoiothermic or poikilothermic thermoregulatory ability of the finger. The differentiation threshold criterion for the poikilo- and homoiothermic group assignment was the minimal rest temperature of the finger equal to 28 degrees C. The correlations in the poikilothermic fingers were very strong and much stronger than in the homoiothermic fingers. Correlations with temperatures were strong both at rest as well as after cooling. Correlations for the sensory nerve potential amplitude likewise for the objective and subjective thresholds were stronger than for the conduction velocity. Sensory nerve potential amplitude increases and subjective and objective thresholds decrease with finger temperature. The obtained results suggest that sensory nerve conduction is related not only to the actual tissue temperature but also to local thermoregulatory ability.

Nucleotide sequence of nine protein-coding genes and 22 tRNAs in the mitochondrial DNA of the sea star Pisaster ochraceus.

We have cloned and sequenced over 9 kb of the mitochondrial genome from the sea star Pisaster ochraceus. Within a continuous 8.0-kb fragment are located the genes for NADH dehydrogenase subunits 1, 2, 3, and 4L (ND1, ND2, ND3, and ND4L), cytochrome oxidase subunits I, II, and III (COI, COII, and COIII), and adenosine triphosphatase subunits 6 and 8 (ATPase 6 and ATPase 8). This large fragment also contains a cluster of 13 tRNA genes between ND1 and COI as well as the genes for isoleucine tRNA between ND1 and ND2, arginine tRNA between COI and ND4L, lysine tRNA between COII and ATPase 8, and the serine (UCN) tRNA between COIII and ND3. The genes for the other five tRNAs lie outside this fragment. The gene for phenylalanine tRNA is located between cytochrome b and the 12S ribosomal genes. The genes for tRNA(glu) and tRNA(thr) are 3' to 12S ribosomal gene. The tRNAs for histidine and serine (AGN) are adjacent to each other and lie between ND4 and ND5. These data confirm the novel gene order in mitochondrial DNA (mtDNA) of sea stars and delineate additional distinctions between the sea star and other mtDNA molecules.

Gene arrangement in sea star mitochondrial DNA demonstrates a major inversion event during echinoderm evolution.

The mitochondrial (mt) DNA from the sea star Pisaster ochraceus has been isolated, restriction-mapped, and cloned into plasmid vectors. Both ribosomal RNA genes, the genes for 12 of the 13 mitochondrial proteins, and 11 of the tRNA genes have been localized by DNA sequence analyses. The sequence arrangement of the genes is markedly different from that seen in sea urchin mitochondrial DNA. A segment of the DNA molecule extending from tRNA(pro), including the tRNA cluster, ND1, ND2, and 16S genes, is inverted in relation to the sea urchin genome. The resulting gene order in the sea star is 12S, 16S, ND2, tRNA cluster, COI. As a result of the inversion, the transcriptional polarity of ND1, ND2, and 16S genes are opposite to that of the 12S and COI genes. The arrangement and transcriptional polarity of the other genes mapped here is the same as seen in urchin.

Relationship between sensory nerve conduction and temperature of the hand.

Electroneurographic and thermographic investigations were carried out in 32 persons. Sensory nerve conduction velocity, the amplitude of sensory nerve potential, subjective and objective electrical threshold were determined during the stimulation of each finger. Thermograms were taken before and after the standard cooling test. Statistical analysis has revealed that all the results of electroneurographic and thermographic parameters obtained from the fingers of the same person as well as of all investigated persons are directly connected. Highly significant (p = 0.001 and 0.01) correlations of second degree were found between electroneurographic and temperature parameters.