Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.      

Evolutionary origin of human and primate malarias: evidence from the circumsporozoite protein gene

We have analyzed the conserved regions of the gene coding for the circumsporozoite protein (CSP) in 12 species of Plasmodium, the malaria parasite. The closest evolutionary relative of P. falciparum, the agent of malignant human malaria, is P. reichenowi, a chimpanzee parasite. This is consistent with the hypothesis that P. falciparum is an ancient human parasite, associated with humans since the divergence of the hominids from their closest hominoid relatives. Three other human Plasmodium species are each genetically indistinguishable from species parasitic to nonhuman primates; that is, for the DNA sequences included in our analysis, the differences between species are not greater than the differences between strains of the human species. The human P. malariae is indistinguishable from P. brasilianum, and P. vivax is indistinguishable from P. simium; P. brasilianum and P. simium are parasitic to New World monkeys. The human P. vivax-like is indistinguishable from P. simiovale, a parasite of Old World macaques. We conjecture that P. malariae, P. vivax, and P. vivax-like are evolutionarily recent human parasites, the first two at least acquired only within the last several thousand years, and perhaps within the last few hundred years, after the expansion of human populations in South America following the European colonizations. We estimate the rate of evolution of the conserved regions of the CSP gene as 2.46 x 10(-9) per site per year. The divergence between the P. falciparum and P. reichenowi lineages is accordingly dated 8.9 Myr ago. The divergence between the three lineages leading to the human parasites is very ancient, about 100 Myr old between P. malariae and P. vivax (and P. vivax-like) and about 165 Myr old between P. falciparum and the other two.      

Oligomeric forms of the membrane-bound acetylcholine receptor disclosed upon extraction of the M(r) 43,000 nonreceptor peptide

Oligomeric forms of the acetylcholine receptor are directly visualized by electron microscopy in receptor-rich membranes from torpedo marmorata. The receptor structures are quantitatively correlated with the molecular species so far identified only after detergent solubilization, and further related to the polypeptide composition of the membranes and changes thereof. The structural identification is made possibly by the increased fragility of the membranes after extraction of nonreceptor peptides and their subsequent disruption upon drying onto hydrophilic carbon supports. Receptor particles in native membranes depleted of nonreceptor peptides appear as single units of 7-8 nm, and double and multiple aggregates thereof. Particle doublets having a main-axis diameter of 19 +/- 3 nm predominate in these membranes. Linear aggregates of particles similar to those observed in rotary replicas of quick-frozen fresh electrolytes (Heuser, J.E. and S. R. Salpeter. 1979, J. Cell Biol. 82: 150-173) are also present in the alkaline-extracted membranes. Chemical modifications of the thiol groups shift the distribution of structural species. Dithiothreitol reduction, which renders almost exclusively the 9S, monomeric receptor form, results in the observation of the 7-8 nm particle in isolated form. The proportion of doublets increases in membranes alkylated with N-ethylmaleimide. Treatment with 5,5’-dithiobis-(nitrobenzoic acid) increases the proportion of higher oligomeric species, and particle aggregates (n=oligo) predominate. The nonreceptor v-peptide (doublet of M(r) 43,000) appears to play a role in the receptor monomer-polymer equilibria. Receptor protein and v-peptide co-aggregate upon reduction and reoxidation of native membranes. In membranes protected ab initio with N- ethylmaleimide, only the receptor appears to self-aggregate. The v-peptide cannot be extracted from these alkylated membranes, though it is easily released from normal, subsequently alkylated or reduced membranes. A stabilization of the dimeric species by the nonreceptor v-peptide is suggested by these experiments. Monospecific antibodies against the v-peptide are used in conjunction with rhodamine- labeled anti-bodies in an indirect immunoflourescence assay to map the vectorial exposure of the v-peptide. When intact membranes, v-peptide depleted and “holey” native membranes (treated with 0.3 percent saponin) are compared, maximal labeling is obtained with the latter type of membranes, suggesting a predominantly cytoplasmic exposure of the antigenic determinants of the v-peptide in the membrane. The influence of the v-peptide in the thiol-dependent interconversions of the receptor protein and the putative topography of the peptide are analyzed in the light of the present results.     

High-throughput screen identifies disulfiram as a potential therapeutic for triple-negative breast cancer cells: Interaction with IQ motif-containing factors

Triple-negative breast cancer (TNBC) represents an aggressive subtype, for which radiation and chemotherapy are the only options. Here we describe the identification of disulfiram, an FDA-approved drug used to treat alcoholism, as well as the related compound thiram, as the most potent growth inhibitors following high-throughput screens of 3185 compounds against multiple TNBC cell lines. The average IC₅₀ for disulfiram was ~300 nM. Drug affinity responsive target stability (DARTS) analysis identified IQ motif-containing factors IQGAP1 and MYH9 as direct binding targets of disulfiram. Indeed, knockdown of these factors reduced, though did not completely abolish, cell growth. Combination treatment with 4 different drugs commonly used to treat TNBC revealed that disulfiram synergizes most effectively with doxorubicin to inhibit cell growth of TNBC cells. Disulfiram and doxorubicin cooperated to induce cell death as well as cellular senescence, and targeted the ESA⁺/CD24^-/low/CD44⁺ cancer stem cell population. Our results suggest that disulfiram may be repurposed to treat TNBC in combination with doxorubicin.     

Pex30p, Pex31p, and Pex32p form a family of peroxisomal integral membrane proteins regulating peroxisome size and number in Saccharomyces cerevisiae

The peroxin Pex23p of the yeast Yarrowia lipolytica exhibits high sequence similarity to the hypothetical proteins Ylr324p, Ygr004p, and Ybr168p encoded by the Saccharomyces cerevisiae genome. Ylr324p, Ygr004p, and Ybr168p are integral to the peroxisomal membrane and act to control peroxisome number and size. Synthesis of Ylr324p and Ybr168p, but not of Ygr004p, is induced during incubation of cells in oleic acid-containing medium, the metabolism of which requires intact peroxisomes. Cells deleted for YLR324w exhibit increased numbers of peroxisomes, whereas cells deleted for YGR004w or YBR168w exhibit enlarged peroxisomes. Ylr324p and Ybr168p cannot functionally substitute for one another or for Ygr004p, whereas Ygr004p shows partial functional redundancy with Ylr324p and Ybr168p. Ylr324p, Ygr004p, and Ybr168p interact within themselves and with Pex28p and Pex29p, which have been shown also to regulate peroxisome size and number. Systematic deletion of genes demonstrated that PEX28 and PEX29 function upstream of YLR324w, YGR004w, and YBR168w in the regulation of peroxisome proliferation. Our data suggest a role for Ylr324p, Ygr004p, and Ybr168p--now designated Pex30p, Pex31p, and Pex32p, respectively--together with Pex28p and Pex29p in controlling peroxisome size and proliferation in Saccharomyces cerevisiae.     

Systematic exploration of essential yeast gene function with temperature-sensitive mutants

Conditional temperature-sensitive (ts) mutations are valuable reagents for studying essential genes in the yeast Saccharomyces cerevisiae. We constructed 787 ts strains, covering 497 (～45%) of the 1,101 essential yeast genes, with ～30% of the genes represented by multiple alleles. All of the alleles are integrated into their native genomic locus in the S288C common reference strain and are linked to a kanMX selectable marker, allowing further genetic manipulation by synthetic genetic array (SGA)-based, high-throughput methods. We show two such manipulations: barcoding of 440 strains, which enables chemical-genetic suppression analysis, and the construction of arrays of strains carrying different fluorescent markers of subcellular structure, which enables quantitative analysis of phenotypes using high-content screening. Quantitative analysis of a GFP-tubulin marker identified roles for cohesin and condensin genes in spindle disassembly. This mutant collection should facilitate a wide range of systematic studies aimed at understanding the functions of essential genes.     

Integrating high-throughput genetic interaction mapping and high-content screening to explore yeast spindle morphogenesis

We describe the application of a novel screening approach that combines automated yeast genetics, synthetic genetic array (SGA) analysis, and a high-content screening (HCS) system to examine mitotic spindle morphogenesis. We measured numerous spindle and cellular morphological parameters in thousands of single mutants and corresponding sensitized double mutants lacking genes known to be involved in spindle function. We focused on a subset of genes that appear to define a highly conserved mitotic spindle disassembly pathway, which is known to involve Ipl1p, the yeast aurora B kinase, as well as the cell cycle regulatory networks mitotic exit network (MEN) and fourteen early anaphase release (FEAR). We also dissected the function of the kinetochore protein Mcm21p, showing that sumoylation of Mcm21p regulates the enrichment of Ipl1p and other chromosomal passenger proteins to the spindle midzone to mediate spindle disassembly. Although we focused on spindle disassembly in a proof-of-principle study, our integrated HCS-SGA method can be applied to virtually any pathway, making it a powerful means for identifying specific cellular functions.     

Characterization of the TRBP domain required for Dicer interaction and function in RNA interference

Background  

Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC). While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP) that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain.