An essential role for the caspase dronc in developmentally programmed cell death in Drosophila

Dronc is a caspase recruitment domain-containing Drosophila caspase that is expressed in a temporally and spatially restricted fashion during development. Dronc is the only fly caspase known to be regulated by the hormone ecdysone. Here we show that ectopic expression of dronc in the developing fly eye leads to increased cell death and an ablated eye phenotype that can be suppressed by halving the dosage of the genes in the H99 complex (reaper, hid, and grim) and enhanced by mutations in diap1. In contrast to previous reports, we show that the dronc eye ablation phenotype can be suppressed by coexpression of the baculoviral caspase inhibitor p35. Dronc also interacts, both genetically and biochemically, with the CED-4/Apaf-1 fly homolog, Dark. Furthermore, extracts made from Dark homozygous mutant flies have reduced ability to process Dronc, showing that Dark is required for Dronc processing. Finally, using the RNA interference technique, we show that loss of Dronc function in early Drosophila embryos results in a dramatic decrease in cell death, indicating that Dronc is important for programmed cell death during embryogenesis. These results suggest that Dronc is a key caspase mediating programmed cell death in Drosophila.     

Extracellular matrix and nuclear localization of beta ig-h3 in human bladder smooth muscle and fibroblast cells

The extracellular matrix (ECM) plays an essential role in bladder structure and function. In this study, expression of beta ig-h3, a recently identified extracellular matrix protein, was investigated in human bladder tissue, and human bladder smooth-muscle (SMC) and fibroblast cells in vitro. SMCs secreted greater than three times the level of this protein compared with fibroblasts. The relative levels of beta ig-h3 mRNA in the two cell types reflected the protein expression. Immunohistochemical analysis demonstrated protein deposition in the ECM as well as cytoplasmic localization and, unexpectedly, nuclei. Anti-beta ig-h3 antibodies also stained the matrix surrounding the detrusor SMCs and nuclei of bladder fibroblasts, SMCs, and urothelium in intact bladder tissue. Western blot analyses of medium and matrix fractions obtained from cells in vitro revealed protein of approximately 70-74 kDa, whereas nuclear extracts contained a 65-kDa reactive protein band. We propose that although this protein is a structural component of bladder ECM, its nuclear localization suggests that it has other regulatory and/or structural functions.     

Reversal of the gating polarity of gap junctions by negative charge substitutions in the N-terminus of connexin 32

Intercellular channels formed by connexins (gap junctions) are sensitive to the application of transjunctional voltage (V(j)), to which they gate by the separate actions of their serially arranged hemichannels (Harris, A. L., D. C. Spray, and M. V. L. Bennett. 1981. J. Gen. Physiol. 77:95-117). Single channel studies of both intercellular and conductive hemichannels have demonstrated the existence of two separate gating mechanisms, termed "V(j)-gating" and "loop gating" (Trexler, E. B., M. V. L. Bennett, T. A. Bargiello, and V. K. Verselis. 1996. Proc. Natl. Acad. Sci. U.S.A. 93:5836-5841). In Cx32 hemichannels, V(j)-gating occurs at negative V(j) (Oh, S., J. B. Rubin, M. V. L. Bennett, V. K. Verselis, and T. A. Bargiello. 1999. J. Gen. Physiol. 114:339-364; Oh, S., C. K. Abrams, V. K. Verselis, and T. A. Bargiello. 2000. J. Gen. Physiol. 116:13-31). A negative charge substitution at the second amino acid position in the N-terminus reverses the polarity of V(j)-gating of Cx32 hemichannels (Verselis, V. K., C. S. Ginter, and T. A. Bargiello. 1994. Nature. 368:348-351;. J. Gen. Physiol. 116:13-31). We report that placement of a negative charge at the 5th, 8th, 9th, or 10th position can reverse the polarity of Cx32 hemichannel V(j)-gating. We conclude that the 1st through 10th amino acid residues lie within the transjunctional electric field and within the channel pore, as in this position they could sense changes in V(j) and be largely insensitive to changes in absolute membrane potential (V(m)). Conductive hemichannels formed by Cx32*Cx43E1 containing a negatively charged residue at either the 8th or 10th position display bi-polar V(j)-gating; that is, the open probability of hemichannels formed by these connexins is reduced at both positive and negative potentials and is maximal at intermediate voltages. In contrast, Cx32*Cx43E1 hemichannels with negative charges at either the 2nd or 5th positions are uni-polar, closing only at positive V(j). The simplest interpretation of these data is that the Cx32 hemichannel can adopt at least two different open conformations. The 1st-5th residues are located within the electric field in all open channel conformations, while the 8th and 10th residues lie within the electric field in one conformation and outside the electric field in the other conformation.     

Anthracenone ABA analogue as a potential photoaffinity reagent for ABA-binding proteins

An anthracenone analogue of abscisic acid (ABA) was synthesized as a potential photoaffinity reagent and tested for biological activity. Reaction between 10,10'-dimethoxy-9-anthrone with two equivalents of the lithiated dianion of cis-3-methylpent-2-en-4-yn-1-ol afforded an acetylenic alcohol key intermediate. Subsequent reduction of the triple bond, functional group manipulation of the side chain alcohol and deprotection of the dimethoxy protected anthrone provided anthracenone ABA analogue 7 as a potential photoaffinity reagent for ABA-binding proteins. The effect of natural ABA and the potential photoaffinity anthracenone ABA 7 on corn cell growth was determined at various concentrations. The results show that anthracenone ABA 7 is perceived as ABA-like, although producing less inhibition than ABA itself. For example, 7 at 33 microM produces approximately the same inhibition as ABA at 10 microM.     

Role of the Ring Methyl Groups in Abscisic Acid Activity in Erucic Acid Accumulation in Oilseed Rape (Brassica napus L.)

Modification of the structure of abscisic acid (ABA) has been reported to result in modification of its physiologic activity. In this study we tested the effect of removing methyl groups from the ring and of chirality of ABA on activity in microspore-derived embryos of oilseed rape (Brassica napus L.). The natural (+)-ABA molecule induced growth inhibition and an increase in the amount of erucic acid accumulated in the oil at medium concentrations less than 1 μm. (−)-ABA showed similar effects. Removing the 7′-methyl group resulted in a dramatic decrease in activity: (+)-7′-demethyl-ABA retained some activity as a growth inhibitor; a 10–100 μm concentration of this compound was needed for a response, and (−)-7′-demethyl-ABA was almost completely inactive. Similar effects were observed with regard to elongase activity, which catalyzes erucic acid biosynthesis from oleic acid. Removal of the 8′- and 9′-methyl groups resulted in a more complex response. These compounds all showed intermediate activity; for growth inhibition, the presence of the 9′-methyl was the more important determinant, whereas chirality dominated the response on erucic acid accumulation, with the (+)-enantiomers being more active. Received July 25, 1997; accepted October 31, 1997     

Characterization of terminal NeuNAcalpha2-3Galbeta1-4GlcNAc sequence in lipooligosaccharides of Neisseria meningitidis

Group B and C Neisseria meningitidis are the major cause of meningococcal disease in the United States and in Europe. N . meningitidis lipooligosaccharide (LOS), a major surface antigen, can be divided into 12 immunotypes of which L1 through L8 were found among Group B and C organisms. Groups B and C but not Group A may sialylate their LOSs with N-acetylneuraminic acid (NeuNAc) at the nonreducing end because they synthesize CMP-NeuNAc. Using sialic acid-galactose binding lectins as probes in an ELISA format, six of the eight LOS immunotypes (L2, L3, L4, L5, L7, and L8) in Groups B and C bound specifically to Maackia amurensis leukoagglutinin (MAL), which recognizes NeuNAcalpha2- 3Galbeta1-4GlcNAc/Glc sequence, but not to Sambucus nigra agglutinin, which binds NeuNAcalpha2-6Gal sequence. The combination of SDS-PAGE and MAL-blot analyses revealed that these six LOSs contained only the NeuNAcalpha2-3Galbeta1-4GlcNAc trisaccharide sequence in their 4.1 kDa LOS components, which have a common terminal lacto-N-neotetraose (LNnT, Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) structure when nonsialylated as shown by previous studies. The LOS-lectin binding was abolished when the LOSs were treated with Newcastle disease viral neuraminidase which cleaves alpha2-->3 linked sialic acid. Methylation analysis of a representative LOS (L2) confirmed that NeuNAc is 2-->3 linked to Gal. Thus, these LOSs structurally mimic certain glycolipids, i.e., paragloboside (LNnT-ceramide) and sialylparagloboside and some glycoproteins in having LNnT and N-acetyllactosamine sequences, respectively, with or without alpha2-->3 linked NeuNAc. The molecular mimicry of the LOSs may play a role in the pathogenesis of N.meningitidis by assisting the organism to evade host immune defenses in man.      

A combined parasitological molecular approach for noninvasive characterization of parasitic nematode communities in wild hosts

Most hosts are concurrently or sequentially infected with multiple parasites; thus, fully understanding interactions between individual parasite species and their hosts depends on accurate characterization of the parasite community. For parasitic nematodes, noninvasive methods for obtaining quantitative, species‐specific infection data in wildlife are often unreliable. Consequently, characterization of gastrointestinal nematode communities of wild hosts has largely relied on lethal sampling to isolate and enumerate adult worms directly from the tissues of dead hosts. The necessity of lethal sampling severely restricts the host species that can be studied, the adequacy of sample sizes to assess diversity, the geographic scope of collections and the research questions that can be addressed. Focusing on gastrointestinal nematodes of wild African buffalo, we evaluated whether accurate characterization of nematode communities could be made using a noninvasive technique that combined conventional parasitological approaches with molecular barcoding. To establish the reliability of this new method, we compared estimates of gastrointestinal nematode abundance, prevalence, richness and community composition derived from lethal sampling with estimates derived from our noninvasive approach. Our noninvasive technique accurately estimated total and species‐specific worm abundances, as well as worm prevalence and community composition when compared to the lethal sampling method. Importantly, the rate of parasite species discovery was similar for both methods, and only a modest number of barcoded larvae (n = 10) were needed to capture key aspects of parasite community composition. Overall, this new noninvasive strategy offers numerous advantages over lethal sampling methods for studying nematode–host interactions in wildlife and can readily be applied to a range of study systems.