Localization of sequences regulating ancestral and acquired sites of esterase6 activity in Drosophila melanogaster

We have broadly defined the DNA regions regulating esterase6 activity in several life stages and tissue types of D. melanogaster using P- element-mediated transformation of constructs that contain the esterase6 coding region and deletions or substitutions in 5' or 3' flanking DNA. Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and the primary sequences regulating its activity lie between -171 and -25 bp relative to the translation initiation site: deletion of these sequences decrease activity approximately 20-fold. Hemolymph activity is also modulated by four other DNA regions, three of which lie 5' and one of which lies 3' of the coding region. Of these, two have positive and two have negative effects, each of approximately twofold. Esterase6 activity is present also in two male reproductive tract tissues; the ejaculatory bulb, which is another ancestral activity site, and the ejaculatory duct, which is a recently acquired site within the melanogaster species subgroup. Activities in these tissues are at least in part independently regulated: activity in the ejaculatory bulb is conferred by sequences between -273 and -172 bp (threefold decrease when deleted), while activity in the ejaculatory duct is conferred by more distal sequences between -844 and -614 bp (fourfold decrease when deleted). The reproductive tract activity is further modulated by two additional DNA regions, one in 5' DNA (-613 to -284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to +2731 bp; threefold decrease when deleted) that probably overlaps the adjacent esteraseP gene. Collating these data with previous studies suggests that expression of EST6 in the ancestral sites is mainly regulated by conserved proximal sequences while more variable distal sequences regulate expression in the acquired ejaculatory duct site.      

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Molecular cloning and primary structure of rat testes metalloendopeptidase EC 3.4.24.15

The complete amino acid sequence of rat testes metalloendopeptidase (EC 3.4.24.15) was deduced from the nucleotide sequence of a cDNA clone isolated by screening a rat testes library with a polyclonal antibody raised against a homogeneous preparation of the rat testes enzyme. The correctness of the sequence was verified by N-terminal amino acid sequence analysis of the isolated enzyme and by partial amino acid sequence analysis of three tryptic peptides located near the N-terminus, the middle, and C-terminus of the native protein. The enzyme is composed of 645 amino acids with a molecular weight of 72,985. This value is close to that of the purified rat testes and brain enzyme as determined by polyacrylamide gel electrophoresis under denaturing and reducing conditions and by molecular sieving chromatography. The enzyme contains the putative active-site sequence -H-E-F-G-H- that is homologous to the sequence in the active site of thermolysin and several other related bacterial enzymes, as well as to active-site sequences of several mammalian zinc metallopeptidases. No amino acid sequence homology, beyond this active site, was found with thermolysin, a bacterial zinc metalloendopeptidase, nor with several mammalian zinc metallopeptidases. Northern blot hybridization analyses showed the presence of mRNA encoding the enzyme in rat testes, but not in other rat tissues in spite of the finding that enzyme activity is widely distributed in all tissues and that relatively high activities are present in rat brain and pituitary.     

The role of snail aestivation in transmission of schistosomiasis in changing climatic conditions

Schistosomiasis vector snails are subjected to extreme seasonal changes, particularly in ephemeral rivers and lentic waterbodies. In the tropics, aestivation is one of the adaptive strategies for survival and is used by snails in times of extremely high temperatures and desiccation. Aestivation therefore plays an important role in maintaining the transmission of schistosomiasis. This review assesses the possible impacts of climate change on the temporal and spatial distribution of schistosomiasis-transmitting snails with special emphasis on aestivation, and discusses the effect of schistosome infection on aestivation ability. The impacts of parasite development on snails, as well as physiological changes, are discussed with reference to schistosomiasis transmission. This review shows that schistosome-infected snails have lower survival rates during aestivation, and that those that survive manage to get rid of the infection. In general, snail aestivation ability is poor and survival chances diminish with time. Longer dry periods result in fewer, as well as uninfected, snails. However, the ability of the surviving snails to repopulate the habitats is high.     

Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.     

Neuroproteomics: Expression Profiling of the Brain's Proteomes in Health and Disease

The term "proteome" describes the protein complement of a genome. Proteomes of cells are dynamic and are directly affected by environmental factors, such as stress and/or drug treatment, or as a result of aging and disease. One of the distinct advantages of proteomic analysis, not attainable with RNA expression data, is the ability to fractionate the cell's proteins into various subpopulations. In neuroscience, "neuromics" (proteomics in the central nervous system) is in its infancy, with a paucity of studies in the context of the brain. One of the objectives of this review is to illustrate the potential of neuromics to profile differences in the distribution of thousands of proteins as a function of disease markers. We have previously used this approach to determine the effects of varied postmortem interval in examining human brain tissue and to identify biomarkers. Here we review proteomic studies of schizophrenia, Alzheimer's disease, and Parkinson's disease. Experimental results regarding Parkinson's disease are presented to illustrate the potential of neuromics to identify pathways of pathogenesis and novel therapeutic targets.     

Involvement of surface cysteines in activity and multimer formation of thimet oligopeptidase

Thimet oligopeptidase is a metalloenzyme involved in regulating neuropeptide processing. Three cysteine residues (246, 248, 253) are known to be involved in thiol activation of the enzyme. In contrast to the wild-type enzyme, the triple mutant (C246S/C248S/C253S) displays increased activity in the absence of dithiothreitol. Dimers, purportedly formed through cysteines 246, 248 and 253, have been thought to be inactive. However, analysis of the triple mutant by native gel electrophoresis reveals the existence of dimers and multimers, implying that oligomer formation is mediated by other cysteines, probably on the surface, and that some of these forms are enzymatically active. Isolation and characterization of iodoacetate-modified monomers and dimers of the triple mutant revealed that, indeed, certain dimeric forms of the enzyme are still fully active, whereas others show reduced activity. Cysteine residues potentially involved in dimerization were identified by modeling of thimet oliogopeptidase to its homolog, neurolysin. Five mutants were constructed; all contained the triple mutation C246S/C248S/C253S and additional substitutions. Substitutions at C46 or C682 and C687 prevented multimer formation and inhibited dimer formation. The C46S mutant had enzymatic activity comparable to the parent triple mutant, whereas that of C682S/C687S was reduced. Thus, the location of intermolecular disulfide bonds, rather than their existence per se, is relevant to activity. Dimerization close to the N-terminus is detrimental to activity, whereas dimerization near the C-terminus has little effect. Altering disulfide bond formation is a potential regulatory factor in the cell owing to the varying oxidation states in subcellular compartments and the different compartmental locations and functions of the enzyme.     

Use of the MIDI-FAME technique to characterize groundwater communities

Fatty acid methyl ester (FAME) profiles were identified directly from groundwater microbial communities concentrated on and extracted with polycarbonate filters. The sensitivity of this direct extraction method was determined using pure cultures of Acinetobacter junii, Pseudomonas putida and Stenotrophomonas maltophilia. A minimum concentration of 107 cells filter-1 was required to identify the predominant fatty acids from each culture. However, at least 3.7 x 109 cells filter-1 were required to obtain fatty acid profiles that matched the signature profiles for pure cultures in a commercial database. While several saturated fatty acids (i.e. 14 : 0, 16 : 0, 18 : 0) were extracted from the polycarbonate filters, they were readily subtracted from microbial fatty acid profiles and did not interfere with the characterization of pure cultures or environmental samples. For the environmental samples, 3 l of groundwater from the Savannah River Site, Aiken, SC, (USA) contained sufficient biomass for direct extraction. A comparative analysis of FAME groundwater profiles demonstrated a qualitative difference among communities sampled from spatially discrete locations, while a groundwater well that was sampled at two time points showed strong similarities over time. Concentration of microbial biomass on polycarbonate filters coupled with the MIDI-FAME extraction of both biomass and filter was a useful technique to characterize microbial communities from groundwater.     

1-Thio-beta-D-galactofuranosides: synthesis and evaluation as beta-D- galactofuranosidase inhibitors

Beta-D-galactofuranosidase is a good chemotherapeutic target for the design of inhibitors, since beta-D-galactofuranose is a constituent of important parasite glycoconjugates but is not present in the host mammals. With this aim, we have synthesized for the first time alkyl, benzyl and aryl 1-thio-beta-D-galactofuranosides by condensation of penta-O-benzoyl-alpha,beta-D-galactofuranose with the corresponding thiols, in the presence of SnCl4as catalyst. The complete chemical and spectroscopical characterization of these compounds showed that the reaction was stereoselective. Debenzoylation with sodium methoxide afforded the beta-S-galactofuranosides in high yield. The thioglycosides were tested as inhibitors of the beta-D- galactofuranosidase of Penicillium fellutanum, using for the first time 4-nitrophenyl-beta-D-galactofuranoside as chromogenic substrate. The 4- aminophenyl-1-thio-beta-D-galactofuranoside, obtained by catalytic hydrogenation of the nitrophenyl derivative, was the best inhibitor being then an adequate ligand for the preparation of an affinity phase aimed at the isolation of beta-d-galactofuranosidases from different sources. Also the inhibitory activity of d-galactono-1, 4-lactone was shown.