Evidence that free GPI glycolipids are essential for growth of Leishmania mexicana.

The cell surface of the parasitic protozoan Leishmania mexicana is coated by glycosylphosphatidylinositol (GPI)-anchored glycoproteins, a GPI-anchored lipophosphoglycan and a class of free GPI glycolipids. To investigate whether the anchor or free GPIs are required for parasite growth we cloned the L.mexicana gene for dolichol-phosphate-mannose synthase (DPMS) and attempted to create DPMS knockout mutants by targeted gene deletion. DPMS catalyzes the formation of dolichol-phosphate mannose, the sugar donor for all mannose additions in the biosynthesis of both the anchor and free GPIs, except for a alpha1-3-linked mannose residue that is added exclusively to the free GPIs and lipophosphoglycan anchor precursors. The requirement for dolichol-phosphate-mannose in other glycosylation pathways in L.mexicana is minimal. Deletion of both alleles of the DPMS gene (lmdpms) consistently resulted in amplification of the lmdpms chromosomal locus unless the promastigotes were first transfected with an episomal copy of lmdpms, indicating that lmdpms, and possibly GPI biosynthesis, is essential for parasite growth. As evidence presented in this and previous studies indicates that neither GPI-anchored glycoproteins nor lipophosphoglycan are required for growth of cultured parasites, it is possible that the abundant and functionally uncharacterized free GPIs are essential membrane components.     

Mutants of Escherichia coli K12 accumulating porphobilinogen: a new locus, hemC.

Mutants of Escherichia coli K12 which accumulated the haem precursor porphobilinogen are described. The mutants grew very slowly on carbon and energy sources which K12 uses only oxidatively, and they had low catalase activities, suggesting that they were deficient in haem. Extracts had one-tenth of the parental activity of the enzyme porphobilinogen deaminase. In transduction, the mutation mapped close to genes ilvD and metE at minute 84. The gene was tentatively identified as hemC, coding for porphobilinogen deaminase. The gene symbol hemC replaces the earlier and temporary symbol popE.     

The species composition,occurrence and temporal stability of submerged aquatic macrophyte patches along the main channel border of pool 5A,Upper Mississippi River

Species composition, relative abundance, distribution and physical habitat associations of submerged aquatic macrophytes in the main channel border (MCB) habitat of Pool 5A, Upper Mississippi River (UMR) were investigated during the summers of 1980 and 1983. The submerged aquatic macrophytes in Pool .5A MCB were a small and stable component of the river ecosystem. Submerged plants occurred primarily in small, monospecific clumps. Clumps in close proximity to each other formed plant patches. Plant patches were stable in location and number between 1980 and 1983; 82.5% of the patches first observed in 1980 were present in 1983. Submerged macrophytes covered about 10–12 ha of the 201 ha MCB in Pool 5A. Submerged plants were most common in the lower two-thirds of the pool. Ten species of aquatic macrophytes occurred on rock channel-training structures and eleven occurred on non-rock substrates in the MCB. The most common submerged plants, in order of abundance, were Vallisneria americana Michx., Heteranthra dubia Jacq., Potamogeton pectinatus L., Ceratophyllum demersum L. and Potamogeton americanus C. & S.     

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.      

Recent developments in the cell biology and biochemistry of glycosylphosphatidylinositol lipids (Review)

Glycosylphosphatidylinositols (GPIs) represent an abundant and ubiquitous class of eukaryotic glycolipids. Although these structures were originally discovered in the form of GPI-anchored cell surface glycoproteins, it is becoming increasingly clear that a significant proportion of the GPI synthetic output of a cell is not directed to protein anchoring. Indeed, pools of nonprotein-linked GPIs can approach 107 molecules per cell in some cell types, especially the protozoa, with a large proportion of these molecules being displayed at the cell surface. Recent studies which form the subject of this review indicate that there is (a) considerable diversity in the range of structural modifications found on GPI glycolipids within and between species and cell types, (b) complexity in the topological arrangement of the GPI biosynthetic pathway in the endoplasmic reticulum, and (c) spatial restriction of the biosynthetic pathway within the endoplasmic reticulum. Furthermore, consistent with additional functional roles for these lipids beyond serving as protein anchor precursors, products of the GPI biosynthetic pathway appear to be widely distributed in the cellular endomembrane system. These studies indicate that there is still much to learn about the organization of glycolipid biosynthetic pathways in eukaryotic cells, the nature and subcellular distribution of the lipid products of these pathways, and the function of these lipids within cells.     

The Influence of Recrystallized Caffeine on Water-Swellable Polymethacrylate Mucoadhesive Buccal Films

The aim of this work was to investigate the influence of particles on the properties of polymethacrylate films intended for buccal delivery. A solvent casting method was used with Eudragit RS and RL (ERS and ERL, respectively) as film-forming rate-controlling polymers, with caffeine as a water-soluble model drug. The physicochemical properties of the model films for a series of formulations with increasing concentrations of caffeine were determined in terms of morphology, mechanical and mucoadhesive properties, drug content uniformity, and drug release and associated kinetics. Typically regarded as non-mucoadhesive polymers, ERS and mainly ERL, were found to be good mucoadhesives, with ERL01 exhibiting a work of mucoadhesion (WoA) of 118.9 μJ, which was about five to six times higher than that observed for commonly used mucoadhesives such as Carbopol^® 974P (C974P, 23.9 μJ) and polycarbophil (PCP, 17.4 μJ). The mucoadhesive force for ERL01 was found to be significantly lower yet comparable to C974P and PCP films (211.1 vs. 329.7 and 301.1 mN, respectively). Inspection of cross-sections of the films indicated that increasing the concentration of caffeine was correlated with the appearance of recrystallized agglomerates. In conclusion, caffeine agglomerates had detrimental effects in terms of mucoadhesion, mechanical properties, uniformity, and drug release at large particle sizes. ERL series of films exhibited very rapid release of caffeine while ERS series showed controlled release. Analysis of release profiles revealed that kinetics changed from a diffusion controlled to a first-order release mechanism.     

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.     

Evolutionary aspects of GPI metabolism in kinetoplastid parasites.

There is a growing, but still very patchy, data base of GPI structure, biosynthesis and function. In this article we speculate freely on the function of GPI anchors, and the origins of GPI-related molecules, primarily with reference to the protozoan parasites Trypanosoma brucei and the Leishmania. The views expressed draw on fairly wild extrapolations and some will, no doubt, not stand the tests of time. Several of the hypotheses presented should therefore be taken with a pinch of salt, some lemon, and large quantities of tequila!