Combining distances of ballistic and myrmecochorous seed dispersal in Adriana quadripartita (Euphorbiaceae)

The separate contributions of different vectors to net seed dispersal curves of diplochorous systems have rarely been characterised. In Australia, myrmecochory is a common seed dispersal syndrome and in the majority of such systems, seeds are initially dispersed ballistically. We measured ballistic and myrmecochorous seed dispersal distances in relation to canopies of Adriana quadripartita (Euphorbiaceae) and used a simulation model to estimate the net dispersal curve. We also compared seed removal rates and ant abundances under, and outside, plant canopies to examine how foraging patterns by ants may affect net dispersal.Overall ant abundance did not show a significant numerical response to seedfall; however, the abundance of the main seed dispersing ant, Rhytidoponera ‘metallica’ did. Despite this, seed removal rates did not differ significantly between canopy and open locations. Rhytidoponera ‘metallica’ account for 93% of observed seed dispersal events. On average, the ants dispersed seeds 1.54 m and in doing so, moved seed a mean radial distance of 0.76 m away from canopy edges. This contribution to net dispersal distance by ants is considerable since ballistic dispersal moved seeds a median distance of 7.5 cm. Our simulation model indicated that the combination of ballistic and ant seed dispersal is expected to result in seeds being transported a median net radial dispersal distance of 1.05 m from the canopy edge.Thus in this system, an important function of diplochory may simply be to move a higher proportion of seeds from under the canopy of parent plants than is possible by ballistic dispersal alone. This ‘dispersal-for-distance’ may result in reduced parent–offspring competition or may increase the probability that seeds reach rare safe sites for germination and recruitment.     

Cloning and characterization of a sialidase from the murine histocompatibility-2 complex: low levels of mRNA and a single amino acid mutation are responsible for reduced sialidase activity in mice carrying the Neula allele

The Neu1 locus, in the S region of the murine histocompatibility-2complex, regulates the sialic acid content of several liverlysosomal enzymes. Three alleles, Neu1^a, Neu1^b, and Neu1^c, havebeen described on the basis of differential sialylation of theenzyme liver acid phosphatase. The Neu1^a allele occurs in asmall number of mouse strains, e.g., SM/J and is associatedwith sialidase deficiency. We recently described G9, a sialidasegene in the human major histocompatibility complex (Milner etal. (1997) J. Biol. Chem., 272, 4549–4558), and we nowreport the characterization of the equivalent gene in mouse.The protein product of the murine G9 gene is 409 amino acidsin length and is 83% identical to its human orthologue. Expressionof the murine G9 protein in insect cells has confirmed thatit is a sialidase, with optimal activity at pH 5. To elucidatethe basis of sialidase deficiency in mouse strains carryingthe Neu1^a allele, we have sequenced the G9 coding regions frommice carrying the three Neu1 alleles and hence defined the aminoacid sequence characteristic of each allotype. Of particularinterest is a Leu-209 to Ile mutation that is unique to theNeu1^a allotype and is associated with reductions in sialidaseactivity of 68% and 88% compared to the Neu1^b and Neu1^c allotypes,respectively, when these three protein variants are expressedin insect cells. Additional factors, such as differential expression,may also influence the activities of the Nen1 allotypes in vivo.We have observed that the level of G9 mRNA is substantiallyreduced in mice carrying the Neu1^a allele compared to the Neu1^b(85–95% reduction) and Neu1^c (70% reduction) alleles. H2 complex MHC Neu1 sialidase     

Purification and characterization of subcomponent C1q of the first component of bovine complement

Bovine C1q, a subcomponent of the first component of complement, was purified in high yield by a combination of euglobulin precipitation, and ion-exchange and molecularsieve chromatography on CM-cellulose and Ultrogel AcA 34. Approx. 12-16mg can be isolated from 1 litre of serum, representing a yield of 13-18%. The molecular weight of undissociated subcomponent C1q, as determined by equilibrium sedimentation, is 430000. On sodium dodecyl sulphate/polyacrylamide gels under non-reducing conditions, subcomponent C1q was shown to consist of two subunits of mol.wts. 69000 and 62000 in a molar ratio of 2:1. On reduction, the 69000-mol.wt. subunit gave chains of mol.wts. 30000 and 25000 in equimolar ratio, and the 62000-mol.wt. subunit decreased to 25000. The amino acid composition, with a high value for glycine, and the presence of hydroxyproline and hydroxylysine, suggests that there is a region of collagen-like sequence in the molecule. This is supported by the loss of haemolytic activity and the degradation of the polypeptide chains of subcomponent C1q when digested by collagenase. All of these molecular characteristics support the structure of six subunits, each containing three different polypeptide chains, with globular heads connected by collagen triple helices as proposed by Reid & Porter (1976) (Biochem. J.155, 19-23) for human subcomponent C1q. Subcomponent C1q contains approx. 9% carbohydrate; analysis of the degree of substitution of the hydroxylysine residues revealed that 91% are modified by the addition of the disaccharide unit Gal-Glc. Bovine subcomponent C1q generates full C1 haemolytic activity when assayed with human subcomponents C1r and C1s.     

Identification of the [FeFe]-Hydrogenase Responsible for Hydrogen Generation in Thermoanaerobacterium saccharolyticum and Demonstration of Increased Ethanol Yield via Hydrogenase Knockout

Three putative hydrogenase enzyme systems in Thermoanaerobacterium saccharolyticum were investigated at the genetic, mRNA, enzymatic, and phenotypic levels. A four-gene operon containing two [FeFe]-hydrogenase genes, provisionally termed hfs (hydrogenase-Fe-S), was found to be the main enzymatic catalyst of hydrogen production. hfsB, perhaps the most interesting gene of the operon, contains an [FeFe]-hydrogenase and a PAS sensory domain and has several conserved homologues among clostridial saccharolytic, cellulolytic, and pathogenic bacteria. A second hydrogenase gene cluster, hyd, exhibited methyl viologen-linked hydrogenase enzymatic activity, but hyd gene knockouts did not influence the hydrogen yield of cultures grown in closed-system batch fermentations. This result, combined with the observation that hydB contains NAD(P)+ and FMN binding sites, suggests that the hyd genes are specific to the transfer of electrons from NAD(P)H to hydrogen ions. A third gene cluster, a putative [NiFe]-hydrogenase with homology to the ech genes, did not exhibit hydrogenase activity under any of the conditions tested. Deletion of the hfs and hydA genes resulted in a loss of detectable methyl viologen-linked hydrogenase activity. Strains with a deletion of the hfs genes exhibited a 95% reduction in hydrogen and acetic acid production. A strain with hfs and ldh deletions exhibited an increased ethanol yield from consumed carbohydrates and represents a new strategy for engineering increased ethanol yields in T. saccharolyticum.Thermophilic anaerobic bacteria have long been of interest for studies of cellulosic biomass conversion due to their native hydrolytic and fermentative abilities (5, 33). However, all thermophilic anaerobes isolated to date have branched fermentation pathways which produce organic acids in addition to solvents such as ethanol (12). For cellulosic fuel production, significant yield loss is likely to be economically unfeasible (11).In their natural environments, saccharolytic fermentative bacteria participate in interspecies hydrogen transfer, producing hydrogen from carbohydrates that is rapidly consumed by methanogens and sulfate-reducing bacteria (30). As a result, the hydrogen partial pressure remains exceedingly low, allowing hydrogen (E₀′, −414 mV) to be produced not only from ferredoxin (E₀′, ∼−400 mV) but also from the less favorable electron source NAD(P)H (E₀′, −320 mV). Fermentative bacteria benefit from hydrogen production, because they are able to coproduce acetic acid and an additional ATP via acetate kinase (23). When grown in pure culture in a closed fermentation vessel, hydrogen is generated primarily from reduced ferredoxin, since generation from NAD(P)H becomes less favorable as the concentration of hydrogen increases (7).We have recently demonstrated high-yield ethanol production in the thermophilic anaerobe Thermoanaerobacterium saccharolyticum JW/SL-YS485 through deletion of the l-lactate dehydrogenase (ldh), phosphate acetyltransferase (pta), and acetate kinase (ack) genes (20). In addition to producing ethanol at high yield, this strain produced significantly less hydrogen, as is required to balance end product electron stoichiometry, although hydrogenase activity in cell extracts remained high. In this study, we used gene knockout to identify gene clusters that are implicated in hydrogenase activity in T. saccharolyticum and to identify the hfs gene operon, which is required for hydrogen production. The hfs operon contains a protein with [FeFe]-hydrogenase and PAS sensory domains that is conserved among a few members of the genera Clostridium and Thermoanaerobacter. Strains with hfs deletions showed decreased acetic acid production, and a strain with hfs and ldh deletions produced ethanol at an increased yield.     

Studies on the maintenance of cytochromes P-450 and b5, monooxygenases and cytochrome reductases in primary cultures of rat hepatocytes

The cytochrome P-450 content of rat hepatocytes declined rapidly over 72 h in culture, due primarily to denaturation to cytochrome P-420. Six different media were investigated for their ability to conserve cytochrome P-450 during culture, and the most successful was a modified Earle's medium. After 72 h culture in this medium, cytochromes P-450 and b5, NADH-cytochrome b5- and NADPH-cytochrome c-reductases were maintained at 40, 100, 35 and 52% of fresh cell values, respectively. Cytochrome P-450 showed differential functional stability during culture with ethoxyresorufin O-deethylation being more stable than either pentoxyphenoxazone O-depentylation or biphenyl 4-hydroxylation. Monooxygenase than did cytochrome P-450 content. This discrepancy was not explained by loss of flavin nucleotides, FMN or FAD.     

Molecular dissection of the Munc18c/syntaxin4 interaction: implications for regulation of membrane trafficking

Sec1p/Munc18 (SM) proteins are believed to play an integral role in vesicle transport through their interaction with SNAREs. Different SM proteins have been shown to interact with SNAREs via different mechanisms, leading to the conclusion that their function has diverged. To further explore this notion, in this study, we have examined the molecular interactions between Munc18c and its cognate SNAREs as these molecules are ubiquitously expressed in mammals and likely regulate a universal plasma membrane trafficking step. Thus, Munc18c binds to monomeric syntaxin4 and the N-terminal 29 amino acids of syntaxin4 are necessary for this interaction. We identified key residues in Munc18c and syntaxin4 that determine the N-terminal interaction and that are consistent with the N-terminal binding mode of yeast proteins Sly1p and Sed5p. In addition, Munc18c binds to the syntaxin4/SNAP23/VAMP2 SNARE complex. Pre-assembly of the syntaxin4/Munc18c dimer accelerates the formation of SNARE complex compared to assembly with syntaxin4 alone. These data suggest that Munc18c interacts with its cognate SNAREs in a manner that resembles the yeast proteins Sly1p and Sed5p rather than the mammalian neuronal proteins Munc18a and syntaxin1a. The Munc18c-SNARE interactions described here imply that Munc18c could play a positive regulatory role in SNARE assembly.     

Measurements of the membrane water permeability (Lp) and its temperature dependence (activation energy) in human fresh and failed-to-fertilize oocytes and mouse oocyte.

The volumetric response of oocytes during rapid alterations of the extracellular osmotic environment were recorded using video microscopy. From these observations, the kinetics of water loss for human and mouse oocytes were determined over the temperature range 37 to 10 degrees C, including 37, 30, 20, and 10 degrees C. The changes in diameter of oocytes were measured over a 5-min period and a computer model was used to derive values for membrane water permeability (Lp) and inactive volume (Vb) and to compare the experimental data to the predicted values. The results for the mouse oocyte Lp were comparable to values determined by other methods. However the human data, for both failed-to-fertilize and fresh oocytes, have a wide range of values with large standard deviations. The Lp values at the various temperatures were used to calculate the Arrhenius activation energy (Ea). An Ea value of 9.48 kcal/mol was found for the fresh mouse oocyte, whereas the activation energy for human oocytes was extremely low, 3.73 kcal/mol for fresh oocytes and 1.93 kcal/mol for failed-to-fertilize oocytes.     

Avanti lipid tools: Connecting lipids,technology, and cell biology

Lipid research is challenging owing to the complexity and diversity of the lipidome. Here we review a set of experimental tools developed for the seasoned lipid researcher, as well as, those who are new to the field of lipid research. Novel tools for probing protein–lipid interactions, applications for lipid binding antibodies, enhanced systems for the cellular delivery of lipids, improved visualization of lipid membranes using gold-labeled lipids, and advances in mass spectrometric analysis techniques will be discussed. Because lipid mediators are known to participate in a host of signal transduction and trafficking pathways within the cell, a comprehensive lipid toolbox that aids the science of lipidomics research is essential to better understand the molecular mechanisms of interactions between cellular components. This article is part of a Special Issue entitled Tools to study lipid functions.     

The Colletotrichum graminicola striatin orthologue Str1 is necessary for anastomosis and is a virulence factor

Striatin family proteins are key regulators in signalling pathways in fungi and animals. These scaffold proteins contain four conserved domains: a caveolin‐binding domain, a coiled‐coil motif and a calmodulin‐binding domain at the N‐terminus, and a WD‐repeat domain at the C‐terminus. Fungal striatin orthologues are associated with sexual development, hyphal growth and plant pathogenesis. In Fusarium verticillioides, the striatin orthologue Fsr1 promotes virulence in the maize stalk. The relationship between fungal striatins and pathogenicity remains largely unexplored. In this study, we demonstrate that the Colletotrichum graminicola striatin orthologue Str1 is required for full stalk rot and leaf blight virulence in maize. Pathogenicity assays show that the striatin mutant strain (Δstr1) produces functional appressoria, but infection and colonization are attenuated. Additional phenotypes of the Δstr1 mutant include reduced radial growth and compromised hyphal fusion. In comparison with the wild‐type, Δstr1 also shows a defect in sexual development and produces fewer and shorter conidia. Together with the fact that F. verticillioides fsr1 can complement Δstr1, our results indicate that C. graminicola Str1 shares five phenotypes with striatin orthologues in other fungal species: hyphal growth, hyphal fusion, conidiation, sexual development and virulence. We propose that fungal striatins, like mammalian striatins, act as scaffolding molecules that cross‐link multiple signal transduction pathways.