Induced Autolysis of Aspergillus oryzae (A. niger group): IV. Carbohydrates

The examination of substances formed during induced autolysis by Aspergillus niger was continued in this work, which dealt in particular with carbohydrates. The autolysate contained a large amount of d-glucose (14 to 20% dry wt) and traces of glycolic aldehyde, dihydroxyacetone, ribose, xylose, and fructose. It also contained glycopeptides (about 10% dry wt), which were split from the cell wall during autolysis and which differed from one another in their level of polymerization and their composition. They were constituted by glucose and mannose, glucose and galactose, or mannose, glucose, and galactose (mannose being the most abundant in this case), and amino acids (chiefly alanine, serine, glutamic acid, and aspartic acid). During autolysis, only a part of the cell wall was dissolved, since it retained its shape. Upon further chemical hydrolysis, it produced mostly glucose and glucosamine, and smaller amounts of mannose, galactose, and amino acids. Presumably, glucomannoproteins and glucogalactoproteins were present in the intact cell as a macromolecular complex, constituting, together with chitin, the major part of the cell wall of Aspergillus.     

Human endothelial cells are target for platelet-activating factor. II. Platelet-activating factor induces platelet-activating factor synthesis in human umbilical vein endothelial cells.

Platelet-activating factor (PAF), a phospholipid mediator with broad and potent biologic activities, is synthesized by several inflammatory cells including endothelial cells (EC). PAF is also an effective stimulating agent for EC leading to increased cell permeability and adhesivity. We examined the synthesis of PAF in human umbilical cord vein EC after stimulation of EC with PAF or with its nonmetabolizable analog 1-O-alkyl-2-N-methyl-carbamyl-sn-glycero-3-phosphocholine (C-PAF). PAF (1 to 100 nM) induced a dose- and time-dependent increase of PAF synthesis as detected by [3H]acetate incorporation into PAF fraction. Stimulation of PAF synthesis occurred via activation of the "remodeling pathway" as the 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (lyso-PAF):acetyl-CoA acetyltransferase was dose-dependently increased after PAF treatment. The de novo pathway of PAF synthesis was not activated under these conditions. C-PAF was able to mimic the effect of authentic PAF on [3H] acetate incorporation. The inactive metabolite lyso-PAF (100 nM) had no influence on PAF synthesis in EC. CV-3988, BN 52021, and WEB 2086, potent and specific antagonists of PAF suppressed PAF effects on the remodeling pathway completely. The PAF- and C-PAF-induced [3H]PAF remained 93% cell-associated and was not degraded up to 10 min after stimulation. Characterization of the [3H]acetate-labeled material co-migrating with authentic PAF revealed that a significant proportion (approximately 57%) was actually 1-acyl-2-acetyl-sn-glycero-3-phosphocholine. PAF-induced PAF synthesis might be an important mechanism for amplifying original PAF signals and potentiating adhesive interactions of circulating cells with the endothelium.     

Melatonin in the seasonal response of the aphid Acyrthosiphon pisum

Aphids display life cycles largely determined by the photoperiod.During the warm long-day seasons.most aphid species reproduce by viviparous parthenogenesis.The shortening of the photoperiod in autumn induces a switch to sexual reproduction.Males and sexual females mate to produce overwintering resistant eggs.In addition to this full life cycle(holocycle),there are anholocyelic lineages that do not respond to changes in photoperiod and reproduce continuously by parthenogenesis.The molecular or hormonal events that trigger the scasonal response(i.c,induction of the sexual phenotypes)are still unknown.Although circadian synthesis of melatonin is known to play a key role in vertebrate photoperiodism,the involvement of the circadian clock and/or of the hor-mone melatonin in insect seasonal responses is not so well established.Here we show that melatonin levels in the aphid Acyrthosiphon pisum are significantly higher in holocyclice aphids reared under short days than under long days,while no differences were found between anholoeyelic aphids under the same conditions.We also found that melatonin is localized in the aphid suboesophageal ganglion(SOG)and in the thoracic ganglionic mass(TGM).In analogy to vertcbrates,insect-type arylalkxylamine N-acetyltransferases(i-AANATs)are thought to play a key role in melatonin synthesis.We measured the expression of four I-AANAT genes identified in A.pisum and localized two of them in situ in the insect central nervous systems(CNS).Levels of expression of these genes were compatible with the quantities of melatonin observed.Moreover,like melatonin,expression of these genes was found in the SOG and the TGM.     

Induction of the Cpx Envelope Stress Pathway Contributes to Escherichia coli Tolerance to Antimicrobial Peptides

Antimicrobial peptides produced by multicellular organisms as part of their innate system of defense against microorganisms are currently considered potential alternatives to conventional antibiotics in case of infection by multiresistant bacteria. However, while the mode of action of antimicrobial peptides is relatively well described, resistance mechanisms potentially induced or selected by these peptides are still poorly understood. In this work, we studied the mechanisms of action and resistance potentially induced by ApoEdpL-W, a new antimicrobial peptide derived from human apolipoprotein E. Investigation of the genetic response of Escherichia coli upon exposure to sublethal concentrations of ApoEdpL-W revealed that this antimicrobial peptide triggers activation of RcsCDB, CpxAR, and σ^E envelope stress pathways. This genetic response is not restricted to ApoEdpL-W, since several other antimicrobial peptides, including polymyxin B, melittin, LL-37, and modified S₄ dermaseptin, also activate several E. coli envelope stress pathways. Finally, we demonstrate that induction of the CpxAR two-component system directly contributes to E. coli tolerance toward ApoEdpL-W, polymyxin B, and melittin. These results therefore show that E. coli senses and responds to different antimicrobial peptides by activation of the CpxAR pathway. While this study further extends the understanding of the array of peptide-induced stress signaling systems, it also provides insight into the contribution of Cpx envelope stress pathway to E. coli tolerance to antimicrobial peptides.     

Lactobacillus gasseri SF1183 Affects Intestinal Epithelial Cell Survival and Growth

It is now commonly accepted that the intestinal microbiota plays a crucial role in the gut physiology and homeostasis, and that both qualitative and quantitative alterations in the compositions of the gut flora exert profound effects on the host’s intestinal cells. In spite of this, the details of the interaction between commensal bacteria and intestinal cells are still largely unknown and only in few cases the molecular mechanisms have been elucidated. Here we analyze the effects of molecules produced and secreted by Lactobacillus gasseri SF1183 on human intestinal HCT116 cells. L. gasseri is a well known species of lactic acid bacteria, commonly associated to the human intestine and SF1183 is a human strain previously isolated from an ileal biopsy of an healthy volunteer. SF1183 produces and secretes, in a growth phase-dependent way, molecule(s) able to drastically interfere with HCT116 cell proliferation. Although several attempts to purify and identify the bioactive molecule(s) have been so far unsuccessful, a partial characterization has indicated that it is smaller than 3 kDa, thermostable and of proteinaceous nature. L. gasseri molecule(s) stimulate a G1-phase arrest of the cell cycle by up-regulation of p21WAF1 rendering cells protected from intrinsic and extrinsic apoptosis. A L. gasseri-mediated reduction of apoptosis and of cell proliferation could be relevant in protecting epithelial barrier integrity and helping in reconstituting tissutal homeostasis.     

Flow‐mediated plasticity in the expression of stickleback nesting glue genes

Nest construction is an essential component of the reproductive behavior of many species, and attributes of nests – including their location and structure – have implications for both their functional capacity as incubators for developing offspring, and their attractiveness to potential mates. To maximize reproductive success, nests must therefore be suited to local environmental conditions. Male three‐spined sticklebacks (Gasterosteus aculeatus) build nests from collected materials and use an endogenous, glue‐like multimeric protein – “spiggin” – as an adhesive. Spiggin is encoded by a multigene family, and differential expression of spiggin genes potentially allows plasticity in nest construction in response to variable environments. Here, we show that the expression of spiggin genes is affected significantly by both the flow regime experienced by a fish and its nesting status. Further, we show the effects of flow on expression patterns are gene‐specific. Nest‐building fish exhibited consistently higher expression levels of the three genes under investigation (Spg‐a, Spg‐1, and Spg‐2) than non‐nesting controls, irrespective of rearing flow treatment. Fish reared under flowing‐water conditions showed significantly increased levels of spiggin gene expression compared to those reared in still water, but this effect was far stronger for Spg‐a than for Spg‐1 or Spg‐2. The strong effect of flowing water on Spg‐a expression, even among non‐nesters, suggests that the increased production of spiggin – or of spiggin rich in the component contributed by Spg‐a – may allow more rapid and/or effective nest construction under challenging high flow conditions.     

Behavioral response of the egg parasitoid <Emphasis Type="Italic">Ooencyrtus telenomicida</Emphasis> to host-related chemical cues in a tritrophic perspective

The response of the generalist egg parasitoid Ooencyrtus telenomicida (Vassiliev) (Hymenoptera: Encyrtidae) to host-related chemical cues from tomato plants, Solanum lycopersicum L., and adults of Nezara viridula (L.) (Heteroptera: Pentatomidae) was investigated in laboratory-based no-choice and paired-choice tests. In Y-tube olfactometer experiments, when female wasps were exposed to volatiles from plants in different conditions, they were attracted only to volatiles produced by N. viridula adult-infested tomato plants. When female wasps were exposed to adults of N. viridula, they were attracted to volatiles from virgin males, and, at a lower level, to volatiles from mated females in preoviposition state. Finally, studies in open arena showed that chemical footprints left by adults of N. viridula did not induce arrestment responses in wasp females. These results are discussed in terms of extrinsic competition with other beneficial egg parasitoids that in field can compete for the same egg mass, since intraguild interactions may affect the success of a biological control program.     

Combined inactivation and expression strategy to study gene function under physiological conditions: application to identification of new Escherichia coli adhesins

In bacteria, whereas disruption methods have been improved recently, the use of plasmid complementation strategies are still subject to limitations, such as cloning difficulties, nonphysiological levels of gene expression, or a requirement for antibiotics as plasmid selection pressure. Moreover, because of the pleiotropic modifications of cell physiology often induced by plasmid-based complementation, these strategies may introduce biases when biological process such as adhesion or biofilm formation are studied. We developed a plasmid-free approach that combines the lambda-red linear DNA recombination method with site-directed insertion of a repression and expression (RExBAD) cassette which places a functional pBAD promoter upstream of a target gene. We showed that this method permits both inactivation and modulation of most Escherichia coli gene expression, including expression of toxin and essential genes. We used this strategy to study adhesion and bacterial biofilms by placing the RExBAD cassette in front of the tra operon, encoding the DNA transfer and pilus genes on the F conjugative plasmid, and in front of flu, the antigen 43 (Ag43) autotransporter adhesin-encoding gene. In silico analysis revealed the existence of 10 genes with homology to the Ag43 gene that were good candidates for genes that encode putative new adhesins in E. coli. We used the RExBAD strategy to study these genes and demonstrated that induction of expression of four of them is associated with adhesion of E. coli to abiotic surfaces. The potential use of the RExBAD approach to study the function of cryptic or uncharacterized genes in large-scale postgenomic functional analyses is discussed.