Use of a real-time polymerase chain reaction thermocycler to study bacterial cell permeabilization by antimicrobial peptides

Antimicrobial peptides are good leads to develop new antibiotics, but knowledge of their mode of action is a prerequisite. Destruction of the microbial membranes through a detergent-like mechanism is one of these modes of action. This is usually studied by using a fluorescent nucleic acid stain such as SYTOX Green, which is impermeable to living cells. Using a simple protocol based on the use of a standard real-time thermocycler, we confirmed that the actions of the antimicrobial peptides LL-37 and magainin 2 on bacterial cells are different.     

Plasmodium falciparum dolichol phosphate mannose synthase represents a novel clade

Dolichol phosphate mannose synthase (DPM) catalyzes the reaction between dolichol phosphate (Dol-P) and guanosine diphosphate mannose (GDP-Man) to form dolichol-phosphate-mannose (Dol-P-Man). This molecule acts as mannose donor for N-glycosylation and glycosylphosphatidylinositol (GPI) biosynthesis. The Plasmodium falciparum DPM1 (Pfdpm1) possesses a single predicted transmembrane region near the N-, but not the C-terminus. Here we show that the cloned Pfdpm1 gene failed to complement a Saccharomyces cerevisiae mutant indicating that the parasite gene does not belong to the baker’s yeast group, as was previously assumed. Furthermore, Pfdpm1 was unable to complement a mouse mutant deficient in DPM but efficiently complements the Schizosaccharomyces pombe fission yeast mutant, indicating a difference between fission yeast and mammalian DPM genes. Therefore, we reanalyzed the hydrophobicity scales of all known DPMs and consequently reclassify the DPM clade into six major novel subgroups. Furthermore, we show that Pfdpm1 represents a unique enzyme among these subgroups.     

Differential binding of chemokines to macrophages and neutrophils in the human inflamed synovium

In chronic inflammatory foci, such as the rheumatoid joint, there is enhanced recruitment of phagocytes from the blood into the tissues. Chemokines are strongly implicated in directing the migration of these cells, although little is known regarding the chemokine receptors that could mediate their chemotaxis into the joint tissue. Therefore the objective of the study was to identify chemokine binding sites on macrophages and neutrophils within the rheumatoid synovium using radiolabeled ligand binding and in situ autoradiography. Specific binding sites for CCL3 (macrophage inflammatory protein-1α), CCL5 (RANTES), CCL2 (monocyte chemoattractant protein-1) and CXCL8 (IL-8) were demonstrated on CD68⁺ macrophages in the subintimal and intimal layers. The number and percentage of intimal cells that bound chemokines were greater in inflamed regions compared to noninflamed regions. The intensity of intimal binding varied between chemokines with the rank order, CCL3 > CCL5 > CCL2 > CXCL8. Neutrophils throughout the synovium bound CXCL8 but did not show any signal for binding CCL2, CCL3 or CCL5.     

Regulation of the mitogen-activated protein kinase signaling pathway by SHP2.

Gab1-SHP2 association is required for Erk mitogen-activated protein kinase activation by several growth factors. Gab1-SHP2 interaction activates SHP2. However, an activated SHP2 still needs to associate with Gab1 to mediate Erk activation. It was unclear whether SHP2 is required to dephosphorylate a negative phosphorylation site on Gab1 or whether SHP2 needs the Gab1 pleckstrin homology (PH) domain to target it to the plasma membrane. We found that expression of a fusion protein consisting of the Gab1 PH domain and an active SHP2 (Gab1PH-SHP2DeltaN) induced constitutive Mek1 and Erk2 activation. Linking the active SHP2DeltaN to the PDK1 PH domain or the FRS2beta myristoylation sequence also induced Mek1 activation. Mek1 activation by Gab1PH-SHP2DeltaN was inhibited by an Src inhibitor and by Csk. Significantly, Gab1PH-SHP2DeltaN induced Src activation. Gab1PH-SHP2DeltaN expression activated Ras, and the Gab1PH-SHP2DeltaN-induced Mek1 activation was blocked by RasN17. These findings suggest that Gab1PH-SHP2DeltaN activated a signaling step upstream of Src and Ras. The SHP2 tyrosine phosphatase activity is essential for the function of the fusion protein. Together, these data show that the Gab1 sequence, besides the PH domain and SHP2 binding sites, is dispensable for Erk activation, suggesting that the primary role of Gab1 association with an activated SHP2 is to target it to the membrane.     

APH-2/Nicastrin Functions in LIN-12/Notch Signaling in the Caenorhabditis elegans Somatic Gonad

Nicastrin is a recently identified member of high-molecular weight complexes containing presenilin. The Caenorhabditis elegans homolog of nicastrin, aph-2, was shown to be required for GLP-1/Notch signaling in the early embryo. In addition to the maternal-effect embryonic lethal phenotype, aph-2 mutant animals also display an egg-laying defect. We show that this latter defect is related to the SEL-12/presenilin egg-laying defect. We also show that aph-2 and sel-12 genetically interact and cooperate to regulate LIN-12/Notch signaling in the development of the somatic gonad. In addition, aph-2 and lin-12/Notch genetically interact. We illustrate a new role for aph-2 in facilitating lin-12 signaling in the somatic gonad, thus providing evidence that APH-2 is involved in both GLP-1/Notch- and LIN-12/Notch-mediated signaling events. Finally, we demonstrate that nicastrin can partially substitute for aph-2, suggesting a conservation of function between these proteins.     

Overnight Dexamethasone Suppression Test: A Reliable Screen for Cushing's Syndrome in the Obese

Objective: Reevaluation of the validity of the 1-mg overnight dexamethasone suppression test (ODST) as a screening test for Cushing's syndrome in obese patients. Research Methods and Procedures: Eighty-six obese patients (body mass index, 30 to 53 kg/m²) that were referred to a general endocrine outpatient clinic for evaluation of simple obesity, diabetes mellitus, hypertension, polycystic ovary disease, or pituitary tumor. One milligram dexamethasone was administered orally at 11:00 pm , and serum cortisol levels were measured the following morning between 8:00 am and 9:00 am . Suppression of serum cortisol to <80 nM (3 μg/dL) was chosen as the cut-off point for normal suppression. Patients with serum cortisol levels ≥80 nM were evaluated for Cushing's syndrome. Results: Suppression of morning cortisol levels to <80 nM occurred in 79 of the 86 obese patients. Seven patients had serum cortisol levels higher than 80 nM; five were eventually diagnosed with Cushing's syndrome and two were considered false positive results in view of normal 24-hour free urinary cortisol and normal suppression on a low dose dexamethasone suppression test (0.5 mg of dexamethasone every 6 hours for 2 days). We found a false positive rate of 2.3% for the ODST using a cut-off serum cortisol of 80 nM. Discussion: The ODST is a valid screening test for Cushing's syndrome in the obese population. The false positive rate was 2.3%, even when using a strict cut-off serum cortisol of 80 nM. Abnormal cortisol suppression in obese patients should be investigated and not be considered false positive results.     

Chemotaxis of Ralstonia eutropha JMP134(pJP4) to the Herbicide 2,4-Dichlorophenoxyacetate

Ralstonia eutropha JMP134(pJP4) and several other species of motile bacteria can degrade the herbicide 2,4-dichlorophenoxyacetate (2,4-D), but it was not known if bacteria could sense and swim towards 2,4-D by the process of chemotaxis. Wild-type R. eutropha cells were chemotactically attracted to 2,4-D in swarm plate assays and qualitative capillary assays. The chemotactic response was induced by growth with 2,4-D and depended on the presence of the catabolic plasmid pJP4, which harbors the tfd genes for 2,4-D degradation. The tfd cluster also encodes a permease for 2,4-D named TfdK. A tfdK mutant was not chemotactic to 2,4-D, even though it grew at wild-type rates on 2,4-D.     

Increased IRP1 and IRP2 RNA binding activity accompanies a reduction of the labile iron pool in HFE-expressing cells.

Iron regulatory proteins (IRPs), the cytosolic proteins involved in the maintenance of cellular iron homeostasis, bind to stem loop structures found in the mRNA of key proteins involved iron uptake, storage, and metabolism and regulate the expression of these proteins in response to changes in cellular iron needs. We have shown previously that HFE-expressing fWTHFE/tTA HeLa cells have slightly increased transferrin receptor levels and dramatically reduced ferritin levels when compared to the same clonal cell line without HFE (Gross et al., 1998, J Biol Chem 273:22068-22074). While HFE does not alter transferrin receptor trafficking or non-transferrin mediated iron uptake, it does specifically reduce (55)Fe uptake from transferrin (Roy et al., 1999, J Biol Chem 274:9022-9028). In this report, we show that IRP RNA binding activity is increased by up to 5-fold in HFE-expressing cells through the activation of both IRP isoforms. Calcein measurements show a 45% decrease in the intracellular labile iron pool in HFE-expressing cells, which is in keeping with the IRP activation. These results all point to the direct effect of the interaction of HFE with transferrin receptor in lowering the intracellular labile iron pool and establishing a new set point for iron regulation within the cell.     

The effects of exposure to seaweed on willingness to mate, oviposition, and longevity in seaweed flies

Abstract  1. Large male seaweed flies (Diptera: Coelopidae) are more likely to mate than smaller males. This is due to sexual conflict over mating, by which females physically resist male attempts to copulate. In some species, large males are simply more efficient at overpowering female resistance.
2. Female reluctance to mate is likely to have evolved due to the costs of mating to females. In many dipterans, males manipulate female behaviour through seminal proteins that have evolved through sperm competition. This behavioural manipulation can be costly to females, for example forcing females to oviposit in sub-optimal conditions and increasing their mortality.
3. Previous work has failed to identify any ubiquitous costs of mating to female coelopids. The work reported here was designed to investigate the effects of exposure to oviposition sites ( Fucus algae) on the reproductive behaviour of four species of coelopid. Algae deposition in nature is stochastic and females mate with multiple males in and around oviposition sites. Spermatogenesis is restricted to the pupal stage and there is last-male sperm precedence. It was predicted that males would avoid wasting sperm and would be more willing to mate, and to remain paired with females for longer, when exposed to oviposition material compared with control males. Females were predicted to incur longevity costs of mating if mating increased their rate of oviposition, especially in the presence of algae.
4. The behaviour of males of all four species concurred with the predictions; however mating did not affect female receptivity, oviposition behaviour, or longevity. Exposure to algae induced oviposition and increased female mortality in all species independently of mating and egg production. The evolutionary ecology of potential costs of mating to female coelopids are discussed in the light of these findings.     

Usefulness of using three different culture media for mold recovery in exposure assessment studies

The choice of culture media when performing exposure assessment for mold quantification is difficult since no standard methods exist and, in many cases, there is no way to predict, before hand, the mold population present and thus, select the most appropriate media. The aim of this project was to compare the efficiency and usefulness of 3 widely used culture media during the recovery of molds in sawmills. Andersen microbial samplers (AMS) and all-glass impingers-30 (AGI-30) were used to sample 51 work sites within 17 sawmills. Rose Bengal Agar (RBA), Czapek Solution Agar (CZA) and Sabouraud Dextrose Agar (SDA) were used directly in AMS and to plate AGI-liquid. The results show that there was no signification difference between the three culture media concerning the counts and the species recovery. However, SDA often lead to non usable counts. The variations obtained with the different culture media when AGI-30 were plated in triplicate was also not significantly different. However, only 19% of the species recovered at one site were present on all culture media. We believe the use of various media increases the chances of obtaining valid counts and an adequate species recovery.