Membrane topology and site-specific mutagenesis of Pseudomonas aeruginosa porin OprD

Pseudomonas aeruginosa OprD is a 420-amino-acid protein that facilitates the uptake of basic amino acids, imipenem and gluconate across the outer membrane. OprD was the first specific porin that could be aligned with members of the non-specific porin super-family. Utilizing multiple alignments in conjugation with structure predictions and amphipathicity calculations, an OprD-topology model was proposed. Sixteen β-strands were predicted, connected by short loops at the periplasmic side. The eight external loops were of variable length but tended to be much longer than the periplasmic ones. Polymerase chain reaction (PCR)-based site-specific mutagenesis was performed to delete separately short stretches (4-8 amino acid residues) from each of the predicted external loops. The mutants with deletions in the predicted external loops L1, L2, L5, L6, L7 and L8 were tolerated in both Escherichia coli and P. aeruginosa. The expressed mutant proteins maintained substantial resistance to trypsin treatment in the context of isolated outer membranes. Proteins with deletions in loops L1, L5, L6, L7 and L8 reconstituted similar imipenem supersusceptibility in a P. aeruginosa OprD::Ω background. The L2-deletion mutant only partially reconstituted supersusceptibility, suggesting that loop L2 is involved in imipenem binding. These data were generally consistent with the topology model.     

Bimodality for plant sizes and spatial pattern in cohorts: the role of competition and site conditions.

The well-known size bimodality in cohorts of plants is revisited with methods emphasizing generic modeling. A link between bimodality and interface growth in nonequilibrium statistical physics is emphasized: the development of bimodality is understood as a phase transition like interface roughening. A specific model is proposed, inspired by gap models. It is used to illustrate generic results, to understand the end point of mean field approximation and aggregation of variables, and to discuss the role of site and competition for the development of a social hierarchy within a forest stand, in a wider ecological context.     

Degradation of human thyroperoxidase in the endoplasmic reticulum involves two different pathways depending on the folding state of the protein

Human thyroperoxidase (hTPO), a type I transmembrane glycoprotein, plays a key role in thyroid hormone synthesis. In a previous paper (Fayadat, L., Niccoli, P., Lanet, J., and Franc, J. L. (1998) Endocrinology 139, 4277-4285) we established that after the synthesis, only 15-20% of the hTPO molecules were recognized by a monoclonal antibody (mAb15) directed against a conformational structure and that only 2% were able to reach the cell surface. In the present study using pulse-chase experiments in the presence or absence of protease inhibitors followed by immunoprecipitation procedures with monoclonal antibodies recognizing unfolded or partially folded hTPO forms we show that: (i) unfolded hTPO forms are degraded by the proteasome and (ii) partially folded hTPO forms are degraded by other proteases. It was also established upon incubating endoplasmic reticulum (ER) membranes in vitro that the degradation of the partially folded hTPO was carried out by serine and cysteine integral ER membrane proteases. These data provide valuable insights into the quality control mechanisms whereby the cells get rid of misfolded or unfolded proteins. Moreover, this is the first study describing a protein degradation process involving two distinct degradation pathways (proteasome and ER cysteine/serine proteases) at the ER level, depending on the folding state of the protein.     

Spontaneously Ig-secreting B-1 cells violate the accepted paradigm for expression of differentiation-associated transcription factors

B-1 cells spontaneously secrete natural Ig that acts as a primary line of defense against infection. A major shortfall in our understanding of this key process centers on the molecular mechanisms regulating natural Ab secretion by B-1 cells. Herein, we demonstrate that secreting B-1 cells use some aspects of the recently recognized plasmacytic differentiation program but deviate from it in important ways. Specifically, we show that key repressors of the plasmacytic program, B cell leukemia/lymphoma-6 and paired box gene 5, are reduced in spontaneously secreting B-1 B cells, as in stimulated differentiated B-2 cells. Surprisingly, we find that key promoters of the plasmacytic program, B lymphocyte inducer of maturation program 1 and X-box binding protein 1, are not up-regulated in secreting B-1 cells, in contrast to secreting B-2 cells. These data demonstrate that B-1 cells operate under a differentiation program that is unique and differs from the paradigm associated with Ig-secreting B-2 cells.     

First blood meal of Ctenocephalides canis (Siphonaptera: Pulicidae) on dogs: time to initiation of feeding and duration

Two experiments were conducted on dogs to evaluate interval to initiation and duration of the first blood meal of Ctenocephalides canis (Curtis). Percentage of fed male and female fleas was calculated for fleas held on dogs for 5, 15, 30, 60 min, 6, and 24 hr. Duration of first blood meal was also measured for individual fleas confined on dogs. When fleas were free in the hair coat, 21.2% had begun blood feeding within 5 min. After 1 hr, 72.5% of fleas had fed. After 6 hr, 95.2% of males and 100% of females had taken a blood meal, and 24 hr after deposition all fleas had fed. There was no significant difference between the 2 sexes. The mean delay between deposition and biting for fleas that began feeding within 15 min was 2 min 52 sec +/- 3 min 2 sec for female fleas and 3 min 8 sec +/- 2 min 45 sec for males. The mean duration of female and male meals was 5 min 3 sec +/- 3 min 41 sec and 6 min 9 sec +/- 6 min 8 sec, respectively. There was no significant difference between the 2 sexes. The dog flea took its blood meal on dogs more slowly than the cat flea did on cats; this meal was significantly longer for Ctenocephalides felis felis (Bouche) than for C. canis.     

The kappa-opioid receptor from human placenta: hydrodynamic characteristics and evidence for its association with a G protein

The kappa nature of opioid binding sites in a brush border membrane (BBM) fraction from human placenta has been confirmed: these sites display considerably higher apparent affinity (KI = 1.2 nM) for the kappa selective ligand U-50488 than they do for the mu and delta selective ligands [D-Ala2, MePhe4, Glyol5] enkephalin (KI = 1.5-2 microM) and [D-Thr2, Leu5] enkephalyl-Thr (KI = 10-15 microM), respectively. The BBM fraction from human placenta was incubated either with the agonist 3H-etorphine or with the antagonist 3H-diprenorphine and subsequently solubilized with digitonin. The solubilized macromolecular radioactivity was found to behave as a homogeneous entity both in molecular exclusion chromatography (app. rs = 6.1 nm) and in linear sucrose gradients (app. S20.w = 12 S). Two lines of evidence indicated that the placental kappa opioid receptor is capable of interacting with a guanine nucleotide regulatory (G) protein: (i) equilibrium binding of the agonist 3H-etorphine in the BBM fraction was clearly inhibited by 5'-guanylylimidodiphosphate (Gpp(NH)p), especially in the presence of Na+ ions while binding of the antagonist 3H-diprenorphine was significantly less so and (ii) the sedimentation velocity of the kappa opioid receptor was decreased down to about 10 S when the BBM fraction was prelabeled with radioligand in the presence of Gpp(NH)p prior to its solubilization with digitonin. The G protein that mediates the effect of Gpp(NH)p might be neither Gs nor Gi since no adenylate cyclase activity could be demonstrated in the BBM fraction from human placenta.     

Parasitism and maintenance of diversity in a fungal vegetative incompatibility system: the role of selection by deleterious cytoplasmic elements

In fungi, horizontal transmission of deleterious cytoplasmic elements is reduced by the vegetative incompatibility system. This self/non-self recognition system may select for greater diversity of fungal incompatibility phenotypes in a frequency-dependent manner but the link between the diversity of fungal phenotypes and the virulence of cytoplasmic parasites has been poorly studied. We used an epidemiological model to show that even when transmission between incompatibility types is permitted, parasite pressure can lead to high levels of polymorphism for vegetative incompatibility systems. Moreover, high levels of polymorphism in host populations can select for less virulent cytoplasmic parasites. This feedback mechanism between parasite virulence and vegetative incompatibility system polymorphism of host populations may account for the general avirulence of most known mycoviruses. Furthermore, this mechanism provides a new perspective on the particular ecology and evolution of the host/parasite interactions acting between fungi and their cytoplasmic parasites.     

Red locust Nomadacris septemfasciata (Serville) upsurges in northern Madagascar between 1998 and 2004

The Red locust (Nomadacris septemfasciata Serville) is commonly found in southern Africa and the Indian Ocean islands. In Madagascar until 1998, only infested crop fields were controlled. However, since 1998 the Red locust has caused considerable crop damage in northern Madagascar, where gregarious individuals were identified for the first time in Madagascar in 2002. In this study, an accurate history of the outbreaks which occurred between 1998 and 2004 is drawn up on the basis of field surveys and anecdotal data. A total area of more than 60,000 ha was infested between 2001 and 2003, at the peak of the outbreak. With these results, we can make out a first biogeographical synthesis for this locust.     

Assessment of Persistence of Bartonella henselae in Ctenocephalides felis

Bartonella henselae (Rhizobiales: Bartonellaceae) is a Gram-negative fastidious bacterium of veterinary and zoonotic importance. The cat flea Ctenocephalides felis (Siphonaptera: Pulicidae) is the main recognized vector of B. henselae, and transmission among cats and humans occurs mainly through infected flea feces. The present study documents the use of a quantitative molecular approach to follow the daily kinetics of B. henselae within the cat flea and its excreted feces after exposure to infected blood for 48 h in an artificial membrane system. B. henselae DNA was detected in both fleas and feces for the entire life span of the fleas (i.e., 12 days) starting from 24 h after initiation of the blood meal.