Characterization of Bacillus thuringiensis ser. jordanica (serotype H71), a novel serovariety isolated in Jordan

The novel strain of Bacillus thuringiensis J112 isolated from a soil sample in Jordan was classified and characterized in terms of toxicity against dipteran and nematode larvae, crystal protein pattern, plasmid profile, and cry gene content. A new name, Bacillus thuringiensis serovariety jordanica (H serotype 71), is proposed for the reference strain J112. The parasporal crystal proteins were toxic to 3(rd) instar larvae of Drosophila melanogaster and to 2(nd) stage juveniles of root knot nematodes Meloidogyne javanica and M. incognita, but showed poor mosquitocidal activity towards Culex pipiens molestus and Culiseta longiareolata larvae. Solubilized and trypsin-digested crystal proteins possessed moderate hemolytic activity against sheep erythrocytes. SDS-polyacrylamide gel electrophoresis revealed that crystals are composed of several polypeptides ranging from 24 to 170 kDa, of which the 20-, 42-, 140-, and 170-kDa proteins were the major components. Analysis of the plasmid pattern of J112 revealed the presence of two large plasmidic bands of about 160 and 205 kbp. PCR with total DNA from strain J112 and specific primers for cry1, cry2, cry3, cry4, and cyt2A genes revealed that cry1, cry3A, cry4, cry5 and cyt2a genes are present.     

Phylogenetic analysis of life-history adaptations in parasitic cowbirds

Parasitic cowbirds lay eggs in the nests of other species anddupe them into caring for their young. Unlike other brood parasites,cowbirds have not developed egg mimicry or bizarre chick morphology.However, most of them parasitize a large number of hosts. Severalfeatures of cowbirds have been proposed as more general adaptationsto brood parasitism. In this study, we used a recent molecularphylogeny as a historical framework to test the possible adaptationsof the parasitic cowbird, including egg size, eggshell thicknessand energy content of the eggs, length of the incubation period,and growth pattern of cowbird nestlings. We used a recentlydeveloped extension of independent contrasts to test whetherthe five cowbird species deviate from general allometric equations.We generated prediction intervals for a nonparasite that evolvedin the place of the cowbirds. By using these prediction intervals,we found that parasitic cowbirds had not reduced weight or energycontent of their eggs, nor their incubation period over evolutionarytime. Cowbird chicks and those of nonparasitic relatives hadsimilar growth pattern. The only characteristic that separatedparasitic cowbirds from their nonparasitic relatives was anincrease in eggshell thickness. All these findings were robustand resisted the use of three models of character evolution.The fact that most traits exhibited by cowbirds were inheritedfrom a nonparasitic ancestor does not rule out that they areadvantageous for parasitism. Future research should focus onsuch traits of cowbird relatives and on how these traits preadapteda particular lineage to become parasites.     

Influence of cytoplasmic deletions on the filopodia-inducing effect of syndecan-3

Syndecans, transmembrane heparan sulfate proteoglycans (HSPG), mediate cell-cell and cell-matrix adhesion thereby controlling cell movement and shape. Syndecan cytoplasmic domains are very short (ca. 30 amino acids) and divided into two constant regions (C1 and C2) separated by one variable (V) region. Here we attempted to map the cytoplasmic region responsible for the filopodia-inducing effect of syndecan-3. We found that only the C1-region was necessary for this effect. In addition, the deletion of the C2-region led to extensive membrane blebbing. Nevertheless, the elimination of the entire cytoplasmic region did not affect delivery of syndecan-3 to the plasma membrane. These results indicate that the different regions of syndecan-3 cytoplasmic domain have different functions probably by binding to distinct proteins.     

Modulation of intestinal urea cycle by dietary spermine in suckling rat

Argininosuccinate synthetase, an ubiquitous enzyme in mammals, catalyses the formation of argininosuccinate, the precursor of arginine. Arginine is recognised as an essential amino acid in foetuses and neonates, but also as a conditionally essential amino acid in adults. Argininosuccinate synthetase is initially expressed in enterocytes during the developmental period, it disappeared from this organ then appeared in the kidneys. Although the importance of both intestinal and renal argininosuccinate synthetases has been recognised for a long time, nutrients have not yet been identified as inducers of the gene expression. In the context of a proteomic screening of intestinal modifications induced by dietary spermine in suckling rats, we showed that argininosuccinate synthetase and carbamoyl phosphate synthase disappeared from enterocytes after this treatment. The disappearance of argininosuccinate synthetase in small intestine was confirmed by immunodetection. Expression of carbamoyl phosphate synthase and argininosuccinate synthetase coding genes decreased also after spermine administration. Expression of other urea cycle enzyme coding genes was modulated by spermine administration: argininosuccinate lyase decreased and arginase increased. Our results fit with the developmental variation of argininosuccinate synthetase and carbamoyl phosphate synthase. Modulation of the gene expression for several urea cycle enzymes suggests a coordination between all the pathway steps and switch toward polyamine (or proline and glutamate) biosynthesis from ornithine.     

Modulation of the host immune response by a transient intracellular stage of Mycobacterium ulcerans: the contribution of endogenous mycolactone toxin

Mycobacterium ulcerans (Mu), the aetiological agent of Buruli ulcer, is an extracellular pathogen producing the macrolide toxin mycolactone. Using a mouse model of intradermal infection, we found that Mu was initially captured by phagocytes and transported to draining lymph nodes (DLN) within host cells. Similar to Buruli ulcers in humans, the infection site eventually became ulcerated with tissue necrosis and extracellular bacteria, at later stages. In contrast to Mycobacterium bovis BCG (BCG), Mu did not disseminate to the spleen. However, mice infected with Mu or BCG developed comparable primary cellular responses to mycobacterial antigens in DLN and spleen. The role of mycolactone in this sequence of events was examined with a mycolactone-deficient (mup045) mutant of Mu. Mup045 bacilli were better internalized than wild-type (wt) bacteria by mouse phagocytes in vitro. Moreover, infection with wt but not mup045 Mu led to inhibition of TNF-alpha expression, upregulation of MIP-2 chemokine, and host cell death within 1 day. Our results suggest that mycolactone expression during the intracellular life of Mu may contribute to immune evasion by inhibiting phagocytosis, provoking apoptosis of antigen presenting cells and altering the establishment of an appropriate inflammatory reaction.     

Cysteine residues are critical for chemokine receptor CXCR2 functional properties

We examined the role of cysteine (Cys) residues present in chemokine receptor CXCR2 for proper surface expression, dimerization, signaling, and chemotaxis. To address this issue, serine or leucine residues were substituted for Cys, generating nine CXCR2 mutants transiently expressed in HEK cells. Single substitution of Cys residues present in the three extracellular loops (C119L, C196L, C286S) or in the seventh-transmembrane (TM) domain (C308L) abolished CXCL8 agonist binding, while no Cys substitution abolished surface receptor expression. We have previously demonstrated that CXCR2 dimerizes under reducing conditions, due to hydrophobic interactions that involve TM3 regions, and here we show that the dimer/monomer CXCR2 ratio drastically increases when analyzed under non-reducing conditions. We report that none of the Cys-deficient CXCR2 mutants abolishes receptor dimerization, demonstrating that Cys-Cys bonds are not the exclusive determinant of CXCR2 dimerization. Furthermore, both wt- and Cys-mutated CXCR2 dimers are expressed at the cell surface, indicating that receptor dimers are efficiently transferred at the plasma membrane. We also show that every Cys substitution in CXCR2, including those that still bind CXCL8, results in an impairment of receptor activity, analyzed as cell chemotaxis and intracellular signaling, suggesting that some structural requirement is likely fulfilled by Cys presence.     

Genetic diversity and genetic differentiation in Daphnia metapopulations with subpopulations of known age

If colonization of empty habitat patches causes genetic bottlenecks, freshly founded, young populations should be genetically less diverse than older ones that may have experienced successive rounds of immigration. This can be studied in metapopulations with subpopulations of known age. We studied allozyme variation in metapopulations of two species of water fleas (Daphnia) in the skerry archipelago of southern Finland. These populations have been monitored since 1982. Screening 49 populations of D. longispina and 77 populations of D. magna, separated by distances of 1.5-2180 m, we found that local genetic diversity increased with population age whereas pairwise differentiation among pools decreased with population age. These patterns persisted even after controlling for several potentially confounding ecological variables, indicating that extinction and recolonization dynamics decrease local genetic diversity and increase genetic differentiation in these metapopulations by causing genetic bottlenecks during colonization. We suggest that the effect of these bottlenecks may be twofold, namely decreasing genetic diversity by random sampling and leading to population-wide inbreeding. Subsequent immigration then may not only introduce new genetic material, but also lead to the production of noninbred hybrids, selection for which may cause immigrant alleles to increase in frequency, thus leading to increased genetic diversity in older populations.