Role of recombination in the evolution of the model plant pathogen Pseudomonas syringae pv. tomato DC3000, a very atypical tomato strain

Pseudomonas syringae pv. tomato strain DC3000 (PtoDC3000) is one of the most intensively studied bacterial plant pathogens today. Here we report a thorough investigation into PtoDC3000 and close relatives isolated from Antirrhinum majus (snapdragon), Apium graveolens (celery), and Solanaceae and Brassicaceae species. Multilocus sequence typing (MLST) was used to resolve the precise phylogenetic relationship between isolates and to determine the importance of recombination in their evolution. MLST data were correlated with an analysis of the locus coding for the type III secreted (T3S) effector AvrPto1 to investigate the role of recombination in the evolution of effector repertoires. Host range tests were performed to determine if closely related isolates from different plants have different host ranges. It was found that PtoDC3000 is located in the same phylogenetic cluster as isolates from several Brassicaceae and Solanaceae species and that these isolates have a relatively wide host range that includes tomato, Arabidopsis thaliana, and cauliflower. All other analyzed tomato isolates from three different continents form a distinct cluster and are pathogenic only on tomato. Therefore, PtoDC3000 is a very unusual tomato isolate. Several recombination breakpoints were detected within sequenced gene fragments, and population genetic tests indicate that recombination contributed more than mutation to the variation between isolates. Moreover, recombination may play an important role in the reassortment of T3S effectors between strains. The data are finally discussed from a taxonomic standpoint, and P. syringae pv. tomato is proposed to be divided into two pathovars.     

Dimorphic expression of uncoupling protein-3 in golden hamster harderian gland: effects of castration and testosterone administration

Hamster (Mesocricetus auratus) harderian gland (HG) is a dimorphic orbital gland producing a copious lipid secretion. Two cell-types are present in hamster HG, type I in both sexes, type II only in males. In hamster HGs, we found a marked sexual dichotomy in the expression of uncoupling protein-3 (UCP3), a mitochondrial protein carrier, that probably exports fatty acid anions and fatty acid peroxides from the mitochondrial matrix. Following castration and/or testosterone treatment: (1) UCP3 levels correlated with the type II-cell percentage, not with testosterone levels, (2) in male HGs, UCP3 was comparable to female levels at 30 days post-castration (when the type II-cell percentage had fallen from 50 to 5%), although testosterone was already near zero at 15 days (when neither the type II-cell percentage nor the UCP3 level had fallen), and testosterone-replacement therapy prevented these changes. Testosterone-treated females possessed type II cells and a UCP3 level about twofold higher than in control females. Males displayed more intense UCP3 immunohistochemical positivity in type I HG cells than females. Hence, testosterone may indirectly control UCP3 expression by regulating the gland's morphological and lipid dimorphism. Straight-chain fatty acids [found in alkyl diacylglycerols (ADGs) in males] are oxidized predominantly in mitochondria, branched-chain fatty acids (abundant in ADGs in females) predominantly in peroxisomes, so we speculate that the higher UCP3 expression in males reflects greater fatty acid flux in HG mitochondria. This is supported by our finding that in female (not male) HGs, the peroxisome-rich fraction contained alpha-methylacyl-CoA racemase (AMACR), an enzyme important in the beta-oxidation of branched-chain fatty acids.     

A vascular endothelial growth factor mimetic accelerates gastric ulcer healing in an iNOS-dependent manner

Angiogenesis is crucial to all types of wound healing, including gastric ulcer healing. The most potent promoter of angiogenesis is vascular endothelial growth factor (VEGF). We hypothesized that a 15-amino acid peptide designed to mimic the angiogenic action of VEGF would accelerate gastric ulcer healing. Gastric ulcers were induced in mice by serosal application of acetic acid. Treatment with the VEGF mimetic accelerated gastric ulcer healing when administered orally or intraperitoneally, at a dose of 50 ng/kg or greater. Such healing was not observed when the reverse sequence pentadecapeptide or the full-length VEGF protein was administered. Contrary to our hypothesis, the VEGF mimetic did not significantly increase angiogenesis in the ulcerated stomach. The enhancement of ulcer healing by the VEGF mimetic occurred independently of cyclooxygenase-2 (COX-2) activity but was blocked by inhibitors of inducible nitric oxide synthase (iNOS). These results demonstrate that a VEGF mimetic is a potent stimulus for gastric ulcer healing, even when given orally. The effects of the mimetic were independent of stimulatory effects on angiogenesis and COX-2 activity but were dependent on iNOS-derived NO production.     

Cystatin B and its EPM1 mutants are polymeric and aggregate prone in vivo

Progressive myoclonus epilepsy type 1 (EPM1) is a neurodegenerative disease correlating with mutations of the cystatin B gene. Cystatin B is described as a monomeric protein with antiprotease function. This work shows that, in vivo, cystatin B has a polymeric structure, highly resistant to SDS, urea, boiling and sensitive to reducing agents and alkaline pH. Hydrogen peroxide increases the polymeric structure of the protein. Mass spectrometry analysis shows that the only component of the polymers is cystatin B. EPM1 mutants of cystatin B transfected in cultured cells are also polymeric. The banding pattern generated by a cysteine-minus mutant is different from that of the wild-type protein as it contains only monomers, dimers and some very high MW bands while misses components of MW intermediate between 25 and 250 kDa. Overexpression of wild-type or EPM1 mutants of cystatin B in neuroblastoma cells generates cytoplasmic aggregates. The cysteine-minus mutant is less prone to the formation of inclusion bodies. We conclude that cystatin B in vivo has a polymeric structure sensitive to the redox environment and that overexpression of the protein generates aggregates. This work describes a protein with a physiological role characterized by highly stable polymers prone to aggregate formation in vivo.     

Zooplankton in the Ligurian Sea: Part II. Exploration of their physical and biological forcing functions during summer 2000

A survey of the biological and physical oceanography of theLigurian Sea was conducted in the late summer of 2000. Forty-onestations were sampled for nutrients, oxygen, fluorescence andhydrographic information. Acoustic backscatter measurementswere used to estimate abundance of small (<5 mm) zooplanktonbiovolume versus depth and the distribution of northern krill,Meganyctiphanes norvegica. Net-tow and underwater video datawere collected to identify the zooplankton present. These datawere used to analyze the Ligurian Sea ecosystem for physicaland biological linkages that control zooplankton abundance anddistribution. Results are compared with those from a similarstudy conducted in 1999. Hydrographic sampling showed a domeof dense water in the southwestern middle of the basin. Thehighest chlorophyll a (Chl a) concentrations were measured inthis area, while small zooplankton biovolume was evenly distributedthroughout the survey. Integrated values of Chl a and smallzooplankton biovolume in 2000 were greater than in 1999. Meganyctiphanesnorvegica, siphonophores and salps were the dominant componentsof the macrozooplankton population in the upper 200 m. In thesampled depth strata, siphonophore abundance did not changeduring the day, while M. norvegica were only caught at night.Acoustic backscatter data show that higher densities of M. norvegicaoccurred in deeper water and in the western and southwesternareas of the Ligurian Sea.     

Selective enzymatic hydrolysis of aromatic diesters using esterase from Brevibacterium imperiale B222

Summary A practical procedure has been developed for the chemoselective microbial hydrolysis of aromatic dicarboxylic esters to give the corresponding monoesters, using cellular lysate and whole cell of Brevibacterium imperiale B222. The produced monoesters can be transformed into hydroxyacids, useful intermediates in the synthesis of polyesters.     

Two new bioluminescent <Emphasis Type="Italic">Henlea</Emphasis> from Siberia and lack of molecular support for <Emphasis Type="Italic">Hepatogaster</Emphasis> (Annelida,Clitellata, Enchytraeidae)

Two bioluminescent enchytraeids, Henlea petushkovi sp. n. and Henlea rodionovae sp. n., are described from the Krasnoyarsk and Irkutsk regions in Eastern Siberia. These large potworms exhibit the typical light-production pattern reported repeatedly in the genus and recently elucidated by Russian researchers in its main biophysical and biochemical aspects. Morphology and DNA indicate that the two species are very closely related, but clearly divergent in the strength of the body wall (thick and opaque in H. petushkovi), structure of the prostomium (in H. rodionovae unprecedentedly wrinkled and mobile), brain shape (almost equilateral in H. petushkovi), size of coelomocytes (60–85 μm in H. petushkovi) and structure of intestinal diverticula (tulip-shaped in H. petushkovi, apple-shaped in H. rodionovae). Limited hybridization seems to occur between them, supported by a single case of conflict between COI and morphology, and a few intermediate morphotypes were noted in greenhouse populations. The new species are phylogenetically distant from all known congeners so far DNA-barcoded, even those that, like them, respond to the diagnosis of the putative subgenus Hepatogaster ?ejka, 1910 (multitubular gut diverticula in VIII, indented brain, dorsal blood vessel from IX, prominent spermathecal glands, and nephridia from 5/6). In fact, our phylogenetic analyses dismiss Hepatogaster as an artificial (polyphyletic) taxon. Issues related to the definition of H. nasuta (Eisen, 1878), H. ochracea (Eisen, 1878) and H. irkutensis Burov, 1929, three species originally described from Siberia, indicate that Henlea taxonomy is still in a state of flux, as regards not only species interrelationships but also species definitions.