Crustacean and rotifer composition of temporary ponds in the Doñana National Park (SW Spain) during floods

The zooplankton of 18 temporary ponds in the Doñana National Park (SW Spain) was studied during floods in February and May 1997. A total of 37 rotifer taxa and 34 crustaceans species were identified (17 cladocerans, 2 ostracans, 4 diaptomids, 7 cyclopodids, 1 harpacticoid, 1 anostracan, 1 notostracan and 1 conchostracan). Zooplankton samples were collected separately from the littoral and the open-water of 12 different ponds. Commonly distributed zooplankton species (frequency of appearance 50%) were not segregated to either the littoral or the open-water according to a chi-square test (P>0.005). The study ponds were divided into seasonal, intermediate and ephemeral ponds according to the length of their hydroperiod. The total numbers of both crustacean and rotifer taxa were highest in the intermediate-hydroperiod ponds (26 and 32 taxa, respectively). The total number of zooplankton taxa collected in both February and May was not significantly correlated to the hydroperiod of the temporary ponds of Doñana during the study period (r=0.165, P=0.526). 21 rotifer taxa and 20 crustacean species were found in the ephemeral ponds; the number of restricted species was also relatively high (3 rotifers and 4 crustaceans). Therefore, the ephemeral ponds held a relatively rich community during floods compared to other temporary ponds of Doñana despite their small size and short wet phase.     

Membrane permeability of fructose-1,6-diphosphate in lipid vesicles and endothelial cells

Fructose-1,6-diphosphate (FDP) is a glycolytic intermediate which has been used an intervention in various ischemic conditions for two decades. Yet whether FDP can enter the cell is under constant debate. In this study we examined membrane permeability of FDP in artificial membrane bilayers and in endothelial cells. To examine passive diffusion of FDP through the membrane bilayer, L-a-phosphatidylcholine from egg yolk (Egg PC) (10 mM) multi-lamellar vesicles were created containing different external concentrations of FDP (0, 0.5, 5 and 50 mM). The passive diffusion of FDP into the vesicles was followed spectrophotometrically. The results indicate that FDP diffuses through the membrane bilayer in a dose-dependent fashion. The movement of FDP through Egg PC membrane bilayers was confirmed by measuring the conversion of FDP to dihydroxyacetone-phosphate and the formation of hydrozone. FDP (0, 0.5, 5 or 50 mM) was encapsulated in Egg PC multilamellar vesicles and placed in a solution containing aldolase. In the 5 and 50 mM FDP groups there was a significant increase in dihydroxyacetone/hydrazone indicating that FDP crossed the membrane bilayer intact. We theorized that the passive diffusion of FDP might be due to disruption of the membrane bilayer. To examine this hypothesis, small unilamellar vesicles composed of Egg PC were created in the presence of 60 mM carboxyfluorescein, and the leakage of the sequestered dye was followed upon addition of various concentrations of FDP, fructose, fructose-6-phosphate, or fructose-1-phosphate (0, 5 or 50 mM). These results indicate that increasing concentrations of FDP increase the leakage rate of carboxyfluorescein. In contrast, no concentration of fructose, fructose-6-phosphate, or fructose-1-phosphate resulted in any significant increase in membrane permeability to carboxyfluorescein. To examine whether FDP could pass through cellular membranes, we examined the uptake of ¹⁴C-FDP by endothelial cells cultured under hypoxia or normoxia for 4 or 16 h. The uptake of FDP was dose-dependent in both the normoxia and hypoxia treated cells, and was accompanied by no significant loss in endothelial cell viability. Our results demonstrate that FDP can diffuse through membrane bilayers in a dose-dependent manner.     

Compound heterozygosity for two different amino-acid substitution mutations in the thrombopoietin receptor (c-mpl gene) in congenital amegakaryocytic thrombocytopenia (CAMT)

Congenital amegakaryocytic thrombocytopenia (CAMT) without physical anomalies is a rare disease, presenting isolated thrombocytopenia and megakaryocytopenia in infancy, which can evolve into aplastic anemia and leukemia. Recently, two heterozygous truncating mutations of the thrombopoietin (TPO) receptor MPL, coded by the c-mpl gene, were identified in a 10-year-old Japanese patient with CAMT transmitted in an autosomal recessive manner. Here, we report for the first time two different MPL amino-acid substitutions in a 2-year-old Italian boy with CAMT and compound heterozygosis for two (c-mpl point mutations. C-to-T transitions were detected on exons 5 and 12 at the 769 and 1904 cDNA nucleotide positions, respectively. The mutation in exon 5 substitutes an arginine with a cysteine (R257C) in the extracellular domain, 11 amino acids distant from the WSXWS motif conserved in the cytokine-receptor superfamily. The mutation in exon 12 substitutes a proline with a leucine (P635L) in the last amino acid of the C-terminal intracellular domain, responsible for signal transduction. As in the Japanese family, the mutations were both transmitted from the parents. TPO plasma levels were highly increased in the patient. The patient's 7-year-old brother, who was a candidate donor for allografting, turned out to be an asymptomatic heterozygous carrier of P635L and showed defective megakaryocyte colony formation from bone-marrow progenitor cells. The present study provides important confirmation that CAMT can be associated with (c-mpl) mutations.     

Volatile Organic Compounds Associated with Microbial Growth in Automobile Air Conditioning Systems

Volatile organic compounds from Penicillium viridicatum and Methylobacterium mesophilicum growing on laboratory media and on component materials of automobile air conditioners were analyzed with gas chromatography and mass spectrometry. P. viridicatum produced compounds such as 4-methyl thiazole, terpenes and alcohols, whereas M. mesophilicum produced dimethyl disulfide, dimethyl trisulfide, and chlorophenol with growth on laboratory media. In comparison with laboratory media, fewer volatiles were detected from colonized foam insulation materials. Biofilms of M. mesophilicum on aluminum evaporator components produced mainly dimethyl disulfide. These biofilms, after inoculation with P. viridicatum, produced offensive smelling alcohols and esters such as 2-methyl propanol, 3-penten-2-ol, and the ethyl ester of butanoic acid. The moisture and substrates innate to the automobile air conditioning systems provided an environment suitable for microbial biofilm development and odor production. Reduction of retained moisture in the air conditioning system coupled with use of less susceptible or antimicrobial substrates are advised for remediation of the noxious odors. Received: 26 February 2000 / Accepted: 2 May 2000     

Functions Encoded by Pyrrolnitrin Biosynthetic Genes from Pseudomonas fluorescens

Pyrrolnitrin is a secondary metabolite derived from tryptophan and has strong antifungal activity. Recently we described four genes, prnABCD, from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin. In the work presented here, we describe the function of each prn gene product. The four genes encode proteins identical in size and serology to proteins present in wild-type Pseudomonas fluorescens, but absent from a mutant from which the entire prn gene region had been deleted. The prnA gene product catalyzes the chlorination of l-tryptophan to form 7-chloro-l-tryptophan. The prnB gene product catalyzes a ring rearrangement and decarboxylation to convert 7-chloro-l-tryptophan to monodechloroaminopyrrolnitrin. The prnC gene product chlorinates monodechloroaminopyrrolnitrin at the 3 position to form aminopyrrolnitrin. The prnD gene product catalyzes the oxidation of the amino group of aminopyrrolnitrin to a nitro group to form pyrrolnitrin. The organization of the prn genes in the operon is identical to the order of the reactions in the biosynthetic pathway.The antibiotic pyrrolnitrin [3-chloro-4-(2′-nitro-3′-chlorophenyl)pyrrole] (PRN) is produced by many pseudomonads and has broad-spectrum antifungal activity (1, 5, 12–14, 17). PRN has been implicated as an important mechanism of biological control of fungal plant pathogens by several Pseudomonas strains (12–14), including P. fluorescens BL915, from which the prn genes were isolated (10).Tryptophan was identified as the precursor for PRN, based on the feeding of cultures with isotopically labeled and substituted tryptophan (2, 7, 8, 17, 25). Biosynthetic pathways were proposed as early as 1967 (7) and have been refined on the basis of tracer studies and the isolation of intermediates (Fig. ​(Fig.1)1) (2, 8, 17, 19, 23, 25). Recently, Hammer et al. (9) described the cloning and characterization of a 5.8-kb DNA region which encodes the PRN biosynthetic pathway. This DNA region confers the ability to produce PRN when expressed heterologously in Escherichia coli and contains four genes, prnABCD, each of which is required for PRN production. In the research described here, we used mutants in which each of the four genes was disrupted and strains which overexpress the individual genes to elucidate the function of each gene product in PRN biosynthesis. Open in a separate windowFIG. 1Biosynthetic pathways for PRN as proposed by van Pée et al. (23) (A) and by Chang et al. (2) (B). The reactions catalyzed by the PRN biosynthetic enzymes encoded by the prnABCD genes are indicated above the appropriate reaction arrows.

Bacterial strains and plasmids.

The bacterial strains and plasmids used in this study are described in Table ​Table1.1. Pseudomonas strains were cultured in Luria-Bertani medium at 28°C. Antibiotics, when used, were added at the following concentrations: tetracycline, 30 μg/ml; and kanamycin, 50 μg/ml. The expression vector pPEH14 consists of the P_tac promoter and rrnB ribosomal terminator from pKK223-3 (Pharmacia, Uppsala, Sweden) cloned into the BglII site of the broad-host-range plasmid pRK290 (4). P_tac is a strong constitutive promoter in Pseudomonas (unpublished data). The PRN biosynthetic genes are the coding regions described by Hammer et al. (9). Each coding region was cloned from the translation initiation codon to the stop codon by PCR with restriction sites added to the ends to facilitate cloning. For prnB, the native GTG initiation codon was changed to ATG. The clones were sequenced after PCR.

TABLE 1

Bacterial strains and plasmids used in this study

Genetic Analysis of the Role of the Transfer Gene, traN, of the F and R100-1 Plasmids in Mating Pair Stabilization during Conjugation

Mating pair stabilization occurs during conjugative DNA transfer whereby the donor and recipient cells form a tight junction which requires pili as well as TraN and TraG in the donor cell. The role of the outer membrane protein, TraN, during conjugative transfer was examined by introduction of a chloramphenicol resistance cassette into the traN gene on an F plasmid derivative, pOX38, to produce pOX38N1::CAT. pOX38N1::CAT was greatly reduced in its ability to transfer DNA, indicating that TraN plays a greater role in conjugation than previously thought. F and R100-1 traN were capable of complementing pOX38N1::CAT transfer equally well when wild-type recipients were used. F traN, but not R100-1 traN, supported a much lower level of transfer when there was an ompA mutation or lipopolysaccharide (LPS) deficiency in the recipient cell, suggesting receptor specificity. The R100-1 traN gene was sequenced, and the gene product was found to exhibit 82.3% overall similarity with F TraN. The differences were mainly located within a central region of the proteins (amino acids 162 to 333 of F and 162 to 348 of R100-1). Deletion analysis of F traN suggested that this central portion might be responsible for the receptor specificity displayed by TraN. TraN was not responsible for TraT-dependent surface exclusion. Thus, TraN, and not the F pilus, appears to interact with OmpA and LPS moieties during conjugation, resulting in mating pair stabilization, the first step in efficient mobilization of DNA.     

Distinct Cytoplasmic and Nuclear Fractions of Drosophila Heterochromatin Protein 1: Their Phosphorylation Levels and Associations with Origin Recognition Complex Proteins

The distinct structural properties of heterochromatin accommodate a diverse group of vital chromosome functions, yet we have only rudimentary molecular details of its structure. A powerful tool in the analyses of its structure in Drosophila has been a group of mutations that reverse the repressive effect of heterochromatin on the expression of a gene placed next to it ectopically. Several genes from this group are known to encode proteins enriched in heterochromatin. The best characterized of these is the heterochromatin-associated protein, HP1. HP1 has no known DNA-binding activity, hence its incorporation into heterochromatin is likely to be dependent upon other proteins. To examine HP1 interacting proteins, we isolated three distinct oligomeric species of HP1 from the cytoplasm of early Drosophila embryos and analyzed their compositions. The two larger oligomers share two properties with the fraction of HP1 that is most tightly associated with the chromatin of interphase nuclei: an underphosphorylated HP1 isoform profile and an association with subunits of the origin recognition complex (ORC). We also found that HP1 localization into heterochromatin is disrupted in mutants for the ORC2 subunit. These findings support a role for the ORC-containing oligomers in localizing HP1 into Drosophila heterochromatin that is strikingly similar to the role of ORC in recruiting the Sir1 protein to silencing nucleation sites in Saccharomyces cerevisiae.     

The Fibronectin Domain ED-A Is Crucial for Myofibroblastic Phenotype Induction by Transforming Growth Factor-β1

Transforming growth factor-β1 (TGFβ1), a major promoter of myofibroblast differentiation, induces α-smooth muscle (sn) actin, modulates the expression of adhesive receptors, and enhances the synthesis of extracellular matrix (ECM) molecules including ED-A fibronectin (FN), an isoform de novo expressed during wound healing and fibrotic changes. We report here that ED-A FN deposition precedes α-SM actin expression by fibroblasts during granulation tissue evolution in vivo and after TGFβ1 stimulation in vitro. Moreover, there is a correlation between in vitro expression of α-SM actin and ED-A FN in different fibroblastic populations. Seeding fibroblasts on ED-A FN does not elicit per se α-SM actin expression; however, incubation of fibroblasts with the anti-ED-A monoclonal antibody IST-9 specifically blocks the TGFβ1-triggered enhancement of α-SM actin and collagen type I, but not that of plasminogen activator inhibitor-1 mRNA. Interestingly, the same inhibiting action is exerted by the soluble recombinant domain ED-A, but neither of these inhibitory agents alter FN matrix assembly. Our findings indicate that ED-A–containing polymerized FN is necessary for the induction of the myofibroblastic phenotype by TGFβ1 and identify a hitherto unknown mechanism of cytokine-determined gene stimulation based on the generation of an ECM-derived permissive outside in signaling, under the control of the cytokine itself.     

The Epstein-Barr Virus BARF1 Gene Encodes a Novel, Soluble Colony-Stimulating Factor-1 Receptor

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus associated with infectious mononucleosis and several tumors. The BARF1 gene is transcribed early after EBV infection from the BamHI A fragment of the EBV genome. Evidence shown here indicates that the BARF1 protein is secreted into the medium of transfected cells and from EBV-carrying B cells induced to allow lytic replication of the virus. Expression cloning identified colony-stimulating factor-1 (CSF-1) as a ligand for BARF1. Computer-assisted analyses indicated that subtle amino acid sequence homology exists between BARF1 and c-fms, the cellular proto-oncogene that is the receptor for CSF-1. Recombinant BARF1 protein was found to be biologically active, and it neutralized the proliferative effects of human CSF-1 in a dose-dependent fashion when assayed in vitro. Since CSF-1 is a pleiotropic cytokine best known for its differentiating effects on macrophages, these data suggest that BARF1 may function to modulate the host immune response to EBV infection.     

Rhodopsin: A Photopigment for Phototaxis in Euglena gracilis

For over 100 years, a major focus of photobiological studies has been the unicellular flagellate, Euglena gracilis, an organism well suited for such investigations by its special complement of organelles that may be considered an ancient, yet complete “visual” system. The possible photoreceptive roles of the cytoplasmic stigma and the photoreceptor (paraflagellar swelling) of E. gracilis are still under debate, because of conflicting interpretations of the results produced so far by the different research groups working on this microorganism. This article deals with our hypothesis, first put forward in the late 1980s, that rhodopsin-like proteins are responsible for photo-detection and that the paraxial rod is involved in the control of flagellar movements. This hypothesis uses oriented dipole and electroconformational coupling mechanisms as the physical phenomena that produce signal transduction. A model for phototaxis is presented.