Characterisation of alpha-1 giardin: an immunodominant Giardia lamblia annexin with glycosaminoglycan-binding activity

Alpha-1 giardin is an immunodominant protein in the intestinal protozoan parasite Giardia lamblia. The Triage((R)) parasite panel, used to detect copro-antigens in stool from giardiasis patients, reacts with an epitope between amino acids 160 and 200 in alpha-1 giardin. This region of the protein is also highly immunogenic during human infections. Alpha-1 giardin is related to annexins and like many other annexins it was shown to be plasma membrane associated. Immunoelectron and immunofluorescence microscopy revealed that some alpha-1 giardin are displayed on the surface of recently excysted cells. Recombinant alpha-1 giardin displayed a Ca(2+)-dependent binding to glycosaminoglycans (GAGs), in particular heparan sulphate, a common GAG in the intestinal tract. Recombinant alpha-1 giardin bound to thin sections of human small intestine, a binding which could be inhibited by adding increasing concentrations of sulphated sugars. A surface associated trypsin activated Giardia lectin (taglin) has been suggested to be important for G. lamblia attachment. In this study we show that a monoclonal antibody that inhibits taglin recognises alpha-1 and alpha-2 giardin. Thus, alpha-1 giardin is a highly immunoreactive GAG-binding protein, which may play a key role in the parasite-host interaction. Our results further show a conserved function of annexins from lower to higher eukaryotes.     

In vivo assay for low-activity mutant forms of Escherichia coli ribonucleotide reductase

Ribonucleotide reductase (RNR) catalyzes the essential production of deoxyribonucleotides in all living cells. In this study we have established a sensitive in vivo assay to study the activity of RNR in aerobic Escherichia coli cells. The method is based on the complementation of a chromosomally encoded nonfunctional RNR with plasmid-encoded RNR. This assay can be used to determine in vivo activity of RNR mutants with activities beyond the detection limits of traditional in vitro assays. E. coli RNR is composed of two homodimeric proteins, R1 and R2. The R2 protein contains a stable tyrosyl radical essential for the catalysis that takes place at the R1 active site. The three-dimensional structures of both proteins, phylogenetic studies, and site-directed mutagenesis experiments show that the radical is transferred from the R2 protein to the active site in the R1 protein via a radical transfer pathway composed of at least nine conserved amino acid residues. Using the new assay we determined the in vivo activity of mutants affecting the radical transfer pathway in RNR and identified some residual radical transfer activity in two mutant R2 constructs (D237N and W48Y) that had previously been classified as negative for enzyme activity. In addition, we show that the R2 mutant Y356W is completely inactive, in sharp contrast to what has previously been observed for the corresponding mutation in the mouse R2 enzyme.     

Giardia lamblia: identification and characterization of Rab and GDI proteins in a genome survey of the ER to Golgi endomembrane system

To investigate the complexity of the endomembrane transport system in the early diverging eukaryote, Giardia lamblia, we characterized homologues of the GTP-binding proteins, Rab1 and Rab2, involved in regulating vesicular trafficking between the endoplasmic reticulum and Golgi in higher eukaryotes, and GDI, which plays a key role in the cycling of Rab proteins. G. lamblia Rab1, 2.1, and GDI sequences largely resemble yeast and mammalian homologues, are transcribed as 0.66-, 0.62-, and 1.4-kb messages, respectively, and are expressed during growth and encystation. Western analyses detected an abundant Rab/GDI complex at approximately 80 kDa, and free GDI (60 kDa) in both trophozoites and encysting cells. Immunoelectron microscopy with antibody to Rab1 localized Rab with ER, encystation secretory vesicles, and lysosome-like peripheral vesicles. GDI associated with these structures, and with small vesicles found throughout the cytoplasm, consistent with GDI's key role in Rab cycling between organelles within the cell.     

Paternity analysis reveals opposing selection pressures on crown coloration in the blue tit (Parus caeruleus)

In socially monogamous species, extra-pair paternity can increase the variance in reproductive success and thereby the potential for sexual selection on male ornaments. We studied whether male secondary sexual ornaments are selected through within- and/or extra-pair reproductive success in the blue tit (Parus caeruleus). Male blue tits display a bright blue crown plumage, which reflects substantially in the ultraviolet (UV) and previously has been indicated to be an important sexual signal. We show that males with a more UV-shifted crown hue were less cuckolded, which probably resulted from female preference for more ornamented mates. By contrast, however, older males and males with a less UV-shifted hue sired more extra-pair young. This probably did not reflect direct female preference, since cuckolders were not less UV-ornamented than the males they cuckolded. Alternatively, a trade-off between UV ornamentation and other traits that enhance extra-pair success could explain this pattern. Our results might reflect two alternative male mating tactics, where more UV-ornamented males maximize within-pair success and less UV-ornamented males maximize extra-pair success. Since crown colour was selected in opposite directions by within-pair and extra-pair paternity, directional selection through extra-pair matings seemed weak, at least in this population and breeding season. Reduced intensity of sexual selection due to alternative mating tactics constitutes a potential mechanism maintaining additive genetic variance of male ornaments.     

Phosphorylation of isocarbostyril- and difluorophenyl-nucleoside thymidine mimics by the human deoxynucleoside kinases

The thymidine mimics isocarbostyril nucleosides and difluorophenyl nucleosides were tested as deoxynucleoside kinase substrates using recombinant human cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK), and mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK). The isocarbostyril nucleoside compound 1-(2-deoxy-beta-D-ribofuranosyl)-isocarbostyril (EN1) was a poor substrate with all the enzymes. The phosphorylation rates of EN1 with TK1 and TK2 were <1% relative to Thd, where as the phosphorylation rates for EN1 were 1.4% and 1.1% with dCK and dGK relative to dCyd and dGuo, respectively. The analogue 1-(2-deoxy-beta-D-ribofuranosyl)-7-iodoisocarbostyril (EN2) showed poor relative-phosphorylation efficiencies (kcat/Km) with both TK1 and dGK, but not with TK2. The kcat/Km value for EN2 with TK2 was 12.6% relative to that for Thd. Of the difluorophenyl nucleosides, 5-(1'-(2'-deoxy-beta-D-ribofuranosyl))-2,4-difluorotoluene (JW1) and 1-(1'-(2'-deoxy-beta-D-ribofuranosyl))-2,4-difluoro-5-iodobenzene (JW2) were substrates for TK1 with phosphorylation efficiencies of about 5% relative to that for Thd. Both analogues were considerably more efficient substrates for TK2, with kcat/Km values of 45% relative to that for Thd. 2,5-Difluoro-4-[1-(2-deoxy-beta-L-ribofuranosyl)]-aniline (JW5), a L-nucleoside mimic, was phosphorylated up to 15% as efficiently as deoxycytidine by dCK. These data provide a possible explanation for the previously reported lack of cytotoxicity of the isocarbostyril- and difluorophenyl nucleosides, but potential mitochondrial effects of EN2, JW1 and JW2 should be further investigated.     

Growth Hormone Treatment of Hypophysectomized Rats Increases Catecholamine-induced Lipolysis and the Number of β-Adrenergic Receptors in Adipocytes: No Differences in the Effects of Growth Hormone on Different Fat Depots

Growth hormone (GH) has a lipolytic effect in adipose tissue but this effect may differ in adipose tissue from various fat depots. This latter possibility was investigated in the present study, in which the effects of GH in vivo on catecholamine-induced lipolysis and the number of β-adrenergic receptors in isolated adipocytes from different fat depots of hypophysectomized rats were investigated. Female and male Sprague-Dawley rats were hypophysectomized or sham-operated at 45 days of age. One week after the operation, hormonal replacement therapy with L-thyroxine and hydrocortisone acetate was given. In addition, groups of rats were treated with GH (1.33 mg/kg per day, given as two daily subcutaneous injections). After 1 week of hormonal treatment, adipocytes were isolated from the parametrial, epididymal and inguinal fat pads, and glycerol release after catecholamine-stimulation and ¹²⁵I-cyanopindolol binding were measured. Hypophysectomy resulted in a marked decrease in the lipolytic response to catecholamines. GH treatment significantly increased catecholamine-induced lipolysis with similar effects in adipocytes from parametrial or epididymal and inguinal fat depots in both female and male rats. There were no differences between norepinephrine compared with isoproterenol-induced responses. ¹²⁵I-cyanopindolol binding was reduced after hypophysectomy and normalized by GH treatment, without differences between parametrial and inguinal adipose tissue regions. We conclude that the lipolytic effects of GH in the rat may partly be mediated by a stimulatory effect on β-adrenergic receptors in adipocytes. In addition, GH exerted similar effect on catecholamine-induced lipolysis and β-adrenergic receptors in adipocytes from parametrial, epididymal and inguinal fat depots.     

Telomere length in relation to colour polymorphism across life stages in the tawny owl

Telomere erosion has been proposed to be tightly associated with senescence, environmental stressors and life history trade‐offs. How telomere dynamics vary across life stages and especially in relation to (heritable) phenotypic traits is still unclear. The tawny owl Strix aluco display a highly heritable melanin‐based colour polymorphism, a grey and a brown morph, linked to several fitness traits including morph‐specific telomere dynamics. As adults, brown tawny owls have shorter relative telomere length (RTL) and exhibit faster telomere shortening rate than grey owls. Here we test if these morph‐specific telomere dynamics emerge already during growth, or if they are induced only in adult life through differential physiological costs associated with the life history of the morphs. We analysed RTL from 287 tawny owl offspring and 81 first breeding adults to evaluate at what life stage morph‐specific patterns emerge. We found no differences in RTL between the two morphs during the nestling period nor at the first breeding attempt. Sex, brood size or size rank in the nest did not affect offspring RTL. Among first‐breeders, females had shorter telomeres than males suggesting a sampling‐time dependent difference in reproductive costs between sexes, due to the prominent sex roles in tawny owls in the early nestling period. The probability to return to breed after the first breeding attempt was not affected by RTL, sex or colour morph. The lack of morph‐specific difference in RTL among nestlings and first breeders suggests that previously observed morph‐specific differences in RTL dynamics in adults emerge at the onset of the breeding career and is likely due to different physiological profiles and life‐history strategies adopted by adults. We conclude that different telomere dynamics and senescence patterns among highly heritable phenotypes (colour morphs) are likely to be a result of differential costs of reproduction and self‐maintenance.     

Sexual differences in biomass and nutrient allocation of first-year Silene dioica plants

Reproductive and somatic biomass, nitrogen (N), and phosphorus (P) pools were compared between females and males in 1st-year plants of Silene dioica. We estimated irretrievable resources allocated to seeds, pollen, flowers, and unrecovered summer leaf investment by collecting plant parts at abscission throughout the season. At the end of the season, we determined resources lost through senescent stems and autumn leaf turnover and resources stored in perennial roots and overwintering buds. Sexual differences in allocation patterns depended on the resource used for comparison, and whether absolute or proportional resource pools were assessed. Total resource pools in terms of biomass and N were similar for females and males. However, male plants acquired relatively more P. The proportional reproductive investment, i.e., reproductive effort, was similar for males and females in terms of biomass and N. In terms of P, male reproductive effort was higher. There was no difference between sexes in the proportional and relative biomass allocated to perennial roots and overwintering buds. However, in terms of absolute and relative N allocation to below-ground parts, females had larger reserves than males. Females, moreover, had a larger proportion of their P in below-ground parts. However, as male total P pools were larger, absolute P reserves did not differ between sexes. The high reproductive effort and N depletion of below-ground parts in males resulted largely from higher flower production compared to females. In females, seeds were the major component of reproductive effort. These results show that if biomass and nutrient allocation are assessed in parallel for dioecious plants, we obtain a more complete view of their sexual differences. Received: 07 May 1998 / Accepted: 30 October 1998     

Enhanced Biofilm Formation and Increased Resistance to Antimicrobial Agents and Bacterial Invasion Are Caused by Synergistic Interactions in Multispecies Biofilms

Most biofilms in their natural environments are likely to consist of consortia of species that influence each other in synergistic and antagonistic manners. However, few reports specifically address interactions within multispecies biofilms. In this study, 17 epiphytic bacterial strains, isolated from the surface of the marine alga Ulva australis, were screened for synergistic interactions within biofilms when present together in different combinations. Four isolates, Microbacterium phyllosphaerae, Shewanella japonica, Dokdonia donghaensis, and Acinetobacter lwoffii, were found to interact synergistically in biofilms formed in 96-well microtiter plates: biofilm biomass was observed to increase by >167% in biofilms formed by the four strains compared to biofilms composed of single strains. When exposed to the antibacterial agent hydrogen peroxide or tetracycline, the relative activity (exposed versus nonexposed biofilms) of the four-species biofilm was markedly higher than that in any of the single-species biofilms. Moreover, in biofilms established on glass surfaces in flow cells and subjected to invasion by the antibacterial protein-producing Pseudoalteromonas tunicata, the four-species biofilms resisted invasion to a greater extent than did the biofilms formed by the single species. Replacement of each strain by its cell-free culture supernatant suggested that synergy was dependent both on species-specific physical interactions between cells and on extracellular secreted factors or less specific interactions. In summary, our data strongly indicate that synergistic effects promote biofilm biomass and resistance of the biofilm to antimicrobial agents and bacterial invasion in multispecies biofilms.     

Ecological advantages of autolysis during the development and dispersal of Pseudoalteromonas tunicata biofilms

In the ubiquitous marine bacterium Pseudoalteromonas tunicata, subpopulations of cells are killed by the production of an autocidal protein, AlpP, during biofilm development. Our data demonstrate an involvement of this process in two parameters, dispersal and phenotypic diversification, which are of importance for the ecology of this organism and for its survival within the environment. Cell death in P. tunicata wild-type biofilms led to a major reproducible dispersal event after 192 h of biofilm development. The dispersal was not observed with a DeltaAlpP mutant strain. Using flow cytometry and the fluorescent dye DiBAC4(3), we also show that P. tunicata wild-type cells that disperse from biofilms have enhanced metabolic activity compared to those cells that disperse from DeltaAlpP mutant biofilms, possibly due to nutrients released from dead cells. Furthermore, we report that there was considerable phenotypic variation among cells dispersing from wild-type biofilms but not from the DeltaAlpP mutant. Wild-type cells that dispersed from biofilms showed significantly increased variations in growth, motility, and biofilm formation, which may be important for successful colonization of new surfaces. These findings suggest for the first time that the autocidal events mediated by an antibacterial protein can confer ecological advantages to the species by generating a metabolically active and phenotypically diverse subpopulation of dispersal cells.