Characterization of the pheromone gene family of an Antarctic and Arctic protozoan ciliate, Euplotes nobilii

Allelic genes encoding water-borne signal proteins (pheromones) were amplified and sequenced from the somatic (macronuclear) sub-chromosomic genome of Antarctic and Arctic strains of the marine ciliate, Euplotes nobilii. Their open reading frames appeared to be specific for polypeptide sequences of 83 to 94 amino acids identifiable with cytoplasmic pheromone precursors (pre-pro-pheromones), requiring two proteolytic steps to remove the pre- and pro-segments and secrete the mature pheromones. Differently from most of the macronuclear genes that have so far been characterized from Euplotes and other hypotrich ciliates, the 5′ and 3′ non-coding regions of all the seven E. nobilii pheromone genes are much longer than the coding regions (621 to 700 versus 214 to 285 nucleotides), and the 5′ regions in particular show nearly identical sequences across the whole set of pheromone genes. These structural peculiarities of the non-coding regions are likely due to the presence of intron sequences and provide presumptive evidence that they are site of basic, conserved activities in the mechanism that regulates the expression of the E. nobilii pheromone genes.     

Regional differences in WT-1 and Tcf21 expression during ventricular development: implications for myocardial compaction

Background

Morphological and functional differences of the right and left ventricle are apparent in the adult human heart. A differential contribution of cardiac fibroblasts and smooth muscle cells (populations of epicardium-derived cells) to each ventricle may account for part of the morphological-functional disparity. Here we studied the relation between epicardial derivatives and the development of compact ventricular myocardium.

Results

Wildtype and Wt1^CreERT2/+ reporter mice were used to study WT-1 expressing cells, and Tcf21^lacZ/+ reporter mice and PDGFRα^-/-;Tcf21^LacZ/+ mice to study the formation of the cardiac fibroblast population. After covering the heart, intramyocardial WT-1+ cells were first observed at the inner curvature, the right ventricular postero-lateral wall and left ventricular apical wall. Later, WT-1+ cells were present in the walls of both ventricles, but significantly more pronounced in the left ventricle. Tcf21-^LacZ + cells followed the same distribution pattern as WT-1+ cells but at later stages, indicating a timing difference between these cell populations. Within the right ventricle, WT-1+ and Tcf21-lacZ+ cell distribution was more pronounced in the posterior inlet part. A gradual increase in myocardial wall thickness was observed early in the left ventricle and at later stages in the right ventricle. PDGFRα^-/-;Tcf21^LacZ/+ mice showed deficient epicardium, diminished number of Tcf21-^LacZ + cells and reduced ventricular compaction.

Conclusions

During normal heart development, spatio-temporal differences in contribution of WT-1 and Tcf21-^LacZ + cells to right versus left ventricular myocardium occur parallel to myocardial thickening. These findings may relate to lateralized differences in ventricular (patho)morphology in humans.     

The metallo-beta-lactamase GOB is a mono-Zn(II) enzyme with a novel active site

Metallo-beta-lactamases (MbetaLs) are zinc-dependent enzymes able to hydrolyze and inactivate most beta-lactam antibiotics. The large diversity of active site structures and metal content among MbetaLs from different sources has limited the design of a pan-MbetaL inhibitor. Here we report the biochemical and biophysical characterization of a novel MbetaL, GOB-18, from a clinical isolate of a Gram-negative opportunistic pathogen, Elizabethkingia meningoseptica. Different spectroscopic techniques, three-dimensional modeling, and mutagenesis experiments, reveal that the Zn(II) ion is bound to Asp120, His121, His263, and a solvent molecule, i.e. in the canonical Zn2 site of dinuclear MbetaLs. Contrasting all other related MbetaLs, GOB-18 is fully active against a broad range of beta-lactam substrates using a single Zn(II) ion in this site. These data further enlarge the structural diversity of MbetaLs.     

Multiple Exposures to Chlorhexidine and Xylitol: Adhesion and Biofilm Formation by Streptococcus mutans

Growing evidence from clinical studies suggests that mothers using xylitol gums or lozenges have decreased levels of Streptococcus mutans (SM) and do not transmit these cariogenic bacteria as readily to their children. To begin to determine mechanisms for these clinical findings and to explore potential synergism of antimicrobial combinations, we studied the effect of multiple exposures of chlorhexidine (CHX) combined with copper gluconate (CG) or zinc gluconate (ZG) followed by xylitol (XYL) on the ability of SM to adhere and form biofilms. Cell suspensions of SM were exposed two times to CHX; CG; CHX plus CG; ZG; and CHX plus ZG, and then four times to XYL. Control cells were exposed six times to water or XYL or received no treatment. For biofilm assessment, glass slides were inoculated with treated cells, and numbers of bacteria were enumerated after 48 hours of incubation. To assess the ability of SM to adhere, microtiter plate wells coated with primary S. sanguinis biofilms grown in sucrose were inoculated with treated SM, and adhesion was determined. Cells exposed to CHX–XYL combinations exhibited significant but transient inhibition of growth. The multiple-exposure regimen groups showed significant decreases in the ability of SM to form biofilms (P < 0.05). However, the CHX–XYL group exhibited a much greater effect than the other treatment groups (P < 0.001). Adhesion studies revealed that none of the multiple-exposure regimens had a significant effect on adhesion of SM to primary biofilms of S. sanguinis. We concluded that significant inhibition of SM growth and subsequent inability to grow as biofilms in the presence of sucrose occurs after a staggered exposure regimen to CHX initially and then to XYL. This may help explain the clinical data showing the decreased levels of SM in mothers treated with CHX and XYL.     

Global genetic diversity of Aedes aegypti

Mosquitoes, especially Aedes aegypti, are becoming important models for studying invasion biology. We characterized genetic variation at 12 microsatellite loci in 79 populations of Ae. aegypti from 30 countries in six continents, and used them to infer historical and modern patterns of invasion. Our results support the two subspecies Ae. aegypti formosus and Ae. aegypti aegypti as genetically distinct units. Ae. aegypti aegypti populations outside Africa are derived from ancestral African populations and are monophyletic. The two subspecies co‐occur in both East Africa (Kenya) and West Africa (Senegal). In rural/forest settings (Rabai District of Kenya), the two subspecies remain genetically distinct, whereas in urban settings, they introgress freely. Populations outside Africa are highly genetically structured likely due to a combination of recent founder effects, discrete discontinuous habitats and low migration rates. Ancestral populations in sub‐Saharan Africa are less genetically structured, as are the populations in Asia. Introduction of Ae. aegypti to the New World coinciding with trans‐Atlantic shipping in the 16th to 18th centuries was followed by its introduction to Asia in the late 19th century from the New World or from now extinct populations in the Mediterranean Basin. Aedes mascarensis is a genetically distinct sister species to Ae. aegypti s.l. This study provides a reference database of genetic diversity that can be used to determine the likely origin of new introductions that occur regularly for this invasive species. The genetic uniqueness of many populations and regions has important implications for attempts to control Ae. aegypti, especially for the methods using genetic modification of populations.     

Differences in defence responses of Pinus contorta and Pinus banksiana to the mountain pine beetle fungal associate Grosmannia clavigera are affected by water deficit

We tested the hypotheses that responses to the mountain pine beetle fungal associate Grosmannia clavigera will differ between the evolutionarily co‐evolved host lodgepole pine (Pinus contorta var. latifolia) and the naïve host jack pine (Pinus banksiana) and that these responses will be influenced by water availability. G. clavigera inoculation resulted in more rapid stem lesion development in lodgepole than in jack pine; water deficit delayed lesion development in both species. Decreased hydraulic conductivity was observed in inoculated lodgepole pine seedlings, likely because of tracheid occlusion by fungal hyphae and/or metabolite accumulation. Drought but not inoculation significantly impacted bark abscisic acid levels. Jasmonic and salicylic acid were implicated in local and systemic responses of both species to G. clavigera, with salicylic acid appearing to play a greater role in jack pine response to G. clavigera than lodgepole pine. Water deficit increased constitutive levels and/or attenuated induced responses to G. clavigera for several monoterpenes in lodgepole but not jack pine. Instead, inoculation of well‐watered but not water deficit jack pine resulted in a greater number of xylem resin ducts. These findings reveal mechanisms underlying differences in G. clavigera‐induced responses between lodgepole and jack pine hosts, and how water availability modulates these responses.     

<Emphasis Type="Italic">Saturnispora serradocipensis</Emphasis> sp. nov. and <Emphasis Type="Italic">Saturnispora gosingensis</Emphasis> sp. nov., two ascomycetous yeasts from ephemeral habitats

Two novel ascomycetous yeast species, Saturnispora serradocipensis and Saturnispora gosingensis, were isolated from leaf detritus in a tropical stream of Southeastern Brazil and a mushroom collected in Taiwan, respectively. Analysis of the D1/D2 domains of the large-subunit of the rRNA gene of these strains showed that these species are related to Saturnispora hagleri, their closest relative. Saturnispora serradocipensis and S. gosingensis differed from S. hagleri, respectively, by seven nucleotide substitutions and two indels and three nucleotide substitutions and three indels in D1/D2 rRNA sequences. The two new species differ from each another by four nucleotide substitutions and one indel in D1/D2 rRNA sequences. However, the ITS sequences of S. serradocipensis, S. gosingensis and S. hagleri were quite divergent, showing that they are genetically separate species. The type strain of S. serradocipensis is UFMG-DC-198^T (=CBS 11756^T = NRRL Y-48717^T), and of S. gosingensis GA4M05^T is (CBS 11755^T = NRRL Y-48718^T).     

Reproductive Behaviour of the Annona Fruit Borer, Cerconota anonella

The Annona fruit borer, Cerconota anonella, causes significant damage to the fruits of Annona squamosa (custard apple) and A. muricata (soursop). The methods currently available for the control of this pest are costly and new techniques, possibly involving the use of pheromones for trapping or disrupting the mating cycle of the insect are required. In order to provide the basic information required for the development of new control systems, the reproductive behaviour of the moth was observed under laboratory conditions. The calling and courtship behaviours exhibited by virgin females and males of C. anonella commenced at the eighth hour of the scotophase and continued for a 3.5‐h period. Males were attracted by conspecific females as young as 1 d old, and showed a courtship behaviour composed of three steps: antennation, wing fanning and short flights. Mating mainly occurred when both males and females were between 2 and 5 d old, but maximum activity was observed on the third day after emergence. Receptive females elevated their wings, showing their abdomens where the abdominal hairpencils were already exposed. As part of the courtship repertoire and immediately prior to copula, males performed pronounced sideways movements of their abdomens, a behaviour that appears to be exclusive to C. anonella.     

Digoxin Derivatives with Enhanced Selectivity for the α2 Isoform of Na,K-ATPase: EFFECTS ON INTRAOCULAR PRESSURE IN RABBITS*

In the ciliary epithelium of the eye, the pigmented cells express the α1β1 isoform of Na,K-ATPase, whereas the non-pigmented cells express mainly the α2β3 isoform of Na,K-ATPase. In principle, a Na,K-ATPase inhibitor with selectivity for α2 could effectively reduce intraocular pressure with only minimal local and systemic toxicity. Such an inhibitor could be applied topically provided it was sufficiently permeable via the cornea. Previous experiments with recombinant human α1β1, α2β1, and α3β1 isoforms showed that the classical cardiac glycoside, digoxin, is partially α2-selective and also that the trisdigitoxose moiety is responsible for isoform selectivity. This led to a prediction that modification of the third digitoxose might increase α2 selectivity. A series of perhydro-1,4-oxazepine derivatives of digoxin have been synthesized by periodate oxidation and reductive amination using a variety of R-NH₂ substituents. Several derivatives show enhanced selectivity for α2 over α1, close to 8-fold in the best case. Effects of topically applied cardiac glycosides on intraocular pressure in rabbits have been assessed by their ability to either prevent or reverse acute intraocular pressure increases induced by 4-aminopyridine or a selective agonist of the A3 adenosine receptor. Two relatively α2-selective digoxin derivatives efficiently normalize the ocular hypertension, by comparison with digoxin, digoxigenin, or ouabain. This observation is consistent with a major role of α2 in aqueous humor production and suggests that, potentially, α2-selective digoxin derivatives could be of interest as novel drugs for control of intraocular pressure.