First evidence for the production of cylindrospermopsin and deoxy-cylindrospermopsin by the freshwater benthic cyanobacterium, Lyngbya wollei (Farlow ex Gomont) Speziale and Dyck

Lyngbya wollei (Farlow ex Gomont) Speziale and Dyck is a common mat-forming benthic cyanobacterium from freshwater habitats. We found that two populations from southeast Queensland (Australia) produce the potent cyanotoxin cylindrospermopsin (CYN) and its analogue, deoxy-cylindrospermopsin (deoxy-CYN). The highest concentrations in environmental samples were 20 and 550 μg g⁻¹ dry weight for CYN and deoxy-CYN, respectively. A sub-sample maintained in culture for over 16 months yielded concentrations of 33 and 308 μg g⁻¹ dry weight for CYN and deoxy-CYN, respectively. The concentration of deoxy-CYN in L. wollei was 10–300 times higher than CYN, suggesting that, unlike many other CYN-producing cyanobacteria, the primary compound produced by L. wollei is deoxy-CYN. The production of CYN and deoxy-CYN by L. wollei represents a potential human health risk and an additional source of these toxins in freshwaters. This is the first report of the production of CYN and deoxy-CYN by L. wollei or any species of the Oscillatoriales.     

Copper transfer from the Cu(I) chaperone, CopZ, to the repressor, Zn(II)CopY: metal coordination environments and protein interactions

Extracellular copper regulates the DNA binding activity of the CopY repressor of Enterococcus hirae and thereby controls expression of the copper homeostatic genes encoded by the cop operon. CopY has a CxCxxxxCxC metal binding motif. CopZ, a copper chaperone belonging to a family of metallochaperones characterized by a MxCxxC metal binding motif, transfers copper to CopY. The copper binding stoichiometries of CopZ and CopY were determined by in vitro metal reconstitutions. The stoichiometries were found to be one copper(I) per CopZ and two copper(I) per CopY monomer. X-ray absorption studies suggested a mixture of two- and three-coordinate copper in Cu(I)CopZ, but a purely three-coordinate copper coordination with a Cu-Cu interaction for Cu(I)2CopY. The latter coordination is consistent with the formation of a compact binuclear Cu(I)-thiolate core in the CxCxxxxCxC binding motif of CopY. Displacement of zinc, by copper, from CopY was monitored with 2,4-pyridylazoresorcinol. Two copper(I) ions were required to release the single zinc(II) ion bound per CopY monomer. The specificity of copper transfer between CopZ and CopY was dependent on electrostatic interactions. Relative copper binding affinities of the proteins were investigated using the chelator, diethyldithiocarbamic acid (DDC). These data suggest that CopY has a higher affinity for copper than CopZ. However, this affinity difference is not the sole factor in the copper exchange; a charge-based interaction between the two proteins is required for the transfer reaction to proceed. Gain-of-function mutation of a CopZ homologue demonstrated the necessity of four lysine residues on the chaperone for the interaction with CopY. Taken together, these results suggest a mechanism for copper exchange between CopZ and CopY.     

Effects of water-level fluctuation and invasive water plants on pheasant-tailed jacana (Hydrophasianus chirugus) at the Annaiwilundawa Ramsar site of northwestern Sri Lanka

The effects of alien invasive water plants (Eichhornia crassipes, Salvinia molesta, Imperata cylindrical and Mikania micrantha) on the pheasant-tailed jacana (Hydrophasianus chirugus) was studied in three reservoirs from June 2004 to May 2005 at the Annaiwilundawa Ramsar site of northwestern Sri Lanka. Jacanas were observed in significantly fewer numbers among invasive plants and open water and in significantly higher numbers among grass and floating leaved plants than predicted from the proportions of the reservoirs covered by these habitat types (Annaiwilundawa χ² = 64.35, P < 0.0001; Suruwila χ² = 45.66, P < 0.0001; Maiyawa χ² = 33.35, P < 0.0001). Feeding was the major activity of the pheasant-tailed jacana during the study period [analysis of variance (ANOVA) F = 79.58, P < 0.0001 in the morning and F = 85.53, P < 0.0001 in the evening]. Jacanas preferred to forage in grass and floating leaved vegetation compared to invasive plants and open water (ANOVA F = 168.63, P < 0.001). The jacana population was significantly positively correlated with water level in all three reservoirs. (Spearman’s rank correlation, Annaiwilundawa z = 2.035, P < 0.05; Suruwila z = 2.19, P < 0.05; Maiyawa z = 2.563, P < 0.05). The findings of this study revealed that the most critical factor for the occupation of the reservoirs by jacanas was the water level and that the spread of the invasive plants in these reservoirs were detrimental to jacanas, since these plants deprived jacanas of useful foraging habitats.     

Structural studies of the Nudix hydrolase DR1025 from Deinococcus radiodurans and its ligand complexes

We have determined the crystal structure, at 1.4A, of the Nudix hydrolase DR1025 from the extremely radiation resistant bacterium Deinococcus radiodurans. The protein forms an intertwined homodimer by exchanging N-terminal segments between chains. We have identified additional conserved elements of the Nudix fold, including the metal-binding motif, a kinked beta-strand characterized by a proline two positions upstream of the Nudix consensus sequence, and participation of the N-terminal extension in the formation of the substrate-binding pocket. Crystal structures were also solved of DR1025 crystallized in the presence of magnesium and either a GTP analog or Ap(4)A (both at 1.6A resolution). In the Ap(4)A co-crystal, the electron density indicated that the product of asymmetric hydrolysis, ATP, was bound to the enzyme. The GTP analog bound structure showed that GTP was bound almost identically as ATP. Neither nucleoside triphosphate was further cleaved.     

Emergence of Epidemic Dengue-1 Virus in the Southern Province of Sri Lanka

BackgroundDengue is a frequent cause of acute febrile illness with an expanding global distribution. Since the 1960s, dengue in Sri Lanka has been documented primarily along the heavily urbanized western coast with periodic shifting of serotypes. Outbreaks from 2005–2008 were attributed to a new clade of DENV-3 and more recently to a newly introduced genotype of DENV-1. In 2007, we conducted etiologic surveillance of acute febrile illness in the Southern Province and confirmed dengue in only 6.3% of febrile patients, with no cases of DENV-1 identified. To re-evaluate the importance of dengue as an etiology of acute febrile illness in this region, we renewed fever surveillance in the Southern Province to newly identify and characterize dengue.Conclusions/SignificanceWe describe the first epidemic of DENV-1 in the Southern Province of Sri Lanka in a population known to be susceptible to this serotype because of prior study. Dengue accounted for 40% of acute febrile illnesses in the current study. The emergence of DENV-1 as the foremost serotype in this densely populated but agrarian population highlights the changing epidemiology of dengue and the need for continued surveillance and prevention.     

Seasonality in Dietary Shifts in Size-Structured Freshwater Fish Assemblages in Three Reservoirs of Sri Lanka

We studied seasonal changes in feeding habits of size-structured fish assemblages in three freshwater reservoirs in Sri Lanka. We obtained fish samples for diet analysis from three reservoirs during the months of rising water level (RIWL) and receding water level (REWL). During RIWL, peripheral areas with terrestrial/semi-terrestrial macrophytes get inundated, which resulted in increased food availability for macrophyte feeding fish. During REWL, detrital food sources increase due to decaying terrestrial plant material, which has inundated. Based on the dietary habits of individual species in the three reservoirs during RIWL and REWL, it is evident that detritivorous and phytoplanktivorous fish species, which belong to low trophic levels do not show significant variation in dietary habits between the two seasons (e.g. Oreochromis mossambicus, Oreochromis niloticus and Amblypharyngodon melettinus). Stenophagous species such as benthic invertebrate predators (Puntius chola and Puntius dorsalis), macrophytophagous species (Etroplus suratensis and Tilapia rendalli) and zooplanktivorous Hemirhamphus limbatus do not exhibit significant variation in dietary habits between seasons. Also their feeding habits do not vary with body size. Euryphagous species such as Puntius filamentosus on the other hand, exhibit variations in dietary habits between seasons as well as with body size. Stenophagous species, which feed on detritus and phytoplankton and euryphagous species, which exhibit temporal dietary plasticity, are therefore abundant in fish assemblages of these reservoirs.     

Slc26a9 Is Inhibited by the R-region of the Cystic Fibrosis Transmembrane Conductance Regulator via the STAS Domain

SLC26 proteins function as anion exchangers, channels, and sensors. Previous cellular studies have shown that Slc26a3 and Slc26a6 interact with the R-region of the cystic fibrosis transmembrane conductance regulator (CFTR), (R)CFTR, via the Slc26-STAS (sulfate transporter anti-sigma) domain, resulting in mutual transport activation. We recently showed that Slc26a9 has both nCl⁻-HCO₃⁻ exchanger and Cl⁻ channel function. In this study, we show that the purified STAS domain of Slc26a9 (a9STAS) binds purified (R)CFTR. When Slc26a9 and (R)CFTR fragments are co-expressed in Xenopus oocytes, both Slc26a9-mediated nCl⁻-HCO₃⁻ exchange and Cl⁻ currents are almost fully inhibited. Deletion of the Slc26a9 STAS domain (a9-ΔSTAS) virtually eliminated the Cl⁻ currents with only a modest affect on nCl⁻-HCO₃⁻ exchange activity. Co-expression of a9-ΔSTAS and the (R)CFTR fragment did not alter the residual a9-ΔSTAS function. Replacing the Slc26a9 STAS domain with the Slc26a6 STAS domain (a6-a9-a6) does not change Slc26a9 function and is no longer inhibited by (R)CFTR. These data indicate that the Slc26a9-STAS domain, like other Slc26-STAS domains, binds CFTR in the R-region. However, unlike previously reported data, this binding interaction inhibits Slc26a9 ion transport activity. These results imply that Slc26-STAS domains may all interact with (R)CFTR but that the physiological outcome is specific to differing Slc26 proteins, allowing for dynamic and acute fine tuning of ion transport for various epithelia.Slc26 genes and proteins have attracted the attention of physiologists and geneticists. Why? Slc26a1 (Sat-1) was characterized as a Na⁺-independent SO₄²⁻ transporter (1). Given the transport characteristics of the founding member of the gene family, Slc26 proteins were assumed to be sulfate transporters. Disease phenotypes, clone characterization, and family additions demonstrate that the Slc26 proteins are anion transporters or channels (2–4). These proteins have varied tissue expression patterns. At one extreme, Slc26a5 in mammals is found in the hair cells of the inner ear (5), whereas Slc26a2 (DTDST) is virtually ubiquitous in epithelial tissues (2).Several Slc26 proteins are found in the epithelia of the lung, intestine, stomach, pancreas, and kidney, usually in apical membranes. Interestingly these are also tissues and membranes in which the cystic fibrosis transmembrane conductance regulator (CFTR)⁵ has been found functionally or by immunohistochemistry. Ko and co-workers (6–8) examined the distribution of Slc26a3 and Slc26a6 in HCO₃⁻ secretory epithelia, and asked if an interaction might occur between these Slc26 proteins and CFTR. In particular, these studies indicate that in expression systems, there is a reciprocal-stimulatory interaction of the STAS (sulfate transporter anti-sigma) domains of Slc26a3 and Slc26a6 with the regulatory region (R-region) of CFTR. These investigators hypothesized that this stimulatory interaction could account for the differences in pancreatic insufficiency and sufficiency observed in cystic fibrosis patients. Nevertheless, knock-out Slc26a6 mouse studies reveal more complicated cell and tissue physiology (see “Discussion”).Slc26a9 has been reported to be a Cl⁻-HCO₃⁻ exchanger (9, 10) or a large Cl⁻ conductance (3, 11, 12). Loriol and co-workers (12) indicated that SLC26A9 has a Cl⁻ conductance that may be stimulated by HCO₃⁻. Two other groups have indicated that the Cl⁻ conductance is not affected by the presence of HCO₃⁻ (10, 11). We have recently demonstrated that Slc26a9 functions as both an electrogenic nCl⁻-HCO₃⁻ exchanger and a Cl⁻ channel (10). Dorwart and colleagues (11) found that WNK kinases inhibited the SLC26A9 Cl⁻ conductance but that this effect was independent of kinase activity. One group has a preliminary report indicating that WNK3 decreased Cl⁻ uptake, whereas WNK4 increased Cl⁻ uptake via Slc26a9 expressed in Xenopus oocytes (13).Slc26a9 and CFTR are also co-expressed in several tissues. Slc26a9 protein has been localized to epithelia of the stomach and lung (9, 10, 14), although mRNA is also detectable in brain, heart, kidney, small intestine, thymus, and ovary (10). The R-region of CFTR was previously shown to increase the activity of Slc26a3 and Slc26a6 by interaction with STAS domains (6, 15, 16). Because Slc26a9 displays several different modes of ion transport, we asked if the R-region of CFTR would also increase the activity of Slc26a9. Our results indicate that the R-region of CFTR does interact with the STAS domain of Slc26a9. However, in the case of Slc26a9 this apparently similar interaction results in inhibition of Slc26a9 ion transport.     

Evaluation of sap flow methods to determine water use by cultivated palms

Sustainable management of water resources allocated to palm crops requires reliable tools for measuring palm water use. Thermometric sap flow methods developed for woody dicot plants hold great potential for use in palms, but there have been few investigations to determine whether such methods can be used successfully in ‘woody’ monocots. Here, we evaluate two sap flow methods for measuring whole-plant water use by potted cocos palms (Syagrus romanzoffiana): the Heat Ratio Method (HRM) and the Compensation Heat Pulse Method (CHPM). Measurements of whole-plant water use from the HRM and the CHPM were compared to gravimetric measurements acquired from an electronic balance. Of the two methods, the HRM gave the most accurate results and it most precisely described patterns of transpiration with respect to environmental conditions and leaf morphology. Estimates of total daily water use from the HRM and the balance were highly correlated (R² = 0.92; P < 0.0001) and very near to a 1:1 relationship—an excellent result given the potential for error associated with each method. As expected, the CHPM was seriously limited at low flow rates, but it agreed well with the HRM at higher flow rates (heat pulse velocity >4 cm h⁻¹). Anatomical investigations revealed that vascular bundles in measured palm fronds were evenly distributed and the distance between bundles was comparable in scale to the diameter of probes of HRM sensors, and most likely an order of magnitude smaller than the probable zone of thermal influence for HRM measurements. This contention was supported by results of mathematical modeling suggesting that HRM heat pulse velocities in palm-like sapwood remain largely unaffected by increases in heterogeneity caused by larger xylem vessels and wider interstitial tissues. Although wounding models for heat pulse sap flow methods have not historically catered for the effects of discrete vascular bundles, they are sufficiently approximate given our empirical validations, and we conclude that palm sapwood is thermally homogenous enough for the HRM and the CHPM to be used without modification. Overall, we present strong evidence that heat pulse methods such as the HRM and the CHPM can be used successfully in woody monocots. Finally, we note that the CHPM is likely to perform better in cases of very high flow rates, while the HRM provides more comprehensive monitoring of the usual range of flow rates including at night, early morning and during water deficit.