Taxonomy, DNA barcoding and phylogeny of three new species of Pythium from Canada.

Three new species of Pythium, namely, P. oopapillum, P. emineosum and P. camurandrum are presented in this paper based on morphological descriptions and molecular phylogenetic characterisation. These new species were isolated from various ecological regions in Canada. They have unique morphological features in the genus Pythium, and form distinct clades in maximum parsimony analyses, which are also supported by maximum likelihood phylogeny using general time reversible model (GTR), and Bayesian inference (BI) phylogeny using Markov Chain Monte Carlo (MCMC) analysis methods. A comparative study of the new species with closely related taxa, their clade positions, and morphological features are described in this paper.

Different levels of COX-1 and COX-2 enzymes in synoviocytes and chondrocytes during joint contracture formation.

OBJECTIVE: To measure the levels of prostaglandin endoperoxide H synthase (PGHS) isozymes (or cyclooxygenase, COX) in vivo during the development of joint contractures secondary to immobilization in rats. METHODS: Rats had one knee joint immobilized for up to 32 weeks. Three groups were compared: 47 rats had knee joints immobilized, 38 animals had sham surgery, and 13 unoperated animals served as controls. Levels of PGHS-1 and PGHS-2 enzymes were characterized in the chondrocytes and synoviocytes of the knee joint by immunohistochemistry. Immunostaining intensity was quantified by microscopy using conventional analysis. RESULTS: PGHS-1 level was lower in synoviocytes of the anterior capsule compared with shams (1.3 vs 2.0; p < 0.05). PGHS-2 level was also lower in synoviocytes of the posterior capsule (1.8 vs 2.3; p < 0.05), but higher in chondrocytes at the anterior aspect of the tibia compared with shams (1.6 vs 0.8; p < 0.05). PGHS-2 staining was increased in chondrocytes at the posterior, opposed, and anterior aspects of the tibia compared with controls (1.1, 0.6, 0.8 vs 0.2, 0.1, 0.2, respectively; all p < 0.05). CONCLUSION: Immobility induced joint contractures are characterized by a contrasting cellular pattern of PGHS enzyme levels: decreased in the synovium and increased in the chondrocytes. These findings suggest that chondrocytic PGHS isoenzymes are important in cartilage degradation of contractured joints.

Alcohol-induced locomotor activation in C57BL/6J, A/J, and AXB/BXA recombinant inbred mice: strain distribution patterns and quantitative trait loci analysis.

RATIONALE: Quantitative trait loci (QTLs) for initial sensitivity to alcohol have been identified in a number of mouse strains (e.g. BXD); however, confirmation is required. OBJECTIVES: The present paper aimed to characterize the C57BL/6J, A/J, and AXB/BXA recombinant inbred (RI) strains of mice for basal and ethanol-induced locomotor activation as measured in an open field and to provide provisional location of QTLs for these phenotypes. METHODS: A/J and C57BL/6J mice were habituated to handling and then randomly assigned to receive one of four alcohol doses (0, 0.5, 1.0, 2.0 g/kg). Subsequently, all available strains of the AXB/BXA RI were tested with the 2 g/kg dose of ethanol or vehicle control. RESULTS: Simple regression and interval mapping were used initially to identify significant gene markers associated with ethanol-induced activation (calculated as total activity on alcohol day-total activity on saline day). Subsequently, composite interval mapping (CIM) was used to increase the accuracy in mapping individual loci. Genetic markers on chromosomes 2, 3, 8, 13, 16, 18 and 19 were associated with ethanol-induced activation. CONCLUSIONS: Three significant markers identified through CIM accounted for 86% of the genetic variance in the ethanol-induced activation. QTLs on chromosome 16 (45.6 cM) and 19 (24 cM) previously associated with alcohol consumption in the AXB/BXA RI mice were found to overlap with QTLs for ethanol-induced activation identified in the present study.

Fate of nitrate acquired by the tubeworm Riftia pachyptila.

The hydrothermal vent tubeworm Riftia pachyptila lacks a mouth and gut and lives in association with intracellular, sulfide-oxidizing chemoautotrophic bacteria. Growth of this tubeworm requires an exogenous source of nitrogen for biosynthesis, and, as determined in previous studies, environmental ammonia and free amino acids appear to be unlikely sources of nitrogen. Nitrate, however, is present in situ (K. Johnson, J. Childress, R. Hessler, C. Sakamoto-Arnold, and C. Beehler, Deep-Sea Res. 35:1723-1744, 1988), is taken up by the host, and can be chemically reduced by the symbionts (U. Hentschel and H. Felbeck, Nature 366:338-340, 1993). Here we report that at an in situ concentration of 40 microM, nitrate is acquired by R. pachyptila at a rate of 3.54 micromol g(-1) h(-1), while elimination of nitrite and elimination of ammonia occur at much lower rates (0. 017 and 0.21 micromol g(-1) h(-1), respectively). We also observed reduction of nitrite (and accordingly nitrate) to ammonia in the trophosome tissue. When R. pachyptila tubeworms are exposed to constant in situ conditions for 60 h, there is a difference between the amount of nitrogen acquired via nitrate uptake and the amount of nitrogen lost via nitrite and ammonia elimination, which indicates that there is a nitrogen "sink." Our results demonstrate that storage of nitrate does not account for the observed stoichiometric differences in the amounts of nitrogen. Nitrate uptake was not correlated with sulfide or inorganic carbon flux, suggesting that nitrate is probably not an important oxidant in metabolism of the symbionts. Accordingly, we describe a nitrogen flux model for this association, in which the product of symbiont nitrate reduction, ammonia, is the primary source of nitrogen for the host and the symbionts and fulfills the association's nitrogen needs via incorporation of ammonia into amino acids.

The ionic composition of the hydrothermal vent tube worm Riftia pachyptila: evidence for the elimination of SO2-4SO and H+ and for a Cl-/HCO-3HCO shift.

Riftia pachyptila is one of the most specialized invertebrate hosts of chemoautotrophic symbionts. Crucial to the functioning of this symbiosis is how these worms cope with fluctuating ion concentrations. Internal sulfate levels in R. pachyptila appear comparable with other benthic marine invertebrates, despite the production of sulfate internally by means of the bacterial oxidation of hydrogen sulfide, suggesting that these worms are able to eliminate sulfate effectively. Internal chloride levels appear comparable; however, coelomic fluid chloride levels decrease significantly as the amount of coelomic fluid bicarbonate increases, demonstrating a 1:1 stoichiometry. We believe this shift in chloride, out of the body fluids, is needed to compensate for changes in electrochemical balance caused by the large increase (up to and greater than 60 mmol L-1) in negatively charged bicarbonate. Riftia pachyptila fits the general pattern of monovalent ion concentrations that is seen in other benthic marine invertebrates, with a high [Na+] : [K+] ratio extracellularly and low [Na+] : [K+] ratio intracellularly. Extracellular pH values of 7.38+/-0.03 and 7.37+/-0. 04 for coelomic fluid and vascular blood, respectively, as well as intracellular pH values of 7.37+/-0.04 and 7.04+/-0.05 for plume and trophosome tissue, respectively, were measured. On the basis of significant decreases in extracellular pH and, in some cases, Na+ and K+, in worms exposed to carbonyl cyanide m-chlorophenylhydrazone, sodium vanadate, and N-ethylmaleimide, we suggest that high concentrations of H+-ATPases, perhaps Na+/H+- or K+/H+-ATPases, are involved in H+ elimination in these animals.

Physiological Functioning of Carbonic Anhydrase in the Hydrothermal Vent Tubeworm Riftia Pachyptila.

On the basis of our experiments, it is clear that carbonic anhydrase (CA) plays an important role in the CO2-concentrating mechanisms in Riftia pachyptila. Plume tissue from freshly collected animals had the highest CA activity, 253.7 +/- 36.0 {mu}mol CO2 min-1 g-1 wet wt, and trophosome activity averaged 109.4 +/- 17.9 {mu}mol CO2 min-1 g-1 wet wt. Exposure of living worms to ethoxyzolamide, a carbonic anhydrase inhibitor, resulted in a 99% decrease in CA activity (from 103.9 +/- 38.6 to 0.7 +/- 0.2 {mu}mol CO2 min-1 g-1 wet wt in the plume tissue and 57.6 +/- 17.9 to 0.04 +/- 0.11 {mu}mol CO2 min-1 g-1 wet wt in the trophosome) and essentially a complete cessation of {Sigma}CO2 uptake. High concentrations of CA appear to facilitate the equilibration between inorganic carbon (Ci) in the external and internal environments, greatly enhancing the diffusion of CO2 into the animal. In summary, R. pachyptila demonstrates very effective acquisition of inorganic carbon from the environment, thereby providing the symbionts with large amounts of CO2. This effective acquisition is made possible by three factors: extremely effective pH regulation, a large external pool of CO2, and, described in this paper, high levels of carbonic anhydrase.

Alcohol preference in AXB/BXA recombinant inbred mice: gender differences and gender-specific quantitative trait loci.

The purpose of the present study was to characterize the C57BL/6J, A/J, and AXB/BXA Recombinant Inbred (RI) strains of mice for voluntary alcohol consumption. Quantitative Trait Locus (QTL) analysis was used to provide provisional location of QTLs for alcohol consumption. The inbred strains were screened for levels of alcohol intake (calculated as alcohol preference and absolute alcohol consumption) by receiving 4 days of forced exposure to a 10% (wt/vol) solution of alcohol, followed by 3 weeks of free choice between water and 10% alcohol. A wide and continuous distribution of values for alcohol consumption and preference was obtained in the AXB/BXA RI strains, confirming polygenic influences on alcohol-related behaviors. Significant gender differences were found for both alcohol preference [F28,651 = 2.12, p < 0.001] and absolute alcohol consumption [F28,647 = 2.57, p < 0.001]. In males, putative QTLs were mapped to chromosomes (Chrs) 2, 5, 7, 10, 11, and 16. Multiple regression analysis indicated that approximately 75% of the genetic variance in alcohol preference in males could be accounted for by three of the QTL regions. Several of the putative QTLs appeared to be male-specific (Tyr on Chr 7; D10Mit126 on Chr 10; D11Mit61 on Chr 11). In females, seven putative QTLs were mapped to Chrs 2, 4, 5, 7, 11, 16, and 19. Approximately 90% of the genetic variance in alcohol preference in females could be accounted for by four QTL regions, as determined by multiple regression. The QTL on Chr 11 near D11Mit35 appeared to be female-specific. This site was close to a female-specific QTL (Alcp2) previously mapped in C57BL/6J x DBA/2J backcrosses by Melo and coworkers (Nat Genet 13, 147, 1996). The QTLs mapped for alcohol preference in the present study must be considered suggestive at the present time, since only D2Mit74 met very strict statistical criteria for significance. However, the concordance across several studies for the loci on Chrs 2, 4, 7, 9, and 11 suggest that some common QTLs influencing alcohol preference have been identified. Confirmation of QTLs mapped in the present study is currently being conducted in a new series of recombinant congenic (RC) strains developed from reciprocal backcrosses between the A/J and C57BL/6J progenitors. The concomitant use of both RI and RC strains developed from the same progenitors should provide a powerful means of detecting, confirming, and mapping QTLs for alcohol-related traits.

Sulfide acquisition by the vent worm Riftia pachyptila appears to be via uptake of HS-, rather than H2S.

Deep-sea hydrothermal vents are home to a variety of invertebrate species, many of which host chemosynthetic bacteria in unusual symbiotic arrangements. The vent tubeworm Riftia pachyptila (Vestimentifera) relies upon internal chemolithoautotrophic bacterial symbionts to support its large size and high growth rates. Because of this, R. pachyptila must supply sulfide to the bacteria, which are far removed from the external medium. Internal H2S ([H2S+HS-+S2-]) can reach very high levels in R. pachyptila (2-12mmoll-1 in the vascular blood), most of which is bound to extracellular hemoglobins. The animal can potentially take up sulfide from the environment via H2S diffusion or via mediated uptake of HS-, or both. It was expected that H2S diffusion would be the primary sulfide acquisition mechanism, paralleling the previously demonstrated preferential uptake of CO2. Our data show, however, that the uptake of HS- is the primary mechanism used by R. pachyptila to obtain sulfide and that H2S diffusion into the worm apparently proceeds at a much slower rate than expected. This unusual mechanism may have evolved because HS- is less toxic than H2S and because HS- uptake decouples sulfide and inorganic carbon acquisition. The latter occurs via the diffusion of CO2 at very high rates due to the maintenance of an alkaline extracellular fluid pH. H2S accumulation is limited, however, to sulfide that can be bound by the hemoglobins, protecting the animal from sulfide toxicity and the symbionts from sulfide inhibition of carbon fixation.

The effects of exposure to ammonia on ammonia and taurine pools of the symbiotic clam

The nutrition of the gutless clam Solemyareidi is supported by the activity of intracellular chemoautotrophic bacteria housed in its gill filaments. Ammonia (the sum of NH3 and NH4+) is utilized as a nitrogen source by the association and is abundant in the clam's environment. In the present study, clams were exposed to 0.01­1.3mmoll-1 ammonia for 22­23h in the presence of thiosulfate as a sulfur substrate. Ammonia exposure increased the ammonia concentration in the tissue pools of the gill, foot and visceral mass from 0.5 to 2µmolg-1wetmass, without added ammonia, to as much as 12µmolg-1wetmass in the presence of 0.7 and 1.3mmoll-1 external ammonia. Gill tissue ammonia concentrations were consistently higher than those in the foot and visceral mass. The elevation of tissue ammonia concentration compared with the medium may be due in part to an ammonia trapping mechanism resulting from a lower intracellular pH compared with sea water and greater permeability to NH3 compared with NH4+. Rates of ammonia incorporation into organic matter (assimilation) were determined using 15N as a tracer. 15N-labeled ammonia assimilation was higher in gill than in foot and increased as a function of 15N-labeled ammonia concentration in the medium. The size of the free amino acid (FAA) pool in the gill also increased as a function of ammonia concentration in the medium. This entire increase was accounted for by a single amino acid, taurine, which was the predominant FAA in both gill and foot tissue. Aspartate, glutamate, arginine and alanine were also abundant but their levels were not influenced by external ammonia concentration. Ammonia assimilation appeared to occur at rates sufficient to account for the observed increase in taurine level. These findings suggest that taurine is a major product of ammonia assimilation.

Inorganic carbon acquisition by the hydrothermal vent tubeworm Riftia pachyptila depends upon high external PCO2 and upon proton-equivalent ion transport by the worm

Riftia pachyptila is the most conspicuous organism living at deep sea hydrothermal vents along the East Pacific Rise. To support its large size and high growth rates, this invertebrate relies exclusively upon internal chemosynthetic bacterial symbionts. The animal must supply inorganic carbon at high rates to the bacteria, which are far removed from the external medium. We found substantial differences in body fluid total inorganic carbon (CO2) both within and between vent sites when comparing freshly captured worms from a variety of places. However, the primary influence on body fluid CO2 was the chemical characteristics of the site from which the worms were collected. Studies on tubeworms, both freshly captured and maintained in captivity, demonstrate that the acquisition of inorganic carbon is apparently limited by the availability of CO2, as opposed to bicarbonate, and thus appears to be accomplished via diffusion of CO2 into the plume, rather than by mediated transport of bicarbonate. The greatly elevated PCO2 measured at the vent sites (up to 12.6 kPa around the tubeworms), which is a result of low environmental pH (as low as 5.6 around the tubeworms), and elevated CO2 (as high as 7.1 mmol l-1 around the tubes) speeds this diffusion. Moreover, despite large and variable amounts of internal CO2, these worms maintain their extracellular fluid pH stable, and alkaline, in comparison with the environment. The maintenance of this alkaline pH acts to concentrate inorganic carbon into extracellular fluids. Exposure to N-ethylmaleimide, a non-specific H+-ATPase inhibitor, appeared to stop this process, resulting in a decline in extracellular pH and CO2. We hypothesize that the worms maintain their extracellular pH by active proton-equivalent ion transport via high concentrations of H+-ATPases. Thus, Riftia pachyptila is able to support its symbionts' large demand for inorganic carbon owing to the elevated PCO2 in the vent environment and because of its ability to control its extracellular pH in the presence of large inward CO2 fluxes.

High lipid content enhances the rate of oxygen diffusion through fish skeletal muscle.

The diffusion coefficient for O2 (Do2) and the solubility constant for O2 (alpha O2) were measured at 15 degrees C in oxidative muscle from striped bass (Morone saxatilis) that had been acclimated to 5 degrees and 25 degrees C. This design allowed us to test the hypothesis that changes in composition of the tissue that are known to occur during thermal acclimation may affect O2 movement. Our measurements permitted calculation of the diffusion constant for O2 (Ko2) through the tissue, which is a primary determinant of capacity for O2 flux. Under isothermal conditions, alpha O2 was 3.59 +/- 0.20 x 10(-2) and 6.64 +/- 0.27 x 10(-2) ml O2.cm-3.atm-1 in tissues from 25 degrees- and 5 degrees C-acclimated animals, respectively. Because O2 is more soluble in lipid than aqueous phase, higher alpha O2 in tissues from cold-acclimated animals can be accounted for by the 13-fold increase in lipid content that is known to occur in oxidative muscle of striped bass during acclimation from 25 degrees to 5 degrees C. When measured under similar isothermal conditions, Do2 showed no significant difference between animals acclimated to warm or cold temperature; Do2 through tissues from 25 degrees- and 5 degrees C-acclimated animals was 2.50 +/- 0.18 and 2.57 +/- 0.40 cm2/s, respectively. Because alpha O2 increases, the calculated KO2 (DO2. alpha O2) is greater in tissue from cold- than from warm-acclimated fish. At physiological temperature, elevated lipid content in oxidative muscle of cold-acclimated striped bass should result in enhanced intracellular movement of O2 and at least partially offset the expected decrease in DO2 at cold temperature.

Background vascular headache: relief with indomethacin.

A patient with long standing sustained unilateral headache ("background vascular") and occasional multiple jabs received prompt and lasting relief from indomethacin. The effectiveness of the drug was tested in a placebo controlled double-blind trial. Indomethacin may be of value in some types of sustained headache.