Cryptic Speciation in Western North America and Eastern Eurasia of the Pathogens Responsible for Laminated Root Rot.

Coniferiporia sulphurascens is a facultative fungal pathogen that causes laminated root rot (LRR) in commercially important coniferous species worldwide. This fungus spreads primarily by way of vegetative mycelium transferring at points of contact between infected and healthy roots. Successful intervention to control LRR requires a better understanding of the population structure and genetic variability of C. sulphurascens. In this study, we investigated the population genetic structure and origin of C. sulphurascens populations in western North America and eastern Eurasia collected from multiple coniferous hosts. By analyzing the small and large mitochondrial ribosomal RNA subunit genes combined with six nuclear loci (internal transcribed spacer region, actin, RNA polymerase II largest subunit, RNA polymerase II second-largest subunit, laccase-like multicopper oxidase, and translation elongation factor 1-α), we observed that none of the alleles among the loci were shared between North American (NA) and Eurasian C. sulphurascens populations. In total, 55 multilocus genotypes (MLGs) were retrieved in C. sulphurascens isolates occurring in these two continental regions. Of these, 41 MLGs were observed among 58 isolates collected from widespread locations in British Columbia (Canada) and the northwestern United States, while 14 MLGs were observed among 16 isolates sampled in Siberia and Japan. Our data showed that the levels of genetic differentiation between the NA and Eurasian populations are much greater than the populations from within each continental region; the two continental populations formed clearly divergent phylogenetic clades or lineages since they were separated approximately 7.5 million years ago. Moreover, the Eurasian population could be the source of the NA population. Our study indicates the existence of cryptic diversity in this pathogen species, and strongly suggests that the NA and Eurasian populations represent two lineages, which have progressively diverged from each other in allopatry.

SLC13 family of Na⁺-coupled di- and tri-carboxylate/sulfate transporters.

The SLC13 family comprises five genes (SLC13A1, SLC13A2, SLC13A3, SLC13A4, and SLC13A5) encoding structurally related multi-spanning transporters (8-13 transmembrane domains) with orthologues found in prokaryotes and eukaryotes. Mammalian SLC13 members mediate the electrogenic Na(+)-coupled anion cotransport at the plasma membrane of epithelial cells (mainly kidney, small intestine, placenta and liver) or cells of the central nervous system. While the two SLC13 cotransporters NaS1 (SLC13A1) and NaS2 (SLC13A4) transport anions such sulfate, selenate and thiosulfate, the three other SLC13 members, NaDC1 (SLC13A2), NaCT (SLC13A5) and NaDC3 (SLC13A3), transport di- and tri-carboxylate Krebs cycle intermediates such as succinate, citrate and α-ketoglutarate. All these transporters play a variety of physiological and pathophysiological roles in the different organs. Thus, the purpose of this review is to summarize the roles of SLC13 members in human physiology and pathophysiology and what the therapeutic perspectives are. We have also described the most recent advances on the structure, expression, function and regulation of SLC13 transporters.

Patterns of colonization and spread in the fungal spruce pathogen Onnia tomentosa.

The basidiomycetous fungus Onnia tomentosa is one of the most widespread root rot pathogens in North America. Although the disease is more severe on spruce and pine trees, this pathogen can infect several coniferous species. To study the population structure of O. tomentosa, we harvested 180 basidiocarps in a 45-year-old white spruce plantation in western Quebec in autumn 1997 and extracted DNA directly from individual basidiocarps. Using a combination of spatial coordinates and molecular data based on the analysis of two mitochondrial and three nuclear loci, we measured the average genet size and molecular diversity and assessed the relative contribution of basidiospores and vegetative growth to the stand colonization. Most of the sampled basidiocarps that clustered spatially belonged to the same genet. A total of 37 discrete multilocus genets of an average size of 3.42 m were obtained. The genet size distribution was skewed towards smaller genets (<3 m) that displayed higher diversity than the larger genets (>3 m). The nuclear loci were in Hardy-Weinberg equilibrium in the larger genets, but not in the smaller genets, which displayed a deficiency of heterozygotes. This suggests a Wahlund effect, whereby different colonization events resulted in expected heterozygosity higher than observed heterozygosity. Using an estimate of the growth rate of the fungus, only a few of the largest genets were approximately the age of the plantation. These observations are consistent with the colonization by basidiospores subsequent to site preparation and tree planting followed by secondary colonization events and vegetative spread.

Inherited epithelial transporter disorders--an overview.

In the late 1990s, the identification of transporters and transporter-associated genes progressed substantially due to the development of new cloning approaches such as expression cloning and, subsequently, to the implementation of the human genome project. Since then, the role of many transporter genes in human diseases has been elucidated. In this overview, we focus on inherited disorders of epithelial transporters. In particular, we review genetic defects of the genes encoding glucose transporters (SLC2 and SLC5 families) and amino acid transporters (SLC1, SLC3, SLC6 and SLC7 families).

First Report of Amylostereum areolatum, the Fungal Symbiont of Sirex noctilio, on Pinus spp. in Canada.

Amylostereum areolatum (Fr.) Boidin (Russulales: Stereaceae) is a white rot fungus that has a symbiotic relationship with several woodwasps including Sirex noctilio Fabricius (Hymenoptera: Siricidae). The vectored fungus together with a phytotoxic mucus, both injected during oviposition by the female S. noctilio, rapidly weaken the host tree, rendering it susceptible to larval development (3). Host trees of A. areolatum include species of Pinus (mainly), Abies, Larix, and Picea and Cryptomeria japonica and Pseudotsuga menziesii (Fungal Databases [online]; USDA). The siricid woodwasp is native to Eurasia and North Africa and has been introduced into New Zealand, Australia, South America, and South Africa (1). In July of 2005, the first established North American population of S. noctilio was reported in Oswego, NY. Prompted by this initial discovery, a trap survey of Ontario counties located along the Canada-U.S. border, close to Upstate New York, was conducted in September and October of 2005. S. noctilio females were captured in four locations in southern Ontario. Two additional locations for S. noctilio were also reported in a survey conducted independently (2). In September and October of 2006, logs of Scots pines showing current Sirex oviposition sites were harvested from the Ontario area bordered by Lakes Huron, Erie, and Ontario to determine the presence of the species-specific fungal symbiont of S. noctilio, A. areolatum. Fungal isolates were obtained by surface sterilizing wood chips showing decay columns followed by incubation at 20°C on 2% malt extract agar. Cultures with morphological characteristics typical of A. areolatum-presence of clamp connections and arthrospores-were used for DNA analysis to confirm species identification. DNA sequences of the internal transcribed spacer (ITS) of the ribosomal RNA gene were queried against the NCBI GenBank database. There was a 99 to 100% match between the ITS sequences from the Ontario isolates and sequences from European and Asian A. areolatum isolates (GenBank Accession Nos. EU249343 and EU249344 versus AF454428, AF506405, AY781245, and AF218389). Matches with A. chailletii (Pers.) Boidin, a native related species, were around 97%. These results confirmed the presence of A. areolatum in the infested material. Cultures were deposited in the National Mycological Herbarium of Canada (DAOM 239280-DAOM 239295). To our knowledge, this represents the first report of A. areolatum in Canada. In its natural range, the insect-fungal complex exists in equilibrium with its host trees and parasites, thus, few negative impacts are observed. However, in the Southern Hemisphere where it has been introduced, it has become a major pest, attacking many important commercial North American species planted as exotics (1). Conifer forests in Canada are threatened by the spread of the S. noctilio/A. areolatum complex, particularly plantations and stands of Pinus banksiana, P. contorta, P. ponderosa, P. resinosa, P. strobus, and P. sylvestris. A survey of Eastern Canada to detect the presence of S. noctilio is on going, and genetics work is being conducted to determine the origin of the introduction of A. areolatum. References: (1) W. M. Ciesla. J. For. 101:18, 2003. (2) P. de Groot et al. Gt. Lakes Entomol. 39:49, 2006. (3) B. Slippers et al. S. Afr. J. Sci. 99:70, 2003.

Molecular epidemiology of white pine blister rust: recombination and spatial distribution.

ABSTRACT Multilocus haplotypes (MLHs) were derived for the spermogonial (monokaryotic haploid) stage of Cronartium ribicola, the causal agent of white pine blister rust. Six random amplified polymorphic DNA loci and three single-strand conformational polymorphism markers were analyzed for 246 rust samples collected from two heavily infected white pine plantations. All cankers sampled were spatially located within the plantations. The hypothesis that spores are not locally disseminated was supported by the absence of any spatial clustering in the distribution of the MLHs. A large number of MLHs was found at both sites and the haplotypic diversity was close to the maximum (one) in both populations. All measures of recombination were not different from expectations under a scenario of sexual recombination. Genetic differentiation between the two sites was very low (theta = 0.023), yet it was significantly different from zero (P < 0.01). This analysis is in agreement with a scenario of extensive sexual recombination followed by some long-distance dispersal.

A nuclear factor other than Sp1 binds the GC-rich promoter of the gene encoding rat poly(ADP-ribose) polymerase in vitro.

Poly(ADP-ribose) polymerase is a nuclear enzyme that has been shown to exert a key role in many important cellular functions, including DNA repair. Its activity was shown to vary substantially between tissues; the testis and the thymus expressed the highest levels of PARP whereas the liver and the kidney (as well as a few other tissues) expressed only low levels of PARP proteins in vivo. The GC-rich nature of its upstream gene promoter, along with the lack of TATA and CAAT boxes, a feature common to most housekeeping genes, is consistent with a major regulatory function played by the positive transcription factor Sp1 in rat PARP gene transcription. Sp1 was indeed recently shown to interact with five distinct GC or GT boxes present in the rat PARP promoter. However, the observation that PARP activity was lower in rat liver than in other tissues was shown not to be the result of reduced Sp1 activity in liver cells but rather suggests the interplay of nuclear proteins other than Sp1 that are required to restrict PARP expression in this organ and maybe in others (such as the kidney). In this study, we investigated this possibility further by defining whether other nuclear proteins might bind the PARP promoter to modulate its transcription in liver cells. As a result, we identified a nuclear factor distinct from Sp1 that binds the PARP promoter at a site overlapping the F2 Sp1 element previously identified. Our results suggest that this protein likely belongs to the CTF-NF1 family of transcription factors.

Transcriptional regulation of the rat poly(ADP-ribose) polymerase gene by Sp1.

Expression of the gene encoding poly(ADP-ribose) polymerase (PARP), although ubiquitous, nevertheless varies substantially between tissues. We have recently shown that Sp1 binds five distinct target sequences (US-1 and F1-F4) in the rat PARP (rPARP) gene promoter. Here we used deletion analyses and site-directed mutagenesis to address the regulatory function played by these Sp1 sites on the basal transcriptional activity directed by the rPARP promoter. Transfection experiments revealed that the most proximal Sp1 site is insufficient by itself to direct any promoter activity. In addition, a weak negative regulatory element was identified between positions -101 and -60. The rPARP promoter directed high levels of chloramphenicol acetyltransferase activity in Jurkat T-lymphoblastoid and Ltk- fibroblast cells but only moderate levels in pituitary GH4C1 and liver HTC cells, correlating with the amounts of PARP detected in these cells by western blot analysis. However, the reduced promoter efficiency in HTC and GH4C1 cells did not result from the lack of Sp1 activity in these cells but suggested that yet uncharacterized regulatory proteins might turn off PARP gene expression by binding negative regulatory elements from the rPARP promoter. Similarly, site-directed mutagenesis on the three most proximal Sp1 elements suggested the influence exerted by Sp1 on the rPARP promoter activity to vary substantially between cell types. It also provided evidence for a basal rPARP promoter activity driven through the recognition of unidentified cis-acting elements by transcription factors other than Sp1.

Optimization of competitor poly(dI-dC).poly(dI-dC) levels is advised in DNA-protein interaction studies involving enriched nuclear proteins.

Procedures used for investigating DNA-protein interactions, such as the electrophoretic mobility shift assay (EMSA) or DNasel footprinting, require that exogenous nucleic acids (or synthetic equivalents) be added to the reaction mixture to prevent or reduce the nonspecific interaction of nuclear proteins with the labeled probe of choice, especially when proteins are obtained from crude nuclear extracts. One of the most potent, and likely the most widely used, non-specific competitor is the synthetic polymer poly(dI-dC).poly(dI-dC). Its addition to the reaction mixture prior to crude nuclear proteins has unquestionably proven very efficient in reducing nonspecific interactions by facilitating detection of the complexes of interest. However, in certain instances, the use of crude extracts alone does not provide adequate answers and the need to further enrich such extracts becomes absolutely necessary. In this study, we provide evidence that amounts of poly(dI-dC).poly(dI-dC) well below those currently described in the literature substantially impair, or even totally prevent, the detection of specific DNA-protein complexes in EMSA when enriched, gel-fractionated or commercially purified nuclear proteins are used, therefore indicating the need to precisely optimize the amount of such a competitor in DNA-protein interaction studies.

The role of adjunctive Rodogyl therapy in the treatment of advanced periodontal disease. A longitudinal clinical and microbiologic study.

Several studies have indicated that the combination of metronidazole and spiramycin is synergistic against anaerobic bacteria and may be effective against oral infections. The present study sought to determine the efficacy and safety of a commercial preparation of these two antibiotics (Rodogyl) when used adjunctively in the treatment of advanced periodontal disease. In a double-blind parallel randomized trial, 56 patients (mean age = 44 years) with advanced periodontitis (50 of whom completed the study) were assigned to either the Rodogyl or placebo group. Both groups were thoroughly scaled and root planned for approximately 6 hours, with one group receiving Rodogyl for 2 weeks and the other a placebo. No other therapy was received during the study period. Two sites in each patient with probing depths of at least 7 mm were selected for study. Plaque level (P1I), gingival inflammation (GI), probing depth (PD), and attachment level (AL) were measured at baseline, 14 days, 1 month, and then at monthly intervals up to 6 months. Subgingival bacteria were monitored with dark-field microscopy. The development of resistant bacteria, as well as side effects to the medications, was also monitored. The Rodogyl group exhibited a greater gain in AL (0.67 mm) from the 2-month interval until the end of the study. Although this difference was statistically significant (P less than 0.05), it was not necessarily of biologic significance. There was a significantly greater decline in the proportion of spirochetes in the Rodogyl group at the 14-day interval, and this difference remained significant (P less than 0.05) at all study intervals. No difference in the proportion of motile organisms was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Reliability of attachment level measurements using the cementoenamel junction and a plastic stent.

Repeated measurements of attachment level appear to be important assessments in periodontal clinical trials, yet the lack of reliability for this assessment creates measurement error which in turn demands increased sample sizes or reduces the power of the test. A plastic occlusal stent has been developed as a fixed reference point to assess changes in probing depths over time and thus reflect differences in attachment levels. The advantages of this system over traditional methods have not been measured. The purpose of this study was to determine intra- and interexaminer reliability for probing depths from the stent and the CEJ. Paired measurements of attachment level using the stent produced correlation coefficients for inter- and intraexaminer readings of 0.84 and 0.76, respectively. For subgingival cementoenamel (CEJ) measurements, lower coefficients of 0.71 and 0.59 were found for inter- and intraexaminer paired readings, respectively. Thus, measurements using the stent appear to be more reliable than subgingival CEJ readings.