Similarities in Restriction Fragment Patterns of Mitochondrial DNAs of PhytophthoraSpecies Strongly Depend on the Restriction Enzyme Used Due to Heterogeneous Base Distribution and Sequence Conservation

Mitochondrial DNAs of six morphologically different Phytophthora species were digested with 15 restriction enzymes. The numbers of restriction fragments obtained differed considerably from those theoretically expected for random base distribution. Enzymes with relatively many G and C in their recognition sequences produced significantly larger numbers of fragments. Moreover, fragments generated by most of these enzymes were more often shared by two or more species than those from enzymes with more A and T in their recognition sequence. It is concluded that base distribution in mitochondrial DNA of Phytophthora is heterogeneous,AT-rich stretches occurring scattered over the mitochondrial genome and GC-rich regions present in conserved sequences, presumably genes. A practical consequence for taxonomic RFLP studies is that optimal enzymes can be selected, depending on the desired level of resolution.     

Tetrapod phylogeny inferred from 18S and 28S ribosomal RNA sequences and a review of the evidence for amniote relationships [published erratum appears in Mol Biol Evol 1991 May;8(3):398]

The 18S ribosomal RNAs of 21 tetrapods were sequenced and aligned with five published tetrapod sequences. When the coelacanth was used as an outgroup, Lissamphibia (living amphibians) and Amniota (amniotes) were found to be statistically significant monophyletic groups. Although little resolution was obtained among the lissamphibian taxa, the amniote sequences support a sister-group relationship between birds and mammals. Portions of the 28S ribosomal RNA (rRNA) molecule in 11 tetrapods also were sequenced, although the phylogenetic results were inconclusive. In contrast to previous studies, deletion or down- weighting of base-paired sites were found to have little effect on phylogenetic relationships. Molecular evidence for amniote relationships is reviewed, showing that three genes (beta-hemoglobin, myoglobin, and 18S rRNA) unambiguously support a bird-mammal relationship, compared with one gene (histone H2B) that favors a bird- crocodilian clade. Separate analyses of four other genes (alpha- crystallin A, alpha-hemoglobin, insulin, and 28S rRNA) and a combined analysis of all sequence data are inconclusive, in that different groups are defined in different analyses and none are strongly supported. It is suggested that until sequences become available from a broader array of taxa, the molecular evidence is best evaluated at the level of individual genes, with emphasis placed on those studies with the greatest number of taxa and sites. When this is done, a bird-mammal relationship is most strongly supported. When regarded in combination with the morphological evidence for this association, it must be considered at least as plausible as a bird-crocodilian relationship.      

Study of Analogues of Thymidine-5′-Monophosphate and Thymidine as Substrates or Inhibitors of Chick Embryo Liver Thymidylate Kinase

Abstract

The phosphorylation of thymidine-5′-monophosphate (dTMP) by chick embryo liver thymidylate kinase (Km (dTMP) =1.2 μM) was inhibited by the 5′-monophosphate derivatives of 5-bromo-2′-deoxyuridine (5-Br-dUMP), 5-iodo-2′-deoxyuridine (5-I-dUMP), 2′,3′-dideoxythymidine (ddTMP), 3′-azido-3′-deoxythymidine (AZT-MP) and the methylene phosphonate analogue of AZT-MP with IC50 values of 8, 24, 14, 5 and 6 μM respectively. 5-Fluoro-2′-deoxyuridine (5-F-dUMP) and dUMP were poor inhibitors (IC50 values > 300 μM). 5-Br-dUMP and 5-I-dUMP were found to be significant substrates of thymidylate kinase with phosphorylation efficiencies (Vmax/Km) of 26 and 6% of that of dTMP, respectively. In contrast, AZT-MP and ddTMP were poor substrates, being phosphorylated 800-fold less efficiently than dTMP. Thymidylate kinase was also significantly inhibited by thymidine and AZT. Our data give a better insight into the topology of the dTMP binding site of this enzyme and show that the 3′-hydroxyl group of dTMP plays a critical role in catalysis.     

Filamentous ascomycetes inhabiting the rhizoid environment of the liverwort Cephaloziella varians in Antarctica are assessed by direct PCR and cloning

We molecularly assesed the ascomycetous fungal communities inhabiting the rhizoid environment of Cephaloziella varians, collected at Rothera Point on the western Antarctic Peninsula. The RFLP-phenotyped and cloned PCR products of a partial small subunit of the ribosomal RNA gene were sequenced and analyzed with neighbor joining (NJ) and maximum parsimony (MP). Both analyses identified four bootstrap-supported groups: (i) a sister group to Onygenales, (ii) a well-supported clade with Phialocephala fortinii, (iii) a large group of clones nested within Chaetothyriales, and (iv) a group nested within Eurotiales with a likely affinity to the genus Aspergillus. An additional marginally supported clade, including helotialean Hymenoscyphus fructigenus, was detected in the NJ analysis. Placement of one clone (possibly helotialean) was not supported by either analysis. We included Hymenoscyphus ericae (Helotiales) in our analyses to test for its presence in clone libraries. None of our clones showed strongly supported affinity to H. ericae. The culture-independent technique proved useful for assessing the composition of rhizoid fungal communities, although it remains unknown whether any of these fungi colonize C. varians tissues. Direct-community assays of this kind might be best combined with traditional isolation techniques to get a more holistic view of fungi occupying plant tissues.     

Bayesian Spatio-Temporal Analysis and Geospatial Risk Factors of Human Monocytic Ehrlichiosis

Variations in spatio-temporal patterns of Human Monocytic Ehrlichiosis (HME) infection in the state of Kansas, USA were examined and the relationship between HME relative risk and various environmental, climatic and socio-economic variables were evaluated. HME data used in the study was reported to the Kansas Department of Health and Environment between years 2005–2012, and geospatial variables representing the physical environment [National Land cover/Land use, NASA Moderate Resolution Imaging Spectroradiometer (MODIS)], climate [NASA MODIS, Prediction of Worldwide Renewable Energy (POWER)], and socio-economic conditions (US Census Bureau) were derived from publicly available sources. Following univariate screening of candidate variables using logistic regressions, two Bayesian hierarchical models were fit; a partial spatio-temporal model with random effects and a spatio-temporal interaction term, and a second model that included additional covariate terms. The best fitting model revealed that spatio-temporal autocorrelation in Kansas increased steadily from 2005–2012, and identified poverty status, relative humidity, and an interactive factor, ‘diurnal temperature range x mixed forest area’ as significant county-level risk factors for HME. The identification of significant spatio-temporal pattern and new risk factors are important in the context of HME prevention, for future research in the areas of ecology and evolution of HME, and as well as climate change impacts on tick-borne diseases.     

Hierarchical Bayesian Spatio–Temporal Analysis of Climatic and Socio–Economic Determinants of Rocky Mountain Spotted Fever

This study aims to examine the spatio-temporal dynamics of Rocky Mountain spotted fever (RMSF) prevalence in four contiguous states of Midwestern United States, and to determine the impact of environmental and socio–economic factors associated with this disease. Bayesian hierarchical models were used to quantify space and time only trends and spatio–temporal interaction effect in the case reports submitted to the state health departments in the region. Various socio–economic, environmental and climatic covariates screened a priori in a bivariate procedure were added to a main–effects Bayesian model in progressive steps to evaluate important drivers of RMSF space-time patterns in the region. Our results show a steady increase in RMSF incidence over the study period to newer geographic areas, and the posterior probabilities of county-specific trends indicate clustering of high risk counties in the central and southern parts of the study region. At the spatial scale of a county, the prevalence levels of RMSF is influenced by poverty status, average relative humidity, and average land surface temperature (>35°C) in the region, and the relevance of these factors in the context of climate–change impacts on tick–borne diseases are discussed.     

Effects of ultraviolet radiation on human epidermal cell alloantigen presentation: initial depression of Langerhans cell-dependent function is followed by the appearance of T6- Dr+ cells that enhance epidermal alloantigen presentation

The effects of ultraviolet radiation (UV) on the immune parameters of human epidermis were studied. We determined the effects of both in vitro and in vivo UV on human epidermal cell surface markers and on epidermal immune function in the allogeneic epidermal cell-lymphocyte reaction (ELR). Epidermal cells obtained immediately after in vitro and in vivo UV exposure exhibited a dose-dependent decrease in alloantigen-presenting function in the ELR. This was not the result of a decrease in the number of T6+ Dr+ Langerhans cells but was due to their being less efficient at alloantigen presentation than equivalent numbers of Langerhans cells from unirradiated skin. The reduced stimulation in the ELR immediately after UV was not reversible by the addition of exogenous IL 1 or indomethacin and thus appeared to be due to a direct effect of UV on the alloantigen-presenting function of Langerhans cells. In contrast to this suppression of the epidermal immune function when epidermal cells were obtained immediately after UV, epidermal cells harvested 24 hr or later after in vivo UV exhibited a dose-dependent enhancement of allostimulatory capacity in the ELR that peaked 3 days after UV. The time course of the enhancement of allostimulation in the ELR after in vivo UV coincided with a decrease in the percentage of Langerhans cells and the appearance within the epidermis of T6- Dr+ cells, which are derived from the bone marrow, as evidenced by their expression of the bone marrow derivation markers HLe 1 and T200. Removal of Dr+ cells but not of T6+ cells from epidermal cell suspensions harvested 3 days after in vivo UV abrogated allostimulation in the ELR, demonstrating that the T6- Dr+ cells were responsible for the observed UV-induced enhancement of alloantigen presentation. Taken together, the results indicate that the timing and dosage of UV exposure are critical factors determining whether suppression or enhancement of epidermal immune function follows UV.