Anti-HEV seroprevalence and rate of viremia in a German cohort of dogs, cats, and horses.

Hepatitis E virus (HEV) genotype 3 infections in Germany are mainly transmitted zoonotically through the consumption of swine meat. Furthermore, there is evidence that pets might come into contact with HEV, but the relevance of companion animals as possible sources of HEV transmission in Germany still needs to be defined. A monitoring study was therefore carried out on dogs, cats, and horses from Germany. In total 365 serum samples from pets (124 dogs, 119 cats, and 122 horses) were tested for HEV by PCR and for anti-HEV antibodies by a commercial ELISA. The HEV seroprevalence determined by the sero-assay varied significantly between dogs (10%), cats (6%), and horses (2%). Liver injury-related enzymes, alanine transaminase (ALT), and aspartate transaminase (AST) showed no differences between HEV-positive or negative animals. None of the pet serum samples tested positive for PCR. This serological study suggests that dogs and cats are significantly exposed to HEV in Germany, while horses are of minor relevance.

Population-based prevalence of multiple radiographically-defined hip morphologies: the Johnston County Osteoarthritis Project.

OBJECTIVE: To provide the first prevalence estimates of different radiographic hip morphologies relevant to dysplasia and femoroacetabular impingement in a well-characterized USA population-based cohort. METHODS: Cross-sectional data were from the baseline examination (1991-1997) of a large population-based prospective longitudinal cohort study (The Johnston County Osteoarthritis Project). HipMorf software (Oxford, UK) was used to assess hip morphology on anteroposterior (AP) pelvis radiographs. Weighted, sex-stratified prevalence estimates and 95% confidence intervals for four key hip morphologies (AP alpha angle, triangular index sign, lateral center edge angle (LCEA), and protrusio acetabula) were derived and further stratified by age, race and body mass index (BMI). RESULTS: A total of 5192 hips from 2596 individuals were included (31% African American, 43% male, mean age 63 years, mean BMI 29 kg/m2). Cam morphology was seen in more than 25% of men and 10% of women. Mild dysplasia was present in about 1/3 of men and women, while pincer morphology was identified in 7% of men and 10% of women. Femoral side (cam) morphologies were more common and more frequently bilateral among men, while pincer morphologies were more common in women; mixed morphologies were infrequent. African-Americans were more likely to have protrusio acetabula than whites. CONCLUSION: We report the first population-based prevalence estimates of radiographic hip morphologies relevant to femoroacetabular impingement (FAI) and dysplasia in the USA. These morphologies are very common, with » men and 1/10 women having cam morphology, 1/3 of all adults having mild dysplasia, and 1/15 men and 1/10 women having pincer morphology in at least one hip.

Measures of hip morphology are related to development of worsening radiographic hip osteoarthritis over 6 to 13 year follow-up: the Johnston County Osteoarthritis Project.

OBJECTIVES: We sought to describe the effect of alterations in hip morphology with respect to worsening hip OA in a community-based sample including African American (AA) and white men and women. METHODS: This nested case-control study defined case hips as Kellgren Lawrence grade (KLG) <3 on baseline supine pelvis radiographs and KLG ≥3 or THR for OA at the 1st or 2nd follow-up visit (mean 6 and 13 years, respectively); control hips had KLG <3 at both visits, with gender/race distribution similar to cases. Hip morphology was assessed using HipMorf software (Oxford, UK). Descriptive means and standard errors were obtained from generalized estimating equation (GEE) models. Sex-stratified GEE regression models (accounting for within-person correlation), adjusted for age, race, BMI, and side were then employed. RESULTS: A total of 120 individuals (239 hips; 71 case/168 control) were included (25% male, 26% AA, mean age 62 years, BMI 30 kg/m(2)). Case hips tended to have greater baseline AP alpha angles, smaller minimum joint space width (mJSW) and more frequent triangular index signs. Adjusted results among men revealed that higher AP alpha angle, Gosvig ratio, and acetabular index were positively associated with case hips; coxa profunda was negatively associated. Among women, greater AP alpha angle, smaller mJSW, protrusio acetabuli, and triangular index sign were associated with case hips. CONCLUSIONS: We confirmed an increased risk of worsening hip OA due to baseline features of cam deformity among men and women, as well as protrusio acetabuli among women, and provide the first estimates of these measures in AAs.

Comparative genomic studies suggest that the cyanobacterial endosymbionts of the amoeba Paulinella chromatophora possess an import apparatus for nuclear-encoded proteins.

Plastids evolved from free-living cyanobacteria through a process of primary endosymbiosis. The most widely accepted hypothesis derives three ancient lineages of primary plastids, i.e. those of glaucophytes, red algae and green plants, from a single cyanobacterial endosymbiosis. This hypothesis was originally predicated on the assumption that transformations of endosymbionts into organelles must be exceptionally rare because of the difficulty in establishing efficient protein trafficking between a host cell and incipient organelle. It turns out, however, that highly integrated endosymbiotic associations are more common than once thought. Among them is the amoeba Paulinella chromatophora, which harbours independently acquired cyanobacterial endosymbionts functioning as plastids. Sequencing of the Paulinella endosymbiont genome revealed an absence of essential genes for protein trafficking, suggesting their residence in the host nucleus and import of protein products back into the endosymbiont. To investigate this hypothesis, we searched the Paulinella endosymbiont genome for homologues of higher plant translocon proteins that form the import apparatus in two-membrane envelopes of primary plastids. We found homologues of Toc12, Tic21 and Tic32, but genes for other key translocon proteins (e.g. Omp85/Toc75 and Tic20) were missing. We propose that these missing genes were transferred to the Paulinella nucleus and their products are imported and integrated into the endosymbiont envelope membranes, thereby creating an effective protein import apparatus. We further suggest that other bacterial/cyanobacterial endosymbionts found in protists, plants and animals could have evolved efficient protein import systems independently and, therefore, reached the status of true cellular organelles.

CD26/dipeptidyl peptidase 4-deficiency alters thymic emigration patterns and leukcocyte subsets in F344-rats age-dependently.

As CD26 (dipeptidyl peptidase 4/DPP4) rapidly truncates incretins N-terminally, including glucagon-like peptide-1, DPP4-inhibitors have been developed for treatment of diabetes type 2. To some extent this is surprising, as CD26/DPP4 is also deeply involved in immune regulation. Long-term pharmacological studies are hampered by off-target inhibition of DPP4-homologues. Therefore, we studied the effects of genetic CD26/DPP4-deficiency by investigating blood, spleen and thymus leucocyte subpopulations of wild-type and CD26-deficient F344-rats at different ages. In young animals at 1 and 3 months of age, there were no differences in leucocyte subsets, while in older animals the T cell composition was changed significantly. From the age of 6 months onwards, reduced numbers of recent thymic emigrants and memory T cells, and consequently an increased amount of naive T cells were observed in CD26-deficient rats. In addition, the architecture of the thymus was altered, as observed by a reduced density of lymphocytes in the medulla. Furthermore, the number of proliferating cells in the thymus was decreased in CD26-deficient rats at a higher age. Moreover, CD26-deficiency resulted in markedly reduced numbers of B cells in later life. Additionally, an age- but not CD26-dependent increase of regulatory T cells and a decrease of natural killer cell numbers were detected in the blood and spleen. Our findings indicate an important role of CD26 in maintaining lymphocyte composition, memory T cell generation and thymic emigration patterns during immunosenescence, with possible implications for using DPP4-inhibitors.

Elevated cytokine expression in the orbitofrontal cortex of victims of suicide.

OBJECTIVE: Based on the reported association between cytokines with depression and suicide, and evidence of increased markers of inflammation in the brain of suicide victims, the present study examined the expression of cytokines in the orbitofrontal cortex of suicide victims. METHOD: In a postmortem sample obtained from the Brodman area 11 of suicides (n = 34) and controls (n = 17), real-time RT-PCR was used to compare the expression of mRNA species for tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta, 4, 5, 6, and 13. RESULTS: Increased expression of IL-4 was found in women suicide victims and IL-13 in men suicide victims. Elevated but not significant cytokine expression was also observed for TNF-alpha in women suicide victims. CONCLUSION: To our knowledge, these results provide the first evidence of the presence of mRNA transcripts of type 2 T-helper cytokines in the human orbitofrontal cortex and their increased expression in the brain of suicides.

Determination of extra renal clearance and generation rate of beta2-microglobulin in hemodialysis patients using a kinetic model.

Beta-2-microglobulin (beta2-m) has been proposed as a marker of middle molecules to assess one aspect of the efficacy of dialysis. Until now, few data have been published about extra renal (metabolic) clearance and generation rates of beta2-microglobulin, which are necessary for calculation of total clearance and mass removal of beta2-m in hemodialysis patients. We have developed a simple method to derive extra renal clearance and generation rates of beta2-m by measuring the pre and post dialysis blood concentrations of beta2-m using kinetic modeling. Ten stable hemodialysis patients were included in this study. Pre and post dialysis concentrations of beta2-m were measured during dialysis with low flux dialyzers (F6 HPS) and after 10 days switching to high flux dialyzers (F60S or Superflux). With a validated two pool model, the generation rate of beta2-m can be determined if extra renal clearance is known. Assuming the generation rate of beta2-m to be constant in each patient, the computer reiterated the calculation of extra renal clearance until the calculated generation rate was equal for both the low flux and the high flux dialyzer. Extra renal clearance was found to be between 1.97 and 4.11 ml/min (average, 3.2 ml/min). Generation rate was found in a rather narrow range between 1.63 and 2.56 mg/kg per day (average, 2.09 mg/kg per day). There was no correlation between extra renal clearance and generation rates. With this simple method, extra renal clearance and generation rates of beta2-m can be determined using data by switching hemodialysis patients from impermeable to permeable membranes.

Transformation of Lotus japonicus using the herbicide resistance bar gene as a selectable marker.

Transgenic plants of the model legume Lotus japonicus were regenerated by hypocotyl transformation using a bar gene as a selectable marker. The bar encodes for Phosphinothricin Acetyl Transferase that detoxifies phosphinothricin (PPT), the active ingredient of herbicides such as Ignite (AgrEvo) and Basta (Hoechst). Transgenic L. japonicus plants resistant to PPT were positive upon PCR by bar gene-specific primers. In 5 out of 7 independent lines tested, PPT resistance segregated as a single dominant allele indicating a single T-DNA insertion into the plant genome. All regenerated plants were fertile and void of visible somaclonal abnormalities contrary to 14% infertility when antibiotic selectable markers were used. The lack of somaclonal variation, ease of PPT application and low cost of PPT makes this protocol an attractive alternative for the regeneration of transgenic L. japonicus. The production of PPT herbicide-resistant L. japonicus plants may have significant commercial applications in crop production.

Are red algae plants? A critical evaluation of three key molecular data sets.

Whether red algae are related to green plants has been debated for over a century. Features present due to their shared photosynthetic habit have been interpreted as support for an evolutionary sisterhood of the two groups but, until very recently, characters endogenous to the host cell have provided no reliable indication of such a relationship. In this investigation, we examine three molecular data sets that have provided key evidence of a possible relationship between green plants and red algae. Analyses of an expanded alignment of DNA-dependent RNA polymerase II largest subunit sequences indicate that their support for independent origins of rhodophytes and chlorophytes is not the result of long-branch attraction, as has been proposed elsewhere. Differences in the pol II C-terminal domain, an essential component of plant mRNA transcription, also suggest different host cell ancestors for the two groups. In contrast, concatenated sequences of two groups of mitochondrial genes, those encoding subunits of NADH-dehydrogenase as well as cytochrome c oxidase subunits plus apocytochrome B, appear to cluster red algal and green plant sequences together because both groups have evolved relatively slowly and share a super-abundance of ancestral positions. Finally, analyses of elongation factor 2 sequences demonstrate a strong phylogenetic signal favoring a rhodophyte/chlorophyte sister relationship, but that signal is restricted to a contiguous segment comprising approximately half of the EF2 gene. These results argue for great caution in the interpretation of phylogenetic analyses of ancient evolutionary events but, in combination, indicate that there is no emerging consensus from molecular data supporting a sister relationship between red algae and green plants.

A bacterial artificial chromosome library of Lotus japonicus constructed in an Agrobacterium tumefaciens-transformable vector.

We constructed a BAC library of the model legume Lotus japonicus with a 6-to 7-fold genome coverage. We used vector PCLD04541, which allows direct plant transformation by BACs. The average insert size is 94 kb. Clones were stable in Escherichia coli and Agrobacterium tumefaciens.

Evolutionary complementation for polymerase II CTD function.

The C-terminal domain (CTD) of the largest subunit (RPB1) of eukaryotic RNA polymerase II is essential for pol II function and has been shown to play a number of important roles in the mRNA transcription cycle. The CTD is composed of a tandemly repeated heptapeptide that is conserved in yeast, animals, plants and several protistan organisms. Some eukaryotes, however, have what appear to be degenerate or deviant CTD regions, and others have no CTD at all. The functional and evolutionary implications of this variation among RPB1 C-termini is largely unexplored. We have transformed yeast cells with a construct consisting of the yeast RPB1 gene with 25 heptads from the primitive protist Mastigamoeba invertens in place of the wild-type CTD. The Mastigamoeba heptads differ from the canonical CTD by the invariable presence of alanines in place of threonines at position 4, and in place of serines at position 7 of each heptad. Despite this double substitution, mutants are viable even under conditions of temperature and nutrient stress. These results provide new insights into the relative functional importance of several of the conserved CTD residues, and indicate that in vivo expression of evolutionary variants in yeast can provide important clues for understanding the origin, evolution and function of the pol II CTD.

National health projections through 2008.

Health spending is expected to resume its rise as a share of gross domestic product (GDP) in the projection period, following 6 years of near stability, increasing from 13.5 percent in 1997 to an estimated 16.2 percent by 2008. This implies an approximate doubling of health spending, from $1.1 trillion in 1997 to $2.2 trillion by 2008. We anticipate a reversal in recent patterns of growth in public and private health spending, with private spending expected to accelerate while Medicare spending slows in response to the implementation of the Balanced Budget Act (BBA) of 1997.

National health expenditures, 1998.

In 1998, national health care expenditures reached $1.1 trillion, an increase of 5.6 percent from the previous year. This marked the fifth consecutive year of spending growth under 6 percent. Underlying the stability of the overall growth, major changes began taking place within the Nation's health care system. Public payers felt the initial effects of the Balanced Budget Act of 1997 (BBA), and private payers experienced increased health care costs and increased premium growth.

Long-branch attraction and the rDNA model of early eukaryotic evolution.

Phylogenetic analyses of ribosomal RNA genes have become widely accepted as a framework for understanding broad-scale eukaryotic evolution. Nevertheless, conflicts exist between the phylogenetic placement of certain taxa in rDNA trees and their expected position based on fossils, cytology, or protein-encoding gene sequences. For example, pelobiont amoebae appear to be an ancient group based on cytologic features, but they are not among the early eukaryotic brances in rDNA analyses. In this report, the derived position of pelobionts in rDNA trees is shown to be unreliable and likely due to long-branch attraction among more deeply branching sequences. All sequences that branch near the base of the tree suffer from relatively high apparent substitution rates and exhibit greater variation in ssu rDNA sequence length. Moreover, the order of the branches leading from the root of the eukaryotic tree to the base of the so-called "crown taxa" is consistent with a sequential attachment, due to "long-branch" effects, of sequences with increasing rates of evolution. These results suggest that the basal eurkaryotic topology drawn from rDNA analyses may be, in reality, an artifact of variation in the rate of molecular evolution among eukaryotic taxa.

A plant regulator controlling development of symbiotic root nodules.

Symbiotic nitrogen-fixing root nodules on legumes are founded by root cortical cells that de-differentiate and restart cell division to establish nodule primordia. Bacterial microsymbionts invade these primordia through infection threads laid down by the plant and, after endocytosis, membrane-enclosed bacteroids occupy cells in the nitrogen-fixing tissue of functional nodules. The bacteria excrete lipochitin oligosaccharides, triggering a developmental process that is controlled by the plant and can be suppressed. Nodule inception initially relies on cell competence in a narrow infection zone located just behind the growing root tip. Older nodules then regulate the number of nodules on a root system by suppressing the development of nodule primordia. To identify the regulatory components that act early in nodule induction, we characterized a transposon-tagged Lotus japonicus mutant, nin (for nodule inception), arrested at the stage of bacterial recognition. We show that nin is required for the formation of infection threads and the initiation of primordia. NIN protein has regional similarity to transcription factors, and the predicted DNA-binding/dimerization domain identifies and typifies a consensus motif conserved in plant proteins with a function in nitrogen-controlled development.

Symbiotic mutants deficient in nodule establishment identified after T-DNA transformation of Lotus japonicus.

Nitrogen-fixing root nodules develop on legumes as a result of an interaction between host plants and soil bacteria collectively referred to as rhizobia. The organogenic process resulting in nodule development is triggered by the bacterial microsymbiont, but genetically controlled by the host plant genome. Using T-DNA insertion as a tool to identify novel plant genes that regulate nodule ontogeny, we have identified two putatively tagged symbiotic loci, Ljsym8 and Ljsym13, in the diploid legume Lotus japonicus. The sym8 mutants are arrested during infection by the bacteria early in the developmental process. The sym13 mutants are arrested in the final stages of infection, and ineffective nodules are formed. These two plant mutant lines were identified in progeny from 1112 primary transformants obtained after Agrobacterium tumefaciens T-DNA-mediated transformation of L. japonicus and subsequent screening for defects in the symbiosis with Mesorhizobium loti. Additional nontagged mutants arrested at different developmental stages were also identified and genetic complementation tests assigned all the mutations to 16 monogenic symbiotic loci segregating recessive mutant alleles. In the screen reported here independent symbiotic loci thus appeared with a frequency of approximately 1.5%, suggesting that a relatively large set of genes is required for the symbiotic interaction.

Amitochondriate amoebae and the evolution of DNA-dependent RNA polymerase II.

Unlike parasitic protist groups that are defined by the absence of mitochondria, the Pelobiontida is composed mostly of free-living species. Because of the presence of ultrastructural and cellular features that set them apart from all other eukaryotic organisms, it has been suggested that pelobionts are primitively amitochondriate and may represent the earliest-evolved lineage of extant protists. Analyses of rRNA genes, however, have suggested that the group arose well after the diversification of the earliest-evolved protists. Here we report the sequence of the gene encoding the largest subunit of DNA-dependent RNA polymerase II (RPB1) from the pelobiont Mastigamoeba invertens. Sequences within RPB1 encompass several of the conserved catalytic domains that are common to eubacterial, archaeal, and eukaryotic nuclear-encoded RNA polymerases. In RNA polymerase II, these domains catalyze the transcription of all nuclear pre-mRNAs, as well as the majority of small nuclear RNAs. In contrast with rDNA-based trees, phylogenetic analyses of RPB1 sequences indicate that Mastigamoeba represents an early branch of eukaryotic evolution. Unlike sequences from parasitic amitochondriate protists that were included in our study, there is no indication that Mastigamoeba RPB1 is attracted to the base of the eukaryotic tree artifactually. In addition, the presence of introns and a heptapeptide C-terminal repeat in the Mastigamoeba RPB1 sequence, features that are typically associated with more recently derived eukaryotic groups, raise provocative questions regarding models of protist evolution that depend almost exclusively on rDNA sequence analyses.