Diversity in and evidence for selection on the mitochondrial genome of Phytophthora infestans

Two extant nomenclature systems were reconciled to relate six mitochondrial DNA (mtDNA) haplotypes of Phytophthora infestans, the oomycete pathogen causing late blight disease on potato and tomato. Carter's haplotypes I-a and I-b were included in Goodwin's haplotype A, while Carter's haplotypes II-a and II-b were included in Goodwin's haplotype B. In addition, haplotypes E and F were included in Carter's haplotype I-b. The mutational differences separating the various haplotypes were determined, and we propose that either haplotype I-b(A) or haplotype I-a(A) is the putative ancestral mtDNA of P. infestans, because either can center all the other haplotypes in a logical stepwise network of mutational changes. The occurrence of the six haplotypes in 548 isolates worldwide was determined. Haplotypes I-a and II-a were associated with diverse genotypes worldwide. As previously suggested, haplotype I-b was found only in the US-1 clonal lineage and its variants (n = 99 isolates from 16 countries on 5 continents), and haplotype II-b was limited to the US-6 clonal lineage and its derivatives (n = 36). In a confirmation of a previous suggestion, the randomly mating population in the Toluca Valley of central Mexico (n = 78) was monomorphic for mtDNA haplotype I-a(A). We hypothesize that selection there may be driving the dominance of that single mtDNA haplotype.     

A comparative evaluation of five typing techniques for determining the diversity of fluorescent pseudomonads

Five typing methods were evaluated, utilising 63 strains of fluorescent pseudomonads, to assess their usefulness as tools to study the bacterial diversity within this complex group. The methods used were Biolog metabolic profiling, restriction fragment length polymorphism ribotyping, PCR ribotyping, and repetitive element sequence-based PCR (rep-PCR) utilising BOX and enterobacterial repetitive intergenic consensus (ERIC) primers. Cluster analysis of the results clearly demonstrated the considerable homogeneity of Pseudomonas aeruginosa isolates and, conversely, the heterogeneity within the other species, in particular P. putida and P. fluorescens, which need further taxonomic investigation. Biolog metabolic profiling enabled the best differentiation among the species. Rep-PCR proved to be highly discriminatory, more so than the other DNA fingerprinting techniques, demonstrating its suitability for the analysis of highly clonal isolates. RFLP ribotyping, PCR ribotyping, and rep-PCR produced specific clusters of P. aeruginosa isolates, which corresponded to their origins of isolation, hence we recommend these methods for intraspecific typing of bacteria.     

Postactivation potentiation response in athletic and recreationally trained individuals

To determine if training status directly impacted the response to postactivation potentiation, athletes in sports requiring explosive strength (ATH; n = 7) were compared to recreationally trained (RT; n = 17) individuals. Over the course of 4 sessions, subjects performed rebound and concentric-only jump squats with 30%, 50%, and 70% 1 RM loads. Jump squats were performed 5 minutes and 18.5 minutes following control or heavy load warm-ups. Heavy load warm-up consisted of 5 sets of 1 repetition at 90% 1 RM back squat. Jump squat performance was assessed with a force platform and position transducer. Heavy load warm-up did not have an effect on the subjects as a single sample. However, when percent potentiation was compared between ATH and RT groups, force and power parameters were significantly greater for ATH (p < 0.05). Postactivation potentiation may be a viable method of acutely enhancing explosive strength performance in athletic but not recreationally trained individuals. Reference Data: Chiu, L.Z.F., A.C. Fry, L.W. Weiss, B.K. Schilling, L.E. Brown, and S.L. Smith. Postactivation potentiation response in athletic and recreationally trained individuals.     

Nek2A kinase stimulates centrosome disjunction and is required for formation of bipolar mitotic spindles

Nek2A is a cell cycle-regulated kinase of the never in mitosis A (NIMA) family that is highly enriched at the centrosome. One model for Nek2A function proposes that it regulates cohesion between the mother and daughter centriole through phosphorylation of C-Nap1, a large coiled-coil protein that localizes to centriolar ends. Phosphorylation of C-Nap1 at the G2/M transition may trigger its displacement from centrioles, promoting their separation and subsequent bipolar spindle formation. To test this model, we generated tetracycline-inducible cell lines overexpressing wild-type and kinase-dead versions of Nek2A. Live cell imaging revealed that active Nek2A stimulates the sustained splitting of interphase centrioles indicative of loss of cohesion. However, this splitting is accompanied by only a partial reduction in centriolar C-Nap1. Strikingly, induction of kinase-dead Nek2A led to formation of monopolar spindles with unseparated spindle poles that lack C-Nap1. Furthermore, kinase-dead Nek2A interfered with chromosome segregation and cytokinesis and led to an overall change in the DNA content of the cell population. These results provide the first direct evidence in human cells that Nek2A function is required for the correct execution of mitosis, most likely through promotion of centrosome disjunction. However, they suggest that loss of centriole cohesion and C-Nap1 displacement may be distinct mitotic events.     

Effect of knee position on hip and knee torques during the barbell squat

Some recommendations suggest keeping the shank as vertical as possible during the barbell squat, thus keeping the knees from moving past the toes. This study examined joint kinetics occurring when forward displacement of the knees is restricted vs. when such movement is not restricted. Seven weight-trained men (mean +/- SD; age = 27.9 +/- 5.2 years) were videotaped while performing 2 variations of parallel barbell squats (barbell load = body weight). Either the knees were permitted to move anteriorly past the toes (unrestricted) or a wooden barrier prevented the knees from moving anteriorly past the toes (restricted). Differences resulted between static knee and hip torques for both types of squat as well as when both squat variations were compared with each other (p < 0.05). For the unrestricted squat, knee torque (N.m; mean +/- SD) = 150.1 +/- 50.8 and hip torque = 28.2 +/- 65.0. For the restricted squat, knee torque = 117.3 +/- 34.2 and hip torque = 302.7 +/- 71.2. Restricted squats also produced more anterior lean of the trunk and shank and a greater internal angle at the knees and ankles. The squat technique used can affect the distribution of forces between the knees and hips and on the kinematic properties of the exercise. PRACTICAL APPLICATIONS: Although restricting forward movement of the knees may minimize stress on the knees, it is likely that forces are inappropriately transferred to the hips and low-back region. Thus, appropriate joint loading during this exercise may require the knees to move slightly past the toes.     

An association between the acute phase response and patterns of antigen induced T cell proliferation in juvenile idiopathic arthritis

The aim of this research was to determine whether all memory T cells have the same propensity to migrate to the joint in patients with juvenile idiopathic arthritis. Paired synovial fluid and peripheral blood mononuclear cell proliferative responses to a panel of antigens were measured and the results correlated with a detailed set of laboratory and clinical data from 39 patients with juvenile idiopathic arthritis. Two distinct patterns of proliferative response were found in the majority of patients: a diverse pattern, in which synovial fluid responses were greater than peripheral blood responses for all antigens tested; and a restricted pattern, in which peripheral blood responses to some antigens were more vigorous than those in the synovial fluid compartment. The diverse pattern was generally found in patients with a high acute phase response, whereas patients without elevated acute phase proteins were more likely to demonstrate a restricted pattern. We propose that an association between the synovial fluid T cell repertoire and the acute phase response suggests that proinflammatory cytokines may influence recruitment of memory T cells to an inflammatory site, independent of their antigen specificity. Additionally, increased responses to enteric bacteria and the presence of αEβ7 T cells in synovial fluid may reflect accumulation of gut associated T cells in the synovial compartment, even in the absence of an elevated acute phase response. This is the first report of an association between the acute phase response and the T cell population recruited to an inflammatory site.     

Lipoprotein lipase- and hepatic triglyceride lipase- promoted very low density lipoprotein degradation proceeds via an apolipoprotein E-dependent mechanism

Apolipoprotein E (apoE) is the primary recognition signal on triglyceride-rich lipoproteins responsible for interacting with low density lipoprotein (LDL) receptors and LDL receptor-related protein (LRP). It has been shown that lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) promote receptor-mediated uptake and degradation of very low density lipoproteins (VLDL) and remnant particles, possibly by directly binding to lipoprotein receptors. In this study we have investigated the requirement for apoE in lipase-stimulated VLDL degradation. We compared binding and degradation of normal and apoE-depleted human VLDL and apoE knockout mouse VLDL in human foreskin fibroblasts. Surface binding at 37 degrees C of apoE knockout VLDL was greater than that of normal VLDL by 3- and 40-fold, respectively, in the presence of LPL and HTGL. In spite of the greater stimulation of surface binding, lipase-stimulated degradation of apoE knockout mouse VLDL was significantly lower than that of normal VLDL (30, 30, and 80%, respectively, for control, LPL, and HTGL treatments). In the presence of LPL and HTGL, surface binding of apoE-depleted human VLDL was, respectively, 40 and 200% of normal VLDL whereas degradation was, respectively, 25 and 50% of normal VLDL. LPL and HTGL stimulated degradation of normal VLDL in a dose-dependent manner and by a LDL receptor-mediated pathway. Maximum stimulation (4-fold) was seen in the presence LPL (1 microgram/ml) or HTGL (3 microgram/ml) in lovastatin-treated cells. On the other hand, degradation of apoE-depleted VLDL was not significantly increased by the presence of lipases even in lovastatin-treated cells. Surface binding of apoE-depleted VLDL to metabolically inactive cells at 4 degrees C was higher in control and HTGL-treated cells, but unchanged in the presence of LPL. Degradation of prebound apoE-depleted VLDL was only 35% as efficient as that of normal VLDL. Surface binding of apoE knockout or apoE-depleted VLDL was to heparin sulfate proteoglycans because it was completely abolished by heparinase treatment. However, apoE appears to be a primary determinant for receptor-mediated VLDL degradation.Our studies suggest that overexpression of LPL or HTGL may not protect against lipoprotein accumulation seen in apoE deficiency.     

Interruption of the fragile X syndrome expanded sequence d(CGG)(n) by interspersed d(AGG) trinucleotides diminishes the formation and stability of d(CGG)(n) tetrahelical structures

Fragile X syndrome is caused by expansion of a d(CGG) trinucleotide repeat sequence in the 5′ untranslated region of the first exon of the FMR1 gene. Repeat expansion is thought to be instigated by formation of d(CGG)_n secondary structures. Stable FMR1 d(CGG)_n runs in normal individuals consist of 6–52 d(CGG) repeats that are interrupted every 9–11 triplets by a single d(AGG) trinucleotide. By contrast, individuals having fragile X syndrome premutation or full mutation present >54–200 or >200–2000 monotonous d(CGG) repeats, respectively. Here we show that the presence of interspersed d(AGG) triplets diminished in vitro formation of bimolecular tetrahelical structures of d(CGG)₁₈ oligomers. Tetraplex structures formed by d(CGG)_n oligomers containing d(AGG) interspersions had lower thermal stability. In addition, tetraplex structures of d(CGG)₁₈ oligomers interspersed by d(AGG) triplets were unwound by human Werner syndrome DNA helicase at rates and to an extent that exceeded the unwinding of tetraplex form consisting of monotonous d(CGG)₁₈. Diminished formation and stability of tetraplex structures of d(AGG)-containing FMR1 d(CGG)_2–50 tracts might restrict their expansion in normal individuals.     

Human Ku antigen tightly binds and stabilizes a tetrahelical form of the Fragile X syndrome d(CGG)n expanded sequence

Hairpin and tetrahelical structures of a d(CGG)(n) sequence in the FMR1 gene have been implicated in its expansion in fragile X syndrome. The identification of tetraplex d(CGG)(n) destabilizing proteins (Fry, M., and Loeb, L. A.(1999) J. Biol. Chem. 274, 12797-12803; Weisman-Shomer, P., Naot, Y., and Fry, M. (2000) J. Biol. Chem. 275, 2231-2238) suggested that proteins might modulate d(CGG)(n) folding and aggregation. We assayed human TK-6 lymphoblastoid cell extracts for d(CGG)(8) oligomer binding proteins. The principal binding protein was identified as Ku antigen by its partial amino acid sequence and antigenicity. The purified 88/75-kDa heterodimeric Ku bound with similar affinities (K(d) approximately 1. 8-10.2 x 10(-9) mol/liter) to double-stranded d(CGG)(8).d(CCG)(8), hairpin d(CGG)(8), single-stranded d(CII)(8), or tetraplex structures of telomeric or IgG switch region sequences. However, Ku associated more tightly with bimolecular G'2 tetraplex d(CGG)(8) (K(d) approximately 0.35 x 10(-9) mol/liter). Binding to Ku protected G'2 d(CGG)(8) against nuclease digestion and impeded its unwinding by the tetraplex destabilizing protein qTBP42. Stabilization of d(CGG)(n) tetraplex domains in FMR1 by Ku or other proteins might promote d(CGG) expansion and FMR1 silencing.