Finding a (pine) needle in a haystack: chloroplast genome sequence divergence in rare and widespread pines.

Critical to conservation efforts and other investigations at low taxonomic levels, DNA sequence data offer important insights into the distinctiveness, biogeographic partitioning and evolutionary histories of species. The resolving power of DNA sequences is often limited by insufficient variability at the intraspecific level. This is particularly true of studies involving plant organelles, as the conservative mutation rate of chloroplasts and mitochondria makes it difficult to detect polymorphisms necessary to track genealogical relationships among individuals, populations and closely related taxa, through space and time. Massively parallel sequencing (MPS) makes it possible to acquire entire organelle genome sequences to identify cryptic variation that would be difficult to detect otherwise. We are using MPS to evaluate intraspecific chloroplast-level divergence across biogeographic boundaries in narrowly endemic and widespread species of Pinus. We focus on one of the world's rarest pines - Torrey pine (Pinus torreyana) - due to its conservation interest and because it provides a marked contrast to more widespread pine species. Detailed analysis of nearly 90% ( approximately 105 000 bp each) of these chloroplast genomes shows that mainland and island populations of Torrey pine differ at five sites in their plastome, with the differences fixed between populations. This is an exceptionally low level of divergence (1 polymorphism/ approximately 21 kb), yet it is comparable to intraspecific divergence present in widespread pine species and species complexes. Population-level organelle genome sequencing offers new vistas into the timing and magnitude of divergence within species, and is certain to provide greater insight into pollen dispersal, migration patterns and evolutionary dynamics in plants.

The use of stock orthoses to assist gait in neuromuscular disorders: a pilot study.

This pilot study compares the effect on walking speed, in eight subjects with neuromuscular conditions, of wearing Ligaflex ankle-foot orthoses (AFO), Leafspring orthoses and shoes or with shoes alone. Range of motion, muscle strength and sensation were tested in the lower leg. Subjects underwent a standardized timed 10-m walking test five times in each of the orthoses and shoes as a measure of gait efficiency. A self-administered questionnaire was used to seek the subjects' perceptions of their functional difficulties and their opinions about the relative comfort and stability of these orthoses. Subjects had reduced ranges and strength of dorsiflexion and eversion. Some had proprioceptive deficiencies. Mean walking speed was 0.99 m/s (Leafspring) and 1.1 m/s (Ligaflex or shoes) compared to about 1.3 m/s for a normal population. Repeated measures ANOVA revealed that subjects were significantly slower in Leafspring compared to Ligaflex or to shoes. Questionnaire results rated the Leafspring as least comfortable and the Ligaflex most stable. Providing stability may be more important than assisting foot clearance when weakness is restricted to distal muscles. Further research is required to evaluate the comfort and effectiveness of orthoses to compensate for ankle instability in people with neuromuscular conditions.

Monoclonal antibodies putatively identifying porcine B cells.

Comparison was made of the binding of 38 test and three standard monoclonal antibodies (mAbs) to B cells from various pig lymphoid tissues by flow cytometry (FCM) and immunohistochemistry. Some mAbs were also tested on B cells from foetal pig tissues. Twenty of the new mAbs bound, though to variable degrees, to porcine B cells but only three were given cluster assignations: C35 (#147) and BB6-11C9 (#167) were assigned to wCD21 and 2F6/8 (#057) was assigned to SWC7.

Changes in swine macrophage phenotype after infection with African swine fever virus: cytokine production and responsiveness to interferon-gamma and lipopolysaccharide.

Cytokines produced by cells of the immune system, including macrophages, can influence inflammatory responses to viral infection. This has been exploited by viruses, which have developed strategies to direct the immune response towards ineffective responses. African swine fever virus (ASFV) is a double-stranded DNA virus that infects macrophages of domestic swine. In this study, primary cells of monocyte macrophage lineage were obtained from the lungs, peritoneum or blood of domestic swine and, after infection with ASFV, supernatants were tested for cytokines using biological assays. The cytokine transforming growth factor-beta (TGF-beta) was detected after infection of macrophage preparations, but tumour necrosis factor (TNF) and interleukin-1 (IL-1) were not detected. ASFV-infected and uninfected macrophage populations were also tested to assess their ability to respond to cytokines by enhancing production of superoxide in the respiratory burst mechanism. Responses to interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) were suppressed in macrophage populations infected with virus, even at low multiplicities of infection. Addition of TGF-beta to uninfected macrophages resulted in a similar suppression of response, but antibody to TGF-beta did not prevent suppression induced by virus. These results are discussed in relation to the pathology of African swine fever.

Monoclonal antibodies defining differentiation antigens of swine lymphoid and myeloid cells.

Monoclonal antibodies recognising swine leucocyte antigens were identified and the corresponding antigens were characterised by determining their tissue distribution and molecular weight as well as immunohistochemical staining. On the basis of these data, we suggest that two antibodies are specific for a molecules within a porcine orthologue of one of the human CDII/CD18 complexes. We suggest that two others recognise swine wCD21, that one may recognise swine wCD6 and that another recognises an antigen designated by the International Swine CD Workshop cluster SWC9. Other antibodies were obtained which are specific for swine macrophages or B cells, but CD or cluster assignments for these antigens have not been made. Detailed analysis of the antibodies is presented. It is proposed that these antibodies will be useful in studies of the pig immune system and of virus infection.

Stimulation of myofibrillar protein synthesis in hindlimb suspended rats by resistance exercise and growth hormone.

The objective of this study was to determine the ability of a single bout of resistance exercise alone or in combination with recombinant human growth hormone (rhGH) to stimulate myofibrillar protein synthesis (Ks) in hindlimb suspended (HLS) adult female rats. Plantar flexor muscles were stimulated with resistance exercise, consisting of 10 repetitions of ladder climbing on a 1 m grid (85 degrees), carrying an additional 50% of their body weight attached to their tails. Saline or rhGH (1 mg/kg) was administered 30' prior to exercise, and Ks was determined with a constant infusion of 3H-Leucine at 15', 60', 180', and 360' following exercise. Three days of HLS depressed Ks approximately 65% and 30-40% in the soleus and gastrocnemius muscles, respectively (p < or = 0.05). Exercise increased soleus Ks in saline-treated rats 149% 60' following exercise (p < or = 0.05), decaying to that of non-exercised animals during the next 5 hours. Relative to suspended, non-exercised rats rhGH+exercise increased soleus Ks 84%, 108%, and 72% at 15', 60' and 360' following exercise (p < or = 0.05). Gastrocnemius Ks was not significantly increased by exercise or the combination of rhGH and exercise up to 360' post-exercise. Results from this study indicate that resistance exercise stimulated Ks 60' post-exercise in the soleus of HLS rats, with no apparent effect of rhGH to enhance or prolong exercise-induced stimulation. Results suggests that exercise frequency may be important to maintenance of the slow-twitch soleus during non-weightbearing, but that the ability of resistance exercise to maintain myofibrillar protein content in the gastrocnemius of hindlimb suspended rats cannot be explained by acute stimulation of synthesis.

Secretion of a macrophage-activating factor distinct from interferon-gamma by human T cell clones.

Supernatants from clones of human T lymphocytes that were responding to a purified Mycobacterium tuberculosis antigen were able to activate macrophages and macrophage-like myeloma cells (U937) to release increased amounts of the microbicidal agent hydrogen peroxide. The activity was not neutralized by monoclonal antibody against interferon-gamma (IFN-gamma), was greater than could be accounted for by the IFN-gamma activity in the supernatants, and was separated from IFN-gamma by high performance liquid chromatography. It is evident that IFN-gamma is not the only macrophage activator released by T lymphocytes responding to microbial antigen, and may not even be the main one to enhance antimicrobial activity in infections such as tuberculosis.

The reaction of the anti-interferon-alpha monoclonal antibody, NK2, with different interferons.

Analysis of the neutralization of human interferon-alpha by the monoclonal antibody NK2 showed, by three different methods, that the avidity constant was about 10(10)M-1. A small decrease in the avidity constant (and in the antibody titre) with increasing interferon concentrations was observed, suggesting that the antibody-interferon complex had a little biological activity. The antibody neutralizes the interferon by preventing the binding of interferon to susceptible cells. It does not react with human-gamma, mouse-alpha, monkey, bovine or rabbit interferons.