Subchondral bone in osteoarthritis: association between MRI texture analysis and histomorphometry.

OBJECTIVE: Magnetic resonance imaging (MRI) texture analysis is a method of analyzing subchondral bone alterations in osteoarthritis (OA). The objective of this study was to evaluate the association between MR texture analysis and ground-truth subchondral bone histomorphometry at the tibial plateau. DESIGN: The local research ethics committee approved the study. All subjects provided written, informed consent. This was a cross-sectional study carried out at our institution between February and August 2014. Ten participants aged 57-84 with knee OA scheduled for total knee arthroplasty (TKA) underwent pre-operative MRI of the symptomatic knee at 3T using a high spatial-resolution coronal T1 weighted sequence. Tibial plateau explants obtained at the time of TKA underwent histological preparation to allow calculation of bone volume fraction (BV.TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and trabecular number (Tb.N). Texture analysis was performed on the tibial subchondral bone of MRI images matched to the histological sections. Regression models were created to assess the association of texture analysis features with BV.TV, Tb.Th, Tb.Sp and Tb.N. RESULTS: MRI texture features were significantly associated with BV.TV (R2 = 0.76), Tb.Th (R2 = 0.47), Tb.Sp (R2 = 0.75) and Tb.N (R2 = 0.60, all P < 0.001). Simple gray-value histogram based texture features demonstrated the highest standardized regression coefficients for each model. CONCLUSION: MRI texture analysis features were significantly associated with ground-truth subchondral bone histomorphometry at the tibial plateau.

The UK breast screening program - what you need to know.

Breast cancer is the most commonly diagnosed cancer in women with a lifetime risk of 1 in 8 in the UK. The disease is associated with considerable morbidity and mortality. The UK breast screening program has been in place for almost 30 years and has evolved with improved diagnostic imaging. The overall benefits of the screening program are subject to continued heated debate with multiple potential harms. The aim of this review is to provide the non-breast specialist health-care professional an overview of the evolution of the breast screening program, a summary of the evidence of the benefits, and a description of the harms. In addition, the diagnostic methods currently employed as well as potential future changes to the screening program and imaging techniques will be covered.

Spatial variation in soil properties and diffuse losses between and within grassland fields with similar short-term management.

One of the major challenges for agriculture is to understand the effects of agricultural practices on soil properties and diffuse pollution, to support practical farm-scale land management. Three conventionally managed grassland fields with similar short-term management, but different ploughing histories, were studied on a long-term research platform: the North Wyke Farm Platform. The aims were to (i) quantify the between-field and within-field spatial variation in soil properties by geostatistical analysis, (ii) understand the effects of soil condition (in terms of nitrogen, phosphorus and carbon contents) on the quality of discharge water and (iii) establish robust baseline data before the implementation of various grassland management scenarios. Although the fields sampled had experienced the same land use and similar management for at least 6 years, there were differences in their mean soil properties. They showed different patterns of soil spatial variation and different rates of diffuse nutrient losses to water. The oldest permanent pasture field had the largest soil macronutrient concentrations and the greatest diffuse nutrient losses. We show that management histories affect soil properties and diffuse losses. Potential gains in herbage yield or benefits in water quality might be achieved by characterizing every field or by area-specific management within fields (a form of precision agriculture for grasslands). Permanent pasture per se cannot be considered a mitigation measure for diffuse pollution. The between- and within-field soil spatial variation emphasizes the importance of baseline characterization and will enable the reliable identification of any effects of future management change on the Farm Platform. HIGHLIGHTS: Quantification of soil and water quality in grassland fields with contrasting management histories.Considerable spatial variation in soil properties and diffuse losses between and within fields.Contrasting management histories within and between fields strongly affected soil and water quality.Careful pasture management needed: the oldest pasture transferred the most nutrients from soil to water.

The North Wyke Farm Platform: effect of temperate grassland farming systems on soil moisture contents, runoff and associated water quality dynamics.

The North Wyke Farm Platform was established as a United Kingdom national capability for collaborative research, training and knowledge exchange in agro-environmental sciences. Its remit is to research agricultural productivity and ecosystem responses to different management practices for beef and sheep production in lowland grasslands. A system based on permanent pasture was implemented on three 21-ha farmlets to obtain baseline data on hydrology, nutrient cycling and productivity for 2 years. Since then two farmlets have been modified by either (i) planned reseeding with grasses that have been bred for enhanced sugar content or deep-rooting traits or (ii) sowing grass and legume mixtures to reduce nitrogen fertilizer inputs. The quantities of nutrients that enter, cycle within and leave the farmlets were evaluated with data recorded from sensor technologies coupled with more traditional field study methods. We demonstrate the potential of the farm platform approach with a case study in which we investigate the effects of the weather, field topography and farm management activity on surface runoff and associated pollutant or nutrient loss from soil. We have the opportunity to do a full nutrient cycling analysis, taking account of nutrient transformations in soil, and flows to water and losses to air. The NWFP monitoring system is unique in both scale and scope for a managed land-based capability that brings together several technologies that allow the effect of temperate grassland farming systems on soil moisture levels, runoff and associated water quality dynamics to be studied in detail. HIGHLIGHTS: Can meat production systems be developed that are productive yet minimize losses to the environment?The data are from an intensively instrumented capability, which is globally unique and topical.We use sensing technologies and surveys to show the effect of pasture renewal on nutrient losses.Platforms provide evidence of the effect of meteorology, topography and farm activity on nutrient loss.

Mean field analysis of a spatial stochastic model of a gene regulatory network.

A gene regulatory network may be defined as a collection of DNA segments which interact with each other indirectly through their RNA and protein products. Such a network is said to contain a negative feedback loop if its products inhibit gene transcription, and a positive feedback loop if a gene product promotes its own production. Negative feedback loops can create oscillations in mRNA and protein levels while positive feedback loops are primarily responsible for signal amplification. It is often the case in real biological systems that both negative and positive feedback loops operate in parameter regimes that result in low copy numbers of gene products. In this paper we investigate the spatio-temporal dynamics of a single feedback loop in a eukaryotic cell. We first develop a simplified spatial stochastic model of a canonical feedback system (either positive or negative). Using a Gillespie's algorithm, we compute sample trajectories and analyse their corresponding statistics. We then derive a system of equations that describe the spatio-temporal evolution of the stochastic means. Subsequently, we examine the spatially homogeneous case and compare the results of numerical simulations with the spatially explicit case. Finally, using a combination of steady-state analysis and data clustering techniques, we explore model behaviour across a subregion of the parameter space that is difficult to access experimentally and compare the parameter landscape of our spatio-temporal and spatially-homogeneous models.

Arginase-1-expressing macrophages are dispensable for resistance to infection with the gastrointestinal helminth Trichuris muris.

Alternatively activated macrophages (AAMs) have key roles in the immune response to a variety of gastrointestinal helminths such as Heligmosomoides bakeri and Nippostrongylus brasiliensis. In addition, AAMs have been implicated in the resolution of infection-induced pathology in Schistosoma mansoni infection. AAMs exert their activity in part via the enzyme arginase-1 (Arg1), which hydrolyses L-arginine into urea and ornithine, and can supply precursor substrate for proline and polyamine production. Trichuris muris is a worm that resides in the large intestine with resistance being characterized by a Th2 T-cell response, which drives alternatively activated macrophage production in the local environment of the infection. To investigate the role of AAMs in T. muris infection, we used independent genetic and pharmacologic models of arginase deficiency. In acute infection and Th2-dominated immunity, arginase-deficient models expelled worms normally. Macrophage-Arg1-deficient mice showed cytokine and antibody levels comparable to wild-type animals in acute and chronic infection. We also found no role for AAMs and Arg1 in infection-induced pathology in the response to T. muris in either chronic (Th1 dominated) or acute (Th2 dominated) infections. Our data demonstrate that, unlike other gastrointestinal helminths, Arg1 expression in AAMs is not essential for resistance to T. muris in effective resolution of helminth-induced inflammation.

Differential growth of the fungus Absidia cylindrospora on 13C/15N-labelled media.

Many studies utilise enrichment of stable isotopes as tracers to follow the interactions occurring within soil food webs and methods have been developed to enrich bacteria, soil fauna and plant litter, Here for the first time we attempt to enrich a soil fungus to 99 atom% with (13)C and (15)N stable isotopes. In this study our objectives were to (a) assess whether the saprotrophic zygomycete fungus Absidia cylindrospora could grow on a medium enriched to 99 atom% with (13)C-glucose and (15)N-ammonium chloride, (b) to determine the level of enrichment obtained, and (c) to examine the change in growth rate of this fungus while it was growing on the dually enriched medium. To achieve this, the fungus was grown on agar enriched with (13)C and (15)N to 99 atom% and its growth rate monitored. The results showed that A. cylindrospora would grow on the highly labelled growth medium, but that its rate of growth was affected compared with the rate on either natural abundance media or media highly enriched with a single isotope ((13)C or (15)N). The implications of these results is that although the fungus is able to utilise these heavier isotopes, the biochemical processes involved in growth are affected, and consideration should be given to these differences when using stable isotope tracers in, for example, soil food web studies.

Tracking the flow of bacterially derived 13C and 15N through soil faunal feeding channels.

The soil food web has been referred to as a 'black box', a 'poor man's tropical rainforest' and an 'enigma', due to its opacity, diversity and the limited insight into feeding specificity. Here we investigate the flow of C and N through the soil food web as a way to gain understanding of the feeding interactions occurring. A bacterium, Pseudomonas lurida, was introduced to soil cores from two different habitats, a grassland and a woodland with the same soil type, enriched to 99 atom% in (13)C and (15)N, to trace the flow of bacterial C and N through the soil food web. Throughout the experiment the soil remained enriched in (13)C and (15)N. Almost all the invertebrates tested gained C and N enrichment indicative of the labelled bacteria, implying that bacterial feeding is a common mechanism within the soil. Only three groups were significantly enriched in both (13)C and (15)N in both habitats. These were Collembola (Entomobryomorpha), Acari (Oribatida), and Nematoda, indicating that these organisms are consuming the most bacteria within both systems. When the invertebrates were grouped into hypothesised trophic levels, those considered secondary decomposers were gaining the most enrichment across all invertebrates tested. This enrichment was also high in the micro-predators within the soil, implying that their main food source was the secondary decomposers, particularly the Collembola. Using an enriched bacterium to track the trophic transfer between organisms within the soil food web is a novel way of empirically showing that interactions are occurring, which normally cannot be seen.

Movement of newly assimilated 13C carbon in the grass Lolium perenne and its incorporation into rhizosphere microbial DNA.

One of the key processes that drives rhizosphere microbial activity is the exudation of soluble organic carbon (C) by plant roots. We describe an experiment designed to determine the impact of defoliation on the partitioning and movement of C in grass (Lolium perenne L.), soil and grass-sterile sand microcosms, using a (13)CO(2) pulse-labelling method. The pulse-derived (13)C in the shoots declined over time, but that of the roots remained stable throughout the experiment. There were peaks in the atom% (13)C of rhizosphere CO(2) in the first few hours after labelling probably due to root respiration, and again at around 100 h. The second peak was only seen in the soil microcosms and not in those with sterilised sand as the growth medium, indicating possible microbial activity. Incorporation of the (13)C label into the microbial biomass increased at 100 h when incorporation into replicating cells, as indicated by the amounts of the label in the microbial DNA, started to increase. These results indicate that the rhizosphere environment is conducive to bacterial growth and replication. The results also show that defoliation had no impact on the pattern of movement of (13)C from plant roots into the microbial population in the rhizosphere.

Measurement at the field scale of soil delta13C and delta15N under improved grassland.

Variations in natural abundance of carbon (C) and nitrogen (N) stable isotopes are widely used as tools for many aspects of scientific research. By examining variations in the ratios of heavy to light stable isotopes, information can be obtained as to what physical, chemical and biological processes may be occurring. The spatial heterogeneity of soil delta(15)N- and delta(13)C-values across a range of scales and under different land use have been described by a number of researchers and the natural abundances of the C and N stable isotopes in soils have been found to be correlated with many factors including hydrology, topography, land use, vegetation cover and climate. In this study the Latin square sampling +1 (LSS+1) sampling method was compared with a simple grid sampling approach for delta(13)C and delta(15)N measurement at the field scale. A set of 144 samples was collected and analysed for delta(15)N and delta(13)C from a 12 x 12 grid (in a 1 ha improved grassland field in south-west England). The dimension of each cell of the grid was approximately 11 x 6 m. The 12 x 12 grid was divided into four 6 x 6 grids and the LSS+1 sampling technique was applied to these and the main 12 x 12 grid for a comparison of sample means and variation. The LSS+1 means from the 12 x 12 grid and the four 6 x 6 grids compared well with the overall grid mean because of the low variation within the field. The LSS+1 strategy (13 samples) generated representative samples from the 12 x 12 grid, and hence would be an acceptable method for sampling similar plots for the measurement of mean isotopic composition.

Reproductive seasonality and rate of increase of wild sambar deer (Rusa unicolor) in a new environment, Victoria, Australia.

Sambar (Rusa unicolor) are the most numerous and rapidly expanding of Australia's six introduced deer species, however, there is little information about the reproductive biology of sambar deer in their natural habitat. To better predict and manage wild sambar populations in Australia it is important to understand their reproductive seasonality and rate of population growth. From results of the present study, there is reporting of field estimates of age at first breeding, reproductive lifespan, juvenile survival, adult bodyweights and fecundity to derive estimates of the current and intrinsic rates of increase for sambar in Victoria, Australia. Mean age of first reproduction was estimated to be 1.8 years, approximately 80 % of hinds calved between April and August, juvenile survival was estimated as 0.81 and age of last reproduction 12.75 years. Seasonality of reproduction is apparently compressed at 36° latitude compared to sambar at the equator indicating a response to photoperiod. Demographic data were used to estimate the current rate of growth of the Victorian population using the two stage Lotke-Euler equation and age-specific schedules of survival and fecundity in a lifetable. These estimates of r were 0.21 and 0.14, respectively, inferring annual rates of population increase of 24 % and 15 %. These data are in the context of a population which, even though there is a marked harvesting, is reportedly growing and dispersing northwards. Suggestions for how this information can inform management decisions directed at the conservation for sustainable use and/or population reduction in Australia are made.

Hormonal contraceptive use modulates the local inflammatory response to bacterial vaginosis.

OBJECTIVES: To compare cervical concentrations of numerous cytokines/chemokines in women with bacterial vaginosis (BV) compared with the levels detected after BV resolution and determine if hormonal contraceptive use modulates the local inflammatory response to BV. METHODS: Cervical secretions from 81 women with BV at enrollment and normal flora at one-month follow-up were analysed for 10 different cytokines/chemokines using multiplexed fluorescent bead-based immunoassays. RESULTS: BV was associated with significantly higher concentrations of IL-1 beta, tumour necrosis factor (TNF), interferon-gamma, IL-2, IL-4, and IL-10 compared with the levels detected in the presence of normal vaginal flora. Analysis of results stratified by contraceptive practice demonstrated significantly lower levels of numerous cytokines among women with BV using hormonal contraceptives compared with those women with BV not using hormonal contraceptives. Hormonal contraceptive use was also associated with a statistically significant lesser change in TNF levels between the two study visits compared with the amount of change detected between visits among women who denied their use. CONCLUSIONS: Despite increases in the levels of both pro and anti-inflammatory cytokines in the lower genital tract of women with BV, the overall balance of these two types of molecules was maintained. The character of this local inflammatory response may help explain the typical absence of overt signs of inflammation among women with BV. In addition, hormonal contraceptive use was associated with significantly lower levels of the pro-inflammatory molecules TNF, interferon-gamma, and granulocyte macrophage colony-stimulating factor in women with BV, but did not significantly reduce the levels of IL-10, a key anti-inflammatory cytokine. These results suggest the possibility of an association between hormonal contraceptive use and altered genital tract immunity.

Roles of instrumented farm-scale trials in trade-off assessments of pasture-based ruminant production systems.

For livestock production systems to play a positive role in global food security, the balance between their benefits and disbenefits to society must be appropriately managed. Based on the evidence provided by field-scale randomised controlled trials around the world, this debate has traditionally centred on the concept of economic-environmental trade-offs, of which existence is theoretically assured when resource allocation is perfect on the farm. Recent research conducted on commercial farms indicates, however, that the economic-environmental nexus is not nearly as straightforward in the real world, with environmental performances of enterprises often positively correlated with their economic profitability. Using high-resolution primary data from the North Wyke Farm Platform, an intensively instrumented farm-scale ruminant research facility located in southwest United Kingdom, this paper proposes a novel, information-driven approach to carry out comprehensive assessments of economic-environmental trade-offs inherent within pasture-based cattle and sheep production systems. The results of a data-mining exercise suggest that a potentially systematic interaction exists between 'soil health', ecological surroundings and livestock grazing, whereby a higher level of soil organic carbon (SOC) stock is associated with a better animal performance and less nutrient losses into watercourses, and a higher stocking density with greater botanical diversity and elevated SOC. We contend that a combination of farming system-wide trials and environmental instrumentation provides an ideal setting for enrolling scientifically sound and biologically informative metrics for agricultural sustainability, through which agricultural producers could obtain guidance to manage soils, water, pasture and livestock in an economically and environmentally acceptable manner. Priority areas for future farm-scale research to ensure long-term sustainability are also discussed.

Feral goats in Australia: A study on the quality and nutritive value of their meat.

The purpose of this study was to determine the quality of fresh and cooked meat, and the nutritive value of this meat from 62 male Australian feral goats. The goats were slaughtered at 5, 10, 20, 30, 40, 50, 60 and 70kg liveweights. Half of the goats were castrated and half were left as intact animals. The quality profiles of meat (e.g. pH, colour, pigment concentrations, cooking loss, shear force value and eating quality of cooked meat) from both castrated and intact feral goats started to decrease when animals were slaughtered at heavier liveweights (e.g. above 40kg). The nutritive value of the meat (chemical compositions, fatty acids and total cholesterol concentrations) changed when animals were castrated and had heavier slaughter weights. Overall, we recommend that 40kg liveweight is the heaviest slaughter weight, since the quality characteristics of meat will be lower when feral goats were slaughtered above 40kg liveweight.

The 'Toolbox' of strategies for managing Haemonchus contortus in goats: What's in and what's out.

A dynamic and innovative approach to managing the blood-consuming nematode Haemonchus contortus in goats is critical to crack dependence on veterinary anthelmintics. H. contortus management strategies have been the subject of intense research for decades, and must be selected to create a tailored, individualized program for goat farms. Through the selection and combination of strategies from the Toolbox, an effective management program for H. contortus can be designed according to the unique conditions of each particular farm. This Toolbox investigates strategies including vaccines, bioactive forages, pasture/grazing management, behavioural management, natural immunity, FAMACHA, Refugia and strategic drenching, mineral/vitamin supplementation, copper Oxide Wire Particles (COWPs), breeding and selection/selecting resistant and resilient individuals, biological control and anthelmintic drugs. Barbervax(®), the ground-breaking Haemonchus vaccine developed and currently commercially available on a pilot scale for sheep, is prime for trialling in goats and would be an invaluable inclusion to this Toolbox. The specialised behaviours of goats, specifically their preferences to browse a variety of plants and accompanying physiological adaptations to the consumption of secondary compounds contained in browse, have long been unappreciated and thus overlooked as a valuable, sustainable strategy for Haemonchus management. These strategies are discussed in this review as to their value for inclusion into the 'Toolbox' currently, and the future implications of ongoing research for goat producers. Combining and manipulating strategies such as browsing behaviour, pasture management, bioactive forages and identifying and treating individual animals for haemonchosis, in addition to continuous evaluation of strategy effectiveness, is conducted using a model farm scenario. Selecting strategies from the Toolbox, with regard to their current availability, feasibility, economical cost and potential ease of implementation depending on the systems of production and their complementary nature, is the future of managing H. contortus in farmed goats internationally and maintaining the remaining efficacy of veterinary anthelmintics.

Defining the requirements for immunological control of mycobacterial infections.

Tuberculosis remains a major public health problem throughout the world. Research into the immunological basis of the host-pathogen relationship has recently benefited from the fascinating convergence of genetic data from mouse models and from humans. Latency - the seemingly quiescent phase of bacterial persistence - is the central problem in controlling tuberculosis and will be the next frontier of research.

Rapid nucleotide sequence analysis of the small subunit ribosomal RNA of Toxoplasma gondii: evolutionary implications for the Apicomplexa.

A method for obtaining a large proportion of the nucleotide sequence of the small subunit ribosomal RNA (srRNA) was applied to the obligate intracellular protozoon Toxoplasma gondii. The method uses reverse transcription of as little as 8 micrograms of total cellular RNA. This fast, efficient method has numerous advantages over traditional gene cloning methods when nucleotide sequences are required for evolutionary studies. A phylogenetic analysis of the srRNA sequence data showed that T. gondii is not especially closely related to any other organism for which srRNA sequences are available, including another member of the Apicomplexa.

Identification, characterisation and genomic cloning of a O-linked N-acetylglucosamine-containing cytoplasmic Leishmania glycoprotein.

Antibodies against Leishmania major wheat germ agglutinin-binding glycoproteins were used to select from a genomic lambda gt11 expression library a clone coding for a L. major glycoprotein. The partial DNA sequence indicated the presence of a mosaic of repetitive sequences. Southern blot hybridisation on genomic DNA using the cloned gene as a probe at high stringency suggested a single gene, which was localised to chromosome band 18. Northern blot analysis of L. major mRNA detected a major transcript of 7.5 kb and a minor 4.0-kb transcript. Antibodies affinity-purified on the fusion protein identified a complex of two water-soluble cytoplasmic polypeptides of approximately 96 kDa and 92 kDa in L. major promastigotes and amastigotes. They also recognised polypeptides in other Leishmania species, in Crithidia lucilliae and very weakly in Leptomonas. The apparent molecular weight of these polypeptides, while conserved within each species, varied between species. A peptide map of the two polypeptides from L. major generated an identical pattern suggesting a close relatedness at the protein level. This protein complex was not hydrolysed by N-glycanase and was not affected by tunicamycin, but treatment with anhydrous hydrogen fluoride suggested that it is O-glycosylated. The glycan moiety appears to be N-acetylglucosamine, and N-acetylglucosamine beta-1,4-galactosyltransferase was capable of adding [3H]galactose to it. This was susceptible to beta elimination and beta-galactosidase treatment. Taken together, the data indicates that gp96/92 belongs to the newly described class of cytoplasmic and nuclear glycoproteins containing O-linked N-acetylglucosamine.

Characterization of a polymorphic family of integral membrane proteins in promastigotes of different Leishmania species.

Antibodies raised against a Leishmania major recombinant promastigote surface antigen 2 (PSA-2) fragment recognized three major polypeptides of approximate M(r) 96,000, 80,000 and 50,000 in promastigotes of three Israeli isolates of L. major including the cloned line LRC-L137-V121, but detected a different array of polypeptides in other L. major isolates. The pattern was different both in number of polypeptides detected and their molecular weight. The antibodies to L. major PSA-2 also recognized polypeptides in L. tropica, L. donovani and very weakly in L. mexicana promastigotes and in Crithidia lucilliae. The number and size of the polypeptides was different in each species. In addition to the membrane-bound PSA-2 polypeptides we identified water-soluble forms of PSA-2 released in promastigote culture supernatants. Peptide maps of the various L. major PSA-2 membrane polypeptides showed they were different from each other. N-terminal amino acid sequence of the three polypeptides expressed by L. major showed they are similar but distinct, consistent with being members of a polymorphic family. Because of the extensive sequence similarity between the PSA-2 genes it has been difficult to assign protein products to individual genes. As a first step towards solving this problem, we have transfected into L. mexicana a genomic clone of a L. major PSA-2 gene and shown that it produces a M(r) 35,000 polypeptide recognized by monoclonal and polyclonal antibodies to L. major PSA-2.

Structure-function studies of human interferons-alpha: enhanced activity on human and murine cells.

To identify functionally important regions of the human interferon (IFN)-alpha molecule, mutagenesis in vitro of human IFN-a genes was used to create analogs with deletions or specific amino acid replacements. These analogs were expressed in vitro using SP6 RNA polymerase and a rabbit reticulocyte lysate protein synthesis system. Deletion of 7 highly conserved hydrophilic amino acids from the C-terminus of human IFN-alpha 4 reduced, but did not abolish, antiviral activity on human cells. However, analogs with deletions of 15 or 25 amino acids from the C-terminus, or 28 amino acids from the N-terminus, had no measurable antiviral activity. The antiviral activity of human IFN-alpha 4 was increased by substitution of cysteine for serine at position 86, and lysine for arginine at position 121. However, other amino acid substitutions at positions 121, 122 or 123 reduced antiviral activity. The size of the side chain of the amino acid residue at position 130 was shown to be important. Replacement of the absolutely conserved leucine residue at position 131 with glutamine had little effect on antiviral activity. However, the introduction of a proline residue at this position abolished antiviral activity, probably due to the formation of a beta turn in the polypeptide chain. The antiviral activity of human IFN-alpha 4 on murine cells was increased by substitutions at positions 86, 121 and 133. This study illustrates the utility of the in vitro mutagenesis and rabbit reticulocyte lysate systems for the investigation of structure-function relationships, and extends our knowledge of the biologically active regions and species specificity of the human IFN-alpha molecule.