Highly diverse heparan sulfate analogue libraries: a novel resource for bioactivity screening of proteins

Introduction Fibroblast growth factors (FGFs) are a multipotent family of growth factors that are important for many biological processes, including development and wound healing. Normal, protease sensitive, prion protein (PrPC) can be converted to the protease resistant, infectious, form (PrPSc) believed to be associated with the pathogenesis of transmissible spongiform encephalopathies. FGFs signal through a family of tyrosine kinase receptors, the FGF receptors (FGFR) with the aid of heparan sulfate (HS), while the role of HS in the biology of PrPC is currently unknown, although depleting cells of HS can prevent production of PrPSc. HS, or its more highly sulfated relation heparin, can exert both positive and negative regulatory activities on a particular FGF‐FGFR combination. The nature of this regulation is determined by the structure of the HS that binds to the proteins. This structure is at least partially determined by the presence of particular sulfate groups along the sugar backbone. Identification of specific sulfate groups that can regulate the activity of proteins has been a long‐term goal in the field. Previously, heparins that had been completely lacking sulfates at specific positions were used to determine the binding and activity requirements for a particular protein. However, this may not necessarily allow for a full examination of the regulatory properties of HS. Here, we present a heparan sulfate analogue library produced by the partial, combinatorial desulfation of heparin. This library was the used to examine the structural properties of heparin required for FGF‐1 signalling through FGFR2c as well as the interactions of HS with PrPC. Materials and methods Porcine intestinal mucosal heparin was subjected to chemical desulfation and enzymatic cleavage. Polysaccharides and oligosaccharides derived from gel filtration chromatography and ion exchange chromatography were tested for their ability to activate FGF signalling through FGF receptors using a BaF3 assay system. Optical biosensors and cell assays were used to study the interaction of PrPC with chemically modified heparin. Results This library possessed vastly more heterogeneity than tissue heparan sulfates, allowing for more systematic screening to help identify those minimal structural features associated with activity. This library was used to examine the different structural features in heparin that support FGF‐1 signalling through FGFR2, showing that HS activity was not strictly dependant on size or charge. In addition, small, low‐sulfated heparins were found to interfere with the PrPC–heparin interaction. Discussion This supports the idea that overall structural features of the HS, rather than just the presence or absence of specific sulfate groups is important for the regulation of protein activity. Future efforts will be focused on further subfractionating the library and identifying specific structural features in HS that support FGF‐1 activity through FGFR2 and other FGFRs as well as the role of HS in the normal function of prion diseases, which may allow for the generation of novel, heparin‐based, therapeutics.     

LcrV plague vaccine with altered immunomodulatory properties

Yersinia pestis, the causative agent of plague, secretes LcrV (low-calcium-response V or V antigen) during infection. LcrV triggers the release of interleukin 10 (IL-10) by host immune cells and suppresses proinflammatory cytokines such as tumor necrosis factor alpha and gamma interferon as well as innate defense mechanisms required to combat the pathogenesis of plague. Although immunization of animals with LcrV elicits protective immunity, the associated suppression of host defense mechanisms may preclude the use of LcrV as a human vaccine. Here we show that short deletions within LcrV can reduce its immune modulatory properties. An LcrV variant lacking amino acid residues 271 to 300 (rV10) elicited immune responses that protected mice against a lethal challenge with Y. pestis. Compared to full-length LcrV, rV10 displayed a reduced ability to release IL-10 from mouse and human macrophages. Furthermore, the lipopolysaccharide-stimulated release of proinflammatory cytokines by human or mouse macrophages was inhibited by full-length LcrV but not by the rV10 variant. Thus, it appears that LcrV variants with reduced immune modulatory properties could be used as a human vaccine to generate protective immunity against plague.     

The relationship between women's subjective and physiological sexual arousal

Previous literature presents discordant results on the relationship between physiological and subjective sexual arousal in women. In this study, the use of hierarchical linear modeling (HLM) revealed a significant concordance between continuous measures of physiological and subjective sexual arousal as assessed during exposure to erotic stimuli in a laboratory setting. We propose that past studies that have found little or no association between the two measures may have been in part limited by the methodology and statistical analyses employed.     

Studies on the production of enterotoxins by Bacillus cereus.

Evidence is presented for the existence of three distinct enterotoxins detected in concentrated cell-free culture filtrates of selected Bacillus cereus strains. The first was a product capable of stimulating the adenylate cyclase-cyclic-AMP system in intestinal epithelial cells and, possibly through this, causing fluid accumulation in ligated ileal sections ("loops") of young rabbits. This was elaborated by a strain isolated from an incident of diarrhoea and which caused diarrhoea in 6 of 10 monkey feedings. The second was tentatively identified as a factor which caused fluid accumulation in rabbit loops but not, apparently, through stimulation of the adenylate cyclase-cyclic-AMP system; this was elaborated by a strain isolated from raw rice which failed to produce symptoms in eight monkey feedings. Together, the behaviour of these two factors indicates that diarrhoea caused by B. cereus enterotoxin may be a cyclic-AMP-mediated event. The third, here referred to as "pyogenic toxin", caused severe tissue damage in the ileal mucosa and was elaborated by a strain isolated from a brain abscess. A factor produced by a strain isolated from an outbreak of vomiting which caused vomiting in 10 of 24 monkey feedings could bot be detected in tests reported here but appears to be a fourth enterotoxin type. Cytopathic effects in tissue cultures, suckling mouse tests, and assays of glycerol production by fat cells were not found to be of value in the detection of any of the enterotoxins.     

Fluorescent amplified fragment length polymorphism genotyping of Campylobacter jejuni and Campylobacter coli strains and its relationship with host specificity, serotyping, and phage typing

Fluorescent amplified fragment length polymorphism (FAFLP) analysis was applied to 276 Campylobacter jejuni strains and 87 Campylobacter coli strains isolated from humans, pigs, cattle, poultry, and retail meats to investigate whether certain FAFLP genotypes of C. jejuni and C. coli are associated with a particular host and to determine the degree of association between FAFLP-defined genotypes and heat-stable serotypes and/or phage types. Within C. coli, the poultry strains clustered separately from those of porcine origin. In contrast, no evidence of host specificity was detected among C. jejuni strains. While C. coli strains show host specificity by FAFLP genotyping, C. jejuni strains that are genotypically similar appear to colonize a range of hosts, rather than being host adapted. Some serotypes and/or phage types (C. jejuni serotype HS18, phage type PT6, and serophage type HS19/PT2 and C. coli HS66, PT2, and HS56/PT2) were the most homogeneous by FAFLP genotyping, while others were more heterogeneous (C. jejuni HS5 and PT39, and C. coli HS24 and PT44) and therefore poor indicators of genetic relatedness between strains. The lack of host specificity in C. jejuni suggests that tracing the source of infection during epidemiological investigations will continue to be difficult. The lack of congruence between some serotypes and/or phage types and FAFLP genotype underlines the need for phenotypic testing to be supplemented by genotyping. This study also demonstrates how, in general, FAFLP generates "anonymous" genetic markers for strain characterization and epidemiological investigation of Campylobacter in the food chain.     

Family history of cardiovascular disease is associated with cardiovascular responses to stress in healthy young men and women.

Heightened cardiovascular stress responsivity is associated with cardiovascular disease, but the origins of heightened responsivity are unclear. The present study investigated whether disturbances in cardiovascular responsivity were evident in individuals with a family history of cardiovascular disease risk. Data were collected from 60 women and 31 men with an average age of 21.4 years. Family history of cardiovascular disease risk was defined by the presence of coronary heart disease, hypertension, diabetes or high cholesterol in participants' parents and grandparents; 75 participants had positive, and 16 had negative family histories. Systolic and diastolic blood pressure (BP), heart rate and heart rate variability were measured continuously for 5 min periods at baseline, during two mental stress tasks (Stroop and speech task) and at 10-15 min, 25-30 min and 40-45 min post-stress. Individuals with a positive family history exhibited significantly greater diastolic BP reactivity and poorer systolic and diastolic BP recovery from the stressors in comparison with family history negative individuals. In addition, female participants with a positive family history had heightened heart rate and heart rate variability reactivity to stressors. These effects were independent of baseline cardiovascular activity, body mass index, waist to hip ratio and smoking status. Family history of hypertension alone was not associated with stress responsivity. The findings indicate that a family history of cardiovascular disease risk influences stress responsivity which may in turn contribute to risk of future cardiovascular disorders.