Purification, characterization and immunological properties of the serotype-specific capsular polysaccharide of Pasteurella haemolytica serotype A7 organisms

The serotype-specific capsular polysaccharide from two strains of Pasteurella haemolytica serotype A7 organisms was purified and characterized by chemical analysis and by 1H and 13C NMR spectroscopy using one- and two-dimensional methods. The polymer has the repeating unit----3)-beta-2-acetamido-2-deoxygalactopyranose-(1----3)-alpha- 2-acetamido- 2-deoxy-6-O-acetyl-glucopyranose-(1-phosphate----. It was immunogenic (capable of eliciting antibodies) for sheep. Chemical removal of O-acetyl groups destroyed both the ability of the polymer to adhere to sheep erythrocytes at neutral pH and the ability to form immune precipitates with specific antisera. Studies using the protein A-gold technique in the electron microscope showed the polysaccharide to be peripherally localized on the bacterial surface.     

Characterization of envelope proteins from Pasteurella haemolytica and Pasteurella multocida

A method was devised for the reproducible isolation of envelopes from Pasteurella haemolytica serotype A2. It was also possible to prepare envelopes from other serotypes of P. haemolytica and Pasteurella multocida using this methodology. Examination of these preparations by SDS-PAGE showed major differences between strains of P. haemolytica and strains of P. multocida which allowed the clear distinction of isolates of these species. Amongst the P. haemolytica serotypes it was possible to distinguish envelope preparations made from A biotype and T biotype organisms easily, but it was not possible to identify individual serotypes from each other. Envelope profiles were sufficiently different between the individual P. multocida serotypes examined to allow each to be identified by its polypeptide profile. Experiments using radiolabelling, antibody absorption, and susceptibility to protease digestion, together with heat modifiability and detergent solubility characteristics indicated that 13 of the envelope proteins were probably surface-located. A high molecular mass immunogenic envelope protein was shown, by immunoblotting, to be present in all strains of P. haemolytica and P. multocida examined.     

DNA binding of a spermine derivative: Spectroscopic study of anthracene-9-carbonyl-n1-spermine with poly[d(G-C)·(d(G-C))] and poly[d(A-T) · d(A-T)]

The binding of polyamines, including spermidine ( 1 ) and spermine ( 2 ), to poly[d(G-C) · d(G-C) ] was probed using spectroscopic studies of anthracene-9-carbonyl-N¹-spermine ( 3 ); data from normal absorption, linear dichroism (LD), and circular dichroism (CD) are reported. Ligand LD and CD for transitions located in the DNA region of the spectrum were used. The data show that 3 binds to DNA in a manner characteristic of both its amine and polycyclic aromatic parts. With poly [(dG-dC) · (dG-dC)], binding modes are occupied sequentially and different modes correspond to different structural perturbations of the DNA. The most stable binding mode for 3 with poly[d(G-C) · d(G-C)] has a site size of 6 ± 1 bases, and an equilibrium binding constant of (2.2 ± 1.1) × 10⁷ M^?1 with the anthracene moiety intercalated. It dominates the spectra from mixing ratios of approximately 133:1 until 6:1 DNA phosphate: 3 is reached. The analogous data for poly [d(A-T) · d(A-T)] between mixing ratios 36:1 and 7:1 indicates a site size of 8.3 ± 1.1 bases and an equilibrium binding constant of (6.6 ± 3.3) × 10⁵ M^?1. Thus, 3 binds preferentially to poly [d(G-C) · d(G-C)] at these concentrations. © 1994 John Wiley & Sons, Inc.     

Psychological and clinical characteristics of female patients with spontaneous coronary artery dissection

Aims

Spontaneous coronary artery dissection (SCAD) is increasingly recognised as a cause of myocardial infarction, but psychological characteristics of patients with SCAD have not yet been extensively investigated. We assessed the prevalence of a broad range of psychological and clinical factors, and their inter-relationships in patients with a history of SCAD. Furthermore, we investigated whether specific clusters of patients with SCAD can be identified.

Methods

Participants were recruited between March and May 2019 from a Dutch SCAD database and completed online questionnaires. Clinical information was verified by review of medical records. Participants were predominantly female (172/183; 94%). Analyses focused on the 172 female patients (mean age 52.0 ± 7.5 years, 37% postmenopausal).

Results

The most common comorbidities of SCAD were migraine (52%), fibromuscular dysplasia (FMD; 29%), chronic pain (29%), and tinnitus (28%). Six women (3%) had pregnancy-associated SCAD. Traditional cardiovascular risk factors were rare (<10%), except for hypertension (31%). Psychological assessment indicated high levels of perceived stress (PSS-10 ≥14; 50%), fatigue (FAS-10 ≥22; 56%), and a frequent history of burnout (25%). The prevalence of depression (9%) and anxiety (12%) was relatively low. Three clusters were identified: (A) FMD and chronic non-ischaemic conditions (tinnitus, chronic pain, and irritable bowel syndrome); (B) migraine; and (C) none of these conditions.

Conclusion

This study shows that perceived stress and fatigue are common in patients with SCAD, in addition to prevalent comorbid FMD, migraine, tinnitus, and non-ischaemic pain conditions. These factors may add to developing tailored rehabilitation programmes for patients with SCAD.

     

Electrochemical monitoring of cell behaviour in vitro: a new technology

This article describes a novel electrochemical technique for the real-time monitoring of changes in the behaviour of adherent human cells in vitro: i.e., a biosensor that combines a biological recognition mechanism with a physical transduction technique, described collectively as Oncoprobe. Confluent viable cells adherent to gold electrodes (sensors) modify the extracellular microenvironment at the cell:sensor interface to produce a change in the electrochemical potential compared to that measured in the absence of cells. The potential was measured as an open circuit potential (OCP) with respect to a saturated calomel reference in the bulk culture medium. Typical OCP values for confluent cultures of human breast carcinoma cells, 8701-BC, approximated -100 mV compared with cell-free values of approximately -15 mV. The OCP for 8701-BC cells was modified in response to temperature changes over the range 32 to 40 degrees C and also to treatments with phytohemagglutinin (PHA, 25 microg/mL), cycloheximide (30 microM) and interleukin-1 beta (IL-1, 0.5 ng/mL) over 24 h. Cultures of synovial fibroblasts also responded to the same treatments with similar responses, producing negative shifts in the OCP signal with PHA and IL-I, but a positive shift in OCP signal with cycloheximide, all relative to the untreated control cultures. From experimental data and theoretical considerations it is proposed that the cell-derived signals are mixed electrode potentials reflecting a "conditioned," more reducing environment at the cell:sensor interface. Only viable cells caused a negative shift in the OCP signal, this being lost when cells were rendered nonviable by formalin exposure. This technology appears unique in its ability to passively "listen in" on cell surface activities, suggesting numerous applications in the fields of drug discovery, chemotherapy, and cell behaviour.     

Novel quantitative phenotypes of exercise training in mouse models

Regular physical exercise has beneficial effects in many human disease states, including cardiovascular diseases, cancer, and depression. Exercise training of genetically modified mouse models may provide insight into the molecular mechanisms that underlie the beneficial effects of exercise. Presently, there is relatively little understanding of the normal physiology of mouse exercise. In this paper, we describe a novel computerized voluntary wheel-running system capable of recording and analyzing individual wheel rotations. Using this system, we demonstrate that C57BL/6 mice run considerable distances during the night in short bouts and at a preferred speed: the cruising speed. We find that the vast majority of running occurs around this cruising speed, which is close to the maximum speed at which the animal can run but is significantly higher than the average speeds recorded by simple digital odometers. We describe how these parameters vary with exercise training and demonstrate marked sex differences in the patterns of voluntary exercise. The results of this study have important implications for the design and interpretation of both voluntary and forced exercise experiments in mouse models. The novel parameters described provide more physiological quantitative measures of voluntary exercise activity and training and will extend the physiological utility of exercise training as a phenotyping tool in genetic mouse models.     

Potent hFPRL1 (ALXR) agonists as potential anti-inflammatory agents

We report the discovery of potent agonists for the human formyl-peptide-like 1 receptor (hFPRL1). These compounds did not act at a closely related receptor denoted human formyl peptide receptor (hFPR) up to 10 microM concentration. Recent studies have indicated that agonizing this receptor may promote resolution of inflammation. In an exploratory study, a novel hFPRL1 agonist showed efficacy in a mouse ear inflammation model following oral administration.     

Defective thymocyte maturation by transgenic expression of a truncated form of the T lymphocyte adapter molecule and Fyn substrate,Sin

Adapter molecules that promote protein-protein interactions play a central role in T lymphocyte differentiation and activation. In this study, we examined the role of the T lymphocyte-expressed adapter protein and Src kinase substrate, Sin, on thymocyte function using transgenic mice expressing an activated, truncated allele of Sin (SinDeltaC). We found that SinDeltaC expression led to reduced numbers of CD4(+) and CD8(+) single-positive cells and reduced thymic cellularity due to increased thymocyte apoptosis. Because the adapter properties of Sin are mediated by tyrosine-based motifs and given that Sin is a substrate for Src tyrosine kinases, we examined the involvement of these kinases in the inhibitory effects of SinDeltaC. We found that in transgenic thymocytes, SinDeltaC was constitutively phosphorylated by the Src kinase Fyn, but not by the related kinase Lck. Using SinDeltaC and fyn(-/-) animals, we also found that the expression of Fyn was required for the inhibitory effect of SinDeltaC on thymocyte apoptosis but not for SinDeltaC-mediated inhibition of T cell maturation. The inhibitory effect of SinDeltaC on thymocyte maturation correlated with defective activation of the mitogen-activated protein kinase extracellular signal-regulated kinase. Our results suggest that the Sin mutant inhibits thymocyte differentiation through Fyn-dependent and -independent mechanisms and that endogenous Sin may be an important regulator of thymocyte development.     

The Time Scale of Evolutionary Innovation

A fundamental question in biology is the following: what is the time scale that is needed for evolutionary innovations? There are many results that characterize single steps in terms of the fixation time of new mutants arising in populations of certain size and structure. But here we ask a different question, which is concerned with the much longer time scale of evolutionary trajectories: how long does it take for a population exploring a fitness landscape to find target sequences that encode new biological functions? Our key variable is the length, of the genetic sequence that undergoes adaptation. In computer science there is a crucial distinction between problems that require algorithms which take polynomial or exponential time. The latter are considered to be intractable. Here we develop a theoretical approach that allows us to estimate the time of evolution as function of We show that adaptation on many fitness landscapes takes time that is exponential in even if there are broad selection gradients and many targets uniformly distributed in sequence space. These negative results lead us to search for specific mechanisms that allow evolution to work on polynomial time scales. We study a regeneration process and show that it enables evolution to work in polynomial time.