A case of sepsis caused by Acidovorax.
This report documents a case of sepsis caused by a recently recognized environmental organism and demonstrates the pathogenicity of this bacterium in the clinical setting.
Blood↗
Biomedical subjects
P Groves
Publications and source records attributed to P Groves.
Characteristics of sex-biased dispersal and gene flow in coastal river otters: implications for natural recolonization of extirpated populations.
River otters (Lontra canadensis) were extirpated from much of their historic distribution because of exposure to pollution and urbanization, resulting in expansive reintroduction programmes that continue today for this and other species of otters worldwide. Bioaccumulation of toxins negatively affects fecundity among mustelids, but high vagility and different dispersal distances between genders may permit otter populations to recover from extirpation caused by localized environmental pollution. Without understanding the influence of factors such as social structure and sex-biased dispersal on genetic variation and gene flow among populations, effects of local extirpation and the potential for natural recolonization (i.e. the need for translocations) cannot be assessed. We studied gene flow among seven study areas for river otters (n = 110 otters) inhabiting marine environments in Prince William Sound, Alaska, USA. Using nine DNA microsatellite markers and assignment tests, we calculated immigration rates and dispersal distances and tested for isolation by distance. In addition, we radiotracked 55 individuals in three areas to determine characteristics of dispersal. Gender differences in sociality and spatial relationships resulted in different dispersal distances. Male river otters had greater gene flow among close populations (within 16-30 km) mostly via breeding dispersal, but both genders exhibited an equal, low probability of natal dispersal; and some females dispersed 60-90 km. These data, obtained in a coastal environment without anthropogenic barriers to dispersal (e.g. habitat fragmentation or urbanization), may serve as baseline data for predicting dispersal under optimal conditions. Our data may indicate that natural recolonization of coastal river otters following local extirpation could be a slow process because of low dispersal among females, and recolonization may be substantially delayed unless viable populations occurred nearby. Because of significant isolation by distance for male otters and low gene flow for females, translocations should be undertaken with caution to help preserve genetic diversity in this species.
Alaska↗
Structural and biochemical characterization of neuronal calretinin domain I-II (residues 1-100). Comparison to homologous calbindin D28k domain I-II (residues 1-93).
This study characterizes the calcium-bound CR I-II domain (residues 1-100) of rat calretinin (CR). CR, with six EF-hand motifs, is believed to function as a neuronal intracellular calcium-buffer and/or calcium-sensor. The secondary structure of CR I-II, defined by standard NMR methods on 13C,15N-labeled protein, contains four helices and two short interacting segments of extended structure between the calcium-binding loops. The linker between the two helix-loop-helix, EF-hand motifs is 12 residues long. Limited trypsinolysis at K60 (there are 10 other K/R residues in CR I-II) confirms that the linker of CR I-II is solvent-exposed and that other potential sites are protected by regular secondary structure. 45Ca-overlay of glutathione S-transferase (GST)-CR(1-60) and GST-CR(61-100) fusion proteins confirm that both EF-hands of CR I-II have intrinsic calcium-binding properties. The primary sequence and NMR chemical shifts, including calcium-sensitive glycine residues, also suggest that both EF-hand loops of CR I-II bind calcium. NMR relaxation, analytical ultracentrifugation, chemical cross-linking and NMR translation diffusion measurements indicate that CR I-II exists as a monomer. Calb I-II (the homologous domain of calbindin D28k) has the same EF-hand secondary structures as CR I-II, except that helix B is three residues longer and the linker has only four residues [Klaus, W., Grzesiek, S., Labhardt, A. M., Buckwald, P., Hunziker, W., Gross, M. D. & Kallick, D. A. (1999) Eur. J. Biochem. 262, 933-938]. In contrast, Calb I-II binds one calcium cation per monomeric unit and exists as a dimer. Despite close homology and similar secondary structures, CR I-II and Calb I-II probably have distinct tertiary structure features that suggest different cellular functions for the full-length proteins.
Amino Acid Sequence↗
Valve disease: Surgery of valve disease: late results and late complications.
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Arrhythmias, Cardiac↗
Cation binding properties of calretinin, an EF-hand calcium-binding protein.
Calretinin (CR) is a neuronal EF-hand protein previously characterized as a calcium (micromolar affinity) binding protein. CR-containing neurons are spared in some neurodegenerative diseases, although it is as yet unconfirmed how CR plays an active role in this protection. Higher levels of some metal cations (e.g. copper and zinc) are associated with these diseases. At the same time, metals such as terbium (NMR and fluorescence) cadmium (NMR) and manganese (EPR) serve as useful calcium analogues in the study of EF-hand proteins. We survey the binding of the above-mentioned metal cations that might affect the structure and function of CR. Competitive 45Ca2+-overlay, competitive terbium fluorescence and intrinsic tryptophan fluorescence are used to detect the binding of metal cations to CR. Terbium and copper (half-maximal effect of 15 microM) bind to CR. Terbium has a similar or greater affinity for the calcium-binding sites of CR than calcium. Copper quenches the fluorescence of terbium-bound CR, and CR tryptophan residues and competes weakly for 45Ca2+-binding sites. Cadmium, magnesium, manganese and zinc bind less strongly (half-maximal effects above 0.1 mM). Therefore, only terbium appears to be a suitable analytical calcium analogue in further studies of CR. The principal conclusion of this work is that copper, in addition to calcium, might be a factor in the function of CR and a link between CR and neurodegenerative diseases.
Animals↗
The activated type 1-polarized CD8(+) T cell population isolated from an effector site contains cells with flexible cytokine profiles.
The capacity of activated T cells to alter their cytokine expression profiles after migration into an effector site has not previously been defined. We addressed this issue by paired daughter analysis of a type 1-polarized CD8(+) effector T cell population freshly isolated from lung parenchyma of influenza virus-infected mice. Single T cells were activated to divide in vitro; individual daughter cells were then micromanipulated into secondary cultures with and without added IL-4 to assess their potential to express type 2 cytokine genes. The resultant subclones were analyzed for type 1 and 2 cytokine mRNAs at day 6-7. When the most activated (CD44(high)CD11a(high)) CD8(+) subpopulation from infected lung was compared with naive or resting (CD44(low)CD11a(low)) CD8(+) cells from infected lung and from normal lymph nodes (LNs), both clonogenicity and plasticity of the cytokine response were highest in the LN population and lowest in the activated lung population, correlating inversely with effector function. Multipotential cells were nevertheless detected among clonogenic CD44(high)CD11a(high) lung cells at 30-50% of the frequency in normal LNs. The data indicate that activated CD8(+) T cells can retain the ability to proliferate and express new cytokine genes in response to local stimuli after recruitment to an effector site.
Animals↗
Use of Pichia pastoris for the expression, purification, and characterization of rat calretinin "EF-hand" domains.
Calretinin (CR) is a calcium-binding, neuronal protein of undefined function. Related proteins either buffer intracellular calcium concentrations or are involved in calcium-signaling pathways. We transformed three CR gene fragment sequences, corresponding to its three complementary domains (I-II, III-IV, and V-VI), into Pichia pastoris. High yields of extracellular expression, of more than 200 mg/liter, were achieved. Simple purification protocols provide high yields of homogenous proteins: dialysis and DEAE-cellulose chromatography for domains I-II and III-IV or ammonium sulfate precipitation and octyl-Sepharose chromatography for domain V-VI. To our knowledge, this is the first report of the expression of an EF-hand protein using P. pastoris. Direct comparison of the purified yields of domain I-II indicates a approximately 20-fold improvement over Escherichia coli. N-terminal amino acid sequencing confirmed our gene products and two anti-calretinin antibodies recognized the appropriate domains. All three CR domains bind (45)Ca and the domain containing EF-hands V and VI seems to have a lower calcium capacity than the other domains. Circular dichroism indicates a high helix content for each of the domains. Calcium-induced structural changes in the first two domains, followed by tryptophan fluorescence, correspond with previous studies, while tyrosine emission fluorescence indicates calcium-induced structural changes also occur in domain V-VI. The methods and expression levels achieved are suitable for future NMR labeling of the proteins, with (15)N and (13)C, and structure-function studies that will help to further understand CR function.
Amino Acid Sequence↗
Single-cell analysis by RT-PCR reveals differential expression of multiple type 1 and 2 cytokine genes among cells within polarized CD4+ T cell populations.
RT-PCR was used to examine the expression of IFN-gamma, IL-2, IL-4, IL-5, IL-6 and IL-10 mRNAs by single murine CD4+ T cells activated either in a strongly type 1-polarized mixed lymphocyte reaction (MLR) or in the type 2-polarized response to immunization with keyhole limpet hemocyanin (KLH) in alum. The frequencies of expression of each cytokine differed markedly between the two responses, consistent with their polarization at the population level. However, most cells expressed only none to three of the six cytokines assayed, few displayed the canonical type 1 profile and none in either response expressed a full type 2 or type 0 profile. A significant fraction of cells co-expressed IFN-gamma with IL-4 and/or other type 2 cytokines at frequencies that suggested that most of these genes were independently regulated. Collectively, these single-cell expression patterns indicate that polarization at the population level can mask substantial intercellular heterogeneity, and show directly that multiple type 1 and 2 cytokines can be expressed simultaneously in an individual T cell.
Animals↗
A model for target protein binding to calcium-activated S100 dimers.
S100 proteins are a family of dimeric calcium-binding proteins implicated in several cancers and neurological diseases. Calbindin D9k is an unusual monomeric member of the S100 family. A calbindin D9k mutant containing a novel calcium-induced helix is characterized. Based on sequence comparison, this helix could be a component of other S100 proteins and a factor in target protein binding. The origin of structural differences between three reported apo S100 dimer structures is verified. We conclude that the differences are a result of modeling rather than a function of different target binding properties. A mechanism for target protein binding is suggested.
Animals↗
A single peripheral CD8+ T cell can give rise to progeny expressing type 1 and/or type 2 cytokine genes and can retain its multipotentiality through many cell divisions.
The lineage relationships between murine CD8(+) T cells with different cytokine profiles were investigated by paired-daughter analysis in the presence and absence of the type 2 cytokine-inducing stimulus, interleukin 4 (IL-4). Single CD8(+) CD44(low) lymph node T cells were activated to divide at high frequency with IL-2 and immobilized antibodies to CD3, CD8, and LFA-1. When these parent cells were subcloned by transferring their daughter or granddaughter cells into secondary cultures with or without IL-4, the subclones expressed diverse combinations of the mRNAs for the type 1 cytokines, interferon gamma (IFN-gamma), and IL-2, and the type 2 cytokines, IL-4, IL-5, IL-6, and IL-10. Frequencies of subclones that expressed IL-4, IL-6, and, to a lesser extent, IL-2, IL-5, and IL-10 were higher among those grown with IL-4, but a significant proportion of those grown without exogenous IL-4 also expressed one or more type 2 cytokines. Subclones within 89% of families displayed different cytokine profiles, indicating that their parent cells were multipotential for this function. Because 98% of parent cells yielded subclones that produced type 1 cytokines and 77% yielded type 2 cytokine producers, we conclude that type 1 and type 2 cytokine-producing CD8(+) T cells can be derived from a common precursor. Similar analyses performed by subcloning after >/=7 or >/=13 cell divisions without IL-4 showed that many CD8(+) T cells retained the potential to shift toward a type 2 cytokine profile in response to IL-4, even after prolonged expansion under conditions that favored type 1 cytokine expression. CD8(+) T cells that express type 1 and/or type 2 cytokines therefore are derived from the same peripheral T cell lineage whose multipotentiality can persist through many cell divisions.
Animals↗
A calbindin D9k mutant containing a novel structural extension: 1H nuclear magnetic resonance studies.
Calbindin D9k is a small, well-studied calcium-binding protein consisting of two helix-loop-helix motifs called EF-hands. The P43MG2 mutant is one of a series of mutants designed to sequentially lengthen the largely unstructured tether region between the two EF-hands (F36-S44). A lower calcium affinity for P43MG was expected on the basis of simple entropic arguments. However, this is not the case and P43MG (-97 kJ.mol-1) has a stronger calcium affinity than P43M (-93 kJ.mol-1), P43G (-95 kJ.mol-1) and even wild-type protein (-96 kJ.mol-1). An NMR study was initiated to probe the structural basis for these calcium-binding results. The 1H NMR assignments and 3JHNH alpha values of the calcium-free and calcium-bound form of P43MG calbindin D9k mutant are compared with those of P43G. These comparisons reveal that little structure is formed in the tether regions of P43MG(apo), P43G(apo) and P43G(Ca) but a helical turn (S38-K41) appears to stabilize this part of the protein structure for P43MG(Ca). Several characteristic NOEs obtained from 2D and 3D NMR experiments support this novel helix. A similar, short helix exists in the crystal structure of calcium-bound wild-type calbindin D9k-but this is the first observation in solution for wild-type calbindin D9k or any of its mutants.
Amino Acid Sequence↗
Cytochrome B sequences suggest convergent evolution of the Asian takin and Arctic muskox.
Relationships of the takin (Budorcas taxicolor) and muskox (Ovibos moschatus) have been speculated upon for many years. Morphological and behavioral similarities between these species have led to suggestions that they are closely related. To test the hypothesis that characteristics shared by the takin and muskox stem from a recent common ancestor, we compared sequences of their mitochondrial cytochrome b genes with those of three other species of Caprinae. We present data that may support rejection of the hypothesis of recent common ancestry and suggest that similarities in behavior and morphology in these two species might be attributed to convergent evolution rather than shared phylogeny.
Animals↗
Right ventricular dysfunction during coronary artery occlusion: pressure-volume analysis using conductance catheters during coronary angioplasty.
OBJECTIVE: To study the effects of coronary artery occlusion on the pressure-volume relations of the right ventricle. DESIGN: Right ventricular pressure-volume cycles were studied using conductance catheters and micromanometers in 19 subjects undergoing coronary angioplasty in a tertiary referral cardiac centre. RESULTS: Catheter occlusions of either the left anterior descending coronary artery or the right coronary artery were associated with a decline in stroke work (mean change (SD): left-13.3 (15.8)%, p = 0.008; right -13.5(16.5)%, p = 0.04). Two patterns of change were evident: an upward shift usually associated with occlusion in the left coronary artery, and a rightward shift in the right coronary artery. In the former there was an increase in maximum ventricular volume (mean change: 3.0(2.7)%, p = 0.004) and in minimum ventricular volume (mean change: 2.3(2.7)%, p = 0.01) and a fall in peak pressure (mean change: -4.8 (5.1)%, p = 0.04). In the latter there was an increase in peak pressure (mean change 9.9(16.3)%, p = 0.04) and an increase in minimum ventricular volume (mean change 3.7(5.0)%, p = 0.02) leading to a fall in stroke volume (mean change -13.3(15.8)%, p = 0.008). CONCLUSIONS: Occlusion of the left anterior descending coronary artery or the right coronary artery is associated with a decline in right ventricular work. However, different patterns of change in indices of preload and afterload lead to different effects on overall right ventricular pump function.
Angioplasty, Balloon, Coronary↗
Phylogenetics of the Caprinae based on cytochrome b sequence.
Relationships within the subfamily Caprinae have never been fully resolved. Phylogenies have been proposed based on morphological, behavioral, ecological, and some molecular comparisons. Because of the relatively poor fossil record of the Caprinae, paleontological evidence has not been extensively used in phylogenetic reconstruction for this group. Traditionally, four tribes: Saigini, Ovibovini, Rupicaprini, and Caprini, have been recognized. We investigated relationships within the Caprinae by comparing sequences of the cytochrome b gene of mitochondrial DNA from 11 species of Caprinae and 1 Bovinae species. Our analyses suggest that revisions to previous phylogenies, including the dissolution of the Ovibovini, are warranted.
Animals↗
Cooperativity and anti-cooperativity between ligand binding and the dimerization of ristocetin A: asymmetry of a homodimer complex and implications for signal transduction.
BACKGROUND: Recent work has indicated that dimerization is important in the mode of action of the vancomycin group of glycopeptide antibiotics. NMR studies have shown that one member of this group, ristocetin A, forms an asymmetric dimer with two physically different binding sites for cell wall peptides. Ligand binding by ristocetin A and dimerization are slightly anti-cooperative. In contrast, for the other glycopeptide antibiotics of the vancomycin group that have been examined so far, binding of cell wall peptides and dimerization are cooperative. RESULTS: Here we show that the two halves of the asymmetric homodimer formed by ristocetin A have different affinities for ligand binding. One of these sites is preferentially filled before the other, and binding to this site is cooperative with dimerization. Ligand binding to the other, less favored half of the dimer, is anti-cooperative with dimerization. CONCLUSIONS: In dimer complexes, anti-cooperativity of dimerization upon ligand binding can be a result of asymmetry, in which two binding sites have different affinities for ligands. Such a system, in which one binding site is filled preferentially, may be a mechanism by which the cooperativity between ligand binding and dimerization is fine tuned and may thus have relevance to the control of signal transduction in biological systems.
Binding Sites↗
Training primary health care workers about drugs: a national survey of UK trainers' perceptions towards training.
Reports have consistently shown that non-specialist drug workers (whose working role is not specifically concentrated on dealing with drug-related issues) are reluctant to work with drug users. A number of explanations have been offered to account for this unwillingness including attitudinal factors, occupational constraints and a lack of motivation to learn about drug-related issues. Previously, it has been shown that training affects commitment to working with substance misusers, although failure to attract particular professional groups (e.g. general practitioners) into training courses has also been reported. No previous research has examined the views of trainers about training primary health care and health-related workers. This study of a (non-probability) sample of UK drug trainers (n = 145) assessed training activity for different health care workers, and trainers' differential perceptions of training needs and methods. GPs were the group least likely to become trained about drug issues. Training in attitudes towards drug using individuals was perceived to be more important than either skills or knowledge training for GPs, practice nurses, other nurses and probation officers. Experiential training methods were perceived to be more important than a didactic approach for training all health groups except GPs for whom lecture type instruction was believed to be equally appropriate. Seventy-nine percent of subjects reported providing training across drugs in alcohol or drugs, alcohol and tobacco. Most trainers who stated that certain professions required independent training believed that GPs should be trained separately from other groups.
Journal Article↗