Root Rot of Pea and Faba Bean in Southern Sweden Caused by Phytophthora pisi sp. nov.

A root rot disease of pea and faba bean caused by a Phytophthora sp. was observed in fields and field soil samples in southern Sweden. Observations of the disease in pea root rot greenhouse assays were systematically recorded, and incidence and geographic distribution data were compared with the pea root rot caused by Aphanomyces euteiches. Following one successful isolation of the pathogen, isolation procedures and selective media were optimized to retrieve more isolates. Phylogenetic analysis showed that the isolates belong to a novel lineage, closely related to Phytophthora sojae, and proposed here as a new species, P. pisi sp. nov. In a collection of 13 isolates from separate fields, intraspecific variation was detected in both nuclear and mitochondrial loci. Pathogenicity tests on a range of crop plants and wild legumes suggest that the host range of the pathogen is restricted to a group of legumes closely related to pea which, in addition to pea, include the crop species faba bean, lentil, common vetch, and chickpea. Morphology, growth requirements, and pathogenicity traits indicate that the species may be identical to the organism previously described as P. erythroseptica var. pisi. The work characterizes a novel Phytophthora sp. causing root rot of legume crops.

[Extensive pneumatization of the posterior skull]. / Ausgedehnte Pneumatisation des hinteren Schädels.

Hyperpneumatization of the craniocervical bones is a very rare disorder and there are only assumptions as to the cause of this potentially dangerous condition. This article reports the case of a 44-year-old patient with severe pneumatization who became symptomatic after a minor skiing accident without any direct trauma.

Explaining the enigmatic K(M) for oxygen in cytochrome c oxidase: a kinetic model.

We present a mathematical model for the functioning of proton-pumping cytochrome c oxidase, consisting of cyclic conversions between 26 enzyme states. The model is based on the mechanism of oxygen reduction and linked proton translocation postulated by Wikström and Verkhovsky (2007). It enables the calculation of the steady-state turnover rates and enzyme-state populations as functions of the cytochrome c reduction state, oxygen concentration, membrane potential, and pH on either side of the inner mitochondrial membrane. We use the model to explain the enigmatic decrease in oxygen affinity of the enzyme that has been observed in mitochondria when the proton-motive force is increased. The importance of the 26 transitions in the mechanism of cytochrome oxidase for the functional properties of cytochrome oxidase is compared through Metabolic Control Analysis. The control of the K(M) value is distributed mainly between the steps in the mechanism that involve electrogenic proton movements, with both positive and negative contributions. Positive contributions derive from the same steps that control enzyme turnover rate in the model. Limitations and possible further applications of the model are discussed.

Low health-related quality of life in adult individuals with multiple limb deficiencies compared with population-based reference values.

BACKGROUND: Knowledge on health-related quality of life (HRQoL) in multiple limb deficiencies (LDs) is limited. OBJECTIVES: To investigate self-reported HRQoL in multiple LDs, assess differences between congenital LD and acquired LD and sex, and to evaluate associations between the types of LDs, demographic variables, and HRQoL. STUDY DESIGN: Cross-sectional cohort study. METHODS: A total of 106 individuals with multiple limb deficiencies treated at the EX-Center were invited by mail to fill out the Short Form-36 survey. RESULTS: Responses from 62 participants, mean age ± SD 49.5 ± 14.2, showed that 43 had congenital LD and 19 had acquired LD. Responders reported reduced HRQoL in all Short Form-36 domains except Role-Emotional, compared with reference values (P < 0.05-<0.001). Individuals with a congenital LD reported worse Bodily Pain than acquired LD (P < 0.05), and women reported lower Physical Function than men (P < 0.05). Sick leave was negatively associated with physical composite score. Living in a rural area was positively associated with Mental Health (P < 0.01), and congenital LD was negatively associated with Vitality (P < 0.05). CONCLUSIONS: Individuals with multiple LDs in Sweden have lower HRQoL compared with reference values. There are significant associations between sick leave and physical function, rural living and mental health, and the type of LD and vitality.

Analysis of genotypic variation in genes associated with virulence in Aggregatibacter actinomycetemcomitans clinical isolates.

BACKGROUND AND OBJECTIVE: Although certain serotypes of Aggregatibacter actinomycetemcomitans are associated more with aggressive periodontitis than are other serotypes, the correlation between distinct lineages and virulence traits in this species is poorly understood. This study aimed to evaluate the polymorphism of genes encoding putative virulence factors of clinical isolates, and to correlate these findings with A. actinomycetemcomitans serotypes, genotypes and periodontal status of the hosts. MATERIAL AND METHODS: Twenty-six clinical isolates from diverse geographic populations with different periodontal conditions were evaluated. Genotyping was performed using pulse-field gel electrophoresis. Polymorphisms in the genes encoding leukotoxin, Aae, ApaH and determinants for serotype-specific O polysaccharide were investigated. RESULTS: The isolates were classified into serotypes a-f, and exhibited three apaH genotypes, five aae alleles and 25 macrorestriction profiles. Two serotype b isolates (7.7%), obtained from Brazilian patients with aggressive periodontitis, were associated with the highly leukotoxic genotype; these isolates showed identical fingerprint patterns and aae and apaH genotypes. Serotype c, obtained from various periodontal conditions, was the most prevalent among Brazilian isolates, and isolates were distributed in two aae alleles, but formed a genetically distinct group based on apaH analysis. Cluster analysis showed a close relationship between fingerprinting genotypes and serotypes/apaH genotypes, but not with aae genotypes. CONCLUSION: Apart from the deletion in the ltx promoter region, no disease-associated markers were identified. Non-JP2-like strains recovered from individuals with periodontal disease exhibited considerable genetic variation regarding aae/apaH genotypes, serotypes and XhoI DNA fingerprints.

Alveolar macrophages and CC chemokines are increased in children with cystic fibrosis.

Airway inflammation is an important component of cystic fibrosis (CF) lung disease. We sought to determine whether alveolar macrophages were involved in early CF lung disease. Children with CF (median age 3.1 yrs) participated in a surveillance programme that included annual bronchoalveolar lavage (BAL). Control samples were obtained from non-CF children (median age 3.1 yrs; n = 24) investigated for persistent respiratory symptoms. Pulmonary infection was detected in 31% (16 out of 51) and 38% (nine out of 24) of children from the CF and non-CF groups, respectively. Alveolar macrophages in BAL were increased in CF compared with non-CF in the absence of infection (223x10(3) versus 85x10(3) cells.mL(-1); p = 0.001) and were associated with elevations in the CC chemokines (macrophage inflammatory protein (MIP)-3alpha (chemokine (C-C motif) ligand (CCL)20; 355.8 versus 46.0 pg.mL(-1); p<0.001), monocyte chemotactic protein-1 (CCL2; 263.5 versus 25.3 pg.mL(-1); p<0.001), MIP-1alpha (CCL3; 38.2 versus 4.9 pg.mL(-1); p<0.001) and MIP-1beta (CCL4; 326.6 versus 27.5 pg.mL(-1); p<0.001)). Total cell counts and neutrophil numbers increased in the presence of infection; however, there was no additional effect of CF. Alveolar macrophages and CC chemokines are elevated in the lungs in young children with CF even in the absence of pulmonary infection. Longitudinal studies are required to determine the clinical relevance of these findings.

Genetic diversity and toxic activity of Aggregatibacter actinomycetemcomitans isolates.

INTRODUCTION: Very little is known of the diversity and expression of virulence factors of serotypes of Aggregatibacter actinomycetemcomitans. Toxic activity on Chinese hamster ovary (CHO) cells and cdt and ltx genotyping were evaluated in A. actinomycetemcomitans serotypes. METHODS: Forty-one A. actinomycetemcomitans isolates were analysed for CHO cell growth inhibition. Genotyping was performed by polymerase chain reactions specific to the ltx promoter region, serotype-specific and cdt region and by sequencing of cdtB. RESULTS: cdtABC was detected in 40 strains. Analysis of the cdtA upstream region revealed 10 cdt genotypes. Toxicity to CHO cells was detected for 92.7% of the isolates; however, no correlation between the toxic activity and the cdt genotype was detected. Serotype c was more prevalent among Brazilian samples (68.0%). Four serotype b isolates from subjects with aggressive periodontitis were associated with high leukotoxin production and exhibited moderate to strong toxic activity in CHO cells, but were classified in different cdt genotypes. High levels of toxicity in CHO cells were not associated with a particular serotype; 57.1% of serotype a isolates presented low toxicity to CHO cells whereas the highly toxic strains belonged to serotypes b and c. Sequencing of cdtB revealed a single nucleotide polymorphism of amino acid 281 but this was not related to the toxic activity in CHO cells. CONCLUSION: Differences in prevalence of the low and highly cytotoxic strains among serotypes reinforce the hypothesis that serotype b and c isolates of A. actinomycetemcomitans are more virulent than serotype a strains.

Mucosal regulatory T cells in airway hyperresponsiveness.

Interest in regulatory T cells (Treg) and their role in immune regulation has grown almost exponentially over the last 10 years, though the notion of a suppressive population of T cells has been in existence since the early 1970s. Recent reports have highlighted the potential role of populations of Treg in control of T-cell-mediated inflammation in tissues, including the lung. In particular, there is now evidence to suggest that Treg form a fundamental part of the regulatory axis operating within the respiratory mucosa and that the number of Treg recruited to the airways may be crucial for the inhibition of airways hyperresponsiveness associated with exacerbations of asthma. A discussion of these concepts is the focus of this chapter.

Microflora in oral ecosystems in subjects with radiation-induced hyposalivation.

AIM: To analyse the microbial flora in specific oral sites in 13 dentate subjects, 6-8 months after completed radiation therapy (RT group) and in 13 matched controls. MATERIAL AND METHODS: The microflora on the tongue, buccal mucosa, vestibulum, supragingival plaque and subgingival region was analysed using duplicate sampling and cultivation technique. A clinical examination was also performed. RESULTS: Candida albicans was found in one or more sites in 54% of the RT subjects and in 15% of the controls. In three RT subjects, C. albicans was found at all four sites analysed. An unexpected finding was that enterococci were found in all RT subjects and in high number in 38%. None of the controls harboured enterococci. In supragingival plaque, Lactobacillus spp. were detected in 92% of the RT subjects and the number and proportion of Lactobacillus spp. were extremely high compared with the controls. Mutans streptococci were detected in high numbers in 31% of the RT subjects, while they were not detected in 23%. CONCLUSION: The microbial results explain why some RT subjects have an increased susceptibility to oral diseases and stress that site-specific microbial analysis is an important diagnostic tool when planning oral health preventive care for RT subjects.

Proton translocation by bacteriorhodopsin and heme-copper oxidases.

Proton translocation is the means by which free energy is conserved from oxygen reduction by the respiratory heme-copper oxidases and from sunlight by bacteriorhodopsin. Three-dimensional structures at atomic resolution of both proteins have aided functional studies of the proton translocation mechanism. A comparison reveals common structural and functional features that may be unique to the primary proton pumps in biology.

Dental plaque pH and micro-organisms during hyposalivation.

We have previously reported that minor gland and whole saliva flow rates and salivary proteins showed differences in individuals with primary Sjögren's syndrome or head and neck radiation therapy, compared with controls (Eliasson et al., 2005). We now hypothesize that pH and number of acidogenic micro-organisms in dental plaque as well as saliva buffering capacity also differ in these individuals. Plaque pH was measured by the microtouch method up to 60 min after a sucrose rinse. Plaque collected from the same sites was analyzed for counts of total and acidic micro-organisms. Compared with their controls, the irradiated group but not the Sjögren's syndrome group displayed significantly lower plaque pH, increased numbers of lactobacilli and Candida species, as well as reduced buffering capacity. Stepwise regression tests suggested that the buccal minor-salivary-gland secretion rate in the test groups and counts of mutans streptococci in the controls were of significant importance for dental plaque pH.

First Report in Sweden of Downy Mildew on Parsley Caused by Plasmopara petroselini.

During September 2004, downy mildew of parsley caused by a species of Plasmopara was observed in an experimental field of parsley (Petroselinum crispum subsp. crispum L. cv. Gigante d'Italia/Hilmar) in Borgeby in southern Sweden. The summer of 2004 was exceptionally wet and humid. Disease became widespread throughout the field in just a few days. Local growers reported that symptoms consistent with downy mildew had appeared in their parsley fields every year since 2001. Plasmopara, under P. nivea, has been reported on parsley in Europe since the middle of the 19th century (4). In recent years, this disease has caused severe damage to parsley grown in several European countries, e.g., France, Germany, Switzerland, and Belgium (1,3). The first symptoms appeared as faint chlorotic spots on the upper surfaces of the leaves. On the corresponding lower surfaces, mycelium and sporangiophores grew profusely and developed a white mat that in part turned dark gray. Eventually, the leaves and stalks became necrotic and died. The sporangiophores were monopodially branched, 248.4 ± 13.36 µm long (n = 17), each branch ending in 2 to 5 ultimate branchlets tapered toward the tip. The trunk diameter measured 7.0 ± 0.77 µm (n = 9) above the basal part and 6.1 ± 0.81 µm just below the first branch. The sporangia were broadly ellipsoidal to ellipsoidal, hyaline, 22.5 ± 0.73 µm long and 16.6 ± 0.48 µm wide (n µ 40). They were mostly nonpapillate when young, although exit pores 4.8 ± 0.32 µm (n = 10) were visible. Mature sporangia exhibited a dehiscence apparatus and a plug in the exit pore. On the basis of the characteristics above, the pathogen was identified as P. petroselini (= P. nivea pro parte [2]). Independent verification of the identity was done by O. Constantinescu at the Botanical Museum, Uppsala, Sweden. A voucher specimen was deposited at the Herbarium UPS, in Uppsala under the number UPS F-118873. To our knowledge, this is the first report of P. petroselini on parsley in Sweden. References: (1) E. Bèliard and J. Thibault. Phytoma 554:2, 2002. (2) O. Constantinescu. Taxon 54:813, 2005. (3) C. Crepel and S. Inghelbrecht. Plant Dis. 87:1266, 2003. (4) A. de Bary, Ann. Sci. Nat. Bot., Sér. 4, 20:5, 1863.

Studies on the role of the oligomeric state and subunit III of cytochrome oxidase in proton translocation.

Anion-exchange fast protein liquid chromatography in the presence of lauryldimethylamine N-oxide (LDAO) was introduced to separate cytochrome oxidase into different complexes that either did or did not contain subunit III. Both kinds of enzyme complex exhibited H+ translocation after reconstitution into phospholipid vesicles, but with a significantly (approx. 50-60%) reduced H+/e- ratio as compared with unchromatographed enzyme. The anion-exchange FPLC fractions of the enzyme (with or without subunit III) sedimented more slowly than the control enzyme upon sucrose gradient centrifugation in the presence of cholate and a high potassium phosphate concentration. When the control enzyme was subjected to the sucrose gradient centrifugation in the presence of LDAO or Triton X-100, instead of cholate, one band containing all subunits was observed, which sedimented slowly like the FPLC fractions. Transfer of this band to cholate medium, and reapplication on the sucrose gradient (with cholate), yielded both a slow- and a fast-migrating band after centrifugation. Enzyme complexes that sedimented slowly or rapidly in the sucrose gradients revealed longer and shorter elution times, respectively, in gel filtration FPLC. This suggests that these complexes corresponds to monomers and dimers of cytochrome oxidase. Solubilization of proteoliposomes and subsequent sucrose gradient centrifugation in cholate yielded one fast-migrating band for the untreated enzyme, but both a fast- and a slow-migrating band for the anion-exchange FPLC-treated enzyme, which was exclusively slow-migrating before reconstitution into liposomes. It is suggested that dimerisation of monomeric cytochrome oxidase may be favoured when the enzyme encounters a membranous milieu, and that the dimeric structure might be necessary for proton translocation.

Principles of coupling between electron transfer and proton translocation with special reference to proton-translocation mechanisms in cytochrome oxidase.

The recent general acceptance of the proton-pumping function of cytochrome oxidase has stimulated discussion and experiment on possible underlying molecular mechanisms. Adequate experimental design requires clear understanding of the theoretical principles governing such a linked function. The increasing structural knowledge of cytochrome oxidase also contributes to a present-day requirement of more precise chemical and physical description of redox-linked proton translocation, which is the fundamental process underlying conservation of energy from aerobic metabolism in all eukaryotes and many bacteria. This essay is based on our original theoretical treatment of this problem, which is expanded here to include discussion of more recent analyses by others, classification of different types of coupling principles, as well as some concrete proposed molecular mechanisms. The latter will be analysed qualitatively, and in some cases quantitatively where this is possible, using a common theoretical framework to help comparison between models. Experimental findings relevant to this problem will be critically reviewed, and some suggestions will be made to stimulate further experiments dedicated to clarify the problem.

A spectral shift in cytochrome a induced by calcium ions.

Ca2+ induces a red shift in the absorption spectrum of ferrocytochrome a when added to uncoupled mitochondria, sub-mitochondrial particles or isolated cytochrome aa3. The shift is identical within experimental error to the previously reported energy-linked shift in intact mitochondria (Wikström, M. K. F. (1972), Biochim. Biophys. Acta 283, 385-390). One mol of calcium produces the shift in one mole of cytochrome a, the KD being approx. 20-30 muM. The calcium-induced shift is readily reversed by chelating agents such as EDTA, ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA) and ATP and is insensitive to uncoupling agents and inhibitors of calcium transport (La3+ and ruthenium red). It is shown that the binding site for calcium that is responsible for the spectral shift is located on the outside of the permeability barrier of the mitochondrial cristae membrane. It is proposed that calcium simulates the energy-linked shift in cytochrome a by binding to a site of cytochrome aa3 that is occupied by protons in energized mitochondria and that is located at the external surface of the mitochondrial membrane.

On the stoichiometry and thermodynamics of proton-pumping cytochrome c oxidase in mitochondria.

Different approaches have been used to evaluate the stoichiometry of proton translocation linked to cytochrome c oxidase in rat liver mitochondria. A mathematical model was designed that successfully describes the kinetics of redox-linked proton translocation provided that the rate of electron transfer is not too high. With ascorbate as reductant, an essentially pH-independent (in the pH range 6--8.5) proton ejection stoichiometry (H+/e-) is obtained from either initial rates of H+ ejection (0.86 +/- 0.12), or the model (0.87 +/- 0.14). Similar results are obtained with either ferrocyanide, N.N.N',N'-tetramethyl-p-phenylenediamine or externally added cytochrome c mediating between ascorbate and cytochrome c in rotenone- and antimycin-inhibited mitochondria. Oxygen pulse experiments with ferrocytochrome c as substrate show fully uncoupler-sensitive redox-linked proton ejection with a stoichiometry of 0.78 +/- 0.14. With murexide to measure Ca2+ uptake during oxidation of ferrocyanide, we found a stoichiometry of two positive charges taken up/electron transferred, confirming earlier findings. These results provide strong evidence that cytochrome c oxidase functions as a redox-linked proton pump with a stoichiometry of one H+ ejected and two charges translocated/electron transferred. The thermodynamic consequences of the proton pump are discussed and a maximal P/O ratio of 1 1/3 for 'site 3' is predicted in agreement with state 4 redox potentials and phosphate potential.

Proton-translocating cytochrome c oxidase in artificial phospholipid vesicles.

The proton translocating properties of cytochrome c oxidase have been studied in artificial phospholipid vesicles into the membranes of which the isolated and purified enzyme was incorporated. Initiation of oxidation of ferrocytochrome c by addition of the cytochrome, or by addition of oxygen to an anaerobic vesicle suspension, leads to ejection of H+ from the vesicles provided that charge compensation is permitted by the presence of valinomycin and K+. Proton ejection is not observed if the membranes have been specifically rendered permeable to protons. The proton ejection is the result of true translocation of H+ across the membrane as indicated by its dependence on the intravesicular buffering power relative to the number of particles (electrons and protons) transferred by the system, and since it can be shown not to be due to a net formation of acid in the system. Comparison of the initial rates of proton ejection and oxidation of cytochrome c yields a H+/e- quotient close to 1.0 both in cytochrome c and oxygen pulse experiments. An approach towards the same stoichiometry is found by comparison of the extents of proton ejection and electron transfer under appropriate experimental conditions. It is concluded that cytochrome c oxidase is a proton pump, which conserves redox energy by converting it into an electrochemical proton gradient through electrogenic translocation of H+.

The plasma membrane potential of human neutrophils. Role of ion channels and the sodium/potassium pump.

Calcium-depleted human neutrophils are depolarised when suspended in calcium-free media containing sodium ions, and are repolarised by extracellular replenishment of Ca2+. The depolarisation is due to a high inward sodium current, which is blocked by calcium and by several other divalent cations, but not by barium. Addition of calcium results in a rise in the cytosolic concentration from approx. 20 nM to the resting level of approx. 130 nM. Calcium influx is strongly accelerated by a voltage-gated calcium channel. This channel might be responsible for the depolarising Na+ current in the absence of divalent cations. In the polarised state the neutrophil membrane has a high intrinsic permeability to K+, which may be low or absent in the depolarised state. Generation of membrane potential from the depolarised state is mainly due to the electrogenic sodium/potassium pump. However, the resting potential of about -75 mV is maintained primarily by the K+ conductance, and only to a small extent by the sodium/potassium pump.

The effect of mitochondrial energization on cytochrome c oxidase kinetics as measured at low temperatures. II. The binding and reduction of dioxygen.

The effect of pre-energization of isolated mitochondria by ATP at room temperature upon the kinetics of oxygen intermediates (measured at very low temperatures) of cytochrome c oxidase has been studied. It was found that "energization" of mitochondria at room temperature had dramatic effects on several partial reactions of cytochrome aa3. Thus, in the "energized" frozen state, the rate of O2 binding to ferrous cytochrome a3 and the subsequent formation of the "peroxy" compound B are accelerated, while oxidation of cytochromes c and c1 is inhibited. These effects of ATP are abolished by oligomycin and uncoupling agents and may, therefore, be reflections of the coupling of the mitochondrial ATP synthetase to the respiratory chain at the level of cytochrome c oxidase, which is the basis of the mechanism of coupling respiration to ATP synthesis and respiratory control.

The mechanism of energy conservation and transduction by mitochondrial cytochrome c oxidase.

Oxidation of ferrocytochrome c by molecular oxygen catalysed by cytochrome c oxidase (cytochrome aa3) is coupled to translocation of H+ ions across the mitochondrial membrane. The proton pump is an intrinsic property of the cytochrome c oxidase complex as revealed by studies with phospholipid vesicles inlayed with the purified enzyme. As the conformation of cytochrome aa3 is specifically sensitive to the electrochemical proton gradient across the mitochondrial membrane, it is likely that redox energy is primarily conserved as a conformational "strain" in the cytochrome aa3 complex, followed by relaxation linked to proton translocation. Similar principles of energy conservation and transduction may apply on other respiratory chain complexes and on mitochondrial ATP synthase.