Disruption of the GDNF binding site in NCAM dissociates ligand binding and homophilic cell adhesion

Most plasma membrane proteins are capable of sensing multiple cell-cell and cell-ligand interactions, but the extent to which this functional versatility is founded on their modular design is less clear. We have identified the third immunoglobulin domain of the Neural Cell Adhesion Molecule (NCAM) as the necessary and sufficient determinant for its interaction with Glial Cell Line-derived Neurotrophic Factor (GDNF). Four charged contacts were identified by molecular modeling as the main contributors to binding energy. Their mutation abolished GDNF binding to NCAM but left intact the ability of NCAM to mediate cell adhesion, indicating that the two functions are genetically separable. The GDNF-NCAM interface allows complex formation with the GDNF family receptor alpha1, shedding light on the molecular architecture of a multicomponent GDNF receptor.     

Imaging of disease dynamics during meningococcal sepsis

Neisseria meningitidis is a human pathogen that causes septicemia and meningitis with high mortality. The disease progression is rapid and much remains unknown about the disease process. The understanding of disease development is crucial for development of novel therapeutic strategies and vaccines against meningococcal disease. The use of bioluminescent imaging combined with a mouse disease model allowed us to investigate the progression of meningococcal sepsis over time. Injection of bacteria in blood demonstrated waves of bacterial clearance and growth, which selected for Opa-expressing bacteria, indicating the importance of this bacterial protein. Further, N. meningitidis accumulated in the thyroid gland, while thyroid hormone T4 levels decreased. Bacteria reached the mucosal surfaces of the upper respiratory tract, which required expression of the meningococcal PilC1 adhesin. Surprisingly, PilC1 was dispensable for meningococcal growth in blood and for crossing of the blood-brain barrier, indicating that the major role of PilC1 is to interact with mucosal surfaces. This in vivo study reveals disease dynamics and organ targeting during meningococcal disease and presents a potent tool for further investigations of meningococcal pathogenesis and vaccines in vivo. This might lead to development of new strategies to improve the outcome of meningococcal disease in human patients.     

Iscoms in parasitological research

During the history of vaccine development, a number of adjuvants and adjuvant formulations have been tested and evaluated for their ability to increase the immunogenicity of different antigens. In this review, Anna Lundén, Karin L?vgren Bengtsson, Anders Sj?lander and Arvid Uggla focus on iscoms (immune stimulating complexes), their characteristics and applications to different types of parasitic antigens.     

Immunoextracted calcitonin in human gastric secretion

Immunoreactive calcitonin (iCT) has been demonstrated in human gastric juice after immunoextraction with immobilized antibodies and subsequent radioimmunoassay. The basal levels were 4.5 +/- 3.1 (mean +/- SD) pg-Eq/ml gastric juice; range 1.2-9.1 pg-Eq/ml; n = 7, and after stimulatory gastric secretion test with pentagastrin 0.3 +/- 0.2 pg-Eq/ml; range 0.1-0.7 pg-Eq/ml; n = 7 (p less than 0.01). The main fraction of iCT from gastric juice eluted in the same region as synthetic human calcitonin (hCT) on Sephadex G-75 gel chromatography. Reverse phase chromatography in a fast protein liquid chromatography (FPLC) system revealed a slightly less hydrophobic character of the iCT from gastric juice compared to synthetic monomeric hCT. The results were further confirmed by using an additional antiserum. In plasma, the calcitonin (CT) levels were after immunoextraction at the basal state 6.6 +/- 1.7 pg-Eq/ml (mean +/- SD); range 5.1-10.1 pg-Eq/ml; n = 7 and after pentagastrin stimulation 9.4 +/- 5.4 pg-Eq/ml; range 6.3-18.5 pg-Eq/ml; n = 7.     

Short‐term soil carbon sink potential of oil palm plantations

Oil palm plantations cover ≈14.6 million ha worldwide and the total area under cultivation is expected to increase during the 21st century . Indonesia and Malaysia together account for 87% of global palm oil production and the combined harvested area in these countries has expanded by 6.5 million ha since 1990. Despite this, soil C cycling in oil palm systems is not well quantified but such information is needed for C budget inventories. We quantified soil C storage (root biomass, soil organic matter (SOM) and microbial biomass) and losses [potential soil respiration (R_s) and soil surface CO₂ flux (F_s)] in mineral soils from an oil palm plantation chronosequence (11–34 years since planting) in Selangor, Malaysia. There were no significant effects of plantation age on SOM, microbial biomass, R_s or F_s, implying soil C was in dynamic equilibrium over the chronosequence. However, there was a significant increase in root biomass with plantation age, indicating a short‐term C sink. Across the chronosequence, R_s was driven by soil moisture, soil particle size, root biomass and soil microbial biomass N but not microbial biomass C. This suggests that the nutrient status of the microbial community may be of equal or greater importance for soil CO₂ losses than substrate availability and also raises particular concerns regarding the addition of nitrogenous fertilizer, i.e. increased yields will be associated with increased soil CO₂ emissions. To fully assess the impact of oil palm plantations on soil C storage, initial soil C losses following land conversion (e.g. from native forest or other previous plantations) must be accounted for. If initial soil C losses are large, our data show that there is no accumulation of stable C in the soil as the plantation matures and hence the conversion to oil palm would probably represent a net loss of soil C.     

The Evolution of the Ribonucleotide Reductases: Much Ado About Oxygen

Ribonucleotide reduction is the only known biological means for de novo production of deoxyribonucleotides, the building blocks of DNA. These are produced from ribonucleotides, the building blocks of RNA, and the direction of this reaction has been taken to support the idea that, in evolution, RNA preceded DNA as genetic material. However, an understanding of the evolutionary relationships among the three modern-day classes of ribonucleotide reductase and how the first reductase arose early in evolution is still far off. We propose that the diversification of this class of enzymes is inherently tied to microbial colonization of aerobic and anaerobic niches. The work is of broader interest, as it also sheds light on the process of adaptation to oxygenic environments consequent to the evolution of atmospheric oxygen.     

Shoulder muscle load and muscle fatigue among industrial sewing-machine operators

Physiological responses to physical work were assessed for 29 female industrial sewing-machine operators during an 8-h working day under ordinary working conditions. During sewing-machine work, the average (left and right) static load in the trapezius muscle was 9% of the maximal electromyogram (EMG) amplitude (% EMG_max), while the average mean load was 15% EMG_max, and the average peak load was 23% EMG_max. The static load level was unrelated to the muscle strength of the sewing-machine operators, which for the group as a whole was within the normal range. The load levels remained unchanged during the working day, while changes in the EMG mean power frequency and zero crossing frequency rate occurred, both indicating the development of muscle fatigue in left and right trapezius muscle during the working day. In line with this, the rating of perceived exertion in the shoulder and neck region increased during. the working day. Dividing the group of sewing-machine operators into two groups, those with the highest frequency and those with the lowest frequency of shoulder/neck troubles showed that the former group had significantly lower muscle strength, despite the fact that no differences in the surface EMG during sewing were found between the two groups. It was concluded that industrial sewing-machine work involves a pattern of shoulder muscle activity which induces fatiguing processes in the shoulder and neck regions. Furthermore, since the static shoulder muscle load was independent of muscle strength, factors other than working posture may be of significance for the static shoulder muscle load.     

Newly diagnosed latent autoimmune diabetes in adults (LADA) is associated with low level glutamate decarboxylase (GAD65) and IA-2 autoantibodies. Diabetes Incidence Study in Sweden (DISS).

A quantitative assay with microSepharose was used to determine GAD65Ab and IA-2Ab levels in 771 population-based patients diagnosed with diabetes mellitus at 15 to 34 years of age, and in 828 matched controls. Among the patients, 587 (76%) were classified with type I, 108 (14%) with type II, and 76 (10%) with unclassifiable diabetes. The levels above normal demonstrated a prevalence of GAD65Ab in 66% of type I diabetes, 50% of type II diabetes and 54% of unclassifiable patients and for IA-2Ab in 40%, 17% and 21%, respectively. Among the autoantibody-positive sera, the LADA patients had a lower GAD65Ab index (median 0.19, p < 0.0001) and IA-2Ab index (median 0.28, p < 0.0001) than the type I patients (median 0.37 and 0.66). Patients with unclassifiable diabetes had a GAD65Ab (median 0.43) or IA-2Ab (median 0.63) index which was not different from the type I diabetes patients. Our data demonstrate that young adult new-onset LADA patients have low level GAD65Ab and IA-2Ab. The low-level autoantibodies may signify a less aggressive beta-cell autoimmunity, which may explain why these patients are often classified with type II or non-insulin-dependent diabetes.     

Proton-coupled structural changes upon binding of carbon monoxide to cytochrome cd1: a combined flash photolysis and X-ray crystallography study

We have investigated dynamic events after flash photolysis of CO from reduced cytochrome cd(1) nitrite reductase (NiR) from Paracoccus pantotrophus (formerly Thiosphaera pantotropha). Upon pulsed illumination of the cytochrome cd(1)-CO complex, at 460 nm, a rapid (<50 ns) absorbance change, attributed to dissociation of CO, was observed. This was followed by a biphasic rearrangement with rate constants of 1.7 x 10(4) and 2.5 x 10(3) s(-1) at pH 8.0. Both parts of the biphasic rearrangement phases displayed the same kinetic difference spectrum in the region of 400-660 nm. The slower of the two processes was accompanied by proton uptake from solution (0.5 proton per active site at pH 7.5-8.5). After photodissociation, the CO ligand recombined at a rate of 12 s(-1) (at 1 mM CO and pH 8.0), accompanied by proton release. The crystal structure of reduced cytochrome cd(1) in complex with CO was determined to a resolution of 1.57 A. The structure shows that CO binds to the iron of the d(1) heme in the active site. The ligation of the c heme is unchanged in the complex. A comparison of the structures of the reduced, unligated NiR and the NiR-CO complex indicates changes in the puckering of the d(1) heme as well as rearrangements in the hydrogen-bonding network and solvent organization in the substrate binding pocket at the d(1) heme. Since the CO ligand binds to heme d(1) and there are structural changes in the d(1) pocket upon CO binding, it is likely that the proton uptake or release observed after flash-induced CO dissociation is due to changes of the protonation state of groups in the active site. Such proton-coupled structural changes associated with ligand binding are likely to affect the redox potential of heme d(1) and may regulate the internal electron transfer from heme c to heme d(1).     

Eosinophilopoiesis in a murine model of allergic airway eosinophilia: involvement of bone marrow IL-5 and IL-5 receptor alpha

The airway inflammation in asthma is dominated by eosinophils. The aim of this study was to elucidate the contribution of newly produced eosinophils in airway allergic inflammation and to determine mechanisms of any enhanced eosinophilopoiesis. OVA-sensitized BALB/c mice were repeatedly exposed to allergen via airway route. Newly produced cells were identified using a thymidine analog, 5-bromo-2'-deoxyuridine, which is incorporated into DNA during mitosis. Identification of IL-5-producing cells in the bone marrow was performed using FACS. Bone marrow CD3+ cells were enriched to evaluate IL-5-protein release in vitro. Anti-IL-5-treatment (TRFK-5) was given either systemically or directly to the airways. IL-5R-bearing cells were localized by immunocytochemistry. Repeated airway allergen exposure caused prominent airway eosinophilia after three to five exposures, and increased the number of immature eosinophils in the bone marrow. Up to 78% of bronchoalveolar lavage (BAL) granulocytes were 5-bromo-2'-deoxyuridine positive. After three allergen exposures, both CD3+ and non-CD3 cells acquired from the bone marrow expressed and released IL-5-protein. Anti-IL-5 given i.p. inhibited both bone marrow and airway eosinophilia. Intranasal administration of anti-IL-5 also reduced BAL eosinophilia, partly via local effects in the airways. Bone marrow cells, but not BAL eosinophils, displayed stainable amounts of the IL-5R alpha-chain. We conclude that the bone marrow is activated by airway allergen exposure, and that newly produced eosinophils contribute to a substantial degree to the airway eosinophilia induced by allergen. Airway allergen exposure increases the number of cells expressing IL-5-protein in the bone marrow. The bone marrow, as well as the lung, are possible targets for anti-IL-5-treatment.