C-reactive Protein Exists in an NaCl Concentration-dependent Pentamer-Decamer Equilibrium in Physiological Buffer

C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca²⁺-dependent manner, and activates complement. Knowledge of its oligomeric state in solution and at surfaces is essential for functional studies. Analytical ultracentrifugation showed that CRP in 2 mm Ca²⁺ exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaCl concentration. The sedimentation coefficients s_20,w⁰ of pentameric and decameric CRP were 6.4 S and in excess of 7.6 S, respectively. In the absence of Ca²⁺, CRP partially dissociates into its protomers and the NaCl concentration dependence of the pentamer-decamer equilibrium is much reduced. By x-ray scattering, the radius of gyration R_G values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged K_D value of 21 μm in solution (140 mm NaCl, 2 mm Ca²⁺) was determined by x-ray scattering and modeling based on crystal structures for the pentamer and decamer. Surface plasmon resonance showed that CRP self-associates on a surface with immobilized CRP with a similar K_D value of 23 μm (140 mm NaCl, 2 mm Ca²⁺), whereas CRP aggregates in low salt. It is concluded that CRP is reproducibly observed in a pentamer-decamer equilibrium in physiologically relevant concentrations both in solution and on surfaces. Both 2 mm Ca²⁺ and 140 mm NaCl are essential for the integrity of CRP in functional studies and understanding the role of CRP in the acute phase response.     

Nucleocytoplasmic Shuttling of p62/SQSTM1 and Its Role in Recruitment of Nuclear Polyubiquitinated Proteins to Promyelocytic Leukemia Bodies

p62, also known as sequestosome1 (SQSTM1), A170, or ZIP, is a multifunctional protein implicated in several signal transduction pathways. p62 is induced by various forms of cellular stress, is degraded by autophagy, and acts as a cargo receptor for autophagic degradation of ubiquitinated targets. It is also suggested to shuttle ubiquitinated proteins for proteasomal degradation. p62 is commonly found in cytosolic protein inclusions in patients with protein aggregopathies, it is up-regulated in several forms of human tumors, and mutations in the gene are linked to classical adult onset Paget disease of the bone. To this end, p62 has generally been considered to be a cytosolic protein, and little attention has been paid to possible nuclear roles of this protein. Here, we present evidence that p62 shuttles continuously between nuclear and cytosolic compartments at a high rate. The protein is also found in nuclear promyelocytic leukemia bodies. We show that p62 contains two nuclear localization signals and a nuclear export signal. Our data suggest that the nucleocytoplasmic shuttling of p62 is modulated by phosphorylations at or near the most important nuclear localization signal, NLS2. The aggregation of p62 in cytosolic bodies also regulates the transport of p62 between the compartments. We found p62 to be essential for accumulation of polyubiquitinated proteins in promyelocytic leukemia bodies upon inhibition of nuclear protein export. Furthermore, p62 contributed to the assembly of proteasome-containing degradative compartments in the vicinity of nuclear aggregates containing polyglutamine-expanded Ataxin1Q84 and to the degradation of Ataxin1Q84.     

A high–performance compact filter system treating domestic wastewater

A new compact wastewater treatment system for use in single houses has been constructed in eastern Norway. The system is based on the principles of sub-surface flow constructed wetlands using various types of Filtralite as filter media. It consists of a septic tank followed by an aerobic biofilter succeeded by an upflow saturated filter. The aerobic biofilter is essential to remove organic matter and achieve nitrification, while the upflow filter polishes the wastewater and removes microorganisms and phosphorus. During the first 3 years of operation, the system has show stable and high removal with the following average values measured from the outlet of septic tank to the outlet of the upflow filter: 97.0%-BOD₇, 30%-N, 99.4%-P, and 70.8%-SS. No Escherichia coli or somatic coliphages have been detected in the effluent. Due to considerable removal of organic mater, nutrients, and pathogens, the effluent will not negatively affect water and soil ecosystems. The system requires low maintenance and is designed to remove phosphorus for 5 years before renewal of the upflow filter media. When saturated with phosphorus, the media is a suitable fertilizer for plant production.     

Isolation of constitutive variants of a subfamily 10 histidine protein kinase (SppK) from Lactobacillus using random mutagenesis

The histidine protein kinase SppK is a peptide pheromone-activated kinase that regulates the production of the bacteriocin sakacin P in Lactobacillus sakei. SppK belongs to subfamily 10 of histidine protein kinases (HPKs), which regulate important processes in Gram-positive bacteria, including virulence, competence and bacteriocin production. To obtain insight into the functional properties of this relatively unknown class of HPKs, we have subjected SppK to random mutagenesis by error-prone PCR, followed by selection for mutants displaying a constitutive phenotype. Most identified mutations were clustered in a predicted coiled coil-like region, which is an important part of the HPK dimer interface and which includes the autophosphorylated histidine. Other mutations were located in the junctions between the dimerization domain and the membrane receptor domain or the catalytic kinase domain. Interestingly, two previously identified constitutive variants of ComD, an SppK homologue involved in competence regulation in Streptococcus pneumoniae, contained single mutations in the same regions.     

Chilled storage of semen from Atlantic halibut, Hippoglossus hippoglossus L. I: optimizing the protocol

Asynchrony in gamete production between females and males, and decrease in semen quality towards the end of reproductive season make chilled short-term storage of Atlantic halibut, Hippoglossus hippoglossus, semen a desirable method to apply for artificial propagation of this fish species. The goal of the present study was to determine the critical physiochemical factors that affect the success of chilled storage of halibut spermatozoa, and to develop a reliable, simple and efficient protocol for the storage. The presence and type of gaseous atmosphere, dilution and type of diluent, dilution ratio, and additional factors including spermatozoa sedimentation and replenishing the storage medium were tested in relation to spermatozoa motility parameters. Also, fertilization tests were performed with stored semen. Normoxia (air atmosphere) conditions were superior to both hyperoxia (pure oxygen) and no gaseous atmosphere for chilled storage. Dilution of semen with a diluent was superior to incubating undiluted semen. A dilution factor of between 6 and 10 times the original semen volume resulted in the longest viability of stored spermatozoa. Preventing spermatozoa sedimentation through daily swirling of the samples was superior to weekly swirling, however the effect was negligible for the first month of storage. Replenishing the storage medium showed no advantage to incubating in unchanged medium. Semen diluted in modified Hanks' balanced salt solution 1:5-1:9, supplemented with antibiotics, and kept at 0-1 degrees C in Ziploc bag filled with air retained its viability for exceptionally long time. A decrease in the percentage of motile spermatozoa was observed after 43 days of storage, and a decrease in curvilinear velocity occurred after 15 days. Samples remained motile for at least 79 days of storage and the fertilization ability was retained for at least 70 days of storage. The results demonstrate a high potential of application of chilled storage of semen into reproduction programs in Atlantic halibut aquaculture.     

Plant volatiles mediate attraction to host and non-host plant in apple fruit moth, Argyresthia conjugella

Plant volatiles mediate host finding in insect herbivores and lead to host fidelity and habitat‐specific mating, generating premating reproductive isolation and facilitating sympatric divergence. The apple fruit moth, Argyresthia conjugella Zeller (Lepidoptera: Argyresthiidae), is a particularly suitable species to study the cues and behavioural mechanisms leading to colonization of a new host: it recurrently oviposits on the non‐host plant, apple Malus domestica Borkh. (Rosaceae), where the larvae cannot complete their development. The larval host of the apple fruit moth (Lepidoptera, Argyresthiidae), is rowan Sorbus aucuparia L. (Rosaceae). Fruit setting in rowan, however, fluctuates strongly over large areas in Scandinavia. Every 2–4 years, when too few rowanberries are available for egg laying in forests, apple fruit moth females oviposit instead on apple in nearby orchards, but not on other fruits, such as pear or plum. This poses the question of which cues mediate attraction to rowan and apple, and how apple fruit moth discriminates rowan from apple. Chemical analysis and antennal recordings showed that 11 out of 15 rowan volatiles eliciting an antennal response in A. conjugella females co‐occur in rowan and apple headspace, in a different proportion. In the field, A. conjugella was attracted to several of these plant volatiles, especially to 2‐phenyl ethanol, methyl salicylate, and decanal. Addition of anethole to 2‐phenyl ethanol had a strong synergistic effect, the 1 : 1 blend is a powerful attractant for A. conjugella males and females. These results confirm that volatiles common to both plants may account for a host switch in A. conjugella from rowan to apple. Some of the most attractive compounds, including 2‐phenyl ethanol, anethole, and decanal, which have been found in several apple cultivars, were not present in the headspace of the apple cultivar, Aroma, which is also susceptible to attack by A. conjugella. This supports the idea that the odour signal from apple is suboptimal for attraction of A. conjugella, but is nonetheless sufficient for attraction, during times when rowan is not available for egg laying.     

Body mass and clutch size may modulate prolactin and corticosterone levels in eiders

Altered body condition, increased incubation costs, and egg loss are important proximate factors modulating bird parental behavior, since they inform the adult about its remaining chances of survival or about the expected current reproductive success. Hormonal changes should reflect internal or external stimuli, since corticosterone levels (inducing nest abandonment) are known to increase while body condition deteriorates, and prolactin levels (stimulating incubation) decrease following egg predation. However, in a capital incubator that based its investment on available body reserves and naturally lost about half of its body mass during incubation, corticosterone should be maintained at a low threshold to avoid protein mobilization for energy supply. This study focused on the regulation of corticosterone and prolactin release in such birds during incubation, when facing egg manipulation (control, reduced, or increased) or a stressful event. Blood samples were taken before and after clutch manipulation and at hatching. Corticosterone levels were determined before and after 30 min of captivity. Female eiders exhibited a high hypothalamic-pituitary-adrenal sensitivity, plasma concentration of corticosterone being increased by four- to fivefold following 30 min of captivity. The adrenocortical response was not modified by body mass loss but was higher in birds for which clutch size was increased. In the same way, females did not show different prolactin levels among the experimental groups. However, when incubation started, prolactin levels were correlated to body mass, suggesting that nest attendance is programmed in relation to the female initial body condition. Moreover, due to an artifactual impact of bird manipulation, increased baseline corticosterone was associated with a prolactin decrease in the control group. These data suggest that, in eiders, body mass and clutch size modification can modulate prolactin and corticosterone levels, which cross-regulate each other in order to finely control incubation behavior.     

Silver ions disrupt K⁺ homeostasis and cellular integrity in intact barley (Hordeum vulgare L.) roots

The heavy metals silver, gold, and mercury can strongly inhibit aquaporin-mediated water flow across plant cell membranes, but critical examinations of their side effects are rare. Here, the short-lived radiotracer (42)K is used to demonstrate that these metals, especially silver, profoundly change potassium homeostasis in roots of intact barley (Hordeum vulgare L.) plants, by altering unidirectional K(+) fluxes. Doses as low as 5 μM AgNO(3) rapidly reduced K(+) influx to 5% that of controls, and brought about pronounced and immediate increases in K(+) efflux, while higher doses of Au(3+) and Hg(2+) were required to produce similar responses. Reduced influx and enhanced efflux of K(+) resulted in a net loss of >40% of root tissue K(+) during a 15 min application of 500 μM AgNO(3), comprising the entire cytosolic potassium pool and about a third of the vacuolar pool. Silver also brought about major losses of UV-absorbing compounds, total electrolytes, and NH(4)(+). Co-application, with silver, of the channel blockers Cs(+), TEA(+), or Ca(2+), did not affect the enhanced efflux, ruling out the involvement of outwardly rectifying ion channels. Taken together with an examination of propidium iodide staining under confocal microscopy, the results indicate that silver ions affect K(+) homeostasis by directly inhibiting K(+) influx at lower concentrations, and indirectly inhibiting K(+) influx and enhancing K(+) efflux, via membrane destruction, at higher concentrations. Ni(2+), Cd(2+), and Pb(2+), three heavy metals not generally known to affect aquaporins, did not enhance K(+) efflux or cause propidium iodide incorporation. The study reveals strong and previously unknown effects of major aquaporin inhibitors and recommends caution in their application.     

A pharmacological analysis of high-affinity sodium transport in barley (Hordeum vulgare L.): a 24Na+/42K+ study

Soil sodium, while toxic to most plants at high concentrations, can be beneficial at low concentrations, particularly when potassium is limiting. However, little is known about Na(+) uptake in this 'high-affinity' range. New information is provided here with an insight into the transport characteristics, mechanism, and ecological significance of this phenomenon. High-affinity Na(+) and K(+) fluxes were investigated using the short-lived radiotracers (24)Na and (42)K, under an extensive range of measuring conditions (variations in external sodium, and in nutritional and pharmacological agents). This work was supported by electrophysiological, compartmental, and growth analyses. Na(+) uptake was extremely sensitive to all treatments, displaying properties of high-affinity K(+) transporters, K(+) channels, animal Na(+) channels, and non-selective cation channels. K(+), NH(4)(+), and Ca(2+) suppressed Na(+) transport biphasically, yielding IC(50) values of 30, 10, and <5 μM, respectively. Reciprocal experiments showed that K(+) influx is neither inhibited nor stimulated by Na(+). Sodium efflux constituted 65% of influx, indicating a futile cycle. The thermodynamic feasibility of passive channel mediation is supported by compartmentation and electrophysiological data. Our study complements recent advances in the molecular biology of high-affinity Na(+) transport by uncovering new physiological foundations for this transport phenomenon, while questioning its ecological relevance.