Reconstitution of contractile actomyosin bundles

Contractile actomyosin bundles are critical for numerous aspects of muscle and nonmuscle cell physiology. Due to the varying composition and structure of actomyosin bundles in vivo, the minimal requirements for their contraction remain unclear. Here, we demonstrate that actin filaments and filaments of smooth muscle myosin motors can self-assemble into bundles with contractile elements that efficiently transmit actomyosin forces to cellular length scales. The contractile and force-generating potential of these minimal actomyosin bundles is sharply sensitive to the myosin density. Above a critical myosin density, these bundles are contractile and generate large tensile forces. Below this threshold, insufficient cross-linking of F-actin by myosin thick filaments prevents efficient force transmission and can result in rapid bundle disintegration. For contractile bundles, the rate of contraction decreases as forces build and stalls under loads of ∼0.5 nN. The dependence of contraction speed and stall force on bundle length is consistent with bundle contraction occurring by several contractile elements connected in series. Thus, contraction in reconstituted actomyosin bundles captures essential biophysical characteristics of myofibrils while lacking numerous molecular constituents and structural signatures of sarcomeres. These results provide insight into nonsarcomeric mechanisms of actomyosin contraction found in smooth muscle and nonmuscle cells.     

Indigenous Bacteria in Hemolymph and Tissues of Marine Bivalves at Low Temperatures

Hemolymph and soft tissues of Pacific oysters (Crassostrea gigas) kept in sand-filtered seawater at temperatures between 1 and 8°C were normally found to contain bacteria, with viable counts (CFU) in hemolymph in the range 1.4 × 10² to 5.6 × 10² bacteria per ml. Pseudomonas, Alteromonas, Vibrio, and Aeromonas organisms dominated, with a smaller variety of morphologically different unidentified strains. Hemolymph and soft tissues of horse mussels (Modiolus modiolus), locally collected from a 6- to 10-m depth in the sea at temperatures between 4 and 6°C, also contained bacteria. The CFU in horse mussel hemolymph was of the same magnitude as that in oysters (mean, 2.6 × 10⁴), and the bacterial flora was dominated by Pseudomonas (61.3%), Vibrio (27.0%), and Aeromonas (11.7%) organisms. In soft tissues of horse mussels, a mean CFU of 2.9 × 10⁴ bacteria per g was found, with Vibrio (38.5%), Pseudomonas (33.0%), and Aeromonas (28.5%) constituting the major genera. After the challenge of oysters in seawater at 4°C to the psychrotrophic fish pathogen Vibrio salmonicida (strains NCIMB 2245 from Scotland and TEO 84001 from Norway) and a commensal Aeromonas sp. isolated from oysters, the viable count in hemolymph increased 1,000-fold to about 10⁵ bacteria per ml. In soft tissues, about a 1,000-fold increase in CFU to 6 × 10⁷ was observed. V. salmonicida NCIMB 2245 invaded hemolymph and soft tissues after 14 days and dominated these compartments after 41 days, whereas strain TEO 84001 did not invade soft tissues to the same extent. Challenge with V. salmonicida NCIMB 2245 resulted in 100% mortality, whereas about 50% of the oysters survived challenge with the Norwegian strain, TEO 84001. The commensal Aeromonas sp. invaded hemolymph and soft tissues and caused 100% mortality. Oyster hemolymph contained agglutinins for Vibrio anguillarum but not for V. salmonicida, whereas we did not find agglutinins for either of these bacteria in horse mussels. Agglutinins for horse and human erythrocytes were found in hemolymph from both animals. We found no differences in agglutinin titers in oysters from different Norwegian locations, and long-term challenge with bacteria in seawater did not result in changes of agglutinin activity. These studies demonstrate that bacteria exist in hemolymph and soft tissues of marine bivalves at temperatures below 8°C. Increased bacterial numbers in seawater at 4°C result in augmented invasion of bacteria in hemolymph and soft tissues. V. salmonicida, a bacterium pathogenic for fish at low temperatures, invades bivalve hemolymph and soft tissues, and thus bivalves may serve as a reservoir for pathogens of fish at low seawater temperatures.     

A Prospective Population Study of Resting Heart Rate and Peak Oxygen Uptake (the HUNT Study,Norway)

Objectives

We assessed the prospective association of resting heart rate (RHR) at baseline with peak oxygen uptake (VO_2peak) 23 years later, and evaluated whether physical activity (PA) could modify this association.

Background

Both RHR and VO_2peak are strong and independent predictors of cardiovascular morbidity and mortality. However, the association of RHR with VO_2peak and modifying effect of PA have not been prospectively assessed in population studies.

Methods

In 807 men and 810 women free from cardiovascular disease both at baseline (1984–86) and follow-up 23 years later, RHR was recorded at both occasions, and VO_2peak was measured by ergospirometry at follow-up. We used Generalized Linear Models to assess the association of baseline RHR with VO_2peak, and to study combined effects of RHR and self-reported PA on later VO_2peak.

Results

There was an inverse association of RHR at baseline with VO_2peak (p<0.01). Men and women with baseline RHR greater than 80 bpm had 4.6 mL·kg⁻¹·min⁻¹ (95% confidence interval [CI], 2.8 to 6.3) and 1.4 mL·kg⁻¹·min⁻¹ (95% CI, −0.4 to 3.1) lower VO_2peak at follow-up compared with men and women with RHR below 60 bpm at baseline. We found a linear association of change in RHR with VO_2peak (p = 0.03), suggesting that a decrease in RHR over time is likely to be beneficial for cardiovascular fitness. Participants with low RHR and high PA at baseline had higher VO_2peak than inactive people with relatively high RHR. However, among participants with relatively high RHR and high PA at baseline, VO_2peak was similar to inactive people with relatively low RHR.

Conclusion

RHR is an important predictor of VO_2peak, and serial assessments of RHR may provide useful and inexpensive information on cardiovascular fitness. The results suggest that high levels of PA may compensate for the lower VO_2peak associated with a high RHR.     

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.     

Genetic and potential non-genetic benefits increase offspring fitness of polyandrous females in non-resource based mating system

Background  

The adaptive significance of female polyandry is currently under considerable debate. In non-resource based mating systems, indirect, i.e. genetic benefits have been proposed to be responsible for the fitness gain from polyandry. We studied the benefits of polyandry in the Arctic charr (Salvelinus alpinus) using an experimental design in which the material investments by the sires and maternal environmental effects were controlled.     

Emergence, spread, persistence and fade-out of sylvatic plague in Kazakhstan

Predicting the dynamics of zoonoses in wildlife is important not only for prevention of transmission to humans, but also for improving the general understanding of epidemiological processes. A large dataset on sylvatic plague in the Pre-Balkhash area of Kazakhstan (collected for surveillance purposes) provides a rare opportunity for detailed statistical modelling of an infectious disease. Previous work using these data has revealed a host abundance threshold for epizootics, and climatic influences on plague prevalence. Here, we present a model describing the local space-time dynamics of the disease at a spatial scale of 20 × 20 km(2) and a biannual temporal scale, distinguishing between invasion and persistence events. We used a Bayesian imputation method to account for uncertainties resulting from poor data in explanatory variables and response variables. Spatial autocorrelation in the data was accounted for in imputations and analyses through random effects. The results show (i) a clear effect of spatial transmission, (ii) a high probability of persistence compared with invasion, and (iii) a stronger influence of rodent abundance on invasion than on persistence. In particular, there was a substantial probability of persistence also at low host abundance.     

Y chromosome haplotype distribution of brown bears (Ursus arctos) in Northern Europe provides insight into population history and recovery

High‐resolution, male‐inherited Y‐chromosomal markers are a useful tool for population genetic analyses of wildlife species, but to date have only been applied in this context to relatively few species besides humans. Using nine Y‐chromosomal STRs and three Y‐chromosomal single nucleotide polymorphism markers (Y‐SNPs), we studied whether male gene flow was important for the recent recovery of the brown bear (Ursus arctos) in Northern Europe, where the species declined dramatically in numbers and geographical distribution during the last centuries but is expanding now. We found 36 haplotypes in 443 male extant brown bears from Sweden, Norway, Finland and northwestern Russia. In 14 individuals from southern Norway from 1780 to 1920, we found two Y chromosome haplotypes present in the extant population as well as four Y chromosome haplotypes not present among the modern samples. Our results suggested major differences in genetic connectivity, diversity and structure between the eastern and the western populations in Northern Europe. In the west, our results indicated that the recovered population originated from only four male lineages, displaying pronounced spatial structuring suggestive of large‐scale population size increase under limited male gene flow within the western subpopulation. In the east, we found a contrasting pattern, with high haplotype diversity and admixture. This first population genetic analysis of male brown bears shows conclusively that male gene flow was not the main force of population recovery.     

DNA-uracil and human pathology

Uracil is usually an inappropriate base in DNA, but it is also a normal intermediate during somatic hypermutation (SHM) and class switch recombination (CSR) in adaptive immunity. In addition, uracil is introduced into retroviral DNA by the host as part of a defence mechanism. The sources of uracil in DNA are spontaneous or enzymatic deamination of cytosine (U:G mispairs) and incorporation of dUTP (U:A pairs). Uracil in DNA is removed by a uracil-DNA glycosylase. The major ones are nuclear UNG2 and mitochondrial UNG1 encoded by the UNG-gene, and SMUG1 that also removes oxidized pyrimidines, e.g. 5-hydroxymethyluracil. The other ones are TDG that removes U and T from mismatches, and MBD4 that removes U from CpG contexts. UNG2 is found in replication foci during the S-phase and has a distinct role in repair of U:A pairs, but it is also important in U:G repair, a function shared with SMUG1. SHM is initiated by activation-induced cytosine deaminase (AID), followed by removal of U by UNG2. Humans lacking UNG2 suffer from recurrent infections and lymphoid hyperplasia, and have skewed SHM and defective CSR, resulting in elevated IgM and strongly reduced IgG, IgA and IgE. UNG-defective mice also develop B-cell lymphoma late in life. In the defence against retrovirus, e.g. HIV-1, high concentrations of dUTP in the target cells promotes misincorporation of dUMP-, and host cell APOBEC proteins may promote deamination of cytosine in the viral DNA. This facilitates degradation of viral DNA by UNG2 and AP-endonuclease. However, viral proteins Vif and Vpr counteract this defense by mechanisms that are now being revealed. In conclusion, uracil in DNA is both a mutagenic burden and a tool to modify DNA for diversity or degradation.     

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.     

Emerging facets of plastid division regulation

Plastids are complex organelles that are integrated into the plant host cell where they differentiate and divide in tune with plant differentiation and development. In line with their prokaryotic origin, plastid division involves both evolutionary conserved proteins and proteins of eukaryotic origin where the host has acquired control over the process. The plastid division apparatus is spatially separated between the stromal and the cytosolic space but where clear coordination mechanisms exist between the two machineries. Our knowledge of the plastid division process has increased dramatically during the past decade and recent findings have not only shed light on plastid division enzymology and the formation of plastid division complexes but also on the integration of the division process into a multicellular context. This review summarises our current knowledge of plastid division with an emphasis on biochemical features, the functional assembly of protein complexes and regulatory features of the overall process.