MOBILIZATION OF β-1,3-GLUCAN AND BIOSYNTHESIS OF AMINO ACIDS INDUCED BY NH4+ ADDITION TO N-LIMITED CELLS OF THE MARINE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)

Mobilization of the reserve β-1,3-glucan (chrysolaminaran) in N-limited cells of the marine diatom Skeletonema costatum (Grev.) Cleve (Bacillariophyceae) was investigated. The diatom was grown in pH-regulated batch cultures with a 14:10-h light:dark cycle until N depletion. In a pulse-chase experiment, the cells were first incubated in high light (200 μmol photons·m ^{− 2}·s ^{− 1}) with ¹⁴C-bicarbonate until dissolved inorganic carbon was exhausted. Unlabeled bicarbonate (1 mM) was then added, and the cells were incubated in the dark and subsequently in low light (20 μmol photons·m ^{− 2}·s ^{− 1}) with additions of 40 μM NH₄ ⁺ . In the ¹⁴C pulse phase with high light and N depletion, β-1,3-glucan accumulated and accounted for 85% of incorporated ¹⁴C. In the subsequent ¹⁴C chase phases, added NH₄ ⁺ was assimilated at an N-specific rate of 0.11 h ^{− 1} in both the dark and low light, and in both cases it caused a significant mobilization of β-1,3-glucan (dark, 26%; low light, 19%). Biochemical fractionation of organic ¹⁴C showed that free amino acids were most rapidly labeled in the early stage of NH₄ ⁺ assimilation, whereas proteins and polysaccharides were labeled more rapidly after 1.2 h. Analysis of the cellular free amino acids strongly indicated that de novo biosynthesis was occurring, with a Gln:Glu ratio increasing from 0.4 to 10 within 1.2 h. After the NH₄ ⁺ was exhausted, the cellular pools of glucan and amino acids became constant or slowly decreased. In another experiment, N-limited cells were first incubated in high light until dissolved inorganic carbon was exhausted and were further incubated in high light with 150 μM NH₄ ⁺ under inorganic carbon limitation. Added NH₄ ⁺ was assimilated at an N-specific rate of 0.023 h ^{− 1}, and cellular β-1,3-glucan decreased by 15% within 6 h. Hence, β-1,3-glucan was mobilized during NH₄ ⁺ assimilation, even though inorganic carbon was modifying the metabolic rates. The results provide new evidence of β-1,3-glucan supplying essential precursors for biosynthesis of amino acids and other components in S. costatum in both the dark and subsaturating light and even saturating light under inorganic carbon limitation.     

Development of an in vivo method for determining material properties of passive myocardium

Calculation of mechanical stresses and strains in the left ventricular (LV) myocardium by the finite element (FE) method relies on adequate knowledge of the material properties of myocardial tissue. In this paper, we present a model-based estimation procedure to characterize the stress-strain relationship in passive LV myocardium. A 3D FE model of the LV myocardium was used, which included morphological fiber and sheet structure and a nonlinear orthotropic constitutive law with different stiffness in the fiber, sheet, and sheet-normal directions. The estimation method was based on measured wall strains. We analyzed the method's ability to estimate the material parameters by generating a set of synthetic strain data by simulating the LV inflation phase with known material parameters. In this way we were able to verify the correctness of the solution and to analyze the effects of measurement and model error on the solution accuracy and stability. A sensitivity analysis was performed to investigate the observability of the material parameters and to determine which parameters to estimate. The results showed a high degree of coupling between the parameters governing the stiffness in each direction. Thus, only one parameter in each of the three directions was estimated. For the tested magnitudes of added noise and introduced model errors, the resulting estimated stress-strain characteristics in the fiber and sheet directions converged with good accuracy to the known relationship. The sheet-normal stress-strain relationship had a higher degree of uncertainty as more noise was added and model error was introduced.     

High-frequency electrically stimulated skeletal muscle contractions increase p70 phosphorylation independent of known IGF-I sensitive signaling pathways

Insulin-like growth factor (IGF-I) is hypothesized to be a critical upstream regulator of mammalian target of rapamycin (mTOR)-regulated protein synthesis with muscle contraction. We utilized a mouse model that expresses a skeletal muscle specific dominant-negative IGF-I receptor to investigate the role of IGF-I signaling of protein synthesis in response to unilateral lengthening contractions (10 sets, 6 repetitions, 100 Hz) at 0 and 3 h following the stimulus. Our results indicate that one session of high frequency muscle contractions can activate mTOR signaling independent of signaling components directly downstream of the receptor.     

Cytochrome c-554 from Methylosinus trichosporium OB3b; a protein that belongs to the cytochrome c2 family and exhibits a HALS-Type EPR signal

A small soluble cytochrome c-554 purified from Methylosinus trichosporium OB3b has been purified and analyzed by amino acid sequencing, mass spectrometry, visible, CD and EPR spectroscopies. It is found to be a mono heme protein with a characteristic cytochrome c fold, thus fitting into the class of cytochrome c(2), which is the bacterial homologue of mitochondrial cytochrome c. The heme iron has a Histidine/Methionine axial ligation and exhibits a highly anisotropic/axial low spin (HALS) EPR signal, with a g(max) at 3.40, and ligand field parameters V/ξ = 0.99, Δ/ξ = 4.57. This gives the rhombicity V/Δ = 0.22. The structural basis for this HALS EPR signal in Histidine/Methionine ligated hemes is not resolved. The ligand field parameters observed for cytochrome c-554 fits the observed pattern for other cytochromes with similar ligation and EPR behaviour.     

Sequence Analysis of the Genome of Piscine Orthoreovirus (PRV) Associated with Heart and Skeletal Muscle Inflammation (HSMI) in Atlantic Salmon (Salmo salar)

Piscine orthoreovirus (PRV) is associated with heart- and skeletal muscle inflammation (HSMI) of farmed Atlantic salmon (Salmo salar). We have performed detailed sequence analysis of the PRV genome with focus on putative encoded proteins, compared with prototype strains from mammalian (MRV T3D)- and avian orthoreoviruses (ARV-138), and aquareovirus (GCRV-873). Amino acid identities were low for most gene segments but detailed sequence analysis showed that many protein motifs or key amino acid residues known to be central to protein function are conserved for most PRV proteins. For M-class proteins this included a proline residue in μ2 which, for MRV, has been shown to play a key role in both the formation and structural organization of virus inclusion bodies, and affect interferon-β signaling and induction of myocarditis. Predicted structural similarities in the inner core-forming proteins λ1 and σ2 suggest a conserved core structure. In contrast, low amino acid identities in the predicted PRV surface proteins μ1, σ1 and σ3 suggested differences regarding cellular interactions between the reovirus genera. However, for σ1, amino acid residues central for MRV binding to sialic acids, and cleavage- and myristoylation sites in μ1 required for endosomal membrane penetration during infection are partially or wholly conserved in the homologous PRV proteins. In PRV σ3 the only conserved element found was a zinc finger motif. We provide evidence that the S1 segment encoding σ3 also encodes a 124 aa (p13) protein, which appears to be localized to intracellular Golgi-like structures. The S2 and L2 gene segments are also potentially polycistronic, predicted to encode a 71 aa- (p8) and a 98 aa (p11) protein, respectively. It is concluded that PRV has more properties in common with orthoreoviruses than with aquareoviruses.     

The relation of emotions to placebo responses

The hypothesis put forth is that expectations of treatment effects reduce negative emotions and thereby reduce symptoms, e.g. pain. Negative emotions increase pain, and it is hypothesized that placebos reduce pain by reducing negative emotions, i.e. feelings of nervousness, fear and anxiety. Placebo analgesia has been shown to be mediated via opioid activity, and relaxation increases opioid activity. The placebo acquires its relaxing effect due to verbal information that pain will be reduced, or due to associations between the placebo and the reduction in pain after effective treatment. Thus, the placebo signals that unpleasantness will be less after administration of the placebo. This involves negative reinforcement which is due to activation of a dopaminergic system that has been found to be activated during placebo analgesia and is involved in positive emotions. The nocebo effect of increased pain is, consistent with this model, because of increased fear and anxiety. The new aspect of the presented model is the hypothesis that expectations reduce negative emotions, and that negative reinforcement that involves the dopaminergic reinforcement system should be a contributor to placebo responses.