A fast dynamic mode of the EF‐G‐bound ribosome

A key intermediate in translocation is an ‘unlocked state’ of the pre‐translocation ribosome in which the P‐site tRNA adopts the P/E hybrid state, the L1 stalk domain closes and ribosomal subunits adopt a ratcheted configuration. Here, through two‐ and three‐colour smFRET imaging from multiple structural perspectives, EF‐G is shown to accelerate structural and kinetic pathways in the ribosome, leading to this transition. The EF‐G‐bound ribosome remains highly dynamic in nature, wherein, the unlocked state is transiently and reversibly formed. The P/E hybrid state is energetically favoured, but exchange with the classical P/P configuration persists; the L1 stalk adopts a fast dynamic mode characterized by rapid cycles of closure and opening. These data support a model in which P/E hybrid state formation, L1 stalk closure and subunit ratcheting are loosely coupled, independent processes that must converge to achieve the unlocked state. The highly dynamic nature of these motions, and their sensitivity to conformational and compositional changes in the ribosome, suggests that regulating the formation of this intermediate may present an effective avenue for translational control.     

Studies on ulcerative dermal necrosis of salmonids

Fourteen fresh run salmon Satmo salar L. with early extant lesions of ulcerative dermal necrosis (UDN) were kept in separate tanks and treated with zinc free malachite green. Ten of the fish were held at 10° C and 4 at 2° C. The treatment precluded infection with Saprolegnia fungus and allowed natural resolution of the lesions. There was a marked difference in rate of healing between warm and cold water conditions.
Histological examination of healing lesions at different stages showed that there was a primary invasion of cuboidal epithelium over the collagen scar followed by a phase of disorganized proliferation which eventually organized itself into normal epithelium. Melanocytes were very obvious in the dermis of healing lesions.     

Drought mildly reduces plant dominance in a temperate prairie ecosystem across years

1. Shifts in dominance and species reordering can occur in response to global change. However, it is not clear how altered precipitation and disturbance regimes interact to affect species composition and dominance.
2. We explored community‐level diversity and compositional similarity responses, both across and within years, to a manipulated precipitation gradient and annual clipping in a mixed‐grass prairie in Oklahoma, USA. We imposed seven precipitation treatments (five water exclusion levels [?20%, ?40%, ?60%, ?80%, and ?100%], water addition [+50%], and control [0% change in precipitation]) year‐round from 2016 to 2018 using fixed interception shelters. These treatments were crossed with annual clipping to mimic hay harvest.
3. We found that community‐level responses were influenced by precipitation across time. For instance, plant evenness was enhanced by extreme drought treatments, while plant richness was marginally promoted under increased precipitation.
4. Clipping promoted species gain resulting in greater richness within each experimental year. Across years, clipping effects further reduced the precipitation effects on community‐level responses (richness and evenness) at both extreme drought and added precipitation treatments.
5. Synthesis: Our results highlight the importance of studying interactive drivers of change both within versus across time. For instance, clipping attenuated community‐level responses to a gradient in precipitation, suggesting that management could buffer community‐level responses to drought. However, precipitation effects were mild and likely to accentuate over time to produce further community change.

     

Translational control during the acute phase response. Ferritin synthesis in response to interleukin-1.

Interleukin-1 (IL-1 beta) increases the synthesis of both heavy and light (L)-ferritin subunits when added to human hepatoma cells (HepG2) grown in culture. RNase protection and Northern blot analysis with L-ferritin probes revealed that no changes in L-ferritin mRNA levels occur after cytokine stimulation. However, the induction coincides with an increased association of the L-subunit mRNA with polyribosomes. Since the recruitment of stored ferritin mRNA onto polyribosomes is seen when iron enters the cell, the effect of IL-1 beta on iron uptake was tested and was found to be unaffected by the lymphokine. Neither transferrin receptor mRNA levels nor the number of receptors displayed on the cell surface was affected by IL-1 beta. However, the action of the cytokine on ferritin translation is inhibited by the action of the intracellular iron chelator deferoxamine. These data indicate that IL-1 beta induces ferritin gene expression by translational control of its mRNA. The pathway of induction is different from iron-dependent ferritin gene expression whereas regulation requires the background presence of cellular iron.     

The β-Hemolysin and Intracellular Survival of Streptococcus agalactiae in Human Macrophages

S. agalactiae (group B streptococci, GBS) is a major microbial pathogen in human neonates and causes invasive infections in pregnant women and immunocompromised individuals. The S. agalactiae β-hemolysin is regarded as an important virulence factor for the development of invasive disease. To examine the role of β-hemolysin in the interaction with professional phagocytes, the THP-1 monocytic cell line and human granulocytes were infected with a serotype Ia S. agalactiae wild type strain and its isogenic nonhemolytic mutant. We could show that the nonhemolytic mutants were able to survive in significantly higher numbers than the hemolytic wild type strain, in THP-1 macrophage-like cells and in assays with human granulocytes. Intracellular bacterial multiplication, however, could not be observed. The hemolytic wild type strain stimulated a significantly higher release of Tumor Necrosis Factor-α than the nonhemolytic mutant in THP-1 cells, while similar levels of the chemokine Interleukin-8 were induced. In order to investigate bacterial mediators of IL-8 release in this setting, purified cell wall preparations from both strains were tested and found to exert a potent proinflammatory stimulus on THP-1 cells. In conclusion, our results indicate that the β-hemolysin has a strong influence on the intracellular survival of S. agalactiae and that a tightly controlled regulation of β-hemolysin expression is required for the successful establishment of S. agalactiae in different host niches.     

Regulated nitrate transport in the cyanobacterium Anacystis nidulans.

Intracellular accumulation of nitrate, indicative of the operation of an active nitrate transport system, has been measured in intact cells of the cyanobacterium Anacystis nidulans. The ability of the cells to accumulate nitrate was effectively hindered by either ammonium addition or selective inhibition of CO2 fixation by DL-glyceraldehyde, with the effect of either compound being prevented by previously blocking ammonium assimilation. The results support the contention that nitrate utilization in cyanobacteria is regulated at the level of nitrate transport through the concerted action of ammonium assimilation and CO2 fixation.     

Identification of major common extracellular proteins secreted by Aeromonas salmonicida strains isolated from diseased fish.

Ten different strains of Aeromonas salmonicida that were isolated from diseased fish were grown under identical conditions (24 h at 25 degree C) in 3% (wt/vol) tryptone soya broth medium supplemented with vitamins and inorganic ions. In each case the extracellular proteins that were formed were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and it was found that there were two significant common components, one with a molecular weight of 70,000 and the other with a weight of 56,000. Application of enzyme purification techniques to the supernatant fraction proteins of a culture of one of the strains resulted in the isolation of a 70-kilodalton (kDa) component, which was found to be a serine protease, and a 56-kDa component, which was hemolytic to trout erythrocytes. Rocket immunoelectrophoresis with rabbit antibodies to the isolated protease and hemolysin showed the same antigenic components in the supernatant fractions of all the cultures. These activities were assayed, and protease activity was found to vary by a factor of three, from 59 to 195 U/ml, while the range of hemolytic activity was over a narrow band, from 28 to 43 U/ml. There was an inconsistency between the immunoelectrophoretic and direct assay data in only one case. This indicated the presence of additional hemolytic activity, in addition to the 56-kDa component. The detection of large amounts of the same protease and hemolysin, two potent degradative activities, in a random series of strains of A. salmonicida suggests that they may be obligatory virulence factors in the development of furunculosis.     

Determination of intracellular nitrate.

A sensitive procedure has been developed for the determination of intracellular nitrate. The method includes: (i) preparation of cell lysates in 2 M-H3PO4 after separation of cells from the outer medium by rapid centrifugation through a layer of silicone oil, and (ii) subsequent nitrate analysis by ion-exchange h.p.l.c. with, as mobile phase, a solution containing 50 mM-H3PO4 and 2% (v/v) tetrahydrofuran, adjusted to pH 1.9 with NaOH. The determination of nitrate is subjected to interference by chloride and sulphate when present in the samples at high concentrations. Nitrite also interferes, but it is easily eliminated by treatment of the samples with sulphamic acid. The method has been successfully applied to the study of nitrate transport in the unicellular cyanobacterium Anacystis nidulans.