Solution structure and backbone dynamics of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin mutant: a molecular analysis of its reduced thermal stability

No general strategy for thermostability has been yet established, because the extra stability of thermophiles appears to be the sum of different cumulative stabilizing interactions. In addition, the increase of conformational rigidity observed in many thermophilic proteins, which in some cases disappears when mesophilic and thermophilic proteins are compared at their respective physiological temperatures, suggests that evolutionary adaptation tends to maintain corresponding states with respect to conformational flexibility. In this study, we accomplished a structural analysis of the K18G/R82E Alicyclobacillus acidocaldarius thioredoxin (BacTrx) mutant, which has reduced heat resistance with respect to the thermostable wild-type. Furthermore, we have also achieved a detailed study, carried out at 25, 45, and 65 degrees C, of the backbone dynamics of both the BacTrx and its K18G/R82E mutant. Our findings clearly indicate that the insertion of the two mutations causes a loss of energetically favorable long-range interactions and renders the secondary structure elements of the double mutants more similar to those of the mesophilic Escherichia coli thioredoxin. Moreover, protein dynamics analysis shows that at room temperature the BacTrx, as well as the double mutant, are globally as rigid as the mesophilic thioredoxins; differently, at 65 degrees C, which is in the optimal growth temperature range of A. acidocaldarius, the wild-type retains its rigidity while the double mutant is characterized by a large increase of the amplitude of the internal motions. Finally, our research interestingly shows that fast motions on the pico- to nanosecond time scale are not detrimental to protein stability and provide an entropic stabilization of the native state. This study further confirms that protein thermostability is reached through diverse stabilizing interactions, which have the key role to maintain the structural folding stable and functional at the working temperature.     

Monocyte Scintigraphy in Rheumatoid Arthritis: The Dynamics of Monocyte Migration in Immune-Mediated Inflammatory Disease

Background

Macrophages are principal drivers of synovial inflammation in rheumatoid arthritis (RA), a prototype immune-mediated inflammatory disease. Conceivably, synovial macrophages are continuously replaced by circulating monocytes in RA. Animal studies from the 1960s suggested that macrophage replacement by monocytes is a slow process in chronic inflammatory lesions. Translation of these data into the human condition has been hampered by the lack of available techniques to analyze monocyte migration in man.

Methods/Principal Findings

We developed a technique that enabled us to analyze the migration of labelled autologous monocytes in RA patients using single photon emission computer tomography (SPECT). We isolated CD14+ monocytes by CliniMACS in 8 patients and labeled these with technetium-99m (^99mTc-HMPAO). Monocytes were re-infused into the same patient. Using SPECT we calculated that a very small but specific fraction of 3.4×10⁻³ (0.95−5.1×10⁻³) % of re-infused monocytes migrated to the inflamed joints, being detectable within one hour after re-infusion.

Conclusions/Significance

The results indicate monocytes migrate continuously into the inflamed synovial tissue of RA patients, but at a slow macrophage-replacement rate. This suggests that the rapid decrease in synovial macrophages that occurs after antirheumatic treatment might rather be explained by an alteration in macrophage retention than in monocyte influx and that RA might be particularly sensitive to treatments targeting inflammatory cell retention.     

3D 13C/1H NMR-based assignments for side-chain resonances of Lactobacillus casei dihydrofolate reductase. Evidence for similarities between the solution and crystal structures of the enzyme

Summary ¹³C-based three-dimensional ¹H–¹H correlation experiments have been used to determine essentially complete ¹³C and ¹H resonance assignments for the amino acid side chains of uniformly ¹³C/¹⁵N labelled L. casei dihydrofolate reductase in a complex with the drug methotrexate. Excellent agreement is observed between these assignments and an earlier set of partial assignments made on the basis of correlating nuclear Overhauser effect and crystal structure data, indicating that the tertiary structure of the enzyme is similar in solution and in the crystal state.To whom correspondence should be addressed.     

Reduced susceptibility to vancomycin and biofilm formation in methicillin-resistant Staphylococcus epidermidis isolated from blood cultures

This study aimed to correlate the presence of ica genes, biofilm formation and antimicrobial resistance in 107 strains of Staphylococcus epidermidis isolated from blood cultures. The isolates were analysed to determine their methicillin resistance, staphylococcal cassette chromosome mec (SCCmec) type, ica genes and biofilm formation and the vancomycin minimum inhibitory concentration (MIC) was measured for isolates and subpopulations growing on vancomycin screen agar. The mecA gene was detected in 81.3% of the S. epidermidis isolated and 48.2% carried SCCmec type III. The complete icaADBC operon was observed in 38.3% of the isolates; of these, 58.5% produced a biofilm. Furthermore, 47.7% of the isolates grew on vancomycin screen agar, with an increase in the MIC in 75.9% of the isolates. Determination of the MIC of subpopulations revealed that 64.7% had an MIC ≥ 4 μg mL^-1, including 15.7% with an MIC of 8 μg mL^-1 and 2% with an MIC of 16 μg mL^-1. The presence of the icaADBC operon, biofilm production and reduced susceptibility to vancomycin were associated with methicillin resistance. This study reveals a high level of methicillin resistance, biofilm formation and reduced susceptibility to vancomycin in subpopulations of S. epidermidis. These findings may explain the selection of multidrug-resistant isolates in hospital settings and the consequent failure of antimicrobial treatment.     

The mode of action of primycin

Primycin, an antibiotic active against Gram-positive microorganisms increased the permeability ofBacillus subtilis cell membranes when used in bacteriostatic concentrations. On addition of the antibiotic to the washed cell suspension, a dose-dependent increase in the conductivity was observed. Furthermore, an enhanced leakage of the nucleotides (measured by the³²P-ATP release from the³²P-labelled culture) could be detected.To get more information about the mechanism of the primycin-membrane interaction, the effect of the antibiotic on the ATPase activity of membrane vesicles prepared from bothBacillus subtilis andEscherichia coli B was studied. Activation was found at about 0.5 nmol antibiotic/g protein and its extent was approximately the same as with sonicated membranes used as controls. Stimulation of ATPase activity was also achieved with vesicles prewashed with 3 mM Tris-HCl buffer.Purified membrane ATPase fromBacillus subtilis could not be activated by primycin at all; above 0.3 nmol/g protein concentration the enzyme was inhibited. When acting on membrane vesicles isolated fromEscherichia coli B, inhibition without previous activation was observed, although sonication caused a substantial activation on the ATPase of these membranes.These observations confirmed our suggestion that the primary target of primycin action is the cell membrane in Gram-positive microorganisms.Abbreviations OD Optical density     

Characterization of "thyroliberin-deamidating enzyme" as a post-proline-cleaving enzyme. Partial purification and enzyme-chemical analysis of the enzyme from anterior pituitary tissue.

An enzyme which catalyzes the deamidation of thyroliberin (TRF; less than Glu-His-Pro-NH2) has been purified 110-fold from extracts of bovine anterior pituitary by ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and gel filtration. This enzyme of 76,000 molecular weight (as estimated by gel filtration) exhibits maximal activity at neutral pH (optimum pH 7.4 to 7.6) in buffers of high ionic strength supplemented with thiol-protecting agents. As indicated by the strong inhibition of the enzymatic activity by N-ethylmaleimide and Hg2+, as well as by the extreme sensitivity toward diisopropyl fluorophosphate, -SH, and -OH residues apparently represent essential functional groups of the enzyme. The stereospecific deamidation of TRF (Km = 4.1 . 10(-4) M) is inhibited competitively by TRF analogues which contain proline or by the proline containing biologically active peptides luliberin (LH-RF), oxytocin, vasopressin, angiotensin II, and Substance P. TRF analogues without proline or peptide amides without proline are ineffective. This enzyme cleaves the appropriate Pro-X bonds in luliberin, angiotensin II, pyroGlu-His-Pro-Gly-NH2, and the collagenase substrate Z-Gly-Pro-Leu-Gly-Pro. Thus, it may be characterized as a post-proline-cleaving enzyme.     

Comparative <Emphasis Type="Italic">in vivo</Emphasis> infection models yield insights on early host immune response to Campylobacter in chickens

Salmonella typhimurium and Campylobacter jejuni pose significant risks to human health and poultry are a major vector for infection. Comparative in vivo infection models were performed to compare the avian host immune response to both bacterial species. Forty-five commercial broiler chickens were orally challenged with either C. jejuni or S. typhimurium whilst 60 similar control birds were mock challenged in parallel. Birds were sacrificed at 0, 6, 20 and 48 h post-infection and cloacal swabs, blood and tissue samples taken. Peripheral blood leukocytes were isolated for flow cytometric analyses and RNA was extracted for gene expression profiling. Colonisation patterns were markedly different between the two bacterial species, with systemic colonisation of Campylobacter outside the gastrointestinal tract. Salmonella infection induced significant changes in circulating heterophil and monocyte/macrophage populations, whilst Campylobacter infection had no effect on the heterophil numbers but caused a significant early increase in circulating monocytes/macrophages. Toll-like receptor 1 (TLR1) gene expression was decreased, and avian β-defensin (AvBD) gene expression (AvBD3, AvBD10 and AvBD12) was significantly increased in response to Salmonella infection (P < 0.05). In contrast, Campylobacter infection induced increased TLR21 gene expression but significantly reduced expression of seven antimicrobial peptide (AMP) genes (AvBD3, AvBD4, AvBD8, AvBD13, AvBD14, CTHL2 and CTHL3; P < 0.05). Considered together, microbiological, cellular and gene expression profiles indicate that the innate immune system responds differently to Salmonella and to Campylobacter infection. Furthermore, reduction in the expression of AMPs may play a role in the persistence of high level colonisation of the host by Campylobacter. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Forager age and foraging state,but not cumulative foraging activity,affect biogenic amine receptor gene expression in the honeybee mushroom bodies

Foraging behavior is crucial for the development of a honeybee colony. Biogenic amines are key mediators of learning and the transition from in-hive tasks to foraging. Foragers vary considerably in their behavior, but whether and how this behavioral diversity depends on biogenic amines is not yet well understood. For example, forager age, cumulative foraging activity or foraging state may all be linked to biogenic amine signaling. Furthermore, expression levels may fluctuate depending on daytime. We tested if these intrinsic and extrinsic factors are linked to biogenic amine signaling by quantifying the expression of octopamine, dopamine and tyramine receptor genes in the mushroom bodies, important tissues for learning and memory. We found that older foragers had a significantly higher expression of Amdop1, Amdop2, AmoctαR1, and AmoctβR1 compared to younger foragers, whereas Amtar1 showed the opposite pattern. Surprisingly, our measures of cumulative foraging activity were not related to the expression of the same receptor genes in the mushroom bodies. Furthermore, we trained foragers to collect sucrose solution at a specific time of day and tested if the foraging state of time-trained foragers affected receptor gene expression. Bees engaged in foraging had a higher expression of Amdop1 and AmoctβR3/4 than inactive foragers. Finally, the expression of Amdop1, Amdop3, AmoctαR1, and Amtar1 also varied with daytime. Our results show that receptor gene expression in forager mushroom bodies is complex and depends on both intrinsic and extrinsic factors.