Analysis of the interaction between single-chain variable fragments and their antigen in a reducing intracellular environment using the two-hybrid system

The coding sequences of three single-chain variable (scFv) fragments (A4, G4 and H3), which bind to dihydroflavonol-4-reductase (DFR) of Petunia hybrida, and the DFR-encoding sequence were cloned in two-hybrid vectors. The vectors were transformed in the yeast strain HF7c (his3-200, trp1-901, leu2-3) and the scFv-DFR interaction was analyzed by measuring yeast growth on medium without histidine. ScFv-G4 and, to a lesser extent, scFv-A4 could interact with DFR in the yeast nucleus. On the contrary, scFv-H3 showed no interaction with its antigen in yeast. The results of a previous expression analysis of the same scFv fragments in the plant cytosol correlate with those of the two-hybrid test. This suggests that it is possible to evaluate the antigen-scFv interaction in a reducing subcellular environment with the two-hybrid test. Therefore, the yeast two-hybrid system can be useful to identify candidate scFv fragments for intracellular antibody applications.     

A Comparison of Disease Burden in Rheumatoid Arthritis,Psoriatic Arthritis and Axial Spondyloarthritis

Objective

The main objective of this study was to compare disease burden in rheumatoid arthritis (RA), psoriatic arthritis (PsA) and axial spondyloarthritis (ax-SpA).

Methods

In this cross-sectional study, all the RA (1093), PsA (365) and ax-SpA (333) patients who visited the out-patient clinic of the Hospital of Southern Norway Trust during the year 2013 were included; the RA patients all had a RA diagnosis verified by the treating rheumatologist, the PsA patients all fulfilled the ClASsification for Psoriatic ARthritis (CASPAR) criteria and the ax-SpA patients all fulfilled the Assessment of SpondyloArthritis international Society (ASAS) classification criteria for ax-SpA. Patient-reported health status, demographic variables, medications, and composite scores of disease activity were assessed. The main analyses were performed using General Linear Models adjusted for age, sex and multiple comparisons. Correlation analyses were performed using Spearman’s rho.

Results

The reported pain, joint pain, patient’s global assessment and fatigue were similar in PsA and ax-SpA, but significantly lower in RA. The 28-joint Disease Activity Score (DAS28) (0.3±0.1, p = 0.003), Clinical Disease Activity Index (CDAI) (1.0±0.4, p = 0.028) and Routine Assessment of Patient Index Data 3 (RAPID3) (0.4±0.1, p = 0.004) were all significantly higher in PsA vs. RA. RAPID3 showed moderate to high correlation with DAS28 (rho = 0.521, p<0.001) and CDAI (rho = 0.768, p<0.001) in RA and PsA, and with Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) (rho = 0.902, p<0.001) and Bath Ankylosing Spondylitis Functional Index (BASFI) (0.865, p<0.001) in ax-SpA and PsA.

Conclusion

In conclusion, patient- reported outcome measures were similar in our population of PsA and ax-SpA patients, but significantly lower for the RA patients. Composite disease activity measures were lower in RA than in PsA and ax-SpA, but the magnitude of these differences was small and probably not of clinical significance. Our study indicates that disease burden in RA, PsA and ax-SpA may be more similar than previously demonstrated.     

LOVely enzymes – towards engineering light‐controllable biocatalysts

Light control over enzyme function represents a novel and exciting field of biocatalysis research. Blue-light photoreceptors of the L ight, O xygen, V oltage (LOV) family have recently been investigated for their applicability as photoactive switches. We discuss here the primary photochemical events leading to light activation of LOV domains as well as the proposed signal propagation mechanism to the respective effector domain. Furthermore, we describe the construction of LOV fusions to different effector domains, namely a dihydrofolate reductase from Escherichia coli and a lipase from Bacillus subtilis. Both fusion partners retained functionality, and alteration of enzyme activity by light was also demonstrated. Hence, it appears that fusion of LOV photoreceptors to functional enzyme target sites via appropriate linker structures may represent a straightforward strategy to design light controllable biocatalysts.     

Enantioselective kinetic resolution of phenylalkyl carboxylic acids using metagenome‐derived esterases

Enantiomerically pure β-arylalkyl carboxylic acids are important synthetic intermediates for the preparation of a wide range of compounds with biological and pharmacological activities. A library of 83 enzymes isolated from the metagenome was searched for activity in the hydrolysis of ethyl esters of three racemic phenylalkyl carboxylic acids by a microtiter plate-based screening using a pH-indicator assay. Out of these, 20 enzymes were found to be active and were subjected to analytical scale biocatalysis in order to determine their enantioselectivity. The most enantioselective and also enantiocomplementary biocatalysts were then used for preparative scale reactions. Thus, both enantiomers of each of the three phenylalkyl carboxylic acids studied could be obtained in excellent optical purity and high yields.     

Binding of human SLBP on the 3'-UTR of histone precursor H4-12 mRNA induces structural rearrangements that enable U7 snRNA anchoring

In metazoans, cell-cycle-dependent histones are produced from poly(A)-lacking mRNAs. The 3′ end of histone mRNAs is formed by an endonucleolytic cleavage of longer precursors between a conserved stem–loop structure and a purine-rich histone downstream element (HDE). The cleavage requires at least two trans-acting factors: the stem–loop binding protein (SLBP), which binds to the stem–loop and the U7 snRNP, which anchors to histone pre-mRNAs by annealing to the HDE. Using RNA structure-probing techniques, we determined the secondary structure of the 3′-untranslated region (3′-UTR) of mouse histone pre-mRNAs H4–12, H1t and H2a–614. Surprisingly, the HDE is embedded in hairpin structures and is therefore not easily accessible for U7 snRNP anchoring. Probing of the 3′-UTR in complex with SLBP revealed structural rearrangements leading to an overall opening of the structure especially at the level of the HDE. Electrophoretic mobility shift assays demonstrated that the SLBP-induced opening of HDE actually facilitates U7 snRNA anchoring on the histone H4–12 pre-mRNAs 3′ end. These results suggest that initial binding of the SLBP functions in making the HDE more accessible for U7 snRNA anchoring.     

Leukotriene C(4) (LTC(4)) does not share a cellular efflux mechanism with cGMP: characterisation of cGMP transport by uptake to inside-out vesicles from human erythrocytes

The transport of cGMP out of cells is energy requiring and has characteristics compatible with an ATP-energised anion pump. In the present study a model with inside-out vesicles from human erythrocytes was employed for further characterisation of the cGMP transporter. The uptake of leukotriene C(4) (LTC(4)), a substrate for multidrug resistance protein (MRP), was concentration-dependently inhibited by the leukotriene antagonist MK571 (IC(50)=110+/-20 nM), but cGMP was unable to inhibit LTC(4) uptake. Oxidised glutathione (GSSG) and glutathione S-conjugates caused a concentration-dependent inhibition of [(3)H]cGMP uptake with IC(50) of 2200+/-700 microM for GSSG, 410+/-210 microM for S-(p-nitrobenzyl)glutathione and 37+/-16 microM for S-decylglutathione, respectively. Antioxidants such as reduced glutathione and dithiothreitol did not influence transport for concentrations up to 100 microM, but both inhibited cGMP uptake with approx. 25% at 1 mM. The cGMP pump was sensitive to temperature without activity below 20 degrees C. The transport of cGMP was dependent on pH with maximal activity between pH 8.0 and 8.5. Calcium caused a concentration-dependent inhibition with IC(50) of 43+/-12 microM. Magnesium gave a marked activation in the range between 1 and 20 mM with maximum effect at 10 mM. The other divalent cations, Mn(2+) and Co(2+), were unable to substitute Mg(2+), but caused some activation at 1 mM. EDTA and EGTA stimulated cGMP transport concentration-dependently with 50% and 100% above control at 100 microM, respectively. The present study shows that the cGMP pump has properties compatible with an organic anion transport ATPase, without affinity for the MRP substrate LTC(4). However, the blockade of the cGMP transporter by glutathione S-conjugates suggests it is one of several GS-X pumps.     

Disulfide bond in Pseudomonas aeruginosa lipase stabilizes the structure but is not required for interaction with its foldase

Pseudomonas aeruginosa secretes a 29-kDa lipase which is dependent for folding on the presence of the lipase-specific foldase Lif. The lipase contains two cysteine residues which form an intramolecular disulfide bond. Variant lipases with either one or both cysteines replaced by serines showed severely reduced levels of extracellular lipase activity, indicating the importance of the disulfide bond for secretion of lipase through the outer membrane. Wild-type and variant lipase genes fused to the signal sequence of pectate lyase from Erwinia carotovora were expressed in Escherichia coli, denatured by treatment with urea, and subsequently refolded in vitro. Enzymatically active lipase was obtained irrespective of the presence or absence of the disulfide bond, suggesting that the disulfide bond is required neither for correct folding nor for the interaction with the lipase-specific foldase. However, cysteine-to-serine variants were more readily denatured by treatment at elevated temperatures and more susceptible to proteolytic degradation by cell lysates of P. aeruginosa. These results indicate a stabilizing function of the disulfide bond for the active conformation of lipase. This conclusion was supported by the finding that the disulfide bond function could partly be substituted by a salt bridge constructed by changing the two cysteine residues to arginine and aspartate, respectively.     

Homozygous mutation in SPATA16 is associated with male infertility in human globozoospermia

Globozoospermia is a rare (incidence <0.1% in male infertile patients) form of teratozoospermia, mainly characterized by round-headed spermatozoa that lack an acrosome. It originates from a disturbed spermiogenesis, which is expected to be induced by a genetic factor. Several family cases and recessive mouse models with the same phenotype support this expectation. In this study, we present a consanguineous family with three affected brothers, in whom we have identified a homozygous mutation in the spermatogenesis-specific gene SPATA16. This is the first example of a nonsyndromic male infertility condition in humans caused by an autosomal gene defect, and it could also mean that the identification of other partners like SPATA16 could elucidate acrosome formation.