Crystal structure of S-ribosylhomocysteinase (LuxS) in complex with a catalytic 2-ketone intermediate

S-Ribosylhomocysteinase (LuxS) is an Fe(2+)-dependent metalloenzyme that catalyzes the cleavage of the thioether bond in S-ribosylhomocysteine (SRH) to produce homocysteine (Hcys) and 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of type II bacterial quorum-sensing molecule. The proposed mechanism involves an initial metal-catalyzed aldose-ketose isomerization reaction, which results in the migration of the ribose carbonyl group from its C1 to C2 position and the formation of a 2-ketone intermediate. A repetition of the isomerization reaction shifts the carbonyl group to the C3 position. Subsequent beta-elimination reaction at the C4 and C5 positions completes the catalytic cycle. In this work, a catalytically inactive mutant (C84A) of Co(2+)-substituted Bacillus subtilis LuxS was cocrystallized with the 2-ketone intermediate and the structure was determined to 1.8 A resolution. The structure reveals that the C2 carbonyl oxygen is directly coordinated with the metal ion, providing strong support for the proposed Lewis acid function of the metal ion during catalysis. Cys-84 and Glu-57 are optimally positioned to act as general acids/bases during the isomerization and elimination reactions. In addition, Ser-6, His-11, and Arg-39 are involved in substrate/ intermediate binding through hydrogen bonding interactions. The above conclusions are further confirmed by site-directed mutagenesis and visible absorption spectroscopic studies.     

Immunocytochemistry on scrape cytology in breast cancer: will it unearth the weaker positives?

OBJECTIVE: To standardize the technique of immunocytochemical (ICC) assessment of estrogen (ER) and progesterone receptor (PR) status in breast cancer by scrape cytology and to compare the results with immunohistochemistry on paraffin blocks. STUDY DESIGN: ICC assessment for ER and PR was done on scrape smears from tissue samples in 200 cases of primary breast cancer. The results were compared to those obtained from immunohistochemical (IHC) evaluation of formalin-fixed paraffin same tissue samples. RESULTS: ER/PR positivity rates as well as staining scores were compared between the scrape smears and tissue sections. The concordance between cytology and histology was 84% for ER and 90% for PR. Both the positivity rates and the staining intensity scores were higher for cytochemistry than for histochemistry. CONCLUSION: The ICC method on scrape smears is a simple test with rapid turnaround time. The sample required is small, and antigen loss due to fixation and processing is minimal. This new method gives a higher yield of hormone receptor positivity and, when used in conjunction with the IHC method, may improve the pickup rate of ER-positive cases, thereby playing an important role in risk stratification and therapeutic decision making in patients with breast cancer.     

Validation of theoretical framework explaining active solute uptake in dynamically loaded porous media

Solute transport in biological tissues is a fundamental process necessary for cell metabolism. In connective soft tissues, such as articular cartilage, cells are embedded within a dense extracellular matrix that hinders the transport of solutes. However, according to a recent theoretical study (Mauck et al., 2003, J. Biomech. Eng. 125, 602–614), the convective motion of a dynamically loaded porous solid matrix can also impart momentum to solutes, pumping them into the tissue and giving rise to concentrations which exceed those achived under passive diffusion alone. In this study, the theoretical predictions of this model are verified against experimental measurements. The mechanical and transport properties of an agarose–dextran model system were characterized from independent measurements and substituted into the theory to predict solute uptake or desorption under dynamic mechanical loading for various agarose concentrations and dextran molecular weights, as well as different boundary and initial conditions. In every tested case, agreement was observed between experiments and theoretical predictions as assessed by coefficients of determination ranging from R²=0.61 to 0.95. These results provide strong support for the hypothesis that dynamic loading of a deformable porous tissue can produce active transport of solutes via a pumping mechanisms mediated by momentum exchange between the solute and solid matrix.     

Activation of the NLRP3 inflammasome by intracellular poly I:C

Several RNA viruses can be detected by the inflammasome, which promotes IL-1β and IL-18 secretion, but the underlying mechanisms of detection remain unclear. Cytosolic dsRNA is a replication intermediate of many RNA viruses. We show here that transfection of the dsRNA analogue poly I:C activates the NLRP3 inflammasome via a pathway requiring endosomal acidification. This detection is independent of the other poly I:C sensors: TLR3 and MDA5. These results suggest a mechanism by which cytosolic dsRNA produced during viral infection could activate the NLRP3 inflammasome.     

Effect of Oxidation of αA- and αB-Crystallins on their Structure, Oligomerization and Chaperone Function

The purpose of this study was to investigate the effect of metal-catalyzed oxidation by H₂O₂ on the structure, oligomerization, and chaperone function of αA- and αB-crystallins. Recombinant αA-and αB-crystallins were prepared by expressing them in E. coli and purifying by size-exclusion chromatography. They were incubated with 1.5 mM H₂O₂ and 0.1 mM FeCl₃ at 37 ^∘C for 24 hrs and the reaction was stopped by adding catalase. Structural changes due to oxidation were ascertained by circular dichroism (CD) measurements and chaperone activity was assayed with alcohol dehydrogenase (ADH) and insulin as target proteins. The oligomeric nature of the oxidized proteins was assessed by molecular sieve HPLC. The secondary structure of the oxidized αA- and αB-crystallins has been substantially altered due to significant increase in random coils, in addition to decrease in β-sheet or α-helix contents. The tertiary structure also showed significant changes indicative of different mode of folding of the secondary structural elements. Chaperone function was significantly compromised as supported by nearly 50% loss in chaperone activity. Oxidation also resulted in the formation of higher molecular weight (HMW) proteins as well as lower molecular weight (LMW) proteins. Thus, oxidation leads to disintegration of the oligomeric structure of αA- and αB-crystallins. Chaperone activity of the HMW fraction is normal whereas the LMW fraction lacks any chaperone activity. So, it appears that the formation of the LMW proteins is the primary cause of the chaperone activity loss due to oxidation.     

Enzymatic esterification of starch using recovered coconut oil

Modification of maize and cassava starches was done using recovered coconut oil and microbial lipase. Microwave esterification was advantageous as it gave a DS 1.55 and 1.1 for maize starch and cassava starch, respectively. Solution state esterification of cassava starch for 36 h at 60 °C gave a DS of 0.08 and semi-solid state esterification gave a DS of 0.43. TGA and DSC studies showed that the higher DS attributed to the thermostability, since onset of decomposition is at a higher temperature (492 °C) than the unmodified (330 °C) and was stable above 600 °C. -Amylase digestibility and viscosity reduced for modified starch.     

Effect of DNA structural flexibility on promoter strength--molecular dynamics studies of E. coli promoter sequences

To study possible correlations between promoter activity and the structural flexibility of the DNA helix, we have carried out unrestrained molecular dynamics simulations of the -10 consensus region sequence and five variants forming the -10 region of various Escherichia coli promoter sequences. Analyses of the trajectories obtained from the simulations show that the consensus sequence has a pattern of two structurally flexible nucleotide steps sandwiched between two stiff steps. In the other sequences, this pattern varies in consonance with the change in the sequence. The variations in the patterns show correlation with the promoter strength.     

Zygomatic implant-retained fixed complete denture for an elderly patient

Dental rehabilitation of a completely edentulous geriatric patient has always been a challenge to the clinician, especially in treating those with higher expectations and demands. Treatment duration and the amount of residual alveolar bone available are often important considerations when planning for dental implant-based fixed treatment for these patients. With the introduction of zygomatic implants, a graftless alternative solution has emerged for deficient maxillary bone with provision for immediate loading. This article describes the treatment of a completely edentulous elderly patient using zygomatic implants in conjunction with conventional implants. The implants were immediately loaded using a definitive acrylic resin fixed denture reinforced with a cast metal framework, to provide function and aesthetics.     

Thermodynamic and kinetic analysis of an RNA kissing interaction and its resolution into an extended duplex

Kissing hairpin interactions form when the loop residues of two hairpins have Watson-Crick complementarity. In a unimolecular context, kissing interactions are important for tertiary folding and pseudoknot formation, whereas in a bimolecular context, they provide a basis for molecular recognition. In some cases, kissing complexes can be a prelude to strand displacement reactions where the two hairpins resolve to form a stable extended intermolecular duplex. The kinetics and thermodynamics of kissing-complex formation and their subsequent strand-displacement reactions are poorly understood. Here, biophysical techniques including isothermal titration calorimetry, surface plasmon resonance, and single-molecule fluorescence have been employed to probe the factors that govern the stability of kissing complexes and their subsequent structural rearrangements. We show that the general understanding of RNA duplex formation can be extended to kissing complexes but that kissing complexes display an unusual level of stability relative to simple duplexes of the same sequence. These interactions form and break many times at room temperature before becoming committed to a slow, irreversible forward transition to the strand-displaced form. Furthermore, using smFRET we show that the primary difference between stable and labile kissing complexes is based almost completely on their off rates. Both stable and labile complexes form at the same rate within error, but less stable species dissociate rapidly, allowing us to understand how these complexes can help generate specificity along a folding pathway or during a gene regulation event.