The cytoplasmic cyclophilin from <Emphasis Type="Italic">Azotobacter vinelandii</Emphasis> interacts with phosphate acetyltransferase isoforms enhancing their in vitro activity

Cyclophilins belong to the peptidyl-prolyl cis/trans isomerase family of enzymes (EC 5.2.1.8), which accelerate protein folding by catalysing the cis/trans isomerisation of proline imidic peptide bonds. In the present study, by a combination of bioinformatics methods, we identify phosphate acetyltransferase isoforms, AvPTA-1 and AvPTA-2, as potential interacting partners of AvPPIB, the cytoplasmic cyclophilin from Azotobacter vinelandii, and demonstrate their physical interaction by co-expression studies. A decrease in AvPPIB PPIase activity, in the presence of AvPTA-1 or AvPTA-2, further confirms each interaction. Phosphate acetyltransferases (EC 2.3.1.8) catalyse the reversible transfer of the acetyl group from acetyl-P to CoA, forming acetyl-CoA and inorganic phosphate. We examined the effect of AvPPIB on the enzymatic activity of both phosphate acetyltransferase isoforms, and noticed an enhancement of the activity, as well as an alteration of the K _m of each isoform, for the reaction substrates, indicating a possible function of AvPPIB in phosphate acetyltransferase activity modulation. Although PPIase activity seems not to be essential for these interactions, since AvPPIB_F99A active site mutant still interacts with both isoforms, it is responsible for the observed phosphate acetyltransferase activity enhancement as AvPPIB_F99A enhanced to a significantly lower extent the phosphate acetyltransferase activity of both isoforms compared with AvPPIB.     

Normal bone turnover markers in a patient with active Paget's disease of bone: response to treatment with zoledronic acid

The treatment of Paget's disease of bone (PDB) aims at the suppression of abnormal bone turnover; bisphosphonates are currently the treatment of choice. Indications for antiresorptive treatment in symptomatic patients with PDB include bone or joint pain, neurological complications, surgery planned at an active pagetic site and hypercalcaemia from immobilisation. The goals of antiresorptive treatment are clinical improvement and biochemical remission, as assessed by the normalisation of bone turnover markers. Clinical deterioration, especially bone pain, should be considered before deciding to treat patients with late sclerotic (burned-out) PDB. Bone scintigraphy may be of importance in these patients, because it depicts increased osteoblastic activity, when bone markers may not. We present a case of late sclerotic PDB with clinical deterioration but normal bone turnover markers, who experienced significant clinical improvement after treatment with zoledronic acid.     

Interactions between subunits of Saccharomyces cerevisiae RNase MRP support a conserved eukaryotic RNase P/MRP architecture

Ribonuclease MRP is an endonuclease, related to RNase P, which functions in eukaryotic pre-rRNA processing. In Saccharomyces cerevisiae, RNase MRP comprises an RNA subunit and ten proteins. To improve our understanding of subunit roles and enzyme architecture, we have examined protein-protein and protein–RNA interactions in vitro, complementing existing yeast two-hybrid data. In total, 31 direct protein–protein interactions were identified, each protein interacting with at least three others. Furthermore, seven proteins self-interact, four strongly, pointing to subunit multiplicity in the holoenzyme. Six protein subunits interact directly with MRP RNA and four with pre-rRNA. A comparative analysis with existing data for the yeast and human RNase P/MRP systems enables confident identification of Pop1p, Pop4p and Rpp1p as subunits that lie at the enzyme core, with probable addition of Pop5p and Pop3p. Rmp1p is confirmed as an integral subunit, presumably associating preferentially with RNase MRP, rather than RNase P, via interactions with Snm1p and MRP RNA. Snm1p and Rmp1p may act together to assist enzyme specificity, though roles in substrate binding are also indicated for Pop4p and Pop6p. The results provide further evidence of a conserved eukaryotic RNase P/MRP architecture and provide a strong basis for studies of enzyme assembly and subunit function.     

Cortical actin filament organization in developing and functioning stomatal complexes of Zea mays and Triticum turgidum

Cortical actin filament (AF) organization was studied in detail in developing stomatal complexes of the grasses Zea mays and Triticum turgidum. AF arrays during the whole stomatal complex development are dynamic, partly following the pattern of cortical microtubule (MT) organization. They also exhibit particular patterns of organization, spatially and temporarily restricted. Among AF arrays, the radial ones that underlie young guard cell (GC) periclinal walls, those that line the bulbous GC ends and the AF ring at the junction between subsidiary cells (SCs) and GCs are described here for the first time. Although many similarities in cortical AF organization exist among the stomatal cells of both plants studied, considerable differences have also been observed between them. Our data reveal that the expanding areas of stomatal cell walls are lined by distinct cortical AF aggregations that probably protect the plasmalemma against mechanical stresses. Experimental AF disruption does not seem to affect detectably stomatal cell morphogenesis. Moreover, the structural and experimental data of this study revealed that, in contrast to the elliptical stomata, in the dumbbell-shaped ones the AFs and MTs seem not to be involved in the mechanism of opening and closing of the stomatal pore.     

Resin-acid derivatives bind to multiple sites on the voltage-sensor domain of the Shaker potassium channel

Voltage-gated potassium (K_V) channels can be opened by negatively charged resin acids and their derivatives. These resin acids have been proposed to attract the positively charged voltage-sensor helix (S4) toward the extracellular side of the membrane by binding to a pocket located between the lipid-facing extracellular ends of the transmembrane segments S3 and S4. By contrast to this proposed mechanism, neutralization of the top gating charge of the Shaker K_V channel increased resin-acid–induced opening, suggesting other mechanisms and sites of action. Here, we explore the binding of two resin-acid derivatives, Wu50 and Wu161, to the activated/open state of the Shaker K_V channel by a combination of in silico docking, molecular dynamics simulations, and electrophysiology of mutated channels. We identified three potential resin-acid–binding sites around S4: (1) the S3/S4 site previously suggested, in which positively charged residues introduced at the top of S4 are critical to keep the compound bound, (2) a site in the cleft between S4 and the pore domain (S4/pore site), in which a tryptophan at the top of S6 and the top gating charge of S4 keeps the compound bound, and (3) a site located on the extracellular side of the voltage-sensor domain, in a cleft formed by S1–S4 (the top-VSD site). The multiple binding sites around S4 and the anticipated helical-screw motion of the helix during activation make the effect of resin-acid derivatives on channel function intricate. The propensity of a specific resin acid to activate and open a voltage-gated channel likely depends on its exact binding dynamics and the types of interactions it can form with the protein in a state-specific manner.     

Wideband Optical Detector of Ultrasound for Medical Imaging Applications

Optical sensors of ultrasound are a promising alternative to piezoelectric techniques, as has been recently demonstrated in the field of optoacoustic imaging. In medical applications, one of the major limitations of optical sensing technology is its susceptibility to environmental conditions, e.g. changes in pressure and temperature, which may saturate the detection. Additionally, the clinical environment often imposes stringent limits on the size and robustness of the sensor. In this work, the combination of pulse interferometry and fiber-based optical sensing is demonstrated for ultrasound detection. Pulse interferometry enables robust performance of the readout system in the presence of rapid variations in the environmental conditions, whereas the use of all-fiber technology leads to a mechanically flexible sensing element compatible with highly demanding medical applications such as intravascular imaging. In order to achieve a short sensor length, a pi-phase-shifted fiber Bragg grating is used, which acts as a resonator trapping light over an effective length of 350 µm. To enable high bandwidth, the sensor is used for sideway detection of ultrasound, which is highly beneficial in circumferential imaging geometries such as intravascular imaging. An optoacoustic imaging setup is used to determine the response of the sensor for acoustic point sources at different positions.     

IgE immunotherapy: A novel concept with promise for the treatment of cancer

The importance of antibodies in activating immune responses against tumors is now better appreciated with the emergence of checkpoint blockade antibodies and with engineered antibody Fc domains featuring enhanced capacity to focus potent effector cells against cancer cells. Antibodies designed with Fc regions of the IgE class can confer natural, potent, long-lived immune surveillance in tissues through tenacious engagement of high-affinity cognate Fc receptors on distinct, often tumor-resident immune effector cells, and through ability to activate these cells under tumor-induced Th2-biased conditions. Here, we review the properties that make IgE a contributor to the allergic response and a critical player in the protection against parasites, which also support IgE as a novel anti-cancer modality. We discuss IgE-based active and passive immunotherapeutic approaches in disparate in vitro and in vivo model systems, collectively suggesting the potential of IgE immunotherapies in oncology. Translation toward clinical application is now in progress.     

Comparative reactivity of human IgE to cynomolgus monkey and human effector cells and effects on IgE effector cell potency

Background: Due to genetic similarities with humans, primates of the macaque genus such as the cynomolgus monkey are often chosen as models for toxicology studies of antibody therapies. IgE therapeutics in development depend upon engagement with the FcεRI and FcεRII receptors on immune effector cells for their function. Only limited knowledge of the primate IgE immune system is available to inform the choice of models for mechanistic and safety evaluations.   Methods: The recognition of human IgE by peripheral blood lymphocytes from cynomolgus monkey and man was compared. We used effector cells from each species in ex vivo affinity, dose-response, antibody-receptor dissociation and potency assays. Results: We report cross-reactivity of human IgE Fc with cynomolgus monkey cells, and comparable binding kinetics to peripheral blood lymphocytes from both species. In competition and dissociation assays, however, human IgE dissociated faster from cynomolgus monkey compared with human effector cells. Differences in association and dissociation kinetics were reflected in effector cell potency assays of IgE-mediated target cell killing, with higher concentrations of human IgE needed to elicit effector response in the cynomolgus monkey system. Additionally, human IgE binding on immune effector cells yielded significantly different cytokine release profiles in each species. Conclusion: These data suggest that human IgE binds with different characteristics to human and cynomolgus monkey IgE effector cells. This is likely to affect the potency of IgE effector functions in these two species, and so has relevance for the selection of biologically-relevant model systems when designing pre-clinical toxicology and functional studies.