Get Phases from Arsenic Anomalous Scattering: de novo SAD Phasing of Two Protein Structures Crystallized in Cacodylate Buffer

The crystal structures of two proteins, a putative pyrazinamidase/nicotinamidase from the dental pathogen Streptococcus mutans (SmPncA) and the human caspase-6 (Casp6), were solved by de novo arsenic single-wavelength anomalous diffraction (As-SAD) phasing method. Arsenic (As), an uncommonly used element in SAD phasing, was covalently introduced into proteins by cacodylic acid, the buffering agent in the crystallization reservoirs. In SmPncA, the only cysteine was bound to dimethylarsinoyl, which is a pentavalent arsenic group (As (V)). This arsenic atom and a protein-bound zinc atom both generated anomalous signals. The predominant contribution, however, was from the As anomalous signals, which were sufficient to phase the SmPncA structure alone. In Casp6, four cysteines were found to bind cacodyl, a trivalent arsenic group (As (III)), in the presence of the reducing agent, dithiothreitol (DTT), and arsenic atoms were the only anomalous scatterers for SAD phasing. Analyses and discussion of these two As-SAD phasing examples and comparison of As with other traditional heavy atoms that generate anomalous signals, together with a few arsenic-based de novo phasing cases reported previously strongly suggest that As is an ideal anomalous scatterer for SAD phasing in protein crystallography

A prospective, randomised comparison of single and three piece acrylic foldable intraocular lenses

Aims: To compare the postoperative performance of single and three piece acrylic foldable intraocular lenses (IOLs). Methods: 20 patients underwent bilateral cataract surgery with a single piece SA30AL IOL in one eye and a three piece MA30BA IOL in the other eye. The eyes were randomly assigned to either a single or three piece lens. The amount of IOL decentration and tilt, area of anterior capsule opening, and degree of posterior capsule opacification were measured using the Scheimpflug anterior segment analysis system (Nidek EAS-1000). Visual acuity and contrast sensitivity were examined. Measurements were performed by masked examiners before and 1 day, 1 week, 1, 3, 6, and 18 months after surgery. Results: There were no significant differences between the two groups (p>0.05, paired t test) in the amount of IOL decentration, IOL tilt, area of anterior capsule opening, degree of posterior capsule opacification, best corrected visual acuity, and contrast sensitivity throughout the 18 month follow up period. Conclusion: The single and three piece acrylic foldable IOLs are equally stable in the eye after surgery

Inhibitors of lysosomal function or serum starvation in control or LAMP2 deficient cells do not modify the cellular levels of Parkinson disease-associated DJ-1/PARK 7 protein

<div><p>Mutations in <i>PARK7/DJ-1</i> gene are associated with familial autosomal recessive Parkinson disease. Recently, lysosomes and chaperone mediated autophagy (CMA) has been reported to participate in the degradation of DJ-1/PARK7 protein. Lamp-2A isoform is considered as the lysosomal receptor for the uptake of proteins being degraded by the CMA pathway. We have used several cell lines with disrupted <i>LAMP2</i> gene expression and their respective control cells to test the possible role of lysosomal degradation and in particular CMA in DJ-1 /PARK7 degradation. Interruption of LAMP-2 expression did not result in an increase of the steady-state protein levels of DJ-1 /PARK7, as it would have been expected. Furthermore, no change in DJ-1 /PARK7 protein levels were observed upon inhibition of lysosomal function with NH₄Cl or NH₄Cl plus leupeptin, or after activation of CMA by serum starvation for 24h. Accordingly, we have not found any evidence that DJ-1 /PARK7 protein levels are regulated via lysosomal degradation or the CMA pathway.</p></div

Full-length p40(phox) structure suggests a basis for regulation mechanism of its membrane binding

The superoxide-producing phagocyte NADPH oxidase is activated during phagocytosis to destroy ingested microbes. The adaptor protein p40(phox) associates via the PB1 domain with the essential oxidase activator p67(phox), and is considered to function by recruiting p67(phox) to phagosomes; in this process, the PX domain of p40(phox) binds to phosphatidylinositol 3-phosphate [PtdIns(3)P], a lipid abundant in the phagosomal membrane. Here we show that the PtdIns(3)P-binding activity of p40(phox) is normally inhibited by the PB1 domain both in vivo and in vitro. The crystal structure of the full-length p40(phox) reveals that the inhibition is mediated via intramolecular interaction between the PB1 and PX domains. The interface of the p40(phox) PB1 domain for the PX domain localizes on the opposite side of that for the p67(phox) PB1 domain, and thus the PB1-mediated PX regulation occurs without preventing the PB1–PB1 association with p67(phox)