Quantitative Characterization of the Amount and Length of (1,3)-β-d-glucan for Functional and Mechanistic Analysis of Fungal (1,3)-β-d-glucan Synthase.

(1,3)-β-d-Glucan synthase (GS) is an essential enzyme for fungal cell wall biosynthesis that catalyzes the synthesis of (1,3)-β-d-glucan, a major and vital component of the cell wall. GS is a proven target of antifungal antibiotics including FDA-approved echinocandin derivatives; however, the function and mechanism of GS remain largely uncharacterized due to the absence of informative activity assays. Previously, a radioactive assay and reducing end modification have been used to characterize GS activity.

Immunological Aspects of Age-Related Macular Degeneration.

Increasing evidence over the past two decades points to a pivotal role for immune mechanisms in age-related macular degeneration (AMD) pathobiology. In this chapter, we will explore immunological aspects of AMD, with a specific focus on how immune mechanisms modulate clinical phenotypes of disease and severity and how components of the immune system may serve as triggers for disease progression in both dry and neovascular AMD. We will briefly review the biology of the immune system, defining the role of immune mechanisms in chronic degenerative disease and differentiating from immune responses to acute injury or infection.

Automated Recognition of Retinal Pigment Epithelium Cells on Limited Training Samples Using Neural Networks.

Purpose: To develop a neural network (NN)-based approach, with limited training resources, that identifies and counts the number of retinal pigment epithelium (RPE) cells in confocal microscopy images obtained from cell culture or mice RPE/choroid flat-mounts.

Methods: Training and testing dataset contained two image types: wild-type mice RPE/choroid flat-mounts and ARPE 19 cells, stained for Rhodamine-phalloidin, and imaged with confocal microscopy. After image preprocessing for denoising and contrast adjustment, scale-invariant feature transform descriptors were used for feature extraction.

Length Specificity and Polymerization Mechanism of (1,3)-β-d-Glucan Synthase in Fungal Cell Wall Biosynthesis.

(1,3)-β-d-Glucan synthase (GS) catalyzes formation of the linear (1,3)-β-d-glucan in the fungal cell wall and is a target of clinically approved antifungal antibiotics. The catalytic subunit of GS, FKS protein, does not exhibit significant sequence homology to other glycosyltransferases, and thus, significant ambiguity about its catalytic mechanism remains. One of the major technical barriers in studying GS is the absence of activity assay methods that allow characterization of the lengths and amounts of (1,3)-β-d-glucan due to its poor solubility in water and organic solvents.

Engineering of New Pneumocandin Side-Chain Analogues from Glarea lozoyensis by Mutasynthesis and Evaluation of Their Antifungal Activity.

Pneumocandins are lipohexapeptides of the echinocandin family that inhibit fungal 1,3-β-glucan synthase. Most of the pathway steps have been identified previously. However, the lipoinitiation reaction has not yet been experimentally verified.

Identification of a cyclic nucleotide as a cryptic intermediate in molybdenum cofactor biosynthesis.

The molybdenum cofactor (Moco) is a redox cofactor found in all kingdoms of life, and its biosynthesis is essential for survival of many organisms, including humans. The first step of Moco biosynthesis is a unique transformation of guanosine 5'-triphosphate (GTP) into cyclic pyranopterin monophosphate (cPMP). In bacteria, MoaA and MoaC catalyze this transformation, although the specific functions of these enzymes were not fully understood.

Atomic force microscopy captures folded ribosome bound nascent chains.

Direct visualization of co-translational folding of nascent polypeptide chains is challenging. Here we present, for the first time, AFM images of large protein constructs based on the membrane binding domain of ankyrin-R, complexed with the ribosome. The characteristic "horse-shoe" shape of ankyrin-R emerging from the ribosome was captured.

Vortex-induced formation of insulin amyloid superstructures probed by time-lapse atomic force microscopy and circular dichroism spectroscopy.

Misfolding and aggregation of proteins are multipathway processes that result in polymorphism of amyloid fibrils. While agitation is one of the most common means of inducing structural variants of fibrils (the so-called 'amyloid strains'), there is as yet no mechanistic explanation for this effect. In this study, time-lapse atomic force microscopy has been employed to probe insulin fibrillation upon intensive agitation.

Noncooperative dimethyl sulfoxide-induced dissection of insulin fibrils: toward soluble building blocks of amyloid.

The enormous molecular weight complicates detailed structural studies of amyloid fibrils and obscures identification of biologically active forms of protein aggregates in amyloid-related diseases. Here we show that aqueous solutions of dimethyl sulfoxide (DMSO) solubilize insulin fibrils while maintaining their beta-pleated structure. This is accompanied by a marked decrease in the fluorescence of thioflavin T.

Detecting solvent-driven transitions of poly(A) to double-stranded conformations by atomic force microscopy.

We report the results of direct measurements by atomic force microscopy of solvent-driven structural transitions within polyadenylic acid (poly(A)). Both atomic force microscopy imaging and pulling measurements reveal complex strand arrangements within poly(A) induced by acidic pH conditions, with a clear fraction of double-stranded molecules that increases as pH decreases. Among these complex structures, force spectroscopy identified molecules that, upon stretching, displayed two distinct plateau features in the force-extension curves.

De novo refolding and aggregation of insulin in a nonaqueous environment: an inside out protein remake.

While thermodynamic penalties associated with protein-water interactions are the key driving force of folding, perturbed hydration of destabilized protein molecules may trigger aggregation, which in vivo often causes cellular and histological damage. Here we show, that the denatured state of an alpha-helical protein, insulin, converts to a non-native beta-sheet-rich structure upon de novo "refolding" in an anhydrous environment. The beta-pleated conformer precipitates from solutions of DMSO-denatured insulin upon dilution with chloroform.

Chiral bifurcation in aggregating insulin: an induced circular dichroism study.

The structural unambiguity of folding is lost when disordered protein molecules convert into beta-sheet-rich fibrils. The resulting polymorphism of protein aggregates has been studied in the context of its biomedical consequences. Events underlying the conformational variance of amyloid fibrils, as well as physicochemical boundaries between folding and misfolding pathways, remain obscure.

Tyrosine side chains as an electrochemical probe of stacked beta-sheet protein conformations.

The in vivo formation of beta-pleated protein aggregates underlies a number of fatal neurodegenerative disorders, such as Alzheimer disease. Since molecular mechanisms of protein misfolding and aggregation remain poorly understood, this has been calling for many diverse biophysical tools capable of addressing different dynamic and conformational aspects of the phenomenon. The two model polypeptides used in this study are poly(l-tyrosine) and insulin.

Conformational indeterminism in protein misfolding: chiral amplification on amyloidogenic pathway of insulin.

Unlike folding, protein aggregation is a multipathway, kinetically controlled process yielding different conformations of fibrils. The dynamics and determinism/indeterminism boundaries of misfolded conformations remain obscure. Here we show that, upon vortexing, insulin forms two distinct types of fibrils with opposite local chiral preferences, which manifest in the opposite twists of bound dye, thioflavin T.

New insights into the self-assembly of insulin amyloid fibrils: an H-D exchange FT-IR study.

The solvent protection of the amide backbone in bovine insulin fibrils was studied by FT-IR spectroscopy. In the mature fibrils, approximately 85 +/- 2% of amide protons are protected. Of those "trapped" protons, a further 25 +/- 2 or 35 +/- 2% is H-D exchanged after incubation for 1 h at 1 GPa and 25 degrees C or 0.

Ethanol-perturbed amyloidogenic self-assembly of insulin: looking for origins of amyloid strains.

A model cosolvent, ethanol, has profound and diversified effects on the amyloidogenic self-assembly of insulin, yielding spectroscopically and morphologically distinguishable forms of beta-aggregates. The alcohol reduces hydrodynamic radii of insulin molecules, decreases enthalpic costs associated with aggregation-prone intermediate states, and accelerates the aggregation itself. Increasing the concentration of the cosolvent promotes curved, amorphous, and finally donut-shaped forms.

Template-controlled conformational patterns of insulin fibrillar self-assembly reflect history of solvation of the amyloid nuclei.

In the presence of ethanol, insulin forms amyloid morphologically distinct from the ambient specimen. Due to stability of fibrils and the autocatalytic character of the process, the two amyloid templates, when seeded, replicate the initial morphologies (and inter-beta-strand hydrogen bonding patterns) regardless of the environmental biases, such as the cosolvent presence. Such "templated memory" effect is advantageous in synthesizing structurally uniform protein nanofibrils under conditions favoring alternative "wild" forms.