Protein conformational diseases, including Alzheimer's, Huntington's, and Parkinson's diseases, result from protein misfolding, giving a distinct fibrillar feature termed amyloid. Recent studies show that only the globular (not fibrillar) conformation of amyloid proteins is sufficient to induce cellular pathophysiology. However, the 3D structural conformations of these globular structures, a key missing link in designing effective prevention and treatment, remain undefined as of yet. By using atomic force microscopy, circular dichroism, gel electrophoresis, and electrophysiological recordings, we show here that an array of amyloid molecules, including amyloid-beta(1-40), alpha-synuclein, ABri, ADan, serum amyloid A, and amylin undergo supramolecular conformational change. In reconstituted membranes, they form morphologically compatible ion-channel-like structures and elicit single ion-channel currents. These ion channels would destabilize cellular ionic homeostasis and hence induce cell pathophysiology and degeneration in amyloid diseases.

A pathological hallmark in brain tissue from patients with Alzheimer's disease (AD) 1 is the accumulation of amyloid ␤ protein (A␤P), a 39 -43-amino acid-long polypeptide, as morphologically heterogeneous neuritic plaques and cerebrovascular deposits (1, 2). A␤P is derived primarily from a proteolytic cleavage of the ␤-amyloid precursor protein (A␤PP), a highly conserved and widely expressed integral membrane protein with a single membrane-spanning polypeptide. The amount and the nature of polypeptides vary considerably among various forms of ADs: A␤P 1-40 and A␤P 1-42 are differentially accumulated in sporadic Alzheimer's disease and non-demented brain samples (3) and a mutation in presenilins is linked with an increased ratio of A␤P 1-42 /A␤P 1-40 in familial Alzheimer's disease (4 -7). The early-onset familial AD has been correlated with an increased level of A␤P 1-42 . However, very little is known about the role of A␤P 1-42 in such pathology and about the mechanism(s) of its action.Accumulating evidence suggests an early and causative role of A␤Ps in the pathogenic cascade (8 -11). Postulated mechanisms of A␤P toxicity include, by its interaction with the tachykinin neuropeptide system, a surface membrane effect (12); by changing cellular ionic concentration via formation of plasma membrane channels (13-15); and by activating oxidative pathways and making cells more responsive to oxidative stress (for review see Refs. 16 and 17). Reactive oxygen species and the antioxidant defenses work probably by altering the lipid peroxidation and membrane composition. However, A␤P polypeptides associated with the reactive oxygen hypothesis have produced conflicting effects on cytoskeletal organization and cell lysis (18 -23).The commonly observed change in the cellular ion concentration involves increased calcium level (24 -26) either indirectly via modulating the existing Ca 2ϩ channel or directly via cation-selective channels formed by A␤Ps. Support for the cation-selective A␤P channels are accumulating. Arispe and his collaborators (13-15, 27) have reported cation-selective channels formed by A␤P 1-40 when reconstituted into lipid bilayers and in the membrane patches excised from hypothalamic gonadotropin-releasing hormone neurons. Kagan and his collaborators (28) have also recorded channel-like activity when A␤P [25][26][27][28][29][30][31][32][33][34][35] was reconstituted in lipid bilayers as well as for both A␤P 1-40 and A␤P 1-42 reconstituted in lipid bilayer, 2 though, with less reliability and reproducibility than the A␤P 25-35 current (28). Whether A␤P 1-42 toxicity is also mediated via A␤P 1-42 forming calcium-permeable ion channel is unclear.The molecular structure of A␤P oligomers, especially as an ion channel, is unknown. Durell et al. (29) have developed theoretical models for the structure of ion channel formed by the membrane-bound A␤P 1-40 . However, no direct structural data from EM, NMR, x-ray diffraction, or other microscopic techniques are available to support the presence of the A␤P channel.We h...

Microcontact printing is a remarkable surface patterning technique. Developed about 10 years ago, it has triggered enormous interest from the surface science community, as well as from engineers and biologists. The last five years have been rich in improvements to the microcontact printing process itself, as well as in new technical innovations, many designed to suit new applications. In this review, we describe the evolution of microcontact printing over the past five years. The review is categorized into three main sections: the improvements made to the technique, new variations, and new applications.

Alzheimer's disease (AD) is a protein misfolding disease. Early hypothesis of AD pathology posits that 39-43 AA long misfolded amyloid beta (Abeta) peptide forms a fibrillar structure and induces pathophysiological response by destabilizing cellular ionic homeostasis. Loss of cell ionic homeostasis is believed to be either indirectly due to amyloid beta-induced oxidative stress or directly by its interaction with the cell membrane and/or activating pathways for ion exchange. Significantly though, no Abeta specific cell membrane receptors are known and oxidative stress mediated pathology is only partial and indirect. Most importantly, recent studies strongly indicate that amyloid fibrils may not by themselves cause AD pathology. Subsequently, a competing hypothesis has been proposed wherein amyloid derived diffusible ligands (ADDLs) that are large Abeta oligomers (approximately >60 kDa), mediate AD pathology. No structural details, however, of these large globular units exist nor is there any known suitable mechanism by which they would induce AD pathology. Experimental data indicate that they alter cell viability by non-specifically changing the plasma membrane stability and increasing the overall ionic leakiness. The relevance of this non-specific mechanism for AD-specific pathology seems limited. Here, we provide a viable new paradigm: AD pathology mediated by amyloid ion channels made of small Abeta oligomers (trimers to octamers). This review is focused to 3D structural analysis of the Abeta channel. The presence of amyloid channels is consistent with electrophysiological and cell biology studies summarized in companion reviews in this special issue. They show ion channel-like activity and channel-mediated cell toxicity. Amyloid ion channels with defined gating and pharmacological agents would provide a tangible target for designing therapeutics for AD pathology.

Hemichannels in the overlapping regions of apposing cells plasma membranes join to form gap junctions and provide an intercellular communication pathway. Hemichannels are also present in the nonjunctional regions of individual cells and their activity is gated by several agents, including calcium. However, their physiological roles are unknown. Using techniques of atomic force microscopy (AFM), fluorescent dye uptake assay, and laser confocal immunofluorescence imaging, we have examined the extracellular calcium-dependent modulation of cell volume. In response to a change in the extracellular physiological calcium concentration (1.8 to ≤1.6 mM) in an otherwise isosmotic condition, real-time AFM imaging revealed a significant and reversible increase in the volume of cells expressing gap-junctional proteins (connexins). Volume change did not occur in cells that were not expressing connexins. However, after the transient or stable transfection of connexin43, volume change did occur. The volume increase was accompanied by cytochalasin D-sensitive higher cell stiffness, which helped maintain cell integrity. These cellular physical changes were prevented by gap-junctional blockers, oleamide and β-glycyrrhetinic acid, or were reversed by returning extracellular calcium to the normal level. We conclude that nongap-junctional hemichannels regulate cell volume in response to the change in extracellular physiological calcium in an otherwise isosmotic situation.

Indirect information on the conformation of highly charged molecular ions may be obtained by monitoring their collisional cross sections and the course of simple gas-phase reactions such as hydrogen-deuterium exchange. In this work, another indirect but more visually oriented approach is explored: electrosprayed protein ions are accelerated toward a highly oriented pyrolytic graphite surface and the resulting single-ion defects are imaged by scanning force and tunneling microscopy. All protein impacts generated shallow hillocks: the shapes depended on the identity and charge state of the incident protein. Lysozyme and myoglobin, both compact, globular proteins in the native state, produced compact, almost circular hillocks. However, hillocks generated by myoglobin that had been denatured in the solution phase were elongated, and the elongation was positively correlated with the charge state of the ion. It appears that structural information about gas-phase multiply charged proteins can be derived from imprints generated by energetic protein impacts on surfaces.

Cisplatin is the most effective cytotoxic agent against many cancers. Its usage, however, is limited due to inefficient uptake by the target cells. A liposomal formulation of cisplatin is reported to partly overcome this limitation. Physicochemical characteristics of the liposome-cisplatin preparation, including its size, stability, encapsulation efficiency, and cytoplasmic internalization efficiency, play a significant role in an effective usage of liposomal formulations. We have used atomic force microscopy (AFM) to determine physicochemical characteristics of cisplatin-encapsulated liposomes, AFM and fluorescence microscopy to examine their cytoplasmic internalization, and Live/Dead assay to examine their cell toxicity. Nonencapsulated cisplatin is globular and 10-50 nm in size. AFM force-dissection and stiffness measurements show that cisplatin-encapsulated liposomes are significantly stiffer ( approximately 100%) and more stable than liposomes without encapsulated cisplatin. Cisplatin-encapsulated liposomes of approximately 250 nm diameter (nanoliposomes) are most efficiently internalized and induce cell toxicity in a time-dependent manner. Liposomes without cisplatin of similar dimensions, although internalized in the cell cytoplasm, do not induce cell toxicity.

We present new results from an energetic surface imprinting method which allows us to outline the general conformation of protein ions in vacuo. Both disulfide-bond-intact and disulfide-bond-reduced gas-phase lysozyme ions were produced by electrospray ionization and were accelerated and impacted onto graphite surfaces. The resulting surface defects, each created by a single incident ion, were imaged with scanning force microscopy. Disulfide-intact lysozyme ions created compact, slightly elliptical hillocks on the surfaces, whereas disulfide-reduced lysozyme produced more oblong, elongated hillocks. By employing a thermal model describing the response of graphite to energy deposited by an elongated incident energetic projectile, we calculated from the hillock sizes for disulfide-reduced lysozyme (Q = 14+) an overall length of 32.1 ± 1.6 nm. This value is close to the length we observe for apomyoglobin (Q = 14+), 35.5 ± 2.4 nm, although apomyoglobin and lysozyme possess significantly different numbers of amino acid residues. Based on these results, we hypothesize that aspects of a protein's native secondary structure are preserved in the gas phase, even if the tertiary structure might be non-native. We have unfolded disulfide-intact lysozyme computationally and find a qualitatively good agreement with the experimentally obtained length of disulfide-intact (Q = 9+) lysozyme.