Defensins comprise a potent class of membrane disruptive antimicrobial peptides (AMPs) with well-characterized broad spectrum and selective microbicidal effects. By using high-resolution synchrotron small angle x-ray scattering to investigate interactions between heterogeneous membranes and members of the defensin subfamilies, α-defensins (Crp-4), β-defensins (HBD-2, HBD-3), and θ-defensins (RTD-1, BTD-7), we show how these peptides all permeabilize model bacterial membranes but not model eukaryotic membranes: defensins selectively generate saddle-splay (‘negative Gaussian’) membrane curvature in model membranes rich in negative curvature lipids such as those with phosphoethanolamine (PE) headgroups. These results are shown to be consistent with vesicle leakage assays. A mechanism of action based on saddle-splay membrane curvature generation is broadly enabling, since it is a necessary condition for processes such as pore formation, blebbing, budding, vesicularization, all of which destabilize the barrier function of cell membranes. Importantly, saddle-splay membrane curvature generation places constraints on the amino acid composition of membrane disruptive peptides. For example, we show that the requirement for generating saddle-splay curvature implies that a decrease in arginine content in an AMP can be offset by an increase in both lysine and hydrophobic content. This ‘design rule’ is consistent with the amino acid compositions of 1,080 known cationic AMPs.

Vaccine-based autoimmune (anti-amyloid) treatments are currently being examined for their therapeutic potential in Alzheimer's disease. In the present study we examined, in a transgenic model of amyloid pathology, the expression of two molecules previously implicated in decreasing the severity of autoimmune responses: TREM2 (triggering receptor expressed on myeloid cells 2) and the intracellular tolerance-associated transcript, Tmem176b (transmembrane domain protein 176b). In situ hybridization analysis revealed that both molecules were highly expressed in plaque-associated microglia, but their expression defined two different zones of plaque-associated activation. Tmem176b expression was highest in the inner zone of amyloid plaques, whereas TREM2 expression was highest in the outer zone. Induced expression of TREM2 occurred co-incident with detection of thioflavine-S-positive amyloid deposits. Transfection studies revealed that expression of TREM2 correlated negatively with motility, but correlated positively with the ability of microglia to stimulate CD4+ T-cell proliferation, TNF (tumour necrosis factor) and CCL2 (chemokine ligand 2) production, but not IFNγ (interferon γ) production. TREM2 expression also showed a positive correlation with amyloid phagocytosis in unactivated cells. However, activating cells with LPS (lipopolysaccharide), but not IFNγ, reduced the correlation between TREM2 expression and phagocytosis. Transfection of Tmem176b into both microglial and macrophage cell lines increased apoptosis. Taken together, these data suggest that, in vivo, Tmem176b+ cells in closest apposition to amyloid may be the least able to clear amyloid. Conversely, the phagocytic TREM2+ microglia on the plaque outer zones are positioned to capture and present self-antigens to CNS (central nervous system)-infiltrating lymphocytes without promoting pro-inflammatory lymphocyte responses. Instead, plaque-associated TREM2+ microglia have the potential to evoke neuroprotective immune responses that may serve to support CNS function during pro-inflammatory anti-amyloid immune therapies.

We investigated the properties and regulation of P2X receptors in immortalized C8-B4 cells of cerebellar microglial origin. Resting C8-B4 cells expressed virtually no functional P2X receptors, but largely increased functional expression of P2X4 receptors within 2–6 h of entering the activated state. Using real-time polymerase chain reaction, we found that P2X4 transcripts were increased during the activated state by 2.4-fold, but this increase was not reflected by a parallel increase in total P2X4 proteins. In resting C8-B4 cells, P2X4 subunits were mainly localized within intracellular compartments, including lysosomes. We found that cell surface P2X4 receptor levels increased by ∼3.5-fold during the activated state. This change was accompanied by a decrease in the lysosomal pool of P2X4 proteins. We next exploited our findings with C8-B4 cells to investigate the mechanism by which antidepressants reduce P2X4 responses. We found little evidence to suggest that several antidepressants were antagonists of P2X4 receptors in C8-B4 cells. However, we found that moderate concentrations of the same antidepressants reduced P2X4 responses in activated microglia by affecting lysosomal function, which indirectly reduced cell surface P2X4 levels. In summary, our data suggest that activated C8-B4 cells express P2X4 receptors when the membrane insertion of these proteins by lysosomal secretion exceeds their removal, and that antidepressants indirectly reduce P2X4 responses by interfering with lysosomal trafficking.

Protein microarray technology possesses some of the greatest potential for providing direct information on protein function and potential drug targets. For example, functional protein microarrays are ideal tools suited for the mapping of biological pathways. They can be used to study most major types of interactions and enzymatic activities that take place in biochemical pathways and have been used for the analysis of simultaneous multiple biomolecular interactions involving protein-protein, protein-lipid, protein-DNA and protein-small molecule interactions. Because of this unique ability to analyze many kinds of molecular interactions en masse, the requirement of very small sample amount and the potential to be miniaturized and automated, protein microarrays are extremely well suited for protein profiling, drug discovery, drug target identification and clinical prognosis and diagnosis. The aim of this review is to summarize the most recent developments in the production, applications and analysis of protein microarrays.

-Defensins are macrocyclic antimicrobial peptides that were previously isolated from leukocytes of a single species, the rhesus macaque. We now report the characterization of baboon -defensins (BTDs) expressed in bone marrow and peripheral blood leukocytes. Four cDNAs encoding -defensin precursors were characterized, allowing for the prediction of 10 theoretical -defensins (BTD-1 to BTD-10) produced by binary, headto-tail splicing of nonapeptides excised from paired precursors. Five of the predicted -defensins were purified from baboon leukocytes, and synthetic versions of each were prepared. Anti--defensin antibody localized the peptides in circulating neutrophils and monocytes and in immature and mature myeloid elements in bone marrow. Each of the BTDs possessed antimicrobial activity against bacterial and fungal test organisms in vitro. Peptide activities varied markedly despite a high degree of sequence conservation among the -defensins tested. Thus, baboons express numerous -defensins which appear to differentially contribute to host defense against diverse pathogens.Antimicrobial peptides (AMPs) are effectors of the innate immune system. AMPs are expressed in cells (epithelia, neutrophils, and macrophages) that come into contact with potentially invasive microorganisms (17). In mammals, the two major classes of AMPs are defensins and cathelicidins. Cathelicidins are characterized by a conserved cathelin prodomain which lies N terminal to highly variable mature peptides that are released by activating proteases (27). Defensins are small, cationic peptides that are composed of three structural subclasses, ␣-, ␤-, and -defensins, differentiated by the spacing and pairing of their six disulfide-bonded cysteines (7, 9, 18). -Defensins are further distinguished by their macrocyclic backbone and as such represent the only known cyclic protein motif expressed in animals (16).The biosynthesis of -defensins requires head-to-tail splicing of two 9-amino-acid sequences derived from -defensin precursors (16). -Defensins were first identified in neutrophils and monocytes of the rhesus monkey (21). Subsequently, Nguyen et al. (15) conducted a phylogenetic survey that revealed the existence of -defensin genes in other Old World monkeys and two apes (the siamang and orangutan), but none in New World monkeys or prosimians. Humans, chimpanzees, bonobos, and gorillas express -defensin pseudogenes in which the precursor mRNA contains a mutation producing a stop codon in the signal sequence, thus preventing translation of the -defensin precursor (15).Rhesus -defensin-1 (RTD-1) is produced from the heterodimeric splicing of two -defensin precursors, proRTD1a and proRTD1b. Homodimeric excision/ligation reactions involving proRTD1a and proRTD1b were revealed by the isolation of RTD-2 and RTD-3 (12, 23). RTD-1, -2, and -3 have potent microbicidal activities against bacteria and fungi (23) and have antiviral activities against human immunodeficiency virus type 1 (3, 24) and herpes simplex virus (26). A synthetic -defensin designed b...