Structural analysis of the NK-lysin-derived peptide NK-2 upon interaction with bacterial membrane mimetics consisting of phosphatidylethanolamine and phosphatidylglycerol.

NK-2 is an antimicrobial peptide derived from helices 3 and 4 of the pore-forming protein of natural killer cells, NK-lysin. It has potent activities against Gram-negative and Gram-positive bacteria, fungi and protozoan parasites without being toxic to healthy human cells. In biophysical assays its membrane activities were found to require phosphatidylglycerol (PG) and phosphatidylethanolamine (PE), lipids which dominate the composition of bacterial membranes. Here the structure and activities of NK-2 in binary mixtures of different PE/PG composition were investigated. CD spectroscopy reveals that a threshold concentration of 50 % PG is needed for efficient membrane association of NK-2 concomitant with a random coil - helix transition. Association with PE occurs but is qualitatively different when compared to PG membranes. Oriented solid-state NMR spectroscopy of NK-2 specifically labelled with 15N indicates that the NK-2 helices are oriented parallel to the PG bilayer surface. Upon reduction of the PG content to 20 mol% interactions are weaker and/or an in average more tilted orientation is observed. Fluorescence spectroscopy of differently labelled lipids is in agreement of an interfacial localisation of both helices where the C-terminal end is in a less hydrophobic environment. By inserting into the membrane interface and interacting differently with PE and PG the peptides probably induce high curvature strain which result in membrane openings and rupture.

Variable antifungal activity of curcumin against planktonic and biofilm phase of different candida species.

OBJECTIVE: To evaluate the in vitro antifungal activity of curcumin against 2 strains of Candida albicans (ATCC 90028 and a clinical isolate - JY strain) and 1 isolate each of 3 nonalbicans - Candida species [Candida parapsilosis (ATCC 22019), C. glabrata (ATCC 90030), and C. dublieniensis (MYA 646)]. MATERIALS AND METHODS: Planktonic MIC of the 4 Candida species was determined using micro broth dilution assay according to CLSI M27-A3 criteria. The biofilm development and sensitivity assay were performed with the 2 C. albicans strains. RESULTS: Curcumin at high concentrations (0.1-2 mg/mL) was effective in inhibiting planktonic organisms of all the 5 tested Candida strains. The planktonic phase and the biofilm phase of C. albicans ATCC 90028 exhibited similar MIC values for curcumin (0.5 mg/mL). Both curcumin and fluconazole were ineffective against the mature biofilms of JY strain. CONCLUSION: Our results reported here for the first time, in particular for the biofilm state of C. albicans, imply that curcumin a natural product could be used as a therapeutic alternative to conventional antifungals although further investigations are required to evaluate its potential.

Inter- and intra-observer variability in three grading systems for oral epithelial dysplasia.

BACKGROUND AND OBJECTIVES: Management of oral premalignant lesions depends on clinical assessment and grading of oral epithelial dysplasia (OED), which remains one of the most important predictors of malignant potential. Numerous grading systems for OED exists with varying sets of assessing criteria and are largely considered subjective. The present study attempted to assess the inter- and intra-observer variability in three grading systems: Binary system, WHO (2005) and Ljubljana systems. METHODOLOGY: Histopathological grading of 63 cases of leukoplakia was performed by two oral pathologists and one general pathologist, who were blinded, using all the three grading systems at different time intervals and was repeated twice. Inter- and intra-observer variability was then evaluated by multivariate kappa analysis. RESULTS: Inter-observer agreement in the two set of observations was found to be "slight" in WHO (k = 0.001 and 0.039), "slight" and "poor" in binary (k = 0.108 and -0.007), "poor" and "slight" in Ljubljana's (k = -0.027 and 0.106) grading systems. Intra-observer agreement ranged from "slight" to "fair" (k = 0.128 and 0.295) in WHO, "fair" to "moderate" (k = 0.224 and 0.420) in binary and "slight" to "fair" (k = 0.161 and -0.353) in Ljubljana's grading systems. CONCLUSIONS: The binary system of classification proved to have an overall better inter- and intra-observer agreement. This study also showed better intra-observer agreement in all the grading systems as well as in individual histopathological parameters. Defining the individual parameters more objectively with the reproducible structuring of the grading systems and training of the pathologists would help reduce the variability in diagnosing dysplasia.

Membrane disruptive antimicrobial activities of human ß-defensin-3 analogs.

Human beta defensin-3 (HßD-3) is a host-defense protein exhibiting antibacterial activity towards both Gram-negative and Gram-positive bacteria. There is considerable interest in the function of this protein due to its increased salt tolerance and activity against Gram-positive Staphylococcus aureus. In this study, analogs of HßD-3 devoid of N and C terminal regions are investigated to determine the influence of specific structural motif on antimicrobial activity and selectivity between Gram-positive and Gram-negative bacteria. Circular dichroism, fluorescence and solid-state NMR experiments have been used to investigate the conformation and mode of action of HßD3 analogs with various model membranes to mimic bacterial inner and outer membranes and also mammalian membranes. Our studies specifically focused on determining four major characteristics: (i) interaction of HßD3 analogs with phospholipid vesicles composed of zwitterionic PC or anionic PE:PG vesicles and LPS; (ii) conformation of HßD3-peptide analogs in the presence of PC or PE:PG vesicles; (iii) ability of HßD3 analogs to permeate phospholipid vesicles composed of PC or PE:PG; and (iv) activities on bacteria cells and erythrocytes. Our results infer that the linear peptide L25P and its cyclic form C25P are more active than L21P and C21P analogs. However, they are less active than the parent peptide, thus pointing towards the importance of the N terminal domain in its biological activity. The variation in the activities of L21P/C21P and L25P/C25P also suggest the importance of the positively charged residues at the C terminus in providing selectivity particularly to Gram-negative bacteria.

Ameloblastic carcinoma of the mandible: Report of a case and review.

Ameloblastic carcinoma is a rare malignant odontogenic neoplasm that can arise either as a de novo lesion or from pre-existing ameloblastoma. Histopathologically, the tumor retains an ameloblastomatous differentiation pattern but shows cytological features of malignancy. Owing to variable biologic behavior and paucity of long-term follow-up cases, there has been no clear consensus on treatment protocol. The present case of ameloblastic carcinoma arose in the mandible of a 24-year-old male. Surgical treatment involved resection of the mandible along with regional lymph nodes. The patient has been on follow up for the past one year without any recurrence or metastases. An update on ameloblastic carcinoma encompassing the histogenesis, immunohistochemical features and treatment aspects are included.

Assessment of tissue eosinophilia as a prognosticator in oral epithelial dysplasia and oral squamous cell carcinoma-an image analysis study.

Association of tissue eosinophilia with oral squamous cell carcinoma has shown variable results ranging from favourable to unfavourable or even having no influence on prognosis. Also, very few studies have been done to know the role of eosinophils in premalignancy. So the present study investigated role of eosinophilic infiltration in oral precancer and cancer and its possible use as a prognosticator. 60 histopathologically proven cases (20 cases each of metastatic and nonmetastatic oral squamous cell carcinoma and oral leukoplakia with dysplasia of various grades) were included. Congo red is used as a special stain for eosinophils. Each specimen slide was viewed under high power in 10 consecutive microscopic fields for counting of eosinophils. As a result, a significant increase in eosinophil count was found in oral carcinomas compared to dysplasia. Nonmetastatic cases showed higher counts than metastatic carcinomas. So, it is concluded that eosinophilia is a favourable histopathological prognostic factor in oral cancer. Moreover, higher eosinophil counts in carcinoma group compared to dysplasia group proved that they might have a role in stromal invasion thus suggesting that quantitative assessment of tissue eosinophilia should become a part of the routine histopathological diagnosis for oral precancer and OSCC.

Helix orientations in membrane-associated Bcl-X(L) determined by 15N-solid-state NMR spectroscopy.

Controlled cell death is fundamental to tissue hemostasis and apoptosis malfunctions can lead to a wide range of diseases. Bcl-x(L) is an anti-apoptotic protein the function of which is linked to its reversible interaction with mitochondrial outer membranes. Its interfacial and intermittent bilayer association makes prediction of its bound structure difficult without using methods able to extract data from dynamic systems. Here we investigate Bcl-x(L) associated with oriented lipid bilayers at physiological pH using solid-state NMR spectroscopy. The data are consistent with a C-terminal transmembrane anchoring sequence and an average alignment of the remaining helices, i.e. including helices 5 and 6, approximately parallel to the membrane surface. Data from several biophysical approaches confirm that after removal of the C-terminus from Bcl-x(L) its membrane interactions are weak. In the presence of membranes Bcl-x(L) can still interact with a Bak BH3 domain peptide suggesting a model where the hydrophobic C-terminus of the protein unfolds and inserts into the membrane. During this conformational change the Bcl-x(L) hydrophobic binding pocket becomes accessible for protein-protein interactions whilst the structure of the N-terminal region remains intact.

LL-37, the only human member of the cathelicidin family of antimicrobial peptides.

Antimicrobial peptides and their precursor molecules form a central part of human and mammalian innate immunity. The underlying genes have been thoroughly investigated and compared for a considerable number of species, allowing for phylogenetic characterization. On the phenotypical side, an ever-increasing number of very varied and distinctive influences of antimicrobial peptides on the innate immune system are reported. The basic biophysical understanding of mammalian antimicrobial peptides, however, is still very limited. This is especially unsatisfactory since knowledge of structural properties will greatly help in the understanding of their immunomodulatory functions. The focus of this review article will be on LL-37, the only cathelicidin-derived antimicrobial peptide found in humans. LL-37 is a 37-residue, amphipathic, helical peptide found throughout the body and has been shown to exhibit a broad spectrum of antimicrobial activity. It is expressed in epithelial cells of the testis, skin, the gastrointestinal tract, and the respiratory tract, and in leukocytes such as monocytes, neutrophils, T cells, NK cells, and B cells. It has been found to have additional defensive roles such as regulating the inflammatory response and chemo-attracting cells of the adaptive immune system to wound or infection sites, binding and neutralizing LPS, and promoting re-epthelialization and wound closure. The article aims to report the known biophysical facts, with an emphasis on structural evidence, and to set them into relation with insights gained on phylogenetically related antimicrobial peptides in other species. The multitude of immuno-functional roles is only outlined. We believe that this review will aid the future work on the biophysical, biochemical and immunological investigations of this highly intriguing molecule.

Topological equilibria of ion channel peptides in oriented lipid bilayers revealed by 15N solid-state NMR spectroscopy.

Ion channel peptides have been prepared by solid-phase peptide synthesis, labeled with 15N at selected sites, and reconstituted into oriented lipid bilayers. The (Leu-Ser-Ser-Leu-Leu-Ser-Leu)3-CONH2 peptide has previously been shown to exhibit well-defined and discrete ionic conductances when investigated by single-channel measurements [Lear, J. D., et al. (1988) Science 240, 1177]. Proton-decoupled 15N solid-state NMR spectroscopy indicates that (Leu-Ser-Ser-Leu-Leu-Ser-Leu)3-CONH2 preferentially aligns parallel to the membrane surface in excellent agreement with its amphipathic helical structure. However, by carefully choosing the conditions of the membrane environment, significant contributions that are indicative of transmembrane alignments become obvious in the 15N chemical shift solid-state NMR spectra. The data thereby provide experimental evidence for an equilibrium between in-plane and transmembrane-oriented helix configurations where the transmembrane and surface-oriented peptide fractions are in slow exchange. Similar topological equilibria are observed when the N-terminus of the LS21 peptide is acetylated. These observations provide experimental support for previous models, suggesting that the channels observed in single-channel conductance measurements are indeed formed by hexameric transmembrane helical bundles. In contrast, the shorter peptide (Leu-Ser-Ser-Leu-Leu-Ser-Leu)2-CONH2 is oriented parallel to the membrane surface under all conditions tested. This peptide exhibits erratic conductance changes when investigated by electrophysiological methods, probably because it is too short to span the lipid bilayer.