No abstract

Human defensins form a family of small, cationic, and Cys-rich antimicrobial proteins that play important roles in innate immunity against invading microbes. They also function as effective immune modulators in adaptive immunity by selectively chemoattracting T lymphocytes and immature dendritic cells. On the basis of sequence homology and the connectivity of six conserved Cys residues, human defensins are classified into ␣ and ␤ families. Structures of several ␤-defensins have recently been characterized, confirming the disulfide connectivity conserved within the family, i.e., Cys 1 -Cys 5 , Cys 2 -Cys 4 , and Cys 3 -Cys 6 . We found that human ␤-defensin 3 (hBD3), a recently described member of the growing ␤ family, did not fold preferentially into a native conformation in vitro under various oxidative conditions. Using the orthogonal protection of Cys 1 -Cys 5 and of Cys 1 -Cys 6 , we chemically synthesized six topological analogs of hBD3 with predefined disulfide connectivities, including the (presumably) native ␤ pairing. Unexpectedly, all differently folded hBD3 species exhibited similar antimicrobial activity against Escherichia coli, whereas a wide range of chemotactic activities was observed with these analogs for monocytes and cells transfected by the chemokine receptor CCR6. Furthermore, whereas substitution of all Cys residues by ␣-aminobutyric acid completely abolished the chemotactic activity of hBD3, the bactericidal activity remained unaffected in the absence of any disulfide bridge. Our findings demonstrate that disulfide bonding in hBD3, although required for binding and activation of receptors for chemotaxis, is fully dispensable for its antimicrobial function, thus shedding light on the mechanisms of action for human ␤-defensins and the design of novel peptide antibiotics.

We developed a kinetic, 96-well turbidimetric procedure that is capable of testing the antimicrobial properties of six human ␣-defensins concurrently on a single microplate. The defensins were prepared by solidphase peptide synthesis and tested against gram-positive bacteria (Staphylococcus aureus and Bacillus cereus) and gram-negative bacteria (Enterobacter aerogenes and Escherichia coli). Analysis of the growth curves provided virtual lethal doses (vLDs) equivalent to conventional 50% lethal doses (LD 50 s), LD 90 s, LD 99 s, and LD 99.9 s obtained from colony counts. On the basis of their respective vLD 90 s and vLD 99 s, the relative potencies of human myeloid ␣-defensins against S. aureus were HNP2 > HNP1 > HNP3 > HNP4. In contrast, their relative potencies against E. coli and E. aerogenes were HNP4 > HNP2 > HNP1 ‫؍‬ HNP3. HD5 was as effective as HNP2 against S. aureus and as effective as HNP4 against the gram-negative bacteria in our panel. HD6 showed little or no activity against any of the bacteria in our panel, including B. cereus, which was highly susceptible to the other five ␣-defensins. The assay described provides a quantitative, precise, and economical way to study the antimicrobial activities of host-defense peptides. Its use has clarified the relative potencies of human ␣-defensins and raised intriguing questions about the in vivo function(s) of HD6.Antimicrobial peptides, such as ␣-defensins, are believed to play substantial roles in the innate host defense against bacterial, fungal, and viral pathogens (3,5,24). Four of these peptides (HNP1 to HNP4) were initially isolated from human leukocytes (13). HNP1 to HNP3 differ only at the N-terminal position, while the other sequences are more diverse. Human defensin 5 (HD5) and HD6, which are synthesized in and secreted by intestinal Paneth cells, were discovered through genomic studies (2,7,8). Because native HNP1, HNP2, and HNP3 are easily purified from leukocytes, they have been widely studied. As the other native ␣-defensin peptides have been recovered in amounts that are small (HNP4), smaller (HD5), or nil (HD6), considerably less is known about their properties.A recent synthesis procedure has made all six human ␣-defensins available for in vitro analysis (22,23). These advances are especially significant for the characterization of HNP4, HD5, and HD6. To study the six ␣-defensins described in this report and to facilitate future studies of selectively modified defensins that we hope to perform in the future, we developed a facile way to assay their antimicrobial properties quantitatively.Broth microdilution methods for the testing of antibiotics are traditionally conducted in 96-well plates, ideally according to guidelines approved by the National Center for Clinical Laboratory Standards (NCCLS) (15). Although such methods are simple to perform, their inherent precision is limited by the use of serial dilutions rather than a continuous calibration curve. Colony counting procedures are considerably more labor intensive to set up and analyze. W...

Six a-defensins have been found in humans. These small arginine-rich peptides play important roles in various processes related to host defense, being the effectors and regulators of innate immunity as well as enhancers of adoptive immune responses. Four defensins, called neutrophil peptides 1 through 4, are stored primarily in polymorphonuclear leukocytes. Major sites of expression of defensins 5 and 6 are Paneth cells of human small intestine. So far, only one structure of human a-defensin (HNP3) has been reported, and the properties of the intestine defensins 5 and 6 are particularly poorly understood. In this report, we present the high-resolution X-ray structures of three human defensins, 4 through 6, supplemented with studies of their antimicrobial and chemotactic properties. Despite only modest amino acid sequence identity, all three defensins share their tertiary structures with other known a-and b-defensins. Like HNP3 but in contrast to murine or rabbit a-defensins, human defensins 4-6 form characteristic dimers. Whereas antimicrobial and chemotactic activity of HNP4 is somewhat comparable to that of other human neutrophil defensins, neither of the intestinal defensins appears to be chemotactic, and for HD6 also an antimicrobial activity has yet to be observed. The unusual biological inactivity of HD6 may be associated with its structural properties, somewhat standing out when compared with other human a-defensins. The strongest cationic properties and unique distribution of charged residues on the molecular surface of HD5 may be associated with its highest bactericidal activity among human a-defensins.

␤-Defensins are small (3 to 5 kDa in size) secreted antimicrobial and antiviral proteins that are components of innate immunity. ␤-Defensins are secreted by epithelial cells, and they are expressed at high levels in several mucosae, including the mouth, where the concentration of these proteins can reach 100 g/ml. Because of these properties, we wondered whether they could be part of the defenses that lower oral transmission of human immunodeficiency virus (HIV) compared to other mucosal sites. Our data show that select ␤-defensins, especially human ␤-defensin 2 (hBD2) and hBD3, inhibit R5 and X4 HIV infection in a dose-dependent manner at doses that are compatible with or below those measured in the oral cavity. We observed that ␤-defensin treatment inhibited accumulation of early products of reverse transcription, as detected by PCR. We could not, however, detect any reproducible inhibition of env-mediated fusion, and we did not observe any modulation of HIV coreceptors following treatment with hBD1 and hBD2, in both resting and phytohemagglutinin-activated cells. Our data instead suggest that, besides a direct inactivation of HIV virions, hBD2 inhibits HIV replication in the intracellular environment. Therefore, we speculate that ␤-defensins mediate a novel antiretroviral mechanism that contributes to prevention of oral HIV transmission in the oral cavity. Immunohistochemical data on hBD2 expression in oral mucosal tissue shows that hBD2 is constitutively expressed, forming a barrier layer across the epithelium in healthy subjects, while in HIV-positive subjects levels of hBD2 expression are dramatically diminished. This may predispose HIV-positive subjects to increased incidence of oral complications associated with HIV infection.

The subject of sol-gel electrochemistry is introduced, starting with a brief account of milestones in its evolution. Then, the types of sol-gel materials that are useful for electrochemistry are presented, followed by a description of recent advances in the various fields of sol-gel electrochemistry. Modified electrodes, solid electrolytes, electrochromic devices, and corrosion protection coatings are described. Emerging fields such as RuO 2 supercapacitors and electrochemical synthesis of sol-gel precursors are also addressed.

No abstract

NiCo2O4-based materials have become a new class of energy storage materials for electrochemical supercapacitors, which manifest remarkable electrochemical performances.