Structure-Activity Determinants in Antifungal Plant Defensins MsDef1 and MtDef4 with Different Modes of Action against Fusarium graminearum

Plant defensins are small cysteine-rich antimicrobial proteins. Their three-dimensional structures are similar in that they consist of an α-helix and three anti-parallel β-strands stabilized by four disulfide bonds. Plant defensins MsDef1 and MtDef4 are potent inhibitors of the growth of several filamentous fungi including Fusarium graminearum. However, they differ markedly in their antifungal properties as well as modes of antifungal action. MsDef1 induces prolific hyperbranching of fungal hyphae, whereas MtDef4 does not. Both defensins contain a highly conserved γ-core motif (GXCX3–9C), a hallmark signature present in the disulfide-stabilized antimicrobial peptides, composed of β2 and β3 strands and the interposed loop. The γ-core motifs of these two defensins differ significantly in their primary amino acid sequences and in their net charge. In this study, we have found that the major determinants of the antifungal activity and morphogenicity of these defensins reside in their γ-core motifs. The MsDef1-γ4 variant in which the γ-core motif of MsDef1 was replaced by that of MtDef4 was almost as potent as MtDef4 and also failed to induce hyperbranching of fungal hyphae. Importantly, the γ-core motif of MtDef4 alone was capable of inhibiting fungal growth, but that of MsDef1 was not. The analysis of synthetic γ-core variants of MtDef4 indicated that the cationic and hydrophobic amino acids were important for antifungal activity. Both MsDef1 and MtDef4 induced plasma membrane permeabilization; however, kinetic studies revealed that MtDef4 was more efficient in permeabilizing fungal plasma membrane than MsDef1. Furthermore, the in vitro antifungal activity of MsDef1, MsDef1-γ4, MtDef4 and peptides derived from the γ-core motif of each defensin was not solely dependent on their ability to permeabilize the fungal plasma membrane. The data reported here indicate that the γ-core motif defines the unique antifungal properties of each defensin and may facilitate de novo design of more potent antifungal peptides

Activation of Sirt1 by Resveratrol Inhibits TNF-α Induced Inflammation in Fibroblasts

Inflammation is one of main mechanisms of autoimmune disorders and a common feature of most diseases. Appropriate suppression of inflammation is a key resolution to treat the diseases. Sirtuin1 (Sirt1) has been shown to play a role in regulation of inflammation. Resveratrol, a potent Sirt1 activator, has anti-inflammation property. However, the detailed mechanism is not fully understood. In this study, we investigated the anti-inflammation role of Sirt1 in NIH/3T3 fibroblast cell line. Upregulation of matrix metalloproteinases 9 (MMP-9), interleukin-1beta (IL-1β), IL-6 and inducible nitric oxide synthase (iNOS) were induced by tumor necrosis factor alpha (TNF-α) in 3T3 cells and resveratrol suppressed overexpression of these pro-inflammatory molecules in a dose-dependent manner. Knockdown of Sirt1 by RNA interference caused 3T3 cells susceptible to TNF-α stimulation and diminished anti-inflammatory effect of resveratrol. We also explored potential anti-inflammatory mechanisms of resveratrol. Resveratrol reduced NF-κB subunit RelA/p65 acetylation, which is notably Sirt1 dependent. Resveratrol also attenuated phosphorylation of mammalian target of rapamycin (mTOR) and S6 ribosomal protein (S6RP) while ameliorating inflammation. Our data demonstrate that resveratrol inhibits TNF-α-induced inflammation via Sirt1. It suggests that Sirt1 is an efficient target for regulation of inflammation. This study provides insight on treatment of inflammation-related diseases

Structural mechanism of muscle contraction

X-ray crystallography shows the myosin cross-bridge to exist in two conformations, the beginning and end of the "power stroke." A long lever-arm undergoes a 60 degrees to 70 degrees rotation between the two states. This rotation is coupled with changes in the active site (OPEN to CLOSED) and phosphate release. Actin binding mediates the transition from CLOSED to OPEN. Kinetics shows that the binding of myosin to actin is a two-step process which affects ATP and ADP affinity. The structural basis of these effects is not explained by the presently known conformers of myosin. Therefore, other states of the myosin cross-bridge must exist. Moreover, cryoelectronmicroscopy has revealed other angles of the cross-bridge lever arm induced by ADP binding. These structural states are presently being characterized by site-directed mutagenesis coupled with kinetic analysis

Characteristics and drug responses of cochlear and vestibular adenylate cyclase

Die Aktivitäten von Adenylatzyklase in isolierten cochlearen und vestibulären Strukturen wurden in Inkubationen mit dem ATP-Analog Adenylylimidodiphosphat gemessen. Hohe Aktivitäten (pro mg protein) fanden sich in Stria vascularis und vestibulären Präparationen, geringere Aktivitäten im Ligamentum spirale, dem 8. Nerven und Cortischem Organ. In allen Präparationen wurde die Enzymaktivität stimuliert durch Fluorid, Guanylylimidodiphosphat oder Mangan(II)ionen und gehemmt durch Ethakrynsäure und Bleiionen. Das ototoxische Zytostatikum cis-Diammin-dichloroplatin hemmte die Zyklase von Stria vascularis signifikant. Adenylate cyclase activity of dissected cochlear and vestibular structures was assayed with the ATP-analog adenylyl imidodiphosphate as substrate. High activities (per mg protein) were found in stria vascularis and in vestibular preparations, lower activities in spiral ligament, VIIIth nerve, and organ of Corti. The enzyme from all structures was stimulated by fluoride, guanylyl imidodiphosphate or manganese(II) ions, and strongly inhibited by ethacrynate and lead ion. The anti-cancer drug cis-diammine-dichloro platinum significantly inhibited adenylate cyclase from stria vascularis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/47263/1/405_2004_Article_BF00454231.pd