Modification of a novel angiogenic peptide, AG30, for the development of novel therapeutic agents

We previously identified a novel angiogenic peptide, AG30, with antibacterial effects that could serve as a foundation molecule for the design of wound-healing drugs. Toward clinical application, in this study we have developed a modified version of the AG30 peptide characterized by improved antibacterial and angiogenic action, thus establishing a lead compound for a feasibility study. Because AG30 has an α-helix structure with a number of hydrophobic and cationic amino acids, we designed a modified AG30 peptide by replacing several of the amino acids. The replacement of cationic amino acids (yielding a new molecule, AG30/5C), but not hydrophobic amino acids, increased both the angiogenic and the antimicrobial properties of the peptide. AG30/5C was also effective against methicillin-resistant Staphylococcus aureus (MRSA) and antibiotic-resistant Pseudomonas aeruginosa. In a diabetic mouse wound-healing model, the topical application of AG30/5C accelerated wound healing with increased angiogenesis and attenuated MRSA infection. To facilitate the eventual clinical investigation/application of these compounds, we developed a large-scale procedure for the synthesis of AG30/5C that employed the conventional solution method and met Good Manufacturing Practice guidelines. In the evaluation of stability of this peptide in saline solution, RP-HPLC analysis revealed that AG30/5C was fairly stable under 5°C for 12 months. Therefore, we propose the use of AG30/5C as a wound-healing drug with antibacterial and angiogenic actions.     

Centriole and PCM cooperatively recruit CEP192 to spindle poles to promote bipolar spindle assembly

The pericentriolar material (PCM) that accumulates around the centriole expands during mitosis and nucleates microtubules. Here, we show the cooperative roles of the centriole and PCM scaffold proteins, pericentrin and CDK5RAP2, in the recruitment of CEP192 to spindle poles during mitosis. Systematic depletion of PCM proteins revealed that CEP192, but not pericentrin and/or CDK5RAP2, was crucial for bipolar spindle assembly in HeLa, RPE1, and A549 cells with centrioles. Upon double depletion of pericentrin and CDK5RAP2, CEP192 that remained at centriole walls was sufficient for bipolar spindle formation. In contrast, through centriole removal, we found that pericentrin and CDK5RAP2 recruited CEP192 at the acentriolar spindle pole and facilitated bipolar spindle formation in mitotic cells with one centrosome. Furthermore, the perturbation of PLK1, a critical kinase for PCM assembly, efficiently suppressed bipolar spindle formation in mitotic cells with one centrosome. Overall, these data suggest that the centriole and PCM scaffold proteins cooperatively recruit CEP192 to spindle poles and facilitate bipolar spindle formation.     

Molecular phylogeny of the tribe Candalidini (Lepidoptera: Lycaenidae): systematics,diversification and evolutionary history

The butterfly tribe Candalidini is geographically restricted to Australia and mainland New Guinea and its adjacent islands. With 60 species and subspecies, it represents a large radiation of Papilionoidea in the Australian region. Although the species-level taxonomy is relatively well understood, the number of genera is uncertain, varying from two to eight. We reconstructed the phylogeny of the Candalidini based on a 13-locus hybrid enrichment probe set (12.8 Kbp: COI, Thiolase, CAD, CAT, DDC, EF1-a, GAPDH, HCL, IDH, MDH, RPS2, RPS5, Wingless), including all previously recognized genera and 76% (28/37) of the species-level diversity of the tribe. Maximum likelihood analysis recovered the Candalidini as a strongly supported monophyletic group. In conjunction with morphological characters, the phylogeny provided a robust framework for a revised classification in which we recognize four genera, 37 species and 23 subspecies. The genus Nesolycaena Waterhouse & R.E. Turner is considered in synonymy with Candalides Hübner, and four other genera are not recognized, namely, Holochila C. Felder, Adaluma Tindale, Zetona Waterhouse and Microscena Tite. Of the four valid genera, the absimilis group (23 species) is placed in the newly described genus Eirmocides Braby, Espeland & Müller gen. nov. (type species Candalides consimilis Waterhouse). The erinus group (six species) is assigned to Erina Swainson, which is reinstated. Chrysophanus cyprotus Olliff is assigned to Cyprotides Tite, which is also reinstated as a monotypic genus. The remaining seven species are placed in Candalides sensu stricto. Overall, we propose 47 new nomenclatural changes at the species and subspecies levels, including the synonymy of Holochila biaka Tite as Eirmocides tringa biaka (Tite) syn. nov. et comb. nov. and recognition of Candalides hyacinthinus gilesi M.R. Williams & Bollam as a distinct species Erina gilesi (M.R. Williams & Bollam stat. rev. et comb. nov. A dated phylogeny using Bayesian inference in BEAST2 and biogeographical and habitat analyses based on the DEC model in BioGeoBEARS indicated that the ancestor of the Candalidini most likely evolved in rainforest habitats of the mesic biome in situ on the Australian plate of Southern Gondwana during the Eocene (c. 43 Ma). A major period of diversification occurred in the Miocene, which coincided with aridification of the Australian continent, followed by a further episode of radiation in montane New Guinea during the Plio-Pleistocene. This published work has been registered on ZooBank by the authors: Michael Braby: http://zoobank.org/urn:lsid:zoobank.org:author:4D3A7605-EBD0-40F6-A5F2-7F67F59E3D60 ; Marianne Espeland: http://zoobank.org/urn:lsid:zoobank.org:author:00D6F9F9-3902-4A8B-846F-720AB32922A6 ; Chris Müller: http://zoobank.org/urn:lsid:zoobank.org:author:15FE5F26-7596-46C2-9697-1FD92A692D0D ; http://zoobank.org/urn:lsid:zoobank.org:pub:47D5CA34-C294-4FBD-84B6-1C2A82B7CADF .     

Crystal structure analysis of human serum albumin complexed with sodium 4-phenylbutyrate

Sodium 4-phenylbutyrate (PB) is an orphan drug for the treatment of urea cycle disorders. It also inhibits the development of endoplasmic reticulum stress, the action of histone deacetylases and as a regulator of the hepatocanalicular transporter. PB is generally considered to have the potential for use in the treatment of the diseases such as cancer, neurodegenerative diseases and metabolic diseases. In a previous study, we reported that PB is primarily bound to human serum albumin (HSA) in plasma and its binding site is drug site 2. However, details of the binding mode of PB to HSA remain unknown. To address this issue, we examined the crystal structure of HSA with PB bound to it. The structure of the HSA–PB complex indicates that the binding mode of PB to HSA is quite similar to that for octanoate or drugs that bind to drug site 2, as opposed to that for other medium-chain length of fatty acids. These findings provide useful basic information related to drug–HSA interactions. Moreover, the information presented herein is valuable in terms of providing safe and efficient treatment and diagnosis in clinical settings.     

Assessment of the Significance of Virulence Factors of Uropathogenic Escherichia coli in Experimental Urinary Tract Infection in Mice

Four Escherichia coli strains, isolated from cystitis patients, belonging to serotype O2:H^? and possessing different combinations of urovirulence factors were examined in an experimental pyelonephritis mouse model to assess the relative importance of virulence factors in causation of urinary tract infections (UTI). The results suggest not only that the each virulence factor has a role in causation of UTI but also that the presence of P fimbriae and production of hemolysin significantly reduced the LD₅₀ and ID₅₀ of the strains in the mouse model. The results also demonstrate that the presence of additional virulence factors acts in an additive or synergetic fashion enhancing the cumulative impact of the strain.     

Purification of endogenous inhibitors of [3H]flunitrazepam binding from bovine brain

Endogenous substances which inhibited the binding of [³H]flunitrazepam ([³H]FNZ) to bovine synaptosomal membranes have been purified from the hot acetic acid extracts of the bovine brain. Three peaks of inhibitory activity were obtained by Sephadex G-10 gel chromatography. Two of the peaks (Peak 2, and Peak 3) which had lower molecular weights that that of peak 1 were identified as inosine and hypoxanthine by TLC methods. Another peak (Peak 1) was further purified to homogeneity using both cation and anion ion-exchange chromatography and the following two-step reversed-phase HPLC. The purified substance inhibited the [³H]FNZ binding dose-dependently and competitively but did not have an effect on the binding of the peripheral-type BZ ligand [³H]Ro 5-4864. It was also shown that the substance was heat-stable and resistant to proteolytic degradation (trypsin, -chymotrypsin, pronase). However, a significant loss of inhibitory activity to [³H]FNZ binding was observed after acid hydrolysis. Molecular weight estimates based on gel filtration methods were less than 500 dalton, and the maximal ultraviolet absorption peak was at 314 nm. These results suggest that this substance is a new endogenous ligand for the central BZ receptor and may play an important role in regulating the GABAergic tone in the central nervous system.