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International audience; The botulinum neurotoxins (BoNTs) are di-chain bacterial proteins responsible for the paralytic disease botulism. Following binding to the plasma membrane of cholinergic motor nerve terminals, BoNTs are internalized into an endocytic compartment. Although several endocytic pathways have been characterized in neurons, the molecular mechanism underpinning the uptake of BoNTs at the presynaptic nerve terminal is still unclear. Here, a recombinant BoNT/A heavy chain binding domain (Hc) was used to unravel the internalization pathway by fluorescence and electron microscopy. BoNT/A-Hc initially enters cultured hippocampal neurons in an activity-dependent manner into synaptic vesicles and clathrin-coated vesicles before also entering endosomal structures and multivesicular bodies. We found that inhibiting dynamin with the novel potent Dynasore analog, Dyngo-4a(TM), was sufficient to abolish BoNT/A-Hc internalization and BoNT/A-induced SNAP25 cleavage in hippocampal neurons. Dyngo-4a also interfered with BoNT/A-Hc internalization into motor nerve terminals. Furthermore, Dyngo-4a afforded protection against BoNT/A-induced paralysis at the rat hemidiaphragm. A significant delay of >30% in the onset of botulism was observed in mice injected with Dyngo-4a. Dynamin inhibition therefore provides a therapeutic avenue for the treatment of botulism and other diseases caused by pathogens sharing dynamin-dependent uptake mechanisms.

In a laboratory-scale combustion reactor, flue-gas samples were collected at two temperatures in the post-combustion zone, 700°C and 400°C, using two different water-cooled sampling probes. The probes were the cooled probe described in the European Standard method EN-1948:1, referred to as the original probe, and a modified probe that contained a salt/ice mixture to assist the cooling, referred to as the sub-zero probe. To determine the efficiency of the cooling probes, internal temperature measurements were recorded at 5cm intervals inside the probes. Flue-gas samples were analyzed for polychlorinated dibenzo-p-dioxin and dibenzofurans (PCDD/Fs). Samples collected at 700°C using the original cooling probe showed higher concentrations of PCDD/Fs compared to samples collected using the sub-zero probe. No significant differences were observed between samples collected at 400°C. The results indicated that artifact formation of PCDD/Fs readily occurs during flue-gas sampling at high temperatures if the cooling within the probe is insufficient, as found for the original probe at 700°C. It was also shown that this problem could be alleviated by using probes with an enhanced cooling capacity, such as the sub-zero probe. Although this may not affect samples collected for regulatory purposes in exit gases, it is of great importance for research conducted in the high-temperature region of the post-combustion zone.

Herein we report the synthesis of discrete iminochromene ("iminodyn") libraries (14-38) as potential inhibitors of dynamin GTPase. Thirteen iminodyns were active (IC(50) values of 260 nM to 100 microM), with N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (17), N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (22), and N,N-(ethane-1,2-diyl)bis(7,8-dihydroxy-2-iminochromene-3-carboxamide) (23) (IC(50) values of 330 +/- 70, 450 +/- 50, and 260 +/- 80 nM, respectively) being the most potent. Five of the most potent iminodyns all inhibited dynamins I and II approximately equally. Iminodyn-22 displayed uncompetitive inhibition with respect to GTP. Selected iminodyns were evaluated for their ability to block receptor mediated endocytosis (RME, mediated by dynamin II) and synaptic vesicle endocytosis (SVE, mediated by dynamin I), with 17 showing no activity while 22 returned RME and SVE IC(50) values of 10.7 +/- 4.5 and 99.5 +/- 1.7 microM, respectively. The iminodyns reported herein represent a new chemical class of the first nanomolar potent dynamin inhibitors that are also effective endocytosis inhibitors.

A series of halogen containing 1,4-naphthoquinon derivatives (1-4,5a-f) were synthesized and studied for their antifungal activities against C. albicans ATCC10231, C. albicans 955, T. mentagrophytes and M. gypseum. The results indicate that compound 2-hydroxy-3-chloro-1,4-naphthoquinone (2), 2-(N-acetyl)- acetamido-3-chloro- 1,4-napthoquinone (3) and 2-(N-acetyl)-acetamido-3-chloro- 1,4- napthoquinone (4) have potent antifungal activity. Among these promising antifungal candidates, 2 and 4 showed better activity than that of clinically antifungal drug clotrimazole (MIC = 8 μg/ml) with MIC = 1 μg/ml and 4 μg/ml, respectively against C.albicans ATCC10231. Compound 2 also exhibited an extremely potent activity (MIC = 0.25 μg/ml) against C.albicans 955 strain compared with clotrimazole (MIC = 16 μg/ml). Structure and activity relationship (SAR) study demonstrated that replacing of 3-position in 1,4- naphthoquinon by a Cl group is essential for antifungal activity. Meanwhile, antifungal activity was decreased considerably when the hydrogen atom at position-2 in naphthoquinone structure were replaced by a bulky group (e.g. diacetyl of phenyl group).

Synaptic vesicles (SVs) are retrieved by more than one mode in central nerve terminals. During mild stimulation, the dominant SV retrieval pathway is classical clathrin-mediated endocytosis (CME). During elevated neuronal activity, activity-dependent bulk endocytosis (ADBE) predominates, which requires activation of the calcium-dependent protein phosphatase calcineurin. We now report that calcineurin dephosphorylates dynamin I in nerve terminals only above the same activity threshold that triggers ADBE. ADBE was arrested when the two major phospho-sites on dynamin I were perturbed, suggesting that dynamin I dephosphorylation is a key step in its activation. Dynamin I dephosphorylation stimulates a specific dynamin I-syndapin I interaction. Inhibition of this interaction by competitive peptides or by site-directed mutagenesis exclusively inhibited ADBE but did not affect CME. The results reveal that the phospho-dependent dynamin-syndapin interaction recruits ADBE to massively increase SV endocytosis under conditions of elevated neuronal activity.

Probing the dynamin binding site: Bis-tyrphostin (1, Bis-T), is a potent inhibitor of the phospholipid-stimulated GTPase activity of dynamin I. Analogues of Bis-T have significant potential as a biological probes for the dissection of endocytic pathways. Bis-T-derived compounds were synthesised and evaluated for their ability to inhibit the GTPase activity of dynamin I. Two analogues (23 and 24) represent the first asymmetrically substituted Bis-T analogues to retain dynamin inhibition.Two azidobenzyl amide (4 and 23) and one 3-trifluoromethyl-3H-diazirin-3-ylphenyl (24) analogues of bis-tyrphostin (1, Bis-T) were synthesised as potential photoaffinity labels for the elucidation of the binding site of compound 1 in dynamin I. Of the two azidobenzyl amide analogues (4 and 23), the terminally substituted 23 retained dynamin I GTPase inhibition (IC(50)=6.4+/-2.8 microM) whilst 4, which was substituted on the central carbon of the amide linker, displayed no activity. Analogue 24 also retained inhibitory activity (IC(50)=36+/-9 microM). Photoaffinity labelling experiments did not unequivocally elucidate the binding pocket of compound 1. However, compounds 23 and 24 represent the first asymmetrically substituted Bis-T analogues to retain dynamin inhibitory activity, providing a new direction for analogue synthesis.

Tetraalkylphosphonium halides play the role of “promoters” for the previously unknown PtII-catalyzed hydroamination of alkenes. The promoting effect of these “cocatalysts” depends only upon their anionic moiety. The PtII/nBu4PBr system exhibits an unprecedented catalytic activity (aerobic conditions) for the hydroamination of ethylene and 1-hexene (regioselectivity > 95 %, Markovnikov).(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

Nowadays, semantic information accompanying 3D graphical data, which is called metadata, has been realized to be essential for several purposes such that available 3D graphical data can be indexed and retrieved by means of its metadata and then other applications can make use of the 3D graphical data efficiently. Even though a number of works have focused on this topic so far, no temporal aspect of 3D graphical data has been taken into account. Especially no complete automatic metadata extraction process has been proposed. A manual process is possible with a small amount of 3D graphical data while impossible with a larger amount. Therefore, the manual process gets heavy and the cost in time and effort is so much required. In such a context, we introduce for 3D graphical data a temporal meta database system where an automatic metadata extraction method takes into consideration the temporal aspect of 3D graphical data. 3D graphical data is arranged in a scene formatted in the standard language VRML. Semantic information of the scene, i.e. metadata, will be automatically achieved. The structure of scene metadata is handled by using the temporal NIAM object oriented conceptual schema model at the abstraction level so that the semantic scene information can get more understandable to users, graphics designers, and application programmers as well. As a result, scene metadata is able to be easily stored and queried by means of some standardized database language such as SQL or OQL. With the aid of the extracted scene metadata, the respective 3D scenes are now exactly and conveniently accessed by other applications such as a virtual guide in transportation, architecture, tourism, etc.

Since 1995, star schema has been popularly utilized to represent warehouse data at the logical level. Information warehouse design mainly focuses on the design of a warehouse star schema. There are many proposals on conceptual data warehouse design of a star schema based on some well-known data models such as the entity relationship model (ERM), and an extension to the Unified Modeling Language (UML). This paper presents the design and implementation of an information warehouse using the object role model (ORM) as the data model both for user modeling of the warehouse and for the underlying implementation model of the system. A warehouse multi-star/star schema is automatically generated from the input schema description based on ORM using a deep-structured natural language interface. Users input simple English sentences together with the structured sections for type declarations and constraints on these sentences. Since the input ORM schema can also be transformed into 5NF operational relational schemas, this system allows warehouse users to generate star schemas from their operational schemas

The proteins of homeobox (HOX) genes are transcription regulators involved in cell type-specific differentiation and patterning of the body plan in vertebrates. Particularly, the HOXD family is involved in limb formation in mice and chicks. There is also some evidence that the HOXD9 gene, a member of the HOXD family, is involved in the pathology of rheumatoid arthritis (RA). The purpose of the present study was to determine if the HOXD9 protein was expressed in RA synovium and then to characterize the HOXD9-expressing cell. Western blotting and immunohistochemical analysis showed that the HOXD9 protein was expressed in the synovium from patients with RA, but not in those from patients with osteoarthritis or healthy individuals. The HOXD9-positive cells were localized in both the lining and sublining areas of the synovium. Furthermore, fluorescent double-staining showed that the HOXD9 protein was expressed in fibroblast-like synoviocytes (FLS). These findings not only indicate that the HOXD9 gene is exclusively expressed in the RA synovium but also suggest that the HOXD9 gene contributes to the pathology of rheumatoid arthritis through the FLS.