The possibility that the strains included within the Mycobacterium avium complex (MAC), but not belonging either to M. avium or to Mycobacterium intracellulare, may be members of undescribed taxa, has already been questioned by several taxonomists. A very homogeneous cluster of 12 strains characterized by identical nucleotide sequences both in the 16S rDNA and in the 16S–23S internal transcribed spacer was investigated. Similar strains, previously reported in the literature, had been assigned either to the species M. intracellulare on the basis of the 16S rDNA similarity or to the group of MAC intermediates. However, several phenotypical and epidemiological characteristics seem to distinguish these strains from all other MAC organisms. The unique mycolic acid pattern obtained by HPLC is striking as it is characterized by two clusters of peaks, instead of the three presented by all other MAC organisms. All of the strains have been isolated from humans and all but one came from the respiratory tract of elderly people. The clinical significance of these strains, ascertained for seven patients, seems to suggest an unusually high virulence. The characteristics of all the strains reported in the literature, genotypically identical to the ones described here, seem to confirm our data, without reports of isolations from animals or the environment or, among humans, from AIDS patients. Therefore, an elevation of the MAC variant was proposed and characterized here, with the name Mycobacterium chimaera sp. nov.; this increases the number of species included in the M. avium complex. The type strain is FI-01069T (=CIP 107892T=DSM 44623T).

Steroidogenic acute regulatory protein-related lipid transfer (StART) domains are ubiquitously involved in intracellular lipid transport and metabolism and other cell-signaling events. In this work, we use a flexible docking algorithm, comparative modeling, and molecular dynamics (MD) simulations to generate plausible three-dimensional atomic models of the StART domains of human metastatic lymph node 64 (MLN64) and steroidogenic acute regulatory protein (StAR) proteins in complex with cholesterol. Our results show that cholesterol can adopt a similar conformation in the binding cavity in both cases and that the main contribution to the protein-ligand interaction energy derives from hydrophobic contacts. However, hydrogen-bonding and water-mediated interactions appear to be important in the fine-tuning of the binding affinity and the position of the ligand. To gain insights into the mechanism of binding, we carried out steered MD simulations in which cholesterol was gradually extracted from within the StAR model. These simulations indicate that a transient opening of loop 61 may be sufficient for uptake and release, and they also reveal a pathway of intermediate states involving residues known to be crucial for StAR activity. Based on these observations, we suggest specific mutagenesis targets for binding studies of cholesterol and its derivatives that could improve our understanding of the structural determinants for ligand binding by sterol carrier proteins.-Murcia, M., J. D. Faráldo-Gómez, F. R. Maxfield, and B. Roux. Modeling the structure of the StART domains of MLN64 and StAR proteins in complex with cholesterol.

Forty-five mycobacterial strains isolated from 23 Colombian HIV-positive patients were identified as members of the Mycobacterium avium complex (MAC) and were characterized using different molecular approaches. Seven of the isolates showed characteristic features that allowed them to be differentiated from other members of the complex. The isolates had a novel 16S-23S rRNA internal transcribed spacer (ITS 1) gene sequence which is described as a new sequevar, MAC-X. All of the seven novel isolates gave a positive result with the MAC-specific AccuProbe (Gen-Probe), but tested negative for Mycobacterium avium and Mycobacterium intracellulare species-specific probes (64 and 100 % of the isolates, respectively). The novel isolates could be differentiated phenotypically from other members of the MAC on the basis of the production of urease and by a consistent mycolic acid pattern. The novel isolates shared some characteristics with M. avium, such as the avium variant I (av-I) pattern of the hsp65 gene as determined by PCR restriction analysis and a positive PCR result for the mig (macrophage-induced) gene. However, the novel isolates showed a unique 16S rRNA gene sequence. DNA-DNA relatedness values, from 24 to 44 %, confirmed the distinction of the novel isolates from other members of the MAC at the genetic level and their status as members of a separate species. The novel isolates are proposed as representatives of a novel species, Mycobacterium colombiense sp. nov., that is closely related to M. avium within the MAC. The type strain is 10B T (=CIP 108962Members of the Mycobacterium avium complex (MAC) are currently identified on the basis of positive results with the commercial MAC-specific probe, AccuProbe (Gen-Probe). Originally, two species were identified within the MAC, namely Mycobacterium avium and Mycobacterium intracellulare. However, recent studies have drawn attention to the wide diversity of isolates that can be detected in the complex (Mijs et al., 2002;Smole et al., 2002;Lebrun et al., 2005). Analysis of the 16S-23S rRNA internal transcribed spacer (ITS 1) sequence, a molecular approach frequently used for the identification of these bacteria, has revealed the existence of more than 30 different sequevars (Tortoli et al., 2004). Only a few of these sequevars belong to members of M. avium (seven sequevars) or M. intracellulare (four sequevars). Recently, members of the MAC represented by sequevar MAC-A were described as the novel species Mycobacterium chimaera. This novel species has similar characteristics to M. intracellulare (Tortoli et al., 2004).During a study to characterize MAC isolates from HIVpositive Colombian patients, a group of distinct strains was identified. These isolates showed several features that allowed them to be differentiated from other members of the MAC and recognized as a separate novel species.Abbreviations: ITS 1, 16S-23S rRNA internal transcribed spacer; MAC, Mycobacterium avium complex; PRA, PCR restriction analysis; RAPD, randomly amplified polymorphic DNA.The GenBank/E...

IntroductionThe platelet ␣IIb␤3 integrin plays a central role in platelet adhesion and aggregation. [1][2][3] Thus, it can support platelet adhesion to immobilized fibrinogen even in the absence of exogenous activators. 4,5 Moreover, when activated, the ␣IIb␤3 heterodimer can bind soluble ligands, including fibrinogen and von Willebrand factor, which can span between platelets to form aggregates. 1,3,6,7 Loss of the receptor or its function on an inherited basis results in the hemorrhagic diathesis Glanzmann thrombasthenia, 8 and inhibitors of the receptor have proven effective in the prevention and treatment of coronary artery thrombosis. 9,10 Biochemical, molecular biologic, and crystallographic evidence indicate that ligands bind to a groove in ␣IIb␤3 that is at the intersection of the ␣IIb ␤ propeller domain and the ␤3 ␤A (I-like) domain. 11 Fibrinogen binds to ␣IIb␤3 via a carboxyl-terminal dodecapeptide sequence in its ␥ chain that contains both a positively charged Lys and a negatively charged Asp (HHLGGAKQAGDV). [12][13][14] The integrin also binds ligands containing the sequence Arg-Gly-Asp (RGD) or Lys-Gly-Asp (KGD), including von Willebrand factor 6,15 and snake venom-derived disintegrins. 16 The drugs eptifibatide and tirofiban, which are patterned after the KGD and RGD sequences, respectively, span the ␣IIb␤3 ligand binding groove with orientations similar to that of an RGD-containing peptide (cilengitide) in the related receptor ␣V␤3 17 ; thus, their positively charged groups interact with ␣IIb Asp224 and their negatively charged carboxyl groups contribute to the coordination of the metal ion in the ␤3 metal ion-dependent adhesion site (MIDAS). 11 Conformational changes in ␣IIb␤3 occur upon receptor activation, and additional changes occur after the binding of ligand to the receptor, leading to the exposure of ligand-induced binding sites (LIBS) that can be detected by LIBS-specific monoclonal antibodies (mAbs). [18][19][20][21] The binding of RGD peptides and both eptifibatide and tirofiban increase the binding of LIBS-specific mAbs. 22 Since ␣IIb␤3 may remain in its high-affinity conformation after dissociation of the competitive inhibitors, transient interactions of these compounds with the receptor may actually facilitate ligand binding by "priming" the receptor. 23 It has been postulated that this effect may have contributed to the increased mortality observed during treatment with orally active inhibitors of ␣IIb␤3 that were administered on a chronic basis. [24][25][26][27][28][29] Moreover, the conformational changes induced by all of the antagonists may contribute to the thrombocytopenia observed with these agents. 30 To identify novel small molecules capable of inhibiting the interaction of fibrinogen with ␣IIb␤3, we used high-throughput screening of several libraries of small molecules, testing the ability of the compounds to inhibit platelet adhesion to fibrinogen. We identified one compound with unique features that provide insights into ␣IIb␤3 structure and function. Methods Monoclonal...