Mapping contacts between regulatory domains of skeletal muscle TnC and TnI by analyses of single-chain chimeras

The troponin (Tn) complex is formed by TnC, TnI and TnT and is responsible for the calcium-dependent inhibition of muscle contraction. TnC and TnI interact in an antiparallel fashion in which the N domain of TnC binds in a calcium-dependent manner to the C domain of TnI, releasing the inhibitory effect of the latter on the actomyosin interaction. While the crystal structure of the core cardiac muscle troponin complex has been determined, very little high resolution information is available regarding the skeletal muscle TnI-TnC complex. With the aim of obtaining structural information regarding specific contacts between skeletal muscle TnC and TnI regulatory domains, we have constructed two recombinant chimeric proteins composed of the residues 1-91 of TnC linked to residues 98-182 or 98-147 of TnI. The polypeptides were capable of binding to the thin filament in a calcium-dependent manner and to regulate the ATPase reaction of actomyosin. Small angle X-ray scattering results showed that these chimeras fold into compact structures in which the inhibitory plus the C domain of TnI, with the exception of residues 148-182, were in close contact with the N-terminal domain of TnC. CD and fluorescence analysis were consistent with the view that the last residues of TnI (148-182) are not well folded in the complex. MS analysis of fragments produced by limited trypsinolysis showed that the whole TnC N domain was resistant to proteolysis, both in the presence and in the absence of calcium. On the other hand the TnI inhibitory and C-terminal domains were completely digested by trypsin in the absence of calcium while the addition of calcium results in the protection of only residues 114-137.     

Context alters the ability of clitoral stimulation to induce a sexually-conditioned partner preference in the rat

We have shown previously that clitoral stimulation (CLS) of female rats induces significant conditioned place preference (CPP), indicating that it is rewarding. The present study asked whether CLS could induce a conditioned partner preference. In the first experiment, sexually naïve females received 10 alternating trials of CLS and No-CLS in the presence of a male rat behind a wire-mesh screen. For one group, CLS was made in the presence of the male scented with almond extract. On alternating trials, those females received sham CLS in the presence of an unscented male behind the screen. The order was reversed for the other group. After 5 trials in each condition, females were placed into an open field with two sexually vigorous males, one scented and the other unscented. Contrary to expectation, females displayed a preference for the male associated with sham CLS. The second experiment examined whether a partner preference could be conditioned by associating CLS with the almond odor alone. A new group of sexually naive females received the same CLS-odor, No-CLS-No Odor pairings as above, but with the odor presented on cotton gauze in the chamber. During the final open field test, those females selectively solicited the scented male. We conclude that CLS that induces CPP also induces conditioned partner preference. However, we propose that CLS in the presence of an inaccessible male created a sexual inhibitory state for female rats.     

A component of the Xanthomonadaceae type IV secretion system combines a VirB7 motif with a N0 domain found in outer membrane transport proteins

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 Å X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7_XAC2622) and its interaction with VirB9. NMR solution studies show that residues 27–41 of the disordered flexible N-terminal region of VirB7_XAC2622 interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7_XAC2622 has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7_XAC2622 oligomerizes through interactions involving conserved residues in the N0 domain and residues 42–49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB7_XAC2622 oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.     

Antibodies to influenza A virus in wild birds across Mongolia, 2006-2009

Wild waterbirds sampled July 2006-September 2009 in Mongolia were tested for antibodies to avian influenza (AI) virus with the use of a commercially available blocking enzyme-linked immunosorbent assay. Antibodies were detected in 25% (572/2,282) of tested birds representing 26 species, and all antibody-positive samples were from 12 species in the orders Anseriformes and Charadriiformes. The highest antibody prevalence was in Ruddy Shelducks (Tadorna ferruginea; 61.7%; n=261; 95% confidence interval [CI] 55.8-67.6%), Whooper Swans (Cygnus cygnus; 38.4%; n=242; 95% CI 32.3-44.5%), Swan Geese (Anser cygnoides; 15%; n=127; 95% CI 8.6-21.4%), Bar-headed Geese (Anser indicus; 13%; n=738; 95% CI 10.3-15.1%), and Mongolian Gulls (Larus mongolicus; 3.9%; n=255; 95% CI 1.3-6.5%). There was no significant temporal or spatial variation in the presence of antibodies in the sampled species. However, Bar-headed Geese and Mongolian Gulls showed spatial variation in antibody prevalence in 2007 and 2008, respectively. Our study provides insights into the hatch year waterbirds' exposure to AI virus at their natal and molting sites in Mongolia.     

Selective delivery of 2-hydroxy APA to Trypanosoma brucei using the melamine motif

Trypanosoma brucei, the parasite that causes human African trypanosomiasis, is auxotrophic for purines and has specialist nucleoside transporters to import these metabolites. In particular, the P2 aminopurine transporter can also selectively accumulate melamine derivatives. In this Letter, we report the coupling of the melamine moiety to 2-hydroxy APA, a potent ornithine decarboxylase inhibitor, with the aim of selectively delivering this compound to the parasite. The best compound described here shows an increased in vitro trypanocidal activity compared with the parent.     

The PilB-PilZ-FimX regulatory complex of the Type IV pilus from Xanthomonas citri

Type IV pili (T4P) are thin and flexible filaments found on the surface of a wide range of Gram-negative bacteria that undergo cycles of extension and retraction and participate in a variety of important functions related to lifestyle, defense and pathogenesis. During pilus extensions, the PilB ATPase energizes the polymerization of pilin monomers from the inner membrane. In Xanthomonas citri, two cytosolic proteins, PilZ and the c-di-GMP receptor FimX, are involved in the regulation of T4P biogenesis through interactions with PilB. In vivo fluorescence microscopy studies show that PilB, PilZ and FimX all colocalize to the leading poles of X. citri cells during twitching motility and that this colocalization is dependent on the presence of all three proteins. We demonstrate that full-length PilB, PilZ and FimX can interact to form a stable complex as can PilB N-terminal, PilZ and FimX C-terminal fragments. We present the crystal structures of two binary complexes: i) that of the PilB N-terminal domain, encompassing sub-domains ND0 and ND1, bound to PilZ and ii) PilZ bound to the FimX EAL domain within a larger fragment containing both GGDEF and EAL domains. Evaluation of PilZ interactions with PilB and the FimX EAL domain in these and previously published structures, in conjunction with mutagenesis studies and functional assays, allow us to propose an internally consistent model for the PilB-PilZ-FimX complex and its interactions with the PilM-PilN complex in the context of the inner membrane platform of the X. citri Type IV pilus.     

Computerized automated morphometric assay including frequency estimation of pentachlorophenol induced nuclear anomalies (micronucleus) in catfish Heteropneustes fossilis

An in vivo study of the effects of pentachlorophenol was carried out with a pre-acclimatized fish species, Heteropneustes fossilis, using four sub-lethal concentrations, 0.1, 0.2, 0.3 and 0.4 ppm, and three sampling times, 48, 72 and 96 h. Cytogenetic preparations were stained by the haematoxylin-eosin technique. The incidence of micronuclei was scored by a manual and an automated method. Small-sized micronuclei appeared in the cytoplasm in addition to the main nucleus. The frequency of micronucleated erythrocytes peaked at 4 days (96 h) exposure. The percentage of single micronuclei increased with longer exposures. The Mann-Whitney U test showed all micronuclei frequencies were significantly different from control (P<0.05). No statistical difference was observed between scores obtained by the manual and automated methods. A linear relationship between the percentage of micronucleated erythrocytes and dose was confirmed at all levels. Computer image analysis of morphological variations of erythrocytes indicated a 1:5 ratio of micronuclei and main nucleus accompanied by a reduction in cell volume by 600 dot units. Pentachlorophenol-mediated genotoxicity was confirmed in this fish for the first time. Possible consequences of genotoxicity and cytotoxicity are discussed.     

Elucidation of the metabolic fate of glucose in the filamentous fungus Trichoderma reesei using expressed sequence tag (EST) analysis and cDNA microarrays

Despite the intense interest in the metabolic regulation and evolution of the ATP-producing pathways, the long standing question of why most multicellular microorganisms metabolize glucose by respiration rather than fermentation remains unanswered. One such microorganism is the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). Using EST analysis and cDNA microarrays, we find that in T. reesei expression of the genes encoding the enzymes of the tricarboxylic acid cycle and the proteins of the electron transport chain is programmed in a way that favors the oxidation of pyruvate via the tricarboxylic acid cycle rather than its reduction to ethanol by fermentation. Moreover, the results indicate that acetaldehyde may be channeled into acetate rather than ethanol, thus preventing the regeneration of NAD(+), a pivotal product required for anaerobic metabolism. The studies also point out that the regulatory machinery controlled by glucose was most probably the target of evolutionary pressure that directed the flow of metabolites into respiratory metabolism rather than fermentation. This finding has significant implications for the development of metabolically engineered cellulolytic microorganisms for fuel production from cellulose biomass.     

Adrenal cortex and stomach lesions associated with stress in wild male African green monkeys (Cercopithecus aethiops) in the post-capture period

The objective of this study was to look for early pathological changes in stress target organs, adrenal glands, and stomachs in captured wild African green monkeys (AGMs). Three wild-caught male AGMs and seven singly housed wild AGMs were euthanized on day 1 and day 45 post-capture, respectively, and compared with four wild males euthanized with a rifle as controls. Morphometric analyses of the adrenal cortices and the cortical zones were done using an image analyzer. By day 45, the confined animals were clinically healthy, but had lost 47% mean body weight despite ad libitum feeding. The width of zona fasciculata in the controls was significantly smaller compared with that of 45-day monkeys (P < 0.05). Numerous acidophilic, hyperplastic and hypertrophic cells were present in the zona fasciculata of the 1-day confined AGMs. In the 45-day monkeys, there was glandular hyperplasia in the zona glomerulosa and the acini were distended and vacuous; yellow, granular pigmentation was distributed in the zona fasciculata. Acute stomach lesions represented by petechiation were seen in one monkey on day 1. Deep, circular, mucosal erosions, one to five in number and measuring from 0.5 to 1 mm in diameter, were present in three monkeys on day 45 post-capture. There were no adrenal cortex or stomach lesions in the rifle-shot monkeys. In conclusion, pathological lesions in the adrenal glands, and stomachs of the wild AGMs and weight loss occurred within the initial 45-day period following capture and confinement.