Substrate-triggered recruitment of the TolC channel-tunnel during type I export of hemolysin by Escherichia coli.

A defining event in type I export of hemolysin by Escherichia coli is the substrate-triggered recruitment of the TolC channel-tunnel by an inner membrane complex. This complex comprises a traffic ATPase (HlyB) and the 478 residue adaptor protein (HlyD), which contacts TolC during recruitment. HlyD has a large periplasmic domain (amino acid residues 81-478) linked by a single transmembrane helix to a small N-terminal cytosolic domain (1-59). Export was disabled by deletion of the ca 60 amino acid residue cytosolic domain of HlyD, even though the truncated HlyD (HlyDDelta45) was, like the wild-type, able to trimerise in the cytosolic membrane, and interact with the traffic ATPase. The mutant HlyB/HlyDDelta45 inner membrane complex engaged the hemolysin substrate, but this substrate-engaged complex failed to trigger recruitment of TolC. Further analyses showed that HlyDDelta45 was specifically unable to bind the substrate. The result suggests that substrate engagement by the traffic ATPase alone is insufficient to trigger TolC recruitment, and that substrate binding to the HlyD cytosolic domain is essential. Analysis of three further N-terminal deletion variants, HlyDDelta26, HlyDDelta26-45 and HlyDDelta34-38, indicated that an extreme N-terminal amphipathic helix and a cytosolic cluster of charged residues are central to the cytosolic domain function. The cytosolic amphipathic helix was not essential for substrate engagement or TolC recruitment, but export was impaired without it. In contrast, when the charged amino acid residues were deleted, the substrate was still engaged by HlyD but engagement was unproductive, i.e. TolC recruitment was not triggered. Our results are compatible with the HlyD cytosolic domain mediating transduction of the substrate binding signal directly, presumably to the HlyD periplasmic domain, to trigger recruitment of TolC and assemble the type I export complex.     

Regeneration and micrografting of lentil shoots

Summary A protocol for regeneration and micrografting of shoots of lentil (Lens culinaris Medik) was developed. Multiple shoots (4–5) were regenerated from cotyledonary node explants on Murashige and Skoog (MS) medium containing 8.8 μM 6-benzylaminopurine. In vitro regenerated shoots were micrografted on rootstocks with 96% efficiency. The successful grafts were transplanted to pots in Redi-earth^TM, hardened off and were grown to maturity with 100% success. The success of the micrografting was independent of the nature and concentration of growth regulator used in shoot initiation medium and the time period for induction of shoots. The protocol was successful with several cultivars of lentil. The advantages of micrografting over in vitro rooting are discussed.     

Bayesian Insight into a Robust Test for Linear Contrast Based on Asymmetric Censored Samples

A robust test for linear contrast using modified maximum likelihood estimators based on symmetrically censored samples proposed by Tiku (1973, 1982a) is studied in this paper from the Bayesian point of view. The effects of asymmetric censoring on this testing procedure is investigated and a good approximation to its posterior distribution in this case is worked out. We also present an example which illustrates the method of obtaining the highest posterior density interval for the linear combination of the unknown location parameters.     

Mapping Interactions between Myosin Relay and Converter Domains That Power Muscle Function

Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile⁵⁰⁸, Asn⁵⁰⁹, and Asp⁵¹¹) in communicating with converter domain residue Arg⁷⁵⁹. We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle.     

Chick embryonic cells as a source for generating in vitro model of muscle cell dystrophy

Chick embryonic cells can be used to develop an easy and economical in vitro model for conducting studies on the disease muscle dystrophy (MD). For this, the limb myoblasts from 11th day chick embryo were isolated and cultured. To this muscle cell culture, anti-dystroglycan antibody (IIH6) was added so as to target the α-dystroglycan and disrupt the connection between the cytoskeletal proteins and the extracellular matrix (which is a characteristic feature of MD). Cells were allowed to differentiate further and the morphometrics and mRNA expression were studied. The IIH6-treated muscle cells displayed changes in morphometry, contractibility, and also atrophy was observed when compared to the control cultures. Concomitant gene expression studies showed an upregulation in TGF-β expression, while the muscle sculpture genes MYOD1, MYF5, LAMA2 and MYOG were downregulated resembling the MD in vivo. This simple and cost-effective method can be useful in studies to further understand the disease mechanism and also in conducting initial studies on effect of novel pharmacological agents.