Alternative Exon 9-Encoded Relay Domains Affect More than One Communication Pathway in the Drosophila Myosin Head

We investigated the biochemical and biophysical properties of one of the four alternative regions within the Drosophila myosin catalytic domain: the relay domain encoded by exon 9. This domain of the myosin head transmits conformational changes in the nucleotide-binding pocket to the converter domain, which is crucial to coupling catalytic activity with mechanical movement of the lever arm. To study the function of this region, we used chimeric myosins (IFI-9b and EMB-9a), which were generated by exchange of the exon 9-encoded domains between the native embryonic body wall (EMB) and indirect flight muscle isoforms (IFI). Kinetic measurements show that exchange of the exon 9-encoded region alters the kinetic properties of the myosin S1 head. This is reflected in reduced values for ATP-induced actomyosin dissociation rate constant (K₁k₊₂) and ADP affinity (K_AD), measured for the chimeric constructs IFI-9b and EMB-9a, compared to wild-type IFI and EMB values. Homology models indicate that, in addition to affecting the communication pathway between the nucleotide-binding pocket and the converter domain, exchange of the relay domains between IFI and EMB affects the communication pathway between the nucleotide-binding pocket and the actin-binding site in the lower 50-kDa domain (loop 2). These results suggest an important role of the relay domain in the regulation of actomyosin cross-bridge kinetics.     

Alternative relay domains of Drosophila melanogaster myosin differentially affect ATPase activity, in vitro motility, myofibril structure and muscle function

The relay domain of myosin is hypothesized to function as a communication pathway between the nucleotide-binding site, actin-binding site and the converter domain. In Drosophila melanogaster, a single myosin heavy chain gene encodes three alternative relay domains. Exon 9a encodes the indirect flight muscle isoform (IFI) relay domain, whereas exon 9b encodes one of the embryonic body wall isoform (EMB) relay domains. To gain a better understanding of the function of the relay domain and the differences imparted by the IFI and the EMB versions, we constructed two transgenic Drosophila lines expressing chimeric myosin heavy chains in indirect flight muscles lacking endogenous myosin. One expresses the IFI relay domain in the EMB backbone (EMB-9a), while the second expresses the EMB relay domain in the IFI backbone (IFI-9b). Our studies reveal that the EMB relay domain is functionally equivalent to the IFI relay domain when it is substituted into IFI. Essentially no differences in ATPase activity, actin-sliding velocity, flight ability at room temperature or muscle structure are observed in IFI-9b compared to native IFI. However, when the EMB relay domain is replaced with the IFI relay domain, we find a 50% reduction in actin-activated ATPase activity, a significant increase in actin affinity, abolition of actin sliding, defects in myofibril assembly and rapid degeneration of muscle structure compared to EMB. We hypothesize that altered relay domain conformational changes in EMB-9a impair intramolecular communication with the EMB-specific converter domain. This decreases transition rates involving strongly bound actomyosin states, leading to a reduced ATPase rate and loss of actin motility.     

Membrane-attached biofilms for VOC wastewater treatment. II: Effect of biofilm thickness on performance

This article reports a study of the performance of membrane-attached biofilms grown in a single tube extractive membrane bioreactor (STEMS) used for the treatment of a synthetic wastewater containing a toxic VOC (1,2-dichloroethane [DCE]). Mass balances show that complete mineralization of DCE was achieved, and that the biofilms were effective in reducing air stripping to negligible levels. Experimental results are presented showing the evolution over time of biofilm thickness and its influence on the flux of DCE across the membrane. It has been found that a trade-off exists between the positive influence of biofilms in reducing air-stripping of DCE, and the negative influence of biofilms in reducing DCE flux across the membrane. These considerations lead to an optimal biofilm thickness in the region of 200 to 400 mum being recommended for this system. (c) 1995 John Wiley & Sons, Inc.     

Functional assignment to maize group 1 LEA protein EMB564 within the cell nucleus using computational analysis

Maize protein EMB564 is a member of group 1 LEA (late embryogenesis abundant) proteins. Currently, the molecular functions of group 1 LEA proteins remain largely unclear. We here report on the functional assignment to EMB564 by computational analysis. EMB564 is predicted as nuclear localization by five different predictors including CELLO, Plant-mPLoc, WoLF PSORT, Predotar and TargetP. EMB564 is found to be remote homologous with DNA/RNA helicases and single-stranded DNA-binding proteins, and their sequences contains similar DNA/RNA binding sites. Furthermore, the three-dimensional (3D) model of EMB564 structurally resembles a variety of nuclear and DNA/RNA-binding proteins, especially those involving in the regulation of cell division, chromosomal replication and DNA unwinding or repairing. Our results reveal that EMB564 protein is most likely to function within the cell nucleus.     

Proteomic identification of differentially expressed proteins in Arabidopsis in response to methyl jasmonate

Jasmonates (JAs) are the well characterized fatty acid-derived cyclopentanone signals involved in the plant response to biotic and abiotic stresses. JAs have been shown to regulate many aspects of plant metabolism, including glucosinolate biosynthesis. Glucosinolates are natural plant products that function in defense against herbivores and pathogens. In this study, we applied a proteomic approach to gain insight into the physiological processes, including glucosinolate metabolism, in response to methyl jasmonate (MeJA). We identified 194 differentially expressed protein spots that contained proteins that participated in a wide range of physiological processes. Functional classification analysis showed that photosynthesis and carbohydrate anabolism were repressed after MeJA treatment, while carbohydrate catabolism was up-regulated. Additionally, proteins related to the JA biosynthesis pathway, stress and defense, and secondary metabolism were up-regulated. Among the differentially expressed proteins, many were involved in oxidative tolerance. The results indicate that MeJA elicited a defense response at the proteome level through a mechanism of redirecting growth-related metabolism to defense-related metabolism.     

Cloning and tissue distribution of three murine alpha/beta hydrolase fold protein cDNAs

We have cloned 3 novel murine cDNAs encoding proteins containing an alpha/beta hydrolase fold; a catalytic domain found in a very wide range of enzymes. These proteins belong to the prosite UPF0017 uncharacterized protein family and we have named them lung alpha/beta hydrolase 1, 2, and 3 (LABH) since they were cloned from lung cDNA. All have 9 coding exons, encoding 412, 425, and 411 residue proteins respectively (46-48 kDa); LABH1 being closely related to LABH3 having 45% identity. All 3 proteins have a single predicted amino-terminus transmembrane domain. An alignment of family members from different phyla enabled the identification of the LABH1 catalytic triad as Ser211, Asp337, and His366. mRNA expression levels of LABH1 and 3 were highest in liver and LABH2 highest in testis. These findings suggest that the LABH proteins consist of a novel family of membrane bound enzymes whose function has yet to be determined.     

Uncovering the Post-embryonic Role of Embryo Essential Genes in Arabidopsis using the Controlled Induction of Visibly Marked Genetic Mosaics: EMB506, an Illustration

The embryo essential gene EMB506 plays a crucial role in the transition of the Arabidopsis embryo from radial symmetry to bilateral symmetry just prior to the early heart stage of development. In addition to influencing embryo development EMB506 also affects chloroplast biogenesis. To further investigate the role of EMB506 gene expression in Arabidopsis we have generated green fluorescent protein (GFP) marked emb506 mosaic sectors at temporally defined stages during embryogenesis and additionally during various stages of vegetative growth, in otherwise phenotypically wild-type plants. We confirm the essential requirement for EMB506 gene expression in chloroplast biogenesis as reflected by the decreased chlorophyll content in emb506 mosaic sectors. We also show that the influence of EMB506 gene expression as it impinges on chloroplast biogenesis is first relevant at an intermediate stage in embryogenesis and that the role of EMB506 gene expression in chloroplast biogenesis is distinct from the essential role of EMB506 gene expression during early embryo development. By inducing emb506 mosaicism after the essential requirement for EMB506 gene expression in embryogenesis and also during vegetative growth we reveal that EMB506 gene expression additionally is required for correct cotyledon-, true leaf- and cauline leaf margin development. The strategy that we describe can be tailored to the mosaic analysis of any cloned EMB gene for which a corresponding mutant exists and can be applied to the mosaic analysis of mutant lethal genes in general.     

Development of a single multiplex amplification refractory mutation system PCR for the detection of rifampin-resistant Mycobacterium tuberculosis

A rapid and simple method for the detection of drug-resistant Mycobacterium tuberculosis is critical for the efficient treatment and control of this pathogen in developing country. Here we developed a single multiplex amplification refractory mutation system (M-ARMS) PCR, in which chimeric-primer and temperature switch PCR (TSP) strategy were included. Using this method, we detected rifampin resistance-associated mutations at codons 511, 516, 526 and 531 in the rifampin resistance-determining region of rpoB gene. The performance of M-ARMS-PCR assay was evaluated with 135 cultured isolates of M. tuberculosis. The sensitivity and specificity were 94.2% and 100%, respectively, compared with direct DNA sequencing, and 86.67% and 89.71%, respectively, compared with culture-based phenotypic drug susceptibility testing. Therefore, this newly-developed M-ARMS-PCR method is useful and efficient with an intended application in provincial Centers for Disease Control and Prevention for rapid detection of rifampin resistance-associated mutations.     

A variable domain near the ATP-binding site in Drosophila muscle myosin is part of the communication pathway between the nucleotide and actin-binding sites

Drosophila expresses several muscle myosin isoforms from a single gene by alternatively splicing six of the 19 exons. Here we investigate exon 7, which codes for a region in the upper 50 kDa domain near the nucleotide-binding pocket. This region is of interest because it is also the place where a large insert is found in myosin VI and where several cardiomyopathy mutations have been identified in human cardiac myosin. We expressed and purified chimeric muscle myosins from Drosophila, each varying at exon 7. Two chimeras exchanged the entire exon 7 domain between the indirect flight muscle (IFI, normally containing exon 7d) and embryonic body wall muscle (EMB, normally containing exon 7a) isoforms to create IFI-7a and EMB-7d. The second two chimeras replaced each half of the exon 7a domain in EMB with the corresponding portion of exon 7d to create EMB-7a/7d and EMB-7d/7a. Transient kinetic studies of the motor domain from these myosin isoforms revealed changes in several kinetic parameters between the IFI or EMB isoforms and the chimeras. Of significance were changes in nucleotide binding, which differed in the presence and absence of actin, consistent with a model in which the exon 7 domain is part of the communication pathway between the nucleotide and actin-binding sites. Homology models of the structures suggest how the exon 7 domain might modulate this pathway.