Myofibrillar proteins assemble to form the highly ordered repetitive contractile structural unit known as a sarcomere. Studies of myogenesis in vertebrate cell culture and embryonic developmental systems have identified some of the processes involved during sarcomere formation. However, isoform changes during vertebrate muscle development and a lack of mutants have made it difficult to determine how these proteins assemble to form sarcomeres. The indirect flight muscles (IFMs) of Drosophila provide a unique genetic system with which to study myofibrillogenesis in vivo. We show in this paper that neither sarcomeric myosin nor actin are required for myoblast fusion or the subsequent morphogenesis of muscle fibres, i.e. fibre morphogenesis does not depend on myofibrillogenesis. However, fibre formation and myofibrillogenesis are very sensitive to the interactions between the sarcomeric proteins. A troponin I (TnI) mutation, hdp3, leads to an absence of TnI in the IFMs and tergal depressor of trochanter (TDT) muscles due to a transcript-splicing defect. Sarcomeres do not form and the muscles degenerate. TnI is part of the thin filament troponin complex which regulates muscle contraction. The effects of the hdp3 mutation are probably caused by unregulated acto-myosin interactions between the thin and thick filaments as they assemble. We have tested this proposal by using a transgenic myosin construct to remove the force-producing myosin heads. The defects in sarcomeric organisation and fibre degeneration in hdp3 IFMs are suppressed, although not completely, indicating the need for inhibition of muscle contraction during muscle development. We show that mRNA and translated protein products of all the major thin filament proteins are reduced in hdp3 muscles and discuss how this and previous studies of thin filament protein mutants indicate a common co-ordinated control mechanism that may be the primary cause of the muscle defects.

During myofibrillogenesis, many muscle structural proteins assemble to form the highly ordered contractile sarcomere. Mutations in these proteins can lead to dysfunctional muscle and various myopathies. We have analyzed the Drosophila melanogaster troponin T (TnT) up 1 mutant that specifically affects the indirect flight muscles (IFM) to explore troponin function during myofibrillogenesis. The up 1 muscles lack normal sarcomeres and contain ''zebra bodies,'' a phenotypic feature of human nemaline myopathies. We show that the up 1 mutation causes defective splicing of a newly identified alternative TnT exon (10a) that encodes part of the TnT C terminus. This exon is used to generate a TnT isoform specific to the IFM and jump muscles, which during IFM development replaces the exon 10b isoform. Functional differences between the 10a and 10b TnT isoforms may be due to different potential phosphorylation sites, none of which correspond to known phosphorylation sites in human cardiac TnT. The absence of TnT mRNA in up 1 IFM reduces mRNA levels of an IFM-specific troponin I (TnI) isoform, but not actin, tropomyosin, or troponin C, suggesting a mechanism controlling expression of TnT and TnI genes may exist that must be examined in the context of human myopathies caused by mutations of these thin filament proteins.

The aim of the present study was to investigate biosurfactant-producing culturable bacteria inhabiting the coast of the Arabian Sea at Karachi. Overall, 15 seawater samples were collected from the Arabian Sea coast of Karachi. Isolation, characterization, and screening for 89 biosurfactant-producing bacterial strains were conducted through 8 conventional screening tests. Through GSP agar plate method 22 strains were found to be Pseudomonas aeruginosa and Gram reaction revealed 70% of the isolates to be gramnegative. Furthermore, 24% of the isolates showed hemolytic activity, 44% exhibited positive results for oil-spreading test, 54% showed emulsification to at least 1 of the 3 hydrocarbons tested, BATH assay results indicated maximum adhesion for hexane, 52.8% produced positive results for CTAB agar plate assay, drop-collapse activity was found in 84% of the isolates, and emulsification assay revealed highest emulsification for xylene. Findings revealed none of the isolates to be negative for every screening test conducted, while only one gram-negative isolate, DGHE65, identified as Pseudomonas aeruginosa, was positive for all the tests for biosurfactant production. Results indicate that these isolates have potential for future environmental friendly applications such as bioremediation and industrial biotechnology.

Loop 1, a flexible surface loop in the myosin motor domain, comprises in part the transducer region that lies near the nucleotide-binding site and is proposed from structural studies to be responsible for the kinetic tuning of product release following ATP hydrolysis (1). Biochemical studies have shown that loop 1 affects the affinity of actin-myosin-II for ADP, motility and the V max of the actin-activated Mg 2؉ -ATPase activity, possibly through P i release (2-8). To test the influence of loop 1 on the mammalian class I myosin, Myo1b, chimeric molecules in which (i) loop 1 of a truncated form of Myo1b, Myo1b 1IQ , was replaced with either loop 1 from other myosins; (ii) loop 1 was replaced with glycine; or (iii) some amino acids in the loop were substituted with alanine and were expressed in baculovirus, and their interactions with actin and nucleotide were evaluated. The steady-state actin-activated ATPase activity; rate of ATP-induced dissociation of actin from Myo1b 1IQ ; rate of ADP release from actin-Myo1b 1IQ ; and the affinity of actin for Myo1b 1IQ and Myo1b 1IQ ⅐ADP differed in the chimeras versus wild type, indicating that loop 1 has a much wider range of effects on the coupling between actin and nucleotide binding events than previously thought. In particular, the biphasic ATP-induced dissociation of actin from actin-Myo1b 1IQ was significantly altered in the chimeras. This provided evidence that loop 1 contributes to the accessibility of the nucleotide pocket and is involved in the integration of information from the actin-, nucleotide-, ␥-P i -, and calmodulin-binding sites and predicts that loop 1 modulates the load dependence of the motor.Myo1b (aka 130-kDa myosin I, MYR 1, and MM1␣) is a mammalian class I myosin that is expressed in many different tissues (9 -11). It is associated with the plasma membrane (12) and in cell protrusions such as lamellipodia and membrane ruffles, suggesting its role in some aspects of cell motility (13). Overproduction of Myo1b or non-functional truncated Myo1b affects the distribution of endocytic compartments, suggesting its role in membrane trafficking (14). Furthermore, evidence indicates that Myo1b mediates lysosome movement (15).The ATP-induced dissociation of actin from actin-Myo1b is much slower than from most other myosins, and unlike skeletal muscle, myosin II is biphasic, consisting of both a fast and a slow phase (16). The fast phase is dependent on ATP concentration and is eliminated by preincubation with ADP. The slow phase is independent of ATP concentration and shares the same rate constant as ADP release but cannot be eliminated by decreasing ADP concentration. We have interpreted these results in conjunction with results from two other approaches: (i) single molecule studies demonstrating that Myo1b exhibits a two-part power stroke (17) and (ii) cryo-electron microscopy studies showing that Myo1b exhibits an ADP-induced conformational change. 1The biphasic nature of the ATP-induced dissociation of actinMyo1b (16, 19); the two-part power stroke (1...

Infantile and childhood-onset cataracts form a heterogeneous group of disorders; among the many genetic causes, numerous pathogenic variants in additional genes associated with autosomal-recessive infantile cataracts remain to be discovered. We identified three consanguineous families affected by bilateral infantile cataracts. Using exome sequencing, we found homozygous loss-of-function variants in DNMBP: nonsense variant c.811C>T (p.Arg271*) in large family F385 (nine affected individuals; LOD score ¼ 5.18 at q ¼ 0), frameshift deletion c.2947_2948del (p.Asp983*) in family F372 (two affected individuals), and frameshift variant c.2852_2855del (p.Thr951Metfs*41) in family F3 (one affected individual). The phenotypes of all affected individuals include infantile-onset cataracts. RNAi-mediated knockdown of the Drosophila ortholog still life (sif), enriched in lens-secreting cells, affects the development of these cells as well as the localization of E-cadherin, alters the distribution of septate junctions in adjacent cone cells, and leads to a 50% reduction in electroretinography amplitudes in young flies. DNMBP regulates the shape of tight junctions, which correspond to the septate junctions in invertebrates, as well as the assembly pattern of E-cadherin in human epithelial cells. E-cadherin has an important role in lens vesicle separation and lens epithelial cell survival in humans. We therefore conclude that DNMBP loss-of-function variants cause infantile-onset cataracts in humans.