Hypertrophy is a fundamental adaptive process employed by postmitotic cardiac and skeletal muscle in response to mechanical load. How muscle cells convert mechanical stimuli into growth signals has been a long-standing question. Using an in vitro model of load (stretch)-induced cardiac hypertrophy, we demonstrate that mechanical stretch causes release of angiotensin II (Ang II) from cardiac myocytes and that Ang II acts as an initial mediator of the stretch-induced hypertrophic response. The results not only provide direct evidence for the autocrine mechanism in load-induced growth of cardiac muscle cells, but also define the pathophysiological role of the local (cardiac) renin-angiotensin system.

Myosin 10 contains a region of predicted coiled coil 120 residues long. However, the highly charged nature and pattern of charges in the proximal 36 residues appear incompatible with coiled-coil formation. Circular dichroism, NMR, and analytical ultracentrifugation show that a synthesized peptide containing this region forms a stable single ␣-helix (SAH) domain in solution and does not dimerize to form a coiled coil even at millimolar concentrations. Additionally, electron microscopy of a recombinant myosin 10 containing the motor, the three calmodulin binding domains, and the fulllength predicted coiled coil showed that it was mostly monomeric at physiological protein concentration. In dimers the molecules were joined only at their extreme distal ends, and no coiled-coil tail was visible. Furthermore, the neck lengths of both monomers and dimers were much longer than expected from the number of calmodulin binding domains. In contrast, micrographs of myosin 5 heavy meromyosin obtained under the same conditions clearly showed a coiled-coil tail, and the necks were the predicted length. Thus the predicted coiled coil of myosin 10 forms a novel elongated structure in which the proximal region is a SAH domain and the distal region is a SAH domain (or has an unknown extended structure) that dimerizes only at its end. Sequence comparisons show that similar structures may exist in the predicted coiled-coil domains of myosins 6 and 7a and MyoM and could function to increase the size of the working stroke.Myosins make up a diverse superfamily of motor proteins (1). The human genome alone contains about 40 myosin genes (2). Of these, about one-third are "conventional" myosins (i.e. the well studied myosin 2), and the rest fall into about 10 different classes. The structure, properties and functions of the majority of myosin classes are poorly characterized and have largely been inferred from sequence comparisons rather than direct experiments on purified proteins (1-3).Muscle myosin 2 dimerizes through its ␣-helical coiled-coil tail. Therefore, it is commonly assumed that any myosin will also be dimeric if it contains a region predicted to be coiled coil. This assumption is dependent on the accuracy of coiled-coil prediction programs, such as COILS (4), PAIRCOIL, or MULTICOIL (5), which are also used by protein-fold prediction sites on the Web such as SMART (6).Although myosin 10 contains a region of predicted coiled coil (Fig. 1A), and is predicted to dimerize, this has not been determined experimentally. We noticed that part of the predicted coiled-coil domain of myosin 10 is highly enriched in charged residues (Fig. 1B). The proximal region consisting of 36 residues is particularly enriched in both positively and negatively charged residues, including the a and d positions of the heptad repeat (a-g) that are canonically hydrophobic residues in coiled coils (Fig. 1B). We suspected that this highly charged sequence is unlikely to form a coiled coil (7), suggesting that this part of myosin 10 may not be able to dimerize....

Quality control mechanisms prevent the cell surface expression of incompletely assembled multisubunit receptors such as the T cell receptor (TCR). The molecular chaperone function of calnexin (IP90, p88), a 90-kilodalton protein that resides in the endoplasmic reticulum (ER), in the retention of representative chains of the TCR-CD3 complex in the ER was tested. Truncation mutants of calnexin, when transiently expressed in COS cells, were exported from the ER and either accumulated in the Golgi or progressed to the cell surface. CD3 epsilon chains cotransfected with the forms of calnexin that were not retained in the ER exited the ER and colocalized with calnexin. Since engineered calnexin determined the intracellular localization of the proteins associated with it, it is concluded that calnexin interacts with incompletely assembled TCR components and retains them in the ER.

Normal mouse lungs lack appreciable numbers of mast cells (MCs) or MC progenitors (MCp's), yet the appearance of mature MCs in the tracheobronchial epithelial surface is a characteristic of allergic, T-cell-dependent pulmonary inflammation. We hypothesized that pulmonary inflammation would recruit MCp's to inflamed lungs and that this recruitment would be regulated by distinct adhesion pathways. Ovalbumin-sensitized and challenged mice had a greater than 28-fold increase in the number of MCp's in the lungs. In mice lacking endothelial vascular cell adhesion molecule 1 (VCAM-1) and in wild-type mice administered blocking monoclonal antibody (mAb) to VCAM-1 but not to mucosal addressin CAM-1 (MadCAM-1), recruitment of MCp's to the inflamed lung was reduced by greater than 75%. Analysis of the integrin receptors for VCAM-1 showed that in ␤7 integrin-deficient mice, recruitment was reduced 73% relative to wild-type controls, and in either BALB/c or C57BL/6 mice, mAb blocking of ␣4, ␤1, or ␤7 IntroductionMast cells (MCs) develop in tissues from bone marrow-derived MC progenitors (MCp's) drawn from the intravascular compartment. The intestine has an abundant supply of MCs, and MCp's constitutively home to this organ. In contrast, the naive mouse lung has few MCp's and lacks mature MCs beyond first-and secondgeneration bronchi. [1][2][3] Nonetheless, MCs accumulate in the epithelial surfaces of both large and small bronchi of mice that are sensitized systemically with ovalbumin (OVA) and challenged repetitively by aerosol inhalation to generate chronic airway inflammation. 3 Furthermore, MCs are required for the development of airway hyperresponsiveness to methacholine in protocols that lack adjuvant or otherwise limit the intensity of sensitization before challenge with OVA. [4][5][6] In humans with bronchial asthma, MCs accumulate and degranulate in both the bronchial epithelium and airway smooth muscle, 7-9 accompanied by increased numbers of MCp's in the blood. 10 Treatment with humanized monoclonal antibody (mAb) that blocks access of serum IgE to the high-affinity Fc⑀RI receptor reduces the frequency of asthma exacerbations. 11 These findings imply a pathophysiologic role for MCs and their expansion both in mouse models of airway inflammation and in the pathogenesis of human bronchial asthma.Although the small airways of the mouse have scant smooth muscle, little lamina propria, and negligible bronchial circulation compared with those of the human, an increment in intraepithelial MCs and leukocyte infiltrates around bronchovascular bundles is consistently observed in inflamed respiratory mucosal surfaces in both species, leading to the suggestion that the site of extravasation is via the associated microvasculature. 12,13 It is thus likely that the pathways needed for incremental MC numbers are conserved. Moreover the low levels of MCp's and scarcity of mature MCs in the naive mouse lung suggest that MCp recruitment accounts for the MC accumulation in the bronchial mucosa with the induction of allergic inflammat...

Myosin V is an unconventional myosin that transports cargo such as vesicles, melanosomes, or mRNA on actin filaments. It is a two-headed myosin with an unusually long neck that has six IQ motifs complexed with calmodulin. In vitro studies have shown that myosin V moves processively on actin, taking multiple 36-nm steps that coincide with the helical repeat of actin. This allows the molecule to "walk" across the top of an actin filament, a feature necessary for moving large vesicles along an actin filament bound to the cytoskeleton. The extended neck length of the two heads is thought to be critical for taking 36-nm steps for processive movements. To test this hypothesis we have expressed myosin V heavy meromyosin-like fragments containing 6IQ motifs, as well as ones that shorten (2IQ, 4IQ) or lengthen (8IQ) the neck region or alter the spacing between 3rd and 4th IQ motifs. The step size was proportional to neck length for the 2IQ, 4IQ, 6IQ, and 8IQ molecules, but the molecule with the altered spacing took shorter than expected steps. Total internal reflection fluorescence microscopy was used to determine whether the heavy meromyosin IQ molecules were capable of processive movements on actin. At saturating ATP concentrations, all molecules except for the 2IQ mutant moved processively on actin. When the ATP concentration was lowered to 10 M or less, the 2IQ mutant demonstrated some processive movements but with reduced run lengths compared with the other mutants. Its weak processivity was also confirmed by actin landing assays.The myosin superfamily consists of at least 18 classes of actin-dependent molecular motors (1). Class V myosins transport cargos such as endoplasmic reticulum in neurons, melanosomes in melanocytes, and mRNA in yeast (2). Similar to other myosins, they are composed of a head that binds ATP and actin and a neck region consisting of calmodulin (CaM) 1 molecules bound to an ␣-helical segment of the myosin heavy chain. The C-terminal tail domain of myosin V has coiled-coil forming motifs that dimerize and create two-headed molecules but do not self-associate into filamentous structures. The IQ motifs of the heavy chain, implicated in the binding of CaM or light chain subunits, have the consensus sequence, IQXXXRGXXXR, where X is any amino acid (3). The neck of mouse myosin V has six IQ motifs, each of which binds CaM, making its neck longer than that of most myosins (4). The six IQ motifs of all myosin V superfamily members are separated by 25-23-25-23-25 amino acids.The well studied myosin II class molecules that power muscle contraction and participate in cytokinesis in nonmuscle cells polymerize into filaments that are interdigitated with actin filaments. Kinetic and mechanical studies have demonstrated that these myosins interact only transiently with actin and typically spend greater than 95% of their kinetic cycle detached from actin. The term "low duty cycle" motor has been used to describe this behavior adopted to allow the sliding of actin filaments past myosin filaments to be unimpede...

Mouse myosin V is a two-headed unconventional myosin with an extended neck that binds six calmodulins. Double-headed (heavy meromyosin-like) and singleheaded (subfragment 1-like) fragments of mouse myosin V were expressed in Sf9 cells, and intact myosin V was purified from mouse brain. The actin-activated MgATPase of the tissue-purified myosin V, and its expressed fragments had a high V max and a low K ATPase . Calcium regulated the MgATPase of intact myosin V but not of the fragments. Both the MgATPase activity and the in vitro motility were remarkably insensitive to ionic strength. Myosin V and its fragments translocated actin at very low myosin surface densities. ADP markedly inhibited the actin-activated MgATPase activity and the in vitro motility. ADP dissociated from myosin V subfragment 1 at a rate of about 11.5 s ؊1 under conditions where the V max was 3.3 s ؊1 , indicating that, although not totally rate-limiting, ADP dissociation was close to the rate-limiting step. The high affinity for actin and the slow rate of ADP release helps the myosin head to remain attached to actin for a large fraction of each ATPase cycle and allows actin filaments to be moved by only a few myosin V molecules in vitro.Fifteen classes of myosins have been defined based on sequence analysis of the conserved motor domain of the molecule (1, 2). There has been extensive biochemical characterization of myosins from class I and II (1). In addition, some of the in vitro properties of chicken brain myosin V have been reported (3, 4). Myosin V is a two-headed molecule composed of two heavy chains weighing 212 kDa. Each heavy chain contains a typical myosin motor domain with a neck domain consisting of six IQ motifs that interact with myosin light chain subunits and calmodulin. Following this is a tail domain that contains regions of coiled-coil interspersed with nonhelical regions. Chicken myosin V copurifies with both calmodulin and essential light chain (5), but the stoichiometry of the various light chains has not been rigorously determined (5). The elongated appearance of the myosin head in rotary shadowed electron micrographic images, however, is consistent with a neck region that has many light chain subunits (4). In addition, chicken brain myosin V contains another low molecular mass subunit (8 kDa) termed the "dynein light chain," which has also been found in association with several other proteins including dynein and nitric-oxide synthase (6).Myosin V genes have been found in humans, rats, mice, chickens, Drosophila, Caenorhabditis elegans, and Saccharomyces cerevisiae (4, 7-13). There are two myosin V genes in yeast and at least two genes for myosin V in mammals, termed Va and Vb. Mutations in yeast and mouse myosin V genes reveal that this myosin likely functions in vesicle transport (see Ref. 2 for review). The dilute gene in mouse encodes myosin Va (9). dilute mutations are characterized by pale coat color, and the severe alleles also display neurological defects. The coat color phenotype is due to defective trafficking...

Cross talk occurs between the human gut and the lung through a gut-lung axis involving the gut microbiota. However, the signatures of the human gut microbiota after active Mycobacterium tuberculosis infection have not been fully understood. Here, we investigated changes in the gut microbiota in tuberculosis (TB) patients by shotgun sequencing the gut microbiomes of 31 healthy controls and 46 patients. We observed a dramatic changes in gut microbiota in tuberculosis patients as reflected by significant decreases in species number and microbial diversity. The gut microbiota of TB patients were mostly featured by the striking decrease of short-chain fatty acids (SCFAs)-producingbacteria as well as associated metabolic pathways. A classification model based on the abundance of three species, Haemophilus parainfluenzae, Roseburia inulinivorans , and Roseburia hominis , performed well for discriminating between healthy and diseased patients. Additionally, the healthy and diseased states can be distinguished by SNPs in the species of B. vulgatus . We present a comprehensive profile of changes in the microbiota in clinical TB patients. Our findings will shed light on the design of future diagnoses and treatments for M. tuberculosis infections.

Attractin is a normal human serum glycoprotein of 175 kDa that is rapidly expressed on activated T cells and released extracellularly after 48-72 hr. We have cloned attractin and find that, as in its natural serum form, it mediates the spreading of monocytes that become the focus for the clustering of nonproliferating T lymphocytes. There are two mRNA species with hematopoietic tissue-specific expression that code for a 134-kDa protein with a putative serine protease catalytic serine, four EGF-like motifs, a CUB domain, a C type lectin domain, and a domain homologous with the ligand-binding region of the common ␥ cytokine chain. Except for the latter two domains, the overall structure shares high homology with the Caenorhabditis elegans F33C8.1 protein, suggesting that attractin has evolved new domains and functions in parallel with the development of cell-mediated immunity.