The role of nitric oxide in small intestinal motility in a model of acute inflammation

Motility responses of the small intestine of iNOS deficient mice (iNOS −/−) and their wildtype littermates (iNOS+/+) to the inflammatory challenge of lipopolysaccharide (LPS) were investigated. LPS administration failed to attenuate intestinal transit in iNOS−/− mice but depressed transit in their iNOS+/+ littermates. Supporting an inhibitory role for sustained nitric oxide (NO) synthesis in the regulation of intestinal motility during inflammation, iNOS immunoreactivity was upregulated in all regions of the small intestine of iNOS+/+ mice. In contrast, neuronal NOS was barely affected. Cyclooxygenase activation was determined by prostaglandin E2 (PGE2) concentration. Following LPS challenge, PGE2 levels were elevated in all intestinal segments in both animal groups. Moreover, COX-1 and COX-2 protein levels were elevated in iNOS+/+ mice in response to LPS, while COX-2 levels were similarly increased in iNOS −/− intestine. However, no apparent relationship was observed between increased prostaglandin concentrations and attenuated intestinal transit. The presence of heme oxygenase 1 (HO-1) in the murine small intestine was also investigated. In both animal groups HO-1 immunoreactivity in the proximal intestine increased in response to treatment, while the constitutive protein levels detected in the middle and distal intestine were unresponsive to LPS administration. No apparent correlation of HO-1 to the suppression of small intestinal motility induced by LPS administration was detected. The presence of S-nitrosylated contractile proteins in the small intestine was determined. γ-smooth muscle actin was basally nitrosylated as well as in response to LPS, but myosin light chain kinase and myosin regulatory chain (MLC20) were not. In conclusion, in a model of acute intestinal inflammation, iNOS-produced NO plays a significant role in suppressing small intestinal motility while nNOS, COX-1, COX-2 and HO-1 do not participate in this event. S-nitrosylation of γ-smooth muscle actin is associated with elevated levels of nitric oxide in the smooth muscle of murine small intestine. ^

Antigen-specific proteolytic antibodies

The antigen recognition site of antibodies is composed of residues contributed by the variable domains of the heavy and light chain subunits (VL and VH domains). VL domains can catalyze peptide bond hydrolysis independent of VH domains (Mei S et al. J Biol Chem. 1991 Aug 25;266(24):15571-4). VH domains can bind antigens noncovalently independent of V L domains (Ward et al. Nature. 1989 Oct 12;341(6242):544-6). This dissertation describe the specific hydrolysis of fusion proteins containing the hepatitis C virus coat protein E2 by recombinant hybrid Abs composed of the heavy chain of a high affinity anti-E2 IgG1 paired with light chains expressing promiscuous catalytic activity. The proteolytic activity was evident from electrophoresis assays using recombinant E2 substrates containing glutathione S-transferase (E2-GST) or FLAG peptide (E2-FLAG) tags. The proteolytic reaction proceeded more rapidly in the presence of the hybrid IgG1 compared to the unpaired light chain, consistent with accelerated peptide bond hydrolysis due to noncovalent VH domain-E2 recognition. An active site-directed inhibitor of serine proteases inhibited the proteolytic activity of the hybrid IgG, indicating a serine protease mechanism. Binding studies confirmed that the hybrid IgG retained detectable noncovalent E2 recognition capability, although at a level smaller than the wildtype anti-E2 IgG. Immunoblotting of E2-FLAG treated with the hybrid IgG suggested a scissile bond within E2 located ∼11 kD from the N terminus of the protein. E2-GST was hydrolyzed by the hybrid IgG at peptide bonds located in the GST tag. The differing cleavage pattern of E2-FLAG and E2-GST can be explained by the split-site model of catalysis, in which conformational differences in the E2 fusion protein substrates position alternate peptide bonds in register with the antibody catalytic subsite despite a common noncovalent binding mechanism. This is the first proof-of principle that the catalytic activity of a light chain can be rendered antigen-specific by pairing with a noncovalently binding heavy chain subunit. ^

Identification and characterization of mutations in SMC contractile genes involved in thoracic aortic aneurysms and dissections

Aortic aneurysms and dissections are the 15th most common cause of death in the United States. Genetic factors contribute to the pathogenesis of thoracic aortic aneurysms and dissections (TAAD). Currently, six loci and four genes have been identified for familial TAAD. Notably, mutations in smooth muscle cell (SMC) contractile genes, ACTA2 and MYH11, are responsible for 15% of familial TAAD, suggesting that proper SMC contraction is important for normal aorta function. Therefore, we hypothesize that mutations in other genes encoding SMC contractile proteins also cause familial TAAD. ^ To test this hypothesis, we used a candidate gene approach to identify causative mutations in SMC contractile genes for familial TAAD. Sequencing DNA in 80 TAAD patients from unrelated families, we identified putative mutations in eight contractile genes. We chose myosin light chain kinase (MLCK ) S1759P for further study for the following reasons: (1) Serine 1759 is conserved between vertebrates and invertebrates. (2) S1759P is predicted to be functionally deleterious by bioinformatics. (3) Low blood pressure is observed in SMC-selective MLCK-deficient mice. ^ In the presence of Ca2+/Calmodulin (CaM), MLCK containing CaM binding and kinase domains are activated to phosphorylate myosin light chain, thereby initiate SMC contraction. The CaM binding sequence of MLCK forms an α-helix structure required for CaM binding. MLCK Serine 1759 is located within the CaM binding domain. S1759P is predicted to decrease the α-helix composition in the CaM binding domain. Hence, we hypothesize that MLCK mutations cause TAAD through disturbing CaM binding and MLCK activity. ^ We further sequenced MLCK in DNA samples from additional 86 probands with familial TAAD. Two more mutations, MLCK A1754T and R1480Stop, were identified, supporting that MLCK mutations cause familial TAAD. ^ To define whether MLCK mutations disrupted CaM binding and MLCK activity, we performed co-immunoprecipitation and kinase assays. Decreased CaM binding and kinase activity was detected in A1754T and S1759P. Moreover, R1480Stop is predicted to truncate kinase and CaM binding domains. We conclude that MLCK mutations disrupt CaM binding and MLCK activity. ^ Collectively, our study is first to show mutations in genes regulating SMC contraction cause TAAD. This finding further highlights the importance of SMC contraction in maintaining aorta function. ^

Role of TRP Channels in Mediating the Calcium Signaling Response of Brain Endothelial Cells to Mechanical Stretch

Traumatic brain injury (TBI) often results in disruption of the blood brain barrier (BBB), which is an integral component to maintaining the central nervous system homeostasis. Recently cytosolic calcium levels ([Ca2+]i), observed to elevate following TBI, have been shown to influence endothelial barrier integrity. However, the mechanism by which TBI-induced calcium signaling alters the endothelial barrier remains unknown. In the present study, an in vitro BBB model was utilized to address this issue. Exposure of cells to biaxial mechanical stretch, in the range expected for TBI, resulted in a rapid cytosolic calcium increase. Modulation of intracellular and extracellular Ca2+ reservoirs indicated that Ca2+ influx is the major contributor for the [Ca2+]i elevation. Application of pharmacological inhibitors was used to identify the calcium-permeable channels involved in the stretch-induced Ca2+ influx. Antagonist of transient receptor potential (TRP) channel subfamilies, TRPC and TRPP, demonstrated a reduction of the stretch-induced Ca2+ influx. RNA silencing directed at individual TRP channel subtypes revealed that TRPC1 and TRPP2 largely mediate the stretch-induced Ca2+ response. In addition, we found that nitric oxide (NO) levels increased as a result of mechanical stretch, and that inhibition of TRPC1 and TRPP2 abolished the elevated NO synthesis. Further, as myosin light chain (MLC) phosphorylation and actin cytoskeleton rearrangement are correlated with endothelial barrier disruption, we investigated the effect mechanical stretch had on the myosin-actin cytoskeleton. We found that phosphorylated MLC was increased significantly by 10 minutes post-stretch, and that inhibition of TRP channel activity or NO synthesis both abolished this effect. In addition, actin stress fibers formation significantly increased 2 minutes post-stretch, and was abolished by treatment with TRP channel inhibitors. These results suggest that, in brain endothelial cells, TRPC1 and TRPP2 are activated by TBI-mechanical stress and initiate actin-myosin contraction, which may lead to disruption of the BBB.

Characterization of the MLC box: A conserved {\it cis\/}-acting DNA element present in the chicken MLC1s/1c gene

The slow/cardiac alkali myosin light chain (MLC1s/1c) is a member of a multigene family whose protein products are essential for activation of the myosin ATPase. In the adult, the MLC1s/1c isoform is expressed in both cardiac and slow-twitch skeletal muscles, while it is expressed by all skeletal muscles during development.^ To elucidate the molecular mechanisms that underlie the transcriptional regulation of MLC1s/1c gene expression, the immediate 5$\sp\prime$ flanking region of the gene was isolated and shown to be capable of directing reporter gene expression. Analysis of this region revealed a 110 bp muscle-specific enhancer that includes a myocyte-specific enhancer-binding factor 2 (MEF-2) site, E-boxes, which are potential binding sites for the basic-helix-loop-helix proteins such as MyoD, and a MLC box. The focus of the thesis was to identify the role of the MLC box in expression of the MLC1s/1c gene.^ The MLC box is a member of the family of CArG box containing cis-acting DNA elements. Mutagenesis showed that the MLC box is necessary, but not sufficient, for the expression of a reporter gene linked to the 5$\sp\prime$ flanking region of the MLC1s/1c gene. Linker scanner and site-directed mutagenesis identified a number of potential sites within the 110 bp muscle-specific enhancer that may cooperate with the MLC box. These are the MEF-2 site, the E-box site, and a 10 bp element located upstream of the MEF-2 site that does not have sequence similarity with any known cis-acting element. The MLC box is capable of binding to factors present in muscle nuclear extracts, as well as to human recombinant serum response factor (SRF). Binding of SRF to the MLC box was correlated with the ability of the 5$\sp\prime$ flanking region of the MLC1s/1c gene to drive reporter gene expression. Results suggest a model in which binding of SRF to the MLC box activates expression of the MLC1s/1c gene while binding of the factors present in the nuclear extracts suppresses the expression of the gene. (Abstract shortened with permission of author.) ^

Low-Intensity Pulsed Ultrasound Improves the Functional Properties of Cardiac Mesoangioblasts

Cell-based therapy is a promising approach for many diseases, including ischemic heart disease. Cardiac mesoangioblasts are committed vessel-associated progenitors that can restore to a significant, although partial, extent, heart structure and function in a murine model of myocardial infarction. Low-intensity pulsed ultrasound (LIPUS) is a noninvasive form of mechanical energy that can be delivered into biological tissues as acoustic pressure waves, and is widely used for clinical applications including bone fracture healing. We hypothesized that the positive effects of LIPUS on bone and soft tissue, such as increased cell differentiation and cytoskeleton reorganization, could be applied to increase the therapeutic potential of mesoangioblasts for heart repair. In this work, we show that LIPUS stimulation of cardiac mesoangioblasts isolated from mouse and human heart results in significant cellular modifications that provide beneficial effects to the cells, including increased malleability and improved motility. Additionally, LIPUS stimulation increased the number of binucleated cells and induced cardiac differentiation to an extent comparable with 5´-azacytidine treatment. Mechanistically, LIPUS stimulation activated the BMP-Smad signalling pathway and increased the expression of myosin light chain-2 together with upregulation of β1 integrin and RhoA, highlighting a potentially important role for cytoskeleton reorganization. Taken together, these results provide functional evidence that LIPUS might be a useful tool to explore in the field of heart cell therapy

Comparison of fusion phage libraries displaying VH or single-chain Fv antibody fragments derived from the antibody repertoire of a vaccinated melanoma patient as a source of melanoma-specific targeting molecules

A single-chain Fv (scFv) fusion phage library derived from random combinations of VH and VL (variable heavy and light chains) domains in the antibody repertoire of a vaccinated melanoma patient was previously used to isolate clones that bind specifically to melanoma cells. An unexpected finding was that one of the clones encoded a truncated scFv molecule with most of the VL domain deleted, indicating that a VH domain alone can exhibit tumor-specific binding. In this report a VH fusion phage library containing VH domains unassociated with VL domains was compared with a scFv fusion phage library as a source of melanoma-specific clones; both libraries contained the same VH domains from the vaccinated melanoma patient. The results demonstrate that the clones can be isolated from both libraries, and that both libraries should be used to optimize the chance of isolating clones binding to different epitopes. Although this strategy has been tested only for melanoma, it is also applicable to other cancers. Because of their small size, human origin and specificity for cell surface tumor antigens, the VH and scFv molecules have significant advantages as tumor-targeting molecules for diagnostic and therapeutic procedures and can also serve as probes for identifying the cognate tumor antigens.

Sequence dependent hypermutation of the immunoglobulin heavy chain in cultured B cells

The variable (V) regions of immunoglobulin heavy and light chains undergo high rates of somatic mutation during the immune response. Although point mutations accumulate throughout the V regions and their immediate flanking sequences, analysis of large numbers of mutations that have arisen in vivo reveal that the triplet AGC appears to be most susceptible to mutation. We have stably transfected B cell lines with γ2a heavy chain constructs containing TAG nonsense codons in their V regions that are part of either a putative (T)AGC hot spot or a (T)AGA non-hot spot motif. Using an ELISA spot assay to detect revertants and fluctuation analysis to determine rates of mutation, the rate of reversion of the TAG nonsense codon has been determined for different motifs in different parts of the V region. In the NSO plasma cell line, the (T)AGC hot spot motif mutates at rates of ≈6 × 10−4/bp per generation and ≈3 × 10−5/bp per generation at residues 38 and 94 in the V region. At each of these locations, the (T)AGC hot spot motif is 20–30 times more likely to undergo mutation than the (T)AGA non-hot spot motif. Moreover, the AGA non-hot spot motif mutates at as high a rate as the hot spot motif when it is located adjacent to hot spot motifs, suggesting that more extended sequences influence susceptibility to mutation.

Isolation and Contraction of the Stress Fiber

Stress fibers were isolated from cultured human foreskin fibroblasts and bovine endothelial cells, and their contraction was demonstrated in vitro. Cells in culture dishes were first treated with a low-ionic-strength extraction solution and then further extracted using detergents. With gentle washes by pipetting, the nucleus and the apical part of cells were removed. The material on the culture dish was scraped, and the freed material was forced through a hypodermic needle and fractionated by sucrose gradient centrifugation. Isolated, free-floating stress fibers stained brightly with fluorescently labeled phalloidin. When stained with anti-α-actinin or anti-myosin, isolated stress fibers showed banded staining patterns. By electron microscopy, they consisted of bundles of microfilaments, and electron-dense areas were associated with them in a semiperiodic manner. By negative staining, isolated stress fibers often exhibited gentle twisting of microfilament bundles. Focal adhesion–associated proteins were also detected in the isolated stress fiber by both immunocytochemical and biochemical means. In the presence of Mg-ATP, isolated stress fibers shortened, on the average, to 23% of the initial length. The maximum velocity of shortening was several micrometers per second. Polystyrene beads on shortening isolated stress fibers rotated, indicating spiral contraction of stress fibers. Myosin regulatory light chain phosphorylation was detected in contracting stress fibers, and a myosin light chain kinase inhibitor, KT5926, inhibited isolated stress fiber contraction. Our study demonstrates that stress fibers can be isolated with no apparent loss of morphological features and that they are truly contractile organelle.

Regulation of the Actin Cytoskeleton by Thrombin in Human Endothelial Cells: Role of Rho Proteins in Endothelial Barrier Function

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.

The Involvement of the Intermediate Chain of Cytoplasmic Dynein in Binding the Motor Complex to Membranous Organelles of Xenopus Oocytes

Cytoplasmic dynein is one of the major motor proteins involved in intracellular transport. It is a protein complex consisting of four subunit classes: heavy chains, intermediate chains (ICs), light intermediate chains, and light chains. In a previous study, we had generated new monoclonal antibodies to the ICs and mapped the ICs to the base of the motor. Because the ICs have been implicated in targeting the motor to cargo, we tested whether these new antibodies to the intermediate chain could block the function of cytoplasmic dynein. When cytoplasmic extracts of Xenopus oocytes were incubated with either one of the monoclonal antibodies (m74–1, m74–2), neither organelle movement nor network formation was observed. Network formation and membrane transport was blocked at an antibody concentration as low as 15 μg/ml. In contrast to these observations, no effect was observed on organelle movement and tubular network formation in the presence of a control antibody at concentrations as high as 0.5 mg/ml. After incubating cytoplasmic extracts or isolated membranes with the monoclonal antibodies m74–1 and m74–2, the dynein IC polypeptide was no longer detectable in the membrane fraction by SDS-PAGE immunoblot, indicating a loss of cytoplasmic dynein from the membrane. We used a panel of dynein IC truncation mutants and mapped the epitopes of both antibodies to the N-terminal coiled-coil domain, in close proximity to the p150Glued binding domain. In an IC affinity column binding assay, both antibodies inhibited the IC–p150Glued interaction. Thus these findings demonstrate that direct IC–p150Glued interaction is required for the proper attachment of cytoplasmic dynein to membranes.

Rho-stimulated Contractility Contributes to the Fibroblastic Phenotype of Ras-transformed Epithelial Cells

Oncogenic transformation of cells alters their morphology, cytoskeletal organization, and adhesive interactions. When the mammary epithelial cell line MCF10A is transformed by activated H-Ras, the cells display a mesenchymal/fibroblastic morphology with decreased cell–cell junctions but increased focal adhesions and stress fibers. We have investigated whether the transformed phenotype is due to Rho activation. The Ras-transformed MCF10A cells have elevated levels of myosin light chain phosphorylation and are more contractile than their normal counterparts, consistent with the activation of Rho. Furthermore, inhibitors of contractility restore a more normal epithelial phenotype to the Ras-transformed MCF10A cells. However, inhibiting Rho by microinjection of C3 exotransferase or dominant negative RhoA only partially restores the normal phenotype, in that it fails to restore normal junctional organization. This result prompted us to examine the effect that inhibiting Rho would have on the junctions of normal MCF10A cells. We have found that inhibiting Rho by C3 microinjection leads to a disruption of E-cadherin cytoskeletal links in adherens junctions and blocks the assembly of new adherens junctions. The introduction of constitutively active Rho into normal MCF10A cells did not mimic the Ras-transformed phenotype. Thus, these results lead us to conclude that some, but not all, characteristics of Ras-transformed epithelial cells are due to activated Rho. Whereas Rho is needed for the assembly of adherens junctions, high levels of activated Rho in Ras-transformed cells contribute to their altered cytoskeletal organization. However, additional events triggered by Ras must also be required for the disruption of adherens junctions and the full development of the transformed epithelial phenotype.

Two Components of Actin-based Retrograde Flow in Sea Urchin Coelomocytes

Sea urchin coelomocytes represent an excellent experimental model system for studying retrograde flow. Their extreme flatness allows for excellent microscopic visualization. Their discoid shape provides a radially symmetric geometry, which simplifies analysis of the flow pattern. Finally, the nonmotile nature of the cells allows for the retrograde flow to be analyzed in the absence of cell translocation. In this study we have begun an analysis of the retrograde flow mechanism by characterizing its kinetic and structural properties. The supramolecular organization of actin and myosin II was investigated using light and electron microscopic methods. Light microscopic immunolocalization was performed with anti-actin and anti-sea urchin egg myosin II antibodies, whereas transmission electron microscopy was performed on platinum replicas of critical point-dried and rotary-shadowed cytoskeletons. Coelomocytes contain a dense cortical actin network, which feeds into an extensive array of radial bundles in the interior. These actin bundles terminate in a perinuclear region, which contains a ring of myosin II bipolar minifilaments. Retrograde flow was arrested either by interfering with actin polymerization or by inhibiting myosin II function, but the pathway by which the flow was blocked was different for the two kinds of inhibitory treatments. Inhibition of actin polymerization with cytochalasin D caused the actin cytoskeleton to separate from the cell margin and undergo a finite retrograde retraction. In contrast, inhibition of myosin II function either with the wide-spectrum protein kinase inhibitor staurosporine or the myosin light chain kinase–specific inhibitor KT5926 stopped flow in the cell center, whereas normal retrograde flow continued at the cell periphery. These differential results suggest that the mechanism of retrograde flow has two, spatially segregated components. We propose a “push–pull” mechanism in which actin polymerization drives flow at the cell periphery, whereas myosin II provides the tension on the actin cytoskeleton necessary for flow in the cell interior.

Galactose-extended glycans of antibodies produced by transgenic plants

Plant-specific N-glycosylation can represent an important limitation for the use of recombinant glycoproteins of mammalian origin produced by transgenic plants. Comparison of plant and mammalian N-glycan biosynthesis indicates that β1,4-galactosyltransferase is the most important enzyme that is missing for conversion of typical plant N-glycans into mammalian-like N-glycans. Here, the stable expression of human β1,4-galactosyltransferase in tobacco plants is described. Proteins isolated from transgenic tobacco plants expressing the mammalian enzyme bear N-glycans, of which about 15% exhibit terminal β1,4-galactose residues in addition to the specific plant N-glycan epitopes. The results indicate that the human enzyme is fully functional and localizes correctly in the Golgi apparatus. Despite the fact that through the modified glycosylation machinery numerous proteins have acquired unusual N-glycans with terminal β1,4-galactose residues, no obvious changes in the physiology of the transgenic plants are observed, and the feature is inheritable. The crossing of a tobacco plant expressing human β1,4-galactosyltransferase with a plant expressing the heavy and light chains of a mouse antibody results in the expression of a plantibody that exhibits partially galactosylated N-glycans (30%), which is approximately as abundant as when the same antibody is produced by hybridoma cells. These results are a major step in the in planta engineering of the N-glycosylation of recombinant antibodies.

Activation of Trp3 by inositol 1,4,5-trisphosphate receptors through displacement of inhibitory calmodulin from a common binding domain

Mammalian homologues of Drosophila Trp form plasma membrane channels that mediate Ca2+ influx in response to activation of phospholipase C and internal Ca2+ store depletion. Previous studies showed that human Trp3 is activated by inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs) and identified interacting domains, one on Trp and two on IP3R. We now find that Trp3 binds Ca2+-calmodulin (Ca2+/CaM) at a site that overlaps with the IP3R binding domain. Using patch-clamp recordings from inside-out patches, we further show that Trp3 has a high intrinsic activity that is suppressed by Ca2+/CaM under resting conditions, and that Trp3 is activated by the following: a Trp-binding peptide from IP3R that displaces CaM from Trp3, a myosin light chain kinase Ca2+/CaM binding peptide that prevents CaM from binding to Trp3, and calmidazolium, an inactivator of Ca2+/CaM. We conclude that inhibition of the inhibitory action of CaM is a key step of Trp3 channel activation by IP3Rs.