Myofunctional therapy Part 2: Prevention of dentofacial disorders.

AirwayCentric approach to prevention of dentofacial disorders. The correct development of functions is important for the prevention of dentofacial disorders from the first days of life. The first and foremost function, to which all others are adapting, is nasal breathing. The AirwayCentric® method focuses on nasal breathing during early development and throughout childhood to promote: Neurobehavioural development and the brain, development of the craniofacial and respiratory complex, correct dental occlusion, proper and restful sleep and to improve performance and life overall. By working at the same time on orofacial functions and structures, many dentofacial disorders can be prevented.

Search for a Light CP-odd Higgs Boson and Low-Mass Dark Matter at the Belle Experiment.

We report on the first Belle search for a light CP-odd Higgs boson, A^{0}, that decays into low mass dark matter, χ, in final states with a single photon and missing energy. We search for events produced via the dipion transition ϒ(2S)→ϒ(1S)π^{+}π^{-}, followed by the on-shell process ϒ(1S)→γA^{0} with A^{0}→χχ, or by the off-shell process ϒ(1S)→γχχ. Utilizing a data sample of 157.3×10^{6} ϒ(2S) decays, we find no evidence for a signal. We set limits on the branching fractions of such processes in the mass ranges M_{A^{0}}<8.97 GeV/c^{2} and M_{χ}<4.44 GeV/c^{2}. We then use the limits on the off-shell process to set competitive limits on WIMP-nucleon scattering in the WIMP mass range below 5 GeV/c^{2}.

Observation of Ξ(1620)

We report the first observation of the double strange baryon Ξ(1620)^{0} in its decay to Ξ^{-}π^{+} via Ξ_{c}^{+}→Ξ^{-}π^{+}π^{+} decays based on a 980 fb^{-1} data sample collected with the Belle detector at the KEKB asymmetric-energy e^{+}e^{-} collider. The mass and width are measured to be 1610.4±6.0(stat)_{-4.2}^{+6.1} (syst) MeV/c^{2} and 59.9±4.8(stat)_{-7.1}^{+2.8}(syst) MeV, respectively. We obtain 4.0σ evidence of the Ξ(1690)^{0} with the same data sample. These results shed light on the structure of hyperon resonances with strangeness S=-2.

Observation of ϒ(4S)→η

We report the first observation of the hadronic transition ϒ(4S)→η^{'}ϒ(1S), using 496 fb^{-1} data collected at the ϒ(4S) resonance with the Belle detector at the KEKB asymmetric-energy e^{+}e^{-} collider. We reconstruct the η^{'} meson through its decays to ρ^{0}γ and to π^{+}π^{-}η, with η→γγ. We measure B(ϒ(4S)→η^{'}ϒ(1S))=[3.43±0.88(stat)±0.21(syst)]×10^{-5}, with a significance of 5.7σ.

Evidence for Isospin Violation and Measurement of CP Asymmetries in B→K

We report the first evidence for isospin violation in B→K^{*}γ and the first measurement of the difference of CP asymmetries between B^{+}→K^{*+}γ and B^{0}→K^{*0}γ. This analysis is based on the data sample containing 772×10^{6}BB[over ¯] pairs that was collected with the Belle detector at the KEKB energy-asymmetric e^{+}e^{-} collider. We find evidence for the isospin violation with a significance of 3.1σ, Δ_{0+}=[+6.2±1.5(stat)±0.6(syst)±1.2(f_{+-}/f_{00})]%, where the third uncertainty is due to the uncertainty on the fraction of B^{+}B^{-} to B^{0}B[over ¯]^{0} production in ϒ(4S) decays. The measured value is consistent with predictions of the standard model. The result for the difference of CP asymmetries is ΔA_{CP}=[+2.4±2.8(stat)±0.5(syst)]%, consistent with zero. The measured branching fractions and CP asymmetries for charged and neutral B meson decays are the most precise to date. We also calculate the ratio of branching fractions of B^{0}→K^{*0}γ to B_{s}^{0}→ϕγ.

Psychosine, a marker of Krabbe phenotype and treatment effect.

Newborn screening (NBS) for Krabbe disease, a rare neurodegenerative disorder caused by deficient galactocerebrosidase (GALC) enzyme activity, has recently been implemented in a number of US states. However, the spectrum of phenotypic manifestations associated with deficient GALC activity complicates the management of screen-positive newborns and underscores the need to identify clinically relevant biomarkers. Earlier studies with a small number of patients identified psychosine, a substrate of the GALC enzyme, as a potential biomarker for Krabbe disease. In this study, we provide, for the first time, longitudinal data on dried blood spot (DBS) psychosine concentrations in different Krabbe disease phenotypes for both untreated patients and those treated with hematopoietic stem cell transplantation (HSCT). Our cohort included patients previously identified by NBS to be at high risk to develop Krabbe disease. Substantially elevated DBS psychosine concentration during the newborn period was found to be a highly specific marker for infantile Krabbe disease. This finding supports the use of DBS psychosine concentration as a second-tier NBS test to aid in the identification of patients who require urgent evaluation for HSCT. In addition, longitudinal assessments showed that both natural disease progression and treatment with HSCT were associated with decreases in DBS psychosine concentrations. Based on these findings we provide recommendations for the interpretation of psychosine concentrations in DBS specimens collected during the first year of life. Future studies should aim to better delineate the relationship between DBS psychosine concentration and disease onset in patients with later-onset forms of Krabbe disease.

Preparation of the Full Set of Recombinant Mouse- and Human-Secreted Phospholipases A2.

A family of 14-20kDa, disulfide-rich, calcium-dependent secreted phospholipases A2 (sPLA2s) that release fatty acids from the sn-2 position of glycerophospholipids can be found in mammals. They have a diverse array of tissue distribution and biological functions. In this chapter we provide detailed protocols for production of nearly all of the mouse and human sPLA2s mainly by expression in bacteria and in vitro refolding or by expression in insect cells. High-resolution mass spectrometry and enzymatic assays were, respectively, used to show that all disulfides are formed and that the enzymes are active, strongly suggesting that each sPLA2 was prepared in the structurally native form. The availability of these proteins has allowed kinetic studies to be carried out, to prepare highly selective antisera, to screen for selective inhibitors, to study receptor binding, and to study the action of each enzyme on mammalian cell membranes and their in vivo biological roles.

Relationship between levels of secreted phospholipase A2 groups IIA and X in the airways and asthma severity.

Background Secreted phospholipase A(2) (sPLA(2) ) may be important mediators of asthma, but the specific sPLA(2) s involved in asthma are not known. Objective To evaluate sPLA(2) group IIA, V, and X proteins (sPLA(2) -IIA, sPLA(2) -V, and sPLA(2) -X) in bronchoalveolar lavage (BAL) fluid, BAL cells, and airway epithelial cells of subjects with and without asthma, and examine the relationship between the levels of specific sPLA(2) enzymes and airway inflammation, asthma severity, and lung function. Methods The expression of sPLA(2) -IIA, sPLA(2) -V, and sPLA(2) -X in BAL cells and epithelial brushings was assessed by qPCR. The levels of these sPLA(2) proteins and sPLA(2) activity with and without group II and group X-specific inhibitors were measured in BAL fluid from 18 controls and 39 asthmatics. Results The airway epithelium expressed sPLA(2) -X at higher levels than either sPLA(2) -IIA or sPLA(2) -V, whereas BAL cells expressed sPLA(2) -IIA and sPLA(2) -X at similar levels. The majority of sPLA(2) activity in BAL fluid was attributed to either sPLA(2) -IIA or sPLA(2) -X. After 10-fold concentration of BAL fluid, the levels of sPLA(2) -X normalized to total protein were increased in asthma and were associated with lung function, the concentration of induced sputum neutrophils, and prostaglandin E(2) . The levels of sPLA(2) -IIA were elevated in asthma when normalized to total protein, but were not related to lung function, markers of airway inflammation or eicosanoid formation. Conclusions and Clinical Relevance These data indicate that sPLA(2) -IIA and sPLA(2) -X are the major sPLA(2) s in human airways, and suggest a link between the levels of sPLA(2) -X in the airways and several features of asthma.

Differential modulation of mediator release from human basophils and mast cells by mizolastine.

BACKGROUND: Basophils and mast cells play a major role in the pathogenesis of allergic disorders by releasing several proinflammatory mediators. Some histamine H1 receptor antagonists exert anti-inflammatory activities by modulating mediator release from basophils and mast cells. OBJECTIVE: To study the in vitro effects of mizolastine, an H1 receptor antagonist, on the release of eicosanoids, histamine and IL-4 from human basophils and lung mast cells. METHODS AND RESULTS: Mizolastine (10(-7)-10(-5) M) concentration-dependently inhibited the release of cysteinyl leukotriene C4 from anti-IgE-stimulated basophils (IC(50): 3.85+/-0.28 microM) and mast cells (IC(50): 3.92+/-0.41 microM). The same concentrations of mizolastine did not affect anti-IgE-induced prostaglandin D2 release from lung mast cells. In contrast, mizolastine enhanced up to 80% IgE-mediated histamine release (EC(50): 4.63+/-0.14 microM) from basophils, but not from mast cells and it significantly potentiated IL-4 release from basophils induced by anti-IgE. Mizolastine did not affect histamine release from basophils induced by formyl peptide, whereas it inhibited cysteinyl leukotriene C4 release (IC(50): 1.86+/-0.24 microM). Blockade of cytosolic phospholipase A2 and arachidonic acid mobilization by pyrrolidine-1 did not alter the effect of mizolastine on histamine release from basophils, thereby excluding accumulation of arachidonic acid metabolic intermediates as the cause of this effect. Mizolastine did not influence anti-IgE-induced activation of extracellular signal-regulated kinase-1 and -2 (ERK-1 and -2) in human basophils. CONCLUSIONS: Mizolastine efficiently inhibits LTC4 synthesis in human basophils and mast cells presumably by interfering with 5-lipoxygenase. In contrast, it enhances histamine and IL-4 release only from anti-IgE-stimulated basophils. Therefore, mizolastine differentially regulates the production of mediators from basophils and mast cells in a cell- and stimulus-specific fashion.

The natural history of embouchure dystonia.

Focal task-specific dystonias are unusual disorders of motor control, often affecting individuals who perform complex repetitive movements. Musicians are especially prone to develop these disorders because of their training regimens and intense practice schedules. Task-specific dystonia occurring in keyboard or string instrumentalists usually affects the hand. In contrast, there have been few descriptions of musicians with task-specific dystonia affecting the muscles of the face and jaw. We report detailed clinical observations of 26 professional brass and woodwind players afflicted with focal task-specific dystonia of the embouchure (the pattern of lip, jaw, and tongue muscles used to control the flow of air into a mouthpiece). This is the largest and most comprehensively studied series of such patients. Patients developed embouchure dystonia in the fourth decade, and initial symptoms were usually limited to one range of notes or style of playing. Once present, dystonia progressed without remission and responded poorly to oral medications and botulinum toxin injection. Patients with embouchure dystonia could be separated by the pattern of their abnormal movements into several groups, including embouchure tremor, involuntary lip movements, and jaw closure. Dystonia not infrequently spread to other oral tasks, often producing significant disability. Effective treatments are needed for this challenging and unusual disorder.

Glycolysis as a target for the design of new anti-trypanosome drugs.

Glycolysis is perceived as a promising target for new drugs against parasitic trypanosomatid protozoa because this pathway plays an essential role in their ATP supply. Trypanosomatid glycolysis is unique in that it is compartmentalized, and many of its enzymes display unique structural and kinetic features. Structure- and catalytic mechanism-based approaches are applied to design compounds that inhibit the glycolytic enzymes of the parasites without affecting the corresponding proteins of the human host. For some trypanosomatid enzymes, potent and selective inhibitors have already been developed that affect only the growth of cultured trypanosomatids, and not mammalian cells.

Functional interaction of calcium-/calmodulin-dependent protein kinase II and cytosolic phospholipase A(2).

Calcium-/calmodulin-dependent protein kinase II (CaM kinase II), a decoder of Ca(2+) signals, and cytosolic phospholipase A(2) (cPLA(2)), an enzyme involved in arachidonate release, are involved in many physiological and pathophysiological processes. Activation of CaM kinase II in norepinephrine-stimulated vascular smooth muscle cells leads to activation of cPLA(2) and arachidonic acid release. Surface plasmon resonance, mass spectrometry, and kinetic studies show that CaM kinase II binds to cPLA(2) resulting in cPLA(2) phosphorylation on Ser-515 and an increase in its enzymatic activity. Phosphopeptide mapping studies with cPLA(2) from norepinephrine-stimulated smooth muscle cells indicates that phosphorylation of cPLA(2) on Ser-515, but not on Ser-505 or Ser-727, occurs in vivo. This novel signaling pathway for arachidonate release is shown to be cPLA(2)-dependent by use of a recently described and highly selective inhibitor of this enzyme.

Design and synthesis of substrate and internal standard conjugates for profiling enzyme activity in the Sanfilippo syndrome by affinity chromatography/electrospray ionization mass spectrometry.

We describe the design and synthesis of substrate and internal standard conjugates for application in profiling enzyme activity of the enzymes alpha-D-2-deoxy-2-N-sulfonamido-glucosamine sulfamidase, alpha-D-2-deoxy-2-N-acetyl-glucosamine hydrolase, acetyl-coenzymeA:alpha-D-2-deoxy-2-amino-glucosamine transferase, and alpha-D-2-deoxy-2-N-acetyl-glucosamine-6-sulfate sulfatase. Deficiency of any one of these enzymes results in a single clinical phenotype known as Sanfilippo syndrome. Such substrates have been proven effective in the confirmation of enzyme deficiency by a combination of affinity chromatography (AC) and electrospray ionization mass spectrometry (ESIMS), which forms the foundation for a new analytical technology (ACESIMS) of general interest and application to clinical and biomedical research.

A pyrrolidine-based specific inhibitor of cytosolic phospholipase A(2)alpha blocks arachidonic acid release in a variety of mammalian cells.

We analyzed a recently reported (K. Seno, T. Okuno, K. Nishi, Y. Murakami, F. Watanabe, T. Matsuur, M. Wada, Y. Fujii, M. Yamada, T. Ogawa, T. Okada, H. Hashizume, M. Kii, S.-H. Hara, S. Hagishita, S. Nakamoto, J. Med. Chem. 43 (2000)) pyrrolidine-based inhibitor, pyrrolidine-1, against the human group IV cytosolic phospholipase A(2) alpha-isoform (cPLA(2)alpha). Pyrrolidine-1 inhibits cPLA(2)alpha by 50% when present at approx. 0.002 mole fraction in the interface in a number of in vitro assays. It is much less potent on the cPLA(2)gamma isoform, calcium-independent group VI PLA(2) and groups IIA, X, and V secreted PLA(2)s. Pyrrolidine-1 blocked all of the arachidonic acid released in Ca(2+) ionophore-stimulated CHO cells stably transfected with cPLA(2)alpha, in zymosan- and okadaic acid-stimulated mouse peritoneal macrophages, and in ATP- and Ca(2+) ionophore-stimulated MDCK cells.

Adenosine analogues as selective inhibitors of glyceraldehyde-3-phosphate dehydrogenase of Trypanosomatidae via structure-based drug design.

In our continuation of the structure-based design of anti-trypanosomatid drugs, parasite-selective adenosine analogues were identified as low micromolar inhibitors of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Crystal structures of Trypanosoma brucei, Trypanosoma cruzi, Leishmania mexicana, and human GAPDH's provided details of how the adenosyl moiety of NAD(+) interacts with the proteins, and this facilitated the understanding of the relative affinities of a series of adenosine analogues for the various GAPDH's. From exploration of modifications of the naphthalenemethyl and benzamide substituents of a lead compound, N(6)-(1-naphthalenemethyl)-2'-deoxy-2'-(3-methoxybenzamido)adenosine (6e), N(6)-(substituted-naphthalenemethyl)-2'-deoxy-2'-(substituted-benzamido)adenosine analogues were investigated. N(6)-(1-Naphthalenemethyl)-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (6m), N(6)-[1-(3-hydroxynaphthalene)methyl]-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (7m), N(6)-[1-(3-methoxynaphthalene)methyl]-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (9m), N(6)-(2-naphthalenemethyl)-2'-deoxy-2'-(3-methoxybenzamido)adenosine (11e), and N(6)-(2-naphthalenemethyl)-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (11m) demonstrated a 2- to 3-fold improvement over 6e and a 7100- to 25000-fold improvement over the adenosine template. IC(50)'s of these compounds were in the range 2-12 microM for T. brucei, T. cruzi, and L. mexicana GAPDH's, and these compounds did not inhibit mammalian GAPDH when tested at their solubility limit. To explore more thoroughly the structure-activity relationships of this class of compounds, a library of 240 N(6)-(substituted)-2'-deoxy-2'-(amido)adenosine analogues was generated using parallel solution-phase synthesis with N(6) and C2' substituents chosen on the basis of computational docking scores. This resulted in the identification of 40 additional compounds that inhibit parasite GAPDH's in the low micromolar range. We also explored adenosine analogues containing 5'-amido substituents and found that 2',5'-dideoxy-2'-(3,5-dimethoxybenzamido)-5'-(diphenylacetamido)adenosine (49) displays an IC(50) of 60-100 microM against the three parasite GAPDH's.

TcRho1, a farnesylated Rho family homologue from Trypanosoma cruzi: cloning, trans-splicing, and prenylation studies.

Rho GTPases are members of the Ras superfamily and are involved in signal transduction pathways, including maintenance of cell morphology and motility, cell cycle progression, and transcription activation. We report the molecular identification in trypanosomatids (Trypanosoma cruzi) of the first member of the Rho family. The cloned Rho protein, TcRho1, shares approximately 40% homology with other members of the Rho family. Southern blot analysis revealed that TcRHO1 is a single copy gene per haploid genome, and Northern blot assays showed a transcript of 1200 nucleotides in length. Mapping the 5'-untranslated region of TcRHO1 transcripts revealed at least five different transcripts derived from differential trans-splicing. Three of the five transcripts contain the trans-splicing site within the coding region of the TcRHO1 gene. TcRho1 also contains the C-terminal sequence CQLF (CAAX motif), which is predicted to direct post-translation prenylation of the cysteine residue. A synthetic peptide containing this C-terminal motif, when tested against Q-Sepharose chromatography fractions from T. cruzi cytosol, was shown to be efficiently farnesylated, but not geranylgeranylated, despite the fact that the CAAX motif with X = Phe specifies geranylgeranylation by mammalian protein geranylgeranyltransferase I. Furthermore, immunoblot analyses of epimastigote protein with anti-S-farnesylcysteine methyl ester and anti-TcRho1 antisera strongly suggested that TcRho1 is farnesylated in vivo. The farnesylation of proteins such as Rho GTPases could be the basis for the selective cytotoxic action of protein farnesyltransferase inhibitors on trypanosomatids versus mammalian cells.

Conformational changes in Leishmania mexicana glyceraldehyde-3-phosphate dehydrogenase induced by designed inhibitors.

The glycolytic enzymes of trypanosomes are attractive drug targets, since the blood-stream form of Trypanosoma brucei lacks a functional citric acid cycle and is dependent solely on glycolysis for its energy requirements. Glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from the pathogenic trypanosomatids T. brucei, Trypanosoma cruzi and Leishmania mexicana are quite similar to each other, and yet have sufficient structural differences compared to the human enzyme to enable the structure-based design of compounds that selectively inhibit all three trypanosomatid enzymes but not the human homologue. Adenosine analogs with substitutions on N-6 of the adenine ring and on the 2' position of the ribose moiety were designed, synthesized and tested for inhibition. Two crystal structures of L. mexicana glyceraldehyde-3-phosphate dehydrogenase in complex with high-affinity inhibitors that also block parasite growth were solved at a resolution of 2.6 A and 3.0 A. The complexes crystallized in the same crystal form, with one and a half tetramers in the crystallographic asymmetric unit. There is clear electron density for the inhibitor in all six copies of the binding site in each of the two structures. The L. mexicana GAPDH subunit exhibits substantial structural plasticity upon binding the inhibitor. Movements of the protein backbone, in response to inhibitor binding, enlarge a cavity at the binding site to accommodate the inhibitor in a classic example of induced fit. The extensive hydrophobic interactions between the protein and the two substituents on the adenine scaffold of the inhibitor provide a plausible explanation for the high affinity of these inhibitors for trypanosomatid GAPDHs.

Direct profiling of multiple enzyme activities in human cell lysates by affinity chromatography/electrospray ionization mass spectrometry: application to clinical enzymology.

We describe a new method for enzyme analysis using affinity capture followed by electrospray ionization mass spectrometry (ACESIMS) for the quantitative determination of the initial velocities of four heparin-modifying enzymes. These enzymes, when defective in affected children, lead to the lysosomal storage disease known as Sanfilippo syndrome. The method relies on substrates and internal standards conjugated to the molecular handle biotin via a heavy isotope-encodable, mass-adjustable linker. Reaction velocities of the Sanfilippo enzymes in a crude lysate prepared from as little as 2500 human skin fibroblasts can be determined. In addition, the ACESIMS method is widely applicable to the simultaneous analysis of multiple enzymes in a complex biological sample by a single analytical technique and will thus serve as a useful tool in basic and clinical biomedical research.

Quantification of cellular acid sphingomyelinase and galactocerebroside beta-galactosidase activities by electrospray ionization mass spectrometry.

BACKGROUND: Diagnosis of Niemann-Pick (A and B) and Krabbe diseases is achieved by measurement of the lysosomal enzymes acid sphingomyelinase (ASM) and galactocerebroside beta-galactosidase (GCG), respectively. Conventional assays use radiolabeled or fluorescent substrates and do not allow simultaneous determination of two or more enzymes in the sample. METHODS: We developed a sensitive and specific method to assay ASM and GCG in skin fibroblast homogenates using biotinylated substrate conjugates. The products were purified by bioaffinity capture on streptavidin-agarose beads and, following release, were analyzed by electrospray ionization mass spectrometry. Quantification was achieved using stable-isotope-labeled internal standards that were chemically identical to the products of the enzymatic reactions. RESULTS: The method demonstrated excellent linearity of ASM and GCG enzymatic product formation with the amount of cellular protein and incubation time. The range of ASM activities in fibroblast lysates from six healthy patients was 39-70 nmol. mg(-1). h(-1) compared with 3.7-5.1 nmol. mg(-1). h(-1) in cell lysates from two patients affected with Niemann-Pick A disease. The GCG activities toward the corresponding substrate conjugate were 4.0-6.8 nmol. mg(-1). h(-1) in cell lysates from healthy patients compared with 0.1-0.2 nmol. mg(-1). h(-1) in cell lysates from two patients affected with Krabbe disease. The amounts of substrate conjugates needed per analysis were 15 nmol (14 microg) for both ASM and GCG. CONCLUSIONS: Electrospray mass spectrometry combined with the use of biotinylated substrate conjugates and bioaffinity purification represents a new approach for the diagnosis of lysosomal storage diseases as demonstrated for Niemann-Pick A and Krabbe diseases. No radioactive substrates are used, and the method uses a single instrumental platform to determine both ASM and GCG in one cell sample.