Upregulation of alpha-skeletal muscle actin and myosin heavy polypeptide gene products in degenerating rotator cuff muscles

Impaired function of shoulder muscles, resulting from rotator cuff tears, is associated with abnormal deposition of fat in muscle tissue, but corresponding cellular and molecular mechanisms, likely reflected by altered gene expression profiles, are largely unknown. Here, an analysis of muscle gene expression was carried out by semiquantitative RT-PCR in total RNA extracts of supraspinatus biopsies collected from 60 patients prior to shoulder surgery. A significant increase of alpha-skeletal muscle actin (p = 0.0115) and of myosin heavy polypeptide 1 (p = 0.0147) gene transcripts was observed in parallel with progressive fat deposition in the muscle, assessed on parasagittal T1-weighted turbo-spin-echo magnetic resonance images according to Goutallier. Upregulation of alpha-skeletal muscle actin and of myosin heavy polypeptide-1 has been reported to be associated with increased muscle tissue metabolism and oxidative stress. The findings of the present study, therefore, challenge the hypothesis that increased fat deposition in rotator cuff muscle after injury reflects muscle degeneration.

The Plasmodium Class XIV Myosin, MyoB, Has a Distinct Subcellular Location in Invasive and Motile Stages of the Malaria Parasite and an Unusual Light Chain

Myosin B (MyoB) is one of the two short class XIV myosins encoded in the Plasmodium genome. Class XIV myosins are characterized by a catalytic "head," a modified "neck," and the absence of a "tail" region. Myosin A (MyoA), the other class XIV myosin in Plasmodium, has been established as a component of the glideosome complex important in motility and cell invasion, but MyoB is not well characterized. We analyzed the properties of MyoB using three parasite species as follows: Plasmodium falciparum, Plasmodium berghei, and Plasmodium knowlesi. MyoB is expressed in all invasive stages (merozoites, ookinetes, and sporozoites) of the life cycle, and the protein is found in a discrete apical location in these polarized cells. In P. falciparum, MyoB is synthesized very late in schizogony/merogony, and its location in merozoites is distinct from, and anterior to, that of a range of known proteins present in the rhoptries, rhoptry neck or micronemes. Unlike MyoA, MyoB is not associated with glideosome complex proteins, including the MyoA light chain, myosin A tail domain-interacting protein (MTIP). A unique MyoB light chain (MLC-B) was identified that contains a calmodulin-like domain at the C terminus and an extended N-terminal region. MLC-B localizes to the same extreme apical pole in the cell as MyoB, and the two proteins form a complex. We propose that MLC-B is a MyoB-specific light chain, and for the short class XIV myosins that lack a tail region, the atypical myosin light chains may fulfill that role.

Anti-idiotypic Fab Fragments Image a Conserved N-terminal Epitope Patch of Grass Pollen Allergen Phl p 1

BACKGROUND AND AIMS: Naturally occurring anti-idiotypic antibodies structurally mimic the original antibody epitope. Anti-idiotypes, therefore, are interesting tools for the portrayal of conformational B-cell epitopes of allergens. In this study we used this strategy particularly for major timothy grass pollen (Phleum pratense) allergen Phl p 1. METHODS AND RESULTS: We used a combinatorial phage display library constructed from the peripheral IgG repertoire of a grass pollen allergic patient which was supposed to contain anti-idiotypic Fab specificities. Using purified anti-Phl p 1 IgG for biopanning, several Fab displaying phage clones could be isolated. 100 amplified colonies were screened for their binding capacity to anti-Phl p 1-specific antibodies, finally resulting in four distinct Fab clones according to sequence analysis. Interestingly, heavy chains of all clones derived from the same germ line sequence and showed high homology in their CDRs. Projecting their sequence information on the surface of the natural allergen Phl p 1 (PDB ID: 1N10) indicated matches on the N-terminal domain of the homo-dimeric allergen, including the bridging region between the two monomers. The resulting epitope patches were formed by spatially distant sections of the primary allergen sequence. CONCLUSION: In this study we report that anti-idiotypic specificities towards anti-Phl p 1 IgG, selected from a Fab library of a grass pollen allergic patient, mimic a conformational epitope patch being distinct from a previously reported IgE epitope area.

Cleavage of IgG1 in gingival crevicular fluid is associated with the presence of Porphyromonas gingivalis

BACKGROUND AND OBJECTIVES: Immunoglobulin (Ig) G1 plays an important role in the adaptive immune response. Kgp, a lysine-specific cysteine protease from Porphyromonas gingivalis, specifically hydrolyses IgG1 heavy chains. The purpose of this study was to examine whether cleavage of IgG1 occurs in gingival crevicular fluid (GCF) in vivo, and whether there is any association with the presence of Porphyromonas gingivalis and other periodontopathogens. MATERIAL AND METHODS: GCF was obtained from nine patients with aggressive periodontitis, nine with chronic periodontitis and five periodontally healthy individuals. The bacterial loads of Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Treponema denticola, Prevotella intermedia and Tannerella forsythia were analysed by real-time polymerase chain reaction, and the presence and cleavage of IgG1 and IgG2 were determined using Western blotting. Kgp levels were measured by ELISA. RESULTS: Cleaved IgG1 was identified in the GCF from 67% of patients with aggressive periodontitis and in 44% of patients with chronic periodontitis. By contrast, no cleaved IgG1 was detectable in healthy controls. No degradation of IgG2 was detected in any of the samples, regardless of health status. Porphyromonas gingivalis was found in high numbers in all samples in which cleavage of IgG1 was detected (P < 0.001 compared with samples with no IgG cleavage). Furthermore, high numbers of Tannerella forsythia and Prevotella intermedia were also present in these samples. The level of Kgp in the GCF correlated with the load of Porphyromonas gingivalis (r = 0.425, P < 0.01). The presence of Kgp (range 0.07-10.98 ng/mL) was associated with proteolytic fragments of IgG1 (P < 0.001). However, cleaved IgG1 was also detected in samples with no detectable Kgp. CONCLUSION: In patients with periodontitis, cleavage of IgG1 occurs in vivo and may suppress antibody-dependent antibacterial activity in subgingival biofilms especially those colonized by Porphyromonas gingivalis.

Focal adhesion kinase is a load-dependent governor of the slow contractile and oxidative muscle phenotype

Striated muscle exhibits a pronounced structural-functional plasticity in response to chronic alterations in loading. We assessed the implication of focal adhesion kinase (FAK) signalling in mechano-regulated differentiation of slow-oxidative muscle. Load-dependent consequences of FAK signal modulation were identified using a multi-level approach after electrotransfer of rat soleus muscle with FAK-expression plasmid vs. empty plasmid-transfected contralateral controls. Muscle fibre-targeted over-expression of FAK in anti-gravitational muscle for 9 days up-regulated transcript levels of gene ontologies underpinning mitochondrial metabolism and contraction in the transfected belly portion. Concomitantly, mRNA expression of the major fast-type myosin heavy chain (MHC) isoform, MHC2A, was reduced. The promotion of the slow-oxidative expression programme by FAK was abolished after co-expression of the FAK inhibitor FAK-related non-kinase (FRNK). Elevated protein content of MHC1 (+9%) and proteins of mitochondrial respiration (+165-610%) with FAK overexpression demonstrated the translation of transcript differentiation in targeted muscle fibres towards a slow-oxidative muscle phenotype. Coincidentally MHC2A protein was reduced by 50% due to protection of muscle from de-differentiation with electrotransfer. Fibre cross section in FAK-transfected muscle was elevated by 6%. The FAK-modulated muscle transcriptome was load-dependent and regulated in correspondence to tyrosine 397 phosphorylation of FAK. In the context of overload, the FAK-induced gene expression became manifest at the level of contraction by a slow transformation and the re-establishment of normal muscle force from the lowered levels with transfection. These results highlight the analytic power of a systematic somatic transgene approach by mapping a role of FAK in the dominant mechano-regulation of muscular motor performance via control of gene expression.

Dynamic patterns of ventricular remodeling and apoptosis in hearts unloaded by heterotopic transplantation

BACKGROUND Mechanical unloading of failing hearts can trigger functional recovery but results in progressive atrophy and possibly detrimental adaptation. In an unbiased approach, we examined the dynamic effects of unloading duration on molecular markers indicative of myocardial damage, hypothesizing that potential recovery may be improved by optimized unloading time. METHODS Heterotopically transplanted normal rat hearts were harvested at 3, 8, 15, 30, and 60 days. Forty-seven genes were analyzed using TaqMan-based microarray, Western blot, and immunohistochemistry. RESULTS In parallel with marked atrophy (22% to 64% volume loss at 3 respectively 60 days), expression of myosin heavy-chain isoforms (MHC-α/-β) was characteristically switched in a time-dependent manner. Genes involved in tissue remodeling (FGF-2, CTGF, TGFb, IGF-1) were increasingly upregulated with duration of unloading. A distinct pattern was observed for genes involved in generation of contractile force; an indiscriminate early downregulation was followed by a new steady-state below normal. For pro-apoptotic transcripts bax, bnip-3, and cCasp-6 and -9 mRNA levels demonstrated a slight increase up to 30 days unloading with pronunciation at 60 days. Findings regarding cell death were confirmed on the protein level. Proteasome activity indicated early increase of protein degradation but decreased below baseline in unloaded hearts at 60 days. CONCLUSIONS We identified incrementally increased apoptosis after myocardial unloading of the normal rat heart, which is exacerbated at late time points (60 days) and inversely related to loss of myocardial mass. Our findings suggest an irreversible detrimental effect of long-term unloading on myocardium that may be precluded by partial reloading and amenable to molecular therapeutic intervention.

MicroRNA miR-199a-5p regulates smooth muscle cell proliferation and morphology by targeting Wnt2 signaling pathway

MicroRNA miR-199a-5p impairs tight junction formation leading to increased urothelial permeability in bladder pain syndrome. Now using transcriptome analysis in urothelial TEU-2 cells we implicate it in the regulation of cell cycle, cytoskeleton remodeling, TGF and Wnt signaling pathways. MiR-199a-5p is highly expressed in the smooth muscle layer of the bladder and we altered its levels in bladder smooth muscle cells (SMC) to validate the pathway analysis. Inhibition of miR-199a-5p with antimiR increased SMC proliferation, reduced cell size and up-regulated miR-199a-5p targets, including Wnt2. Overexpression of Wnt2 protein or treating SMCs with recombinant Wnt2 closely mimicked the miR-199a-5p inhibition, whereas down-regulation of Wnt2 in antimiR-expressing SMCs with shRNA restored cell phenotype and proliferation rates. Overexpression of miR-199a-5p in the bladder SMCs significantly increased cell size and up-regulated SM22, SM alpha-actin and SM myosin heavy chain mRNA and protein levels. These changes, as well as increased expression of ACTG2, TGFB1I1, and CDKN1A were mediated by up-regulation of smooth muscle-specific transcriptional activator myocardin at mRNA and protein levels. Myocardin-related transcription factor (MRTF-A) downstream targets Id3 and MYL9 were also induced. Up-regulation of myocardin was accompanied by down-regulation of Wnt-dependent inhibitory Kruppel-like transcription factor 4 (KLF4) in miR-199a-5p overexpressing cells. In contrast, KLF4 was induced in antimiR-expressing cells following the activation of Wnt2 signaling, leading to repression of myocardin-dependent genes. MiR-199a-5p plays a critical role in the Wnt2-mediated regulation of proliferative and differentiation processes in the smooth muscle and may behave as a key modulator of smooth muscle hypertrophy, relevant for organ remodeling.

Pi release from myosin: a simulation analysis of possible pathways

The release of phosphate (Pi) is an important element in actomyosin function and has been shown to be accelerated by the binding of myosin to actin. To provide information about the structural elements important for Pi release, possible escape pathways from various isolated myosin II structures have been determined by molecular dynamics simulations designed for studying such slow processes. The residues forming the pathways were identified and their role was evaluated by mutant simulations. Pi release is slow in the pre-powerstroke structure, an important element in preventing the powerstroke prior to actin binding, and is much more rapid for Pi modeled into the post-rigor and rigor-like structures. The previously proposed backdoor route is dominant in the pre-powerstroke and post-rigor states, whereas a different path is most important in the rigor-like state. This finding suggests a mechanism for the actin-activated acceleration of Pi release.

Identification of 48 homologues of phosphatidylethanol in blood by LC-ESI-MS/MS

Phosphatidylethanol (PEth) is an abnormal phospholipid carrying two fatty acid chains. It is only formed in the presence of ethanol via the action of phospholipase D (PLD). Its use as a biomarker for alcohol consumption is currently under investigation. Previous methods for the analysis of PEth included high-performance liquid chromatography (HPLC) coupled to an evaporative light scattering detector (ELSD), which is unspecific for the different homologues--improved methods are now based on time of flight mass spectrometry (TOF-MS) and tandem mass spectrometry (MS/MS). The intention of this work was to identify as many homologues of PEth as possible. A screening procedure using multiple-reaction monitoring (MRM) for the identified homologues has subsequently been established. For our investigations, autopsy blood samples collected from heavy drinkers were used. Phosphatidylpropanol 16:0/18:1 (internal standard) was added to the blood samples prior to liquid-liquid extraction using borate buffer (pH 9), 2-propanol and n-hexane. After evaporation, the samples were redissolved in the mobile phase and injected into the LC-MS/MS system. Compounds were separated on a Luna Phenyl Hexyl column (50 mm x 2 mm, 3 microm) by gradient elution, using 2 mM ammonium acetate and methanol/acetone (95/5; v/v). A total of 48 homologues of PEth could be identified by using precursor ion and enhanced product ion scans (EPI).

Heavy metal cations permeate the TRPV6 epithelial cation channel

TRPV6 belongs to the vanilloid family of the transient receptor potential channel (TRP) superfamily. This calcium-selective channel is highly expressed in the duodenum and the placenta, being responsible for calcium absorption in the body and fetus. Previous observations have suggested that TRPV6 is not only permeable to calcium but also to other divalent cations in epithelial tissues. In this study, we tested whether TRPV6 is indeed also permeable to cations such as zinc and cadmium. We found that the basal intracellular calcium concentration was higher in HEK293 cells transfected with hTRPV6 than in non-transfected cells, and that this difference almost disappeared in nominally calcium-free solution. Live cell imaging experiments with Fura-2 and NewPort Green DCF showed that overexpression of human TRPV6 increased the permeability for Ca(2+), Ba(2+), Sr(2+), Mn(2+), Zn(2+), Cd(2+), and interestingly also for La(3+) and Gd(3+). These results were confirmed using the patch clamp technique. (45)Ca uptake experiments showed that cadmium, lanthanum and gadolinium were also highly efficient inhibitors of TRPV6-mediated calcium influx at higher micromolar concentrations. Our results suggest that TRPV6 is not only involved in calcium transport but also in the transport of other divalent cations, including heavy metal ions, which may have toxicological implications.