Chlorotoxin binds to both matrix metalloproteinase 2 and neuropilin 1.

Chlorotoxin (CTX), a scorpion venom-derived 36-residue miniprotein, binds to and is taken up selectively by glioblastoma cells. Previous studies provided controversial results concerning target protein(s) of CTX. These included CLC3 chloride channel, matrix metalloproteinase 2 (MMP-2), regulators of MMP-2, annexin A2, and neuropilin 1 (NRP1). The present study aimed at clarifying which of the proposed binding partners can really interact with CTX using biochemical methods and recombinant proteins. For this purpose, we established two new binding assays based on anchoring the tested proteins to microbeads and quantifying the binding of CTX by flow cytometry. Screening of His-tagged proteins anchored to cobalt-coated beads indicated strong interaction of CTX with MMP-2 and NRP1, whereas binding to annexin A2 was not confirmed. Similar results were obtained with fluorophore-labeled CTX and CTX-displaying phages. Affinity of CTX to MMP-2 and NRP1 was assessed by the "immunoglobulin-coated bead" test, in which the proteins were anchored to beads by specific antibodies. This assay yielded highly reproducible data using both direct titration and displacement approach. The affinities of labeled and unlabeled CTX appeared to be similar for both MMP-2 and NRP1 with estimated KD values of 0.5 to 0.7 µM. Contrary to previous reports, we found that CTX does not inhibit the activity of MMP-2 and that CTX not only with free carboxyl end but also with carboxamide terminal end binds to NRP1. We conclude that the presented robust assays could also be applied for affinity-improving studies of CTX to its genuine targets using phage display libraries.

Diffuse mesangial sclerosis in a PDSS2 mutation-induced coenzyme Q10 deficiency.

BACKGROUND: A 7-month-old male infant was admitted because he was suffering from nephrotic syndrome, along with encephalomyopathy, hypertrophic cardiomyopathy, clinically suspected deafness and retinitis pigmentosa, and an elevated serum lactate level. METHODS: Coenzyme Q10 supplementation was started because of the clinical suspicion of primary CoQ10 deficiency. Despite intensive efforts, he passed away 4 weeks after admission. RESULTS: The results of genetic tests, available postmortem, explored two hitherto undescribed mutations in the PDSS2 gene. Both were located within the polyprenyl synthetase domain. Clinical exome sequencing revealed a heterozygous missense mutation in exon 3, and our in-house joint-analysis algorithm detected a heterozygous large 2923-bp deletion that affected the 5 prime end of exon 8. Other causative defects in the CoQ10 and infantile nephrosis-related genes examined were not found. A postmortem histological, immunohistochemical, and electron microscopic evaluation of the glomeruli revealed collapsing-sclerosing lesions consistent with diffuse mesangial sclerosis. The extrarenal alterations included hypertrophic cardiomyopathy and diffuse alveolar damage. A histological evaluation of the central nervous system and skeletal muscles did not demonstrate any obvious abnormality. CONCLUSIONS: Until now, the clinical features and the mutational status of 6 patients with a PDSS2 gene defect have been reported in the English literature. Here, we describe for the first time detailed kidney morphology features in a patient with nephrotic syndrome carrying mutations in the PDSS2 gene.

Novel possible pharmaceutical research tools: stem cells, gene delivery and their combination.

Both stem cell research and gene delivery are very promising fields of today's biomedical research. In the present review we first attempt to summarize the state of the art in stem cell research. We describe the major categories of stem cells based on cell sources: embryonic, fetal, postnatal and induced pluripotent stem cells. We then present new data on stem cell cultures of dental pulp origin as examples of the progress of postnatal stem cell research. Afterwards, we briefly summarize the most promising achievements in the field of gene delivery. As an example of such advances, we describe novel in vitro and in vivo gene delivery studies to demonstrate that salivary glands are highly potential targets for gene therapy: they can be used to produce therapeutic peptides delivered either into the oral cavity or into the systemic circulation. Finally, we describe and compare studies combining the use of stem cells and gene delivery. We conclude that stem cell therapy and gene delivery alone are both very exciting research areas, and they may act in synergy when used in combination.

Integration of neuronally predifferentiated human dental pulp stem cells into rat brain in vivo.

Pluripotency and their neural crest origin make dental pulp stem cells (DPSCs) an attractive donor source for neuronal cell replacement. Despite recent encouraging results in this field, little is known about the integration of transplanted DPSC derived neuronal pecursors into the central nervous system. To address this issue, neuronally predifferentiated DPSCs, labeled with a vital cell dye Vybrant DiD were introduced into postnatal rat brain. DPSCs were transplanted into the cerebrospinal fluid of 3-day-old male Wistar rats. Cortical lesion was induced by touching a cold (-60°C) metal stamp to the calvaria over the forelimb motor cortex. Four weeks later cell localization was detected by fluorescent microscopy and neuronal cell markers were studied by immunohistochemistry. To investigate electrophysiological properties of engrafted, fluorescently labeled DPSCs, 300 µm-thick horizontal brain slices were prepared and the presence of voltage-dependent sodium and potassium channels were recorded by patch clamping. Predifferentiated donor DPSCs injected into the cerebrospinal fluid of newborn rats migrated as single cells into a variety of brain regions. Most of the cells were localized in the normal neural progenitor zones of the brain, the subventricular zone (SVZ), subgranular zone (SGZ) and subcallosal zone (SCZ). Immunohistochemical analysis revealed that transplanted DPSCs expressed the early neuronal marker N-tubulin, the neuronal specific intermediate filament protein NF-M, the postmitotic neuronal marker NeuN, and glial GFAP. Moreover, the cells displayed TTX sensitive voltage dependent (VD) sodium currents (I(Na)) and TEA sensitive delayed rectifier potassium currents (K(DR)). Four weeks after injury, fluorescently labeled cells were detected in the lesioned cortex. Neurospecific marker expression was increased in DPSCs found in the area of the cortical lesions compared to that in fluorescent cells of uninjured brain. TTX sensitive VD sodium currents and TEA sensitive K(DR) significantly increased in labeled cells of the cortically injured area. In conclusion, our data demonstrate that engrafted DPSC-derived cells integrate into the host brain and show neuronal properties not only by expressing neuron-specific markers but also by exhibiting voltage dependent sodium and potassium channels. This proof of concept study reveals that predifferentiated hDPSCs may serve as useful sources of neuro- and gliogenesis in vivo, especially when the brain is injured.

In vivo secretion of the mouse immunoglobulin G Fc fragment from rat submandibular glands.

BACKGROUND: Salivary glands have been proposed as target organs for gene therapy. They secrete endogenous, as well as transgenic proteins, in a polarized manner. Transgene-encoded regulated pathway proteins primarily follow the regulated pathway in rat salivary glands and are secreted into saliva in an exocrine manner. Conversely, constitutive pathway proteins generally are secreted more basolaterally and thus follow the endocrine route. In the present study, we studied in vivo the sorting of the mouse immunoglobulin G2b Fc fragment, which is physiologically secreted via the constitutive pathway. METHODS: Adenoviral vectors encoding the Fc fragment and human growth hormone were delivered into rat and mouse submandibular glands in vivo to compare their serum-to-saliva distribution. We also compared the intracellular localization of the Fc fragment and growth hormone by confocal microscopy. RESULTS: We found that the Fc fragment was secreted almost entirely into the bloodstream from rat and mouse submandibular glands via a constitutive or constitutive-like pathway. This sorting behaviour is clearly different from that of transgenic human growth hormone, which is secreted in a regulated pathway, both in neuroendocrine cells and as a transgenic protein from salivary gland cells. We also found that simultaneously expressed human growth hormone and the mouse Fc fragment do not appear to influence each other's sorting behaviour. The Fc fragment showed a primarily basal localization, whereas growth hormone showed an apical localization, in rat submandibular gland acinar cells. CONCLUSIONS: The results obtained in the present study indicate that the mouse Fc fragment is a useful model protein for examining the basolateral versus apical secretory pathways employed by transgenic secretory proteins in salivary glands.

Myotubular/centronuclear myopathy and central core disease.

The term congenital myopathy is applied to muscle disorders presenting with generalized muscle weakness and hypotonia from early infancy with delayed developmental milestones. The congenital myopathies have been classified into various categories based on morphological findings on muscle biopsy. Although the clinical symptoms may seem homogenous, the genetic basis is remarkably variable. This review will focus on myotubular myopathy, centronuclear myopathy, central core disease, and congenital neuromuscular disease with uniform Type 1 fiber, myopathies that are subjects of our ongoing examinations.

Possible role of duration of PKC-induced ERK activation in the effects of agonists and phorbol esters on DNA synthesis in Panc-1 cells.

Protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) have been implicated in the effects of regulatory peptides on proliferation. We studied how ERK was activated by PKC following regulatory peptide or phorbol ester stimulation and we also investigated the effect of ERK activation on proliferation in Panc-1 cells. Panc-1 cells transfected with CCK1 receptors were treated with cholecystokinin (CCK), neurotensin (NT), or phorbol 12-myristate 13-acetate (PMA). DNA synthesis was studied by measuring tritiated thymidine incorporation. PKC isoforms were selectively inhibited with Gö6983 and 200 nM Ro-32-0432, their translocation was detected by confocal microscopy and by subcellular fractionation followed by immunoblotting. ERK cascade activation was detected with phosphoERK immunoblotting and inhibited with 20 microM PD98059. PMA and CCK inhibited, NT stimulated DNA synthesis. These effects were inhibited by Ro-32-0432 but not by Gö6983 suggesting the involvement of PKCepsilon in proliferation control. Confocal microscopy and subcellular fractionation demonstrated that PMA, CCK, and NT caused cytosol to membrane translocation of PKCepsilon and ERK activation that was inhibited by Ro-32-0432 but not by Gö6983. ERK activation was prolonged following PMA and CCK, but transient after NT treatment. PMA, CCK, and NT all activated cyclinD1, while p21CIP1 expression was increased by only PMA and CCK, but not by NT; each of these effects is inhibited by PD98059. In conclusion, our results provide evidence for PKCepsilon-mediated differential ERK activation and growth regulation in Panc-1C cells. Identification of the mechanisms by which these key signaling pathways are modulated could provide a basis for the development of novel therapeutic interventions to treat pancreatic cancer.

Intermittent spontaneous breathing protects the rat diaphragm from mechanical ventilation effects.

OBJECTIVE: Short-term mechanical ventilation has been proven to reduce diaphragm force and fiber dimensions. We hypothesized that intermittent spontaneous breathing during the course of mechanical ventilation would minimize the effects of mechanical ventilation on diaphragm force and expression levels of transcription factors (MyoD and myogenin). DESIGN: Randomized, controlled experiment. SETTING: Animal basic science laboratory. SUBJECTS: Male Wistar rats, weighing 350-500 g. INTERVENTIONS: Anesthetized and tracheotomized rats were submitted to either 24 hrs of spontaneous breathing (SB, n = 5), 24 hrs of continuous controlled mechanical ventilation (CMV, n = 7), or controlled mechanical ventilation with intermittent spontaneous breathing: 60 mins every 5 hrs of mechanical ventilation repeated four times (ISB60, n = 8) or 5 mins every 5 hrs 55 mins of mechanical ventilation repeated four times (SB5, n = 9). They were compared with control animals free from intervention (C, n = 5). MEASUREMENTS AND MAIN RESULTS: The profile of the diaphragm force-frequency curve of the controls and SB group was significantly different from that of the ISB and CMV groups; especially, the mean asymptotic force was less in the ISB and CMV compared with controls and SB. CMV resulted in a significant decrease in the diaphragm type I (-26%, p < .05 vs. C) and type IIx/b (-39%, p < .005 vs. C and SB) cross-sectional area, whereas this was not observed in the ISB groups. Diaphragm MyoD protein expression was significantly decreased after ISB60 (-35%, p < .0001 vs. C and SB) and even more after CMV (-73%, p < .0001 vs. others). The same pattern was observed with myogenin protein levels. Positive relationships between diaphragm MyoD and myogenin protein levels and diaphragm force were observed. CONCLUSIONS: The data demonstrated that intermittent spontaneous breathing during the course of mechanical ventilation may minimize the deleterious effect of controlled mechanical ventilation on diaphragm force, fiber dimensions, and expression of transcription factors.

Expression and distribution of the nitric oxide synthases in idiopathic inflammatory myopathies.

Different immune effector mechanisms have been characterised in the idiopathic inflammatory myopathies (IIM): in polymyositis (PM) and sporadic inclusion body myositis (sIBM), T-cell-mediated cytotoxicity targets nonnecrotic muscle fibres, whereas in dermatomyositis (DM) the complement-mediated immune response is directed against the microvasculature. As nitric oxide (NO) has an important function in cell signalling and in the cytotoxicity displayed by activated macrophages, it is potentially involved in the immunopathogenesis of IIM. Using immunohistochemical, in situ hybridisation and Western blotting techniques, we visualised the three isoforms of NO synthase (NOS) in muscle tissues from normal controls and from patients diagnosed with IIM. The levels of both constitutive isoforms of NOS (endothelial, i.e., eNOS, and neuronal, i.e., nNOS) were unchanged in IIM as compared with normal muscle. Both protein and mRNA of the inducible form (iNOS) were detected in half of the control biopsies. Constant and increased iNOS protein expression was found in endomysial infiltrates of PM and sIBM, whereas perimysial inflammatory cells in DM were largely negative. We developed a quantitative Western blotting protocol which confirmed the constitutive nature of nNOS and eNOS and the significant induction of iNOS in PM. Our results appoint iNOS with a dual function: a limited and transient role in normal muscle physiology and an active cytotoxic role in PM and sIBM.

Localization of the alpha-chemokine SDF-1 and its receptor CXCR4 in idiopathic inflammatory myopathies.

We studied the distribution of stromal cell-derived factor 1 isoforms alpha and beta, and their receptor CXCR4, in polymyositis, sporadic inclusion body myositis and dermatomyositis using in situ hybridization, immunohistochemistry, immunofluorescence and Western blotting. In control muscle, polymyositis and sporadic inclusion body myositis, stromal cell-derived factor-1alpha expression was noted in muscle fibers, while stromal cell-derived factor-1beta and CXCR4 were predominantly localized to capillaries and arterioles. In dermatomyositis, stromal cell-derived factor-1beta immunoreactivity of blood vessels was focally increased. The vast majority of inflammatory cells in idiopathic inflammatory myopathies were CXCR4 positive. A subset of helper T-cells and macrophages expressed stromal cell-derived factor-1alpha, while only rare inflammatory cells expressed stromal cell-derived factor-1beta. A significant increase of stromal cell-derived factor-1alpha and CXCR4 was observed in protein extracts of idiopathic inflammatory myopathies in comparison with normal controls. The abundance of both CXCR4 and its ligand stromal cell-derived factor-1 implicates their interaction in the pathogenesis of idiopathic inflammatory myopathies and identifies these proteins as possible targets for selective immune therapy.

Early changes in rat diaphragm biology with mechanical ventilation.

To better characterize the effects of 24-hour mechanical ventilation on diaphragm, the expression of myogenic transcription factors, myosin heavy chains, and sarcoplasmic/endoplasmic reticulum calcium-ATPase pumps was examined in rats. In the diaphragm of mechanically ventilated animals, the mRNA of MyoD, myosin heavy chain-2a and -2b, and sarcoplasmic/endoplasmic reticulum calcium-ATPase-1a decreased, whereas myogenin mRNA increased. In the diaphragm of anesthetized and spontaneously breathing rats, only the mRNA of MyoD and myosin heavy chain-2a decreased. MyoD and myogenin protein expression followed the changes at the mRNA, whereas the myosin heavy chain isoforms did not change. Parallel experiments involving the gastrocnemius were performed to assess the relative contribution of muscle shortening versus immobilization-induced deconditioning on muscle regulatory factor expression. Passive shortening produced no additional effects compared with immobilization-induced deconditioning. The overall changes followed a remarkably similar pattern except for MyoD protein expression, which increased in the gastrocnemius and decreased in the diaphragm while its mRNA diminished in both muscles. The early alterations in the expression of muscle protein and regulatory factors may serve as underlying molecular basis for the impaired diaphragm function seen after 24 hours of mechanical ventilation. Whether immobilization-induced deconditioning and/or passive shortening play a role in these alterations could not be fully unraveled.