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  • Dry needling at myofascial trigger spots of rabbit skeletal muscles modulates the biochemicals associated with pain, inflammation, and hypoxia. 23346198

    Background and Purpose. Dry needling is an effective therapy for the treatment of pain associated with myofascial trigger point (MTrP). However, the biochemical effects of dry needling that are associated with pain, inflammation, and hypoxia are unclear. This study investigated the activities of β-endorphin, substance P, TNF-α, COX-2, HIF-1α, iNOS, and VEGF after different dosages of dry needling at the myofascial trigger spots (MTrSs) of a skeletal muscle in rabbit. Materials and Methods. Dry needling was performed either with one dosage (1D) or five dosages (5D) into the biceps femoris with MTrSs in New Zealand rabbits. Biceps femoris, serum, and dorsal root ganglion (DRG) were sampled immediately and 5 d after dry needling for β-endorphin, substance P, TNF-α, COX-2, HIF-1α, iNOS, and VEGF immunoassays. Results. The 1D treatment enhanced the β-endorphin levels in the biceps femoris and serum and reduced substance P in the biceps femoris and DRG. The 5D treatment reversed these effects and was accompanied by increase of TNF-α, COX-2, HIF-1α, iNOS, and VEGF production in the biceps femoris. Moreover, the higher levels of these biochemicals were still maintained 5 d after treatment. Conclusion. Dry needling at the MTrSs modulates various biochemicals associated with pain, inflammation, and hypoxia in a dose-dependent manner.
    Document Type:
    Reference
    Product Catalog Number:
    AB5382
    Product Catalog Name:
    Anti-Nitric Oxide Synthase II Antibody - (Anti-Nitric Oxide Synthase II Antibody)

  • A biochemical and functional protein complex involving dopamine synthesis and transport into synaptic vesicles. 19903816

    Synaptic transmission depends on neurotransmitter pools stored within vesicles that undergo regulated exocytosis. In the brain, the vesicular monoamine transporter-2 (VMAT(2)) is responsible for the loading of dopamine (DA) and other monoamines into synaptic vesicles. Prior to storage within vesicles, DA synthesis occurs at the synaptic terminal in a two-step enzymatic process. First, the rate-limiting enzyme tyrosine hydroxylase (TH) converts tyrosine to di-OH-phenylalanine. Aromatic amino acid decarboxylase (AADC) then converts di-OH-phenylalanine into DA. Here, we provide evidence that VMAT(2) physically and functionally interacts with the enzymes responsible for DA synthesis. In rat striata, TH and AADC co-immunoprecipitate with VMAT(2), whereas in PC 12 cells, TH co-immunoprecipitates with the closely related VMAT(1) and with overexpressed VMAT(2). GST pull-down assays further identified three cytosolic domains of VMAT(2) involved in the interaction with TH and AADC. Furthermore, in vitro binding assays demonstrated that TH directly interacts with VMAT(2). Additionally, using fractionation and immunoisolation approaches, we demonstrate that TH and AADC associate with VMAT(2)-containing synaptic vesicles from rat brain. These vesicles exhibited specific TH activity. Finally, the coupling between synthesis and transport of DA into vesicles was impaired in the presence of fragments involved in the VMAT(2)/TH/AADC interaction. Taken together, our results indicate that DA synthesis can occur at the synaptic vesicle membrane, where it is physically and functionally coupled to VMAT(2)-mediated transport into vesicles.
    Document Type:
    Reference
    Product Catalog Number:
    PP40
    Product Catalog Name:
    IgG, Goat - (IgG, Goat)

  • Ontogeny of biochemical, morphological and functional parameters of synaptogenesis in primary cultures of rat hippocampal and cortical neurons. 25757474

    Synaptogenesis is a critical neurodevelopmental process whereby pre- and postsynaptic neurons form apposed sites of contact specialized for chemical neurotransmission. Many neurodevelopmental disorders are thought to reflect altered patterns of synaptic connectivity, including imbalances between excitatory and inhibitory synapses. Developing rapid throughput approaches for assessing synaptogenesis will facilitate toxicologic and drug screening studies of neurodevelopmental disorders. The current study describes the use of high-content imaging to quantify the ontogeny of excitatory and inhibitory synapses using in vitro models of neurodevelopment. These data are compared to biochemical and functional measures of synaptogenesis.The ontogenetic patterns of synapse formation were compared between primary rodent hippocampal and cortical neurons over 28 days in vitro (DIV). As determined by ELISA, the increase in synaptophysin expression levels as cultures matured was similar between hippocampal and cortical cultures. High-content imaging of immunoreactivity of excitatory and inhibitory synaptic biomarkers demonstrated an overall greater number of synapses in hippocampal relative to cortical neurons with marked differences in the pattern of inhibitory synapse development between these two neuronal cell types. Functional assays revealed that both the mean firing rates and mean bursting rates were significantly increased in cortical cultures relative to hippocampal cultures. This difference may reflect decreased inhibitory synaptic tone in cortical versus hippocampal cultures.These data demonstrate differences and similarities in the ontogeny of synaptogenesis between hippocampal and cortical neurons, depending on the biological level examined. Assessment of synaptophysin protein levels by ELISA showed a general increase in synapse formation in both cell types with increasing time in culture, while high-content imaging was able to delineate cell type-dependent differences in formation of excitatory versus inhibitory synapses. The functional significance of differences in the balance of excitatory to inhibitory synapses was confirmed by the assessment of network activity using microelectrode arrays. These results suggest that high-content imaging and microelectrode arrays provide complementary approaches for quantitative assessment of synaptogenesis, which should provide a robust readout of toxicologic and pharmacologic effects on this critical neurodevelopmental event.
    Document Type:
    Reference
    Product Catalog Number:
    MAB368

  • Biochemical characterization of protein quality control mechanisms during disease progression in the C22 mouse model of CMT1A. 24175617

    Charcot-Marie-Tooth disease type 1A (CMT1A) is a hereditary demyelinating neuropathy linked with duplication of the peripheral myelin protein 22 (PMP22) gene. Transgenic C22 mice, a model of CMT1A, display many features of the human disease, including slowed nerve conduction velocity and demyelination of peripheral nerves. How overproduction of PMP22 leads to compromised myelin and axonal pathology is not fully understood, but likely involves subcellular alterations in protein homoeostatic mechanisms within affected Schwann cells. The subcellular response to abnormally localized PMP22 includes the recruitment of the ubiquitin-proteasome system (UPS), autophagosomes and heat-shock proteins (HSPs). Here we assessed biochemical markers of these protein homoeostatic pathways in nerves from PMP22-overexpressing neuropathic mice between the ages of 2 and 12 months to ascertain their potential contribution to disease progression. In nerves of 3-week-old mice, using endoglycosidases and Western blotting, we found altered processing of the exogenous human PMP22, an abnormality that becomes more prevalent with age. Along with the ongoing accrual of misfolded PMP22, the activity of the proteasome becomes compromised and proteins required for autophagy induction and lysosome biogenesis are up-regulated. Moreover, cytosolic chaperones are consistently elevated in nerves from neuropathic mice, with the most prominent change in HSP70. The gradual alterations in protein homoeostatic response are accompanied by Schwann cell de-differentiation and macrophage infiltration. Together, these results show that while subcellular protein quality control mechanisms respond appropriately to the presence of the overproduced PMP22, with aging they are unable to prevent the accrual of misfolded proteins.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Biochemical and mechanical environment cooperatively regulate skeletal muscle regeneration. 22415307

    During forelimb regeneration in the newt Notophthalmus viridescens, the dynamic expression of a transitional matrix rich in hyaluronic acid, tenascin-C, and fibronectin controls muscle cell behavior in vivo and in vitro. However, the influence of extracellular matrix (ECM) remodeling on tissue stiffness and the cellular response to mechanical variations during regeneration was unknown. By measuring the transverse stiffness of tissues in situ, we found undifferentiated regenerative blastemas were less stiff than differentiated stump muscle (13.3±1.6 vs. 16.6±1.2 kPa). To directly determine how ECM and stiffness combine to affect skeletal muscle fragmentation, migration, and fusion, we coated silicone-based substrates ranging from 2 to 100 kPa with matrices representative of transitional (tenascin-C and fibronectin) and differentiated environments (laminin and Matrigel). Using live-cell imaging, we found softer tenascin-C-coated substrates significantly enhanced migration and fragmentation of primary newt muscle cells. In contrast, stiffer substrates coated with laminin, Matrigel, or fibronectin increased differentiation while suppressing migration and fragmentation. These data support our in vivo observations that a transitional matrix of reduced stiffness regulates muscle plasticity and progenitor cell recruitment into the regenerative blastema. These new findings will enable the determination of how biochemical and mechanical cues from the ECM control genetic pathways that drive regeneration.
    Document Type:
    Reference
    Product Catalog Number:
    AB19013
    Product Catalog Name:
    Anti-Tenascin Antibody - (Anti-Tenascin Antibody)

  • Biochemical characterization of CD1d expression in the absence of beta2-microglobulin. 10092605

    CD1d is a major histocompatibility complex class I-like molecule that exhibits a distinct antigen processing pathway that functions in the presentation of hydrophobic antigens to T cells. CD1d has been previously shown to be expressed on the cell surface of human intestinal epithelial cell lines in vivo and a transfected cell line in vitro independently of beta2-microglobulin (beta2m). To define the relationship between CD1d and beta2m and characterize the biochemical structure of CD1d in the absence of beta2m, we have used a newly generated series of CD1d transfectants and CD1d-specific antibodies. These studies show that in the absence of beta2m, CD1d is expressed on the cell surface as a 45-kDa glycoprotein that is sensitive to endoglycosidase-H and is reduced to 37-kDa after N-glycanase digestion. In contrast, in the presence of beta2m, CD1d is expressed on the cell surface as a 48-kDa endoglycosidase-H-resistant glycoprotein. Pulse-chase metabolic labeling studies demonstrate that acquisition of endoglycosidase-H resistance of CD1d is observed in the presence of beta2m but not in the absence of beta2m even after a 24-h chase period. Thus, CD1d is able to be transported to the cell surface independently of beta2m; however, in the absence of beta2m, the glycosylation pattern of CD1d is altered and consistent with an immature glycoprotein.
    Document Type:
    Reference
    Product Catalog Number:
    MABC983
    Product Catalog Name:
    Anti-CD1d Antibody, clone D5 - (Anti-CD1d Antibody, clone D5 )

  • Functional and biochemical analysis of endothelial (dys)function and NO/cGMP signaling in human blood vessels with and without nitroglycerin pretreatment. 11889009

    In experimental animal models, long-term in vivo treatment with nitroglycerin (NTG) induces both endothelial dysfunction and tolerance to nitrates. However, it is still controversial whether nitrate tolerance in humans is associated with both endothelial dysfunction and impaired vascular response to nitrovasodilator-derived NO.
    Document Type:
    Reference
    Product Catalog Number:
    05-611
    Product Catalog Name:
    Anti-phospho-VASP (Ser239) Antibody, clone 16C2 - (Anti-phospho-VASP (Ser239) Antibody, clone 16C2)

  • Biochemical mechanism of caffeic acid phenylethyl ester (CAPE) selective toxicity towards melanoma cell lines. 20685355

    In the current work, we investigated the in vitro biochemical mechanism of Caffeic Acid Phenylethyl Ester (CAPE) toxicity and eight hydroxycinnamic/caffeic acid derivatives in vitro, using tyrosinase enzyme as a molecular target in human SK-MEL-28 melanoma cells. Enzymatic reaction models using tyrosinase/O(2) and HRP/H(2)O(2) were used to delineate the role of one- and two-electron oxidation. Ascorbic acid (AA), NADH and GSH depletion were used as markers of quinone formation and oxidative stress in CAPE induced toxicity in melanoma cells. Ethylenediamine, an o-quinone trap, prevented the formation of o-quinone and oxidations of AA and NADH mediated by tyrosinase bioactivation of CAPE. The IC(50) of CAPE towards SK-MEL-28 melanoma cells was 15muM. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased CAPE's toxicity towards SK-MEL-28 cells indicating quinone formation played an important role in CAPE induced cell toxicity. Cyclosporin-A and trifluoperazine, inhibitors of the mitochondrial membrane permeability transition pore (PTP), prevented CAPE toxicity towards melanoma cells. We further investigated the role of tyrosinase in CAPE toxicity in the presence of a shRNA plasmid, targeting tyrosinase mRNA. Results from tyrosinase shRNA experiments showed that CAPE led to negligible anti-proliferative effect, apoptotic cell death and ROS formation in shRNA plasmid treated cells. Furthermore, it was also found that CAPE selectively caused escalation in the ROS formation and intracellular GSH (ICG) depletion in melanocytic human SK-MEL-28 cells which express functional tyrosinase. In contrast, CAPE did not lead to ROS formation and ICG depletion in amelanotic C32 melanoma cells, which do not express functional tyrosinase. These findings suggest that tyrosinase plays a major role in CAPE's selective toxicity towards melanocytic melanoma cell lines. Our findings suggest that the mechanisms of CAPE toxicity in SK-MEL-28 melanoma cells mediated by tyrosinase bioactivation of CAPE included quinone formation, ROS formation, intracellular GSH depletion and induced mitochondrial toxicity.
    Document Type:
    Reference
    Product Catalog Number:
    05-647
    Product Catalog Name:
    Anti-Tyrosinase Antibody, clone T311 - (Anti-Tyrosinase Antibody, clone T311)