Mammalian small heat shock proteins (sHSP) form polydisperse and dynamic oligomers that undergo equilibrium subunit exchange. Current models of their chaperone activity hypothesize that recognition ...and binding of protein non-native states involve changes in the oligomeric state. The equivalent thermodynamic representation is a set of three coupled equilibria that includes the sHSP oligomeric equilibrium, the substrate folding equilibrium, and the equilibrium binding between the sHSP and the substrate non-native states. To test this hypothesis and define the binding-competent oligomeric state of human Hsp27, we have perturbed the two former equilibria and quantitatively determined the consequences on binding. The substrate is a set of T4 lysozyme (T4L) mutants that bind under conditions that favor the folded state over the unfolded state by 10(2)-10(4)-fold. The concentration-dependent oligomer equilibrium of Hsp27 was perturbed by mutations that alter the relative stability of two major oligomeric states including phosphorylation-mimicking mutations that result in the dissociation to a small multimer over a wide range of concentrations. Correlation of binding isotherms with size exclusion chromatography analysis of the Hsp27 oligomer equilibrium demonstrates that the multimer is the binding-competent state. Binding occurs through two modes, each characterized by different affinity and number of binding sites, and results in T4L.Hsp27 complexes of different hydrodynamic properties. Mutants of the Hsp27 phosphorylation mimic that reverse the reduction in oligomer size also reduce the extent of T4L binding. Taken together, these results suggest a central role for the oligomeric equilibrium in regulating the chaperone activity of sHSP. The mutants identify sequence features important for modulating this equilibrium.
Indian cobra (Naja naja) venoms from different geographical locations vary in their composition, biochemical, and pharmacological properties. Venom samples from eastern, western and southern India ...are compared in this study. The venom from eastern region was found to be the most lethal of the three regional venoms. Monovalent antivenom (NNEV-IgG) prepared against the eastern venom was found to cross-react with the other two regional venoms. NNEV-IgG at an Ag:Ab ratio of 1:25 completely neutralized the lethality of eastern venom. At this ratio, it did not neutralize the other two venoms, but the survival time of experimental mice was extended significantly. Commercially available polyvalent antivenom neutralized the lethality of western venom at an Ag:Ab ratio of 1:60 and increased the survival time of experimental mice injected with eastern and southern venoms marginally. Further, NNEV-IgG neutralized the tested pharmacological and enzymatic activities of all the three venom samples dose dependently, with neutralization potency varying with the geographic origin of the tested venoms. Thus, the present study demonstrates the diversity in the immunological properties of venom from different geographical regions and underscores the importance of developing region-specific antivenoms for therapeutic purpose.
Therapeutic use and function of recombinant molecules can be studied by the expression of foreign genes in mice. In this study, we have expressed human Fcγ receptor-Ig fusion molecules (FcγR-Igs) in ...mice by administering FcγR-Ig plasmid DNAs hydrodynamically and compared their effectiveness with purified molecules in blocking immune-complex (IC)-mediated inflammation in mice. The concentration of hydrodynamically expressed FcγR-Igs (CD16A(F)-Ig, CD32A(R)-Ig and CD32A(H)-Ig) reached a maximum of 130 μg ml(-1) of blood within 24 h after plasmid DNA administration. The in vivo half-life of FcγR-Igs was found to be 9-16 days and western blot analysis showed that the FcγR-Igs were expressed as a homodimer. The hydrodynamically expressed FcγR-Igs blocked 50-80% of IC-mediated inflammation up to 3 days in a reverse passive Arthus reaction model. Comparative analysis with purified molecules showed that hydrodynamically expressed FcγR-Igs are more efficient than purified molecules in blocking IC-mediated inflammation and had a higher half-life. In summary, these results suggest that the administration of a plasmid vector with the FcγR-Ig gene can be used to study the consequences of blocking IC binding to FcγRs during the development of inflammatory diseases. This approach may have potential therapeutic value in treating IC-mediated inflammatory autoimmune diseases such as lupus, arthritis and autoimmune vasculitis.
Hyaluronidase, ubiquitous enzyme in snake venoms, known originally as “spreading factor”, has not been well studied. The present study describes the purification and characterization of hyaluronidase ...from Indian cobra (
Naja naja) venom and provides systematic evaluation of the spreading property of the enzyme. Hyaluronidase (NNH1) has been purified through gel permeation and ion exchange chromatography. The molecular mass was found to be 70.406 kDa by MALDI-TOF mass spectrometry and with the
pi p
I of 9.2. The amino acid sequence of the N-terminus was found to be NEQSTHGAYV. The enzyme shows absolute specificity for hyaluronan and belongs to the group of neutral active enzymes. Tetrasaccharides are the final product of hyaluronan digestion. The enzyme cleaves β 1,4-glycosidic linkage and belongs to a group of endo-β-
N-acetyl hexosaminidases. Hyaluronidase indirectly potentiates the myotoxicity of VRV-PL-VIII, a phospholipolytic myotoxin, and also the hemorrhagic potency of a hemorrhagic complex-I. Localization of hyaluronan in human skin section and selective degradation by venom hyaluronidase (NNH1) corroborate the plausible in vivo degradation of hyaluronan in the extracellular matrix (ECM) resulting in easy dissemination of VRV-PL-VIII myotoxin and hemorrhagic complex-I.
CM-Sephadex C-25 column chromatography profile of Indian cobra (Naja naja) venom from eastern region showed a distinct and a dominant phospholipase peak, peak-10, while it was not seen in either ...southern or western venom samples. Peak-10 was subjected to CM-Sephadex C-25 and Sephadex G-50 column chromatography to isolate NN-X-PLA(2). NN-X-PLA(2) is a single chain protein with the relative molecular weight of 10kDa by SDS-PAGE. It was toxic to mice with an LD(50) value 0.098 mg/kg body weight (i.p.) and the mice exhibited acute neurotoxic symptoms. Upon indirect stimulation, it inhibited the twitching of frog's gastrocnemius muscle in a dose dependent manner. NN-X-PLA(2) was weakly anticoagulant and devoid of cytotoxicity, myotoxicity, hemorrhage, edema inducing, and directlytic activities and effects on platelet aggregation process. Upon chemical modification independently with p-bromophenacyl bromide and acetic anhydride, NN-X-PLA(2) lost both enzymatic and toxic properties.
Indian cobra (
Naja naja) venom from different geographical locations varied in its composition and biochemical, pharmacological and immunological properties. Recently it has been shown that the ...variation in composition of venom from different geographical origin of Indian peninsula is due to the quantitative difference in the same components and also the presence of different biochemical entities with respect to their origin. This disparity in venom composition may be due to several environmental factors. However, very little is known about the systemic effects on vital organs caused by the venom due to regional variation. In the present investigation, the venom samples procured from eastern, western and southern regions were compared for histopathological effects on skeletal muscle and some vital organs (heart, lungs, liver and kidney) in the mouse model. All the three venom samples damaged vital organs such as cardiac muscle, gastrocnemius muscle, liver, lungs and kidneys; however, the extent of damage varied greatly. Eastern venom predominantly damaged cardiac muscle and kidney, western venom injured the liver and the southern venom affected the lung. In addition, the eastern venom caused the recruitment of a flux of inflammatory cells in the skeletal muscle unlike southern and western venom samples. These results suggest the diversity of target-specific toxins in all the three regional venoms. Thus, the study explores the possible variations in the pathological effects of cobra (
Naja naja) venom samples on vital organs due to geographical distribution in the Indian subcontinent. It also emphasizes the importance of intra-specific variation of venom samples for the production of efficacious and region-specific therapeutic antivenom.
Despite the recent advances in anti-cancer therapies, breast cancer accounts for the highest percentage of estimated new cases among female cancer patients. The anti-cancer drug Ukrain, a ...plant-derived semi-synthetic compound, has been shown to be effective in a variety of tumor models including colon, brain, ovarian, melanoma and lymphoma. However, the direct cytotoxic effects of Ukrain have yet to be investigated in breast cancer models.
Herein, we investigated the in vitro and in vivo cytotoxicity of Ukrain using murine (4T07 and TUBO) and human (SKBR-3) breast cancer cell lines.
Cells were treated with varying concentrations of Ukrain for up to 72 h and analyzed for viability by trypan blue exclusion, apoptosis by intracellular caspase 3 and Annexin V staining, and proliferative potential by a clonogenic assay. Female BALB/c mice were challenged subcutaneously (s.c.) with 4T07-RG cells and administered 5 mg/kg or 12.5 mg/kg body weight Ukrain intravenously (i.v.) on the same day and 3 days later. Protective immune responses were determined following re-challenge of tumor-free mice 35 days post primary challenge.
Ukrain exposure induced apoptosis in a dose and time-dependent manner with 50 µg/mL Ukrain leading to >50% cell death after 48 h exposure for all three breast cancer cell lines. Ukrain administration (12.5 mg/kg) led to significant inhibition of 4T07 tumor growth in vivo and sustained protective anti-tumor immunity following secondary challenge.
Our findings demonstrate the in vitro and in vivo cytotoxic effects of Ukrain on breast cancer cells and may provide insight into designing Ukrain-based therapies for breast cancer patients.
Indian cobra (Naja naja naja) venom obtained from three different geographical regions was studied in terms of electrophoretic pattern, biochemical and pharmacological activities. SDS-PAGE banding ...pattern revealed significant variation in the protein constituents of the three regional venoms. The eastern venom showed highest indirect hemolysis and hyaluronidase activity. In contrast, western and southern venoms were rich in proteolytic activity. All the three regional venoms were devoid of p-tosyl-L-arginine methyl ester hydrolysing activity. The eastern venom was found to be most lethal among the three regional venoms. The lethal potency varied as eastern > western > southern regional venoms. In addition, all the three regional venoms showed marked variations in their ability to induce symptoms/signs of neurotoxicity, myotoxicity, edema and effect on plasma coagulation process. Polyclonal antiserum prepared against the venom of eastern region cross-reacted with both southern and western regional venoms, but varied in the extent of cross-reactivity by ouchterlony immunodiffusion and ELISA.
Abstract
Va14Ja18 natural T (iNKT) cells rapidly elicit a robust effector response to different glycolipid Ags, with distinct functional outcomes. Biochemical parameters controlling iNKT cell ...function are partly defined. However, the impact of iNKT cell receptor β-chain repertoire and how α-galactosylceramide (α-GalCer) analogues induce distinct functional responses have remained elusive. Using altered glycolipid ligands, we discovered that the Vb repertoire of iNKT cells impacts recognition and Ag avidity, and that stimulation with suboptimal avidity Ag results in preferential expansion of high-affinity iNKT cells. iNKT cell proliferation and cytokine secretion, which correlate with iNKT cell receptor down-regulation, are induced within narrow biochemical thresholds. Multimers of CD1d1-αGalCer- and αGalCer analogue-loaded complexes demonstrate cooperative engagement of the Va14Ja18 iNKT cell receptor whose structure and/or organization appear distinct from conventional αβ TCR. Our findings demonstrate that iNKT cell functions are controlled by affinity thresholds for glycolipid Ags and reveal a novel property of their Ag receptor apparatus that may have an important role in iNKT cell activation.
Acetaminophen (APAP) overdose is the most common cause of Acute Liver Failure (ALF). Following APAP overdose, toxicity is initiated by the conversion of APAP to its reactive metabolic ...N‐acetyl‐p‐benzoquinone imine (NAPQI) by CYP2E1/CYP3A4. NAPQI binds covalently to mitochondrial proteins forming 3‐(cysteine‐S‐yl)‐acetaminophen (APAP‐Cys). This generates initial hepatotoxicity in the form of centrilobular necrosis and sterile inflammation. Lipocalin‐2 (LCN2), is an acute‐phase innate immune protein, upregulated during tissue injury in various organs including the liver. Recently we have demonstrated that LCN2 is pro‐inflammatory and elevated during APAP overdose in mouse model. Interestingly, LCN2 knockout (LCN2 KO) mice exhibit protection from APAP overdose compared to its wild type (WT) littermates. During APAP overdose there is necrosis of hepatocytes, these hepatocytes release Damage Associated Molecular Patterns (DAMPS) that further exacerbate the inflammatory process. One such DAMP is High Mobility Group Box 1 (HMGB1). However, the role of LCN2 and HMGB1 in APAP overdose has not yet been investigated.
Present study investigates the molecular mechanism of LCN2 during the progression of acute liver injury in APAP overdose model. We hypothesize that during APAP induced ALF, LCN2 induces the expression and translocation of HMGB1 which in turn mediates inflammation and progression of injury. An in vitro model of HepG2 cells as well as an in vivo model of WT and LCN2 KO mice will be utilized to test our hypothesis. HepG2 cells will be treated with recombinant LCN2 to investigate whether exogenous LCN2 can cause induction and translocation of HMGB1. Translocation of HMGB1 will be assessed by immunofluorescence staining. Serum and tissues samples of WT and LCN2 KO mice subjected to APAP overdose will be assessed for expression of HMGB1. APAP‐cys formation will be measured in WT and LCN2 KO using the High‐Performance Liquid Chromatography method to ensure comparable bioactivation based injury between the WT and LCN2 KO mice. Lastly, histopathology will be performed on liver samples from both groups to assess liver pathology. It is anticipated that LCN2 mediates progression of liver injury in APAP overdose via HMGB1.
Support or Funding Information
Philadelphia College of Osteopathic Medicine‐Division of Research
This is from the Experimental Biology 2018 Meeting. There is no full text article associated with this published in The FASEB Journal.
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