Anti-serpin Antibody-mediated Regulation of Proteases in Autoimmune Diabetes

Secretion of anti-serpin B13 autoantibodies in young diabetes-prone nonobese diabetic mice is associated with reduced inflammation in pancreatic islets and a slower progression to autoimmune diabetes. Injection of these mice with a monoclonal antibody (mAb) against serpin B13 also leads to fewer inflammatory cells in the islets and more rapid recovery from recent-onset diabetes. The exact mechanism by which anti-serpin activity is protective remains unclear. We found that serpin B13 is expressed in the exocrine component of the mouse pancreas, including the ductal cells. We also found that anti-serpin B13 mAb blocked the inhibitory activity of serpin B13, thereby allowing partial preservation of the function of its target protease. Consistent with the hypothesis that anti-clade B serpin activity blocks the serpin from binding, exposure to exogenous anti-serpin B13 mAb or endogenous anti-serpin B13 autoantibodies resulted in cleavage of the surface molecules CD4 and CD19 in lymphocytes that accumulated in the pancreatic islets and pancreatic lymph nodes but not in the inguinal lymph nodes. This cleavage was inhibited by an E64 protease inhibitor. Consequently, T cells with the truncated form of CD4 secreted reduced levels of interferon-γ. We conclude that anti-serpin antibodies prevent serpin B13 from neutralizing proteases, thereby impairing leukocyte function and reducing the severity of autoimmune inflammation.     

The 14-3-3 protein rad24p modulates function of the cdc14p family phosphatase clp1p/flp1p in fission yeast

Schizosaccharomyces pombe cells divide through the use of an actomyosin-based contractile ring. In response to perturbation of the actomyosin ring, S. pombe cells delay in a "cytokinesis-competent" state characterized by continuous repair and maintenance of the actomyosin ring and a G2 delay. This checkpoint mechanism requires the function of the Cdc14p-family phosphatase Clp1p/Flp1p and the septation initiation network (SIN). In response to cytokinetic defects, Clp1p, normally nucleolar in interphase, is retained in the cytoplasm until completion of cell division in a SIN-dependent manner. Here, we show that a phosphorylated form of Clp1p binds the 14-3-3 protein Rad24p and is retained in the cytoplasm in a Rad24p-dependent manner in response to cytokinesis defects. This physical interaction depends on the function of the SIN component, Sid2p. In the absence of Rad24p, cells are unable to maintain SIN signaling and lose viability upon mild cytokinetic stress. The requirement of Rad24p in this checkpoint is bypassed by ectopic activation of the SIN. Furthermore, SIN-dependent nuclear exclusion of Clp1p is dependent on Rad24p function. We conclude that Rad24p-mediated cytoplasmic retention of Clp1p/Flp1p is important for cell viability upon stress to the division apparatus.     

Development of a hybrid <Emphasis Type="Italic">delta</Emphasis>-endotoxin and its expression in tobacco and cotton for control of a polyphagous pest <Emphasis Type="Italic">Spodoptera litura</Emphasis>

A hybrid -endotoxin protein was designed against a polyphagous lepidopteran insect pest Spodoptera litura, which is tolerant to most of the known -endotoxins. The hybrid -endotoxin was created by replacing amino acid residues 530–587 in a poorly active natural Cry1Ea protein, with a highly homologous 70 amino acid region of Cry1Ca in domain III. The truncated -endotoxins Cry1Ea, Cry1Ca and the hybrid protein Cry1EC accumulated in Escherichia coli to form inclusion bodies. The solubilised Cry1EC made from E. coli was 4- fold more toxic to the larvae of S. litura than Cry1Ca, the best known -endotoxin against Spodoptera sp. None of the two truncated toxins, solubilised from E. coli caused larval mortality. However, trypsinised Cry1Ca protoxin obtained from E. coli and solubilised from inclusion bodies caused mortality of S. litura with LC₅₀ 513 ng/ml semi synthetic diet. A synthetic gene coding for the hybrid$-endotoxin Cry1EC was designed for high level expression in plants, taking into consideration several features found in the highly expressed plant genes. Transgenic, single copy plants of tobacco as well as cotton were developed. The selected lines expressed Cry1EC at 0.1–0.7% of soluble leaf protein. Such plants were completely resistant to S. litura and caused 100% mortality in all stages of larval development. Hence, unlike in E. coli, the hybrid -endotoxin folded into a functionally active conformation in both tobacco and cotton leaves. The truncated Cry1EC expressed in tobacco leaves was about 8-fold more toxic (LC₅₀ 58 ng/ml diet) compared to expression in E. coli.     

IQGAP-related Rng2p organizes cortical nodes and ensures position of cell division in fission yeast

Correct positioning of the cell division machinery is crucial for genomic stability and cell fate determination. The fission yeast Schizosaccharomyces pombe, like animal cells, divides using an actomyosin ring and is an attractive model to study eukaryotic cytokinesis. In S. pombe, positioning of the actomyosin ring depends on the anillin-related protein Mid1p. Mid1p arrives first at the medial cortex and recruits actomyosin ring components to node-like structures, although how this is achieved is unknown. Here we show that the IQGAP-related protein Rng2p, an essential component of the actomyosin ring, is a key element downstream of Mid1p. Rng2p physically interacts with Mid1p and is required for the organization of other actomyosin ring components into cortical nodes. Failure of localization of Rng2p to the nodes prevents medial retention of Mid1p and leads to actomyosin ring assembly in a node-independent manner at nonmedial locations. We conclude that Mid1p recruits Rng2p to cortical nodes at the division site and that Rng2p, in turn, recruits other components of the actomyosin ring to cortical nodes, thereby ensuring correct placement of the division site.     

Pyruvate Dehydrogenase Kinase-mediated Glycolytic Metabolic Shift in the Dorsal Root Ganglion Drives Painful Diabetic Neuropathy

The dorsal root ganglion (DRG) is a highly vulnerable site in diabetic neuropathy. Under diabetic conditions, the DRG is subjected to tissue ischemia or lower ambient oxygen tension that leads to aberrant metabolic functions. Metabolic dysfunctions have been documented to play a crucial role in the pathogenesis of diverse pain hypersensitivities. However, the contribution of diabetes-induced metabolic dysfunctions in the DRG to the pathogenesis of painful diabetic neuropathy remains ill-explored. In this study, we report that pyruvate dehydrogenase kinases (PDK2 and PDK4), key regulatory enzymes in glucose metabolism, mediate glycolytic metabolic shift in the DRG leading to painful diabetic neuropathy. Streptozotocin-induced diabetes substantially enhanced the expression and activity of the PDKs in the DRG, and the genetic ablation of Pdk2 and Pdk4 attenuated the hyperglycemia-induced pain hypersensitivity. Mechanistically, Pdk2/4 deficiency inhibited the diabetes-induced lactate surge, expression of pain-related ion channels, activation of satellite glial cells, and infiltration of macrophages in the DRG, in addition to reducing central sensitization and neuroinflammation hallmarks in the spinal cord, which probably accounts for the attenuated pain hypersensitivity. Pdk2/4-deficient mice were partly resistant to the diabetes-induced loss of peripheral nerve structure and function. Furthermore, in the experiments using DRG neuron cultures, lactic acid treatment enhanced the expression of the ion channels and compromised cell viability. Finally, the pharmacological inhibition of DRG PDKs or lactic acid production substantially attenuated diabetes-induced pain hypersensitivity. Taken together, PDK2/4 induction and the subsequent lactate surge induce the metabolic shift in the diabetic DRG, thereby contributing to the pathogenesis of painful diabetic neuropathy.     

The secretome signature of reactive glial cells and its pathological implications

Glial cells are non-neuronal components of the central nervous system (CNS). They are endowed with diverse functions and are provided with tools to detect their own activities and those of neighboring neurons. Glia and neurons are in continuous reciprocal communication under both physiological and neuropathological conditions, and glia secrete various guidance factors or proteinaceous signals that service vital neuronal–glial interactions in health and disease. Analysis and profiling of glial secretome, especially of microglia and astrocytes, have raised new expectations for the diagnosis and treatment of CNS disorders, and the availability of a catalog of glia-secreted proteins might provide an origin for further research on the complex extracellular signaling mediated by glial cells. Components of the glial secretome play important roles as mediators and modulators of brain structure and function during neuroprotection and neurodegeneration. Therapeutic hypothermia has been acclaimed an effective modulator of brain injury via its substantial effect on the protein expression profiles of glia. Furthermore, emerging proteomic tools and methodologies make feasible the documentation of the reactive glial secretome signature. This review focuses on reactive glial cells and the uniqueness of their secretome during diverse neuropathological conditions. This article is part of a Special Issue entitled: An Updated Secretome.     

Synthesis, characterization and applications of graft copolymer (κ-carrageenan-g-vinylsulfonic acid)

The synthesis of graft copolymer (κ-carrageenan-g-vinylsulfonic acid) is carried out in nitrogen atmosphere using potassium peroxymonosulfate (PMS) and malonic acid (MA) as redox system. The effect of reaction variables including the concentration of vinylsulfonic acid 1.3×10(-2) to 6.7×10(-2) mol dm(-3), PMS 4×10(-3) to 20×10(-3) mol dm(-3), MA 1.6×10(-3) to 4.8×10(-3) mol dm(-3), sulfuric acid 1×10(-3) to 8×10(-3) mol dm(-3), κ-carrageenan 0.4-1.8 g dm(-3) as well as time duration 60-180 min and temperature 25-45 °C has been studied. The water swelling capacity of graft copolymer is investigated. Flocculation property for both coking and non-coking coals is studied for the treatment of coal mine waste water. The graft copolymer has been characterized by FTIR and thermogravimetric analysis.     

Improved clonal propagation of <Emphasis Type="Italic">Spilanthes</Emphasis><Emphasis Type="Italic">acmella</Emphasis> Murr. for production of scopoletin

A reproducible protocol for clonal propagation of Spilanthes acmella has been established. Routinely, the cultures were established in spring (January–April) season because of the highest aseptic culture establishment and high frequency shoot proliferation. Incorporation of 5 μM N⁶-benzyladenine (BA) to Murashige and Skoog (MS) basal medium showed 100% bud-break and promoted multiple shoot proliferation in cultures. Interestingly, a higher concentration of BA (7–15 μM) promoted stunted shoots with pale leaves while a lower concentration (1–3 μM) resulted in shoots with long internodes and excessive adventitious root proliferation from all over their surface. For recurrent shoot multiplication, single node segments from in vitro-developed shoots were excised and cultured on MS + BA (5 μM) medium where 20.3-fold shoot multiplication was achieved every 5 weeks. Finally, these shoots were successfully rooted on half-strength MS medium (major salts reduced to half-strength) with 50 g l⁻¹ sucrose, with a frequency of 100%. Transplantation survival of micropropagated plants was 88.9%. Additionally, accumulation of scopoletin, a phytoalexin, was revealed for the first time in the uninfected leaves of Spilanthes. Further, the quantitative estimation by HPLC with a fluorescence detector showed that the amounts of scopoletin content (0.10 μg g⁻¹ DW) in the leaves of micropropagated plants are comparable to those of field-grown mother plants. The study thus signifies the effectiveness of in vitro methodology for true-to-type plant regeneration of Spilanthes and their later utility for biosynthesis and constant production of scopoletin throughout the year.