IgG is a tetrameric protein composed of two copies each of the light and heavy chains. The four-chain structure is maintained by strong noncovalent interactions between the amino-terminal half of pairs of heavy-light chains and between the carboxyl-terminal regions of the two heavy chains. In addition, interchain disulfide bonds link each heavy-light chain and also link the paired heavy chains. An engineered human IgG4 specific for human tumor necrosis factor-cy (CDP571) is similar to human myeloma IgG4 in that it is secreted as both disulfide bonded tetramers (approximately 75% of the total amount of IgG) and as tetramers composed of nondisulfide bonded half-IgG4 (heavy chain disulfide bonded to light chain) molecules. However, when CDP571 was genetically engineered with a proline at residue 229 of the core hinge region rather than serine, CDP57 I(S229P), or with an IgGl rather than IgG4 hinge region, CDP571(yl), only trace amounts of nondisulfide bonded half-lgG tetramers were observed. Trypsin digest reversephase HPLC peptide mapping studies of CDP57 1 and CDP57 1 (y I ) with on-line electrospray ionization mass spectroscopy supplemented with Edman sequencing identified the chemical factor preventing inter-heavy chain disulfide bond formation between half-IgG molecules: the two cysteines in the IgG4 and IgGl core hinge region KPSCP and GPPCP, respectively) are capable of forming an intrachain disulfide bond. Conformational modeling studies on cyclic disulfide bonded CPSCP and CPPCP peptides yielded energy ranges for the low-energy conformations of 31-33 kcallmol and 40-42 kcal/mol, respectively. In addition, higher torsion and angle bending energies were observed for the CPPCP peptide due to backbone constraints caused by the extra proline. These modeling results suggest a reason why a larger fraction of intrachain bonds are observed in IgG4 rather than IgGl molecules: the serine in the core hinge region of IgG4 allows more hinge region flexibility than the proline of IgGl and thus may permit formation of a stable intrachain disulfide bond more readily.
Morinda officinalis is beneficial for the treatment of inflammatory bowel disease (IBD). The hairy root with higher genetic and biochemical stability cultured from M. officinalis might have similar effects to treat IBD. In this study, the main chemical composition of the root extracts of M. officinalis (MORE) native plant and the hairy root extract of M. officinalis (MOHRE) was compared by quantitative HPLC. The difference of their therapeutic effects and potential mechanism was evaluated using 3% dextran sodium sulfate-induced chronic colitis in mice and T lymphocytes in vitro. The results found that MOHRE possesses many specific peaks unobserved in the chromatogram of native plant. The content of iridoids in the MORE (3.10%) and MOHRE (3.01%) is somewhat similar but quite different for their anthraquinones’s content (0.14 and 0.66%, respectively). Despite all this, treatment with both MORE and MOHRE significantly attenuated the symptoms of colitis, including diarrhea, body weight loss, colon shortening, histological damage, and decreased inflammatory cytokine levels. In addition, they dose-dependently increased the apoptosis of T lymphocyte in vivo and in vitro. And, the differences for treatment effects on ulcerative colitis (UC) between them both in this study were mostly insignificant. The results demonstrated that the effects of MORE and MOHRE for the treatment of UC are similar, although there are a few difference on their chemical composition, indicating the hairy root cultured from M. officinalis might be able to replace its native plant on treatment of UC. The successful derivation of a sustainable hairy root culture provides a model system to study the synthetic pathways for bioactive metabolites, which will make the use of bioreactors to largely produce traditional medicine become reality.
A chemically defined medium (MB-DF) has been developed for the propagation of the rat prostate adenocarcinoma cell line (PA-III). The medium (MB-DF) was supplemented with fetuin, insulin, transferrin, dexamethasone, and 5 alpha - dihydrotestosterone, but require no serum supplement. The adapted line, PA-IIIf, has been grown in this serum-free medium for 30 passages over a period of 12 months without losing the malignant or other characteristics of the parent line. When transplanted into syngeneic rats, the PA-IIIf cells reconstituted the adenocarcinoma and metastasized spontaneously via ipsilateral lymphatic channels to the lungs. This cell line will be of use in further analyses of enzymes associated with biological properties of the tumor cells.
Asthma is one of the most common chronic and inflammatory respiratory diseases, which is estimated to affect 1-10% of the population in different regions across the world. Previous studies have shown that recombinant Ling-Zhi 8 (rLZ-8), an immunoregulatory protein originally extracted from Ganoderma lucidum, plays multiple roles in regulating murine immune cells, including T cells. Here, we examined whether rLZ-8 would ameliorate pulmonary inflammation in a model of asthma-like mice. We found that rLZ-8 significantly inhibited the lung inflammation and reduced infiltration of inflammatory cells, including dendritic cells and eosinophils, in OVAinduced asthmatic mice. It also deceased IL-17A level but increased IL-10 level in bronchoalveolar lavage fluid (BALF) while reducing ROR t mRNA expression and enhancing Foxp3 mRNA level in the lung tissue. Flow cytometry studies demonstrated that rLZ-8 remarkably down-regulated Th17 cells but upregulated Foxp3 + regulatory T (Treg) cells, rather than influencing Th1 versus Th2 cells. Experiments in vitro also showed that rLZ-8 suppressed murine CD3 + T cell proliferation and reduced the frequency of Th17 cells while promoting the differentiation of CD4 + Foxp3 + Tregs. Moreover, rIL-8 similarly altered human Th17/Treg generation or their balance in vitro. Finally, we found that rLZ-8 suppressed signaling pathways of both STAT3 and NF-B (P100/P52) in murine lung tissue as well as cultured T cells. Thus, we have demonstrated that rLZ-8 attenuates pulmonary inflammation through regulating the balance of Th17/Treg cells in OVA-induced asthmatic mice and that rLZ-8 may be a potential therapeutic agent for the treatment of asthma in clinic. K E Y W O R D S herbal ingredient, pulmonary inflammation, Th cell, treg 1 INTRODUCTION Asthma, a chronic inflammatory airway disease associated with reversible airflow obstruction and persistent airway hyperresponsiveness, is a major public health problem that affects 300 million people worldwide. Although asthma is relatively well controlled by the most Abbreviations: BALF, bronchoalveolar lavage fluid; DC, dendritic cell; DEX, dexamethasone; rLZ-8, recombinant Ling-Zhi protein-8; Treg, regulatory T cell. effective first-line treatment, including inhaled corticosteroids, an estimated 40% of asthmatics fail to respond to corticosteroid and show no improvement in airway function. 1 In addition, various side effects resulting from the long-term use of these agents necessitate alternative therapeutic options. Allergen-specific CD4 + T cells have been shown to play a key role in the development of asthma. 2 In patients with the mild to moderate asthma, the classic mode of onset is that specific IgE Abs of B cells induced by CD4 + Th2 cells mediate immune responses and
Objectives To explore the effect of recombinant LZ-8 (rLZ-8) on streptozocin (STZ)-induced diabetic rats and further illustrate its underlying mechanism. Methods Rats were intraperitoneally injected with single-dose STZ 50 mg/kg for induction of type 1 diabetes (T1D), and then, the diabetic rats were treated with rLZ-8 for 3 months. The clinical symptoms, fasting blood glucose, insulin, cytokines, histopathology, flow cytometry and immunofluorescence were used to evaluate the therapeutic effect and underlying mechanism of rLZ-8 on alleviating diabetes mellitus (DM). Key findings Treatment with rLZ-8 obviously alleviated the clinical symptoms of T1D and dose-dependently reduced the levels of blood glucose, blood lipid and haemoglobin A1c (HbA1c) in diabetic rat model. Meanwhile, rLZ-8 markedly increased insulin secretion and protected against STZ-induced pancreatic tissue injury. Additionally, rLZ-8 dramatically inhibited the levels of TNF-α and IL-1β, and obviously increased the level of IL-10 in serum and pancreas. Further investigation indicated that rLZ-8 treatment significantly increased the number of regulatory T cells (Tregs) and up-regulated the expression of Foxp3 to restore balance between anti-inflammatory and inflammatory cytokines. Conclusions These data suggest that rLZ-8 can antagonize STZ-induced T1D, and its mechanism may be related to inhibit inflammation and enhance Tregs generation.
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