Differences in N-glycan structures foundon recombinant IgA1 and IgA2 producedin murine myeloma and CHO cell lines

The development and production of recombinant monoclonal antibodies is well established. Although most of these are IgGs, there is also great interest in producing recombinant IgAs since this isotype plays a critical role in providing immunologic protection at mucosal surfaces. the choice of expression system for production of recombinant antibodies is crucial because they are glycoproteins containing at least one N-linked carbohydrate. these glycans have been shown to contribute to the stability, pharmacokinetics and biologic function of antibodies. We have produced recombinant human IgA1 and all three allotypes of IgA2 in murine myeloma and CHo cell lines to systematically characterize and compare the N-linked glycans. Recombinant IgAs produced in murine myelomas differ significantly from IgA found in humans in that they contain the highly immunogenic Galα(1,3)Gal epitope and N-glycolylneuraminic acid residues, indicating that murine myeloma is not the optimal expression system for the production of human IgA. In contrast, IgAs produced in CHo cells contained glycans that were more similar to those found on human IgA. expression of IgA1 and IgA2 in Lec2 and Lec8 cell lines that are defective in glycan processing resulted in a less complex pool of N-glycans. In addition, the level of sialylation of rIgAs produced in murine and CHo cells was significantly lower than that previously reported for serum IgA1. these data underscore the importance of choosing the appropriate cell line for the production of glycoproteins with therapeutic potential.Key words: recombinant antibody, IgA, glycosylation, expression system, mass spectrometry     

Local kallikrein�Ckinin system is involved in podocyte apoptosis under diabetic conditions

The kallikrein-kinin system (KKS) serves as the physiologic counterbalance to the renin-angiotensin system. This study was conducted to examine the changes in the expression of KKS components in podocytes under diabetic conditions and to elucidate the functional role of bradykinin (BK) in diabetes-associated podocyte apoptosis. Thirty-two rats were injected with either diluent (n = 16, C) or with streptozotocin intraperitoneally (n = 16, DM), and 8 rats from each group were treated with BK infusion for 6 weeks. Immortalized mouse podocytes were cultured in media containing 5.6 mmol/l glucose (NG), NG + 10(-7) mol/l AII (AII), or 30 mmol/l glucose (HG) with or without 10(-8) mol/l BK. Urinary albumin excretion was significantly higher in DM rats, and this increase was ameliorated by BK. Not only kininogen, kallikrein, and BK B1- and B2-receptor expression but also BK levels were significantly decreased in DM glomeruli and in cultured podocytes exposed to HG. The changes in the expressions of apoptosis-related molecules and the increase in the number of apoptotic cells in DM glomeruli as well as in HG- and AII-stimulated podocytes were significantly abrogated by BK. The suppressed KSS within podocytes under diabetic condition was associated with podocyte apoptosis, suggesting that BK may be beneficial in preventing podocyte loss in diabetic nephropathy.     

Synthesis and antibacterial activities of novel oxazolidinones having cyclic sulfonamide moieties

The synthesis of a new series of oxazolidinones having cyclic sulfonamide moieties is described. Their in vitro antibacterial activities against both Gram-positive and Gram-negative bacteria were tested and the effect of substituents on the oxazolidinone ring was investigated. A particular compound 15g having [1,2,5]thiadiazolidin-1,1-dioxide moiety showed the most potent antibacterial activity.     

Inositol 1,4,5-Trisphosphate Receptor in Chromaffin Secretory Granules and Its Relation to Chromogranins

The inositol 1,4,5-trisphosphate (IP₃)-mediated intracellular Ca²⁺ releases in secretory cells play vital roles in controlling not only the intracellular Ca²⁺ concentrations but also the Ca²⁺-dependent exocytotic processes. Of intracellular organelles that release Ca²⁺ in response to IP₃, secretory granules stand out as the most prominent organelle and are responsible for the majority of IP₃-dependent Ca²⁺ releases in the cytoplasm of chromaffin cells. Bovine chromaffin granules were the first granules that demonstrated the IP₃-mediated Ca²⁺ release as well as the presence of the IP₃ receptor (IP₃R) in granule membranes. Secretory granules contain all three (type 1, 2, and 3) IP₃R isoforms, and 58–69% of total cellular IP₃R isoforms are expressed in bovine chromaffin granules. Moreover, secretory granules contain large amounts (2–4 mM) of chromogranins and secretogranins; chromogranins A and B, and secretogranin II being the major species. Chromogranins A and B, and secretogranin II are high-capacity, low-affinity Ca²⁺ binding proteins, binding 30–93 mol of Ca²⁺/mol of protein with dissociation constants of 1.5–4.0 mM. Due to this high Ca²⁺ storage properties of chromogranins secretory granules contain ~40 mM Ca²⁺. Furthermore, chromogranins A and B directly interact with the IP₃Rs and modulate the IP₃R/Ca²⁺ channels, i.e., increasing the open probability and the mean open time of the channels 8- to 16-fold and 9- to 42-fold, respectively. Coupled chromogranins change the IP₃R/Ca²⁺ channels to a more ordered, release-ready state, whereby making the IP₃R/Ca²⁺ channels significantly more sensitive to IP₃.     

Protective effect of ginseng sapogenins against 2,2'-azobis (1-aminopropane) dihydrochloride (AAPH)-induced LLC-PK₁ cell damage

The effect of ginseng sapogenins, aglycone parts of ginsenosides, against oxidative damage by radical generator, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), in renal epithelial LLC-PK(1) cells was investigated to identify the structural characteristics of sapogenins to have renoprotective effects. Of the tested sapogenins, Δ(20(21))-protopanaxatriol showed the strongest protective effect against the AAPH-induced LLC-PK(1) cell damage. Based on the structure and stronger activity of Δ(20(21))-protopanaxatriol than the other sapogenins, the hydroxyl group in C-6 and double bond in C-20(21) position were important for renoprotective effect of sapogenin against oxidative stress.     

Hydrazinocurcumin,a novel synthetic curcumin derivative,is a potent inhibitor of endothelial cell proliferation

Curcumin and some of its derivatives were known as in vivo inhibitors of angiogenesis. In present study, a novel curcumin derivative, named hydrazinocurcumin (HC) was synthesized and examined for its biological activities. HC potently inhibited the proliferation of bovine aortic endothelial cells (BAECs) at a nanomolar concentration (IC(50)=520 nM) without cytotoxicity. In vivo and in vitro angiogenesis experiments showed HC as a new candidate for anti-angiogenic agent.     

Genetic diversity and structure of the endangered species Megaleranthis saniculifolia in Korea as revealed by allozyme and ISSR markers

The genetic diversity and structure of 12 populations of Megaleranthis saniculifolia, a rare endemic Korean plant, were analyzed using 14 allozyme loci coding 10 enzymes and 78 ISSR loci using seven primers. The genetic diversity of M. saniculifolia at the species level was similar to that observed in out-crossing and long-lived perennials, while at the population level, it was significantly low. The high F _IS value of many populations as well as homozygote excess occurred relatively evenly in many populations in relation to the Hardy-Weinberg expectation, suggesting that inbreeding was occurring within the M. saniculifolia populations. The degree of genetic differentiation based on the two markers was high, and there was no correlation between geographic and genetic distance. Bayesian cluster analysis did not reveal any remarkable geographic trends. Positive correlations were observed between genetic diversity (H _e and h) and population size. Therefore, low genetic diversity within the population and high population differentiation of M. saniculifolia were closely related to the influence of genetic drift, particularly in highly isolated populations. In addition, the fixation of the main alleles at several loci in the opposite direction provided good evidence for genetic drift. The genetic diversity of M. saniculifolia could be compromised if the distribution area or the size of the population were further reduced. In particular, the isolated populations that are fragmented within an area could be at high risk of extinction due to accelerated inbreeding or genetic drift. Considering this, a close monitoring of the population size and of the changes in the genetic structure must be performed. Some practical measures for genetic conservation are also proposed.     

Inhibition of P‐Glycoprotein‐Induced Multidrug Resistance by a Clerodane‐Type Diterpenoid from Sindora sumatrana

The aim of the present study was to investigate the effects of di‐ and sesquiterpenoids isolated from the pods of Sindora sumatrana Miq. (Leguminosae) on P‐glycoprotein (P‐gp) function in an adriamycin‐resistant human breast cancer cell line, MCF‐7/ADR. Over‐expression of P‐gp is known to be one of the mechanisms involved in multidrug resistance (MDR), which is a major obstacle in clinical cancer treatment. Among six di‐ and sesquiterpenoids extracted from S. sumatrana, (+)‐7β‐acetoxy‐15,16‐epoxycleroda‐3,13(16),14‐trien‐18‐oic acid ( 1 ) showed a strong P‐gp inhibitory effect, as great as that of verapamil, a representative P‐gp inhibitor. Compound 1 enhanced daunomycin accumulation more than fourfold and significantly decreased daunomycin efflux compared with control, resulting in a decrease in the IC₅₀ value for daunomycin. These results suggest that compound 1 inhibits the functioning of P‐gp and, therefore, can be developed as an MDR‐reversing agent.