Further investigation of the light chain shifting phenomenon: Light chain replacement through secondary rearrangement induced by lectin stimulation in the hybridoma cell line HB4C5

We found that when the hybridoma cell line HB4C5 was stimulated with wheat germ agglutinin (WGA), loss of production of the original λ light chain occurred, followed by production of new light chain, which mirrored the reaction when stimulated with concanavalin A (ConA). We previously reported that the RAG genes are expressed not only in HB4C5 and its ConA-treated variant subclones, but also in the in the parental Namalwa cells, which are known to be in the plasma state. However, the new λ light chains were expressed only in the HB4C5 cells and not in the parental Namalwa cells. Here we found that the RAG genes are expressed in HB4C5 cells after continuous stimulation with WGA. To further investigate the mechanism of this loss of original λ light chain production by stimulation with lectins in HB4C5 cells, which leads to a sIg-negative subpopulation, we analyzed the differences between HB4C5 and Namalwa cells. In this present study, we found that a 70 kDa phosphorylated protein in HB4C5 cells became undetectable after stimulation with lectins (WGA and ConA), and was not detected in Namalwa cells before or after lectin stimulation. It has been believed that the RAG genes and loss of original λ light chain production are required to induce expression of a new λ light chain in the HB4C5 cells. We suggested that the phosphorylated 70 kDa protein in HB4C5 cells play important roles in regulating the production of new λ light chains which is induced by lectins. This revised version was published online in June 2006 with corrections to the Cover Date.     

Generation of specificity-variant antibodies by alteration of carbohydrate in light chain of human monoclonal antibodies.

A hybridoma line, C5TN, produced human monoclonal antibody of which light chain had N-linked carbohydrate chain within the variable region. Some molecular-weight variants of light chain of the antibody were produced by C5TN variants resistant to cytotoxic effect of concanavalin A. The variant antibodies significantly altered the original cross-reactivity with antigens or lost the ability of antigen binding. The variants variously trimmed their carbohydrate chains by glycosidases, showed the changed reactivity or acquired the ability to bind for antigens. The carbohydrate-deficient antibodies from tunicamycin-treated C5TN and the variant clones behaved in a similar manner on antigen-binding reactivity. Furthermore, comparison of antibodies of which light chains have carbohydrate chains sensitive and resistant to some glycosidases showed that carbohydrate chain in variable region of light chain can influence their reactivity with antigen.     

Mechanism of the inhibition of cell growth by N1,N12-bis(ethyl)spermine.

The mechanism of the antiproliferation effect of N1,N12-bis(ethyl)spermine (BESPM) was studied in detail using mouse FM3A cells, since this polyamine analogue mimics the functions of spermine in several aspects [Igarashi, K., Kashiwagi, K., Fukuchi, J., Isobe, Y., Otomo, S. & Shirahata, A. (1990) Biochem. Biophys. Res. Commun. 172, 715-720]. Our results indicate that not only the decrease in sperimine and spermine caused by BESPM but also its accumulation play important roles on the inhibition of cell growth by BESPM, since BESPM accumulated in cells at a concentration fivefold that of spermidine in control cells. In comparison with the polaymine-deficient cells caused by alpha-difluoromethylornithine, an inhibitor of ornithine decarboxylase, and ethylglyoxal bis(guanylhydrazone), an inhibitor of S-adenosylmethionine decarboxylase, the behavior of polyamine-deficient cells caused by BESPM was different as follows: the inhibition of cell growth by BESPM was not abrogated by spermine or spermidine; polyamine uptake, which is stimulated during polyamine deficiency, was greatly inhibited, while spermidine/spermine N1-acetyltransferase activity, which is inhibited during polyamine deficiency, was enhanced in BESPM-treated cells; thymidine kinase activity did not decrease in BESPM-treated cells; inhibition of cell growth and macromolecule synthesis by BESPM correlated with the swelling of mitochondria and the decrease in ATP content; BESPM caused cell death when incubated together for several days. The role of BESPM accumulation on inhibition of cell growth is discussed.     

Simvastatin enhances induction of inducible nitric oxide synthase in 3T3-L1 adipocytes

The present study was designed to determine whether hydroxymethylglutaryl-CoA reductase inhibitors (statins) modulate the NO production via iNOS in adipocytes stimulated by lipopolysaccharide (L) and tumour necrosis factor-α (T). Well-differentiated 3T3-L1 adipocytes significantly produced NO by LT-treatment. Pre-incubation with simvastatin, a lipophilic statin, pravastatin, a hydrophilic one, or Y27632, an inhibitor of Rho kinase, further enhanced the production of NO. The effect of simvastatin was offset by mevalonate and geranylgeranyl pyrophosphate (GGPP) but not by squalene. The mRNA level for iNOS parallelled the NO production. The NF-κB was activated by the LT-treatment and was further enhanced by simvastatin, pravastatin or Y27632 addition. Mevalonate and GGPP completely offset the effect of simvastatin. Statins and Y27632 also further increased the interleukin-6 secretion in the LT-treated 3T3-L1 adipocytes. These results suggest that statins, especially lipophilic type, enhance induction of iNOS by inhibiting the small GTP-binding protein signal in adipocytes.     

Establishment and characterization of monoclonal antibodies against Chuzan virus K-47

We established sixteen mouse monoclonal antibodies reactive to Chuzan virus K-47 strain using P3-X63-Ag8-U1 cells as fusion partner cells. Among them, CG53/2/4 recognized a 100K structural protein of the virus. The 100K antigen lost it's antigenicity for CG53/2/4 after mild periodate oxidation treatment, suggesting that the 100K viral antigen is a glycoprotein. In addition, CG53/2/4 neutralized the viral infectivity. This indicates that the 100K glycoprotein is essential for the infection of the virus. The other monoclonal antibodies reacted with a 41K antigen of the virus. Especially CG1/1 showed the highest reactivity to the virus. Forward step sandwich assay using CG1/1 and biotinylated CG53/2/4 could detect the virus at 10TCID₅₀/ml. Therefore, these monoclonal antibodies can evantually predict the virus infection to the animals before their sideration.     

In vitro immunization can elicit the expansion of diverse repertoire of B cells from peripheral blood mononuclear cells

We previously developed an in vitro immunization (IVI) protocol of human peripheral blood mononuclear cells (PBMC) for generating antigen-specific human antibodies. In order to clarify whether IVI protocolinduces antigen-specific B cell responses in PBMC, we analyzed family gene usage and sequence of the variable region gene of immunoglobulin heavy chain (VH gene) of the antibody produced from the in vitro immunized PBMC. Sequence homology analyses of VH gene demonstrated that a larger repertoire of B cells can be sensitized with mite-extract than with cholera toxin B subunit and rice allergen. Further, antigen-specific B cells were efficiently expanded by using CpG oligodeoxynucleotide as adjuvant. These results suggest that appropriate combination of sensitizing antigen and adjuvant is primarily important for expansion of antigen-specific B cells in IVI protocol.     

Epidemiological and serological survey of brucellosis in Mongolia by ELISA using sarcosine extracts

Brucellosis is an important zoonosis, and serological surveillance is essential to its control. However, cross-reactions of attenuated live cells of Brucella abortus strain S-19 and B. melitensis strain Rev-1 with Yersinia enterocolitica O9 or vaccinated animal sera interfere with accurate serological diagnosis by the Rose Bengal test (RBT). Therefore, we used ELISA with sarcosine extracts from the virulent B. abortus strain 544 to eliminate false-positives among RBT positive-sera. A total of 697 serum samples were collected in Mongolia from humans and animals in 23 nomadic herds. The herds were classified into three groups as brucellosis-endemic (BE), brucellosis-suspected (BS), or Brucella-vaccinated (BV). The number of 295 animals (43.0%) was positive by RBT, but 206 (69.8%) of these were positive according to ELISA; therefore, 30.2% of the RBT-positive sera were found to be false positives. The false positive samples for RTB represent 4.1%, 27.4%, and 68.2% of the animals from the BE, BS, and BV herds, respectively. In addition, 32% of RBT-positive human sera were also false positives. Thus, our ELISA would be more specific than RTB and useful for epidemiological surveillance for brucellosis.     

Expressions of the prepro-orexin and orexin type 2 receptor genes in obese rat

We examined the expressions of the prepro-orexin gene in the lateral hypothalamic area (LHA), the genes of the neuropeptide Y (NPY) and proopiomelanocortin (POMC) in the arcuate nucleus (ARC), the orexin type 1 receptor (OX1R) gene in the ventromedial hypothalamic nucleus (VMH) and the orexin type 2 receptor (OX2R) gene in the paraventricular nucleus (PVN) in 6-, 12- and 18-week-old male lean (Fa/?) and obese (fa/fa) Zucker rats, using in situ hybridization histochemistry. The fa/fa rats showed hyperglycemia at 12- and 18-week-old. The prepro-orexin mRNA level in fa/fa rats at 18-week-old and the OX2R mRNA level in fa/fa rats at 12- and 18-week-old were significantly decreased compared to controls. The NPY mRNA levels in fa/fa rats at each time point were significantly increased compared to controls, but the POMC mRNA levels were decreased. Prepro-orexin and OX2R mRNA levels in fa/fa rats pretreated with insulin normalized to the levels found in Fa/? rats. These results suggest that the regulation of prepro-orexin gene expression might be independent of the regulation of the NPY and POMC genes in the ARC in fa/fa rats.