Shuichi Hashizume

Identification of lactoferrin as an essential growth factor for human lymphocytic cell lines in serum-free medium

A serum-free medium supplemented with growth factor(s) was devised to grow human lymphocytic cell lines. The medium was developed using human lymphocytic cell line, Bri 7 cells. In the process of constructing the medium, human lactoferrin was found to be an essential growth factor for the cell line. Human lactoferrin has higher growth stimulatory activity than human transferrin, and was sensitive to heat. Long-term cultivation of the cells was achieved in the defined medium supplemented with human lactoferrin only. The defined medium specifically supported the growth of various other human B- and T-lymphocytic cell lines but not the growth of various mouse lymphocytic cell lines. In lactoferrin-supplemented medium, the growth of some human cell lines were further stimulated by the addition of a combination of insulin, ethanolamine and selenium, or another combination of 2-mercaptoethanol and the above three factors. Bovine lactoferrin could be substituted for human lactoferrin.

Elevated expression levels of the 14-3-3 family of proteins in lung cancer tissues

Immunochemical staining of lung cancer sections with a murine monoclonal anti-14-3-3 antibody showed a sharp discrimination of the cancer tissue from neighboring normal counterparts in 88 of 121 primary lung cancer tissue specimens of all four major lung cancer histologies; specifically, 32 of 48 adenocarcinomas, 36 of 44 squamous cell carcinomas, 10 of 13 large cell carcinomas, and 10 of 16 small cell carcinomas, respectively, were stained positively. Sets of the 10,000 x g supernatants of normal and cancerous lung tissue homogenates, each set prepared from surgically dissected tissues of the cancer and its surrounding normal part, were assayed for 14-3-3 proteins by the sandwich enzyme-linked immunosorbent assay using two different monoclonal antibodies to 14-3-3 proteins. The results of the assay demonstrated 7.2 times higher 14-3-3 protein content in the lung cancer tissue (378 +/- 200 ng ml-1) as compared with the normal lung (54 +/- 35 ng ml-1). These results indicate that the 14-3-3 family of proteins can be an effective marker for lung cancer diagnosis such as sputum cytodiagnosis and that 14-3-3 proteins might be involved in the development of lung cancers.

Oral Streptococci Exhibit Diverse Susceptibility to Human β-Defensin-2: Antimicrobial Effects of hBD-2 on Oral Streptococci

We examined the antimicrobial effects of human β-defensin-2 (hBD-2) on 17 species of oral streptococci to investigate the involvement of antimicrobial peptide activity in oral microflora development and the clinical use of the antimicrobial peptide for oral microflora control. Oral streptococci exhibit diverse levels of susceptibility to human β-defensin-2 (hBD-2). Two major cariogenic bacterial species, Streptococcus mutans (S. mutans) and S. sobrinus, were found to be susceptible to the peptide, indicating that it is a potential therapeutic agent for preventing dental caries. S. mitis exhibited the lowest susceptibility to the peptide. S. mitis is a major indigenous bacterium in the oral microflora, and our results suggest that it might possess a certain resistance mechanism against hBD-2.

HO-323, a human B-lymphoblastoid cell line useful for making human-human hybridomas.

A 6-thioguanine-resistant human B-lymphoblastoid cell line, HO-323, was isolated for making human-human hybridomas with high efficiency. Fusions with peripheral blood lymphocytes of systemic lupus erythematosus (SLE) patients and lymphocytes isolated from lymph nodes of lung and breast cancer patients yield constantly more than one hybridoma clones per 10(5) HO-323 cells plated. HO323 cells also fused with lymphocytes from normal peripheral blood to give hybridomas in the same efficiency. The HO-323 cells were diploid with 46 chromosomes and non-secretors of immunoglobulins. This parent cells doubled every 15 hr and could proliferate in serum-containing medium, even if they were plated at low cell density of less than 10(3) cells/ml. The cells could grow in serum-free medium as well as in serum-containing medium, and the resultant human-human hybridomas could also grow in the same media.

[27]Cell culture assay of biological activity of lactoferrin and transferrin

Publisher Summary This chapter describes the methods for cell culture in serum free medium supplemented with lactoferrin, and compares the biological activities of lactoferrin with those of transferrin. Lactoferrin shares with transferrin the property of reversibly binding two atoms of iron, and presents structural and functional homology with transferrin. Both are monomeric glycoproteins, having two similar oligosaccharide chains, with molecular weights of about 8 × 10 4 . Both molecules possess two independent metal binding sites, each of which can bind a ferric ion together with a bicarbonate anion. The complete amino acid sequence of human serum transferrin ∼6 and partial amino acid sequence of lactoferrin have been reported. It has been reported that lactoferrin may play a role in iron transport in the intestine, and transferrin may not, though lactoferrin and transferrin are similar as described above. It has also been reported that lactoferrin binds to a specific macrophage receptor without competition with transferrin.

In vitro immunization of human peripheral blood lymphocytes: Establishment of B cell lines secreting IgM specific for cholera toxin B subunit from lymphocytes stimulated with IL-2 and IL-4

In vitro immunization (IVI) techniques have a great potential in the production of human monoclonal antibodies (MAbs) against various antigens. An IVI method of human peripheral blood lymphocytes (PBL) has been developed with a human lung adenocarcinoma cell line in our laboratory. Although several cancer specific human MAbs were successfully generated by using this IVI method, it was not available for soluble antigens, which prompted us to improve the method for generation of human MAbs against soluble antigens. IVI with soluble antigens was effectively caused by the addition of muramyl dipeptides, interleukin-2 and interleukin-4. It was found that the difference of sensitivity of lymphocytes depending upon donors could be overcome by finding the optimal concentrations of IL-2 and IL-4. IVI of human PBL was performed with cholera toxin B subunit (CTB) and the immunized B cells were transformed by Epstein-Barr virus. Anti-CTB antibody was detected using an indirect ELISA. B cells producing anti-CTB antibodies were directly cloned by a soft agar cloning method.

Stimulation of monoclonal antibody production by human-human hybridoma cells with an elevated concentration of potassium or sodium phosphate in serum-free medium

Potassium or sodium phosphate was found to stimulate the production of human monoclonal antibody by human-human hybridoma HB4C5. The addition of 15 mM Na-phosphate (pH 7.4) into serum-free culture medium increased the antibody production up to 4-fold, when seeded at cell density of 1×105 cells/ml in dishes. At the higher cell density of 5×105 cells/ml, K-phosphate was more effective than Na-phosphate, at the same concentration. In large-scale continuous culture, the addition of 10 mM Na-phosphate into serum-free culture medium stimulated antibody production by HB4C5 cells 6-fold.

Effects of an aqueous extract of cocoa on nitric oxide production of macrophages activated by lipopolysaccharide and interferon-γ

Objective Macrophages are the primary targets of bacterial lipopolysaccharide (LPS). The effects of cocoa extract on production of nitric oxide (NO) by murine J774.1 macrophages activated by LPS and interferon-γ (IFN-γ) were examined. Methods Cocoa was suspended in heated water and centrifuged, and the supernatant was then filtered. Nitrite was measured as a quantitative indicator of NO by spectrophotometry. LPS (1.0 mg/mL) and IFN-γ (100 U/mL) were added to cultured macrophages with 0.05% cocoa extract, 0.25% cocoa extract, or pure water. NO synthesis by macrophages was significantly inhibited by cocoa extract (P < 0.01). Results The inhibitory effect increased with concentration of the extract (P < 0.01). IFN-γ (100 U/mL) and, later, LPS (100 μg/mL) were added, together with 2.0% cocoa or pure water, to cultured macrophages. An inhibitory effect on NO production was observed on addition of only IFN-γ, but more significant effects were obtained with addition of LPS (P < 0.01) and addition of both was most effective (P < 0.01). Conclusions These data suggested that cocoa extract contains a suppressor of NO production in murine macrophages activated by LPS and IFN-γ. This effect does not appear to be caused merely by neutralization of LPS.

Activity enhancement of a lung cancer-associated human monoclonal antibody HB4C5 by N-deglycosylation.

It has been known that the lung cancer-associated human monoclonal antibody HB4C5 comprises two lambda light chains of 30 and 32 kD and that the 30 kD species is exclusively responsible for the antibody activity. This study demonstrates that the two light chains were both N-glycosylated with glycosyl residues of different sizes, one of which was sensitive to neuraminidase and the other insensitive. Our unpublished data of DNA sequence for the light chain of this monoclonal antibody indicated that the light chain contains only one possible site for N-glycosylation, which located in the CDR-1. N-Deglycosylation of this monoclonal antibody under the denaturing condition resulted in the complete conversion of the two light chains into one identical polypeptide chain of 26 kD. Activity of this monoclonal antibody was found to be significantly enhanced by N-deglycosylation. All the facts described above consistently indicate that the activity of this monoclonal antibody is interfered with by the attachment of bulky glycosyl residues at the antigen binding site on the light chain. The N-deglycosylated antibody was stable under the storage conditions employed, suggesting that the glycosyl residues attached to the light chain do not play any important biological role except for interference with the antibody activity of binding antigen.

Electrophoretic recovery of proteins from polyacrylamide gel

Abstract Polyacrylamide gel electrophoresis, which can effectively resolve a mixture of proteins into individual constituents with a simple apparatus, has been widely and routinely used for analytical purposes. However, its application for preparative purposes is limited owing to the lack of a universal method that permits the recovery of general proteins from the gel in high yields. In early days, the electrophoretically separated protein in gel was recovered by extraction or solubilization of the excised gel, and later by electrophoretic elution. Regarding the electrophoretic recovery of proteins, a number of methods have been reported, which can be divided into two categories: (1) “electroelution”, which recovers the protein of interest from excised gel electrophoretically, and (2) “continuous elution” of applie proteins from a preparative-scale gel during electrophoresis, where the electrophoretically separated proteins that migrate through the gel into a buffer stream at the gel bottom are fractionated consecutively. Characteristic features of the electroelution method reside in its simple requirements for the elution apparatus and that microgram amounts of protein can be quantitatively recovered in the concentrated form. The major drawbacks of electroelution are that the method requires manipulations for locating the protein in the gel, followed by the excision and re-electrophoresis of the relevant part of gel for eluting the protein. The continuous elution method, on the other hand, has advantages in its high loading capacity of sample protein and easy monitoring of the elution process. This method, however, requires expensive equipment and gives low concentrations of the recovered protein, the purity of which is sometimes poor. For those who are trying the electrophoretic recovery of protein from polyacrylamide gel for the first time, the electroelution method would be the first choice in view of the easy manipulation, the small amount of protein required for loading and the satisfactory recovery yield with least expense without using of any costly equipment.