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We examined radiation effects on the relationship between diabetes development and genetic background in atomic-bomb (A-bomb) survivors. Our main aim in this study was to shed light on the role of genetic background in diabetes onset among A-bomb survivors by studying possible relationships between human leukocyte antigen (HLA) genotypes and the diabetes in patients and controls. We examined the effects of different HLA haplotypes on type 2 diabetes development by determining the DQA1 and DRB1 alleles of Hiroshima A-bomb survivors (111 diabetic patients and 774 controls) using the polymerase chain reaction-sequence specific oligonucleotide probes (PCR-SSOP) method. We noted an increased risk of diabetes in the higher dose group among these patients (trend p = 0.001). The risk of the most heavily exposed group was significantly higher than that of the unexposed group or the low-dose group especially in survivors with the DQA1*03-DRB1*09 or DQA1*0401-DRB1*08 haplotypes (trend p = 0.002 or p = 0.05, respectively). By contrast, in people with other haplotypes, the risk did not increase significantly with increasing dose. These results suggest that individuals with specific HLA haplotypes may have an increased risk of diabetes with increased-dose categories.

We found previously that the peripheral CD4 T-cell populations of heavily exposed A-bomb survivors contained fewer naïve T cells than we detected in the corresponding unexposed controls. To determine whether this demonstrable impairment of the CD4 T-cell immunity of A-bomb survivors was likely to affect the responsiveness of their immune systems to infection by common pathogens, we tested the T cells of 723 survivors for their ability to proliferate in vitro after a challenge by each of the Staphylococcus aureus toxins SEB, SEC-2, SEC-3, SEE and TSST-1. The results presented here reveal that the proliferative responses of T cells of A-bomb survivors became progressively weaker as the radiation dose increased and did so in a manner that correlated well with the decreasing CD45RA-positive (naïve) [but not CD45RA-negative (memory)] CD4 T-cell percentages that we found in their peripheral blood lymphocyte (PBL) populations. We also noted that the T cells of survivors with a history of myocardial infarction tended to respond poorly to several (or even all) of the S. aureus toxins, and that these same individuals had proportionally fewer CD45RA-positive (naïve) CD4 T cells in their PBL populations than we detected in survivors with no myocardial infarction in their history. Taken together, these results clearly indicate that A-bomb irradiation led to an impairment of the ability of exposed individuals to maintain their naïve T-cell pools. This may explain why A-bomb survivors tend to respond poorly to toxins encoded by the common pathogenic bacterium S. aureus.

Background: Epidemiological studies conducted among atomic bomb (A-bomb) survivors by the Radiation Effects Research Foundation (RERF) have indicated a significant radiation-dose-dependent increase in risks of certain cancers among survivors. The mechanisms for different organ susceptibilities to radiation have remained unclear. Epidemiological and biological data show that colorectal cancer (CRC) risk factors may vary by anatomic subsite. We categorized CRC into two subtypes: Proximal colon cancer (cecum, ascending colon, and transverse colon) and distal CRC (descending colon, sigmoid colon, rectosigmoid junction, and rectum). CD14 is known to enhance the inflammatory response in the colorectum by acting as a co-receptor to detect bacterial lipopolysaccharide and activating the production of inflammatory cytokine. In this study, we examined the association between polymorphisms of the cytokine CD14 gene and risks of CRC subtypes. Methods: We analyzed the follow-up data from the RERF Immunogenome Study Cohort of 4,673 A-bomb survivors at the baseline covering 1981-2005: a total of 222 CRC cases, comprising 81 proximal colon cancer cases and 131 distal CRC cases. We focused on the single nucleotide polymorphisms (SNPs) in the 5′-untranstated region of CD14 (CD14-C/A) and genotyped all cohort subjects. Relative risks (RRs) for CRC and subtypes were estimated. Results: RRs of all-subtypes-combined CRC or either of the subtypes significantly increased with increasing radiation dose. When dividing the subjects into 2 groups by genotypes (CD14-A/A homozygote and CD14-C/C homozygote combined with CD14-C/A heterozygote), A-bomb survivors with CD14-A/A homozygote revealed significantly higher RRs of all-subtype-combined CRC (1.52, 95%CI: 1.15-2.01) and distal CRC (1.76, 95% CI: 1.21-2.57) than those with CD14-C/C homozygote combined with CD14-C/A heterozygote. However, there was no significant difference between 2 genotype groups in proximal colon cancer (RR=1.24, 95% CI: 0.79-1.95). When categorizing radiation dose into 3 levels (< 5 mGy defined as “non-exposed”, 5-700 mGy, and >700 mGy), A-bomb survivors with CD14-A/A who were exposed to the highest radiation dose (>700 mGy) were at significantly higher risk of all-subtype-combined CRC (RR=2.53, 95%CI: 1.58-4.04) and distal CRC (RR=3.26, 95%CI: 1.71-6.23) than “non-exposed” survivors with CD14-C/C combined with CD14-C/A. Conclusion: These results suggest the potential involvement of a CD14-mediated inflammatory response in the development of radiation-associated CRC, specifically distal CRC, but not proximal colon cancer, among A-bomb survivors. This possibly contributes to the different susceptibilities of the proximal colon and distal colorectum to radiation exposure. Citation Format: Yiqun Hu, Kengo Yoshida, Junko Kajimura, Seishi Kyoizumi, Yoichiro Kusunoki, John Cologne, Waka Ohishi, Ikue Hayashi, Kei Nakachi, Tomonori Hayashi. CD14 gene polymorphisms associated with development of colorectal cancer subtypes among atomic bomb survivors in Japan. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2199. doi:10.1158/1538-7445.AM2014-2199

Reactive oxygen species (ROS) play an important role in cell-mediated immune responses, although overproduction and excessive accumulation of ROS may also enhance risks of inflammation-related diseases. To address these concerns, we have developed an assay system to determine ROS (H2O2 and O2.-) levels in blood cells to investigate the effects age and of radiation exposure on ROS levels in the blood cells obtained from 2,789 Japanese atomic-bomb survivors, as well as associations between intracellular ROS levels and immune-/inflammation-related gene polymorphisms. The results showed that H2O2 levels in monocyte and granulocyte fractions increased with age (P

Increased frequencies of cells carrying mutations at several loci have been found in the blood cells of atomic-bomb (A-bomb) survivors upon testing four or five decades after the bombing. Interestingly, though, we have been unable to demonstrate any radiation-associated increases in the frequencies of mutant blood cells in which human leukocyte antigen (HLA)-A expression has been disrupted; this is true both of preliminary tests on the T cells of a small subset of A-bomb survivors and of the much more extensive study reported here in which we screened a much larger group of survivors for HLA-A2 loss mutations in B cells and granulocytes as well as in T cells. In attempting to explain our inability to detect any increases in HLA-A2-negative cell numbers in HLA-A2 heterozygous individuals exposed to A-bomb irradiation, we decided to test the hypothesis that HLA-A mutant lymphocytes might well have been induced by radiation exposure in much the same way as every other type of mutant we encountered, but may subsequently have been eliminated by the strong negative selection associated with their almost inevitable exposure to autologous natural killer (NK) cells in the bloodstream of each of the individuals concerned. We now report that mutant B lymphocyte cell lines that have lost the ability to express the HLA-A2 antigen do indeed appear to be much more readily eliminated than their parental heterozygous counterparts during co-culture in vitro with autologous NK cells. We make this claim first because we have observed that adding autologous NK cells to in vitro cultures of HLA-A2 heterozygous B or T cell lines appeared to cause a dose-dependent decrease in the numbers of HLA-A2-negative mutants that could be detected over a period of 3 days, and second because when we used peripheral blood HLA-A2 heterozygous lymphocyte cultures from which most of the autologous NK cells had been removed we found that we were able to detect newly-arising HLA-A2 mutant T cells in substantial numbers. Taken together, these results strongly support the hypothesis that autologous NK cells are responsible for eliminating mutant lymphocytes that have lost the ability to express self-HLA class I molecules in vivo, and may well therefore explain why we have been unable to detect increased frequencies of HLA-A2 mutants in samples from any of the 164 A-bomb survivors whose HLA-A2 heterozygote status made their lymphocytes suitable for our tests.

Mutant cells generated in vivo can be eliminated when mutated gene products are presented as altered MHC/peptide complexes and recognized by T cells. Diminished expression of MHC/peptide complexes enables mutant cells to escape recognition by T cells. In the present study, we tested the hypothesis that mutant lymphocytes lacking expression of MHC class I molecules are eliminated by autologous NK cells. In H-2b/k F1 mice, the frequency of H-2Kb-negative T cells was higher than that of H-2Kk-negative T cells. The frequency of H-2K-deficient T cells increased transiently after total body irradiation. During recovery from irradiation, H-2Kk-negative T cells disappeared more rapidly than H-2Kb-negative T cells. The disappearance of H-2K-deficient T cells was inhibited by administration of Ab against asialo-GM1. H-2Kk-negative T cells showed higher sensitivity to autologous NK cells in vitro than H-2Kb/k heterozygous or H-2Kb-negative T cells. Adding syngeneic NK cells to in vitro cultures prevented emergence of mutant cells lacking H-2Kk expression but had little effect on the emergence of mutant cells lacking H-2Kb expression. Results in the H-2b/k F1 strain correspond with the sensitivity of parental H-2-homozygous cells in models of marrow graft rejection. In H-2b/d F1 mice, there was no significant difference between the frequencies of H-2Kb-negative and H-2Kd-negative T cells, although the frequencies of mutant cells were different after radiation exposure among the strains examined. H-2b/d F1 mice also showed rapid disappearance of the mutant T cells after irradiation, and administration of Ab against asialo-GM1 inhibited the disappearance of H-2K-deficient T cells in H-2b/d F1 mice. Our results provide direct evidence that autologous NK cells eliminate mutant cell populations that have lost expression of self-MHC class I molecules.

In multicellular organisms, the role of gap junction intercellular communication (GJIC) in the regulation of cell proliferation, cell differentiation, and apoptosis is becoming increasingly recognized as one of the major cellular functions from the start of the fertilized egg, through normal development of the embryo and fetus, to the sexual maturation of the adult and ultimately to the maintenance of health of the aging adult. Given that the function of this membrane-associated protein channel is to synchronize electrotonic or metabolic functions, differential regulation of function at the transcriptional, translational, and posttranslational levels of a family of highly evolutionarily conserved genes (connexins) needs to be considered. Both inherited mutations and environmental modulation of GJIC could, in principle, affect the function of gap junctions to control cell proliferation, cell differentiation, and apoptosis, thereby leading to a wide variety of pathologies. We review a few techniques used to characterize the ability of stem and progenitor cells to perform GJIC.

Even after more than 60 years have passed, exposure to atomic-bomb (A-bomb) radiation continues to have long-term health effects in survivors, whose risks of inflammation-related diseases increase with increasing radiation dose. It is known that persistent inflammation underlies several cancers through increased production of reactive oxygen species (ROS) and endogenous tumor promoters (e.g., TNF-α). In our previous study, plasma levels of inflammatory cytokines and biomarkers, IL-6, TNF-α, CRP, IFN-γ, Igs, and erythrocyte sedimentation rate (ESR), increased significantly with radiation dose. We have recently developed an automated plasma ROS assay system based on use of a clinical chemistry analyzer. In this study we investigated association between radiation dose, plasma ROS levels, and sub-clinical inflammatory status in A-bomb survivors in Hiroshima. Excluding patients with history of cancer, rheumatoid arthritis, chronic bronchitis, or myocardial infarction, we randomly selected 442 study subjects from the Adult Health Study population, consisting of A-bomb survivors and unexposed people. We observed statistically significant radiation-associated increase in plasma ROS levels. ROS levels were positively associated with the inflammatory biomarkers IL-6, TNF-α, CRP, and ESR ( P It is evident that total ROS production increased with enhanced inflammatory status, suggesting a plausible mechanistic pathway between radiation-induced persistent inflammation and cancer.

Since radiation exposure affects the immune system as well as risk of various cancers, we have commenced an “immunogenome” study to look at genetic susceptibility to radiation-associated cancers. HLA class I molecules are known to regulate various immune and inflammatory responses. In this study, we examined effects of radiation exposure on individual susceptibility to gastric cancer based on HLA Class I ( HLA-A, -B, -C ) polymorphisms. This study comprised 150 gastric cancer patients and 300 controls. HLA genotype distribution among the controls not exposed to atomic-bomb radiation was almost identical to that in the general Japanese population. Comparison between non-exposed gastric cancer patients and non-exposed controls showed no statistically significant relationship between gastric cancer risk and HLA-B or HLA-C genotypes. However, frequency of HLA-A*2601 , one of the HLA-A genotypes, was significantly lower among cases (3.7%) than controls (18.3%), with P HLA-A*2601 among the gastric cancer patients (average radiation dose: 0.8 Gy) (18.8%) was almost the same as frequency among the controls (15.8%). Risk elevation by radiation was most prominent in HLA-A*2601 , compared with other genotypes. These results suggest that HLA-A*2601 may be involved in decreased gastric cancer risk among non-exposed people and that, among those with the HLA-A*2601 allele, radiation exposure may increase gastric cancer risk. On the basis of this finding, we conducted further analysis of association between the HLA-A*2601 allele and plasma levels of various inflammatory markers along with analyses of pathological typing and H. pylori infection.

The objective of this study was to isolate and partially characterize several rat liver epithelial cell clones containing myc, ras and myc/ras oncogenes in order to study their roles in apoptosis and to test the hypothesis that gap junctional intercellular communication is necessary for apoptosis in solid tissues and that the loss of junctional communication leads to tumorigenesis. The co-transfection of the myc and ras oncogenes in the normal rat liver epithelial cell line (WB-F344) resulted in a loss of functional channels and normal growth regulation; cell–cell communication was significantly decreased and tumorigenicity determined in adult male F344 rats was induced. We examined cell growth properties, gap junctional intercellular communication (GJIC), using the scrape-loading–dye transfer and fluorescence-redistribution-after-photobleaching assays, and tumorigenicity in a series of normal and v-myc-, c-Ha-ras- and v-myc/c-Ha-ras-transfected WB-F344 cell lines. The c-Ha-ras- and the v-myc/c-Ha-ras-transduced cell lines appeared distinctly different from the other lines, having spindle-shaped morphology, shorter generation time and contact insensitivity. On the other hand, the normal WB-F344 cell line and the v-myc-transduced cell line showed excellent GJIC. Moreover, the c-Ha-ras-transduced cell lines displayed decreasing levels of GJIC associated with their increasing tumorigenicity. The v-myc/c-Ha-ras-transformed cell lines showed the lowest levels of GJIC and were also the most tumorigenic. These findings suggest that the reduction of GJIC in c-Ha-ras- and v-myc/c-Ha-ras-transformed WB-F344 cells is linked to their tumorigenic potential. These cell lines should provide valuable tools to study the role of GJIC in apoptosis during tumorigenesis.

Many studies have suggested a relationship between certain alleles of the human leukocyte antigen (HLA) and the prevalence of some diseases or the immunological responsiveness to certain antigens. Furthermore, our studies in the past have demonstrated decreased immune function among atomic-bomb survivors who were exposed to high doses of radiation. However, no studies have addressed the possibility of various degrees of radiation-induced immune suppression being dependent on HLA type. To investigate the possibility of differing frequency distributions of HLA type in the Hiroshima atomic bomb survivors, HLA-DQA1 alleles and HLA-DR antigens were typed for 291 survivors in a high-dose group (>1.5 Gy), 339 survivors in an intermediate-dose group (0.005-1.5 Gy), and 388 in a distally exposed control group (

The effect of linoleic acid (LA) on gap-junction permeability, connexin 43 mRNA level, protein level, and phosphorylation, and the numbers of gap-junctional membrane plaques were studied in the rat liver epithelial cell line WB-F344 to determine whether changes in these parameters correlated with the enhanced cell growth and the inhibition of gap-junction function. When cultured in a medium with low serum (1%), these cells exhibited a slower growth rate than in the high serum medium (7%). Addition of linoleic acid (0.01-3 mg/ml) to the low serum medium increased the growth rate and inhibited gap junctional intercellular communication (GJIC) in a dose-dependent manner. In a comparison of short-term and long-term treatments with LA, GJIC in short-term treated (1 h) WB cells was inhibited at 3 mg/ml LA but readily recovered by washing and removing LA from cells, whereas GJIC in long-term treated (6 days) WB cells did not recover by washing and removing LA from WB cells. Western blot analysis of connexin 43 showed that a short-term incubation with linoleic acid increased the relative amount of unphosphorylated connexin 43 protein, but a long-term incubation with linoleic acid decreased the amount of unphosphorylated connexin 43 protein and increased the relative amount of hyperphosphorylated connexin 43 protein. Connexin 43 and p53 mRNA levels decreased in a time- and dose-dependent manner in linoleic acid-treated cells. These results suggest that growth stimulation and gap junctional intercellular communication inhibition of rat liver epithelial cells by linoleic acid may be mediated in part through modulation of p53 expression and function.

Increased gap junctional intercellular communication induced by agents that stimulate the adenylyl cyclase/cAMP pathway was observed in the GnRH-secreting neuronal cell line, GT1-7, and possible underlying mechanisms were examined. A 24-hour treatment of GT1-7 neurons with 100 microM dibutyryl cAMP + 100 microM IBMX or with 2 microM forskolin increased by greater than 2-fold the percentage of cells that were dye coupled, using the noninvasive dye coupling assay, fluorescent recovery after photobleaching (FRAP). Longer treatment times (48 h) and higher concentrations of dibutyryl cAMP (500 microM) did not further increase the percentage of dye-coupled cells, while there was no increase in dye coupling observed between untreated cells and cells treated for 2 h or less. The increase in dye coupling induced by dibutyryl cAMP/IBMX was inhibited by octanol or dieldrin, agents known to block gap junction-mediated intercellular coupling in other cell types. Western blot analysis of total protein or membrane protein-enriched extracts revealed no apparent difference in the cellular levels of connexin 26, a connexin subtype previously shown to be expressed by GT1-7 cells, between untreated cells and cells treated for 24 h with dibutyryl cAMP/IBMX or forskolin. In addition, expression of connexin 32 or 43 protein before or after treatment was not detected. On the other hand, a dramatic increase in both the number of neurites and neurites that immunostained positive for connexin 26 was observed in dibutyryl cAMP/IBMX-treated cells. We hypothesize that the observed increase in dye coupling between GT1-7 neurons following stimulation of the adenylyl cyclase/cAMP pathway results from an augmentation of cell-cell contacts due to an increased number of neurites containing gap junctional plaques, possibly through an effect on cellular differentiation.

Based on the concern of organochlorides in the environment and in human tissue, this study was designed to determine whether various noncytotoxic levels of heptachlor and heptachlor epoxide could inhibit, reversibly, gap junctional intercellular communication in human breast epithelial cells (HBEC). Cytotoxicity and gap junctional intercellular communication (GJIC) were evaluated by lactate dehydrogenase assay and fluorescence redistribution after photobleaching analysis, respectively. Both heptachlor and heptachlor epoxide were noncytotoxic up to 10 microg/ml. At this concentration, heptachlor and heptachlor epoxide inhibited GJIC of normal human breast epithelial cells after 1 h treatment. Within a 24 h treatment with heptachlor and heptachlor epoxide at 10 microg/ml, recovery of GJIC had not returned. GJIC completely recovered after a 12 h treatment of 1 microg/ml heptachlor epoxide, but it did not recover after a 24 h treatment of 1 microg/ml heptachlor. RT-PCR and Western blots were analyzed to determine whether the heptachlor or heptachlor epoxide might have altered the steady-state levels of gap junction mRNA and/or connexin protein levels or phosphorylation state. No significant difference in the level of connexin 43 (Cx43) message between control and heptachlor-treated cells was observed. Western blot analyses showed hypophosphorylation patterns in cells treated with 10 microg/ml heptachlor and heptachlor epoxide for 1 h with no recovery within 24 h. Immunostaining of Cx43 protein in normal HBEC indicated that heptachlor and heptachlor epoxide caused a loss of Cx43 from the cell membranes at noncytotoxic dose levels. Taken together, these results suggest that heptachlor and heptachlor epoxide can alter GJIC at the post-translational level, and that, under the conditions of exceeding a threshold concentration in the breast tissue containing 'initiated' cells for a long time and not being counteracted by anti-tumor-promoting chemicals, they could act as breast tumor promoters.

A new HaeIII polymorphism, which is found in the first intron of the human p53 gene, provides a genetic marker for tumor suppressor p53 gene alterations.

Past epidemiology studies reported that atomic-bomb (A-bomb) radiation exposure enhanced risk of colon cancer, but not rectum cancer, among the survivors. The reason for this different sensitivity to radiation remains to be elucidated. Colorectal cancer is a multi-factorial disease, the onset of which is attributed to both environmental and genetic factors, with animal experiments showing that intestinal bacterial toxins accelerate carcinogenesis. It was recently reported that single nucleotide polymorphisms (SNPs) identified in the promoter region of CD14, encoding a receptor for the lipopolysaccharide component of the outer membrane of Gram-negative bacteria, regulate CD14 gene expression and thereby affect susceptibility to inflammatory diseases such as atopic dermatitis, coronary artery disease, and hepatic diseases. This study investigated relationship between a novel CD14 gene polymorphism and the development of radiation-associated colon and rectum cancers among an A-bomb survivor cohort, in terms of a case-cohort study. On the basis of this T/G polymorphism, we determined CD14 genotypes with 210 colorectal cancer cases and with a subcohort of 2,160 individuals who were randomly selected from the cohort. Our results showed that the individuals exposed to A-bomb radiation (≥5 mGy) were at an increased risk of colon cancer, but not rectum cancer, with relative risk (RR) of 1.19/Gy (95%CI: 1.04-1.36). When we divided the study subjects into three groups for radiation dose and into two groups for CD14 genotypes, significantly high risk of colon cancer was found with RR of 2.40 (95%CI: 1.37-4.23) for the combined category of CD14-T/T homozygote and the highest dose (≥0.7Gy), compared with the baseline category (CD14-T/G or G/G and no dose). These results suggest that CD14-related inflammatory response might be involved in the development of radiation-associated colon cancer, not rectum cancer, among A-bomb survivors, and that the CD14 genotyping might be involved in individual risk of colon cancer together with radiation dose. To find a functional significance of this polymorphism, we are currently working on ELISA-based assay to measure serum levels of soluble CD14 among healthy individuals in relation to the CD14 genotyping. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4715.

A simple and rapid method for identification of alleles at the human leucocyte antigen (HLA)-DQA1 locus is described. The polymorphic second exon of the HLA-DQA1 locus was amplified by the polymerase chain reaction (PCR) method. The amplified DNA was analyzed by single-strand conformation polymorphism (SSCP) and restriction enzyme cleavage assay. Using this method, the eight known DQA1 alleles could be distinguished from each other. This paper suggests that the method can be used for quick genotyping of DQA1 alleles, but detecting point mutations at various positions in a fragment as well as new HLA-DQA1 genotypes should also be possible.

RERF epidemiology studies found that risks of certain cancers among atomic-bomb (A-bomb) survivors, including gastric cancer, increased with past exposure dose to atomic radiation, and that these risks remain high even today. RERF immunology studies implied that radiation exposure greatly affected host immune systems of A-bomb survivors, further providing for the possibility that altered immune response in this population, specifically inflammatory response and immunological host defense, might be involved in the development of various cancers. In fact, we have reported that the enhancement of persistent inflammation with increased age was further accelerated among people exposed to A-bomb radiation. In this study, we examined relationship between gastric cancer risk and radiation dose based on 43 SNPs of inflammation-related genes ( IL1B, IL6, IL8, IL10, BAT1, NFKBIL1, LTA, TNF, and LST1 ), in a case-control study of 238 cases and 1757 controls within a subcohort of the RERF Adult Health Study. We found statistically significant association between IL10 , LTA , and TNF SNPs and risk of gastric cancer, but not between other SNPs and risk of gastric cancer. Three SNPs on the LTA-TNF loci were associated with elevated risk of gastric cancer. Additionally, our genome analysis identified a single haplotype block within the IL10 gene locus, which was indicated by four htSNPs (generating two major alleles, IL10-ATTA and IL10-GGCG ). We found that risk of gastric cancer significantly varied by IL-10 haplotype in both non-exposed and exposed individuals, and that risk-enhancing effects of radiation were evident in all the haplotypes. Our findings offer further evidence that variation in the IL10 and TNF loci affects gastric cancer risk.

We synthesized a temperature-responsive polymer, N-(isopropylacrylamide)-methacrylic acid copolymer, to which poly-l-lysine was introduced. The synthesized polymer as well as the parent polymer showed reversible soluble-insoluble changes in response to temperature changes across the lower critical solution temperature at 32 degree C in an aqueous solution. We found that the polymer efficiently captured acidic bio-macromolecules such as RNA, glycosaminoglycans and mucin-type glycoproteins in biological samples, and the captured molecules were recovered using aqueous NaCl solutions at high concentration. The target acidic molecules thus obtained will be employed for further studies such as structural analysis after brief desalting procedure. The proposed method does not require any chromatographic separations, but only needs a small volume of an aqueous salt solution for releasing captured molecules. Overall procedures are quite easy and simple, and are completed at least within 1 h. We show a few examples for capturing RNA and glycosaminoglycans from cultured cells using the polymer.

A cloned synthetic DNA fragment coding for 27-desamidosecretin (DAS), which differs from natural porcine secretin by the lack of the amide group at the carboxyl-terminal valine residue, was fused to the β-galactosidase gene ( lacZ ) at the EcoRI site near the 3'-terminus of the gene. The fusion gene was efficiently expressed in Escherichia coli , yielding 1.15 × 10 6 fused-protein molecules per cell. After the treatment of the fused protein with cyanogen bromide, DAS was purified by a combination of ion-exchange column chromatography and reverse-phase high-performance liquid chromatography (HPLC). The structure of bacterial DAS was confirmed by tryptic mapping and amino acid composition analyses. The bacterial DAS was not amidated at its carboxyl-terminal valine residue. Nevertheless, it stimulated pancreatic secretion in rats as does authentic porcine secretin, indicating that the carboxyl-terminal amide is not essential to secretin activity.

By immunizing rabbits with purified human epidermal growth factor (hEGF), which was synthesized by Escherichia coli constructed by a genetic engineering technique, high titer antiserum for hEGF was obtained. The antiserum has an excellent specificity to hEGFs {hEGF, hEGF [1-51], hEGF [1-47] and [Leu21] hEGF} and has almost no cross-reactivity with other biological peptides, such as mouse EGF and human platelet derived growth factor. Using radioiodinated hEGF, a radioimmunoassay (RIA) for hEGF was established by a double antibody-polyethylene glycol method. As little as 10 pg/tube of hEGF was successfully determined by the present RIA methods. The intra- and inter-assay coefficients of variation for hEGF in human plasma or serum were 2.4-4.9% and 3.7-6.2%, respectively. The hEGF levels in human body fluids determined by the present method were as follows : urine (11-100 ng/ml), serum (1.0-1.8 ng/ml), plasma (0.2-0.8 ng/ml), sperm (17-47 ng/ml), saliva (0.9-3.0 ng/ml) and tears (9.5-27 ng/ml).

An automated HPLC method was proposed for the determination of a human epidermal growth factor, hEGF[1-53], and its fragments, hEGF[1-51] and hEGF[1-47], in the cultured medium of E, coli. The cultured medium was directly injected into the HPLC system which was composed of a short protein-coated octadecylsilane (ODS) precolumn for deproteinization and trapping of hEGFs and a usual ODS analytical column. By a column-switching technique, hEGFs trapped in the precolumn were transferred to the analytical column to allow their separation. The detection limits of hEGFs were ca. 100ng by UV detection at 210nm and were ca. 10ng by native fluorescene detection (Ex 287nm, Em 340nm). The recovery of the spiked hEGFs in the present method was almost quantitative (99.5-101.4%) with good reproducibility (less than 3% of RSD, within each run). The present method is simple and reproducible enough for the routine analysis of cultured medium for hEGFs.

A high performance liquid chromatographic method for the determination of human epidermal growth factors (hEGFs) in human urine is described. Using an anti-hEGF antibody column as a precolumn and an ODS column as an analytical column, each urine sample was directly injected onto the precolumn with aqueous mobile phase. With 0.2% trichloroacetic acid solution, the trapped hEGFs were transferred to the analytical column to allow their separation by reversed phase mode. The present method revealed that human urine contains hEGF[1-50], [1-51] and [1-52] in addition to hEGF[1-53]; the content determined by native fluorescence detection coincided with that by enzyme immunoassay. In the present method, the relative standard deviation (RSD) was less than 3% (n=6) for 25ng/ml of hEGF[1-53] and the detection limit was ca. 3ng for each hEGF, and the loading capacity of hEGF[1-53] on the anti-hEGF antibody precolumn was unchanged during analyses of 500 human urine samples.

The biliary and urinary excretions of immunoreactive human epidermal growth factor (hEGF) after intravenous administration were determined in rats. At intravenous doses of 50, 75, 100 and 500 μg/kg of hEGF, the same maximal biliary concentrations of immunoreactive hEGF were observed. The maximal concentration of immunoreactive hEGF in bile (about 20 ng/ml) sustained for 2 h after injection at a dose of 500 (μg/kg). Above findings indicate the presence of saturable biliary secretion system. However, the extent of total biliary excretion of the dose was less than 0.12% in all cases. On the other hand, no saturable urinary excretion of immunoreactive hEGF was observed. The extent of total urinary excretion of the dose was less than 1.5%. It was concluded that biliary and urinary excretions would not contribute to the marked dose-dependent pharmacokinetics of immunoreactive hEGF observed after intravenous administration in rats.

“Protein-coated ODS”をプレカラムとする自動試料前処理装置を用いて,血しょう試料の直接注入逆相高速液体クロマトグラフィーにより,血しょう中のアドリアマイシン&びその代謝産物の定量法を確立した.分離法は,検出法によって異なり,アドリアマイシン及びその代謝産物の特異的な蛍光で検出する場合には,簡単な2段階溶出法で分離定量が行えたが,特異性の低い紫外検出法(290nm)の場合には,洗浄ステップを増やし,アドリアマイシン&びその代謝産物のピークが,血しょう中の他の物質の妨害を受けないよう設定する必要があった.本法は,除タンパクしてから高速液体クロマトグラフ分析する方法に比べて,添加薬物の回収率が定量的(100士2%)であり,簡易かつ,再現性のよい(相対標準偏差2%以下)血しょう中の総量(遊離型+タソパク結合型)のアドリアマイシン類の定量法として有用であると考えられた.又,必ずしも内標準物質を必要とせず,血しょうを直接分析するため,容器などへのアドリアマイシン類の吸着が無視できる特徴もあった.特サギにアドリアマイシンを3.8mg/kg静注後の血しょう中アドリアマイシン類の定量に本法が応用できた.

A sensitive enzyme immunoassay for human epidermal growth factor (hEGF) is described. The anti-hEGF antibody was prepared by immunizing rabbits with hEGF, which was synthesized by Escherichia coli using the genetic engineering technique. The present assay system was based on the sandwiching of an antigen between anti-hEGF F(ab')2 precoated on a 96-well polystyrene plate and beta-D-galactosidase-labeled anti-hEGF Fab'. The range of measurable hEGF by this assay was 0.1-100 pg/well. Recoveries of hEGF added to serum and urine ranged between 94 and 108%. The intra- and inter-assay coefficients of variation were less than 6 and 8%, respectively. The results obtained by this assay method correlated well with those obtained by the radioimmunoassay method. By using this assay, the time course of serum hEGF levels in mice after the various administrations were also examined.