Genetic characterization and conservation priorities of chicken lines.

Molecular markers are a useful tool for evaluating genetic diversity of chicken genetic resources. Seven chicken lines derived from the Plymouth Rock breed were genotyped using 40 microsatellite markers to quantify genetic differentiation and assess conservation priorities for the lines. Genetic differentiation between pairs of the lines (pairwise FST) ranged from 0.201 to 0.422. A neighbor-joining tree of individuals, based on the proportion of shared alleles, formed clearly defined clusters corresponding to the origins of the lines. In Bayesian model-based clustering, most individuals were clearly assigned to single clusters according to line origin and showed no admixture. These results indicated that a substantial degree of genetic differentiation exists among the lines. To decide priorities for conservation, the contribution of each line to the genetic diversity was estimated. The result indicated that a loss of 4 of the 7 lines would lead to a loss from 1.14 to 3.44% of total genetic diversity. The most preferred line for conservation purposes was identified based on multilocus microsatellite analysis. Our results confirmed that characterization by means of molecular markers is helpful for establishing a plan for conservation of chicken genetic resources.

Effect of Arachidonic Acid-enriched Oil Diet Supplementation on the Taste of Broiler Meat.

To elucidate the relationship between the arachidonic acid (AA) content and the taste of broiler meat, the effects of AA-enriched oil (AAO) supplements on the fatty acid content and sensory perceptions of thigh meat were evaluated. Four types of oil, including corn oil (CO), a 1:1 mixture of AAO and palm oil (PO) (1/2 AAO), a 1:3 mixture of AAO and PO (1/4 AAO), and a 1:7 mixture of AAO and PO (1/8 AAO) were prepared. Each type of oil was mixed with silicate at a ratio of 7:3, and added to the diet at a final proportion of 5% of fresh matter. Broiler chickens were fed these diets for 1 wk before slaughter. In thigh meat, the AA content of the 1/2 and 1/4 AAO groups was significantly higher than that of the CO group. The AA content in thigh meat (y, mg/g) increased linearly with increasing dietary AAO content (x, g/100 g of diet), according to the equation y = 0.5674+0.4596× (r(2) = 0.8454). The content of other fatty acids was not significantly different among the 4 diet groups. Sensory evaluation showed that the flavor intensity, umami (L-glutamate taste), kokumi (continuity, mouthfulness, and thickness), and aftertaste of the 1/2 and 1/4 AAO groups were significantly higher than that of the CO group. There were significant positive correlations between AA content in thigh meat and the flavor intensity, total taste intensity, umami, and aftertaste. These data suggest that the taste of broiler meat can be improved by the amount of dietary AA supplementation.

Supplemental arachidonic acid-enriched oil improves the taste of thigh meat of Hinai-jidori chickens.

The Hinai-dori is a breed of chicken native to the Akita prefecture in Japan. A cross between the Hinai-dori and Rhode Island Red breeds has been commercialized as the Hinai-jidori chicken, one of the most popular brands in Japan. High arachidonic acid (AA) content is a characteristic feature of Hinai-jidori chicken meat. To elucidate the relationship between AA content and the palatability of the Hinai-jidori chicken, we examined the effects of palm oil (PO), corn oil (CO), and arachidonic acid-enriched oil (AAO) diet supplementation on the fatty acid content and sensory perceptions in thigh meat. Each oil and silicate was mixed at a ratio of 7:3, 5% of fresh matter was added to the finisher diet, and Hinai-jidori chickens were fed these diets for 2 wk before slaughter. In thigh meat, the AA content of the AAO group was significantly more than 2-fold higher than that of the PO and CO groups. Other fatty acid contents were not significantly different among the groups. Sensory evaluation showed that the total taste intensity, umami (l-glutamate taste), kokumi (continuity, mouthfulness, and thickness), and aftertaste of the AAO group were significantly higher than those of PO and CO groups in both chicken soup and steamed minced meat. These data suggest that the palatability of chicken meat can be improved by dietary AA supplementation.

An efficient method of early caponization in slow-growing meat-type chickens.

We developed a new tool for testectomy and investigated the efficiency with regard to caponizing time and growth performance in Hinai-jidori chickens, a popular breed of chicken in Japan. Hinai-jidori chicks were divided into 2-, 4-, and 8-wk caponized groups and an intact male group (20 birds/group) at 2 wk of age and were raised until 26 wk of age. The testes of the male chicks caponized at 8 wk of age were surgically removed from both sides using a Japanese traditional tool, whereas those of male chicks caponized at 2 and 4 wk of age were surgically removed from only one side using the new tool. Using the traditional method, caponization of an 8-wk-old chick was achieved in 324.6 s (5 min 24 s), whereas using the new method, caponization of 2- and 4-wk-old chicks was achieved in only 35.9 and 28.4 s, respectively. Moreover, at 10 and 18 wk of age, the chicks caponized at 4 wk of age were significantly heavier than the chicks caponized at 8 wk of age. The data suggest that the decrease in the daily weight gain caused by caponization at a younger age was less than that at an older age. We concluded that early caponization shortens the caponizing time significantly and improves the decrease in the daily weight gain after caponization, thereby enabling efficient capon production from slow-growing meat-type chickens at early stages of development.

A method for discriminating a Japanese brand of chicken, the Hinai-jidori, using microsatellite markers.

The Hinai-dori is a native breed of chicken from the Akita Prefecture in Japan. A cross between the Hinai-dori and Rhode Island Red breeds has been commercialized as the Hinai-jidori chicken, one of the most popular brands of chicken in Japan. Here, a method of discriminating between the Hinai-jidori and other chickens is described. Individuals (555) of the Hinai-dori breed were analyzed by using 37 microsatellite markers on the Z chromosome. Fourteen of the marker loci (ABR1003, ADL0250, ABR0241, ABR0311, ABR1004, ABR1013, ABR0633, ABR1005, ABR0089, ABR1007, ABR1001, ABR1009, ABR1010, and ABR1011) were fixed in the Hinai-dori breed. So, the Hinai-jidori chicken, F(1) of the Hinai-dori breed, must have at least one of the alleles with all fixed loci. When these alleles on 14 loci from the Hinai-dori breed were not detected in meat samples, it would be judged that the samples were not the Hinai-jidori chicken. Thus, the use of these 14 microsatellite markers provides a practical method of accurately discriminating the Hinai-jidori chicken from other chickens on the market.

Correlation between hyperproliferation and suprabasal integrin expression in human epidermis reconstituted in culture.

In normal epidermis integrin expression is largely confined to the basal layer. However, during wound healing and in psoriatic lesions suprabasal expression is observed. Although the potential importance of suprabasal integrin expression in the pathogenesis of psoriasis has been established, the cause of suprabasal expression is unknown. We now describe changes in integrin expression that occur with time when normal human keratinocytes are grown on two types of dermal equivalent, de-epidermized dermis and collagen gels containing fibroblasts. We show that suprabasal integrin expression is correlated with suprabasal expression of the EGF receptor, but not with expression of keratin 10 or keratin 16. By quantitating the proportion of basal keratinocytes expressing the proliferation marker Ki-67 we could show that suprabasal integrin expression is correlated with high proliferative activity within the basal layer. Taken together with our earlier work, these results suggest that suprabasal integrin expression is linked to hyperproliferation and not to abnormal terminal differentiation or to inflammation; they also establish dermal equivalent cultures as useful experimental models with which to manipulate keratinocyte integrin expression.

Evaluation of serum concentration of parathyroid hormone-related protein and its implication in hypercalcemia in squamous cell carcinoma of the head and neck.

Hypercalcemia is a common and serious complication associated with squamous cell carcinoma (SCC) and is considered to be caused by a tumor-derived factor, parathyroid hormone-related protein (PTHrP). However, the correlation between serum levels of calcium and PTHrP and the kinetics of PTHrP in SCC of the head and neck is unknown, because the behavior of the circulating form of PTHrP in patients has not been determined. In the present study, the PTHrP concentrations in serum samples from 54 patients (37 with SCC and 17 with benign tumors) were measured by a recently developed radioimmunoassay directed toward the C-terminal region of PTHrP, and the laboratory data including those calcium levels in corresponding samples were reviewed retrospectively. Results showed hypercalcemia in four patients with advanced cancer and in whom elevation of the serum PTHrP concentration was observed simultaneously. The regression analysis also revealed the linear relationship of the calcium level to the PTHrP concentration, but not to the concentration of phosphorus or creatinine, suggesting that monitoring of serum PTHrP level is useful for prediction of hypercalcemia associated with head and neck cancer.

Simultaneous production of G- and M-CSF by an oral cancer cell line and the synergistic effects on associated leucocytosis.

We established a novel cell line, TSU, from a oral cancer patient with marked leucocytosis. The culture supernatant of TSU cells promoted granulocytic colony formation by mouse bone marrow cells, indicating that TSU produced granulocyte-colony stimulating factor (G-CSF). The concentration of G-CSF was 2.45 micrograms/mg protein, measured by enzyme-linked immunosorbent assay (ELISA). The maximum number of colonies induced by TSU culture supernatant was more than that achieved with recombinant human G-CSF (rhG-CSF) and the size of the colonies induced by TSU supernatant was obviously larger than those achieved with rhG-CSF. The activity of TSU supernatant was completely inhibited by antihuman G-CSF and macrophage-colony stimulating factor (M-CSF) antibodies, but was only partially inhibited by antihuman G- or M-CSF antibody alone. These results indicate that not only G-CSF but also M-CSF, both of which could be produced by TSU cells, are involved in causing leucocytosis; the results suggest that the synergistic production of G- and M-CSF could play an important role in the leucocytosis associated with oral cancer.

Identification and characterization of autocrine-motility-factor-like activity in oral squamous-cell-carcinoma cells.

A human oral squamous-cell-carcinoma cell line, HOC313, was found to produce a factor which stimulates cell motility in an autocrine manner. The motility factor of HOC313 cells also promoted the locomotory activity of B16 murine melanoma cells reported to be sensitive to autocrine motility factor (AMF). HOC313 cells were found to express a large amount of AMF-receptor mRNA. In addition, the cell motility activity of HOC313 cells was completely blocked by pertussis toxin, a known inhibitor of AMF activity, suggesting that the motility factor of HOC313 cells may be AMF or a closely related factor. Immunocytochemical analysis has revealed that the AMF-like factor of HOC313 cells diminishes the cell-surface expression of adhesive molecule E-cadherin. These results suggest that down-regulation of E-cadherin may be involved in the cell-motility activity induced by the AMF-like factor of HOC313 cells.

The p53 tumor-suppressor gene and ras oncogene mutations in oral squamous-cell carcinoma.

The frequencies of mutations in the p53 tumor-suppressor gene and ras proto-oncogenes were investigated systematically in surgically resected oral squamous-cell carcinomas (SCCs) using single-strand conformation polymorphism (SSCP) and/or dot-blot hybridization analysis of DNA fragments which had been amplified by the polymerase chain reaction (PCR). p53 gene mutations, within the region of exons 5 to 8, were detected in 17 out of 27 (63%) tumor specimens. The role of p53 mutations in cell-line establishment was investigated. p53 gene mutations were detected in 5 out of 6 tissue samples from which cell lines were established and in 4 out of 5 specimens from which cell lines could not be established, suggesting that the presence of p53 gene mutations is not by itself sufficient for cell-line establishment. Tumor samples were also analyzed for point mutational activation of the ras proto-oncogenes. One out of 30 (3%) tumors showed an activating point mutation in codon 12 of H-ras, this being consistent with reports from Europe and USA but not with any from India. Compared to frequencies of the other genetic changes so far reported for oral SCC, the p53 mutations have been observed most often to undergo genetic change. p53 gene mutation is thus intimately involved in the genesis of oral SCC and consequently should be useful as a marker for the diagnosis of this neoplasm.

Gene amplification and overexpression of epidermal growth factor receptor in squamous cell carcinoma of the head and neck.

The degree of gene amplification for epidermal growth factor receptor (EGFR) and its expression levels were examined in 4 cases of tumor lesions and their cell lines of human squamous cell carcinoma (SCC) of the oral cavity. The amplification was detected in 1 case (ZA), but not significantly in 3 other cases (HOC605, HOC815, and HOC927) in which the amplification did not occur during the cell line establishment. In those 3 cases, levels of EGFR synthesis and human EGF (hEGF) binding capacity were varied: HOC605 and HOC815 had slightly increased levels of hEGF binding capacity and EGFR synthesis, respectively. While HOC927 had the lowest levels of both, the hEGF binding capacity was elevated in the tumor lesion when compared with the normal counterpart of the same patient. These results suggest that the increased capacity for EGF binding plays a more important role than does gene amplification on the tumorigenesis of SCC of the head and neck.

[Rapidly progressive periodontitis combined with plasma cell gingivitis: a case report].

A case of rapidly progressive periodontitis combined with plasma cell gingivitis with marked enlargement of the gingiva was presented. Clinically, in the plasma cell gingivitis, the gingiva appear red, friable and bleed easily; usually it does not induce loss of attachment. Histologically, a dense infiltration of the normal plasma cells in the connective tissue is a common finding. A hypersensitivity reaction to some antigens, often flavorings or spices, is generally recognized. In this case, a rapidly progressive loss of attachment was observed, so rapidly progressive periodontitis was diagnosed. Differential diagnosis of the plasma cell gingivitis could be determined by histological and ultrastructural examination. Allergens, however, could not be identified. Conventional periodontal therapy, including intensive plaque control, could not cure the plasma cell gingivitis completely but recurrence of gingival enlargement and loss of attachment could be well controlled.

Growth of the malignant and nonmalignant human squamous cells in a protein-free defined medium.

A novel protein-free synthetic medium has been developed for the culture of human squamous cell carcinoma cells. This medium, designated PF86-1, supports the serial subcultivation of six out of nine human squamous cell carcinoma cell lines in a protein-free, chemically defined condition without the adapting culture from serum-containing conditions. These cell lines growing in PF86-1 exhibited nearly equal potency to grow in massive culture without noticeable changes in morphology but presented a significantly decreased level of colony forming efficiency when compared with the cells cultured in serum-containing media, suggesting the implication of some autocrine mechanism. Interestingly, this medium supported the growth of normal human squamous cells of oral mucosa and skin for more than 2 mo. in the primary explant culture in spite of high levels of calcium ion concentration, where the overgrowth of fibroblasts as contaminant was not observed. These results suggest that PF86-1 supports the growth of cells derived from epidermal tissues selectively and provides the same defined condition for growth of malignant and nonmalignant human squamous cells. It seems, therefore, that PF86-1 allows investigations on the products of squamous cell carcinoma cells or on the differences of growth mechanisms between normal and neoplastic human squamous cells.

Epidermal growth factor stimulates the anchorage-independent growth of human squamous cell carcinomas overexpressing its receptors.

We examined the effects of epidermal growth factor (EGF) on the anchorage-dependent and -independent growth of four human squamous carcinoma cell lines that overexpress EGF receptors. While EGF inhibited anchorage-dependent growth, it stimulated anchorage-independent growth of all four cell lines tested. The results suggest that the proliferative responses to EGF are characterized by a preference for anchorage-independent, rather than -dependent growth, in cells overexpressing EGF receptors. Moreover, as EGF has been shown to stimulate the in vivo growth of squamous carcinoma cells overexpressing EGF receptors, it is also suggested that the in vitro EGF responsiveness of these cells in soft agar, but not in monolayer, better correlates with the in vivo EGF responsiveness.

A study of an in vitro model for invasion of oral squamous cell carcinoma.

An in vitro model for studying the invasion mechanism of oral squamous cell carcinoma (SCC) was developed using a specific culture matrix composed of collagen gel combined with human fibroblasts. Five SCC cell lines cultured on collagen-only gels showed stratified growth on the gels. However, all five cell lines showed invasive growth into the matrix when cultured on the fibroblast-incorporated collagen gels. Moreover, fibroblast-conditioned medium was shown to promote the invasion of HSC-3 cells into the collagen gels. These results suggest that fibroblasts play an important role in the invasion of oral SCC cells in vitro. Four cell lines, which were newly established in our laboratory, were tested in this assay system. These cell lines cultured on fibroblast-incorporated collagen gels expressed morphologic and biologic characteristics in vitro, similar to those in vivo.

Activation of oncogenes in human oral cancer cells: a novel codon 13 mutation of c-H-ras-1 and concurrent amplifications of c-erbB-1 and c-myc.

By NIH3T3 transfection assay in conjunction with in vitro transient neomycin selection, activated c-H-ras-1 oncogenes were detected in two squamous cell carcinoma cell lines, ZA and HOC-313, newly established from human oral cancer patients. ZA had a point mutational activation at the 13th codon, this activation of c-H-ras-1 being novel in human cancer cells, while HOC-313 appeared to have an activation at the 12th codon. In ZA, 16- to 32-fold amplification of the EGF receptor gene, c-erbB-1 and a few-fold amplification of c-myc were detected. The significance of these findings is discussed in relation to multistep carcinogenesis in human cells.

Biosynthesis of the epidermal growth factor receptor in human squamous cell carcinoma lines: secretion of the truncated receptor is not common to epidermal growth factor receptor-hyperproducing cells.

The biosynthesis of the EGF receptor was examined in the epidermoid carcinoma cell line A431 and five novel cell lines from human squamous cell carcinomas possessing high numbers of EGF receptors. Newly synthesized EGF receptors were visualized by labeling with [35S]methionine and immunoprecipitation with a monoclonal anti-EGF receptor antibody. In addition, the processing of the EGF receptor and its intracellular transport was analyzed by distinguishing cell surface receptors from intracellular receptors and by treating cells with inhibitors such as tunicamycin, monensin and brefeldin A. These analyses revealed that in all the tumor cell lines the EGF receptor is synthesized as a glycosylated protein of Mr 160,000 which is converted to the receptor of Mr 170,000 through posttranslational glycosylation. The receptors of Mr 160,000 and 170,000 appeared to possess high mannose type oligosaccharide chains because endoglycosidase H treatment reduced their molecular weights by approximately 30,000. A431 was the only tumor cell line studied that secreted the truncated EGF receptor of Mr 110,000. In A431 cells, the truncated EGF receptor was generated from a protein of Mr 60,000 through tunicamycin- and monensin-sensitive glycosylation. A431 cells treated with monensin secreted the truncated receptor as a Mr 95,000 form.

Growth-inhibitory effects of epidermal growth factor and overexpression of its receptors on human squamous cell carcinomas in culture.

The role of epidermal growth factor (EGF) and its receptors in human cancers was studied using 24 human cell cultures including 15 of squamous cell carcinoma (SCC) of the skin, oral cavity, and esophagus. EGF was found to inhibit the growth and colony formation of all the SCC cells at doses that are mitogenic in many other cells, including epidermal keratinocytes and dermal fibroblasts. This inhibitory effect of EGF on SCCs was specific, because EGF did not inhibit and in some cases slightly stimulated the growth of other tumor cells, such as adenocarcinomas of the stomach, cervix, and breast and sarcomas. The amounts of EGF receptors on these SCC cells were measured by immunoprecipitation of labeled proteins with anti-EGF receptor polyclonal antibody and binding assay of membrane preparations using 125I-EGF. Of 13 SCC cell cultures tested, all except 3 of esophageal SCC showed higher levels of EGF receptor than normal epidermal keratinocytes, which contain 1.5 X 10(5) binding sites/cell. In general, SCCs of the skin and oral cavity had large amounts of EGF receptor on the order of 10(6)/cell, whereas the receptor of esophageal SCCs was on the order of 10(5)/cell. Some SCC cells had about twice as many EGF receptors as A431 cells. The values for the equilibrium dissociation constant (Kd) of these cells were on the order of nM. The sensitivity to the inhibitory effect of EGF correlated well with the elevated level of EGF receptors in 12 SCC cell lines, and higher significance was obtained when data on esophageal SCCs were excluded. The present observations suggest that EGF and EGF receptors play a role in the development of SCCs.

High incidence of amplification of the epidermal growth factor receptor gene in human squamous carcinoma cell lines.

Southern blot-hybridization analysis of DNAs from human tumors demonstrated amplification of the epidermal growth factor (EGF) receptor gene in 10 of 12 squamous cell carcinoma cell lines tested and in none of 18 tumor cell lines of nonsquamous cell carcinomas. The degree of amplification in the squamous cells varied from 2- to 50-fold relative to the epidermal keratinocyte. Hybridization analysis of the RNA showed that the amplification of the EGF receptor gene is accompanied with an increase of the 5.6 kilobases of EGF receptor mRNA. Scatchard plot analysis and sodium dodecyl sulfate-polyacrylamide gel analysis of the EGF receptor revealed that the synthesis of the EGF receptor is also greater in the cells with amplified EGF receptor gene. In contrast, Southern blot analysis of DNAs of primary tumors showed that incidence of amplification of the EGF receptor gene in squamous cells (1 of 6) was almost as frequent as in nonsquamous cells (1 of 4). These results show that amplification of the EGF receptor gene is commonly found in various tumors. In addition, our data suggest that primary squamous cell carcinomas with amplified EGF receptor gene may readily adapt to growth in tissue culture.