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We investigated the biological activity of IL-4 to murine connective tissue-type mast cells (CTMC). When purified peritoneal mast cells, typical CTMC, were incubated with pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) in methylcellulose, about one-fifth of mast cells showed clonal growth. Recombinant IL-4 alone did not stimulate the clonal growth, and purified IL-3 alone induced development of a small number of tiny clusters. In contrast, addition of IL-4 to IL-3 increased the number of clusters by a factor of 10. The number and size of clusters induced by the combination of IL-3 and IL-4 were comparable to those of mast cell clusters induced by PWM-SCM. The present results indicate that IL-4 is an essential factor for in vitro clonal growth of CTMC.

Effects of bacterial lipopolysaccharide (LPS) and X-irradiation on CSF production and granulopoiesis in long-term bone marrow cultures were studied. Levels of colony-stimulating factor (CSF) increased soon after the refeeding of the culture, but the activity was undetectable at day 7. Addition of LPS induced a significant increase in CSF levels in the culture, followed by an elevated granulopoiesis. The increase in CSF levels was suppressed when culture medium that had been harvested at refeeding on day 7 was added. Although irradiation did not increase CSF production, granulopoiesis was markedly stimulated shortly after irradiation. Thus granulopoiesis in long-term bone marrow culture may also be regulated by humoral factors such as CSF, and the culture system may represent the in vivo response to haemopoietic stimuli.

We have established a new T-cell line (CL-8313) that produces interleukin-3 from the spleen cells of mice with a radiation-induced myeloproliferative disorder. IL-3 activity was detected at an extremely high level in the culture medium of the CL-8313 cell line in the absence of any exogenous stimulator. A large amount of IL-3 transcript also was demonstrated in CL-8313 cells. BPA- and CSF-activity was detected at a high level in the culture medium of the CL-8313 cell line. Southern blot analysis of high molecular weight DNA from the CL-8313 cells showed they had unique rearrangement of the antigen receptor beta chain gene. Therefore, we concluded that CL-8313 cells belonged to T-lineage lymphocytes and constitutively produced a high level of IL-3.

We designed a short-term in vitro assay for detecting tumor promoters, utilizing the activation of Epstein-Barr virus (EBV) expression in EBV genome-carrying human lymphoblastoid cells. This system is composed of EBV-non-producer Raji cells as the indicator, n-butyrate as the EBV-inducer, and the test substance. After addition of the latter 2 components to the culture medium, the cells are cultivated for 48 h at 37 degrees C and the ratio of EBV early antigen (EA)-expressing cells was assessed using immunofluorescence. This assay system allows for a rapid detection of the activity of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and its related compounds and also of the Euphorbiaceae plant extracts containing such active principles. Among several microbial products tested, teleocidin, an indole-alkaloid produced by a Streptomyces species, was also detected and had an activity level comparable to that of TPA. Other promoters, such as anthralin, phenol, Tween 60 and 80 and the carcinogenic ("initiator") substances including benzopyrene, did not react with the system. The test is simple to perform, reproducible and should be applicable for mass-screening of promoter substances in the environment.

Mutant mice of Sl/Sld genotype are deficient in melanocytes, erythrocytes, mast cells and germ cells. Deficiency of melanocytes, erythrocytes and mast cells is not attributable to an intrinsic defect in their precursor cells but to a defect in the tissue environment that is necessary for migration, proliferation and/or differentiation. We investigated the mechanism of germ cell deficiency in male Sl/Sld mice by producing aggregation chimaeras from Sl/Sld and +/ + embryos. Chimaeric mice with apparent white stripes were obtained. Two of four such chimaeras were fertile and the phenotypes of resulting progenies showed that some Sl/Sld germ cells had differentiated into functioning sperms in the testis of the chimaeras. In cross sections of the testes of chimaeras, both differentiated and nondifferentiated tubules were observed. However, the proportions of type A spermato-gonia to Sertoli cells in both types of tubules were comparable to the values observed in differentiated tubules of normal +/+ mice. We reconstructed the whole length of four tubules from serial sections. Differentiated and nondifferentiated segments alternated in a single tubule. The shortest differentiated segment contained about 180 Sertoli cells and the shortest nondifferentiated segment about 150 Sertoli cells. These results suggest that Sertoli cells of either Sl/Sld or +/ + genotype make discrete patches and that differentiation of type A spermatogonia does not occur in patches of Sl/Sld Sertoli cells.

A Paleomagnetic method for detecting fireplaces in Pre-ceramic sites was established, and its availability was tested in detail.At first, a bonfire experiment was carried out with the purpose of evaluating magnetic property changes of soils through a baking process. When soils were baked, the remanent magnetizations changed (1) to become stronger in intensity by one to two orders, (2) to become more stable in both intensity and direction against alternating-field demagnetization, and (3) to be directed more nearly in an identical direction than before being backed. These magnetic property changes may have been caused by chemical change (dehydration) from goethite to hematite or magnetite in soils through the baking process. These differences before and after baking were adopted as criteria to detect fireplaces and to determine their positions in Pre-ceramic sites (Paleomagnetic method).Secondly, the availability of these criteria was tested using soils in two archeological sites, Tamatu-Tanaka site, Hyogo (Yayoi age) and Ichi-no-Kubo site, Oita (early Jomon age-Preceramic age), where fireplaces have been definitely recognized with the naked eye. The results showed that remanent magnetizations of backed soils sampled from the fireplaces in these sites satisfy the above-mentioned criteria, and that those of unbaked soils sampled in the same sites do not. However, differences between the magnetic properties of the baked and the unbaked soils were not so significant as seen in the results of the bonfire experiment. This is attributed to chemical alteration of magnetic particles induced through a chemical process over a long period. Consequently, it is important to utilize all the above-mentioned criteria with significant care, in order to correctly detect fireplaces in Pre-ceramic sites.

For the purpose of clarifying the fracture toughness and microfracture mechanism of fiber reinforced plastics composites at cryogenic temperature 4K, fracture toughness tests were performed for a short glass fiber reinforced polyester composite. Acoustic emission signals were also detected during the fracture toughness tests to obtain a reasonable explanation of the microfracture mechanism. The results are summarized, as follows:(1) The maximum load on the load-displacement curve at 4K increases by about three times that at room temperature 297K.(2) The AE activity for the specimen at 4K is extremely higher than that at 297K.(3) The fracture toughness KAE, which is defined as the critical stress intensity factor corresponding to the abrupt increase of AE energy, becomes larger with decreasing temperature below room temperature.(4) The fracture toughness obtained by the 5% offset procedure of ASTM standard does not agree with the fracture toughness KAE.(5) The AE signals were discriminated into two types by the spectrum analysis and the microfracture mechanism was discussed.

Giant lysosomal granules of bgJ/bgJ mutant mice were used as a marker to investigate the histological composition of kidney proximal tubule cells. Embryos of the bgJ/bgJ genotype and those of the +/+ genotype were aggregated, and lysosomes of their proximal tubule cells were histochemically stained with the β-glucuronidase activity. Tubules composed of bgJ/bgJ-type epithelial cells alone, tubules composed of +/+-type epithelial cells alone and tubules containing both types of epithelial cells were observed in cross sections. When the long straight portion of proximal tubules was reconstructed from serial sections, most of the tubules were of the mixed type, and distinct patches of bgJ/bgJ-type or +/+-type epithelial cells were detectable. These patches appeared to extend to the longitudinal rather than the circumferential direction. This suggests the clonal proliferation followed the mixing of embryonic progenitor cells for proximal tubule cells. Estimation from proportions of bgJ/bgJ-type proximal tubules in total examined proximal tubules gave a pool size of approximately four primordial precursor cells for proximal tubules in both right and left kindeys. The fact that the proportion of bgJ/bgJ-type components was comparable between the right and left kidneys of each chimera suggests that all proximal tubule cells in both right and left kidneys may originate from common primordial precursor cells.

Injection of Staphylococcal enterotoxin A (SEA) into WBB6F1-W/WV mice genetically deficient in mast cells resulted in a 10-fold increase in the histidine decarboxylase [HDC, L-histidine carboxylase, EC 4.1.1.22] activity of their spleen. The nature of the spleen cells responsible for this increased HDC activity was studied. The HDC induction by SEA was abolished on day 1 after X-ray irradiation of the mice at 400 rad and restored by transplantation of bone marrow cells from normal WBB6F1-+/+ littermates into the X-ray irradiated WBB6F1-W/WV mice. Transplantation of cells from other organs of the normal mice, such as the thymus, mesenteric lymph node and spleen, did not restore the HDC increase significantly. Transplantation of cultured mast cells also did not restore the increase. Moreover, the high HDC activity of spleen cells induced by SEA was not affected by their treatment with anti-Thy-1,2 antibody and complement. Depletion of phagocytes from the spleen by treatment with carbonyl iron resulted in decrease in HDC activity. These results suggested that phagocytic cells derived from haemopoietic stem cells of the bone marrow were responsible for the increase in HDC activity induced by SEA.

The paper is concerned with the fracture toughness and microfractures of a sheet molding compound polyester composite at room and low temperatures. Fracture toughness tests were performed by using compact tension specimens of the composite at room temperature and liquid nitrogen temperature, 77K. Acoustic emission signals were monitored during the fracture toughness tests. The microscopic observation of the fractured surfaces and the spectrum analysis of the acoustic emission signals were made in order to obtain a reasonable explanation of the fracture mechanism. The results are summarized as follows:(1) The load-crack mouth displacement curves at liquid nitrogen and room temperatures became nonlinear at about the same load level and the maximum load observed at liquid nitrogen temperature increased by about three times that observed at room temperature. The acoustic emission activity for the specimen tested at liquid nitrogen temperature was higher than that at room temperature.(2) The fracture toughness KAE at liquid nitrogen temperature, obtained as the stress intensity factor which corresponds to the onset of abrupt increase of the accumulated acoustic emission energy, was extremely larger than that at room temperature.(3) At room temperature the fracture toughness KAE was in good agreement with the fracture toughness KQ obtained by the 5% offset procedure of ASTM E399, and at liquid nitrogen temperature KAE was larger than KQ.(4) From the microscopic observation, it was found that the fracture of a sheet molding compound composite accompanies the microfractures of four types, i.e. fiber breakage, fiber debonding, resin cracking and delamination.(5) The spectrum analysis indicated that the acoustic emission signals could be classified into three types for the specimens tested at room temperature and four types for the specimens tested at liquid nitrogen temperature. An attempt was made to assign each type of the frequency spectra to the microfracture, and the fracture mechanism of the SMC composite was discussed.

Ras-oncogene activation was investigated in the bladder tumors of F344 male rats given N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water. DNA from one of the nine transitional cell carcinomas contained an H-ras oncogene detectable by the NIH/3T3 transfection assay. Analysis of p21 ras proteins suggested that the activating mutation resided within codon 61 of the H-ras gene and that such activating mutations were not present in other tumors. In contrast to mutational activation of ras genes, enhanced expression of p21 was observed in all tumors examined by immunohistochemical techniques with the use of Formalin-fixed paraffin-embedded tissue sections and an anti-ras p21 antibody, RAP-5. Further histochemical analysis of bladder tissues at various stages of the BBN-induced carcinogenic process indicated that the enhanced expression of p21 appeared early; the reactivity with RAP-5 was observed in diffuse hyperplastic epithelia after 5 weeks of exposure to BBN. The frequency of ras oncogenes, activated either by point mutations or overexpression of p21, in BBN-induced rat bladder carcinomas has thus been shown to be similar to that observed in human bladder carcinomas.

L-8313 is a murine T-cell leukemia cell line the cells of which constitutively produce interleukin-3 (IL-3). S2-8313 is a unique subline of L-8313 which has lost IL-3 producing activity. Although the growth of S2-8313 cells in the bone marrow is comparable to that of L-8313 cells, the survival time of C3H mice grafted with S2-8313 cells is significantly shorter than that of C3H mice grafted with L-8313 cells. The accelerated death observed in C3H mice bearing S2-8313 cells is attributable to early development of granulocytopenia and thrombocytopenia, which resulted from the loss of the IL-3 producing activity.

To detect mutationally activated ras oncogenes, we analyzed electrophoretic mobilities of ras p21 proteins utilizing the fact that many ras oncogenes produce abnormal p21 proteins that migrate at SDS/polyacrylamide gel electrophoresis as a fast-moving or slow-moving species in comparison to a normal p21 depending on the kind of mutation. Of 18 human tumor cell lines analyzed, four (SW480, SW620 and SW403 colon cancers, and SW626 ovary cancer) produced p21 belonging to the slow-moving species, suggesting a point mutation within codon 12 of a member of the three ras genes, H-, Ki- and N-ras. Subsequent DNA transfection analysis using NIH/3T3 cells as recipients identified activated Ki-ras oncogenes in the same four but not in other 14 cell lines. Thus, the analysis of p21 might serve as a rapid primary method to screen a large number of tumor materials for the presence of certain types of mutationally activated ras oncogenes.

�ABSTRACT About half of the chimeras produced by aggregation of two mouse embryos are sex chimeras composed of both XX and XY cells. We developed a fast and easy method to identify sex chimeras by using elect ropho retic bimorphism of an X-linked enzyme, phosphoglycerate kinase-1 (PGK-1), as a marker. When embryos resulting from the crossing of a Pgk-lb/Pgk-lb female and a Pgk�1a/Y male are aggregated, the genotype of sex chimeras is Pgk�1b/Pgk�1a�-Pgk�1b/Y. Most of these were identifiable from the PGK-1 electrophoretic pattern of blood cells (i.e., AB type) and the appearance of genitalia (male type or apparently abnormal). Genotypes of functional sperm in the testes of the male-type sex chimeras were also identifiable from the PGK-1 electrophoretic pattern of progenies. Examination of gonads of the sex chimeras revealed that a considerable proportion was hermaphroditic. With this method, reasonable numbers of male-type sex chimeras and hermaphrodites may be selected and used as material for investigating sexual differentiation.

To determine possible migratory responses to LPS of haemopoietic stem cells (CFUs) in LPS-nonresponsive (C3H/HeJ) mice, changes in the number of CFUs and granulocyte-macrophage progenitor cells (CFUc) after LPS-treatment where studied in splenectomized C3H/ HeJ mice. Splenectomy abrogated the decrease in bone marrow stem cells (CFUs and CFUc) induced by LPS in both LPS-responsive and nonresponsive mice. Recovery of bone marrow stem cells after irradiation was greatly enhanced by splenectomy in C3H/slc mice, but such enhancement was not observed in C3H/HeJ mice. These data suggest that the increase in the number of haemopoietic stem cells in the spleen in C3H/HeJ mice depends on the migration of haemopoietic stem cells from marrow to the spleen. Thus, the expansion of CFUs does not correlate with increase in the number of whole body haemopoietic stem cells. The migrationinducing activity of LPS seems to differ from its haemopoietic stimulatory activity, since the LPS-nonresponsive C3H/HeJ mice respond to the former activity of LPS.

Murine monoclonal antibody (MAb) B72.3, prepared against a membrane-enriched extract of metastatic carcinoma and reactive with a high-molecular-weight determinant, designated tumor-associated glycoprotein (TAG)-72, was shown to be reactive immunohistochemically with 97% of a variety of primary adenocarcinomas of the stomach (n = 40). All "early" gastric carcinomas were reactive with MAb B72.3, although the average percentage cellular reactivity was lower than in "advanced" carcinomas. TAG-72 antigen was detected in benign lesions (i.e. adenomatous polyps and hyperplastic polyps) with intestinal metaplasia. Dysplastic lesions characterized by cellular atypia, abnormal differentiation, and disorganized mucosal architecture demonstrated higher TAG-72 expression than non-dysplastic epithelia. In contrast, normal gastric mucosa was generally non-reactive with MAb B72.3. Assays using serial sections of normal, benign and malignant gastric tissues with two MAbs (B1.1 and COL-6) directed against distinct epitopes of carcinoembryonic antigen (CEA) demonstrated differential reactivity, confirming that TAG-72 and CEA are distinct, non-coordinately expressed antigens. Our results suggest that TAG-72 antigen may be expressed in malignant and dysplastic epithelial cells, as well as in intestinalized epithelium of the stomach which has been closely related to subsequent carcinoma development. Hence, MAb B72.3 may be a useful immunohistochemical adjunct for detecting early foci of adenocarcinomas and premalignant lesions of the stomach.

Two modes of mast cell growth are present, one dependent on diffusible growth factors (interleukins [IL] 3 and 4) and another dependent on contact with fibroblasts. The 3T3 fibroblast cell lines derived from WCB6F1-+/+ mouse embryos supported the proliferation of cultured mast cells (CMC), whereas the 3T3 fibroblast cell lines from WCB6F1-Sl/Sld mouse embryos did not. To investigate the relationship between growth factor-dependent and fibroblast-dependent growths of mast cells, we cocultured CMC and 3T3 fibroblasts in the presence of diffusible growth factors. WCB6F1-+/+ mouse embryo-derived 3T3 cells did not affect the growth factor-dependent proliferation of CMC, but WCB6F1-Sl/Sld mouse embryo-derived 3T3 cells significantly suppressed the proliferation. Close cell-to-cell contact was necessary for the suppression. The NWS1 fibroblast cell line was established from the spleen cells of an adult WBB6F1-+/+ mouse. Although the NWS1 cell line had no supporting effect on the proliferation of CMC in the absence of diffusible growth factors, it did not suppress the proliferation of CMC induced by the growth factors. The present result suggests that a product of mutant Sl genes may be involved in the suppressive activity of WCB6F1-Sl/Sld mouse embryo-derived 3T3 cells.

The paper is concerned with the fracture toughness of glass fiber reinforced plastics at low temperature. By the use of compact tension specimens of a random chopped-strand SMC composite, fracture toughness tests were performed at temperatures, 298K, 200K, 77K and 4K. The main results are summarized as follows : (1) At the lower temperatures, the nonlinearity of load-crack mouth displacement curve becomes remarkable and the acoustic emission activity becomes higher. (2) From the microscopic observation, it was found that the zone of damage consisting of fiber debonding, matrix cracking and fiber breakage is formed in the vicinity of the notch tip. (3) The fracture toughness value KAE, obtained as the stress intensity factor which corresponds to the onset of abrupt increase of the accumulated acoustic emission energy, is larger, as the temperature is lower. (4) At a low temperature, the fracture toughness value KQ obtained by the 5% offset procedure of ASTM E 399 is extremely smaller than the fracture toughness value KAE at the same temperature. Thus, the value KQ could be inappropriate for the fracture toughness at a low temperature. (5) The equation involving the tensile strength of fibers is derived for the fracture toughness of the random chopped-strand SMC composite at a low temperature.

Purpose of review Pluripotent stem cells (PSCs) have the capacity to differentiate into various types of cells, and are promising cell sources for regenerative therapy and drug screening. However, to realize the clinical application of PSCs, a large number of highly qualified target cells must be stably prepared with low cost. To achieve this, great improvements in the reprogramming, differentiation, and elimination of residual PSCs will be necessary. In this review, we summarize the updated knowledge about metabolism in PSCs and its application. Recent findings Recent studies have shown that PSCs have distinct metabolic profiles compared to differentiated cells. The metabolic profiles of PSCs are indispensable for the maintenance of pluripotency, self-renewal, differentiation capacity, and cell survival. Summary Metabolic approaches show improved simplicity, scalability, and lower cost than conventional methods for differentiation and elimination of residual PSCs. Thus, manipulation of PSC metabolism will lead to new technologies to improve their efficiencies.

Hepatocellular carcinoma (HCC) is the fifth most common cancer in the world. We constructed subtracted cDNA libraries enriched with genes overexpressed in HCCs. Among the 17 genes identified were molecular chaperones, Hsp110, Hsp90B, and Hsp70-1. Expression of the Hsp110 family members was further analyzed, and increased transcript levels of Hsp110 and Apg-2, but not Apg-1, were found in 12 and 14, respectively, of 18 HCCs. Immunohistochemical analysis demonstrated the overexpression of the proteins in tumor cells. Apg-2 had chaperone ability similar to Hsp110 in a thermal denaturation assay using luciferase, and showed anti-apoptotic activity. These results suggest that the Hsp110 family members play important roles in hepatocarcinogenesis through their chaperoning activities.

Tissue distribution and cellular localization of rhophilin, a 71 kDa Rho-binding protein, were examined in mice. Rhophilin mRNA was highly expressed in adult testis, but was absent in the testis of W/WV mice deficient in germ cells. An anti-rhophilin antibody detected a band of an expected size in sperm extracts, which was enriched in the tail fraction. Immunofluorescence analysis revealed two lines of striated staining running in parallel in the principal piece of the sperm tail. These results suggest that rhophilin is expressed in germ cells and localized in the fibrous sheath of the sperm tail.

Mild hypothermia shows protective effects on patients with brain damage and cardiac arrest. To elucidate the molecular mechanisms underlying these effects, we analyzed the effects of low culture temperature (32 degrees C) and cold-inducible RNA-binding protein (Cirp) expression on apoptosis in vitro. In BALB/3T3 cells treated with tumor necrosis factor (TNF)-alpha and cycloheximide, the down-shift in temperature from 37 degrees C to 32 degrees C increased the expression of Cirp and suppressed the apoptosis. Activation of caspase-8 was suppressed, and the level of phosphorylated extracellular signal-regulated kinase (ERK) was increased. Transduction of Cirp into the Cirp-deficient mouse fibroblasts increased the level of phosphorylated ERK and suppressed the TNF-alpha-induced apoptosis both at 37 degrees C and 32 degrees C. The ERK-specific inhibitor PD98059 decreased the cytoprotective effect of Cirp as well as that of low culture temperature. These data suggest that mild hypothermia protects cells from TNF-alpha-induced apoptosis, at least partly, via induction of Cirp, and that Cirp protects cells by activating the ERK pathway.

A cDNA clone encoding the human motilin precursor was isolated from an intestinal library using synthetic oligonucleotide probes. The predicted amino acid sequence indicates that the motilin precursor consists of 115 amino acids and includes a 25-residue N-terminal signal peptide followed by the 22-amino-acid motilin sequence and a long, 68-residue C-terminal peptide. The amino acid sequence of human motilin predicted from the cDNA sequence is indentical to its porcine counterpart, which has been determined by protein sequencing. Proteolytic processing of promotilin to motilin occurs at the sequence, Lys-Lys, this being the first reported instance of processing occurring at a pair of Lys residues. In other precursors it occurs at Lys-Arg, Arg-Arg, Arg, or very rarely Lys.

DNA-mediated gene transfer (transfection) studies using NIH 3T3 cells as recipients have demonstrated the presence of transforming genes (oncogenes) in diverse human tumours. A large proportion of oncogenes so far detected by DNA transfection are related to the Ha-ras onc gene of Harvey (and BALB) murine sarcoma viruses (MSV), Ki-ras, the oncogene of Kirsten MSV, and a third member of the ras gene family, N-ras. Individual tumours of many different organs have been associated with the activation of members of the ras gene family. We now present the first systematic survey of human urinary tract tumours processed immediately after surgery, as well as normal tissues from the same patients, to detect the presence of such genes. We demonstrate activation of Ha-ras as an oncogene in around 10% of randomly selected urinary tract tumours as well as direct evidence that oncogene activation is the result of a somatic event which is selected for within the tumour cell population.

DNA prepared from the 3-methylcholanthrene (3MC)-transformed human 312H cell line induced foci on NIH/3T3 cells, whereas DNAs prepared from 7,12-dimethylbenz[a]-anthracene-transformed and the dimethylsulfoxide control 312H cell lines failed to induce foci. The transformed gene from the 3MC-transformed 312H cells was identified as an activated form of the human cellular transforming H-ras oncogene. Analysis of the ras oncogene p21 product in this transformant by immunoprecipitation and gel electrophoresis suggested that this gene was activated by mutation in the 61st codon. These findings demonstrate that activation of a member of the ras gene family can occur in a chemically transformed human cell line.

Efforts to investigate the progression of events that lead human cells of epithelial origin to become neoplastic in response to carcinogenic agents have been aided by the development of tissue culture systems for propagation of epithelial cells. In the present study, nontumorigenic human epidermal keratinocytes immortalized by adenovirus 12 and simian virus 40 (Ad 12-SV40) were transformed by treatment with the chemical carcinogens N-methyl-N'-nitro-N-nitrosoguanidine or 4-nitroquinoline-1-oxide. Such transformants showed morphological alterations and induced carcinomas when transplanted into nude mice, whereas primary human epidermal keratinocytes treated with these chemical carcinogens failed to show any evidence of transformation. This in vitro system may be useful in assessing environmental carcinogens for human epithelial cells and in detecting new human oncogenes.

症例は49才の女性で心タンポナーデを初発症状として来院.入院時より弾性硬に腫大した甲状腺腫と可動性不良のリンパ節を両鎖骨上部に触知した.検査にて甲状腺機能低下およびCEA値の軽度上昇を認めた.頸部CTおよび甲状腺シンチ所見より原発性の甲状腺癌を疑つたが,組織的には甲状腺生検でムチン産生像が見られ,コロイド形成は認められなかつた事よりむしろ低分化腺癌の甲状腺転移の可能性が示唆された.頻回の心膜穿刺にて心タンポナーデに対処し化学療法と放射線照射を行なつたが,入院7カ月目に気管切開部よりの出血にて死亡した.剖検では頸部より縦隔洞,肺門部にかけて軟部組織とリンパ節にびまん性の腫瘍浸潤が認められた.抗サイログロブリン抗体によるPAP法染色にて腫瘍細胞に一致して陽性所見が得られ,またムチン産生像も確認された事より本例はムチン産生性甲状腺癌と診断された.本疾患は従来きわめてまれとされ本邦での報告はまだないが,最近これをくつがえす国外の報告もあり,文献的考察を加えて報告した.

A clinical and pathological analysis was done on 87 autopsy cases of carcinoma of the bladder. Of the 87 cases 68 were men and 19 were women, with a mean age of 64 years. The average survival time was 39 months from the onset of symptoms to death. The patients with multiple tumors had a longer average survival time than the patients with single tumors (51 compared to 40 months), and the average survival times were definitely more favorable for men than for women (43 compared to 23 months). Histological examination revealed transitional cell carcinoma in 77 patients, squamous cell carcinoma in 4, vesical adenocarcinoma in 3 and urachal adenocarcinoma in 3. Squamous cell carcinoma and vesical adenocarcinoma seemed to be more malignant neoplasms. Metastasis was noted in 58 of 87 cases (66.7 per cent) and occurred most frequently in lymph nodes (37.9 per cent), liver (29.9 per cent), lungs (29.9 per cent) and bones (24.1 per cent). Of the 87 cases of bladder carcinoma 11 had primary carcinoma in organs other than the bladder.

We reviewed 41 cases of transitional cell carcinoma of the renal pelvis and ureter with special reference to the coexistence or subsequent development of bladder cancer. Bladder cancer was associated with an upper urinary tract neoplasm in 20 of the 41 cases (48 per cent). Most of these patients (15 of 20) were treated by radical total cystectomy or 1-stage nephroureterocystectomy. The incidence of ureteral stump cancer after nephrectomy alone or incomplete nephroureterectomy was 64 per cent (7 of 11 patients).Twelve surgically removed specimens of the renal pelvis, ureter and/or bladder were examined extensively for the histological association of pre-neoplastic disease, such as atypical hyperplasia and carcinoma in situ. Every specimen had these changes of the urothelium adjacent to and remote from obvious tumors.

Mast cells are a unique class of blood cell. Unlike most blood cells, undifferentiated precursors of mast cells migrate in the bloodstream, invade tissues, proliferate there and then differentiate. Even after differentiation, some mast cells may proliferate extensively. Differentiation of mast cells is regulated by both diffusible growth factors and direct contact with fibroblasts.

In spite of the apparent depletion of mast cells in tissues of mutant mice of W/Wv genotype, cells with many features of mast cells do develop when bone marrow cells of W/Wv mice are cultured in the presence of pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM). In order to resolve this discrepancy and facilitate the analysis of the W mutation, we attempted to establish an in vitro system in which the in vivo defect of W/Wv mice can be reproduced. Cultured mast cells (CMC) were developed from bone marrow cells of either W/Wv or congenic +/+ mice, and then co-cultured with NIH/3T3 mouse fibroblasts in media supplemented only with fetal calf serum (i.e., in the absence of PWM-SCM). Under this condition, CMC from +/+ mice continued to divide and were maintained for more than 4 weeks. The supportive effect of NIH/3T3 cells required close-range interactions with CMC and was not due to synthesis of the known mast cell growth factors, interleukins 3 and 4. By contrast, CMC from W/Wv mice were not maintained, and the number of mast cells remaining after 4 weeks of co-culture was only 1% of the normal +/+ counterparts. Thus, the humoral factor-independent and cell contact-dependent system presented here revealed the intrinsic defects in growth and differentiation of CMC derived from W/Wv mice and might be useful for biochemical and molecular analysis of the gene product(s) encoded at the W locus.

To define the extent of involvement of ras oncogenes in human gastric cancers, we surveyed for the presence of ras oncogenes, activated by either point mutations within their coding sequences or overexpression of ras protein p21, by the combined use of several analytic techniques. Primary gastric cancers were first analyzed by deoxyribonucleic acid transfection assay using NIH/3T3 cells as recipients and by restriction enzyme analysis, which detects point mutations at codon 12 of the H-ras gene. None of seven tumors analyzed scored as positive. Furthermore, none of them had ras p21 with altered electrophoretic mobility on immunoprecipitation and Western blotting, confirming the absence of ras oncogenes activated by point mutations in these tumors. However, in 6 of 7 tumors, the amounts of p21 exceeded that in human placenta. Amplification of the K-ras gene was found in 1 of 11 (including the 7 described above) gastric cancers. Immunohistochemical analysis of ras p21 expression in these 11 tumors was then carried out using the anti-ras p21 monoclonal antibody RAP-5. All cancers showed more reactivity with RAP-5 than did normal mucosa adjacent to the cancers, indicating increased expression of ras p21. These results indicated that transformation of the stomach mucosa from the normal to the malignant phenotype is rarely associated with activation of ras genes by point mutations, but is frequently associated with enhanced expression of ras p21.

Information about the differentiation of mast cells has increased remarkably in the past ten years. This progress has resulted from the introduction of techniques which developed in other fields of experimental hematology. Once mast cells were recognized as a progeny of multipotential hematopoietic stem cells, their unique differentiation processes were clarified. Although most of the progeny of stem cells leave the hematopoietic tissue after maturation, undifferentiated precursors of mast cells leave the hematopoietic tissue. Morphologically, unidentifiable precursors migrate in the bloodstream, invade the connective tissues or the mucosa of the alimentary canal, proliferate, and differentiate into mast cells. Even after their morphological differentiation, some mast cells retain an extensive proliferative potential. There are at least two subpopulations of mast cells: a connective-tissue type and a mucosal type. Connective tissue-type and mucosal mast cells can be distinguished by histochemical, electron microscopical, biochemical and immunological criteria; however, these two types can interchange, and their phenotypes are determined by the anatomical microenvironment in which their final differentiation occurs. Although biochemical natures of the anatomical microenvironment are unknown, molecules that support proliferation and differentiation of mast cells in vitro have been characterized, i.e., interleukin 3 and interleukin 4. In the next ten years, increased information about the differentiation processes will probably induce further understanding of mast cell functions.

Members of the ras gene family are activated as oncogenes in many different human cancers. To systematically determine the frequency at which such genes might be involved in the neoplastic process affecting a specific target tissue, urothelial cells, we surveyed a large series of urinary tract tumors for ras oncogenes by DNA transfection and by molecular genetic analysis. Harvey (Ha)-ras oncogenes were detected in 2 of 38 tumors by transfection, molecularly cloned in biologically active form, and shown to contain single base changes at codon 61 leading to substitutions of arginine and leucine, respectively, for glutamine at this position. One additional Ha-ras oncogene was identified in a bladder carcinoma by restriction polymorphisms at codon 12. In one of 21 tumors, we observed a 40-fold amplification of the Kirsten (Ki)-ras gene. No amplification of other ras genes was detected in any of the tumors analyzed. Our findings strengthen the conclusion that codons 12 and 61 are the major "hot spots" of ras oncogene activation and suggest that quantitative alterations in expression due to gene amplification may provide an alternative mechanism for ras gene activation in primary human tumors.

The therapeutic effect of an aromatic retinoic acid analogue (Ro 10-9359) and mitomycin C (MMC) on rats with bladder tumours induced by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) was examined. Eight-week-old female Wistar rats were given 0.05% BBN in drinking water for 8 weeks. Therapy was started at week 26 and all rats were killed at week 30. MMC at a dose of 0.3 mg/kg twice a week ip for 3 weeks significantly reduced the incidence and the mean number of tumours. With oral Ro 10-9359 at a dose of 100 mg/kg once weekly for 4 weeks, no significant effect was observed. The combination of MMC and Ro 10-9359 significantly reduced the mean number, but not the incidence, of tumours. The difference between the effect of MMC alone and that of MMC given in combination with Ro 10-9359 was not statistically significant. Thus no favorable effect of the retinoid could be demonstrated either alone or in combination with MMC.

The human motilin gene has been isolated and characterized. The gene spans about 9 kilobase pairs (kb) and the 0.7 kb motilin mRNA is encoded by five exons. The 22-amino-acid motilin sequence is encoded by exons 2 and 3. The human motilin gene was mapped to the p21.2→p21.3 region of chromosome 6 by hybridization of the cloned cDNA to DNAs from a panel of reduced human-mouse somatic cell hybrids and by in situ hybridization to human prometaphase chromosomes. RNA blotting using RNA prepared from various regions of the human gastrointestinal tract revealed high levels of motilin mRNA in duodenum and lower levels in the antrum of the stomach; motilin mRNA could not be detected by this procedure in the esophagus, cardia of the stomach, descending colon or gallbladder.

Sl/Sld mutant mice are profoundly deficient in tissue mast cells as a result of a defect in the microenvironment promoting the development of these cells. To facilitate the analysis of the Sl mutation, we attempted to establish an in vitro system in which the in vivo defect of Sl/Sld mice could be reproduced. 3T3 cell lines were established from 17-day-old embryos of Sl/Sld and congenic +/+ genotypes and were cocultured with mast cells obtained in vitro from the bone marrow of +/+ mice. All eight 3T3 cell lines derived from +/+ of T-cell-derived growth factors. By contrast, none of eight 3T3 cell lines from Sl/Sld embryos supported mast cells under similar conditions. The defect in Sl/Sld 3T3 cells was further characterized as a failure to induce the G1-to-S transition in synchronized mast cells upon contact, suggesting that the Sl gene product is indispensable for this activity. When 3T3 cells of +/+ genotype, grown on pieces of cellulose acetate membrane, were transplanted into the peritoneal cavity of Sl/Sld mice, mast cells appeared locally in the transplanted 3T3 cell layers. These results suggested an essential role of fibroblasts in vivo as the tissue microenvironment promoting the development of mast cells and that they are defective in Sl/Sld mice. The present coculture system duplicated mast-cell deficiency of Sl/Sld mice in vitro and should prove useful for analysis of the Sl gene product.

Coronary artery diseases (CAD) are the most commonly occurring disorders in the developed countries. Development of new therapeutics and their success in clinical studies require confirmation of therapeutic effects in large animal models. Swine is an ideal animal model because their anatomical features are similar to humans. However, sometimes their large body size hampers translational research, particularly in cell or tissue transplantation procedures for adult stage and long-term observation studies. We have been developing the smallest experimental pig, called micro-mini pigs (MMPs), for regenerative medicine. Five- to 14-month-old mature MMPs were used (n = 15, body weight: 13.4 ± 2.14 kg). In the acute myocardial infarction (AMI) model, AMI was induced by cryoinjury (CI). In the ischemic heart disease (IHD) model, IHD was induced using an ameroid constrictor (AC) that was applied to the left anterior descending artery, with the diagonal branches ligated. Cardiac function in both models was assessed by magnetic resonance imaging (MRI) with late gadolinium enhancement, and coronary angiography (CAG) was performed to evaluate the collateral arteries. Animals were sacrificed 4 weeks after procedures to evaluate the pathological changes. On MRI, the ejection fraction in CI-induced AMI models decreased from 57.7 ± 3.2% to 35.8 ± 4.7% (Δ62.2%, p = 0.012) (n = 8). In contrast, AC also impaired cardiac function, but some pigs did not develop heart failure due to the development of collateral branches (pre: 54.4 ± 4.4%, post: 41.1 ± 8.0%, Δ75.7%, p = 0.028 [n = 7]). Gadolinium contrast-enhanced MRI and pathological examination confirmed scarring in both models. The proportion of scar area in the left ventricular region of CI-induced AMI models vs. AC-induced IHD models was 19.4% vs. 10.3% (p = 0.046) (MRI) and 17.6% vs. 9.2% (p = 0.046) (pathology). Immunohistological analysis also showed the presence of marked neovascularization in AC models, which led to greater variation in the impairment of cardiac function. MMPs are the smallest available swine for experimental use that are suitable for translational research. They allow for long-term observation of adult pathology. Both models of AMI and IHD were successfully established in MMPs. The MMP preclinical heart failure model may accelerate further development of new treatments for CAD.

Transcription Around the Clock The biological clock that controls daily rhythms in mammalian physiology and behavior is thought to be regulated in large part by transcriptional events (see the Perspective by Doherty and Kay ). Koike et al. (p. 349 ; published online 30 August) produced a comprehensive analysis of these transcriptional events across the entire mouse liver genome over a 24-hour period. Only ∼22% of cycling messenger RNA transcripts were driven by de novo transcription, suggesting that posttranscriptional events also play an important regulatory role in the mammalian clock. Biological timing in organisms can also respond to rhythmic cues from the environment. Morf et al. (p. 379 , published online 23 August) explored how one such cue, cycles in ambient temperature, influence circadian timing in mammalian cells. Cold-inducible RNA–binding protein (CIRP) accumulates when body temperature is low. A systematic search for binding partners of CIRP identified RNA encoding core components of the circadian clock. Loss of CIRP decreased the amplitude of circadian gene expression and cells lacking CIRP adapted more quickly to temperature cycles.

28 anticancer agents have been surveyed for Epstein-Barr virus (EBV) activating potency. Two vinca alkaloids with antitubulin activity, vinblastine (VLB) and vincristine (VCR), enhanced the expression of EBV early antigens (EA) in a latently infected human lymphoblastoid cell line (Raji), when used in combination with n-butyrate. Other antitubulin agents, colchicine, colcemid, and podophyllotoxin, had the same effect, although their effects were less than that of the potent tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA).

Carbon particles enhance hemopoiesis in irradiated mice. Serum from carbon-treated mice stimulated macrophage colony formation, and inhibited granulocyte colony formation. The finding suggests that carbon-treatment modulates the hemopoietic environment through the monocyte-macrophage system.

To prevent hemorrhagic cystitis induced by ifosfamide, continuous bladder irrigation with 3,000 ml. of saline per day was performed in 8 patients. Ifosfamide was given by an intravenous infusion one to two hours at a dose of either 1,600 to 1,950 mg./m2 daily for three days, or 1,430 to 1,480 mg./m2 daily for five days. Gross hematuria was induced in none and microscopic hematuria in one during 24 courses of treatment. There was no complication associated with catheter placement, other than urethral discomfort in 1 patient. Thus this appears to be a useful and effective method.

Nude mice were inoculated with BMA1 cells. These are cells which produce granulocyte-macrophage colony stimulating factor (GM-CSF); They are derived from mouse bone marrow stromal cells transfected with adenovirus 5 DNA. Progressive neutrophilia developed as the tumor grew, but disappeared quickly after local tumor excision. Media conditioned with tumor cells had GM-CSF but neither erythropoietin, nor burst-promoting activity. In all the tumors which developed, focal areas of bone formation were found among fibrosarcomatous tissues.