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Murine adrenal tumor cells (Y-1 clone) were stimulated by adrenocorticotropic hormone (ACTH) and cyclic adenosine 3',5'-monophosphate (cyclic AMP) to produce steroid hormone (delta 4, 3-keto steroids). The steroids were secreted into the medium immediately after synthesis. The optimum concentrations of ACTH and cyclic AMP for stimulation of steroid production were 10(-2) U/ml and 1.0 mM, respectively. In serum-free medium, ACTH and cyclic AMP stimulated steroidogenesis in Y-1 cells, but the amount of steroid hormone in the culture medium was low. However, a high level of steroid production was maintained with medium containing 10 mg/ml bovine serum albumin (BSA). In culture medium containing a higher concentration of BSA, Y-1 cells did not become spherical as is usually the case when steroid production is stimulated by ACTH or cyclic AMP. The morphological changes did not always correlate with steroid secretion by Y-1 cells.

To study the mechanism of DNA excision repair, a DNA repair system employing permeable mouse sarcoma (SR-C3H/He) cells was established and characterized. SR-C3H/He cells were permeabilized with a 0.0175% Triton X-100 solution. The permeable cells were treated with 1 mM ATP and 0.11 mM bleomycin, and then washed thoroughly to remove ATP and bleomycin. Repair DNA synthesis occurred in the bleomycin-damaged, permeable SR-C3H/He cells when incubated with ATP and four deoxyribonucleoside triphosphates. The repair nature of the DNA synthesis was confirmed by the BrdUMP density shift technique, and by the reduced sensitivity of the newly synthesized DNA to Escherichia coli exonuclease III. The DNA synthesis was optimally enhanced by addition of 0.08 M NaCl. Studies using selective inhibitors of DNA synthesis showed that aphidicolin-sensitive DNA polymerase (DNA polymerase alpha and/or delta) and DNA polymerase beta were involved in the repair process. The present DNA repair system is thought to be useful to study nuclear DNA damage by bleomycin, removal of the damaged ends by an exonuclease, repair DNA synthesis by DNA polymerases and repair patch ligation by DNA ligase(s).

Suppression of the cellular immune system appears to be a prerequisite for the manifestation of adult T-cell leukemia (ATL). In other words, ATL will develop when impairment of the immune system is caused by the infection of human T-lymphotropic virus type I (HTLV-I). This defect of immune surveillance against virus-infected cells may be a result of the impairment of the function of cytotoxic T-cells (CTLs) specific for the HTLV-I-infected cells. The manifestation of ATL could be predicted by examining the function of CTLs in HTLV-I carriers. A new strategy of prevention and therapy for ATL would include an attempt to restore and fortify the CTL function of the host.

Non-radioactive hybridization probes were prepared using the M13 phage vector and the universal sequencing primer. The probe sequence to be used was first cloned into the M13 vector, and the minus strand of the template DNA was then synthesized with the Klenow fragment of E. coli DNA polymerase I in the presence of the biotinylated nucleotide, biotin-11-dUTP, as a label. Resultant DNA was heavily biotinylated, and made up of the entire minus strand of the template DNA. The long tag sequence derived from the M13 vector may increase the sensitivity of the detection. The biotinylated hybrids were visualized with the streptavidin-alkaline phosphatase conjugate and chromogenic substrates. As shown by Southern hybridization, the probe prepared in this way could be used to detect less than 1 pg of target sequence and a single copy gene sequence in human genomic DNA within several hours of signal development.

Dynamic ergometer exercise in a supine position was applied to 64 patients more than 1 year after valvular heart surgery, and the left ventricular reserve was evaluated echocardiographically. The left ventricular reserve declined in the mitral stenosis-mitral valve replacement group, while it was better maintained in the mitral stenosis-mitral commissurotomy, aortic regurgitation and aortic stenosis groups. The patients were divided into 3 groups depending on whether the percentage increase during exercise of stroke index, an index of left ventricular pump function, increased, unchanged, or decreased. The percentage increase of mean velocity of circumferential fibre shortening (y) and that of left ventricular end-diastolic diameter (x) during exercise were plotted for each group. The increased group was isolated from the unchanged group by the line of y = -5.02x + 30.1; the unchanged group was isolated from the decreased group by that of y = -5.68x-10.0, and the increased and unchanged groups were clearly isolated from the decreased group by that of y = -6.86x-4.76. We conclude that dynamic ergometer exercise echocardiography is useful for evaluating the left ventricular reserve of postoperative patients with valvular heart disease. It was also thought that the subclinical state of cardiac failure can be effectively detected by the present method.</P>

We studied the in vivo antitumor effects of natural human tumor necrosis factor-alpha (nHuTNF-alpha) and natural human interferon-alpha (nHuIFN-alpha), both of which were produced by HVJ (hemagglutinating virus of Japan)-stimulated acute lymphatic B cell leukemia line, BALL-1 cells. To clarify the interaction between nHuTNF-alpha and nHuIFN-alpha, we used novel experimental models of lung metastasis and intraabdominal carcinomatosis which we developed in nude mice using a human tumor line, RPMI 4788. While the intravenous administration of nHuTNF-alpha or nHuIFN-alpha alone inhibited lung metastasis, the two cytokines given in combination synergistically inhibited lung metastasis. In a comparative study, nHuTNF-alpha and recombinant human interferon-gamma (rHuIFN-gamma) in combination also synergistically inhibited lung metastasis. Treatment with nHuTNF-alpha and nHuIFN-alpha combined significantly prolonged the survival of nude mice with intraabdominal carcinomatosis. Complete regression of five different human tumor xenografts was achieved by the simultaneous intratumoral injection of nHuTNF-alpha and nHuIFN-alpha. Histological examination revealed that tumor cell lysis occurred 24 h after the intratumoral administration of the cytokines. No significant signs of toxicity to nude mice were observed at any dose tested. The synergism of nHuTNF-alpha and nHuIFN-alpha may allow treatment at a relatively low dose range, thus minimizing side effects. The wide range of anticancer activity of these agents may provide better therapeutic efficacy. The in vivo assay systems which we have developed are useful for the analysis of the biological activities and interactions of cytokines and chemotherapeutic drugs.

An attempt was made to isolate the cell proliferation stimulation factors in the supernatant of embryo carcases and adult muscles of chickens. Evidence was obtained for the presence of at least two or more stimulating factors in both the embryonic and adult muscular supernatants. These factors did not require a supplement of sera or other supporting agents. Furthermore, the use of the salting-out method with ammonium sulfate revealed two or more growth stimulants in the supernatant of chick cells.

The effect of an intravenous injection of squid-ink (sepia-melanin) solution on adult mouse spheroid alveolar epithelial cells was observed by the electron microscope. Sepia-melanin particles were seen in all alveolar wall cells examined that seems to suggest the entrance of sepia-melanin particles into the spheroid alveolar epithlial cells from the alveolar blood capillary. In cases of large penetrations of sepia-melanin particles into spheroid alveolar epithelial cells, a greater increase was found in the intramitochondrial granules. In addition, the so-called inclusion body believed to be formed by the degeneration of mitochondria had very high electron density and its quantity was abundant. On the contrary, in cases where the quantity of sepia-melanin entrance into the spheroid alveolar epithelial cell was small, neither an increase of intramitochondrial granules, an increase of the electron density nor an increase in the quantity of specific inclusion body was found.

Forty-five patients with acute leukemia were compared on cellular immunity measures versus prognosis. The patients were treated according a multicombination therapy protocol. The purified protein derivative (PPD) test and dinitrochlorobenzene (DNCB) test on admission indicated low positive percentages. In acute non-lymphocytic leukemia (ANLL) patients, the 50% survival durations were 11 months in the PPD positive group and 6 months in the PPD negative group. In acute lymphocytic leukemia (ALL) patients, the 50% survival durations were 21 months in the PPD positive group and 13 months in the PPD negative group. Peripheral lymphocyte blastogenesis by phytohemagglutinin (PHA) stimulation was examined at various clinical stages. The stimulation indices were generally low, and no correlation was found between the PHA test and clinical stages. These cellular immunity measures appeared to reflect one aspect of the clinical condition in acute leukemia patients, and further studies are needed for predicting prognosis.

Electron microscope observations were conducted on the relationship between mitochondria and inclusion body in mice spheroid alveolar epithelial cells after injection of trypan blue, an acidic dye and Alcian blue 8GS, a basic dye, by vital staining procedures. When both dyes were injected, the mitochondria of the spheroid alveolar epithelial cell became degenerated; however, in injection of only trypan blue, the cristae showed an increase in electron density. In injection on only Alcian blue 8GS, the cristae showed negative contrast. In most cases the trypan blue particles did not enter into mitochondria, whereas particles of Alcian blue 8GS sometimes entered into the mitochondria. When trypan blue particles entered mitochondria, deposits were not evident in the inclusion body, whereas when Alcian blue particles entered mitochondria deposits were seen in the inclusion body. In both of these cases only a few inclusion bodies were formed so that only traces or no inclusion bodies with vacuolar appearance were observed. From these findings it is suggested that mitochondria maybe convert to inclusion bodies.

The relationship between alveolar macrophages and spheroid alveolar epithelial cells was studied with the electron microscope after injection of squid-ink solution into the trachea of the mouse. At 20 hours after injection of squid-ink solution slight degeneration was evident in alveolar macrophages with sepia-melanin particles being phagocytized with partial digestion by lysosmes. Furthermore, hardly any changes were seen in mitochondria and inclusion bodies of the spheroid alveolar epithelial cells. In contrast, at one week after injection of squid-ink solution, almost all alveolar macrophages were degenerated with destruction of the ectoplasm in which the ingested sepia-melanin particles were digested by lysosomes into fine particles, and the mitochondria of spheroid alveolar epithelial cells were degenerated and the inclusion bodies were hardly formed. At three weeks after injection of squid-ink solution, alveolar macrophages as well as speroid alveolar epithelial cells showed almost complete recovery of functional structure. As the phagocyte in the alveolar space, neutrophile leucocytes were also observed in addition to the so-called alveolar macrophage.

Tissue localization of a subcomponent of the first component of complement (CLq) was examined in one postmortem case of HBs antigen (HBs Ag) positive hepatocellular carcinoma and in six cases of chronic hepatitis from liver biopsy specimens. The direct immunofluorescent method was used after fixation with 2% para-formaldehyde in concentrated ammonium sulfate. CLq localization was found in collagen fibers and the cytoplasm of fibroblasts in the connective tissues of specimens examined. The localization was particularly marked in the region of the fundal glands of the gastric wall. Apart from collagen fibers, other sites of localization included the surface membrane of lymphocytes, especially those cells of the mesenteric lymph nodes. In HBs Ag positive specimens, immune deposit-like substances appeared localized intra-hepatically and in the renal glomeruli. Since C3 and C4 were identified concomitantly, it indicates that these substances were indeed immune diposits. Despite the finding that C3 and C4 were identified together in the hepatic cell cytoplasm, C1q itself was not demonstrated in all hepatic cell cytoplasms.

Today Vitallium is used for surgical implants. It is a casting alloy which, with advances in casting technology, is also used commercially for making instruments of fairly complex shape. Because of its expense, however, it is not widely used in Japan. Instead, a series of 18-8 Mo alloys are used in Japan even though of insufficient strength. Used over a long period of time in the body, especially for the purpose of preserving structual functions as part of the human skeleton, it often corrodes, resulting in either abnormalities in tissue cells or, because of its insufficient strength, danger of bending and breaking with aging. In spite of a marked advance in fracture treatment, we have hardly any suitable materials for making instruments appropriate to the internal fixation of fractures in Japan. We, therefore, conducted various experiments to develop an alloy with sufficient corrosive resistance and strength that could be formed into a complex shape to take the place of Vitallium alloy, finally succeeding in developing an alloy we call "COP". The characteristic properties of COP may be summarized as follows: 1. The main components are 20% Cr, 20% Ni, 20% Co and 4% Mo aside from 0.2% P. 2. As it contains "P", it shows a marked age-hardening. In its molten state its machinability is excellent, and later it can readily be hardened by heat-treatment. 3. It has not only a marked yield point and tensile strength but also has toughness in elongation and reduction of area, showing a strength which surpasses Vitallium. 4. Its corrosive resistance is great. 5. Its cost is far cheaper than Vitallium.

The present study was conducted to investigate the usefulness of the direct leucocyte migration agarose method for studying cell-mediated immunity in vitro. Comparative studies of the purified protein derivative (PPD) skin test and the leucocyte migration inhibition test (LMIT) in which PPD was used as test antigen indicated a significant qualitative and a weak quantitative correlation between these two tests. Furthermore a positive correlation was found between the LMIT and the macrophage migration inhibition test (MIT) using ultrasonicated authochthonous tumor antigen. Comparative studies of the LMIT, MIT, PPD skin and DNCB tests on the same patients showed that cases responding positively to the the PPD skin and DNCB tests tended to respond positively to the LMIT and MIT. Patients with digestive organ cancers were examined by the LMIT. With the advance of cancer, decreased positive test test rates were found. After gastric cancer operations the LMIT findings were divided into two groups: one type changed from positive to negative, and the other type changed from negative to positive. The former response was suggestive of a successful operation, and the latter response was suggestive of a non-curative operation. These results indicated that the direct leucocyte migration inhibition agarose test was useful investigating cell-mediated immunity.

1. Three cases of acute rejection were detected by macrophage migration inhibition tests (MIT) conducted directly on seven patients who had received renal allografts. The macrophage migration inhibitory factor (MIF) activity was positive in all cases 1-2 days before the appearance of acute rejection. 2. After the administration of a high dose of Solu-Medrol (1g/day for 3 days) to suppress the acute rejection, MIF activity recovered to its normal level 3 days later. These findings seem to indicate that MIT yields immunologically useful criteria for the early detection of an acute rejection.

The surface of Gross virus-induced murine lymphoblasts and C-type virus particles budding from these cells were investigated under the scanning electron microscope (SEM). The cells appeared spindle-shaped or roughly-rounded with extensive surface features consisting of microvilli, blebs and ruffled membranes. C-type virus particles were detected on the cell membrane as small spherical particles, distinguishable from the microvilli. Clustered virions were observed in some cases. However, the distribution of virions appeared to be random. The surface of the virion was smooth and had no globular units at high magnification. These morphological observations were confirmed in ultrathin sections.

Salmonella typhimurium was invariably isolated from our J strain murine leukosis. Immunization of D103 mice with either inactivated Salmonella typhimurium or the cell-free extract of leukosis inhibited the transplantation of leukosis. The adoptive immunization of D103 mice with spleen cells of Strong A mice immunized with either Salmonella or the cell-free extract of leukosis inhibited the transplantation of leukosis. The addition of either Salmonella or the cell-free extract of leukosis inhibited the migration of macrophages of leukosis spleen in tissue culture. Strong A mice is non-susceptible to J strain leukosis. However, inoculation of neonatal Strong A mice with the cell-free extract of leukosis produced a susceptibility to the transplantation of leukosis. These results suggest that both a filtrable agent and Salmonella typhimurium are present in cells of this leukosis and might be etiologically related to the leukosis.

Viral nucleoprotein complexes were extracted from nuclei of permissive cells (CV-1) infected with simian virus 40 (SV40) and examined by electron microscopy. SV40 nucleoprotein complexes (SV40 chromatin) showed nucleosomes in linear bead-like arrangements along the extended closed circular DNA. The contour length of the SV40 chromatin was only 1.0-1.8 times shorter than that of viral DNA obtained after deproteinization. The data suggest that the circular DNA in SV40 chromatin can be extended to nearly its full length without detachment of the histone complexes.