Michio Fujisawa

Activation of the c-K-ras oncogene in a human pancreas carcinoma

The human pancreas carcinoma cell line T3M-4 contains activated c-Kirsten (K)-ras oncogene detectable by the DNA-mediated gene transfer technique using NIH/3T3 cells. DNA fragments containing coding lesions have been cloned, and nucleotide sequence analysis suggests that the T3M-4 oncogene has been activated by a single nucleotide transition from A to C in the second exon, which results in the substitution of histidine for glutamine in coden 61 of the predicted amino acid sequence. The quantity analysis of c-K-ras oncogene in the DNA and RNA of T3M-4 cells revealed that the c-K-ras gene was amplified and overexpressed in T3M-4 cells. These findings indicate that the T3M-4 c-K-ras oncogene is activated by different mutational events.

Increased angiotensin-converting enzyme in peripheral blood monocytes from patients with sarcoidosis.

Angiotensin-converting enzyme (ACE) activity was measured in isolated peripheral blood monocytes and culture medium from 28 patients with sarcoidosis and compared with values obtained from monocytes of 25 normal control subjects. ACE activity was determined by radioimmunoassay of angiotensin II produced from angiotensin I. While there was no measurable ACE activity in monocytes or culture medium from normal controls under the conditions of our study, monocytes from patients with sarcoidosis all showed activity both in cells and culture medium. The mean ACE activity of monocytes from patients with sarcoidosis was 2.0 pg angiotensin II formed/min per 10(5) cells, and that released into medium over a 24-h interval was 30.4 pg angiotensin II/min per 10(5) cells. The monocyte ACE from patients with sarcoidosis was activated by chloride ions and inhibited by EDTA, captopril, and rabbit antiserum to purified human plasma ACE, indicating that enzymatic activity was effected specifically by ACE. Thus, our studies show a significant elevation and release of ACE by peripheral blood monocytes of patients with sarcoidosis under conditions where monocytes of normal control subjects do not demonstrate ACE activity.

Establishment of a human colony-stimulating-factor-producing cell line from an undifferentiated large cell carcinoma of the lung.

Two different types of lung cancer, a squamous cell carcinoma designated as OTUK, and an undifferentiated large cell carcinoma named as T3M‐10, were investigated in vitro; both produced a moderate neutrophilia in each patient. In order to analyze this phenomenon, primary cultures were performed. Epithelial cells attached and grew in both of the cultures. The conditioned medium from both tumor cells revealed high colony‐stimulating‐factor (CSF) activities. From T3M‐10 tumor cultures, a new CSF‐producing cell line has been established. T3M‐10 cells have been continuously propagated during the last 28 months and produced CSF. Chromosomal analysis revealed the cell line to be a human aneuploid one with a near‐diploid mode. These results indicate that the two different types of lung cancers produce CSF, which may have stimulated granulopoiesis in vivo in the patients as well.

Induction by fibroblast growth factor of angiotensin converting enzyme in vascular endothelial cells invitro

Induction of vascular endothelial cells with pituitary fibroblast growth factor (FGF) provoked an increase in angiotensin converting enzyme activity. The stimulatory effect of FGF on ACE activity was dose-dependent (ED50 = 1.0 ng/ml). Our results suggest a possible role for pituitary FGF in regulation of ACE production in vascular endothelial cells.

Sarcoid granulomas metabolize 25-hydroxyvitamin D3invitro

Sarcoid granulomas metabolized 25-hydroxyvitamin D3 to two unidentified metabolites during in vitro incubation. A two-step high pressure liquid chromatography system revealed two unique elution positions of these sarcoid-derived metabolites that exactly comigrated with the elution positions of 5(Z)-19-nor-10-oxo-25(OH)D3 and 5(E)-19-nor-10-oxo-25(OH)D3, respectively. These unique metabolites did not bind specifically to a protein receptor for 1,25(OH)2D3.

Human myeloid leukemia cells metabolize 25-hydroxyvitamin D3 in vitro

Human promyelocytic leukemia cells (HL-60) converted 25-hydroxyvitamin D3 to two more polar metabolites during in vitro incubations. A two-step high pressure liquid chromatography system revealed two unique elution positions of those leukemic cell-derived metabolites that exactly co-migrated with the elution positions of 5(Z)-19-nor-10-oxo-25-hydroxyvitamin D3 and 5(E)-19-nor-10-oxo-25-hydroxyvitamin D3, respectively. These unique metabolites did not bind specifically to a protein receptor for 1,25-dihydroxyvitamin D3.

High-cholesterol diet-induced lipoproteins stimulate lipoprotein lipase secretion in cultured rat alveolar macrophages

We have previously shown that cultured rat alveolar macrophages synthesize and secrete lipoprotein lipase into the medium. The purpose of the present experiments is to examine whether cholesterol-enriched lipoproteins from cholesterol-fed animals have any effects on the lipoprotein lipase secretion and the lipid accumulation in macrophages. Macrophages incubated with the VLDL obtained from rats fed a normal diet secreted 2-fold higher amounts of lipoprotein lipase than those without lipoproteins. Intermediate-, low- and very-low-density lipoproteins from rats fed a high-cholesterol diet also enhanced the lipoprotein lipase secretion. Normal high- and low-density lipoproteins, and high-density lipoproteins from hypercholesterolemic animals did not cause any increase in the lipoprotein lipase secretion. The lipoproteins which stimulated the lipoprotein lipase secretion caused intracellular accumulation of both triacylglycerol and cholesterol. It is speculated that macrophages residing in the environment rich in lipoproteins, especially hypercholesterolemic lipoproteins, take them up and accumulate lipids intracellularly, and that this process links with the lipoprotein lipase secretion. The secreted lipoprotein lipase could facilitate, by degrading lipoproteins, the uptake of lipoprotein lipase-modified lipoproteins. Probably such a series of events is of importance in the foam cell formation of macrophages.

Corticosteroid receptor in type II pneumocytes of the rat

Glucocorticoid receptor in rat type II pneumocytes has been characterized. The Scatchard plot analysis of 3H-dexamethasone binding to type II cells showed a single class of binding sites. The apparent Kd of 3H-dexamethasone binding by a whole cell assay was 9.1 nM and the maximal binding capacity was 78.0 f mol/10(6) cells (0.31 pmol/mg cytosol protein).

Successful treatment of spinocerebellar ataxia 6 with medicinal herbs

A case of familial spinocerebellar ataxia 6 with typical symptoms is presented. A 60‐year‐old Japanese female suffered from gait disturbance, ataxia and dizziness. Head magnetic resonance imaging revealed a typical atrophic image in cerebellum. Genetic tests revealed an expanded allele of 22 CAG repeats at the spinocerebellar ataxia type 6 locus. She was diagnosed with spinocerebellar ataxia 6. Her mother was also diagnosed with the same disease. A mixture of 18 medicinal herbs (modified Zhengan Xifeng Tang) was given according to the differential diagnosis based on the guidelines of traditional Chinese medicine. All of the symptoms were remarkably improved after 60 days of the herbal treatment. One year after discontinuation of the treatment, she complained of gait ataxia. She was treated with the modified Zhengan Xifeng Tang for 60 days. Gait ataxia was markedly improved by the second treatment. Fifteen months after discontinuation of the second treatment, she complained of gait ataxia again. The same remedy was given for 60 days. Gait ataxia was remarkably reduced again. The results may imply therapeutic potential of the medicinal herbs for spinocerebellar ataxia 6.

Chapter 27 Brain-associated small cell lung cancer antigen (BASCA) has at least three epitopes

Abstract Brain-Associated Small Cell Lung Cancer Antigen (BASCA) is a peptide with a molecular weight of 124 kDalton recognised by murine monoclonal antibody TFS-4. BASCA is selectively expressed on the cell membrane of small cell lung cancer among lung cancers, and on human brain among normal tissues. To elucidate the molecular characteristics of this molecule, monoclonal antibodies raised against small cell lung cancer were investigated in terms of reactivity with affinity-purified BASCA. Competitive inhibition assays of binding, and surface immunofluorescence studies on live small cell lung cancer cells, revealed that BASCA has at least three independent epitopes; two on the cell surface and one inaccessible from outside.