GEP oncogene promotes cell proliferation through YAP activation in ovarian cancer.

G-protein-coupled receptors (GPCRs) and their ligands function in the progression of human malignancies. Gα12 and Gα13, encoded by GNA12 and GNA13, respectively, are referred to as the GEP oncogene and are implicated in tumor progression. However, the molecular mechanisms by which Gα12/13 activation promotes cancer progression are not fully elucidated. Here, we demonstrate elevated expression of Gα12/13 in human ovarian cancer tissues. Gα12/13 activation did not promote cellular migration in the ovarian cancer cell lines examined. Rather, Gα12/13 activation promoted cell growth. We used a synthetic biology approach using chimeric G proteins and GPCRs activated solely by artificial ligands to selectively trigger signaling pathways downstream of specific G proteins. We found that Gα12/13 promotes proliferation of ovarian cancer cells by activating the transcriptional coactivator YAP, a critical component of the Hippo signaling pathway. Furthermore, we reveal that inhibition of YAP by short hairpin RNA or a specific inhibitor prevented the growth of ovarian cancer cells. Therefore, YAP may be a suitable therapeutic target in ovarian cancer.

Synthesis of kanamycin A analogs containing a 6-amino-6-deoxyglycofuranose moiety.

Three kanamycin A analogs containing 6-amino-6-deoxyglycofuranoses have been prepared as candidates for potential activity against resistant bacteria producing 6'-N-acetyltransferase. They are 4-O-(6-amino-3,5,6-trideoxy-alpha-D-, -beta-D-, and -beta-L-erythro -hexofuranosyl)-6-O-(3-amino-3-deoxy-alpha-D-glucopyranosyl)-2,5-dideoxy-5-epi-5-fluorostreptamine. Structure-activity relationships of these compounds are discussed.

Amino acids dissolved in stream water as possible home stream odorants for masu salmon.

It is well established that salmon return to their home stream by sensing the odors of the stream water. In this study we have attempted to identify the home stream odorants used by masu salmon in Lake Toya. The salmon in Lake Toya return to the home stream which flows into the lake after lake life for 2-3 years. Besides water from the home stream, waters from two other streams which flow into Lake Toya were also used in the experiments. We analyzed the compositions of amino acids, inorganic cations and bile acids in waters from the three streams. Application of mixtures of inorganic cations or bile acids, reconstituted based on the compositions of the stream waters, to the olfactory epithelium induced only very small responses. On the other hand, application of mixtures of amino acids induced large responses. The response to artificial stream water reconstituted based on the compositions of amino acids and salts closely resembles that to the corresponding stream water. Cross-adaptation experiments with three combinations of the mixtures were carried out. The response pattern for each combination closely resembled that to the corresponding combination of stream waters. Based on the results obtained, we concluded that amino acids dissolved in the home stream water are possible home stream odorants.

Relationship between N-acetyltransferase (NAT) activity and liver protoporphyrin level in experimental porphyria.

The results of our previous studies demonstrated that isonicotinic acid hydrazide (INH) can aggravate griseofulvin (GF)-induced protoporphyria in mice. To elucidate this phenomenon, we studied the relationship between liver protoporphyrin (PP) levels and N-acetyltransferase (NAT) activity, which is known to be a major catabolic enzyme of INH metabolism in the liver. The results revealed a significant correlation between liver PP levels and NAT activity in the mice fed 0.1% GF and 0.05% INH. In this group, mice with high NAT activity developed severe protoporphyria. There was no correlation, however, between liver PP levels and NAT activity in the mice fed GF alone or INH alone. The result suggested that INH metabolites formed by NAT enhance the hepatotoxicity of GF in mice.

Relationship between porphyria cutanea tarda (PCT) and viral hepatitis.

Recent reports have revealed the high prevalence of serological markers of viral hepatitis in porphyria cutanea tarda (PCT). We present two cases of PCT associated with hepatitis C and discuss the relationship between PCT and viral hepatitis. Case 1: A 50-year-old Japanese male noticed blisters, erosions, and fragility on sun-exposed areas of his skin in November of 1990. He had no history of excessive alcohol intake. He had been taking analgesics for eighteen years. Case 2: A 64-year-old Japanese male was referred in October of 1989 because of pigmentation on sun-exposed areas of his skin. He had been drinking alcohol excessively for 43 years. The hepatitis C virus (HCV) antibody was present in each case. Tests for the HCV antibody and hepatitis B serological markers were run in 5 other patients. HCV antibody was present in 3 of them. The two cases negative for the HCV antibody exhibited the hepatitis B antibody. We speculated that viral hepatitis infection may play an important role in precipitating PCT in cases with a history of a long term excessive intake of alcohol or chemicals.

Alzheimer's amyloid precursor protein mRNA without exon 15 is ubiquitously expressed except in the rat central nervous system.

The expression of L-beta A4 amyloid precursor protein (L-APP) mRNA, which is a splicing product excluding exon 15 of the APP gene, was investigated in various tissues of adult rats by a polymerase chain reaction analysis of reverse-transcribed RNA (RT-PCR). L-APP mRNA was ubiquitously expressed in all the examined tissues including the liver, kidney, heart, skeletal muscle, spleen, thymus, adrenal, stomach, submandibular gland, testis and ovary, except for the central nervous system (CNS) tissues such as the brain and spinal cord. The DNA sequence analysis of the RT-PCR products from adult rat liver showed an L-APP cDNA form, in which exon 14 was spliced from exon 14 to exon 16, and exon 15 of the APP gene was excluded. In addition, regarding as the brain and liver, L-APP mRNA expression was examined during the development of the embryonic stage. In the brain, no L-APP mRNA expression was detected even in the embryonic stage, whereas L-APP mRNA expression of the liver was still found in the embryonic stage. These results suggest that the splicing event excluding exon 15, which is exactly adjacent to exon 16 and exon 17 encoding the beta A4 protein, would probably occur very rarely in the CNS and that the splicing of L-APP might already be regulated in the embryonic stage.

The rat central nervous system expresses Alzheimer's amyloid precursor protein APP695, but not APP677 (L-APP form).

A novel splicing form of beta A4 amyloid precursor protein (APP) lacking exon 15, corresponding to 18 residues, was first reported in leukocytes and then in ubiquitous organs. To determine which APP molecules (APP695, APP751, or APP770) either with (N-APP) or without (L-APP; leukocyte-derived APP) exon 15 were expressed in various organs, we investigated the alternative splicing at exon 15 in the rat brain, kidney, heart, and testis by a PCR analysis of reverse-transcribed RNA and Southern blot analysis. Regarding APP695 without exons 7 and 8, L-APP was either seldom or never expressed in the brain, whereas both N- and L-APP were expressed in other organs. On the other hand, regarding APP751/770 containing exon 7, which codes for the Kunitz-type serine protease inhibitor domain, both N- and L-APP were expressed in all the organs examined, including the brain. These results suggest that a particular alternative regulation system related to exon 15 might be present in only APP695 of the brain and influence the proteolytic processing of APP.

Immunohistochemical distribution of amyloid precursor protein during normal rat development.

This study focused on the immunohistochemical identification of the beta/A4 amyloid precursor protein (APP) in various developmental stages of both the rat central nervous system (CNS) and the peripheral nervous system (PNS). A comparative study with myelin basic protein (MBP) and synaptophysin (SYP) facilitated the understanding of neuronal maturation and synaptogenesis on both prenatal and postnatal development. Our immunohistochemical study revealed APP to be widely distributed through the nervous system while existing mainly in the cytoplasm, dendrites and axons of the neurons. However, immunoreactivity was also observed in either the ependymal cells or the choroid plexus epithelial cells. Our immunostaining was carried out by the hydrated autoclaving method and revealed the expression of APP at embryonic day 15 in the neuron of the mesencephalic nucleus of the trigeminal nerve and the anterior horn of the spinal cord, trigeminal and spinal ganglion, ependymal cells and the choroid plexus. We thus observed dramatic changes of APP expression in the cerebellum from the embryonic stage. The maturation of synaptogenesis in the cerebellar molecular layer was parallel to the extension of the dendrites of Purkinje cells, which revealed immunoreactivity for APP. These findings suggested that APP played an important role in neuronal maturation and synaptogenesis. Thus, APP is considered to be a useful marker for neuronal development.

Experimental murine protoporphyria induced by griseofulvin (GF): the relationship between hepatic porphyrin levels and liver function test values in mice treated with GF.

To investigate the hepatic abnormalities accompanying experimental protoporphyria due to griseofulvin (GF), liver function test values and porphyrin levels in mice were assayed at days 2, 4, 8, and 16 after starting the administration of 0.5% GF feed. Furthermore, in an attempt to elucidate the harmful effects of GF on liver functions, the above mentioned assay was also performed after the feed was discontinued in mice given 0.5% GF feed for 16 days. The hepatic protoporphyrin (PP) level had already risen by day 2, but the erythrocytic PP level was within normal limits at that time. Hepatic PP levels increased gradually, followed by an increase in erythrocytic PP levels. The variation in liver function test values roughly paralleled the porphyrin levels. Over the time span of the response to GF, the variations in the serum glutamate oxaloacetate transaminase (S-GOT) levels, serum glutamate pyruvate transaminase (S-GPT) levels, and leucine amino peptidase (LAP) levels resembled those in hepatic PP. On the other hand, the changes in alkaline phosphatase (ALP) levels paralleled those of the erythrocytic PP levels. Erythrocytic and fecal protoporphyrin levels decreased to the normal level one month after the discontinuation of GF administration, but the hepatic protoporphyrin level still was 53.6 times higher than the normal level two months after switching to normal feed. The values of liver function tests had returned to within the normal range after one month. By the fourth day after the administration of GF, a brown pigmented material could be observed around the hepatocytes and the Glisson sheath; the amount of this material increased day by day.(ABSTRACT TRUNCATED AT 250 WORDS)

Polychlorinated biphenyls (PCBs) and polychlorinated quaterphenyls (PCQs) concentrations in skin surface lipids and blood of patients with yusho.

The relationship between PCBs and PCQs concentrations in the skin surface lipids and blood was investigated in this study. PCBs and PCQs concentrations in thirty two patients with PCB poisoning (Yusho) and 20 normal controls were analyzed. The collection of skin surface lipids was performed by the method of cotton pad with 70% ethyl alcohol. The alkaline decomposition method described in the official standard analytical methods for the isolation of PCBs and PCQs fractions was used. In the blood of control group, mean value of PCBs concentration was 2.1 ng/g, and that of PCQs concentration was too low to be detected by our analytical method. On the other hand, the PCBs concentration in the Yusho group were two times higher than those in the control group. The mean value of PCQs concentration was 0.90 ng/g in Yusho group, while it was not detectable in the control group. In the skin surface lipids of patients with Yusho, the mean value of PCBs concentration was 580.0 ng/g, but was 324.4 ng/g in the control group. The mean value of PCQs concentration in the skin surface lipids of Yusho patients was 25.7 ng/g, although it was not detected in the control group. PCBs and PCQs levels in the skin surface lipids were higher than those in the blood. This means that cutaneous sebaceous system is one of the excreted systems of polyhalogenated chemicals, such as PCBs or PCQs, when these chemicals are precipitated in human or mammalians.

The synergistic effect of chlorinated chemicals and low concentration of griseofulvin on porphyrin metabolism--the effect of 0.05% hexachlorobenzene and 0.1% griseofulvin on young and old mice.

The difference in the synergistic effect of 0.05% hexachlorobenzene (HCB) and 0.1% Griseofulvin (GF) on porphyrin metabolism between young (4 weeks old) and old (8 weeks old) mice was investigated. These mice were divided into four groups, group A was treated with feeds containing 0.05% HCB and 0.1% GF, group B with 0.05% HCB, group C with 0.1% GF and group D with normal feed. The treatments were continued for 8 and 16 days, after which porphyrins in the erythrocytes, feces and liver were analyzed by a chromatographic method. Hepatic protoporphyrin levels had risen by 8th day in the young mice treated with 0.05% HCB and 0.1% GF, and by 16th day in the young mice treated with 0.1% GF, but no rise in the hepatic protoporphyrin levels in the old mice was seen. Hepatic protoporphyrin level by 16th day in the young mice treated with 0.1% GF had significantly risen compared to that in the old mice with same treatment. Fecal coproporphyrin level had risen at 8th day in the young mice treated with 0.05% HCB and 0.1% GF and by 16th day in the young mice treated with 0.05% HCB. It had also risen by 16th day in the old mice treated with 0.05% HCB and 0.1% GF. Fecal protoporphyrin level in the young mice treated with 0.05% HCB and 0.1% GF, 0.1% GF alone was three to four times higher than those in normal mice. Erythrocytic coproporphyrin and protoporphyrin levels in the young mice treated with 0.05% HCB and 0.1% GF, 0.05% HCB or 0.1% GF alone were generally higher than those in the old mice.(ABSTRACT TRUNCATED AT 250 WORDS)

[In vitro analysis for cellular toxicity of polychlorinated biphenyls (PCBs) and 2,3,4,7,8-pentacholorodibenzofuran (PCDF) on PLC/PRF/5 cell proliferation (II)--The effects of ursodeoxycholic acid and chenodeoxycholic acid on cell toxicity].

We investigated the cell toxicity of polychlorinated biphenyls (PCBs) and 2,3,4,7,8-pentachlorodibenzofuran (PCDF) as two kinds of indicator of the quantity of secreting protein, which is an HBV surface antigen (HBsAg) in PLC/PRF/5 cells, and the DNA of those cells was counted the radioactivity for dot hybridization method, respectively. Furthermore, the reductive action of ursodeoxycholic acid (UDCA) and chenodeoxycholic acid (CDCA) on the PCBs and PCDF toxicity was investigated. HBsAg titer increased to 10 to 15% with the addition of CDCA at the concentration of 0.02 x 2(-6)%. However, the slope of the curve of DNA synthesis of HeLa cells at the presence of PCDF was gradually increased at the concentration of 0.02 x 2(-4)% of UDCA and CDCA, and it became to overlap with a control group while PCBs did not. These results mean that PCDF cell toxicity was suppressed a little by UDCA and CDCA, but the case of PCBs did not.

Alzheimer's amyloid precursor protein accumulates within axonal swellings in human brain lesions.

By using a monoclonal antibody and polyclonal antibodies to human beta amyloid precursor protein (APP), we immunohistochemically examined axonal swellings (spheroids). The immunoreactivity of APP was observed in numerous spheroids, which were found around the areas of old cerebral infarctions, in the globus pallidus with Hallervorden-Spatz disease as well as in or around brain abscesses. The axonal swellings were not immunolabeled with beta protein. These results implied that APP accumulated in spheroids regardless of beta protein depositions.

Alzheimer's amyloid precursor protein-positive degenerative neurites exist even within kuru plaques not specific to Alzheimer's disease.

To clarify the relationship between amyloid formation and amyloid precursor protein (APP), the brain sections from eight patients with Alzheimer's disease (AD) and four with Gerstmann-Sträussler Syndrome (GSS) were investigated immunohistochemically by the double-immunostaining method. In AD, most APP-positive senile plaques belong to classical plaques or primitive plaques, whereas in diffuse plaques, APP-positive neuritic components are rarely observed. The authors documented that anti-APP-labeled degenerative neurites surrounding kuru plaques in all four GSS patients. These kuru plaques were verified by double immunostaining using anti-prion protein and anti-APP. The APP-positive structures in kuru plaques were almost identical with those seen in the classical plaques in AD. The authors concluded that APP-positive degenerative neurites are not an early event in the amyloid formation of senile plaques. It is therefore postulated that depositions of beta/A4 and prion proteins are primary events that may involve the surrounding microenvironment and result in the secondary formation of APP-positive degenerative neurites, not specific to AD.

A case of erythropoietic protoporphyria with severe liver dysfunction suggesting a close relationship between erythrocyte protoporphyrin levels and those of gamma-GTP.

A case of erythropoietic protoporphyria (EPP) with severe acute abdominal pain and jaundice was reported. Erythrocyte protoporphyrin (PP) levels were constantly high, and liver histology showed a slight fibrosis with inflammatory infiltration. During the investigation period of 18 months, erythrocyte PP levels closely paralleled those of serum gamma-GTP.

The synergistic effect of chlorinated chemicals (trichlorosalicylanilid, 4-chloro-m-cresol, trichlorophenoxyacetic acid, trichloroethanol, trichloromethiazide, trichlorofon and trichloroacetaldehyde) and low concentrations of griseofulvin on porphyrin metabolism.

The synergistic effect of chlorinated chemicals and 0.1% griseofulvin (GF) on porphyrin metabolism was investigated. Drinking water containing 0.03% trichlorosalicylanilid, 0.1% 4-chloro-m-cresol, 0.1% trichlorophenoxyacetic acid, 0.3% trichloroethanol, 0.1% trichloromethiazide, 0.3% trichlorofon and 0.1% trichloroacetaldehyde was given separately to dd-y strain mice. Each group was divided into two subgroups, one treated with feed containing 0.1% GF and the other treated with normal feed. The treatments were continued for 45 to 165 days, after which porphyrins in the erythrocytes, feces and liver were analyzed by a chromatographic method. In the 0.03% trichlorosalicylanilid and 0.1% GF group, 0.1% 4-chloro-m-cresol and 0.1% GF group, 0.1% trichlorophenoxyacetic acid and 0.1% GF group and 0.3% trichloroethanol and 0.1% GF group, a slight elevation of fecal coproporphyrin and protoporphyrin was seen. There was no elevation of hepatic and erythrocytic porphyrins. This result shows that the chemicals used in this study did not have a potential for porphyria, but that they are capable of inducing slight porphyrin abnormalities in a synergistic reaction with 0.1% GF.

In vitro analysis of polychlorinated biphenyls (PCBs) and 2,3,4,7,8-pentachlorodibenzofuran (PCDF) cellular toxicity in PLC/PRF/5 cell proliferation--the effect of ursodeoxycholic acid, inchin-gorei-san and shou-saiko-to on cell toxicity.

We investigated the cell toxicity of polychlorinated biphenyls (PCBs) and 2,3,4,7,8-pentachlorodibenzofuran (PCDF) as an indicator of the quantity of m-RNA, which synthesizes the HBV core antigen region, and secreting protein, which is an HBV surface antigen in PLC/PRF/5 cells. The determination of m-RNA was conducted according to the methods of reverse transcriptase and polymerase chain reaction. Furthermore, the reductive action of ursodeoxycholic acid, shou-saiko-to and inchin-gorei-san on the PCBs and PCDF toxicity was investigated. The cell number of 1 x 10(5)/ml and concentrations of 200 micrograms/ml for PCBs and 500 microM for PCDF were used in these experiments. The titer of HBsAg was gradually increased from 1 in 1 x 10(3)/ml of cell numbers to 100 in 1 x 10(6)/ml of cell numbers. The curve became a plateau in 1 x 10(6)/ml of cell numbers. The cell number of 1 x 10(5)/ml was used in the experiments. The titer of HBsAg decreased following in the increase of concentration of PCBs. The HBsAg, even in the PCBs concentration of 1000 micrograms/ml showed a titer of 22.5%. However, the highest concentration of PCDF in this study, that is, 500 microM of PCDF, did not show any decrease of HBsAg activity. The concentrations of 200 micrograms/ml for PCBs and 500 microM for PCDF were used in the investigation of drug effects. A high titer HBsAg was observed in high concentrations of shou-saiko-to in comparison with a control group. Ursodeoxycholic acid and inchin-gorei-san exhibited a similar tendency.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparative study on the concentrations of polychlorinated biphenyls (PCBs) and polychlorinated quaterphenyls (PCQs) in the blood and hair of "Yusho" patients and inhabitants of Nagasaki Prefecture.

The relationship between polychlorinated biphenyl (PCB) and polychlorinated quaterphenyl (PCQ) concentrations in the blood and hair was investigated. The materials in this study consisted of 49 blood samples and hair obtained from 27 patients with PCB poisoning (Yusho) and 22 normal controls. The alkaline decomposition method described in the official standard analytical methods for the isolation of PCB and PCQ fractions was used. In the blood of the control group, the mean concentration of PCBs was 2.25 ppb, while the concentration of PCQs was too low to be detected by our analytical method. On the other hand, the mean concentration of PCBs in the Yusho group was about 2.8 times higher than that in the control group. The mean concentration of PCQs in the blood was 0.61 ppb in the Yusho group, but PCQs were not detectable in the control group. In the hair, the mean concentration of PCBs was 25.85 ppb and 9.41 ppb in the Yusho group and control group respectively. The mean concentration of PCQs in the hair of Yusho patients was 0.44 ppb, but PCQs were not detected in the control group. The PCB level in the hair was higher than that in the blood, but the PCQ level in the hair was lower than that in the blood. These results suggest a difference of excretory system among polyhalogenated compounds such as PCBs or PCQs. We consider that PCBs display a greater affinity than PCQs for the pilosebaceous system of the human skin.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased senile plaques without microglia in Alzheimer's disease.

To clarify the association of microglia with senile plaques, the brains from 13 patients with Alzheimer's disease (AD) and 23 nondemented aged controls were investigated immunohistochemically by a double-labeling method using anti-beta-protein antiserum and anti-ferritin antibody, which is a recently reported microglia marker. In addition, a quantitative analysis was performed. The senile plaques which appeared initially in the nondemented aged controls consisted of a diffuse type without any amyloid cores and these were found in the group aged 50-59 years. The great majority of them were found to contain no ferritin-positive microglia. The number and proportion (percentage) of microglia-containing diffuse plaques increased with age. Classical and compact plaques began to appear in the brains of the group aged 70 years and over, and practically all of them contained microglia. These results suggest that microglia are not associated with initial plaque formation, but correlate with amyloid core formation. In AD, the most prominent feature was that the diffuse plaques, which contained either no or only a few ferritin-positive microglia, increased markedly.

A comparative study on polychlorinated biphenyls (PCB) and polychlorinated quaterphenyls (PCQ) concentrations in subcutaneous fat tissue, blood and hair of patients with yusho and normal control in Nagasaki prefecture.

The relationship between PCB and PCQ concentrations in the blood, subcutaneous fat tissue and hair was investigated in this study. PCB and PCQ concentrations in twenty four patients with PCB poisoning (yusho) and 59 normal controls were analyzed. The alkaline decomposition method described in the official standard analytical methods for the isolation of PCB and PCQ fractions was used. The mean value of PCB concentrations was 2.43 ppb, CB% ratio was 0.69 and the PCQ concentration in the blood of the control group was too low to be detected by our analysis. On the other hand, the PCB concentration and CB% ratio in the yusho group were two times higher than those in the control group. The mean value of PCQ concentration was 1.34 ppb in the yusho group although it was below the level of detection in the control group. The mean PCB concentration in the hair of patients with yusho was 28.92 ppb, and 8.06 ppb in the control group. CB% ratio in the hair of patients with yusho was two times higher than that in the control group. The mean value of PCQ concentration in the hair of patients with yusho was 0.55 ppb although it was not detected in the control group. The PCB and PCQ concentrations in the hair were not greatly elevated when compared with those in the subcutaneous fat tissue. However, the hair is an excellent sample for the detection of these chemicals because it can be collected simply without operation.