Induced Pluripotent Stem Cell-Derived Hematopoietic Embryoid Bodies Improve Mouse Status in Septic Peritonitis.

We examined the efficacy of embryoid bodies from 6-day induced pluripotent stem cells an in vivo sepsis model. Injection of embryoid bodies to septic mice improved the condition of their lungs and significantly increased their survival rate. Although embryoid bodies secretedsphingosine-1-phosphate in vitro, its serum levels in mouse plasma were significantly reduced compared to that in the control (untreated mice receiving PBS). Low concentrations of sphingosine-1-phosphate protected endothelial cells, while high concentrations disrupted endothelial barrier integrity. Therefore, exogenous sphingosine-1-phosphate secreted by embryoid bodies during early stage of sepsis might down regulate endogenous production of sphingosine-1-phosphate. Inhibition of excessive sphingosine-1-phosphate release protects against endothelial injury and suppresses a vicious cycle of inflammatory reactions. The obtained results open new prospects in induced pluripotent stem cells-based therapy for sepsis.

Induced Pluripotent Stem Cell-Derived Hematopoietic Embryoid Bodies Secrete Sphingosine-1-Phosphate and Revert Endothelial Injury.

The possibility of sphingosine-1-phosphate production by induced pluripotent stem cells is examined to assess their potential in treatment of sepsis. The hematopoietic embryoid bodies were derived from the culture of 6-day-old differentiated induced pluripotent stem cells. These embryoid bodies secreted sphingosine-1-phosphate, an important bioactive lipid that regulates integrity of the pulmonary endothelial barrier, prevents elevation of its permeability, and impedes the formation of stress fibers in human endotheliocytes derived from umbilical vein. The data attest to potentiality of induced pluripotent stem cells in treatment of sepsis.

Association of novel promoter single nucleotide polymorphisms in vasopressin V1a receptor gene with essential hypertension in nonobese Japanese.

We studied the association between four novel single nucleotide polymorphisms (SNPs) in the promoter region of V1aR gene and essential hypertension in 620 Japanese subjects (365 hypertensives and 255 healthy). A significant association was found between one of the genotypes and alleles at SNP -6951 and hypertension in a subsample of nonobese individuals. This association demonstrated an independent risk for nonobese hypertension.

IgM secretion and absorption in the materno-fetal interface of a viviparous teleost, Neoditrema ransonneti (Perciformes; Embiotocidae).

We investigated maternal IgM secretion in the ovary and the absorption of IgM by fetuses in a viviparous fish, Neoditrema ransonneti (Embiotocidae). Serum IgM, whose molecular weight was approx. 820k, was purified by two steps of gel filtration chromatography. Immunohistochemistry and Western blotting revealed that IgM was secreted from the epithelia of the ovigerous lamellae of pregnant females into ovarian cavity fluid. The IgM-secreting activity of ovigerous folds showed notable changes according to the reproductive stage. In fetuses, IgM was absorbed as macromolecules by enterocytes of the hypertrophied hindgut. IgM in the fetal blood was also demonstrated, although its concentration remained low during gestation. These findings suggest that IgM was transported from the maternal tissues to embryos via a unique pathway in N. ransonneti.

Elevation of both cyclooxygenase-2 and prostaglandin E2 receptor EP3 expressions in rat placenta after uterine artery ischemia-reperfusion.

Intrauterine growth restriction (IUGR) has a multifactorial pathogenesis and is an important cause of perinatal mortality. The relationship between fetal weight and placental blood flow in an animal model of IUGR has been investigated, showing that fetal growth is regulated by placental blood flow. The aim of the present study was to determine whether ischemia-reperfusion (I/R) injury stimulates the prostaglandin E2 (PGE2) system or the vascular endothelial growth factor (VEGF) system in the placenta of a rat IUGR model. COX-2 is reported to be involved in ischemic damage in many organs. There are 4 types of PGE2 receptor (EP1, EP2, EP3 and EP4). It is well known that EP1 and EP3 is associated with vasoconstriction. In the present study, vessels were occluded in the right uterine horn on day 17 of pregnancy in rats, and the clamps were removed after 30 min of ischemia. At 24h, 48 h, and 5 days after I/R injury, the live fetuses and placentas were obtained by cesarean section. This study revealed that I/R injury caused IUGR 5 days after the treatment. COX-2 expression and EP3 receptor expression were significantly elevated at 24h after I/R injury, but VEGF mRNA expression was not altered in the placenta from the ischemic horn compared with the non-ischemic horn. These results suggested that induction of the COX-2-EP3 system in the placenta may be one of the causes of IUGR induced by uterine ischemia, because the EP3 receptor and PGE2 are well known to mediate vasoconstriction in many organs.

New monoclonal antibody, 1C5, reactive with human cervical adenocarcinoma of the uterus, with immunodiagnostic potential.

A murine monoclonal antibody, 1C5, was produced by fusion of spleen cells obtained from mice immunized with CAC-1, a human cell line of adenocarcinoma derived from uterine cervix, and NS/1 myeloma cells. 1C5 can be used for the staining of routine formalin-fixed and paraffin-embedded tissue sections. 1C5-defined antigen was found to have a molecular weight of 26,000. The 1C5-defined antigen was resistant to neuraminidase and trypsin treatment, but sensitive to periodate treatment, indicating that an epitope of the 1C5-defined antigen is a carbohydrate moiety. Immunohistochemical study using immunoperoxidase staining demonstrated that 1C5 reacted with 87% of adenocarcinomas of the uterine cervix, 39% of endometrial carcinomas of the uterus, 100% of ovarian mucinous cystadenocarcinomas, 43% of ovarian serous cystadenocarcinomas, 45% of adenocarcinomas of the colon, and 40% of gastric adenocarcinomas, thus showing the broad reactivity to adenocarcinoma cells of various origins. However, 1C5 did not show any reactivity to ectocervix epithelium, cervical intraepithelial neoplasia, or squamous cell carcinoma of the uterine cervix. In addition, adenocarcinoma of the uterine cervix exhibited strong cytoplasmic reactivity with 1C5, whereas endometrial carcinoma of the uterus showed the luminal reactivity. 1C5 also reacts with 95% ethanol-fixed malignant cells in cervical smears.

Clinical implications of K-ras mutations in malignant epithelial tumors of the endometrium.

Tumorigenesis in humans and experimental animals appears to involve the activation of ras protooncogenes for a number of organ systems and seems to be important to the development of the metastatic phenotype in several model systems. Clinically, the presence of activated ras protooncogenes has been reported to be a negative prognostic factor in the myelodysplastic syndrome and in adenocarcinoma of the lung. In the present study we examined 49 cases of endometrial carcinoma for mutations in the first exon of K-ras using the polymerase chain reaction and direct sequencing. Mutations in codon 12 or 13 of K-ras were detected in 6 of 49 cases (12.2%). These six cases consisted of five endometrioid endometrial carcinomas, each of which had a mutation in codon 12, and one case of clear cell carcinoma, which had a mutation in codon 13. In our study the presence of mutations in K-ras appeared to be an unfavorable prognostic factor. Three of six patients with the mutation died during follow-up, while only 7% of the 43 patients without K-ras mutations expired during this same period. In multivariate analysis using the Cox proportional hazard model, K-ras activation appeared to be an independent risk factor when compared with clinical stage, depth of myometrial invasion, and patient age. Thus, our findings support the hypothesis that K-ras protooncogene activation plays an important role in determining the aggressiveness of endometrial carcinoma.

Fine-scale deletion mapping of the distal long arm of chromosome 6 in 70 human ovarian cancers.

To define a small region on chromosome 6q containing a putative tumor suppressor gene for ovarian cancer, we examined loss of heterozygosity in 70 ovarian tumors of three histological types with nine restriction fragment length polymorphism markers located at 6q24-27. Among 33 cancers of serous type that were informative at one or more loci, 17 showed allelic loss at a few or all loci examined, whereas only 1 of 15 mucinous-type tumors and 2 of 12 clear-cell tumors revealed loss of heterozygosity. This result supported our earlier suggestion that alteration of a gene on chromosome 6q may play an important role during development of serous ovarian tumors (Sato et al., Cancer Res., 51: 5118-5122, 1991). Frequent losses were observed between loci defined by CI6-119 (D6S195) at 6q26 and CI6-49 (D6S161) at 6q27. A detailed deletion map indicated a commonly deleted region between loci defined by CI6-111 (D6S193) and CI6-24 (D6S149); these two markers are estimated to be 1.9 cM apart on the basis of linkage analysis. Our results further define a region containing a tumor suppressor gene involved in ovarian carcinoma within an approximately 2-megabase-long segment of chromosome 6q.

Correlation between expression of the matrix metalloproteinase-1 gene in ovarian cancers and an insertion/deletion polymorphism in its promoter region.

Matrix metalloproteinases (MMPs), a family of closely related enzymes that degrade the extracellular matrix, are likely to be involved in invasion and metastasis of tumor cells. A guanine (G) insertion/deletion polymorphism within the promoter region of MMP-1 influences the transcription of this gene; i.e., the 2G (insertion-type) promoter possesses greater transcriptional activity than the 1G (deletion-type) promoter. To investigate whether this feature contributes to cancer development and/or progression, we genotyped 163 ovarian cancer patients for the polymorphism and then analyzed levels of expression of the MMP-1 gene in their tumors. The proportion of patients who were either heterozygotes or homozygotes for the 2G allele was significantly higher than that observed among 150 individuals without cancer (P = 0.028). Moreover, the levels of MMP-1 expression in cancer tissues among the patients carrying 2G alleles were elevated significantly in comparison with 1G homozygotes (P = 0.0038). By stimulating degradation of extracellular matrix, an excess of MMP-1 production may enhance development and/or rapid progression of ovarian cancers.

Detailed deletion mapping of chromosome 17q in ovarian and breast cancers: 2-cM region on 17q21.3 often and commonly deleted in tumors.

Using 11 restriction fragment length polymorphism markers, we examined loss of heterozygosity on the long arm of chromosome 17, where one or more genes responsible for hereditary breast and ovarian cancers may be present, in sporadic forms of 94 ovarian and 246 breast cancers. Loss of heterozygosity was observed in 33 of 84 (39.3%) ovarian and in 88 of 214 (41.1%) breast cancers that were informative with at least one marker. Detailed deletion mapping of chromosome 17q in these cancers identified two distinct, commonly deleted regions. One was located between 17q12 and 17q21.3 and the other between 17q25.1 and 17q25.3. In breast cancers, the proximal commonly deleted region was between two loci defined by markers CI17-701 and CI17-730 at 17q21.3, which are 2.4 cM apart. This segment overlaps the region that includes the putative gene for hereditary breast and ovarian carcinomas. The results suggest that at least two tumor suppressor genes associated with sporadic ovarian and breast cancers are present on chromosome 17q and that one of them may be the same gene that is responsible for the hereditary form.

Definition of a commonly deleted region in ovarian cancers to a 300-kb segment of chromosome 6q27.

Allelic deletions of chromosome 6q that occur frequently in ovarian cancers imply the presence of a putative tumor suppressor gene in this chromosomal vicinity. We analyzed DNA from 32 patients with ovarian carcinomas for loss of heterozygosity at loci on the distal portion of chromosome 6q and constructed a detailed deletion map. The map indicated a commonly deleted region between loci D6S149 (defined by CI6-24) and A2, which are estimated to be 300 kb apart on the basis of our cosmid contig map. By means of exon trapping, we found that the human AF-6 gene, which is disrupted in acute myeloid leukemia cells that carry a (6;11)(q27;q23) translocation, is located within the commonly deleted region. Subsequent screening of the AF-6 gene in ovarian carcinomas revealed no mutations. However, our mapping results, which narrowed the region containing the putative tumor suppressor gene to a 300-kb segment of 6q27, will facilitate further efforts to identify a gene associated with ovarian cancer.

Ethanol attenuates vasorelaxation via inhibition of inducible nitric oxide synthase in rat artery exposed to interleukin-1ß.

Nitric oxide produced by inducible nitric oxide synthase (iNOS) regulates sepsis-induced hypotension. During septic shock, interleukin (IL)-1ß is synthesized in endothelial cells and smooth muscle cells by endotoxin. Ethanol (EtOH) suppresses endotoxin-induced hypotension. The present study aimed to elucidate the effect of EtOH on gradual relaxation and iNOS expression induced by IL-1ß in isolated rat superior mesenteric arteries (SMAs). Exposure to IL-1ß-induced contraction in SMA rings, followed by a gradual relaxation of phenylephrine precontracted tone. Contraction was abolished by indomethacin (IM), cycloheximide (Chx), and endothelium denudation. In contrast, the gradual relaxation was abolished by NOS inhibitors, Chx, endothelium denudation, and inhibited by EtOH (50 and 100 mM). However, IM had no effect on relaxation. Western blot analysis demonstrated that iNOS expression was induced by IL-1ß and was inhibited by EtOH and endothelium denudation. Furthermore, messenger RNA expression of iNOS, but not endothelial NOS, was inhibited by EtOH. These data suggest that IL-1ß-induced contraction is mediated by thromboxane A2, whereas IL-1ß-induced relaxation occurs via NO derived from iNOS. The endothelium plays an important role in vasorelaxation. Taken together, EtOH inhibits IL-1ß-mediated vasorelaxation by suppressing endothelium iNOS expression. This study provides the first evidence of EtOH -induced inhibition of IL-1ß-mediated vasorelaxation.

Phospholipase D activity of human amnion cells stimulated with phorbol ester and bradykinin.

We investigated the activity of phospholipase D (PLD) in human amnion cells labeled with [3H]oleate. The PLD activity was detected as signal-induced synthesis of phosphatidic acid (PA) and in the presence of ethanol, phosphatidylethanol (PEt). The PLD was shown to be activated by phorbol, 12-myristate, 13-acetate (PMA), calcium ionophore A23187, oxytocin, bombesin and bradykinin, but not by platelet-activating factor (PAF) and epidermal growth factor (EGF). The amniotic PLD thus appeared to be activated by a variety of agonists but with a certain specificity to stimulators. We examined the mode of the PLD activation using PMA (20 nM) and bradykinin (1 microM) as model stimulators. PMA and bradykinin elicited a rapid and sustained response with the peaks of PA-labeling attained at 5 and < 1 min after stimulation, respectively. In both cases, there was a concomitant rise of diacylglycerol (DG), and the PA accumulation was suppressed by ethanol at the expense of labeling of PEt. The PA synthesis caused by the two stimulators was similarly inhibited by staurosporine and by a chronic treatment with PMA (100 nM for 24 h), suggesting that the activation of PLD is linked to the action of protein kinase C. With the cells labeled with radioactive choline and ethanolamine, we found that the amniotic PLD hydrolyzed almost equally phosphatidylcholine and phosphatidylethanolamine. Although bradykinin and PMA stimulated cellular PLD to a comparable extent, prostaglandin (PG)E2 release was not stimulated by bradykinin in contrast to the marked effect by PMA. Further work is thus needed to clarify the significance of the novel PLD signaling pathway in the function of amnion cells.

Lipid peroxidation in the rat brain after CO inhalation is temperature dependent.

We reported previously that 7-hydroperoxycholesterols, 7 alpha- and 7 beta-hydroperoxycholest-5-en-3 beta-ol (7 alpha-OOH and 7 beta-OOH), indicated lipid peroxidation. In the present study, we measured not only 7-hydroperoxycholesterols but also oxysterols (7 alpha- and 7 beta-hydroxycholesterol, 7 alpha-OH, and 7 beta-OH) and 3 beta-hydroxycholest-5-en-7-one (7-keto) in the brains of rats that underwent either a sham operation (control), hypoxia, or CO inhalation (1005 ppm) at 37 degrees C for 90 min followed by 48 h of recovery. The levels of 7-hydroperoxycholesterols, 7 beta-OH, and 7-keto were low in the hypoxia group, while the levels were unaltered in the CO group compared with the controls. Among the three groups of CO inhalation, these levels were high in the hyperthermia group (39 degrees C), and the 7-hydroperoxycholesterols were low in the hypothermia group (32 degrees C), compared with the control group. The blood O(2) saturation was almost normal in the hypothermia group, while it was similarly low in the hyperthermia and normothermia groups. The temperature-dependent lipid peroxidation in the brain after CO inhalation and recovery can not be explained by hypoxia due to CO-hemoglobin formation, but may contribute to the delayed neuronal death following CO inhalation. Hypothermia may be applicable to treat patients after CO inhalation.

Activities of antioxidant enzymes and lipid peroxidation in endometrial cancer.

Antioxidant enzyme activities and lipid peroxidation were analysed in normal endometrium and endometrial cancer tissues from Finnish and Japanese patients. The catalase and glutathione peroxidase activities of normal endometrium were significantly lower in Finns than in Japanese. Lipid peroxidation was slightly higher in endometrial cancer as compared with normal endometrium both in the Finns and in the Japanese. When cancer tissues were compared with normal endometrium both in Finns and Japanese the activity of superoxide dismutase was significantly lower in cancer tissue than in normal endometrium. In Finns glutathione S-transferase activity was also lower in endometrial cancer tissue than in normal endometrium, and a similar tendency was also found in Japanese. This study suggests that endometrial cancer tissue is associated with an impaired enzymic antioxidant defence system.

Serum lipids in Finnish and Japanese postmenopausal women.

Serum lipids were measured in 30 Finnish and Japanese postmenopausal women. Total cholesterol, HDL cholesterol, LDL cholesterol, apo B and the HDL cholesterol/apo A1 ratio were higher in Finnish than in Japanese women. The LDL cholesterol/apo B and apo A1/apo B ratios were lower in Finns than in Japanese. In serum phospholipids the percentage of arachidonic acid was higher and the percentage of the n - 3 fatty acids, eicosapentaenoic acid and docosahexaenoic acid, and the eicosapentaenoic acid/arachidonic acid ratio were lower in Finnish than in Japanese women. No significant correlations were found between serum oestrone levels and lipid parameters.

Cloning and characterization of a cDNA fragment coding beta-casein-like protein preferentially expressed in cervical adenocarcinoma cell line CAC-1.

We generated new murine monoclonal antibodies against bovine beta-casein. One of eight new monoclonal antibodies generated, 12G2, specifically reacted not only with beta-casein, but also with human cervical adenocarcinoma cell line CAC-1. To identify the gene encoding the putative protein (BCLP) defined by monoclonal antibody (mAb) 12G2, a complementary DNA library was constructed. We obtained the inserted cDNA fragment that showed high homology to reported DNA sequences of unknown function in normal human placenta. We think that BCLP was overexpressed or changed its structure by carcinogenesis.

Demonstration of selective protein complexes of p53 with 73 kDa heat shock cognate protein, but not with 72 kDa heat shock protein in human tumor cells.

It has been demonstrated that p53, especially, mutant p53 (mp53), makes protein complexes with major heat shock proteins hsp72/hsc73. However, there is no direct evidence showing whether hsp72 or hsc73 could bind preferentially to p53. In the present study, using TYKnu human ovarial carcinoma cells and monoclonal antibodies reacting specifically to hsp72/hsc73, we were able to find the selective protein complex formation with p53, presumably mp53, and hsc73, but not in the case of p53 and hsp72. The p53-hsc73 protein complexes dissociate with the addition of ATP, indicating that the dissociation is dependent upon the ATP-hydrolysis. These data suggest that hsc73 rather than hsp72 plays an important role in the yet undefined mechanism of disregulated cell growth control by mp53.

Telomerase activity in malignant ovarian tumors with deregulation of cell cycle regulatory proteins.

Using a semiquantitative telomeric repeat amplification protocol assay, telomerase-positive frequencies and enzyme levels were measured. Out of 95% of 49 human ovarian tumors, the highest level of telomerase activity was observed in malignant tumors. Furthermore, by immunohistochemical staining of cell cycle regulatory proteins (pRB, p16, cyclin D1, cyclin E and p53) at the G1 checkpoint, we evaluated the relation between each protein alterations and the levels of telomerase activity. We could not demonstrate a clear relation with each molecule except for cyclin E, but suggesting that aberrant accumulation of these proteins was considered as a reason for telomerase deregulation, which may play an essential role in the pathway of telomerase regulation.

Gonadotropin-releasing hormone agonist has the ability to induce increased matrix metalloproteinase (MMP)-2 and membrane type 1-MMP expression in corpora lutea, and structural luteolysis in rats.

Gonadotropin-releasing hormone (GnRH) and its agonist analog (GnRHa) are well known to have luteolytic effects. We previously reported that prolactin (PRL) stimulated matrix metalloproteinase (MMP)-2 activity to degrade collagen type IV as a mechanism of structural luteolysis. The effects of GnRHa treatment on developed corpora lutea are unknown. In this study we assessed the effect of GnRH on MMP expression and induction of structural involution of developed corpora lutea of superovulated rats using GnRHa. Pregnant mare serum gonadotropin-human chorionic gonadotropin (hCG)-synchronized ovulation and luteinization were induced in immature female rats, followed by daily treatment with GnRHa from 5 days after hCG treatment. GnRHa-induced involution of corpora lutea was evident 3 days after the treatment, as shown by their markedly smaller size (60% of the control weight). Nine days after hCG injection, serum progesterone and 20alpha-dihydroprogesterone concentrations were as low as those associated with structural luteolysis. These findings revealed that GnRHa has the ability to induce structural luteolysis in superovulated rats in the same way that PRL does. To gain information on mechanisms of luteal involution induced by GnRHa, we performed gelatin zymography. This showed a significant increase in the active form of MMP-2 in the luteal extract of GnRHa-treated rats (more than twofold that of the control). Activation of pro-MMP-2 by membrane type-MMP (MT-MMP) is reported to be a rate-limiting step for catalytic function. Another function of MT-MMP is to degrade collagen types I and III. The plasma membrane fraction of corpora lutea of GnRHa-treated rats activated pro-MMP-2 of fetal calf serum, resulting in a marked shift of the 68-kDa band to the 62-kDa band in the zymogram. A Northern hybridization study also revealed simultaneous significant increases in expression of MMP-2 mRNA and MT1-MMP mRNA in corpora lutea of GnRHa-treated rats (more than threefold the control level). In summary, hormonal and histological features of corpora lutea of GnRHa-treated superovulated rats correspond to those of structural luteolysis. GnRHa stimulated the expression of MMP-2 and MT1-MMP in developed corpora lutea associated with involution. These findings support the conclusion that MMP-2, activated by MT1-MMP, and MT1-MMP itself, remodel the extracellular matrix during structural luteolysis induced by GnRHa.