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Regulation of N-myc oncogene expression is an important determinant of the biological behavior of neuroblastoma. The N-myc promoter contains several potential binding sites for transcription factors of the Sp1 family. Mutation of a CT-box motif contained within a 26bp region required for N-myc downregulation by retinoic acid decreased basal transcriptional activity and altered DNA–protein interactions of the promoter, while mutations flanking this motif did neither. On super-shift, this region was shown to recruit Sp1 and Sp3 transcription factor proteins, while a functionally significant CT-box mutation resulted in their replacement by NF-1 transcription factor. Lysates from Drosophila S2 cells expressing exogenous Sp1, Sp3, and NF-1 proteins were able to partially mimic gel shift complexes seen with neuroblastoma nuclear extract and either wild type or mutant probes. Transient transfections of S2 cells showed that both individually and together, Sp1 and Sp3 were able to trans-activate a wild type CT-box-driven luciferase reporter construct in a dose-dependent manner. Transfection of the wild type but not mutant CT-box oligonucleotide was able to decrease endogenous N-myc expression in neuroblastoma cells. Together these results suggest that the CT-box element serves a critically functional role, and in the basal state, allows for N-myc trans-activation by Sp1 and Sp3. Moreover when mutated, the CT-box may still function as a binding motif for alternate transcription factors such as NF-1 that can allow persistent N-myc expression.

Sustained CR of refractory aggressive natural killer cell leukemia following unrelated cord blood transplantation

The relationship between insulin-like growth factor I (IGF-I), a hormone which has potent metabolic effects and stimulates protein synthesis, and prolactin and oestradiol was examined to investigate a possible mechanism for the luteal cell hypertrophy that is responsible for the increase in size of the corpus luteum. A luteal cell line (GG-CL) derived from large luteal cells of the pregnant rat corpus luteum was used. IGF-I, IGF-I receptor and oestrogen receptor \g=b\mRNA contents were determined by semiquantitative RT\p=n-\PCR.The results revealed that prolactin upregulates the expression of IGF-I mRNA in luteal cells, but not that of its receptor. IGF-I had no effect on the expression of its receptor but caused a dose-related increase in the expression of oestrogen receptor \g=b\.Furthermore, whereas IGF-I upregulated oestrogen receptor \g=b\ expression, oestradiol downregulated expression of mRNA for both IGF-I and its receptor. This effect of oestradiol is not mediated through progesterone which is stimulated by oestradiol in the corpus luteum. The developmental studies indicate that mRNA for IGF-I and its receptor are not expressed in tandem throughout pregnancy. Whereas the receptor mRNA is expressed at higher concentrations in early pregnancy, that of its ligand is highly expressed close to parturition. Collectively, the results indicate that prolactin stimulates luteal IGF-I production, which in turn acts on the luteal cell to stimulate expression of oestrogen receptor \g=b\.Luteal cells with increased oestrogen receptor \g=b\can respond fully to oestradiol, leading to cell hypertrophy.

The corpus luteum expresses two enzymes that scavenge superoxide radicals and protect the cells from their toxic activities: cytosolic copper, zinc-superoxide dismutase (Cu,Zn-SOD) and mitochondrial manganese-SOD (Mn-SOD). The present study was undertaken to investigate whether the mRNA expression of each of these enzymes is regulated by luteotropic hormones. Cu,Zn-SOD and Mn-SOD mRNA levels were determined by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). We first examined the effects of prolactin (PRL) on Cu,Zn-SOD and Mn-SOD mRNA expression in the corpus luteum. Hypophysectomy of Day 3 pregnant rats caused a sharp decline in both Cu,Zn-SOD and Mn-SOD mRNA levels, which was completely reversed by PRL administration. To further examine the effects of PRL and rat placental lactogen (rPL) on the expression of these enzymes, either primary luteinized granulosa cells or temperature-sensitive simian virus-40 transformed luteal cells (GG-CL) were cultured with different doses of PRL or rPL. These hormones induced a remarkable increase in Cu,ZnSOD and Mn-SOD mRNA levels in both primary luteinized granulosa cells and GG-CL cells. Interestingly, whereas PRL up-regulated the expression of the SOD in luteal cells, other luteotropic hormones such as estradiol and dexamethasone inhibited both SOD mRNA expression while progesterone had no effect. In conclusion, PRL and PRL-like hormones induce a protective ability against toxic oxygen radicals by stimulating the expression of SODs, a phenomenon that may play an important role in maintaining luteal cell integrity and steroidogenic capacity.

Interleukin (IL)-6, a multifunctional cytokine originally described as a T cell-derived factor, is also produced by different cell types, and it influences a wide variety of physiological and pathophysiological processes. Recent studies further suggest that IL-6 has a role in down-regulating hormone production by endocrine organs and can negatively affect the steroidogenic capacity of both ovaries and testes. Thus, the aims of this investigation were to examine whether IL-6 plays a role in luteolysis and, more specifically, to determine whether luteal cells express the IL-6 gene, whether this expression is developmentally and hormonally regulated in pregnancy, and whether the corpus luteum could be a target for IL-6 action. Using semiquantitative RT-PCR, messenger RNA (mRNA) encoding both components of the IL-6 receptor [the ligand-binding subunit (IL-6 R) and the IL-6 R-associated signal transducer (gp130)] were found to be highly expressed in corpora lutea throughout pregnancy. In contrast, IL-6 mRNA expression was barely detectable from day 4 through the end of pregnancy, whereas a sharp and abrupt expression of IL-6 mRNA occurred immediately after parturition. Although the corpus luteum does not express IL-6 mRNA during most of pregnancy, it could be induced to express this gene with an in vivo injection of the bacterial endotoxin, lipopolysaccharide. In addition, when corpora lutea from day-15 pregnant rats were isolated and maintained in culture, IL-6 mRNA that was undetectable at 0 h increased in a time-related manner and reached significant levels after 4 h of incubation, followed by a similar increase in IL-6 protein secreted in the culture media. Isolation of the small and large luteal cells by elutriation indicated that both cell populations can secrete IL-6 in culture. The apparent ability of luteal cells to spontaneously express IL-6 in vitro, together with the lack of IL-6 expression during most of pregnancy, led us to examine whether the IL-6 gene is silenced throughout pregnancy by luteotropic hormones. Corpora lutea from day-15 pregnant rats were cultured in the presence of different doses of progesterone; the synthetic glucocorticoid, dexamethasone; 17beta-estradiol; and PRL. Progesterone and dexamethasone markedly inhibited IL-6 mRNA expression, whereas 17beta-estradiol had a minimal inhibitory effect, and PRL did not affect IL-6 mRNA expression. In summary, results of this investigation have revealed that the rat corpus luteum expresses the IL-6 receptor system and that luteal cells are able to secrete IL-6. However, IL-6 gene expression is silenced during most of pregnancy, probably by the high levels of progesterone locally produced in the corpus luteum. The salient finding that progesterone and glucocorticoid strongly inhibit the expression of IL-6 in the corpus luteum suggests that one important luteotropic role of progesterone and glucocorticoids could be to prevent the expression of IL-6, which might have a deleterious effect on luteal function.

This study was undertaken to investigate the regulation of mitochondrial manganese superoxide dismutase (Mn-SOD) and cytosolic copper-zinc SOD (Cu,Zn-SOD) in the corpus luteum by inflammatory cytokines. We first examined the developmental expression of both SOD mRNAs in the rat corpus luteum throughout pregnancy. SOD mRNA levels were determined by semiquantitative reverse transcription-polymerase chain reaction. Whereas Cu,Zn-SOD mRNA levels decreased during late pregnancy, Mn-SOD mRNA levels remained elevated. We secondly examined the effects of inflammatory reaction on luteal SODs. Rats received injections of lipopolysaccharide (LPS; 5 mg, i.p.) on Day 15 of pregnancy, and corpora lutea were removed 2 h later. LPS caused an increase in Mn-SOD mRNA levels in the corpus luteum and a decrease in serum progesterone levels, but neither in levels of Cu,Zn-SOD mRNA. To further study the effects of LPS or LPS-induced cytokines, we incubated either whole corpora lutea obtained on Day 15 of pregnancy or a temperature-sensitive simian virus-40 transformed luteal cell line (GG-CL; derived from large luteal cells of the corpus luteum of pregnant rats) in serum-free medium with LPS, interleukin-1a (IL-1a), IL-b, IL-6, and tumor necrosis factor a. LPS and these cytokines induced a remarkable increase in Mn-SOD mRNA levels in both corpora lutea and GG-CL cells but had no effect on Cu,Zn-SOD mRNA expression. In conclusion, Cu,Zn-SOD and Mn-SOD mRNAs are differently expressed and regulated in the corpus luteum of pregnancy. Mn-SOD mRNA, but not Cu,ZnSOD mRNA, is highly induced by inflammatory cytokines and may play an important role in protecting luteal cells from inflammation-mediated oxidative damage.

Estradiol, together with PRL and placental lactogens, regulates steroidogenesis and cell hypertrophy in the rat corpus luteum of pregnancy. Although binding experiments have demonstrated the presence of estrogen-binding sites, no evidence exists as to whether the rat corpus luteum of pregnancy expresses the estrogen receptor (ER) genes. In this investigation, we have analyzed the expression of the two ER genes (ERα and ERβ) (by RT-PCR and in situ hybridization) in the rat corpus luteum, studied their developmental changes throughout pregnancy, and investigated the regulation of ERα and ERβ messenger RNA (mRNA) expression by PRL and placental lactogens. The RT-PCR studies showed that both ER mRNA species (ERα and ERβ) are coexpressed in the rat corpus luteum during pregnancy. Whereas ERα mRNA increased from early pregnancy, reached a maximum at midpregnancy, and had a remarkable decline before parturition; ERβ mRNA remained constant throughout pregnancy, with a significant decline at parturition. Examinati...

The primary culture of rat luteal cells and their long-term maintenance have been difficult. Low cellular yields have limited the possibility for the study of gene regulation in luteal cells. The goal of this study was to develop a cell line to serve as a model by which to study the expression and regulation of various genes specific to luteal cells. We attempted to develop a luteal cell line by transformation of large luteal cells through infection with a temperature-sensitive simian virus (SV-40 tsA209) mutant that has a temperature-sensitive mutation required for the maintenance of cell transformation. We report here the successful establishment of such a cell line, designated GG-CL cells. Large luteal cells were purified to homogeneity by flow cytometry from corpora lutea of day 14 pregnant rats, cultured for 24 h, and then infected with the SV-40 tsA209 mutant virus. Transformed cells were maintained at the permissive temperature (33 C) until colonies were identified. Several colonies of transformed ...

In this study, we investigated whether progesterone exerts an intraluteal action despite the lack of progesterone receptors (PR) in the rat corpus luteum and whether progesterone acts through the glucocorticoid receptor (GR) to enhance its own levels by down-regulating the expression of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD). We first established that the corpus luteum constitutively expresses the GR throughout pregnancy and after parturition. We also generated a temperature sensitive SV-40 transformed luteal cell line (GG-CL) that expresses the GR and 20alpha-HSD but lacks the PR. Treatment with different doses of either progesterone or dexamethasone caused a dose-related decrease in 20alpha-HSD mRNA in both cultured corpora lutea and in the luteal cell line. RU486, a PR/GR antagonist, completely blocked both the progesterone and the dexamethasone mediated inhibition of 20alpha-HSD expression in GG-CL cells. In summary, this report provides the first evidence that despite the absence of the PR in the rat corpus luteum, progesterone can act through the GR to down-regulate the expression of 20alpha-HSD, an enzyme that catabolizes progesterone and reduces progesterone secretion by the corpus luteum.

The deployment of Wi-Fi network has been growing rapidly due to its ease of implementation and affordability in prize. The increase in number of users and applications results in increment of multimedia traffic in Wi-Fi networks. There are lots of performance issues in Wi-Fi network with respect to Quality of Services (QoS), especially with voice and video applications. In this paper we propose the adaptive channel access mechanism to improve the performance of real time traffic under high traffic environment. The proposed adaptive channel access mechanism improves the performance of voice and video traffic by 15% to 20% in terms of packet delay and throughput under high traffic load environment.

Image registration is the process of mapping coordinates between one image and another, in order to achieve a correct correspondence. Medical image is one of research field that have benefited from image registration technique. Registration of pre-operative image dataset and intra-operative images owns great value of efficiency to analyze the differences between the images, especially, in the field of radiation surgery and neurosurgery. For this purpose, we studied the appropriate mapping between CT and ultrasound image, in order to observe the possibility to perform the image guide surgery. We applied Least Square Support Vector Machine which is a tool based on a statistical learning theory, as the key technique in mapping the different data, to minimize the mapping time in the registration process. The experimental results show that the LS-SVM based image registration method yields high accuracy with simplicity of practical application.

Background Scant evidence has been reported on the evaluation of quality-of-life (QOL) in patients who had undergone surgical treatment due to pelvic floor prolapse including cystocele. The aim of this study is to evaluate the impact of surgical intervention on patients' QOL before and after surgery. Methods Between 1997 and 2007, 135 patients (median age: 66.6 years) with pelvic floor prolapse including cystocele underwent bladder neck suspension with anterior/posterior colporrhaphy. The follow-up period was 39.6 months. Seventy-two patients (53 %) had urinary incontinence. The cystocele was graded as mild (grade 2), moderate (grade 3), and severe (grade 4) in 35, 60, and 40, respectively, according to the Baden-Walker classification. A urodynamic study was performed in 69 patients (51 %) who had obstructive symptoms with 100 ml or more of postvoid residual urine. Postoperative QOL was longitudinally assessed in 114 patients by scoring three disease-specific items (sensation of vaginal bulging, obstructive symptoms, urinary incontinence), and one overall health-related QOL (HR-QOL), and compared with corresponding baseline scores. Results A longitudinal study demonstrated that a significant improvement in these symptoms was sustained at a median follow-up of 62.2 months. HR-QOL was significantly associated with vitality assessed by SF 36 (p = 0.036). Multivariate analysis revealed that update urinary incontinence, pre-operative HR-QOL was independent prognostic factors for predicting postoperative patient's satisfaction. Conclusions Although surgical repair of pelvic floor prolapse can achieve acceptable results with intermediate-term durability as well as improving the QOL, preoperative patients' HR-QOL may be considered in the decision making process for treatment.

We report a case of uterine angiomyolipoma confirmed with molecular-genetic analysis by fluorescence in situ hybridization (FISH). A 25-year-old nulliparous woman visited Yamaguchi University Hospital with a complaint of lower abdominal pain. Magnetic resonance imaging demonstrated an ill-bordered uterine tumor and exploratory laparotomy revealed a myometrial elastic-soft tumor at the anterior wall of the uterine corpus. Histopathologically, the tumor consisted of fascicles of smooth muscle cells with intermingled adipocytes and small to medium-sized arterial blood vessels surrounded by epithelioid cells of clear cytoplasm. FISH examination revealed chromosome X trisomy, which was comparable to a previously reported molecular-genetic finding of PEComa family tumors including angiomyolipoma. Although the tumor was immunohistochemically negative for HMB-45 antigen, the histological and FISH findings were compatible with angiomyolipoma.

Cycle based architecture simulation environment framework dasiaMICSpsila is proposed. The framework proposes the abstracted interfaces for system components, so that a new simulation environment for a system can be built only by implementing additional components required in its architecture under Mics interface definition. Mics interface also allows to use existing simulation tools as system components. By use of this Mics environment, system designers may try various architectures even at the early phase of system development. And optimization methods for high-speed simulation are described. For an example system design issue, simulation environment for several architectures are built, and their simulating speed and overhead are evaluated.

Eight monoclonal antibodies directed against Squamous Cell Carcinoma Antigens (A1 and A2) were collected and evaluated by three working groups. Recombinant antigens, fusion proteins and native antigens from normal tissue were used to evaluate antibody specificity. Five antibodies reacted with both A1 and A2. Two of these antibodies (K123 and K131) showed related binding characteristics, whereas SCC140, K182 and SCC111 demonstrated unique epitope specificity and were not related to the reference antibodies included (F1H3, F2H7 and SCC107). SCC111 reacted particularly well with antigen on Western blot, indicating that the epitope was partly hidden when the antigen was in solution. Two antibodies (SCC103 and SCC109) reacted only with A2 and the fusion protein A1/A2, indicating that they recognized an A2 epitope in exon 8. The A2-specific antibodies are unique in their binding to A2 and are different from the reference antibodies included (SCC104 and K122). SCC103 is probably the best A2-specific antibody available. One antibody, K136, was A1-specific and is related to reference antibody K135. The new antibodies can be used to establish immunometric assays for specific measurement of A1, A2 or both A1 and A2 together.

We evaluated the usefulness of cefpodoxime proxetil (CPDX-PR) in the treatment of puerperal infection and obtained the following results. (i) The susceptibilities of 124 clinical isolates from 85 uterine lochia samples were determined. The MIC at which the growth of Streptococcus agalactiae, Escherichia coli, and Bacteroides fragilis isolates was inhibited by 90% (MIC90) was 0.39 micrograms/ml or less. The MIC90 for Staphylococcus aureus was 3.13 micrograms/ml. (ii) Seven puerperal women received 200 mg of CPDX-PR orally. The CPDX concentration in the lochias in the uterine cavity was not statistically different from that in the vagina, suggesting that the vaginal samples, which can be obtained more safely and aseptically, can be substituted for the uterine samples. The CPDX concentration in cubital venous blood reached a peak of 1.61 micrograms/ml at 3 hours after CPDX-PR administration. The CPDX concentration in the lochias gradually increased and reached a peak of 1.20 micrograms/ml in the uterine cavity and 1.27 micrograms/ml in the vagina at 5 h after drug administration and gradually declined thereafter. These results suggest that CPDX-PR, with its good transfer to the lochia and its potent antimicrobial activity, is a promising drug for the prophylactic and therapeutic treatment of puerperal infections caused by susceptible organisms.

A novel unified phase compiler framework for embedded VLIWs and DSPs is shown. In this compiler, a given program is represented in 3D representation space, which enables to estimate required resources and elapsed time quantatively. Transformation of 3D representation graph which corresponds to an optimization method for specific processor architecture is also proposed. The proposal compiler and the optimization methods are compared with ordinary compiler in terms of their generated codes, and hence they are proved to be effective.

Memory address allocation method for DSPs of indirect addressing with indexed auto-modification is proposed. At first, address auto-modification amounts for a given program are analyzed. And then address allocation of program variables are moved and shifted, so that both indexed and simple auto-modifications are effectively exploited. For further reduction in overhead codes, memory address allocation method coupled with computational reordering is proposed. The proposed methods are applied to the existing compiler, and generated codes prove their effectiveness.

In this article, a new design method of nonuniform filter banks with rational sampling factors is presented. In the method, supremum of absolute error between input and output is used as an error criteria. By minimizing this error criteria, the method gives the synthesis bank for a given analysis bank. By use of this method, strict filter specifications in other existing methods are no longer required. Furthermore, an iterative design method is extended, so that the whole filter bank system has better performance in the defined error criteria.

This paper discusses the nature of the paradigm shift that will be accelerated as the next-generation Internet (NGI) pervades society. First, we define the NGI society as one in which the network is so ubiquitous that no one will be conscious of connecting with it. Then we investigate trends and expectations of current NGI research and development projects worldwide. Secondly, we have performed comprehensive questionnaire-based market research into new technologies. This reveals that Japan can be expected to create global markets by developing NGI applications in home-electronic appliances, mobile assistants, transportation systems, and animation games. Furthermore, we generalize the discussion on the nature of the paradigm shift as follows. Although modern western rationalism produced the astonishing growth of the industrialized countries in the 19th-20th centuries, this approach alone no longer offers the prospect of much further advance. The 21st century paradigm for science and technology, for social, economic and governmental systems, and for industrial structures and working styles will necessarily shift towards autonomous and cooperative decentralization. The Internet is a decentralized computer system that is operated autonomously and cooperatively by individuals and small organizations. The NGI will further accelerate this paradigm shift. We conclude that this approach is essentially similar to the traditional oriental philosophy of harmony, and urge that it should be adopted in positioning Japan within the global, information-oriented network society of the 21st century.

We have evaluated the effectiveness of project based distance learning (PBDL) as a means of expanding the number of business persons who take post-graduate courses, particularly in entrepreneurial studies. We established that for learning based on entrepreneurial projects, the two-way distribution of text and still images via the IP network, and of audio-visual transmissions at 64 kbps speeds via low-cost ISDN or ADSL lines, are practical for communications between users. Again, the audio-visual information made a major contribution to the mutual trust between business persons and university people, who had had little or no previous contact with each other, which is necessary in carrying out such an entrepreneurial project. In entrepreneurial project based real time distance learning, the hybrid video conference system proved effective as a low cost network environment for the stable distribution of audio-visual information and documents. On the other hand, for non-real time distance learning, there is an urgent need to develop an expert exchange system that will make it possible to exchange information with experts possessing advanced entrepreneurial information and to access a database formed from this advanced information.

Urocortin is a member of the CRF neuropeptide family and has a 43% homology to CRF in amino acid sequence. Urocortin has been found to bind with high affinity to CRF receptors. CRF has been detected in the human ovary and has been demonstrated to suppress ovarian steroidogenesis in vitro. In this study we examined urocortin and CRF receptor expression in normal cycling human ovaries, using immunohistochemistry and RT-PCR. Normal cycling human ovaries were obtained at oophorectomy and hysterectomy from patients who underwent surgery for cervical cancer or myoma uteri. Intense urocortin immunoreactivity was detected in luteinized thecal cells of regressing corpora lutea, in which only luteinized thecal cells have the capacity for steroidogenesis. Immunoreactive urocortin was also detected in luteinized granulosa and thecal cells of functioning corpora lutea, in which both cell components are capable of producing steroids. RT-PCR analyses revealed that messenger ribonucleic acid levels for urocortin, CRF, and CRF receptor type 1 and type 2alpha were significantly higher in the regressing corpus luteum than in the functioning corpus luteum. The spatial and temporal immunolocalization patterns of CRF receptor were similar to those of urocortin. These results suggest that urocortin is locally synthesized in steroidogenic luteal cells and acts on them as an autocrine and/or paracrine regulator of ovarian steroidogenesis, especially during luteal regression.

Serum melatonin concentrations were studied in normal pregnant women and in women with several types of pathologic pregnancies, e.g., twins, preeclampsia or intrauterine growth retardation (IUGR). Blood samples were collected from the maternal antecubital vein at 14:00 hr (daytime) and 02:00 hr (nighttime) during pregnancy, and also from the umbilical vein and artery immediately after delivery. Serum melatonin concentrations were measured by radioimmunoassay. Daytime serum melatonin levels in normal (single fetus; singleton) pregnancies were low. While the levels showed an increasing tendency toward the end of pregnancy, no statistically significant changes occurred. On the other hand, the nighttime serum melatonin levels increased after 24 weeks of gestation, with significantly (P0.01) high levels after 32 weeks; these values decreased to non-pregnant levels on the 2nd day of puerperium. Nighttime serum melatonin levels were significantly (P0.05) higher in twin pregnancies after 28 weeks of gestation than in singleton pregnancies, whereas the patients with severe preeclampsia showed significantly (P0.05) lower serum melatonin levels than the mild preeclampsia or the normal pregnant women after 32 weeks of gestation. Melatonin concentrations in umbilical vessels showed a higher tendency in neonates who were born during at night compared with the other neonates; moreover, those in the umbilical artery were generally higher than those in the umbilical vein. The present results indicate that in humans, the maternal serum melatonin levels show a diurnal rhythm, which increases until the end of pregnancy, reflecting some pathologic states of the feto-placental unit. Fetuses may produce melatonin with a circadian rhythm.

The present study was undertaken to investigate the role of vascular endothelial growth factor (VEGF) in luteal angiogenesis and the regulation of VEGF in the corpus luteum (CL) during mid-pregnancy in rats. Protein concentrations and mRNA levels of VEGF in the CL significantly increased from Day 9 to Day 12 and remained at the same level as Day 12 until Day 15. To study whether estradiol is involved in VEGF expression between Day 12 and Day 15, rats undergoing hypophysectomy-hysterectomy on Day 12 were treated with estradiol until Day 15. Protein concentrations and mRNA levels of VEGF in the CL were significantly decreased by hypophysectomy-hysterectomy, and this inhibitory effect was completely reversed by estradiol treatment. Changes in vascular density in the CL were parallel to those in VEGF expression. To examine whether the effect of estradiol is mediated by VEGF, anti-VEGF antibody was administered to hypophysectomized-hysterectomized rats simultaneously with estradiol. The recovery in the vascular density, CL weight, and serum progesterone concentration caused by estradiol was significantly inhibited by the anti-VEGF antibody treatment. In conclusion, the present study has demonstrated that VEGF contributes to luteal angiogenesis, CL development, and progesterone production during mid-pregnancy in rats and that luteal VEGF expression is increased by estradiol.

Nitric oxide (NO) is known to be generated from L-arginine and may regulate glomerular filtration, tubular sodium reabsorption, and renin secretion. Impairment of renal function might influence NO production secondary to endothelial dysfunction, decreased NO synthesis and increased activity of arginine analogues inhibiting NO synthase. In this study, we evaluated the effect of L-arginine on the blood pressure and urinary sodium excretion in patients with chronic renal failure. A 300-ml dose of 10% L-arginine solution was administered intravenously over 30 min and blood pressure was monitored every 10 min under basal conditions and for 120 min after infusion. The patients were divided into two groups based on the reduction in mean blood pressure (dMBP) following infusion, namely non-responders (dMBP10 mmHg) and responders (dMBP10 mmHg). Urine and blood samples were collected to determine electrolytes, urinary NO2 + NO3 by the Griess method, urinary cGMP, plasma renin activity (PRA), and the plasma aldosterone concentration (PAC). L-arginine significantly decreased MBP in 8 patients and caused no significant change in 10 patients. Urinary sodium excretion and the NO2 + NO3 level were significantly increased following L-arginine infusion and the increment of fractional excretion of sodium was higher in responders. However, there were no significant changes in PRA, PAC, and cGMP. Our findings suggest that a vasodilator effect of NO induced by L-arginine loading may, at least in part, be associated with increased renal sodium excretion in patients with chronic renal failure.

The superoxide radical and its scavenger, superoxide dismutase (SOD), play important roles in the regulation of corpus luteum function. The present study was undertaken to investigate whether SOD is related to pregnancy-induced maintenance of corpus luteum function. Placentae obtained from rats on Day 12 of pregnancy were incubated for 24 h, and the supernatant was used as placental luteotropins. Pseudopregnant rats were given the placental incubation medium from Day 9 to Day 12 of pseudopregnancy. The treatment significantly increased serum progesterone concentrations on Day 12 of pseudopregnancy. Both activities and mRNA levels of copper-zinc SOD (Cu,Zn-SOD) and manganese SOD (Mn-SOD) in the corpus luteum were also increased on Day 12 of pseudopregnancy. Treating the placental incubation medium with charcoal significantly eliminated the stimulatory effects of placental incubation medium on serum progesterone concentrations and luteal Mn-SOD expression, but not on Cu,Zn-SOD expression. The inhibitory effect of the charcoal treatment on luteal Mn-SOD expression was reversed by supplementation with testosterone or dihydrotestosterone (DHT), but serum progesterone concentrations were recovered only by DHT. Testosterone or DHT alone had no effect on serum progesterone concentrations and luteal SOD expression. In conclusion, placental luteotropins increased SOD expression in the corpus luteum and stimulated progesterone production, suggesting that SOD is involved in the maintenance of the corpus luteum function by placental luteotropins. In addition, androgen, with other placental luteotropins, acted to stimulate progesterone production and Mn-SOD expression in pseudopregnant rats.

Immature rats receiving equine chorionic gonadotropin (eCG) and human CG (hCG) were used to study the time course changes in nitric oxide synthase (NOS) activity in the ovary during ovulation. To study the role of NO in ovulation, the effects of intrabursal injection of L-N(G)-monomethylarginine (L-NMMA, 125 microg/20 microl/bursa), a NOS inhibitor, on the number of ova shed were also examined. Rats were sacrificed at -48, 0, 3, 6, 9, 12, and 24 h after hCG injection, and the ovaries were collected for the NOS activity assay, Western blotting, NADPH-diaphorase histochemistry and immunohistochemistry. Total NOS and constitutive NOS activities in the ovary increased significantly at 9 h after hCG injection and the values remained high thereafter. Inducible NOS (iNOS) activity was detectable as a small peak at 3 and 6 h after hCG injection. Endothelial NOS (eNOS) protein production increased after hCG injection with a peak at 12 h, whereas iNOS protein production decreased at 12 and 24 h after hCG injection. NADPH-diaphorase positive cells increased at the thecae of growing follicles after hCG injection, appeared at mural granulosa cells before ovulation, and were detected in newly formed corpora lutea, which coincided with the results in eNOS positive cells by immunohistochemistry. L-NMMA given to rats at 5 or 7 h after hCG was most effective in reducing the number of ova shed. These results indicate that the NOS activity and NOS positive cells increased after hCG injection, and that eNOS was likely the main NOS increasing in the ovary during ovulation. It is concluded that NO produced between 5 and 9 h after hCG might play a supportive role in ovulation.

Superoxide radicals are known to inhibit progesterone production by luteal cells and have also been reported to cause apoptosis in various cells. The corpus luteum has an antioxidant enzyme to scavenge superoxide radicals: copper-zinc superoxide dismutase (Cu, Zn-SOD). However, it remains unknown how the decrease in intracellular Cu,Zn-SOD activity influences luteal function. This study was therefore undertaken to investigate whether suppression of intracellular Cu,Zn-SOD activity inhibits progesterone production by rat luteal cells and causes apoptosis. To suppress intracellular Cu, Zn-SOD activity, dispersed rat luteal cells were incubated with Cu, Zn-SOD antisense oligonucleotides. The 48-h treatment with antisense oligonucleotides (10 microM) inhibited Cu,Zn-SOD activity by 50% and Cu,Zn-SOD mRNA level by 30%, whereas sense oligonucleotides used as the control had no effect. Progesterone concentration in the medium was significantly decreased by the 48-h treatment with antisense oligonucleotides in the presence of hCG, and this inhibitory effect was completely blocked by the simultaneous addition of N-acetyl-L-cysteine, an antioxidant. Treatment with antisense oligonucleotides caused no significant change in the percentage of apoptotic cells as morphologically evaluated by the nuclear staining with Hoechst dye. In conclusion, the decrease in intracellular Cu, Zn-SOD activities inhibits progesterone production by rat luteal cells, which may be mediated by superoxide radicals, suggesting that intracellular Cu,Zn-SOD plays important roles in the regulation of luteal function.

A procedure for the isolation of highly purified lysosomes from normal rat kidney is described.The method depends on the swelling of mitochondria when the postnuclear supernatant fraction is incubated with 2 mM Ca2+. The lysosomes can then be separated from the swollen mitochondria by Percoll density gradient centrifugation.The lysosomal fraction obtained by our method was enriched more than 30-fold in terms of marker enzymes with a yield of about 11%. Electron microscopic examination and the measurement of the activities of marker enzymes for various subcellular organelles indicated that our lysosomal preparation was essentially free from contamination by other organelles.We believe that this procedure for isolating kidney lysosome will be useful in the study of the mechanisms of specific modification, processing and catabolism of proteins.

Estradiol, together with PRL and placental lactogens, regulates steroidogenesis and cell hypertrophy in the rat corpus luteum of pregnancy. Although binding experiments have demonstrated the presence of estrogen-binding sites, no evidence exists as to whether the rat corpus luteum of pregnancy expresses the estrogen receptor (ER) genes. In this investigation, we have analyzed the expression of the two ER genes (ER alpha and ER beta) (by RT-PCR and in situ hybridization) in the rat corpus luteum, studied their developmental changes throughout pregnancy, and investigated the regulation of ER alpha and ER beta messenger RNA (mRNA) expression by PRL and placental lactogens. The RT-PCR studies showed that both ER mRNA species (ER alpha and ER beta) are coexpressed in the rat corpus luteum during pregnancy. Whereas ER alpha mRNA increased from early pregnancy, reached a maximum at midpregnancy, and had a remarkable decline before parturition; ER beta mRNA remained constant throughout pregnancy, with a significant decline at parturition. Examination of ER alpha and ER beta mRNA expression at the cellular level, by in situ hybridization, showed ER alpha expressed in both follicles and corpus luteum, with maximal expression at midpregnancy. In parallel with the RT-PCR studies, ER beta mRNA was similarly expressed throughout pregnancy in the corpus luteum, but it was less abundant when compared with small and growing follicles. Western blot analysis revealed two ER immunoreactive proteins in the nuclear fraction obtained from pregnant rat corpus luteum: a 67-kDa moiety, highly expressed at midpregnancy but barely detectable in early and late gestation; and a 61-kDa form that remained developmentally unchanged. Hypophysectomy, performed early in pregnancy, induced a sharp decline in ER alpha mRNA expression but a less-marked reduction in ER beta mRNA levels. PRL treatment reverted the inhibition induced by hypophysectomy in both receptor subtypes. When primary luteinized cells were used to test the effect of PRL, rat placental lactogen I, and rat placental lactogen II on the expression of ER alpha and ER beta mRNA, all these lactogenic hormones stimulated both ER mRNA species in a dose-dependent manner. The regulation of ER mRNA expression was further evaluated in a luteal cell line, termed GG-CL, which apparently expresses only the ER beta mRNA species. Culture of the GG-CL cells, in the presence of PRL, resulted in a dose-related up-regulation of ER beta mRNA expression. In addition, PRL treatment enhanced the binding activity of GG-CL cell nuclear proteins to a classical estrogen response element. Furthermore, in these cells, estradiol treatment induced a dose-dependent up-regulation of the mRNA encoding protein kinase C delta isoform, a well-known estrogen target gene in the corpus luteum of the pregnant rat.

The primary culture of rat luteal cells and their long-term maintenance have been difficult. Low cellular yields have limited the possibility for the study of gene regulation in luteal cells. The goal of this study was to develop a cell line to serve as a model by which to study the expression and regulation of various genes specific to luteal cells. We attempted to develop a luteal cell line by transformation of large luteal cells through infection with a temperature-sensitive simian virus (SV-40 tsA209) mutant that has a temperature-sensitive mutation required for the maintenance of cell transformation. We report here the successful establishment of such a cell line, designated GG-CL cells. Large luteal cells were purified to homogeneity by flow cytometry from corpora lutea of day 14 pregnant rats, cultured for 24 h, and then infected with the SV-40 tsA209 mutant virus. Transformed cells were maintained at the permissive temperature (33 C) until colonies were identified. Several colonies of transformed cells were isolated and passaged. They multiplied at 33 C and formed multilayers. At the nonpermissive temperature (40 C), cells reverted to the normal differentiated phenotype similar to the primary luteal cells in culture. To determine whether GG-CL cells express the genes found in normal luteal cells, messenger RNA (mRNA) expression was examined by either Northern analysis or RT-PCR with primers specific to each mRNA. GG-CL cells were found to express receptors for interleukin-6 and glucocorticoid, as well as the newly discovered estrogen receptor-beta (ER-beta) and the orphan nuclear receptor nur 77. No receptors for ER-alpha, progesterone, LH, or PRL could be detected. This cell line also expressed 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), but not cholesterol side-chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase, or aromatase cytochrome P450 (P450arom). Although the cells did not express the PRL receptor, they did express Janus kinase (JAK2) and signal transducers and activators of transcription (Stat5b), and, when transfected with the PRL receptor, they responded to PRL with a marked inhibition in 20alpha-HSD mRNA expression. In addition, estradiol enhanced ER-beta expression in a dose-dependent manner whereas cAMP stimulation caused a marked and rapid increase in the expression of the orphan receptor nur 77. In summary, a temperature-sensitive cell line was successfully established from the large luteal cells of rat corpora lutea. These cells express key genes encoding enzymes and receptors inherent to this defined luteal cell population and respond to stimulation by PRL, estradiol, and cAMP.

In an attempt to determine appropriate diet in nephrotic syndrome, nephrotic rats, induced by puromycin aminonucleoside, were nourished by total parenteral nutrition fluid containing the same energy, but three different levels (1.65, 3.3, and 6.6%) of amino acids for 7 d. The fractional rate of total protein synthesis in the liver was determined by injecting a flooding dose of [3H]phenylalanine. The proportion of newly synthesized proteins retained and exported by the liver was estimated by injecting a tracer dose of [14C]leucine and then measuring the protein radioactivity remaining in the liver and present in the plasma after secretion was completed. Nephrotic animals synthesized more protein than control animals. Although the absolute synthesis rates of total protein in liver were increased with increasing amino acid administration, the absolute rates of synthesis of albumin were higher in the 3.3% group than in the other groups in nephrotic rats. However, kidney protein synthesis in nephrotic rats was higher in the 1.65% group than in the 3.3% group. Interestingly, the 3.3% group revealed the smallest urinary excretion of total protein and albumin. In addition, in the 3.3% group, plasma concentrations of total protein and albumin were higher, and plasma concentrations of total cholesterol and triglyceride were lower than in other groups. It was concluded that the 3.3% group, corresponding to a normal protein diet, has the greatest salutary effect on urinary protein excretion, followed by protein and lipid metabolism, in nephrotic rats. Not only protein intake but also the energy:protein ratio are important for diet therapy in nephrotic animals. The technique of total parenteral nutrition may be useful in defining the factors involved in glomerular permeability or permselectivity and intracellular protein metabolism.

Ephemerality and prolongation of luteal function have been matters of great concern in reproduction for many years. However, their control mechanisms are very complex and differ among mammals. Recently, evidence has indicated that reactive oxygen species may play important roles in the regulation of luteal function. Reactive oxygen species are present in most somatic cells and are involved in apoptosis, or 'physiological cell death'. In the corpus luteum, reactive oxygen species also exert luteolytic effects as well as some paradoxical luteotrophic effects. This paper discusses the possible roles of reactive oxygen species in the control of luteal function.

To study the relationship between follicular atresia, apoptosis, and nitric oxide (NO) generation in follicular development, steroidogenesis, NO levels in follicular fluid and apoptosis were analysed in the various sized follicles of women receiving ovarian stimulation with human menopausal gonadotrophin (HMG)-human chorionic gonadotrophin (HCG) treatments for in-vitro fertilization (IVF)-embryo transfer. The follicles were divided into three groups by diameter: large follicle,or = 18 mm; medium follicle,or = 12 andor = 15 mm; small follicle,or = 10 mm. Follicular fluid was obtained from 20 women 34 h after HCG administration, and the concentrations of oestradiol, progesterone and testosterone, and nitrite, nitrate, arginine and citrulline were measured. Granulosa cells obtained from each group of follicular fluid were stained with Hoechst dye, and nuclear morphology was examined by a fluorescence microscopy. Oestradiol and progesterone concentrations in large follicles were significantly (P0.01) higher than those in medium or small follicles, and testosterone concentrations in small follicles were significantly (P0.01) higher than those in large follicles. There were no significant differences in the concentrations of nitrite, nitrate, arginine and citrulline among three groups. The percentage of apoptotic cells with nuclear fragmentation was significantly (P0.01) higher in small follicles than in large follicles. The present results suggested that small follicles with poor response to HMG may undergo atresia through apoptosis. No significant difference in the follicular NO level between large and small follicles led us to speculate on a different responsiveness to NO in these two types of follicles.

The effects of prostaglandin E1 (PGE1) on transient forebrain ischemia were studied in the gerbil. The animals were randomly divided into six groups of 6 each according to the method of administration as follows: group 1, no administration; group 2, subcutaneous administration with 5 ml.kg-1 of physiological saline, 30 min prior to ischemia; group 3, the same method with 3 mg.kg-1 of PGE1; group 4, 3 mg.kg-1 of PGE1, during 24 hours (12 hours prior to ischemia, and 12 hours following ischemia); group 5, 3 mg.kg-1 of PGE1, during 96 hours (12 hours prior to ischemia and 84 hours following ischemia); and group 6, sham operation. They were anesthetized with isoflurane and transient forebrain ischemia was induced by occluding bilateral common carotid arteries for 5 min. The extracranial electroencephalogram (EEG) was recorded from the electrodes placed at the vertex. During the experimental procedures, temperatures at tympanic membrane and rectum were maintained at 37.0 +/- 0.2 degrees C by means of a heating mat and control of the air temperature in all groups. After 6 days of survival, they were sacrificed, and the brain tissues were fixed for the immunohistochemical and histopathological analyses. The hippocampal CA 1 regions were stained for monoclonal anti microtubule-associated protein 2 (MAP 2), and hematoxylin and eosin. In the 4th group, EEG recovery was recognized earlier than the other groups. Immunoreactivities for MAP 2 and the number of surviving pyramidal cells after ischemia in the CA 1 regions were also well maintained. These results suggest the PGE1, has protective effects against degradation of cytoskeletal proteins and delayed neuronal death in the gerbil, and it might be due to direct protective action of cell membrane in addition to its microcirculatory improvement.

The effect of prostaglandin F2 alpha (PGF2 alpha) on superoxide radical production by macrophages was studied in pseudopregnant rats. Peritoneal macrophages prepared on day 7 or 13 of pseudopregnancy (psp) were incubated with various doses of PGF2 alpha for 90 min, and the production of superoxide radical was measured by the cytochrome C reduction method. PGF2 alpha significantly stimulated superoxide radical production by macrophages on day 13 of psp, but not on day 7 of psp. The pretreatment of macrophages with an inhibitor of protein kinase C (H7), Ca2+ channel blocker (Verapamil), Ca2+ chelators (EGTA, BAPTA), and an inhibitor of GTP-binding protein (pertussis toxin) prevented the stimulatory effects of PGF2 alpha on superoxide radical production. In conclusion, PGF2 alpha stimulated superoxide radical production by macrophages through the intracellular signal transduction pathway including activation of protein kinase C through the GTP-binding protein and Ca2+ influx, which would play important roles in the luteolytic process in psp rats.

The effect of ulinastatin on delayed neuronal death was studied in the gerbil. The animals were divided into 2 groups according to the temperatures of the temporalis muscle, tympanic membrane, and the rectum. One group consisted of 36 animals in whom the temperature was maintained at 36.5 degrees C and the another was at 38 degrees C. They were anesthetized with isoflurane and injected intravenously with 5 ml.kg-1 of normal saline solution, 100 mg.kg-1 of alpha-tocopherol, 250,000 units.kg-1 of ulinastatin, or 500,000 units.kg-1 of ulinastatin, respectively. Transient ischemia was then induced by occluding bilateral common carotid arteries for 5 min. Six days later, they were sacrificed, and the brain tissues were fixed and stained for histopathological analysis of hippocampal CA1 regions. alpha-Tocopherol and ulinastatin prevented delayed neuronal death in the 36.5 degrees C group, but did not in the 38 degrees C group. alpha-tocopherol is recognized as an intrinsic radical scavenger, and known to prevent delayed neuronal death. Compared with 100 mg.kg-1 of alpha-tocopherol, 500,000 units.kg-1 of ulinastatin had the same protective effect on delayed neuronal death at 36.5 degrees C. We concluded that administration of ulinastatin prevented delayed neuronal death at 36.5 degrees C.

Contraction of the pre- and postglomerular arteries play an important role in the regulation of glomerular blood flow. Mesangial cells may also be involved in the mechanism of this regulation, but it has not been clarified yet whether or not mesangial cells and vascular smooth muscles show an identical phenotype, especially in terms of their contractile proteins. In this study, in order to elucidate any difference in the cellular phenotypes between mesangial cells and renal vascular smooth muscles, we investigated the localization of myosin heavy chain isoforms using a monoclonal antibody against SM1 and SM2. Both SM1 and SM2 are specific to smooth muscles. SM1 is specifically expressed in smooth muscles from early development and SM2 appears after birth. In normal renal tissues, SM1 and SM2 were expressed only in the smooth muscle cells of the arterioles and small arteries. However, glomerular cells, including mesangial cells, were not stained with either anti-SM1 antibody or anti-SM2 antibody. Localization of SM1 and SM-2 was similar to that of alpha-smooth muscle actin (SM alpha-actin). Staining for SM-1 was not observed in the mesangial areas of renal tissues with glomerular disease. These results clearly indicate that mesangial cells have a different phenotype from that of vascular smooth muscle cells in terms of their contractile proteins.

An involvement of reactive oxygen species in CL regression has been reported. We have shown that a decrease in serum progesterone concentrations coincides with a decrease in superoxide dismutase (SOD) activities and an increase in lipid peroxide levels in the CL after Day 15 of pregnancy. Recently it has been found that ischemia-reperfusion stimulates reactive oxygen species production and causes tissue damage in various organs. We therefore tested the effect of ischemia-reperfusion in the ovary on CL function in pregnant rats. On Day 15 of pregnancy, after clamping of the bilateral ovarian vessels for 30 min, the ovaries were reperfused for 90 min by declamping. The ischemia-reperfusion decreased serum progesterone concentration and SOD activity in the CL and increased lipid peroxide in the CL 90 min after reperfusion. The effects of ischemia-reperfusion, including the decrease in serum progesterone concentrations, were completely blocked by simultaneous injection of SOD and catalase, but not by indomethacin administration. The present study shows that CL function was inhibited by reactive oxygen species produced by ischemia-reperfusion in the ovary and that the effect was not mediated through prostaglandins.