Local regulation of corpus luteum development and regression in the cow: Impact of angiogenic and vasoactive factors.

The corpus luteum (CL) of the estrous cycle in the cow is a dynamic organ which has a life time of approximately 17-18 days. The main function of the CL is to secrete a large amount of progesterone (P) thereby supporting the achievement of pregnancy. As the CL matures, the steroidogenic cells establish contact with many capillaries and the matured CL is composed of many vascular endothelial cells that account for up to 50% of all CL cells. The bovine CL produces several major angiogenic and vasoactive foctors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoietin-1 and -2 (ANPT-1 and -2), prostaglandin F(2alpha) (PGF(2alpha)), endothelin-1 (EDN1), angiotensin II (Ang II) and nitric oxide (NO). These factors regulate P secretion directly and/or indirectly within the CL. Moreover, different actions of PGF(2alpha) in the early cycle CL (non-luteolytic) and the mid cycle CL (luteolytic) may provide insight into the luteolysis cascade in the cow. The aim of the present review is to describe the current concepts of the local mechanisms for the cascade of development and regression of the bovine CL as regulated by luteal angiogenic and vasoactive factors.

Constant light housing attenuates circadian rhythms of mPer2 mRNA and mPER2 protein expression in the suprachiasmatic nucleus of mice.

Constant light (LL) or constant dark (DD) environmental lighting conditions cause a free-running period and activity reduction in the rodent behavioral circadian rhythm. In order to understand the molecular process underlying behavioral rhythms in LL or DD housing conditions, we examined the circadian profile of mPer2 mRNA and mPER2 in the suprachiasmatic nucleus (SCN), a main oscillator, of free-running mice. The circadian expression rhythm of mPer2 in the SCN was dampened under 7-day LL conditions, whereas that of mPER2 protein was moderately attenuated and its expression peak delayed. The circadian expression of mPer2 and its product was slightly attenuated and advanced by 7-day DD conditions. With arrhythmic behavioral activity caused by long-term LL housing, mPER2 protein lost its rhythmicity in the SCN. On the other hand, LL or DD housing did not affect the mPer2 gene and its product in the cerebral cortex. The present results suggest that mPER2 circadian expression in the SCN corresponds well with behavioral circadian oscillation under LL or DD conditions. Thus, the behavioral circadian rhythm seems to correlate with molecular clock works in the SCN.

Measurement of positron production efficiency from a tungsten monocrystalline target using 4- and 8-GeV electrons.

Intense positron sources are being widely investigated for the next-generation linear colliders and B factories. A new method utilizing an axially oriented crystal as a positron-production target is one of the bright schemes, since it provides a powerful photon source through channeling and coherent bremsstrahlung processes when high-energy electrons penetrate the target. A series of positron-production experiments with tungsten crystals hit by 4- and 8-GeV single-bunch electron beams were carried out at the KEKB 8-GeV injector linac. Three tungsten crystals with different thicknesses (2.2, 5.3, and 9.0 mm) and those combined with amorphous tungsten plates were tested on a precise goniometer. The positron-production yields were measured with a magnetic spectrometer in the positron momentum (P(e(+))) range from 5 to 20 MeV/c. The angle of the <111> crystal axis with respect to the electron-beam direction was controlled by measuring the relative intensities of the produced positrons as a function of the rotational angle of the goniometer. The results show that the enhancements of the positron yield from crystal targets compared to amorphous targets of the same thickness at P(e(+))=20 MeV/c are from 1.5 to 3.7 and from 1.8 to 5.1, depending upon the target thickness for 4- and 8-GeV electrons, respectively.

Pressure effect on denaturant-induced unfolding of hen egg white lysozyme.

The influence of hydrostatic pressure (< or =100 MPa) on denaturant-induced unfolding of hen egg white lysozyme was investigated by means of ultraviolet spectroscopy at various temperatures. Assuming a two-state transition model, the dependence of Gibbs free-energy change of unfolding on the denaturant concentration was calculated. Under applied hydrostatic pressure, these data were interpreted as suggesting that a two-state model is not applicable in a restricted temperature range; the dominant effect of hydrostatic pressure is to affect the cooperativity in protein unfolding due to a chemical equilibrium shift in the direction of the reduction in the system volume. The deviation from the two-state transition model appears to be rationalized by assuming that applied pressure induces an intermediate conformation between the native and unfolded states of the protein. The implication of the thermodynamic stability of protein under pressure was discussed.

Trandolaprilat, an angiotensin-converting enzyme inhibitor, is not excreted in bile via an ATP-dependent active transporter (cMOAT).

Trandolapril is the prodrug of an angiotensin-converting enzyme (ACE) inhibitor. It has been proposed that its active metabolite, trandolaprilat, is mainly excreted in bile, but this has not been clearly demonstrated. Recently it has been reported that temocaprilat, an active metabolite of the ACE inhibitor temocapril, is effectively excreted in bile via an ATP-dependent active transporter (canalicular multispecific organic anion transporter: cMOAT). To investigate whether trandolaprilat has the pharmacological ability to affect the cMOAT system in a manner similar to temocaprilat. The lipophilicity of trandolaprilat and temocaprilat was measured to determine the n-octanol-water partition coefficients. The dose-dependent inhibition of the up-take of [3H]-estradiol-17beta-D-glucuronide and [3H]-2,4-dinitrophenyl-S-glutathione, which are good substrates for cMOAT, in canalicular membrane vesicles (CMVs) prepared from Sprague-Dawley rats was determined in the presence of trandolaprilat and temocaprilat. The partition coefficient of trandolaprilat (log Po/w - 1.1) was about 30 times higher than that of temocaprilat (log Po/w - 2.5). The uptake of [3H]-estradiol-17beta-D-glucuronide and [3H]-2,4-dinitrophenyl-S-glutathione was dose-dependently inhibited by the presence of temocaprilat, but trandolaprilat had no effect on the transport of [3H]-estradiol-17beta-D-glucuronide or [3H]-2,4-dinitrophenyl-S-glutathione into CMVs even at concentrations as high as 200 microM. It could be concluded that trandolaprilat has a higher lipophilicity than temocaprilat. But the hepatobiliary excretion system via cMOAT may not contribute to the excretion of trandolaprilat in bile.

A rapid and simple method for sex identification by heteroduplex analysis, using denaturing high-performance liquid chromatography (DHPLC).

A novel method for sex identification, using a denaturing high-performance liquid chromatography (DHPLC) system, is described. Among many methods for identifying sex, the most popular and credible system has been the polymerase chain reaction (PCR) method, using nucleotide primer sets of the amelogenin gene, which is shared on both the X and Y chromosomes. With this conventional method, the judgment depends on detection of the size difference between the PCR products derived from the X and Y chromosomes. In this study, we adopted DHPLC to detect the difference by checking heteroduplex formation between the products, which enabled us to shorten the PCR products to 45bp and the separation time to within a period of 8min per sample. This new system may have wide applications in many different fields, such as forensic medicine, prenatal diagnosis, inbreeding of animals, and anthropology.

Obstructive jaundice facilitates hepatic metastasis of B16F1 mouse melanoma cells: participation of increased VCAM-1 expression in the liver.

Obstructive jaundice facilitates experimental liver metastasis in the rat model, but the detailed mechanisms of this facilitation remain unclear. This study aimed to evaluate the contribution of vascular cell adhesive molecules-1 (VCAM-1) to augmented hepatic metastasis in cases of obstructive jaundice. Obstructive jaundice was induced in male C57BL/6 mice by common bile duct obstruction for 5 days using a Surgiclip. For the biliary decompression, obstructive jaundice was induced for 5 days, followed by removal of the Surgiclip. Liver specimens and blood samples were obtained from animals 5 days after biliary obstruction (OJ5) or sham operation and 2, 5, 11, 14 days after biliary decompression. The expression of VCAM-1 mRNA was increased in the livers from the OJ5 group. Western blot analysis demonstrated increased expression of VCAM-1 protein in the livers of the OJ5 group, in contrast with low VCAM-1 expression in the sham group. The expression of VCAM-1 protein was sustained at high levels at 2 days and decreased at 5 days after biliary decompression (BD5). For the induction of experimental hepatic metastasis, male C57BL/6 mice were randomized to three groups (sham, OJ5, BD5) of six animals each. B16F1 melanoma cells were introduced into the animals by an intraportal injection. Metastatic colonies in the livers were investigated 13 days after inoculation. The mean number of metastatic colonies was significantly increased in the OJ5 group (70.5+/-51.2) compared to that of the sham group (7.2+/-7.9) (p<0.05). This augmentation of hepatic metastasis was abrogated in the BD5 group (16.8+/-20.3). In conclusion, our results suggest that augmented hepatic metastasis in cases of obstructive jaundice are partly mediated through VCAM-1/VLA-4 interaction.

Time-dependent promotion activity of 17beta-estradiol on uterine carcinogenesis in mice initiated with N-ethyl-N-nitrosourea.

The time-dependent promotion activity of 17beta-estradiol (E2) by initiation with N-ethyl-N-nitrosourea (ENU) on induction of mouse uterine endometrial proliferative lesions was examined. Illumination-induced persistent estrous female CD-1 mice were divided into five groups at 9 weeks of age. At 10 weeks of age, mice in all groups (n=25) were given a single intra-uterine administration of ENU (50 mg/kg), dissolved in polyethylene glycol. Animals in Groups 2 to 5 were then implanted s.c. with an E2 pellet at 9, 11, 14 and 17 weeks of age. The implants were left in place for 8 weeks and then taken out. At the termination of the experiment (week 15 after the ENU-treatment), all surviving mice were killed and the development of uterine proliferative lesions were assessed. All groups demonstrated endometrial hyperplasias and adenocarcinomas and the incidences of the latter in ENU plus E2 treated animals (Groups 2 to 5; 36, 48, 35 and 36%, respectively) were significantly higher compared to 8% for Group 1, without any variation with the age at E2 treatment. However, the incidences of adenocarcinomas plus severe hyperplasias increased from Groups 1 to 5 (28, 40, 56; P<0.05, 61; P<0.05 and 80%; P<0.01, respectively), indicating that promotion effects of E2 on induction of uterine proliferative lesions in the uterine endometrium become more pronounced with the interval after ENU initiation.

Partially unfolded equilibrium state of hen lysozyme studied by circular dichroism spectroscopy.

Equilibrium unfolding of hen egg white lysozyme as a function of GdnCl concentration at pH 0.9 was studied over a temperature range 268.2-303.2 K by means of CD spectroscopy. As monitored by far- and near-UV CD at 222 and 289 nm, the lack of coincidence between two unfolding transition curves was observed, which suggests the existence of a third conformational species in addition to native and unfolded states. The three-state model, in which a stable intermediate is populated, was employed to estimate the thermodynamic parameters for the GdnCl-induced unfolding. It was found that the transition from the native to intermediate states proceeds with significant changes in enthalpy and entropy due to an extremely cooperative process, while the transition from the intermediate to unfolded states shows a low cooperativity with small enthalpy and entropy changes. These results indicate that the highest energy barrier for the GdnCl-induced unfolding of hen lysozyme is located in the process from the native state to the intermediate state, and this process is largely responsible for the cooperativity of protein unfolding.

Molecular cloning and expression analysis of a putative nuclear protein, SR-25.

We cloned a full-length mouse cDNA and its human homologue encoding a novel protein designated as "SR-25." In Northern blot analysis, SR-25 mRNA was expressed in all organs tested, and relatively abundant in testis and thymus. Deduced amino acid sequences of mouse SR-25 and human SR-25 showed 77.7% identity. SR-25 has a serine-arginine repeat (SR repeat) and two types of amino acid clusters: a serine cluster and a highly basic cluster. Based on the presence of many nuclear localizing signals and a similarity to RNA splicing proteins, SR-25 is strongly suggested to be a nuclear protein and may contribute to RNA splicing.

Role of energy metabolism in drug-induced acute gastric mucosal injuries in humans.

BACKGROUND: In various drug-induced gastric mucosal injuries, it has been speculated that changes in gastric mucosal energy metabolism differ according to the cause of injury. This study was conducted to investigate the role of energy metabolism in two drug-induced gastric mucosal injuries in humans. METHODS: The subjects were patients with acute gastric mucosal injury due to non-steroidal anti-inflammatory drugs (NSAID) or steroids, and normal controls. Two sets of tissue specimens from the antrum and corpus of the stomach were obtained. One specimen from each area was used for histological analysis and the other was stored in liquid nitrogen. The stored tissues were homogenized. Adenosine triphosphate (ATP), adenosine diphosphate (ADP) and adenosine monophosphate (AMP) were measured by the luciferin-luciferase method. RESULTS AND CONCLUSIONS: In the NSAID group, the ATP levels and the total adenine nucleotide (TAN) levels in both the antrum and corpus were significantly lower than in the control group. In the steroid group, no significant differences were observed in either the ATP or TAN levels. The NSAID decreased energy metabolism in the entire stomach while the steroid had a negligible effect on energy metabolism.

Molecular cloning and tissue-specific expression of a new member of the regenerating protein family, islet neogenesis-associated protein-related protein.

Islet neogenesis-associated protein (INGAP) is a protein expressed during islet neogenesis. We have cloned a novel cDNA having a similar sequence to INGAP cDNA. The cDNA encodes 175 amino acids designated INGAP-related protein (INGAPrP). INGAP is expressed in cellophane-wrapped pancreas, but not in normal pancreas, whereas INGAPrP was abundantly expressed in normal pancreas.

Transplacental administration of diethylstilbestrol (DES) causes lesions in female reproductive organs of Donryu rats, including endometrial neoplasia.

The effects of transplacental administration of diethylstilbestrol (DES) on female reproductive organs were investigated using Donryu rats. The animals were given subcutaneous injections of DES dissolved in olive oil at doses of 0.01 or 0.1 mg/kg on days 17 and 19 of gestation. In female offspring, clinical signs, body weights and estrous cycles were continuously assessed until all survivors were killed at month 18. A low mean litter size and shortening of period of pregnancy were recognized in the 0.1 mg/kg group. Disorder and/or suspension of the estrous cycle (so called persistent estrus) also appeared very early in the 0.1 mg/kg group. Macroscopically, the incidences of hypoplasia of the oviduct, cystic dilatation of the uterus and small size of the uterine cervix were higher in the 0.1 mg/kg group than those in the control group. Histologically, in the ovary, the incidence and degree of atrophy were increased in both 0.01 and 0.1 mg/kg groups. In the uterus, total incidences of endometrial hyperplasias were about the same in all groups. However, endometrial adenocarcinomas were dose-dependently increased in the treated groups, the incidence in the 0.1 mg/kg group being significant, compared to that in the control. In the vagina, mucification was more prominent in the treated animals, especially at the higher dose, but no tumors were observed. The present results indicate that prenatal exposure to DES can produce uterine adenocarcinomas in rats, as reported earlier for mice, although its carcinogenic activity is not so strong. Increase of endometrial adenocarcinoma incidence might depend on hormonal imbalance resulting from the ovarian atrophy due to transplacental treatment of DES.

The volume and compressibility changes of lysozyme associated with guanidinium chloride and pressure-assisted unfolding.

The apparent specific volumes and isentropic compressibilities of hen egg white lysozyme were measured in aqueous guanidinium chloride solutions at 25 degrees C by means of a vibrational densimeter and a sing-around ultrasonic velocimeter. Little transition attributable to a protein unfolding was detected in the partial specific volume, while the partial specific isentropic compressibility decreased slightly around the transition region. The pressure-assisted unfolding was also investigated in aqueous guanidinium chloride solutions by means of ultraviolet spectroscopy. Assuming a two-state transition model, it was found that the free energy change of unfolding depends almost linearly on pressure and the unfolding reaction is accompanied by a small decrease in volume. The compressibility behavior is in conflict with the notion that a protein structure is almost completely unfolded by guanidinium chloride and most of the amino acid residues in the protein interior are exposed to solvent. These results support the current view that globular proteins have some residual structures even in the unfolded state induced by a strong denaturant.

Pressure-induced unfolding of lysozyme in aqueous guanidinium chloride solution.

The pressure-induced unfolding of lysozyme was investigated in an aqueous guanidinium chloride solution by means of ultraviolet spectroscopy. Assuming a two-state transition model, volume changes were calculated from the slope of free energy vs. pressure plots over a temperature range of 10 to 60 degrees C. Between 25 and 60 degrees C, almost constant volume changes were observed in the transition region, which was reflected in almost identical slopes of the free energy change vs. pressure plots. On the other hand, the different slopes were observed in the pressure dependence of free energy change at temperatures lower than 25 degrees C. These data were interpreted as suggesting that a two-state model is not appropriate at low temperature, but instead one or more intermediates are present under these conditions. The volume changes for unfolding became less negative at temperatures higher than 25 degrees C.

Splenic artery aneurysm associated with systemic lupus erythematosus: report of a case.

We herein report on a 64-year-old Japanese female patient who presented with a splenic artery aneurysm (SAA) associated with systemic lupus erythematosus (SLE). The saccular aneurysm, which measured 3 cm in diameter, was located in the proximal third of the splenic artery from the pancreas with a portosystemic shunt. A double ligation of the splenic artery (the distal and proximal sides of the aneurysm) was performed without a splenectomy. The postoperative course showed acute pancreatitis without either splenic infarction or portal thrombus. To our knowledge, the closed association of SLE with an aneurysmal dilatation of the splenic artery has not been previously reported. Both the pathogenesis and the management of SAA associated with SLE are discussed following the presentation of this case. This is the first reported case of SAA associated with SLE.

Sucrose permeability as a means of detecting diseases of the upper digestive tract.

The healthy gastric epithelium will not allow easy permeation of a disaccharide-sized molecule such as sucrose. However, during gastric damage, intact sucrose can pass the gastric epithelium and ultimately appear in the urine. We examined the relationship between total urinary sucrose excretion and various diseases. We used 149 patients (105 had upper gastrointestinal disease, 12 had gastric cancer and 32 were normal). Subjects were given a solution containing 100 g sucrose in 450 c.c. water. All urine was collected for 7.5 h. The urinary sucrose concentration was determined by anion exchange high-performance liquid chromatography. Total urinary sucrose excretion was significantly higher in patients with gastric ulcer and those with gastric cancer than in endoscopically normal controls. In the 34 patients with gastric ulcer, the total sucrose excretion was closely correlated with ulcer size. Ulcer location did not affect urinary sucrose excretion. A strong correlation was also observed between sucrose excretion and lesion size in the 12 patients with gastric cancer. The sucrose permeability test may be a relatively sensitive method to detect gastric disease.

An automated method for the simultaneous determination of pravastatin and its main metabolite in human plasma by high-performance liquid chromatography/atmospheric pressure chemical ionization mass spectrometry.

A new method for the determination of pravastatin, a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, and its main metabolite (R-416) in human plasma using high-performance liquid chromatography/atmospheric pressure (negative ion) chemical ionization mass spectrometry (LC/APCI-MS) is described. Pravastatin and R-416 in human plasma were isolated using solid phase extraction technique and analyzed by LC/APCI-MS. Selected ion monitoring was employed for selectivity and sensitivity, which enabled the quantification over a range of 0.625-80 mg/mL with acceptable precision and accuracy. No derivatization was required for these polar molecules. The retention times of the pravastatin, R-416 and the internal standard (R-1437) were 2.1, 2.5 and 3.9 min, respectively, with a total analysis time of 5 min. This method was validated and compared with the automated gas chromatography/negative ion chemical ionization mass spectrometry procedure.

Granular cell foci of the uterus in Donryu rats.

Eleven cases of uterine granular cell foci were observed in a total of 855 female Donryu rats. All the lesions were microscopical and focal or multifocal in nature, and composed of uniformly large cells with abundant granulated eosinophilic cytoplasm. Histopathologically, immunohistochemically and ultrastructurally they resembled granular cell tumours of the uterus reported in rats and mice. The development of granular cell foci may be the result of hormonal stimulation.

Initiating and promoting effects of concurrent oral administration of ethylenethiourea and sodium nitrite on uterine endometrial adenocarcinoma development in Donryu rats.

The effects of oral administration of ethylenethiourea (ETU) and sodium nitrite on endometrial adenocarcinoma development was investigated in female Donryu rats. At 10 week of age, groups 1 and 3 received a single dose of polyethylene glycol (PEG) into the uterine cavity, while groups 2 and 4 were given N-ethyl-N-nitrosourea (ENU) (15 mg/kg) solution, dissolved in PEG, in the same manner. ETU (80 mg/kg) and sodium nitrite (56 mg/kg) dissolved in distilled water were orally given to animals in groups 3 and 4 once a week from 11 to 51 weeks of age. Groups 1 and 2 received the vehicle alone. At termination (52 weeks of age), endometrial adenocarcinomas were observed in 29, 13 and 57% of rats in groups 2, 3 and 4, respectively, but not in group 1. Persistent estrus appeared earlier in groups 3 and 4 than in group 1. In groups 3 and 4, hyperplastic and neoplastic lesions of the digestive tract, especially the forestomach, were also observed. The results indicate that N-nitroso ETU formed in the stomach by concurrent oral administration of ETU and sodium nitrite, itself induces endometrial adenocarcinomas by its mutagenic action, as well as promoting their development after ENU-initiation, presumably by influencing the hormonal balance.