Endometrial epithelial-mesenchymal transition (EMT) by menstruation-related inflammatory factors during hypoxia.

Endometriosis is characterised by inflammation and fibrotic changes. Our previous study using a mouse model showed that proinflammatory factors present in peritoneal haemorrhage exacerbated inflammation in endometriosis-like grafts, at least in part through the activation of prostaglandin (PG) E2 receptor and protease-activated receptor (PAR). In addition, hypoxia is a well-known inducer of fibrosis that may be associated with epithelial-mesenchymal transition (EMT). However, the complex molecular interactions between hypoxia and proinflammatory menstruation-related factors, PGE2 and thrombin, a PAR1 agonist, on EMT in endometriosis have not been fully characterised. To explore the effects of hypoxia and proinflammatory factors on EMT-like changes in endometrial cells, we determined the effects of PGE2 and thrombin (P/T) on EMT marker expression and cell migration in three dimensional cultured human endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs). Treatment of EECs with P/T under hypoxia stimulated cell migration, increased the expression of mesenchymal N-cadherin, vimentin and C-X-C chemokine receptor type 4 (CXCR4), and reduced the expression of epithelial E-cadherin. Furthermore, treatment with C-X-C motif chemokine ligand 12 (CXCL12), a ligand for CXCR4, increased EMT marker expression and cell migration. In ESCs, P/T or oestrogen treatment under hypoxic conditions increased the expression and secretion of CXCL12. Taken together, our data show that hypoxic and proinflammatory stimuli induce EMT, cell migration and inflammation in EECs, which was increased by CXCL12 derived from ESCs. These data imply that inflammatory mediators in retrograde menstrual fluid contribute to ectopic endometrial EMT and migration in the presence of peritoneal hypoxia.

Hydrothermal Ethanol Flames in Co-Flow Jets.

Results on the autoignition and stabilization of ethanol hydrothermal fames in a Supercritical Water Oxidation (SCWO) reactor operating at constant pressure are reported. The flames are observed as luminous reaction zones occurring in supercritical water; i.e., water at conditions above its critical point (approximately 22 MPa and 374 °C). A co-flow injector is used to inject fuel (inner flow), comprising an aqueous solution ranging from 20 %-v to 50 %-v ethanol, and air (annular flow) into a reactor filled with supercritical water at approximately 24.3 MPa and 425 °C. Results show hydrothermal fames are autoignited and form diffusion flames which exhibit laminar and/or turbulent features depending upon flow conditions. Two orthogonal camera views are used; one providing a backlit shadowgraphic image of the co-flow jet and the other providing color images of the flame. In addition, spectroscopic measurements of flame emissions in the UV and visible spectrum are discussed.

Inhibitory effect of hydrocortisone on cerebral salt wasting after subarachnoid hemorrhage in rats.

Cerebral salt wasting (CSW) frequently occurs concomitantly with subarachnoid hemorrhage (SAH). CSW induces excessive natriuresis and osmotic diuresis, reduces total blood volume, aggravates cerebral vasospasm and causes cerebral ischemia after SAH. This study examined the inhibitory effect of hydrocortisone on CSW in rat SAH models. Hydrocortisone had an inhibitory effect on CSW because hydrocortisone functioned in a dose-dependent manner to inhibit the increase in sodium excretion and sodium/potassium ratio after SAH onset. We conclude that hydrocortisone is a useful drug for the treatment of CSW after SAH.

Characterization of cerebral salt wasting after subarachnoid hemorrhage model induced by endovascular puncture.

Cerebral salt wasting (CSW) frequently occurs concomitantly with an aneurysmal subarachnoid hemorrhage (SAH). CSW induces excessive natriuresis and osmotic diuresis, and reduces the total volume of blood. We previously reported that a rat model with SAH induced by endovascular puncture (EP) exhibited CSW. Therefore, we investigated the relationship between the spread of bleeding in the subarachnoid space and the intensity of CSW. We also investigated the development of CSW in different SAH models. SAH was induced by EP or by 0.3 mL of blood injection (BI) into the cisterna magna. To evaluate the occurrence of CSW, urine was cumulatively collected at the onset of SAH to 6 h later and analyzed for sodium (Na) excretion. SAH was classified from grade 1 (no bleeding) to grade 4 (severe bleeding) based on the spread of bleeding in the subarachnoid space. In the EP model (SAH grade > 2) as the SAH grade increased, the volume of urine and Na excretion also significantly increased. Although the BI model rats exhibited SAH of grade 4, the volume of urine and Na excretion did not change. Therefore, our conclusion is that the spread of bleeding in the subarachnoid space may not cause CSW.

New approach for chronic renal failure model by direct kidney injection of doxorubicin in rats.

Several experimental chronic renal failure (CRF) models are available for testing new drugs. A CRF model induced by the intravenous injection of 2 mg/kg of doxorubicin (DXR) twice during a 20-day interval reportedly results in pathological characteristics similar to glomerular sclerosis seen clinically. However, it normally takes more than 16 weeks to create this CRF model. We used three methods of direct drug injection into the kidney of rats to determine the method that would induce CRF within 4 weeks; Method A: DXR was injected directly into both kidneys; Method B: DXR was injected directly into the left kidney immediately after right nephrectomy; Method C: DXR was injected directly into the left kidney 1 week before right nephrectomy, and DXR was injected again directly into the left kidney. As a result, urinary protein, blood urea nitrogen (BUN), creatinine and creatinine clearance were significantly changed >1 week after the injection of DXR by Method C. Quantification of tissue transforming growth factor-beta1 (TGF-beta1), which is a prime fibrogenic cytokine in renal fibrosis, significantly increased in the kidney. A light microscopic image showed glomerular decrement, tubular dilation and atrophy and vacuolation of parenchyma. In conclusion, the results of this study demonstrate that the DXR model using Method C develops CRF within 4 weeks.

Changes in charge density of heparan sulfate isolated from cancerous human liver tissue.

Heparan sulfate fractions were isolated from three normal human livers and three cancerous human liver tissues, and their polyanionic properties were examined using electrophoresis, sequential partition fractionation, and chemical analyses. More than 60% of total glycosaminoglycans from normal human liver and about 30% from cancerous liver tissue were found to be heparan sulfate from their resistance to exhaustive digestion with chondroitinase ABC and their susceptibility to nitrous acid treatment. The heparan sulfate isolated from cancerous liver tissue afforded a lower sulfate/uronic acid molar ratio (0.58 to 0.65) than did normal human liver heparan sulfate (0.76 to 0.80). Also, the former showed lower electrophoretic mobility in 0.1 M HCl and a different partition fractionation profile in comparison with the latter. These differences in charge density of the macromolecule were not detected on the chondroitin sulfate and/or dermatan sulfate fractions isolated from normal human liver and cancerous liver tissue.

The glycosaminoglycans in human hepatic cancer.

A method is proposed for the analysis of glycosaminoglycans that were isolated from human liver, combining cellulose acetate electrophoresis and enzymatic digestion with mucopolysaccharidases. The major constituent of glycossaminoglycans in the healthy liver is heparin sulfate and/or heparin (about 65%), with approximately equal quantities of dermatan sulfate and hyalauronic acid (about 13.5 and 13%, respectively) and a small amount of chondroitin sulfate. These components, especially chondroitin sulfate and hyaluronic acid, are markedly increased in hepatic carcinomas.

Glycosaminoglycans in 3'-methyl-4-dimethylaminoazobenzene-induced rat hepatic cancer.

The changes in glycosaminoglycans in livers of rats with 3'-methyl-4-dimethylaminoazobenzene-induced hepatic cancer were examined and compared with those in fetal liver. The incorporation of 35S into sulfated glycosaminoglycans in hepatic cancer tissue was also studied after i.p. injection of Na(2)35SO4. The major component of glycosaminoglycans in healthy adult rat liver was heparan sulfate (61.7%), with hyaluronic acid (21.1%), dermatan sulfate (13.1%), and chondroitin sulfate (4.0%) as minor components. The quantities of all of the examined glycosaminoglycans were higher in tumors than in normal liver, but chondroitin sulfate and hyaluronic acid were more prevalent in the tumors (about 51 and 7 times higher, respectively). As a result, the share of heparan sulfate was decreased (37.1%) in the tumors. The high content of chondroitin sulfate (about 30 times) and the decreased share of heparan sulfate (29.0%) were also observed in fetal liver. The incorporation of 35S into individual glycosaminoglycans varied markedly. Approximately 90% of the label in glycosaminoglycans of healthy liver was found in a heparan sulfate fraction 4 hr after injection. In hepatic cancer tissue, however, 35S incorporation into both chondroitin sulfate and dermatan sulfate fractions was increased about 6.5- and 5.6-fold, respectively, of those in healthy liver.

Changes in the cellular glycosaminoglycans of cultured mastocytoma cells induced by sodium butyrate.

The effect of sodium butyrate on the cellular glycosaminoglycans of cultured mastocytoma p-815-4 cells was investigated using enzymic digestion, electrophoresis, nitrous acid degradation, and sequential partition fractionation. The average cellular glycosaminoglycan content of mastocytoma p-815-4 cells grown in the presence of 2 mM sodium butyrate was ten times as much as that of the control p-815-4 cells. Approximately 90% of the glycosaminoglycans isolated from the control cells and 70% from the butyrate-treated cells were found to be chondroitin 4-sulfate by enzymic digestion. The remainders were chondroitinase ABC-resistant. Hyaluronic acid and dermatan sulfate were not detected in either control cells or butyrate-treated cells. The chondroitinase ABC-resistant fraction of glycosaminoglycans from butyrate-treated cells showed a molar ratio of sulfate to uronic acid of more than 2.0, and provided some physicochemical properties characteristic to reference bovine lung heparin.

Differential interaction of cations with the thiamine and biotin transport proteins of Lactobacillus casei.

Lactobacillus casei cells contain separate and specific binding proteins which mediate the cellular uptake of thiamine and biotin. In buffered salt solutions, these proteins exhibit a very high affinity for their vitamin substrate. Dissociation constants (Kd values) at pH 7.5 are 0.03 and 0.15 nM for thiamine and biotin, respectively. Optimal binding of biotin requires the presence of cations. This cation dependence is substantial since the Kd for biotin is 60-fold higher in a buffer containing 0.1 mM K-Hepes, compared with a buffer composed of 50 mM K-Hepes and 5 mM MgCl2. Measurements of Kd versus cation concentration showed that Mg2+ is 300-fold more effective than K+ in promoting biotin binding. The extent of cation dependence decreases as the pH is reduced from 7.5 to 5.0, suggesting that protons can partially fulfill the cation requirement. In contrast, binding of thiamine to the thiamine transport protein shows no dependence on the ionic composition of the medium. These results suggest that the transport protein for the anionic vitamin, biotin, contains a binding site for cations. Cotransport of both the vitamin and cation into the cell might then occur during the normal transport cycle, allowing the cellular uptake of the vitamin to occur against the membrane potential. Conversely, the cationic vitamin, thiamine, does not appear to be transported into the cell as a complex with other ions.

Experimental model for investigating hyponatremia after subarachnoid hemorrhage in rats.

Hyponatremia is a common complication in patients with aneurysmal subarachnoid hemorrhage (SAH). Such patient demonstrates excessive natriuresis and an increased risk of symptomatic cerebral vasospasm. However, the precise mechanisms underlying SAH induced hyponatremia remain unclear. In the present study, in order to establish an experimental model of hyponatremia following SAH, we induced SAH in rats, and evaluated the serum sodium (Na) levels, Na excretion and physiological parameters. Twenty-four male Wistar rats were used. SAH was induced by an endovascular puncture method. The mean arterial pressure (MAP), intracranial pressure (ICP), and cerebral blood flow (CBF) were monitored continuously. The urine was collected cumulatively for 12 hours after SAH, and the urine Na concentration was determined with a spectrophotometer. The serum Na levels were measured at 12 hrs, 2 and 4 days following the SAH induction. The mean (+/- standard deviation) baseline ICP was 3.5 +/- 2.6 mmHg, and increased to 67.4 +/- 17.6 mmHg immediately following induction of SAH. CBF decreased rapidly, and then gradually recovered to 70-80% of baseline. The urine volume and total Na excretion were significantly increased in comparison to those of the sham (P < 0.05). The serum Na level was significantly decreased at 4 days following SAH (P < 0.05). The present results demonstrated for the first time that rats with SAH exhibited excessive natriuresis. The endovascular puncture model is suitable for investigating hyponatremia that occurs concomitantly with natriuresis and diuresis after SAH.

Pathophysiological relevance of the CD14 receptor in surgical patients: biological activity of endotoxin is regulated by the CD14 receptor.

Endotoxins (lipopolysaccharides, LPSs) are potent bacterial poisons, and they are always present in the intestine in considerable numbers. Stress, such that as a resulting from multiple injuries, burns, hypovolemia, hypoxia, intestinal ischemia, and surgery can lead to a breakdown of the gut barrier, allowing endotoxins to enter the systemic circulation via translocation. However, estimating the biological activity of translocated circulating endotoxins and identification of the mechanisms regulating their biological activities remain complex problems. CD14 has been found to exist as a soluble protein in the serum and as a glycosylphosphatidylinositol (GPI)-anchored protein of myeloid lineage cells. It plays key roles in both LPS-induced activation and in LPS internalization by cells. In this article, we outline: (i) the biological activity of circulating endotoxin; and (ii) the role of membrane and/or soluble CD14 regulating the bioactivity of circulating endotoxin in a human model of postoperative endotoxemia.

Significance of lymphoscintigraphic mapping with Tc-99m human serum albumin and tin colloid in sentinel lymph node biopsy in breast cancer.

Sentinel lymph node biopsy (SLNB) in breast cancer is considered in order to spare node-negative patients from axillary lymph node dissection. To assess the clinical significance of lymphoscintigraphic mapping in SLNB, we analyzed the lymphatic drain to the sentinel lymph nodes (SLNs) in terms of the pattern and direction of the hot spot. Twenty-three breast cancer patients were enrolled for SLNB. Before surgery, lymphoscintigraphic mapping of SLN was performed using Tc-99m human serum albumin (HSA) and tin colloids, and the hot spot was marked. The Tc-99m HSA and tin colloids were subcutaneously injected above the tumor and peritumor sites, respectively, and lymphoscintigraphic scanning was monitored every 5 to 10 min, for up to 2 h after injection. The SLN was identified using a combination of a blue dye, indigocalmine, and a gamma probe during surgery. The hot spot pattern and direction of the lymphatic drains were evaluated in 21 of 23 cases. Two cases did not have a hot spot. Single, double, and multiple hot spots were observed in 12 cases (52.1%), 8 cases (34.7%), and 1 case (4.3%), respectively. The positions of the hot spots were: axillary (n=17, 80.9%), axillary and sternal (n=3, 14.2%), and phrenic (n=1, 4.7%). The sensitivity and specificity rates in SLNB were 66.6% and 100%, respectively, and the overall predictive rate was 85.7%. Lymphoscintigraphy produced false negatives in three cases (33.3%), including one on the phrenic side. Lymphoscintigraphic mapping with Tc-99m HSA and tin colloids is useful for determining the SLN, and avoiding a false negative. The pattern and direction of the lymphatic drain to the SLN in scintigraphy need to be considered for the elimination of axillary lymph node dissection in node-negative patients with breast cancer.

Tonic activity during visuo-oculomotor behavior in the monkey superior colliculus.

The purpose of this study was to examine whether the superior colliculus is involved in intermediary cognitive processes such as memory, movement preparation, and peripheral attention. To answer this question, we recorded single cell activities in the superior colliculus of monkeys trained to perform a series of visuo-oculomotor tasks: delayed saccade task (SACD), saccade task with overlap target (SACO), and attention task (ATT). We recorded 141 neurons showing tonic activities related to the tasks. Depending on the predominance of the activities among the three tasks, we classified the tonic neurons into four types: (1) visuomotor (greater activity in SACO), (2) mnemonic motor (SACD dominant), (3) attention (ATT), and (4) nonspecific. Among 108 neurons recorded in the intermediate layer, 13 were of a visuomotor type, 15 were of a mnemonic motor type, and 13 were of an attention type. The other 67 neurons were of a non-specific type. Of the 33 neurons in the superficial layer, many neurons were of the non-specific type. These results suggested that the tonic activities in the superior colliculus are related to memory of the target location, preparation of saccades and peripheral attention.

A pre-operative chemoembolization therapy using lipiodol, cisplatin and gelatin sponge for hepatocellular carcinoma.

The significance of pre-operative transcatheter arterial chemoembolization therapy using lipiodol, cisplatin and gelatin sponge (Gelfoam) for the prevention of the recurrence of hepatocellular carcinoma (HCC) was evaluated. On the 103 patients who underwent radical operations for HCC with a tumor size less than 10 cm, 52 patients received no pre-operative therapy (group C), and 51 patients received pre-operative chemoembolization using lipiodol, a chemotherapeutic agent and Gelfoam. Of these 51 patients, 37 patients received a combination of lipiodol, cisplatin and Gelfoam (group A), while the remaining 14 patients received lipiodol, adriamycin and Gelfoam (group B). The disease-free survival rates after surgery were compared between group A, group B and group C. The 2-year disease-free survival rates in group A, group B and group C were 72%, 46% and 54%, respectively. These rates therefore suggest that pre-operative chemoembolization using lipiodol, cisplatin and Gelfoam is a useful method to prevent the recurrence of HCC after surgery.

Excitotoxic lesions of the pedunculopontine tegmental nucleus produce contralateral hemiparkinsonism in the monkey.

Dopaminergic nigrostriatal neurons, degeneration of which causes Parkinson's disease, are known to receive excitatory input almost exclusively from the pedunculopontine tegmental nucleus (PPN). We report here that excitotoxic lesions of the PPN produce abnormal motor signs relevant to hemiparkinsonism in the macaque monkey. Under the guidance of extracellular unit recordings, the electrophysiologically identified PPN was injected unilaterally with kainic acid. These PPN-lesioned monkeys exhibited mild to moderate levels of flexed posture and hypokinesia in the upper and lower limbs contralateral to the lesion. In most of the monkeys, such pathophysiological events were gradually improved and became stationary in 1-2 weeks. The hemiparkinsonian symptoms observed after PPN destruction might be ascribed to a decrease in nigrostriatal neuron activity due to excitatory input ablation.

Exogenous microglia enter the brain and migrate into ischaemic hippocampal lesions.

We compared migration of systemically injected microglia into normal brain vs. ischaemic brain using a model of ischaemic hippocampal lesion. Microglia were labeled by a fluorescent dye using our standard phagocytosis procedure of microscopic particles and then injected intra-arterially into Mongolian gerbils subjected to ischaemia reperfusion neuronal injury. Delayed death of pyramidal neurons was confirmed by conventional histological analysis and dUTP nick end labeling (TUNEL) method. Clusters of dye-tagged cells migrating into the hippocampal ischaemic lesions were confirmed histochemically to be microglia. Since peripherally injected microglia exhibit specific affinity for ischaemic brain lesions and does not exacerbate ischaemic neuronal injury in the present model, we suggest that microglia may have a potential to be used as a piggy-back ride to deliver therapeutic genes and/or drugs for CNS repair following transitory global ischaemic insult.

Electrically controlled proliferation of human carcinoma cells cultured on the surface of an electrode.

Human carcinoma cells, MKN45, were cultured on the surface of a metal-coated plastic plate electrode the potential of which was controlled. The proliferation rate and cell morphology were altered depending on the applied potential. Cell proliferation was halted in the potential range above 0.4 V vs. Ag/AgCl, although cells started to proliferate again when the applied potential was shifted from 0.4 V to 0.1 V vs. Ag/AgCl. Fluorescence probe studies indicated that the fluidity of plasma membrane decreased in association with halting of cell proliferation. These results suggest that electrical stimulation causes cells to temporarily halt proliferation, and that cell proliferation was reversibly controlled by electrode potential. The mechanism is interpreted in relation to the change of plasma membrane structure represented by membrane fluidity.

Glycylproline dipeptidyl aminopeptidase and gamma-glutamyl transpeptidase in human hepatic cancer and embryonal tissues.

Changes of glycylproline dipeptidyl aminopeptidase (GPDA) and gamma-glutamyl transpeptidase (gamma-GTP) activities and their subcellular distributions were compared in human hepatic cancer and embryonal tissues. The activity of GPDA in cancer tissues was significantly higher than that found in healthy liver, though there were no significant differences between fetal and adult livers. The placenta, however, had the highest GPDA activity. The activity of gamma-GTP, on the other hand, was increased significantly not only in cancer tissues but also in live tissues adjacent to the tumor, and it was higher in the fetal liver but much lower in the placenta. Subcellular distribution of GPDA was also different from that of gamma-GTP in cancer tissues, especially after postmortem changes. These results suggest the possibility that GPDA has carcinoembryonic characters similar to gamma-GTP, but the mechanisms, whereby serum activities of these two enzymes were increased in hepatocellular carcinoma patients, are different from each other.