Species differences in androgen receptor expression in the medial preoptic and anterior hypothalamic areas of adult male and female rodents.

The medial preoptic and anterior hypothalamic areas (MPO/AH) are important androgen targets regulating homeostasis, neuroendocrinology and circadian rhythm as well as instinctive and sociosexual behaviors. Although species differences between rats and mice have been pointed out in terms of morphology and physiology, detailed distributions of androgen receptor (AR) have never been compared between the two rodents. In the present study, AR distribution was examined immunohistochemically in serial sections of the MPO/AH and compared for adult rats and mice. Western blotting and immunohistochemistry clearly demonstrated that AR expression in the brain was stronger in mice than in rats and was stronger in males than in females. In addition, we found (1) an "obliquely elongated calbindin-ir cell island" in mice medial preoptic nucleus (MPN) expressed AR intensely, as well as the sexually dimorphic nucleus in the MPN (SDN-MPN) in rats, strongly supporting a "putative SDN-MPN" previously proposed in mice; (2) AR expression in the suprachiasmatic nucleus (SCN) was much more prominent in mice than in rats and differed in localization between the two species; (3) a mouse-specific AR-ir cell cluster was newly identified as the "tear drop nucleus (TDN)", with male-dominant sexual dimorphism; and (4) two rat-specific AR-ir cell clusters were also newly identified as the "rostral and caudal nebular islands", with male-dominant sexual dimorphism. The present results may provide basic morphological evidence underlying species differences in androgen-modified psychological, physiological and endocrinergic responses. Above all, the findings of the mouse-specific TDN and differing AR expression in the SCN might explain not only species difference in gonadal modification of circadian rhythm, but also distinct structural bases in the context of transduction of SCN oscillation. The current study could also serve as a caution that data on androgen-sensitive functions obtained from one species should not always be directly applied to others among rodents.

Establishment of feeder-independent cloned caprine trophoblast cell line which expresses placental lactogen and interferon tau.

A feeder-independent cloned trophoblast cell line, HTS-1, was established from a mature placenta of Shiba goat (Capra hircus). During the growth phase, single HTS-1 cells exhibited ruffled membranes or lamellipodia often accompanied by elongated cell shape, indicating highly motile nature of the cells. At or near confluence, HTS-1 cells formed monolayers with few sign of cellular overlapping. Binucleate cells were found at a high frequency especially in the peripheral regions of monolayers. In small colonies and the monolayers, majority of HTS-1 cells assumed polygonally shaped cobble-stone like morphology characteristic to epithelial cells, although considerable variations in cellular morphology were observed despite of repeated cloning. Time-lapse video recordings of HTS-1 cells during culture revealed that not only the small colonies but also the monolayers near or at confluence were remarkably motile, often causing extreme elongation of the cells within them. The extremely plastic nature of HTS-1 cells in vitro is likely to be the reflection of the extraordinary capacity of caprine trophoblast cells to be stretched to extreme thinness in vivo as shown by electron microscopy. HTS-1 cells cultured on matrigel are highly invasive, and express MT1-MMP which, in the mouse, has been known to be expressed at the invasive edge of trophoblast both in vitro and in vivo. HTS-1 cells express placental lactogen (PL) and interferon-tau (IFNtau), as confirmed by immunocytochemistry, Western blotting and RT-PCR analysis. Both PL and IFNtau expression in the cells appeared to be down-regulated by cell-cell contact. In the medium conditioned by HTS-1 cells, the presence of secretory form of PL and IFNtau was confirmed by Western blotting. The HTS-1 cell line will serve as a useful in vitro model for the analysis of the molecular and/or cellular mechanisms underlying synepitheliochorial placentation in bovidae animals.

Efficient production of a heterologous peroxidase, DyP from Geotrichum candidum Dec 1, on solid-state culture of Aspergillus oryzae RD005.

The productivity of a peroxidase (DyP) originating from Geotrichum candidum Dec 1 was enhanced in the solid-state culture using Aspergillus oryzae RD005. When the humidity, water content, and temperature were adjusted to 60%, 50% and 27 degrees C, respectively, the productivity of DyP reached 5.3 g per kilogram wheat bran, which was used as the solid medium. The yield of 5.3 g per kg wheat bran corresponded to the yield of a 56 kg submerged culture. The productivity per gram carbon of the medium in the solid-state culture was 4.1-fold that in the submerged culture.

Basal nitric oxide production is enhanced by hydraulic pressure in cultured human periodontal ligament fibroblasts.

To understand orthodontic tooth movement and determine optimal orthodontic force from a biological viewpoint, nitric oxide production in cultured human periodontal ligament fibroblasts was measured at varying levels of hydraulic pressure. The fibroblasts in a culture flask were exposed to the controlled change in hydraulic pressure, and intracellular nitric oxide levels were measured in real time by a nitric oxide-binding fluorescent dye, diaminofluorescein-2. The fibroblasts produced a significantly larger amount of nitric oxide at the pressure of 75 and 100 mmHg, compared with the pressure of 0, 25, and 50 mmHg (P <.0001, one-way ANOVA, and P <.05, Tukey-Kramer test). Immunohistochemically, the cultured fibroblasts expressed brain nitric oxide synthase. The pressure level to enhance nitric oxide production was comparable to the magnitude of clinically used orthodontic force (80 g/cm(2)). Nitric oxide might be a key regulator in orthodontic tooth movement.

Improvement of bond strength in metal-ceramic systems using a gold intermediate layer.

The mechanism of bonding between metal and ceramic in systems using the functionally graded method with pure gold and gold mixture as a primer was examined. Four types of samples, porcelain, porcelain-gold, porcelain-metal and porcelain-gold-metal were prepared. The gold intermediate layer was fired at 1000 degrees C. For porcelain and metal, low-fusing opaque, body porcelain and palladium alloy were used. The intermediate layer was composed of three layers; pure gold, gold-palladium and gold-porcelain layer. During the bending test of each sample, the porcelain peeled away from the porcelain-metal system, while porcelain with the gold intermediate layer remained on the metal surface even after maximal loading. The bond strength of the porcelain-gold-metal system was much higher than that of the porcelain-metal system, and the toughness of the former was much greater than that of the latter. Laser microscopy and scanning electron microscopy (SEM) showed a smooth interface between the intermediate layer and the metal which suggested proper chemical bonding, and no gap was observed. At the interface between the porcelain and the gold intermediate alloy, a good mechanical anchor lock was observed. Electron probe microanalysis (EPMA) showed a clear distribution of each element (e.g. Si, Au and Pd) in the porcelain, gold intermediate layer and metal frame.

[Sudden syncope during spinal block under sitting position: a report of three cases].

We reported 3 cases of sudden syncope during saddle block under sitting position. Patients were healthy and had no history of fainting. Syncope occurred following hypotension and bradycardia during difficult lumbar dural punctures under sitting position. Patients were treated successfully by changing position to supine, elevating both legs and giving vasopressors. Neurocardiac syncope due to the activation of afferent cardiac C-fiber has been suggested as a possible explanation in sudden syncope, which follows head-up position or emotional change. The first sign of syncope was hypotension and bradycardia due to cardiac C-fiber reflex. How to prevent this NCS under saddle block are as follows; 1. vigorous search for history of syncope, 2. pay attention to the patients during spinal tap, 3. skillful technique in spinal tap, and 4. proper premedication including anticholinergic agents. Treatments include 1. changing position to supine, 2. elevation of both legs to increase ventricular end-diastolic pressure, and 3. use of vasopressors including phenylephrine.

Intracellular calcium response to hydraulic pressure in human periodontal ligament fibroblasts.

Human periodontal ligament fibroblasts in culture were exposed to the controlled change in hydraulic pressure and were monitored continuously with an electric pressure gauge, and the concentration of intracellular calcium was measured in real time by a calcium-binding fluorescent dye, fluo-3. The elevation of hydraulic pressure to a level ranging from 20 to 50 mm Hg induced transient elevation of the intracellular calcium concentration in about 10% of the fibroblasts observed, indicating that these cells could respond to the pressure change. The results supported further an idea that periodontal ligament fibroblasts, responding to the pressure exerted by orthodontic force, would initiate the chain of events in orthodontic tooth movement, including alveolar bone remodeling. The threshold level of pressure (27 to 68 g/cm2) obtained in this experiment, at which the fibroblasts started to respond, would provide a biochemical basis to determine the optimal magnitude of stress for clinical orthodontics.

Production and characterization of single-chain tissue-type plasminogen activator produced by an established cell line from human uterine muscle.

This report describes the purification and characterization of single-chain tissue-type plasminogen activator (sct-PA) present in tissue culture medium of a cell line established from human uterine muscle. The cell line used for the experiment, KW, had estrogen receptor. The PA fraction (KW-PA) was purified from the tissue culture medium of KW employing several steps of affinity chromatography and gel filtration in the presence of aprotinin. The final product (KW-PA) of purification, which predominantly contained the inactive form of sct-PA as well as active sct-PA to a lesser extent, revealed a single band with a molecular weight of 70,000 on sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis both in the absence and presence of reducing agent. Electrophoretic enzymography demonstrated a single lytic zone at Mr 70,000. When KW-sct-PA was treated with plasmin, SDS-polyacrylamide gel electrophoresis revealed two bands of Mr 37,000 and 33,000 under reduced conditions. Such plasmin treatment of KW-sct-PA enhanced the enzymatic activity as well as the [3H]DFP incorporation significantly. The KW-sct-PA demonstrated a higher affinity for lysine than did melanoma-t-PA, but the fibrin affinity of KW-sct-PA was identical with that of melanoma-t-PA. Circular dichroism (CD) analysis showed that the CD spectra of KW-sct-PA were different from those of melanoma-t-PA. These results suggest that the single-chain inactive form of t-PA which was obtained from the tissue culture medium of the cell line from human uterine muscle is activated to a two-chain form on plasmin treatment, with an accompanying significant increase in enzymatic activity.

Characterization of plasminogen activator produced by an established cell line from human ovary.

An established cell line (OC-1) was obtained from human ovarian tissue, which yielded a high concentration of plasminogen activator (PA) in the culture medium. The PA (OC-1-PA) produced by the cell line was purified and compared with urokinase (UK), proform of UK (pro-UK), and tissue-type PA (t-PA) purified from human melanoma cells (Bowes). OC-1-PA was purified by Zn chelate-Sepharose affinity chromatography followed by high-performance liquid chromatography with a Zn chelate-5PW column and with a p-amino-benzamidine-5PW column, giving a yield of 58.3% and a purification factor of 15,439. This purified material revealed a single band of Mr 55,000 on sodium dodecylsulfate polyacrylamide gel electrophoresis in the presence or absence of reducing agents. Electrophoretic enzymography demonstrated that the Mr 55,000 protein band had a plasminogen-dependent fibrinolytic activity. Treatment with plasmin did not change the Mr even in the presence of reducing agents. These results suggest that OC-1-PA has a single-chain structure protected from protease degradation, which is completely different from UK. The activator had higher affinities for lysine and fibrin than those of UK or pro-UK. An immunological study demonstrated that OC-1-PA cross-reacted with anti-UK IgG but not with anti-t-PA IgG. All these findings indicate that OC-1-PA belongs immunologically to the UK type, but its structure differs from that of UK.

New type of plasminogen activator produced by an established cell line from human ovary.

An established cell line (OC-1) was obtained from human ovarian tissue, which yielded a high concentration of plasminogen activator (PA) in the culture medium. The PA (OC-1-PA) produced by the cell line was purified and compared with urokinase (u-PA), proform of UK (scu-PA), and tissue-type PA (t-PA) purified from human melanoma cells (Bowes). OC-1-PA was purified by Zn chelate-Sepharose affinity chromatography followed by high performance liquid chromatography with a Zn chelate-5PW column and with a p-aminobenzamidine-5PW column, giving a yield of 58.3% and a purification factor of 15,439. This purified material revealed a single band of Mr 55,000 on sodium dodecylsulfate polyacrylamide gel electrophoresis in the presence or absence of reducing agents. Electrophoretic enzymography demonstrated that the Mr 55,000 protein band had a plasminogen-dependent fibrinolytic activity. Treatment with plasmin did not change the Mr even in the presence of reducing agents. These results suggest that OC-1-PA has a single-chain structure protected from protease degradation, which is completely different from UK. The activator had higher affinities for lysine and fibrin than those of u-PA or scu-PA. An immunological study demonstrated that OC-1-PA cross-reacted with anti-u-PA IgG but not with anti-t-PA IgG. All these findings indicate that OC-1-PA belongs immunologically to the u-PA type, but its structure differs from that of u-PA.

Urinary excretion rate of antithrombin III related antigen in cerebral stroke.

The urinary excretion rate of antithrombin III related antigen (AT III RA) was examined in cerebral stroke. The excretion rate of AT III RA in cerebral hemorrhage (CH) was 12.33 +/- 1.61 X 10(-4) ml/min. The patients with CH were further classified into two groups: in group CH-I, whose consciousness state was stupor or further deteriorated including coma on admission, the excretion rate of AT III RA was 18.08 +/- 2.50 X 10(-4) ml/min. In group CH-II, whose consciousness state was clear on admission, the excretion rate of AT III RA was significantly lower than that in CH-I (6.20 +/- 1.56 X 10(-4) ml/min). The excretion rate in cerebral thrombosis (CT) was 1.96 +/- 0.25 X 10(-4) ml/min, which was significantly lower than that in CH. The excretion rate of AT III RA in both CH and CT was significantly higher than that in the healthy control group (0.29 +/- 0.04 X 10(-4) ml/min). Thus, AT III may change dynamically in cerebral stroke.

Characterization of the biological activity of antithrombin III in patients with hepatic cirrhosis.

A discrepancy was found in hepatic cirrhosis patients between two forms of biological activities, i.e. progressive antithrombin activity (prog AT) and heparin cofactor activity (heparin AT). A reduced level of heparin AT was obtained in comparison with that of prog AT, despite the fact that both activities correlated well in normal controls. The binding ability of AT III to heparin was observed by crossed immunoelectrophoresis, and showed a reduced second peak of AT III with a faster AT III with a qualitative defect in its binding ability to heparin due to impaired protein synthesis.

Determination of tissue plasminogen activator by an enzyme-immunoassay method.

The sensitive assay method of tissue plasminogen activator was established by an enzyme-immunoassay method, and discriminates tissue (nonurokinase) type plasminogen activator from urokinase. The sensitivity was 0.1 ng/assay tube, and the plasma concentration of tissue plasminogen activator in normal healthy subjects was 1.22 +/- 0.25 ng/ml. Distribution of tissue plasminogen activator was examined in normal tissue. A melanoma cell line was employed as cell culture medium for determination of tissue plasminogen activator.

Production of plasminogen activator in a melanoma cell line.

A melanoma cell line (Bowes) was found to produce plasminogen activator even on its growing phase, and the rate of plasminogen activator production was rather constant. The production of plasminogen activator was proportional to the cell number. Morphologically, no specific features for plasminogen activator production were seen. Plasminogen activator was observed in the lysate of this cell line only when the cell number was large. The extracellular plasminogen activator activity was higher than the intracellular plasminogen activator activity, suggesting the existence of a secretion mechanism for the plasminogen activator.

Production of plasminogen activator in a melanoma cell line.

A melanoma cell line (Bowes) was found to produce plasminogen activator even on its growing phase, and the rate of plasminogen activator production was rather constant. The production of plasminogen activator was proportional to the cell number. Morphologically, no specific features for plasminogen activator production were seen. Plasminogen activator was observed in the lysate of this cell line only when the cell number was large. The extracellular plasminogen activator activity was higher than the intracellular plasminogen activator activity, suggesting the existence of a secretion mechanism for the plasminogen activator.