Alteration of leukotriene C4 levels in experimental spinal cord injury]

Leukotriene C4 (LTC4) production in the guinea pig spinal cord following compression injury was determined by radioimmunoassay, in the same way thromboxane B2 (TXB2), a stable metabolite of thromboxane A2 (TXA2), was also measured. When the spinal cords were compressed under a 20 gram weight for 10 minutes, LTC4 levels reached peak values (2.2 +/- 0.4 pmol/g cord) 10 minutes after release, then gradually decreased until being undetectable 60 minutes later. TXB2 levels reached peak values (146.8 +/- 6.2 pmol/g cord) 5 minutes after the release from compression, the TXB2 level then gradually decreased, but remained at about 1/2 of the peak level even 60 minutes after the release. When the spinal cords were compressed with various weights, TXB2 production depended on the degree of compression, while LTC4 production was not affected by the compression injury. The LTC4 production confirmed in the injured spinal cords is suggestive of its relation to secondary disorders after spinal cord injury, spinal edema, in particular.     

Tachykinin receptor cross-talk. Immunological cross-reactivity between the external domains of the substance K and substance P receptors.

In the present study, we have chemically synthesized peptides which correspond to the four putative extracellular domains of the predicted substance K (SK) receptor protein and raised specific polyclonal antibodies against these peptides. These antibodies were then tested for both structural and functional recognition of epitopes on the substance P (SP) receptor on rat AR42J pancreatic cells and human IM9 lymphoblasts, which express the SP receptor, but not the SK receptor. Antibodies directed against the first, second, and fourth external domains of the predicted SK receptor recognized a 58-kDa protein on AR42J cells. This protein has a molecular weight similar to the previously demonstrated SP receptor on both AR42J cells and IM9 cells. Furthermore, antibodies against the second and fourth extracellular domains significantly inhibited specific 125I-SP binding on both AR42J and IM9 cells, and also significantly inhibited SP-induced mobilization of [Ca2+]i on AR42J cells. These data suggest that the second and fourth extracellular domains of the SK and SP receptors may share common structural motifs for ligand binding and signaling mechanism.     

Solubilization and characterization of the pyrilamine-binding protein from cultured smooth muscle cells

The cultured smooth muscle cell line DDT1MF-2 expresses a large number (9.7 x 10(6) receptors/cell) of functional histamine H1-type receptors [J. Cell. Physiol. 134:367-375 1988]. Two different binding assays, gel filtration and polyethylene glycol precipitation, indicated that the [3H]pyrilamine binding activity was solubilized by 1% digitonin with binding characteristics similar to those of intact cells. The solubilized proteins were then purified by sequential gel filtration, chromatofocusing, and reverse phase high pressure liquid chromatography. The calculated molecular weight of this purified pyrilamine-binding protein was 38-40 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. [3H]Pyrilamine binding to these 38-40-kDa proteins indicated a single class of binding site with a Kd of 288 nM, which is equivalent to that of intact cells and digitonin-solubilized proteins. The computer analysis Scatfit also indicated that one molecule of [3H]pyrilamine bound to one molecule of purified protein. Furthermore, a polyclonal antibody raised against the purified protein recognized the 38-40-kDa band by Western blotting techniques, specifically bound to the cell surface of DDT1MF-2 cells, and inhibited [3H]pyrilamine binding to these cells in a dose-dependent manner. These data strongly suggest that the purified 38-40-kDa protein is part of an antagonist binding domain on the histamine H1 receptor on DDT1MF-2 cells.     

Climbing exercise increases bone mass and trabecular bone turnover through transient regulation of marrow osteogenic and osteoclastogenic potentials in mice.

To investigate the relationship between the effects of bone turnover and bone marrow cell development in bone cells, we developed a mouse voluntary climbing exercise model. Climbing exercise increased bone volume and transient osteogenic potential of bone marrow. This model would be suitable for investigating the mechanistic roles of mechanical loading. INTRODUCTION: The relationship between bone mass gain and local bone formation and resorption in mechanically loaded bone is not well understood. MATERIALS AND METHODS: Sixty-five C57BL/6J mice, 8 weeks of age, were assigned to five groups: a baseline control and two groups each of ground control and climbing exercise mice for 2 and 4 weeks. Mice were housed in a 100-cm tower and had to climb toward a bottle placed at the top to drink water. RESULTS: Compared with the ground control, bone mineral density of the left femur increased in the climbing mice at 4 weeks. At 2 and 4 weeks, bone formation rate (BFR/BS) of periosteal surface, the cross-sectional area, and moment of inertia were increased in the climbing mice, whereas BFR/BS and eroded surface (ES/BS) of endosteal surface did not differ. The trabecular bone volume (BV/TV) of the proximal tibia increased in climbing mice, and osteoclast surface (Oc.S/BS) and osteoclast number decreased at 2 weeks. At 4 weeks, there were increases in BV/TV and parameters of bone formation, including mineralized surface, mineral apposition rate, and bone formation rate. In marrow cell cultures from the tibia, the number of alkaline phosphatase+ colony forming units-fibroblastic and the area of mineralized nodule formation in climbing mice were increased, and the number of osteoclast-like TRACP+ multinucleated cells was lower at 2 weeks. At 4 weeks, these parameters recovered to the levels of the ground controls. CONCLUSION: Our results indicate that climbing increased trabecular bone volume and reduced bone resorption, with a subsequent increase in bone formation. Intermittent climbing downregulates marrow osteoclastogenic cells and upregulates osteogenic cells initially, but further exercise seemed to desensitize them. Cortical envelopes were enlarged earlier, but the response seems to differ from trabecular bone.     

Accumulation of filamentous tau in the cerebral cortex of human tau R406W transgenic mice

Missense mutations of the tau gene cause autosomal dominant frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), an illness characterized by progressive personality changes, dementia, and parkinsonism. There is prominent frontotemporal lobe atrophy of the brain accompanied by abundant tau accumulation with neurofibrillary tangles and neuronal cell loss. Using a hamster prion protein gene expression vector, we generated several independent lines of transgenic (Tg) mice expressing the longest form of the human four-repeat tau with the R406W mutation associated with FTDP-17. The TgTauR406W 21807 line showed tau accumulation beginning in the hippocampus and amygdala at 6 months of age, which subsequently spread to the cortices and subcortical areas. The accumulated tau was phosphorylated, ubiquitinated, conformationally changed, argyrophilic, and sarcosyl-insoluble. Activation of GSK-3beta and astrocytic induction of mouse tau were observed. Astrogliosis and microgliosis correlated with prominent tau accumulation. Electron microscopic examination revealed the presence of straight filaments. Behavioral tests showed motor disturbances and progressive acquired memory loss between 10 to 12 months of age. These findings suggested that TgTauR406W mice would be a useful model in the study of frontotemporal dementia and other tauopathies such as Alzheimer's disease (AD).     

Evaluation of time-resolved fluorometric immunoassay of CA 50

Serum CA 50 was determined by a time resolved fluorometric immunoassay (TR-FIA) with CANAG CA-50 DELFIA kit. Evaluation of the assay system gave satisfactory results in its sensitivity, accuracy, reproducibility, dynamic range and easy handling. No prozone phenomenon was observed up to 347,000 U/ml. From a histogram of 134 normal sera, the cut off point was determined at 34 U/ml. CA 50 in 202 patients' sera was determined with this assay. Nineteen of 20 patients pancreatic cancer, 6 of 21 gastric cancer, 14 of 25 hepatoma gave positive values. In comparison with CA 19-9, higher values and higher rates of positive CA 50 were observed in benign and malignant liver diseases, suggesting its non-cancerous origin in the liver. A high correlation was observed between the level of CA 50 and CA 19-9 of 157 patients' sera. Serum CA 50 was completely correlated with CA 19-9 in the clinical course of patients with pancreatic cancer, but not in patients with hepatoma. Thus we conclude that the CANAG CA-50 DELFIA System is useful for the diagnosis and monitoring cancer patients but must be used with care because of its elevation in benign liver diseases.