Gastric inhibitory polypeptide as an endogenous factor promoting new bone formation after food ingestion

Calcium plays a fundamental role as second messenger in intracellular signaling and bone serves as the body's calcium reserve to tightly maintain blood calcium levels. Calcium in ingested meal is the main supply and inadequate calcium intake causes osteoporosis and bone fracture. Here, we describe a novel mechanism of how ingested calcium is deposited on bone. Meal ingestion elicits secretion of the gut hormone gastric inhibitory polypeptide (GIP) from endocrine K cells in the duodenum. Bone histomorphometrical analyses revealed that bone formation parameters in the mice lacking GIP receptor (GIPR(-/-)) were significantly lower than those of wild-type (GIPR(+/+)) mice, and that the number of osteoclasts, especially multinuclear osteoclasts, was significantly increased in GIPR(-/-) mice, indicating that GIPR(-/-) mice have high-turnover osteoporosis. In vitro examination showed the percentage of osteoblastic cells undergoing apoptosis to be significantly decreased in the presence of GIP. Because GIPR(-/-) mice exhibited an increased plasma calcium concentration after meal ingestion, GIP directly links calcium contained in meal to calcium deposition on bone.     

Purification and spectral characterization of a 121-kilodalton phytochrome from etiolated pea (Pisum sativum L.) seedlings

Procedures for the purification of native phytochrome from etiolatedpea seedlings without the use of immuno-purification techniquesare described. Phytochrome (in the P_FR form) was purified bypolyethyleneglycol fractionation, adsorption to pentyl agaroseand batch elution, chromatography on DEAE-Sepharose, adsorptionto phenyl Toyopearl and batch elution, and chromatography onRed Toyopearl. The resulting phytochrome had specific absorbanceratios (SAR = A₆₆₆/A₂₈₀ of P_R) that ranged from 0.55 to 0.6.The subsequent chromatography on Sephacryl S-300 yielded verypure phytochrome with a SAR of 0.98. P_R and P_FR peaks in thedifference spectrum of the phytochrome were centered at 665and 730 nm, respectively. The spectral change ratio (Ar/Afr)of the difference spectrum was unchanged after the chromatographyon phenyl Toyopearl, and the value was 1.05–1.08, indicatingthat the spectral properties of this preparation were intact.The absorption spectra indicated that the peak absorbance ofP_FR was at 728–730 nm and that of P_R was at 666–667nm. These peak positions were essentially same as those obtainedwith the undegraded oat phytochrome. Incubation of the samplepurified on Sephacryl S-300 at 25?C for 5 h in either the P_Ror P_FR form did not result in degradation of the molecule. Therate of dark reversion of P_FR observed with the purified peaphytochrome was similar to that observed in vivo. The additionof dithionite had no effect on the reversion rate. ²Present address: Fuji-Gotenba, Research Lab. of Chugai PharmaceuticalCo. Ltd., Gotenba, Shizuoka, 412 Japan (Received February 22, 1990; Accepted May 28, 1990)     

Kinetochore stretching-mediated rapid silencing of the spindle-assembly checkpoint required for failsafe chromosome segregation

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Electromyographic characterization of walking behavior initiated spontaneously in crayfish

Crayfish initiate walking behavior not only reflexively in response to external stimuli but also spontaneously in the absence of any specific stimulus. In order to analyze the initiation mechanism underlying these different types of walking, we made simultaneous electromyographic (EMG) recordings from thoracic legs when animals initiated walking, either reflexively or spontaneously, and video recorded their movements synchronously with the EMG recording. Two different stimuli, mechanical and chemical, were used to reflexively induce walking. A non-rhythmic, sustained activation of leg muscles was found to precede the behavioral initiation of either type of walking. The duration of this non-rhythmic muscle activation was significantly longer in the spontaneously initiated walking than in the mechanical stimulus-evoked walking, although no difference was observed between the spontaneous and chemical stimulus-evoked walking. EMG recordings from all eight legs revealed that their non-rhythmic muscle activation occurred almost simultaneously prior to initiation of rhythmical stepping movements. When an animal was suspended without a leg substratum, the timing of muscle activation was more variable among the legs than in the free condition on the substratum. When the circumesophageal commissures were both severed to eliminate signals descending from the brain to the thoracic ganglia, the bilaterally coordinated rhythmic burst activity was not observed in the walking legs. These findings suggest that the spontaneous initiation of walking behavior requires sensory feedback signals from leg proprioceptors, subserved by a different descending activation mechanism from that for stimulus-driven initiation of walking.     

GEP100 links epidermal growth factor receptor signalling to Arf6 activation to induce breast cancer invasion

Epidermal growth factor (EGF) receptor (EGFR) signalling is implicated in tumour invasion and metastasis. However, whether there are EGFR signalling pathways specifically used for tumour invasion still remains elusive. Overexpression of Arf6 and its effector, AMAP1, correlates with and is crucial for the invasive phenotypes of different breast cancer cells. Here we identify the mechanism by which Arf6 is activated to induce tumour invasion. We found that GEP100/BRAG2, a guanine nucleotide exchanging factor (GEF) for Arf6, is responsible for the invasive activity of MDA-MB-231 breast cancer cells, whereas the other ArfGEFs are not. GEP100, through its pleckstrin homology domain, bound directly to Tyr1068/1086-phosphorylated EGFR to activate Arf6. Overexpression of GEP100, together with Arf6, caused non-invasive MCF7 cells to become invasive, which was dependent on EGF stimulation. Moreover, GEP100 knockdown blocked tumour metastasis. GEP100 was expressed in 70% of primary breast ductal carcinomas, and was preferentially co-expressed with EGFR in the malignant cases. Our results indicate that GEP100 links EGFR signalling to Arf6 activation to induce invasive activities of some breast cancer cells, and hence may contribute to their metastasis and malignancy.     

Preparation of a conjugate of 2',5'-triadenylate 5'-triphosphate and biotinylated firefly luciferase and its use for sensitive bioluminescent enzyme immunoassay

A bioluminescent enzyme immunoassay (BLEIA) for 2',5'-oligoadenylate 5'-triphosphate (pppA2'p5'A2'Ap5'A, 2-5A) was developed using a conjugate of 2-5A and firefly luciferase as a probe. The conjugate was prepared from a 2-5A derivative bearing a thiol group at the 2'(or 3') end, and streptavidin (SA) bearing maleimide via a linker-arm and biotinylated luciferase. The thiol group of the 2-5A derivative was relatively stable under aerobic conditions and remained 100% and 56% intact at 25 degrees C after 1 and 96 h, respectively, without dimerization by aerobic oxidation. The thiol-modified 2-5A was coupled with the SA-maleimide conjugate, followed by complex formation with biotinylated firefly luciferase. BLEIA using this conjugate allowed for the direct analysis of 2-5A in a range of 5-500 fmol (0.1-10 nM in a 50 microL sample) and in a reaction time of 15 min. The measurement of microquantities of 2-5 A from various sources is easy to perform by this BLEIA, because no radioisotope labeled 2-5A was required as a probe.     

Genetic analysis of rooting ability of transplanted rice (Oryza sativa L.) under different water conditions

In order to assess the benefits of superior rooting ability of rice (Oryza sativa L.) for growth after transplanting under water-limiting conditions, genetic differences in the rooting ability of rice seedlings 30 d after sowing, with their visible roots either pruned or not pruned, were quantified by several root parameters 4 d after transplanting (DAT), under flooded or non-flooded paddy fields (four treatments in total), together with production traits at maturity. Ninety-eight recombinant inbred lines from the two japonica ecotypes, a lowland variety Otomemochi, and an upland variety Yumenohatamochi, were genotyped with 107 simple sequence repeat (SSR) markers. Otomemochi in general produced more adventitious roots, partitioned a greater proportion of biomass to roots, and had a greater increment of root dry weight (DeltaRW) at 4 DAT than Yumenohatamochi, but these variety differences were less clear under non-flooded conditions without root pruning. Several quantitative trait loci (QTLs) associated with rooting ability were identified mainly in chromosomes 1, 4, and 6 across the four treatments or in combined analysis. On the other hand, QTLs for DeltaRW around RM2357 in chromosome 5 and for maximum new root length in RM215-RM205 in chromosome 9 were found only under root pruning treatments and under non-flooded conditions without root pruning, respectively. Greater DeltaRW was associated with higher head dry weight per hill at maturity only in the non-flooded and root-pruning treatments. This study suggests the importance of rooting ability after transplanting and, possibly, other mechanisms for adaptation to non-flooded conditions.