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81.
Pituitary adenylate cyclase-activating polypeptide (PACAP) potentiates glucose-induced insulin release and increases cytosolic Ca2+ concentration ([Ca2+]i) in islet beta-cells in a concentration-dependent manner with two peaks at 10(-13) and 10(-9) M. PAC1 receptor (PAC1-R) and VPAC2 receptor (VPAC2-R) are expressed in pancreatic beta-cells and thought to be involved in insulin release. We aimed to determine the receptor types involved in the [Ca2+]i responses to 10(-13) and 10(-9) M PACAP. We measured [Ca2+]i in beta-cells and examined comparative effects of PAC1-R-selective agonist maxadilan, its antagonist M65, VPAC2-R-selective agonist Ro25-1553, and native ligands of PACAP and VIP. In the presence of 8.3 mM glucose, maxadilan, Ro25-1553, PACAP, and VIP at 10(-13) and 10(-9) M all increased [Ca2+]i. PACAP and maxadilan elicited greater effects at 10(-9) M than at 10(-13) M both in the incidence and amplitude of [Ca2+]i responses. For VIP and Ro25-1553, in contrast, the effects at 10(-9) and 10(-13) M were comparable. Furthermore, the amplitude of [Ca2+]i responses to 10(-9) M PACAP, but not 10(-13) M PACAP, was suppressed by M65. The results suggest that VPAC2-R and PAC1-R contribute equally to [Ca2+]i responses to sub-picomolar concentrations of PACAP, while PAC1-R has greater contribution to [Ca2+]i responses to nanomolar concentrations of this peptide. 相似文献
82.
Mochizuki S Miyasaka T Goto M Ogasawara Y Yada T Akiyama M Neishi Y Toyoda T Tomita J Koyama Y Tsujioka K Kajiya F Akasaka T Yoshida K 《Biochemical and biophysical research communications》2003,306(2):505-508
Intra-aortic measurement of nitric oxide (NO) would provide valuable insights into NO bioavailability in systemic circulation and vascular endothelial function. In the present study, we thus developed a catheter-type NO sensor to measure intra-aortic NO concentration in vivo. An NO sensor was encased and fixed in a 4-Fr catheter. The sensor was then located in the thoracic aorta via the femoral artery through a 7-Fr catheter to measure intra-aortic plasma NO concentration in vivo in anesthetized dogs. Infusion of acetylcholine (10 microg/kg) increased base-to-peak plasma NO level in the aorta by 2.4+/-0.4 nM (n=7). After 20-min infusion of N(G)-methyl-L-arginine (NO synthase inhibitor), changes in plasma NO concentration in response to acetylcholine were attenuated significantly (1.8+/-0.4 nM, P<0.003, n=7). In conclusion, the newly developed catheter-type NO sensor successfully measured acetylcholine-induced changes in intra-aortic plasma concentration of endothelium-derived NO in vivo and demonstrated applicability to direct evaluation of intravascular NO bioavailability. 相似文献
83.
A structure-function study was undertaken to determine the effects of N-terminal mutations in pepsin designed to introduce the Lys-X-Tyr motif and increase N-terminal flexibility. At pH 7.0, E7K/T12A/E13Q pepsin was inactivated more slowly compared to WT, whereas the mutants E7K and T12A/E13Q were not stabilized. Far-UV circular dichroism revealed that changes in secondary structure accompanied the inactivation process, and that the structural changes occurred at approximately the same rate as inactivation. All of the inactivated pepsin forms showed retention of substantial secondary structure, more than previously determined for pepsin denatured at pH 7.2 and 8.0, suggesting the presence of a structural intermediate at pH 7.0. The coupled mutations at positions 12 and 13 impacted the pH dependence of activity at pH 0.9, lowered affinity for a synthetic substrate, and lowered the turnover number. The introduction of Lys at position 7 apparently destabilized the interaction between prosegment-enzyme body as evidenced by activation at higher pH (>or= 4.0) compared to WT, but showed no change for pH dependence of activity, nor a statistically significant change in affinity for the synthetic substrate. 相似文献
84.
In the presentstudy, we examined the ability of adenosine 3',5'-cyclicmonophosphate (cAMP) to reduce elevated levels of cytosolicCa2+ concentration([Ca2+]i)in pancreatic -cells.[Ca2+]iand reduced pyridine nucleotide, NAD(P)H, were measured in rat single-cells by fura 2 and autofluorescence microfluorometry. Sustained[Ca2+]ielevation, induced by high KCl (25 mM) at a basal glucose concentration (2.8 mM), was substantially reduced by cAMP-increasing agents, dibutyryl cAMP (DBcAMP, 5 mM), an adenylyl cyclase activatorforskolin (10 µM), and an incretin glucagon-likepeptide-1-(7-36) amide (109 M), as well as byglucose (16.7 mM). The[Ca2+]i-reducingeffects of cAMP were greater at elevated glucose (8.3-16.7 mM)than at basal glucose (2.8 mM). An inhibitor of protein kinase A (PKA),H-89, counteracted[Ca2+]i-reducingeffects of cAMP but not those of glucose. Okadaic acid, a phosphataseinhibitor, at 10-100 nM also reduced sustained [Ca2+]ielevation in a concentration-dependent manner. Glucose, but not DBcAMP,increased NAD(P)H in -cells.[Ca2+]i-reducingeffects of cAMP were inhibited by 0.3 µM thapsigargin, an inhibitorof the endoplasmic reticulum (ER)Ca2+ pump. In contrast,[Ca2+]i-reducingeffects of cAMP were not altered by ryanodine, an ERCa2+-release inhibitor,Na+-free conditions, or diazoxide,an ATP-sensitive K+ channelopener. In conclusion, the cAMP-PKA pathway reduces[Ca2+]ielevation by sequestering Ca2+ inthapsigargin-sensitive stores. This process does not involve, but ispotentiated by, activation of -cell metabolism. Together with theknown[Ca2+]i-increasingaction of cAMP, our results reveal dual regulation of -cell[Ca2+]iby the cAMP-signaling pathway and by a physiological incretin. 相似文献
85.
Nobuhiko Okamoto Hide-aki Chiyo Katsumi Imai Kazumasa Otani Yasuyuki Futagi 《Human genetics》1994,93(5):605-606
The Costello syndrome is characterized by dwarfism, unique cutaneous lesions, distinct facial gestalt, and mental retardation. We present a Japanese patient with the Costello syndrome. She showed high serum IgM level during the early infantile period. Nissen's fundplication was carried out to treat severe gastroesophageal reflux. Endocrinological investigations revealed a partial deficiency of growth hormone. 相似文献
86.
Hideki Takanari Joji Morita Hidetaka Yamanaka Kenichiro Yada Asahiko Takahashi Kosaku Izutsu 《Biology of the cell / under the auspices of the European Cell Biology Organization》1994,82(1):51-57
Summary— The pH-related change in morphology of vinblastine (VLB)-induced paracrystals formed in Chinese hamster ovary (CHO) cells was examined immunohistochemically in order to determine both the mechanism of tubulin crystallization and the influence of acidic pHs on cytoskeletal microtubules. Lowering the extracellular pH (pHe) rapidly reduced the intracellular pH (pHi) in CHO cells. Lowering the pHi to near the neutral range significantly accelerated the growth of VLB-induced paracrystals, compared to that of paracrystals formed at a physiological pHe. However, further cytoplasmic acidification caused by the addition of sodium azide into the culture medium induced the disappearance of typical paracrystals and the appearance of a highly organized meshwork of tubulin appearing as short, thick filaments at the light microscopic level. Treatments using different concentrations of VLB at different pHe's showed that low pHi's (6.7 and 6.3) suppressed paracrystal-formation at lower concentrations of VLB (5×10?6 M and 10?5 M). At higher concentrations of VLB (5×10?5 M and 10?4 M), only short filaments were formed at pHi 6. 3. Electron microscopy revealed that the filaments had a ladder-like structure probably consisting of a stacked series of fused rings. This indicates that paracrystals may be modified by extremely low pH. These results show that paracrystals are unstable in living cells and that their formation is regulated by environmental pH. 相似文献
87.
Contribution of a prosegment lysine residue to the function and structure of porcine pepsinogen A and its active form pepsin A. 总被引:1,自引:0,他引:1
A conserved lysine residue, Lys36p, on the prosegment of pepsinogen was replaced with a positively charged arginine (K36pR), a negatively charged glutamic acid (K36pE), and a neutral side chain methionine (K36pM). K36pM and K36pE mutants were extremely unstable and degraded rapidly, especially K36pE, which was inactivated during purification. This instability was confirmed by microcalorimetry where the denaturing temperatures for K36pM and K36pE were 6 degrees C and 10 degrees C lower than the wild-type, respectively. As a function of pH, K36pM and K36pR were activated over a broader range of pH as compared with wild-type. The mutant pepsinogens were activated faster than wild-type with K36pM being activated approximately 10 times faster. The activated pepsins from the various mutant pepsinogens showed lower kinetic efficiency than wild-type enzyme. Catalytic rate constants were reduced by half. The results suggested Lys36p is important for the correct folding of the active-centre residues. The molecular modeling calculation suggested that the position of Asp215 was substantially altered. In conclusion, the above results would suggest that Lys36p was important not only for stability of the prosegment and pepsinogen, but also for the correct alignment of the active-centre residues. 相似文献
88.
89.
Matsumoto M Yada M Hatakeyama S Ishimoto H Tanimura T Tsuji S Kakizuka A Kitagawa M Nakayama KI 《The EMBO journal》2004,23(3):659-669
Insoluble aggregates of polyglutamine-containing proteins are usually conjugated with ubiquitin in neurons of individuals with polyglutamine diseases. We now show that ataxin-3, in which the abnormal expansion of a polyglutamine tract is responsible for spinocerebellar ataxia type 3 (SCA3), undergoes ubiquitylation and degradation by the proteasome. Mammalian E4B (UFD2a), a ubiquitin chain assembly factor (E4), copurified with the polyubiquitylation activity for ataxin-3. E4B interacted with, and thereby mediated polyubiquitylation of, ataxin-3. Expression of E4B promoted degradation of a pathological form of ataxin-3. In contrast, a dominant-negative mutant of E4B inhibited degradation of this form of ataxin-3, resulting in the formation of intracellular aggregates. In a Drosophila model of SCA3, expression of E4B suppressed the neurodegeneration induced by an ataxin-3 mutant. These observations suggest that E4 is a rate-limiting factor in the degradation of pathological forms of ataxin-3, and that targeted expression of E4B is a potential gene therapy for SCA3. 相似文献
90.
Tomoko?Sagai Hiroshi?Masuya Masaru?Tamura Kunihiko?Shimizu Yukari?Yada Shigeharu?Wakana Yoichi?Gondo Tetsuo?Noda Toshihiko?ShiroishiEmail author 《Mammalian genome》2004,15(1):23-34
Polarized expression of the Sonic hedgehog (Shh) gene in the posterior mesenchyme is essential for pattern formation in the appendages of higher vertebrates, from teleost fins to tetrapod limb buds. We report on a sequence in intron 5 of the Lmbr1 gene, which resides approximately 1 Mb from the Shh coding region in the mouse genome and is highly conserved among teleost fishes and throughout the tetrapod lineage. Positional cloning revealed that two mouse mutations, Hx and M100081, characterized by mirror-image digit duplication and ectopic anterior Shh expression, have base substitutions in this sequence. Absence of the conserved sequence in limbless reptiles and amphibians and a cis-trans test using the Hx and Shh KO alleles suggest that the sequence is a cis-acting regulator that controls the polarized expression of Shh.
The nucleotide sequence data reported in this paper have been submitted to GenBank and have been assigned the accession number: AB092986 to AB093004, AB093207, and AB114903. 相似文献