钙在无花果细胞盐诱导脯氨酸积累中的作用

接种于含ＮａＣｌ 培养基的无花果愈伤组织细胞生长极显著受抑,Ｎａ＋ 含量增加,Ｋ／Ｎａ 比值下降,游离脯氨酸积累。培养基中添加一定量ＣａＣｌ２ 不仅在一定程度上缓解盐分对生长的抑制作用,增加Ｋ＋／Ｎａ ＋ 比,而且明显促进游离脯氨酸积累。如果在添加钙的同时再添加细胞钙调素活性抑制剂盐酸氯丙嗪（ＣＰＺ） 或盐酸三氟拉嗪（ＴＦＰ） ,均使钙促进的脯氨酸积累受到明显抑制,表明盐胁迫诱导的脯氨酸积累可能涉及细胞ＣａＣａＭ系统。     

The amino terminus of high-voltage-activated calcium channels

Voltage-gated calcium channels (VGCCs), calmodulin (CaM), and calmodulin kinase II (CaMKII) are essential for various nervous system functions. CaM and CaMKII differentially regulate calcium dependent facilitation (CDF) and calcium dependent inactivation (CDI) of the Cav1 and Cav2 families of VGCCs. It is generally accepted that conserved structures in the C-terminus of these channels regulate CDF and CDI, and yet recent evidence indicates that other intracellular regions may be involved. We recently discovered that N-terminal sequences in Cav1.2 bind CaM and CaMKII, and function to regulate CDI as well as surface expression and open probability, respectively. Cav1 and Cav2 share significant portions of N-terminal sequence and therefore we explored whether homologous binding sites might exist in Cav2.1. Here, we show that like the proximal N-terminus of Cav1.2, the homologous region of Cav2.1 contains sequences which interact either directly or indirectly with CaM.     

Touch-induced gene (IbTCH1) from sweet potato [Ipomoea batatas (L.) Lam.]: molecular cloning and functional analysis

The cDNA of the touch-induced genes (TCH) of the sweet potato [Ipomoea batatas (L.) Lam.] has been cloned and analyzed. IbTCH1, which exists as at least two-copy genes in the genome of the sweet potato, encodes for 148-amino acid polypeptides, and harbors four conversed Ca²⁺-binding motif EF-hands. IbTCH1 was shown to be expressed in the flower, leaf, thick pigmented root, and particularly in the white fibrous root, but expressed only weakly in the petiole. IbTCH1 is upregulated upon exposure to environmental stresses, dehydration, and jasmonic acid. Furthermore, IbTCH1 is developmentally regulated in the leaf and root. These results strongly indicate that the gene performs functions in both plant development and in defense/stress-signaling pathways.     

Phosrestide-1, a peptide derived from the Drosophila photoreceptor protein phosrestin I, is a potent substrate for Ca/calmodulin-dependent protein kinase II from rat brain

Multifunctional Ca²⁺/calmodulin-dependent protein kinase type II (CaMK II) plays a crucial role in mediation of cellular responses to rising cytosolic Ca²⁺ levels. We find that the novel peptide substrate PGTIEKKRSNAMKKMKSIEQHR serves as a highly potent substrate for CaMK II enzymes purified from both Drosophila and rat. The peptide is derived from a photoreceptor-specific protein, phosrestin I, of the Drosophila compound eye and is designated as phosrestide-1. Using saturating substrate concentrations, the enzymes from both species transfer the γ-phosphoryl group of ATP to phosrestide-1 at a level three to ten times greater than to the commercially available mammalian-derived CaMK II substrates, autocamtide-3 and syntide-2. This indicates a conservation of substrate preferences for CaMK II derived from distantly related species, a dipteran fly and a mammal. Although phosrestide-1 contains two potential serine residues for CaMK II phosphorylation, we find that only the C-terminal serine is phosphorylated by rat CaMK II. However, removal of the upstream sequence containing the N-terminal serine substantially reduced the potency of phosrestide-1 as a CaMK II substrate to a level comparable to that of syntide-2 or autocamtide-3. We also find that a peptide representing the N-terminal segment of phosrestide-1 does not inhibit either CaMK II. Therefore, the enhanced potency of phosrestide-1 as a CaMK II substrate is likely to be due to a preferred conformation of the peptide induced by the N-terminal segment rather than to a specific binding of the enzymes to the N-terminus of the peptide. To the best of our knowledge, phosrestide-1 is the first CaMK II substrate which is designed based on an invertebrate sequence. The high phosphorylation level of phosrestide-1 by CaMK II of mammalian origin may reflect highly conserved CaMK II signaling cascades between vertebrates and invertebrates.     

Calmodulin tagging provides a general method of using lanthanide induced magnetic field orientation to observe residual dipolar couplings in proteins in solution

A general method is presented for magnetic field alignment of proteins in solution. By tagging a target protein with calmodulin saturated with paramagnetic lanthanide ions it is possible to measure substantial residual dipolar couplings (RDC) whilst minimising the effects of pseudocontact shifts on the target protein. A construct was made consisting of a calmodulin-binding peptide (M13 from sk-MLCK) attached to a target protein, dihydrofolate reductase in this case. The engineered protein binds tightly to calmodulin saturated with terbium, a paramagnetic lanthanide ion. By using only a short linker region between the M13 and the target protein, some of the magnetic field alignment induced in the CaM(Tb^{3 +})₄ is effectively transmitted to the target protein (DHFR). ¹H-¹⁵N HSQC IPAP experiments on the tagged complex containing ¹⁵N-labelled DHFR-M13 protein and unlabelled CaM(Tb^{3 +})₄ allow one to measure RDC contributions in the aligned complex. RDC values in the range +4.0 to –7.4 Hz were measured at 600 MHz. Comparisons of ¹H-¹⁵N HSQC spectra of ¹⁵N-DHFR-M13 alone and its complexes with CaM(Ca^{2 +})₄ and CaM(Tb³⁺)₄ indicated that (i) the structure of the target protein is not affected by the complex formation and (ii) the spectra of the target protein are not seriously perturbed by pseudocontact shifts. The use of a relatively large tagging group (CaM) allows us to use a lanthanide ion with a very high magnetic susceptibility anisotropy (such as Tb³⁺) to give large alignments while maintaining relatively long distances from the target protein nuclei (and hence giving only small pseudocontact shift contributions).     

八肽胆囊收缩素拮抗NDAP对大鼠脊髓钙调蛋白的作用

为探讨ＣＣＫ－８抗阿片作用的受体后分子机理，本实验观察了ＣＣＫ－８和κ受体激动剂ＮＤＡＰ对大鼠脊髓背柱突触小体钙调蛋白活性的影响。结果表明：（１）１０ｎｍｏｌ／Ｌ－１μｍｏｌ／ＬＮＤＡＰ显著低大鼠髓背侧部突触小体的ＣａＭ 活性（Ｐ均＜０．０保ǎ亮吭黾樱种谱饔貌欢霞忧浚庵肿饔每杀惶匾煨驭适芴遄瓒霞粒危铮颍拢危桑ǎ宝蹋恚铮欤蹋┩耆瓒稀＃ǎ玻茫茫耍冈诘团ǘ仁保ǎ保埃保埃埃睿恚铮欤蹋     

Identification and Characterization of Higher Plant Myosins Responsible for Cytoplasmic Streaming

in vitro using these myosins and of localization studies using antiserum raised against each heavy chain, we suggested that both myosins are molecular motors for generating the motive force for cytoplasmic streaming in higher plant cells. The 170-kDa myosin is expressed not only in somatic cells but also in germinating pollen. In contrast, the 175-kDa myosin is distributed only in somatic cells. In the tip region of growing pollen tubes, it has been demonstrated that a tip-focused Ca²⁺ gradient is indispensable for growth and tube orientation. Cytoplasmic streaming in this region has been shown to be inactivated by high concentrations of Ca²⁺. The motile activity in vitro of 170-kDa myosin is suppressed by low (μM) levels of Ca²⁺ through its CaM light chain, suggesting that this suppression is one of the mechanisms for inactivating cytoplasmic streaming near the tip region of pollen tubes. The motile activity in vitro of 175-kDa myosin is also inhibited by Ca²⁺ at concentrations higher than 10⁻⁶M. It has been revealed that the elevation of cytosolic Ca²⁺ concentrations causes the cessation of cytoplasmic streaming even in somatic cells. Therefore, Ca²⁺-sensitivity of the motile activity of myosin appears to be a general molecular basis for Ca²⁺-induced cessation of cytoplasmic streaming. Received 6 September 2000/ Accepted in revised form 7 October 2000     

Characterization and Ca2+-induced expression of calmodulin (CaM) in marine dinoflagellates

Calmodulin (CaM) is one of the major Ca²⁺-binding proteins in the cells, and it plays multiple roles in several Ca²⁺ signaling pathways and regulating the activities of other proteins. In the present study, we characterized CaM genes from the marine dinoflagellates Amphidinium carterae, Cochlodinium polykrikoides, Prorocentrum micans, and P. minimum, and examined their expression patterns upon the addition and chelation of calcium. Their cDNAs had same ORF length (450 bp) and encoded the same protein, but with few nucleotide differences in the ORF and different 3′- and 5′ untranslated regions (UTRs). The four CaM proteins consist of four EF-hand Ca²⁺-binding motifs, two N-terminal domains and two C-terminal domains, and they were highly conserved within eukaryotes. The CaM gene expressions in the tested species increased by calcium treatments; however, they were significantly down-regulated by the calcium-chelator EGTA. The CaM genes of the test species were inducible and regulated by different calcium doses, suggesting their major role in calcium regulation in dinoflagellates.