Cooperation between PKC-alpha and PKC-epsilon in the regulation of JNK activation in human lung cancer cells

Phorbol esters can induce activation of two mitogen-activated protein kinase (MAPK) pathways, the extracellular signal-regulated kinase (ERK) pathway and the c-Jun N-terminal kinase (JNK) pathway. Unlike ERK activation, JNK activation by phorbol esters is somehow cell-specific. However, the mechanism(s) that contribute to the cell-specific JNK activation remain elusive. In this study, we found that phorbol 12-myristate 13-acetate (PMA) induced JNK activation only in non-small cell lung cancer (NSCLC) cells, but not in small cell lung cancer (SCLC) cells, whereas ERK activation was detected in both cell types. In NSCLC cells, PMA induced JNK activation in a time- and dose-dependent manner. JNK activation was attenuated by protein kinase C (PKC) down-regulation through prolonged pre-treatment with PMA and significantly inhibited by PKC inhibitors G?6976 and GF109203X. Subcellular localization studies demonstrated that PMA induced translocation of PKC-alpha, -betaII, and -epsilon isoforms, but not PKC-delta, from the cytosol to the membrane. Analysis of various PKC isoforms revealed that PKC-epsilon was exclusively absent in the SCLC cell lines tested. Ectopic expression of PKC-epsilon in SCLC cells restored PMA activation of JNK signaling only in the presence of PKC-alpha, suggesting that PKC-alpha and PKC-epsilon act cooperatively in regulating JNK activation in response to PMA. Furthermore, using dominant negative mutants and pharmacological inhibitors, we define that a putative Rac1/Cdc42/PKC-alpha pathway is convergent with the PKC-epsilon/MEK1/2 pathway in terms of the activation of JNK by PMA.     

New 2(3-->20)abeotaxane and 3,11-cyclotaxane from needles of Taxus cuspidata

Two new taxoid metabolites, 2alpha,7beta,10beta-triacetoxy-5alpha,13alpha-dihydroxy-2(3-->20)abeotaxa-4(20),11-dien-9-one (1) and 2alpha-acetoxy-5alpha-cinnamoyloxy-9alpha,10beta-dihydroxy-3,11-cyclotax-4(20)-en-13-one (2), were isolated from the methanol extract of needles of the Japanese yew, Taxus cuspidata.     

The calcium-dependent activity of large-conductance, calcium-activated K+ channels is enhanced by Pyk2- and Hck-induced tyrosine phosphorylation

Recent results showing that large-conductance, calcium-activated K⁺ (BK_Ca) channels undergo direct tyrosine phosphorylation in the presence of c-Src tyrosine kinase have suggested the involvement of these channels in Src-mediated signaling pathways. Given the important role for c-Src in integrin-mediated signal transduction, we have examined the potential regulation of BK_Ca channels by proline-rich tyrosine kinase 2 (Pyk2), a calcium-sensitive tyrosine kinase activated upon integrin stimulation. Transient coexpression of murine BK_Ca channels with either wild-type Pyk2 or hematopoietic cell kinase (Hck), a Src-family kinase, led to an enhancement of BK_Ca channel activity over the range of 1–10 µM free calcium, whereas coexpression with catalytically inactive forms of either kinase did not significantly alter BK_Ca gating compared with channels expressed alone. In the presence of either wild-type Pyk2 or Hck, BK_Ca -subunits were found to undergo tyrosine phosphorylation, as determined by immunoprecipitation and Western blotting strategies. However, tyrosine phosphorylation of the BK_Ca -subunit was not detected for channels expressed alone or together with inactive forms of either Pyk2 or Hck. Interestingly, wild-type, but not inactive, Pyk2 was also present in BK_Ca channel immunoprecipitates, suggesting that Pyk2 may coassociate with the BK_Ca channel complex after phosphorylation. Collectively, the observed modulation and phosphorylation of BK_Ca channels by Pyk2 and a Src-family kinase may reflect a general cellular mechanism by which G protein-coupled receptor and/or integrin activation leads to the regulation of membrane ion channels. BK channels; tyrosine kinase; calcium; immunoprecipitation     

针刺对去卵巢大鼠脑内胆碱乙酰转移酶基因表达的影响

本工作旨在探讨雌激素对脑内乙酰胆碱生成的影响和电针刺激“足三里”穴对去卵巢大鼠脑内乙酰胆碱生成的调整作用。实验选用成年Wistar雌性大鼠,将动物分为正常对照组(INT)、去卵巢组(OVX)和去卵巢针刺组(OVX AC)。用放射免疫分析方法测定血中雌二醇含量,采用RT-PCR方法获得大鼠脑内胆碱乙酰转移酶(ChAT)mRNA的逆转录表达产物——cDNA,用琼脂糖凝胶电泳方法检测,并通过原位杂交方法观察海马ChAT mRNA阳性神经元的表达,然后用计算机图像分析系统进行统计分析。实验结果显示：去卵巢组大鼠体内雌激素水平明显降低,脑内ChAT mRNA的RT-PCR产物和海马ChAT mRNA阳性表达产物的平均面积、平均积分光度值均明显减少,与对照组和针刺组比较有显著性差异;去卵巢针刺“足三里”穴组与去卵巢组相比,大鼠血中雌激素水平明显升高,脑内ChAT mRNA RT-PCR产物明显增多,海马的ChAT mRNA表达阳性神经元增多。以上结果提示：脑内ChAT基因表达与体内雌激素水平有密切关系,去卵巢后针刺“足三里”穴对ChAT的调节作用可能是针刺增强脑内乙酰胆碱含量的机制之一。     

Genotypes of Cryptosporidium species infecting fur-bearing mammals differ from those of species infecting humans

Of 471 specimens examined from foxes, raccoons, muskrats, otters, and beavers living in wetlands adjacent to the Chesapeake Bay, 36 were positive for five types of Cryptosporidium, including the C. canis dog and fox genotypes, Cryptosporidium muskrat genotypes I and II, and Cryptosporidium skunk genotype. Thus, fur-bearing mammals in watersheds excreted host-adapted Cryptosporidium oocysts that are not known to be of significant public health importance.     

Genetic diversity of Cryptosporidium spp. in captive reptiles

The genetic diversity of Cryptosporidium in reptiles was analyzed by PCR-restriction fragment length polymorphism and sequence analysis of the small subunit rRNA gene. A total of 123 samples were analyzed, of which 48 snake samples, 24 lizard samples, and 3 tortoise samples were positive for Cryptosporidium: Nine different types of Cryptosporidium were found, including Cryptosporidium serpentis, Cryptosporidium desert monitor genotype, Cryptosporidium muris, Cryptosporidium parvum bovine and mouse genotypes, one C. serpentis-like parasite in a lizard, two new Cryptosporidium spp. in snakes, and one new Cryptosporidium sp. in tortoises. C. serpentis and the desert monitor genotype were the most common parasites and were found in both snakes and lizards, whereas the C. muris and C. parvum parasites detected were probably the result of ingestion of infected rodents. Sequence and biologic characterizations indicated that the desert monitor genotype was Cryptosporidium saurophilum. Two host-adapted C. serpentis genotypes were found in snakes and lizards.