大肠杆菌脂多糖对人红细胞蛋白激酶C活性的影响

正常成人红细胞与大肠杆菌脂多糖温育后,胞膜蛋白激酶C活性显著增加,脂多糖这种作用呈现剂量和时间依赖性。结果提示脂多糖能激活红细胞蛋白激酶C。     

Acute neurite retraction triggered by lysophosphatidic acid: timing of the inhibitory effects of genistein

Acute neurite retraction, elicited by diverse agents in several neuronal cell types, has been reported to be inhibited by genistein, a kinase antagonist that is relatively (though not absolutely) selective for tyrosine kinases. It was hypothesized that genistein acts upon ssome final common pathway that integrates multiple extrinsic and intrinsic signals to regulate whether neurites will execute a retraction response (J. Neurochem., 61 (1993) 340–343). To define this pathway in more detail, a quantitative study of NG108-15 cell rapid-onset neurites was carried out as they retract in response to lysophosphatidic acid (LPA, 10 μM). Following the application of LPA, most neurites exhibited early morphologic changes between 0.5 and 1.5 min, followed by progressive shortening and eventual retraction, with 50% of neurites completely retracted by 5 min and 80% gone by 10 min. Genistein did not inhibit the formation of subcortical F-actin, nor its functional competence in several assays. Genistein protected neurites when added at any time prior to the onset of the earliest morphologic changes, but failed to block progression when added to neurites that were already undergoing retraction. These findings imply that the final common pathway (i.e. the critical target(s) for genistein) must be activated late, after the increase in F-actin levels has peaked and just before retraction is initiated.     

雌激素对内膜癌细胞中FAK基因转录的影响

目的定量观察雌激素刺激后内膜癌细胞的焦点粘连激酶（Focal—adhesion kinase，FAK）转录水平。方法不同浓度的17β-雌二醇体外刺激雌激素敏感的内膜癌细胞株RL95—2。加用三苯氧胺或ICI 182，780雌激素拮抗剂的细胞及未加雌激素的细胞设为阴性对照。经不同作用时间后，采用实时RT—PCR方法测定处理后的细胞内FAK的mRNA水平。结果在给予不同浓度的雌激素共培养1d或5d后，细胞的FAK基因的转录水平对比阴性对照细胞无明显改变。结论雌激素对内膜癌细胞的FAK转录可能无显著影响。FAK基因在内膜癌组织中的过度表达的机理需要进一步深入研究。     

蛋白激酶C亚型在慢性"炎症性"肺动脉高压大鼠肺动脉中的表达

目的 研究慢性"炎症性"肺动脉高压大鼠在肺动脉高压形成过程中肺动脉蛋白激酶C(PKC)亚型的表达.方法 建立野百合碱诱导的慢性"炎症性"肺动脉高压大鼠模型,应用Western blot技术检测肺动脉高压形成过程中大鼠肺动脉四种PKC亚型(PKCα、PKCβⅡ、PKCδ和PKCε)的表达变化.结果 PKCα、PKCβⅡ和PKCδ亚型在正常和肺动脉高压大鼠肺动脉中均有表达,而PKCε亚型未检测到.在肺动脉高压形成过程中,大鼠肺动脉胞浆和胞膜组分表达的PKCα均逐渐上升,到第14天达到高峰后略有下降,且胞膜表达量的升高远比胞浆明显.胞浆PKCβⅡ和PKCδ表达量均在第8天达最高,而胞膜中二者均表现出持续升高的趋势.结论 PKCα、PKCβⅡ和PKCδ亚型可能参与了慢性"炎症性"肺动脉高压的形成,其表达变化可能与其转位有关.     

A novel compound heterozygous dysferlin mutation in Miyoshi myopathy siblings responding to dantrolene

Miyoshi myopathy (MM) is an autosomal recessive distal muscular dystrophy characterized by mutations of the dysferlin gene. Although several pairs of homozygous/heterozygous mutations have been reported, few effective treatments of MM are available. We had observed the decreased serum creatine kinase (CK) before and after administration of dantrolene in the elder brother and the increased serum CK before and after discontinuance of the drug on suspicion of drug-induced hepatopathy in the younger sister. We report a novel pair of heterozygous mutations in the 3'-splicing site of exon 26 and the translation site of exon 28 of the dysferlin gene in two siblings, and effective treatment of their MM with dantrolene.     

Screening for SNCA and LRRK2 mutations in Greek sporadic and autosomal dominant Parkinson's disease: identification of two novel LRRK2 variants

Mutations in SNCA and LRRK2 genes, encoding alpha-synuclein and leucine-rich repeat kinase 2, respectively, cause autosomal dominant Parkinson's disease (AdPD). The LRRK2 G2019S (c.6055G > A) and R1441G (c.4321C > G) mutations have also been identified in sporadic PD (sPD). We studied 55 unrelated patients with AdPD, 235 patients with sPD, and 235 healthy age- and gender-matched controls all of Greek origin. Patients with AdPD were screened for SNCA and LRRK2 mutations by direct sequencing. SNCA gene dosage analysis was also performed for AdPD using quantitative duplex polymerase chain reaction of genomic DNA. In addition, we investigated the frequency of the LRRK2 G2019S mutation in sPD. We found no missense mutations or multiplications in the SNCA gene. Here we report two novel variants, A211V (c.632C > T) and K544E (c.1630A > G) in LRRK2 gene in two patients with AdPD that was not present in controls. We identified only one patient with sPD (1/235; 0.4%) carrying the G2019S mutation. LRRK2 mutations are present in AdPD and sPD patients of Greek origin.     

Protein kinase C�� expression in breast cancer as measured by real-time PCR, western blotting and ELISA

The protein kinase C (PKC) family of genes encode serine/threonine kinases that regulate proliferation, apoptosis, cell survival and migration. Multiple isoforms of PKC have been described, one of which is PKCδ. Currently, it is unclear whether PKCδ is involved in promoting or inhibiting cancer formation/progression. The aim of this study was therefore to investigate the expression of PKCδ in human breast cancer and relate its levels to multiple parameters of tumour progression. Protein kinase Cδ expression at the mRNA level was measured using real-time PCR (n=208) and at protein level by both immunoblotting (n=94) and ELISA (n=98). Following immunoblotting, two proteins were identified, migrating with molecular masses of 78 and 160 kDa. The 78 kDa protein is likely to be the mature form of PKCδ but the identity of the 160 kDa form is unknown. Levels of both these proteins correlated weakly but significantly with PKCδ concentrations determined by ELISA (for the 78 kDa form, r=0.444, P<0.005, n=91 and for the 160 kDa form, r=0.237, P=0.023, n=91) and with PKCδ mRNA levels (for the 78 kDa form, r=0.351, P=0.001, n=94 and for the 160 kDa form, r=0.216, P=0.037, n=94). Protein kinase Cδ mRNA expression was significantly higher in oestrogen receptor (ER)-positive compared with ER-negative tumours (P=0.007, Mann–Whitney U-test). Increasing concentrations of PKCδ mRNA were associated with reduced overall patient survival (P=0.004). Our results are consistent with a role for PKCδ in breast cancer progression.     

Protein kinase C isozymes that mediate enhancement of neurite outgrowth by ethanol and phorbol esters in PC12 cells

Using PC12 cells to study ethanol's effects on growth of neural processes, we found that ethanol enhances NGF- and basic FGF-induced neurite outgrowth. Chronic ethanol exposure selectively up-regulates δ and ε protein kinase C (PKC) and increases PKC-mediated phosphorylation in PC12 cells. Since PKC regulates differentiation, we investigated the role of PKC in enhancement of neurite outgrowth by ethanol. Like ethanol, 0.3–10 nM phorbol 12-myristate, 13-acetate (PMA) increased NGF-induced neurite outgrowth. However, higher concentrations did not, and immunoblot analysis demonstrated that 100 nM PMA markedly depleted cells of β, δ and ε PKC. PMA (100 nM) also down-regulated β, δ and ε PKC in ethanol-treated cells and completely prevented enhancement of neurite outgrowth by ethanol. In contrast, the cAMP analogue 8-bromoadenosine cAMP did not completely mimic the effectsof ethanol on neurite outgrowth, and ethanol was able to enhance neurite formation in mutant PC12 cells deficient in protein kinase A (PKA). These findings implicate β, δ or εPKC, but not PKA, in the neurite-promoting effects of ethanol and PMA. Since chronic ethanol exposure up-regulates δ and ε, but not βPKC, these findings suggest that δ or εPKC regulate neurite outgrowth.     

Cloning and characterisation of the adenosyl phosphosulphate kinase gene from Aspergillus nidulans

The sD gene of Aspergillus nidulans has been cloned by heterologous screening of rationally selected cosmids. Co-transformation of the sD50 mutant JMP1 confirmed the presence of a functional gene. Sequence analysis determined this gene to be 680 bp in length, containing a 59-bp intron and encoding a protein of 206 amino acids. A protein-sequence comparison revealed a similarity to the C-terminal region of ATP sulphurylase, the sC gene product. Further sequence comparison revealed differences in a consensus sequence ATP-binding motif, indicating non-functionality of the APS kinase-like domain of ATP sulphurylase, and confirms sD as the gene encoding APS kinase in A. nidulans. Received: 17 April / 29 August 1997