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     

Prognostic Factors in Myeloma: What They Tell Us About the Pathophysiology of the Disease

Prognostic factors in myeloma are not only important for allowing comparisons to be made between therapeutic protocols but they also provide us with an insight into the pathophysiology of the disease and important mechanisms which result in disease progression. Prognostic factors in myeloma relate to the inherent proliferative capacity of the malignant clone, tumor bulk, renal function and other factors which reflect tumor host and host tumor interactions. The highly significant effect of the labelling index (LI) suggests that the clonogenic cell is ontologically very close to the malignant plasma cell on which the labelling index is derived. The explanation for the important role of the β2-microglobulin (β2M) level over and above its reflection of renal function is as yet unclear.     

Effect of low glycogen on glycogen synthase in human muscle during and after exercise

Subjects cycled at a work load calculated to elicit 75% of maximal oxygen uptake on two occasions: the first to fatigue (34.5 ± 5.3 min; mean ± SE), and the second at the same workload and for the same duration as the first. Biopsies were obtained from the quadriceps femoris muscle before and immediately after exercise, and 5 min post-exercise. Before the first experiment, muscle glycogen was lowered by a combination of exercise and diet, and before the second, experiment muscle glycogen was elevated. In the low glycogen condition (LG), muscle glycogen decreased from 169 ± 15 mmol glucosyl units kg^-1dry wt at to rest to 13 ± 6 after exercise. In the high glycogen condition (HG) glycogen decreased from 706 ± 52 at rest to 405 ± 68 after exercise. Glycogen synthase fractional activity (GSF) was always higher during the LG treatment. During exercise in the HG condition, those subjects who cycled for < 35 min (n= 3) had GSF values in muscle which were lower than at rest, whereas those subjects who cycled for > 35 min (n= 4) had values which were similar to or higher than at rest. Thus the change in GSF in muscle during HG was positively related to the exercise duration (r= 0.94; y = 254–17x + 0.3x²; P < 0.001) and negatively related to the glycogen content at the end of exercise (r=–0.82; y= 516–2x + 0.001x²; P < 0.05). During LG exercise GSF remained constant. GSF increased markedly after 5 min post-exercise in both HG and LG conditions. cAMP dependent protein kinase activity increased similarly during both LG and HG exercise and reverted to the preexercise values 5 min post-exercise. It is concluded that muscle contraction decreases GSF, but low glycogen levels can attenuate or abolish the decrease in GSF. The rapid increase of GSF during recovery from exercise does not require glycogen depletion during the exercise.     

The effects of interferon-beta on phorbol ester or calcium ionophore-induced intercellular adhesion molecule-I expression in epidermal carcinoma cells.

Keratinocyte intercellular adhesion molecule (ICAM)-I expression is induced by interferon (IFN)-gamma. It has been previously reported that IFN-beta suppresses IFN-gamma-induced ICAM-I expression in A431 cells, a human squamous cell carcinoma cell line. In this study, the suppression mechanisms were investigated at the post second messenger level. Both 12-O-tetradecanoylphorbol-13-acetate (TPA) and calcium ionophore (A23187) induce ICAM-I expression in A431 cells. ICAM-I expression induced by either was not suppressed with cotreatment with IFN-beta. Furthermore, IFN-beta did not inhibit the translocation of protein kinase C (PKC) by TPA. It appears that the pathways involved in ICAM-I expression induced by activation of PKC or increased in intracellular Ca++ are not affected by IFN-beta.     

Attenuation of myocardial injury due to oxygen free radicals(OFR) by pretreatment with OFR or calcitonin gene-related peptide

目的：研究氧自由基（ＯＦＲ）及降钙素基因相关肽（ＣＧＲＰ）预处置对ＯＦＲ所致离体大鼠心脏损伤的拮抗作用．方法：Ｌａｎｇｅｎｄｏｒｆ法灌流心脏，电解ＫＨ液产生ＯＦＲ．结果：ＣＧＲＰ或ＯＦＲ预处置减轻ＯＦＲ所致心脏收缩功能下降，冠脉流量减少和肌酸激酶（ＣＫ）释放增加．蛋白激酶Ｃ（ＰＫＣ）抑制剂Ｈ７可取消ＯＦＲ预处置的心脏保护作用（对照组，ＯＦＲ损伤组，ＯＦＲ预处置组，Ｈ７加ＯＦＲ预处置组及Ｈ７组的ＣＫ释放量分别是１１０±７，２１５±２３，１６９±１４，２４０±３０，１１３±１９Ｕ·Ｌ－１）．结论：ＯＦＲ或ＣＧＲＰ预处置对ＯＦＲ所致心肌损伤具有拮抗作用，该作用与ＰＫＣ激活有关．     

实验性视网膜光化学损伤中的蛋白激酶C

目的：研究实验性视网膜光化学损伤中蛋白激酶C(protein kinase C，PKC)活性的改变；探索地塞米松(dexamethasone，DXM)对视网膜中PKC活性的影响。 方法；48只SD大鼠分为对照组和DXM组，后者于光照前3天开始，连续5天腹膜腔注射DXM 1mg/(kg·d)。经(1 900±106.9)Ix的绿色荧光灯(λ＝510nm～560nm)连续照射24小时后的6小时和1、3、7、14天，进行视网膜PKC活性检测。 结果：光化学损伤后视网膜中的PKC活性在短暂的上升后即出现持续性的下降；DXM对PKC的活性改变无明显作用。 结论：光化学损伤中的视网膜功能障碍可能与PKC活性的持续降低有关。 （中华眼底病杂志，1997，13：78-80）     

-2-Hydroxyglutaric acid inhibits mitochondrial creatine kinase activity from cerebellum of developing rats

L-2-Hydroxyglutaric acid (LGA) is the biochemical hallmark of patients affected by the neurometabolic disorder known as L-2-hydroxyglutaric aciduria (LHGA). Although this disorder is predominantly characterized by severe neurological findings and pronounced cerebellum atrophy, the neurotoxic mechanisms of brain injury are virtually unknown. In the present study, we investigated the effect of LGA, at 0.25-5mM concentrations, on total creatine kinase (tCK) activity from cerebellum, cerebral cortex, cardiac muscle and skeletal muscle homogenates of 30-day-old Wistar rats. CK activity was measured also in the cytosolic (Cy-CK) and mitochondrial (Mi-CK) fractions from cerebellum. We verified that tCK activity was significantly inhibited by LGA in the cerebellum, but not in cerebral cortex, cardiac muscle and skeletal muscle. Furthermore, CK activity from the mitochondrial fraction was inhibited by LGA, whereas that from the cytosolic fraction of cerebellum was not affected by the acid. Kinetic studies revealed that the inhibitory effect of LGA on Mi-CK was non-competitive in relation to phosphocreatine. Finally, we verified that the inhibitory effect of LGA on tCK was fully prevented by pre-incubation of the homogenates with reduced glutathione (GSH), suggesting that this inhibition is possibly mediated by oxidation of essential thiol groups of the enzyme. Considering the importance of creatine kinase activity for energy homeostasis, our results suggest that the selective inhibition of this enzyme activity by increased levels of LGA could be possibly related to the cerebellar degeneration characteristically found in patients affected by L-2-hydroxyglutaric aciduria.