Inhibitory effect of ketamine on phosphorylation of the extracellular signal-regulated kinase1/2 following brain ischemia and reperfusion in rats with hyperglycemia

To determine if the inhibitory effects of ketamine on the extracellular signal-regulated kinase (ERK) 1/2 are involved in reduction of the hyperglycemia-exaggerated cerebral ischemic lesion, rats with normoglycemia, hyperglycemia, or hyperglycemia supplemented with ketamine were subjected to 15 min of forebrain ischemia, and then, reperfusion for 0.5, 1, and 3h. Phosphorylation of ERK1/2 in the brain tissues was assessed by immunohistochemistry and Western blot analysis. In rats with normoglycemia, we demonstrated a moderate increase of the ERK1/2 phosphorylation in the cingulum cortex and hippocampus CA3 following an ischemic intervention. It quickly dropped to control levels after reperfusion for 0.5h. In rats with hyperglycemia, however, the increase of the ERK1/2 phosphorylation in these areas was significantly higher in all animals reperfused. The neuronal death, detected by the TdT-mediated-dUTP nick end labeling assays, was found in the cingulum cortex (5.23+/-2.34, per high power feild) and hippocampus CA3 areas (6.29+/-3.68, per 1mm(2)) in hyperglycemic group after reperfusion for 3h. With ketamine treatment, the ERK1/2 phosphorylation in cingulum cortex and hippocampus CA1 and CA3 areas was found to be the same as that in normoglycemia rats. Our results suggest that hyperglycemia may increase the ischemic insult through modulation of the signal transduction pathways involving ERK1/2. The inhibitory effects of ketamine on the hyperglycemia-activated ERK1/2 phosphorylation are probably through inhibition of the N-methyl d-aspartate-mediated calcium influx, which subsequently reduce the hyperglycemia-exaggerated cerebral damage.     

Computational biology insights into genotype-clinical phenotype-protein phenotype relationships between novel SLC26A2 variants identified in inherited skeletal dysplasias

BackgroundPathogenic variants in the transmembrane sulfate transporter protein SLC26A2 are associated with different phenotypes of inherited chondrodysplasias. As limited data is published from India, in this study we sought to elucidate the molecular basis of inherited chondrodysplasias in an Indian cohort.MethodsMolecular screening of 32 fetuses with antenatally diagnosed lethal skeletal dysplasia was performed by next generation sequencing and Sanger sequencing. The genotype-protein phenotype characterization was done using computational biology techniques like homology modelling, stability and pathogenicity predictions.ResultsWe identified five rare autosomal recessive SLC26A2 [NM_000112.4] variants, including three homozygous c.796dupA(p.Thr266Asnfs*12), c.1724delA(p.Lys575Serfs*10), and c.1375_1377dup(p.Val459dup) and two heterozygous variants (c.532C > T(p.Arg178*)) and (c.1382C > T(p.Ala461Val)) in compound heterozygous form in a total of four foetuses. Genotype-protein phenotype annotations highlighted that the clinically severe achondrogenesis 1B causative c.796dupA(p.Thr266Asnfs*12) and c.1724delA(p.Lys575Serfs*10)variants impact SLC26A2 protein structure by deletion of the protein core and transmembrane STAS domains, respectively. In clinically moderate atelosteogenesis type 2 phenotype, the c.1382C > T(p.Ala461Val) variant is predicted to distort alpha helix conformation and alter the bonding properties and free energy dynamics of transmembrane domains and the c.532C > T(p.Arg178*) variant results in loss of both core transmembrane and STAS domains of the SLC26A2 protein. The c.1375_1377dup(p.Val459dup) variant identified in clinically milder atelosteogenesis type II-diastrophic dysplasia spectrum lethal phenotype is predicted to decrease the Qualitative Model Energy Analysis (QMean), which affects major geometrical aspects of the SLC26A2 protein structure.ConclusionWe expand the spectrum of SLC26A2 related lethal chondrodysplasia and report three novel variants correlating clinical severity and protein phenotype within the lethal spectrum of this rare dysplasia. We demonstrate the relevance of structural characterization to aid novel variant reclassification to provide better prenatal management and reproductive options to families with lethal antenatal skeletal disorder.     

利用穿梭质粒快速构建含乙型肝炎病毒表面抗原（HBsAg …

目的 应用新型杆状病毒表达系统快速构建含有ＨＢｓＡｇ基因的重组杆状病毒，高效表达ＨＢｓＡｇ，为ＨＢＶ诊断试剂、疫苗及治疗研究提供依据。方法 构建含有ＨＢｓＡｇ基因的供体质粒ｐＦＢ－ＢＳ，转化Ｂａｃ－ｔｏ－Ｂａｃ杆状病毒表达试剂盒中的ＤＨ１０Ｂａｃ致敏菌，利用其含有的细菌Ｔｎ７转座繁忙将ＨＢｓＡｇ基因重组至穿梭质粒Ｂａｃｍｉｄ上，快速筛选出含有ＨＢｓＡｇ基因的重组杆状病毒。结果 此重组病毒能在昆虫细     

Involvement of protein kinase c in responses of rat dorsal horn neurons to mechanical stimuli and periaqueductal gray descending inhibition

 The effects of a protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), on the activity and periaqueductal gray (PAG)-induced inhibition of rat dorsal horn neurons of the lumbar spinal cord were tested. A microdialysis fiber was placed through the dorsal horn for the purpose of local application of pharmacological agents. Extracellular single-unit recordings from dorsal horn neurons were made near the microdialysis fiber. TPA was tested on nociceptive dorsal horn cells. There was a significant increase in the background activity and responses to ”brush”, with no changes in responses to pressure and pinch stimuli. TPA also significantly blocked the PAG-induced inhibition of responses to brush, press, and pinch. These effects were eliminated by coadministration of the PKC inhibitor NPC-15437. The solvent, which contained dimethyl sulfoxide, was also tested for its effect on the responses to peripheral mechanical stimuli and PAG-induced inhibition of the dorsal horn neurons. There were no significant changes. This experiment suggests that activation of the PKC second messenger system might increase the activity of dorsal horn neurons and their responses to peripheral stimuli; in addition, the phorbol ester attenuated the PAG-induced descending inhibition of the dorsal horn neuron activity. Received: 15 May 1996 / Accepted: 14 November 1996     

CD40-mediated lymphotoxin α expression in human B cells is tyrosine kinase dependent

The cytokine lymphotoxin (LT)α is known to play a role in B cell activation. As the engagement of the B cell antigen CD40 is known to lead to B cell proliferation and differentiation, we studied LTα expression in human B cells after CD40 ligation. We demonstrate that anti-CD40 monoclonal antibody (mAb) induces strong LTα mRNA and surface expression in human tonsil B cells. Induction of LTα mRNA and surface expression by CD40 ligation is inhibited by the protein tyrosine kinase (PTK) inhibitors herbimycin and genistein in a dose-dependent manner. The protein kinase C (PKC)-specific inhibitors sphingosine and bisindolylmaleimide caused negligible inhibition of anti-CD40-induced LTα mRNA and surface expression. No inhibition is observed with the protein kinase (PKA) inhibitors H89 and HA1004. Cross-linking of the transmembrane phosphatase CD45 to CD40 by using goat-anti-mouse F(ab')₂ fragments strongly inhibits CD40-mediated LTα expression in human B cells, confirming the role of PTK activation in CD40-mediated induction of LTα expression. Inhibitors of the serine/threonine protein phosphatases PP1 and PP2A, okadaic acid and calyculin induce LTα mRNA expression. In contrast, cyclosporin A, an inhibitor of the serine/threonine phosphatase calcineurin has no effect on anti-CD40-induced LTα expression. These results suggest that induction of LTα expression in B cells following engagement of CD40 involves activation of protein tyrosine kinases.     

Protein kinase C in IL- 2 signal transduction

Interleukin-2 (IL-2), secreted principally by activated helper T-cells, plays a pivotal role in the generation and regulation of the immune response. The various biologic functions of IL-2 have been the focus of intensive study over the years and have been well worked out. By contrast, an understanding of the intracellular signals coupled to the IL-2 receptor and responsible for mediating IL-2 effects in T-cells is far less developed, and the role that protein kinase C (PKC) may play in the various cellular responses to IL-2 receptor activation is unclear. In this article we will discuss IL-2, its receptors, and IL-2 signal transduction in relation to the physiological roles PKC activation may play in IL-2-mediated activation of T-cells and other hematopoietic cells.     

L-tryptophan injection fails to alter nutrient selection by rats

Nutritional studies on rats given a choice between two diets differing in protein content have led to the proposal that brain 5-HT content regulates protein intake [2]. Pharmacologic studies under similar conditions of dietary self-selection suggest that brain 5-HT controls carbohydrate intake [41]. We tested the effect of elevating brain 5-HT via tryptophan injection (100 mg/kg) on short-term food intake and selection by rats choosing between two diets differing in protein and carbohydrate content. Under these conditions neither total food intake nor protein and carbohydrate selection were affected despite increases of 50% in brain concentrations of 5-HT and 5-HIAA. The effect of Trp administration was selective to serotonin metabolism as brain concentrations of NE, DA and DOPAC were not affected. These results suggest that alterations in brain 5-HT content which may occur following meal ingestion may not be of physiological importance in regulating nutrient intake and selection.     

中国版纳猪MHCI类P1分子全长的原核表达与纯化

目的：获得原核表达的中国版纳猪ＳＬＡＩ类Ｐ１蛋白质分子。方法：ＰＣＲ扩增去信号肽的ＳＬＡＩ类Ｐ１ｃＤＮＡ序列，亚克隆至ｐＧＥＭＴ载体，测序。将亚克隆的Ｐ１　ｃＤＮＡ片段插入表达载体ｐＥＴ４２ｂ（＋），构建重组表达质粒ｐＥＴ－４２ｂ（＋）／ｓｌａ－ｐｌ，转化Ｅ·ｃｏｌｉ表达菌 ＢＬ２１－ＣｏｄｏｎＰｌｕｓ（ＤＥ３）－ＲＩＬ，ＩＰＴＧ诱导 Ｐ１－８ ｘ ｈｉｓ融合蛋白表达，经包涵体洗涤，８ ｍｏｌ／Ｌ尿素变性溶解，Ｎｉ２＋亲和层析，梯度透析后，定量保存。ＳＤＳ－ＰＡＧＥ、ｗｅｓｔｅｒｎ－ｂｌｏｔｔｉｎｇ鉴定目的蛋白的表达与纯化。结果：目的蛋白（分子量３９．５ ｋＤ）表达量占细菌总蛋白 １５％，每升表达菌获得纯度９５％的目的蛋白 ４０ ｍｇ～６０ｍｇ。结论：成功建立猪 ＳＬＡ分子全长原核表达、纯化体系，为建立间接识别猪移植抗原ＳＬＡＩ类分子的人Ｔ细胞系及表位分析打下基础。     

PKC在成年大鼠脊髓背角C-纤维诱发电位长时程增强的诱导和维持中的作用

目的：探讨蛋白激酶C (PKC) 在大鼠脊髓背角C-纤维诱发电位长时程增强（LTP）的诱导和维持中的作用。方法： 细胞外记录技术在脊髓腰膨大部记录背角浅层神经元C-纤维诱发电位。 结果：(1) PKC的选择性抑制剂chelerythrine（200 μmol/L）或G 6983（100 μmol/L）对脊髓背角C-纤维诱发电位的基础电位没有影响，但可完全阻断脊髓背角LTP的诱导。(2) Chelerythrine或G 6983呈时间依赖性翻转脊髓背角LTP。在LTP 诱导后15 min，脊髓局部给予chelerythrine（200 μmol/L）后，LTP逐渐降低，于给药后70 min降至对照水平；而G 6983（100 μmol/L）产生同chelerythrine相似的效应，在用药后110 min，LTP降至对照水平。但同样浓度的chelerythrine或G 6983在LTP 诱导后3 h，均不能翻转业已建立的LTP。结论： PKC参与脊髓背角C-纤维诱发电位LTP的诱导和早期维持，而不影响晚期LTP的维持。     

类金刚石薄膜蛋白吸附与碳相成分关系的定量分析研究

运用灰色系统理论中的 T型关联度分析方法 ,对类金刚石 (DL C)薄膜、富石墨相 DL C薄膜和富金刚石相 DL C薄膜三种 DL C薄膜进行了碳相成分对其白蛋白 (HSA)、纤维蛋白原 (HFG)、免疫球蛋白 (Ig G)三种血浆蛋白吸附量影响的定量分析研究。合理地解释了三种材料蛋白吸附量随碳相成分变化的实验结果 ,并得出如下重要的分析结论 :(1)石墨和 C- H相对 HSA的吸附影响较大 ,随着二者的增加 ,HSA的吸附量下降 ;(2 )与 HFG吸附有较强关联的碳相成分是 DL C相和 C- O相 ,前者呈负相关 ,后者为正相关 ;(3)各碳相成分对 Ig G的吸附均有性质不尽相同的影响 ,但程度有限 ,且彼此间相差不大 ;(4 ) DL C碳相具有增强 HSA吸附、抑制 HFG、Ig G吸附的双重功效 ,其对 DL C薄膜血液相容性的影响远较其它碳相成分更为重要。