肿瘤浸润性γδT细胞（γδTIL）CDR3δ1区的序列分析

目的对肿瘤浸润性γδT细胞（γδTIL）CDR3δ1区进行序列分析。方法用固相包被抗体,体外扩增14例肿瘤组织（胃癌、肾癌、卵巢癌、膀胱癌、食管癌、肺癌、嗜铬细胞瘤）和2例肿瘤腹水（浆乳癌）的γδ肿瘤浸润淋巴细胞（tumor infiltration lymphocytes,γδTIL）,RT-PCR法扩增CDR3δ1基因片段,测序分析其序列特征。结果 16例γδTIL的CDR3δ1确实存在优势序列,不完全相同。其中4例胃癌来源的γδTIL中3例的CDR3δ1优势序列相同,3例食管癌来源的γδTIL中2例的CDR3δ1优势序列相同。结论体外扩增培养的γδTIL的TCR CDR3δ1序列具有相对优势特征。不同个体的同种组织来源的γδTIL的CDR3δ1优势序列趋于相同。     

Biochemical characterization of tau protein and its associated syndapin 1 and protein kinase Cɛ for their functional regulation in rat brain

Background

We recently reported that both sulfatide and cholesterol-3-sulfate (SCS) function as potent stimulators for the GSK-3β-mediated phosphorylation of tau protein (TP) in vitro [J. Biochem. 143 (2008) 359–367].

Methods

By means of successive gel filtration on a Superdex 200 pg column and three distinct ion-exchange column chromatographies, TP and its associated proteins were highly purified from the extract of rat brain.

Results

We found that (i) syndapin 1 and novel protein kinase C? (nPKC?) were identified as the TP-associated proteins; (ii) SCS highly stimulated the phosphorylation of TP and syndapin 1 by nPKC? as well as CK1; (iii) the full phosphorylation of TP and syndapin 1 by nPKC? in the presence of sulfatide resulted in their dissociation; (iv) TP primed by CK1 functioned as an effective phosphate acceptor for GSK-3β; (v) syndapin 1 highly stimulated the GSK-3β-mediated phosphorylation of TP; and (vi) TP isoforms were highly expressed in aged brain, whereas syndapin 1 was consistently detected in adult brain, but not in newborn brain.

General significance

These results provided here suggest that (i) TP-associated nPKC? suppresses the GSK-3β-mediated phosphorylation of TP through the phosphorylation of GSK-3β by the kinase in vitro; and (ii) SCS act as effective sole mediators to induce the GSK-3β-mediated high phosphorylation of both TP and its associated syndapin 1 involved in the biochemical processes of neuronal diseases, including Alzheimer's disease.     

Diverse regulation of sensory signaling by C. elegans nPKC-epsilon/eta TTX-4

Molecular and pharmacological studies in vitro suggest that protein kinase C (PKC) family members play important roles in intracellular signal transduction. Nevertheless, the in vivo roles of PKC are poorly understood. We show here that nPKC-epsilon/eta TTX-4 in the nematode Caenorhabditis elegans is required for the regulation of signal transduction in various sensory neurons for temperature, odor, taste, and high osmolality. Interestingly, the requirement for TTX-4 differs in different sensory neurons. In AFD thermosensory neurons, gain or loss of TTX-4 function inactivates or hyperactivates the neural activity, respectively, suggesting negative regulation of temperature sensation by TTX-4. In contrast, TTX-4 positively regulates the signal sensation of ASH nociceptive neurons. Moreover, in AWA and AWC olfactory neurons, TTX-4 plays a partially redundant role with another nPKC, TPA-1, to regulate olfactory signaling. These results suggest that C. elegans nPKCs regulate different sensory signaling in various sensory neurons. Thus, C. elegans provides an ideal model to reveal genetically novel components of nPKC-mediated molecular pathways in sensory signaling.     

Neocarzinostatin chromophore: Presence of a cyclic carbonate subunit and its modification in the structure of other biologically active forms

On the basis of spectroscopic evidence, opening of a five-membered cyclic carbonate ring (1,3-dioxolan-2-one) in the C₁₅-subunit of the previously determined partial structure $\text{1}$ (Fig. 1) of the major neocarzinostatin chromophore (NCS-Chrom $\text{A}$), is proposed to account for its base-catalyzed methanolysis to NCS-Chrom $\text{C}$. NCS-Chrom $\text{B}$, apparently an authentic natural product present as a minor component in all preparations of NCS studied, was found to be formally equivalent to the hydrolysis/decarboxylation product of the cyclic carbonate functionality in NCS-Chrom $\text{A}$. The mercaptan-dependent DNA strand-scission activity, equivalent for NCS-Chrom $\text{A}$, $\text{B}$ and $\text{C}$, is independent of the integrity of the cyclic carbonate ring system and implicates a secondary site in the C₁₅-substructure for mercaptan activation.     

Natal origins and timing of migration of two passerine species through the southern Alps: inferences from multiple stable isotopes (δ2H, δ13C, δ15N, δ34S) and ringing data

Understanding spatial linkages between areas used by migratory animals during the annual cycle is fundamental to their conservation. Stable isotope measurements of animal tissues can be a valuable tool in understanding spatial connectivity and migration phenology of migratory wildlife. We inferred natal origins of two migratory passerines, European Pied Flycatcher Ficedula hypoleuca and European Robin Erithacus rubecula, captured during autumn migration in the Italian Alps, by combining feather δ²H (δ²H_f) and ring recovery data. We used a spatially explicit likelihood-based method to assign individuals to a precipitation δ²H surface calibrated to represent feather δ²H, together with the directional probability of origin from ring recoveries. The highest probabilities of origin for most individuals of both species were in central and north-eastern Europe. Seasonal trends in δ²H_f, which described the species’ migratory phenology through the Italian Alps, were correlated with feather δ¹³C, δ¹⁵N and δ³⁴S values, indicating strong spatial discrimination related to continental patterns for these isotopes. We demonstrate how this combined information can define catchment areas and migratory connectivity of birds intercepted in the Alps. We highlight the importance of ringing data in defining directional priors to define Bayesian-based probability surfaces using continental δ²H_f isoscapes, and how such information can be used to inform estimates of migratory connectivity.     

Casein Kinase 1δ Stabilizes Mature Axons by Inhibiting Transcription Termination of Ankyrin

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In vivo CRISPR screens identify the E3 ligase Cop1 as a modulator of macrophage infiltration and cancer immunotherapy target

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Protein kinase Cdelta participates in insulin-induced activation of PKB via PDK1

PKCdelta has been shown to be activated by insulin and to interact with insulin receptor and IRS. PKB(Akt) plays an important role in glucose transport and glycogen synthesis. In this study, we investigated the possibility that PKCdelta may be involved in insulin-induced activation of PKB. Studies were conducted on primary cultures of rat skeletal muscle. PKB was activated by insulin stimulation within 5min and reached a peak by 15-30min. Insulin also increased the physical association between PKCdelta with PKB and with PDK1. The insulin-induced PKCdelta-PKB association was PI3K dependent. PKB-PKCdelta association was accounted for by the involvement of PDK1. Overexpression of dominant negative PKCdelta abrogated insulin-induced association of PKCdelta with both PKB and PDK1. Blockade of PKCdelta also decreased insulin-induced Thr308 PKB phosphorylation and PKB translocation. Moreover, PKCdelta inhibition reduced insulin-induced GSK3 phosphorylation. The results indicate that insulin-activated PKCdelta interacts with PDK1 to regulate PKB.