Lentiviral vector-mediated expression of GFP or Kir2.1 alters the electrophysiology of neonatal rat ventricular myocytes without inducing cytotoxicity

Recombinant lentiviral vectors (LVs) are capable of transducing neonatal rat ventricular myocytes (NRVMs) and providing stable, long-term transgene expression. The goal of the present study was to comprehensively test whether transduction of NRVMs by LVs results in cytotoxicity and to examine the electrophysiological consequences of gene modification of NRVM monolayers by two vectors: one encoding a putatively inert enhanced green fluorescent protein (eGFP) and the other a major ion channel protein, inward rectifier K(+) channel (Kir) 2.1. Freshly isolated NRVMs were transduced and cultured in monolayers. Immunohistochemistry, Trypan blue exclusion, annexin V binding followed by flow cytometry (FCM), and terminal transferase dUTP nick-end labeling assays were performed to assess for cytotoxicity. Optical mapping studies of action potential propagation in NRVM monolayers were performed to characterize the electrophysiological alterations following transduction. The cytotoxicity assays revealed that transduction had no adverse effects on NRVM cultures. However, eGFP-transduced monolayers exhibited a decrease in conduction velocity (CV) and action potential duration (APD) compared with monolayers transduced with LVs encoding LacZ or devoid of a transgene. In addition, small interfering RNA-mediated knockdown of eGFP expression corrected this phenotype. In contrast, Kir2.1 gene-modified monolayers showed an increase in CV and a predictable decrease in APD. This study demonstrates that LVs transduce NRVMs without cytotoxic effects. However, eGFP has a significant effect on APD and CV in this experimental system and calls into question the widely held belief that GFP is physiologically inert. In addition, LV-mediated overexpression of Kir2.1 opens up the prospect of studying the functional role of inward rectifier K(+) current in cardiac arrhythmias.     

Spitting out information: Trigona bees deposit saliva to signal resource locations

Stingless bees of the species Trigona spinipes (Fabricius 1793) use their saliva to lay scent trails communicating the location of profitable food sources. Extracts of the cephalic labial glands of the salivary system (not the mandibular glands, however) contain a large amount (approx. 74%) of octyl octanoate. This ester is also found on the scent-marked substrates at the feeding site. We demonstrate octyl octanoate to be a single compound pheromone which induces full trail following behaviour. The identification of the trail pheromone in this widely distributed bee makes it an ideal organism for studying the mechanism of trail following in a day flying insect.     

Modulating membrane protein stability and association by design

Membrane proteins perform crucial communication functions across biological membranes. They represent important targets for therapies and have unique properties for biotechnological applications. Recently, the number of high-resolution membrane protein structures has significantly increased and new insights into the sequence/structure relationships of transmembrane helical assemblies have been gained. Together with new experimental techniques, these advances have improved our understanding of membrane protein folding, stability, and recognition. Consequently, new design strategies are emerging that aim to target and stabilize simple transmembrane helical interfaces.     

Closing in on the resting state of the Shaker K(+) channel

Membrane depolarization causes voltage-gated ion channels to transition from a resting/closed conformation to an activated/open conformation. We used voltage-clamp fluorometry to measure protein motion at specific regions of the Shaker Kv channel. This enabled us to construct new structural models of the resting/closed and activated/open states based on the Kv1.2 crystal structure using the Rosetta-Membrane method and molecular dynamics simulations. Our models account for the measured gating charge displacement and suggest a molecular mechanism of activation in which the primary voltage sensors, S4s, rotate by approximately 180 degrees as they move "outward" by 6-8 A. A subsequent tilting motion of the S4s and the pore domain helices, S5s, of all four subunits induces a concerted movement of the channel's S4-S5 linkers and S6 helices, allowing ion conduction. Our models are compatible with a wide body of data and resolve apparent contradictions that previously led to several distinct models of voltage sensing.     

Fetal and early post-natal development of the human spleen: from primordial arterial B cell lobules to a non-segmented organ

Immunohistological analysis of 31 human spleens from the 11th week of gestation to the early postnatal period suggested that fetal organ development may be preliminarily divided into four stages. At stage 0 the organ anlage contained erythrocyte precursors, few macrophages and almost no lymphocytes. Fetal spleens of stage I exhibited arterial vascular lobules and lymphocytes just began colonizing the organ. At stage II, B and T lymphocytes formed periarteriolar clusters. B cell clusters predominated, because B cells aggregated around the more peripheral branches of splenic arterioles, while T cells occupied the more centrally located parts of the vessels. The vascular lobules of stage I and II consisted of central arterioles surrounded by B cells, capillaries and peripheral venules. The lobular architecture slowly dissolved at late stage II when sinuses grew out from the peripheral venules into the centre of the lobule. Interestingly, the B cell accumulations around peripheral arterioles did not represent the precursors of follicles, but apparently persisted as periarteriolar B cell clusters in the adult splenic red pulp, while follicles containing FDCs developed at late stage II from B cells in direct contact to T cell clusters around larger arterial vessels. At stage III before birth the lobular architecture was no longer recognized. The chemokine CXCL13 was already present in vascular smooth muscle and adjacent stromal cells at stage I before B cells immigrated. CCL21, on the contrary, was only demonstrated in fibroblast-like cells supporting T cell clusters from stage II onwards.     

The differential expression of ribosomal 18S RNA paralog genes from the chaetognath <Emphasis Type="Italic">Spadella cephaloptera</Emphasis>

Chaetognaths constitute a small marine phylum of approximately 120 species. Two classes of both 18S and 28S rRNA gene sequences have been evidenced in this phylum, even though significant intraindividual variation in the sequences of rRNA genes is unusual in animal genomes. These observations led to the hypothesis that this unusual genetic characteristic could play one or more physiological role(s). Using in situ hybridization on the frontal sections of the chaetognath Spadella cephaloptera, we found that the 18S Class I genes are expressed in the whole body, with a strong expression throughout the gut epithelium, whereas the expression of the 18S Class II genes is restricted to the oocytes. Our results could suggest that the paralog products of the 18S Class I genes are probably the "housekeeping" 18S rRNAs, whereas those of class II would only be essential in specific tissues. These results provide support for the idea that each type of 18S paralog is important for specific cellular functions and is under the control of selective factors.     

酵母双杂交系统筛选与RapGAP相互作用的蛋白质

目的筛选与Rap GAP相互作用的蛋白质,为进一步研究人源Rap1GAP介导的信号转导通路、揭示其与肿瘤的关系提供实验依据。方法选用与Rap1GAP同源的来自美丽线虫的Rap GAP作为饵蛋白,以来源于美丽线虫的c DNA文库作为靶蛋白,应用p PC97、p PC86组成的酵母双杂交系统筛选c DNA文库中与Rap GAP相互作用的蛋白质。结果通过营养缺陷平板(-LTH)筛选出63个拟似阳性菌落。经过Lac Z鉴定,19个菌落为阳性,其中7个为强阳性。提取来自19个酵母菌落中的重组DNA,经PCR扩增,12个菌落出现阳性结果。将该19个重组DNA分别电转化入DH5α细菌,涂板培养后,每板挑取4~10个克隆,通过Sal I和Not I双酶切鉴定进行阳性克隆筛选。将阳性克隆的重组DNA进行序列测定。测序结果与Gen Bank比较,其中4个克隆的DNA片段为Y39b6a基因片段、2个为Rap GAP、1个为苯丙氨酸-4-羟化酶、1个为细胞色素C氧化酶,还有1个DNA片段编码美丽线虫特有的小分子蛋白的基因片段,其余11个DNA片段不编码已知蛋白质。结论初步筛选出与Rap GAP相互作用的蛋白质,特别是其中有2个克隆为Rap GAP,提示Rap GAP可能以二聚体的方式存在。     

Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family

Background

It is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5''-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions.

Results

Phylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5''-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate.

Conclusions

Like the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine oxidases as well as by pyridoxal 5''-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1584-3) contains supplementary material, which is available to authorized users.     

SNAP-tag technology mediates site specific conjugation of antibody fragments with a photosensitizer and improves target specific phototoxicity in tumor cells

Cancer cells can be killed by photosensitizing agents that induce toxic effects when exposed to nonhazardous light, but this also causes significant damage to surrounding healthy cells. The specificity of photodynamic therapy can be increased by conjugating photosensitizing agents to antibodies and antibody fragments that bind specifically to tumor cell antigens. However, standard conjugation reactions produce heterogeneous products whose targeting specificity and spectroscopic properties can be compromised. In this study, we used an antibody fragment (scFv-425) that binds to the epidermal growth factor receptor (EGFR) as a model to investigate the use of SNAP-tag fusions as an improved conjugation strategy. The scFv-425-SNAP-tag fusion protein allowed the specific conjugation of a chlorin e6 photosensitizer modified with O(6)-benzylguanine, generating a homogeneous product that was delivered specifically to EGFR(+) cancer cells and resulted in significant, tumor cell-specific cytotoxicity. The impact of our results on the development of photodynamic therapy is discussed.     

Mouse PRDM9 DNA-binding specificity determines sites of histone H3 lysine 4 trimethylation for initiation of meiotic recombination

Meiotic recombination generates reciprocal exchanges between homologous chromosomes (also called crossovers, COs) that are essential for proper chromosome segregation during meiosis and are a major source of genome diversity by generating new allele combinations. COs have two striking properties: they occur at specific sites, called hotspots, and these sites evolve rapidly. In mammals, the Prdm9 gene, which encodes a meiosis-specific histone H3 methyltransferase, has recently been identified as a determinant of CO hotspots. Here, using transgenic mice, we show that the sole modification of PRDM9 zinc fingers leads to changes in hotspot activity, histone H3 lysine 4 trimethylation (H3K4me3) levels, and chromosome-wide distribution of COs. We further demonstrate by an in vitro assay that the PRDM9 variant associated with hotspot activity binds specifically to DNA sequences located at the center of the three hotspots tested. Remarkably, we show that mutations in cis located at hotspot centers and associated with a decrease of hotspot activity affect PRDM9 binding. Taken together, these results provide the direct demonstration that Prdm9 is a master regulator of hotspot localization through the DNA binding specificity of its zinc finger array and that binding of PRDM9 at hotspots promotes local H3K4me3 enrichment.