571 Physiologie und verwandte Themen
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An optochemokine tandem was developed to control the release of calcium from endosomes into the cytosol by light and to analyze the internalization kinetics of G-protein coupled receptors (GPCRs) by electrophysiology. A previously constructed rhodopsin tandem was re-engineered to combine the light-gated Ca\(^{2+}\)-permeable cation channel Channelrhodopsin-2(L132C), CatCh, with the chemokine receptor CXCR4 in a functional tandem protein tCXCR4/CatCh. The GPCR was used as a shuttle protein to displace CatCh from the plasma membrane into intracellular areas. As shown by patch-clamp measurements and confocal laser scanning microscopy, heterologously expressed tCXCR4/CatCh was internalized via the endocytic SDF1/CXCR4 signaling pathway. The kinetics of internalization could be followed electrophysiologically via the amplitude of the CatCh signal. The light-induced release of Ca\(^{2+}\) by tandem endosomes into the cytosol via CatCh was visualized using the Ca\(^{2+}\)-sensitive dyes rhod2 and rhod2-AM showing an increase of intracellular Ca\(^{2+}\) in response to light.
The Venus flytrap Dionaea muscipula counts prey-induced action potentials to induce sodium uptake
(2016)
Carnivorous plants, such as the Venus flytrap (Dionaea muscipula), depend on an animal diet when grown in nutrient-poor soils. When an insect visits the trap and tilts the mechanosensors on the inner surface, action potentials (APs) are fired. After a moving object elicits two APs, the trap snaps shut, encaging the victim. Panicking preys repeatedly touch the trigger hairs over the subsequent hours, leading to a hermetically closed trap, which via the gland-based endocrine system is flooded by a prey-decomposing acidic enzyme cocktail. Here, we asked the question as to how many times trigger hairs have to be stimulated (e.g., now many APs are required) for the flytrap to recognize an encaged object as potential food, thus making it worthwhile activating the glands. By applying a series of trigger-hair stimulations, we found that the touch hormone jasmonic acid (JA) signaling pathway is activated after the second stimulus, while more than three APs are required to trigger an expression of genes encoding prey-degrading hydrolases, and that this expression is proportional to the number of mechanical stimulations. A decomposing animal contains a sodium load, and we have found that these sodium ions enter the capture organ via glands. We identified a flytrap sodium channel DmHKT1 as responsible for this sodium acquisition, with the number of transcripts expressed being dependent on the number of mechano-electric stimulations. Hence, the number of APs a victim triggers while trying to break out of the trap identifies the moving prey as a struggling Na\(^+\)-rich animal and nutrition for the plant.
The animal diet of the carnivorous Venus flytrap, Dionaea muscipula, contains a sodium load that enters the capture organ via an HKT1-type sodium channel, expressed in special epithelia cells on the inner trap lobe surface. DmHKT1 expression and sodium uptake activity is induced upon prey contact. Here, we analyzed the HKT1 properties required for prey sodium osmolyte management of carnivorous Dionaea. Analyses were based on homology modeling, generation of model-derived point mutants, and their functional testing in Xenopus oocytes. We showed that the wild-type HKT1 and its Na\(^+\)- and K\(^+\)-permeable mutants function as ion channels rather than K\(^+\) transporters driven by proton or sodium gradients. These structural and biophysical features of a high-capacity, Na\(^+\)-selective ion channel enable Dionaea glands to manage prey-derived sodium loads without confounding the action potential-based information management of the flytrap.
Background: In the phase III AVAGAST trial, the addition of bevacizumab to chemotherapy improved progression-free survival (PFS) but not overall survival (OS) in patients with advanced gastric cancer. We studied the role of Angiopoietin-2 (Ang-2), a key driver of tumour angiogenesis, metastasis and resistance to antiangiogenic treatment, as a biomarker.
Methods: Previously untreated, advanced gastric cancer patients were randomly assigned to receive bevacizumab (n = 387) or placebo (n = 387) in combination with chemotherapy. Plasma collected at baseline and at progression was analysed by ELISA. The role of Ang-2 as a prognostic and a predictive biomarker of bevacizumab efficacy was studied using a Cox proportional hazards model. Logistic regression analysis was applied for correlations with metastasis. Results: Median baseline plasma Ang-2 levels were lower in Asian (2143 pg ml\(^-\)\(^1\)) vs non-Asian patients (3193 pg ml\(^-\)\(^1\)), P<0.0001. Baseline plasma Ang-2 was identified as an independent prognostic marker for OS but did not predict bevacizumab efficacy alone or in combination with baseline VEGF. Baseline plasma Ang-2 correlated with the frequency of liver metastasis (LM) at any time: Odds ratio per 1000 pg ml\(^-\)\(^1\) increase: 1.19; 95% CI 1.10-1.29; P<0.0001 (non-Asians) and 1.37; 95% CI 1.13-1.64; P = 0.0010 (Asians).
Conclusions: Baseline plasma Ang-2 is a novel prognostic biomarker for OS in advanced gastric cancer strongly associated with LM. Differences in Ang-2 mediated vascular response may, in part, account for outcome differences between Asian and non-Asian patients; however, data have to be further validated. Ang-2 is a promising drug target in gastric cancer.
Polygonum cuspidatum (Japanese knotweed, also known as Huzhang in Chinese), a plant that produces bioactive components such as stilbenes and quinones, has long been recognized as important in traditional Chinese herbal medicine. To better understand the biological features of this plant and to gain genetic insight into the biosynthesis of its natural products, we assembled a draft genome of P. cuspidatum using Illumina sequencing technology. The draft genome is ca. 2.56 Gb long, with 71.54% of the genome annotated as transposable elements. Integrated gene prediction suggested that the P. cuspidatum genome encodes 55,075 functional genes, including 6,776 gene families that are conserved in the five eudicot species examined and 2,386 that are unique to P. cuspidatum. Among the functional genes identified, 4,753 are predicted to encode transcription factors. We traced the gene duplication history of P. cuspidatum and determined that it has undergone two whole-genome duplication events about 65 and 6.6 million years ago. Roots are considered the primary medicinal tissue, and transcriptome analysis identified 2,173 genes that were expressed at higher levels in roots compared to aboveground tissues. Detailed phylogenetic analysis demonstrated expansion of the gene family encoding stilbene synthase and chalcone synthase enzymes in the phenylpropanoid metabolic pathway, which is associated with the biosynthesis of resveratrol, a pharmacologically important stilbene. Analysis of the draft genome identified 7 abscisic acid and water deficit stress-induced protein-coding genes and 14 cysteine-rich transmembrane module genes predicted to be involved in stress responses. The draft de novo genome assembly produced in this study represents a valuable resource for the molecular characterization of medicinal compounds in P. cuspidatum, the improvement of this important medicinal plant, and the exploration of its abiotic stress resistance.
A comparative view on sex differentiation and gametogenesis genes in lungfish and coelacanths
(2018)
Gonadal sex differentiation and reproduction are the keys to the perpetuation of favorable gene combinations and positively selected traits. In vertebrates, several gonad development features that differentiate tetrapods and fishes are likely to be, at least in part, related to the water-to-land transition. The collection of information from basal sarcopterygians, coelacanths, and lungfishes, is crucial to improve our understanding of the molecular evolution of pathways involved in reproductive functions, since these organisms are generally regarded as “living fossils” and as the direct ancestors of tetrapods. Here, we report for the first time the characterization of >50 genes related to sex differentiation and gametogenesis in Latimeria menadoensis and Protopterus annectens. Although the expression profiles of most genes is consistent with the intermediate position of basal sarcopterygians between actinopterygian fish and tetrapods, their phylogenetic placement and presence/absence patterns often reveal a closer affinity to the tetrapod orthologs. On the other hand, particular genes, for example, the male gonad factor gsdf (Gonadal Soma-Derived Factor), provide examples of ancestral traits shared with actinopterygians, which disappeared in the tetrapod lineage.
In the mammalian host, the Trypanosoma brucei cell surface is covered with a densely packed protein coat of a single protein, the variant surface glycoprotein (VSG). The VSG is believed to shield invariant surface proteins from host antibodies but there is limited information on how far antibodies can penetrate into the VSG monolayer. Here, the VSG surface coat was probed to determine whether it acts as a barrier to binding of antibodies to the membrane proximal VSG C-terminal domain. The binding of C-terminal domain antibodies to VSG221 or VSG118 was compared with antibodies recognising the cognate whole VSGs. The C-terminal VSG domain was inaccessible to antibodies on live cells but not on fixed cells. This provides further evidence that the VSG coat acts as a barrier and protects the cell from antibodies that would otherwise bind to some of the other externally disposed proteins.
Low field NMR has been successfully used for the evaluation of seed composition and quality, but largely only in crop species. We show here that 1.5T NMR provides a reliable means for analysing the seed lipid fraction present in a wide range of species, where both the seed size and lipid concentration differed by >10 fold. Little use of high field NMR has been made in seed research to date, even though it potentially offers many opportunities for studying seed development, metabolism and storage. Here we demonstrate how 17.5T and 20T NMR can be applied to image seed structure, and analyse lipid and metabolite distribution. We suggest that further technical developments in NMR/MRI will facilitate significant advances in our understanding of seed biology.
Sex-specific markers are a prerequisite for understanding reproductive biology, genetic factors involved in sex differences, mechanisms of sex determination, and ultimately the evolution of sex chromosomes. The Western mosquitofish, Gambusia affinis, may be considered a model species for sex-chromosome evolution, as it displays female heterogamety (ZW/ZZ), and is also ecologically interesting as a worldwide invasive species. Here, de novo RNA-sequencing on the gonads of sexually mature G. affinis was used to identify contigs that were highly transcribed in females but not in males (i.e., transcripts with ovary-specific expression). Subsequently, 129 primer pairs spanning 79 contigs were tested by PCR to identify sex-specific transcripts. Of those primer pairs, one female-specific DNA marker was identified, Sanger sequenced and subsequently validated in 115 fish. Sequence analyses revealed a high similarity between the identified sex-specific marker and the 3' UTR of the aminomethyl transferase (amt) gene of the closely related platyfish (Xiphophorus maculatus). This is the first time that RNA-seq has been used to successfully characterize a sex-specific marker in a fish species in the absence of a genome map. Additionally, the identified sex-specific marker represents one of only a handful of such markers in fishes.