春小麦对骤旱的响应特征及其阈值分析

与缓慢发展的干旱过程不同,骤旱具有发生速度快,短期内可致害的特点。目前,关于作物骤旱致害的临界阈值及其调控机制尚不清楚。以春小麦为供试作物,通过桶栽试验,模拟研究骤旱过程中小麦受旱致害的过程特征及其控制因素。结果发现,发生骤旱时土壤含水量下降呈先快后慢的变化趋势,叶片水分和叶水势则呈先慢后快的指数变化趋势。叶片光合生理指标对土壤水分的下降存在明显的阈值响应,且不同生理指标的阈值并不完全相同,其中净光合速率与表征叶片光合能力的指标（最大羧化速率）对土壤有效含水量的响应阈值为0.4,气孔导度和蒸腾速率对土壤有效含水量的响应阈值分别为0.5和0.4。而小麦光合生理指标对叶片水分和叶水势的阈值响应并不明显。同时依据各生理指标相关和通径分析结果得出,骤旱发生时引起小麦叶片净光合速率快速降低的主导因子为非气孔因素,而并不是以往作物受旱研究中的气孔因素。本研究结果有望丰富干旱影响认知,并可为科学应对干旱提供依据。     

Adipose Tissue Lipolysis Promotes Exercise-induced Cardiac Hypertrophy Involving the Lipokine C16:1n7-Palmitoleate

Endurance exercise training induces substantial adaptive cardiac modifications such as left ventricular hypertrophy (LVH). Simultaneously to the development of LVH, adipose tissue (AT) lipolysis becomes elevated upon endurance training to cope with enhanced energy demands. In this study, we investigated the impact of adipose tissue lipolysis on the development of exercise-induced cardiac hypertrophy. Mice deficient for adipose triglyceride lipase (Atgl) in AT (atATGL-KO) were challenged with chronic treadmill running. Exercise-induced AT lipolytic activity was significantly reduced in atATGL-KO mice accompanied by the absence of a plasma fatty acid (FA) increase. These processes were directly associated with a prominent attenuation of myocardial FA uptake in atATGL-KO and a significant reduction of the cardiac hypertrophic response to exercise. FA serum profiling revealed palmitoleic acid (C16:1n7) as a new molecular co-mediator of exercise-induced cardiac hypertrophy by inducing nonproliferative cardiomyocyte growth. In parallel, serum FA analysis and echocardiography were performed in 25 endurance athletes. In consonance, the serum C16:1n7 palmitoleate level exhibited a significantly positive correlation with diastolic interventricular septum thickness in those athletes. No correlation existed between linoleic acid (18:2n6) and diastolic interventricular septum thickness. Collectively, our data provide the first evidence that adipose tissue lipolysis directly promotes the development of exercise-induced cardiac hypertrophy involving the lipokine C16:1n7 palmitoleate as a molecular co-mediator. The identification of a lipokine involved in physiological cardiac growth may help to develop future lipid-based therapies for pathological LVH or heart failure.     

应用核酶体外切割马铃薯卷叶病毒外壳蛋白基因的研究

针对马铃薯卷叶病毒外壳蛋白基因第356～358位点“GUC”．设计、合成了一种“锤头状”核酶。将核酶基因克隆在体外转录载体PSPT19的SP6启动子下游;同时将PLRVCPcDNA亚克隆在体外转录载体pSPT18的SP6启动子下游。利用SP6RNA聚合酶分别体外转录,获得核酶分子和靶RNA序列。在41℃保温进行核酶切割反应,检测到预期大小且被切开的两个RNA短片段。     

The Toll-Like Receptor 4 (TLR4) Variant rs2149356 and Risk of Gout in European and Polynesian Sample Sets

Deposition of crystallized monosodium urate (MSU) in joints as a result of hyperuricemia is a central risk factor for gout. However other factors must exist that control the progression from hyperuricaemia to gout. A previous genetic association study has implicated the toll-like receptor 4 (TLR4) which activates the NLRP3 inflammasome via the nuclear factor-κB signaling pathway upon stimulation by MSU crystals. The T-allele of single nucleotide polymorphism rs2149356 in TLR4 is a risk factor associated with gout in a Chinese study. Our aim was to replicate this observation in participants of European and New Zealand Polynesian (Māori and Pacific) ancestry. A total of 2250 clinically-ascertained prevalent gout cases and 13925 controls were used. Non-clinically-ascertained incident gout cases and controls from the Health Professional Follow-up (HPFS) and Nurses Health Studies (NHS) were also used. Genotypes were derived from genome-wide genotype data or directly obtained using Taqman. Logistic regression analysis was done including age, sex, diuretic exposure and ancestry as covariates as appropriate. The T-allele increased the risk of gout in the clinically-ascertained European samples (OR = 1.12, P = 0.012) and decreased the risk of gout in Polynesians (OR = 0.80, P = 0.011). There was no evidence for association in the HPFS or NHS sample sets. In conclusion TLR4 SNP rs2143956 associates with gout risk in prevalent clinically-ascertained gout in Europeans, in a direction consistent with previously published results in Han Chinese. However, with an opposite direction of association in Polynesians and no evidence for association in a non-clinically-ascertained incident gout cohort this variant should be analysed in other international gout genetic data sets to determine if there is genuine evidence for association.     

A cryptochrome-like protein is involved in the regulation of photosynthesis genes in Rhodobacter sphaeroides

Blue light receptors belonging to the cryptochrome/photolyase family are found in all kingdoms of life. The functions of photolyases in repair of UV-damaged DNA as well as of cryptochromes in the light-dependent regulation of photomorphogenetic processes and in the circadian clock in plants and animals are well analysed. In prokaryotes, the only role of members of this protein family that could be demonstrated is DNA repair. Recently, we identified a gene for a cryptochrome-like protein (CryB) in the α-proteobacterium Rhodobacter sphaeroides. The protein lacks the typical C-terminal extension of cryptochromes, and is not related to the Cry DASH family. Here we demonstrate that CryB binds flavin adenine dinucleotide that can be photoreduced by blue light. CryB binds single-stranded DNA with very high affinity ( K _d∼10⁻⁸ M) but double-stranded DNA and single-stranded RNA with far lower affinity ( K _d∼10⁻⁶ M). Despite of that, no in vitro repair activity for pyrimidine dimers in single-stranded DNA could be detected. However, we show that CryB clearly affects the expression of genes for pigment-binding proteins and consequently the amount of photosynthetic complexes in R. sphaeroides . Thus, for the first time a role of a bacterial cryptochrome in gene regulation together with a biological function is demonstrated.     

Pertussis Toxin-sensitive Signaling of Melanocortin-4 Receptors in Hypothalamic GT1-7 Cells Defines Agouti-related Protein as a Biased Agonist

Melanocortin-4 receptor (MC4R)-induced anorexigenic signaling in the hypothalamus controls body weight and energy homeostasis. So far, MC4R-induced signaling has been exclusively attributed to its coupling to G_s proteins. In line with this monogamous G protein coupling profile, most MC4R mutants isolated from obese individuals showed a reduced ability to activate G_s. However, some mutants displayed enhanced G_s coupling, suggesting that signaling pathways independent of G_s may be involved in MC4R-mediated anorexigenic signaling. Here we report that the G_s signaling-deficient MC4R-D90N mutant activates G proteins in a pertussis toxin-sensitive manner, indicating that this mutant is able to selectively interact with G_i/o proteins. Analyzing a hypothalamic cell line (GT1-7 cells), we observed activation of pertussis toxin-sensitive G proteins by the wild-type MC4R as well, reflecting multiple coupling of the MC4R to G_s and G_i/o proteins in an endogenous cell system. Surprisingly, the agouti-related protein, which has been classified as a MC4R antagonist, selectively activates G_i/o signaling in GT1-7 cells. Thus, the agouti-related protein antagonizes melanocortin-dependent G_s activation not only by competitive antagonism but additionally by initiating G_i/o protein-induced signaling as a biased agonist.The melanocortin system has been shown to play a pivotal role in food intake and energy homeostasis. Therefore, dysfunction of the melanocortin system inevitably leads to an obese phenotype in mammals. Accordingly, targeted disruption of the melanocortin-4 receptor (MC4R)² gene in mice causes an obesity-diabetes syndrome characterized by hyperphagia, hyperinsulinemia, and hyperglycemia (1). The importance of MC4R signaling in the regulation of human metabolism has been highlighted by the finding that mutations in the MC4R gene are the most frequent monogenic cause of severe obesity (2–7).Signaling pathways involved in MC4R-mediated regulation of energy homeostasis have been attributed to its coupling to G_s proteins and the resulting activation of the protein kinase A pathway (8, 9). Agouti and agouti-related protein (AGRP) are the only known endogenously occurring neuropeptides that block GPCR activity and are, therefore, classified as MCR antagonists. AGRP has been shown to block melanocortin signaling at MC3R and MC4R subtypes (10). In addition, it has been proposed that AGRP decreases basal as well as forskolin-promoted adenylyl cyclase activity, thus also acting as an inverse agonist on basal MCR activity (11). However, recent studies revealed that the effects of AGRP on appetite control are independent of melanocortin signaling (12, 13). For example, in mice deficient of the melanocortin precursor proopiomelanocortin starvation after AGRP neuron ablation is independent of melanocortin signaling (13). Thus, the orexigenic effects induced by AGRP appear to be mediated in a melanocortin-independent manner by a so far unknown mechanism.Interestingly, the aforementioned MC4R mutants isolated from obese patients exerted inconsistent effects on G_s signaling. For example, the MC4R-G181D or -S94R mutants showed a loss-of-function phenotype, whereas the MC4R-P78L or -R165W variants exhibited reduced function, whereas other mutants (MC4R-G253S, -I317T, -I251L) showed no functional alterations. Even more surprisingly, some mutants (MC4R-S127L, -P230L) constitutively increased G_s-dependent adenylyl cyclase activity (5). Therefore, no clear correlation could be drawn between the cellular phenotype resulting from these mutations and obesity observed in vivo.Melanocortin-independent actions of AGRP and non-uniform effects of obesity-associated mutations on G_s signaling suggest that the MC4R receptor may interact with G proteins other than G_s. The D90N mutation of the MC4R has also been associated with severe early onset obesity (14). This MC4R variant binds melanocortins with unchanged high affinity, but agonist binding does not initiate G_s signaling (14). Thus, the D90N variant represents an excellent tool to analyze putative G_s-independent signaling pathways of the MC4R.Directly measuring incorporation of GTPγ³⁵S, we show herein that the wild-type MC4R and the MC4R-D90N mutant activate pertussis toxin (PTX)-sensitive G_i/o proteins. Multiple coupling of the MC4R to G_s and G_i/o proteins in HEK293 cells is reflected by cAMP accumulation, as treatment of cells with PTX significantly increased melanocortin-induced cAMP accumulation, indicative of simultaneous activation of adenylyl cyclase stimulating and inhibiting pathways. Using a hypothalamic cell line (GT1-7 cells) endogenously expressing MC4R, we demonstrate PTX-sensitive melanocortin-induced GTPγ³⁵S incorporation and an increase in MC4R-mediated cAMP accumulation in response to toxin treatment. In addition, we show that AGRP, assumed to be an antagonist of the MC4R, promotes PTX-sensitive signaling in GT1-7 cells and, thus, exhibits biased agonistic actions on MC4R in hypothalamic cells. These data may explain melanocortin-independent AGRP signaling and define the MC4R as an interface integrating anorexigenic and orexigenic signaling depending on the stimulus received.     

Electrical protein array chips for the detection of staphylococcal virulence factors

A new approach for the detection of virulence factors of Staphylococcus aureus and Staphylococcus epidermidis using an electrical protein array chip technology is presented. The procedure is based on an enzyme-linked sandwich immunoassay, which includes recognition and binding of virulence factors by specific capture and detection antibodies. Detection of antibody-bound virulence factors is achieved by measuring the electrical current generated by redox recycling of an enzymatically released substance. The current (measured in nanoampere) corresponds to the amount of the target molecule in the analyzed sample. The electrical protein chip allows for a fast detection of Staphylococcus enterotoxin B (SEB) of S. aureus and immunodominant antigen A homologue (IsaA homologue) of S. epidermidis in different liquid matrices. The S. aureus SEB virulence factor could be detected in minimal medium, milk, and urine in a concentration of 1 ng/ml within less than 23 min. Furthermore, a simultaneous detection of SEB of S. aureus and IsaA homologue of S. epidermidis in a single assay could be demonstrated.