Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

High production of laccase by a new basidiomycete, <Emphasis Type="Italic">Trametes</Emphasis> sp

A new basidiomycete, Trametes sp. 420, produced laccase at 6,810 U l⁻¹ (268 mg, 25.4 U mg⁻¹ protein for guaiacol) in glucose medium and 7,870 U l⁻¹ (310 mg) in cellobiose medium with induction by 0.5 mM Cu²⁺ and 6 mM o-toluidine. Laccase isozyme E (LacE) was the sole laccase in the fermentation products. It was stable at pH 5–9 and below 70°C over 30 min. The K _m values of LacE for four substrates (guaiacol ABTS, 2,6-dimethoxyphenol and syringaldazine) varied from 5 to 245 μM. The activity of LacE was strongly inhibited by NaN₃ but not by EDTA or dimethylsulfoxide. LacE at 0.5 U l⁻¹ could decolorize industrial dyes. The open reading frame of the lacE gene was 2,130 bp and was interrupted by 10 introns. It displayed a high homology to laccases from other fungi. Pingui Tong and Yuzhi Hong contributed equally to the study     

Cd2+、Cu2+胁迫对黑藻(Hydrilla verticillata)的生长及光合荧光特性的影响

以黑藻(Hydrilla verticillata)为供试材料,采用Cd2+和Cu2+等两种重金属分别在5个浓度梯度水平下的河砂水培方法,研究Cd2+或Cu2+不同浓度胁迫对黑藻株高、生物量、成活率和叶绿素含量的影响,以及对黑藻叶片最小荧光(Fo)、最大荧光(Fm),PSⅡ最大量子产率(QYmax,)、稳态下的PSⅡ反应中心关闭程度(1-Qp_Lss)、稳态下的非光化学淬灭(NPQ_Lss)等光合荧光参数及其荧光成像的影响,探讨各个参数分别随镉、铜浓度递增的变化规律。研究结果表明,Cd2+胁迫下黑藻的株高显著下降,说明Cd2+可能对黑藻叶片的维管束结构产生伤害作用;Cd2+和Cu2+胁迫对黑藻鲜重均无显著影响,说明与水生植物自由水含量存在一定关系;Cd2+和Cu2+胁迫均使黑藻干重显著下降,其中Cu2+胁迫对黑藻干重的影响更显著,说明Cu2+胁迫对黑藻累积生物量的影响远大于Cd2+;Cd2+和Cu2+胁迫下叶绿素各值均呈下降趋势,而Cu2+胁迫对叶绿素的影响更大,说明Cu2+对黑藻叶绿体的毒害比Cd2+更大。随着Cd2+或Cu2+胁迫浓度梯度的增加,黑藻的叶绿素荧光参数(Fo、Fm、QYmax)呈显著下降趋势,(1-Qp_Lss)呈上升趋势,而NPQ_Lss先上升后下降。黑藻在不同重金属胁迫下的生理指标、荧光参数及成像特征等方面所表现出的变化差异性,反映出同等浓度下黑藻受重金属胁迫的影响程度为:Cd2+胁迫Cu2+胁迫;黑藻可以在Cu2+浓度低于1 mg/L的环境下具有正常的光合活性,可推测将黑藻用于低浓度Cu污染水域的修复;在Cu2+浓度达3 mg/L以上环境下黑藻即无法长时间生存,可推测黑藻可以作为Cd污染水环境的指示种。     

Regulation of MMP-2 expression and activity by β-1,3-<Emphasis Type="Italic">N</Emphasis>-acetylglucosaminyltransferase-8 in AGS gastric cancer cells

β-1,3-N-acetylglucosaminyltransferase-8(β3Gn-T8) catalyzes the transfer of GlcNAc to the non-reducing terminus of the Galβ1-4GlcNAc of tetraantennary N-glycan in vitro. It has been reported to be involved in malignant tumors, but a comprehensive understanding of how the glycolsyltransferase correlates with the invasive potential of human gastric cancer is not currently available. Therefore, we investigated the ability and possible mechanism involved with β3Gn-T8 in modulating matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in AGS gastric cancer cells. Here, we found out that siRNA-mediated suppression of the β3Gn-T8 could directly reduce the MMP-2 expression and activity as observed in RT-PCR, western blot and gelatin zymography analysis. Meanwhile, TIMP-2 expression had been increased. Cell invasion assay using matrigel matrix-coated transwell inserts showed that the invasive property was greatly suppressed in β3Gn-T8 siRNA transfected cells. Furthermore, cells overexpressing β3Gn-T8 gene (when transfected with pEGFP-C1 plasmid) also expressed MMP-2 gene, but TIMP-2 expression had been inhibited. The invasive ability of these cells was also enhanced. Protein–protein interaction analysis using STRING database showed that β3Gn-T8 and MMP-2 may have related signal pathway. In summary, our results reveal a new mechanism by which β3Gn-T8 can regulate MMP-2 and TIMP-2. We suggest that β3Gn-T8 can be used as a novel therapeutic target for human gastric treatment.     

Personalized medicine and proteomics: lessons from non-small cell lung cancer

Personalized medicine allows the selection of treatments best suited to an individual patient and disease phenotype. To implement personalized medicine, effective tests predictive of response to treatment or susceptibility to adverse events are needed, and to develop a personalized medicine test, both high quality samples and reliable data are required. We review key features of state-of-the-art proteomic profiling and introduce further analytic developments to build a proteomic toolkit for use in personalized medicine approaches. The combination of novel analytical approaches in proteomic data generation, alignment and comparison permit translation of identified biomarkers into practical assays. We further propose an expanded statistical analysis to understand the sources of variability between individuals in terms of both protein expression and clinical variables and utilize this understanding in a predictive test.     

Lipoyl synthase requires two equivalents of S-adenosyl-L-methionine to synthesize one equivalent of lipoic acid

Lipoyl synthase (LipA) catalyzes the formation of the lipoyl cofactor, which is employed by several multienzyme complexes for the oxidative decarboxylation of various alpha-keto acids, as well as the cleavage of glycine into CO(2) and NH(3), with concomitant transfer of its alpha-carbon to tetrahydrofolate, generating N(5),N(10)-methylenetetrahydrofolate. In each case, the lipoyl cofactor is tethered covalently in an amide linkage to a conserved lysine residue located on a designated lipoyl-bearing subunit of the complex. Genetic and biochemical studies suggest that lipoyl synthase is a member of a newly established class of metalloenzymes that use S-adenosyl-l-methionine (AdoMet) as a source of a 5'-deoxyadenosyl radical (5'-dA(*)), which is an obligate intermediate in each reaction. These enzymes contain iron-sulfur clusters, which provide an electron during the cleavage of AdoMet, forming l-methionine in addition to the primary radical. Recently, one substrate for lipoyl synthase has been shown to be the octanoylated derivative of the lipoyl-bearing subunit (E(2)) of the pyruvate dehydrogenase complex [Zhao, S., Miller, J. R., Jian, Y., Marletta, M. A., and Cronan, J. E., Jr. (2003) Chem. Biol. 10, 1293-1302]. Herein, we show that the octanoylated derivative of the lipoyl-bearing subunit of the glycine cleavage system (H-protein) is also a substrate for LipA, providing further evidence that the cofactor is synthesized on its target protein. Moreover, we show that the 5'-dA(*) acts directly on the octanoyl substrate, as evidenced by deuterium transfer from [octanoyl-d(15)]H-protein to 5'-deoxyadenosine. Last, our data indicate that 2 equiv of AdoMet are cleaved irreversibly in forming 1 equiv of [lipoyl]H-protein and are consistent with a model in which two LipA proteins are required to synthesize one lipoyl group.     

Identification of sparse neural functional connectivity using penalized likelihood estimation and basis functions

One key problem in computational neuroscience and neural engineering is the identification and modeling of functional connectivity in the brain using spike train data. To reduce model complexity, alleviate overfitting, and thus facilitate model interpretation, sparse representation and estimation of functional connectivity is needed. Sparsities include global sparsity, which captures the sparse connectivities between neurons, and local sparsity, which reflects the active temporal ranges of the input-output dynamical interactions. In this paper, we formulate a generalized functional additive model (GFAM) and develop the associated penalized likelihood estimation methods for such a modeling problem. A GFAM consists of a set of basis functions convolving the input signals, and a link function generating the firing probability of the output neuron from the summation of the convolutions weighted by the sought model coefficients. Model sparsities are achieved by using various penalized likelihood estimations and basis functions. Specifically, we introduce two variations of the GFAM using a global basis (e.g., Laguerre basis) and group LASSO estimation, and a local basis (e.g., B-spline basis) and group bridge estimation, respectively. We further develop an optimization method based on quadratic approximation of the likelihood function for the estimation of these models. Simulation and experimental results show that both group-LASSO-Laguerre and group-bridge-B-spline can capture faithfully the global sparsities, while the latter can replicate accurately and simultaneously both global and local sparsities. The sparse models outperform the full models estimated with the standard maximum likelihood method in out-of-sample predictions.     

Radiosynthesis and evaluation of a fluorine-18 labeled radioligand targeting vesicular acetylcholine transporter

Vesicular acetylcholine transporter (VAChT) is a reliable biomarker for assessing the loss of cholinergic neurons in the brain that is associated with cognitive impairment of patients. 5-Hydrotetralin compound (±)-5-OH-VAT is potent (K_i?=?4.64?±?0.32?nM) and selective for VAChT (>1800-fold and 398-fold for σ₁ and σ₂ receptor, respectively) with favorable hydrophilicity (LogD?=?1.78), while (?)-5-OH-VAT originally serves as the radiolabeling precursor of (?)-[¹⁸F]VAT, a promising VAChT radiotracer with a logD value of 2.56. To evaluate (?)-5-OH-[¹⁸F]VAT as a radiotracer for VAChT, we performed in vitro binding assay to determine the potency of the minus enantiomer (?)-5-OH-VAT and plus enantiomer (+)-5-OH-VAT, indicating that (?)-5-OH-VAT is a more potent VAChT enantiomer. Radiosynthesis of (?)-5-OH-[¹⁸F]VAT was explored using three strategies. (?)-5-OH-[¹⁸F]VAT was achieved with a good yield (24?±?6%) and high molar activity (～37?GBq/µmol, at the end of synthesis) using a microwave assisted two-step one-pot procedure that started with di-MOM protected nitro-containing precursor (?)-6. MicroPET studies in the brain of nonhuman primate (NHP) suggest that (?)-5-OH-[¹⁸F]VAT readily penetrated the blood brain barrier and specifically accumulated in the VAChT-enriched striatum with improved washout kinetics from striatum compared to [¹⁸F]VAT. Nevertheless, the lower target to non-target ratio may limit its use for in vivo measurement of the VAChT level in the brain.