Molecular and biochemical characterization of benzalacetone synthase and chalcone synthase genes and their proteins from raspberry (Rubus idaeus L.)

Two new members of the polyketide synthase (PKS) gene family (RiPKS4 and RiPKS5) were cloned from raspberry fruits (Rubus idaeus L., cv Royalty) and expressed in Escherichia coli. Characterization of the recombinant enzyme products indicated that RiPKS4 is a bifunctional polyketide synthase producing both 4-hydroxybenzalacetone and naringenin chalcone. The recombinant RiPKS4 protein, like the native protein from raspberry fruits [W. Borejsza-Wysocki, G. Hrazdina, Plant Physiol. 1996;110: 791-799] accepted p-coumaryl-CoA and ferulyl-CoA as starter substrates and catalyzed the formation of both naringenin chalcone, 4-hydroxy-benzalacetone and 3-methoxy-4-hydroxy-benzalacetone. Although activity of RiPKS4 was higher with ferulyl-CoA than with p-coumaryl-CoA, the corresponding product, 3-methoxy-4-hydroxy phenylbutanone could not be detected in raspberries to date. Sequence analysis of the genes and proteins suggested that this feature of RiPKS4 was created by variation in the C-terminus due to DNA recombination at the 3′ region of its coding sequence. RiPKS5 is a typical chalcone synthase (CHS) that uses p-coumaryl-CoA only as starter substrate and produces naringenin chalcone exclusively as the reaction product.     

Dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia exhibits activity against a substrate containing a 4-hydroxyproline residue

The crystal structure of dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia was determined at 2.8-A resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space group P4(3)2(1)2, with unit cell parameters a = b = 105.9 A and c = 161.9 A. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal beta-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes. Stenotrophomonas dipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In the Stenotrophomonas enzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.     

Pin1-dependent prolyl isomerization modulates the stress-induced phosphorylation of high molecular weight neurofilament protein

Aberrant phosphorylation of neuronal cytoskeletal proteins is a key pathological event in neurodegenerative disorders such as Alzheimer disease (AD) and amyotrophic lateral sclerosis, but the underlying mechanisms are still unclear. Previous studies have shown that Pin1, a peptidylprolyl cis/trans-isomerase, may be actively involved in the regulation of Tau hyperphosphorylation in AD. Here, we show that Pin1 modulates oxidative stress-induced NF-H phosphorylation. In an in vitro kinase assay, the addition of Pin1 substantially increased phosphorylation of NF-H KSP repeats by proline-directed kinases, Erk1/2, Cdk5/p35, and JNK3 in a concentration-dependent manner. In vivo, dominant-negative (DN) Pin1 and Pin1 small interfering RNA inhibited epidermal growth factor-induced NF-H phosphorylation. Because oxidative stress plays an important role in the pathogenesis of neurodegenerative diseases, we studied the role of Pin1 in stressed cortical neurons and HEK293 cells. Both hydrogen peroxide (H(2)O(2)) and heat stresses induce phosphorylation of NF-H in transfected HEK293 cells and primary cortical cultures. Knockdown of Pin1 by transfected Pin1 short interference RNA and DN-Pin1 rescues the effect of stress-induced NF-H phosphorylation. The H(2)O(2) and heat shock induced perikaryal phospho-NF-H accumulations, and neuronal apoptosis was rescued by inhibition of Pin1 in cortical neurons. JNK3, a brain-specific JNK isoform, is activated under oxidative and heat stresses, and inhibition of Pin1 by Pin1 short interference RNA and DN-Pin1 inhibits this pathway. These results implicate Pin1 as a possible modulator of stress-induced NF-H phosphorylation as seen in neurodegenerative disorders like AD and amyotrophic lateral sclerosis. Thus, Pin1 may be a potential therapeutic target for these diseases.     

Variation on Composition and Bioactivity of Essential Oils of Four Common Curcuma Herbs

Chemical compositions, antioxidative, antimicrobial, anti‐inflammatory, and cytotoxic activities of essential oils extracted from four common Curcuma species (Curcuma longa, Curcuma phaeocaulis, Curcuma wenyujin, and Curcuma kwangsiensis) rhizomes in P. R. China are comparatively studied. In total, 47, 49, 35, and 30 compounds are identified in C. longa, C. phaeocaulis, C. wenyujin, and C. kwangsiensis essential oils by GC/MS, and their richest compounds are ar‐turmerone (21.67%), elemenone (19.41%), curdione (40.23%) and (36.47%), respectively. Moreover, C. kwangsiensis essential oils display the strongest DPPH (2,2‐diphenyl‐1‐picrylhydrazyl) radical‐scavenging activity (IC₅₀, 3.47 μg/ml), much higher than ascorbic acid (6.50 μg/ml). C. phaeocaulis oils show the best antibacterial activities against Escherichia coli (MIC, 235.54 μg/ml), Pseudomonas aeruginosa (391.31 μg/ml) and Staphylococcus aureus (378.36 μg/ml), while C. wenyujin and C. kwangsiensis oils show optimum activities against Candida albicans (208.61 μg/ml) and Saccharomyces cerevisiae (193.27 μg/ml), respectively. C. phaeocaulis (IC₅₀, 4.63 μg/ml) and C. longa essential oils (73.05 μg/ml) have the best cytotoxicity against LNCaP and HepG2, respectively. C. kwangsiensis oils also exhibit the strongest anti‐inflammatory activities by remarkably down‐regulating expression of COX‐2 and TNF‐α. Therefore, due to their different chemical compositions and bioactivities, traditional Chinese Curcuma herbs should be differentially served as natural additives for food, pharmaceutical, and cosmetic.     

西安地区现代人颅骨非测量性研究

本文对西安地区140例(男70、女70)出土成人颅骨进行了非测量性变异类型的研究,计算出了25项形态的常见变异类型出现率、以及性差显著和非常显著的项目和类型,并进行了讨论。     

玉米根系分布空间和空间密度分布的数学模型

应用重积分研究了土壤层中散根型和直根型玉米根系的分布空间,同时研究了玉米根系的空间密度分布．     

Identification and Characterization of Shiga Toxin Type 2 Variants in Escherichia coli Isolates from Animals, Food, and Humans

There is considerable heterogeneity among the Shiga toxin type 2 (Stx2) toxins elaborated by Shiga toxin-producing Escherichia coli (STEC). One such Stx2 variant, the Stx2d mucus-activatable toxin (Stx2dact), is rendered more toxic by the action of elastase present in intestinal mucus, which cleaves the last two amino acids of the A2 portion of the toxin A subunit. We screened 153 STEC isolates from food, animals, and humans for the gene encoding Stx2dact by using a novel one-step PCR procedure. This method targeted the region of stx_2dact that encodes the elastase recognition site. The presence of stx_2dact was confirmed by DNA sequencing of the complete toxin genes. Seven STEC isolates from cows (four isolates), meat (two isolates), and a human (one isolate) that carried the putative stx_2dact gene were identified; all were eae negative, and none was the O157:H7 serotype. Three of the isolates (CVM9322, CVM9557, and CVM9584) also carried stx₁, two (P1332 and P1334) carried stx₁ and stx_2c, and one (CL-15) carried stx_2c. One isolate, P1130, harbored only stx_2dact. The Vero cell cytotoxicities of supernatants from P1130 and stx₁ deletion mutants of CVM9322, CVM9557, and CVM9584 were increased 13- to 30-fold after treatment with porcine elastase. Thus, Stx2dact-producing strains, as detected by our one-step PCR method, can be isolated not only from humans, as previously documented, but also from food and animals. The latter finding has important public health implications based on a recent report from Europe of a link between disease severity and infection with STEC isolates that produce Stx2dact.     

MicroRNA研究进展

近年来,在许多真核生物中发现了一类能时序调控发育的、长度约为22个核苷酸（nt）的非编码小分子RNA,称为microRNA(miRNA)。它由RNaseⅢ蛋白Drosha和Dicer共同酶解作用产生,由Exportin-5转运出核,通过与small interfering RNA(siRNA)相似的机制,导致mRNA降解,或者与mRNA的3’端非翻译区结合,阻遏转录后翻译。miRNA在发育和某些人类疾病发生中有着重要作用。     

电刺激右后背海马诱导双侧前背海马网络癫痫

目的 :急性强直电刺激右侧后背HPC诱导双侧HPC癫痫电网络形成的细胞机制。方法 :强直电刺激 (6 0Hz,2s,0 .4～ 0 .6mA)大鼠右后背HPCCA1基树突区 ,每隔 10min刺激一次 ,施加 10个刺激串。结果 :①分别抑制双侧CA1神经元单位放电频率 ,对侧的抑制效应更明显 (对侧 :6 2 .94 %± 3.6 8% ;同侧 :36 .6 1%± 3.14 % ,P <0 .0 1) ,出现抑制后爆发式放电。随着刺激串数的增加 ,抑制作用逐渐减弱。②同步原发性网络和单位后放电 ,以同侧CA1多见 (P<0 .0 1)。③ 90Hz或 12 0Hz原发性或继发性网络后放电仅仅累及同侧CA1。④对侧CA3基树突区网络与下托神经元单位放电出现同步继发性后放电 ,反复发作 ,持续约数小时。结论 :电刺激诱导的对侧HPC抑制后爆发式放电和长时程、反复发作的网络与单个神经元同步继发性后放电可能是跨半球癫痫网络形成的重要表现形式。