OC-IΔD86 (Oryzacystatin-IΔD86)基因的原核表达及活性分析

根据OC-IΔD86基因序列, 设计合成了7条寡核苷酸片段, 通过重叠延伸PCR技术合成了OC-IΔD86基因, 利用设计好的BamH I/ Xho I酶切位点将OC-IΔD86基因克隆到原核表达载体pet21b中, 在1 mmol/L的IPTG 诱导后5 h, OC-IDD86融合基因在大肠杆菌中得到表达, 表达产物处于可溶状态, 其表达量占总蛋白的11.4%, 可溶性蛋白的16.4%; 利用Ni-NTA系统纯化该蛋白并经PEG20000浓缩后, 活性分析表明该蛋白酶抑制剂在体外表现出对木瓜蛋白酶明显的抑制作用。这为转OC-IΔD86基因的抗根结线虫植物基因工程抗体制备, 以及进一步体内的抗根结线虫研究奠定了基础。     

Diversification of substrate specificities in teleostei Fads2: characterization of Δ4 and Δ6Δ5 desaturases of Chirostoma estor

Currently existing data show that the capability for long-chain PUFA (LC-PUFA) biosynthesis in teleost fish is more diverse than in other vertebrates. Such diversity has been primarily linked to the subfunctionalization that teleostei fatty acyl desaturase (Fads)2 desaturases have undergone during evolution. We previously showed that Chirostoma estor, one of the few representatives of freshwater atherinopsids, had the ability for LC-PUFA biosynthesis from C₁₈ PUFA precursors, in agreement with this species having unusually high contents of DHA. The particular ancestry and pattern of LC-PUFA biosynthesis activity of C. estor make this species an excellent model for study to gain further insight into LC-PUFA biosynthetic abilities among teleosts. The present study aimed to characterize cDNA sequences encoding fatty acyl elongases and desaturases, key genes involved in the LC-PUFA biosynthesis. Results show that C. estor expresses an elongase of very long-chain FA (Elovl)5 elongase and two Fads2 desaturases displaying Δ4 and Δ6/Δ5 specificities, thus allowing us to conclude that these three genes cover all the enzymatic abilities required for LC-PUFA biosynthesis from C₁₈ PUFA. In addition, the specificities of the C. estor Fads2 enabled us to propose potential evolutionary patterns and mechanisms for subfunctionalization of Fads2 among fish lineages.     

Impaired thymic selection and abnormal antigen-specific T cell responses in Foxn1(Δ/Δ) mutant mice

Background

Foxn1^Δ/Δ mutant mice have a specific defect in thymic development, characterized by a block in TEC differentiation at an intermediate progenitor stage, and blocks in thymocyte development at both the DN1 and DP cell stages, resulting in the production of abnormally functioning T cells that develop from an atypical progenitor population. In the current study, we tested the effects of these defects on thymic selection.

Methodology/Principal Findings

We used Foxn1^Δ/Δ; DO11 Tg and Foxn1^Δ/Δ; OT1 Tg mice as positive selection and Foxn1^Δ/Δ; MHCII I-E mice as negative selection models. We also used an in vivo system of antigen-specific reactivity to test the function of peripheral T cells. Our data show that the capacity for positive and negative selection of both CD4 and CD8 SP thymocytes was reduced in Foxn1^Δ/Δ mutants compared to Foxn1^+/Δ control mice. These defects were associated with reduction of both MHC Class I and Class II expression, although the resulting peripheral T cells have a broad TCR Vβ repertoire. In this deficient thymic environment, immature CD4 and CD8 SP thymocytes emigrate from the thymus into the periphery. These T cells had an incompletely activated profile under stimulation of the TCR signal in vitro, and were either hypersensitive or hyporesponsive to antigen-specific stimulation in vivo. These cell-autonomous defects were compounded by the hypocellular peripheral environment caused by low thymic output.

Conclusions/Significance

These data show that a primary defect in the thymic microenvironment can cause both direct defects in selection which can in turn cause indirect effects on the periphery, exacerbating functional defects in T cells.     

Identification of the Metabolite (MΔ1) of 2-tert-Butyl-4-(2,4-dichloro-5-isopropoxyphenyl)-Δ21,3, 4-oxadiazolin-5-one (Oxadiazon) in Rice Plants

Out of metabolites of 2-tert-butyl-4-(2,4-dichloro-5-isopropoxyphenyl)-Δ²-1,3,4-oxadiazolin-5-one (oxadiazon) in rice plants one of the unidentified compounds nominated as M–1 was found much in head parts as compared with the parent compound and other metabolites. Identification of M–1 was made by means of thin-layer chromatography, gas-liquid chromatography, mass spectrometry and coincidence by the synthetic compound.

M–1 was identified as 1-(2,4-dichloro-5-isopropoxyphenyl)-1-methoxycarbonyl-2-trimethyl-acetyl-hydrazine and a pathway of cleavage of oxadiazolin ring of oxidiazon in rice plants was confirmed.     

花生Δ~(12)脂肪酸脱氢酶基因高效表达载体构建与原核表达(英文)

花生是世界范围内广泛种植的重要油料作物之一,其种子中富含油酸和亚油酸。△12脂肪酸脱氢酶(FAD2)是亚油酸合成的关键酶,催化油酸(18:1)在△12位上脱氢生成亚油酸(18:2),但由于△12脂肪酸脱氢酶本身的特性,目前还没有有效的方法将其纯化并在蛋白水平作进一步的研究,尚需对其结构和功能之间以及表达调控进行更深入全面的研究。本文利用从花生中克隆的△12脂肪酸脱氢酶基因(GenBank接受号为AY1006)构建高效表达载体,把花生?12脂肪酸脱氢酶基因全长序列插入到大肠杆菌高效表达载体pRSETB中,构建了pRSET/HO-A融合表达载体,并转化到大肠杆菌表达菌BL21(DE3)pLysS中,在IPTG诱导下,pRSET/HO-A融合表达载体在BL21(DE3)pLysS菌株中高效表达了?12脂肪酸脱氢酶。利用Clon-Tech蛋白纯化Kit进一步分离了目的蛋白,同时加入外源性底物油酸在20℃温育6h后,进行脂肪酸甲酯化处理,通过气相色谱(GC)和气相色谱/质谱(GC-MS)分析表明,所编码的酶具有?12脂肪酸脱氢酶的活性,能将外源性的底物油酸转化为亚油酸,转化率为11.8%。花生△12脂肪酸脱氢酶基因的原核表达目前国内外还未见报导,本实验为其进一步的大量纯化和结构功能分析奠定了基础。     

Δ~(12)-脂肪酸脱氢酶基因在大肠杆菌中的表达

把高山被孢霉 (Mortierellaalpina)和深黄被孢霉 (Mortierellaisabellina)的Δ1 2 脂肪酸脱氢酶基因亚克隆到大肠杆菌表达载体pET2 1a中 ,获得重组表达载体pMACL1 2和pMI CL1 2 ,并用氯化钙方法将重组表达载体转化到大肠杆菌BL2 1 (DE3)中。筛选阳性克隆进行培养 ,然后分离其细胞膜蛋白 ,并构建体外表达体系 ,同时加入外源性底物油酸进行表达。经气相色谱 (GC)分析表明 ,分别有 1 7 87%和 1 7 60 %的油酸转化为亚油酸     

The free energies of metabolic reactions (ΔG) are not positive

Biochemistry textbooks often erroneously state that some metabolic reactions have positive free energies (ΔG), which suggests that they are spontaneous in the reverse direction. This incorrectly implies that reverse fluxes can occur through some steps in a metabolic pathway while the overall flux through the pathway is in the forward direction. In fact, at steady state all the reactions of a metabolic pathway proceed in the forward direction with the same flux. During periods of transition between steady states a positive free energy for any one reaction in a pathway means that the pathway is not actually proceeding from beginning to end.     

Barramundi (Lates calcarifer) desaturase with Δ6/Δ8 dual activities

Barramundi is a commercially farmed fish in Australia. To examine the potential for barramundi to metabolise dietary α-linolenic acid (ALA, 18:3 n-3), the existence of barramundi desaturase enzymes was examined. A putative fatty acid Δ6 desaturase was cloned from barramundi liver and expressed in yeast. Functional expression revealed Δ6 desaturase activity with both the 18 carbon (C(18)) and C(24) n-3 fatty acids, ALA and 24:5 n-3 as well as the C(18) n-6 fatty, linoleic acid (LA, 18:2 n-6). Metabolism of ALA was favoured over LA. The enzyme also had Δ8 desaturase activity which raises the potential for synthesis in barramundi of omega-3 (n-3) long chain polyunsaturated fatty acids from ALA via a pathway that bypasses the initial Δ6 desaturase step. Our findings not only provide molecular evidence for the fatty acid desaturation pathway in the barramundi but also highlight the importance of taking extracellular fatty acid levels into account when assessing enzyme activity expressed in Saccharomyces cerevisiae.     

15-Deoxy-Δ(12,14)-prostaglandin J(2) attenuates the biological activities of monocyte/macrophage cell lines

Monocytes/macrophages link the innate and adaptive immune systems, and in inflammatory disorders their activation leads to tissue damage. 15-Deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural peroxisome proliferator-activated receptor gamma (PPARγ) ligand, has garnered much interest because it possesses anti-inflammatory properties in a number of experimental models. However, whether it regulates monocytes/macrophage pathophysiology is still unknown. This study was designed to examine the effects of 15d-PGJ(2) on the phagocytosis, proliferation and inflammatory cytokines generation in mouse monocyte/macrophage cell line RAW264.7 and J774A.1 cells upon lipopolysaccharide challenge. Our results showed that 15d-PGJ(2) inhibited the phagocytic activity and cell proliferation in a dose-dependent manner, and suppressed proinflammatory cytokines expression, such as tumor necrosis factor-α, transforming growth factor-β1, interleukin-6, and monocyte chemotactic protein-1. These effects were independent of PPARγ, because PPARγ agonist (troglitazone or ciglitazone) and PPARγ antagonist (GW9662) did not affect these activities mentioned above in cells. Treatment of 15d-PGJ(2) also did not modulate expression and distribution of PPARγ. However, these effects of 15d-PGJ(2) were abrogated by antioxidant N-acetylcysteine. Moreover, treatment of 15d-PGJ(2) induced a significant increase in reactive oxygen species production in RAW264.7 and J774A.1 cells. In conclusion, 15d-PGJ(2) attenuates the biological activities of mouse monocyte/macrophage cell line cells involving oxidative stress, independently of PPARγ. These data further underline the anti-inflammation potential of 15d-PGJ(2).     

Differential gene expression signatures for cell wall integrity found in chitin synthase II (chs2Δ) and myosin II (myo1Δ) deficient cytokinesis mutants of Saccharomyces cerevisiae

Background

As the hepatitis B genotyping is important for assessing its clinical implications and geographical distribution, the sub-genotypes have been found useful for determination of specific genomic markers related to hepatocarcinogenesis. In Pakistan, there is no reported data on molecular evolutionary analysis of HBV. A study was, therefore, much needed to evaluate the spectra of mutations present in the strains prevalent here.

Findings

to confirm specificity of PCR typing, phylogenetic analysis of the pre-S1 region and the divergence was studied through 13 sequences of 362 bp (accession number EF432765 – EF432777). A total of 315 serum samples, selected from HBsAg positive patients representing the major ethnic groups, residing in Karachi, Sindh were tested for genotyping. Genotype D (219/315) was found to be the most prevalent (70%) amongst our patients. The rest of the genotypes A and a mixture of A and D (AD) were distributed as 20%, and 10% respectively. Phylogenetic tree demonstrated clustering of 11 samples with subgenotype D1 sequences and the remaining two strains on a branch within D3 samples. All samples intermixed with strains from other countries and were found to be closely related to Indian, Iranian and Egyptian HBV strains with 98.7 – 99.0% homology.

Conclusion

This study confirms the predominance of genotype D in southeastern Asia and presence of subgenotypes DI and D3 in the Pakistani infected patients. More studies are required to investigate the reason for fewer inclusions of D3 compared to the D1 in Pakistani HBV strains.     

Modulation of inflammatory response and parasitism by 15-Deoxy-Δ(12,14) prostaglandin J(2) in Trypanosoma cruzi-infected cardiomyocytes

Trypanosoma cruzi infection produces an intense inflammatory response in diverse tissues including the heart. The inflammatory reaction is critical for the control of the parasites’ proliferation and evolution of Chagas disease. 15-Deoxy-Δ^12,14 prostaglandin J₂ (15dPGJ2) can repress the inflammatory response in many experimental models. However, the precise role of peroxisome proliferator-activated receptor γ (PPARγ) ligands in T. cruzi infection or in Chagas disease is poorly understood. This work reports the first evidence that 15dPGJ2 treatment increases the number of intracellular parasites as shown by fluorescence microscopy and it is also able to inhibit the expression and activity of different inflammatory enzymes such as inducible nitric oxide synthase (NOS-2), matrix metalloproteinases 2 and 9 (MMP-2, MMP-9), as well as pro-inflammatory cytokine (TNF-α and IL-6) mRNA expression in neonatal mouse cardiomyocytes after T. cruzi infection. Transfection of cardiomyocytes with small interfering RNA (siRNA) induces silencing of PPARγ and impairs the effects of 15dPGJ2 on the modulation of pro-inflammatory enzymes. Moreover, transfection restores the ability of these cells to control the intracellular growth of T. cruzi. We also found that PPARγ-independent pathways are involved, since 15dPGJ2 also exerts its effect through extracellular signal-regulated kinases-mitogen-activated protein kinase (Erk-MAPK) and nuclear factor-κB (NF-κB). The use of specific pharmacological inhibitors confirmed these findings. Our data point out that 15dPGJ2 is a potent modulator of the inflammatory process and regulator of parasites growth through PPARγ-dependent and independent (Erk-MAPK- and NF-κB) pathways in T. cruzi infected neonatal cardiac cells.     

pH dependence of the enantioselective excited-state quenching of Λ,Δ-Tb(III) and Λ,Δ-Eu(III)tris(pyridine-2,6-dicarboxylate) chelates by ferricytochrome c from horse heart and ferricytochrome c-550 from Paracoccus versutus

 The pH dependence of the dynamic quenching of the luminescence from Tb(III) and Eu(III) tris(pyridine-2,6-dicarboxylate≡DPA) chelates by the title proteins is studied. For Tb(DPA)₃ ^3– also the quenching by the Lys 14→Glu and Lys99→Glu mutants of cytochrome c-550 (cytc-550) is investigated. The rate constants for quenching of the electronically excited Λ and Δ enantiomers of the luminophore by equine cytochrome c show a sharp decrease upon increasing the pH from 7 to 10, which can be described phenomenologically by deprotonation of a single acidic group with pK _a of 9.2±0.1 for Eu and 9.4±0.1 for Tb. These values are similar to that found for the alkaline transition of the protein. The alkaline conformer(s) of the protein at pH>10 is found to be a very inefficient quencher of the lanthanide luminescence. For Tb, but not for Eu, a significant lowering of the degree of enantioselectivity (E _q) in the quenching is found along with a reduction of the quenching rates. For cytc-550, the decrease of the quenching rate constants with increasing pH is described by pK _a=9.8±0.1 and for the two mutants the same value is obtained. For the cytc-550 proteins the change of the quenching rates does not correlate with the alkaline transition, for which a pK _a of 11.2 has been reported by other workers. For all proteins, the reduction of the quenching rates at high pH is ascribed to a reduction of the binding affinity of the excited lanthanide complex to the surface area of the protein near the exposed heme edge, caused by deprotonation of (presumably) several lysine residues. Received: 3 April 1998 / Accepted: 15 June 1998     

15-Deoxy-Δ(12,14)-prostaglandin J(2) interferes inducible synthesis of prostaglandins E(2) and F(2α) that suppress subsequent adipogenesis program in cultured preadipocytes

Cultured preadipocytes enhance the synthesis of prostaglandin (PG) E(2) and PGF(2α) involving the induction of cyclooxygenase (COX)-2 during the growth phase upon stimulation with a mixture of phorbol 12-myristate 13-acetate, a mitogenic factor, and calcium ionophore A23187. Here, we studied the interactive effect of 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) on the inducible synthesis of the endogenous PGs in cultured preadipocytes and its implication in adipogenesis program. 15d-PGJ(2) interfered significantly the endogenous synthesis of those PGs in response to cell stimuli by suppressing the induction of COX-2 following the attenuation of NF-κB activation. In contrast, Δ(12)-PGJ(2) and troglitazone had almost no inhibitory effects, indicating a mechanism independent of the activation of peroxisome proliferator-activated receptor γ for the action of 15-PGJ(2). Pyrrolidinedithiocarbamate (PDTC), an NF-κB inhibitor, effectively inhibited on the inducible synthesis of those PGs in preadipocytes. Endogenous PGs generated by preadipocytes only during the growth phase in response to the cell stimuli autonomously attenuated the subsequent adipogenesis program leading to the differentiation and maturation of adipocytes. These effects were prevented by additional co-incubation of preadipocytes with either 15d-PGJ(2) or PDTC although 15d-PGJ(2) alone has no stimulatory effect. Moreover, 15d-PGJ(2) did not block the inhibitory effects of exogenous PGE(2) and PGF(2α) on the adipogenesis program in preadipocytes. Taken together, 15d-PGJ(2) can interfere the COX pathway leading to the induced synthesis of endogenous PGs that contribute to negative regulation of adipogenesis program in preadipocytes.     

Nonenzymatic free radical-catalyzed generation of 15-deoxy-Δ(12,14)-prostaglandin J₂-like compounds (deoxy-J₂-isoprostanes) in vivo

15-Deoxy-Δ^12,14-prostaglandin J₂ (15-d-PGJ₂) is a reactive cyclopentenone eicosanoid generated from the dehydration of cyclooxygenase-derived prostaglandin D₂ (PGD₂). This compound possesses an α,β-unsaturated carbonyl moiety that can readily adduct thiol-containing biomolecules such as glutathione and cysteine residues of proteins via the Michael addition. Due to its reactivity, 15-d-PGJ₂ is thought to modulate inflammatory and apoptotic processes and is believed to be an endogenous ligand for peroxisome proliferator-activated receptor-γ. However, the extent to which 15-d-PGJ₂ is formed in vivo and the mechanisms that regulate its formation are unknown. Previously, we have reported the formation of PGD₂ and PGJ₂-like compounds, termed D₂/J₂-isoprostanes (D₂/J₂-IsoPs), produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid (AA). Based on these findings, we investigated whether 15-d-PGJ₂-like compounds are also formed via this nonenzymatic pathway. Here we report the generation of novel 15-d-PGJ₂-like compounds, termed deoxy-J₂-isoprostanes (deoxy-J₂-IsoPs), in vivo, via the nonenzymatic peroxidation of AA. Levels of deoxy-J₂-IsoPs increased 12-fold (6.4 ± 1.1 ng/g liver) in rats after oxidant insult by CCl₄ treatment, compared with basal levels (0.55 ± 0.21 ng/g liver). These compounds may have important bioactivities in vivo under conditions associated with oxidant stress.     

Primary acute dengue and the deletion in chemokine receptor 5 (CCR5Δ32)

Dengue virus is a significant arboviral pathogen that is continuing to spread due to human travel and invasion of the mosquito vectors into new regions. Chemokine receptor 5 (CCR5) has a truncated 32 base pair deletion form (CCR5Δ32), which has been associated with resistance to HIV but increased severity in some flaviviral diseases. If CCR5Δ32 is associated with dengue, European carriers of this mutation may be at increased risk. In a Western Australian population with the same frequency of CCR5Δ32 (0.08) as that found in southern Europe there was no significant difference in CCR5Δ32 allele frequency between returned travellers with and without dengue (p = 0.82, OR = 0.86, 95% CI = 0.35–2.1).     

High level accumulation of gamma linolenic acid (C18:3Δ6.9,12 cis) in transgenic safflower (Carthamus tinctorius) seeds

Gamma linolenic acid (GLA; C18:3Δ6,9,12 cis), also known as γ-Linolenic acid, is an important essential fatty acid precursor for the synthesis of very long chain polyunsaturated fatty acids and important pathways involved in human health. GLA is synthesized from linoleic acid (LA; C18:2Δ9,12 cis) by endoplasmic reticulum associated Δ6-desaturase activity. Currently sources of GLA are limited to a small number of plant species with poor agronomic properties, and therefore an economical and abundant commercial source of GLA in an existing crop is highly desirable. To this end, the seed oil of a high LA cultivated species of safflower (Carthamus tinctorius) was modified by transformation with Δ6-desaturase from Saprolegnia diclina resulting in levels exceeding 70% (v/v) of GLA. Levels around 50% (v/v) of GLA in seed oil was achieved when Δ12-/Δ6-desaturases from Mortierella alpina was over-expressed in safflower cultivars with either a high LA or high oleic (OA; C18:1Δ9 cis) background. The differences in the overall levels of GLA suggest the accumulation of the novel fatty acid was not limited by a lack of incorporation into the triacylgylcerol backbone (>66% GLA achieved), or correlated with gene dosage (GLA levels independent of gene copy number), but rather reflected the differences in Δ6-desaturase activity from the two sources. To date, these represent the highest accumulation levels of a newly introduced fatty acid in a transgenic crop. Events from these studies have been propagated and recently received FDA approval for commercialization as Sonova?400.     

1-Bromo-3-(1',1'-dimethylalkyl)-1-deoxy-Δ(8)-tetrahydrocannabinols: New selective ligands for the cannabinoid CB(2) receptor

Δ(8)-Tetrahydrocannabinol (26), 3-(1',1'-dimethylbutyl)- (12), 3-(1',1'-dimethylpentyl)- (13), 3-(1',1'-dimethylhexyl)- (14) and 3-(1',1'-dimethylheptyl)-Δ(8)-tetrahydrocannabinol (15) have been converted into the corresponding 1-bromo-1-deoxy-Δ(8)-tetrahydrocannabinols (25, 8-11). This was accomplished using a protocol developed in our laboratory in which the trifluoromethanesulfonate of a phenol undergoes palladium mediated coupling with pinacolborane. Reaction of this dioxaborolane with aqueous-methanolic copper(II) bromide provides the aryl bromide. The affinities of these bromo cannabinoids for the cannabinoid CB(1) and CB(2) receptors were determined. All of these compounds showed selectivity for the CB(2) receptor and one of them, 1-bromo-1-deoxy-3-(1',1'-dimethylhexyl)-Δ(8)-tetrahydrocannabinol (10), exhibits 52-fold selectivity for this receptor with good (28nM) affinity.