Partial purification of potato tuber invertase and its proteinaceous inhibitor

Improved purification of potato tuber invertase was achieved by utilizing a form of affinity chromatography between the enzyme and Concanavalin A (Con A) bound to Sepharose. Twenty-fold increases in specific activity were routinely obtained with this step and the enzyme was purified 190-fold over that found in the crude homogenate. The Con A-Sepharose chromatography step gave a greater purification than any other step in the invertase isolation procedure. There was up to 170% recovery of the activity loaded onto the column. α-Methyl-d-mannoside, sucrose, d-glucose and d-fructose eluted the enzyme from the Con A-Sepharose column with similar recoveries, although the volume of eluent required varied with the sugar. This unusually high recovery of invertase activity was obtained with some batches of tubers but not with others. There was evidence to suggest that the high recovery, or activation, may be due to the release of an inhibitor from the enzyme in the presence of Con A-Sepharose. Adsorption of invertase to Con A-Sepharose could be eliminated by incubation of the enzyme with α-mannosidase and β-glucosidase, indicating that potato tuber invertase is a glycoprotein. Proteinaceous inhibitor purification was improved by treatment of the tuber extract at low pH.     

Increasing intracellular cAMP and cGMP inhibits cadmium-induced oxidative stress in rat submandibular saliva

The effect of cadmium on induction of oxidative stress in rat submandibular saliva and protective role of increasing intracellular cAMP and cGMP by use of specific phosphodiesterase inhibitors, theophylline and sildenafil were investigated. Pure submandibular saliva was collected intraorally by micro polyethylene cannula from anaesthetized rats using pilocarpine as secretagogue. Acute administration of cadmium (10 mg/kg) caused significant oxidative stress by increasing lipid peroxidation by-products (thiobarbituric reactive substances, TBARS) and decreasing total thiols and total antioxidant power of the saliva. Concurrent therapy of rats by theophylline (25 mg/kg) and sildenafil (5 mg/kg) prevented cadmium-induced oxidative stress in saliva. Theophylline and sildenafil inhibited cadmium-induced increase in lipid peroxidation and decrease in total thiols and antioxidant power. It is concluded that cadmium administration results in oxidative stress in rat submandibular saliva, which can be protected by concurrent administration of specific cyclic nucleotide phosphodiesterase inhibitors.     

The sulfamide moiety affords higher inhibitory activity and oral bioavailability to a series of coumarin dual selective RAF/MEK inhibitors

Introducing a sulfamide moiety to our coumarin derivatives afforded enhanced Raf/MEK inhibitory activity concomitantly with an acceptable PK profile. Novel sulfamide 17 showed potent HCT116 cell growth inhibition (IC₅₀ = 8 nM) and good PK profile (bioavailability of 51% in mouse), resulting in high in vivo antitumor efficacy in the HCT116 xenograft (ED₅₀ = 4.8 mg/kg). We confirmed the sulfamide moiety showed no negative impact on tests run on the compound to evaluate DMPK (PK profiles in three animal species, CYP inhibition and CYP induction) and the safety profile (hERG and AMES tests). Sulfamide 17 had favorable properties that warranted further preclinical assessment     

番茄转化酶抑制子基因克隆及其表达分析

从普通栽培型番茄品种‘桃太郎’、野生醋栗番茄和潘那利番茄的幼苗中采用RT-PCR方法,扩增出转化酶抑制子基因cDNA序列的部分片段。经测序表明：不同基因型番茄的转化酶抑制子基因同源性高达97.97%以上。采用半定量RT-PCR方法对‘桃太郎’苗期根、茎、叶和花后功能叶、花期子房以及果实不同发育阶段不同部位的表达进行检测,结果表明：INH在根中表达较弱,茎中有强烈表达,从幼叶到衰老叶表达逐渐减弱;同一时期INH在果肉中表达相对较强,维管束次之,胶质胎座相对较弱;花期子房中INH的表达逐渐增强,花后3d左右达到最大,花后5～8d表达量迅速下降。     

Regulatory proteins of F1F0-ATPase: Role of ATPase inhibitor

An intrinsic ATPase inhibitor inhibits the ATP-hydrolyzing activity of mitochondrial F₁F₀-ATPase and is released from its binding site on the enzyme upon energization of mitochondrial membranes to allow phosphorylation of ADP. The mitochondrial activity to synthesize ATP is not influenced by the absence of the inhibitor protein. The enzyme activity to hydrolyze ATP is induced by dissipation of the membrane potential in the absence of the inhibitor. Thus, the inhibitor is not responsible for oxidative phosphorylation, but acts only to inhibit ATP hydrolysis by F₁F₀-ATPase upon deenergization of mitochondrial membranes. The inhibitor protein forms a regulatory complex with two stabilizing factors, 9K and 15K proteins, which facilitate the binding of the inhibitor to F₁F₀-ATPase and stabilize the resultant inactivated enzyme. The 9K protein, having a sequence very similar to the inhibitor, binds directly to F₁ in a manner similar to the inhibitor. The 15K protein binds to the F₀ part and holds the inhibitor and the 9K protein on F₁F₀-ATPase even when one of them is detached from the F₁ part.     

Design,synthesis and anti-melanogenic effect of cinnamamide derivatives

Pigmentation disorders are attributed to excessive melanin which can be produced by tyrosinase. Therefore, tyrosinase is supposed to be a vital target for the treatment of disorders associated with overpigmentation. Based on our previous findings that an (E)-β-phenyl-α,β-unsaturated carbonyl scaffold can play a key role in the inhibition of tyrosinase activity, and the fact that cinnamic acid is a safe natural substance with a scaffolded structure, it was speculated that appropriate cinnamic acid derivatives may exhibit potent tyrosinase inhibitory activity. Thus, ten cinnamamides were designed, and synthesized by using a Horner-Emmons olefination as the key step. Cinnamamides 4 (93.72% inhibition), 9 (78.97% inhibition), and 10 (59.09% inhibition) with either a 2,4-dihydroxyphenyl, or 4-hydroxy-3-methoxyphenyl substituent showed much higher mushroom tyrosinase inhibition at 25?µM than kojic acid (18.81% inhibition), used as a positive control. Especially, the two cinnamamides 4 and 9 having a 2,4-dihydroxyphenyl group showed the strongest inhibition. Docking simulation with tyrosinase revealed that these three cinnamamides, 4, 9, and 10, bind to the active site of tyrosinase more strongly than kojic acid. Cell-based experiments carried out using B16F10 murine skin melanoma cells demonstrated that all three cinnamamides effectively inhibited cellular tyrosinase activity and melanin production in the cells without cytotoxicity. There was a close correlation between cellular tyrosinase activity and melanin content, indicating that the inhibitory effect of the three cinnamamides on melanin production is mainly attributed to their capability for cellular tyrosinase inhibition. These results imply that cinnamamides having the (E)-β-phenyl-α,β-unsaturated carbonyl scaffolds are promising candidates for skin-lighting agents.     

Identification of a D-amino acid decapeptide HIV-1 entry inhibitor

Entry of human immunodeficiency virus type 1 (HIV-1) virion into host cells involves three major steps, each being a potential target for the development of entry inhibitors: gp120 binding to CD4, gp120-CD4 complex interacting with a coreceptor, and gp41 refolding to form a six-helix bundle. Using a D-amino acid decapeptide combinatorial library, we identified peptide dC13 as having potent HIV-1 fusion inhibitory activity, and effectively inhibiting infection by several laboratory-adapted and primary HIV-1 strains. While dC13 did not block binding of gp120 to CD4, nor disrupt the gp41 six-helix bundle formation, it effectively blocked the binding of an anti-CXCR4 monoclonal antibody and chemokine SDF-1alpha to CXCR4-expressing cells. However, because R5-using primary viruses were also neutralized, the antiviral activity of dC13 implies additional mode(s) of action. These results suggest that dC13 is a useful HIV-1 coreceptor antagonist for CXCR4 and, due to its biostability and simplicity, may be of value for developing a new class of HIV-1 entry inhibitors.     

枇杷种质资源的离体保存研究Ⅱ生长抑制剂的影响

在枇杷种质资源离体保存中,加入植物生长抑制剂可以延缓枇杷试管苗的生长速度,延长保存时间。实验结果表明,有利于枇杷种质保存的3种生长抑制剂浓度分别是:多效唑(PP₃₃₃)2~5mg/L、矮壮素(CCC)25mg/L、脱落酸(ABA)10mg/L;其中浓度为5mg/L的多效唑效果最为理想。高浓度的甘露醇(>5g/L)对试管苗会造成毒害,不利于枇杷种质的保存;5g/L甘露醇保存效果较好     

Directed mutagenesis confirms the functional importance of positively selected sites in polygalacturonase inhibitor protein

Polygalacturonase inhibitor proteins (PGIPs) protect plants against invasion by diverse microbial and invertebrate enemies that use polygalacturonase (PG) to breach the plant cell wall. Directed mutagenesis has identified specific natural mutations conferring novel defensive capability in green bean PGIP against a specific fungal PG. These same sites are identified as positively selected by phylogenetic codon-substitution models, demonstrating the utility of such models for connecting retrospective comparative analyses with contemporary, ecologically relevant variation.     

Effect of potential amine prodrugs of selective neuronal nitric oxide synthase inhibitors on blood–brain barrier penetration

Several prodrug approaches were taken to mask amino groups in two potent and selective neuronal nitric oxide synthase (nNOS) inhibitors containing either a primary or secondary amino group to lower the charge and improve blood–brain barrier (BBB) penetration. The primary amine was masked as an azide and the secondary amine as an amide or carbamate. The azide was not reduced to the amine under a variety of in vitro and ex vivo conditions. Despite the decrease in charge of the amino group as an amide and as carbamates, BBB penetration did not increase. It appears that the uses of azides as prodrugs for primary amines or amides and carbamates as prodrugs for secondary amines are not universally effective for CNS applications.