Plasmodium yoelii: the effect of second blood meal and anti-sporozoite antibodies on development and gene expression in the mosquito vector, Anopheles stephensi

The sporogonic development of the malaria parasite takes place in the mosquito and a wide range of factors modulates it. Among those, the contents of the blood meal can influence the parasite development directly or indirectly through the mosquito response to the infection. We have studied the effect of a second blood meal in previously infected mosquitoes and the effect of anti-sporozoite immune serum on parasite development and mosquito response to the infection. The prevalence and intensity of infection and gene expression of both Plasmodium yoelii and Anopheles stephensi was analyzed. We verified that a second blood meal and its immune status interfere with parasite development and with Plasmodium and mosquito gene expression.     

Scintillometric determination of DNA repair in human cell lines: A critical appraisal

The ability of a variety of chemical and physical agents to stimulate DNA repair synthesis in human cell cultures was tested by a simplified scintillometric procedure, with the use of hydroxyurea (HU) to suppress DNA replicative synthesis. After incubation with [³H]thymidine, the radioactivity incorporated in to DNA was determined in controls (C) and treated (T) cultures and in the corresponding HU series (C_HU, T_HU). The ratios T_HU/C_HU and T_HU/T:C_HU/C, indicating absolute and relative increases of DNA radioactivity, were calculated. When both ratios were significantly higher than 1, they were taken as indices of DNA repair stimulation, whereas, no stimulation in inferred when both of them are ?1. The scintillometric estimate of DNA repair was always in agreement with the autoradiographic observations, so that the procedure adopted can be used as a rapid test for screening investigations.Agents giving a relative but no an absolute increase of DNA radioactivity are generally not inducers of repair synthesis as estimated by autoradiography. However, the same scintillometric results are also occasionally observed with DNA repair inducers, such as methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), owing to alterations of thymidine pool radioactivity. These chemicals, besides affecting the levels of labelled precursors in the intracellular pool in the T series, differently modified the increase of pool radioactivity which is a regular effect of HU. With such chemicals, DNA repair synthesis can be detected only after normalization of th DNA radioactivity on the basis of pool alterations.The quantitative value of the autoradiographic estimate of DNA repair is also affected by the changes in the radioactivity of the thymidine pool although autoradiography retains its qualitative value.Dimethylnitrosamine, mitomycin C and potassium dichromate, described by other authors as inducers of DNA repair, also gave negative results by the scintillometric procedure after normalization of DNA radioactivities. However, in our hands, these agents were unable to stimulated repair synthesis, according to the results of autoradiography and isopynic centrifugation.The proposed scintillometric procedure is effective in indicating false negative inducers of DNA repair, not giving rise to false positives.     

Effects of p-fluorophenylalanine on the induction of mutations in bacteriophage T4: I. 5-Bromouracil mutagenesis

The amino acid analogue p-fluorophenylalanine (PFPA) was found to have no mutagenic activity in the r system of bacteriophage T4. However, under standard conditions for 5-bromouracil (5-BU) mutagenesis, PFPA depressed the induced frequencies for both forward and reverse mutations. When the folate antagonist sulphanilamide (SU) was omitted from the mutagenic treatment medium or when it was replaced by Trimethoprim (TM), another folate antagonist, this depressive effect was abolished. It was proposed that PFPA alleviated the inhibitory action of SU.     

Polymorphism p.Val231Ile alters substrate selectivity of drug-metabolizing arylamine N-acetyltransferase 2 (NAT2) isoenzyme of rhesus macaque and human

Arylamine N-acetyltransferases (NATs) are polymorphic enzymes mediating the biotransformation of arylamine/arylhydrazine xenobiotics, including pharmaceuticals and environmental carcinogens. The NAT1 and NAT2 genes, and their many polymorphic variants, have been thoroughly studied in humans by pharmacogeneticists and cancer epidemiologists. However, little is known about the function of NAT homologues in other primate species, including disease models. Here, we perform a comparative functional investigation of the NAT2 homologues of the rhesus macaque and human. We further dissect the functional impact of a previously described rhesus NAT2 gene polymorphism, causing substitution of valine by isoleucine at amino acid position 231. Gene constructs of rhesus and human NAT2, bearing or lacking non-synonymous polymorphism c.691G>A (p.Val231Ile), were expressed in Escherichia coli for comparative enzymatic analysis against various NAT1- and NAT2-selective substrates. The results suggest that the p.Val231Ile polymorphism does not compromise the stability or overall enzymatic activity of NAT2. However, substitution of Val231 by the bulkier isoleucine appears to alter enzyme substrate selectivity by decreasing the affinity towards NAT2 substrates and increasing the affinity towards NAT1 substrates. The experimental observations are supported by in silico modelling localizing polymorphic residue 231 close to amino acid loop 125–129, which forms part of the substrate binding pocket wall and determines the substrate binding preferences of the NAT isoenzymes. The p.Val231Ile polymorphism is the first natural polymorphism demonstrated to affect NAT substrate selectivity via this particular mechanism. The study is also the first to thoroughly characterize the properties of a polymorphic NAT isoenzyme in a non-human primate model.     

Purification by p-aminobenzoic acid (PABA)-affinity chromatography and the functional reconstitution of the nateglinide/H+ cotransport system in the rat intestinal brush-border membrane

(-)-N-(trans-4-isopropylcyclohexanecarbonyl)-D-phenylalanine (nateglinide) is a novel oral hypoglycemic agent possessing a peptide-type bond and a carboxyl group in its structure. Recently, we have shown that nateglinide transport occurs via the ceftibuten/H+ cotransport system, which is distinct from PepT1, and that the fluorescein/H+ cotransport system is involved in the uptake of nateglinide. The aim of this study was to characterize the functional properties of the intestinal nateglinide transporter. In the first part of this study, we demonstrated that the ceftibuten/H+ cotransport system is identical to the fluorescein/H+ cotransport system. We succeeded in purification of the nateglinide transporter from brush-border membranes of the rat small intestine using p-aminobenzoic acid (PABA)-affinity chromatography. We then investigated the functional properties of the nateglinide transporter using proteoliposomes prepared from the PABA-affinity chromatography elute. We demonstrated that nateglinide, ceftibuten, and fluorescein are transported by the same transporter in the intestine.     

Xyloglucan xyloglucosyl transferases from barley (Hordeum vulgare L.) bind oligomeric and polymeric xyloglucan molecules in their acceptor binding sites

Background

Xyloglucan xyloglucosyl transferases (EC 2.4.1.207), known as xyloglucan endotransglycosylases (XETs) use a disproportionation reaction mechanism and modulate molecular masses of xyloglucans. However, it is not known precisely how these size modulations and transfer reactions occur with polymeric acceptor substrates.

Methods

cDNAs encoding three barley HvXETs were expressed in Pichia pastoris and reaction mechanism and molecular properties of HvXETs were investigated.

Results

Significant differences in catalytic efficiencies (k_cat·K_m⁻¹) were observed and these values were 0.01, 0.02 and 0.2 s⁻¹·mg⁻¹·ml for HvXET3, HvXET4 and HvXET6, respectively, using tamarind xyloglucan as a donor substrate. HPLC analyses of the reaction mixtures showed that HvXET6 followed a stochastic reaction mechanism with fluorescently or radioactively labelled tamarind xyloglucans and xyloglucan-derived oligosaccharides. The analyses from two successive reaction cycles revealed that HvXET6 could increase or decrease molecular masses of xyloglucans. In the first reaction cycle equilibrium was reached under limiting donor substrate concentrations, while xyloglucan mass modulations occurred during the second reaction cycle and depended on the molecular masses of incoming acceptors. Deglycosylation experiments indicated that occupancy of a singular N-glycosylation site was required for activity of HvXET6. Experiments with organic solvents demonstrated that HvXET6 tolerated DMSO, glycerol, methanol and 1,4-butanediol in 20% (v/v) concentrations.

Conclusions

The two-phase experiments demonstrated that large xyloglucan molecules can bind in the acceptor sites of HvXETs.

General significance

The results characterise donor and acceptor binding sites in plant XET, report that HvXETs act on xyloglucan donor substrates adsorbed onto nanocrystals and that HvXETs tolerate the presence of organic solvents.     

Identification of bottlenecks in Escherichia coli engineered for the production of CoQ(10)

In this work, Escherichia coli was engineered to produce a medically valuable cofactor, coenzyme Q₁₀ (CoQ₁₀), by removing the endogenous octaprenyl diphosphate synthase gene and functionally replacing it with a decaprenyl diphosphate synthase gene from Sphingomonas baekryungensis. In addition, by over-expressing genes coding for rate-limiting enzymes of the aromatic pathway, biosynthesis of the CoQ₁₀ precursor para-hydroxybenzoate (PHB) was increased. The production of isoprenoid precursors of CoQ₁₀ was also improved by the heterologous expression of a synthetic mevalonate operon, which permits the conversion of exogenously supplied mevalonate to farnesyl diphosphate. The over-expression of these precursors in the CoQ₁₀-producing E. coli strain resulted in an increase in CoQ₁₀ content, as well as in the accumulation of an intermediate of the ubiquinone pathway, decaprenylphenol (10P-Ph). In addition, the over-expression of a PHB decaprenyl transferase (UbiA) encoded by a gene from Erythrobacter sp. NAP1 was introduced to direct the flux of DPP and PHB towards the ubiquinone pathway. This further increased CoQ₁₀ content in engineered E. coli, but decreased the accumulation of 10P-Ph. Finally, we report that the combined over-production of isoprenoid precursors and over-expression of UbiA results in the decaprenylation of para-aminobenzoate, a biosynthetic precursor of folate, which is structurally similar to PHB.     

对氨基苯甲酸对牙龄卟啉单胞菌胰酶样蛋白酶活性的影响

目的：测定不同浓度PABA对牙龄卟啉单胞菌胰酶样蛋白酶（TLP）活性的影响,以探讨血链球菌在龈下菌微生态平衡中的作用。方法：在1/2浓度的BH培养基中加入不同浓度的PABA和一定浊度的P.gingivalis,行常规培养。测定培养物上清液和菌细胞中TLP活性及蛋白质含量,计算TLP的比活。结果：PABA对P.gingivalis TLP的比活产生促进作用。结论：PABA影响P.gingivalis TLP的活性提示血链球菌可能对龈下菌斑的微生态平衡具调节作用。     

Folate-dependent hydrolysis of acetyl-coenzyme A by recombinant human and rodent arylamine N-acetyltransferases

Arylamine N-acetyltransferases (NATs) are drug and xenobiotic metabolizing enzymes that catalyze the N-acetylation of arylamines and hydrazines and the O-acetylation of N-hydroxy-arylamines. Recently, studies report that human NAT1 and mouse Nat2 hydrolyze acetyl-coenzyme A (AcCoA) into acetate and coenzyme A in a folate-dependent fashion, a previously unknown function. In this study, our goal was to confirm these findings and determine the apparent Michaelis–Menten kinetic constants (V_max and K_m) of the folate-dependent AcCoA hydrolysis for human NAT1/NAT2, and the rodent analogs rat Nat1/Nat2, mouse Nat1/Nat2, and hamster Nat1/Nat2. We also compared apparent V_max values for AcCoA hydrolysis and N-acetylation of the substrate para-aminobenzoic acid (PABA). Human NAT1 and its rodent analogs rat Nat2, mouse Nat2 and hamster Nat2 catalyzed AcCoA hydrolysis in a folate-dependent manner. Rates of AcCoA hydrolysis were between 0.25–1% of the rates for N-acetylation of PABA catalyzed by human NAT1 and its rodent orthologs. In contrast to human NAT1, human NAT2 and its rodent analogs rat Nat1, mouse Nat1, and hamster Nat1 did not hydrolyze AcCoA in a folate-dependent manner. These results are consistent with the possibility that human NAT1 and its rodent analogs regulate endogenous AcCoA levels.