Facilitated isolation of rare recombinants by ligase chain reaction: selection for intragenic crossover events in the Drosophila optomotor-blind gene

Ligase chain reaction (LCR) was evaluated as a tool for the detection of point mutations. For the mutation studied, the specificity of the method is sufficient to detect the mutant allele in the presence of a 200-fold molar excess of the wild-type sequence. LCR was therefore employed in a genetic recombination experiment as a probe for a recessive lethal point mutation. LCR greatly facilitated the isolation of a rare recombinant originating from a crossover event in the 40 kb interval separating the lethal mutation and an enhancer trap insertion in the optomotor-blind locus. The recombinant will allow the study of gene control in situ, in a largely unperturbed regulatory environment.     

Cloning and expression of a cDNA encoding mouse kidney -amino acid oxidase

A cDNA encoding -amino acid oxidase (DAO;EC 1.4.3.3) has been isolated from a BALB/c mouse kidney cDNA library by hybridization with the cDNA for the porcine enzyme. Analysis of the nucleotide (nt) sequence of the clone revealed that it has a 1647-nt sequence with a 5′-terminal untranslated region of 68 nt that encodes 345 amino acids (aa), and a 3′-terminal untranslated region of 544 nt that contains the polyadenylation signal sequence ATTAAA. The deduced aa sequence showed 77 and 78% aa identity with the porcine and human enzymes, respectively. Two catalytically important aa residues, Tyr²²⁸ and His³⁰⁷, of the porcine enzyme, were both conserved in these three species. RNA blot hybridization analysis indicated that a DAO mRNA, of 2 kb, exists in mouse kidney and brain, but not liver. Synthesis of a functional mouse enzyme in Escherichia coli was achieved through the use of a vector constructed to insert the coding sequence of the mouse DAO cDNA downstream from the tac promoter of plasmid pKK223-3, which was designed so as to contain the lac repressor gene inducible by isopropyl-β- -thiogalactopyranoside. Immunoblot analysis confirmed the synthesis and induction of the mouse DAO protein, and the molecular size of the recombinant mouse DAO was found to be identical to that of the mouse kidney enzyme. Moreover, the maximum activity of the mouse recombinant DAO was estimated to be comparable with that of the porcine DAO synthesized in E. coli cells.     

PCR 扩增人型结核杆菌 DNA 及结核杆菌属 DNA 探针的制备

用多聚酶链反应(PCR)方法扩增人型、牛型结核杆菌基因组 DNA,获得特异的158 bpDNA 片段,而从另外十三种分枝杆菌未见到特异的扩增产物.回收158 bpDNA 片段作探针,它除与人型、牛型结核杆菌有特异的杂交信号外,与金黄色葡萄球菌、绿脓杆菌及一些分枝杆菌皆没有杂交反应.结果表明,PCR 可用于检测结核杆菌基因组 DNA,扩增产物158 bp DNA 片段可作为探针用于检测人型、牛型结核杆菌并鉴别结核杆菌与其它分枝杆菌.     

In vitro glucose uptake activity of Aegles marmelos and Syzygium cumini by activation of Glut-4, PI3 kinase and PPARγ in L6 myotubes

The purpose of the present study is to investigate the effect of methanolic extracts of Aegles marmelos and Syzygium cumini on a battery of targets glucose transporter (Glut-4), peroxisome proliferator activator receptor gamma (PPARgamma) and phosphatidylinositol 3' kinase (PI3 kinase) involved in glucose transport. A. marmelos and S. cumini are anti-diabetic medicinal plants being used in Indian traditional medicine. Different solvent extracts extracted sequentially were analysed for glucose uptake activity at each step and methanol extracts were found to be significantly active at 100ng/ml dose comparable with insulin and rosiglitazone. Elevation of Glut-4, PPARgamma and PI3 kinase by A. marmelos and S. cumini in association with glucose transport supported the up-regulation of glucose uptake. The inhibitory effect of cycloheximide on A. marmelos- and S. cumini-mediated glucose uptake suggested that new protein synthesis is required for the elevated glucose transport. Current observation concludes that methanolic extracts of A. marmelos and S. cumini activate glucose transport in a PI3 kinase-dependent fashion.     

Damage and mutagenesis of E. coli and bacteriophage λ induced by oxathiolane and aziridinyl steroids

λ-Escherichia coli complexes exhibited remarkable sensitivity to the treatment with test steroidal derivatives in the presence of Cu(II). The decline in plaque-forming units after steroid treatment was more pronounced in complexes with some of the irradiation repair-defective mutants of E. coli K-12, i.e., recA, lexA and polA, as compared to uvrA and wild-type strains. The red gene of λ phage and recA gene of E. coli seem to have a complementary effect on the steroid-induced lesions. An enhanced level of mutagenesis was observed when steroid-treated E. coli cells were transformed with steroid-treated pBR322 plasmid DNA. A remarkable degree of c mutation was also observed when steroid I-treated phage particles were allowed to adsorb on steroid-treated wild-type bacteria. Moreover, the oxathione steroid treatment of λcI857-E. coli lysogen resulted in prophage induction in nutrient broth even at 32°C. Thus on the basis of these results, the role of SOS repair system in steroid-induced mutagenesis and repair of DNA lesions in E. coli and bacteriophage λ has been suggested.     

Determination of the sequence of an expressible cDNA clone encoding ERp60/calregulin by the use of a novel nested set method

An analysis of the N-terminal sequence of the luminal endoplasmic reticulum protein, ERp60, showed that it was identical to the well-characterized Ca2+-binding protein, calregulin. A full-length, expressible cDNA clone encoding this protein was isolated from a mouse fibroblast cDNA library. A novel nested set strategy for the production of overlapping fragments for DNA sequencing was used to determine the complete nucleotide (nt) sequence of both strands of the ERp60 clone. This method utilizes a series of nonspecific deletion primers in conjunction with a specific site primer to generate the nested set fragments. This procedure possesses several advantages over other nested set techniques, since it does not require (i) the re-cloning of the DNA insert into other vectors, (ii) any prior knowledge of the restriction sites of the nt sequence, or (iii) the transformation and analysis of bacterial subclones. ERp60 has a 17-amino acid (aa) signal sequence and the mature protein contains 399 aa with a calculated M(r) of 46,347.     

Identification of five Orius species in Japan by multiplex polymerase chain reaction

We developed multiplex polymerase chain reaction methods to identify five Orius (Heteroptera: Anthocoridae) species that occur commonly in Japan: Orius sauteri, Orius minutus, Orius strigicollis, Orius nagaii, and Orius tantillus. The method amplified internal transcribed spacer 1 of the nuclear ribosomal DNA by using five primers simultaneously and produced species-specific banding patterns upon agarose gel electrophoresis. Reliability of the method was tested for 350 individuals of 23 strains, and consistent results were obtained. Dichotomous keys are also provided for easy and quick species identification.     

Efficient, galactose-free production of Candida antarctica lipase B by GAL10 promoter in Δgal80 mutant of Saccharomyces cerevisiae

An efficient yeast gene expression system with GAL10 promoter that does not require galactose as an inducer was developed using Δgal80 mutant strain of Saccharomyces cerevisiae. We constructed several combinations of gal mutations (Δgal1, Δgal80, Δmig1, Δmig2, and Δgal6) of S. cerevisiae and tested for their effect on efficiency of recombinant protein production by GAL10 promoter using a lipase, Candida antarctica lipase B (CalB), as a reporter. While the use of Δgal1 mutant strain required the addition of a certain amount of galactose to the medium, Δgal80 mutant strain did not require galactose. Furthermore, it was found that the recombinant CalB could be produced more efficiently (1.6-fold at 5 L-scale fermentation) in Δgal80 mutant strain than in the Δgal1 mutant. The Δgal80 mutant strain showed glucose repressible mode of expression of GAL10 promoter. Using Δgal80 mutant strain of S. cerevisiae, CalB was efficiently produced in a glucose-only fermentation at volumes up to 500 L.     

Fusarium roseum and Aspergillus oryzae-mediated enantioselective reduction of benzils to benzoins

Aspergillus oryzae OUT5048 and Fusarium roseum OUT4019 were found to be effective biocatalysts in the reduction of benzils to optically active benzoins. Easily available symmetrical benzil derivatives were reduced to the corresponding benzoins [(S)-2-hydroxy-1,2-diphenylethanones] by A. oryzae OUT5048 with up to 94% ee and by F. roseum OUT4019 with up to 98% ee, respectively. In this work, first general method for whole-cell-mediated selective reduction of benzils to benzoins is reported. It is also shown that this method is applicable for benzils with both electron-withdrawing and electron-donating groups.     

Hydroxylation of naringin by Trichoderma harzianum to dramatically improve its antioxidative activity

Transforming naringin using the mycelium of Trichoderma harzianum CGMCC 1523 produces two metabolites, 3′,4′,5,7-tetrahydroxy flavanone-7-rhamnoglucoside (3′-OHN) and 3′,4′,5′,5,7-pentahydroxy flavanone-7-rhamnoglucoside (3′,5′-DOHN), both of which were characterized by ESI–MS, ¹H NMR and ¹³C NMR analyses. The time course of the biotransformation by T. harzianum showed that 3′-OHN and 3′,5′-DOHN appeared simultaneously at 6 h, and the conversion yield (32.6%) of 3′,5′-DOHN was higher (10.6%) than that of 3′-OHN at 56 h. The optimal biotransformation temperature was 30 °C, the optimal pH was 5.0, and the optimal concentration of naringin was 400 mg/l. The bigger volume of biotransformation mixture and lower shaking speed did not favor hydroxylation reactions. The radical scavenging activity of naringin at 2000 μM was 11.1%, whereas activity of 3′-OHN at 100 μM could reach 38.4%, which is 68.6 times more than naringin. Antioxidative activity of 3′,5′-DOHN was increased 13.5% at 100 μM compared to 3′-OHN.     

Genetic evidence that the elevated levels of Escherichia coli helicase II antagonize recombinational DNA repair

Some phages survive irradiation much better upon multiple than upon single infection, a phenomenon known as multiplicity reactivation (MR). Long ago MR of UV-irradiated λ red phage in E. coli cells was shown to be a manifestation of recA-dependent recombinational DNA repair. We used this experimental model to assess the influence of helicase II on the type of recombinational repair responsible for MR. Since helicase II is encoded by the SOS-inducible uvrD gene, SOS-inducing treatments such as irradiating recA⁺ or heating recA441 cells were used. We found: i) that MR was abolished by the SOS-inducing treatments; ii) that in uvrD background these treatments did not affect MR; and iii) that the presence of a high-copy plasmid vector carrying the uvrD⁺ allele together with its natural promoter region was sufficient to block MR. From these results we infer that helicase II is able to antagonize the type of recA-dependent recombinational repair acting on multiple copies of UV-damaged λ DNA and that its antirecombinogenic activity is operative at elevated levels only.     

Enzymatic synthesis of mannobioses and mannotrioses by reverse hydrolysis using α-mannosidase from Aspergillus niger

Various manno-oligosaccharides including and

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were formed when a highly concentrated mannose solution was incubated in the presence of α-mannosidase from Aspergillus niger. and

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were isolated by activated carbon chromatography followed by high performance liquid chromatography using an amino-silica column. In addition to the above oligosaccharides,

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, and

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were also isolated.     

Essential oils composition of Eupatorium species growing wild in the Amazon

The essential oils of six Eupatorium species were obtained by hydrodistillation and analysed by GC-MS. The oil of E. macrophyllum was rich in sabinene (46.7%) and limonene (23.3%). The oil of E. laevigatum was mainly constituted by a mixture of aristolone+laevigatin (23.6%), globulol (16.2%) and germacrene D (8.6%). The principal constituents of the oils of the chemotypes A and B of E. squalidum, E. amygdalinum and E. conyzoides were caryophyllene oxide (17.4–30.1%), globulol (25.1%), germacrene D (10.4–21.6%), spathulenol (14.2%) and β-caryophyllene (7.1–12.3%). The oils of the chemotypes A and B of E. marginatum were dominated by α-zingiberene (57.5%), α-gurjunene (19.5%), germacrene D (14.8%), (E)-8-bisabolene (9.7%) and α-selinene (9.0%).     

IL2-330G polymorphic allele is associated with decreased risk of Helicobacter pylori infection in adulthood

We evaluated whether polymorphisms in genes coding molecules linked to the innate and adaptive immune response are associated with susceptibility to Helicobacter pylori infection. IL1B-511C → T, IL1B-31 T → C, IL1RN allele 2, IL2-330 T → G, TNFA-307 G → A, TLR2Arg677Trp, TLR2Arg753Gln, TLR4Asp299Gly, and TLR5^392STOP polymorphisms were determined in 541 blood donors. IL2-330 T → G allele carriers had a decreased H. pylori infection risk (OR = 0.63, 95% CI = 0.43–0.93) after adjustment for demographic and environmental factors. Hence, we investigated whether the polymorphism is functional by evaluating IL-2 serum concentration in 150 blood donors and 100 children. IL-2 pro-inflammatory and anti-inflammatory properties were indirectly investigated by determining serum IFN-γ and IL-10/TGF-β levels. The polymorphism was associated with increased mean IL-2 levels in H. pylori-positive adults (2.65 pg/mL vs. 7.78 pg/mL) and children (4.19 pg/mL vs. 8.03 pg/mL). Increased IL-2 was associated with pro-inflammatory activity in adults (IFN-γ = 18.61 pg/mL vs. 25.71 pg/mL), and with anti-inflammatory activity in children (IL-10 = 6.99 vs. 14.17 pg/mL, TGF-β = 45.88 vs. 93.44 pg/mL) (p < 10⁻³ for all). In conclusion, in the context of H. pylori infection, IL2-330 T → G polymorphism is functional and is associated with decreased risk of infection in adults.     

β-Carotene, vitamin C and vitamin E content of the marine microalga Dunaliella tertiolecta cultured with different nitrogen sources

Variations in the β-carotene, vitamin C and vitamin E content of D. tertiolecta have been shown to result from the nitrogen source used in the culture medium. Differences of 101%, 38% and 69% have been found in β-carotene, ascorbic acid and tocopherol content in mg/g of dry matter, respectively, and differences of 147%, 63% and 37% occurred in β-carotene, vitamin C and E concentrations in mg/litre of culture, respectively. Considering the β-carotene, vitamin C and vitamin E content in mg/g of chlorophyll a, maximum variations occurred in β-carotene content, with differences of 145% among the different nitrogen sources. Maximum β-carotene and vitamin C values were found in urea cultures, whereas urea cultures showed the minimum values for vitamin E.     

In vitro antimicrobial activity of alpha-melanocyte stimulating hormone against major human pathogen Staphylococcus aureus

Alpha-melanocyte stimulating hormone (α-MSH) is an endogenous anti-inflammatory peptide reported to possess antimicrobial properties, however their role as antibacterial peptides is yet to be established. In the present study, we examined in vitro antibacterial activity of α-MSH against S. aureus strain ISP479C and several methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) S. aureus strains. Antibacterial activity was examined by varying several parameters, viz., bacterial cell densities, growth phase, pH, salt concentration, and temperature. Antibacterial activity was also examined in complex biomatrices of rat whole blood, plasma and serum as well as in biofilm form of bacteria. Our results showed that α-MSH possessed significant and rapid antibacterial activity against all the studied strains including MRSA (84% strains were killed on exposure to 12 μM of α-MSH for 2 h). pH change from 7.4 to 4 increased α-MSH staphylocidal activity against ISP479C by 21%. Antibacterial activity of α-MSH was dependent on bacterial cell density and independent of growth phase. Moreover, antimicrobial activity was retained when α-MSH was placed into whole blood, plasma, and serum. Most importantly, α-MSH exhibited antibacterial activity against staphylococcal biofilms. Multiple membrane permeabilization assays suggested that membrane damage was, at least in part, a major mechanism of staphylocidal activity of α-MSH. Collectively the above findings suggest that α-MSH could be a promising candidate of a novel class of antimicrobial agents.     

Transformation of C19-steroids and testosterone production by sterol-transforming strains of Mycobacterium spp.

Redox conversions of exogenic C₁₉-steroids (androstenedione (AD), androstadienedione (ADD), testosterone and 1(2)-dehydrotestosterone) were studied using a mutant strain of Mycobacterium sp. Et1 with high level of 17β-hydroxysteroid dehydrogenase (17-HSD) activity. Factors affecting target 17β-reduction and side reactions by resting and growing cells were estimated. The effects of glucose supplementation, pH, mode of substrate addition were identified. The results confirmed that double reduction of androstadienedione, both of 17-keto group and 1(2)-double bond, is more effective for testosterone formation than a single reduction of 17-keto group of AD. These findings argued for the application of the strain capable of sterol side chain degradation and expressed 17-HSD, 3-ketosteroid-1(2)-dehydrogenase and 1-ene-reductase activities, for testosterone obtaining from sitosterol. Under optimal conditions, the conversion of sitosterol (5 g/l) by Mycobacterium sp. VKM Ac-1816D in laboratory fermenter resulted in 50–55% molar yield of testosterone.