Double lesions are produced in DNA oligomer by ionizing radiation and by metal-catalyzed H2O2 reactions

It was demonstrated previously that double lesions are produced in DNA by ionizing radiation. These double lesions consist of adjacent nucleotides each bearing a modified base. The goal of the present investigation was to determine whether Fenton chemistry can generate the same kind of lesions. DNA oligomers were exposed to metal-catalyzed H(2)O(2) reactions, and the products were characterized by chromatography and by mass spectrometry. Double lesions are produced by this treatment in which deoxyguanosine is oxidized to 8-oxo-7,8-dihydrodeoxyguanosine and an adjacent pyrimidine nucleoside is degraded to a formamido remnant.     

Detection and characterization of formamido lesions in DNA by liquid chromatography-mass spectrometry

DNA X-irradiated in oxygenated aqueous solution produces the formamido lesion from the breakdown of pyrimidine nucleosides. This pyrimidine breakdown product inhibits the hydrolysis by nuclease P1 of the phosphoester bond 3' to the damaged nucleoside. Consequently, the lesion can be obtained from an enzymatic digest of the DNA as a modified dinucleoside monophosphate in which the 5' nucleoside contains the lesion. In this form, the formamido lesion can be detected with good sensitivity by liquid chromatography-mass spectrometry (LC-MS). Nucleosides that have lost the base moiety also inhibit nuclease P1. Together, the formamido and abasic lesions account for all of the substantial peaks in the LC-MS ion current profile.     

PAK4 promotes kinase-independent stabilization of RhoU to modulate cell adhesion

P21-activated kinase 4 (PAK4) is a Cdc42 effector protein thought to regulate cell adhesion disassembly in a kinase-dependent manner. We found that PAK4 expression is significantly higher in high-grade human breast cancer patient samples, whereas depletion of PAK4 modifies cell adhesion dynamics of breast cancer cells. Surprisingly, systematic analysis of PAK4 functionality revealed that PAK4-driven adhesion turnover is neither dependent on Cdc42 binding nor kinase activity. Rather, reduced expression of PAK4 leads to a concomitant loss of RhoU expression. We report that RhoU is targeted for ubiquitination by the Rab40A–Cullin 5 complex and demonstrate that PAK4 protects RhoU from ubiquitination in a kinase-independent manner. Overexpression of RhoU rescues the PAK4 depletion phenotype, whereas loss of RhoU expression reduces cell adhesion turnover and migration. These data support a new kinase-independent mechanism for PAK4 function, where an important role of PAK4 in cellular adhesions is to stabilize RhoU protein levels. Thus, PAK4 and RhoU cooperate to drive adhesion turnover and promote cell migration.     

The mitospecific domain of Mrp7 (bL27) supports mitochondrial translation during fermentation and is required for effective adaptation to respiration

We demonstrate here that mitoribosomal protein synthesis, responsible for the synthesis of oxidative phosphorylation (OXPHOS) subunits encoded by the mitochondrial genome, occurs at high levels during glycolysis fermentation and in a manner uncoupled from OXPHOS complex assembly regulation. Furthermore, we provide evidence that the mitospecific domain of Mrp7 (bL27), a mitoribosomal component, is required to maintain mitochondrial protein synthesis during fermentation but is not required under respiration growth conditions. Maintaining mitotranslation under high-glucose-fermentation conditions also involves Mam33 (p32/gC1qR homologue), a binding partner of Mrp7’s mitospecific domain, and together they confer a competitive advantage for a cell’s ability to adapt to respiration-based metabolism when glucose becomes limiting. Furthermore, our findings support that the mitoribosome, and specifically the central protuberance region, may be differentially regulated and/or assembled, under the different metabolic conditions of fermentation and respiration. On the basis of our findings, we propose that the purpose of mitotranslation is not limited to the assembly of OXPHOS complexes, but also plays a role in mitochondrial signaling critical for switching cellular metabolism from a glycolysis- to a respiration-based state.     

Nucleic acid sequence-based amplification (NASBA) detection of medically important Candida species.

Nucleic Acid Sequence Based Amplification (iNASBA), an isothermal amplification technique for nucleic acids, was evaluated for the identification of medically important Candida species using primers selected from 18S rRNA sequences conserved in fungi. An RNA fragment of 257 nucleotides was amplified for Candida albicans. Nineteen different fungi were tested for rRNA amplification with the NASBA. All were positive when analyzed on agarose gel, whereas human RNA was negative. For the identification of Candida species, NASBA amplification products were analyzed in an enzyme bead-based detection format, using species-specific biotinylated probes and a generic Candida HRPO probe or a membrane-based system using biotinylated probes and avidin-HPRO. Discrimination of the major human pathogenic Candida spp. was based on a panel of biotinylated probes for C. krusei, C. tropicalis, C. albicans, C. glabrata, and C. lusitaniae. Using rRNA dilutions obtained from pure cultures of C. albicans, the combination of NASBA and the enzymatic bead-based detection yielded a sensitivity equivalent to 0.01 CFU. In a model system using 1 ml of artificially contaminated blood as few as 1-10 CFU of C. albicans could be detected. Testing of 68 clinical blood samples from patients suspected of candidemia showed that eight samples were positive for C. albicans and one for C. glabrata. Testing of 13 clinical plasma samples from patients suspected of fungemia identified the presence of C. albicans in two specimens. The whole procedure of sample preparation, amplification and identification by hybridization can be performed in 1 day. This speed and the observed sensitivity of the assay make the NASBA a good alternative to PCR for the detection of candidemia.     

Transacylase activity of lactating bovine mammary fatty acid synthase

An assay for the transacylation reaction catalyzed by fatty acid synthase was developed which does not require model substrates or labelled acyl-derivatives of CoA. It involves the transfer of the acyl group from unlabelled CoA to [³H]CoA. This assay shows the occurrence of transacylation at a relatively high rate with a variety of substrates that the enzyme is able to utilize. The activity is unaffected by dissociation of the enzyme or modification by iodoacetamide or 2-chloroacetyl-CoA.     

Analysis of the meiosis in the F_1 hybrids of Longiflorum × Asiatic(LA) of lilies(Lilium) using genomic in situ hybridization

Longiflorum and Asiatic lilies of the genus Lilium of the family Liliaceae are two important groups of modern lily cultivars.One of the main trends of lily breeding is to realize introgression between these groups.With cut style pollination and embryo rescue,distant hybrids between the two groups have been obtained.However,the F1 hybrids are highly sterile or some of them could produce a small number of 2n gametes,and their BC1 progenies are usually triploids.Dutch lily breeders have selected many cultivars...     

Survey of Genomics Approaches to Improve Bioenergy Traits in Maize,Sorghum and Sugarcane

Bioenergy crops currently provide the only source of alternative energy with the potential to reduce the use of fossil transportation fuels in a way that is compatible with existing engine technology,including in developing countries.Even though bioenergy research is currently receiving considerable attention,many of the concepts are not new,but rather build on intense research efforts from 30 years ago.A major difference with that era is the availability of genomics tools that have the potential to acceler...     

Screening for asymptomatic bowel cancer in general practice.

General practitioners screened 4284 asymptomatic people aged over 40 to compare the incidence of large bowel cancer and polyps with a control general practice (4288 patients). Compliance was best in young women (60%), and overall it was 42%. Twenty six patients who had a positive Haemoccult test result (1.5% of those screened) were examined by colonoscopy and 10 had polyps. The incidence of cancers in the two groups was similar but in the control (unscreened patients) practice no polyps were found.