Effect of pH on cytochromes b in ATP-Mg submitochondrial particles

Addition of ATP to anaerobic, succinate-reduced phosphorylating submitochondrial particles (ATP-Mg particles) causes reduction of cytochromes b absorbing at 558 and 566 nm in the pH range 5.5–9.0. The extent of the reduction of both cytochromes induced by ATP is maximal at pH 7.4–7.5. On the other hand, addition of ATP to anaerobic, NADH-reduced particles causes oxidation of b₅₆₂ at high pH, while it causes reduction of cytochromes absorbing at 558 and 566 nm at low pH. The optimal pH for the oxidation of cytochromes b is in the region 8.5–9.0. Partial reduction of the cytochromes absorbing at 558 and 566 nm can be brought about non-energetically by lowering the potential of the substrate redox couple or by making the reaction mixture alkaline. Addition of the electron-transfer mediator, phenazine methosulphate, to anaerobic, NADH-reduced particles causes complete reduction of cytochromes b absorbing at 558 and 566 nm in the pH range 5.5–9.0. The findings are interpreted in terms of a pH-induced removal of an accessibility barrier (structural or kinetic) that interferes with the redox equilibrium between NADH and cytochrome b.     

Adherence of LR white sections to glass slides for silver enhancement immunogold labeling

A continuing problem in immunogold labeling of 1 microns LR White sections for light microscopy is the lack of adherence of the sections to the glass microscope slides during silver enhancement. A technique is described to overcome this problem using a 2% Formvar solution to coat the glass.     

Biosynthesis of the red antibiotic, prodigiosin, in Serratia: identification of a novel 2-methyl-3-n-amyl-pyrrole (MAP) assembly pathway, definition of the terminal condensing enzyme, and implications for undecylprodigiosin biosynthesis in Streptomyces

The biosynthetic pathway of the red-pigmented antibiotic, prodigiosin, produced by Serratia sp. is known to involve separate pathways for the production of the monopyrrole, 2-methyl-3-n-amyl-pyrrole (MAP) and the bipyrrole, 4-methoxy-2,2'-bipyrrole-5-carbaldehyde (MBC) which are then coupled in the final condensation step. We have previously reported the cloning, sequencing and heterologous expression of the pig cluster responsible for prodigiosin biosynthesis in two Serratia sp. In this article we report the creation of in-frame deletions or insertions in every biosynthetic gene in the cluster from Serratia sp. ATCC 39006. The biosynthetic intermediates accumulating in each mutant have been analysed by LC-MS, cross-feeding and genetic complementation studies. Based on these results we assign specific roles in the biosynthesis of MBC to the following Pig proteins: PigI, PigG, PigA, PigJ, PigH, PigM, PigF and PigN. We report a novel pathway for the biosynthesis of MAP, involving PigD, PigE and PigB. We also report a new chemical synthesis of MAP and one of its precursors, 3-acetyloctanal. Finally, we identify the condensing enzyme as PigC. We reassess the existing literature and discuss the significance of the results for the biosynthesis of undecylprodigiosin by the Red cluster in Streptomyces coelicolor A3(2).     

Mechanisms of inactivation of HSV-2 during storage in frozen and lyophilized forms

The structural integrity of herpes simplex virus 2 (HSV-2) during freezing, thawing, and lyophilization has been studied using scanning and transmission electron microscopy. Viral particles should be thawed quickly from -80 to 37 degrees C to avoid artifacts of thawing. To avoid freezing damage, the virus should be rapidly frozen (>20 K s(-1)) rather than slowly frozen as occurs on the shelf of a lyophilizer (<1 K s(-1)). Fast freezing and thawing allows six cycles of freeze thaw with no loss of viral titer TCID50. Viral particles were characterized using immunogold labeling methods. Freshly thawed virus had 19 +/- 4 polyclonal immunogold particles virus(-1); virus stored at -80 degrees C for at least 4 months had 17 +/- 3 particles virus(-1); virus stored for 1 week at 4 degrees C had 8 +/- 4 particles virus(-1). By bulk lyophilization the number of particles was 4 +/- 4, but by fast freezing and lyophilization the number of gold particles improved to 12 +/- 5. The loss of viral membrane was directly observed, and the in vitro loss was demonstrated to occur through three possible pathways, including (i) simultaneous release of tegument and membrane, (ii) sequential release of membrane and then tegument, and (iii) release like by in vivo infection. The capsids were not further degraded as indicated by the lack of free DNA, which was only released by boiling the viral samples with 1% SDS, followed by a dilution to 0.001% w/v SDS for the real-time PCR reaction.     

Affinity capture of a biotinylated retrovirus on macroporous monolithic adsorbents: towards a rapid single-step purification process

A streptavidin derivitised macroporous monolith was developed to enable single-step capture of chemically biotinylated Moloney Murine Leukaemia Virus (MoMuLV) from crude, unclarified cell culture supernatant. Monoliths were prepared by aqueous cryopolymerisation of acrylamide with N,N'-methylene-bis (acrylamide) and glycidyl methacrylate (Arvidsson et al. [2003] J Chrom A 986:275-290). Streptavidin was immobilised to the epoxy functionalised monoliths. Particulate-containing cell culture supernatant was passed through the monolith without preclarification of the feedstock and adsorption capacities of 2 x 10(5) cfu/ml of adsorbent were demonstrated (cf. Fractogel streptavidin, at 3.9 x 10(5) cfu/ml of adsorbent). The specific titre of the recovered fraction was increased by 425-fold; however, recoveries of less than 8% were achieved. Adsorption of nonbiotinylated MoMuLV on the streptavidin-coated monolith was not observed.     

High-resolution identification of chromosomal abnormalities using oligonucleotide arrays containing 116,204 SNPs

Mutation of the human genome ranges from single base-pair changes to whole-chromosome aneuploidy. Karyotyping, fluorescence in situ hybridization, and comparative genome hybridization are currently used to detect chromosome abnormalities of clinical significance. These methods, although powerful, suffer from limitations in speed, ease of use, and resolution, and they do not detect copy-neutral chromosomal aberrations--for example, uniparental disomy (UPD). We have developed a high-throughput approach for assessment of DNA copy-number changes, through use of high-density synthetic oligonucleotide arrays containing 116,204 single-nucleotide polymorphisms, spaced at an average distance of 23.6 kb across the genome. Using this approach, we analyzed samples that failed conventional karyotypic analysis, and we detected amplifications and deletions across a wide range of sizes (1.3-145.9 Mb), identified chromosomes containing anonymous chromatin, and used genotype data to determine the molecular origin of two cases of UPD. Furthermore, our data provided independent confirmation for a case that had been misinterpreted by karyotype analysis. The high resolution of our approach provides more-precise breakpoint mapping, which allows subtle phenotypic heterogeneity to be distinguished at a molecular level. The accurate genotype information provided on these arrays enables the identification of copy-neutral loss-of-heterozygosity events, and the minimal requirement of DNA (250 ng per array) allows rapid analysis of samples without the need for cell culture. This technology overcomes many limitations currently encountered in routine clinical diagnostic laboratories tasked with accurate and rapid diagnosis of chromosomal abnormalities.