Improved protein identification through the use of unstained gels

In this work, a method for improved protein identification of low-abundance proteins using unstained gels, in combination with robotics and matrix-assisted laser desorption/ionization tandem mass spectrometry, has been developed and evaluated. Omitting the silver-staining process resulted in increased protein identification scores, an increase in the number of peptides observed in the MALDI mass spectrum, and improved quality of the tandem mass spectrometry data.     

Molecular Diversity of Rumen Methanogens from Sheep in Western Australia

The molecular diversity of rumen methanogens in sheep in Australia was investigated by using individual 16S rRNA gene libraries prepared from the rumen contents obtained from six merino sheep grazing pasture (326 clones), six sheep fed an oaten hay-based diet (275 clones), and five sheep fed a lucerne hay-based diet (132 clones). A total of 733 clones were examined, and the analysis revealed 65 phylotypes whose sequences (1,260 bp) were similar to those of cultivated methanogens belonging to the order Methanobacteriales. Pasture-grazed sheep had more methanogen diversity than sheep fed either the oaten hay or lucerne hay diet. Methanobrevibacter strains SM9, M6, and NT7 accounted for over 90% of the total number of clones identified. M6 was more prevalent in grazing sheep, and SM9, despite being found in 16 of the 17 sheep, was more prevalent in sheep fed the lucerne-based diet. Five new species were identified. Two of these species exhibited very little sequence similarity to any cultivated methanogens and were found eight times in two of the six sheep that were grazing pasture. These unique sequences appear to represent a novel group of rumen archaea that are atypical for the rumen environment.     

HIV Reactivation from Latency after Treatment Interruption Occurs on Average Every 5-8 Days—Implications for HIV Remission

HIV infection can be effectively controlled by anti-retroviral therapy (ART) in most patients. However therapy must be continued for life, because interruption of ART leads to rapid recrudescence of infection from long-lived latently infected cells. A number of approaches are currently being developed to ‘purge’ the reservoir of latently infected cells in order to either eliminate infection completely, or significantly delay the time to viral recrudescence after therapy interruption. A fundamental question in HIV research is how frequently the virus reactivates from latency, and thus how much the reservoir might need to be reduced to produce a prolonged antiretroviral-free HIV remission. Here we provide the first direct estimates of the frequency of viral recrudescence after ART interruption, combining data from four independent cohorts of patients undergoing treatment interruption, comprising 100 patients in total. We estimate that viral replication is initiated on average once every ≈6 days (range 5.1- 7.6 days). This rate is around 24 times lower than previous thought, and is very similar across the cohorts. In addition, we analyse data on the ratios of different ‘reactivation founder’ viruses in a separate cohort of patients undergoing ART-interruption, and estimate the frequency of successful reactivation to be once every 3.6 days. This suggests that a reduction in the reservoir size of around 50-70-fold would be required to increase the average time-to-recrudescence to about one year, and thus achieve at least a short period of anti-retroviral free HIV remission. Our analyses suggests that time-to-recrudescence studies will need to be large in order to detect modest changes in the reservoir, and that macaque models of SIV latency may have much higher frequencies of viral recrudescence after ART interruption than seen in human HIV infection. Understanding the mean frequency of recrudescence from latency is an important first step in approaches to prolong antiretroviral-free viral remission in HIV.     

pHG165: A pBR322 copy number derivative of pUC8 for cloning and expression

During the construction of the Messing pUC plasmid series, the rop(rom) gene of pBR322 which mediates the activity of RNAI was deleted. This has resulted in an elevated copy number for the pUC plasmids which makes the expression of beta-galactosidase activity constitutive in a host containing the Iqtss lac repressor. We describe the construction of a new series of vectors which retain the pUC multiple cloning site (MCS) but in which copy number control has been recovered. In addition, the lac alpha/lac promoter expression region has been inserted into a HpaI cassette. This facilitates the movement of recombinant DNA clones within the MCS. It also increases the complementation activity of the lac alpha peptide by an order of magnitude, allowing selection of recombinants by their Lac- phenotype on MacConkey agar.     

Dissecting the telomere region of barley chromosome 5HL using rice genomic sequences as references: new markers for tracking a complex region in breeding

The terminal region of barley chromosome 5HL controls malt extract, diastatic power, free amino acid nitrogen, alpha-amylase activity, seed dormancy and pre-harvest sprouting. Comparative analysis of the barley and rice maps has established that the terminal region of barley chromosome 5HL is syntenic to rice chromosome 3L near the telomere end. The rice BAC (Bacterial Artificial Chromosome) sequences covering the region of chromosome 3L were used to search barley expressed sequenced tags database. Thirty-three genes were amplified by PCR (polymerase chain reaction) with the primers designed from barley ESTs (expressed sequence tag). Comparison of the sequences of the PCR generated DNA fragments revealed polymorphisms including single nucleotide polymorphism (SNP), insertions or deletions between the barley varieties. Seven new PCR based molecular markers were developed and mapped within 10 cM in three doubled haploid barley populations (Stirling × Harrington, Baudin × AC Metcalfe and Chebec × Harrington). The mapped genes maintain the micro-syntenic relationship between barley and rice. These gene specific markers provide simple and efficient tools for germplasm characterization and marker-assisted selection for barley malting quality, and ultimately lead to isolation and identification of the major gene(s) controlling multiple quality traits on barley chromosome 5HL.     

Purification, characterisation, and inhibition by monoterpenes of acetylcholinesterase from the waxmoth, Galleria mellonella (L.)

Acetylcholinesterase (AChE) was purified from the brain of the waxmoth, Galleria mellonella (L.) by affinity chromatography followed by anion exchange chromatography. It resolved as a single band by polyacrylamide gel electrophoresis (PAGE) both non-denaturing and SDS (silver stained), and as a single peak by high pressure liquid chromatography (HPLC), in an overall yield of 32% representing 283-fold purification. This was a true acetylcholinesterase, with no activity as a non-specific cholinesterase (butyrylcholinesterase). The molecular weight determined by PAGE in the absence and presence of sodium dodecyl sulphate (SDS) was ca. 240,000 Da and 60,000 Da respectively, indicating an arrangement of tetrameric subunits. 2-Heptanone, the honeybee alarm pheromone, reversibly and competitively inhibited the purified Galleria AChE with a K_i value of 1.34×10⁻³ M. Furthermore, five monoterpenes associated with plant defence and representing a range of functional groups, also were reversible competitive inhibitors of the purified AChE from Galleria, which is consistent with previous data for electric eel AChE (Ryan, M.F., Byrne, O., 1988. Plant-insect coevolution and inhibition of acetylcholinesterase. Journal of Chemical Ecology 14, 1965-1975).     

Serum bactericidal activities of moxifloxacin and levofloxacin against aerobic and anaerobic intra-abdominal pathogens

We studied the serum bactericidal activity (SBA) of moxifloxacin and levofloxacin against common pathogens associated with complicated intra-abdominal infections. Ten healthy volunteers received a single dose of moxifloxacin (400 mg) and levofloxacin (750 mg) and serum samples were collected at 2, 4, 8, 12, and 24h after the dose of each drug. Bactericidal titers in serum over time were determined for aerobic gram-negative bacilli (Escherichia coli, Klebseilla pneumoniae, and Enterobacter cloacae) and anaerobic bacteria (Bacteroides fragilis, Bacteroides thetaiotaomicron, Prevotella bivia, and Finegoldia magna). Both fluoroquinolones provided rapid (2h) attainment and prolonged (24h) SBA (titers > or = 1:8) against each of the aerobic bacilli studied. SBA was observed for at least 12h against B. fragilis strains with MICs < or = 2 microg/ml to moxifloxacin and < or = 4 microg/ml to levofloxacin. Prolonged (12h) SBA (titers > or = 1:2) was also observed against isolates of B. thetaiotaomicron, P. bivia, and F. magna with moxifloxacin < or = MICs 2 microg/ml.     

Coencapsulation of irinotecan and floxuridine into low cholesterol-containing liposomes that coordinate drug release in vivo

A liposomal delivery system that coordinates the release of irinotecan and floxuridine in vivo has been developed. The encapsulation of floxuridine was achieved through passive entrapment while irinotecan was actively loaded using a novel copper gluconate/triethanolamine based procedure. Coordinating the release rates of both drugs was achieved by altering the cholesterol content of distearoylphosphatidylcholine (DSPC)/distearoylphosphatidylglycerol (DSPG) based formulations. The liposomal retention of floxuridine in plasma after intravenous injection was dramatically improved by decreasing the cholesterol content of the formulation below 20 mol%. In the case of irinotecan, the opposite trend was observed where increasing cholesterol content enhanced drug retention. Liposomes composed of DSPC/DSPG/Chol (7:2:1, mole ratio) containing co-encapsulated irinotecan and floxuridine at a 1:1 molar ratio exhibited matched leakage rates for the two agents so that the 1:1 ratio was maintained after intravenous administration to mice. The encapsulation of irinotecan was optimal when copper gluconate/triethanolamine (pH 7.4) was used as the intraliposomal buffer. The efficiency of irinotecan loading was approximately 80% with a starting drug to lipid molar ratio of 0.1/1. Leakage of floxuridine from the liposomes during irinotecan loading at 50 degrees C complicated the ability to readily achieve the target 1:1 irinotecan/floxuridine ratio inside the formulation. As a result, a procedure for the simultaneous encapsulation of irinotecan and floxuridine was developed. This co-encapsulation method has the advantage over sequential loading in that extrusion can be performed in the absence of chemotherapeutic agents and the drug/drug ratios in the final formulation can be more precisely controlled.     

Enzyme‐immunoassay for the measurement of luteinizing hormone in the serum of African elephants (Loxodonta africana)

Circulating patterns of progesterone and luteinizing hormone (LH) in the elephant have been well characterized, and routine monitoring of these hormones is now viewed as a valuable tool for making informed decisions about the reproductive management of elephants in captivity. Currently, LH monitoring in elephants is done with radio‐immunoassays (RIAs); unfortunately, the use of radioactive materials in RIAs limits their application to institutions with laboratory facilities equipped for the storage and disposal of radioactive waste. Enzyme‐immunoassays (EIAs) offer an inexpensive and more zoo‐friendly alternative to RIA. This work reports on an EIA capable of quantifying circulating LH in African elephants. The EIA employs a biotin label and microtiter plates coated with goat anti‐mouse gamma globulin. LH surges in African elephants (n=3) increased fivefold over baseline concentrations (1.00±0.1 ng/ml vs. 0.2±0.1 ng/ml) and occurred 19.3±0.2 days apart. Ovulatory LH surges were associated with an increase in serum progestogens from 4.8±0.4 ng/ml to 11.7±0.4 ng/ml. The ability to quantify reproductive hormones in elephants via EIA is an important step in the process of making endocrine monitoring more accessible to zoos housing these species. Zoo Biol 21:403–408, 2002. © 2002 Wiley‐Liss, Inc.