A procedure for axenic isolation of the marine microalga Isochrysis galbana from heavily contaminated mass cultures

Isochrysis galbana, one of the most widely usedmarine microalgae in the rearing of finfish and shellfish larvae, is masscultured frequently in outdoor tanks. Under prolonged and repeated culture,severe contamination occurs. Axenic isolation of I.galbanafrom such cultures was best achieved by using a ternary procedure involvingpercoll-gradient centrifugation, treatment with antibiotics, and growth on agarmedium. Protozoa and other algae were removed most effectively by isolation ofI. galbana at the 30–40% density layer on apercoll-gradient. Removal of bacteria was accomplished using a mixture of 5antibiotics (250 g mL^–1 ampicillin, 50g mL^–1 gentamycin, 100 gmL^–1 kanamycin, 500 gmL^–1 neomycin, 50 gmL^–1 streptomycin). Axenic colonies were isolated fromasolid medium prepared from 1% purified agar. The ternary procedure isconsideredapplicable to the isolation of other axenic single-celled microalgae fromheavily contaminated cultures.     

Antibiotic and biocide resistance in bacteria: introduction

Drug resistance in bacteria is increasing and the pace at which new antibiotics are being produced is slowing. It is now almost commonplace to hear about methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant enterococci (VRE), multi-drug resistance in Mycobacterium tuberculosis (MDRTB) strains and multi-drug-resistant (MDR) gram-negative bacteria. So-called new and emerging pathogens add to the gravity of the situation. Reduced susceptibility to biocides is also apparently increasing, but is more likely to be low level in nature and to concentrations well below those used in hospital, domestic an industrial practice. A particular problem, however, is found with bacteria and other micro-organisms present in biofilms, where a variety of factors can contribute to greater insusceptibility compared with cells in planktonic culture. Also of potential concern is the possibility that widespread usage of biocides is responsible for the selection and maintenance of antibiotic-resistant bacteria. The basic mechanisms of action of, and bacterial resistance to, antibiotics are generally well documented, although data continue to accumulate about the nature and importance of efflux systems. In contrast, the modes of action of most biocides are poorly understood and consequently, detailed evaluation of bacterial resistance mechanisms is often disappointing. During this Symposium, the mechanisms of bacterial resistance to antibiotics and biocides are discussed at length. It is hoped that this knowledge will be used to develop newer, more effective drugs and biocides that can be better and perhaps, on occasion, more logically used to combat the increasing problem of bacterial resistance.     

Beverage specific alcohol intake in a population-based study: Evidence for a positive association between pulmonary function and wine intake

Background  

Lung function is a strong predictor of cardiovascular and all-cause mortality. Previous studies suggest that alcohol exposure may be linked to impaired pulmonary function through oxidant-antioxidant mechanisms. Alcohol may be an important source of oxidants; however, wine contains several antioxidants. In this study we analyzed the relation of beverage specific alcohol intake with forced expiratory volume in one second (FEV₁) and forced vital capacity (FVC) in a random sample of 1555 residents of Western New York, USA.     

The complex nature of protein phosphatases

Protein phosphatases are integrally associated with the regulation of cellular signaling. The mechanisms underlying the specific regulatory roles are likely to be unique to each cell system. Nevertheless, analysis of phosphatase regulation in a number of systems has identified phosphatase targeting through association with a wide range of binding partners to be a fundamental mechanism of regulation. Using protein phosphatase 2A (PP2A) as an example, this snapshot summarizes these fundamental mechanisms of protein phosphatase regulation.     

Characterization of a novel parainfluenza virus,caviid parainfluenza virus 3, from laboratory guinea pigs (Cavia porcellus)

A novel Respirovirus was isolated from nasopharyngeal swab specimens from clinically normal laboratory guinea pigs, and was characterized and named caviid parainfluenza virus 3 (CavPIV-3). The CavPIV-3 is enveloped, is 100 to 300 nm in diameter, and has a characteristic 15-nm-diameter chevron-shaped virus ribonucleocapsid protein. Sequence analysis of the fusion glycoprotein of CavPIV-3 revealed it to be 94% identical to human and guinea pig parainfluenza 3 (PIV-3) viruses and 80% identical to bovine PIV-3. To determine whether CavPIV-3 causes clinical disease in laboratory guinea pigs and to compare the serologic response of guinea pigs to CavPIV-3 and to other paramyxoviruses, an infection study was performed, in which groups of guinea pigs were inoculated with CavPIV-3, Sendai virus, simian virus 5 (SV-5), murine pneumonia virus (PVM), or bovine PIV-3 virus. During the course of the study, guinea pigs were maintained in an infectious disease suite, housed in Micro-Isolator cages, and were only manipulated under a laminar flow hood. Clinical signs of disease were not observed in any of the paramyxovirus-inoculated guinea pigs during the eight-week course of the study, and histologic signs of disease were not evident at necropsy eight weeks after inoculation. Guinea pigs inoculated with CavPIV-3, Sendai virus, PVM, and bovine PIV-3 developed robust homologous or heterologous serologic responses. In contrast, guinea pigs inoculated with SV-5 developed modest or equivocal serologic responses, as assessed by use of an enzyme-linked immunosorbent assay. Further, use of the SV-5 enzyme-linked immunosorbent assay resulted in the highest degree of non-specific reactivity among all of the paramyxovirus assays. In summary, CavPIV-3 is a novel guinea pig Respirovirus that subclinically infects laboratory guinea pigs, resulting in a robust serologic response, but no observed clinical or histologic disease. The CavPIV-3 fusion glycoprotein gene sequence is available from GenBank as accession No. AF394241, and the CavPIV-3 virus is available from the American Type Culture Collection as accession No. DR-1547.     

Peak capacity of ion mobility mass spectrometry: separation of peptides in helium buffer gas

Advances in the field of proteomics depend upon the development of high-throughput separation methods. Ion mobility-mass spectrometry is a fast separation method (separations on the millisecond time-scale), which has potential for peptide complex mixture analysis. Possible disadvantages of this technique center around the lack of orthogonality between separation based on ion mobility and separation based on mass. In order to examine the utility of ion mobility-mass spectrometry, the peak capacity (phi) of the technique was estimated by subjecting a large dataset of peptides to linear regression analysis to determine an average trend for tryptic peptides. This trend-line, along with the deviation from a linear relationship observed for this dataset, was used to define the separation space for ion mobility-mass spectrometry. Using the maximum deviation found in the dataset (+/-11%) the peak capacity of ion mobility-mass spectrometry is approximately 2600 peptides. These results are discussed in light of other factors that may increase the peak capacity of ion mobility-mass spectrometry (i.e. multiple trends in the data resulting from multiple classes of compounds present in a sample) and current liquid chromatography approaches to complex peptide mixture analysis.     

Targeted transgene insertion into human chromosomes by adeno-associated virus vectors

Efficient methods are needed for the precise genetic manipulation of diploid human cells, in which cellular senescence and low conventional gene targeting rates limit experimental and therapeutic options. We have shown previously that linear, single-stranded DNA vectors based on adeno-associated virus (AAV) could accurately introduce small (<20 bp) genetic modifications into homologous human chromosomal sequences. Here we have used AAV vectors to introduce large (>1 kb) functional transgene cassettes into the hypoxanthine phosphoribosyl transferase (HPRT) and Type I collagen (COL1A1) loci in normal human fibroblasts. The transgene cassettes are inserted at high frequencies (1% of the total cell population under optimal conditions) and without secondary mutations. Selection for the inserted transgene cassette can be used to enrich for targeting events, such that >70% of surviving cells have undergone gene targeting with an appropriately designed vector. This approach should prove useful both for functional genomic analysis in diploid human cells and for therapeutic gene targeting.