Effects of Fixation on Cell Volume of Marine Planktonic Protozoa

The effects of fixation on the cell volume of marine heterotrophic nanoflagellates and planktonic ciliates were investigated. Decreases in cell volume depended on the combination of the protozoan taxa and the particular fixative. For a particular fixative and protozoan species, degree of shrinkage was independent of physiological state. The volume of fixed cells was found to be approximately 20 to 55% lower than the cell volume of live organisms. For the heterotrophic microflagellates, the fixatives ranked, in order of decreasing effect on cell volume, as glutaraldehyde, formaldehyde, acid Lugol's solution, and modified van der Veer solution. With oligotrichous ciliates and a tintinnid ciliate, formaldehyde caused less shrinkage than glutaraldehyde or acid Lugol's solution. With the aldehyde fixatives, the microflagellates were found to shrink more than the ciliates. Differential effects of fixation on cell volumes may result in an underestimation of the biomass of certain protozoan taxa in natural samples.     

Purification and Characterization of a Central Cholinergic Enhancing Factor from Rat Brain: Its Identity as Phosphoethanolamine

A compound that can enhance the apparent synthesis of acetylcholine in cultured explants of the medial septal nucleus has been purified from rat brain and identified as phosphoethanolamine. Acetylcholine synthesis is stimulated two- to threefold in cultures grown for 5 days in the presence of phosphoethanolamine, ethanolamine, or cytidine 5'-diphosphoethanolamine at concentrations above 100 microM. This effect appears to result from an increase in the accumulation of choline via the high-affinity, sodium-dependent uptake mechanism. The development of choline acetyltransferase activity is not affected. Phosphoethanolamine and ethanolamine seem to enhance the ability of developing cholinergic neurons to utilize choline accumulated via the sodium-dependent high-affinity choline uptake mechanism for the preferential production of acetylcholine without increasing the general metabolism of the cultures. Choline itself and its related derivatives are not stimulatory for these effects.     

Ontogeny of glyoxysomes in maturing and germinated cotton seeds—a morphometric analysis

Morphometric procedures were used with light and electron microscopy to examine glyoxysome number, volume, shape and distribution as well as mesophyll cell volume, in cotyledons of mature (50 d postanthesis), imbibed (5h) and germinated (24 and 37 h) cotton (Gossypium hirsutum L.) seeds. Additionally, activities of five glyoxysomal marker enzymes in cotyledon extracts were assayed at each of the above ages. Cell volume was determined from photomicrographs of Epon-embedded sections by the point-counting procedure. Analysis of variance showed that cell volume was not different among the tissue segments studied. Glyoxysomes were cytochemically stained for catalase (EC 1.11.1.6) activity with the 3,3-diaminobenzidine-tetrahydrochloride procedure. Analyses involving both phase and electron microscopy, and two separate sterologic calculations for determining the number of glyoxysomes per cell, indicate that glyoxysomes are numerous in mature seeds, persist through desiccation and imbibition, then increase dramatically in volume (seven fold) but not number (a maximum of 1.5-fold), when enzyme activities increase two to six times (depending on the enzyme). During the entire period of increase in glyoxysomal enzyme activities, no ultrastructural evidence was found for glyoxysome formation or destruction. Our data, in contrast to some proposals in the literature, indicate that cottonseed glyoxysomes form during seed maturation, then develop following seed imbibition into pleomorphic organelles by posttranslational accumulation of proteins from the cytosol and transfer of membrane components probably from the endoplasmic reticulum.Abbreviations DAB 3,3-diaminobenzidine tetrahydrochloride - DPA days postanthesis - ER endoplasmic reticulum     

Dark Anaerobic Inactivation of Photosynthetic Oxygen Evolution by Chlamydomonas reinhardtii

When intact cells of Chlamydomonas reinhardtii were anaerobicallyincubated in the dark, rapid inactivation of oxygen evolutionwith benzoquinone as the Hill oxidant occurred. Measurementsof electron transport using thylakoids isolated after anaerobictreatment showed that the inactivation occurred at, or before,the secondary electron acceptor of PS II, whereas PS I activitywas largely unaffected. In addition, after anaerobic treatmentfluorescence transients measured with no addition or with dibromomethylisopropylbenzoquinonepresent were virtually the same as those obtained with DCMUpresent. When 10 mM NaHCO₃ was added to inactivated cells, partof the oxygen evolution capacity was restored rapidly. However,almost complete recovery (within 20 to 25 min) required theaddition of oxygen as well. This recovery was not light-dependentand was faster in the presence of 1 mM KCN. We suggest thatthe in activation of benzoquinone-dependent oxygen evolutionwas due to both bicarbonate depletion and reduction of the plastoquinonepool. ¹Present address: Institute of Molecular Biophysics, FloridaState University, Tallahassee, Florida 32306, U.S.A. (Received January 17, 1984; Accepted February 25, 1984)     

The generation of stable,high MAb expressing CHO cell lines based on the artificial chromosome expression (ACE) technology

The manufacture of recombinant proteins at industrially relevant levels requires technologies that can engineer stable, high expressing cell lines rapidly, reproducibly and with relative ease. Commonly used methods incorporate transfection of mammalian cell lines with plasmid DNA containing the gene of interest. Identifying stable high expressing transfectants is normally laborious and time consuming. To improve this process, the ACE System has been developed based on pre‐engineered artificial chromosomes with multiple recombination acceptor sites. This system allows for the targeted transfection of single or multiple genes and eliminates the need for random integration into native host chromosomes. To illustrate the utility of the ACE System in generating stable, high expressing cell lines, CHO based candidate cell lines were generated to express a human monoclonal IgG1 antibody. Candidate cell lines were generated in under 6 months and expressed over 1 g/L and with specific productivities of up to 45 pg/cell/day under non‐fed, non‐optimized shake flask conditions. These candidate cell lines were shown to have stable expression of the monoclonal antibody for up to 70 days of continuous culture. The results of this study demonstrate that clonal, stable monoclonal antibody expressing CHO based cell lines can be generated by the ACE System rapidly and perform competitively with those cell lines generated by existing technologies. The ACE System, therefore, provides an attractive and practical alternative to conventional methods of cell line generation. Biotechnol. Bioeng. 2009; 104: 540–553 © 2009 Wiley Periodicals, Inc.     

Optimization of 7-alkene-3-quinolinecarbonitriles as Src kinase inhibitors

The 7-alkene-3-quinolinecarbonitrile 20, a potent inhibitor of Src enzymatic and cellular activity with IC₅₀ values of 2.1 and 58 nM, respectively, had comparable efficacy to bosutinib in a colon tumor xenograft study.     

Auditioning of CHO host cell lines using the artificial chromosome expression (ACE) technology

In order to maximize recombinant protein expression in mammalian cells many factors need to be considered such as transfection method, vector construction, screening techniques and culture conditions. In addition, the host cell line can have a profound effect on the protein expression. However, auditioning or directly comparing host cell lines for optimal protein expression may be difficult since most transfection methods are based on random integration of the gene of interest into the host cell genome. Thus it is not possible to determine whether differences in expression between various host cell lines are due to the phenotype of the host cell itself or genetic factors such as gene copy number or gene location. To improve cell line generation, the ACE System was developed based on pre‐engineered artificial chromosomes with multiple recombination acceptor sites. This system allows for targeted transfection and has been effectively used to rapidly generate stable CHO cell lines expressing high levels of monoclonal antibody. A key feature of the ACE System is the ability to isolate and purify ACEs containing the gene(s) of interest and transfect the same ACEs into different host cell lines. This feature allows the direct auditioning of host cells since the host cells have been transfected with ACEs that contain the same number of gene copies in the same genetic environment. To investigate this audition feature, three CHO host cell lines (CHOK1SV, CHO‐S and DG44) were transfected with the same ACE containing gene copies of a human monoclonal IgG1 antibody. Clonal cell lines were generated allowing a direct comparison of antibody expression and stability between the CHO host cells. Results showed that the CHOK1SV host cell line expressed antibody at levels of more than two to five times that for DG44 and CHO‐S host cell lines, respectively. To confirm that the ACE itself was not responsible for the low antibody expression seen in the CHO‐S based clones, the ACE was isolated and purified from these cells and transfected back into fresh CHOK1SV cells. The resulting expression of the antibody from the ACE newly transfected into CHOK1SV increased fivefold compared to its expression in CHO‐S and confirmed that the differences in expression between the different CHO host cells was due to the cell phenotype rather than differences in gene copy number and/or location. These results demonstrate the utility of the ACE System in providing a rapid and direct technique for auditioning host cell lines for optimal recombinant protein expression. Biotechnol. Bioeng. 2009; 104: 526–539 © 2009 Wiley Periodicals, Inc.     

A selective medium for Fusobacterium spp.

J.S. BRAZIER, D.M. CITRON AND E.J.C. GOLDSTEIN, 1991. A new selective medium (JVN) for the isolation of Fusobacterium spp. from clinical material is described. The medium incorporates josamycin, vancomycin and norfloxacin (at 3, 4 and 1 μg/ml, respectively) as the selective agents, plus 5% defibrinated horse blood in Fastidious Anaerobe Agar Base (Lab M). This formula allowed luxuriant growth of all 82 strains (eight recognized species) of fusobacteria tested, while significantly inhibiting 51/51 (100%) strains of facultative anaerobes and 45/51 (88%) strains of other obligate anaerobes. JVN medium allowed the successful isolation of strains of Fusobacterium naviforme, F. nucleatum and F. necrophorum from the gingivae of 9/16 healthy volunteers, and strains of F. varium and F. mortiferum from faecal suspensions seeded with these organisms.