Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation.

DNA-calcium phosphate co-precipitates arise spontaneously in supersaturated solutions. Highly effective precipitates for transfection purposes, however, can be generated only in a very narrow range of physico-chemical conditions that control the initiation and growth of precipitate complexes. The concentrations of calcium and phosphate are the main factors influencing characteristics of the precipitate complex, but other parameters, such as temperature, DNA concentration and reaction time are important as well. An example for this is the finding that almost all of the soluble DNA in the reaction mix can be bound into an insoluble complex with calcium phosphate in <1 min. Extending the reaction time to 20 min results in aggregation and/or growth of particles and reduces the level of expression. With improved protocols we gained better reproducibility and higher efficiencies both for transient and for stable transfections. Up to 60% of cells stained positive for beta-gal and transient production of secreted proteins was improved 5- to 10-fold over results seen with transfections using standard procedures. Similar improvements in efficiency (number of recombinant cell colonies) were observed with stable transfections, using co-transfected marker plasmids for selection. Transient expression levels 2 days after DNA transfer and titers obtained from stable cell lines, emerging weeks later, showed strong correlation.     

Radiological evaluation of bone status in the jaw and in the vertebral column in a group of women

Vertebral bone mineral content was determined in a group of 56 women, ages 30–62. These measurements were compared with the status of supporting bone in the jaws (alveolar, molar and bicuspid) and with gingival health. There was a significant decline in vertebral bone mineral content from the pre- to post-menopausal group. Molar and bicuspid measurements were highly correlated. There was some association between lumbar bone mineral content and molar bone status for postmenopausal women. For postmenopausal women, the cases of greatest percent bone loss in alveolar crest were associated with lower lumbar bone mineral content. Gingival health did not confound the bone status measurements. The 56 subjects did not exhibit the degree of reduction in bone density that is observed in the general population. Further investigation using these radiographic techniques may reveal a link between substantial bone loss in the jaw and moderate to severe bone loss in the lumbar vertebrae.     

Comparison of the cellular internalization of antibodies used either as immunotoxins or in ADEPT

The internalization into tumor cells of two antibodies (C242 and 454A12), which make potent immunotoxins when linked to ricin A-chain, and an antibody (A5B7), which does not make a potent immunotoxin but has proven useful in ADEPT, was evaluated. The 454A12 antibody was rapidly taken into the cells, 50% of the antibody being internalized after 2 h. The C242 antibody was internalized more slowly, approx 50% being taken up by the cells in 24 h. With A5B7, less than 10% of the antibody was internalized after 24 h. Internalization of the C242 antibody was accompanied by the appearance of antibody degradation products in the cell medium after 2 h, and this degradation could be inhibited by addition of a metabolic inhibitor that prevented cell internalization. In contrast, minimal degradation of the A5B7 antibody could be detected up to 24 h after binding to the cells. In conclusion, both 454A12 and C242 antibodies, which make potent immunotoxins, were internalized into tumor cels. The A5B7 antibody, which does not make a potent immunotoxin, was not internalized, and this property may be one reason why A5B7 has proved useful for delivery of enzymes in ADEPT.     

ThechlL (frxC) gene: Phylogenetic distribution in vascular plants and DNA sequence fromPolystichum acrostichoides (Pteridophyta) andSynechococcus sp. 7002 (Cyanobacteria)

We examinedchlL (frxC) gene evolution using several approaches. Sequences from the chloroplast genome of the fernPolystichum acrostichoides and from the cyanobacteriumSynechococcus sp. 7002 were determined and found to be highly conserved. A complete physical map of the fern chloroplast genome and partial maps of other vascular plant taxa show thatchlL is located primarily in the small single copy region as inMarchantia polymorpha. A survey of a wide variety of non-angiospermous vascular plant DNAs shows thatchlL is widely distributed but has been lost in the pteridophytePsilotum and (presumably independently) within the Gnetalean gymnosperms.The namefrxC was originally used to denote a gene encoding a product with probable Fe : S cluster binding activity. This activity was postulated due to the amino acid sequence similarity between this product and the Fe : S-binding nitrogenase iron proteinnifH. Fe : S-binding is a property shared by ferredoxins, which are denoted by the prefix frx. However, this gene does not encode a ferredoxin. It is much larger than any known ferredoxin, it binds its Fe : S cluster between two halves of a homodimer (Fujita & al. 1989,Burke & al. 1993 a, c) instead of within a single subunit, and it lacks the pattern of clustered cysteines present in all ferredoxins (Meyer 1988). Therefore, we use the namechlL to recognize the sequence and functional similarities to the bacterial PChlide reductase subunit,bchL. Similar usage has been adopted for this (Suzuki & Bauer 1992) and other (Choquet & al. 1992,Burke & al. 1993b) PChlide reductase subunits.     

Process intensification strategies toward cell culture-based high-yield production of a fusogenic oncolytic virus

We present a proof-of-concept study for production of a recombinant vesicular stomatitis virus (rVSV)-based fusogenic oncolytic virus (OV), rVSV-Newcastle disease virus (NDV), at high cell densities (HCD). Based on comprehensive experiments in 1 L stirred tank reactors (STRs) in batch mode, first optimization studies at HCD were carried out in semi-perfusion in small-scale cultivations using shake flasks. Further, a perfusion process was established using an acoustic settler for cell retention. Growth, production yields, and process-related impurities were evaluated for three candidate cell lines (AGE1.CR, BHK-21, HEK293SF)infected at densities ranging from 15 to 30 × 10⁶ cells/mL. The acoustic settler allowed continuous harvesting of rVSV-NDV with high cell retention efficiencies (above 97%) and infectious virus titers (up to 2.4 × 10⁹ TCID₅₀/mL), more than 4–100 times higher than for optimized batch processes. No decrease in cell-specific virus yield (CSVY) was observed at HCD, regardless of the cell substrate. Taking into account the accumulated number of virions both from the harvest and bioreactor, a 15–30 fold increased volumetric virus productivity for AGE1.CR and HEK293SF was obtained compared to batch processes performed at the same scale. In contrast to all previous findings, formation of syncytia was observed at HCD for the suspension cells BHK 21 and HEK293SF. Oncolytic potency was not affected compared to production in batch mode. Overall, our study describes promising options for the establishment of perfusion processes for efficient large-scale manufacturing of fusogenic rVSV-NDV at HCD for all three candidate cell lines.     

Cavity-nesting birds are limited by nesting habitat in Neotropical agricultural landscapes

Most studies comparing biodiversity between natural and human-modified landscapes focus on patterns in species occurrence or abundance, but do not consider how different habitat types meet species' breeding requirements. Organisms that use or nest in tree cavities may be especially threatened by habitat conversion due to the loss of their nesting sites. Although cavity-nesting bird diversity is highest in the tropics, little is known about how tropical birds use cavities, how agriculture affects their reproductive biology, and how effective nest boxes could be as a conservation strategy in tropical agriculture. Here, we explored how habitat conversion from tropical forests to pasture affects the abundance, nesting habitat availability, and nest success of cavity-nesting birds in Northwest Ecuador. We conducted bird surveys and measured natural cavity availability and use in forest and agriculture. We also added artificial nest boxes to forest and agriculture to see whether cavity limitation in agriculture would elicit higher use of artificial nest boxes. We found evidence of cavity limitation in agriculture—there were many more natural cavities in forest than in agriculture, as well as more avian use of nest boxes placed in agriculture as compared to forest. Our results suggest that it is important to retain remnant trees in tropical agriculture to provide critical nesting habitat for birds. In addition, adding nest boxes to tropical agricultural systems could be a good conservation strategy for certain species, including insectivores that could provide pest-control services to farmers. Abstract in Spanish is available with online material.     

MULTIVARIATE ANALYSIS OF SELECTION IN EXPERIMENTAL POPULATIONS DERIVED FROM HYBRIDIZATION OF TWO ECOTYPES OF THE ANNUAL PLANT DIODIA TERES W. (RUBIACEAE)

Morphologically variable F₂ genotypes derived from hybridization of coastal and inland ecotypes of the annual plant Diodia teres were used to identify selection on morphological traits in the natural habitat of each ecotype. These ecotypes occur in very different habitats, and have evolved pronounced morphological differentiation. Selection analysis can suggest whether present patterns of selection can explain morphological differences between ecotypes. F₂ genotypes were characterized morphologically, clonally replicated, and transplanted into the habitat of each ecotype. Selection was measured on six morphological traits. Directional and stabilizing selection occurred on many traits; direction and strength of selection varied sharply at different stages of growth, as revealed by a path-analysis approach that divided selection into a set of independent components. Directional selection favored traits of the native population at the coastal habitat, but less so at the inland habitat. Selection was of sufficient strength to create the observed morphological differences between ecotypes in 25–100 generations, given constant selection and sufficient genetic variation. In effects on fitness, most traits were neither independent nor consistently interactive with other traits. Rather, many traits entered into strong but evanescent interactions affecting particular components of fitness. Observed interactions did not support the hypothesis that the morphology of each ecotype was functionally integrated to a high degree.     

Mitotic HeLa cells contain a CENP-E-associated minus end-directed microtubule motor.

A minus end-directed microtubule motor activity from extracts of HeLa cells blocked at prometaphase/metaphase of mitosis with vinblastine has been partially purified and characterized. The motor activity was eliminated by immunodepletion of Centromere binding protein E (CENP-E). The CENP-E-associated motor activity, which was not detectable in interphase cells, moved microtubules at mean rates of 0.46 micron/s at 37 degrees C and 0.24 micron/s at 25 degrees C. The motor activity co-purified with CENP-E through several purification procedures. Motor activity was clearly not due to dynein or to kinesin. The microtubule gliding rates of the CENP-E-associated motor were different from those of dynein and kinesin. In addition, the pattern of nucleotide substrate utilization by the CENP-E-associated motor and the sensitivity to inhibitors were different from those of dynein and kinesin. The CENP-E-associated motor had an apparent native molecular weight of 874,000 Da and estimated dimensions of 2 nm x 80 nm. This is the first demonstration of motor activity associated with CENP-E, strongly supporting the hypothesis that CENP-E may act as a minus end-directed microtubule motor during mitosis.     

Peroxynitrite-Induced Cytotoxicity in PC12 Cells: Evidence for an Apoptotic Mechanism Differentially Modulated by Neurotrophic Factors

Abstract: Peroxynitrite is a powerful oxidant formed by the near-diffusion-limited reaction of nitric oxide with superoxide. Large doses of peroxynitrite (>2 m M ) resulted in rapid cell swelling and necrosis of undifferentiated PC12 cells. However, brief exposure to lower concentrations of peroxynitrite (EC₅₀ = 850 µ M ) initially (3–4 h) caused minimal damage to low-density cultures. By 8 h, cytoplasmic shrinkage with nuclear condensation and fragmentation became increasingly evident. After 24 h, 36% of peroxynitrite-treated cells demonstrated these features associated with apoptosis. In addition, 46% of peroxynitrite-treated cells demonstrated DNA fragmentation (by terminal-deoxynucleotide transferase-mediated dUTP-digoxigenin nick end-labeling) after 7 h, which was inhibited by posttreatment with the endonuclease inhibitor aurintricarboxylic acid. Serum starvation also resulted in apoptosis in control cells (23%), the percentage of which was not altered significantly by peroxynitrite treatment. Although peroxynitrite is known to be toxic to cells, the present study provides a first indication that peroxynitrite induces apoptosis. Furthermore, pretreatment of cells with nerve growth factor or insulin, but not epidermal growth factor, was protective against peroxynitrite-induced apoptosis. However, both acidic and basic fibroblast growth factors greatly increased peroxynitrite-initiated apoptosis, to 63 and 70%, respectively. Thus, specific trophic factors demonstrate differential regulation of peroxynitrite-induced apoptosis in vitro.