Characterization of palindromic loop mismatch repair tracts in mammalian cells

Single- and multi-base (loop) mismatches can arise in DNA by replication errors, during recombination, and by chemical modification of DNA. Single-base and loop mismatches of several nucleotides are efficiently repaired in mammalian cells by a nick-directed, MSH2-dependent mechanism. Larger loop mismatches (> or =12 bases) are repaired by an MSH2-independent mechanism. Prior studies have shown that 12- and 14-base palindromic loops are repaired with bias toward loop retention, and that repair bias is eliminated when five single-base mismatches flank the loop mismatch. Here we show that one single-base mismatch near a 12-base palindromic loop is sufficient to eliminate loop repair bias in wild-type, but not MSH2-defective mammalian cells. We also show that palindromic loop and single-base mismatches separated by 12 bases are repaired independently at least 10% of the time in wild-type cells, and at least 30% of the time in MSH2-defective cells. Palindromic loop and single-base mismatches separated by two bases were never repaired independently. These and other data indicate that loop repair tracts are variable in length. All tracts extend at least 2 bases, some extend <12 bases, and others >12 bases, on one side of the loop. These properties distinguish palindromic loop mismatch repair from the three known excision repair pathways: base excision repair which has one to six base tracts, nucleotide excision repair which has approximately 30 base tracts, and MSH2-dependent mismatch repair, which has tracts that extend for several hundred bases.     

Effects of early rearing experience on adult behavior and nesting in captive Hawaiian crows (Corvus hawaiiensis)

Behavioral data were collected during the breeding season on eight pairs of Hawaiian crows (Corvus hawaiiensis) housed at two facilities on different islands. All data were collected from videocamera time‐lapse recordings of the nesting platforms. Behaviors included frequency of nest cup manipulation, percent of time spent on nest, allopreening, play, and stereotypy. The number of breeding pairs increased from four in 1996 to six in 1997, to seven in 1998, and to eight in 1999. Five of the older birds (three males and two females) were solitary‐reared for most if not all of their first year, while the remaining 12 birds were all socially reared. Significant differences were found between isolate‐ and socially‐reared birds, with isolate‐reared birds having higher rates of solo play (P = 0.0041) and stereotypies (P = 0.0090). Pairs that were comprised of at least one isolate‐reared bird engaged in significantly less allopreening (P = <0.0001) than pairs in which both birds were socially reared. From 1996 to 1999, 87 eggs were laid, with a mean of 1.88 ± 0.24 SEM eggs per clutch. Only three females produced clutches every year, and they were responsible for 85.1% of the eggs laid. Although not significant, the mean number of clutches produced per pair decreased from 2.50 ± 0.65 in 1996 to 0.87 ± 0.99 in 1999. Age of females does not appear to be a critical factor in the decrease in clutch production. New pairing combinations are under way in an effort to improve propagation in this highly endangered species. Zoo Biol 21:59–75, 2002. © 2002 Wiley‐Liss, Inc.     

Effect of salinity on the distribution, growth, and toxicity of Karenia spp.

The first recorded bloom of Karenia spp., resulting in brevetoxin in oysters, in the low salinity waters of the Northern Gulf of Mexico (NGOMEX) occurred in November 1996. It raised questions about the salinity tolerance of Karenia spp., previously considered unlikely to occur at salinities <24 psu, and the likelihood that the bloom would reoccur in the NGOMEX. Salinity was investigated as a factor controlling Karenia spp. abundance in the field, using data from the NGOMEX 1996 bloom and Florida coastal waters from 1954 to 2004, and growth and toxin production in cultures of Karenia brevis (Davis) G. Hansen and Moestrup. During the NGOMEX bloom, Karenia spp. occurred much more frequently at low salinities than in Florida coastal waters over the last 50 years. The data suggest that the NGOMEX bloom started on the NW Florida Shelf, an area with a higher frequency of Karenia spp. at low salinities than the rest of Florida, and was transported by an unusual westward surface current caused by Tropical Storm Josephine. The minimum salinity at which growth occurred in culture ranged between 17.5 and 20 psu, but the optimal salinity ranged between low values of 20 or 25 and high values of 37.5–45 psu, depending on the clone. The effect of salinity on toxin production in one clone of K. brevis was complex, but at all salinities brevetoxin levels were highest during the stationary growth phase, suggesting that aging, high density blooms may pose the greatest public health threat. The results demonstrate that Karenia spp. can be a public health threat in low salinity areas, but the risk in the NGOMEX is relatively low. No bloom has occurred since the 1996 event, which was probably associated with a special set of conditions: a bloom along the Florida Panhandle and a tropical storm with a track that set up a westward current.     

Conditional osmotic stress in yeast: a system to study transport through aquaglyceroporins and osmostress signaling

The accumulation and transport of solutes are hallmarks of osmoadaptation. In this study we have employed the inability of the Saccharomyces cerevisiae gpd1Delta gpd2Delta mutant both to produce glycerol and to adapt to high osmolarity to study solute transport through aquaglyceroporins and the control of osmostress-induced signaling. High levels of different polyols, including glycerol, inhibited growth of the gpd1Delta gpd2Delta mutant. This growth inhibition was suppressed by expression of the hyperactive allele Fps1-Delta1 of the osmogated yeast aquaglyceroporin, Fps1. The degree of suppression correlated with the relative rate of transport of the different polyols tested. Transport studies in secretory vesicles confirmed that Fps1-Delta1 transports polyols at increased rates compared with wild type Fps1. Importantly, wild type Fps1 and Fps1-Delta1 showed similarly low permeability for water. The growth defect on polyols in the gpd1Delta gpd2Delta mutant was also suppressed by expression of a heterologous aquaglyceroporin, rat AQP9. We surmised that this suppression was due to polyol influx, causing the cells to passively adapt to the stress. Indeed, when aquaglyceroporin-expressing gpd1Delta gpd2Delta mutants were treated with glycerol, xylitol, or sorbitol, the osmosensing HOG pathway was activated, and the period of activation correlated with the apparent rate of polyol uptake. This observation supports the notion that deactivation of the HOG pathway is closely coupled to osmotic adaptation. Taken together, our "conditional" osmotic stress system facilitates studies on aquaglyceroporin function and reveals features of the osmosensing and signaling system.     

Secretion of a functional single-chain Fv protein in transgenic tobacco plants and cell suspension cultures

A synthetic gene encoding an anti-phytochrome single-chain Fv (scFv) antibody bearing an N-terminal signal peptide has been used to transform tobacco plants. Immunoblot analysis showed that transformed plants accumulate high levels of scFv protein, accounting for up to 0.5% of the total soluble protein fraction, which could be extracted by simple infiltration and centrifugation of leaf tissue. A substantial proportion of the scFv protein extracted in this way was found to possess antigen-binding activity. Callus cell suspension cultures derived from transformed plants secrete functional scFv protein into the surrounding medium. Compared with the levels of scFv protein observed in plants expressing the native scFv gene, the incorporation of an N-terminal signal peptide, to target the scFv to the apoplast, results in elevated accumulation of the protein.     

A scaling approach for quantifying the net CO2 flux of the Kuparuk River Basin,Alaska

Net CO₂ flux measurements conducted during the summer and winter of 1994–96 were scaled in space and time to provide estimates of net CO₂ exchange during the 1995–96 (9 May 1995–8 May 1996) annual cycle for the Kuparuk River Basin, a 9200 km² watershed located in NE Alaska. Net CO₂ flux was measured using dynamic chambers and eddy covariance in moist‐acidic, nonacidic, wet‐sedge, and shrub tundra, which comprise 95% of the terrestrial landscape of the Kuparuk Basin. CO₂ flux data were used as input to multivariate models that calculated instantaneous and daily rates of gross primary production (GPP) and whole‐ecosystem respiration (R) as a function of meteorology and ecosystem development. Net CO₂ flux was scaled up to the Kuparuk Basin using a geographical information system (GIS) consisting of a vegetation map, digital terrain map, dynamic temperature and radiation fields, and the models of GPP and R. Basin‐wide estimates of net CO₂ exchange for the summer growing season (9 May?5 September 1995) indicate that nonacidic tundra was a net sink of ?31.7 ± 21.3 GgC (1 Gg = 10⁹ g), while shrub tundra lost 32.5 ± 6.3 GgC to the atmosphere (negative values denote net ecosystem CO₂ uptake). Acidic and wet sedge tundra were in balance, and when integrated for the entire Kuparuk River Basin (including aquatic surfaces), whole basin summer net CO₂ exchange was estimated to be in balance (?0.9 ± 50.3 GgC). Autumn to winter (6 September 1995–8 May 1996) estimates of net CO₂ flux indicate that acidic, nonacidic, and shrub tundra landforms were all large sources of CO₂ to the atmosphere (75.5 ± 8.3, 96.4 ± 11.4, and 43.3 ± 4.7 GgC for acidic, nonacidic, and shrub tundra, respectively). CO₂ loss from wet sedge surfaces was not substantially different from zero, but the large losses from the other terrestrial landforms resulted in a whole basin net CO₂ loss of 217.2 ± 24.1 GgC during the 1995–96 cold season. When integrated for the 1995–96 annual cycle, acidic (66.4 + 25.25 GgC), nonacidic (64.7 ± 29.2 GgC), and shrub tundra (75.8 ± 8.4 GgC) were substantial net sources of CO₂ to the atmosphere, while wet sedge tundra was in balance (0.4 + 0.8 GgC). The Kuparuk River Basin as a whole was estimated to be a net CO₂ source of 218.1 ± 60.6 GgC over the 1995–96 annual cycle. Compared to direct measurements of regional net CO₂ flux obtained from aircraft‐based eddy covariance, the scaling procedure provided realistic estimates of CO₂ exchange during the summer growing season. Although winter estimates could not be assessed directly using aircraft measurements of net CO₂ exchange, the estimates reported here are comparable to measured values reported in the literature. Thus, we have high confidence in the summer estimates of net CO₂ exchange and reasonable confidence in the winter net CO₂ flux estimates for terrestrial landforms of the Kuparuk river basin. Although there is larger uncertainty in the aquatic estimates, the small surface area of aquatic surfaces in the Kuparuk river basin (≈ 5%) presumably reduces the potential for this uncertainty to result in large errors in basin‐wide CO₂ flux estimates.     

Characterization and implications of host-cell protein aggregates in biopharmaceutical processing

In the production of biopharmaceuticals such as monoclonal antibodies (mAbs) and vaccines, the residual amounts of host-cell proteins (HCPs) are among the critical quality attributes. In addition to overall HCP levels, individual HCPs may elude purification, potentially causing issues in product stability or patient safety. Such HCP persistence has been attributed mainly to biophysical interactions between individual HCPs and the product, resin media, or residual chromatin particles. Based on measurements on process streams from seven mAb processes, we have found that HCPs in aggregates, not necessarily chromatin-derived, may play a significant role in the persistence of many HCPs. Such aggregates may also hinder accurate detection of HCPs using existing proteomics methods. The findings also highlight that certain HCPs may be difficult to remove because of their functional complementarity to the product; specifically, chaperones and other proteins involved in the unfolded protein response (UPR) are disproportionately present in the aggregates. The methods and findings described here expand our understanding of the origins and potential behavior of HCPs in cell-based biopharmaceutical processes and may be instrumental in improving existing techniques for HCP detection and clearance.     

The Proapoptotic F-box Protein Fbxl7 Regulates Mitochondrial Function by Mediating the Ubiquitylation and Proteasomal Degradation of Survivin

Fbxl7, a component of the Skp1·Cul1·F-box protein type ubiquitin E3 ligase, regulates mitotic cell cycle progression. Here we demonstrate that overexpression of Fbxl7 in lung epithelia decreases the protein abundance of survivin, a member of the inhibitor of apoptosis family. Fbxl7 mediates polyubiquitylation and proteasomal degradation of survivin by interacting with Glu-126 within its carboxyl-terminal α helix. Furthermore, both Lys-90 and Lys-91 within survivin serve as ubiquitin acceptor sites. Ectopically expressed Fbxl7 impairs mitochondrial function, whereas depletion of Fbxl7 protects mitochondria from actions of carbonyl cyanide m-chlorophenylhydrazone, an inhibitor of oxidative phosphorylation. Compared with wild-type survivin, cellular expression of a survivin mutant protein deficient in its ability to interact with Fbxl7 (E126A) and a ubiquitylation-resistant double point mutant (KK90RR/KK91RR) rescued mitochondria to a larger extent from damage induced by overexpression of Fbxl7. Therefore, these data suggest that the Skp1·Cul1·F-box protein complex subunit Fbxl7 modulates mitochondrial function by controlling the cellular abundance of survivin. The results raise opportunities for F-box protein targeting to preserve mitochondrial function.