Improved fermentation processes for NS0 cell lines expressing human antibodies and glutamine synthetase

To meet the increasing requirement for therapeutic antibodies to conduct clinical trials, an enhanced culture medium and fed-batch process was developed for GS-NS0 cell lines. This process was shown to produce high concentrations of monoclonal antibodies for several cell lines expressing different antibodies. Cells were adapted to growth in a glutamine- and serum-free medium containing bovine serum albumin (BSA), cholesterol, and transferrin. A number of amino acids were found to be depleted during cell culture. The concentrations of these amino acids were increased, and further cell culture analyses were performed. This process of cell growth and analysis was repeated over multiple cycles until no depletion was detected. This resulted in an amino acid supplement that was shown to be generic and enhanced antibody productivity up to 5-fold for the three cell lines tested. Transferrin was replaced using tropolone, a lipophilic iron chelator and ferric ammonium citrate. Cell growth was equivalent to that in transferrin-containing medium over the wide ranges tested. A concentrated feed solution, based on the amino acid supplement and the components of the serum- and protein-free supplements, was formulated. Addition of this feed in response to metabolic requirements resulted in a harvest titer a further 2-fold higher than the enhanced culture medium. Harvest antibody titers of up to 600 mg/L were achieved for three cell lines expressing different antibodies, representing an increase of 10-fold over the starting concentrations.     

Electrophysiological and morphological characterization of rat embryonic motoneurons in a defined system

In an attempt to integrate biological components with silicon-based devices and systems, artificial silane surfaces have been successfully used to grow motoneurons in a defined environment. In this study we characterized the morphology and electrophysiology of purified rat embryonic (E14) motoneurons grown on a self-assembled monolayer (SAM) of N-1[3-(trimethoxysilyl)propyl]diethylenetriamine (DETA) versus that on ornithine/laminin surfaces in serum-free media. On DETA motoneurons were flat and grew more processes, whereas on ornithine/laminin they tended to aggregate. The membrane time constant, a characteristic associated with electrotonic compactness, was significantly longer for motoneurons grown on DETA. Other electrophysiological parameters were similar for the motoneurons on the different surfaces. This is the first study where purified ventral horn motoneurons were cultured in a completely defined (nonbiological surface, serum-free) environment.     

Applications of a bilateral denoising filter in biological electron microscopy

Due to the sensitivity of biological sample to the radiation damage, the low dose imaging conditions used for electron microscopy result in extremely noisy images. The processes of digitization, image alignment, and 3D reconstruction also introduce additional sources of noise in the final 3D structure. In this paper, we investigate the effectiveness of a bilateral denoising filter in various biological electron microscopy applications. In contrast to the conventional low pass filters, which inevitably smooth out both noise and structural features simultaneously, we found that bilateral filter holds a distinct advantage in being capable of effectively suppressing noise without blurring the high resolution details. In as much, we have applied this technique to individual micrographs, entire 3D reconstructions, segmented proteins, and tomographic reconstructions.     

Molecular phylogeny and phylogeography of free-living Bryozoa (Cupuladriidae) from both sides of the Isthmus of Panama

Genetic data were used to identify Recent species of free-living bryozoans (Cupuladriidae) from both sides of the Isthmus of Panama, and to examine their phylogenetic relationships, species richness, and population structures. An approximately 480bp fragment of the 16S mitochondrial rRNA gene was sequenced from 182 individuals from Panama, the Gulf of Mexico, and El Salvador. Ten haplotype groups (Cupuladria 4, 5, and 6; Discoporella 1, 2, 3A, 3B, 3C, 7, and 8) were identified. Genetic distances between haplotype groups (3.2-26.5%; K2P+Gamma) were 1-2 orders of magnitude greater than within groups (0.1-1.4%). Seven of the haplotype groups represent morphologically distinct species; Discoporellas 3A-C appear to be cryptic species. Phylogenetic analyses identified two pairs of transisthmian sister clades. An average divergence rate derived from other taxa suggests that Cupuladrias 4 and 5 diverged approximately 7Ma, a Discoporella 7 clade diverged from a 3A-C clade approximately 11Ma, and the 3A-C clade radiated approximately 6-4Ma; these events all predated final closure of the isthmus? 3Ma. The Caribbean side of the isthmus, with 5 species, is only marginally richer in cupuladriids than the Pacific side, with 4, but has greater phylogenetic depth. The Caribbean retains lineages stemming from a New World Miocene radiation that are not represented in the eastern Pacific; extant eastern Pacific cupuladriids share most recent common ancestry with only two of the Caribbean lineages. Species in the eastern Pacific tend to show shallow population structures, with high levels of gene flow between geographically separate populations, whereas Caribbean species tend to show deeper populations structures, with indications of restricted gene flow between Bocas del Toro/Gulf of Mosquitos and Costa Arriba/San Blas. The population structures derive from Pleistocene histories and may be of limited value in interpreting the macroevolutionary pattern, as our results provide no evidence of speciation on either side of the isthmus following closure in the late Pliocene.     

The effects of amino acid supplementation on muscular performance during resistance training overreaching

The purpose of this study was to examine the effects of amino acid supplementation on muscular strength, power, and high-intensity endurance during short-term resistance training overreaching. Seventeen resistance-trained men were randomly assigned to either an amino acid (AA) or placebo (P) group and underwent 4 weeks of total-body resistance training consisting of two 2-week phases of overreaching (phase 1: 3 x 8-12 repetitions maximum [RM], 8 exercises; phase 2: 5 x 3-5 RM, 5 exercises). Muscle strength, power, and high-intensity endurance were determined before (T1) and at the end of each training week (T2-T5). One repetition maximum squat and bench press decreased at T2 in P (5.2 and 3.4 kg, respectively) but not in AA, and significant increases in 1 RM squat and bench press were observed at T3-T5 in both groups. A decrease in the ballistic bench press peak power was observed at T3 in P but not AA. The fatigue index during the 20-repetition jump squat assessment did not change in the P group at T3 and T5 (fatigue index = 18.6 and 18.3%, respectively) whereas a trend for reduction was observed in the AA group (p = 0.06) at T3 (12.8%) but not T5 (15.2%; p = 0.12). These results indicate that the initial impact of high-volume resistance training overreaching reduces muscle strength and power, and it appears that these reductions are attenuated with amino acid supplementation. In addition, an initial high-volume, moderate-intensity phase of overreaching followed by a higher intensity, moderate-volume phase appears to be very effective for enhancing muscle strength in resistance-trained men.     

Structural basis of heroin and cocaine metabolism by a promiscuous human drug-processing enzyme

We present the first crystal structures of a human protein bound to analogs of cocaine and heroin. Human carboxylesterase 1 (hCE1) is a broad-spectrum bioscavenger that catalyzes the hydrolysis of heroin and cocaine, and the detoxification of organophosphate chemical weapons, such as sarin, soman and tabun. Crystal structures of the hCE1 glycoprotein in complex with the cocaine analog homatropine and the heroin analog naloxone provide explicit details about narcotic metabolism in humans. The hCE1 active site contains both specific and promiscuous compartments, which enable the enzyme to act on structurally distinct chemicals. A selective surface ligand-binding site regulates the trimer-hexamer equilibrium of hCE1 and allows each hCE1 monomer to bind two narcotic molecules simultaneously. The bioscavenger properties of hCE1 can likely be used to treat both narcotic overdose and chemical weapon exposure.     

Drugs for the control of parasitic diseases: current status and development in schistosomiasis

The morbidity caused by schistosomiasis has been controlled in China, Egypt and the Philippines mainly by the widespread use of the safe and efficacious drug praziquantel (PZQ), and by oxamniquin and PZQ in Brazil. To date, there is no evidence of development of clinically relevant resistance. Thanks to the commitment of national governments at the World Health Assembly in 2001, and the emergence of significant funding for control, it is predicted that there will soon be more widespread use of PZQ in sub-Saharan Africa, where morbidity due to schistosomiasis is most prevalent. There are currently no available alternative drugs to PZQ (with the possible exception of oxamniquin), although perhaps PZQ analogues could be developed. Artemether, used to control malaria, is effective against immature schistosomes, but is less effective against adult worms. The efficacy of myrrh, recently marketed as an antischistosomacide in Egypt, has not been independently confirmed.     

Photochemical transformations and bacterial utilization of high-molecular-weight dissolved organic carbon in a southern Louisiana tidal stream (Bayou Trepagnier)

UVirradiation of dissolved organic carbon (DOC) in the laboratory can producesmall, labile organic compounds utilizable by microbes, but few studies haveattempted to document this process in situ. ¹³Cnuclear magnetic resonance (NMR) was used to examine the bulk chemicalcomposition of natural and laboratory-irradiated high-molecular-weight DOC(HMW-DOC) from shaded (150 mol m^–2s^–1 average light in surface water) and open (1500mol m^–2 s^–1) field sitesoverone and a half years. ¹³C NMR revealed only small differences incarbon functional groups between laboratory irradiated and non-irradiatedHMW-DOC. However, bacterial protein productivity per cell (BPP) was enhanced innaturally irradiated samples of HMW-DOC in a field mesocosm experiment (p <0.05), suggesting that bacterial growth was enhanced by photochemicalproductionof labile DOC substrates. Absorbance characteristics such as spectral slope,absorbance at 350 nm, and the absorbance ratio 250nm/365 nm revealed that HMW-DOC was photoreactive,yetno differences in these values were found between samples irradiated with andwithout UV-B. In experiments conducted with simulated solar radiation in thelaboratory and with natural light in the field mesocosm experiment, UV-A(320–400 nm) and photosynthetically active radiation (PAR;400–700 nm) were more effective than UV-B (280–320nm) in HMW-DOC photolysis.     

Psychophysical study of image orientation perception

The experiment reported here investigates the perception of orientation of color photographic images. A collection of 1000 images (mix of professional photos and consumer snapshots) was used in this study. Each image was examined by at least five observers and shown at varying resolutions. At each resolution, observers were asked to indicate the image orientation, the level of confidence, and the cues they used to make the decision. The results show that for typical images, accuracy is close to 98% when using all available semantic cues from high-resolution images, and 84% when using only low-level vision features and coarse semantics from thumbnails. The accuracy by human observers suggests an upper bound for the performance of an automatic system. In addition, the use of a large, carefully chosen image set that spans the 'photo space' (in terms of occasions and subject matter) and extensive interaction with the human observers reveals cues used by humans at various image resolutions: sky and people are the most useful and reliable among a number of important semantic cues.