The distribution of repeating [Gal beta 1,4GlcNAc beta 1,3] sequences in asparagine-linked oligosaccharides of the mouse lymphoma cell lines BW5147 and PHAR 2.1

The occurrence and distribution of the repeating disaccharide [Gal beta 1,4GlcNAc beta 1,3] in the different types of Asn-linked oligosaccharides in mouse lymphoma BW5147 cells have been studied. Glycopeptides were prepared from cells grown in medium containing [6-3H]galactose, and the bi-, tri-, and tetraantennary Asn-linked oligosaccharides were fractionated by serial lectin affinity chromatography on concanavalin A-Sepharose, pea lectin -Sepharose, leukoagglutinating phytohemagglutinin-agarose, and Datura stramonium agglutinin-agarose. As described in this report, the latter lectin binds glycopeptides that contain either the repeating N-acetyllactosamine sequence or an outer mannose residue substituted at C-2 and C-6 by N-acetyllactosamine. The isolated glycopeptides were subjected to methylation analysis, specific exoglycosidase treatments, and digestion with Escherichia freundii endo-beta-galactosidase. Our data indicate that approximately two-thirds of the tetraantennary and one-half of the triantennary Asn-linked oligosaccharides contain repeating N-acetyllactosamine sequences in at least one branch. Many of the repeating sequences contain an additional galactose residue linked alpha 1,3 to a penultimate galactose residue. By contrast, less than 10% of the biantennary oligosaccharides contain the repeating disaccharide. The distribution of the repeating N-acetyllactosamine unit was also examined in a cell line ( PHAR 2.1) that is deficient in UDP-GlcNAc:alpha-mannoside beta 1,6-N-acetylglucosaminyltransferase. These cells are unable to synthesize tetraantennary and certain triantennary species and instead accumulate biantennary oligosaccharides. The total content of repeating N-acetyllactosamine units is greatly decreased in this line, and those that are present are found predominantly in triantennary Asn-linked oligosaccharides. These results demonstrate that the repeating N-acetyllactosamine sequence occurs commonly in complex-type Asn-linked oligosaccharides in BW5147 cells but is confined primarily to tri- and teraantennary species.     

THE EVOLUTION OF GENETIC CORRELATIONS: AN ANALYSIS OF PATTERNS

The genetic correlation is a central parameter of quantitative genetics, providing a measure of the rate at which traits respond to indirect selection (i.e., selection that does not act upon the traits under study, but some other trait with which they have genes in common). In this paper, I review the pattern of variation among four combinations of traits: life history × life history (L × L), morphological × morphological (M × M), life history × morphological (L × M), and behavioral × behavioral (B × B). A few other combinations were investigated, but insufficient data were obtained for separate analysis. A total of 1798 correlations, distributed over 51 different animal and plant species, were analyzed. The analysis was conducted at two levels: first by dividing the data set solely by trait combination, and second by blocking the data by trait combination and species. Because selection will tend to fix alleles that show positive correlations with fitness traits faster than those that are negative and because the latter are expected to arise more frequently by mutation, correlations between life-history traits are predicted to be more often negative than those between morphological traits. This prediction was supported, with the ranking in decreasing proportion of negative correlations being: L × L > L × M > B × B > M × M. The mean magnitude of the genetic correlation shows little variation among morphological and life-history combinations, and the distribution of values is remarkably flat. However, the estimated standard errors and the coefficient of variation (SE/r_G) are large, making it difficult to separate biological factors influencing the pattern of dispersion from experimental error. Analysis of the phenotypic and genetic correlations suggest that for the combinations M × M and L × M, but not L × L or B × B, the phenotypic correlation is an adequate estimate of the genetic correlation.     

Observing the Temperature Dependent Transition of the GP2 Peptide Using Terahertz Spectroscopy

The GP2 peptide is derived from the Human Epidermal growth factor Receptor 2 (HER2/nue), a marker protein for breast cancer present in saliva. In this paper we study the temperature dependent behavior of hydrated GP2 at terahertz frequencies and find that the peptide undergoes a dynamic transition between 200 and 220 K. By fitting suitable molecular models to the frequency response we determine the molecular processes involved above and below the transition temperature (T _D). In particular, we show that below T _D the dynamic transition is dominated by a simple harmonic vibration with a slow and temperature dependent relaxation time constant and that above T _D, the dynamic behavior is governed by two oscillators, one of which has a fast and temperature independent relaxation time constant and the other of which is a heavily damped oscillator with a slow and temperature dependent time constant. Furthermore a red shifting of the characteristic frequency of the damped oscillator was observed, confirming the presence of a non-harmonic vibration potential. Our measurements and modeling of GP2 highlight the unique capabilities of THz spectroscopy for protein characterization.     

Operation of a Benchtop Bioreactor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to carefully control temperature, pH, and dissolved oxygen concentrations in particular makes them essential to efficient large scale growth and expression of fermentation products. This video will briefly describe the advantages of the fermentor over the shake flask. It will also identify key components of a typical benchtop fermentation system and give basic instruction on setup of the vessel and calibration of its probes. The viewer will be familiarized with the sterilization process and shown how to inoculate the growth medium in the vessel with culture. Basic concepts of operation, sampling, and harvesting will also be demonstrated. Simple data analysis and system cleanup will also be discussed.