A method for the facile solid-phase synthesis of gramicidin S and its analogs

Summary A simple method is described for the facile synthesis of gramicidin S and six other analogs, using standard solidphase synthetic technology and a single solution-phase cyclization step. The peptides were purified to homogeneity and characterized by plasma desorption time-of-flight mass spectrometry and NMR spectroscopy. Complete ¹H NMR assignments for all seven peptides (in aqueous solution) are presented. Unlike previous approaches, the presented method is simple, automatable, rapid (less than three days), high-yielding, requires no side-chain protection during cyclization, and appears to be generally applicable to the preparation of a variety of related head-to-tail cyclic peptides.Abbreviations Boc t-butyloxycarbonyl - BOP benzotriazoyl N-oxytris(dimethylamino)phosphonium hexafluorophosphate - Bzl benzyl - DCC N,N-dicyclohexylcarbodiimide - DCM dichloromethane - DIEA N,N-diisopropylethylamine - DMF N,N-dimethylformamide - DQF-COSY double-quantum-filtered correlation spectroscopy - DSS 2,2-dimethyl-2-silapentane-5-sulfonate, sodium salt - EDAC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide - HBTU 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate - HOBt 1-hydroxybenzotriazole - 4-MeBzl 4-methylbenzyl - NHS N-hydroxysuccinimide - NOESY nuclear Overhauser effect spectroscopy - PAM phenylacetamidomethyl (resin) - RP-HPLC reversed-phase high-performance liquid chromatography - TFA trifluoroacetic acid - TOCSY total correlation spectroscopy - Tos tosyl - Troc 2,2,2-trichloroethylcarbamate.     

Provisional mapping of the gene for a cell surface marker, GA-1, in the red-necked wallaby Macropus rufogriseus

A series of M. rufogriseus-mouse somatic cell hybrids was constructed and analysed cytologically, enzymatically and immunologically. A monoclonal antibody, GA-1, was prepared against an M. rufogriseus cell surface antigen on an M. rufogriseus-mouse somatic cell hybrid. A gene determining the expression of this antigen was provisionally assigned to the long arm of the M. rufogriseus chromosome 3. The monoclonal antibody also reacted with an M. rufus (red kangaroo)-mouse somatic cell hybrid containing only the M. rufus chromosome 5, the G-banded chromosome identical to M. rufogriseus 3q. The results also suggest synteny of the genes for the marsupial enzymes hypoxanthine phosphoribosyltransferase and phosphoglycerate kinase-A.     

Estimation of pristane levels in murine ascites fluid and in purified monoclonal antibody preparations

A simple method for the rapid analysis of pristane has been designed using a heptane extract of the aqueous sample followed by separation and detection by gas chromatography. Ascites fluid lots from three monoclonal antibodies (MAbs) and their purified preparations were analysed using the procedure. The highest detected level of pristane was 600 μg/mL in a single lot of ascites fluid with other values less than 140 μg/ml. For the purified samples, no pristane was detected down to the demonstrated limit of 4 μg/mg of MAb.     

Structure and dynamics of detergent-solubilized M13 coat protein (an integral membrane protein) determined by 13C and 15N nuclear magnetic resonance spectroscopy

The major coat protein of the filamentous bacteriophage M13 is inserted as an integral protein in the inner membrane of the Escherichia coli host upon infection. M13 coat protein is an ideal model membrane protein and has been the target of many biophysical studies. An overview is presented here of the application of nuclear magnetic resonance spectroscopy to the study of the structure and dynamics of M13 coat protein in several lipid-mimetic environments. The coat protein may be biosynthetically enriched with 13C- and 15N-labelled amino acids, allowing the resolution and assignment of individual nuclei. Structural fluctuations at selected sites have been monitored using 13C relaxation and isotope-detected amide hydrogen exchange kinetics. A model is proposed for the structure of a coat protein dimer in detergent micelles.     

Rapid Assessment of Lignin Content and Structure in Switchgrass (Panicum virgatum L.) Grown Under Different Environmental Conditions

Switchgrass (Panicum virgatum L.) is a candidate feedstock in bioenergy, and plant breeding and molecular genetic strategies are being used to improve germplasm. In order to assess these subsequent modifications, baseline biomass compositional data are needed in a relevant variety of environments. In this study, switchgrass cv. Alamo was grown in the field, greenhouse, and growth chamber and harvested into individual leaf and stem tissue components. These components were analyzed with pyrolysis vapor analysis using molecular beam mass spectrometry, Fourier transform infrared, and standard wet chemistry methods to characterize and compare the composition among the different growth environments. The details of lignin content, S/G ratios, and degree of cross-linked lignin are discussed. Multivariate approaches such as projection to latent structures regression found a very strong correlation between the lignin content obtained by standard wet chemistry methods and the two high throughput techniques employed to rapidly assess lignin in potential switchgrass candidates. The models were tested on unknown samples and verified by wet chemistry. The similar lignin content found by the two methods shows that both approaches are capable of determining lignin content in biomass in a matter of minutes.     

High species mobility in species-rich plant communities: An intercontinental comparison

Using results from a long-term study of fine-scale dynamics in grasslands in four widely separated study areas from two continents, we provide further evidence to support the idea of the carousel model as an aid to describe the high fine-scale temporal and spatial species mobility found in grassland communities. Cumulative species numbers on small subplots in plots situated in stable plant communities, determined as the sum of species appearing in these subplots in one or more years over a period of time, are very high. In floristically different species-rich grasslands, varying from moist pine savannas in North Carolina and Mississippi, to humid chalk grassland in the Netherlands and seasonally dry limestone grassland in Sweden, average species numbers on subplots of 0.01 m² in plots of 2.5 m² over the period 1985–1989 were similar, most plots falling in the range 10.8–13.2. The total cumulative species numbers were similar as well, most plots falling in the range 17.4 and 20.9. Yearly average species numbers remained relatively constant. Considerable species turnover is occurring in all these communities; on average three species appear and three disappear each year in each 0.01 m² subplot. Total species accumulation on 0.01 m² subplots over the period 1985–1989 varied considerably, from 4.1 to 11.6, and is correlated with the cumulative species total on the plot, the latter figure being considered as correlated with the size of the species pool.