Evolution of phage with chemically ambiguous proteomes

Background  

The widespread introduction of amino acid substitutions into organismal proteomes has occurred during natural evolution, but has been difficult to achieve by directed evolution. The adaptation of the translation apparatus represents one barrier, but the multiple mutations that may be required throughout a proteome in order to accommodate an alternative amino acid or analogue is an even more daunting problem. The evolution of a small bacteriophage proteome to accommodate an unnatural amino acid analogue can provide insights into the number and type of substitutions that individual proteins will require to retain functionality.     

Clean technology: industry and environment, a viable partnership?

The industrial sector is becoming increasingly interested in eliminating potential pollution at source and reducing energy use. Biotechnology provides cheaper, cleaner alternatives to a wide range of traditional processes--but its adoption has been slower than expected. If industry is to become truly compatible with the environment, companies and the public will have to be convinced of the ecological and economic value of clean technology.     

Multiple Molecular Mechanisms Underlying Subdiagnostic Variants of Marfan Syndrome

Mutations in the FBN1 gene, which encodes fibrillin-1, cause Marfan syndrome (MFS) and have been associated with a wide range of milder, overlap phenotypes. The factors that modulate phenotypic severity, both between and within families, remain to be determined. This study examines the relationship between the FBN1 genotype and phenotype in families with extremely mild phenotypes and in those that show striking clinical variation among apparently affected individuals. In one family, clinically similar but etiologically distinct disorders are segregating independently. In another, somatic mosaicism for a mutant FBN1 allele is associated with subdiagnostic manifestations, whereas germ-line transmission of the identical mutation causes severe and rapidly progressive disease. A third family cosegregates mild mitral valve prolapse syndrome with a mutation in FBN1 that can be functionally distinguished from those associated with the classic MFS phenotype. These data have immediate relevance for the diagnostic and prognostic counseling of patients and their family members.     

Novel rhodococci and other mycolate actinomycetes from the deep sea

A large number of mycolate actinomycetes have been recovered from deep-sea sediments in the NW Pacific Ocean using selective isolation methods. The isolates were putatively assigned to the genus Rhodococcus on the basis of colony characteristics and mycolic acid profiles. The diversity among these isolates and their relationship to type strains of Rhodococcus and other mycolate taxa were assessed by Curie point pyrolysis mass spectrometry (PyMS). Three major (A, C, D) and two minor (B, E) groups were defined by PyMS. Cluster A was a large group of isolates recovered from sediment in the Izu Bonin Trench (2679 m); Cluster C comprised isolates from both the Izu Bonin Trench (6390 and 6499 m) and from the Japan Trench (4418, 6048 and 6455 m). These Cluster C isolates showed close similarity to Dietzia maris and this was subsequently confirmed using molecular methods. Cluster D contained isolates recovered from a sediment taken from a depth of 1168m in Sagami Bay and were identified as members of the terrestrial species Rhodococcus luteus. Clusters B and E had close affinities with members of the genera Gordonia and Mycobacterium. The presence of Thermoactinomyces in certain of the deep-sea sediments studied was indicative of the movement of terrestrial material into the ocean depths.16S ribosomal RNA gene sequence analyses produced excellent definition of most genera of the mycolata, and indicated that the among the deep sea isolates (1) were novel species of Corynebacterium, Gordonia and Mycobacterium, and (2) a Sea of Japan isolate the phylogenetic depth of which suggests the possibility of a new genus. Polyphasic taxonomic analysis revealed considerable diversity among the deep sea rhodococci and evidence for recently diverged species or DNA groups.     

Monitoring recombinant human interferon-gamma N-glycosylation during perfused fluidized-bed and stirred-tank batch culture of CHO cells

Chinese hamster ovary cells producing recombinant human interferon-gamma were cultivated for 500 h attached to macroporous microcarriers in a perfused, fluidized-bed bioreactor, reaching a maximum cell density in excess of 3 x 10(7) cells (mL microcarrier)-1 at a specific growth rate (mu) of 0.010 h-1. During establishment of the culture, the N-glycosylation of secreted recombinant IFN-gamma was monitored by capillary electrophoresis of intact IFN-gamma proteins and by HPLC analysis of released N-glycans. Rapid analysis of IFN-gamma by micellar electrokinetic capillary chromatography resolved the three glycosylation site occupancy variants of recombinant IFN-gamma (two Asn sites occupied, one Asn site occupied and nonglycosylated) in under 10 min per sample; the relative proportions of these variants remained constant during culture. Analysis of IFN-gamma by capillary isoelectric focusing resolved at least 11 differently sialylated glycoforms over a pI range of 3.4 to 6.4, enabling rapid quantitation of this important source of microheterogeneity. During perfusion culture the relative proportion of acidic IFN-gamma proteins increased after 210 h of culture, indicative of an increase in N-glycan sialylation. This was confirmed by cation-exchange HPLC analysis of released, fluorophore-labeled N-glycans, which showed an increase in the proportion of tri- and tetrasialylated N-glycans associated with IFN-gamma during culture, with a concomitant decrease in the proportion of monosialylated and neutral N-glycans. Comparative analyses of IFN-gamma produced by CHO cells in stirred-tank culture showed that N-glycan sialylation was stable until late in culture, when a decline in sialylation coincided with the onset of cell death and lysis. This study demonstrates that different modes of capillary electrophoresis can be employed to rapidly and quantitatively monitor the main sources of glycoprotein variation, and that the culture system and operation may influence the glycosylation of a recombinant glycoprotein.     

Enhanced vigilance in monogamous pairs of the lizard, Tiliqua rugosa

The Australian sleepy lizard, Tiliqua rugosa, forms monogamouspairs for up to 8 weeks each spring before mating. We observedthat males had food in their mouths significantly less oftenwhen they were in pain than when they were alone. Females hadfood in the mouth independent of the presence or absence ofmales. Among females that had been feeding, indicated by foodin the mouth, we observed them feeding, as we approached, lessoften when they were in pairs than when they were alone. Amongfemales in pairs with food in their mouths, we observed themfeeding less often when their male partners were not feedingthan when their partners were feeding. This suggests that femalesare alerted to approaching danger earlier when they are in apair, and alerted earlier in a pair when their male partneris not feeding. Enhanced vigilance may be one function of pairingbehavior.     

Riparian zones as havens for exotic plant species in the central grasslands

In the Central Grasslands of the United States, we hypothesized that riparian zones high in soil fertility would contain more exotic plant species than upland areas of low soil fertility. Our alternate hypothesis was that riparian zones high in native plant species richness and cover would monopolize available resources and resist invasion by exotic species. We gathered nested-scale vegetation data from 40 1 m²subplots (nested in four 1000 m² plots) in both riparian and upland sites at four study areas in Colorado, Wyoming, and South Dakota (a total of 320 1 m² subplots and 32 1000 m² plots). At the 1 m² scale, mean foliar cover of native species was significantly greater (P<0.001) in riparian zones (36.3% ± 1.7%) compared to upland sites (28.7% ± 1.5%), but at this small scale there were no consistent patterns of native and exotic species richness among the four management areas. Mean exotic species cover was slightly higher in upland sites compared to riparian sites (9.0% ± 3.8% versus 8.2% ± 3.0% cover). However, mean exotic species richness and cover were greater in the riparian zones than upland sites in three of four management areas. At the 1000 m² scale, mean exotic species richness was also significantly greater (P<0.05) in riparian zones (7.8 ± 1.0 species) compared to upland sites (4.8 ± 1.0 species) despite the heavy invasion of one upland site. For all 32 plots combined, 21% of the variance in exotic species richness was explained by positive relationships with soil % silt (t =1.7, P=0.09) and total foliar cover (t = 2.4, P=0.02). Likewise, 26% of the variance in exotic species cover (log₁₀ cover) was explained by positive relationships with soil % silt (t =2.3, P=0.03) and total plant species richness (t = 2.5, P=0.02). At landscape scales (four 1000 m² plots per type combined), total foliar cover was significantly and positively correlated with exotic species richness (r=0.73, P<0.05) and cover (r=0.74, P<0.05). Exotic species cover (log₁₀ cover) was positively correlated with log₁₀% N in the soil (r=0.61, P=0.11) at landscape scales. On average, we found that 85% (±5%) of the total number of exotic species in the sampling plots of a given management area could be found in riparian zones, while only 50% (±8%) were found in upland plots. We conclude that: (1) species-rich and productive riparian zones are particularly invasible in grassland ecosystems; and (2) riparian zones may act as havens, corridors, and sources of exotic plant invasions for upland sites and pose a significant challenge to land managers and conservation biologists.     

Viral ion channels: molecular modeling and simulation

In a number of membrane-bound viruses, ion channels are formed by integral membrane proteins. These channel proteins include M2 from influenza A, NB from influenza B, and, possibly, Vpu from HIV-1. M2 is important in facilitating uncoating of the influenza A viral genome and is the target of amantadine, an anti-influenza drug. The biological roles of NB and Vpu are less certain. In all cases, the protein contains a single transmembrane alpha-helix close to its N-terminus. Channels can be formed by homo-oligomerization of these proteins, yielding bundles of transmembrane helices that span the membrane and surround a central ion-permeable pore. Molecular modeling may be used to integrate and interpret available experimental data concerning the structure of such transmembrane pores. This has proved successful for the M2 channel domain, where two independently derived models are in agreement with one another, and with solid-state nuclear magnetic resonance (NMR) data. Simulations based on channel models may yield insights into possible ion conduction and selectivity mechanisms.     

Cortisol Is Not Associated with Telomere Shortening or Chromosomal Instability in Human Lymphocytes Cultured under Low and High Folate Conditions

Chronic psychological stress and nutritional deficiencies are factors that impact negatively on human health and disease risk. Chronic stress has been associated with accelerated leukocyte telomere shortening in numerous cohorts, however, a mechanistic link has proven elusive. This study tested the hypotheses that chronic exposure to the stress hormone, cortisol, causes telomere shortening and chromosome instability (CIN) in vitro, and that these effects would be further exacerbated by folate (vitamin B9) deficiency. Primary human lymphocytes were maintained in vitro for 12 days in medium containing either 25 nM folic acid (FA(low)) or 100 nM FA (FA(high)), together with either 0, 400, 1000 or 3500 nM cortisol. The interactive effects of cortisol and FA were examined by comparing telomere length (TL), biomarkers of DNA damage, and cytostasis. At day 12 TL was 5-17% longer in lymphocytes cultured in FA(low) conditions (mean ± SD;10.2% ± 1.6), compared with those in FA(high) medium (9.1% ± 1, p = 0.02). Refuting the hypothesis, TL was consistently greater in the presence of cortisol. The effect of FA deficiency on the frequency of DNA damage was significant for nucleoplasmic bridges, circular nuclei, micronuclei and nuclear buds, (p < 0.0001 – 0.001). The effect of cortisol, however, was negligible, only reaching statistical significance for the frequency of fused nuclei (p = 0.04). Cortisol was significantly associated with reduced cell division and growth and had an apparent protective effect on cell viability in the FA(low) conditions. Conclusions: Both chronic cortisol exposure, and folate deficiency, resulted in telomere elongation, however, the effect of cortisol was marginal relative to that of folate. Cortisol was not associated with increased chromosomal instability, but caused a significant reduction in cell division and growth. Together these results indicate that cortisol is not directly genotoxic and that the telomere shortening associated with increased psychological stress in vivo may not be explained by a direct effect of cortisol.