Temporal changes in PTHrP, Bcl-2, Bax, caspase, TGF-beta, and FGF-2 expression following growth plate irradiation with or without radioprotectant.

This study examined temporal changes in growth plate apoptosis molecules and growth factors in an animal model of radiation injury with and without a radioprotectant. Thirty weanling 5-week Sprague-Dawley rats underwent right knee irradiation with single-fraction 17.5 Gy while the left served as internal control. Six animals each were sacrificed at 0.5, 1, 2, 3, or 4 weeks after irradiation. Half of the animals received pretreatment with amifostine (WR-2721) radioprotectant. Immunohistochemical staining for PTHrP, Bcl-2, Bax, caspase-3, FGF-2, and TGF-beta was performed. PTHrP decreased to a nadir at 1 week after irradiation but rebounded to above control levels at 2 weeks in the reserve and transitional zones. The radioprotectant amifostine blunted the decrease in PTHrP but kept PTHrP expression lower than controls during the rebound phase in untreated irradiated animals. Hypertrophic zone Bax expression was decreased by amifostine in both irradiated and non-irradiated limbs at 1 and 2 weeks. FGF, TGF-beta, Bcl-2, and caspase levels generally decreased at 1 week and returned thereafter toward control levels. These findings underscore the importance of PTHrP in response to growth plate irradiation and show the novel finding of a decrease in Bax expression with amifostine pretreatment.     

Bioenergetics and solute uptake under extreme conditions

The ion and particularly the proton and sodium ion permeabilities of cytoplasmic membranes play crucial roles in the bioenergetics of microorganisms. The proton and sodium permeabilities of membranes increase with temperature. Psychrophilic and mesophilic bacteria and mesophilic, (hyper)thermophilic, and halophilic archaea are capable of adjusting the lipid composition of their membranes in such a way that the proton permeability at the respective growth temperature remains constant (homeoproton permeability). Thermophilic bacteria are an exception. They rely on the less permeable sodium ions to generate a sodium motive force, which is subsequently used to drive energy-requiring membrane-bound processes. Transport of solutes across bacterial and archaeal membranes is mainly catalyzed by primary ATP-driven transport systems or by proton- or sodium-motive-force-driven secondary transport systems. Unlike most bacteria, hyperthermophilic bacteria and archaea prefer primary uptake systems. Several high-affinity ATP-binding cassette (ABC) transporters for sugars from hyperthermophiles have been identified and characterized. The activities of these ABC transporters allow these organisms to thrive in their nutrient-poor environments.     

Human milk provides peptides highly stimulating the growth of bifidobacteria.

The large intestine of breast-fed infants is colonized predominantly by bifidobacteria, which have a protective effect against acute diarrhea. In this study we report for the first time the identification of human milk peptides that selectively stimulate the growth of bifidobacteria. Several bifidogenic peptides were purified chromatographically from pepsin-treated human milk and identified as proteolytically generated fragments from the secretory component of the soluble polyimmunoglobulin receptor and lactoferrin; both of these proteins exhibit antimicrobial effects. Hydrolysis of the identified peptides with the gastrointestinal proteases pepsin, trypsin and chymotrypsin did not lead to the loss of bifidogenic activity, indicating their potential function in vivo. Sequential comparison revealed a similar structural motif within the identified peptides. A correspondingly designed small peptide (prebiotic lactoferrin-derived peptide-I, PRELP-I) was found to stimulate the growth of bifidobacteria as effectively as the native peptides. The combination of antimicrobial and bifidobacterial growth stimulatory activity in human milk proteins leads to highly specific compounds capable of regulating the microbial composition of infants' large intestine.     

Functional Expression of a Fungal Laccase in Saccharomyces cerevisiae by Directed Evolution

Laccase from Myceliophthora thermophila (MtL) was expressed in functional form in Saccharomyces cerevisiae. Directed evolution improved expression eightfold to the highest yet reported for a laccase in yeast (18 mg/liter). Together with a 22-fold increase in k_cat, the total activity was enhanced 170-fold. Specific activities of MtL mutants toward 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and syringaldazine indicate that substrate specificity was not changed by the introduced mutations. The most effective mutation (10-fold increase in total activity) introduced a Kex2 protease recognition site at the C-terminal processing site of the protein, adjusting the protein sequence to the different protease specificities of the heterologous host. The C terminus is shown to be important for laccase activity, since removing it by a truncation of the gene reduces activity sixfold. Mutations accumulated during nine generations of evolution for higher activity decreased enzyme stability. Screening for improved stability in one generation produced a mutant more stable than the heterologous wild type and retaining the improved activity. The molecular mass of MtL expressed in S. cerevisiae is 30% higher than that of the same enzyme expressed in M. thermophila (110 kDa versus 85 kDa). Hyperglycosylation, corresponding to a 120-monomer glycan on one N-glycosylation site, is responsible for this increase. This S. cerevisiae expression system makes MtL available for functional tailoring by directed evolution.     

Data exploration in phylogenetic inference: scientific, heuristic, or neither

The methods of data exploration have become the centerpiece of phylogenetic inference, but without the scientific importance of those methods having been identified. We examine in some detail the procedures and justifications of Wheeler's sensitivity analysis and relative rate comparison (saturation analysis). In addition, we review methods designed to explore evidential decisiveness, clade stability, transformation series additivity, methodological concordance, sensitivity to prior probabilities (Bayesian analysis), skewness, computer‐intensive tests, long‐branch attraction, model assumptions (likelihood ratio test), sensitivity to amount of data, polymorphism, clade concordance index, character compatibility, partitioned analysis, spectral analysis, relative apparent synapomorphy analysis, and congruence with a “known” phylogeny. In our review, we consider a method to be scientific if it performs empirical tests, i.e., if it applies empirical data that could potentially refute the hypothesis of interest. Methods that do not perform tests, and therefore are not scientific, may nonetheless be heuristic in the scientific enterprise if they point to more weakly or ambiguously corroborated hypotheses, such propositions being more easily refuted than those that have been more severely tested and are more strongly corroborated. Based on common usage, data exploration in phylogenetics is accomplished by any method that performs sensitivity or quality analysis. Sensitivity analysis evaluates the responsiveness of results to variation or errors in parameter values and assumptions. Sensitivity analysis is generally interpreted as providing a measure of support, where conclusions that are insensitive (robust, stable) to perturbations are judged to be accurate, probable, or reliable. As an alternative to that verificationist concept, we define support objectively as the degree to which critical evidence refutes competing hypotheses. As such, degree of support is secondary to the scientific optimality criterion of maximizing explanatory power. Quality analyses purport to distinguish good, reliable, accurate data from bad, misleading, erroneous data, thereby assessing the ability of data to indicate the true phylogeny. Only the quality analysis of character compatibility can be judged scientific—and a weak test at that compared to character congruence. Methods judged to be heuristic include Bremer support, long‐branch extraction, and safe taxonomic reduction, and we underscore the great heuristic potential of a posteriori analysis of patterns of transformations on the total‐evidence cladogram. However, of the more than 20 kinds of data exploration methods evaluated, the vast majority is neither scientific nor heuristic. Given so little demonstrated cognitive worth, we conclude that undue emphasis has been placed on data exploration in phylogenetic inference, and we urge phylogeneticists to consider more carefully the relevance of the methods that they employ. [T]he cult of impressive technicalities or the cult of precision may get the better of us, and interfere with our search for clarity, simplicity, and truth [Popper, 1983, p. 60. Empirical papers chosen for publication are judged to be of interest to a broad systematics audience because they represent exemplary case studies involving some important contemporary issue or issues. These may be unusually thorough explorations of data, applications of new methodology, illustrations of fundamental principles, and/or investigations of interesting evolutionary questions. [Systematic Biology: Instructions for authors, 2002; italics added]