In Vitro Characterization of Peptidoglycan-Associated Lipoprotein (PAL)–Peptidoglycan and PAL–TolB Interactions

The Tol-peptidoglycan-associated lipoprotein (PAL) system of Escherichia coli is a multiprotein complex of the envelope involved in maintaining outer membrane integrity. PAL and the periplasmic protein TolB, two components of this complex, are interacting with each other, and they have also been reported to interact with OmpA and the major lipoprotein, two proteins interacting with the peptidoglycan. All these interactions suggest a role of the Tol-PAL system in anchoring the outer membrane to the peptidoglycan. Therefore, we were interested in better understanding the interaction between PAL and the peptidoglycan. We designed an in vitro interaction assay based on the property of purified peptidoglycan to be pelleted by ultracentrifugation. Using this assay, we showed that a purified PAL protein interacted in vitro with pure peptidoglycan. A peptide competition experiment further demonstrated that the region from residues 89 to 130 of PAL was sufficient to bind the peptidoglycan. Moreover, the fact that this same region of PAL was also binding to TolB suggested that these two interactions were exclusive. Indeed, the TolB-PAL complex appeared not to be associated with the peptidoglycan. This led us to the conclusion that PAL may exist in two forms in the cell envelope, one bound to TolB and the other bound to the peptidoglycan.     

Identification of a Streptococcus pneumoniae Gene Locus Encoding Proteins of an ABC Phosphate Transporter and a Two-Component Regulatory System

The Escherichia coli Pst system belongs to the family of ABC transporters. It is part of a phosphate (PHO) regulon which is regulated by extracellular phosphate. Under conditions of phosphate limitation, the response regulator PhoB is phosphorylated by the histidine kinase PhoR and binds to promoters that share a consensus PHO box. Under conditions of phosphate excess, PhoR, Pst, and PhoU downregulate the PHO regulon. Screening of a library of pneumococcal mutants with defects in exported proteins revealed a putative two-component regulatory system, PnpR-PnpS, and a downstream ABC transporter, similar to the Pst system in E. coli including a gene encoding a PhoU protein. Similar to E. coli, mutagenesis of the ATP-binding cassette gene, pstB, resulted in decreased uptake of phosphate. The effects of the loss of the pneumococcal Pst system extended to decreased transformation and lysis. Withdrawal of phosphate led to transformation deficiency in the parent strain R6x but not to penicillin tolerance, suggesting that reduced bacterial death was independent of phosphate. None of these phenotypes was observed in the pneumococcal loss-of-function mutant phoU. By using a lacZ reporter construct, it was demonstrated that expression of the two-component regulatory system PnpR-PnpS was not influenced by different concentrations of phosphate. These results suggest a more complex role of the Pst system in pneumococcal physiology than in that of E. coli.     

Analysis of Synthesis, Stability, Phosphorylation, and Interacting Polypeptides of the 34-Kilodalton Product of Open Reading Frame 6 of the Early Region 4 Protein of Human Adenovirus Type 5

The 34-kDa early-region 4 open reading frame 6 (E4orf6) product of human adenovirus type 5 forms complexes with both the cellular tumor suppressor p53 and the viral E1B 55-kDa protein (E1B-55kDa). E4orf6 can inhibit p53 transactivation activity, as can E1B-55kDa, and in combination these viral proteins cause the rapid turnover of p53. In addition, E4orf6-55kDa complexes play a critical role at later times in the regulation of viral mRNA transport and shutoff of host cell protein synthesis. In the present study, we have further characterized some of the biological properties of E4orf6. Analysis of extracts from infected cells by Western blotting indicated that E4orf6, like E1A and E1B products, is present at high levels until very late times, suggesting that it is available to act throughout the infectious cycle. This pattern is similar to that of E4orf4 but differs markedly from that of another E4 product, E4orf6/7, which is present only transiently. Synthesis of E4orf6 is maximal at early stages but ceases completely with the onset of shutoff of host protein synthesis; however, it was found that unlike E4orf6/7, E4orf6 is very stable, thus allowing high levels to be maintained even at late times. E4orf6 was shown to be phosphorylated at low levels. Coimmunoprecipitation studies in cells lacking p53 indicated that E4orf6 interacts with a number of other proteins. Five of these were shown to be viral or virally induced proteins ranging in size from 102 to 27 kDa, including E1B-55kDa. One such species, of 72 kDa, was shown not to represent the E2 DNA-binding protein and thus remains to be identified. Another appeared to be the L4 100-kDa nonstructural adenovirus late product, but it appeared to be present nonspecifically and not as part of an E4orf6 complex. Apart from p53, three additional cellular proteins, of 84, 19, and 14 kDa were detected by using an adenovirus vector that expresses only E4orf6. The 19-kDa species and a 16-kDa cellular protein were also shown to interact with E4orf6/7. It is possible that complex formation with these viral and cellular proteins plays a role in one or more of the biological activities associated with E4orf6 and E4orf6/7.     

Stratification based on reproductive state reveals contrasting patterns of age-related variation in demographic parameters in the kittiwake

Heterogeneity in individual quality can be a major obstacle when interpreting age‐specific variation in life‐history traits. Heterogeneity is likely to lead to within‐generation selection, and patterns observed at the population level may result from the combination of hidden patterns specific to subpopulations. Population‐level patterns are not relevant to hypotheses concerning the evolution of age‐specific reproductive strategies if they differ from patterns at the individual level. We addressed the influence of age and a variable used as a surrogate of quality (yearly reproductive state) on survival and breeding probability in the kittiwake. We found evidence of an effect of age and quality on both demographic parameters. Patterns observed in breeders are consistent with the selection hypothesis, which predicts age‐related increases in survival and traits positively correlated with survival. Our results also reveal unexpected age effects specific to subgroups: the influence of age on survival and future breeding probability is not the same in nonbreeders and breeders. These patterns are observed in higher‐quality breeding habitats, where the influence of extrinsic factors on breeding state is the weakest. Moreover, there is slight evidence of an influence of sex on breeding probability (not on survival), but the same overall pattern is observed in both sexes. Our results support the hypothesis that age‐related variation in demographic parameters observed at the population level is partly shaped by heterogeneity among individuals. They also suggest processes specific to subpopulations. Recent theoretical developments lay emphasis on integration of sources of heterogeneity in optimization models to account for apparently “sub‐optimal” empirical patterns. Incorporation of sources of heterogeneity is also the key to investigation of age‐related reproductive strategies in heterogeneous populations. Thwarting “heterogeneity's ruses” has become a major challenge: for detecting and understanding natural processes, and a constructive confrontation between empirical and theoretical studies.     

Inferences about nested subsets structure when not all species are detected

Comparisons of species composition among isolated ecological communities of different size have often provided evidence that the species in communities with lower species richness form nested subsets of the species in larger communities. In the vast majority of studies, the question of nested subsets has been addressed using information on presence‐absence, where a “0” is interpreted as the absence of a given species from a given location. Most of the methodological discussion in earlier studies investigating nestedness concerns the approach to generation of model‐based matrices corresponding to the null hypothesis of a nonnested pattern. However, it is most likely that in many situations investigators cannot detect all the species present in the location sampled. The possibility that zeros in incidence matrices reflect nondetection rather than absence of species has not been considered in studies addressing nested subsets, even though the position of zeros in these matrices forms the basis of earlier inference methods. These sampling artifacts are likely to lead to erroneous conclusions about both variation over space in species richness, and the degree of similarity of the various locations. Here we propose an approach to investigation of nestedness, based on statistical inference methods explicitly incorporating species detection probability, that take into account the probabilistic nature of the sampling process. We use presence‐absence data collected under Pollock's robust capture‐recapture design, and resort to an estimator of species richness originally developed for closed populations to assess the proportion of species shared by different locations. We develop testable predictions corresponding to the null hypothesis of a nonnested pattern, and an alternative hypothesis of perfect nestedness. We also present an index for assessing the degree of nestedness of a system of ecological communities. We illustrate our approach using avian data from the North American Breeding Bird Survey collected in Florida Keys.     

On the use of parataxonomy in biodiversity monitoring: a case study on wild flora

Monitoring programs that assess species-richness and turnover are now regarded as essential to document biodiversity loss worldwide. Implementation of such programs is impeded by a general decrease in the number of skilled naturalists. Here we studied how morphotypes, instead of species, might be used by unskilled participants (referred to as “volunteers”) to survey common plant communities. Our main questions were: (1) Can morphotypes be used as a robust estimator of species-richness (α-diversity) and assemblage turnover (β-diversity)? and (2) What is the robustness (reproducibility and repeatability) of such methods? Double inventories were performed on 150 plots in arable field margins, one by a non-expert using morphotypes, the other by a taxonomist using species. To test the robustness of morphotype identification among participants, 20 additional plots were surveyed by eight volunteers using the same protocol. We showed that (1) the number of morphotypes identified by unskilled volunteers in a plot was always strongly correlated with species-richness. (2) Morphotypes were sensitive to differences among habitats but were less accurate than species to detect these differences. (3) Morphotype identification varied significantly within and between volunteers. Due to this lack of repeatability and reproducibility, parataxonomy cannot be considered a good surrogate for taxonomy. Nevertheless, assuming that morphotypes are identified with standardized methods, and that results are used only to evaluate gross species-richness but not species turnover, parataxonomy might be a valuable tool for rapid biodiversity assessment of common wild flora.     

Bacterial L-arabinose isomerases: industrial application for D-tagatose production

D-tagatose is a natural monosaccharide with a low caloric value and has an anti-hyperglycemiant effect. This hexose has potential applications both in pharmaceutical and agro-food industries. However, the use of D-tagatose remains limited by its production cost. Many production procedures including chemical and biological processes were developed and patented. The most profitable production way is based on the use of L-arabinose isomerase which allows the manufacture of D-tagatose with an attractive rate. Future developments are focused on the generation of L-arabinose isomerases having biochemical properties satisfying the industrial applications. This report provides a brief review of the most recent patents that have been published relating to this area.     

N-cadherin deficiency impairs pericyte recruitment, and not endothelial differentiation or sprouting, in embryonic stem cell-derived angiogenesis

Endothelial cells express two classical cadherins, VE-cadherin and N-cadherin. VE-cadherin is absolutely required for vascular morphogenesis, but N-cadherin is thought to participate in vessel stabilization by interacting with periendothelial cells during vessel formation. However, recent data suggest a more critical role for N-cadherin in endothelium that would regulate angiogenesis, in part by controlling VE-cadherin expression. In this study, we have assessed N-cadherin function in vascular development using an in vitro model derived from embryonic stem (ES) cell differentiation. We show that pluripotent ES cells genetically null for N-cadherin can differentiate normally into endothelial cells. In addition, sprouting angiogenesis was unaltered, suggesting that N-cadherin is not essential for the early events of angiogenesis. However, the lack of N-cadherin led to an impairment in pericyte covering of endothelial outgrowths. We conclude that N-cadherin is necessary neither for vasculogenesis nor proliferation and migration of endothelial cells but is required for the subsequent maturation of endothelial sprouts by interacting with pericytes.