Use of Physcomitrella patens actin 5′ regions for high transgene expression: importance of 5′ introns

We have isolated four actin (Act) genes from Physcomitrella patens and used their corresponding 5′ regions for recombinant expression of the human vascular endothelial growth factor (rhVEGF₁₂₁) in transiently transformed Physcomitrella protoplasts and in stable transformed lines. In the transient system, we found up to 11-fold activity of the corresponding 5′ regions as compared with that of the plant constitutive 35S promoter. Moreover, the use of an optimised expression vector in which the human VEGF signal peptide was exchanged with a plant signal peptide resulted in an additional 7-fold increase in secreted rhVEGF. We found that the 5′ introns of PpAct1, PpAct5 and PpAct7 are essential for high expression. The enhancing mechanisms of the introns, however, seem to be different since in the case of PpAct1, the expression level is stimulated only in the presence of the endogenous promoter, whereas the 5′ introns of PpAct5 and PpAct7 stimulate expression also in combination with the 35S promoter. Beyond this, the isolated 5′ regions are shown to be useful for high expression levels in transgenic moss lines with values of secreted rhVEGF up to 96 μg g^-1 dry weight.A. Weise and M. Rodriguez-Franco have contributed equally to this work.     

Hexapeptides that interfere with HIV-1 fusion peptide activity in liposomes block GP41-mediated membrane fusion

Upon receptor-mediated activation, the gp41 hydrophobic, conserved fusion peptide inserts into the target membrane and promotes the kind of perturbations required for the progression of the HIV-cell fusion reaction. Using a synthetic combinatorial library we have identified all d-amino acid hexapeptide sequences that inhibited the fusion peptide capacity of perturbing model membranes. Two hexapeptides that effectively inhibited the fusion peptide in these systems were subsequently shown to inhibit cell-cell fusion promoted by gp41 expressed at cell surfaces. These observations might be of importance for understanding the mechanisms underlying fusion peptide activity and suggest new strategies for screening compounds that target these viral sequences.     

In Vivo Himar1-Based Transposon Mutagenesis of Francisella tularensis

Francisella tularensis is the intracellular pathogen that causes human tularemia. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of entry. We report the development of a Himar1-based random mutagenesis system for F. tularensis (HimarFT). In vivo mutagenesis of F. tularensis live vaccine strain (LVS) with HimarFT occurs at high efficiency. Approximately 12 to 15% of cells transformed with the delivery plasmid result in transposon insertion into the genome. Results from Southern blot analysis of 33 random isolates suggest that single insertions occurred, accompanied by the loss of the plasmid vehicle in most cases. Nucleotide sequence analysis of rescued genomic DNA with HimarFT indicates that the orientation of integration was unbiased and that insertions occurred in open reading frames and intergenic and repetitive regions of the chromosome. To determine the utility of the system, transposon mutagenesis was performed, followed by a screen for growth on Chamberlain's chemically defined medium (CDM) to isolate auxotrophic mutants. Several mutants were isolated that grew on complex but not on the CDM. We genetically complemented two of the mutants for growth on CDM with a newly constructed plasmid containing a nourseothricin resistance marker. In addition, uracil or aromatic amino acid supplementation of CDM supported growth of isolates with insertions in pyrD, carA, or aroE1 supporting the functional assignment of genes within each biosynthetic pathway. A mutant containing an insertion in aroE1 demonstrated delayed replication in macrophages and was restored to the parental growth phenotype when provided with the appropriate plasmid in trans. Our results suggest that a comprehensive library of mutants can be generated in F. tularensis LVS, providing an additional genetic tool to identify virulence determinants required for survival within the host.     

Singlet oxygen generation by UVA light exposure of endogenous photosensitizers

UVA light (320-400 nm) has been shown to produce deleterious biological effects in tissue due to the generation of singlet oxygen by substances like flavins or urocanic acid. Riboflavin, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), beta-nicotinamide adenine dinucleotide (NAD), and beta-nicotinamide adenine dinucleotide phosphate (NADP), urocanic acid, or cholesterol in solution were excited at 355 nm. Singlet oxygen was directly detected by time-resolved measurement of its luminescence at 1270 nm. NAD, NADP, and cholesterol showed no luminescence signal possibly due to the very low absorption coefficient at 355 nm. Singlet oxygen luminescence of urocanic acid was clearly detected but the signal was too weak to quantify a quantum yield. The quantum yield of singlet oxygen was precisely determined for riboflavin (PhiDelta = 0.54 +/- 0.07), FMN (PhiDelta = 0.51 +/- 0.07), and FAD (PhiDelta = 0.07 +/- 0.02). In aerated solution, riboflavin and FMN generate more singlet oxygen than exogenous photosensitizers such as Photofrin, which are applied in photodynamic therapy to kill cancer cells. With decreasing oxygen concentration, the quantum yield of singlet oxygen generation decreased, which must be considered when assessing the role of singlet oxygen at low oxygen concentrations (inside tissue).     

Temporal regulation of enhancer function in intestinal epithelium: a role for Onecut factors

An intestine-specific gene regulatory region was previously identified near the second exon of the human adenosine deaminase (ADA) gene. In mammalian intestine, ADA is expressed at high levels only along the villi of the duodenal epithelium, principally if not exclusively in enterocytes. Within the ADA intestinal regulatory region, a potent duodenum-specific enhancer was identified that controls this pattern of expression. This enhancer has been shown to rely on PDX-1, GATA factors, and Cdx factors for its function. Upstream of the enhancer, a separate temporal regulatory region was identified that has no independent enhancer capability but controls the timing of enhancer activation. DNase I footprinting and electrophoretic mobility shift assays were used to begin to characterize temporal region function at the molecular level. In this manner, binding sites for the Onecut (OC) family of factors, YY1, and NFI family members were identified. Identification of the OC site was especially interesting, because almost nothing is known about the function of OC factors in intestine. In transgenic mice, mutation of the OC site to ablate binding resulted in a delay of 2-3 weeks in enhancer activation in the developing postnatal intestine, a result very similar to that observed previously when the entire temporal region was deleted. In mammals, the OC family is comprised of OC-1/HNF-6, OC-2, and OC-3. An examination of intestinal expression patterns showed that all three OC factors are expressed at detectable levels in adult mouse duodenum, with OC-2 predominant. In postnatal day 2 mice only OC-2 and OC-3 were detected in intestine, with OC-2 again predominant.     

Managing Variability: Herding Strategies in Communal Rangelands of Semiarid Namaqualand, South Africa

Herd mobility is a tool for managing environmental variability in African pastoral systems. This study examines the monthly mobility patterns of 24 herds over six years in the 20,000 ha communal area of Paulshoek, Namaqualand, and assesses the social, economic, and ecological factors affecting the livestock movement of individual herds and all herds combined. When the mobility pattern of all herds was considered, no seasonal or between-year differences in response to rainfall were evident. An analysis of individual herd mobility patterns showed that half of the herds were relatively sedentary over the study period while the other half were regularly mobile. Although herders used mobility to manage their herds in the unpredictable semiarid environment, their daily decisions were often made in response to their social, economic, or personal situations. There was no significant difference in livestock production between herding strategies, but sedentary herders had a greater localized impact on the rangeland than mobile herders. Our analysis suggests that non-environmental factors play a significant role in herd mobility and may consequently affect the efficiency of livestock production and environmental management.     

A high-density consensus map of barley linking DArT markers to SSR,RFLP and STS loci and agricultural traits

Background

Wheat is an excellent species to study freezing tolerance and other abiotic stresses. However, the sequence of the wheat genome has not been completely characterized due to its complexity and large size. To circumvent this obstacle and identify genes involved in cold acclimation and associated stresses, a large scale EST sequencing approach was undertaken by the Functional Genomics of Abiotic Stress (FGAS) project.

Results

We generated 73,521 quality-filtered ESTs from eleven cDNA libraries constructed from wheat plants exposed to various abiotic stresses and at different developmental stages. In addition, 196,041 ESTs for which tracefiles were available from the National Science Foundation wheat EST sequencing program and DuPont were also quality-filtered and used in the analysis. Clustering of the combined ESTs with d2_cluster and TGICL yielded a few large clusters containing several thousand ESTs that were refractory to routine clustering techniques. To resolve this problem, the sequence proximity and "bridges" were identified by an e-value distance graph to manually break clusters into smaller groups. Assembly of the resolved ESTs generated a 75,488 unique sequence set (31,580 contigs and 43,908 singletons/singlets). Digital expression analyses indicated that the FGAS dataset is enriched in stress-regulated genes compared to the other public datasets. Over 43% of the unique sequence set was annotated and classified into functional categories according to Gene Ontology.

Conclusion

We have annotated 29,556 different sequences, an almost 5-fold increase in annotated sequences compared to the available wheat public databases. Digital expression analysis combined with gene annotation helped in the identification of several pathways associated with abiotic stress. The genomic resources and knowledge developed by this project will contribute to a better understanding of the different mechanisms that govern stress tolerance in wheat and other cereals.     

Iterleukin 1 alpha is a marker of endothelial cellular senescent

The number of old and oldest old patients undergoing surgery of varying severity is increasing. Ageing is a process that changes the performances of most physiological systems and increases susceptibility to diseases and death; accordingly, host responses to surgical stress are altered with ageing and the occurrence of age-related increase in susceptibility to post-operative complications has been claimed. Twenty-four male patients undergoing Lichtenstein (LH) hernioplasty for unilateral inguinal hernia were included in this study and divided in two groups (Young and Old respectively), according to their age. As expression of the acute phase response, we measured changes in concentration of pro-inflammatory cytokines Tumor necrosis factor-α and Interleukin-1β, leukocytes, acute phase proteins C-reactive protein and α 1-antitrypsin. Elderly humans showed prolonged and strong inflammatory activity compared to younger subjects in response to surgical stress, indicating that the acute-phase response to surgical stress of elderly humans varies from that of the young, showing initial hyperactivity and a delayed termination of the response. Thus, the acute phase response to surgical stress is higher in old subjects, but the clinical significance of this remains unclear. It is not known whether a causal relationship exists between this stronger acute phase response and the increases in susceptibility to post-operative complications observed in aged patients.     

Iterleukin 1 alpha is a marker of endothelial cellular senescent

Background  

The functional changes associated with endothelial senescence may be involved in human aging and age-related vascular disorders. Since the inflammatory cytokine interleukin (IL-)1 inhibits endothelial growth, we evaluated the expression of IL-1α, IL-1β and their antagonist, the IL-1 receptor antagonist (IL-1ra), in endothelial in vitro senescence and quiescence. We also examined the expression of IL-1α in human senescent and progeric fibroblasts.