Upregulation of the immune protein gene hemolin in the epidermis during the wandering larval stage of the Indian meal moth, Plodia interpunctella

Expression of hemolin, which generates an immune protein, was up-regulated in wandering fifth instar larval stage of Plodia interpunctella. The mRNA level peaked in the middle of the wandering stage. Major expression was in the epidermis, rather than in the fat body or gut. To test a possible ecdysteroid effect on hemolin induction we treated with RH-5992, an ecdysteroid agonist, and KK-42, which inhibits ecdysteroid biosynthesis in both feeding and wandering fifth instar larvae. When feeding larvae were treated with RH-5992 the hemolin mRNA level was increased. When wandering larvae were treated with KK-42 its level was reduced. In addition, when KK-42-treated larvae were subsequently treated with RH-5992 the hemolin mRNA level was recovered. These results strongly suggest that ecdysteroid up-regulates the expression of hemolin mRNA. Hormonal and bacterial effects on hemolin induction were further analyzed at the tissue level. Major induction of hemolin mRNA was detected following both RH-5992 treatment and bacterial injection in the epidermis of both feeding and wandering larvae. Minor induction of hemolin was detected in the fat body following a bacterial injection, but not RH-5992 treatment. We infer that in P. interpunctella larvae, the epidermis is the major tissue for hemolin induction in na?ve insects and in insects manipulated with bacterial and hormonal treatments.     

PD-1-dependent mechanisms maintain peripheral tolerance of donor-reactive CD8+ T cells to transplanted tissue

Peripheral mechanisms of self-tolerance often depend on the quiescent state of the immune system. To what degree such mechanisms can be engaged in the enhancement of allograft survival is unclear. To examine the role of the PD-1 pathway in the maintenance of graft survival following blockade of costimulatory pathways, we used a single-Ag mismatch model of graft rejection where we could track the donor-specific cells as they developed endogenously and emerged from the thymus. We found that graft-specific T cells arising under physiologic developmental conditions at low frequency were actively deleted at the time of transplantation under combined CD28/CD40L blockade. However, this deletion was incomplete, and donor-specific cells that failed to undergo deletion up-regulated expression of PD-1. Furthermore, blockade of PD-1 signaling on these cells via in vivo treatment with anti-PD-1 mAb resulted in rapid expansion of donor-specific T cells and graft loss. These results suggest that the PD-1 pathway was engaged in the continued regulation of the low-frequency graft-specific immune response and thus in maintenance of graft survival.     

Inhibitory action of NoxA1 on dual oxidase activity in airway cells

Imbalance between pro- and antioxidant mechanisms in the lungs can compromise pulmonary functions, including blood oxygenation, host defense, and maintenance of an anti-inflammatory environment. Thus, tight regulatory control of reactive oxygen species is critical for proper lung function. Increasing evidence supports a role for the NADPH oxidase dual oxidase (Duox) as an important source for regulated H(2)O(2) production in the respiratory tract epithelium. In this study Duox expression, function, and regulation were investigated in a fully differentiated, mucociliary airway epithelium model. Duox-mediated H(2)O(2) generation was dependent on calcium flux, which was required for dissociation of the NADPH oxidase regulatory protein Noxa1 from plasma membrane-bound Duox. A functional Duox1-based oxidase was reconstituted in model cell lines to permit mutational analysis of Noxa1 and Duox1. Although the activation domain of Noxa1 was not required for Duox function, mutation of a proline-rich domain in the Duox C terminus, a potential interaction motif for the Noxa1 Src homology domain 3, caused up-regulation of basal and stimulated H(2)O(2) production. Similarly, knockdown of Noxa1 in airway cells increased basal H(2)O(2) generation. Our data indicate a novel, inhibitory function for Noxa1 in Duox regulation. This represents a new paradigm for control of NADPH oxidase activity, where second messenger-promoted conformational change of the Nox structure promotes oxidase activation by relieving constraint induced by regulatory components.     

Chromosomal gfp labelling of Pseudomonas aeruginosa using a mini-Tn7 transposon: application for studies of bacteria-host interactions

Analysis of bacterial interactions with host cells using multiple techniques is essential for studies on microbial pathogenesis and for the development of new antimicrobial therapies. Pseudomonas aeruginosa is an important opportunistic pathogen that can cause severe, often life-threatening pulmonary infections in individuals with impaired host defense mechanisms. Using a mini-Tn7 transposon delivery system, we have chromosomally labelled the strain P. aeruginosa PAK with a green fluorescent protein gene (gfp) and tested PAKgfp as a research tool for studies of bacteria-host interactions. We were able to reliably and rapidly measure the interactions of PAKgfp with A549 human lung epithelial cells by using flow cytometry, a fluorometric microplate reader-based assay, and fluorescence microscopy. With these analytical tools, we have demonstrated the adhesion of PAKgfp to the extracellular matrix protein fibronectin and the involvement of fibronectin in PAKgfp-A549 cell interactions. PAKgfp can be successfully used to explore the effects of various pharmacological compounds on P. aeruginosa - host cell interactions in both in vitro and in vivo systems, with potentially important medical applications.     

The bactericidal effect of ultraviolet and visible light on Escherichia coli

The bactericidal radiation dosages at specific wavelengths in the ultraviolet (UV)-visible spectrum are not well documented. Such information is important for the development of new monochromatic bactericidal devices to be operated at different wavelengths. In this study, radiation dosages required to cause mortality of an Escherichia coli strain, ATCC 25922, at various wavelengths between 250 and 532 nm in the UV and visible spectrum were determined. Radiation at 265 nm in the UV region was most efficient in killing the E. coli cells and 100% mortality was achieved at a dose of 1.17 log mJ/cm(2). In the visible spectrum, the radiation dosages required for a one-log reduction of the E. coli cell density at 458 and 488 nm were 5.5 and 6.9 log mJ/cm(2), respectively. However, at 515 and 532 nm, significant killing was not observed at radiation dosage up to 7 log mJ/cm(2). Based on the cell survival data at various radiation dosages between 250 and 488 nm, a predictive equation for the survival of E. coli cells is derived, namely log(S/S(0)) = -(1.089 x 10(7) e(-0.0633lambda))D. The symbols, S(0), S, lambda, and D, represent initial cell density, cell density after irradiation, wavelength of the radiation and radiation dosage, respectively. The proportion of the surviving E. coli cells decreases exponentially with the increase in radiation dosage at a given wavelength. In addition, the radiation dose required for killing a certain fraction of the E. coli cells increases exponentially as the wavelength of radiation increases.     

The xthA gene product of Archaeoglobus fulgidus is an unspecific DNase.

A thermostable enzyme from the hyperthermophilic sulphate-reducing archaeon, Archaeoglobus fulgidus, was expressed and characterized on the assumption that it is homologous to exonuclease III from Escherichia coli. Sequence similarity database searches were performed based on the amino acid sequence of exonuclease III. The 774 bp long gene was isolated from a culture sample and cloned into different vectors. Expression proved successful by transforming pET28_Af_Exo in Origami B(DE3) containing a tRNA plasmid with extra copies of argU, ileY and leuW tRNA genes as a host strain. The lack of thioredoxin reductase (trxB) and glutathione reductase (gor) in Origami B(DE3) allowed formation of disulfide bridges in the cytosol. Purification was performed by heat treatment of the soluble fraction at 80 degrees C for 30 min followed by a two-step ion exchange chromatography. The activity of the enzyme could be maintained. Optimal activity was achieved at 80 degrees C and at a pH of 7. Within the characterization of the protein we could not find any data verifying exonucleolytic activity in the presence of Mg2+ as described [Ankenbauer, W., Laue, F., Sobek, H., & Greif, M. (2000), patent number WO2001023583]. Instead strong DNA binding properties of the enzyme and nicking activities of double stranded DNA comparable to unspecific DNases could be observed. In contrast to exonuclease III from Escherichia coli, the xthA gene product of Archaeoglobus fulgidus is able to degrade supercoiled plasmids and shows no preferences for blunt or recessed 3'-termini of linear double stranded DNA. The enzyme is inhibited by EDTA and shows only weak activity when replacing Mg2+ with Ca2+ ions.     

Identification of microsatellite markers for fragrance in rice by analysis of the rice genome sequence

Several chemical constituents are important to the fragrance of cooked rice. However, the chemical compound 2-acetyl-1-pyrroline (AP) is regarded as the most important component of fragrance in the basmati- and jasmine-style fragrant rices. AP is found in all parts of the plant except the roots. It is believed that a single recessive gene is responsible for the production of fragrance in most rice plants. The detection of fragrance can be carried out via sensory or chemical methods, although each has their disadvantages. To overcome these difficulties, we have identified an (AT)₄₀ repeat microsatellite or simple sequence repeat (SSR) marker for fragrant and non-fragrant alleles of the fgr gene. Identification of this marker was facilitated through use of both the publicly available and restricted access sequence information of the Monsanto rice sequence databases. Fifty F₂ individuals from a mapping population were genotyped for the polymorphic marker. This marker has a high polymorphism information content (PIC = 0.9). Other SSR markers linked to fragrance could be identified in the same way of use in other populations. This study demonstrates that analysis of the rice genome sequence is an effective option for identification of markers for use in rice improvement.     

Cell cycle: how, when and why cells get rid of cyclin A.

Sequences outside the 'destruction box' direct the degradation of cyclin A to completion before the metaphase-anaphase transition; cyclin A that escapes timely degradation can block the metaphase-anaphase transition, impede anaphase and telophase, and impair a cell's ability to arrest in G1 of the next cell cycle.