Saline culture of crops: a genetic approach.

Increasing salinity of soil and water threatens agriculture in arid and semiarid regions. By itself, the traditional engineering approach to the problem is no longer adequate. Genetic science offers the possibility of developing salt-tolerant crops, which, in conjunction with environmental manipulation, could improve agricultural production in saline regions and extend agriculture to previously unsuited regions.

The phospholipid flippase DnfD localizes to late Golgi and is involved in asexual differentiation in Aspergillus nidulans.

The maintenance of cell shape requires finely tuned and robust vesicle trafficking in order to provide sufficient plasma membrane materials. The hyphal cells of filamentous fungi are an extreme example of cell shape maintenance due to their ability to grow rapidly and respond to the environment while keeping a relatively consistent shape. We have previously shown that two phospholipid flippases, which regulate the asymmetry of specific phospholipids within the plasma membrane, are important for hyphal growth in Aspergillus nidulans. Here, we examine the rest of the phospholipid flippases encoded by A. nidulans by obtaining single and double deletions of all four family members, dnfA, dnfB, dnfC, and dnfD. We find that deleting dnfC does not impart a noticeable phenotype, by itself or with other deletions, but that dnfD, the homolog of the essential yeast gene neo1, is important for conidiation. dnfD deletion mutants form misshapen conidiophore vesicles that are defective in metulae formation. We localize DnfD to late Golgi equivalents, where it appears just before dissociation of this organelle. We propose that DnfD functions in a trafficking process that is specifically required for the morphological changes that take place during conidiation.

Regulation of adenovirus-mediated elafin transgene expression by bacterial lipopolysaccharide.

Lipopolysaccharide (LPS) is a mediator of inflammatory lung injury. Selective augmentation of host defense molecules such as elafin (an elastase inhibitor with antimicrobial activity) at the onset of pulmonary inflammation is an attractive potential therapeutic strategy. The aim of this study was to determine whether elafin expression could be induced by LPS administered after transfection with adenovirus (Ad) encoding human elafin downstream of the murine cytomegalovirus (CMV) promoter (known to be potentially responsive to LPS). In addition, we aimed to determine the effect of local elafin augmentation on neutrophil migration to the lung. LPS significantly up-regulated elafin expression from pulmonary epithelial cells transfected with Ad-elafin in vitro. In murine airways expression of human elafin was achieved using doses low enough (3 x 10(7) plaque forming units) to circumvent overt vector-induced inflammation. LPS significantly up-regulated human elafin secretion in murine airways treated with Ad-elafin [117 ng/ml in bronchoalveolar lavage fluid (BALF) after LPS administration, 5.9 ng/ml after PBS, p < 0.01)]. Over-expression of elafin significantly augmented LPS-mediated neutrophil migration into the airways in vivo (1.30 x 10(6) neutrophils in BALF after Ad-elafin/LPS treatment, 0.54 x 10(6) after Ad-lacZ/LPS (p < 0.05), 0.63 x 10(6) after PBS/LPS (p < 0.05)) and significantly enhanced human neutrophil migration in vitro. These data suggest novel functions for elafin in neutrophil migration, and that judicious selection of promoters may allow single, low-dose adenoviral administration to effect inflammation-specific expression of potentially therapeutic transgenes.

Restricted spatiotemporal expression of lactoferrin during murine embryonic development.

Lactoferrin is a member of the transferrin family of iron-binding glycoproteins. Lactoferrin is induced by estrogen in the mouse uterus during early pregnancy. However, the expression and function, if any, of lactoferrin in the preimplantation embryo during this developmental period has not been investigated. In the current study, the spatiotemporal expression of lactoferrin during murine embryogenesis was examined using in situ hybridization and immunohistochemical analyses. Lactoferrin expression was first detected in the 2-4 cell fertilized embryo and continued until the blastocyst stage of development. Interestingly, at the 16-cell stage, coinciding with the first major differentiation step in the embryo, lactoferrin messenger RNA (mRNA) is synthesized by the inner cells, whereas the protein is selectively taken up by the outside cells. This differential pattern of lactoferrin messenger RNA and protein localization continues until the blastocyst stage, with expression almost absent in the hatched blastocyst. Lactoferrin expression does not resume in the embryo until the latter half of gestation, where it is first detected in neutrophils of the fetal liver at embryonic day 11.5 and later in epithelial cells of the respiratory and digestive systems. Our results show that lactoferrin is expressed in a tightly regulated spatiotemporal manner during murine embryogenesis and suggest a novel paracrine role for this protein in the development of the trophoectodermal lineage during preimplantation development.

Human neutrophil elastase regulates the expression and secretion of elafin (elastase-specific inhibitor) in type II alveolar epithelial cells.

Elafin is a low molecular weight antiproteinase believed to be important in the regulation of elastase mediated tissue damage. The expression of elafin is known to be regulated by proinflammatory cytokines such as interleukin-1 beta and tumour necrosis factor but little was known regarding the effect of human neutrophil elastase (HNE). Employing a chloramphenicol acetyltransferase reporter construct of the human elafin gene, reverse transcription PCR from total cellular RNA and ELISA techniques, we have examined the effect of human neutrophil elastase on the transcription and secretion of human elafin in the pulmonary epithelial A549 cell line. Stimulation with HNE at concentrations of 10(-10) and 10(-11) M resulted in a significant upregulation of elafin promoter activity. Similarly, transcription of the endogenous human elafin gene was upregulated with HNE concentrations ranging from 10(-10) to 10(-12) M. In addition, we demonstrate that stimulation with HNE at concentrations ranging from 10(-9) and 10(-12) M resulted in a significant reduction in the secreted elafin protein as measured in the cell supernatant. These results provide further evidence for a role of elafin in the regulation of HNE driven proteolysis of the extracellular matrix.

Murine strongyloidiasis: the effects of cyclosporin A and thiabendazole administered singly and in combination.

A single Cyclosporin A (CsA) dose of 30 mg kg-1 given orally at day 4 post-infection (p.i.) to Sprague-Dawley rats infected with Strongyloides ratti, reduced the faecal larval count by 46.8 +/- 1.2%. CsA was equally effective when the same dose rate was administered subcutaneously at day 4 p.i., reducing the faecal larval count by 41.6 +/- 8.6%. Thiabendazole (TBZ) given orally at 5 or 10 mg kg-1 (single dose at day 4 p.i.) reduced the faecal larval counts by 57.1 +/- 4.1% and 69.0 +/- 9.6%, respectively. Orally administered CsA was less effective than 5 mg TBZ kg-1 (at day 4 p.i.) Co-administration of 5 mg TBZ kg-1 and CsA did not elicit synergy or additive efficacy, indicating that CsA did not antagonise the anti-strongyloides activity of TBZ. The data suggests that for patients with current, historical or serological evidence of strongyloidiasis, CsA may be used where immunosuppressive therapy is required for other concurrent reasons or when TBZ is contraindicated.

A system for production of commercial quantities of human lactoferrin: a broad spectrum natural antibiotic.

We previously reported the production of limited quantities of biologically active recombinant human lactoferrin in the filamentous fungus Aspergillus oryzae. In the present study, we report a modification of this production system combined with a classical strain improvement program that has enabled production of levels of recombinant human lactoferrin in excess of 2 g/l. The protein was expressed in Aspergillus awamori as a glucoamylase fusion polypeptide which was secreted into the growth medium and processed to mature human lactoferrin by an endogenous KEX-2 peptidase. The recombinant protein retains full biological activity in terms of its ability to bind iron and human enterocyte receptors. Furthermore, the recombinant protein functions as a potent broad spectrum antimicrobial protein.

Structural organization of the mouse lactoferrin gene.

The complete structure of the mouse lactoferrin gene is presented. Mouse lactoferrin (mLF) is encoded by a single copy gene of approximately 30 kilobases (kb) in size. The gene is organized into 17 exons separated by 16 introns. The exons range in size from 48 base pairs (bp) to 190 bp whereas the introns range from 0.2 kb to 4.3 kb. Structural analysis of the mouse lactoferrin gene reveals that this gene shares a similar intron-exon distribution pattern with both human transferrin and chicken ovotransferrin.

Regulation of pulmonary and systemic bacterial lipopolysaccharide responses in transgenic mice expressing human elafin.

The control of lung inflammation is of paramount importance in a variety of acute pathologies, such as pneumonia, the acute respiratory distress syndrome, and sepsis. It is becoming increasingly apparent that local innate immune responses in the lung are negatively influenced by systemic inflammation. This is thought to be due to a local deficit in cytokine responses by alveolar macrophages and neutrophils following systemic bacterial infection and the development of a septic response. Recently, using an adenovirus-based strategy which overexpresses the human elastase inhibitor elafin locally in the lung, we showed that elafin is able to prime lung innate immune responses. In this study, we generated a novel transgenic mouse strain expressing human elafin and studied its response to bacterial lipopolysaccharide (LPS) when the LPS was administered locally in the lungs and systemically. When LPS was delivered to the lungs, we found that mice expressing elafin had lower serum-to-bronchoalveolar lavage ratios of proinflammatory cytokines, including tumor necrosis factor alpha (TNF-alpha), macrophage inflammatory protein 2, and monocyte chemoattractant protein 1, than wild-type mice. There was a concomitant increase in inflammatory cell influx, showing that there was potential priming of innate responses in the lungs. When LPS was given systemically, the mice expressing elafin had reduced levels of serum TNF-alpha compared to the levels in wild-type mice. These results indicate that elafin may have a dual function, promoting up-regulation of local lung innate immunity while simultaneously down-regulating potentially unwanted systemic inflammatory responses in the circulation.

Hospital outbreak traced to a case of Norwegian scabies.

A hospital outbreak, traced to a case of Norwegian scabies, resulted in 45 and 32 symptomatic cases in staff and patients, respectively. A total of 228 asymptomatic in-patient contacts and over 500 staff required prophylactic treatment with either 1% gamma benzene hexachloride lotion or 5% permethrin cream. Recurrences occurred in seven staff members and five patients. The outbreak was contained within four months.