The concerted action of SEPT9 and EPLIN modulates the adhesion and migration of human fibroblasts.

Septins are cytoskeletal proteins that participate in cell adhesion, migration, and polarity establishment. The septin subunit SEPT9 directly interacts with the single LIM domain of epithelial protein lost in neoplasm (EPLIN), an actin-bundling protein. Using a human SEPT9 KO fibroblast cell line, we show that cell adhesion and migration are regulated by the interplay between both proteins. The low motility of SEPT9-depleted cells could be partly rescued by increased levels of EPLIN. The normal organization of actin-related filopodia and stress fibers was directly dependent on the expression level of SEPT9 and EPLIN. Increased levels of SEPT9 and EPLIN enhanced the size of focal adhesions in cell protrusions, correlating with stabilization of actin bundles. Conversely, decreased levels had the opposite effect. Our work thus establishes the interaction between SEPT9 and EPLIN as an important link between the septin and the actin cytoskeleton, influencing cell adhesion, motility, and migration.

An Interaction Network of the Human SEPT9 Established by Quantitative Mass Spectrometry.

Septins regulate the organization of the actin cytoskeleton, vesicle transport and fusion, chromosome alignment and segregation, and cytokinesis in mammalian cells. SEPT9 is part of the core septin hetero-octamer in human cells which is composed of SEPT2, SEPT6, SEPT7, and SEPT9. SEPT9 has been linked to a variety of intracellular functions as well as to diseases and diverse types of cancer. A targeted high-throughput approach to systematically identify the interaction partners of SEPT9 has not yet been performed. We applied a quantitative proteomics approach to establish an interactome of SEPT9 in human fibroblast cells. Among the newly identified interaction partners were members of the myosin family and LIM domain containing proteins. Fluorescence microscopy of SEPT9 and its interaction partners provides additional evidence that SEPT9 might participate in vesicle transport from and to the plasma membrane as well as in the attachment of actin stress fibers to cellular adhesions.

Deletion of the Aspergillus fumigatus lysine biosynthesis gene lysF encoding homoaconitase leads to attenuated virulence in a low-dose mouse infection model of invasive aspergillosis.

Aspergillus fumigatus is an important pathogen of the immunocompromised host, causing pneumonia and invasive disseminated disease with high mortality. In order to determine the importance of lysine biosynthesis for growth and pathogenicity, the A. fumigatus lysF gene, encoding a homologue of the A. nidulans homoaconitase LysF, was cloned and characterized. Cosmid cosGTM encoding lysF complemented a lysF mutant of Aspergillus nidulans. A. fumigatus lysF was deleted, resulting in a lysine-auxotroph. This phenotype was complemented to the wild-type by supplementation of the medium with both L-lysine and alpha-aminoadipic acid, or transformation using cosmid cosGTM. To study the virulence of the lysF deletion mutant of A. fumigatus, a low-dose intranasal mouse infection model of invasive aspergillosis was optimized for immunosuppressed BALB/c mice, allowing the application of an infection dose as low as 5 x 10(3) conidia per mouse. In this murine model, the Delta lysF mutant was avirulent, suggesting that lysine biosynthesis, or at least a functional homoaconitase, is important for survival of A. fumigatus in vivo and a potential target for antifungal drugs.

Pulmonary inflammation in rats after intratracheal instillation of quartz, amorphous SiO2, carbon black, and coal dust and the influence of poly-2-vinylpyridine-N-oxide (PVNO).

Effects of poly-2-vinylpyridine-N-oxide (PVNO) were investigated in numerous in vivo and in vitro studies published in the nineteen sixties and seventies. These studies showed that PVNO inhibited development of fibrosis from quartz dust and improved lung clearance of quartz after inhalation exposure. Ameliorating effects of PVNO were observed also for pulmonary damage from colloidal SiO2 and organic substances, and the fibrogenic inflammation caused by carrageenan. Although it is not proven that silicosis is a precondition for quartz-induced lung tumours, we investigated the hypothesis that PVNO could reduce the lung tumour risk from quartz in rats. A carcinogenicity study was therefore started in rats with the main focus on the quantitative relationships among pulmonary inflammation, fibrosis and neoplasia caused by intratracheal instillation of 3 mg quartz DQ 12 with or without additional subcutaneous PVNO treatment. Other study groups were treated with multiple dust instillations, i.e. 30 instillations of 0.5 mg amorphous SiO2 at intervals of 2 weeks, 10 instillations of 0.5 mg of ultrafine carbon black or 1 mg coal at weekly intervals. The analyses of the bronchoalveolar lavage fluid (BALF) 9 months after start of the life-time study showed that the aim of producing similar levels of increased enzyme concentrations in the four groups treated with quartz/PVNO, amorphous SiO2, carbon black and coal was achieved. A 2.5- to 7.7-fold increase for lactate dehydrogenase (LDH), total protein, alkaline phosphatase and gamma-glutamyl transferase (gamma-GT) was found in these groups as compared to the control. In contrast, quartz treatment without PVNO increased the LDH level up to 24-fold and of total protein to 13-fold. However, the cell counts in the BALF were not so much different in all five groups, i.e. quartz without PVNO (leukocytes: 480.000, PMN: 190.000), quartz with PVNO (leukocytes: 300.000, PMN: 100.000), amorphous SiO2 (leukocytes: 570.000, PMN: 315.000), carbon black (leukocytes: 390.000, PMN: 150.000) and coal (leukocytes: 200.000, PMN: 65.000). Histopathological investigations after four weeks and three months revealed that the used PVNO sample was active in the quartz and amorphous SiO2 groups and markedly reduced the incidences or severity of several pulmonary changes such as macrophage accumulation, inflammatory cell infiltration, interstitial fibrosis, bronchiolo-alveolar hyperplasia, alveolar lipoproteinosis and amorphous SiO2 -induced granulomatous alveolitis/interstitial fibrotic granulomas. Also in the lung-associated lymph nodes (LALN), PVNO treatment significantly reduced the incidence and severity of inflammation in both quartz and amorphous SiO2 groups as evidenced by the presence of well-circumscribed aggregates of intact particle-laden macrophages without signs of degeneration and accompanying granulocytic infiltration and fibrosis. Immunological investigations at the 9 months timepoint on the in vitro production of reactive nitrogen (RNI) or oxygen (ROI) intermediates and tumour necrosis factor (TNF-alpha) from BALF-derived cells indicated a diminished responsiveness to LPS in all particle treatment groups. A diminished production of ROI was also found in the quartz, carbon black, and coal dust groups, respectively, as compared to the values seen in the quartz/PVNO- and amorphous SiO2 treated groups. Treatment with quartz plus PVNO restored the capability of the cells to respond to LPS as compared to the treatment with quartz alone. TNF-alpha production was diminished in the groups treated with quartz, carbon black, and coal dust alone whereas in the quartz/PVNO- and amorphous SiO2-treated groups an elevated TNF-alpha production was seen. These results led to the conclusion that only amorphous SiO2 did not affect the "normal" ability of the cells to respond to LPS and that PVNO protected the cells from a toxic effect of the quartz particles.

Noninvasive measurement of midexpiratory flow indicates bronchoconstriction in allergic rats.

This study was designed to evaluate the value and applicability of tidal breathing pattern analysis to assess bronchoconstriction in conscious rats. Using noninvasive, head-out body plethysmography and the decrease in tidal midexpiratory flow (EF(50)), we measured airway responsiveness (AR) to inhaled acetylcholine and allergen in conscious Brown-Norway rats, followed by invasive determination of pulmonary conductance (GL) and EF(50) in anesthetized rats. Dose-response studies to acetylcholine showed that noninvasively recorded EF(50) closely reflected the dose-dependent decreases observed with the invasive monitoring of simultaneously measured GL and EF(50). After sensitization and intratracheal boost to ovalbumin or saline, rats were assessed for early and late AR to aerosolized ovalbumin. Ovalbumin aerosol challenge resulted in early and late AR in allergen-sensitized rats, whereas controls were unresponsive. The allergen-specific AR, as measured noninvasively by EF(50), was similar in degree compared with invasively recorded EF(50) and GL and was associated with enhanced IgE and airway inflammation. We conclude that EF(50) is a noninvasive and physiologically valid index of bronchoconstriction in a rat model of asthma.