Evaluation of an ATP Assay to Quantify Bacterial Attachment to Surfaces in Reduced Gravity

Aim: To develop an assay to quantify the biomass of attached cells and biofilm formed on wetted surfaces in variable-gravity environments. Methods and Results: Liquid cultures of Pseudomonas aeruginosa were exposed to 30-35 brief cycles of hypergravity (< 2-g) followed by free fall (i.e., reduced gravity) equivalent to either lunar-g (i.e., 0.17 normal Earth gravity) or micro-g (i.e., < 0.001 normal Earth gravity) in an aircraft flying a series of parabolas. Over the course of two days of parabolic flight testing, 504 polymer or metal coupons were exposed to a stationary-phase population of P. aeruginosa strain ERC1 at a concentration of 1.0 x 10(exp 5) cells per milliliter. After the final parabola on each flight test day, half of the material coupon samples were treated with either 400 micro-g/L ionic silver fluoride (microgravity-exposed cultures) or 1% formalin (lunar-gravity-exposed cultures). The remaining sample coupons from each flight test day were not treated with a fixative. All samples were returned to the laboratory for analysis within 2 hours of landing, and all biochemical assays were completed within 8 hours of exposure to variable gravity. The intracellular ATP luminescent assay accurately reflected cell physiology compared to both cultivation-based and direct-count microscopy analyses. Cells exposed to variable gravity had more than twice as much intracellular ATP as control cells exposed only to normal Earth gravity

Comparison of Sample and Detection Quantification Methods for Salmonella Enterica from Produce

The purpose of this study was to identify and optimize fast and reliable sampling and detection methods for the identification of pathogens that may be present on produce grown in small vegetable production units on the International Space Station (ISS), thus a field setting. Microbiological testing is necessary before astronauts are allowed to consume produce grown on ISS where currently there are two vegetable production units deployed, Lada and Veggie

Increased glomerular cell heparan sulfates in vitro by ciclosporin A: a Possible explanation of Its beneficial effect in idiopathic nephrotic syndrome

BACKGROUND/AIM: In idiopathic nephrotic syndrome (INS), ciclosporin A (CsA) was shown to decrease proteinuria, an effect explained by its immunologic and hemodynamic actions. In order to determine whether CsA could have a direct action on glomerular cells, we studied the effect of CsA on glomerular cells in vitro, particularly on glycosaminoglcycans (GAG) and heparan sulfates (HS) which are decreased in INS patients. METHODS: Human glomerular epithelial cells and rat mesangial cells were cultured at various concentrations of CsA. HS were quantified using a cationic membrane after metabolic labeling. RESULTS: Mesangial cell GAG and HS and epithelial cell HS increased significantly when cells were cultured with CsA. For both cell types this increase was prevailing on the secreted fraction of HS in comparison with the cellular fraction. CsA induced also an increase in cellular cAMP levels, but the effect of CsA was not transduced via a cAMP pathway. CONCLUSIONS: CsA is able to increase glomerular GAG and HS in vitro. As this effect of CsA was the opposite effect on glomerular cells to the effect of plasma from INS patients, we conclude that this direct action of CsA on glomerular cells could explain in part the effect of this drug in decreasing proteinuria in INS

Synthesis of anticoagulantly active heparan sulfate proteoglycans by glomerular epithelial cells involves multiple 3-O-sulfotransferase isoforms and a limiting precursor pool.

Endothelial and other select cell types synthesize a subpopulation of heparan sulfate (HS) proteoglycans (HSPGs), anticoagulant HSPGs (aHSPGs) that bear aHS-HS chains with the cognate 3-O-sulfated pentasaccharide motif that can bind and activate anti-thrombin (AT). Endothelial cells regulate aHSPG production by limiting levels of HS 3-O-sulfotransferase-1 (3-OST-1), which modifies a non-limiting pool of aHS-precursors. By probing kidney cryosections with (125)I-AT and fluorescently tagged AT we found that the glomerular basement membrane contains aHSPGs, with the staining pattern implicating synthesis by glomerular epithelial cells (GECs). Indeed, cultured GECs synthesized aHS with high AT affinity that was comparable with the endothelial product. Disaccharide analyses of human GEC (hGEC) HS in conjunction with transcript analyses revealed that hGECs express predominantly 3-OST-1 and 3-OST-3(A). aHS production has not been previously examined in cells expressing multiple 3-OST isoforms. This unusual situation appears to involve novel mechanisms to regulate aHS production, as HS structural analyses suggest hGECs exhibit excess levels of 3-OST-1 and an extremely limiting pool of aHS-precursor. A limiting aHS-precursor pool may serve to minimize aHS synthesis by non-3-OST-1 isoforms. Indeed, we show that high in vitro levels of 3-OST-3(A) can efficiently generate aHS. Non-3-OST-1 isoforms can generate aHS in vivo, as the probing of kidney sections from 3-OST-1-deficient mice revealed GEC synthesis of aHSPGs. Surprisingly, Hs3st1(-/-) kidney only expresses 3-OST isoforms having a low specificity for aHS synthesis. Thus, our analyses reveal a cell type that expresses multiple 3-OST isoforms and produces minimal amounts of aHS-precursor. In part, this mechanism should prevent aHS overproduction by non-3-OST-1 isoforms. Such a role may be essential, as 3-OST isoforms that have a low specificity for aHS synthesis can generate substantial levels of aHSPGs in vivo

In vitro decrease of glomerular heparan sulfate by lymphocytes from idiopathic nephrotic syndrome patients

BACKGROUND: Lymphocytes are involved in the physiopathologic mechanism of idiopathic nephrotic syndrome (INS). We have recently demonstrated that plasma from patients with INS decreases human glomerular epithelial cell (GEC) glycosaminoglycans (GAGs), particularly heparan sulfates (HS) in vitro. In this study we investigate the effect of peripheral blood lymphocytes (PBL) from INS patients on glomerular cell GAG and HS. METHODS: Human GECs were cultured with total peripheral blood mononuclear cells (PBMCs), PBL, and monocytes from patients and controls. The amounts of GAG and HS were assessed using a cationic membrane after metabolic labeling. RESULTS: In coculture with GECs, mononuclear cells from controls decreased total epithelial cell GAG (-30% with PBMC, P &lt; 0.05; -25% with PBL, P &lt; 0.02; -19% with monocytes, P &lt; 0.05). Particularly HSs were decreased (-36% with PBMC, P &lt; 0.05; -27% with PBL, P &lt; 0.02; and -19% with monocytes, P &lt; 0.05). When GECs were in coculture with PBL from INS patients, the decrease in GAG and HS was significantly greater in comparison to control PBL (-10%, P &lt; 0.02; -10%, P &lt; 0.02, respectively, for GAG and HS). Moreover, supernatants of stimulated PBMCs from patients decreased also GAG and HS in comparison with controls (-13%, P &lt; 0.02; -15%, P &lt; 0.02, respectively, for GAG and HS). CONCLUSION: These data provide direct evidence that PBLs from INS patients are able to decrease GEC HS as previously shown with plasma from patients. This might be instrumental in the onset of albuminuria

Effect of plasma from patients with idiopathic nephrotic syndrome on proteoglycan synthesis by human and rat glomerular cells

In vivo and in vitro findings have shown that plasma of patients with idiopathic nephrotic syndrome (INS) contain factors that increase glomerular permeability to proteins. The effects of these factors on proteoglycan synthesis by glomerular cells are unknown. To investigate the effect of plasma from patients with INS (n = 23) and other glomerulopathies (n = 12) on the amount of proteoglycans synthesized by cultured rat mesangial cells and human glomerular epithelial cells, glomerular cells were cultured for 24 h with plasma from patients or control subjects, and incorporation of Na2(35)SO4 in chondroitin dermatan sulfate and heparan sulfate was assessed using a cationic nylon membrane. The mean ratio of glycosaminoglycan produced by rat mesangial cells when in contact with plasma (5%) from INS patients to the amount produced when in contact with control plasma was 0.70+/-0.06. The mean ratio of heparan sulfate was 0.58+/-0.08. The decrease of heparan sulfate production was present in the cellular and in the extracellular fraction. It was observed when the cells were in contact with plasma from patients in relapse but not when in remission. No decrease of heparan sulfate production was observed with four of the five patients with membranous glomerulonephritis (ratio of 1.27+/-0.03), IgA nephropathy (n = 5, ratio of 1.27+/-0.03), and membranoproliferative glomerulonephritis (n = 2, ratio of 1.39+/-0.34). When human glomerular epithelial cells were exposed to 5% plasma from INS patients in relapse (n = 9), the mean ratio of heparan sulfate was 0.62+/-0.06 in the cellular fraction and 0.72+/-0.08 in the medium. When in contact with plasma from patients in remission, no difference of glycosaminoglycan production was observed. A factor present in plasma from patients with INS during initial episodes or relapses is able to decrease the proteoglycan production of glomerular cells