Green fluorescent protein-propidium iodide (GFP-PI) based assay for flow cytometric measurement of bacterial viability.

BACKGROUND: Several staining protocols have been developed for flow cytometric analysis of bacterial viability. One promising method is dual staining with the LIVE/DEAD BacLight bacterial viability kit. In this procedure, cells are treated with two different DNA-binding dyes (SYTO9 and PI), and viability is estimated according to the proportion of bound stain. SYTO9 diffuses through the intact cell membrane and binds cellular DNA, while PI binds DNA of damaged cells only. This dual-staining method allows effective separation between viable and dead cells, which is far more difficult to achieve with single staining. Although SYTO9-PI dual staining is practical for various bacterial viability analyses, the method has a number of disadvantages. Specifically, the passage of SYTO9 through the cell membrane is a slow process, which is significantly accelerated when the integrity of the cell membrane is disrupted. As a result, SYTO9 binding to DNA is considerably enhanced. PI competes for binding sites with SYTO9 and may displace the bound dye. These properties diminish the reliability of the LIVE/DEAD viability kit. In this study, we investigate an alternative method for measuring bacterial viability using a combination of green fluorescent protein (GFP) and PI, with a view to improving data reliability. METHODS: Recombinant Escherichia coli cells with a plasmid containing the gene for jellyfish GFP were stained with PI, and green and red fluorescence were measured by FCM. For comparison, cells containing the plasmid from which gfp was removed were stained with SYTO9 and PI, and analyzed by FCM. Viability was estimated according to the proportion of green and red fluorescence. In addition, bioluminescence and plate counting (other methods to assess viability) were used as reference procedures. RESULTS: SYTO9-PI dual staining of bacterial cells revealed three different cell populations: living, compromised, and dead cells. These cell populations were more distinct when the GFP-PI combination was used instead of dual staining. No differences in sensitivity were observed between the two methods. However, substitution of SYTO9 with GFP accelerated the procedure. Bioluminescence and plate counting results were in agreement with flow cytometric viability data. CONCLUSIONS: In bacterial viability analyses, the GFP-PI combination provided better distinction between current viability stages of E. coli cells than SYTO9-PI dual staining. Additionally, the overall procedure was more rapid. No marked differences in sensitivity were observed.     

Isolation and Characterization of a Hantavirus from Lemmus sibiricus: Evidence for Host Switch during Hantavirus Evolution

A novel hantavirus, first detected in Siberian lemmings (Lemmus sibiricus) collected near the Topografov River in the Taymyr Peninsula, Siberia (A. Plyusnin et al., Lancet 347:1835-1836, 1996), was isolated in Vero E6 cells and in laboratory-bred Norwegian lemmings (Lemmus lemmus). The virus, named Topografov virus (TOP), was most closely related to Khabarovsk virus (KBR) and Puumala viruses (PUU). In a cross focus reduction neutralization test, anti-TOP Lemmus antisera showed titers at least fourfold higher with TOP than with other hantaviruses; however, a rabbit anti-KBR antiserum neutralized TOP and KBR at the same titer. The TOP M segment showed 77% nucleotide and 88% amino acid identity with KBR and 76% nucleotide and 82% amino acid identity with PUU. However, the homology between TOP and the KBR S segment was disproportionately higher: 88% at the nucleotide level and 96% at the amino acid level. The 3' noncoding regions of KBR and the TOP S and M segments were alignable except for 113- and 58-nucleotide deletions in KBR. The phylogenetic relationships of TOP, KBR, and PUU and their respective rodent carriers suggest that an exceptional host switch took place during the evolution of these viruses; while TOP and KBR are monophyletic, the respective rodent host species are only distantly related.     

Basic FGF regulates interstitial collagenase gene expression in human smooth muscle cells

Basic fibroblast growth factor (bFGF) is a mitogenic factor that is implicated in smooth muscle cell growth in atherosclerosis and vascular restenosis. In this study, we examined the effect of bFGF on the expression of the interstitial collagenase gene in human vascular smooth muscle cells. Results from Northern transfer analysis showed that bFGF increased collagenase mRNA levels greater than threefold as early as 24 h. Collagenase pre-mRNA levels were elevated approximately threefold by bFGF, according to RT-PCR analysis. Transient transfections of the smooth muscle cells with a 4.4-kb human collagenase promoter-CAT reporter gene, however, failed to show upregulation of the promoter activity by bFGF. Interestingly, transfections with deleted fragments containing promoter sequences from -1047 to -2271 resulted in modest stimulation of the collagenase-CAT promoter activity by bFGF. bFGF did not alter the stability of the collagenase mRNA, as demonstrated by degradation studies. The enhanced collagenase mRNA levels elicited by bFGF were reflected in increased amounts of collagenase protein that were detected by Western blot analysis. In summary, bFGF upregulates the interstitial collagenase expression, resulting in turnover of the extracellular matrix, an event that could facilitate smooth muscle cell migration and proliferation during the early stages of atherosclerosis and restenosis. J. Cell. Biochem. 65:32–41. © 1997 Wiley-Liss, Inc.     

Body Weight and Health Status: Importance of Socioeconomic Position and Working Conditions

Objective: To examine the association between relative body weight and health status and the potential modifying effects of socioeconomic position and working conditions on this association. Research Methods and Procedures: The data were derived from three identical cross‐sectional surveys conducted in 2000, 2001, and 2002. Respondents to postal surveys were middle‐aged employees of the City of Helsinki (7148 women and 1799 men, response rate 67%). BMI was based on self‐reported weight and height. Health status was measured by the Short‐Form 36 subscales and component summaries. Results: Body weight was inversely associated with physical health, but in mental health, differences between BMI categories were small and inconsistent. In women, physical health deteriorated monotonically with increasing BMI, whereas in men, poor physical health was found among the obese only. Socioeconomic position did not modify the association between BMI and health. In women, the association between body weight and physical health became stronger with decreasing job control and increasing physical work load, whereas in men, a similar modifying effect was found for high job demands. Discussion: Body weight was associated with physical health only. Lower levels of relative weight in women than in men may be associated with poor physical health. High body weight combined with adverse working conditions may impose a double burden on physical health.     

Responses of ground vegetation species to clear-cutting in a boreal forest: aboveground biomass and nutrient contents during the first 7 years

Rapid growth of ground vegetation following clear-cutting is important to site productivity because vegetation retains nutrients in the ecosystem and can decrease nutrient leaching prior to stand re-establishment. Aboveground biomass, nutrient contents (N, P, K and Ca) and species composition of ground vegetation were determined 1 year before and for 7 years after clear-cutting of a mixed forest dominated by Norway spruce [Picea abies (L.) H. Karst.] in eastern Finland. The biomass of the feather mosses [Pleurozium schreberi Brid. and Hylocomium splendens (Hedw.) B. S.& G.] and the dwarf shrubs (Vaccinium myrtillus L. and V. vitis-idaea L.), which had dominated the ground vegetation in the mature forest, significantly decreased after clear-cutting. However, with the exception of H. splendens, these species had recovered within 3–5 years. The biomass of Deschampsia flexuosa (L.) Trin. considerably increased soon after clear-cutting, and Epilobium angustifolium L. appeared 3–5 years after cutting. These species contributed to the retention of nutrients not simply because of their biomass but also because of higher nutrient concentrations in their tissues. Total biomass and nutrient contents of the ground vegetation exceeded those of the pre-cutting levels. The proportion of ground vegetation biomass and nutrient contents represented by mosses decreased after cutting, while V. myrtillus, although reduced after cutting, remained a marked nutrient sink. The results suggest that H. splendens is the most sensitive species to cutting, but the biomass of P. schreberi, V. myrtillus and V. vitis-idaea return to initial levels soon after clear-cutting as do the nutrient contents of ground vegetation.     

Changes in the Above- and Below-ground Biomass and Nutrient Pools of Ground Vegetation After Clear-cutting of a Mixed Boreal Forest

Ground vegetation may act as a sink for nutrients after clear-cutting and thus decrease leaching losses. Biomass and nutrient (N, P, K, Ca) pools of ground vegetation (mosses, roots and above-ground parts of field layer) were determined one year before and five years after clear-cutting of a Norway spruce (Picea abies (L.) H. Karst.) dominated boreal mixed forest stand in eastern Finland (63°51′ N, 28°58′ E). Before clear-cutting the average biomass of ground vegetation was 5307 kg ha⁻¹, with nutrient contents of 46.9 kg N ha⁻¹1, 4.1 kg P ha⁻¹1, 16.2 kg K ha⁻¹1 and 13.9 kg Ca ha⁻¹1. The biomass and nutrient pools decreased after clear-cutting being lowest in the second year, the biomass decreasing by 46–65% in the cut plots. The nutrient pools decreased as follows: N 54–72%, P 36–68%, K 51–71% and Ca 57–74%. The decrease in ground vegetation nutrient uptake, and the observed reduced depth of rooting may decrease nutrient retention after clear-cutting and decomposing dead ground vegetation is a potential source of leached nutrients. These negative effects of clear-cutting on the nutrient binding capacity of ground vegetation was short-lived since the total biomass and nutrient pools returned to pre-cutting levels or were even greater by the end of the 5-year study period.     

Mutations of Factor H Impair Regulation of Surface-bound C3b by Three Mechanisms in Atypical Hemolytic Uremic Syndrome

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy associated with mutations in complement proteins, most frequently in the main plasma alternative pathway regulator factor H (FH). The hotspot for the FH mutations is in domains 19–20 (FH19–20) that are indispensable for FH activity on C3b bound covalently to host cells. In aHUS, down-regulation of cell-bound C3b by FH is impaired, but it is not clear whether this is due to an altered FH binding to surface-bound C3b or to cell surface structures. To explore the molecular pathogenesis of aHUS we tested binding of 14 FH19–20 point mutants to C3b and its C3d fragment, mouse glomerular endothelial cells (mGEnC-1), and heparin. The cell binding correlated well, but not fully, with heparin binding and the cell binding site was overlapping but distinct from the C3b/C3d binding site that was shown to extend to domain 19. Our results show that aHUS-associated FH19–20 mutants have different combinations of three primary defects: impaired binding to C3b/C3d, impaired binding to the mGEnC-1 cells/heparin, and, as a novel observation, an enhanced mGEnC-1 cell or heparin binding. We propose a model of the molecular pathogenesis of aHUS where all three mechanisms lead eventually to impaired control of C3b on the endothelial cell surfaces. Based on the results with the aHUS patient mutants and the overlap in FH19–20 binding sites for mGEnC-1/heparin and C3b/C3d we conclude that binding of FH19–20 to C3b/C3d is essential for target discrimination by the alternative pathway.Atypical hemolytic uremic syndrome (aHUS)2 is a familial disease characterized by erythrocyte fragmentation and hematuria, damaged renal endothelium, vascular microthrombi, and thrombocytopenia (1). The syndrome leads ultimately to end-stage renal disease with a high mortality rate (2). In aHUS cases point mutations have been found in complement components C3, factor B, CD46, factor I, and factor H (FH), all of which play a role in the activation or control of the alternative pathway (3–8). More than half of the mutations have been found to originate in the HF1 gene that encodes FH and FH-like protein 1.The alternative pathway is initiated spontaneously by hydrolysis of C3 to C3_H2O that forms the C3-convertase C3_H2OBb (9, 10). This enzyme complex converts numerous C3 molecules to C3b that are covalently bound onto practically any nearby surface (11). On a so-called activator surface, such as a microbe, the surface-bound C3b molecules are not efficiently eliminated and therefore new C3bBb complexes are formed leading to more C3b depositions and eventually effective opsonization or damage of the target cell. On non-activator surfaces, such as viable self (host) cells, factor I cleaves C3b to inactive C3b (iC3b) in the presence of one of the cofactors (CD46, CD35, FH, and FHL-1) (12–16). FH is the only one of these cofactors that mediates recognition of self-surfaces making the alternative pathway capable of discriminating between activating and non-activating surfaces (17–19).The two main functions of FH are to prevent the alternative pathway activation in plasma and on self-surfaces. This 150-kDa glycoprotein consists of 20 tandemly arranged short consensus repeat domains that are composed of ∼60 amino acids. Domains 1–4 are essential for the cofactor and decay accelerating activity (20). In the middle region of FH (domains 5–15) there are two binding sites for C-reactive protein (21), one or two sites for glycosaminoglycans (GAGs) (22–25), and one site for C3c part of C3b (C3b/C3c) (25, 26). The C-terminal domains 19–20 (FH19–20) possess binding sites for the thiol ester domain of C3b (C3d or C3dg, TED domain) and GAGs (26, 27).The most common types of mutations found in aHUS are FH missense mutations located within FH19–20 that was recently solved as crystal and NMR structures (2, 28, 29). The C terminus of FH is crucial in self-cell protection as demonstrated by the severity of the aHUS cases and also in a recent mouse model of aHUS where domains 16–20 had been deleted (30, 31). Histopathology of aHUS in these mice had all the characteristics of human aHUS being concordant with the similarity of binding sites for C3b, heparin, and human umbilical vein endothelial cells between human and mouse FH domains 18–20 (32). Binding of mouse or human FH to glomerular endothelial cells has not been characterized despite the fact that in aHUS damage occurs mainly in the small vessels, especially in the glomeruli.The molecular pathogenesis leading to the clinical aHUS in patients with FH mutations remains elusive. The suggested molecular mechanisms for some aHUS-associated mutations include defective binding of the mutated FH to GAGs, endothelial cells, or C3b/C3d (28, 29, 33, 34). The aim of this study was to define the effects of nine aHUS-associated FH mutations and five other structurally closely located mutations on binding of FH19–20 to C3b, C3d, mouse glomerular endothelial cells, and heparin. We identified three primary defects of the mutants: impaired C3b/C3d binding, enhanced mGEnC-1/heparin binding, and impaired mGEnC-1/heparin binding that could lead via three mechanisms to incapability of FH to eliminate C3b on plasma-exposed self-cells. The results clarify the mechanism of target discrimination of the alternative pathway by the C terminus of FH.     

Long‐term temporal and spatial trends in eutrophication status of the Baltic Sea

Much of the Baltic Sea is currently classified as ‘affected by eutrophication’. The causes for this are twofold. First, current levels of nutrient inputs (nitrogen and phosphorus) from human activities exceed the natural processing capacity with an accumulation of nutrients in the Baltic Sea over the last 50–100 years. Secondly, the Baltic Sea is naturally susceptible to nutrient enrichment due to a combination of long retention times and stratification restricting ventilation of deep waters. Here, based on a unique data set collated from research activities and long‐term monitoring programs, we report on the temporal and spatial trends of eutrophication status for the open Baltic Sea over a 112‐year period using the HELCOM Eutrophication Assessment Tool (HEAT 3.0). Further, we analyse variation in the confidence of the eutrophication status assessment based on a systematic quantitative approach using coefficients of variation in the observations. The classifications in our assessment indicate that the first signs of eutrophication emerged in the mid‐1950s and the central parts of the Baltic Sea changed from being unaffected by eutrophication to being affected. We document improvements in eutrophication status that are direct consequences of long‐term efforts to reduce the inputs of nutrients. The reductions in both nitrogen and phosphorus loads have led to large‐scale alleviation of eutrophication and to a healthier Baltic Sea. Reduced confidence in our assessment is seen more recently due to reductions in the scope of monitoring programs. Our study sets a baseline for implementation of the ecosystem‐based management strategies and policies currently in place including the EU Marine Strategy Framework Directives and the HELCOM Baltic Sea Action Plan.     

Spiny-cheek crayfish Orconectes limosus carry a novel genotype of the crayfish plague pathogen Aphanomyces astaci

The oomycete Aphanomyces astaci causes mass mortalities of European crayfish. Different species of North American crayfish, original hosts of this parasite, seem to carry different strains of A. astaci. So far, four distinct genotype groups have been recognised using Random Amplification of Polymorphic DNA (RAPD-PCR). We succeeded in isolating A. astaci from the spiny-cheek crayfish Orconectes limosus, a widespread invader in Europe, and confirmed that this species carries a novel A. astaci genotype. Improving knowledge on the diversity of this parasite may facilitate identification of genotypes in mass mortalities of European crayfish, thus tracing the sources of infection.