Activation of Plasmin in Mastitic Milk

Milk and whey samples from healthy and inflamed udder quarters of 10 Ayrshire cows were analyzed for proteolytic activity using radial caseolysis procedures, a fluorogenic coumaryl peptide substrate, and casein agarose zymography. Free lysosomal enzyme activity (N–acetyl–beta–D–glucosaminidase) was used as the criterion for inflammation. All mastitic milk samples had proteolytic activity, tentatively identified as plasmin (comigration at Mr 83 000 and characteristic fragmentation). The plasmin activities in mastitic milk were on average 2.9 μg/ml (range 0.5–12.5) as measured by radial caseolysis. Milk or whey specimens from healthy quarters were all negative except 1 in which an activity of 0.1 μg/ml was found in both specimens. The caseolytic activities were totally inhibited by 50 KITJ/ml of aprotinin, a serine proteinase inhibitor from bovine lung. No free plasminogen activator (PA) activity was found in any of the samples. Howewer, according to zymographic analyses PA molecules corresponding to urokinase were found in healthy and especially in mastitic specimens. Analysis of plasmin may provide an alternative means of screening for mastitic milk samples.     

Tubulin and actin protein patterns in Scots pine (Pinus sylvestris) roots and developing ectomycorrhiza with Suillus bovinus

The role of tubulin and actin in the development of Scots pine ( Pinus sylvestris ) roots and in the formation of the ectomycorrhiza with the basidiomycete Suillus bovinus was studied by immunoblotting of 2D-gels with anti-tubulin and anti-actin antibodies. In the short roots the α-tubulin pattern was different from that in the other root types due to the more acidic pI of the two α-tubulins. During the formation of the ectomycorrhiza, two new α-tubulins were detected in the acidic α-tubulin cluster. No such variation occurred in the plant β-tubulin patterns. The fungal tubulins dominated in the ectomycorrhiza, but no changes in tubulin polypeptide patterns from those in the S. bovinus mycelium were observed. Contrary to the tubulins, plant actin dominated in the mycorrhiza. The specific α-tubulin patterns of uninfected and infected short roots indicate that α-tubulin is involved in the morphogenesis of Pinus sylvestris short roots. The high level of plant actin at early stage of the mycorrhiza formation suggests a significant role of this protein in the interaction between plant cells and fungal hyphae.     

The central role of septa in the basidiomycete Schizophyllum commune hyphal morphogenesis

The purpose of the present research was to observe in the filamentous basidiomycete Schizophyllum commune, the connection between the nuclear division and polymerization of the contractile actin ring with subsequent formation of septa in living hyphae. The filamentous actin was visualized using Lifeact-mCherry and the nuclei with EGFP tagged histone 2B (H2B). Time-lapse fluorescence microscopy confirmed that in monokaryotic and dikaryotic hyphae, the first signs of the contractile actin ring occur at the site of the nuclear division, in one to two minutes after division. At this stage, the telophase nuclei have moved tens of micrometers from the division site. The actin ring is replaced by the septum in six minutes. The apical cells treated with filamentous actin disrupting drug latrunculin A, had swollen tips but the cells were longer than in control samples due to the absence of the actin rings. The nuclear pairing and association with clamp cell development as well as the clamp cell fusion with the subapical cell was disrupted in latrunculin-treated dikaryotic hyphae, indicating that actin filaments are involved in these processes, also regulated by the A and B mating-type genes. This suggests that the actin cytoskeleton may indirectly be a target for mating-type genes.     

Seasonal fluctuations in macrophyte cover and water transparency of four brown-water lakes: implications for crustacean zooplankton in littoral and pelagic habitats

The dynamics of crustacean zooplankton in the littoral and pelagic zones of four forest lakes having variable water qualities (colour range 130–340 mg Pt l⁻¹, Secchi depth 70–160 cm) were studied. The biomass of zooplankton was higher in the littoral zone than in the pelagic zone only in the lake having the highest transparency. In the three other lakes, biomass was significantly higher in the pelagic zone than in the littoral zone. In the two lakes with highest transparency, the littoral biomass of cladocerans significantly followed the development of macrophyte vegetation, and cladoceran biomass reached the maximum value at the time of highest macrophyte coverage. In lakes with lowest transparency, littoral zooplankton biomass developed independently of macrophyte density and decreased when macrophyte beds were densest. The seasonal development of the littoral copepod biomass did not follow the development of macrophytes in any of the lakes. The mean size of cladocerans in the pelagic zone decreased with increasing Secchi depth of the lake, whereas in the littoral zone no such phenomenon was detected. Seasonally, when water transparency increased temporarily in two of the lakes, the mean size of cladocerans in the pelagic zone decreased steeply. For copepods, no relationship between water transparency and body size was observed. The results suggested that in humic lakes the importance of the littoral zone as a refuge decreases with decreasing transparency of the water and that low water transparency protects cladocerans from fish predation. All the observed between-lake differences could not be explained by fish predation, but were probably attributed to the presence of chaoborid larvae with variable densities. Feeding efficiency of chaoborids is not affected by visibility and thus they can obscure the relationship between water quality, fish density, and the structure of crustacean zooplankton assemblages. Handling editor: S. I. Dodson     

Effect of prey type and inorganic turbidity on littoral predator–prey interactions in a shallow lake: an experimental approach

Predation often represents the prevailing process shaping aquatic ecosystems. As foraging and antipredatory behaviour frequently relate to vision, turbidity may often impair the interactions between the predator and its prey, depending on prey type and source and level of turbidity. We studied the effect of inorganic turbidity (0–30 NTU) on the effectiveness of fish feeding on two types of prey in different habitats: free-swimming cladoceran (Daphnia pulex) in open water and plant-associated cladoceran (Sida crystallina) attached to Nuphar lutea leaves. For the planktivore, we used vision-oriented perch (Perca fluviatilis) common in the littoral zone of temperate lakes. In our study, increasing inorganic turbidity did not appear to initiate any significant change in the feeding efficiency of perch on free-swimming Daphnia pulex. However, we saw a markedly different feeding efficiency when perch targeted plant-attached Sida crystallina. Our results substantiate that floating-leaved macrophytes in turbid lakes may provide a favourable habitat for plant-attached cladocerans.     

Methyl-β-cyclodextrin Suppresses Hyaluronan Synthesis by Down-regulation of Hyaluronan Synthase 2 through Inhibition of Akt

Hyaluronan synthases (HAS1–3) are integral plasma membrane proteins that synthesize hyaluronan, a cell surface and extracellular matrix polysaccharide necessary for many biological processes. It has been shown that HAS is partly localized in cholesterol-rich lipid rafts of MCF-7 cells, and cholesterol depletion with methyl-β-cyclodextrin (MβCD) suppresses hyaluronan secretion in smooth muscle cells. However, the mechanism by which cholesterol depletion inhibits hyaluronan production has remained unknown. We found that cholesterol depletion from MCF-7 cells by MβCD inhibits synthesis but does not decrease the molecular mass of hyaluronan, suggesting no major influence on HAS stability in the membrane. The inhibition of hyaluronan synthesis was not due to the availability of HAS substrates UDP-GlcUA and UDP-GlcNAc. Instead, MβCD specifically down-regulated the expression of HAS2 but not HAS1 or HAS3. Screening of signaling proteins after MβCD treatment revealed that phosphorylation of Akt and its downstream target p70S6 kinase, both members of phosphoinositide 3-kinase-Akt pathway, were inhibited. Inhibitors of this pathway suppressed hyaluronan synthesis and HAS2 expression in MCF-7 cells, suggesting that the reduced hyaluronan synthesis by MβCD is due to down-regulation of HAS2, mediated by the phosphoinositide 3-kinase-Akt-mTOR-p70S6K pathway.     

Immunoreactivity to cell surface syndecans in cytoplasm and nucleus: tubulin-dependent rearrangements

Syndecans are transmembrane proteoglycans implicated in the regulation of cell growth and differentiation, by interacting with growth factors. Although syndecans play a major role in regulating cell morphology, little is known about their subcellular distribution and in vivo association with the cytoskeleton. To address this question, we investigated the subcellular distribution and dynamic rearrangement of syndecans-1, -2, and -4, using confocal laser microscopy. Furthermore, we monitored the spatial relation of syndecans to tubulin in both mitotic and interphase cells. Initially, the reactivity to syndecans was confined to the cytoplasm, staining of the cell membranes appearing later. Syndecan-1 also seems to translocate to the nucleus in a time-dependent manner. The mitotic spindle shows unexpectedly more syndecans than that found in interphase cells. After vinblastine treatment, both syndecan-1 and tubulin were recovered as paracrystalline occlusion bodies, and the nuclear reactivity to syndecan-1 disappeared, suggesting tubulin-mediated nuclear transport of this proteoglycan. Plasma membrane staining reappeared in the postmitotic cells. Nuclear translocation predominantly affected syndecan-1, whereas syndecan-2 and -4 remained in cytoplasm and cell membrane. This is the first report on regulated nuclear translocation and the presence of syndecan-1 in the mitotic spindle, where it may stabilize the mitotic machinery. The syndecan-1/tubulin complex may also act as a vehicle for the transport of protein growth factors to the cell nucleus.     

Mercury-binding capacity of organic and inorganic selenium in rat blood and liver

The mercury-binding capacity of seleno-DL-methionine and selenium dioxide was assessed in male Wistar rats. Mercury was supplied as fish loaves made of northern pike or rainbow trout. We used a selenium concentration of 3.4 mg/kg fish, about sixfold compared to the equivalent quantity of mercury. Seleno-DL-methionine had a tendency to increase both methyl mercury and total mercury in blood, although it also seemed to reduce the proportion of methyl mercury of total mercury. Selenium dioxide lowered mercury levels by 24–29% both in the blood and in the liver of rats that were fed with northern pike.