Binding and bioactivity of insulin in primary cultures of carp (Cyprinus carpio) hepatocytes

Isolated carp hepatocytes cultured in serum-free, chemically defined medium were used to investigate within the same cell preparation characteristics of the binding of insulin as well as effects of insulin on cellular metabolism. The binding of human [¹²⁵I]-insulin to carp hepatocytes was studied in kinetic, saturation and displacement experiments. A dependency of insulin binding on the collagenase used for cell isolation was demonstrated. Insulin binding decreased during the first 12h of culture but remained constant during the following 12h. The kinetic experiments revealed that [¹²⁵I]-insulin binding reached a steady state within 20–30 min of incubation. The mathematical analysis of the saturation experiments demonstrated the existence of two populations of binding sites, one with high affinity (K_d1 = 5.5 pM) and low capacity (B_max1 = 0.14 fmol/mg protein or 77 binding sites/cell) and one with low affinity (K_d2 = 2.4 nM) and high capacity (B_max2 = 17.6 fmol/mg protein or 9623 binding sites/cell). In competition experiments, 312 pM [¹²⁵I]-insulin was displaced by cold insulin, IGF-I and IGF-II with IC₅₀ values of 2.2, 7.9 and 20.3 nM, respectively. Glucagon was without effect. Binding of insulin to carp hepatocytes resulted in a significant reduction of glucose release and a significant increase of protein synthesis as of de novo fatty acid synthesis. dedicated to Prof. Dr. W. Hanke on the occasion of his 65^th birthday.     

Residential runoff – The role of spatial density and surface cover,with a case study in the Höjeå river catchment,southern Sweden

Depending on the spatial density of built cover and location within a catchment, residential areas make varying contributions to surface runoff throughout different rainfall events. This study examined these contributions and the associated effect of different surface covers and sustainable drainage systems on runoff generation. The Soil Conservation Service curve number (SCS-CN) method was applied to analyse urban development in the Höjeå river catchment in southern Sweden.The results indicated that identical amounts of surface runoff are generated by low-density residential areas on heavy clay soils and high-density residential areas on sandy soils. However, increasing the density of built-up areas on sandy soils is likely to be more disruptive to the hydrological balance and to generate a greatly increasing difference in runoff as building density and impermeable surface area increase. A similar pattern is likely to occur if rainfall intensity increases. It may therefore be appropriate to apply different planning considerations to residential developments depending on the existing soil group, e.g. conserving existing vegetated surfaces on sandy soil and incorporating permeable paving materials and sub-surface infiltration beds in development on clayey soils. Increased area of impermeable surface cover will increase surface runoff in all residential areas, irrespective of building density, soil group and rainfall intensity. However, adapting a systems approach in combining vegetative structures and permeable paving materials with subsurface infiltration beds can help mitigate the impact of surface runoff, particularly in urban developments on clay-rich soils.     

The impact of small-scale design interventions on the behaviour patterns of the users of an urban wasteland

Urban derelict space can form a valuable complementary element to the formal green spaces of a city: wastelands are often biologically diverse and their unregulated status can provoke innovative spatial activities by a wide range of users. Using the case of an urban brownfield in Tallinn, Estonia, this paper examines how such a space is used and evaluates the magnitude of the impact of minor design interventions – so-called urban acupuncture – on the activities carried out by the users. The study used field observations and behaviour mapping to compare the spatial pattern of the users before and after small design interventions. Although there was widespread use beforehand there was both an increase in use and a different pattern afterwards, which was clearly detectable from the composite behaviour maps of both arrangements, where users follow the properties of the particular environment. The small improvements tested showed the largest effect on the female and the older user groups, raising the number of overall visits and increasing the occurrence of active behaviour almost five times. The research demonstrates that small, inexpensive and possibly temporary interventions can have a major positive effect.     

Alteration of certain blood parameters of freshwater teleost <Emphasis Type="Italic">Mystus vittatus</Emphasis> after chronic exposure to Metasystox and Sevin

Indiscriminate use of pesticides has elevated the risk of contamination of environment and aquatic habitat. Considering the above fact, the present study has been under taken to investigate the alteration of some blood parameters of freshwater teleost Mystus vittatus after chronic exposure to sublethal concentrations of Metasystox (4 ppm.) and Sevin (7 ppm.) individually. The main alterations observed in certain haematological parameters were clotting time (CT), erythrocyte sedimentation rate (ESR), haemoglobin percentage (Hb%), red blood corpuscles (RBCs), white blood corpuscles (WBCs), pack cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC). Alterations in organic constituents of blood, such as glucose, blood urea, total plasma protein and cholesterol, and in inorganic constituents, such as calcium, iron, magnesium, and phosphorus, were also studied. Alteration of enzymes such as glucose-6-phosphatase (G-6-pase), fructose-1-6-diphosphatase (F-1-6-dipase), serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) were also studied, so that the changes in the blood biochemistry due to the pesticidal stress could be understood. Results indicated that CT, WBCs, MCH, MCHC, glucose, blood urea, cholesterol, magnesium and SGOT were increased, whereas other parameters were found to be decreased in both cases. Possible reasons for the above elevation are discussed in the light of available literature.     

Minimising aerobic respiratory demands could form the basis to sub-lethal copper tolerance by rainbow trout gill epithelial cells in vitro

Mechanisms of Cu tolerance were investigated in respiratory epithelial cell cultures, from rainbow trout gills, by studying O₂ consumption and protein synthesis rates, intracellular Na concentration and TER. The lowest concentration found to reduce O₂ consumption was 25 M Cu. This did not affect either protein synthesis rate or intracellular Na concentration and was interpreted in terms of copper tolerance; i.e., how these two energetically demanding processes are maintained despite a reduction in aerobic ATP supply. The relationship between protein synthesis rate and synthesis cost is exponential and the cost of protein synthesis in gill cells was found to be minimal (i.e., this cell occupies a position on the asymptotic section of the protein synthesis rate/synthesis cost model) and unaffected by 25 M Cu. Thus protein synthesis rates could be maintained since any reduction would represent an insignificant energy saving. Intracellular Na concentrations and O₂ consumption rates were linearly correlated suggesting reducing intracellular maintenance costs would have a greater significance in terms of overall energetic conservation. Intracellular Na maintenance costs, calculated from O₂ consumption rates and intracellular Na concentrations, were found to decline after exposure to 25 M Cu. Since TER was unaffected this implied the reduced costs arose from membrane `channel arrest'. Thus the Na/K ATPase energy demands, associated with maintaining intracellular Na concentration, could be reduced by decoupling metabolic demand and membrane function. Therefore this study may demonstrate how the flexibility of cellular energetics enables gill epithelial cells to tolerate sub-lethal Cu.     

Development of a whole-body cortisol extraction procedure for determination of stress in golden shiners, <Emphasis Type="Italic">Notemigonus crysoleucas</Emphasis>

Baitfish such as golden shiners are subjected to stress during harvesting, grading, and transport. Their small size makes it difficult to measure the stress response with the biological indicator cortisol using conventional assay methods for plasma. This paper examines the development and validation of methods for whole-body cortisol extraction from individual baitfish. Three types of extracts were tested: (1) an ethyl ether unaltered extract (UA); (2) an extract reconstituted in phosphate buffered saline (PBS); (3) an extract that had been increased in volume by the addition of food-grade vegetable oil (VO). These extracts were evaluated using validation tests with radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISA). The UA extract produced inadequate volumes of extract for multiple assays and could not be used for the determination of cortisol in a single fish. The PBS reconstitution method failed the precision recovery of serial dilutions (62.3%), linearity (R ²: 0.7864), and parallelism validation tests. The VO volume-boosting method passed all validation tests [intra-assay coefficent of variation (%CV): 16.3 for ELISA and 5.9 for RIA; inter-assay %CV: 10.3; spiked recovery: 102.0%; dilution recovery: 93.0%; linearity R ²: 0.9435; log of serial dilutions was parallel] and provided enough extract for multiple assays from an individual baitfish. Based on these results, we conclude that the VO volume-boosting method presents a means for determining cortisol from individual baitfish using either RIA or ELISA assays.