Binding of the J 1 Adhesion Molecules to Extracellular Matrix Constituents

The J1 glycoproteins can be obtained in multiple forms in the soluble fraction of developing and adult mouse brain tissue. They are recovered as two forms of apparent molecular weights of 160,000 and 180,000 (J1-160) from adult mouse brain and as forms of apparent molecular weights of 200,000 and 220,000 (J1-220) from developing brain. J1-160 and J1-220 share common epitopes but are considered as separate entities, with J1-220 being immunochemically closely related if not identical to tenascin. Based on the observation that J1 immunoreactivity appears on basement membrane and interstitial collagens after denervation of the neuromuscular junction in adult rodents, we became interested in investigating the binding properties of J1 glycoproteins to extracellular matrix constituents in vitro. Both J1-160 and J1-220 bound to collagens type I-VI and IX but not to laminin, fibronectin, bovine serum albumin, or gelatin under hypotonic buffer conditions. Under isotonic buffer conditions, J1-220 bound to all collagen types, whereas J1-160 bound only to collagen types V and VI with values that could be examined by Scatchard analysis. Binding of J1-220 to collagens displayed two binding constants (KD) between 1.5 and 4.4 X 10(-9) and 1.8 and 5.5 X 10(-8) M, respectively, under hypotonic buffer conditions and a single KD of 2.1-8.0 X 10(-8) M under isotonic buffer conditions. Binding of J1-160 to collagens had an apparent KD of 1.9-8.0 X 10(-9) M under hypotonic buffer conditions. Under isotonic buffer conditions, binding constants of J1-160 to collagen types V and VI were approximately 2 X 10(-8) M. Binding of J1-220 to collagen type I could be inhibited by J1-220, J1-160, and collagen type VI but not by fibronectin or gelatin. Conversely, binding of J1-160 was inhibited by J1-220, J1-160, and collagen type VI (in order of decreasing efficacy of competition). J1-160 and J1-220 were retained on a heparin-agarose column and eluted in a salt gradient at approximately 0.5 M NaCl. The formation of the J1-heparin complexes was inhibited 100-fold more efficiently by heparin than by chondroitin sulfate. These experiments show that J1 glycoproteins resemble in many respects the extracellular matrix constituents fibronectin, laminin, vitronectin, and von Willebrand factor.     

Mathematical modelling of runoff and material transport from drainage areas into recipient water bodies

This paper describes a set of mathematical models to calculate runoff, soil erosion and chemical substance yields for drainage areas. The models represent different degrees of complexity and details of describing hydrological and hydrochemical processes. The availability of initial information and the requirements of the task determine the choice of a model for each particular case. These models have been written as PC/AT programs, to enable their use by a wide range of specialists. The models have been tested in different physical and geographical conditions, e.g. in some small watersheds on the Karelian Isthmus, west of Lake Ladoga, and in experimental catchment areas located in the Valdai Hills area in the southern part of Lake Ladoga drainage area. In further applications the drainage area models have been combined with models of the recipient water body, to obtain a single model of a watershed-lake system. The results of tests have proved that the models can be used for evaluation, simulation and forecasting of the processes of rainfall and snowmelt runoff, infiltration, evaporation, water erosion and pollutant wash-off from drainage areas.     

Vertical distribution of benthic nematodes in an oligotrophic lake: seasonality, species and age segregation

The vertical distribution of nematodes up to 20 cm sediment depth was studied over a one-year period in the alpine, oligotrophic lake Königssee. Ten water depths were examined, four of which correspond to the littoral (1 m, 2 m, 5 m, 10 m), three to the littoriprofundal (15 m, 20 m, 30 m) and three to the profundal (60 m, 120 m, 190 m). The sediment was devided into four layers (0–2 cm, 2–5 cm, 5–10 cm and 10–20 cm). (1) The highest proportion of nematodes was found in the top layer. Out of 45 263 nematodes, 89% were found in the first 5 cm of the sediment and only 1 % deeper than 10 cm. (2) The proportion of nematodes in the top layer increased along with water depth. Water depth was a better predictor of percentage of nematodes in the top layer than particle size or content of organic carbon in the sediment. (3) There were considerable differences among species in their vertical distribution in the sediment. (4) There was a significant trend for juveniles to occupy more superficial layers than adults across various species. (5) There are time related vertical preferences among adults and juveniles of several nematode species across the year, suggesting vertical migration in the sediment (e.g. Aphanolaimus aquaticus Daday, Eumonhystera longicaudatula Gerlach & Riemann, Tobrilus gracilis Bastian, Monhystera paludicola de Man, Ethmolaimus pratensis de Man and Ironus tenuicaudatus de Man). The factors determining the vertical variation in nematode abundance in freshwater systems across space and time are still unknown.     

The other kind of biological NMR—Studies of enzyme-substrate interactions

NMR spectroscopy has proved to be a valuable tool in the study of the interactions between enzymes and their substrates. The kinds of structural and dynamic information which can be obtained are illustrated by studies of three enzymes involved in drug metabolism. Cytochromes P₄₅₀ play a crucial role in metabolism of a wide range of exogenous chemicals. NMR has been used to measure distances from the haem iron of the cytochrome to protons of the bound substrate, leading to detailed structural models for the enzyme-substrate complexes. The other two enzymes, chloramphenicol acetyltransferase and β-lactamase, are responsible for bacterial resistance to specific antibiotics. In chloramphenicol acetyltransferase, NMR has been used to determine the conformation of coenzyme A bound to the enzyme, while in the case of β-lactamase the pK of a specific lysine residue at the active site has been determined, providing valuable information on the catalytic mechanism. Special issue dedicated to Dr. Herman Bachelard.     

Microbial α-mannosidases

Microbial α-mannosidases are used in the analysis of glycopeptides and the developmental regulation of lysosomal enzymes. This survey presents comparison of properties of this high molecular weight, oligomeric protein from a number of microbial sources.     

The origin and recycling of sedimented biogenic debris in a subalphine eutrophic lake (Lake Bled, Slovenia)

The areal distribution of organic C contents, ¹³C values, total N and P and biogenic Si contents in surficial sediments were used to study the distribution, origin and diagenetic transformations of sedimented biogenic debris in the eutrophic subalpine Lake Bled (Slovenia), which for most of the yearhas an anoxic hypolimnion. The influence of an allochthonous input, restricted to the western basin, was clearly traced by higher organic C and total N and P contents, higher ¹³C values, and higher sedimentation rate in comparison to the eastern basin. The low ¹³C values of sedimentary organic matter in the major part of the lake, lower than the ¹³C values of different types of organic matter, suggest that this sedimentary organic matter is most probably the product of a microbial community and not a residue of primary production.The temporal variation of benthic diffusive fluxes of NH₄, Si and PO₄, derived from modelling the pore water profiles, was related to sedimentation of phytoplanktonic blooms, while the PO₄ fluxes were also dependent on changing redox conditions at the sediment-water interface in the period of the winter-spring overtum. The removal of PO₄ in pore waters is probably due to the adsorption of phosphate and precipitation of apatite and vivianite. The budget of C, N and P at the sediment-water interface revealed a high recycling efficiency (>70%), also confirmed by the rather uniform (or only slightly decreasing) vertical profiles of organic C, total N and P in sediment cores and C/N and C/P ratios. The percentage of biogenic Si recycling is low (<10%), suggesting its removal in sediments.     

Sediment quality assessment in the delta of rivers Rhine and Meuse based on field observations,bioassays and food chain implications

The quality of sediment was assessed in 46 sites in the delta of the rivers Rhine and Meuse (The Netherlands) by means of physical-chemical analysis, field observations on the macrobenthic community structure, accumulation studies and bioassays using Chironomus riparius, Daphnia magna and Photobacterium phosphoreum. The results of chemical analyses were classified using national criteria for sediment quality. Results of field studies and bioassays were classified using criteria derived from research in reference areas or based on data from literature. Risk assessment was carried out according to the sediment quality triad and by means of multi criteria analysis (MCA). The Triad approach was used to demonstrate causal relations between effects on the macrozoobenthos community structure, effects demonstrated in bioassays and sediment pollution. This was done by making a comparison of sediment contamination levels with toxicity data from literature for the test organisms of the bioassays. Using the MCA method, for each site a numerical value was derived for total environmental risk in the present situation, based on observed effects. In this way, a relative risk ranking of all sites was realized. The MCA values for the present situation were also compared with MCA scores based on estimated risks after remediation in 1995, in order to set priorities for sites where remediation is expected to cause a significant reduction in environmental risk. In most of the 46 sites studied so far, the macrofauna community was poorly developed, judged by a low number of benthic species, low abundances and a high dominance of species regarded as relatively tolerant to chemical pollution. In bioassays high sediment toxicity was demonstrated for 24 sites. Using the sediment quality triad approach, 25 sites were identified as areas where pollution can be held responsible for the effects observed in the field. From a comparison of contaminant concentrations in different types of food with maximum tolerable risk levels, and the application of a bioaccumulation model it was concluded that the sediment pollution also implies high risks for plant-, benthos- or fish-eating birds (secondary poisoning of top predators). In the Nieuwe Merwede highest MCA risk scores were found for shallow parts where highly polluted sediments are found. It is concluded that the sediment quality triad and the MCA provide additional information which can be used to establish priorities for remedial action. Based on an ecotoxicological evaluation of the improved quality of new sediments that will be deposited after removal of the polluted sediments in the Nieuwe Merwede, it is concluded that in this upstream part of the Rhine delta remedial action will be effective.     

Fluoranthene as a model toxicant in sediment studies with Chironomus riparius

The bioavailability of fluoranthene (as a model toxicant) in freshwater sediment tests with Chironomus riparius was investigated in relation to the partitioning of the chemical between water and sediment. Effects on survival and successful development of newly hatched larvae through to adult emergence were measured over a range of sediment concentrations. Radiolabelled fluoranthene was employed to enable sediment, aqueous and tissue concentrations to be determined. A significant effect on emergence was obtained at a measured sediment concentration of 80 mg kg^-1 (dry weight). The measured concentration in the overlying water was 88 g l^-1. Although effects at 170 mg kg^-1 (dry weight) were more severe, the measured concentration in the overlying water was similar (89 g l^-1), probably reflecting the approach towards the solubility limit. Bioaccumulation of fluoranthene, determined by analysis of emerging adults, was approximately proportional to the sediment concentration. Although the aqueous phase concentration appeared to be the principal factor controlling bioavailability, the increasing toxicity, when water concentrations had reached a plateau, suggested that other mechanisms may also be involved. Different sediment spiking procedures resulted in broadly similar partitioning of the chemical between the sediment and the overlying water. There were only minor differences in toxicity between the procedures, two of which allowed the carrier solvent used for spiking to be eliminated before addition of the test organisms. It was concluded that, for most purposes, all four procedures were equally acceptable, and by retaining a flexible approach, even for standardised regulatory tests, spiking procedures may be tailored to the physico-chemical properties of the test material.     

Human immunodeficiency virus protein gp120 interferes with β-adrenergic receptor-mediated protein phosphorylation in cultured rat cortical astrocytes

Summary 1. We have previously shown that acute exposure to the HIV coat protein gp120 interferes with the -adrenergic regulation of astroglial and microglial cells (Leviet al., 1993). In particular, exposure to 100 pM gp120 for 30 min depressed the phosphorylation of vimentin and glial fibrillary acidic protein (GFAP) induced by isoproterenol in rat cortical astrocyte cultures. In the present study we have extended our analysis on the effects of gp120 on astroglial protein phosphorylation.2. We found that chronic (3-day) treatment of the cells with 100 pM gp120 before exposure to isoproterenol was substantially more effective than acute treatment in depressing the stimulatory effect of the -adrenergic agonist on vimentin and GFAP phosphorylation.3. Even after chronic treatment with gp120, no differences were found in the levels and solubility of these proteins.4. Besides stimulating the phosphorylation of intermediate filament proteins, isoproterenol inhibited the incorporation of³²P into a soluble acidic protein of 80,000M _r , which was only minimally present in Triton X-100-insoluble extracts.5. Treatment of astrocytes with a phorbol ester or exposure to³H-myristic acid indicated that the acidic 80,000M _r protein is a substrate for protein kinase C (PKC) and is myristoylated, thus suggesting that it is related to the MARCKS family of PKC substrates.6. Acute (30-min) treatment with 100 pM gp120 totally prevented the inhibitory effect of isoproterenol on the phorphorylation of the 80,000M _r MARCKS-like protein.7. Our studies corroborate the hypothesis that viral components may contribute to the neuropathological changes observed in AIDS through the alteration of signal transduction systems in glial cells.     

Substrate and inhibitor specificities of the thermostable alcohol dehydrogenase allozymes ADH-71k and ADH-FCh.D. ofDrosophila melanogaster

Purified thermostable alcohol dehydrogenase allozymes ADH-71k and ADH-FCh.D. ofDrosophila melanogaster have been compared with the two common enzyme forms ADH-F and ADH-S. Enzyme kinetic parameters for various primary and secondary alcohols were determined under standard conditions used previously. Both ADH-71k and ADH-FCh.D. show ADH-S-like reaction kinetics andK _m values, due to retrograde evolution at site 214, Pro Ser. Inhibition studies with alcohol dehydrogenase inhibitors pyrazole, 4-methylpyrazole, and cibacron blue 3GA were also performed. Activity measurements on crude extracts of larvae and flies from isogenic lines of ADH-FCh.D. revealed a consistently higher activity than in ADH-71k-containing strains, in contrast to the original strains.K.Th.E is indebted to the Royal Norwegian Council for Technological and Scientific Research for their postdoctoral fellowship. Prof. J. S. McKinley-McKee gave me the opportunity to work in his laboratory. I thank Dr. Knut Sletten of the Biochemical Institute for the kind gift of 2-methoxyethanol and amino acid analysis of some samples. The Biological Institute, Oslo, Section of General Genetics, is gratefully acknowledged for enabling me to use their fly-breeding facilities. Dr. John B. Gibson provided us with a sample of FCh.D. flies for the construction of isogenic lines in which Dr. Johan Hageman participated, owing to Postdoctoral Grant 436-931-P from the Foundation of Biological Research (BION), which is subsidized by the Netherlands Organization for Scientific Research (NWO). J. H. and Paula Truyens were involved in the measurements on the crude extracts. Work at Victoria University was supported by the VUW Internal Grant Committee.