The localization of heparin-binding fragments on human C4b-binding protein

C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the C system. C4BP interacts with C C4b on a domain located in a 48-kDa chymotryptic fragment. We now demonstrate that C4BP contains heparin-binding fragments, which are located within the C4b binding domain. We have used an assay using heparin coupled to Sepharose CL-6B to show that 125I-C4BP binds to heparin in a time-dependent, saturable, and reversible manner. Binding could be inhibited by purified 48-kDa fragments and direct binding on the 48-kDa fragments to heparin-Sepharose was demonstrated by SDS-PAGE. mAb against native C4BP and the isolated 160-kDa central core fragment were evaluated for their ability to block the binding of 125I-C4BP to heparin and C4b. The relative efficacy of mAb against intact C4BP in blocking C4BP binding to heparin-Sepharose was similar to that for blocking 125I-C4BP binding to C4b. In addition, heparin blocked the binding of 125I-C4BP to C4b and vice versa. It is therefore likely that the heparin-binding fragments are localized on or close to the C4b-binding site of C4BP.     

Single-stranded circular DNA generated from broad host range plasmid R1162 and its derivatives

Extraction of R1162 plasmid DNA with the alkaline lysis method yields considerable amounts of single-stranded circular plasmid DNA. Destabilization of plasmid DNA is stimulated by the R1162 mob region in cis. The formation of single-stranded circular DNA is initiated at a specific site on the plasmid, presumably the origin of transfer (oriT).     

Effect of weather on asthmatic children in the Eastern part of the Netherlands

A biometeorological analysis of the daily asthma complaints, classified in 5 degrees of severity, of 50 boys and 25 girls, 7 to 16 years old, at the asthma center Eykeloord (Eastern Netherlands), during 1964 has shown that in winter and early spring the number of asthma attacks in both sexes increased during periods of cooling. The total rise in asthma attacks increased with the length of the cooling period and decreased in relation to the length of the period of warming up. During the summer months June and August these meteorotropic effects were not statistically significant. The observations confirmed the previous findings at an asthma center in the Western Netherlands.

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Zusammenfassung Eine biometeorologische Analyse der täglichen Asthmabeschwerden, die in 5 Schweregraden klassifiziert wurden, bei 50 Knaben und 25 Mädchen im Alter von 7 bis 16 Jahren in der Asthmaklinik Eykeloord (Ost-Holland)während des Jahres 1964 hat gezeigt, dass im Winter und Vorfrühling die Anzahl der Asthmaanfälle bei Kälteeinbrüchen zunimmt. Asthmaanfälle nehmen an Zahl mit der Länge der Kälteperioden zu und mit der Länge der Wärmeperioden ab.Während der Sommermonate Juni bis August waren diese Beziehungen zwar auch nachweisbar aber nicht signifikant. Die Beobachtungen bestätigten früheren Ergebnisse in einer Asthmaklinik in West-Holland.

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Resume Une analyse biométéorologique des accès d'asthme a montré qu'en hiver et au début du printemps le nombre des dits accès augmente au moment d'une advection d'air froid. Pour ce faire, on s'est basé sur l'observation de 50 garçons et de 25 fillettes de 7 à 16 ans répartis en 5 classes d'acuité de la maladie. Tous étaient en traitement durant l'année 1964 à la clinique pour asthmatiques d'Eykeloord(Hollande orientale). Les accès d'asthme sont d'autant plus nombreux que la période froide est plus longue et leur nombre diminue parallèlement à la durée des périodes plus chaudes. La même relation a pu être établie durant les mois d'été (de juin à août), mais pas de façon significative. Ces observations confirment celles qui avaient été faites antérieurement dans une clinique pour asthmatiques de l'ouest de la Hollande.

     

Bio-availability of phosphorus in sediments of the western Dutch Wadden Sea

The purpose of this study was to make a prognosis of the effects of extended purification of terrestrial waste water, reaching the Wadden Sea by the River Rhine and Lake IJssel, on the phosphate concentration in the western Wadden Sea.The quantities of different phosphorus fractions in intertidal and subtidal sediments of the Marsdiep tidal basin (western Dutch Wadden Sea) were measured. Different methods are applied to determine the amount of phosphorus that can be released from these sediments. The direct bioavailability is determined by inoculating sediment suspensions with a natural mixture of precultured micro-organisms from the sampling area. A second approach is the measurement of the phosphate release under different redox conditions. Sequential extraction of sediment samples with different solvents is also applied. Under the present conditions and compared to the nutrient loads from fresh water (Lake IJssel) and from the North Sea, the phosphorus stored in the sediments of the western Dutch Wadden Sea plays a minor role in the total supply to micro-algae and bacteria. The bulk of the biologically available phosphorus in the sediments originates from the metal-associated fraction. Releasable phosphate may contribute to the local annual primary production to an extent of ca 45 to ca 150 g C m^–2 a^–1. The total amount of phosphorus in the sediment (mainly calcite associated) is twice to 6 times the biologically available amount.     

Seagrass ecosystem multifunctionality under the rise of a flagship marine megaherbivore

Large grazers (megaherbivores) have a profound impact on ecosystem functioning. However, how ecosystem multifunctionality is affected by changes in megaherbivore populations remains poorly understood. Understanding the total impact on ecosystem multifunctionality requires an integrative ecosystem approach, which is especially challenging to obtain in marine systems. We assessed the effects of experimentally simulated grazing intensity scenarios on ecosystem functions and multifunctionality in a tropical Caribbean seagrass ecosystem. As a model, we selected a key marine megaherbivore, the green turtle, whose ecological role is rapidly unfolding in numerous foraging areas where populations are recovering through conservation after centuries of decline, with an increase in recorded overgrazing episodes. To quantify the effects, we employed a novel integrated index of seagrass ecosystem multifunctionality based upon multiple, well-recognized measures of seagrass ecosystem functions that reflect ecosystem services. Experiments revealed that intermediate turtle grazing resulted in the highest rates of nutrient cycling and carbon storage, while sediment stabilization, decomposition rates, epifauna richness, and fish biomass are highest in the absence of turtle grazing. In contrast, intense grazing resulted in disproportionally large effects on ecosystem functions and a collapse of multifunctionality. These results imply that (i) the return of a megaherbivore can exert strong effects on coastal ecosystem functions and multifunctionality, (ii) conservation efforts that are skewed toward megaherbivores, but ignore their key drivers like predators or habitat, will likely result in overgrazing-induced loss of multifunctionality, and (iii) the multifunctionality index shows great potential as a quantitative tool to assess ecosystem performance. Considerable and rapid alterations in megaherbivore abundance (both through extinction and conservation) cause an imbalance in ecosystem functioning and substantially alter or even compromise ecosystem services that help to negate global change effects. An integrative ecosystem approach in environmental management is urgently required to protect and enhance ecosystem multifunctionality.     

Phospho-beta-glucosidase from Fusobacterium mortiferum: purification, cloning, and inactivation by 6-phosphoglucono-delta-lactone.

6-Phosphoryl-beta-D-glucopyranosyl:6-phosphoglucohydrolase (P-beta-glucosidase, EC 3.2.1.86) has been purified from Fusobacterium mortiferum. Assays for enzyme activity and results from Western immunoblots showed that P-beta-glucosidase (Mr, 53,000; pI, 4.5) was induced by growth of F. mortiferum on beta-glucosides. The novel chromogenic and fluorogenic substrates, p-nitrophenyl-beta-D-glucopyranoside-6-phosphate (pNPbetaGlc6P) and 4-methylumbelliferyl-beta-D-glucopyranoside-6-phosphate (4MUbetaGlc6P), respectively, were used for the assay of P-beta-glucosidase activity. The enzyme hydrolyzed several P-beta-glucosides, including the isomeric disaccharide phosphates cellobiose-6-phosphate, gentiobiose-6-phosphate, sophorose-6-phosphate, and laminaribiose-6-phosphate, to yield glucose-6-phosphate and appropriate aglycons. The kinetic parameters for each substrate are reported. P-beta-glucosidase from F. mortiferum was inactivated by 6-phosphoglucono-delta-lactone (P-glucono-delta-lactone) derived via oxidation of glucose 6-phosphate. The pbgA gene that encodes P-beta-glucosidase from F. mortiferum has been cloned and sequenced. The first 42 residues deduced from the nucleotide sequence matched those determined for the N terminus by automated Edman degradation of the purified enzyme. From the predicted sequence of 466 amino acids, two catalytically important glutamyl residues have been identified. Comparative alignment of the amino acid sequences of P-beta-glucosidase from Escherichia coli and F. mortiferum indicates potential binding sites for the inhibitory P-glucono-delta-lactone to the enzyme from F. mortiferum.     

Effect of endurance training and seasonal fluctuation on coagulation and fibrinolysis in young sedentary men

Van den Burg, P. J. M., J. E. H. Hospers, M. Van Vliet, W. L. Mosterd, B. N. Bouma, and I. A. Huisveld. Effect of endurance training and seasonal fluctuation on coagulation and fibrinolysis inyoung sedentary men. J. Appl. Physiol.82(2): 613-620, 1997.The effect of 12 wk of submaximal trainingon hemostatic variables was studied in 20 young sedentary men (Tr) and19 nontraining matched controls (Con). After training, a morepronounced increase in factor VIII coagulant activity(P < 0.01), reflected in a decrease in activated partial thromboplastin time(P < 0.01) during maximal exercise,was seen. Both basal plasminogen activator inhibitor 1 antigen (PAI-1Ag) and activity (PAI-1 Act; P < 0.05), as well as basal and exercise-induced tissue-type plasminogenactivator antigen (t-PA Ag; P < 0.05), were decreased after training. The overall effect onfibrinolysis was reflected in an increase in the t-PA Act/t-PA Ag ratioin the Tr group. In contrast, during the same period (February-June),the Con group demonstrated an increase in basal PAI-1 Ag and PAI-1 Act(P < 0.05), together with anincrease in basal and exercise-induced t-PA Ag(P < 0.05). Both basal andexercise-induced t-PA Act were unchanged, but t-PA Act/t-PA Ag wasdecreased (P < 0.05) in the Congroup. We conclude that physical training promotes both coagulation andfibrinolytic potential during exercise and may reverse unfavorableseasonal effects on fibrinolysis.

     

O-(4-Diazo-3,5-di[125I]iodobenzoyl)sucrose, a novel radioactive label for determining organ sites of catabolism of plasma proteins.

A method is described for radiolabelling proteins with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose (DD125IBS). When proteins so labelled were degraded within lysosomes, the radioactive fragments were largely retained within the organelle. High specific radioactivities were obtained without changing the properties of the protein. The validity of the method was demonstrated in vivo in rats using the short-lived protein lactate dehydrogenase, isoenzyme M4, and the long-lived protein bovine serum albumin. Derivatization with DD125IBS did not alter the clearance of either protein. Uptake of DD125IBS-labelled lactate dehydrogenase, isoenzyme M4, by liver and spleen of rats was determined. Radioactivity in these tissues increased up to about 2 h after injection (at this time the protein has been almost completely cleared from the blood) and subsequently declined with a half-life of approx. 20 h. After differential fractionation of liver, radioactivity was largely found in the mitochondrial and lysosomal fraction. The results of these studies establish that DD125IBS covalently coupled to plasma proteins should be a useful radioactive tracer for identifying the tissue and cellular sites of catabolism of relatively long-lived circulating proteins.     

Endocytosis of lactate dehydrogenase isoenzyme M4 in rats in vivo. Experiments with enzyme labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose.

1. Pig lactate dehydrogenase isoenzyme M4 was labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose and injected intravenously into rats. Previous work has shown that this label does not influence the clearance of the enzyme (half-life about 26 min) and that it is retained within the lysosomes for several hours after endocytosis and breakdown of the protein [De Jong, Bouma & Gruber (1981) Biochem. J. 198, 45--51]. 2. The distribution of the radioactivity over a large number of tissues was determined 2 h after injection. A high percentage of the injected dose was found in liver (41%), spleen (10%) and bone including marrow (21%). 3. Autoradiography indicated uptake of the enzyme mainly by Kupffer cells of the liver, by spleen macrophages and by bone marrow macrophages. 4. Liver cells were isolated 1 h after injection of the enzyme. Kupffer cells, endothelial cells and parenchymal cells were found to endocytose the enzyme at rates corresponding to 4230, 35 and 25 ml of plasma/day per g of cell protein, respectively. 5. Previous injection of carbon particles greatly reduced the uptake of the enzyme by liver and spleen, but the uptake by bone marrow was not significantly changed.