Determination of proton dissociation constants by ion-exchange high-performance liquid chromatography

The common methods to determine dissociation constants of solutes, e.g., uv spectrophotometry, potentiometry, and conductimetry, are accurate but require at least 1 nmol of compound. High-performance liquid chromatography (HPLC) allows 1 pmol of a uv-absorbing compound to be detected. By adjusting the polarity of the mobile phase, reverse and normalphase properties of an ion-exchanger can be minimized, resulting in a high correlation between charge and retardation of the solute. Thus, the degree of ionization of several compounds was monitored in mobile-phase compositions of different pH values using cation exchange. The pK values of several pterin derivatives corresponded to those obtained by other methods. In addition, pK values of two unidentified pterin derivatives were determined, using only 20 pmol of each.     

Macrofaunal community types in ponds and small lakes (Overijssel,The Netherlands)

A survey was carried out at 157 sites, situated in pools and small lakes in the province of Overijssel (The Netherlands), to describe the macro-invertebrate community and their environment. A total of 61 environmental viriables was measured at each sampling site. The main aim was to described a regional ecological typology of ponds and small lakes. Multivariate analysis techniques are appropriate in data analysis for typological purposes. Different multivariate analysis techniques (FLEXCLUS, NODES, DCCA, PCA) were used in combination with ecological information on individual taxa to derive and describe site groups in terms of taxon composition and mean environmental conditions. The resulting site groups were termed cenotypes. Nine cenotypes were distinguished among the ponds and small lakes. The main differences between the cenotypes were related to duration of drought, acidity, morphology and nutrient load. In particular, the four cenotypes within the group of stagnant, pH-neutral ponds/lakes showed an overlap in taxon composition. These cenotypes represent a web-shaped continuum dominated by dimensions (relation of width to depth), nutrient load, and bottom composition (especially mesotrophic peat). The most important anthropogenic processes are acidification, eutrophication, and changes in the original hydrology.     

Effect of exogenous hormones on leaf yellowing in cut flowering branches of Alstroemeria pelegrina L.

Inclusion of IAA in the vase water had little effect on leaf yellowing in cut flowering branches of Alstroemeria pelegrina L. while kinetin delayed leaf yellowing at 10^-4M (continuous treatment). Chlorophyll was effectively retained by 10^-7M gibberellic acid (GA) in the vase water or by a 20h pulse at 5°C with 10^-5/10^-4M GA. After 16h of ¹⁴C-GA, uptake at 20°C relatively high levels of ¹⁴C were found in leaves and low levels in stems and flowers. After this treatment about half of the ¹⁴C-GA, in leaves was metabolized into unknown compounds. Corrigendum. Owing to an error in the proofreading process, the article was published incorrectly. The article as it should have been published is presented here.     

The binding of human lipoprotein lipase treated VLDL by the human hepatoma cell line HepG2.

It has been suggested that besides the LDL-receptor, hepatocytes possess an apo E or remnant receptor. To evaluate which hepatic lipoprotein receptor is involved in VLDL remnant catabolism, we studied the binding of VLDL remnants to HepG2 cells. Native VLDL was obtained from type IIb hyperlipidemic patients and treated with bovine milk lipoprotein lipase (LPL). This LPL-treated VLDL (LPL-VLDL) was used as representative for VLDL remnants. Our results show that LPL-VLDL binds with high affinity to HepG2 cells. Competition experiments showed that the binding of 125I-labelled LPL-VLDL is inhibited to about 30% of the control value by the simultaneous addition of an excess of either unlabelled LDL or LPL-VLDL. Preincubation of HepG2 cells with LDL resulted in a reduction of the binding of LDL and LPL-VLDL to 34 and 55% of the control value, whereas preincubation of the cells with heavy HDL (density between 1.16 and 1.21 g/ml) stimulated the binding of LDL and LPL-VLDL to about 230% of the control value. Preincubation of the cells with insulin (250 nM/l) also stimulated the binding of both LDL and LPL-VLDL (175 and 143% of the control value, respectively). We conclude that LPL-VLDL binds to the LDL-receptor of HepG2 cells and that no evidence has been obtained for the presence on HepG2 cells of an additional receptor that is involved in the binding of VLDL remnants.     

Loss of cardiac contractility and severe morphologic changes by acutely lowering the pH of the perfusion medium: protection by fatty acids

When the pH of the perfusion medium of rat Langendorff heart, paced at a rate of 300 beats/min, is abruptly lowered from pH 7.5 to 7.0, the hearts stop beating within 6 min in more than half of the cases. Reperfusion with pH 7.5 medium after 10 min pH 7.0 perfusion does not cause contractility to resume within 5 min. The causative factor is intracellular acidosis, resulting in severe morphological alterations of plasma membrane and mitochondria. It is probably initiated by the loss of membrane-bound calcium. Oleate, complexed with albumin included in the perfusion media, protects the hearts. This may be explained by maintenance of capillary flow and limitation of cellular acidosis.     

Translation of mouse interferon mRNA in Xenopus laevis oocytes and in rabbit reticulocyte lysates

Mouse interferon mRNA, extracted from NDV (Newcastle disease virus)-induced L-929 cells has been translated with high efficiency in $\text{Xenopus laevis}$ oocytes and rabbit reticulocyte lysates. The translational efficiency of a crude RNA extract was 10 640 interferon units/mg RNA/hour for the $\text{Xenopus}$ oocytes and 4 012 interferon units/mg RNA/hour for the reticulocyte lysates. The translation product fulfilled the usual criteria for mouse interferon, $\text{viz.}$ species specificity and neutralization by specific anti-mouse interferon antiserum. Upon injection of crude interferon mRNA into $\text{Xenopus}$ oocytes, interferon activity appeared both in the oocyte homogenates and the oocyte incubation medium. When analyzed by velocity sedimentation in formamidesucrose, the mouse interferon mRNA showed a rather sharp peak halfway between the 4 S and 18 S RNA markers, as could be expected from a mRNA which codes for a 20,000 dalton protein.     

Intercellular exchange of lysosomal enzymes: enzyme assays in single human fibroblasts after co-cultivation.

Intercellular exchange of N-acetyl-β-D-glucosaminidase (EC 3.2.1.30) β-galactosidase (EC 3.2.1.23) and acid α-glucosidase (EC 3.2.1.20) was studied after cocultivation of normal and enzyme deficient human fibroblasts in confluent cultures. Enzyme activities were measured in single cells using microchemical procedures. After co-cultivation of normal control fibroblasts and those from a patient with Sandhoff's disease an increase of activity of N-acetyl-β-D-glucosaminidase was found in Sandhoff cells, together with a decrease of activity in normal control cells. After co-cultivation of normal fibroblasts and those from patients with glycogenosis II and GM1-gangliosidosis, no indication was found for intercellular transfer of acid α-glucosidase and β-galactosidase respectively. The significance of the results is discussed in respect of the hypothesis of Hickman and Neufeld about secretion and uptake of lysosomal enzymes.