Simplified luciferase assay of NAD+ applied to microsamples from liver,kidney and pancreatic islets

A new single-step procedure for the bioluminescence assay of NAD⁺, permitting measurements on the pmol level (10^?12 mol) is described. Acid extracts of NAD⁺ were prepared in different tissues. The acidification destroys reduced pyridine nucleotides and most enzymes which are present in the tissue sample. After neutralization the extract is added to a light-yielding solution, and the luminescence is measured with a photomultiplier. The maximal height of the signal is measured by means of a digital voltmeter. The light yielder is bacterial luciferase with appropriate additives and supplemented with malate and malate dehydrogenase.The modified light-yielding solution provides for continuous formation of NADH resulting in a durable level of light emission. The cycle involved was shown not to operate with NADP⁺. The slow fading of the emission permits simplification of the measuring procedure. Rapid injection in front of the phototube can thus be omitted and replaced by ordinary mixing before the reaction cell is positioned for the measurement. Furthermore, the instrumentation required is less elaborate than in photokinetic assay, since it is not necessary to record and integrate the time course of the emission. To test the applicability of the method, analyses of pmol amounts were performed in the islets of Langerhans and in tissue samples of much smaller size than fine needle biopsies.     

Isolation and characterization of three different carbohydrate chains from melanoma tissue plasminogen activator

Electrophoretic analysis of endoglycosidase-treated tissue plasminogen activator obtained from human melanoma cells showed that the heterogeneity observed for the protein in these preparations is caused by an N-glycosidically linked N-acetyllactosamine type of carbohydrate chain which is present in about 50% of the molecules. An oligomannose type and an N-acetyllactosamine type of glycan is present in all molecules. Three glycopeptides were isolated and characterized by 1H-NMR, sugar determination, methylation analysis and amino acid determination. The exact attachment site for each of the three glycans could be deduced from the amino acid compositions of the glycopeptides. Asn-117 carries the oligomannose type of glycan, the structure of which was completely determined. Asn-184 is the site where the presence or absence of a biantennary N-acetyllactosamine type of glycan causes the size heterogeneity. The third N-glycosylation site, Asn-448, was found to carry a triantennary or tetraantennary N-acetyllactosamine type of carbohydrate chain.     

Divalent Cation Effects on the Shaker K Channel Suggest a Pentapeptide Sequence as Determinant of Functional Surface Charge Density

The effects of the divalent cations strontium and magnesium on Shaker K channels expressed in Xenopus oocytes were investigated with a two-electrode voltage-clamp technique. 20 mm of the divalent cation shifted activation (conductance vs. potential), steady-state inactivation and inactivation time constant vs. potential curves 10–11 mV along the potential axis. The results were interpreted in terms of the surface charge theory, and the surface charge density was estimated to be −0.27 e nm⁻². A comparison of primary structure data and experimental data from the present and previous studies suggests that the first five residues on the extracellular loop between transmembrane segment 5 and the pore region constitutes the functional surface charges. The results further suggest that the surface charge density plays an important role in controlling the activation voltage range. Received: 12 November 1997/Revised: 1 June 1998     

Dytiscus latissimus and D. circumcinctus (Coleoptera,Dytiscidae) larvae as predators on three case-making caddis larvae

The predacious behaviour of Dytiscus circumcinctus and D. latissimus larvae was studied experimentally. When offered different prey simultaneously, D. latissimus larvae preferred cased caddis larvae relative to mayfly nymphs and isopods, whereas in D. circumcinctus the preference order was reversed. Notonectid nymphs and tadpoles were consumed in higher numbers by D. circumcinctus than by D. latissimus larvae. D. circumcinctus larvae and instar III larvae of D. latissimus most frequently captured caddis larvae through the case wall, whereas the instar I and II larvae of D. latissimus normally attacked the thorax of the exposed larva from above the front opening of the case. Limnephilus borealis, L. nigriceps and L. rhombicus caddis larvae differed in case structure, and they were all successfully captured by D. latissimus and D. circumcinctus instar II and III larvae. Neither capture success nor ingestion efficiency varied significantly between the two Dytiscus species or between different prey species. Instar II and III D. circumcinctus larvae had shorter reaction times than those of D. latissimus. The larger L. borealis and L. rhombicus larvae were preferred by the two last Dytiscus larval instars, and the handling time of these two prey was longer than that of L. nigriceps larvae.     

Size-selective predation by fourhorn sculpin,Myoxocephalus quadricornis (L.) (Pisces) on Mesidotea entomon (L.) (Crustacea,Isopoda)

During three different seasons Mesidotea entomon specimens from fourhorn sculpin stomachs were analyzed, and compared with the M. entomon population in the field. The field samplings were carried out in the northern Bothnian Sea. Feeding experiments revealed fourhorn sculpins to be highly selective when feeding on M. entomon.The fourhorn sculpin most preferably selected large M. entomon in the field as well as in the laboratory experiments. The preference for large M. entomon remained after correction for availability of differently sized M. entomon. In summer the actual sizes of M. entomon eaten by fourhorn sculpin were smaller than in autumn and winter. During all three seasons the oldest M. entomon were the ones most preferred. Ultimate effects of predation by fourhorn sculpin on the life-history of M. entomon are discussed.     

Metabolism of chylomicron arachidonic and linoleic acid in the rat

Chyle and chylomicrons, obtained after feeding thoracic duct cannulated rats [3H]arachidonic (20:4) and [14C]linoleic acid (18:2) in cream, were injected i.v. into recipient animals. 7.5-15 min after injection, the 14C/3H ratio of the triacylglycerols remaining in plasma was about half of that in the injected chylomicrons, indicating that the chylomicron remnants formed retained relatively more [3H]20:4 than [14C]18:2. The 14C/3H ratio of plasma diacylglycerols was about 6-fold lower than that of plasma free fatty acids. The proportion of [3H]20:4 found in plasma cholesteryl esters was several-fold higher than that of [14C]18:2. Inhibition of hepatic lipase by a specific antiserum did not significantly influence the clearance of triacylglycerols, but increased the amount of 3H in plasma diacylglycerols. It also prevented the rapid clearance of phosphatidylethanolamine from plasma. The liver uptake of [3H]20:4 exceeded that of [14C]18:2. Antiserum against hepatic lipase diminished the difference. In contrast, the 14C/3H ratio of adipose tissue was higher than that of the injected chyle lipoproteins.     

An attempt to predict the start,peak and end of a pollen season

The different phases, start, peak and end of the pollen season were predicted using a cumulated activity method. This procedure is a development of the temperature sum method. The temperature was transformed to activity (A):

A = 2**[(T+g)/(10*h)] - k.

T = daily mean temperature over the treshold.

g,h,k = coefficients.

Other weather parameters are also to be included in the future.

The activity is cumulated day by day and the sum is used as an indicator of a specific phase. A cumulated activity model is defined by the above formula with specified values of the three coefficients g, h and k. Four conditions stating criteria for valid cumulation days and the cumulated target sum for the phase in question are to be defined. The model was based on pollen and weather data in Stockholm 1973–1988. The cumulated activity method was found to be best applicable to Ulmus, Betula and Pinus, but less useful for Alnus, Corylus and Quercus. The model was not satisfactory for Poaceae and Artemisia.