Phosphates of the naphthol AS series in the quantitative determination of alkaline and acid phosphatase activities “in situ” studied in polyacrylamide membrane model systems and by cytospectrophotometry

Summary Histochemical media for the demonstration of alkaline and acid phosphatases using phosphates of naphthol AS series as the substrates and various diazonium salts as the couplers were tested in the capability of reflecting various levels of enzyme activities.Polyacrylamide membranes with incorporated enzymes (various concentrations of purified enzymes as well as of sonicated leucocytes, macrophages and of sonicated homogenates of various organs) were used as model systems in which the activity was estimated both with biochemical and with histochemical methods. Parallel experiments were performed in sedimentation chamber preparations of guinea-pig leucocytes and macrophages in which the activity was demonstrated with the same media as in polyacrylamide films. The quantitative measurements were performed in a cytospectrophotometer using the two-wavelength method.Increasing the substrate concentration which in standard histochemical media has been 1/8 mg per ml more azo-dye is produced in the reactions for both phosphatases. If the substrate concentration is higher than 1/2 mg per ml the standard concentration of the diazonium salt (1 mg per ml) becomes insufficient for an effective capturing of the released naphthol AS in the reaction for alkaline phosphatase. Due to a very high inhibitoty effect in the case of most commercially available diazonium salts the increase of their concentration annules the beneficial action of an increased substrate concentration on the azo-dye production. 4-amino-diphenylamine diazonium sulfate has an exceptional position because it was not inhibitory even in the concentration of 4 mg/ml.In the case of acid phosphatase the higher substrate concentration was incompatible with the use of Past Red Violet LB. Hexazo-p-rosanilin was an efficient and the most chromogenic coupler used in simultaneous as well as in postincubation coupling. With the latter localization is possible on the cellular (not subcellular) level.More chromogenic combinations are generally better for the cytospectrophotometrical measurement. The shape of extinction curves of azo-dyes produced with combinations studied was similar in models and in smears. In many combinations it was dependent on the presence of lipoproteins. A too steep decline of some curves prevented the use of some combinations in alkaline phosphatase determination with the two-wavelength method, even if they are very good in the qualitative studies and might be suitable for scanning cytospectrophotometry. p]The shape of extinction curves of azo-dyes produced in the reaction for acid phosphatase using hexazo-p-rosanilin as the coupling agent was independent of the presence of lipoproteins.The curves of azo-dyes produced in simultaneous coupling are not exactly the same as the curves obtained by postincubation coupling.In receipt of a fellowship of Netherlands Organization for the Advancement of Pure Research (Z.W.O.). Abbreviations used: AN-naphthol AS-AN phosphate; AS-naphthol AS-phosphate; B-Fast Blue B salt; BB-Fast Blue BB salt; BI-naphthol AS-BI phosphate; CL-naphthol AS-CL phosphate; DS-diazonium salt; GR-naphthol AS-GR phosphate; HP-hevazo-p-rosanilin; LB-Fast Red Violet LB salt; MX-naphthol AS-MX phosphate; S-substrate; TR-naphthol AS-TR phosphate (in the first half of the abbreviation), Fast Red TR salt (in the second half of the abbreviation); VB-4-amino-diphenylamine diazonium sulfate.     

A Rapid Screening Test for Aflatoxin-synthesizing Aspergilli of the flavus-oryzae Group

A rapid test for the recognition of aflatoxin-synthesizing strains of the Aspergillus flavus–oryzae group is described. For this purpose the strains are cultivated on Czapek–Dox agar enriched with an aqueous extract of groundnuts, and in which sodium nitrate is replaced by ammonium chloride. Toxin production is observed by the production of a bright blue fluorescence in the medium when placed under an ultraviolet lamp.     

Growth Inhibition of Staphylococci by Sodium Thiosulphate

The addition of sodium thiosulphate to a medium as neutralizer of an iodine antiseptic resulted in unexpected growth inhibition of various strains of staphylococci and micrococci. The minimum growth inhibiting concentration varied with different strains. The inhibitory effect of sodium thiosulphate was more pronounced in media with low pH values than in those with high pH values, and was diminished by the addition of Tween 80. The action was also found to depend on the concentration of l -cystine in the medium. It is suggested that the use of sodium thiosulphate be avoided in growth media designed to neutralize iodine in disinfection efficiency tests when staphylococci or micrococci are used as test organisms.     

A comparison of sperm morphology and silver nitrate staining characteristics in the domestic ferret and the black-footed ferret

Ejaculated sperm from the domestic ferret (Mustela putorius furo) and the black-footed ferret (Mustela nigripes) were compared for differences in morphological abnormalities and argentophilic protein distribution. Thawed domestic ferret sperm was also compared to fresh sperm to determine whether there were any effects on cell morphology due to cryopreservation. There were statistically significant differences between the two species of ferret in two of the categories scored. The domestic ferret had a higher frequency of cells that were bent in the midpiece and in the principal piece, and a higher frequency of headless and tailless cells when compared to the black-footed ferret. There were no statistically significant differences in cell morphology between the fresh and cryopreserved ejaculates of the domestic ferret employing a standard egg yolk cryoextender. Silver nitrate staining distribution was different between the two species in both the head and tail region.     

Microporosity of the substratum regulates differentiation of MDCK cells in vitro

Summary We have analyzed the ability of the physical substratum to modulate both the ultrastructural and protein synthetic characteristics of the Madin-Darby canine kidney (MDCK) renal cell line. When MDCK cells were seeded on Millipore Millicell CM microporous membrane cell culture inserts they demonstrated a more columnar organization with an increase in cell density sixfold greater than the same cells seeded on conventional plastic substrata. After 1 wk postseeding on the microporous membrane a partial basal lamina was noted, with a contiguous basement membrane being apparent after 2 wk. One-dimensional sodium dodecyl sulfate gel electrophoresis was used to analyze detergent-solubilized proteins from MDCK cells maintained on plastic substrata vs. microporous membranes. When proteins were pulse-labeled with [³⁵S]methionine, a 55 kDa protein was evident in the cytosolic extract of cells grown on collagen, laminin, and nontreated plastic substrata; but this labeled protein was not evident in similar extracts from cells grown on collagen and laminin-coated microporous membranes. To test if the polarized, basement-membrane secreting phenotype of the MDCK cells could be generated on a microporous membrane without pretreatment with any extracellular matrix (ECM) components, cells were seeded on the Millipore Millicell HA (cellulosic) microporous membrane. This type of substrata does not need a coating of ECM components for cell attachment. A partial basement membrane was formed below cells where the basal surface of the cell was planar, but not in areas where the cell formed large cytoplasmic extensions into the filter. This led us to the conclusion that the microporous nature of the substrata can dictate both ultrastructural and protein synthetic activities of MDCK cells. Furthermore, we suggest that both the planar nature of the basal surface and the microporosity of the substrate are corequisites for the deposition of the basement membrane.     

Twenty years of numerical syntaxonomy

The development of numerical syntaxonomy during its first 20 yr is reviewed. The use of methods of numerical classification and ordination is the dominating feature of the development. National and local phytosociological data banks were established, large data sets handled and many important vegetation monographs were methodically based on multivariate data analysis. Particularly the development in Italy, the Netherlands, Czechoslovakia, and Sweden contributed to new theoretical elements of numerical syntaxonomy. Ordination became a common tool of searching for reticulate synsystematic relations between community types. The most popular ordination techniques have been Principal Components Analysis and Detrended Correspondence Analysis. Hierarchical agglomerative techniques of clustering still prevail in classification, although the divisive strategy of TWINSPAN has also become an effective tool for phytosociological clustering and table sorting. Extensive program packages, also for personal computers have now become standard equipment for many vegetation scientists.     

A new dissimilarity measure and a new optimality criterion in phytosociological classification

A new dissimilarity measure, Uppsala dissimilarity, is proposed. It is a Manhattan-type measure in between the Canberra and Gower measures, based on the differences between scores in relevés compared, but it also takes both the sums of scores and the difference between maximum and minimum score into account. The measure is considered realistic for phytosociological material.A new optimality criterion has been developed after unsatisfactory results had been obtained with the DOL criterion (Popma et al. 1983) which was developed previously by our group. Problems with DOL were especially met when the criterion was applied to the distribution of only one species over the cluster array obtained. The new criterion takes both internal cluster homogeneity and between-cluster dissimilarity into account. Between-cluster dissimilarity is calculated for all other clusters and not only for the nearest neighbour, as in DOL. The new criterion has both an unweighted form: SOM, and a form with weighting for cluster size: SWOM.This new criterion was successfully applied to the evaluation of the sharpness of distribution of individual species over cluster arrays, under the name of SIM: species indication measure and SWIM, species weighted indication measure.The measures were applied to some test data. Differences between the unweighted and weighted forms were found which could not be easily interpreted.Some remarks are made on the coherence of d-SAHN and h-SAHN approaches in agglomerative clustering within the new strategy proposed.Abbreviations DOL = Detection of Optimal Level - S(W)IM = Species (Weighted) Indication Measure - S(W)OM = Standardized (Weighted) Optimality Measure - UD = Uppsala Dissimilarity measure - WPGMA = Weighted Pair-Group Method Average linking clustering - SAHN = Sequential Agglomerative Hierarchical Non-overlapping clustering     

Monitoring juvenile stocks of flatfish in the Wadden Sea and the coastal areas of the southeastern North Sea

Results are presented of an on-going monitoring program, started in 1970, of the demersal fish population in the Wadden Sea, Scheldt estuary, and along the continental coast between the Belgian-French border and Esbjerg. Particular attention is given to long-term trends in overall abundance and annual variations in spatial distribution of 0- and 1-group plaice and sole in relation to year-class strength and to variations in growth rate. Recruitment level in both plaice and sole appeared to be higher in the 1980s than in the 1970s. The most important nursery areas for plaice are the German Bight and the Wadden Sea, and the relative contributions of these areas to the total plaice stock in different years are relatively stable. In contrast, the most important nurseries for sole are along the continental coast, and the contribution of different parts of these nurseries vary considerably from year to year. Abundant year-classes of sole can originate from northern as well as from southern parts of the nurseries. Relations between survey results and year-class estimates from Virtual Population Analysis (VPA) for plaice are better compared to sole. No trends have been observed in mean length at age 0 and 1 during the years the surveys have been carried out. It is concluded that the quality of the continental coastal area and the Wadden Sea as nursery area for plaice and sole has not declined during the period of investigation. Presented at the VI International Wadden Sea Symposium (Biologische Anstalt Helgoland, Wattenmeerstation Sylt, D-2282 List, FRG, 1–4 November 1988)     

Effects of Solar Radiation on Photoorientation,Motility and Pigmentation in a Freshwater Cryptomonas

The effects of solar radiation on motility, photoorientation and pigmentation have been studied in a freshwater Cryptomonas species. The diaphototactic orientation performed by the cells is impaired within about 90 min of solar radiation. Likewise, the percentage of motile cells within the population and the average velocity of the swimming cells decreases within about the same exposure time. This effect is not due to a thermal stress but rather seems to be caused by the solar UV-B component, since decreasing short wavelength UV radiation by means of an artificial ozone filter or UV cut-off filters increased the tolerated exposure time. Solar radiation also bleached the photosynthetic pigments of the cells as shown by absorption difference spectra.     

Semisynthesis of Arg15, Glu15, Met15, and Nle15-aprotinin involving enzymatic peptide bond resynthesis

The semisynthesis of homologues of aprotinin, the bovine pancreatic trypsin inhibitor, is described. The P₁ lysine¹⁵ residue was replaced by two methods. The first procedure, which consisted of two enzymatic steps for the incorporation of other amino acids has previously been described. The second approach consisted of six steps of both enzymatic and chemical nature. The modified inhibitor, in which the lysine¹⁵-alanine¹⁶ peptide bond is hydrolyzed, was used as the starting material. All carboxyl groups of the modified inhibitor were esterified with methanol; the lysine¹⁵ methylester group was then selectively hydrolyzed. Afterward, lysine¹⁵ itself was split off. Arginine, glutamic acid, methionine, andl-2-aminohexanoic acid (norleucine, Nle) were incorporated using water-soluble carbodiimide combined with an acylation catalyst. The methylester group was used to prevent polymerization. The reactive-site peptide bonds were resynthesized using either chymotrypsin or trypsin.