Positive colloidal thorium dioxide as an electron microscopical contrasting agent for glycosaminoglycans,compared with ruthenium red and positive colloidal iron

Summary In electron microscopy Thorotrast has been used as a specific contrasting agent for acid glycosaminoglycans. Because of its high atomic number, thorium (Z=90) gives good contrast in the electron microscope, but at present it is less frequently used for this purpose. We prepared a positive colloidal solution of ThO₂ without stabilizers to compare its properties with those of ruthenium red and positive colloidal iron for contrasting fetal mouse epiphyseal cartilage. The results indicate that colloidal ThO₂, which is easy to prepare in any laboratory, gives better results than ruthenium red and colloidal iron do in this kind of cartilage. Furthermore, as judged from data in the literature and obtained in our laboratory, it penetrates this tissue better than Thorotrast does, probably because of the absence of stabilizers.     

Human Dermal Fibroblasts Demonstrate Positive Immunostaining for Neuron- and Glia- Specific Proteins

In stem cell cultures from adult human tissue, undesirable contamination with fibroblasts is frequently present. The presence of fibroblasts obscures the actual number of stem cells and may result in extracellular matrix production after transplantation. Identification of fibroblasts is difficult because of the lack of specific fibroblast markers. In our laboratory, we isolate and expand neural-crest-derived stem cells from human hair follicle bulges and investigate their potential to differentiate into neural cells. To establish cellular identities, we perform immunohistochemistry with antibodies specific for glial and neuronal markers, and use fibroblasts as negative control. We frequently observe that human adult dermal fibroblasts also express some glial and neuronal markers. In this study, we have sought to determine whether our observations represent actual expression of these markers or result from cross-reactivity. Immunohistochemistry was performed on human adult dermal fibroblasts using acknowledged glial and neuronal antibodies followed by verification of the data using RT-qPCR. Human adult dermal fibroblasts showed expression of the glia-specific markers SOX9, glial fibrillary acidic protein and EGR2 (KROX20) as well as for the neuron-specific marker class III β-tubulin, both at the protein and mRNA level. Furthermore, human adult dermal fibroblasts showed false-positive immunostaining for S100β and GAP43 and to a lower extent for OCT6. Our results indicate that immunophenotyping as a tool to determine cellular identity is not as reliable as generally assumed, especially since human adult dermal fibroblasts may be mistaken for neural cells, indicating that the ultimate proof of glial or neuronal identity can only be provided by their functionality.     

An estimation of drag in front crawl swimming

Propulsive arm forces of twelve elite male swimmers during a front crawl swimming-like activity were measured. The swimmers pushed off against grips which are attached to a 23 m tube at 0.8 m under the water surface. The tube was fixed to a force transducer. Since at constant speed, mean propulsive force equals mean drag force this method also provides the mean active drag on a moving swimmer. The mean propulsive force at a speed of v = 1.48 m s-1 appeared to be 53.2 +/- 5.8 N which is two to three times smaller than what is reported by other authors for active drag but which is in agreement with values reported for passive drag on a (towed) swimmer who is not moving. Discrepancies with indirect active drag measurements are discussed.     

Perturbation of cultured human vascular endothelial cells by phorbol ester or thrombin alters the cellular von Willebrand factor distribution

We have studied the influence of perturbation of cultured human umbilical vein endothelial cells on the distribution of the von Willebrand factor. As shown previously, short-term (less than 1 hr) treatment of endothelial cells with the phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) or thrombin resulted in the release of cellular stored von Willebrand factor. Long-term treatment with PMA or thrombin evoked a distinct change in the endothelial cell distribution of von Willebrand factor, evident 24 to 48 hrs after exposure. Whereas the contents of the von Willebrand factor storage sites in the cells were gradually restored within 48 hrs, enhanced amounts of von Willebrand factor were secreted into the medium. However, PMA did not increase the endothelial cell contents of mRNA encoding for von Willebrand factor. The number as well as the size of von Willebrand factor storage granules in the endothelial cells increased after exposure to the phorbol ester, as determined by immunofluorescence microscopy. A second treatment with PMA or thrombin, 48 hrs after cells had been stimulated with these agents, resulted again in the instantaneous release of von Willebrand factor. PMA and thrombin caused a decrease in the von Willebrand factor contents of the extracellular matrix. Pulse-chase experiments revealed that PMA blocked the deposition of von Willebrand factor in the subendothelium, whereas PMA did not affect the degradation of matrix von Willebrand factor. Thus, perturbation of endothelial cells changes the cellular distribution of von Willebrand factor.     

Single-stranded DNA used as an efficient new vehicle for transformation of plant protoplasts

In relation to the question which DNA form (single- or double-stranded) is transferred by Agrobacterium tumefaciens to plant cells, we studied the behaviour of single-stranded DNA, as compared to double-stranded DNA, when it is introduced into plant protoplasts by electroporation. To this end, we cloned a construct with a plant NPTII gene as well as a CAT gene in the M13 vectors tg130 and tg131. We found that both complementary single-stranded molecules gave rise to substantial CAT activity in plant protoplasts, suggesting that single-stranded DNA is converted into double-stranded DNA by the plant cell replication machinery. Unexpectedly, we found that single-stranded DNA leads to a 3–10 fold higher frequency of stable transformation (selection for kanamycin resistance) than double-stranded DNA. These results indicate that the use of single-stranded DNA might be considered in experiments in which optimal transformation frequencies are needed, e.g. with protoplasts form recalcitrant plant species.Abbreviations ss single-stranded - ds double-stranded - CAT chloramphenicol acetyl transferase - NPTII neomycin phosphotransferase II - RT room temperature     

Genes involved in the biosynthesis of PQQ fromAcinetobacter calcoaceticus

From a gene bank of theAcinetobacter calcoaceticus genome a plasmid was isolated that complements four different classes of PQQ^- mutants. Subclones of this plasmid revealed that the four corresponding PQQ genes are located on a fragment of 5 kilobases. The nucleotide sequence of this 5 kb fragment was determined and by means of Tn5 insertion mutants the reading frames of the PQQ genes could be identified. Three of the PQQ genes code for proteins of M_r 29700 (gene I), M_r 10800 (gene II) and M_r 43600 (gene III) respectively. In the DNA region where gene IV was mapped however the largest possible reading frame encodes for a polypeptide of only 24 amino acids. A possible role for this small polypeptide will be discussed. Finally we show that expression of the four PQQ genes inAcinetobacter lwoffi andEscherichia coli lead to the synthesis of the coenzyme in these organisms.     

Propelling efficiency of front-crawl swimming

In this study the propelling efficiency (ep) of front-crawl swimming, by use of the arms only, was calculated in four subjects. This is the ratio of the power used to overcome drag (Pd) to the total mechanical power (Po) produced including power wasted in changing the kinetic energy of masses of water (Pk). By the use of an extended version of the system to measure active drag (MAD system), Pd was measured directly. Simultaneous measurement of O2 uptake (VO2) enabled the establishment of the relationship between the rate of the energy expenditure (PVO2) and Po (since when swimming on the MAD system Po = Pd). These individual relationships describing the mechanical efficiency (8-12%) were then used to estimate Po in free swimming from measurements of VO2. Because Pd was directly measured at each velocity studied by use of the MAD system, ep could be calculated according to the equation ep = Pd/(Pd + Pk) = Pd/Po. For the four top class swimmers studied, ep was found to range from 46 to 77%. Total efficiency, defined as the product of mechanical and propelling efficiency, ranged from 5 to 8%.     

Viability measurements of hybridoma cells in suspension cultures

Several methods were applied to determine the viability of hybridoma cells in suspension. These methods include dye inclusion and exclusion assays such as the classical trypan blue exclusion assay, the propidium iodide (PI) exclusion assay and the fluorescein diacetate (FDA) inclusion assay. Furthermore, the relation was studied between release of lactate dehydrogenase (LDH) by hybridoma cells and their viability. Also the ATP content of the cells and cellular heterogeneity as measured with a flow cytometer were determined in relation to cellular viability. The dye inclusion and exclusion assays using trypan blue, FDA, PI were shown to be useful methods to determine cellular viability. With the FDA and PI methods it was possible to obtain additional information about cells which are in a transition state between viable and non-viable. The viability according to the scatter properties of the cells appears to reflect the overall condition of the cells, although interpretation of the results is difficult. Measurement of LDH release in the culture fluid or the cytoplasmic ATP content could not be used as parameters for cell viability.