Biomedical subjects

C F Verkoelen

Publications and source records attributed to C F Verkoelen.

28 records · Page 2Linked to original sources

PubMed1994

Glycosaminoglycans and other sulphated polysaccharides in calculogenesis of urinary stones.

Naturally occurring glycosaminoglycans (GAGs) and other, semisynthetic, sulphated polysaccharides are thought to play an important role in urolithiasis. Processes involved in urinary stone formation are crystallization and crystal retention. Oxalate transport and renal tubular cell injury are determining factors in these processes. In this article experimental results concerning the possible mechanisms of action of GAGs and other sulphated polysaccharides are reviewed. GAGs are inhibitors of crystal growth and agglomeration and possibly also of nucleation. They can prevent crystal adherence, correct an abnormal oxalate flux and prevent renal tubular cell damage.

Animals↗

PubMed1994

Etiology of experimental calcium oxalate monohydrate nephrolithiasis in rats.

In a rat-model system, tubular crystal retention as a possible mechanism for the etiology of nephrolithiasis in man, was studied by conventional transmission electron microscopy. The animals were supplied for nine days with a crystal-inducing diet, with ethylene glycol plus NH4Cl in their drinking-water. After this induction period, a two day regime with fresh drinking-water was included, to allow crystals to be removed by spontaneous crystalluria. After aldehyde fixation of the rat kidneys, large crystals were seen inside the tubular lumen. The crystals were attached to cell surfaces and covered by neighboring epithelial cells. Some crystals were overgrown by several epithelial cells and underwent a process of so-called exotubulosis, resulting in free or cell-surrounded crystals in the interstitium, and possibly in crystals in Giant cells. To investigate the fate of the retained crystals, some animals were additionally exposed to a low-oxalate challenge from drinking water containing 0.1 volume per cent of ethylene glycol for 12 or 30 days, respectively. It was assumed that this would interfere with the retained intratubular or interstitial crystals, and allow the crystals to grow into mini-stones. This was not observed. After the oxalate challenge, no crystals were found to be retained in the tubules (free or covered by cells). Interstitial crystals were observed, but it remains to be demonstrated whether such crystals actually grow into mini-stones or that they are removed by the sterile inflammation process observed.

Ammonium Chloride↗

PubMed1993

Absence of a transcellular oxalate transport mechanism in LLC-PK1 and MDCK cells cultured on porous supports.

Transepithelial oxalate transport across polarized monolayers of LLC-PK1 cells, grown on collagen-coated microporous membranes in Transwell culture chambers, was studied in double-label experiments using [14C]-oxalate together with [3H]-D-mannitol as an extracellular marker. The [14C]-labeled glucose analog alpha-methyl-glucoside (alpha-MG) was used as functional marker for active proximal tubular sugar transport. Cellular uptake of oxalate and alpha-MG at both the apical and basolateral plasma membrane was determined. When added to the upper compartment, alpha-MG was actively taken up at the apical membrane, directed through the cells to the basolateral membrane and transported to the lower compartment, indicating functional epithelial sugar transport by LLC-PK1 cells. In LLC-PK1 cells, the uptake of alpha-MG at the apical membrane was approximately 50 times higher than that at the basolateral membrane. In contrast to this active transport of sugar, LLC-PK1 cells did not demonstrate oxalate uptake either at the apical or basolateral plasma membrane. The apical-to-basolateral (A- > B) flux of oxalate in LLC-PK1 cells was identical to the basolateral-to-apical (B- > A) oxalate flux in these cells. Moreover these flux characteristics were similar to those found for D-mannitol, indicating paracellular movement for both compounds. From these data, it is concluded that, under the experimental conditions used, LLC-PK1 cells do not exhibit a specific transcellular transport system for oxalate.

Animals↗

PubMed1992

Distinction of two different classes of small-cell lung cancer cell lines by enzymatically inactive neuron-specific enolase.

Neuron specific enolase (NSE) is widely used as a neuro-endocrine marker. However the presence of NSE in many non-neuroendocrine tissues has raised questions on the specificity of NSE. We have investigated NSE immunoreactivity (NSA-ag), gamma-enolase activity and total enolase activity in small cell lung cancer (SCLC) cell lines. During well-controlled exponential growth comparison of NSE-ag content and gamma-enolase activity with the doubling-time (Td) and NSE-ag content with gamma-enolase and total enolase activity led to a clear distinction of two types of cell line: variant cell lines plus part of the classic cell lines (type I) and the remaining classic cell lines (type II). The distinction was based upon both an abrupt 6-fold increase of gamma-enolase activity and an 18-fold increase of NSE-ag, which for the larger part was enzymatically inactive. Within each group the increase of NSE-ag content was significantly correlated with the increase of gamma-enolase activity and both NSE-ag content and gamma-enolase activity increased linearly with Td. It is concluded that gamma-enolase seems to be associated with the regulation of growth rate and that a compound with the gamma-enolase antigen but without enzyme activity can distinguish two different classes of SCLC cell lines. Furthermore the demonstration that NSE-ag can represent the active enzyme as well as an enzymatically inactive compound may explain why a controversy about neuron- or non-specificity of NSE exists.

Carcinoma, Small Cell↗

PubMed1988

Quantitation of polyamines in cultured cells and tissue homogenates by reversed-phase high-performance liquid chromatography of their benzoyl derivatives.

A rapid and simple method, originally described by Redmond and Tseng [J. Chromatogr., 170 (1979) 479] was applied to the analysis of di- and polyamines in cultured human tumour cells and human tumour xenografts. Optimization of the procedures and evaluation of the characteristic features of the assay are described. The (modified) procedure employs precolumn derivatization with benzoyl chloride, extraction of the derivatives by chloroform, separation by reversed-phase high-performance liquid chromatography under isocratic conditions and detection by ultraviolet absorbance measurement at 229 nm. The complete analysis was accomplished within 10 min per sample. The detection limit was ca. 1 pmol. The intra- and inter-assay coefficients of variation were 2.5-4.4% and 3.4-13.1%, respectively. The presence of well known inhibitors of polyamine biosynthesis, such as DL-alpha-difluoromethylornithine and methylglyoxal bis(guanylhydrazone), did not interfere with the assay, and disturbance by cyclohexylamine could be avoided by changing the polarity of the mobile phase. The method proved to be very suitable because it is rapid, simple, requires a minimum of sample pretreatment, and still provides sufficient sensitivity to quantitate polyamines in relatively small amounts of cells (10(5) cells) or tumour tissues (less than 1 mg), even after treatment with inhibitors of polyamine biosynthesis.

Animals↗

PubMed1988

Application of the MTT assay to human prostate cancer cell lines in vitro: establishment of test conditions and assessment of hormone-stimulated growth and drug-induced cytostatic and cytotoxic effects.

In this study the usefulness of the MTT assay for the quantitation of growth modulating effects on cultured prostate cancer lines (PC-3, PC-93, and LNCaP) was investigated. The MTT test is based on the enzymatic reduction of the tetrazolium salt MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide++ +] in living, metabolically active cells but not in dead cells. The reaction is carried out in situ in multiwell plates, and the reaction product, a purple-colored formazan soluble in dimethylsulfoxide, is measured colorimetrically, using a multiwell plate reader. Optimal test conditions were established for each of the cell lines used. With the hormone-sensitive cell line LNCaP, and stimulatory effect of the synthetic androgen R1881 was demonstrable by the MTT test. A sharp optimum occurred at a concentration of 10(-10) M R1881. Treatment of cells of either cell line with antineoplastic agents resulted in a dose-dependent reduction of MTT converting activity, reflecting the impaired survival of the drug-treated cells. Good correlations of the results obtained with the MTT-test, as compared with a thymidine incorporation assay or with direct DNA measurements, were observed. As the MTT test offers a high degree of precision and is easy to do, it is suitable for the purpose of (large-scale) chemosensitivity testing. Moreover, serial measurements might easily be performed in order to provide additional information on the mode of action of the drugs tested, i.e., to discriminate between cytostatic and cytotoxic drug effects.

Androgens↗

PubMed1987

Problems of pharmacokinetic studies on alpha-difluoromethylornithine in mice.

The pharmacokinetics of alpha-difluoromethylornithine (DFMO), an irreversible inhibitor of polyamine biosynthesis, were investigated in BALB/c (nude) mice after i.p. injection and after oral administration of radiolabeled drug. After i.p. injection the compound was rapidly cleared from the serum (t1/2 alpha = 14 min; t1/2 beta = 2.1 h) and from tissues such as muscle, liver and kidney (t1/2 alpha = 30-60 min; t1/2 beta = 2.1 h). DFMO concentrations were proportional to the administered dose (10-2000 mg/kg) in both serum and tissues. Oral administration of DFMO was carried out by dissolving the compound in drinking water at a concentration of 20 g/l. Studies on the distribution showed that DFMO did not accumulate preferentially in any particular tissue. An extremely wide variation in the dose actually achieved in different animals was observed; this ranged from 350 to 2800 mg/kg for a 14-h treatment period. A significant correlation (r = 0.83-0.92) between the dose of DFMO, calculated from the consumption of drinking water for each individual animal, and the DFMO concentrations in serum, muscle, spleen, liver and kidney was found. Similarly, it was shown that oral administration of DFMO during the daytime resulted in 10- to 15-fold lower levels than administration during the night. After discontinuation of treatment DFMO levels in serum and tissues decreased by 50% in approximately 6 h. From these results it is concluded that the optimal treatment schedule of mice with DFMO (or other drugs with similar pharmacodynamic properties) consists in a combination of oral administration via the drinking water and additional i.p. injection (during the daytime). Furthermore, the drug intake of the individual animals should be monitored to check whether the experimental requirements are actually fulfilled.

Administration, Oral↗

PubMed1986

Measurement of the survival of human tumor cells after implantation in athymic nude mice.

A method has been developed to determine the survival of human tumor cells after implantation in athymic nude mice. The method involves the labelling of cells of the prostatic cancer cell line PC-3 with tritiated thymidine after attachment of the cells to Millipore filter disks. The amount of radioactive label incorporated in the cells remained constant for at least 48 hr during in vitro incubation. After s.c. implantation of the filter disks in nude mice, however, the fraction of surviving cells, reflected by the amount of radioactivity persisting on the filters, rapidly decreased with time. Only a relatively small fraction (approx. 10%) of the cells survived for more than 24 hrs under these conditions. Pretreatment of the mice with cyclophosphamide (100 mg/kg, 24 hr prior to implantation) resulted in a 2-fold increase of tumor-cell survival. Likewise, the frequency of tumor takes after injection of PC-3 cells was enhanced following cyclophosphamide pretreatment. These results indicate that immune defense mechanisms might be partly (but not completely) responsible for tumor-cell rejection in nude mice. The technique described here might be a valuable tool for the rapid evaluation of possible effects of various types of pretreatment of the host animal on tumor cell survival and, thereby, on tumor growth.

Animals↗

PubMed1986

Species-dependent differences of the biochemical properties of diamine oxidase.

Diamine oxidase (DAO) from tissues of mice, rats and humans showed different properties with respect to stability and kinetic parameters. DAO-activities in homogenates of rat or human tissues, but not of mouse tissues, rapidly decreased upon storage at -20 degrees C. The Km-value for putrescine was 90 microM in mouse kidney or intestine. In rats different Km-values were observed before (272 microM) and after freezing (102 microM). A similar effect was observed with DAO in human kidney (321 and 39 microM, respectively). Treatment of rats with heparin resulted in a depletion of intestinal DAO and the concomitant appearance of DAO in blood. The enzyme remaining in the intestine showed the lower Km-value.

Amine Oxidase (Copper-Containing)↗

PubMed1984

Determination of the growth rate of human prostatic cells in primary culture by a morphometric technique.

A morphometric technique, based on the measurement of the area of individual cell colonies and of its increase in time, was applied to study the rate of proliferation of human prostatic cells in vitro. The reliability of the method was checked by determination of the growth rate of cultures of the continuous cell line PC93 by the morphometrical technique as well as by counting of the cell number. No significant difference was found in the population doubling times measured by either of these methods. It was therefore concluded that the morphometrical technique could be applied also to study the growth rate of primary cultures of prostatic epithelial cells, in which counting of the cell number is generally impossible. The results showed that, with primary cultures derived from hyperplastic prostates and prostatic carcinomas as well as from the prostatic tumor line PC82, rapid growth occurred during the first two or three days of culture; measurements performed at a later time appeared to be less reliable. It was demonstrated by the effect of serum deprivation on the growth of PC82 cells that the technique described here is, in principle, suitable to monitor the effect of various agents on the growth of cells in primary culture. The method is non-destructive and requires minimal amounts of tissue; it may be applied especially to cultures that cannot be dispersed easily into single cell suspensions.

Adenocarcinoma↗