George M. Padilla

Molecular cytogenetic analysis of the bladder carcinoma cell line BK-10 by spectral karyotyping.

The bladder cancer cell line BK‐10 was established from a grade III–IV transitional cell carcinoma (TCC). BK‐10 is near‐tetraploid (±4n) and consists of two subclones with 20–25 structural aberrations. Here we report the cytogenetic analysis of BK‐10 by G‐banding, spectral karyotyping (SKY), and FISH. SKY refers to the hybridization of 24 differentially labeled chromosome painting probes and the simultaneous visualization of all human chromosomes using spectral imaging. SKY enabled us to confirm 12 markers in BK‐10 previously described by G‐banding, redefine 11 aberrations, and detect 4 hidden chromosomal rearrangements, 2 of which had been identified as normal or deleted copies of chromosome 20 and 1 as a normal chromosome 3. Twenty out of 21 translocations identified were unbalanced. FISH analysis of BK‐10 using chromosome arm‐specific paints, centromere probes, and oncogene/tumor suppressor gene‐specific probes revealed a deletion of CDKN2A (p16) in all copies of chromosome 9, a low‐level amplification of MYC (five copies), and loss of one copy of TP53; detected the presence of the Y chromosome in a hidden translocation; and detected four copies of ERBB‐2. A probe set for BCR and ABL verified breakpoints for all translocations involving chromosomes 9 and 22. A new karyotype presentation, “SKY‐gram,” is introduced by combining data from G‐banding, SKY, and FISH analysis. This study demonstrates the approach of combining molecular cytogenetic techniques to characterize fully the multiple complex chromosomal rearrangements found in the bladder cancer cell line BK‐10, and to refine the chromosomal breakpoints for all translocations. Genes Chromosomes Cancer 25:53–59, 1999 Published 1999 Wiley‐Liss, Inc.

Determination of free Ca ion concentrations with an ion-selective electrode in the presence of chelating agents in comparison with calculated values.

Experimentally determined free Ca ion concentrations, measured with a Ca-selective electrode, were compared with values calculated with a computer program utilizing stability constants of the chelating agents: NTA, EDTA, and EGTA used to set the free ion concentration in the range of 10−3 to 10−6m. In the presence of 0.1 m KCl, 2 mm MgCl2, 20 mm Hepes (pH 7.4), 2 mm ATP, 0.1 mm CaCl2 (total concentration), and various ligand concentrations the measured free Ca2+ levels were found to be approximately six to seven times greater than the computer-derived values. Apparent stability constants for Ca-ATP, Ca-EDTA, and Ca-EGTA were determined under these experimental conditions.

Hemolysis induced by Prymnesium parvum toxin kinetics and binding

Abstract Rates of hemolysis of rabbit erythrocytes induced by Prymnesium parvum toxin (prymnesin) have been measured colorimetrically at 25.5°. The data have been treated as consecutive first-order rate processes associated with the prolytic and lytic period for which two specific rate constants were obtained ( k′ and k ψ , respectively). Both are a function of concentration and temperature, though only the first constant has a linear Arrhenius relationship. The variation of the second constant with concentration of prymnesin follows Michaelis-Menten kinetics. Both constants are decreased by inhibitors (cholesterol and cephalin (approx. 10 μM)) at a given concentration of prymnesin, and the effect is ascribed to reduction in the effective concentration of prymnesin owing to the formation of a toxin-inhibitor complex. The binding of prymnesin to erythrocyte membranes during the prolytic period was investigated using tritium-labeled toxin, and it was found that about 10% of the labeled material is loosely bound and about 30% is more firmly bound.

Effect of Gymnodinium breve toxin on hemolysis induced by Prymnesium parvum toxin

Abstract A toxin fraction has been isolated from axenic cultures of the Florida red-tide organism, Gymnodinium breve and purified by gel filtration chromatography, using Sephadex LH 20 and chloroform. The hemolytic activity of this toxin fraction has been compared with that of purified prymnesin, the toxin isolated from the euryhaline chrysomonad Prymnesium parvum (Carter). The hemolytic activity, using rabbit erythrocytes in isotonic buffer at pH 5·5 and expressed as hemolytic units (HD50), of G. breve toxin is slight in comparison with that of prymnesin. The G. breve toxin fraction, however, appears to inhibit the hemolytic activity of prymnesin, principally by extending the prolytic phase of hemolysis and not the lytic phase.

Quantitative contribution of factors regulating rat colonic crypt epithelium: role of parenteral and enteral feeding, caloric intake, dietary cellulose level and the colon carcinogen DMH

Abstract. To elucidate the role and quantitative contribution of several exogenous factors which may regulate colon crypt mitotic activity, proliferative zone height (PZH) and crypt height, groups of rats were subjected to various feeding regimens both with and without treatment with the colon carcinogen, 1,2‐dimethylhydrazine (DMH). The rats were divided into two major groups and one group was given eight weekly injections of DMH base at 9·5 mg kg‐1 body weight. Throughout this period and for two additional weeks the rats were isocalorically fed either a defined nutritionally complete diet with different levels of dietary cellulose or they were parenterally (i.v.) fed a nutritionally complete liquid formula with different caloric levels. The rats were then injected with colchicine 3 h prior to sacrifice to arrest and to collect dividing cells at metaphase. The results of multiple regression analysis of all data were interpreted to indicate that parenteral feeding caused dramatic suppression of the colon crypt height (CH) and of the number of metaphase figures per crypt (MC). Increased cellulose intake stimulated CH but suppressed MC. The CH was also stimulated by DMH. CH was positively correlated to PZH and MC. The MC was suppressed by cellulose intake and negatively correlated to PZH but was positively correlated to CH. The PZH was positively correlated to CH. These findings were related to the role of luminal food, functional workload, kcal intake and treatment with DMH on the measured colon crypt parameters. A quantitative assessment of factors that regulate the measured colonic crypt parameters was accomplished.

Synchronous Cell Differentiation

Publisher Summary Synchronous cell differentiation refers to the occurrence of simultaneous cellular transformations throughout a population of cells. Although the induction of cell division synchrony may be a prerequisite for such an experimental situation, it need not be the primary concern of the investigator. In fact, the motivating force for studying a synchronously differentiating system is that it is only through the amplification of specific biochemical and morphological changes that analytical methods of cell biology are successfully employed. The chapter focuses mainly on those cellular communities in which synchrony has been induced experimentally throughout the entire population. In order to achieve an understanding of the process of differentiation one must look beyond the question of genome replication. The possibly common feature in the systems described in the chapter has been the intimate involvement of extranuclear elements in morphogenetic changes. Membrane complexes, such as the bacterial chromosome “initiator” and the cortical organelles in protoza, are strategically localized to assume more than an incidental relationship with nuclear elements as a shift in the cell state is taking place. One must examine a specific event of differentiation not merely as a further example of the molecular virtuosity of cells but as an indication of the mechanisms which control such specialization.

Aromatase activity in microsomes from rat ventral prostate and dunning R3327H rat prostatic adenocarcinoma

We have measured aromatase activity in microsomes obtained from rat ventral prostate, using the 3H2O release method as described by Weisz. Production of 3H2O from 1 beta-[3H]androstenedione correlated with estrogen production measured by RIA and by TLC. The assay was optimized for incubation time and protein concentration, and used to determine the aromatase activity of ventral prostate microsomes from rats of varying age. Aromatase activity per mg microsomal protein increased from an average of 4 pmol/mg protein X h in 3-month old rats to 68 pmol/mg protein X h in 8-month old rats. Aromatase activity was also measured in microsomes from the Dunning R3327H rat prostatic adenocarcinoma, and was increased in tumors removed 225 days after implantation compared to tumors removed 141 days after implantation. Tumors removed 225 days after implantation from rats which had been treated with DES for 14 days displayed increased aromatase activity compared to untreated tumors. The presence of aromatase activity in the rat ventral prostate and rat prostatic adenocarcinoma would allow regulation of estrogen levels independent of circulating estrogen. Thus, in situ changes in estrogen production with age may contribute to the development of prostatic disease.

Hemolytically active components from P. parvum and G. breve toxins

Abstract Hemolytically active fractions were isolated from the toxins produced by the red-tide dinoflagellate Gymnodinium breve (GBTX) and the chrysomonad Prymnesium parvum (PPTX). High pressure liquid chromatography through bonded phase (ODS) silica columns using a gradient of methanol in chloroform yielded 6 major fractions from GBTX, 3 of which were hemolytic (HD 50 =0.3−0.56 μg ·ml −1 ). None were ichthyotoxic. Of the 6 fractions obtained from PPTX, 4 were hemolytic (HD 50 =0.013−2.8 μg ·ml −1 ) but only one (fraction 6) was ichthyotoxic. This fraction was ∼ 2000 times more hemolytic than the crude PPTX (HD 50 =33.2 μg ·ml −1 ). Analysis of their UV spectra indicates that the fractions within each group are closely related.

Purification of the ichthyotoxic component of Gymnodinium breve (red tide dinoflagellate) toxin by high pressure liquid chromatography

High pressure liquid chromatography of a chloroform extract of the red tide dinoflagellate, Gymnodinium breve, was used to separate the ichthyotoxic component from the hemolytic and non-toxic fractions. The best separation was achieved by gradient elution with an increasingly polar mixture of chloroform and methanol through a micro particle silica column. Unlike preparative TLC, high pressure liquid chromatography achieved approximately 100-fold purification of G. breve toxin as determined by a probit analysis of dose-survival curves of its ichthyotoxicity. It was found that the purified toxin was unstable when stored in the dark at 4°C in the dry state as judged by its chromatographic behavior and u.v. and fluorescence spectra. The results of these analyses indicate that G. breve toxin contains a single ichthyotoxic component.

Hemolysis induced by Prymnesium parvum toxin calorimetric studies

The relationship between binding of the hemolytic toxin (prymnesin) to bovine erythrocytes and the amount of heat liberated was examined as a function of pH using a flow microcalorimeter and 3H-labelled toxin isolated from the euryhaline alga Prymnesium parvum. A high degree of correlation (correlation coefficient = 0.986) was found between the amount of heat generated and the quantity of toxin that was allowed to interact with the erythrocytes. No significant binding of toxin was observed at pH 7 but it increased linearly as the pH was reduced to 5.5. Maximum heat and binding occured at a pH range 4.5–5.5. The same pattern was followed in terms of the amount of heat liberated and the hemolytic activity of the toxin. The differences in the maximum binding and heat production as a function of pH was independent of the average red cell volume which remained constant at pH 5.5 and 6.2 (102.4 and 102.6 μm3, respectively).