Paula T. Beall

Clomiphene protects against osteoporosis in the mature ovariectomized rat

SummaryClomiphene citrate, a mixed estrogen agonist-antagonist, protects mature ovariectomized breeder rats from changes in total body calcium and from deterioration of femur structure. Over 6 months, mature ovariectomized rats took up calcium at the rate of 0.7 ± 0.5 mg/day, while normal controls gained 2.5±0.7 mg/day (mean±SE) as measured by whole body neutron activation analysis. Injections of clomiphene (20 mg/kg/week) kept ovariectomized rats in positive calcium balance at 2.0±0.5 mg/day. Reductions in total femur calcium content, cortical thickness, and visible trabeculae of femurs in ovariectomized animals were prevented by chronic clomiphene administration. These results in animals suggest a possible new line of investigation of the use of antiestrogenic drugs as therapeutic agents for hormone-dependent osteoporosis in animals and humans.

Practical methods for biological NMR sample handling

The ability of NMR parameters T1 and T2 to be used to distinguish one tissue from another and diseased from normal tissues has wide application in diagnostic medicine. Measurement of such values in vitro on excised tissues and fluids was the basis for fundamental discoveries which provided the impetus for the development of in vivo clinical whole body NMR imagers. Therefore, as in vivo imaging grows, the need for screening and testing of new ideas in vitro will grow. The success of data collection in vitro depends greatly on the care and understanding with which biological samples are processed. This paper summarizes proven methods for handling of soft and firm biopsy material, blood components, body fluids, and culture cells. The effects of temperature variation and storage parameters are discussed for animal and human samples. The paper is a practical guide to the hows and whys of biological sample handling for NMR.

The “Systemic Effect” of Elevated Tissue and Serum Relaxation Times for Water in Animals and Humans with Cancers

In this decade, following the Damadian discovery (Science 1971) that the nuclear resonance signal would non-invasively detect disease, nuclear magnetic resonance (NMR) spectroscopy has shown potential as a tool in medical diagnosis. One area in which NMR has been utilized successfully is in the exploration of the physical properties of water in disease states, especially cancer. This chapter is specificially aimed at one phenomenon called the “systemic effect”, whereby the physical properties of water and the concentration of water are changed in distant organs and sera of animals bearing tumors. An understanding of this phenomenon and our knowledge of it can be approached from a historical perspective coupled with a presentation of the latest experimental data in this field.

Inhibition of active sodium transport by cytochalasin B in rat jejunum in vitro

The effects of cytochalasin B on electrophysiological properties and sodium transport in rat jejunum in vitro are described. Stripped paired rat jejunal segments were maintained in Ussing chambers with Leibovitzs (L-15) tissue culture medium bubbled with 100% oxygen. L-15 medium contains galactose as the only sugar, and an assortment of amino acids and cofactors to nourish the tissue. Electrophysiological parameters of short-circuit current (Isc) and transepithelial potential difference could be maintained for up to 4 h in control tissues. Upon application of cytochalasin B (20 micrograms/ml), on the mucosal side, Isc and potential difference fell within 1 h from 1.93 +/- 0.12 to 1.09 +/- 0.14 (mean +/- S.E.) muequiv./cm2 per h and from 5 to 2.5 mV. Tissue resistance remained unchanged at approx. 110 omega X cm2 for up to 4 h. 22Na net flux was 4.1 +/- 0.9 muequiv./cm2 per h during the last control period and fell to zero within 1 h after cytochalasin B treatment. Transmission electron micrographs revealed no gross morphological changes at this dose. Absorptive junctional morphology was apparently not altered by cytochalasin B treatment, a finding which was consistent with the stable transepithelial electrical resistance observed during exposure to this drug. Active sodium transport processes coupled to hexose, amino acid, and chloride movements are all possible in L-15 medium. However, following exposure to 20 micrograms/ml cytochalasin B, all net sodium transport is completely inhibited. The data are consistent with the hypothesis of a common regulator for active sodium transport processes which is modulated through structural changes in cytoskeletal organization.

Cell Cycle Phase-Specific Changes in Relaxation Times and Water Content in HeLa Cells

The ubiquity of water molecules and their essential role in life processes is well known. Although a number of quantitative methods are now available for the study of water, the structure and function of this simple molecule in solutions and in biological systems is not well understood. Much of the early work with water in biological tissues was confined primarily to simple measurements of water content of different tissues. In 1913, it was noted that the percentage of water in some tumors is higher than that of the host tissues (1). Cramer, in 1916, presented evidence supporting the notion that the percentage of water in tumors is directly related to the rate of tumor growth (2). McEwen et al. (3) later reported that the increased percentage of water in tumors was not due to the loss of dry solids. Evidence demonstrating a systemic effect of tumors on the water content of other organs was reported in 1932 by Schlottman and Rubenow (4). By 1943, this systemic effect of tumors on the percentage of water in organs was well known (1). After the 1940’s, interest in the water content of tumors seemed to wane.

The microtubule cytoskeleton in normal and transformed cells in vitro.

Cell transformation in vitro is usually accompanied by alterations in morphology and growth properties which may be related directly or indirectly to malignancy. Although the initial lesion of transformation probably involves the interaction of a carcinogen with the cell genome, subsequent expression of the mutagenic event is manifested in altered cell shape, alterations in motility, spreading, and adhesive properties, loss of density-dependent growth control, loss of anchorage dependence and changes in cell surface properties. Recent evidence has suggested that many aspects of transformation may relate to changes in a delicate system of cytoplasmic microfilaments and microtubules known collectively as the cytoskeleton (Edelman 1976; Nicolson 1976). Components of the cytoskeleton play a major role in the regulation of cell form (Porter 1966; Nicolson 1976), and both microtubules and microfilaments have been shown to be involved in cell motility (Porter 1966; Allison 1973) and substrate attachment (Goldman and Follett 1970; Pollack and Rifkin 1975; Miller et al. 1977). Also, the modulation of cell surface proteins is regulated to a large extent by submembranous assemblies of microtubules and microfilaments (Edelman 1976).

Microtubule complexes correlated with growth rate and water proton relaxation times in human breast cancer cells

Ten established human breast cancer cell lines display patterns of microtubule organization which are characterized by growth rate of the cell populations and the freedom of mobility of cellular water molecules measured by nuclear magnetic resonance spectroscopy. Cell lines with population-doubling times of 1 to 2 days demonstrate rapid mobility of water molecules by proton spin-lattice and spin-spin relaxation times (T1 greater than 750 msec, T2 greater than 120 msec) and have diffuse patterns of tubulin immunofluorescent antibody staining. Moderately fast dividing cells (population-doubling times of 3 to 7 days) have T1 values of 600 to 750 msec and show approximately 50% organized complexes of polymerized microtubules in the cytoplasm. Slow-growing cell lines demonstrate more restricted mobility of water molecules (T1 values of 500 to 600 msec) and contain abundant networks of polymerized microtubules. The three-way correlation of the physical parameter of water proton relaxation times, the structural parameter of microtubule organization, and the physiological parameter of growth suggest a close interaction of water molecules with the cytoplasmic macromolecular network in the performance of physiological function.

NMR relaxation times of water protons in human colon cancer cell lines and clones

Established lines of human colon cancer cells from several sources (LS180, LS174T, HT29, SW480, SW1345) had water proton nuclear magnetic resonance (NMR) spin-lattice relaxation times (T1) of 460 +/- 45 msec to 982 +/- 9 msec and spin-spin relaxation times (T2) of 83 +/- 6 msec to 176 +/- 6 msec. Two clones derived from single cells of line LS174T were similar in T1 and T2 to the parent line. Differences among the cell lines were not totally a function of cellular hydration. Normal adult and fetal human primary colon cells were wetter and had higher T1 and T2 values than established cell lines. Relaxation times in this study substantiate variations seen for human colon tumors in earlier studies. Established cell lines maintained water relaxation times similar to tumor tissue values. Along with other morphological and biochemical criteria, the relaxation times suggest that these established human colon cancer cell lines may serve as a good experimental model for the study of human colon cancer.