Ivan L. Cameron

Interactions of the DNA intercalator acridine orange, with itself, with caffeine, and with double stranded DNA

Caffeine (CAF) inhibits the intercalation of acridine orange (AO) into cellular DNA. Optical absorption and fluorescence spectroscopy were employed to determine the molecular interactions of AO with itself, with CAF, and with double stranded herring sperm DNA (dsDNA). AO dimerization was observed at concentrations >2 micromol. The sharp increase in fluorescence (lambda(em)=530 nm) at 5 micromol of AO was attributed to AO multimer formation. From 0.5 to 5.0 micromol, the AO self-association binding constant (K(assoc)) was determined to be 38620 mol(-1), however, the presence of 150 mmol NaCl increased K(assoc) to 118000 mol(-1) attributed to the charge neutralization. The K(assoc) for AO with CAF was confirmed to be 256 mol(-1). K(assoc) for the binding of AO with 20 micromol DNA ranged from, 32000 mol(-1) at 2 micromol AO, to approximately 3700 mol(-1) at 10 micromol AO, in the absence of NaCl. This AO concentration dependency of K(assoc) value with DNA was attributed to AO intercalation into dsDNA at high dsDNA/AO ratios, and electrostatic binding of AO to dsDNA at low AO ratios. The findings provide information used to explain fluorescence intensity values at lambda(em) at 530 nm from studies that combine AO, caffeine, and dsDNA.

Pathogenesis of degenerative joint disease in the human temporomandibular joint.

The wide range of disease prevalences reported in epidemiological studies of temporomandibular degenerative joint disease reflects the fact that diagnoses are frequently guided by the presence or absence of non-specific signs and symptoms. Treatment is aimed at alleviating the disease symptoms rather than being guided by an understanding of the underlying disease processes. Much of our current understanding of disease processes in the temporomandibular joint is based on the study of other articular joints. Although it is likely that the molecular basis of pathogenesis is similar to that of other joints, additional study of the temporomandibular joint is required due to its unique structure and function. This review summarizes the unique structural and molecular features of the temporomandibular joint and the epidemiology of degenerative temporomandibular joint disease. As is discussed in this review, recent research has provided a better understanding of the molecular basis of degenerative joint disease processes, including insights into: the regulation of cytokine expression and activation, arachidonic acid metabolism, neural contributions to inflammation, mechanisms of extracellular matrix degradation, modulation of cell adhesion in inflammatory states, and the roles of free radicals and heat shock proteins in degenerative joint disease. Finally, the multiple cellular and molecular mechanisms involved in disease initiation and progression, along with factors that may modify the adaptive capacity of the joint, are presented as the basis for the rational design of new and more effective therapy.

Characterization of proton NMR relaxation times in normal and pathological tissues by correlation with other tissue parameters

To help understand which tissue parameters best account for the water proton NMR relaxation times, the longitudinal relaxation time (T1), the transverse relaxation time (T2), and the water content of 16 tissues from normal adult rats were measured at 10.7 MHz and 29 degrees C. Regression analyses between the above and other tissue parameters were performed. These other tissue parameters included: the amounts of various organic and inorganic components, protein synthetic rate, oxygen consumption rate, and morphological composition. In addition, the differences in T1, T2, and water content values between normal liver and malignant tumor (Morris #7777 a transplantable hepatoma) were studied to help understand how a disease state can be detected and characterized by NMR spectroscopy. The results of this study and information from the literature allow the following generalizations to be made about tissue T1 and T2 values: (1) Each normal tissue has rather consistent and characteristic T1 and T2 relaxation times which are always shorter than the T1 and T2 of bulk water; (2) tissues with higher water content tend to have longer T1 relaxation times; (3) tissue T2 values are not, however, as well correlated with water content as T1 values; (4) tissues with shorter T1 values have higher calculated hydration fractions, greater amounts of rough endoplasmic reticulum, and a greater rate of protein synthetic activity; (5) tissues with higher lipid content, associated with intracellular non-membrane bounded lipid droplets, tend to have longer T2 values; (6) tissues with greater overall surface area, whether in the form of cellular membranes or intracellular or extracellular fibrillar macromolecules, tend to have shorter T2 values; (7) the differences between T1 and T2 values between tumor and normal tissues correlated with differences in the volume fraction (amounts) of extracellular fluid volumes and in the amounts of membrane and fibrillar surface area in the cells. The above generalizations should be useful in predicting T1 and T2 changes associated with specific tissue pathologies.

Fish oil supplementation enhanced CPT-11 (irinotecan) efficacy against MCF7 breast carcinoma xenografts and ameliorated intestinal side-effects.

SummaryThe cancer chemotherapeutic efficacy of the topoisomerase I inhibitor, CPT-11 (irinotecan) is often limited by the induction of severe delayed diarrhoea. In animal studies, CPT-11 use is associated with histopathological damage to the mucosa of the small and large intestines. Results from the present study demonstrate that 60 mg CPT-11 per kg body weight (i.v. q4d × 6) halted the growth, but did not cause significant regression, of MCF7 human breast carcinoma xenografts in mice fed a diet containing 7% corn oil. However, when the diet of the MCF7-bearing mice was supplemented with 3% or 6% fish oil, the same CPT-11 treatment caused significant regression of the MCF7 xenograft. Histomorphometric analyses of intestinal mucosa of mice treated with CPT-11 and fed the diet containing 7% corn oil indicated that treatment with CPT-11 induced structural changes in the intestinal mucosa which persisted at least 5 days after the last dose of CPT-11. The intestinal mucosal architecture of mice that were treated with CPT-11 and fed the diets containing fish oil was largely unchanged from the architecture of the group of mice which did not receive CPT-11. These findings indicate that fish oil supplements may be a useful adjunct to CPT-11 treatment.

The pineal gland of the gerbil, Meriones unguiculatus. II. Morphometric analysis over a 24-hour period.

SummaryBy means of morphometric analytical procedures, a diurnal rhythm in the cellular volume of gerbil pinealocytes was determined. This rhythm has been attributed primarily to a change in the cytoplasmic volume of the pinealocytes which is low during the daylight hours and increases to reach a peak during the middle of the dark period. At the ultrastructural level, six cytoplasmic components of the pinealocytes were found to exhibit a rhythm: free cytoplasm, smooth endoplasmic reticulum (SER), rough endoplasmic reticulum (RER) and ribosomes, secretory vesicles, microtubules, and mitochondria. The presumptive secretory vesicles and the microtubules reached a peak in volume one hour before lights-off. It is suggested that lights-on and lights-off both signal a decrease in size and/or number of the secretory vesicles. The SER and RER/ribosomes reached their peak volume one hour after lights-off which is interpreted as indicating a peak in indoleamine synthesis and protein synthesis, respectively. The volume of free cytoplasm exhibits two peaks; one occurs one hour before lights-off while the second peak occurs in the middle of the dark phase. It is suggested that, although part of the secretory product of the pinealocyte may be present in dense-cored vesicles, other locations could include the free cytoplasm and clear secretory vesicles.

Non-steroidol anti-inflammatory drug effect on crypt cell proliferation and apoptosis during initiation of rat colon carcinogenesis

Sustained use of non-steroidal anti-inflammatory drugs (NSAIDs) may prevent colorectal cancer. However, the optimal drug, period of efficacy and mechanism(s) of action are unknown. Experiments were undertaken to determine which of several NSAIDs would modulate colon crypt cell proliferation or apoptosis when given during the initiation phase of 1,2-dimethylhydrazine (DMH)-induced rat colon cancer. Colon crypts located both away from and over an aggregate of lymphoid nodules (ALN) were examined. Rats were injected with aspirin, indomethacin, nabumetone, sodium salicylate, 16,16-dimethyl prostaglandin E2 or saline for 3 days and DMH or DMH vehicle on day 4 of each week for 8 weeks, then killed 3 days after the last DMH injection. At the time of killing, DMH had significantly increased crypt cell proliferation but not apoptosis. There was significantly more cell proliferation and apoptosis in crypts over the ALN than away from the ALN. Aspirin and salicylate increased proliferation and apoptosis in crypts over the ALN. Finally, the distributional peaks of cell proliferation and apoptosis were shifted significantly closer together after DMH. Thus, DMH increases proliferation and alters the distribution of proliferating and apoptotic cells in colon crypts early in carcinogenesis. Aspirin may suppress tumour incidence via salicylate by enhancing apoptosis in carcinogen-initiated cells.

Relationship between cellular ATP, potassium, sodium and magnesium concentrations in mammalian and avian erythrocytes

Intracellular K+/Na+ ratios of erythrocytes of various mammalian species are known to differ markedly. We have measured ATP, K+, Na+, Mg2+, H2O contents of erythrocytes of twelve mammalian and three avian species. Our results indicate that the intracellular ATP concentration in erythrocytes of different species is in close positive correlation with the K+/Na+ ratios (linear correlation coefficient, r = 0.852). Furthermore, ATP levels in erythrocytes of individual sheep with different potassium concentrations correspond with their K+/Na+ ratios (r = 0.747). Intracellular magnesium concentrations also correlate with ATP concentrations in erythrocytes of different species (r = 0.629) and in different sheep (r = 0.549).

A MECHANISTIC VIEW OF THE NON-IDEAL OSMOTIC AND MOTIONAL BEHAVIOR OF INTRACELLULAR WATER

It is commonly assumed that essentially all of the water in cells has the same ideal motional and colligative properties as does water in bulk liquid state. This assumption is used in studies of volume regulation, transmembrane movement of solutes and electrical potentials, solute and solution motion, solute solubility and other phenomena. To get at the extent and the source of non‐ideally behaved water (an operational term dependent on the measurement method), we studied the motional and colligative properties of water in cells, in solutions of amino acids and glycine peptides whose surface characteristics are known, and in solution of bovine serum albumin, hemoglobin and some synthetic polypeptides. Solutions of individual amino acids with progressively larger hydrophobic side chains showed one perturbed water molecule (structured‐slowed in motion) per nine square angstroms of hydrophobic surface area. Water molecules adjacent to hydrophobic surfaces form pentagonal structural arrays, as shown by X‐ray diffraction studies, that are reported to be disrupted by heat, electric field, hydrostatic pressure and phosphorylation state. Hydrophilic amino acids demonstrated water destructuring (increased motion) that was attributed to dielectric realignment of dipolar water molecules in the electric field between charge groups. In solutions of proteins, several methods indicate the equivalent of 2–8 layers of structured water molecules extending beyond the protein surface, and we have recently demonstrated that induced protein conformational change modifies the extent of non‐ideally behaved water. Water self‐diffusion rate as measured in three different cell types was about half that of bulk water, indicating that most of the water in these cells was slower in motion than bulk water. In different cell types the extent of osmotically perturbed water ranged from less than half to almost all of the intracellular water. The assumption that essentially all intracellular water has ideal osmotic and motional behavior is not supported by the experimental findings. The non‐ideality of cell water is an operational term. Therefore, the amount of non‐ideally behaving water is dependent on the characteristics of water targeted, i.e. the measurement method, and a large fraction of it is explainable in mechanistic terms at a molecular level based on solute—solvent interactions.

Responses of hepatoma-bearing rats to total parenteral hyperalimentation and to ad libitum feeding

Abstract BUF rats with a transplantable Morris hepatoma No. 7777 were given three feeding regimens, (i) solid food ad libitum, (ii) total parenteral hyperalimentation, and (iii) liquid diet ad libitum. Those rats on solid or liquid food ad libitum undergo body weight loss or cancer cachexia with decreased food intake. Total parenteral hyperalimentation prevented the loss in body weight but stimulated tumor growth when compared to those fed ad libitum. Although there was no significant difference in survival time in the tumorous rats in the different feeding groups, the parenterally fed rats appeared to die with fluid overload while the ad libitum fed rats died of inanition or starvation.

Effects of iron supplementation and ET-18-OCH3 on MDA-MB 231 breast carcinomas in nude mice consuming a fish oil diet.

Lipid peroxidation products can be cytotoxic. Our objectives were (1) to use two pro-oxidants (iron and a pro-oxidative drug) to selectively increase lipid peroxidation in the implanted human breast tumours of mice consuming fish oil and (2) to kill the cancer cells without harming normal host tissues. The theoretical basis for selective cytotoxicity is that normal cells are better able to handle oxidative stress than cancer cells. Male athymic nude mice, consuming an AIN-76 diet, were injected s.c. with MDA-MB 231 human breast carcinoma cells. Three weeks later, all mice had palpable tumours, 3-10 mm in diameter, and diets were changed to modified AIN-76 diets containing 19% menhaden fish oil and 1% corn oil with or without supplemental 0.3% ferric citrate. After 2 weeks, half of the mice on each diet (19% fish oil with or without supplemental ferric citrate) were injected (three times per week for 2 weeks) with the ether-lipid drug edelfosine (ET-18-OCH3). The concentration of lipid peroxidation products in tumours (as measured by thiobarbituric acid-reactive substances, TBARS) was significantly increased by both ferric citrate and ET-18-OCH3. The TBARS in livers were not increased, nor was there evidence of other harmful side-effects to the host mice. The addition of iron enhanced tumour cell death whereas ET-18-OCH3 suppressed tumour cell mitosis. The use of iron supplementation combined with ET-18-OCH3 resulted in the slowest growth rate, lowest mitotic index, highest level of lipid peroxidation products and increased the cytotoxic index in tumours without detectable harm to the host. That iron supplementation increased tumour suppression beyond that expected from the increase in the concentration of TBARS in the tumour merits further investigation.