Metabolic and ultrastructural changes in the frog ovarian follicle in response to pituitary stimulation.

Frog ovarian fragments were prevented from ovulating in vitro by the addition of actinomycin D up to 3 hr following pituitary stimulation; but addition of Actinomycin D 6 hr after stimulation was far less effective. Puromycin, on the other hand, effectively inhibited ovulation when added as late as 6 hr after pituitary stimulation. Although actinomycin D reduced uptake of uridine-(3)H, and puromycin reduced uptake of leucine-(3)H and lysine-(14) by pituitary-stimulated ovarian tissue minus oocytes (OTMO) in vitro, it was found that pituitary stimulation did not significantly increase uptake of these compounds by OTMO. Radioautographs of ovarian follicles fixed 6 hr after the addition of pituitary extract and uridine-(3)H in vitro revealed increased RNA synthesis in the peritoneal surface epithelium, compared with unstimulated controls, while the ovarian sac epithelium showed no increase. Gross ultrastructural changes occurred in the peritoneal area of ovarian follicles following pituitary stimulation in vivo, including loss of collagen fibrils, and general disorganization of the connective tissue theca. Changes in the rough endoplasmic reticulum of the peritoneal epithelial cells, while frequently encountered, were less pronounced. None of these changes was observed in the ovarian sac area, or in the interfollicular region. The above data are consistent with the hypothesis that pituitary stimulation of the frog ovary results in increased synthesis of RNA and protein by the peritoneal epithelial cells, and that the protein may be collagenase.

Hypophyseal control of genetic expression during chick feather and skin differentiation.

The normal sequence of molecular events which occurs during feather and skin differentiation in the chick embryo is interrupted by pituitary gland ablation. The characteristic pattern of development is reestablished in hypophysectomized embryos treated with pituitary extract and in hypophysectomized embryos in parabiotic union with their twins within a double-yolked egg. These results suggest that genetic expression in differentiating chick feather and skin after day 12 of incubation is regulated by hormones.

Polysome morphology: evidence for endocrine control during chick embryogenesis.

Surgical removal of the pituitary gland had little apparent effect on the distribution or morphology of skin and feather polysomes in chicken embryos incubated for 12 days. However, polysome patterns obtained from 15-day-old hypophysectomized embryos differed markedly from those of their controls. In addition, a tetrad-shaped polysome, characteristic of the 158S peak of the 12-day-old embryo but not of the 15-day-old control, is still retained in the operated embryo at 15 days. Therefore, it appears that after day 12 of embryogenesis the structure of the four-ribosome polysome from skin and feathers is contingent on a functional hypophysis.

pH-sensitive liposomes: possible clinical implications.

When pH-sensitive molecules are incorporated into liposomes, drugs can be specifically released from these vesicles by a change of pH in the ambient serum. Liposomes containing the pH-sensitive lipid palmitoyl homocysteine (PHC) were constructed so that the greatest pH differential (6.0 to 7.4) of drug release was obtained near physiological temperature. Such liposomes could be useful clinically if they enable drugs to be targeted to areas of the body in which pH is less than physiological, such as primary tumors and metastases or sites of inflammation and infection.

Hyperthermia and local anesthetics: potentiation of survival of tumor-bearing mice.

Lidocaine infusion of a CA755 mammary adenocarcinoma growing in the hind leg of BDF1 mice results in a significant increase in the animals' survival when combined with heating for 1 hour in a 43.5 degrees C water bath. This ability of local anesthetics to prolong survival following hyperthermia is consistent with the hypothesis that increases in membrane fluidity influence sensitivity to heat. In view of the extensive clinical experience with local anesthetics, the delay between clinical application and the observation that they potentiate the action of hyperthermia in animals may be reduced.

Design of liposomes for enhanced local release of drugs by hyperthermia.

Liposomes can be designed to release an entrapped drug preferentially at temperatures attainable by mild local hyperthermia. In a test system in vitro, protein synthesis by Escherichia coli is inhibited and killing of the cells is enhanced by heating neomycin-containing liposomes to their phase transition temperature to maximize drug release. In the presence of serum the ratio of release at 44 degrees C to that at 37 degrees C can be made greater than 100:1, suggesting possible applications in the treatment of tumors or local infection.

Liposomes and local hyperthermia: selective delivery of methotrexate to heated tumors.

Liposomes with phase transitions a few degrees above physiological temperature delivered more than four times as much methotrexate to murine tumors heated to 42 degrees C as to unheated control tumors. Most of the accumulated drug appeared to be intracellular and bound to dihydrofolate reductase, the enzyme blocked by methotrexate in its role as an antineoplastic agent.

Concentration-dependent increase of murine P388 and B16 population doubling time by the acyclic monoterpene geraniol.

Geraniol, an acyclic end product of a plant isoprene pathway and a pyrophosphorylated intermediate in plant and animal pathways, caused a concentration-dependent increase in the population doubling time of murine P388 leukemia cells in suspension culture and of B16 melanoma cells in monolayer culture. The suppression of the growth of P388 cells by geraniol (0-0.9 mM) and by mevinolin (0-0.25 microM), a competitive inhibitor of mevalonate biosynthesis, was reversed by the addition of 0.5 mM mevalonolactone to the growth medium. Flow cytometry of asynchronous B16 cells grown with geraniol (0-0.15 mM) revealed a population characterized by larger cells with altered nuclear characteristics. Over the course of four studies, dietary geraniol increased the 50% survival time of mice by 10, 29, 33, and 50% following the i.p. transfer of P388 cells. The results of the latter study showed that, following the i.p. transfer of 1 x 10(5) P388 cells, the control group of female C57BL x DBA/2 F1 mice had a 50% survival time of 24 days and a maximum survival of 27 days. Mice fed a diet containing 0.1% geraniol for 14 days prior to and following the P388 cell transfer had a 50% survival time of 36 days, and 20% of the mice remained free of tumors during the 50-day trial. These studies support the possibility that monoterpenes and other isoprenoid products of plant metabolism are in part responsible for the anticarcinogenic actions of diverse fruits, vegetables, and cereal products.

Structural changes in murine cancer associated with hyperthermia and lidocaine.

Hyperthermia alone and hyperthermia with lidocaine cause changes in the fine structure of the CA755 tumor cell as well as the breakdown of the tumor vasculature. The first structural change, observed immediately after termination of hyperthermia of 43.5 degrees for 1 hr, is the vesiculation of the Golgi apparatus. Other structural changes occur later but with variable times of onset. The changes appear to be unrelated to the presence of lidocaine. Vascular breakdown results in hemorrhaging within the tumor, and its onset and intensity appear to vary directly with the size of the tumor. Breakdown of the tumor cell plasmalemma and degenerative changes of the cytoplasm and nucleoplasm are seen more frequently in large tumors and in the interior of small tumors at any given time after the end of hyperthermia. The vesiculation of the Golgi persists in treated cells for as long as 30 hr. This modification may represent an intensification in the function of the Golgi apparatus; however, it closely corresponds to that found in a variety of other cells treated with a class of compounds, including lidocaine, that specifically inhibits the function of the Golgi apparatus. The effect of these compounds is rapidly reversible, unlike hyperthermia. Since the Golgi apparatus probably is crucial in repairing any deleterious effects of hyperthermia, any impairment of its normal function would place most treated tumor cells in a difficult position. The rate of tumor destruction may ultimately depend on the breakdown of the tumor vasculature following hyperthermia and lidocaine.

Systemic lidocaine enhancement of hyperthermia-induced tumor regression in transplantable murine tumor models.

Previously, we reported that local lidocaine infusion of a CA 755 mammary adenocarcinoma growing in C57BL X DBA/2 F1 mice, when combined with local heating for 1 hr in a 43.5 degrees water bath, significantly increased survival and inhibited tumor growth more than heating alone. Because of its clinical implications, systemic lidocaine was tested in the above model system and in a murine fibrosarcoma tumor model. An equivalent supraadditive, tumor-inhibitory effect of heat and lidocaine was obtained with both systemically and intratumor-administered lidocaine. The serum levels of lidocaine necessary to achieve tumor regression were within the therapeutic range for the control of arrhythmia in humans. Several treatment schedules, varying the mode of drug delivery, were evaluated. The effects of treatment on tumor growth characteristics were analyzed using an extension of the Cox survival model.

Selective delivery of liposome-associated cis-dichlorodiammineplatinum(II) by heat and its influence on tumor drug uptake and growth.

In an attempt to optimize the chemotherapeutic treatment of mouse tumor Sarcoma 180, liposomes containing cis-dichlorodiammineplatinum(II) (PDD), having transition temperatures of few degrees higher than the rectal temperature of mice, were used in combination with local hyperthermia. The uptake of radioactive PDD by tumors heated for 1 hr at 42 degrees was almost four-fold greater when the drug was associated in liposomes than if administered as free drug. Uptake of liposome-administered radioactive platinum by liver was twice that obtained with free PDD, whereas its incorporation by the kidney was the same by either method of drug administration. The effect of various combinations of hyperthermia, drug-containing liposomes, and free PDD on tumor growth was also studied. Treatment with liposome-associated PDD plus local heating resulted in a dose-modifying factor of 7 when compared with free drug and no hyperthermia. The dose-modifying factor was 2.5 when PDD liposomes and heat were compared within free drug and heat. Thus, PDD could be specifically released from liposomes by heat and resulted in both a greater drug uptake and a delayed tumor growth following treatment. Potential normal tissue toxicity problems, however, still need to be resolved before clinical application of this combined modality will be possible.

Influence of membrane-lipid composition on translocation of nascent proteins in heated Escherichia coli.

In studies using Escherichia coli we have shown that new protein species appear in the outer membrane fraction with concomitant losses of nascent proteins from the soluble and inner membrane fractions following heat exposure. Of the various explanations for this phenomenon, temperature-induced membrane disorganization appeared the most likely. It is suggested that heat mimics the action of the signal sequence of a protein on the lipid bilayer allowing non-signal-sequence-containing proteins to be translocated. To test this hypothesis we grew E. coli K1060 cells, an unsaturated fatty acid requiring auxotroph, supplemented during growth with fatty acids of varying chain length in an attempt to determine whether biological membranes of varying ability to maintain their bilayer configuration could be constructed. The rationale being that such membranes would allow us to determine whether differences in translocation would occur in cells grown at the same temperature supplemented with either 16:1 or 20:1 unsaturated fatty acids when the cells were subjected to a series of thermal insults. Protein translocation occurred to a greater extent and at lower temperatures in cells supplemented with the longer chain fatty acid. Treatment of outer membranes with either 1 M salt, 6 M urea or high pH and studies determining fluorescent polarization values by scanning up and down through a series of temperatures ranging from 15 to 49 degrees C indicate that the proteins translocated by heat to the outer membrane are integral. Protein translocation may represent an adaptive response to an altered environment enabling the cell to respond to stress by stabilizing its outer membrane.

Influence of dietary protein levels on survival of rats following kidney irradiation.

Concern about radiation induced nephropathy results in a dose limiting constraint in some applications of radiation therapy. An understanding of the etiology of radiation therapy. An understanding of the etiology of radiation nephropathy is essential if attempts to alter the time course or extent of the pathology are to be successful. In an attempt to gain a better understanding of this disease process, and to see if it could be altered by dietary manipulation, young adult male Sprague-Dawley rats were unilaterally nephrectomized, after which the remaining kidney was exteriorized and exposed to 14 Gy of X rays. Non-irradiated control animals had their remaining kidney exteriorized for a comparable length of time. Five days after irradiation, the animals were switched from standard lab rations to isocaloric diets of differing protein content. Diets used included 4%, 20% and 50% protein and the 4% and 20% diets given in combination with 0.9% NaCl drinking water. For all the diet groups, irradiated animals had median survival times shorter than their corresponding non-irradiated controls. Within the irradiated groups, the ranking of the median survival times was: 4% + 0.9% NaCl greater than 4% greater than 20% + 0.9% NaCl = 20% greater than 50%. The differences in survival among the irradiated groups were significant at the 0.01 level. These data indicate that kidney response to irradiation can be altered by manipulation of dietary protein levels. Such information may have clinical application.

Influence of protein nutrition on dose-survival relationship following rat kidney irradiation.

Immediately following unilateral nephrectomy the remaining kidney of juvenile male Sprague-Dawley rats was sham irradiated or irradiated to doses of 14-30 Gy. Following irradiation the animals were placed on isocaloric diets of either 20 or 4% protein. Median life spans for the animals on the low protein diet were significantly increased compared to the median life spans on the 20% protein diet. Serum urea nitrogen (SUN) levels were periodically measured in rats from each of the experimental groups. SUN levels in the irradiated rats fed the 20% protein diet increased significantly over unirradiated controls as a function of time. In contrast animals fed the 4% protein diet showed no significant changes in SUN levels irrespective of the size of radiation dose and time post irradiation. Renal protective factors calculated as the ratio of 80% survival times for animals fed the 20% protein diet compared to animals fed the 4% protein diet can be calculated to be 2.3 at 18 Gy and 2.8 at 22 Gy. Likewise, a SUN protective factor calculated as the ratio of percentage of nonirradiated control SUN values for the two diets (SUN 20% irradiated) (SUN 20% nonirradiated) (SUN 4% irradiated) (SUN 4% nonirradiated) is 2.4 for 18 Gy and 3.9 for 22 Gy.

Hyperthermic sensitivity and growth stage in Escherichia coli.

Hyperthermic sensitivities of Escherichia coli B/r and Bs-1 were determined for lag-, midlog-, and stationary-phase cells at 47, 48, and 49 degrees C. In both strains midlog-phase cells were strikingly more heat sensitive (100-fold greater killing after 4 h at 48 degrees C) than stationary-phase cells, with intermediate sensitivity for lag-phase cells. In contrast to the reported difference in the radiation sensitivity between these two strains, very little difference in heat sensitivity was seen. Patterns of fatty acid composition of both strains were very similar at each phase of growth. From midlog to stationary phase, 16:1 and 18:1 unsaturated fatty acids decrease from 16 and 30% to 0.5 and 3%, respectively, while the C17 and C19 cyclopropane fatty acids increase from 7 and 3% to 22 and 25%, respectively. Concomitant with these changes in fatty acid composition, substantially higher membrane microviscosity values were recorded for stationary-phase cells. Total membrane microviscosity was positively associated with the C17 and C19 cyclopropane fatty acid composition and with cell survival following hyperthermia. In contrast to hyperthermic sensitivity, radiation survival differences between B/r and Bs-1 are little affected by growth stage. We propose that these results are consistent with a critical influence of membrane lipids on cellular hyperthermic sensitivity and further that the target sites for radiation and hyperthermia are different in these cells.

Clinical prospects for liposomes.

The use of liposomes has recently been the subject of considerable attention as a promising and versatile approach to drug delivery. Particularly intriguing is the possibility of targeting liposomes to specific areas of the body such as tumors or sites of inflammation or parasitic invasion for either local accumulation or release of associated drugs. This review focuses mainly on recent in vivo work having clinical potential. An extensive discussion of liposome preparation and entrapment of drugs for controlled release in vivo is also included. The stability of liposomes in biological fluids is a major problem. The mode of administration, either intraperitoneal, subcutaneous, local, oral, or respiratory, is closely related to the life of the liposomes in vivo. Following in vivo administration the lifetime of a liposome is critically dependent on its composition, size, and charge. Liposome toxicity appears to be minimal, but should be considered when administering liposomes to patients. Tissues such as the liver, spleen, and lungs, because of macrophage ingestion of liposomes, become potential sites of drug toxicity. The use of liposomes to deliver antiparasitic drugs in the treatment of malaria and leishmaniasis is promoting; so it is the use of surfactant-carrying liposomes in the treatment of respiratory distress syndrome in premature babies. Recent cancer studies utilizing liposomes both in vivo and in vitro have shown promise. In tumor-bearing animals a liposome drug delivery system has caused a regression, delayed tumor growth, and increased survival time. Although the clinical use of liposomes is only in its infancy, its potential in future therapy appears promising.

Cholesteryl hemisuccinate alters membrane fluidity, angiotensin receptors, and responses in adrenal glomerulosa cells.

We examined the effects of cholesteryl hemisuccinate on membrane fluidity and angiotensin II (AII) actions in bovine adrenal glomerulosa cells. Incubating cells with cholesteryl hemisuccinate decreased membrane fluidity and markedly inhibited AII binding. The effect on binding was characterized by a decrease in AII receptor number. The effects of AII on phosphatidyl inositol turnover and calcium fluxes, proposed intermediaries of AII actions on aldosterone secretion, were less impaired than AII binding by cholesteryl hemisccinate. AII stimulation of aldosterone secretion was preserved despite the decrease in AII binding after cholesteryl hemisuccinate treatment. These results indicate that AII binding can be dissociated from its effects on aldosteronogenesis by a reagent that alters membrane fluidity.

An approach to AIDS therapy using hyperthermia and membrane modification.

Altering the biophysical characteristics of cell membranes by diet and membrane perturbing agents markedly influences thermosensitivity of cells. Likewise, manipulation of viral envelopes either by altering their lipid composition by diet or by the use of agents that perturb the lipid envelope influence infectivity of enveloped viruses and the progression of viral disease. The use of hyperthermia and envelope modification as a combined approach to treat AIDS has until now neither been suggested nor attempted. On the basis of my previous work and a review of the literature, I theorize that the combination of hyperthermia with procedures designed to alter the viral envelope will likely result in an increased viral sensitivity and be useful clinically for treatment of patients with enveloped viral diseases such as AIDS.

Differential response to heat of metastatic and non-metastatic rat mammary tumors.

Metastasizing and non-metastasizing transplantable mammary tumors were implanted into female W/Fu rats. A pair of tumors were employed, the SMT-2A and MT-W9B. When these tumors were exposed to water bath heating at 43.5 degrees C for 60 minutes, a significantly longer tumor-free growth delay was obtained in the metastasizing tumor compared to its non-metastasizing counterpart. The protein to phospholipid ratio and the content of arachidonic acid was lower in the metastasizing tumor than in the non-metastasizing one. By way of apparent compensation, the metastasizing tumor contained more linoleic and stearic acid. These observations suggest a relation between metastasizing capacity, thermal sensitivity, and membrane composition.

Extracorporeal whole body hyperthermia treatments for HIV infection and AIDS.

Whole body hyperthermia therapy (WBHT) is the elevation of the core body temperature to 42 degrees C. In vitro studies have confirmed that 42 degrees C is cytocidal for virally infected lymphocytes, and even more effective when heating is repeated 4 days later. The safety and efficacy of two successive sessions of WBHT (4 days apart) was evaluated in 30 patients with AIDS (not on protease inhibitors), randomized to: 1) untreated controls, 2) low temperature WBHT for 1 hour at 40 degrees C and repeated 96 hours later, and 3) high temperature WBHT for 1 hour at 42 degrees C and repeated 96 hours later. The sorbent suspension in the ThermoChem System (HemoCleanse, West Lafayette, IN) system automatically controlled blood phosphate, calcium, and other electrolyte concentrations during WBHT. In 1 year of follow-up after WBHT, there were positive effects of the therapy on frequency of AIDS defining events, Karnofsky score, and weight maintenance. However, effects on plasma HIV RNA and CD4 counts were transient. Two successive WBHT treatments were performed in four patients who were on protease inhibitor/triple drug therapy, but had suboptimal response. In follow-up for 6 months, plasma HIV RNA and CD4 improved after WBHT, and the patients remained clinically well. This WBHT may have specific advantages in patients with suboptimal response to protease inhibitor therapy.