Intracellular Na+:K+ ratios in human cancer cells as revealed by energy dispersive x-ray microanalysis.

Intranuclear sodium, potassium, and chloride contents were measured by energy-dispersive x-ray microanalysis in freeze-fractured, freeze-dried, bulk-tumor samples taken from 10 patients suffering from invasive urogenital cancers. Human biopsies were carried out during the first diagnostic interventions before any cytostatic treatment had been applied. Pathohistological diagnosis established the malignancy in each case. The cancers were classified in three types: keratinizing, transitional cell, and hypernephroid carcinoma. More than 250 cell nuclei were measured from each type of cancer. The results were compared with those obtained in intact human urothelium taken from patients having no malignant processes. Proximal and distal tubular epithelial cell nuclei representing the origin of human hypernephroid cancer were also measured in rat kidney because corresponding healthy human material cannot be obtained. The analyses revealed, in all three types of cancer cells, that the average intranuclear sodium content increased more than three-fold, the potassium content decreased 32, 16, and 13%, respectively; meanwhile the chloride content increased, but to a lesser extent than did the sodium. The intranuclear Na+:K+ ratios were more than five-fold higher in the cancer cells on the average, and their distribution histograms were much broader than in the normal human urothelium and in the tubular cell nuclei of the rat kidney. The results obtained fit well with the theory of Cone, C. D., Jr. 1971. J. Theor. Biol. 30: 151-181 according to which the sustained depolarization of the cell membrane may be of mitogenic effect.

Age-dependence of heat-induced strand separation of DNA in situ in postmitotic cells of rat brain as revealed by acridine orange microfluorimetry.

Acridine orange microfluorimetry as well as Feulgen microfluorimetry were applied on smears of cerebral and cerebellar cortex of young (2-month-old) and old (24-27-month-old) female Wistar rats. The strand separation of DNA caused by thermal denaturation was measured between 50 degrees and 90 degrees C in the nuclei of cortical nerve cells and cerebellar granular cells. In the young nerve cell nuclei a considerable DNA-loss was observed after thermal denaturation, therefore, corresponding corrections should have been made on the heat denaturation curves. The old nerve cell nuclei of brain cortex showed a much higher heat stability than the young ones, whereas in the cerebellar granular cells the age-dependent difference was much less expressed. Our observations on chromatin in situ show the same tendency as do the findings obtained on isolated chromatin by others. The results are discussed first from a methodical point of view then the arguments are listed suggesting a possible role of interprotein cross-links in the age-dependent stabilization of the chromatin gel.

The effect of caloric restriction on the lateral diffusion constant of hepatocyte membrane proteins in C57BL/6 male mice of different ages: FRAP studies on liver smears.

The lateral mobility of proteins in hepatocyte plasma membranes was compared in calorically restricted and ad libitum (AL)-fed C57BL/6 male mice in age groups from 7 to 28 months. Caloric restriction was achieved by means of the every-other-day (EOD) feeding regimen, maintained for various periods from 1 to 15 months. Protein lateral diffusion constant (D) in hepatocyte membranes was measured by means of fluorescence recovery after photobleaching (FRAP) in liver smears. The peroxide-induced autofluorescence (PIAF) was utilized as a fluorescent label. A mild (1 mM for 10 min) H2O2 treatment of liver smears produces oxidation of riboflavin that is bound to all proteins of the cell membrane. Using this technique, the average lateral diffusion constant (D) and the fractional recovery (FR) of these proteins can be measured. EOD feeding resulted in a significant decrease in body weights and also a significant increase in the values of D in all age groups after 1 month of EOD feeding. After 3.5 months of EOD feeding a further increase of D was observed (up to about 15%). Nevertheless no further change in D occurred if the EOD feeding was maintained for 6.5 or even 15 months. The negative linear age correlation of D observed in the AL-fed animals was present also under the EOD feeding; however, the whole regression equation shifted towards higher values. These experiments indicate that caloric restriction influences the lateral diffusion constant of membrane proteins in hepatocytes. The results are interpreted as a result of an increased protein turnover caused by the caloric restriction.

Semiconduction of proteins as an attribute of the living state: the ideas of Albert Szent-Györgyi revisited in light of the recent knowledge regarding oxygen free radicals.

Oxyradicals have been considered as harmful byproducts causing molecular damage during aging. However, evidence is accumulating to show that the actual situation is more complex: the living state implicitly involves the production of oxyradicals. (1) Blast type cells produce much less oxyradicals than the differentiated ones, and an increased production of OH radicals induce differentiation of various lines of leukemia cells; meanwhile, their superoxide dismutase expression increases to a very high extent. (2) The supramolecular organization of the cells is developed by means of "useful" crosslinking effects OH radicals. (3) Repiratory inhibition of oxyradical production (KCN-intoxication, suffocation, etc.) would kill living organisms prior to the exhaustion of energy reserves. It is assumed that the continuous flux of OH radicals is a prerequisite for a electron delocalization on the proteins, which is a semiconduction of p-type, proposes already in 1941 by Albert Szent-Györgyi, and refuted on a "theoretical" basis. It has become clear by now that the carbon-based semiconduction is possible because diamond transistors are known to exist. The recently developed atomic force microscopy offers some real possibilities for experimental testing of this assumption. This concept may lead us to new horizons in interpretation of living functions, such as the basic memory mechanisms in brain cells and their impairment during aging.

On the true role of oxygen free radicals in the living state, aging, and degenerative disorders.

Oxyradicals are generally considered harmful byproducts of oxidative metabolism, causing molecular damage in living systems. They are implicated in various processes such as mutagenesis, aging, and series of pathological events. Although all this may be justified, evidence is accumulating that it is an oversimplified view of the real situation. We can assume nowadays that the living state of cells and organisms implicitly requires the production of oxyradicals. This idea is supported by experimental facts and arguments as follows. (1) Complete inhibition of the oxyradical production by KCN (or by any block of respiration) kills the living organisms much before the energy reserves would be exhausted. (2) Construction of the supramolecular organization of the cells (especially of their membranous compounds) requires the cross-linking effect of oxyradicals, particularly that of OH* radicals. (3) Blast type cells produce much fewer oxyradicals than do differentiated ones, and interventions increasing the production of OH* radicals induce differentiation of various lines of leukemic (HL-60 and K562) and normal (fibroblasts, chondroblasts, etc.) cells, while SOD expression increases greatly. (4) It is reasonable to assume that the continuous flux of OH* radicals is prerequisite to maintenance of constant electron delocalization on the proteins, which is a semiconductive phenomenon suggested in 1941 by Szent-Györgyi, but it has never been proven experimentally. It is theoretically possible to describe the function of the synapses as that of a single p-n-p transistor, assuming that the free radical flux maintains electron movements on the subsynaptic structures, while the actual membrane potential is governing the electron flux. This theoretical approach may open completely new possibilities for our understanding of the normal functions of living organisms, such as basic memory mechanisms in brain cells, their aging processes, and therapeutic approaches to many degenerative disorders, such as various types of dementia.

Intracellular water and dry mass content as measured in bulk specimens by energy-dispersive X-ray microanalysis.

Intracellular water content (IWC) was measured in freeze-fractured biological bulk specimens by means of energy-dispersive X-ray microanalysis. The method is based on the concentration differences of certain elements (potassium and phosphorus) between frozen-hydrated and frozen-dried states of the tissues as applied formerly to sectioned material by others. A new mathematical formula has been derived giving rather precise figures for IWC. No elemental standards are necessary for the measurement: one has to obtain only the peak to background ratios in wet and dry states of the cells. the method is sensitive enough to reveal age-dependent as well as drug-induced changes of IWC in liver and brain cells. The values obtained are quite comparable with the theoretically expected one. Technical problems of the application of this method are discussed in detail.

Cytochemical studies on the fibroblast-preadipocyte relationships in cultured fibroblast cell lines.

The theoretical basis of adipogenesis has always been a matter of debate. One concept suggests that all types of adipocytes are derived from undifferentiated connective tissue cells, whereas another concept suggests that adipocytes develop from specialized cells only that are able to accumulate fat. Many conflicting data have been published with respect to the transition of fibroblasts into preadipocytes. For example, this transition has been declared as impossible for dermal and perimysial fibroblasts. The present study analysed spontaneous accumulation of fat in various types of fibroblasts from different origin (retroocular, skin, NIH/3T3, and L929). It was found that intense Oil Red O-positive triglyceride-containing droplets accumulated in practically all types of fibroblasts provided that the cells were cultured on glass surface. When the cells were cultured on plastic surfaces, lipid staining was inhibited in a variable manner: inhibition was virtually complete in skin fibroblasts, whereas in other types of fibroblasts, inhibition was only partial. It is concluded that all types of fibroblasts can accumulate fat spontaneously, and thus can be considered as preadipocytes. Therefore, interpretations of data obtained with cultures of fibroblasts with respect to adipogenesis have to be reconsidered.

[The effect of some solvents on the epidermis (author's transl)]. / Die Wirkung einiger Lösungsmittel aug die Epidermis

The ultrastructure of epidermis pretreated with 0,1 N sofium hydroxide, ether-ethanol mixture and 0,5 M sodium thioglycolate was studied. Sodium hydroxide dissolves the keratohyalin granules and the inter fibrillar material in the keratin. It has no effect on the epidermal and keratin fibers and on the marginal dense band of the horny cells. It "produces" a keratin pattern in the psoriatic horny layer. After sodium thioglycolate treatment the keratohyalin disappears, the epidermal and keratin fibers show a periodicity and the cementing material in the horny cells is more opaque. After lipid extraction the structure of keratohyalin becomes inhomogenous. The effect is somewhat similar to that of sodium thioglycolate but there is no periodicity in the horny layer. The intercellular contact layers of desmosomes as well as the Selby-Odland bodies disappear after each kind of treatment, The different effect of sodium thioglycolate on the horny layer and on the keratohyalin implies that the material of the keratohyalin and of the cementing substance are not identical.

Virus-like structures in lupus erythematosus discoides.

Biopsy specimens from typical skin lesions of DLE patients were studied by means of electron microscopy. Virus-like structures were discovered in the cytoplasm of fibrocytes, endothelial cells and keratinocytes. After prolonged chloroquine treatment a decrease in the frequency and size of these structures was observed.

Age-dependent alterations of the rate of RNA synthesis in rat brain cell nuclei.

Endogenous Mn2+ and Mg2+ activated RNA polymerase activity was measured in isolated cell nuclei of brain from rats of different age-groups. It was established that the activity of the nucleoplasmic RNA polymerase is maintained at the level of young animals up to an age of 16 months but is decreased after 24 months. The nucleolar RNA polymerase activity decreases already at 16 months and a higher ratio of the Mn2+:Mg2+ activated RNA polymerase activities has been found to be characteristic for the older animals. By means of stepwise sucrose density gradient centrifugation fractions were obtained from the nuclear preparations highly enriched in cell nuclei of neuronal and glial origin respectively. By measuring the activity of the nucleoplasmic and nucleolar RNA polymerases in these fractions it was found that the elevated ratio of the nucleoplasmic to nucleolar RNA polymerase activity at 16 months of age is a characteristic of the neuronal nuclei while the glial nuclei behave by the opposite manner. A parallelism existing between the age-dependent change of the endogenous RNA polymerase activity and that of perichromatic granules of rat brain cortical cells is discussed.