Rab10-Positive Tubular Structures Represent a Novel Endocytic Pathway That Diverges From Canonical Macropinocytosis in RAW264 Macrophages.

Using the optogenetic photo-manipulation of photoactivatable (PA)-Rac1, remarkable cell surface ruffling and the formation of a macropinocytic cup (premacropinosome) could be induced in the region of RAW264 macrophages irradiated with blue light due to the activation of PA-Rac1. However, the completion of macropinosome formation did not occur until Rac1 was deactivated by the removal of the light stimulus. Following PA-Rac1 deactivation, some premacropinosomes closed into intracellular macropinosomes, whereas many others transformed into long Rab10-positive tubules without forming typical macropinosomes. These Rab10-positive tubules moved centripetally towards the perinuclear Golgi region along microtubules. Surprisingly, these Rab10-positive tubules did not contain any endosome/lysosome compartment markers, such as Rab5, Rab7, or LAMP1, suggesting that the Rab10-positive tubules were not part of the degradation pathway for lysosomes. These Rab10-positive tubules were distinct from recycling endosomal compartments, which are labeled with Rab4, Rab11, or SNX1. These findings suggested that these Rab10-positive tubules may be a part of non-degradative endocytic pathway that has never been known. The formation of Rab10-positive tubules from premacropinosomes was also observed in control and phorbol myristate acetate (PMA)-stimulated macrophages, although their frequencies were low. Interestingly, the formation of Rab10-positive premacropinosomes and tubules was not inhibited by phosphoinositide 3-kinase (PI3K) inhibitors, while the classical macropinosome formation requires PI3K activity. Thus, this study provides evidence to support the existence of Rab10-positive tubules as a novel endocytic pathway that diverges from canonical macropinocytosis.

[The rationale for the application of laserophoresis of biologically active compounds].

Laserophoresis is a technique for the transcutaneous administration of biologically active compounds by means of low-intensity laser radiation (LFLR). It is currently regarded as a most promising method for the integrated application of a pharmaceutical substance and a physical factor. At present laserophoresis of various medicinal preparations is successfully used after preliminary experimental studies of their phoretic properties for the treatment of various inflammatory and dystrophic conditions as well as for the prevention of skin ageing. The most important route for the administration of the majority of drug preparations is through the shunts provided by perspiratory glands and hair follicles. Another essential factor determining the potential possibility of drug penetration through the skin is the characteristic of the substance chosen for the administration, such as its molecular weight, chemical structure, conformation, and hydrophilic properties. However, the most likely mechanism underlying the transport of the substance through the glandular cells of perspiratory glands and epithelial cells of hair follicles is pinocytosis, i.e. the process integrating exocytosis and endocytosis. To-day, the majority of the researchers lay emphasis on thermodynamic triggering of Ca2+-dependent processes as the primary mechanism behind the biological action of low-intensity laser radiation. Both exocytosis and endocytosis being the Ca2+-dependent processes, the liberation of Ca2+-ions under the influence of LFLR causes the activation of pinocytosis as a whole.

Effect of static electric fields in root cells of Vicia faba (Fabaceae).

Serial electron microscopic sections were prepared from half-ripened meristematic root cells of Vicia faba (Fabaceae) which had been exposed gradually to 700, 1000, 2500, 3500, and 5000 V/m static electric fields during seven days with and without Zn and Cd electrodes. At the end of five weeks, wall loosenings and very small nuclei were observed in those root cells which were exposed to static electric currents from the lower side of the medium without electrodes, while abnormalities in cell formation, e.g., two cells with one nucleus, and GER occurrence were present in an electrolytic (Cd upward and Zn downward) medium. The cells exposed to a static current from the upper side of the medium had small nuclei and abnormal cell divisions in the electrolyte, but in a non-electrolyte very large nuclei and thicker cell walls were observed, the cytoplasm was dense with GER, pinocytosis was seen filled with mitochondria, and protoplast formation with big nuclei was seen in exocytosis.

Ultrastructural changes in thyroid perifollicular capillaries during normal postnatal development and after infrared laser radiation.

This study was carried out on the perifollicular capillaries of the thyroid gland during postnatal (PN) development in normal conditions and after irradiation (46.8 J/cm(2)) with an infrared (IR) laser (904 nm). This was done using 11-, 21-, and 35-day-old Wistar rats. The changes in the capillaries were determined using quantitative methods as well as electron microscopy. During normal PN development the most relevant changes were an increase in the size of the capillaries, especially the lumen, thinning of the endothelium and an increase in the size of pinocytotic vesicles. Our results suggest that during PN development, the capillaries undergo some growth and maturation processes until they reach the optimal morphological conditions for their exchange functions. IR laser irradiation seems to stimulate the growth and maturation of endothelial cells in the youngest rats, while in older ones it causes irregular thickening of the endothelium and a reduction of the capillary lumen. These changes could be a sign of functional alterations in follicular cells caused by exposure to IR laser.

Microwave irradiation of rats at 2.45 GHz activates pinocytotic-like uptake of tracer by capillary endothelial cells of cerebral cortex.

Far-field exposures of male albino rats to 2.45-GHz microwaves (10-microseconds pulses, 100 pps) at a low average power density (10 mW/cm2; SAR approximately 2 W/kg) and short durations (30-120 min) resulted in increased uptakes of tracer through the blood-brain barrier (BBB). The uptake of systemically administered rhodamine-ferritin complex by capillary endothelial cells (CECs) of the cerebral cortex was dependent on power density and on duration of exposure. At 5 mW/cm2, for example, a 15-min exposure had no effect. Near-complete blockade of uptake resulted when rats were treated before exposure to microwaves with a single dose of colchicine, which inhibits microtubular function. A pinocytotic-like mechanism is presumed responsible for the microwave-induced increase in BBB permeability.

An ultrastructural study of salivary gland radiosensitivity after alpha-adrenergic stimulation.

The submandibular glands in 32 rats were exposed to either fractionated irradiation comparable to 50 Gy or a single fraction of 50 Gy. Prior to irradiation half the rats were given cyclocytidine, an alpha-adrenergic agonist which depletes serous cell granules. The results suggest that irradiation causes a significant, irreversible destruction of serous cells. Visible morphological changes were: intracytoplasmic vacuoles, destroyed mitochondria with disrupted cristae and derangement of the endoplasmic reticulum. Animals treated with cyclocytidine had diminished radiation injury. This drug may act as radioprotector, depleting secretory heavy metal granules, reducing radiation damage in the submandibular salivary glands.

ATPase and morphologic changes induced by UVB on Langerhans cells in guinea pigs.

We have devised, in guinea pigs, an improved ATPase technique which enables one to proceed from light to electron microscope study while preserving, on the ultrastructural level, the various membranous structures, in particular the Langerhans cell (LC) granules. Using this method, we have been able to confirm the action of acute, low-dose UVB on the surface enzymatic marker, ATPase. Moreover, this study has shown that the ATPase-negative LC contain abnormal LC granules or, more often, are deficient in LC granules. In a previous work, we have shown that, after epicutaneous application of a hapten, one successively observes an extensive adsorptive pinocytosis process, the disappearance of the membranous ATPase system, and the appearance of LC granules in the cytoplasm. Therefore we may suppose that, after UVB irradiation, the disappearance of the ATPase system and/or the possible alteration of the adsorptive pinocytosis process interrupts or alters the formation of LC granules. These successive events might play a vital role in the formation of the hapten--carrier protein-Ia antigen complex. In their absence in a large number of LC, following UV irradiation, epicutaneous application of a hapten would lead to the development of a state of immune tolerance.

Reversible microwave effects on the blood-brain barrier.

Low level microwave exposure of Chinese hamsters resulted in reversible permeability of the blood-brain barrier (BBB) to horseradish peroxidase (HRP). Lesions were grossly visible in random areas of the brain immediately following exposure, but were not as common following a 1 h recovery period and were absent after a 2 h recovery period. The apparent route of increased permeability was via endothelial vesicular transport, since reaction product was not seen passing through the endothelial tight junctions. In addition, endothelial flooding of HRP, platelet aggregation and perivascular edema were observed only in experimental animals. Possible mechanisms of the enhanced vesicular transport are discussed.

[Action of x-rays and neuraminidase on pinocytotic activity and microtubule-microfilament system of Ehrlich ascites tumor cells in monolayer culture (author's transl)]. / Einwirkung von Röntgenstrahlen und Neuraminidase auf Pinozytoseaktivität und Mikrotubulus-Mikrofilament-System von Ehrlich-Aszites-Tumorzellen in Monolayerkultur.

Monolayer cultures of Ehrlich ascites tumor cells in exponential growth phase were treated with X-rays and neuraminidase alone or in combination. A radiation dose of 2 Gy (200 rd) and higher effected a significant inhibition of DNA synthesis and cell proliferation. Neuraminidase treatment in addition to irradiation did not modify the growth-inhibitory irradiation effect. Cells pretreated for 0.5 to 1.0 hours with neuraminidase (in Earle's salt solution) and cultured thereafter (4h) in serum-containing growth medium exhibited an enhanced development of the microtubule-microfilament-system. Morphometric analysis of microtubules on electronmicroscopic sections revealed a nearly 4-fold increased number in neuraminidase treated cells while the average length of microtubules was similar to controls. Pinocytosis as measured by the ultrastructural uptake of ferritin appeared to be increased following neuraminidase treatment. A connection between neuraminic acid containing components (glycoproteins or glycolipids?) of the cell membrane or cell surface, on the one hand, and the cytoskeleton and the endocytotic process of these tumor cells, on the other hand, is suggested.

[Ultrastructure of skeletal muscle tissue of microwave damaged chick embryos]. / Ul'trastruktura skeletnoi myshechnoi tkani kurinykh émbrionov pri mikrovolnovom porazhenii.

The effect of ultrahigh-frequency energy of electromagnetic field of low intensity on certain morphometric indices of intracellular organoids and on cellular ultrastructure were studied in the developing skeletal muscular tissue of chick embryos. In the skeletal muscles of irradiated embryos a limited cellular destruction, structural disorders in myonic organoids were revealed. Reactive-recovery processes manifested themselves in hyperplasy and hypertrophy of organoids, in activation of protein synthesis, in increasing amount of myosatellites. At early stages after irradiation peripheral mitochondria are subjected to greater changes. After hatching, central mitochondria in myons suffer more. Quantitative changes in myonic organoids and degree of their destruction seem to depend on.a peculiar differentiation of the muscular tissue, blood supply and innervation of the muscle as an organ.

Cytoplasmic effects of X-irradiation on cultured cells in a nondividing stage. 3. Alterations in plasma membrane motility.

Cultured, density-dependent growth inhibited human glia cells were exposed to X-radiation, generated by an 8-MeV linear accelerator, at a dose of 200 Gy. Phase contrast microscopy, time-lapse cinemicrography and scanning electron microscopy showed the irradiated cells to have increased ruffling activity of plasma membranes and enhanced macropinocytosis with a maximum approximately 24 hours after irradiation. "Atypical" central ruffles arising from the upper cell surface were demonstrated on some irradiated cells. The turnover of plasma membranes was supposed to be increased in the irradiated cells resulting in the formation of the observed branched, thread-like cells. The diminished cell surface area was believed to result from an unbalance between degradation and renewal of the plasma membranes in the irradiated cells.

Effect of ultraviolet radiation on pinocytosis in Amoeba proteua.

Ultraviolet (UV) irradiation (4 000-10 000 erg X mm(-2) decreased membrane potential and input resistance of Amoeba proteus and induced formation of pinocytotic channels. Submaximal pinocytosis induced by UV light was additive to pinocytosis induced by K+ or Na+ and stimulated in the presence of EGTA. It was not inhibited by the presence of La+++ or by pretreatment with dibucaine. In these respects and with respect to optimum pH and pCa, UV induced pinocytosis. Accumulation of K+ in the amoeba membrane after a dose of radiation may explain the similarity between pinocytosis induced by UV light and potassium salts. Ca++ present during the period of irradiation inhibited the effect of UV light. Instead Ca++ applied after irradiation (1-20 mM) increased channel formation. This effect was stimulated the presence of local anesthetic drugs. It is suggested that high doses of UV light may induce channel formation by releasing Ca++ from the cell membrane into the cell (UV induced pinocytosis). Ca++ may be released at the moment of absorption of UV light in the membrane as well as during the period of depolarization which follows irradiation. Low doses of UV light may permit extracellular Ca++ to enter the cell and stimulate channel formation (calcium induced pinocytosis). Dithiotreitol (1 mM) applied after irradiation depressed both UV and calcium induced pinocytosis so these may be the result of the same structural change which involves the formation of disulphide bonds in the membrane.

Effect of near ultraviolet and visible light on amoeba.

The near ultraviolet and visible light (VL) impinging at an intensity of 2-5 x 10(2) J s-1 m-2 for 2-5 h kills the mitotic and the early S-phase (0- to 15-min-old) amoebae. At the mid- and late S-period only a fraction of cells are killed by VL and G2 phase cells are quite resistant. Amoebae of all cell cycle stages show a delay in the first mitotic division. DNA synthesis, as measured by [3H]thymidine incorporation, is depressed in the VL-exposed early-S amoebae. A concurrent but temporary inhibition in [3H]leucine incorporation also occurs in these cells. However, no significant change in [3H]uridine incorporation has been found. To localize the site of lethal damage, nuclear transplantation studies were undertaken between the control amoebae and the amoebae treated with VL. The nucleus of a VL-exposed early S-phase cell recovers when transplanted immediately after VL exposure into an enucleate G2 cytoplasm but dies if grafted into an enucleat S-phase cytoplasm. The therapeutic effect of the G2 cytoplasm, although at a lower level, is also evident even when the treated early S-phase nucleus is implanted 20 h later, but not after 48 h, into the G2 cytoplasm. The amoeba cytoplasm shows resistance to VL-irradiation, can accept a control nucleus from any cell cycle stage, and function normally. The G2 nucleus also remains apparently unaffected to VL exposure and can survive when it is transfered to the control cytoplasm of any cell-cycle phase. All these findings are discussed in the light of the possible existence of a repair system against VL-induced damage in the G2-phase amoeba.

The effect of cyclophosphamide, methotrexate and X-irradiation on the ultrastructure and endocytic capacity of murine peritoneal macrophages.

100 mg/kg cyclophosphamide given subcutaneously daily for 5 days and whole-body X-irradiation (500 r) had an appreciable effect on the phagocytosis of E. coli by mouse peritoneal macrophages. Less bacteria were ingested by cells from the treated mice as compared with control cells. In contrast, whole-body X-irradiation had a stimulatory effect on pinocytosis as compared with cells from cyclophosphamide treated mice or control mice. Morphological changes in the cells from animals treated with cyclophosphamide or X-rays were evident. The cells were larger than normal macrophages and contained large amounts of lipid and phagolysosomes and had more vacuoles as compared with control cells. Many giant cells were present in the treated mice. Treatment of mice with methotrexate for 5 days, although affecting the cells morphologically in that they were larger than normal cells and had many vacuoles did not appear to have any effect on either phagocytosis or pinocytosis. Cells from methotrexate treated animals did not contain the large quantities of lipid seen in cells from the cyclophosphamide treated and X-irradiated mice.

An ultrastructural quantitative method for the evaluation of the permeability to horseradish peroxidase of cerebral cortex endothelial cells of the rat.

A quantitative method for the evaluation of the importance of pinocytotic phenomena in cerebral cortex capillaries is proposed. The method involved the counting of labeled "caveolae intracellulares" in endothelia cells with the use of horseradish peroxidase as a tracer molecule. A mean pinocytotic value per capillary is then computed. It is demonstrated that the value obtained after irradiation of the brain is higher than in control animals. This information may provide an ultrastructural quantitative approach to the analysis of the blood brain barrier and of other problems related to vascular permeability of the cerebral cortex.