The Cell Clinic: Closable Microvials for Single Cell Studies

We present the development of a cell clinic. This is a micromachined cavity, or microvial, that can be closed with a lid. The lid is activated by two polypyrrole/Au microactuators. Inside the microvials two Au electrodes have been placed in order to perform impedance studies on single or a small number of cells. We report on impedance measurements on Xenopus leavis melanophores. We could measure a change in the impedance upon cell spreading and identify intracellular events such as the aggregation of pigment granules. The electrical data is correlated to optical microscopy.     

Pathomorphological signs of damage to capillary endothelium in skeletal muscle after traumatic injury to or replantation of a limb

Several months after an extensive crush injury to or replantation of a limb in rats, capillary endotheliocytes of its skeletal muscle are seen to undergo ultrastructural changes of both “dark” and “light” types. Destructive/degenerative changes of the “light” type are accompanied by intracellular edema and cytoplasmic homogenization and can eventually result in monocellular colliquative necrosis. Changes of the “dark” type are characterized by signs of functional overstrain in the endotheliocytes, succeeded by destructive/degenerative cytoplasmic changes, increased electron density of intracellular organelles, loss of distinct boundaries by the nucleus and intracellular structures, rupture of cell membranes, endothelial desquamation, and some other changes, which eventually lead to coagulation necrosis, followed by breakdown of the cell into small fragments. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N^no 10, pp. 378–383, October, 1994     

In vivo electrical characteristics of human skin, including at biological active points

The aim is to compare the mean values of the in vivo electrical characteristics of bioiogical active points (BAPs) with those of the surrounding human skin. The impedance measurements at BAPs and on the surrounding skin are carried out in vivo on ten young, healthy people. The results of the measurements show that the BAP resistance R_P is smaller, and the capacitance C_P is higher, than the corresponding values for skin, R_S and C_S, respectively, these differences are larger at low frequencies (at f=3 Hz, R_S/R_P=3.19 and C_P/C_S=3.2). The mean values of the impedance measurements at the BAPs are different from those measured on the skin. The dependence of R_P and C_P on the pressing force, in the range of about 1–5 N, for the BAPs, has a smaller slope than that observed for the surrounding skin. An equivalent circuit for the BAPs is proposed that describes sufficiently well the experimental results obtained. These results show that the large dispersion in the observed impedance characteristics of the human body measurements in different body regions can be related to the influence of the BAPs present under the electrodes.     

Skin lipids in rats administered with melatonin

Effects of the pineal hormone melatonin on skin lipids in Wistar rats were studied by thinlayer chromatography. The reaction was shown to be delayed. Contents of total lipids and the majority of fractions increased over 24 h after administration of melatonin. Concentrations of triglycerides and phospholipids decreased, while the contents of cholesterol, cholesterol esters, and free fatty acids increased by the end of the second day. Our findings indicate that the blood and subcutaneous fat, as well as changed metabolic interrelations of skin lipids are involved in the skin response to increased melatonin concentration. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 4, pp. 463–465, April, 1999     

Pathogenesis of leptospirosis: interaction of Leptospira interrogans with in vitro cultured mammalian cells

Interactions of virulent Leptospira interrogans with murine monocyte-macrophage-like J774A.1 cells and Vero (African green monkey kidney fibroblasts) cells from attachment to internalization were investigated by a series of morphological analysis. Fontana silver staining revealed that only the pathogenic leptospires were able to attach to host cells and the attachment pattern varied depending on cell types that they interacted with. Transmission electron microscopy (TEM) analysis confirmed the formation of the leptospires-containing phagosomes and their colocalization with lysosomes in macrophages were verified by confocal microscopic analysis. Results of F-actin rearrangements examination indicated that virulent leptospires invaded host cells via a microfilament-independent pathway.     

Differentiation of mammalian skeletal muscle cells cultured on microcarrier beads in a rotating cell culture system

The growth and repair of adult skeletal muscle are due in part to activation of muscle precursor cells, commonly known as satellite cells or myoblasts. These cells are responsive to a variety of environmental cues, including mechanical stimuli. The overall goal of the research is to examine the role of mechanical signalling mechanisms in muscle growth and plasticity through utilisation of cell culture systems where other potential signalling pathways (i.e. chemical and electrical stimuli) are controlled. To explore the effects of decreased mechanical loading on muscle differentiation, mammalian myoblasts are cultured in a bioreactor (rotating cell culture system), a model that has been utilised to simulate microgravity. C₂C₁₂ murine myoblasts are cultured on microcarrier beads in a bioreactor and followed throughout differentiation as they form a network of multinucleated myotubes. In comparison with three-dimensional control cultures that consist of bioreactor exhibit an attenuation in differentiation. This is demonstrated by reduced immunohistochemical staining for myogenin and α-actinin. Western analysis shows a decrease, in bioreactor cultures compared with control cultures, in levels of the contractile proteins myosin (47% decrease, p<0.01) and tropomyosin (63% decrease, p<0.01). Hydrodynamic measurements indicate that the decrease in differentiation may be due, at least in part, to fluid stresses acting on the myotubes. In addition, constraints on aggregate size imposed by the action of fluid forces in the bioreactor affect differentation. These results may have implications for muscle growth and repair during spaceflight.     

Surface mapping of thoracic impedance cardiograms

A method is desbribed for mapping the electrical impedance cardiac signal on the anterior thoracic surface in man. A sinusoidal current was injected through electrodes placed around the neck and lower abdomen and voltage differences were measured with a single exploratory electrode at 30 well defined position on the thorax and upper abdomen. The expected interbeat variability was minimised by digital processing. Different pattern of dZ/dt, the first derivative of the cardiac impedance change were obtained at different points on the thoracic showing the additional information and discrimination provided by impedance surface mapping. The results of this work suggest that classical impedance cardiography detects mainly blood flow changes in the large upper thoracic vessels of the clavicular region. With the present method it is possible to visualise the effect of changes in heart volume and shape on the local impedance distribution and the spreading of blood flow related impedance changes across the thoracic and upper abdominal surfaces.     

Interdigitated microelectrode-based microchip for electrical impedance spectroscopic study of oral cancer cells

In this study, electric/electrochemical impedance spectroscopy and cyclic voltammetry were used to study the cellular activities of oral cancer cell line CAL 27, including the kinetics of cell adhesion, spreading, and cell proliferation on interdigitated microelectrodes (IMEs). Impedance spectra of CAL 27 cells on IMEs electrodes were obtained in cell growth medium and in 0.1 M PBS with 50 mM [Fe(CN)₆]^3−/4− as redox probe. Equivalent circuits were used to model both cases. In cell growth medium, impedance spectra allowed us to analyze the changes in capacitance and resistance due to cell attachment on the IMEs over the entire experiment period. It was found that cell spreading caused the most significant decrease in capacitance component and slight increase in resistance component. Impedance change at given frequencies (between 10 kHz to 100 kHz) was found to be linearly increased with increasing cell number of CAL 27 on the IMEs. In comparison with non-cancer oral epithelial cells (Het-1A), at equal cell number, cancer cells always generated impedance several folds higher than that of non-cancer cells. In the presence of [Fe(CN)₆]^3−/4−, impedance spectra allowed us to analyze the change in electron transfer resistance of IMEs due to cell attachment, in which an increase trend was observed at 24 h with increasing cell number from 2500 cells to 10000 cells on IMEs. Double layer capacitance was also affected by cell attachment, and a decrease in double layer capacitance was observed with increasing cell number on the electrodes. Cyclic voltammetric measurements correlated well with the impedance results. The results of this study demonstrated the use of electrochemical approaches to obtain and understand cellular behaviors/activities of oral cancer cells, potentially providing useful tools for cancer cell research.     

Mode of attachment and histopathological effects of Macrogyrodactylus clarii, a monogenean gill parasite of the catfish Clarias gariepinus, with a report on host response

A detailed account is given about the mode of attachment and histopathological effects of Macrogyrodactylus clarii Gussev, 1961, a viviparous monogenean from the gills of the catfish Clarias gariepinus. Most parasites attach their haptors to the proximal region of the gill filament (primary gill lamellae), while few specimens were seen attached to the distal region. Attachment of the haptor was achieved mainly by the blade of the hamuli, but no evidence was found indicating the participation of marginal hooklets in the attachment. The hamuli of M. clarii penetrate into the interlamellar epithelium of the gill tissue. Some evidences were found to indicate that M. clarii may also utilize suction force during haptoral attachment. The pathological effects of M. clarii are manifested by breakdown of the coating epithelium, necrosis of the epithelial cells, vacuolations inside and outside the host cells, fusion of the gill lamellae, rupture of blood capillaries, infiltration of erythrocytes and degeneration and fibrosis of the interlamellar epithelium. The host response includes the appearance of lymphocytes, mucoid secretions and hyperplasia of the tissue at the site of attachment.     

Dynamics of changes in skin lipids after stress: Effects of exogenous melatonin

Changes in the skin lipid composition induced by water-immersion stress in rats treated and untreated with melatonin were studied by thin-layer chromatography. Skin lipids showed a delayed reaction to stress. Melatonin exerted a protective effect which was manifested on the 2nd day after treatment in restoration of the level of total lipids and the absolute content of the majority of lipid fractions. The data suggest modification of, the metabolic relationships between skin lipids as well as lipids of the blood and subcutaneous adipose tissue. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 5, pp. 519–522, May, 1999     

Analysis of the O-wave in acute right ventricular apex impedance measurements with a standard pacing lead in animals

Modern pacemakers (implantable devices used for maintaining an appropriate heart rate in patients) can use an intracardiac ventricular impedance signal for physiological cardiac stimulation control. Intracardiac ventricular impedance from nine animal subjects is analysed and presented (seven sheep: 49.0±6.5 kg, sinus rhythm 100.3±16.5 beats min⁻¹, average impedance 629.8±72.6Ω; and two dogs: 30 kg each, sinus rhythm 86.0 beats min⁻¹, 862.1Ω and 134.0 beats min⁻¹, 1114.6Ω, respectively). The averaged curve and standard deviation curve of the impedance in sinus rhythm were analysed in MATLAB to clarify and study consistent impedance shape over one heart cycle. In eight of nine (89%) animal subjects, a consistent impedance slope change (notch) was observed in the early stage of the cardiac filling phase. This result was reproduced in an additional subject with simultaneous echocardiographical measurements of mitral valve blood flow. The notch occured soon after rapid early filling (E-wave in mitral flow) but prior to ventricular filling caused by atrial contraction, indicating that the impedance notch was caused by rapid ventricular filling and that it might be a sensed feature of diagnostic value. The intracardiac impedance notch in the present study had similar features to the non-invasive transthoracic impedance O-wave reported by others, and it is shown here that an O-wave is found in intracardiac impedance signals, strongly suggesting that the non-invasive O-wave is caused by cardiac events.     

Effect of recombinant forms of urokinase plasminogen activator on platelet aggregation and intracellular calcium accumulation

Urokinase caused plasmin-dependent inhibition of platelet aggregation in platelet-rich plasma, while its proteolytically inactive form had no such effect. Both forms potentiated the increase in platelet calcium concentration induced by aggregation inductors and facilitated aggregation of washed platelets. In contrast to full-length urokinase molecule, its aminoterminal fragment inhibited platelet aggregation and the corresponding elevation of intracellular calcium. These data suggest that urokinase exerts a plasmin-independent effect on platelet activity. This effect depends on urokinase structure. Translated formByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 9, pp. 339–343, September 1999     

The role of melatonin in parasite biology

Regarded as the circadian hormone in mammals, melatonin is a highly conserved molecule, present in nearly all species. In this review, we discuss the role of this indolamine and its precursors in the cell biology of parasites and the role of the molecule in the physiology of the host. In Plasmodium, melatonin can modulate intracellular concentrations of calcium and cAMP, which in turn can regulate kinase activity and cell cycle. In Trypanosoma infections, modulation of the immune system by melatonin is extremely important in controlling the parasite population. Melatonin also contributes to the inflammatory response to Toxoplasma gondii infection. Thus, there are a number of unique adaptations involving intricate connections between melatonin and the biology of the parasite-host relationship.     

Stimulation of neutrophil actin polymerization and degranulation by opsonized and unopsonized Candida albicans hyphae and zymosan.

We previously showed that unopsonized Candida albicans hyphae stimulated a delayed rise in the putative neutrophil second messengers Ca2+ and inositol 1,4,5-trisphosphate and subsequent O2- release, as compared with opsonized hyphae or zymosan. Therefore, cytoskeletal and degranulation temporal responses to these stimuli were examined. Unopsonized zymosan elicited no neutrophil responses under the experimental condition used. Neutrophil actin polymerization (quantitated by fluorescent measurements of NBD phallacidin) was rapid after stimulation by opsonized hyphae or zymosan (peaking at 1 and 2 min, respectively). This corresponded to observed changes in microscopic actin polymerization, measured with rhodamine phalloidin, which progressed from initially diffuse to collarlike to cylinderlike staining patterns surrounding the hyphae. Compared with opsonized hyphae, unopsonized hyphae resulted in a delayed appearance of the last two visible patterns (P less than 0.05) and in quantitative actin polymerization despite similarly rapid initial contact and spreading over the hyphae by neutrophils. Unlike other neutrophil responses, degranulation did not follow the delayed patterns of responses to stimulation with unopsonized hyphae. In the absence of the release of the cytoplasmic marker lactate dehydrogenase, the release of beta-glucuronidase, an azurophil granule marker, gradually and progressively rose in response to all of the stimuli but unopsonized zymosan. The low but significant levels observed were within a range consistent with published results for degranulation responses to particulate stimuli without cytochalasin B. A quantitative immunoassay of lactoferrin, a specific granule marker, detected no release into supernatants, and immunofluorescent staining indicated concomitant depletion of lactoferrin from neutrophil granules and binding to hyphal and neutrophil surfaces after stimulation by unopsonized hyphae. Thus, the delayed actin polymerization response to unopsonized hyphae occurred subsequent to neutrophil attachment and spreading and resembled the temporal sequence of other neutrophil responses linked to the respiratory burst. In contrast, the degranulation responses to all stimuli appeared to begin and progress gradually after observed attachment and spreading of the neutrophil over hyphal surfaces without a clear temporal relationship to rises in cytoplasmic Ca2+ or F-actin. In addition, the avid binding of released lactoferrin to cell surfaces eliminates its value as a quantitative marker of enzyme release but raises the possibility that it might participate in fungicidal activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

On the origin of the electrostatic surface potential of Aspergillus niger spores in acidic environments

The electrostatic surface potential of fungal spores is generally regarded as potentially influencing spore aggregation and pellet formation in submerged cultures of filamentous fungi. Spores of Aspergillus niger are typically characterized by negative zeta potentials over a wide range of pH values. In this study, this particular behavior is ascribed to the presence of an extensive melanin coating. It is proposed on the basis of zeta potential and pigment extraction experiments that this outermost layer affects the pH-dependent surface potential in two manners: (i) by the addition of negative charges to the spore surface and (ii) by the pH-dependent release of melanin pigment. Chemical analyses revealed that deprotonation of melanin-bound carboxyl groups is most probably responsible for pigment release under acidic conditions. These findings were incorporated into a simple model which has the ability to qualitatively explain the results of zeta potential experiments and, moreover, to provide the basis for quantitative investigations on the role of electrostatics in spore aggregation.     

Extrapineal melatonin and its role in the neuroendocrine regulation of homeostasis

The hormone of melatonin is the main regulator of biological rhythms. It was first found in the pineal gland in 1958. Melatonin is involved in the regulation of many vital physiological processes: maturation and development of genitalias, metabolism of pigments and free radicals, immune response, mood and sleep, and cell proliferation and differentiation. The pineal gland is not the only organ synthesizing melatonin. Extrapineal melatonin is widely dis-tributed in humans and animals. Melatonin-producing cells are found in the gastrointestinal and respiratory tracts, pancreas, adrenal and thyroid glands, thymus, cerebellum, urogenital system, placenta, and other organs. Melatonin is intensely synthesized in non-endocrine cells: mast cells, natural killers, eosinophilic leukocytes, platelets, and endotheliocytes. Such a wide distribution of melatonin reflects its key role as an intercellular neuroendocrine regulator and coordinator of many complex and interrelated biological processes. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 127, No. 4, pp. 364–730, April, 1999     

The Role of F-Actin in Hypo-Osmotically Induced Cell Volume Change and Calcium Signaling in Anulus Fibrosus Cells

Loading of the spine induces dynamic changes in the osmotic environment of the intervertebral disc (IVD) due to the exudation and recovery of tissue water. Cells from the anulus fibrosus (AF) respond to osmotic stress with altered biosynthesis through a pathway that may involve calcium (Ca2+) as a second messenger. We examined the hypothesis that AF cells respond to hypo-osmotic stress by swelling and initiating regulatory volume decrease (RVD). Further, the role of F-actin disruption and transient increases in intracellular calcium concentration ([Ca2+]i) in volume adaptation were studied. In response to hypo-osmotic stress, AF cells swelled, disrupted F-actin, and exhibited [Ca2+]i transients in proportion to the magnitude of the stress. The transient disruption of F-actin was dependent on the presence of extracellular Ca2+. After swelling, AF cells underwent RVD at all magnitudes of hypo-osmotic stress. The extent of RVD was diminished significantly by F-actin breakdown using cytochalasin D or by inhibition of swelling-induced F-actin disruption by removing extracellular Ca2+. Swelling-induced disruption of F-actin facilitated RVD, as evidenced by a more rapid volume recovery with increased F-actin breakdown. In conclusion, our findings suggest that the F-actin network plays an important role in the response of AF cells to osmotic stress.     

Influence of fibrinolysis system on ADP- and serotonin-induced aggregation of human platelets

Urokinase, a component of the fibrinolytic system, induces a time-dependent decrease in platelet aggregation activated by ADP and serotonin. Significant inhibition of ADP-induced aggregation was observed on the 30th–60th min and serotonin-induced on the 3th–10th min of preincubation with urokinase and depended on urokinase concentration. The plasmin inhibitor aprotinin partially abolished urokinase-induced reduction of the amplitude and rate of ADP-induced aggregation and had no effect on serotonin-induced aggregation. Our results favor multiple mechanisms of urokinase influence on platelet activity. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 2, pp. 158–161, February, 1998     

Specific amino acid restriction inhibits attachment and spreading of human melanoma via modulation of the integrin/focal adhesion kinase pathway and actin cytoskeleton remodeling

We had previously found that selective restriction of amino acids inhibits invasion of human A375 melanoma. Integrins, cell surface receptors for the components of extracellular matrix (ECM), are activated during cell adhesion and spreading, and initiate signaling pathways that control growth and invasion of tumor cells. We examined the effect of tyrosine (Tyr) and phenylalanine (Phe), methionine (Met) or glutamine (Gln) restriction on attachment and spreading of A375 and MeWo melanoma cell lines on fibronectin and laminin. In A375 cells, restriction of Tyr/Phe or Met inhibited attachment to and spreading on laminin and fibronectin, inhibited alpha3 and alpha4 integrin expression, and inhibited accumulation of FAK-Tyr397 and F-actin at leading edges of cell protrusions. Tyr/Phe restriction also inhibited attachment-induced autophosporylation of FAK-Tyr397. In MeWo cells, the order of inhibition by amino acid restriction on cell attachment and spreading was as follows: Gln > Tyr/Phe > Met. Restriction of Gln reduced alpha5 integrin expression. All amino acid restrictions similarly inhibited phosphorylation of FAK-Tyr397, FAK-Tyr577, FAK-Tyr861 and paxillin-Tyr31. Gln restriction exhibited the strongest inhibition of actin cytoskeleton remodeling during the cell spreading. The present study reveals that specific amino acid restriction inhibits attachment and spreading of melanoma via inhibition of specific integrin expression, inhibition of integrin-mediated FAK phosphorylation, and modulation of actin cytoskeleton remodeling. These data provide additional understanding of the mechanism by which specific amino acid restriction controls invasion and migration of melanoma.     

Factors affecting the measurement of bone impedance

Factors which affect the measurement of the electrical properties of bone were examined. Environmental conditions such as the pH of the preserving solution and time of exposure of the sample to air, which controls the moisture content of the specimen, significantly affected the bone impedance. A change in pH of ×2 from the neutral solution (pH=7) caused a change in the resistance R. conductance β and capacitance C values of 70 per cent, 125 per cent and 190 per cent respectively. A 5 min exposure to air increased the resistance by 92 per cent and reduced the capacitance by 35 per cent of their original values. A loss of 8 per cent moisture content by weight resulted in 400 per cent increase in R and 65 per cent decrease in C. Significant errors were introduced in the measurement of bone impedance owing to stray and wiring capacitances; these were avoided by using a differential impedance measurement technique. Further, it is shown that it is essential to measure the phase angle while measuring the bone impedance. Finally, it is illustrated that when normalising for different sized bone specimens, both the resistive component and capacitive component have to be considered while computing the impedance per unit volume.