Changes In Intracellular Activity Research Articles

Design of an ultrasound chamber for cellular excitation and observation.

In this work, a design of integrating ultrasonic transduction with live cell imaging chamber is introduced. The principle of a metal-incident-glass-output acoustic path was used to deliver a uniform energy profile into the imaging/incubation chamber in the form of leaky Lamb waves. The design was applied to examine living mouse mammary gland epithelial cells (EpH4). Significant changes in intracellular activities were observed even at a very low energy intensity level (1 MHz, ISATA = 1 mW/cm2, continuous wave). Live imaging with ultrasonic stimulation provides a different paradigm to interrogate cellular mechanosensitive responses in real time.

Biodynamic imaging of live porcine oocytes, zygotes and blastocysts for viability assessment in assisted reproductive technologies.

The success of assisted reproductive technologies relies on accurate assessment of reproductive viability at successive stages of development for oocytes and embryos. The current scoring system used to select good-quality oocytes relies on morphologically observable traits and hence is indirect and subjective. Biodynamic imaging may provide an objective approach to oocyte and embryo assessment by measuring physiologically-relevant dynamics. Biodynamic imaging is a coherence-gated approach to 3D tissue imaging that uses digital holography to perform low-coherence speckle interferometry to capture dynamic light scattering from intracellular motions. The changes in intracellular activity during cumulus oocyte complex maturation, before and after in vitro fertilization, and the subsequent development of the zygote and blastocyst provide a new approach to the assessment of preimplant candidates.

Arabidopsis COBRA‐LIKE 10, a GPI‐anchored protein, mediates directional growth of pollen tubes

Successful reproduction of flowering plants requires constant communication between female tissues and growing pollen tubes. Female cells secrete molecules and peptides as nutrients or guidance cues for fast and directional tube growth, which is executed by dynamic changes of intracellular activities within pollen tubes. Compared with the extensive interest in female cues and intracellular activities of pollen tubes, how female cues are sensed and interpreted intracellularly in pollen is poorly understood. We show here that COBL10, a glycosylphosphatidylinositol (GPI)-anchored protein, is one component of this pollen tube internal machinery. Mutations in COBL10 caused gametophytic male sterility due to reduced pollen tube growth and compromised directional sensing in the female transmitting tract. Deposition of the apical pectin cap and cellulose microfibrils was disrupted in cobl10 pollen tubes. Pollen tube localization of COBL10 at the apical plasma membrane is critical for its function and relies on proper GPI processing and its C-terminal hydrophobic residues. GPI-anchored proteins are widespread cell sensors in mammals, especially during egg-sperm communication. Our results that COBL10 is critical for directional growth of pollen tubes suggest that they play critical roles in cell-cell communications in plants.

Biphasic intracellular expression of Staphylococcus aureus virulence factors and evidence for Agr-mediated diffusion sensing.

Staphylococcus aureus invades a variety of mammalian cells and escapes from the endosome to multiply in the cytoplasm. We had previously hypothesized that the molecular events leading to escape of S. aureus from the endosome involved the Agr virulence factor regulatory system. In this report we demonstrate that temporal changes in intracellular activation of the Agr regulon correlates with expression of membrane active toxins. Also, the initial expression of Agr by even small numbers of staphylococci resulted in the permeabilization of the endosomal membrane and the eventual escape of bacteria into the cytoplasm by 3 h post invasion. After Agr downregulation, a second peak of expression coincided with increased permeability of the host cell membrane. In contrast to the parental strain, an Agr-mutant was unable to escape into the cytoplasm and was observed in intact endosomes as late as 5 h post invasion. These data provide evidence that staphylococcal virulence factor production during invasion of host cells is mediated by an Agr-dependent process that is most accurately described in the context of diffusion sensing.

Ethanol increases hepatocyte water volume.

Mouse hepatocytes respond to osmotic stress with adaptive changes in transmembrane potential, Vm, such that hypotonic stress hyperpolarizes cells and hypertonic stress depolarizes them. These changes in Vm provide electromotive force for redistribution of ions such as Cl-, and this comprises part of the mechanism of hepatocyte volume regulation. We conducted the present study to determine whether ethanol administered in vitro to mouse liver slices increases hepatocyte water volume, and whether this swelling triggers adaptive changes in the Vm. Cells in mouse liver slices were loaded with tetramethylammonium ion (TMA). Changes in hepatocyte water volume were computed from measurements with ion sensitive microelectrodes of changes in intracellular activity of TMA (a1TMA) that resulted from water fluxes. Ethanol (70 mM) increased hepatocyte water volume immediately, and this peaked at 17% by 7 to 8 min, by which time a plateau was reached. Liver slices also were obtained from mice treated 12 hr prior with 4-methylpyrazole (4 mM). The effect of ethanol on their hepatocyte water volume was identical to that from untreated mice, except that the onset and peak were delayed 2 min. Hepatocyte Vm showed no differences between control or ethanol-treated cells during the course of volume changes. In contrast, hyposmotic stress, created by dropping external osmolality 50 mosm, increased Vm from -30 mV to -46 mV. Ethanol did not inhibit this osmotic stress-induced hyperpolarization, except partially at high concentrations of 257 mM or greater. We infer that ethanol-induced swelling of hepatocytes differs from that resulting from hyposmotic stress.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in membrane potential, membrane resistance, and intracellular H +, K +, Na +, and Cl − activities during the progesterone-induced maturation of urodele amphibian oocytes

Changes in intracellular activities of H +, K +, Na +, and Cl − ions were recorded with ion-selective microelectrodes during progesterone-induced maturation of full-grown oocytes of the urodele amphibians Ambystoma mexicanum and Pleurodeles waltlii. The membrane potential ( E m) and electrical resistance ( R m) were also determined. During the first hours after initiation of maturation, the oocytes slowly depolarized and R m gradually increased. By the end of maturation of Pleurodeles oocytes E m had stabilized at about −10 mV and R m had increased from 410 to 1760 kΩ. The same initial pattern was observed for Ambystoma, but in most oocytes a rapid transition occurred at about the time of germinal vesicle breakdown (GVBD): E m spontaneously shifted from about −15 to about +30 mV; simultaneously R m dropped from 1230 down to 100 kΩ (i.e., less than the initial 270 kΩ resistance). The internal K + activity did not show any important variation during maturation of Ambystoma and Pleurodeles oocytes. Na + activity increased slightly at the onset of GVBD in Ambystoma; a further marked increase of Na +, accompanied by an increase in Cl − activity, was observed as soon as E m shifted to a positive value. In Pleurodeles sodium activity was also more elevated in matured than in immature oocytes. The average pH of Ambystoma immature oocytes was 7.48 ± 0.05 (external pH 7.5). A transient alkalinization to 7.64 ± 0.04 took place during the first 4–6 hr postprogesterone. Cytoplasmic pH was restored to 7.50 ± 0.07 between 10 and 12 hr postprogesterone, before the onset of GVBD and the shift of E m. The difference between the measured oocyte pH and the calculated equilibrium pH decreases during the course of maturation, due partly to the depolarization of E m.

Cellular and transepithelial responses of goldfish intestinal epithelium to chloride substitutions

In goldfish intestine chloride was substituted by large inorganic anions (gluconate or glucuronate) either mucosally, serosally or bilaterally. Changes in intracellular activities of chloride (a i Cl−), sodium (a i Na+) and potassium (a i K+), pHi, relative volume, membrane and transepithelial potentials, transepithelial resistance and voltage divider ratio were measured. Control values were:a i Cl−=35 meq/liter, a i Na+=11 meq/liter and a i K+=95 meq/liter. During bilateral substitution the latter two did not change while a i Cl− dropped to virtually zero.

An intracellular analysis of gamma‐aminobutyric‐acid‐associated ion movements in rat sympathetic neurones.

Double-barrelled ion-sensitive micro-electrodes were used to measure the changes of the intracellular activities of Cl-, K+, and Na+ (aiCl, aiK, aiNa) in neurones of isolated rat sympathetic ganglia during the action of gamma-aminobutyric acid (GABA). The membrane potential of some of the neurones was manually 'voltage clamped' by passing current through the reference barrel of the ion-sensitive micro-electrode. This enabled us to convert the normal depolarizing action of GABA into a hyperpolarization. A GABA-induced membrane depolarization was accompanied by a decrease of aiCl, aiK and no change in aiNa, whereas a GABA-induced membrane hyperpolarization resulted in an increase of aiCl, aiK and also no change in aiNa. GABA did not change the free intracellular Ca2+ concentration, as measured with a Ca2+-sensitive micro-electrode, whereas such an effect was seen during the action of carbachol. pH-sensitive electrodes, on the other hand, revealed a small GABA-induced extracellular acidification. The inward pumping of Cl- following the normal, depolarizing action of GABA required the presence of extracellular K+ as well as Na+, whereas CO2/HCO3--free solutions did not influence the uptake process. Furosemide, but not DIDS, blocked the inward pumping of Cl-. In conclusion, our data show that only changes in intracellular activities of K+ and Cl- are associated with the action of GABA. Furthermore, they indicate that a K+/Cl- co-transport, and not a Cl-/HCO3- counter-transport, may be involved in the homoeostatic mechanism which operates to restore the normal transmembrane Cl- distribution after the action of GABA.

Effect of temperature on extracellular accumulation of β-D-N-acetylglucosaminidase in I-cell disease fibroblast cultures

The activities of most lysosomal enzymes are elevated in the culture fluid of skin fibroblasts derived from patients with I-cell disease with a corresponding reduction in the intracellular activities when the cells are cultured at 37°C. When I-cell fibroblast cultures are incubated at 27°C for 8–24 hr, the β-D-N-acetylglucosaminidase (EC 3.2.1.30) activity accumulated in the culture fluid is reduced to approximately 25% of the activity in 37°C control cultures without a corresponding change in intracellular activity. No significant effect of temperature is observed on the intra- and extracellular distribution of β-D-N-acetylglucosaminidase in non-I-cell fibroblast cultures. These findings suggest the existence of two lysosomal enzyme pools in I-cell fibroblasts, one of which is temperature-dependent and destined for excretion while the other remains intracellular and appears to be unaffected by temperature.

Changes in intracellular activities of lysosomal enzymes in tissues of rats during aging

A comparison of results obtained from studies of the intracellular fractions of the tissues of liver, brain and heart of “young” (1–2 months), “old” (24–27 months) and “senile” (34–37 months) rats showed that the ratios of three enzymes, acid phosphatase, β- N-acetylglucosaminidase and acid RNase of the liver and heart were very similar and their activities decreased with age. On the other hand, the protein content in the supernatant of the liver, and acid DNase activities in the supernatant of the brain increased significantly with age. When the 24–27 month and 34–37 month old rats were compared, the ratios of the total activities of liver β- N-glucosaminidase and brain acid DNase in the supernatant and the specific activity of brain β- N-glucosaminidase in the microsomal fraction increased significantly.

Changes in electrical activity of the myocardial fibers after subcutaneous transplantation of the heart into the mouse ear

The intracellular resting potential (RP) and the action potential (AP) of the transplanted mouse heart on the third to fourth day after transplantation were lower than the intracellular potentials of newborn mice. Starting from the fifth to sixth day there was a gradual increase in the amplitude of the intracellular potentials. On the first 7–8 days after isologous and heterologous transplantation the dynamics of the changes in intracellular activity was the same. Later, in the case of the heterograft, a second decrease in amplitude of RP and AP of the myocardial fibers of the graft took place, probably in connection with the development of a rejection reaction. In the isografts the amplitude of the intracellular potentials toward the end of the first month after transplantation was close to the values of RP and AP of the fibers of the recipient's heart.

Effects of metabolites present during growth of Tetrahymena pyriformis on the subsequent secretion of lysosomal hydrolases

Tetrahymena were grown in proteose-peptone medium supplemented with glucose, mannose, fructose, galactose, acetate, succinate, or pyruvate and then washed and resuspended in a non-nutrient salt solution and the amounts of 7 acid hydrolases secreted into the medium in a one hour incubation were measured. Cells that had been grown in the presence of glucose secreted about half the amounts of acid phosphatase, beta-N-acetylglucosaminidase and acid protease as did control cells grown in unsupplemented medium. Pyruvate was about as effective as glucose and both were slightly more effective than acetate or fructose. Succinate had little effect. Similar experiments showed that alpha-mannosidase, beta-fucosidase, and beta-galactosidase are secreted into the salt solution and the secretion is reduced by prior growth of the cells in medium supplemented with glucose or mannose but not galactose. Except for alpha-mannosidase, these reductions in amounts of hydrolase secreted were not accompanied by appreciable changes in intracellular activity, and therefore demonstrate a persistent effect of growth in the presence of certain metabolites on the subsequent secretion of lysosomal hydrolases. Since the inhibition of subsequent secretion depended on both the individual metabolite and the particular hydrolase examined, it appears that the effect of metabolites is not limited to a general inhibition of secretion but may differentially alter some properties of lysosomal subpopulations. A preliminary characterization of the secreted acid protease of Tetrahymena suggests that there may be two acid proteases released, since up to 25% of the activity was not inhibited by high concentrations of pepstatin, leupeptin, or chymostatin.

Intracellular and extracellular enzyme patterns during microcyst germination in the cellular slime mold Polysphondylium pallidum

Microcysts of Polysphondylium pallidum, formed in modified Sussman's (1963) medium, germinate synchronously without activation in 4 hr in a sterile nonnutrient medium. Germination is cycloheximide-sensitive only within the first 2 hr. In this early period, cycloheximide stops emergence of amoebae but does not prevent the initial swelling phase. Actinomycin D and nucleic acid analogs at high concentrations have no evident effects on the germination process. Of nine hydrolytic enzymes studied, only alkaline phosphatase shows a large intracellular increase in activity. The increase in activity is cycloheximide-sensitive, indicating a requirement for coincident protein synthesis. Additional experiments show the lack of activators or inhibitors of alkaline phosphatase activity at different stages of the germination process. Changes in the phosphatase isozyme population could not be detected and the enzyme is not excreted. Several lysosomal glycosidases show little change in intracellular activity but their activity increases dramatically in the extracellular medium. The extracellular increases in these enzymes correlate well with the morphological loss of the microcyst wall, suggesting a possible role for certain of these enzymes in wall digestion.

The lysosome: a role in disease.

Increasing interest in elucidating the role of the lysosome in intracellular metabolic economy has resulted in a deeper understanding of some inborn errors of metabolism, as well as a clearer definition of the intracellular vacuolar apparatus and the role of lysosomes in intracellular drug action and possible specific chemotherapeutic actions. The lysosomal system plays a key role in Pompe's disease (glycogenosis type II). The effect of a selective deficiency of lysosomal acid maltase (acid α-glucosidase) on muscle morphology and biochemistry is shown. The activity of other lysosomal acid hydrolases in muscle is significantly increased. Change in intracellular activity and translocation of primary and secondary lysosomes after membrane activation by steroids are discussed, with data showing the intranuclear migration of lysosomal-like particles, suggesting a mechanism for template activation. Lysosomes play a decisive role in initiating nucleocytoplasmic exchange of information, and they may play a role in genetic derepression, with obvious repercussions in normal and abnormal cell growth. The lysosome model in reference to acid hydrolase induction, especially endocytic and phagolysosomal activity, has been examined.

Facilitation of reactions of cortical neurons to cortical and peripheral stimulation after tetanization of the brain surface

The electrical activity of neurons of the sensomotor cortex of an unanesthetized rabbit was investigated. Conditioning tetanization of the cortex was carried out through surface electrodes located close to the site of the lead. Test stimuli were supplied through electrodes 2.5–12 mm more caudally on the cortical surface. In addition, peripheral test stimuli were applied. Impulse reactions to previously ineffective stimuli develop after conditioning tetanization and prolonged (up to 1 min) intensification of exciting postsynaptic potentials (EPSP) to cortical and peripheral test stimuli is observed. Facilitation of the reactions is especially clear during tetanization superthreshold for evoking epileptiform afterdischarges. It continued after the conclusion of these discharges and could also be observed during tetanization subthreshold for evoking afterdischarges. The time course of the facilitation was similar to the time course of the post-tetanic intensification of reactions of single stimuli applied with the electrodes used for tetanization. An analysis of the changes in intracellular activity makes it possible to assume that the mechanism of post-tetanic potentiation (PTP) lies at the basis of the described facilitation, which is considered as the "cellular analog" of the dominant focus which develops as a result of tetanization of the cortical surface.