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PubMed1998

Role of nitric oxide and cyclooxygenase products in controlling vascular tone in uterine microvessels of rats.

The importance of nitric oxide (NO) and dilator prostaglandins in uterine resistance arterioles was investigated. In pentobarbital anaesthetized rats at dioestrus-2, the uterine microcirculation in vivo was transilluminated by a fibreoptic probe and microvessels (circumferential arterioles) viewed by video microscopy. Arteriolar diameters were measured while increasing concentrations of acetylcholine (ACh), serotonin (5-HT), phenylephrine (PE), or angiotensin II (AII) were applied topically (suffused) over the uterus. Agonists were applied alone or with ibuprofen (IBU; cyclooxygenase inhibitor), N omega-nitro-L-arginine (L-NA; nitric oxide synthase inhibitor) or both. Circumferential arterioles were dilated by ACh and 5-HT (10(-8)-10(-4) mol l-1) and constricted by PE (10(-8)-10(-5) mol l-1) and AII (10(-11)-10(-7) mol l-1). Suffusion of L-NA or L-NA with ibuprofen (10(-4) mol l-1 each) abolished ACh-induced dilation; ibuprofen alone blocked dilation at higher ACh concentrations. Serotonin-induced relaxation was significantly attenuated by L-NA alone or in combination with ibuprofen. Vasoconstriction induced by PE was enhanced by L-NA alone and L-NA with ibuprofen, but ibuprofen alone had no effect. In contrast, AII-induced constriction was enhanced significantly by ibuprofen or L-NA and further enhanced when both ibuprofen and L-NA were present. These results suggest that ACh can release either nitric oxide (NO) or cyclooxygenase products to cause uterine arteriolar dilation and that 5-HT-induced uterine microvascular relaxation is mediated via NO only. They also suggest that PE-induced vasoconstriction is attenuated by the release of NO but not cyclooxygenase products and that constrictor responses evoked by AII are attenuated by both NO and dilator prostaglandin release. Thus, both nitric oxide and dilator prostaglandins are important in the control of uterine microvessels.

Acetylcholine↗
PubMed2000

Peptidomimetic antagonists of alphavbeta3 inhibit bone resorption by inhibiting osteoclast bone resorptive activity, not osteoclast adhesion to bone.

Osteoclasts are actively motile on bone surfaces and undergo alternating cycles of migration and resorption. Osteoclast interaction with the extracellular matrix plays a key role in the osteoclast resorptive process and a substantial body of evidence suggests that integrin receptors are important in osteoclast function. These integrin receptors bind to the Arg-Gly-Asp (RGD) sequence found in a variety of extracellular matrix proteins and it is well established that the interaction of osteoclast alpha v beta 3 integrin with the RGD motif within bone matrix proteins is important in osteoclast-mediated bone resorption. In this study, we characterized the effects of two synthetic peptidomimetic antagonists of alpha v beta 3, SC-56631 and SC-65811, on rabbit osteoclast adhesion to purified matrix proteins and bone, and on bone resorption in vitro. SC-56631 and SC-65811 are potent inhibitors of vitronectin binding to purified alpha v beta 3. Both SC-56631 and SC-65811 inhibited osteoclast adhesion to osteopontin- and vitronectin-coated surfaces and time-lapse video microscopy showed that osteoclasts rapidly retract from osteopontin-coated surfaces when exposed to SC-56631 and SC-65811. SC-56631 and SC-65811 blocked osteoclast-mediated bone resorption in a dose-responsive manner. Further analysis showed that SC-65811 and SC-56631 reduced the number of resorption pits produced per osteoclast and the average pit size. SC-65811 was a more potent inhibitor of bone resorption and the combination of reduced pit number and size led to a 90% inhibition of bone resorption. Surprisingly, however, osteoclasts treated with SC-65811, SC-56631 or the disintegrin echistatin, at concentrations that inhibit bone resorption did not inhibit osteoclast adhesion to bone. These results suggest that alphavbeta3 antagonists inhibited bone resorption by decreasing osteoclast bone resorptive activity or efficiency but not by inhibiting osteoclast adhesion to bone per se.

Aniline Compounds↗
PubMed2004

Caspase activity inhibition delays programmed spermatogenic cell death in vitro.

Programmed cell death or apoptosis was analyzed in rat Sertoli-spermatogonial cell cocultures prepared from 2-9 day old rats using time-lapse video microscopy, a cell viability fluorescence microscopy assay, immunocytochemical markers, and cell-permeable caspase inhibitory peptides with reversible and irreversible effects. We show that apoptosis can initially affect a single member of a spermatogonial cell cohort and that single non-viable spermatogonial cells can remain conjoined to viable spermatogonial cells. The integrity of the cytoskeletal F-actin network and the presence on Bcl-2 immunoreactivity are valuable markers of spermatogonial cell viability. Apoptotic bodies released into the culture medium are generally eliminated after culture medium replenishment; however, spermatogonial apoptotic cell remnants can be taken up by Sertoli cells, which are known to represent a phagocytic somatic population within the seminiferous epithelium. Cell permeable caspase-1 and caspase-4 inhibitory peptides with reversible and irreversible action were supplemented to a serum-free hormone-growth factor-supplemented medium. In the absence of the caspase inhibitory peptide, the viability of spermatogonial cells decreases gradually with time in coculture. However, the addition of caspase inhibitory peptides causes a significant accumulation of spermatogenic cells per unit surface area. Although inhibition of caspases, the executors of spermatogonial cell death, results in a substantial increase of spermatogonial cells in the cocultures, it remains to be determined what the differentiation potential of caspase-inhibited spermatogonial cell cohorts is.

Actins↗
PubMed2006

Encapsulation of attached ectoparasitic glochidia larvae of freshwater mussels by epithelial tissue on fins of naive and resistant host fish.

To metamorphose into juveniles and subsequently mature into adults, the glochidia larvae of freshwater mussels in the order Unionoida must temporarily parasitize the gills, fins, or other external structures of fish. Once attached to the fish, the glochidium is encapsulated by host fish epithelial tissue. The migration of epithelial cells of the bluegill sunfish Lepomis macrochirus over glochidia of Utterbackia imbecillis was examined by time-lapse video microscopy, and the morphology was examined by scanning electron microscopy. Initially, the leading edge epithelial cells migrating over the larvae became rounded and the cells moved as a sheet until the attached glochidium was completely covered. Cyst formation on host fish that had been repeatedly exposed to mussel larvae was significantly delayed and morphologically irregular compared to that on naïve fish. Cyst formation on other species of fish that are less successful as hosts was examined. In general, it took longer for glochidia to become encapsulated on these less suitable potential hosts. The delay and irregularities in cyst formation on resistant fish and nonhost fish species may result in increased mortality and reduced success of metamorphosis of glochidia.

Animals↗
PubMed1994

Active and passive mechanical properties of isolated arterioles from STZ-induced diabetic rats. Effect of aminoguanidine treatment.

Studies were performed to examine the effect of experimental diabetes (4-6 weeks duration) on both the passive elastic and active myogenic properties of isolated skeletal muscle arterioles. Studies were conducted on untreated streptozotocin (60 mg/kg)-induced diabetic rats and in similar rats treated daily with either amino-guanidine (25 mg/kg) or methylguanidine (25 mg/kg). First-order cremaster muscle arterioles were isolated, cannulated, and pressurized in the absence of intraluminal flow. Video microscopy was used to determine relationships between arteriolar diameter and intraluminal pressure both in the active and passive (o mmol/l Ca(2+)-2 mmol/l EGTA superfusated) tes. The measurements were used to calculate active myogenic responses, arteriolar distensibility, and stress-strain relationships. Under passive conditions, arterioles from untreated diabetic animals appeared to be stiffer and less distensible compared with similar arterioles from control animals. Under active conditions, i.e., in the presence of extracellular Ca2+, arterioles from the untreated diabetic group showed impaired myogenic reactivity as evidenced by a significant (P < 0.001) reduction in the negative slope of the pressure-diameter relationship over a physiological range of intraluminal pressures. Chronic treatment with aminoguanidine prevented the diabetes-induced changes in the active and passive properties of the isolated arterioles while treatment with methylguanidine appeared ineffective. Vasodilator responses to topically applied acetylcholine (10(-8) to 5 x 10(-6) mol/l) were significantly impaired in diabetic animals irrespective of treatment with aminoguanidine. The data indicate that experimental diabetes is associated with a decreased passive distensibility, or stiffening, of skeletal muscle arterioles that, in addition, may contribute to impaired active myogenic responses.

Acetylcholine↗
PubMed2003

Replication increases beta-cell vulnerability to human islet amyloid polypeptide-induced apoptosis.

Type 2 diabetes is characterized by a relative beta-cell deficit as a result of increased beta-cell apoptosis and islet amyloid derived from the beta-cell peptide islet amyloid polypeptide (IAPP). Human IAPP (h-IAPP) but not mouse IAPP (m-IAPP) induces apoptosis when applied to cells in culture, a property that depends on the propensity of h-IAPP to oligomerize. Since beta-cell mass is regulated, the question arises as to why it is not adaptively increased in response to insulin resistance and hyperglycemia in type 2 diabetes. This adaptation might fail if dividing beta-cells preferentially underwent apoptosis. We tested the hypothesis that beta-cells are preferentially vulnerable to h-IAPP-induced apoptosis. We established a microculture environment to perform time-lapse video microscopy (TLVM) and studied beta-cells (RIN) and HeLa cells undergoing replication or apoptosis. Sequential images (every 10 min for 36 h in RIN or 24 h in HeLa cells) of cells in vivo were analyzed, and each mitotic and apoptotic event was documented. Freshly dissolved h-IAPP caused a dose-dependent increased rate of apoptosis (P < 0.0001) in both cell types. At low and medium levels of toxicity, cells that had previously undergone mitosis were more vulnerable to h-IAPP-induced apoptosis than nondividing cells (P < 0.05). In the first 3 h after mitosis (full cell cycle length 26 +/- 0.6 h), beta-cells were particularly susceptible to h-IAPP-induced apoptosis (P < 0.05). Neither m-IAPP nor mature amyloid aggregates of h-IAPP were cytotoxic (P = 0.49). To corroborate these cell culture studies, we examined sections of human pancreatic tissue (five cases of type 2 diabetes) and human islets incubated for 48 h +/- h-IAPP. Both were stained for apoptosis with the transferase-mediated dUTP nick-end labeling method and analyzed for the presence of paired apoptotic cells anticipated in the event of postmitotic apoptosis. In human pancreatic tissue 26 +/- 5% (single plane of examination) and in human islets incubated with h-IAPP 44 +/- 4% of apoptotic islet cells were paired. In conclusion, replicating beta-cells are preferentially vulnerable to h-IAPP-induced apoptosis in cell culture. Postmitotic apoptosis was also documented in humans with type 2 diabetes and in human islet tissue. We postulate that beta-cell deficiency in type 2 diabetes may result in part from failure to adaptively increase beta-cell mass due to increased vulnerability of replicating beta-cells to undergo apoptosis. If this postulate is correct, then inhibition of apoptosis should allow recovery of beta-cell mass in type 2 diabetes.

Amyloid↗
PubMedDate not supplied

Transient domains induced by influenza haemagglutinin during membrane fusion.

During low pH-induced fusion of influenza virus with erythrocytes we have observed differential dispersion of viral lipid and haemagglutinin (HA) into the erythrocyte membrane, and viral RNA into the erythrocyte using fluorescence video microscopy. The movement of both viral lipid and HA from virus to cell was restricted during the initial stages of fusion relative to free diffusion. This indicates the existence of relatively long-lived barriers to diffusion subsequent to fusion pore formation. Fluorescence anisotropy of phospholipid analogues incorporated into the viral membrane decreased when the pH was lowered to levels required for optimum fusion. This indicates that the restricted motion of viral membrane components was not due to rigidification of membrane lipids. The movement of HA from the fusion site was also assessed by photosensitized labelling by means of a fluorescent substrate (NBD-taurine) passing through the band 3 sialoglycoprotein (the erythrocyte anion transporter). We also examined the flow of lipid and aqueous markers during fusion of HA-expressing cells with labelled erythrocytes. During this cell-cell fusion, movement of lipid between fusing membranes begins before the fusion pore is wide enough to allow diffusion of aqueous molecules (M(r) > 500). The data indicate that HA is capable of creating domains in the membrane and controlling continuity of aqueous compartments which are bounded by such domains.

Cell Fusion↗
PubMed2001

Abnormal migration phenotype of mitogen-activated protein kinase-activated protein kinase 2-/- neutrophils in Zigmond chambers containing formyl-methionyl-leucyl-phenylalanine gradients.

Time-lapsed video microscopy and confocal imaging were used to study the migration of wild-type (WT) and mitogen-activated protein kinase-activated protein kinase 2 (MK2-/-) mouse neutrophils in Zigmond chambers containing fMLP gradients. Confocal images of polarized WT neutrophils showed an intracellular gradient of phospho-MK2 from the anterior to the posterior region of the neutrophils. Compared with WT neutrophils, MK2-/- neutrophils showed a partial loss of directionality but higher migration speed. Immunoblotting experiments showed a lower protein level of p38 mitogen-activated protein kinase and a loss of fMLP-induced extracellular signal-related kinase phosphorylation in MK2-/- neutrophils. These results suggest that MK2 plays an important role in the regulation of neutrophil migration and may also affect other signaling molecules.

Actins↗
PubMed2002

Live-cell dynamics and the role of costimulation in immunological synapse formation.

Using transfected fibroblasts expressing both wild-type I-E(k) and green fluorescent protein-tagged I-E(k) with covalently attached antigenic peptide, we have monitored movement of specific MHC:peptide complexes during CD4(+) T cell-APC interactions by live-cell video microscopy. Ag recognition occurs within 30 s of T cell-APC contact, as shown by a sharp increase in cytoplasmic calcium ion concentration. Within 1 min, small MHC:peptide clusters form in the contact zone that coalesce into an immunological synapse over 3-20 min. When T cells conjugated to APC move across the APC surface, they appear to drag the synapse with them. This system was used to examine the role of costimulation in the formation of the immunological synapse. Blocking CD80/CD28 or ICAM-1/LFA-1 interactions alters synapse morphology and reduces the area and density of accumulated complexes. These reductions correlate with reduced T cell proliferation, while CD69 and CD25 expression and TCR down-modulation remain unaffected. Thus, costimulation is essential for normal mature immunological synapse formation.

Actins↗
PubMed2005

Astrocyte-derived ATP induces vesicle shedding and IL-1 beta release from microglia.

ATP has been indicated as a primary factor in microglial response to brain injury and inflammation. By acting on different purinergic receptors 2, ATP is known to induce chemotaxis and stimulate the release of several cytokines from these cells. The activation of purinergic receptors 2 in microglia can be triggered either by ATP deriving from dying cells, at sites of brain injury or by ATP released from astrocytes, in the absence of cell damage. By the use of a biochemical approach integrated with video microscopy experiments, we investigated the functional consequences triggered in microglia by ATP released from mechanically stimulated astrocytes, in mixed glial cocultures. Astrocyte-derived ATP induced in nearby microglia the formation and the shedding of membrane vesicles. Vesicle formation was inhibited by the ATP-degrading enzyme apyrase or by P2X(7)R antagonists. Isolation of shed vesicles, followed by IL-1beta evaluation by a specific ELISA revealed the presence of the cytokine inside the vesicular organelles and its subsequent efflux into the extracellular medium. IL-1beta efflux from shed vesicles was enhanced by ATP stimulation and inhibited by pretreatment with the P2X(7) antagonist oxidized ATP, thus indicating a crucial involvement of the pore-forming P2X(7)R in the release of the cytokine. Our data identify astrocyte-derived ATP as the endogenous factor responsible for microvesicle shedding in microglia and reveal the mechanisms by which astrocyte-derived ATP triggers IL-1beta release from these cells.

Adenosine Triphosphate↗
PubMed1999

Mechanisms of endothelin-induced venoconstriction in isolated guinea pig mesentery.

In the present study, endothelin (ET) agonists and receptor selective antagonists were used to characterize ET receptors mediating constriction in guinea pig mesenteric veins (250-300 micrometers diameter) in vitro. The contribution of ET-evoked vasodilator release to venous tone was also explored. Computer-assisted video microscopy was used to monitor vein diameter. Endothelin-1 (ET-1), endothelin-3 (ET-3), and sarafotoxin 6c (S6c) produced sustained concentration-dependent contractions with a rank order agonist potency of ET-1 = S6c > ET-3. Indomethacin (1 microM) and Nomega-nitro-L-arginine (100 microM) enhanced ET-1 and S6c responses. The ETA selective antagonists BQ-610 (100 nM) and PD156707 (10 nM) shifted ET-1 concentration-response curves rightward and decreased maximal ET-1 responses, without changing S6c responses. The ETB selective antagonist BQ-788 (100 nM) shifted S6c responses rightward but produced no change in ET-1 responses. Combined application of BQ-788 and BQ-610 or BQ-788 and PD 156707 produced a rightward shift in ET-1 responses that was greater than shifts produced by BQ-610 or PD 156707 alone. In conclusion, smooth muscle in guinea pig mesenteric veins expresses ETA and ETB receptors coupled to contractile mechanisms. Activation of endothelial ETB receptors results in release of vasodilators, primarily nitric oxide.

Animals↗
PubMed1999

Influence of fibrillar collagen structure on the mechanisms of platelet thrombus formation under flow.

We have used real-time video microscopy to study the mechanisms of platelet adhesion to type I collagen fibrils of distinct structure exposed to flowing blood. Electron microscopy analysis by surface replication demonstrated morphological differences between acid-insoluble fibrils, displaying a regularly repeating striated pattern (banded collagen), and acid-soluble fibrils generated by pepsin treatment of insoluble collagen, smaller in size with a helical configuration (nonbanded collagen). These structural differences proved to be related to the role of platelet integrin alpha(2)beta(1) in stabilizing adhesion to collagen under a variety of flow conditions. Blocking alpha(2)beta(1) function with a monoclonal antibody had no effect on platelet adhesion to insoluble type I collagen coated at high density on a glass surface, whereas there was an absolute dependence of alpha(2)beta(1) function for the initial permanent arrest of platelets and subsequent thrombus formation on pepsin-solubilized type I collagen under the same conditions. In contrast, reconstituted, banded fibrils prepared from pepsin-solubilized type I collagen supported platelet adhesion and thrombus development even when platelet alpha(2)beta(1) function was blocked, a process that was greatly accelerated by pre-exposure of this substrate to autologous plasma under flow. These results implicate a collagen receptor(s) on platelets other than alpha(2)beta(1) that can selectively engage domains in banded, but not nonbanded type I collagen when alpha(2)beta(1) function is blocked. In addition, collagen structure may regulate the extent and affinity of the binding under flow of plasma components such as von Willebrand factor and/or other alpha(IIb)beta(3) ligands.

Adult↗
PubMed2000

The influence of age on nailfold capillary dimensions in childhood.

OBJECTIVE: To identify the influence of age on nailfold capillary dimensions in children between 6 and 15 years. METHODS: Capillary density (the number of capillaries in a 3 mm length of the distal row) and capillary dimensions were measured in 110 healthy children using the technique of nailfold video microscopy. The age groups studied were as follows: 6 to 7 years (17 children), 8 to 9 years (15 children), 10 to 11 years (34 children), 12 to 13 years (24 children), and 14 to 15 years (20 children). RESULTS: There was a significant trend for the arterial and venous diameters to rise with age. However, this trend was not present for apical or loop diameters, nor for the capillary density. Results did not differ between males and females. CONCLUSION: In studies incorporating capillary dimensions in children, results should ideally be age adjusted, and dimensions should not be categorized as normal or abnormal without taking the child's age into account.

Adolescent↗
PubMed2000

The dynamics of neuronal death: a time-lapse study in the retina.

Using time-lapse video microscopy, we have followed the process of neuronal death in an intact region of the mammalian nervous system. We show here the fast dynamics of nuclear fragmentation, which is over in <1 hr for neurons undergoing apoptosis in the living rat retina. Nuclear fragmentation is accompanied by a progressive raise of intracellular calcium and followed by erratic movement of the apoptotic cells, documenting their loss of adhesion.

Amidines↗
PubMedDate not supplied

The effect of irradiation on mitotic and apoptotic frequency in head and neck cancer cell lines, the correlation to p53 mutations and clonogenic survival.

It was our intention to enlighten the controversy between the mainstream of studies and our previous results showing a correlation between the intrinsic radiosensitivity and the p53 allele status of 20 human head and neck squamous cell carcinoma (HNSCC) cell lines. In our study cell lines carrying a wild-type (WT) p53 allele were significantly more radioresistant than cell lines which lacked a WT gene. We observed nine HNSCC cell lines with known intrinsic radiosensitivity and p53 allele status with time-lapse video microscopy after irradiation with 2 and 3 Gy. We studied the mitotic and apoptotic frequencies and scored the apoptoses as to whether they occurred morphologically in mitosis or in interphase. Irradiation with 2 or 3 Gy did not induce apoptosis in the cell lines studied. As expected the mitotic frequency was reduced by the irradiation. This was statistically significant in the cell lines which were radiosensitive when measured with a clonogenic assay. p53 allele status did not have an independent effect on the cell lines, except that the irradiation favoured the apoptosis in mitosis in the cell lines with WT p53 and the apoptosis in the interphase in the cell lines with a mutated or non-functional p53 gene. We conclude that although the apoptosis is not induced by irradiation with 2 Gy or 3 Gy, the p53 suppressor gene seems to influence the process of apoptosis after irradiation in the cell lines studied.

Acridine Orange↗
PubMed2000

Microcirculatory dysfunction in chronic venous insufficiency (CVI).

The elevated ambulatory pressure in the peripheral venous system of chronic venous insufficiency (CVI) patients manifests itself not only in the form of disturbed macrocirculation but also and particularly in microangiopathic changes. For this reason, it is closely correlated with trophic disorders of the skin and can ultimately lead to ulceration. Using microcirculation research techniques, we are able to provide clear evidence of a typical microangiopathy in chronic venous insufficiency. Fifty CVI in Widmer stages I, II, and III were examined with fluorescence video microscopy, intravital video capillaroscopy, transcutaneous oxygen partial pressure measurement, TcpO2 and laser Doppler flowmetry. The effects of compression therapy with individually fitted compression stockings on capillary morphology were studied over a period of 4 weeks in 20 CVI patients in Widmer stages I and II. The capillary pressure was measured during simulated muscle contraction using a servo-null micropressure system. We periodically drew blood from the dorsalis pedis vein and a brachial vein of 11 healthy test persons and 8 patients with stage III CVI during experimental venous hypertension in order to evaluate the expression pattern of leukocyte adhesion molecules involved in inflammation: LFA-1 (CD11a), Mac-1 (CD11b), p150,95 (CD11c), CD18, VLA-4 (CD49d), and L-selectin (CD62L). In the same patients, we used immunohistochemical methods to examine clinically unaffected skin and the skin near an ulcer, focusing on the adhesion molecules ICAM-1, VCAM-1, and E-selectin. The microangiopathic changes observed with worsening clinical symptoms include a decrease in the number of capillaries, glomerulus-like changes in capillary morphology, a drop in the oxygen content (tcpO2) of the skin, increased permeability of the capillaries to low-molecular-weight substances, increased laser Doppler flux reflecting elevated subcutaneous flow, and diminished vascular reserve. These microangiopathic changes worsen in linear proportion to the clinical severity of chronic venous insufficiency. In patients with venous ulcerations, the baseline expression of LFA-1 and VLA-4 on lymphocytes, Mac-1 expression on the myeloid cell line, and L-selectin expression on all three cell lines was not significantly different form that in healthy controls. During orthostatic stress, there was a significant reduction in the expression of L-selectin in blood cells collected at foot level in the controls (p=0.002), but not in the patients. Clinical improvement by compression therapy was accompanied by an increase in the number of nutritive capillaries, while the diameter of the capillaries and the dermal papillae was reduced. When ulcers healed in a short period (<6 weeks), we observed a concomitant increase in the number of capillaries (p<0.05). Microangiopathy appears before tropic disorders of the skin develop. Even trophically normal skin areas may have dilated nutritive capillaries, an early sign of disturbed skin perfusion. These changes represent a plausible explanation for the development and to recurrency tendency of venous ulcers. The reduced expression of lymphocytic L-selectin in healthy controls during the orthostatic stress test may be an indication that the cells are activated by venous stasis. Clinically effective therapeutic measures improve the impaired microcirculation of the skin in the ankle area.

Bandages↗
PubMed1992

Time-lapse analysis of gravitropism in Ceratodon protonemata.

The tip cell of the protonema of the moss Ceratodon purpureus (Hedw.) Brid. is negatively gravitropic when grown in the dark on supplemented agar. Gravitropism, plastid distribution, and plastid movement were studied in living cells using time-lapse video microscopy and infrared light. A wrong-way (downward) curvature preceded upward curvature and was detected as early as 2 minutes after reorientation. Upward curvature began 30-45 minutes after reorientation to the horizontal. Cell division temporarily reversed upward curvature, but did not inhibit wrong-way curvature. Since significant amyloplast sedimentation always occurred before the start of upward curvature, it is possible that these amyloplasts function as statoliths for upward curvature. However, no significant amyloplast sedimentation occurred before wrong-way curvature. Thus, this early phase of gravitropism cannot require plastid sedimentation for gravity sensing. Most plastids moved within and between zones, and plastid zonation was highly dynamic. Plastids moved toward the apex and toward the base of the cell at rates much slower than cytoplasmic streaming. Despite the dynamic nature of plastid movement and zonation, during upward curvature the distance between sedimented plastids and the apex stayed constant. Time-lapse analysis has revealed intriguing events not readily seen previously using destructive sampling.

Cell Cycle↗
PubMed2001

Volume-activated chloride currents contribute to the resting conductance and invasive migration of human glioma cells.

We used an in vitro model for glioma cell invasion (transwell migration assay) and patch-clamp techniques to investigate the role of volume-activated Cl(-) currents (I(Cl,Vol)) in glioma cell invasion. Hypotonic solutions ( approximately 230 mOsm) activated outwardly rectifying currents that reversed near the equilibrium potential for Cl(-) ions (E(Cl)). These currents (I(Cl,Vol)) were sensitive to several known Cl(-) channel inhibitors, including DIDS, tamoxifen, and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB). The IC(50) for NPPB inhibition of I(Cl,Vol) was 21 microm. Under isotonic conditions, NPPB (165 microm) blocked inward currents (at -40 mV) and increased input resistance in both standard whole-cell recordings and amphotericin perforated-patch recordings. Reducing [Cl(-)](o) under isotonic conditions positively shifted the reversal potential of whole-cell currents. These findings suggest a significant resting Cl(-) conductance in glioma cells. Under isotonic and hypotonic conditions, Cl(-) channels displayed voltage- and time-dependent inactivation and had an I(-) > Cl(-) permeability. To assess the potential role of these channels in cell migration, we studied the chemotactic migration of glioma cells toward laminin or vitronectin in a Boyden chamber containing transwell filters with 8 microm pores. Inhibition of I(Cl,Vol) with NPPB reduced chemotactic migration in a dose-dependent fashion with an IC(50) of 27 microm. Time-lapse video microscopy during patch-clamp recordings revealed visible changes in cell shape and/or movement that accompanied spontaneous activation of I(Cl,Vol), suggesting that I(Cl,Vol) is activated during cell movement, consistent with the effects of NPPB in migration assays. We propose that I(Cl,Vol) contributes to cell shape and volume changes required for glioma cell migration through brain tissue.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗