Cancer microcell initiation and determination.

BACKGROUND: Cancer remains one of the leading causes of death worldwide, despite the possibilities to detect early onset of the most common cancer types. The search for the optimal therapy is complicated by the cancer diversity within tumors and the unsynchronized development of cancerous cells. Therefore, it is necessary to characterize cancer cell populations after treatment has been applied, because cancer recurrence is not rare. In our research, we concentrated on small cancer cell subpopulation (microcells) that has a potential to be cancer resistance source. Previously made experiments has shown that these cells in small numbers form in specific circumstances after anticancer treatment. METHODS: In experiments described in this research, the anticancer agents' paclitaxel and doxorubicin were used to stimulate the induction of microcells in fibroblast, cervix adenocarcinoma, and melanoma cell lines. Mainly for the formation of microcells in melanoma cells. The drug-stimulated cells were then characterized in terms of their formation efficiency, morphology, and metabolic activity. RESULTS: We observed the development of cancer microcells and green fluorescent protein (GFP) transfection efficiency after stress. In the time-lapse experiment, we observed microcell formation through a renewal process and GFP expression in the microcells. Additionally, the microcells were viable after anticancer treatment, as indicated by the nicotinamide adenine dinucleotide hydrogen phosphate (NADPH) enzyme activity assay results. Taken together, these findings indicate that cancer microcells are viable and capable of resisting the stress induced by anticancer drugs, and these cells are prone to chemical substance uptake from the environment. CONCLUSION: Microcells are not only common to a specific cancer type, but can be found in any tumor type. This study could help to understand cancer emergence and recurrence. The appearance of microcells in the studied cancer cell population could be an indicator of the individual anticancer therapy effectiveness and patient survival.

E. coli nitroreductase NfsA is a reporter gene for non-invasive PET imaging in cancer gene therapy applications.

The use of reporter genes to non-invasively image molecular processes inside cells has significant translational potential, particularly in the context of systemically administered gene therapy vectors and adoptively administered cells such as immune or stem cell based therapies. Bacterial nitroreductase enzymes possess ideal properties for reporter gene imaging applications, being of non-human origin and possessing the ability to metabolize a range of clinically relevant nitro(hetero)cyclic substrates. Methods: A library of eleven Escherichia coli nitroreductase candidates were screened for the ability to efficiently metabolize 2-nitroimidazole based positron emission tomography (PET) probes originally developed as radiotracers for hypoxic cell imaging. Several complementary methods were utilized to detect formation of cell-entrapped metabolites, including various in vitro and in vivo models to establish the capacity of the 2-nitroimidazole PET agent EF5 to quantify expression of a nitroreductase candidate. Proof-of-principle PET imaging studies were successfully conducted using 18F-HX4. Results: Recombinant enzyme kinetics, bacterial SOS reporter assays, anti-proliferative assays and flow cytometry approaches collectively identified the major oxygen-insensitive nitroreductase NfsA from E. coli (NfsA_Ec) as the most promising nitroreductase reporter gene. Cells expressing NfsA_Ec were demonstrably labelled with the imaging agent EF5 in a manner that was quantitatively superior to hypoxia, in monolayers (2D), multicellular layers (3D), and in human tumor xenograft models. EF5 retention correlated with NfsA_Ec positive cell density over a range of EF5 concentrations in 3D in vitro models and in xenografts in vivo and was predictive of in vivo anti-tumor activity of the cytotoxic prodrug PR-104. Following PET imaging with 18F-HX4, a significantly higher tumor-to-blood ratio was observed in two xenograft models for NfsA_Ec expressing tumors compared to the parental tumors thereof, providing verification of this reporter gene imaging approach. Conclusion: This study establishes that the bacterial nitroreductase NfsA_Ec can be utilized as an imaging capable reporter gene, with the ability to metabolize and trap 2-nitroimidazole PET imaging agents for non-invasive imaging of gene expression.

Effects of sinapic acid on hepatic cytochrome P450 3A2, 2C11, and intestinal P-glycoprotein on the pharmacokinetics of oral carbamazepine in rats: Potential food/herb-drug interaction.

Dietary supplements, herbal medicines, and other foods may affect the pharmacokinetics and/or pharmacodynamics of carbamazepine (CBZ), which may possibly lead to potential drug-drug/herb-drug interactions, as CBZ has a narrow therapeutic window. Sinapic acid (SA) is a bioactive phytoconstituent used as a dietary supplement for the treatment of epilepsy. This study determined the effects of SA on the pharmacokinetics of CBZ and proposed a possible interaction mechanism in twenty-four male wistar rats (180-210 g). A single CBZ dose (80 mg/kg) was administered orally to rats with or without SA pretreatment (20 mg/kg p.o. per day for 7 days, n = 6). The CBZ concentration in plasma samples was determined by using a sensitive reversed-phase high-performance liquid chromatography assay. The pharmacokinetic parameters were calculated by using non-compartmental analysis. Significance was determined through Dunnett's multiple comparison test or one-way analysis of variance as appropriate; p < 0.05 were considered significant. The change in the pharmacokinetic parameters (Cmax, Tmax, AUC0-t, AUC0-∞, T½, and kel) of CBZ was evaluated after the administration of CBZ alone or after CBZ co-administration with SA pretreatment. The plasma concentration of CBZ was higher after SA pretreatment than that without pretreatment. The pharmacokinetics of orally administered CBZ were found to be significantly altered (p < 0.05) in rats pretreated with SA compared to those in rats administered CBZ alone. The increases in the Cmax, AUC0-t, T1/2, and MRT of CBZ were 29.79%, 57.18%, 77.18%, and 58.31%, respectively, whereas the kel and apparent oral CL/F were significantly reduced (p < 0.05) in rats pretreated with SA compared to those in rats not pretreated with SA (43.87% and 42.50%, respectively). However, no significant change was observed in the Tmax of CBZ in rats pretreated with SA compared to that in rats that did not receive pretreatment. The enhancement in Cmax, AUC0-t, T1/2, and MRT and the reduction in Kel and CL/F values resulted from the significant inhibition of CYP3 A2, the CYP2C11-mediated metabolism of CBZ in the liver, and the inhibition of intestinal P-glycoprotein/MDR1, which enhanced the rate of CBZ absorption. Further studies are required to determine the clinical relevance of these observations.

EBF recommendation on practical management of critical reagents for PK ligand-binding assays.

Critical reagents play a crucial role in ligand-binding assays; the robustness and reliability of an assay is defined by the quality and long-term availability of these reagents. However, neither regulatory guidelines nor relevant scientific papers provide clear directions for set-up, life cycle management and, more importantly, the acceptance criteria required for the testing of the critical reagents for pharmacokinetic, biomarker and immunogenicity assays. The ambiguity from current guidelines can be a challenge for the bioanalytical community. Members of the European Bioanalysis Forum community undertook a more pragmatic approach on how to assess the impact of critical reagents. In this paper, a review and corresponding gap analysis of the current guidelines and relevant papers will be provided as well as decision trees proposed for lot-to-lot changes of critical reagents for pharmacokinetic assays.

Predictive Value of Indocyanine Green Plasma Disappearance Rate on Liver Function and Complications After Liver Transplantation.

BACKGROUND The aim of this study was to investigate the correlation between indocyanine green plasma disappearance rate (ICG-PDR) and allograft function as well as postoperative complications after liver transplantation. MATERIAL AND METHODS In this prospective study, 115 cases of adult liver transplantation performed from 1 June 2016 to 1 December 2016 were enrolled. These 115 patients were divided into a group of PDR <18%/min (50 cases) and a group of PDR ≥18%/min (65 cases). The rates of liver recovery, postoperative complications, and survival were compared between these 2 groups. RESULTS Among the total of 115 patients, 111 patients recovered well and were discharged, whereas 4 patients died during the first month after the operation. Between the 2 groups, significant differences were observed in terms of the model for end-stage liver disease (MELD) score, intraoperative bleeding volume, and the level of hemoglobin (Hb), pre-albumin (PA) and total bilirubin (TB) the first week after the operation. Overall, the incidence of hepatic arterial complications and pneumonia was much higher in the PDR<18%/min group (P<0.05). CONCLUSIONS The early postoperative value of ICG-PDR was closely related to graft function and could act as a good predictor for the incidence of postoperative arterial complications.

Intracellular trafficking and exocytosis of a multi-component siRNA nanocomplex.

Despite the importance of siRNA delivery systems, understanding of their intracellular fate remains elusive. We recently developed a multi-component siRNA nanocomplex to deliver siRNA to hepatic stellate cells (HSCs). The objective of this study is to study post-internalization trafficking of this siRNA nanocomplex and its multiple components like siRNA, protamine, and streptavidin, in HSCs. After internalization, the nanocomplex entrapped in early endosomes undergoes three possible routes including endosomal escape, exocytosis, and entrapment in lysosomes. Significant amount of siRNA dissociates from the nanocomplex to exert silencing activity. After escaping from endosomes, protamine dissociates from the nanocomplex and stays inside the cytoplasm. Golgi complex plays an important role in exocytosis of the nanocomplex. We also demonstrate that exocytosis is one of the major reasons accounting for the transient silencing activity of nonviral siRNA delivery. Incorporation of exocytosis inhibitors in nonviral siRNA delivery systems may extend the silencing activity of siRNA.

High Throughput Danio Rerio Energy Expenditure Assay.

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

Hepatic insulin sensitivity in healthy and prediabetic subjects: from a dual- to a single-tracer oral minimal model.

Recently, a model was proposed to assess hepatic insulin sensitivity during a meal, i.e., the ability of insulin to suppress glucose production (EGP), SI (P). The model was developed on EGP data obtained from a triple-tracer meal and the tracer-to-tracee clamp technique and validated against the euglycemic hyperinsulinemic clamp. The aim of this study was to assess whether SI (P) can be obtained from plasma concentrations measured after a single-tracer meal by incorporating the above EGP model into the oral glucose minimal model by describing both glucose production and disposal (OMM(PD)). Triple-tracer meal data of two databases (20 healthy and 60 healthy and prediabetic subjects) were used. Virtually model-independent EGP estimates were calculated. OMM(PD) was identified on exogenous and endogenous glucose concentrations, providing indices of SI (P), disposal insulin sensitivity (SI (D)), and EGP. The model fitted the data well, and SI (P) and SI (D) were estimated with precision in both databases (SI (P) = 5.48 ± 0.54 10(-4) dl·kg(-1)·min(-1) per µU/ml and SI (D) = 9.93 ± 2.18 10(-4) dl·kg(-1)·min(-1) per µU/ml in healthy; SI (P) = 5.41 ± 3.55 10(-4) dl·kg(-1)·min(-1) per µU/ml and SI (D) = 5.34 ± 6.17 10(-4) dl·kg(-1)·min(-1) per µU/ml, in healthy and prediabetic subjects). Estimated SI (P) and that derived from the triple-tracer EGP model were very similar on average. Moreover, the time course of EGP normalized to basal EGP (EGPb), and EGP/EGPb agreed with the results obtained using the triple-tracer method. In this study, we have demonstrated that SI (P), SI (D), and EGP/EGPb time course can be estimated reliably from a single-tracer meal protocol in both healthy and prediabetic subjects.

Tracer-based estimates of protein flux in cases of incomplete product renewal: evidence and implications of heterogeneity in collagen turnover.

The synthesis of various molecules can be estimated by measuring the incorporation of a labeled precursor into a product of interest. Unfortunately, a central problem in many studies has been an inability to estimate the intracellular dilution of the precursor and therein correctly calculate the synthesis of the product; it is generally assumed that measuring the true product labeling is straightforward. We initiated a study to examine liver collagen synthesis and identified an apparent problem with assumptions regarding measurements of the product labeling. Since it is well known that collagen production is relatively slow, we relied on the use of [(2)H]H2O labeling (analogous to a primed infusion) and sampled animals over the course of 16 days. Although the water labeling (the precursor) remained stable and we observed the incorporation of labeled amino acids into collagen, the asymptotic protein labeling was considerably lower than what would be expected based on the precursor labeling. Although this observation is not necessarily surprising (i.e., one might expect that a substantial fraction of the collagen pool would appear "inert" or turn over at a very slow rate), its implications are of interest in certain areas. Herein, we discuss a novel situation in which tracers are used to quantify rates of flux under conditions where a product may not undergo complete replacement. We demonstrate how heterogeneity in the product pool can lead one to the wrong conclusions regarding estimates of flux, and we outline an approach that may help to minimize errors surrounding data interpretation.

Role of mitochondrial calcium uniporter in regulating mitochondrial fission in the cerebral cortexes of living rats.

The mitochondrial calcium uniporter (MCU) transports Ca2+ from the cytoplasm to the mitochondrial matrix and thus maintains Ca2+ homeostasis. Previous studies have reported that inhibition of MCU by ruthenium red (RR) protects the brain from ischemia/reperfusion (I/R) injury and that mitochondrial fission plays an important role in I/R injury. However, it is still not known whether MCU affects mitochondrial fission. In the present study, treatment with RR was found to decrease the concentration of free calcium in the mitochondria, calcineurin enzyme activity and dynamin-related protein 1 expression, and treatment with spermine was found to have the opposite effect in organisms subjected to occlusion of the middle cerebral artery lasting 2 h followed by 24 h reperfusion. These results indicate that MCU may be related to mitochondrial fission via modulating mitochondrial Ca2+ uptake and this relationship between MCU and mitochondrial fission may protect the brain from I/R injury.

Full recovery from a potentially lethal dose of mercuric chloride.

INTRODUCTION: Mercuric chloride poisoning is rare yet potentially life-threatening. We report a case of poisoning with a potentially significant amount of mercuric chloride which responded to aggressive management. CASE REPORT: A 19-year-old female presented to the Emergency Department with nausea, abdominal discomfort, vomiting of blood-stained fluid, and diarrhea following suicidal ingestion of 2-4 g of mercuric chloride powder. An abdominal radiograph showed radio-opaque material within the gastric antrum and the patient's initial blood mercury concentration was 17.9 µmol/L (or 3.58 mg/L) at 3 h post-ingestion. Given the potential toxicity of inorganic mercury, the patient was admitted to the intensive care unit and chelation with dimercaprol was undertaken. Further clinical effects included mild hemodynamic instability, acidosis, hypokalemia, leukocytosis, and fever. The patient's symptoms began to improve 48 h after admission and resolved fully within a week. DISCUSSION: Mercuric chloride has an estimated human fatal dose of between 1 and 4 g. Despite a reported ingestion of a potentially lethal dose and a high blood concentration, this patient experienced mild to moderate poisoning only and she responded to early and appropriate intervention. Mercuric chloride can produce a range of toxic effects including corrosive injury, severe gastrointestinal disturbances, acute renal failure, circulatory collapse, and eventual death. Treatment includes close observation and aggressive supportive care along with chelation, preferably with 2,3-dimercapto-1-propane sulfonate or 2,3-meso-dimercaptosuccinic acid.

Safety and pharmacokinetics of sodium nitrite in patients with subarachnoid hemorrhage: a phase IIa study.

OBJECT: Intravenous sodium nitrite has been shown to prevent and reverse cerebral vasospasm in a primate model of subarachnoid hemorrhage (SAH). The present Phase IIA dose-escalation study of sodium nitrite was conducted to determine the compound's safety in humans with aneurysmal SAH and to establish its pharmacokinetics during a 14-day infusion. Methods In 18 patients (3 cohorts of 6 patients each) with SAH from a ruptured cerebral aneurysm, nitrite (3 patients) or saline (3 patients) was infused. Sodium nitrite and saline were delivered intravenously for 14 days, and a dose-escalation scheme was used for the nitrite, with a maximum dose of 64 nmol/kg/min. Sodium nitrite blood levels were frequently sampled and measured using mass spectroscopy, and blood methemoglobin levels were continuously monitored using a pulse oximeter. RESULTS: In the 14-day infusions in critically ill patients with SAH, there was no toxicity or systemic hypotension, and blood methemoglobin levels remained at 3.3% or less in all patients. Nitrite levels increased rapidly during intravenous infusion and reached steady-state levels by 12 hours after the start of infusion on Day 1. The nitrite plasma half-life was less than 1 hour across all dose levels evaluated after stopping nitrite infusions on Day 14. CONCLUSIONS: Previous preclinical investigations of sodium nitrite for the prevention and reversal of vasospasm in a primate model of SAH were effective using doses similar to the highest dose examined in the current study (64 nmol/kg/min). Results of the current study suggest that safe and potentially therapeutic levels of nitrite can be achieved and sustained in critically ill patients after SAH from a ruptured cerebral aneurysm.

Quantitative analysis of vitreous and plasma concentrations of brilliant blue g after use as a surgical adjuvant in chromovitrectomy.

PURPOSE: To measure the concentration of brilliant blue G (BBG) in vitreous and plasma after use as a surgical adjuvant for staining and peeling of the internal limiting membrane to determine potential systemic adverse effects. METHODS: This study was designed as a prospective, interventional, clinical, case series. Five eyes from five patients with macular hole or epiretinal membrane underwent BBG-assisted internal limiting membrane and epiretinal membrane removal. The vitreous samples were obtained and stored at the end of surgery in all five cases. The plasma specimens were extracted and stored at the end of the operation, after 4 hours, and after 7 days post operation. For BBG analysis of plasma and vitreous, high-performance liquid chromatography coupled with tandem mass spectrometric detection was used. RESULTS: Brilliant blue G was not detected in plasma from all five cases at the three points of measurement. The mean vitreous BBG concentration was 34.5 ± 23.7 ng/mL (range, 11.3-70.9 ng/mL). Postoperative progress was good, and adverse effects were not observed in any of the five cases. CONCLUSION: Brilliant blue G, which remained at low levels in the vitreous cavity, was not found in the systemic blood flow after the operation. Thus, any adverse effects of systemic BBG would be avoided.

The passage of a diffusible indicator through a microvascular system.

THE AIM: (1) To develop a mathematical model of the passage of a diffusible indicator through microcirculation based on a stochastic description of diffusion and flow; (2) To use Goresky transform of the dilution curves of the diffusible indicators for the estimation of the permeability of a tissue-capillary barrier. THE METHOD: We assume that there are two causes for flow to be stochastic: (a) All microvessels are divided between open and closed microvessels. There exists random exchange between the two groups. (b) The flow through open microvessels is also random. We assume that each diffusing tracer has a probability to leave the intravascular space, and has a probability to return. We also assume that all considered processes are stationary (stability of microcirculation). CONCLUSION: (a) The distribution of the time to pass microcirculation by diffusing indicator is given by a compound Poisson distribution; (b) The permeability of tissue-capillary barrier can be obtained from the means, delay, and dispersions of the dilutions of intravascular and diffusing traces.

Staining internal limiting membrane with a mixture of BBG and sodium hyaluronate.

AIMS: To evaluate the intraoperative applicability and safety of a mixture of brilliant blue G and sodium hyaluronate (visco-BBG) for staining the inner limiting membrane (ILM). METHODS: A retrospective consecutive case series. Seventy-four eyes that had undergone ILM peeling were studied. During vitrectomy, ILM peeling with visco-BBG (visco-BBG group) was performed on 40 eyes; 12 with a macular hole (MH), 26 with an epimacular membrane (ERM) and 2 with a retinal detachment due to a MH (MHRD). ILM peeling with BBG dissolved in balanced salt solution (BSS-BBG group) was performed on 34 eyes; 9 with a MH, 23 with an ERM and 2 with a MHRD. The main outcome measures were the distribution of the dye within the vitreous cavity and the retinal sensitivity in the MH patients of the two groups by microperimetry. RESULTS: The visco-BBG was injected over the retina where the ILM was intended to be peeled, and it stained the ILM in all cases. It did not disperse throughout the vitreous cavity or into the subretinal space. The BSS-BBG dispersed throughout the vitreous cavity, and its distribution was difficult to control. The two solutions did not stain the epiretinal membranes or any residual posterior hyaloid membrane. The difference in the retinal sensitivity between the two patients with MH of two groups was not significant. No complications were found in the visco-BBG group, although an accidental retinal perforation was found in one eye of the BSS-BBG group. Transmission electron microscopy confirmed that the membrane peeled was the ILM. CONCLUSIONS: Visco-BBG can be a useful method to assist macular surgery and can overcome some of the disadvantages of conventional BBG solutions dissolved in BSS.

Calcium rubies: a family of red-emitting functionalizable indicators suitable for two-photon Ca2+ imaging.

We designed Calcium Rubies, a family of functionalizable BAPTA-based red-fluorescent calcium (Ca(2+)) indicators as new tools for biological Ca(2+) imaging. The specificity of this Ca(2+)-indicator family is its side arm, attached on the ethylene glycol bridge that allows coupling the indicator to various groups while leaving open the possibility of aromatic substitutions on the BAPTA core for tuning the Ca(2+)-binding affinity. Using this possibility we now synthesize and characterize three different CaRubies with affinities between 3 and 22 µM. Their long excitation and emission wavelengths (peaks at 586/604 nm) allow their use in otherwise challenging multicolor experiments, e.g., when combining Ca(2+) uncaging or optogenetic stimulation with Ca(2+) imaging in cells expressing fluorescent proteins. We illustrate this capacity by the detection of Ca(2+) transients evoked by blue light in cultured astrocytes expressing CatCh, a light-sensitive Ca(2+)-translocating channelrhodopsin linked to yellow fluorescent protein. Using time-correlated single-photon counting, we measured fluorescence lifetimes for all CaRubies and demonstrate a 10-fold increase in the average lifetime upon Ca(2+) chelation. Since only the fluorescence quantum yield but not the absorbance of the CaRubies is Ca(2+)-dependent, calibrated two-photon fluorescence excitation measurements of absolute Ca(2+) concentrations are feasible.

Subchronic thyroid toxicity evaluation of 4,4'-methylenebis(N,N'-dimethyl)aniline in Fischer 344 rats.

Female F344 rats were exposed to 4,4'-methylenebis(N,N'-dimethyl)aniline (MDA) by dietary feed at concentrations of 0, 50, 200, 375, 500, or 750 ppm for 5 d, 2 wk, 4 wk, and 13 wk duration. Endpoints evaluated included clinical observations, body weights, thyroid weights, serum thyroid hormones, blood MDA, gross pathology, and thyroid histopathology. There were no MDA exposure-related clinical signs of toxicity. Mean body weight decreased 5% compared to control in the 750 ppm group during study wk 6 through 13. Serum TSH increased and serum T4 and T3 levels decreased with increasing feed concentrations of MDA and time of exposure. Thyroid weight increases were both concentration- and exposure time-dependent and statistically significant at ≥375 ppm. Incidence and severity of decreased colloid, follicular cell hypertrophy and follicular cell hyperplasia were also related to MDA concentration and exposure time. A no-observed-adverse-effect level (NOAEL) of 200 ppm was selected based on the statistically significant increase in incidence of follicular cell hyperplasia at concentrations ≥375 ppm.

A model for transit time distributions through organs that accounts for fractal heterogeneity.

It has been shown that density functions of organ transit time distributions of vascular markers (washout curves) are characterized by a power-law tail, reflecting the fractal nature of the vascular network. Yet, thus far, no closed-form model is available that can be fitted to such organ outflow data. Here we propose a model that accounts for the existing data. The model is a continuous mixture of inverse Gaussian densities, implying flow heterogeneity in the organ. It has been fitted to outflow data from the rabbit heart and rat liver. The power-law decay with exponent -3 observed in the heart, corresponds to an intra-organ flow distribution with a relative dispersion of about 35%.

Tuning electrical conduction along endothelial tubes of resistance arteries through Ca(2+)-activated K(+) channels.

RATIONALE: Electrical conduction through gap junction channels between endothelial cells of resistance vessels is integral to blood flow control. Small and intermediate-conductance Ca(2+)-activated K(+) channels (SK(Ca)/IK(Ca)) initiate electrical signals in endothelial cells, but it is unknown whether SK(Ca)/IK(Ca) activation alters signal transmission along the endothelium. OBJECTIVE: We tested the hypothesis that SK(Ca)/IK(Ca) activity regulates electrical conduction along the endothelium of resistance vessels. METHODS AND RESULTS: Freshly isolated endothelial cell tubes (60 µm wide; 1-3 mm long; cell length, ≈35 µm) from mouse skeletal muscle feed (superior epigastric) arteries were studied using dual intracellular microelectrodes. Current was injected (±0.1-3 nA) at site 1 while recording membrane potential (V(m)) at site 2 (separation distance=50-2000 µm). SK(Ca)/IK(Ca) activation (NS309, 1 µmol/L) reduced the change in V(m) along endothelial cell tubes by ≥50% and shortened the electrical length constant (λ) from 1380 to 850 µm (P<0.05) while intercellular dye transfer (propidium iodide) was maintained. Activating SK(Ca)/IK(Ca) with acetylcholine or SKA-31 also reduced electrical conduction. These effects of SK(Ca)/IK(Ca) activation persisted when hyperpolarization (>30 mV) was prevented with 60 mmol/L [K(+)](o). Conversely, blocking SK(Ca)/IK(Ca) (apamin+charybdotoxin) depolarized cells by ≈10 mV and enhanced electrical conduction (ie, changes in V(m)) by ≈30% (P<0.05). CONCLUSIONS: These findings illustrate a novel role for SK(Ca)/IK(Ca) activity in tuning electrical conduction along the endothelium of resistance vessels by governing signal dissipation through changes in membrane resistance. Voltage-insensitive ion channels can thereby tune intercellular electrical signaling independent from gap junction channels.

Visualizing the effect of hypoxia on fluorescence kinetics in living HeLa cells using the fluorescent ubiquitination-based cell cycle indicator (Fucci).

Fluorescent proteins are widely used for the direct visualization of events such as gene expression and subcellular localization in mammalian cells. It is well established that oxygen is required for formation of functional chromophore; however, the effect of hypoxia on fluorescence emission has rarely been studied. For this purpose, under hypoxic conditions, we investigated the kinetics of red and green fluorescence in HeLa cells from two fluorescent proteins, monomeric Kusabira Orange 2 (mKO2) and monomeric Azami Green (mAG), respectively, using the fluorescent ubiquitination-based cell cycle indicator (Fucci). In this system, cells in G1 or other phases emit red or green fluorescence, respectively. We found that hypoxia abrogated both red and green fluorescence about ~10h after the treatment, although their protein levels were almost maintained. The treatment did not significantly affect fluorescence in cells constitutively expressing the same fluorescent proteins lacking the ubiquitin ligase-binding domains. The abrogation of fluorescence resulted from a combination of ubiquitination-dependent degradation of pre-existing functional proteins during specific cell cycle phases, and the expression of newly synthesized non-fluorescent proteins containing non-oxidized chromophore during hypoxic treatment. Indeed, non-fluorescent cells after hypoxic treatment gradually developed fluorescence after reoxygenation in the presence of cycloheximide; kinetics of recovery were much faster for mAG than for mKO2. Using the Fucci system, we could clearly visualize for the first time the effect of hypoxia on the fluorescence kinetics of proteins expressed in living mammalian cells.