Dual‐wavelength excitation for fluorescence‐based quantification of zinc protoporphyrin IX and protoporphyrin IX in whole blood

Quantification of erythrocyte zinc protoporphyrin IX (ZnPP) and protoporphyrin IX (PPIX), individually or jointly, is useful for the diagnostic evaluation of iron deficiency, iron‐restricted erythropoiesis, lead exposure, and porphyrias. A method for simultaneous quantification of ZnPP and PPIX in unwashed blood samples is described, using dual‐wavelength excitation to effectively eliminate background fluorescence from other blood constituents. In blood samples from 35 subjects, the results of the dual‐wavelength excitation method and a reference high performance liquid chromatography (HPLC) assay were closely correlated both for ZnPP (r_s = 0.943, p < 0.0001; range 37–689 μmol ZnPP/mol heme, 84–1238 nmol/L) and for PPIX (r_s = 0.959, p < 0.0001; range 42–4212 μmol PPIX/mol heme, 93–5394 nmol/L). In addition, for ZnPP, the proposed method is compared with conventional single‐wavelength excitation and with commercial front‐face fluorimetry of washed erythrocytes and whole blood. We hypothesize that dual‐wavelength excitation fluorimetry will provide a new approach to the suppression of background fluorescence in blood and tissue measurements of ZnPP and PPIX. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

The effect of the administration of cobaltic protoporphyrin IX on drug metabolism, carbon tetrachloride activation and lipid peroxidation in rat liver microsomes

The effects of cobaltic protoporphyrin IX (CPP) administration on hepatic microsomal drug metabolism, carbon tetrachloride activation and lipid peroxidation have been investigated using male Wistar rats. CPP (125 mumol/kg, 72 h before sacrifice) profoundly decreased the levels of hepatic microsomal heme, particularly cytochrome P-450. Consequently, the associated mixed-function oxidase systems were equally strongly depressed. An unexpected finding was that CPP administration also greatly decreased the activity of NADPH/cytochrome c reductase, a result not generally found with the administration of the more widely used cytochrome P-450 depleting agents, cobaltous chloride. Activation of carbon tetrachloride, measured as covalent binding of [14C] CCl4, spin-trapping of CCl3 and CCl4-stimulated lipid peroxidation, was much lower in liver microsomes from CPP-treated rats. Other microsomal lipid peroxidation systems, utilising cumene hydroperoxide or NADPH/ADP-Fe2+, were also depressed in parallel with the decrease in microsomal enzyme activities.     

405 nm versus 633 nm for protoporphyrin IX excitation in fluorescence‐guided stereotactic biopsy of brain tumors

Fluorescence diagnosis may be used to improve the safety and reliability of stereotactic brain tumor biopsies using biopsy needles with integrated fiber optics. Based on 5‐aminolevulinic‐acid‐induced protoporphyrin IX (PpIX) fluorescence, vital tumor tissue can be localized in vivo during the excision procedure to reduce the number of necessary samples for a reliable diagnosis. In this study, the practical suitability of two different PpIX excitation wavelengths (405 nm, 633 nm) was investigated on optical phantoms. Violet excitation at 405 nm provides a 50‐fold higher sensitivity for the bulk tumor; this factor increases up to 100 with decreasing fluorescent volume as shown by ray tracing simulations. Red excitation at 633 nm, however, is noticeably superior with regard to blood layers obscuring the fluorescence. Experimental results on the signal attenuation through blood layers of well‐defined thicknesses could be confirmed by ray tracing simulations. Typical interstitial fiber probe measurements were mimicked on agarose‐gel phantoms. Even in direct contact, blood layers of 20–40 µm between probe and tissue must be expected, obscuring 405‐nm‐excited PpIX fluorescence almost completely, but reducing the 633‐nm‐excited signal only by 25.5%. Thus, 633 nm seems to be the wavelength of choice for PpIX‐assisted detection of high‐grade gliomas in stereotactic biopsy.

PpIX signal attenuation through clinically relevant blood layers for 405 nm (violet) and 633 nm (red) excitation.     

Accumulation of protoporphyrin IX in light-sensitive mutants of Escherichia coli.

The accumulation of protoporphyrin IX (Proto IX) in light-sensitive mutants of Escherichia coli was detected by spectrofluorimetry. Fluorescence emission and excitation spectra were recorded from extracts of bacterial cells. Proto IX clearly accumulated in cells with mutations in the visA (hemH) gene but not in the wild-type strain CA274 or in visA mutants that had been rendered light-resistant by introduction of the wild-type visA⁺ gene. Accumulation of Proto IX was also not observed in cells with a mutation in the visB gene. These results confirm the hypothesis that the sensitivity of the visA mutants to light is caused by the abnormal accumulation of Proto IX, a substrate of ferrochelatase, as the result of a genetic defect in the gene for ferrochelatase.     

Proliferation and morphology of chick embryo cells cultured in the presence of horse serum and hemoglobin

Summary We have shown previously that hemoglobin greatly stimulates chick embryo cell proliferation in Eagle's minimal essential medium supplemented with horse serum. In the present study we compared the effects of horse serum plus 10 μM hemoglobin to those of fetal bovine serum on subcultures of chick embryo cells serially propagated at high cell densities. The cells became elongated in the presence of fetal bovine serum and their rate of proliferation progressively decreased, whereas they became polygonal in the presence of horse serum plus hemoglobin and proliferated well in successive cell passages. The polygonal cell obtained in the presence of horse serum plus hemoglobin rapidly elongated if cultured at low cell densities in the presence of fetal bovine serum, but, in contrast, elongated cells did not yield polygonal cells if cultured at low densities in the presence of horse serum plus hemoglobin. It is possible that the polygonal and elongated cells are undifferentiated cells and differentiating myogenic cells, respectively.     

Preliminary investigation of the use of high frequency ultrasound imaging in the chick embryo

BACKGROUND: The purpose of this study was to investigate the feasibility of using high frequency ultrasound to study the chick embryo in a noninvasive and longitudinal fashion. METHODS: A total of 10 SPF White Leghorn chick embryos (GDs 11-17; Hamburger and Hamilton stage 37-43) were consecutively examined with a GE Logiq 400 ProSeries ultrasound unit using an 11-MHz small parts ultrasound probe. Access for ultrasound visualization of the embryos was accomplished by opening a 2-3-cm window either in the air cell over the blunt end of the egg or laterally over the embryo-dependent side of the egg. Warmed ultrasound coupling gel was used for imaging, and thermal regulation was maintained with infant heel warmers. The ultrasound images were recorded directly on digital video using a Sony TRV 900 DV camcorder. The images were directly converted to jpeg and mjeg2 files for further analysis. RESULTS: Effective visualization of each embryo was possible on each day of the study period. The embryos were best visualized through the opening made in the air cell at the blunt end of the egg. The extent of the anatomic survey of the chick embryo was dependent upon the position of the embryo in the egg relative to the opening in the air cell. Doppler color flow mapping studies were obtained of the embryonic and extraembryonic circulation. CONCLUSIONS: This preliminary investigation clearly shows the feasibility of high frequency ultrasound imaging to study chick embryo development in a longitudinal and noninvasive fashion. Further studies are presently ongoing regarding earlier embryo development, as well as to determine the stability and dynamics of the methodology.     

The influence of light and darkness on cutaneous fluorescence in mice.

The present work was carried out to investigate the role of light and darkness on the endogenous biosynthesis of porphyrins in mammalian skin (hairless BALB/c mouse) in vivo. In the skin of mice that were constantly kept in darkness (DD), increased endogenous porphyrin fluorescence was observed, which mainly originated from protoporphyrin IX (PpIX). No significant increase in the porphyrin levels was observed in mice that were kept under a normal day-night cycle (LD 12:12 h). The presence of cutaneous PpIX together with ambient light may comprise a photosensitizing mechanism by which PpIX may be a photomessenger between ambient light and internal rhythms.     

Cyclic strain induces proliferation of cultured embryonic heart cells

Summary Embryonic heart cells undergo cyclic strain as the developing heart circulates blood to the embryo. Cyclic strain may have an important regulatory role in formation of the adult structure. This study examines the feasibility of a computerized cell-stretching device for applying strain to embryonic cardiocytes to allow measurement of the cellular response. A primary coculture of myocytes and a secondary culture of nonmyocytes from stage-31 (7 d) embryonic chick hearts were grown on collagen-coated membranes that were subsequently strained at 2 Hz to 20% maximal radial strain. After 24 h, total cell number increased by 37±6% in myocyte cocultures and by 26±6% in nonmyocyte cultures over unstrained controls. Lactate dehydrogenase and apoptosis assays showed no significant differences in cell viabilities between strained and unstrained cells. After 2 h strain, bromodeoxyuridine incorporation was 38±1.2% versus 19±0.2% (P<0.01) in strained versus unstrained myocyte cocultures, and 35±2.1% versus 16±0.2% (P=0.01) in nonmyocyte cultures. MF20 antibody labeling and periodic acid-Schiff (PAS) staining estimated the number of myocytes in strained wells as 50–67% larger than in control wells. Tyrosine phosphorylation may play a role in the cellular response to strain, as Western blot analysis showed an increase in tyrosine phosphorylation of two proteins with approximate molecular weights of 63 and 150 kDa within 2 min of strain. The results of this study indicate that embryonic chick cardiocytes can be cultured in an active mechanical environment without significant detachment and damage and that increased proliferation may be a primary response to strain.     

Development of the interferon system. I. In chicken cells development in ovo continues on time in vitro

Summary When confluent monolayers of cells derived from chicken embryos of different gestational age were cultured for several days without a medium change, a condition termed in vitro aging, the cells' developed an increased capacity to express the interferon (IFN) system. The capacity to both produce IFN and to respond to its antiviral action were enhanced up to 1000- and 100-fold, respectively. Remarkably, the programmed development of the IFN system in these cells seemed to continue virtually uninterrupted after monodispersion of the cells and seeding at high cell density. Cells prepared from young embryos required more time to develop the IFN system than cells from older embryos with the yield of IFN, and sensitivity to its action, related directly to the total in ovo and in vitro age of the cells in culture. For example, essentially the same yields of IFN were obtained from cell cultures made from 5-d-old embryos “aged” for 10 d in vitro, as were obtained from 10-d-old embryos whose cells were aged in vitro for 5 d. In contrast, inducibility of 2′–5′ oligoadenylate synthetase by IFN and the induction of heat shock genes by elevated temperature are not enhanced with in vitro aging. The programmed development of the IFN system that starts in ovo seems to continue on schedule in vitro, making the development of the IFN system in chick embryo cells appear as a time-dependent process. This study was supported by the grant RO1 AI18381 from the national Institute of Allergy and Infectious Diseases, Bethesda, MD, and benefited from services of the Cell Culture Facility of the Biotechnology Center at The Univeristy of Connecticut.     

Development of coordinated movement in chicks: II. Temporal analysis of hindlimb muscle synergies at embryonic day 10 in embryos with spinal gap transections

Spinal neural circuits can recruit muscles to produce organized patterns of activity early in embryonic development. In a previous study, using multichannel electromyographic (EMG) recordings, we characterized burst parameters for these patterns in the legs of chick embryos during spontaneous motility in ovo at embryonic days (E) 9 and E10 (Bradley and Bekoff, 1990). Results of the study suggested both neural and biomechanical factors play an important role in the development of coordinated limb movements. In this study, to explore the contribution of descending neural inputs to the control of leg movements during motility, we applied similar methods to characterize motor patterns produced by the spinal cord in the absence of descending inputs. Thoracic spinal gap transections were performed at E2 and EMG patterns were recorded at E10. Several EMG features for chronic spinal embryos were similar to those for normal embryos and demonstrate that lumbar spinal circuits can be correctly assembled to control limb movements in the absence of connectivity with more rostral neural structures during early differentiation processes. However, certain aspects of the EMG patterns in chronic spinal embryos were different from patterns in normal embryos and provide support for conclusions drawn earlier by Oppenheim (1975). Specifically, our data support the view that propriospinal and/or supraspinal inputs function to regulate the timing of cyclic limb movements controlled by spinal neural circuits. Finally, we consider the possible long-term effects of chronic spinal gap transections as compared to acute spinal transections on the development of motility. © 1992 John Wiley & Sons, Inc.     

Fluorescence spectroscopy of 9-anthroyloxy fatty acids in solvents

A series of anthroyloxy fatty acid (AF) fluorescent probes, with the anthroyloxy group covalently linked at various positions along the alkyl chain, were studied in solvents exhibiting a wide range of polarity and hydrogen-bond donor (H_d) and acceptor (H_a) ability. These probes were sensitive to the solvent polarity as reflected by the Stokes' shift observed in steady state fluorescence. As determined by multi-linear regression analysis of the observed Stokes' shift and solvent parameters, such as orientation polarizability (Δf), H_d and H_a of the solvents, all the probes were sensitive to the H_d of solvents but were not affected by the H_a of solvents except the 2-AF. Due to the proximity of the polar headgroup to the fluorophore, it appears that some intramolecular hydrogen-bonding is present in 2-AF, an interaction that is sensitive to the pH of the solvent, but is less sensitive to the H_d and H_a of the solvents. Fluorescence lifetimes measured by the multi-frequency phase-modulation technique in mixtures of hexane and ethanol reflect a modified Stern-Volmer behavior suggesting the second solvent, ethanol, specifically interacts with the probe, in part through collisional quenching. Also, the lifetime data were sensitive to very low concentrations of the second solvent (0–0.1%, by vol.). The results from this study provide insight into the intrinsic differences between the different AF positions that must be taken into consideration while investigating the dynamics of lipid bilayer systems. Moreover, this study illustrates the utility and resolving power of lifetime based measurements needed for the interpretation of heterogeneous biophysical environments.     

Fluorescence correlation spectroscopy in the nanosecond time range: rotational diffusion of bovine carbonic anhydrase B

A fluorescence correlation experiment for measurement of rotational diffusion in the nanosecond time scale is described. Using this method, the rotational diffusion coefficient of bovine carbonic anhydrase B labelled with tetramethylrhodamine isothiocyanate was estimated to be D _r=(1.14±0.15)×10⁷ s^-1 at 22°C. The experiment is based on a cw argon ion laser, a microfluorimeter with local solution flow inside the sample cell, and two photon detectors. The fluorescence intensity autocorrelation function in the nanosecond time range is computed with the help of a time-to-amplitude converter and a multichannel pulse-amplitude analyser.     

Growth and metabolism of marine fish Chinook salmon embryo cells: response to lack of glucose and glutamine

A peculiar phenomenon, differing from the response of mammalian cells, occurred when Chinook salmon embryo (CHSE) cells were passaged in the medium lacking of both glucose and glutamine. To elucidate metabolic mechanism of CHSE cells, the metabolism parameters, key metabolic enzymes, and ATP levels were measured at different glucose and glutamine concentrations. In the glutamine-free culture, hexokinase activity kept constant, and lactate dehydrogenase (LDH) activity decreased. This indicated that lack of glutamine did not expedite glucose consumption but made it shift to lower lactate production and more efficient energy metabolism. The results coincided with the experimental results of unaltered specific glucose consumption rate and decreased yield coefficients of lactate to glucose. In the glucose-free culture, simultaneous increase of glutaminase activity and of specific ammonia production rate suggested an increased flux into the glutaminolysis pathway, and increases of both glutamate dehydrogenase activity and yield coefficient of ammonia to glutamine showed an increased flux into deamination pathway. However, when glucose and glutamine were both lacking, the specific consumption rates of most of amino acids increased markedly, together with decrease of LDH activity, indicating that pyruvate derived from amino acids, away from lactate production, remedied energy deficiency. When both glucose and glutamine were absent, intracellular ATP contents and the energy charge remained virtually unaltered.Revisions requested 16 December 2004; Revisions received 24 January 2005     

Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging

Although methods for light microscopy of chromatin are well established, there are no quantitative data for nucleosome concentrations in vivo. To establish such a method we used a HeLa clone expressing the core histone H2B fused to the enhanced yellow fluorescent protein (H2B-EYFP). Quantitative gel electrophoresis and fluorescence correlation spectroscopy (FCS) of isolated oligonucleosomes show that 5% of the total H2Bs carry the fluorescent tag and an increased nucleosome repeat length of 204 bp for the fluorescent cells. In vivo, the mobility and distribution of H2B-EYFP were studied with a combination of FCS and confocal imaging. With FCS, concentration and brightness of nascent molecules were measured in the cytoplasm, while in the nucleoplasm a background of mobile fluorescent histones was determined by continuous photobleaching. Combining these results allows converting confocal fluorescence images of nuclei into calibrated nucleosome density maps. Absolute nucleosome concentrations in interphase amount up to 250 microM locally, with mean values of 140(+/-28)microM, suggesting that a condensation-controlled regulation of site accessibility takes place at length scales well below 200 nm.     

9-Hydroxybenfluron induced inhibition of proliferation and metabolism in hela cells

The highest concentration of 9-hydroxybenfluron (HBF) tested, namely 4·05 μmol1^?1, induced immediate cytotoxic effects which were manifested by total inhibition of cell proliferation after only 24 h of culture. In a certain proportion of the cells cytolytic effects were observed at longer times of culture. Lower concentrations of HBF induced toxicity that was concentration- and time-dependent. The toxic effect appeared to occur in two phases. Cells, which had lost their ability to divide, did not stop their metabolism in which glutamine was the main source of energy. The results suggest that HBF primarily interferes with one of the phases of the cell cycle and only secondarily influences energy processes. Because benfluron (BF) and HBF have similar effects, it is suggested that the cytotoxicity of BF can be ascribed to the influence of its metabolite, HBF.     

Activities of Enzymes Involved in the Metabolism of Platelet-Activating Factor in Neural Cell Cultures During Proliferation and Differentiation

Platelet-Activating Factor (PAF) is a potent lipid mediator involved in physiological and pathological events in the nervous tissue where it can be synthesized by two distinct pathways. The last reaction of the de novo pathway utilizes CDPcholine and alkylacetylglycerol and is catalyzed by a specific phosphocholinetransferase (PAF-PCT) whereas the remodelling pathway ends with the reaction catalyzed by lyso-PAF acetyltransferase (lyso-PAF AcT) utilizing lyso-PAF, a product of phospholipase A₂ activity, and acetyl-CoA. The levels of PAF in the nervous tissue are also regulated by PAF acetylhydrolase that inactivates this mediator. We have studied the activities of these enzymes during cell proliferation and differentiation in two experimental models: 1) neuronal and glial primary cell cultures from chick embryo and 2) LA-N-1 neuroblastoma cells induced to differentiate by retinoic acid (RA). In undifferentiated neuronal cells from 8-days chick embryos the activity of PAF-PCT was much higher than that of lyso-PAF AcT but it decreased during the period of cellular proliferation up to the arrest of mitosis (day 1–3). During this period no significant changes of lyso-PAF AcT activity was observed. Both enzyme activities increased during the period of neuronal maturation and the formation of cellular contacts and synaptic-like junctions. The activity of PAF acetylhydrolase was unchanged during the development of the neuronal cultures. PAF-PCT activity did not change during the development of chick embryo glial cultures but lyso-PAF AcT activity increased up to the 12th day. RA treatment of LA-N-1 cell culture in proliferation decreased PAF-PCT activity and had no significant effect on lyso-PAF AcT and PAF acetylhydrolase indicating that the synthesis of PAF by the enzyme catalyzing the last step of the de novo pathway is inhibited when the LA-N-1 cells are induced to differentiate. These data suggest that: 1) in chick embryo primary cultures, both pathways are potentially able to contribute to PAF synthesis during development of neuronal cells particularly when they form synaptic-like junctions whereas, during development of glial cells, only the remodelling pathway might be particularly active on synthesizing PAF; 2) in LA-N-1 neuroblastoma cells PAF-synthesizing enzymes coexist and, when cells start to differentiate the contribution of the de novo pathway to PAF biosynthesis might be reduced.     

Non-invasive label-free monitoring the cardiac differentiation of human embryonic stem cells in-vitro by Raman spectroscopy

Background

Online label-free monitoring of in-vitro differentiation of stem cells remains a major challenge in stem cell research. In this paper we report the use of Raman micro-spectroscopy (RMS) to measure time- and spatially-resolved molecular changes in intact embryoid bodies (EBs) during in-vitro cardiogenic differentiation.

Methods

EBs formed by aggregation of human embryonic stem cells (hESCs) were cultured in defined medium to induce differentiation towards cardiac phenotype and maintained in purpose-built micro-bioreactors on the Raman microscope for 5 days (between days 5 and 9 of differentiation) and spatially-resolved spectra were recorded at 24 h intervals.

Results

The Raman spectra showed that the onset of spontaneous beating of EBs at day 7 coincided with an increase in the intensity of the Raman bands at 1340 cm^− 1, 1083 cm^− 1, 937 cm^− 1, 858 cm^− 1, 577 cm^− 1 and 482 cm^− 1. The spectral maps corresponding to these bands had a high positive correlation with the expression of the cardiac-specific α-actinin obtained by immuno-fluorescence imaging of the same EBs. The spectral markers obtained here are also in agreement with previous studies performed on individual live hESC-derived CMs.

Conclusions

The intensity profile of these Raman bands can be used for label-free in-situ monitoring of EBs to estimate the efficacy of cardiogenic differentiation.

General significance

As the acquisition of the time-course Raman spectra did not affect the viability or the differentiation potential of the hESCs, this study demonstrates the feasibility of using RMS for on-line non-invasive continuous monitoring of such processes inside bioreactor culture systems.     

Neuroethological approaches to the study of motor development in chicks: Achievements and challenges

Chicks and chick embryos provide a useful model system for the study of related to the development of motor behaviors. EMG and kinematic analyses of leg movements have been used to provide new data on the organization of embryonic motility. These data suggest that the circuitry needed to produce a basic, coordinated motor pattern is available early in development. This circuitry then appears to be retained throughout life. Evidence from analysis of EMG patterns and leg deafferentation studies suggest that the output of this basic circuit can be modulated by sensory input to produce the motor patterns of later behaviors, such as hatching and walking. If the same circuitry is present throughout life, then mechanisms for initiation and termination of particular behaviors must be available to ensure that specific behaviors are turned on and off at appropriate times. For example, hatching can be turned on by a specific sensory signal: proprioceptive signals from the bent neck. In addition to reviewing current research on the development of chick motor behaviors, methodological considerations and suggestions for future research are presented. © 1992 John Wiley & Sons, Inc.