Origin and fate of rat plasma cholesterol in vivo. Modelling of cholesterol movements between plasma and organs

A cholesterol system model was developed in the rat following a single injection of red cells containing free (unesterified) [3H]cholesterol. The radioactivity of free and esterified cholesterol in the different parts of the system was measured during the 48 h following tracer introduction. The model consisted of seven compartments (red cell free cholesterol, plasma and liver free and esterified cholesterol, total cholesterol in the rapidly and slowly exchangeable carcass pools). The model was validated by the similarity between simulated and experimental values during the 48 h following tracer introduction. Both the fractional rate of cholesterol esterification in the plasma (0.44 h-1) and liver (0.01 h-1) and the fractional exchange rate of free cholesterol from the plasma towards the various organs (particularly 3 h-1 towards the liver for a total of 7 h-1) can be estimated with this model. The results show that cholesterol movements between the plasma and the different organs take place mainly through intense free cholesterol exchanges, resulting in a low net flux.     

A mutation in glyceraldehyde 3-phosphate dehydrogenase alters endocytosis in CHO cells

The CHO cell mutant FD 1.3.25 exhibits both increased accumulation and altered distribution of endocytosed fluid phase tracers. Neither the rate of tracer internalization nor the kinetics of recycling from early endosomes was affected, but exocytosis from late endocytic compartments appeared to be decreased in the mutant. Endocytosed tracer moved more rapidly to the cell poles in FD1.3.25 than in wild type cells. An abundant 36-kD polypeptide was found associated with taxol-polymerized microtubules in preparations from wild type and mutant; in the former but not the latter this polypeptide could be dissociated by incubation of the microtubules in ATP or high salt. The 36-kD polypeptide co- electrophoresed in two dimensions with the monomer of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Analysis of cDNA clones showed that the mutant is heterozygous for this enzyme, with approximately 25% of the GAPDH RNA containing a single nucleotide change resulting in substitution of Ser for Pro234, a residue that is conserved throughout evolution. Stable transfectants of wild type cells expressing the mutant monomer at approximately 15% of the total enzyme exhibited the various changes in endocytosis observed in FD1.3.25.     

Monosynaptic restriction of transsynaptic tracing from single, genetically targeted neurons

There has never been a wholesale way of identifying neurons that are monosynaptically connected either to some other cell group or, especially, to a single cell. The best available tools, transsynaptic tracers, are unable to distinguish weak direct connections from strong indirect ones. Furthermore, no tracer has proven potent enough to label any connected neurons whatsoever when starting from a single cell. Here we present a transsynaptic tracer that crosses only one synaptic step, unambiguously identifying cells directly presynaptic to the starting population. Based on rabies virus, it is genetically targetable, allows high-level expression of any gene of interest in the synaptically coupled neurons, and robustly labels connections made to single cells. This technology should enable a far more detailed understanding of neural connectivity than has previously been possible.     

Functionalized thymidine derivatives as carriers for the γ-emitter technetium tricarbonyl moiety

Nuclear medicine imaging of cell proliferation has gained broad interest in clinical oncology. A good tracer to image cell proliferation, possibly associated to tumour progression, should rapidly and specifically be incorporated into growing DNA. Following this idea, we have singled out thymidine as a potential biological carrier for delivery of the organometallic fragment [M(CO)₃]⁺ (M = “cold” Re, γ-emitter ^99mTc) to DNA. In the present paper we report the two-step synthesis of thymidine N-3 derivatives and their coordination complexes. Thymidine could be alkylated without protecting the sugar alcoholic functions. Methyl-2,2′-diaminodiethylamine thymidine derivative was quickly reacted with rhenium and technetium carbonyls in almost quantitative yield. The resulting ^99mTc complex represents a convenient radiopharmaceutical for single photon emission tomography (SPECT).     

Effects of red cell permeability on transcapillary tracer transport: The case of negligible back diffusion

Estimates of capillary tracer permeability calculated using multiple indicator data depend upon the particular model adopted to describe blood tissue exchange. The model proposed by Crone (1963) is appropriate when some of the injected tracer diffuses into the tissue but does not return appreciably to the bloodstream before data collection is terminated. Under these conditions extraction of tracer by the tissue depends on a single dimensionless parameter, α_cap, defined as the ratio of capillary permeability surface area to water flow. The effects of finite red cell tracer permeability on the Crone model estimate of capillary permeability are examined in the present study. The results indicate that even when back diffusion from the extravascular space is negligible, significant errors in the Crone model estimate can be expected when capillary permeability is relatively high and the ratio of red cell to capillary permeability is less than unity. However, when an aliquot of blood is equilibrated with tracer prior to injection and the dimensionless capillary permeability is relatively low (i.e. α_cap ≦ 0.25 for a haematocrit≦50%), the whole blood Crone model estimate of α_cap will be within 10% of the actual value, irrespective of red cell permeability. Red cell-plasma exchange for commonly used tracer-organ combinations should not significantly affect Crone estimates of capillary permeability under normal physiological conditions, but may be important in low flow situations. Supported by Grant No. HL 19153 (SCOR in Pulmonary Vascular Diseases). This work was done during Dr. Roselli’s tenure as an NHLBI Training Grant Fellow (NHLBI Training Grant No. 07123).     

Light Sheet Microscopy for Single Molecule Tracking in Living Tissue

Single molecule observation in cells and tissue allows the analysis of physiological processes with molecular detail, but it still represents a major methodological challenge. Here we introduce a microscopic technique that combines light sheet optical sectioning microscopy and ultra sensitive high-speed imaging. By this approach it is possible to observe single fluorescent biomolecules in solution, living cells and even tissue with an unprecedented speed and signal-to-noise ratio deep within the sample. Thereby we could directly observe and track small and large tracer molecules in aqueous solution. Furthermore, we demonstrated the feasibility to visualize the dynamics of single tracer molecules and native messenger ribonucleoprotein particles (mRNPs) in salivary gland cell nuclei of Chironomus tentans larvae up to 200 µm within the specimen with an excellent signal quality. Thus single molecule light sheet based fluorescence microscopy allows analyzing molecular diffusion and interactions in complex biological systems.     

The horseradish peroxidase technique for cell lineage studies.

The identification of cell lineage for a given cell type of a particular tissue is an important step in understanding how this process contributes to histogenesis. The importance in understanding cell lineage has relevance for both theoretical and practical reasons. For example, delineating and identifying histogenic principals is required to advance stem cell research and tissue regeneration. To document cell lineage in a given experimental preparation, a number of techniques have been developed. This typically requires the injection of a tracer compound into a founder cell. As this cell produces progeny, the tracer is passed on to the daughter cells. By detecting the tracer in the progeny or daughter cells the investigator can determine which cells originated from the cell that was originally injected with the tracer. By using such an approach it is possible to trace the developmental path from the first cells of the embryo to the specialized cells making the tissue of the adult. A number of tracer compounds have been used with good results in lineage tracing. One of these tracer compounds is horseradish peroxidase (HRP). Several variations of the technique are available depending on what species are studied or what histological requirements are made by the study. A basic technique that can be adapted to individual needs is presented. Included in this protocol on lineage tracing are the procedures for injection, fixation, and the microscope evaluation of labelled cells in the Helobdella triseralis embryo. A brief discussion of the technique will note its advantages and disadvantages. Examples of labelled cell preparations are given to illustrate the technique.     

Axonal and dendritic transport in Purkinje cells of cerebellar slice cultures studied by microinjection of horseradish peroxidase

Axonal and dendritic transport in single Purkinje neurons of cerebellar slice cultures was quantified as single transport distances. Examination of the cells within a vital tissue was regarded as being an approach to the in situ condition. The Purkinje cells were organotypically integrated in the in vitro tissues and extended long axonal projections connecting synapses to the target neurons. The tracer horseradish peroxidase (HRP) was applied via microinjection to the somata of the Purkinje cells and the injected neurons were incubated thereafter for defined time-intervals. The tracer was transported anterogradely into the neuron processes. The measurements on both the axonal and the dendritic transport of microinjected HRP revealed continuous transportation with increasing times of postincubation. This transport was reduced by the use of microtubule-depolymerizing drugs. The axonal transport of the tracer was either retarded in colchicine-treated cells or continuously reduced for up to 50% in vinblastine-treated neurons. Thus, a correlation of axonal transport to the microtubules was demonstrated. The dendrites were filled with the tracer after 60 min of postincubation. Dendritic transport was reduced by the use of vinblastine, and not significantly by colchicine. The results strongly support the dependence of neuronal transport on microtubules as a component of the cytoskeleton. Received: 26 April 1998 / Accepted: 18 August 1998     

Conditional Genetic Transsynaptic Tracing in the Embryonic Mouse Brain

Anatomical path tracing is of pivotal importance to decipher the relationship between brain and behavior. Unraveling the formation of neural circuits during embryonic maturation of the brain however is technically challenging because most transsynaptic tracing methods developed to date depend on stereotaxic tracer injection. To overcome this problem, we developed a binary genetic strategy for conditional genetic transsynaptic tracing in the mouse brain. Towards this end we generated two complementary knock-in mouse strains to selectively express the bidirectional transsynaptic tracer barley lectin (BL) and the retrograde transsynaptic tracer Tetanus Toxin fragment C from the ROSA26 locus after Cre-mediated recombination. Cell-specific tracer production in these mice is genetically encoded and does not depend on mechanical tracer injection. Therefore our experimental approach is suitable to study neural circuit formation in the embryonic murine brain. Furthermore, because tracer transfer across synapses depends on synaptic activity, these mouse strains can be used to analyze the communication between genetically defined neuronal populations during brain development at a single cell resolution. Here we provide a detailed protocol for transsynaptic tracing in mouse embryos using the novel recombinant ROSA26 alleles. We have utilized this experimental technique in order to delineate the neural circuitry underlying maturation of the reproductive axis in the developing female mouse brain.     

A clonal analysis of spinal cord development in the zebrafish

 We describe the results of a clonal analysis of spinal cord development in the zebrafish. The data were obtained from embryos in which fluorescent lineage tracer was injected into single cells in the neural plate at the two-somite stage. Injected animals were allowed to survive until either 4 days or 2 weeks postfertilization. In other experiments, bromodeoxy uridine (BrdU) was injected intraperitoneally at 30 h postfertilization (hpf) after lineage tracer injection in the neural plate at the two-somite stage, and the embryos fixed at 38 hpf. We restricted our experiments to the thoracic region of the spinal cord. Our experiments were aimed at answering questions regarding the proliferative abilities of neuroepithelial cells during embryonic development (as deduced from the size of the clones), the modes of cell division (as deduced from the uptake of BrdU into clone cells), positional differences in the proliferation of cells within the neural plate itself, the cellular composition of the clones, and cell dispersion (deduced from the regional distribution of clone cells). Received: 30 December 1994 / Accepted: 9 March 1997     

Biodegradation processes in a laboratory-scale groundwater contaminant plume assessed by fluorescence imaging and microbial analysis

Flow reactors containing quartz sand colonized with biofilm were set up as physical model aquifers to allow degrading plumes of acetate or phenol to be formed from a point source. A noninvasive fluorescent tracer technique was combined with chemical and biological sampling in order to quantify transport and biodegradation processes. Chemical analysis of samples showed a substantial decrease in carbon concentration between the injection and outflow resulting primarily from dilution but also from biodegradation. Two-dimensional imaging of the aqueous oxygen [O2(aq)] concentration field quantified the depletion of O2(aq) within the contaminant plume and provided evidence for microbial respiration associated with biodegradation of the carbon source. Combined microbiological, chemical, and O2(aq) imaging data indicated that biodegradation was greatest at the plume fringe. DNA profiles of bacterial communities were assessed by temperature gradient gel electrophoresis, which revealed that diversity was limited and that community changes observed depended on the carbon source used. Spatial variation in activity within the plume could be quantitatively accounted for by the changes observed in active cell numbers rather than differences in community structure, the total biomass present, or the increased enzyme activity of individual cells. Numerical simulations and comparisons with the experimental data were used to test conceptual models of plume processes. Results demonstrated that plume behavior was best described by growth and decay of active biomass as a single functional group of organisms represented by active cell counts.     

Cell-free plasma layer in cerebral microvessels.

Two diameters of vessel and red cell column in cerebral microvessels (> 29.8 microns in diameter) of cat were measured together with red cell velocity, using a two fluorescent tracer method. A fluorescein isothiocyanate (FITC)-labeled red cell was adopted as a flow tracer to measure the cell velocity with a dual window technique. Based on the fluorescence image, the red cell column diameter was measured. Plasma was stained with rhodamine-B isothiocyanate (RITC)-labeled dextran to measure the vessel diameter. The thickness of the cell-free plasma layer could be determined from the difference of the two diameters. The obtained thickness of the cell-free layer was not described by a simple function of vessel diameter or red cell velocity; it was dependent on the pseudo shear rate defined by the ratio of cell velocity to vessel radius. The layer thickness increased with a decrease in the pseudo shear rate.     

Synthesis and preliminary evaluation of mono-[123I]iodohypericin monocarboxylic acid as a necrosis avid imaging agent

Hypericin monocarboxylic acid was synthesized in an overall yield of 25% in four steps and radiolabelled with iodine-123 in good yield (>75%). The resulting mono-[(123)I]iodohypericin monocarboxylic acid was evaluated in normal mice and in rats with ethanol induced liver necrosis. In this model, tracer concentration in necrotic liver tissue was 14 times higher than in the viable liver tissue as quantified by autoradiography at 24h post injection. The results indicate the feasibility of visualization of necrotic tissue with the novel tracer.     

Role of apical tubules in endocytosis in nonciliated cells of the ductuli efferentes of the rat: a kinetic analysis

The apical region of nonciliated cells of the ductuli efferentes of the rat contains tubular coated pits (TCP) connected to the apical plasma membrane, apical tubules (AT) which occasionally show a partial coat, and endosomes which are often continuous with one or more apical tubules. To investigate the formation and fate of TCP and AT, a quantitative analysis was performed on the labeling indices of these structures at various time intervals (0.5-120 min) after a single injection of a tracer, cationic ferritin (CF), into the lumen of the rete testis. The labeling indices of both TCP and AT exhibited similar cyclical patterns, first reaching a peak at 25 min, then dropping to a minimum at 35 min, then rising to a second peak at 60 min. Since TCP were well labeled at 30 sec while AT were not, the tracer must rapidly enter TCP and thence AT. However, since tracer was virtually absent from the lumen by 30 min, it was not possible to reconcile the second peak of labeling index of TCP and AT by this mechanism. In another experiment, rats were injected once as before, injected again at 30 min, and then sacrificed at 30 min following the second injection. The results from this experiment showed that the labeling index of TCP and AT did not drop but was similar to that of the 60-min peak after a single injection. The interpretation is that there was recycling of tracer, which had already migrated from TCP to AT to endosomes, back to the apical plasma membrane via apical tubules. Moreover, when rats were injected once, injected again at 30 min, and sacrificed 3 min following the second injection, the labeling index for TCP and AT was significantly higher (P less than .05) than at the 30-min time interval after a single injection, indicating that recycled apical tubules were functionally capable of binding further CF. Morphological observations on images of transition between TCP and AT and the fact that AT were often found connected to endosomes suggest that TCP detach from the cell surface to give rise to AT, which in turn fuse to form endosomes. The kinetic analysis demonstrates in quantitative terms that a portion of the AT, which fuses to form endosomes, recycles back to the apical plasma membrane and contributes to the formation of new TCP.     

Optimization of 13C isotopic tracers for metabolic flux analysis in mammalian cells

Mammalian cells consume and metabolize various substrates from their surroundings for energy generation and biomass synthesis. Glucose and glutamine, in particular, are the primary carbon sources for proliferating cancer cells. While this combination of substrates generates static labeling patterns for use in (13)C metabolic flux analysis (MFA), the inability of single tracers to effectively label all pathways poses an obstacle for comprehensive flux determination within a given experiment. To address this issue we applied a genetic algorithm to optimize mixtures of (13)C-labeled glucose and glutamine for use in MFA. We identified tracer combinations that minimized confidence intervals in an experimentally determined flux network describing central carbon metabolism in tumor cells. Additional simulations were used to determine the robustness of the [1,2-(13)C(2)]glucose/[U-(13)C(5)]glutamine tracer combination with respect to perturbations in the network. Finally, we experimentally validated the improved performance of this tracer set relative to glucose tracers alone in a cancer cell line. This versatile method allows researchers to determine the optimal tracer combination to use for a specific metabolic network, and our findings applied to cancer cells significantly enhance the ability of MFA experiments to precisely quantify fluxes in higher organisms.     

Distribution of 125I-insulin in cardiocytes at steady state binding conditions at 37 degrees C

The distribution of 125I-insulin in cardiocytes was analyzed by light microscope autoradiography. Semithin sections were used to distinguish between surface-bound and internalized tracer. At 37 degrees C, when steady state binding conditions were reached, 40 to 60% of the cell-bound tracer was located in the plasma membrane region and the remainder was in the cell interior. Autoradiograms of whole cells were used to study the distribution of tracer molecules on the cell surface. Because Poisson distributions of silver grains were observed on 90% of the cells, it was concluded that the distribution of the insulin-receptor complexes was close to random. In contrast to the findings of Schlessinger et al., no aggregation of insulin-receptor complexes into patches was observed.     

Single channel gating events in tracer flux experiments I. Theory

Tracer ion flux measurements are a commonly used method for studying ion transport through membranes of cellular systems, where the rate of ion flow is determined by gating processes which control the opening and closing of transmembrane channels. Due to recent advances in the theoretical analysis of tracer flux from or into closed membrane structures (CMS), the mechanism of gating reactions can, in principle, be derived from flux data. A physically well founded analysis is presented for the dependence of the total tracer ion content of a collection of CMS on the gating processes. For functionally uncoupled gating units a mean single channel flux contribution [equation, see text] can be defined, where k is the intrinsic single channel flux coefficient, t the time over which flux is measured, and p(tau,t) is the probability that a given channel was open for a total period tau during t. This quantity reflects the mean time course of the tracer content due to flux through a single channel. Expressions for are derived that explicitly take into account a distribution in the lifetime of open channels. On the basis of the results, kinetic and thermodynamic parameters of multiphasic gating reactions can be determined from the time course of the overall tracer content in a colleciion of CMS.     

Interpretation of Tracer Washout Curves from a Population of Muscle Fibers

Consideration is given to the role of a population of muscle fibers of distributed diameters in the observed washout of a tracer, with particular reference to radioisotopic K and muscle fibers. It is concluded that if washout of tracer from a single fiber is described as first order, then washout of tracer from a population of fibers is apt to appear as first order.