A novel fluorescence ratiometric method confirms the low solvent viscosity of the cytoplasm.

Two homologous indocyanine dyes, Cy3.18 and Cy5.18, can be used as a ratio pair for fluorometric determination of solvent viscosity. Succinimidyl ester derivatives of these dyes can be attached to inert carrier macromolecules, such as Ficoll 70, for measurement of intracellular or intravesicular solvent viscosity. When the viscosity of the solvent was varied by various methods, the fluorescence intensity ratio (Cy3/Cy5) in a mixture of Cy3.18-Ficoll 70 (Cy3F70) and Cy5.18-Ficoll 70 (Cy5F70) in solution was found to be solely a function of solvent viscosity and was insensitive to other solvent parameters such as dielectric constant, temperature, and the ability of the solvent to form hydrogen bonds. Most important, it was insensitive to the presence of large macromolecules, such as proteins, which increase the shear viscosity but have little effect on solvent viscosity. Following microinjection into the cytoplasm of living tissue culture cells, no binding of Cy3F70 or Cy5F70 to intracellular components was detected by fluorescence recovery after photobleaching. Fluorescence intensity ratio imaging of Cy3F70 and Cy5F70 in non-motile interphase CV1 and PtK1 cells showed that the solvent viscosity of cytoplasm was not significantly different from water and showed no spatial variation.     

Differentiation of silver-enhanced mercury and gold in tissue sections of rat dorsal root ganglia

Summary Autometallography was used in conjunction with light and electron microscopy to detect traces of gold and mercury in the dorsal root ganglia of rats treated with sodium aurothiomalate and mercuric chloride. In order to differentiate between gold and mercury in tissue sections, the gold accumulations were removed by potassium cyanide, leaving mercury sulphides/selenides as the only possible catalysts for autometallographic development. With this technique, it is now possible to differentiate between all tissue metals capable of initiating the autometallographic process, i.e. gold, vesicular zinc, and sulphides and selenides of mercury and silver.     

Direct enantiospecific HPLC bioanalysis of (R,S)-atenolol on a chiral stationary phase

The simultaneous determination of the enantiomers of the β₁-selective adrenergic antagonist atenolol in human plasma and urine is described. After an alkaline preextraction atenolol is extracted from biological material at pH 12.3 using dichloromethane/propan-2-ol. The separation of the underivatized enantiomers is achieved by high-performance liquid chromatography on a chiral stationary phase (Chiralcel OD, cellulose tris-3, 5-dimethylphenylcarbamate, coated on silica gel) with fluorimetric detection. (?)-(S)-Pindolol is used as an internal standard. The detection limits of 5 ng/ml enantiomer in plasma and 50 ng/ml enantiomer in urine are sufficient for pharmacokinetic studies after therapeutic doses. © 1993 Wiley-Liss, Inc.     

Continuous control of water activity during biocatalysis in organic media

Summary This paper describes a newly developed technique to adjust and control the water activity in enzymatic reactions in organic media. A saturated salt solution of known water activity is circulated inside a silicone tube, submerged into the reaction medium. The circulating solution can both absorb and release water. Water activity control during lipase catalyzed esterification was demonstrated with diisopropyl ether as solvent.     

Congo red inhibition of scrapie agent replication.

Congo red inhibits the accumulation of protease-resistant PrP in scrapie-infected mouse neuroblastoma cells. Here we show that Congo red also inhibits the replication of scrapie infectivity in these cells. This observation is consistent with the idea that protease-resistant PrP is a vital component of the scrapie agent or that agent replication depends on the presence of protease-resistant PrP in the cell.     

Lateral hydraulic conductivity of early metaxylem vessels in Zea mays L. roots

The hydraulic conductivity of the lateral walls of early metaxylem vessels (Lp_x in m · s^–1 · MPa^–1) was measured in young, excised roots of maize using a root pressure probe. Values for this parameter were determined by comparing the root hydraulic conductivities before and after steam-ringing a short zone on each root. Killing of living tissue virtually canceled its hydraulic resistance. There were no suberin lamellae present in the endodermis of the roots used. The value of Lp_x ranged between 3 · 10^–7 and 35 · 10^–7 m · s^–1 · MPa^–1 and was larger than the hydraulic conductivity of the untreated root (Lp_r = 0.7 · 10^–7 to 4.0 · 10^–7 m · s^–1 · MPa^–1) by factor of 3 to 13. Assuming that all flow through the vessel walls was through the pit membranes, which occupied 14% of the total wall area, an upper limit of the hydraulic conductivity of this structure could be given(Lp_pm=21 · 10^–7 to 250 · 10^–7 m · s^–1 · MPa^–1). The specific hydraulic conductivity (Lp_cw) of the wall material of the pit membranes (again an upper limit) ranged from 0.3 · 10^–12 to 3.8 · 10^–12 m² · s^–1 · MPa^–1 and was lower than estimates given in the literature for plant cell walls. From the data, we conclude that the majority of the radial resistance to water movement in the root is contributed by living tissue. However, although the lateral walls of the vessels do not limit the rate of water flow in the intact system, they constitute 8–31% of the total resistance, a value which should not be ignored in a detailed analysis of water flow through roots.Abbreviatations and Symbols k_wr (T ₁ ^2/W ) rate constant (half-time) of water exchange across root (s^–1 or s, respectively) - Lp_cw specific hydraulic conductivity of wall material (m² · s^–1 · MPa^–1) - Lp_pm hydraulic conductivity of pit membranes (m · s ^–1 · MPa^–1) - Lp_r hydraulic conductivity of root (m · s^–1 · MPa^–1) - Lp_x lateralhydraulic conductivity of walls of root xylem (m · s ^–1 · MPa^–1) This research was supported by a grant from the Bilateral Exchange Program funded jointly by the Natural Sciences and Engineering Research Council of Canada and the Deutsche Forschungsgemeinschaft to C.A.P., and by a grant from the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 137, to E.S. The expert technical help of Mr. Burkhard Stumpf and the work of Ms. Martina Murrmann and Ms. Hilde Zimmermann in digitizing chart-recorder strips is gratefully acknowledged.     

Functional and biochemical association of Hck with the LIF/IL-6 receptor signal transducing subunit gp130 in embryonic stem cells.

The role played by the Src-related tyrosine kinase, Hck, in embryonic stem (ES) cell differentiation was investigated by replacing a conserved C-terminally located tyrosine with phenylalanine by gene targeting. Targeted ES cells display a 7- to 9-fold elevation in constitutive Hck kinase activity and require approximately 15 times less leukaemia inhibitory factor (LIF) than parental ES cells to maintain their stem cell character in vitro. We also demonstrate a rapid and transient increase in Hck tyrosine kinase activity in parental ES cells stimulated by LIF and, finally, show that Hck is physically associated with gp130, an affinity converter and signal transducing component of the LIF receptor. Thus, these results provide biological and biochemical evidence that Hck participates in signal transduction from the LIF receptor.     

Nitric oxide synthase-containing nerve fibers and neurons in the genital tract of the female mouse

Nitric oxide (NO) is generated intracellularly from L-arginine by the action of the enzyme nitric oxide synthase (NOS). The present investigation demonstrates immunoreactivity against NOS and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity in nerve cells and fibers of the reproductive system of the female mouse. The density of nerve fibers staining for NOS varied among different genital organs. The ovary and Fallopian tube were devoid of NOS-positive nerves. The uterine horns received sparse innervation by NOS-containing nerve fibers. The most abundant NOergic innervation was found in the uterine cervix and vagina, where the nerve fibers ran parallel to the smooth muscle bundles and beneath the epithelium; they also accompanied intramural blood vessels. The vaginal muscular wall contained single or groups of NOS-reactive nerve cells. Clusters of NOS-containing neurons were located in Frankenhäuser's ganglion at the cervico-vaginal junction. NO may therefore act as a transmitter in the nervous control of the female reproductive tract.     

A model for the neuronal implementation of selective visual attention based on temporal correlation among neurons

We propose a model for the neuronal implementation of selective visual attention based on temporal correlation among groups of neurons. Neurons in primary visual cortex respond to visual stimuli with a Poisson distributed spike train with an appropriate, stimulus-dependent mean firing rate. The spike trains of neurons whose receptive fields donot overlap with the focus of attention are distributed according to homogeneous (time-independent) Poisson process with no correlation between action potentials of different neurons. In contrast, spike trains of neurons with receptive fields within the focus of attention are distributed according to non-homogeneous (time-dependent) Poisson processes. Since the short-term average spike rates of all neurons with receptive fields in the focus of attention covary, correlations between these spike trains are introduced which are detected by inhibitory interneurons in V4. These cells, modeled as modified integrate-and-fire neurons, function as coincidence detectors and suppress the response of V4 cells associated with non-attended visual stimuli. The model reproduces quantitatively experimental data obtained in cortical area V4 of monkey by Moran and Desimone (1985).