The adrenergic innervation of the renal artery and vein of the rat

Summary The adrenergic innervation of the extrarenal blood vessels of the rat left kidney was investigated by fluorescence histochemistry and by electron microscopy. The trunk of the renal artery proximal to the aorta is elastic and appears to be very sparsely innervated. In contrast, near the kidney the renal artery—which divides into 3 to 4 large branches of the muscular type possesses a dense adrenergic innervation. The adrenergic terminal axons are situated in the adventitia close to the external elastic lamella, but only rarely in close contact with smooth muscle cells. In most instances several terminal axons are grouped and enclosed by a Schwann cell, single axons being rare. All terminal axons are able to take up and to store 5-hydroxydopamine which strongly suggests that they are adrenergic. The innervation of the renal vein is more sparse than that of the muscular arteries but somewhat denser than that of the elastic artery. In addition, close to the origin of the renal artery the presence of small intensively fluorescent (SIF) cells as well as of some adrenergic ganglion cells is noted. The latter are situated in the adrenergic nonterminal axon bundles, which run parallel to the blood vessels.It is concluded that the uneven adrenergic innervation along the artery as well as individual variations in the branching of the artery are the main causes of the unusually high individual variations of the NA content of this organ such as used in pharmacological experiments.     

Brain tissue transplanted to the anterior chamber of the eye

Summary Knowing the ontogenesis of the central monoamine neurons of the rat it is possible to obtain, by free-hand dissection from embryos and newly born animals, pieces containing dopamine (DA), noradrenaline (NA), and 5-hydroxytryptamine (5-HT) neurons that are small enough to permit homologous transplantation to the anterior chamber of the eye of adult animals. With this technique it was established that all three types of immature monoamine neurons are able to survive in the anterior chamber. Fluorescence histochemical analysis of whole mount preparations of the sympathetically denervated host irides revealed that both the catecholamine- and the 5-HT-neurons are able to partly reinnervate the irides, forming networks of varicose nerve terminals similar to the normally present sympathetic adrenergic ground plexus.Monoamine nerve cell bodies are attached to the irides but the majority of fluorescent nerve cell bodies is located within the transplants. Serial sectioning of these transplants showed rather well organized brain tissue, containing groups of fluorescent and non-fluorescent cell bodies, many areas being innervated by monoamine nerve terminals. When brain tissue was transplanted before the normal appearance of fluorescent neuroblasts (embryos with a crown-rump length less than 8 mm) monoamine neurons developed and matured within the eye.The amount of newly formed nerves of central origin recovered on the irides increased with time between the 2nd and 4th postoperative week and persisted after 2 months. The yield of new fibers was better using transplants from embryos with a crown-rump length between 15 and 30 mm than using transplants from larger embryos and newly born animals.If embryonic brain tissue known to be devoid of monoamine nerve cell bodies but containing monoamine nerve terminals in the adult state (cortex cerebri and cerebelli, spinal cord) was transplanted to sympathetically non-denervated eyes, the sympathetic adrenergic fibers seemed to be able to innervate the transplants.This work was supported by grants from the Swedish Medical Research Council (14×–3185), Karolinska Institutets fonder, and Magnus Bergvalls Stiftelse. We thank Miss Monica Eliasson, Mrs. Ulla Flyger, Mrs. Barbro Norstedt and Miss Ingrid Strömberg for skilful technical assistance. The generous gifts of Nialamide, Pfizer, and Pargyline, Abbott are gratefully acknowledged.     

The reaction of the preoptic nucleus of Xenopus laevis tadpoles to osmotic stimulation

Summary The development of the preoptic nucleus of Xenopus laevis tadpoles during metamorphosis was studied and the effect of osmotic stimulation on this process investigated. The development of this region was not affected by treatment for one or more days in hypertonic media. It was found that at the end of metamorphosis the neurosecretory cells in the preoptic nucleus are localized in three regions: the rostro-dorsal, the caudo-dorsal and the ventral region. After osmotic stimulation only the neurosecretory cells of the caudo-dorsal region appeared to have reacted, as indicated by their loss of neurosecretory (PIC positive) material. It is concluded that the cells of this region may be involved in the synthesis of the posterior lobe hormones.The author thanks Prof. Dr. J. C. van de Kamer and Dr. F. C. G. van de Veerdonk for their interest and many helpful discussions, Dr. L. Boomgaart and Dr. A. P. van Overbeeke for correcting the English text and Miss C. M. G. van Bemmel for technical assistance.     

Determination of cytoplasmic calcium concentration inDryopteris spores

Germination of Dryopteris spores is mediated by the physiologically active, far-red-absorbing form of phytochrome, Pfr, and external Ca²⁺ is necessary for the transduction of the light signal. Because knowledge about the cytoplasmic calcium ion concentration, [Ca²⁺]_i, is of great importance for understanding the role of calcium during signal transduction, this value was measured using fura-2 in fern spores undergoing the normal developmental progression into germination. Fura-2 was loaded into the spores by electroporation, which does not disrupt the normal process of germination. The intensity of the fluorescence emission of the loaded fura-2 was analysed by a microspectrophotometric assay of single spores, and successful loading could be obtained by the application of ten electrical pulses (field strength 7.5 kV · cm^–1, half-life (time constant) 230 s). Fura-2 was alternately excited by light of wavelengths 355 and 385 nm through an inverted fluorescence microscope, and the emitted fura-2 fluorescence was collected by a silicon-intensified video camera. The cytoplasmic calcium ion concentration was calculated from the ratio of the camera output obtained for both wavelengths and displayed by a pseudo-color technique. Spores responded to changes of the extracellular Ca²⁺ concentration, and this observation is considered as evidence that fura-2 is loaded into the cytoplasm. The substitution of a low external [Ca²⁺] (1 mM ethyleneglycol-bis(2-aminoethyl-ether) {ie166-01},N-tetraacetic acid (EGTA)) by 1 mM CaCl₂ caused a fast increase of [Ca²⁺]_i from approx. 50 nM to above 500 nM. In contrast, the subsequent substitution of CaCl₂ by EGTA decreased [Ca²⁺]_i again below 100 nM within 0.5 h. Furthermore, the application of ionomycin could initiate a change in [Ca2+]_i according to the Ca²⁺ gradient established between the extracellular medium and cytoplasm. In spores sown on a Ca²⁺-free medium, [Ca²⁺]_i, analysed in a buffer containing EGTA, was found to be around 50 nM during the first days of cultivation, independent of the irradiation protocol. However, if spores were grown in darkness on a Ca²⁺-containing medium and analysed in EGTA, [Ca²⁺]_i was significantly higher ( 500 nM). In red-light-irradiated spores, [Ca²⁺]_i was found to decrease with increasing time after irradiation, and was determined to be less than 100 nM when analysis was done 44 h after germination was initiated by the light treatment.Dedicated to Professor H. Mohr on the occasion of his 60th birthday     

Action spectra of the light-growth response in three behavioral mutants of Phycomyces

Null-point action spectra of the light-growth response were measured for three mutants of Phycomyces blakesleeanus (Burgeff) and compared with the action spectrum of the wild type (WT). The action spectrum for L150, a recently isolated night-blind mutant, differs from the WT spectrum. The L150 action spectrum has a depression near 450 nm and small alterations in its long-wavelength cutoff, the same spectral regions where its photogravitropism action spectrum is altered. This indicates that the affected gene product influences both phototropism and the light-growth response. For L85, a hypertropic (madH) mutant, the light-growth-response action spectrum is very similar to that of WT even though the photogravitropism action spectrum of L85 has been shown previously to be altered in the near-UV region. The affected gene product in this mutant appears to affect phototropic transduction but not light-growth-response transduction. The action spectrum of C110, a stiff (madE) mutant, differs significantly from the WT spectrum near 500 nm, the same spectral region where sporangiophores of madE mutants have been shown to have small alterations in second-derivative absorption spectra. This indicates that the madE gene product may be physically associated with a photoreceptor complex, as predicted by system-analysis studies.Abbreviations SE standard error of the mean - UV ultraviolet light - Wt wild type I dedicated to Masaki Furuya on the occasion of his 65th birthdayWe thank H. Reiner Schaefer for performing some of the experiments and for help in data analysis, David Durant for computer programming, and Benjamin Horwitz for helpful discussions. This work was supported by a grant from the National Institutes of Health (GM29707) to E.D. Lipson.     

Migration of the cell nucleus during the amoebo-flagellate transformation ofPhysarum polycephalum is mediated by an actin-generated force that acts on the centrosome

Summary The mechanism of cell-nuclear migration during the amoebo-flagellate transformation inPhysarum polycephalum was examined by fluorescence microscopy after staining with a tubulinspecific antibody, rhodamine-conjugated phalloidin and 4,6-diamidino-2-phenylindole (DAPI). While the round amoeba cells changed to comma-shaped swarm cells within 20min after suspension in buffer, the cell nuclei moved from the central region of each cell to the periphery, each forming a sharp projection in the direction of movement. A centrosome also migrated from the center of the cell to the cell periphery. Since the centrosome was in close contact with the tip that protruded from the cell nucleus throughout the cellnuclear migration, the migration of the cell nucleus and the centrosome could be recognized as comigration. Then the flagella began to elongate from the centrosome and the cells became slender and polarized, adopting the so-called comma-shape. On the basis of these observations, the transformation process was classified into three steps: cell-nuclear migration, flagella formation and swarm maturation. The comigration of the cell nucleus and the centrosome was not inhibited by the anti-microtubule drug nocodazole (4 M) but it was inhibited by the anti-microfilament drug cytochalasin A (4 M), suggesting that the force of migration is generated by microfilaments. To investigate the role of the centrosome in this comigration in detail, we identified two aberrant strains, defective in swimming ability, from among various laboratory strains. The two strains, TM 4 and J, were found to have defects in cell-nuclear migration. Strain TM 4 had two types of irregular swarm cells: in one, only a part of the cell nucleus projected a thin filamentous structure; and in the other, no cell-nuclear migration occurred. Strain J had two centrosomes per cell and such swarm cells exhibited an attempt of cell-nuclear migration at two sites which corresponded to the position of the centrosome. The characteristics of these two strains indicate that the centrosome is essential for cell-nuclear migration. Our observations suggest that the cell-nuclear migration is mediated by actin-generated forces that act on the centrosome rather than on the cell nucleus itself.Abbreviations FITC fluorescein isothiocyanate - DAP 4,6-diamidino-2-phenylindole - PBS phosphate-buffered saline - KPB potassium phosphate buffer - MTOC microtubule organizing center     

Role of conserved proline residues in stabilizing tryptophan synthase alpha subunit: analysis by mutants with alanine or glycine

To study the role of Pro residues in the conformation and conformational stability of a protein, nine mutant alpha subunits of tryptophan synthase from Escherichia coli, in which Ala or Gly was substituted for each of six Pro residues (positions 28, 57, 62, 96, 132, and 207) that are conserved in 10 microorganisms, were constructed by means of site-directed mutagenesis. The far-ultraviolet (UV) CD spectra of five mutant alpha subunits with Ala in place of Pro were identical to the spectrum of the wild-type protein, the exception being the mutant at position 207 (P207A). CD values in the far-UV region were less negative for P207A, indicating that the Pro residue at position 207 plays a role in maintaining the intact structure of the alpha subunit. The negative CD values of the Gly mutants examined (P28G, P96G, and P132G) were also decreased. Calorimetric measurements showed that the two mutants at position 28 (P28G and P28A) gave two peaks in the excess heat capacity curve, whereas the wild type and other Pro mutants had only a single peak. The stability of each mutant protein relative to that of the wild type was about the same for P57A, less for P62A and P132A, and markedly decreased for P96A and P207A, which are substituted at less mobile positions. The changes of denaturation entropy (delta delta dS) at the denaturation temperature of the wild-type protein (54.1 degrees C at pH 9.0) were positive for P57A, P62A, and P132A, but negative for P96A, P207A, and P132G.(ABSTRACT TRUNCATED AT 250 WORDS)     

Complexes of mycobactin fromMycobacterium smegmatis with scandium,yttrium and lanthanum

Summary The interaction of cations of group IIIb elements (Sc, Y, La) with mycobactin S in ethanol leads to the formation of 1:1 complexes which closely resemble the known aluminium compound with respect to ultraviolet absorption and fluorescence emission spectra. Determination of molar stoichiometry by spectrophotometry shows that this method can be conveniently applied to the estimation of purity in mycobactin samples. Hydrolytic dissociation measurements based on aqueous extraction of the labelled complexes in heterogeneous phase indicate a pronounced gradation in cationbinding stability, which increases from La (rapid and complete dissociation) to Sc ( 24% dissociation under similar conditions). The observed properties of the complexes are rationalized by semi-empirical model calculations, which suggest that ionic radius effects resulting from interaction of the IIIb cations with mycobactin S would not favour octahedral coordination of these elements as in the stable Fe(III) complex.     

Lateral heterogeneity in human platelet plasma membrane and lipids from the time-resolved fluorescence of trans-parinaric acid

We have investigated the complex behaviour of the time resolved fluorescence intensity and anisotropy of trans-parinaric acid, incorporated into fragments of the plasma membrane of human platelets and in multibilayers of lipids extracted from that membrane. It is shown that the observation of anisotropies that increase at long times can be satisfactorily interpreted by assuming two populations of the fluorescence probe with distinct lifetimes, rotational relaxation times and order parameters. The heterogeneous probe distribution was correlated with a similar heterogeneity in the lipid composition of the bilayer, modulated by temperature. Below 35°C an important fraction of the lipids of the plasma membrane are apparently in the form of solid-like domains (20% at 20°C). However, in the physiological temperature range that solid/fluid heterogeneity is almost negligible. Since these effects were also observed in multibilayers of lipids from the platelet membrane, the formation of solid-like clusters appears to arise from lipid-lipid interactions only, and most probably involving cholesterol. These results support the previous finding of a lateral phase separation for temperatures less than 37°C described by Gordon et al. (1983) in a spin-probe study of the platelet plasma membrane.Abbreviations DPH 1,6-diphenyl-1,3,5-hexatriene - DPPC dipalmitoyl-phosphatidylcholine - tPnA trans-parinaric acid Offprint requests to: A. U. Acuña     

Growth and photosynthetic activities of wild-type and antenna-deficient mutant strains of Rhodobacter capsulatus

Cells of Rhodobacter capsulatus wild-type strains (37b4, B 10) and mutant strains, lacking lightharvesting (LH) complex II (B800–850) and defective in formation of LH I (B870) complex [U 43 (pTXB 87), U43 (pTXA6-10)] were grown photosynthetically at high and low light intensities in a turbidostate. The mutant strain U43 (pTXA6-10), lacking any LH system, was able to grow at high and low light intensities with doubling times of 4.6 and 9.8 h, respectively. In this mutant the concentration of photochemical reaction centers (RC) per cell and per membrane protein was several times higher than in wild type cells, but the bacteriochlorophyll content, the size of the photosynthetic unit and the rate of photophosphorylation were lower than in wild type cells. Reversible bleaching of reaction center and photophosphorylation were measured under different excitation light intensities. The charge recombination in the RC between the primary donor and Q_B was very slow in the mutant strains. Two membrane fractions differing in absorption spectra and light saturation behaviour of reversible bleaching and photophosphorylation were isolated from the mutant strains. The experimental data indicate that photosynthetic units of different composition and/or organization are present in the mutant cells.Abbreviations DSM Deutsche Sammlung von Mikroorganismen, Braunschweig