New species of Roccellinastrum with an emendation of the genus

The genus Roccellinastrum Follm. emend. Henssen & Vobis is placed in the Lecideaceae. Two new species are described, R. epiphyllum Henssen & Vobis, from Chile and R. neglectum Henssen & Vobis, from New Zealand and Tasmania. The new combination R. candidum (Mull. Arg.) Henssen is proposed. Roccellinastrum is characterized by apothecia of diverse shape without a proper margin, and a developmental morphology corresponding to that of the Lecanorales, by small amyloid asci, branched paraphyses and excipular hyphae, a byssoid–spon–giose thallus composed of thick–walled hyphae, and members of the Chlorococcales as phycobionts. The principal chemical constituent is protocetraric acid. The species have different spore septation and chemistry. Summary in Spanish.     

Measurements of pH in the apoplast of sunflower leaves by means of fluorescence

A method was elaborated by which the pH in leaf apoplast can be measured. The technique is based on the pH dependent fluorescence of 5-carboxyfluorescein (5-CF) or fluorescein isothiocyanate (FITC). The fluorescein isothiocyanate is coupled with a macromolecular dextran molecule (FITC-dextran). For eliminating the effect of the absolute dye concentration the dual excitation technique was applied. It was shown that the ratio of fluorescence excited by light of 491 nm and 463 nm was virtually independent of the concentration of 5-CF and that this fluorescence ratio was related to the pH. The plasmalemma is practically impermeable to FITC-dextran and in the test we carried out over a period of 6 h not the slightest indication was found that it may penetrate the plasma membrane. For 5-CF this cannot be ruled out completely. It is possible that at pH values below 4.5 it may penetrate biological membranes at low rates.
Experiments with leaves of sunflower ( Helianthus animus cv. Erika) perfused with 5-carboxyfluorescein and supplied with different nitrogen forms showed that NH⁺₄ application resulted in a decrease and NO⁺₃ application in an increase of the leaf apoplast pH. Leaf spraying with fasicoccin was followed by a pH decrease, while leaf spraying with the protonophores p -trifluoromethoxy carbonytcyanide phenylhydra-zon (FCCP) or nigericin resulted in neutral apoplastic pH. These results provide evidence that the method is well suited for measuring the response of the leaf apoplast pH to changing physiological conditions.     

The TraM protein of plasmid R1 is a DNA-binding protein

The TraM protein of the resistance plasmid R1 was purified to homogeneity and used for DNA-binding studies. Both gel retardation- and footprint experiments showed that TraM specifically binds to DNA of plasmid R1 comprising the region between the origin of transfer and the traM gene. Several TraM molecules bind and, according to the footprint experiments, two distinct sites of specific binding exist. The two sites are separated from each other by 12 nucleotides and each contains an inverted repeat. DNase I protection assays showed that the initial TraM binding occurs at these palindromic sequences. At higher protein concentrations the lengths of the DNA segments protected by TraM were increased towards the traM gene. In one region this extension leads to binding of TraM protein at its own promoters.     

Adaptations of Goldner's Masson Trichrome Stain for the Study of Undecalcified Plastic Embedded Bone

Specialized adaptations for application of Goldner's Masson trichrome stain to plastic embedded undecalcified bone specimens are presented. This stain can be used successfully on methyl-glycol methacrylate, glycol methacrylate and Spurr embedded bones. The stain affords the advantage of good cellular staining due to the hematoxylin component with concomitant sharp discrimination of mature bone matrix which stains green, immature new bone matrix which stains red, and calcified cartilage which stains very pale green. Use of red filters during photomicrography aids in bone-osteoid discrimination in black and white photographs.     

Evidence for a driving role of ingrowing axons for the shifting of older retinal terminals in the tectum of fish

In amphibians and teleosts, retina and tectum grow incongruently. In order to maintain the retinotopy of the retinotectal projection, Gaze, Keating, and Chung (1974) postulated a shifting of terminals throughout growth. In order to test the possibility that ingrowing retinal fibers are the driving force for this shifting, we induced a permanent retinal projection into the ipsilateral tectum in juveniles of the cichlid fish Haplochromis burtoni. The surface of the tectum had increased (11–18 months later) 2.5–5.8 times, and the surface of the retina 8.6–14 times. Filling of ganglion cells with horseradish peroxidase (HRP) retrogradely from the tectum showed ipsilaterally regenerating ganglion cells only in the center of the retina. The position of ganglion cells indicated that the ipsilateral projection derived only from axotomized and regenerating retinal ganglion cells but not from those newly born. Ipsilaterally projecting retinal fibers showed terminals only in the rostral half of the tectum. Comparison of area of terminations of ipsilaterally projecting ganglion cells at various times after the crush provided no evidence for expansion or a shift into caudal tectal areas throughout the period of growth. These findings are compatible with the idea that newly ingrowing fibers induce older terminals to move caudally.     

Increases in arterial blood oxygen during exercise in the lemon shark (Negaprion brevirostris)

Summary Polyethylene cannulae were implanted in pre- and post-branchial blood vessels allowing nonstressful blood sampling over a variety of activity ranges in an active tropical elasmobranch, the lemon shark (Negaprion brevirostris). TheP ₅₀ was found to be 11.8 Torr at 24°C and pH of 7.7. A Bohr shift of –0.36 was also found. BloodP _{o 2} and oxygen content were measured during rest, routine swimming, and exercise in unanesthetized, free swimming juveniles. Under all conditions venous oxygen levels were low with venousP _{o 2} of 7.1±2.7 Torr, and venous oxygen content ( ) of 0.56±0.4 vol%. However, a large variability was found in arterial blood measurements. ArterialP _{o 2} ranged from 7 to 80 Torr, while arterial oxygen content (Ca_{o 2}) varied from 1.6 vol% to 6.8 vol% among ten experimental animals. A significant increase in arterialP _{o 2}, oxygen content, and hematocrit was noted during increased activity. Since the venous system provides little or no oxygen reserve, increased oxygen extraction from the blood ( ) appears to be met by an increase inCa_{o 2} rather than a decrease in . Mechanisms to accomplish this may include increasing hematocrit and vacular gill shunts.     

Dermorphins, Opioid Peptides from Amphibian Skin, Act on Opioid Receptors of Mouse Neuroblastoma x Rat Glioma Hybrid Cells

Dermorphin and its Hyp6 analogue are opiate-like heptapeptides originally discovered in frog skin and characterized by the presence of a D-Ala2 residue in their sequence. They were assayed for their capacity to compete with [3H]Leu-enkephalin for binding to opioid receptors in membranes of neuroblastoma x glioma hybrid cells. In the presence of 7 nM-[3H]Leu-enkephalin, the concentrations at which they caused 50% inhibition of [3H]enkephalin binding (IC50 values) are 0.1 micro M and 0.3 micro M, respectively. In contrast, the synthetic L-Ala2-dermorphin shows very low affinity for the opioid receptors. In addition, like other opioid peptides, dermorphin and hyp6-dermorphin inhibit the elevation by prostaglandin E1 (PGE1) of the level of adenosine 3':5'-cyclic monophosphate (cyclic AMP) (IC50 values 0.2 micro M and 0.4 micro M, respectively). The inhibition is prevented by the opiate antagonist naloxone, L-Ala2-dermorphin is at least three orders of magnitude less potent in inhibiting the PGE1-evoked increase in the level of cyclic AMP. The results show that peptides with an amino acid sequence quite different from that of the enkephalins can bind to opioid receptors of the hybrid cells.     

Endocytosis and breakdown of ribonuclease oligomers by sinusoidal rat liver cells in vivo. I. Effect of size.

Aspects of protein structure determining endocytosis of proteins by sinusoidal rat liver cells in vivo have been studied, using cross-linked or aggregated derivatives of bovine pancreatic ribonuclease A (labelled with 125I) as probes. Ribonuclease was cross-linked by reaction with dimethylsuberimidate, a way of modification that does not change the charge of the protein. Monomer, dimer and polymer fractions were isolated by gel filtration and characterized in respect of size and number of amino groups modified. Maintenance of enzyme activity, stability of disulfide bonds, and lack of susceptibility to endoproteases showed that the cross-linking procedure did not result in gross conformational changes of the ribonuclease molecules. Monomer, dimer and polymer fractions were injected into nephrectomized rats and plasma clearance and uptake in liver and spleen were determined. About 30% of the injected polymer fraction was found in liver 15 min after injection; for dimer and monomer fractions values of 6% and 2% of the dose were found. Similar differences were found in spleen. Autoradiography, cell isolation, and subcellular fractionation showed that in liver the radioactive proteins were taken up in lysosomes of sinusoidal cells. Similar results were obtained with fractions of aggregated ribonuclease prepared by freeze-drying the protein from 50% acetic acid. Our results demonstrate that the rate of uptake of the ribonuclease derivatives is positively correlated with the size of the molecules. Similarity of the results obtained with cross-linked and aggregated fractions suggests that the number of ribonuclease 'subunits'/molecule, rather than the procedures used to prepare the polymers, determine the rate of uptake by liver and spleen.