The role of phenylalanine in differentiating amphibian melanocytes

To see whether phenylalanine serves as a substrate in melanogenesis, hanging drop explants of neural crest from amphibian (Ambystoma maculatum and A. mexicanum) embryos were subjected on the seventh day in vitro to treatment with phenylalanine-³H and studied by means of light microscopic radioautography. All melanin-containing cells showed label. On the other hand, when puromycin, an inhibitor of protein synthesis, together with the labeled amino acid was administered to the cultures, no radioactivity was incorporated by pigmented cells. Comparable results were obtained when leucine was substituted for phenylalanine. In control experiments, puromycin and labeled tyrosine or 3,4-dihydroxyphenylalanine (DOPA), both known precursors for melanin synthesis, were administered to the neural crest cultures. In these experiments, puromycin had no effect on the incorporation of label by pigmented cells. Our data strongly indicate that in differentiating amphibian melanocytes with functional pigment-forming systems, phenylalanine is used in protein synthesis, but does not serve as a substrate for the tyrosine-tyrosinase system.In another series of experiments, explants of neuroepithelium (neural crest anlage) were grown from the time of explantation to the seventh day in vitro in the presence of phenyllactic acid, an analog of phenylalanine. Pigment cells developed normally.These results suggest that phenylalanine plays little or no role in pigment cell differentiation.     

Endocytosis in Chinese hamster fibroblasts : Inhibition by glucose

Endocytosis in Chinese hamster ovary fibroblasts was investigated by measuring the rate of uptake of ³H-sucrose, which is known to enter cells only by endocytosis. Serum, polyvinylpyrrolidone (PVP), adenosine triphosphate, insulin, and cyclic 3′,5′-adenosine monophosphate, all of which are known to increase the rate of endocytosis by other cell systems, had no effect on Chinese hamster fibroblasts. However, medium in which these cells had been maintained for several days, referred to as conditioned medium, had a profound effect on endocytosis. These cells endocytosed 3 to 5 times as rapidly in conditioned medium as in fresh medium. A logarithmic inhibition of this effect was observed with increasing -glucose concentrations, however, glucose-free medium did not produce as great an effect as conditioned medium. This suggests that these cells may endocytose in response to their nutritional requirements.     

The respiratory chain of plant mitochondria. XV. Equilibration of cytochromes c549, b553, b557 and ubiquinone in mung bean mitochondria: Placement of cytochrome b557 and estimation of the midpoint potential of ubiquinone

1. 1. Cycles of oxidation followed by reduction at pH 7.2 have been induced in uncoupled anaerobic mung bean mitochondria treated with succinate and malonate by addition of oxygen-saturated medium. Under the conditions used, cytochromes b₅₅₇, b₅₅₃, c₅₄₉ (corresponding to c₁ in mammalian mitochondria) and ubiquinone are completely oxidized in the aerobic state, but become completely reduced in anaerobiosis.

2. 2. The time course of the transition from fully oxidized to fully reduced in anaerobiosis was measured for cytochromes c₅₄₉, b₅₅₇, and b₅₅₃. The intramitochondrial redox potential (IMP_h) was calculated as a function of time for each of the three cytochromes from the time course of the oxidized-to-reduced transition and the known midpoint potentials of the cytochromes at pH 7.2. The three curves so obtained are superimposable, showing that the three cytochromes are in redox equilibrium under these conditions during the oxidized-to-reduced transition.

3. 3. This result shows that the slow reduction of cytochrome b₅₅₇ under these conditions, heretofore considered anomalous, is merely a consequence of its more negative midpoint potential of +42 mV at pH 7.2, compared to +75 mV for cytochrome b₅₅₃ and +235 mV for cytochrome c₅₄₉. Cytochrome b₅₅₇ is placed on the low potential side of coupling site II and transfers electrons to cytochrome c₅₄₉ via the coupling site.

4. 4. The time course of the transition from fully oxidized to fully reduced was also measured for ubiquinone. Using the change in intramitochondrial potential IMP_h with time obtained from the three cytochromes, the change in redox state of ubiquinone with IMP_h was calculated. When replotted as IMP_h versus the logarithm of the ratio (fraction oxidized)/(fraction reduced), two redox components with n = 2 were found. The major component is ubiquinone with a midpoint potential E_m7.2 = + 70 mV. The minor component has a midpoint potential E_m7.2 = − 12 mV; its nature is unknown.

The mechanism of cobalamin binding to hog intrinsic factor

Chicken brain Arylsulfatase A (E.C.3.1.6.1) was immobilized by interaction with Concanavalin A. The immobilized enzyme retained its catalytic activity and this enzyme can be reused without appreciable loss of activity. The storage stability of bound and soluble enzymes was comparable and binding of enzyme to Concanavalin A increases its thermal stability. Kinetic studies indicated that bound enzyme shows similar anomalous kinetics as that of free enzyme but slight change was observed in relation to pH optima, Km value and activation energy.     

Functional impairment of MSC induced by transient warming events: Correlation with loss of adhesion and altered cell size

Background

We recently showed that transient warming effects decreased the functional and adhesion properties of mesenchymal stromal cells (MSC) while post-thaw viability remained high. In an attempt to better predict functional impairment of cryopreserved MSC, we further analysed the correlation between viability, immunosuppressive activity and adhesion of cells exposed or not to warming events.

The presence of heartwood decay,a precursor to hollow formation,within Eucalyptus camaldulensis in relation to stem size

River Red Gums (Eucalyptus camaldulensis Dehnh.) are large trees that dominated many of continental Australia's riparian areas, providing abundant hollows for nesting and denning fauna. Land clearing and forestry have reduced the availability of large trees, and much of the remaining forest is dominated by small regrowth trees that lack visible hollow entrances. Inspection of recently felled River Red Gums revealed that heartwood decay, a precursor of hollow formation, can be common in relatively small trees. Internal decay and voids remain inaccessible until stochastic damage exposes them, which may not occur until the tree is old, and hence probably large. This discrepancy indicates that there is likely to be a size range of trees into which artificial entrances can be added to quickly increase hollow availability in landscapes undergoing active restoration as fauna habitat.