Control of erythroid differentiation: asynchronous expression of the anion transporter and the peripheral components of the membrane skeleton in AEV- and S13-transformed cells

Chicken erythroblasts transformed with avian erythroblastosis virus or S13 virus provide suitable model systems with which to analyze the maturation of immature erythroblasts into erythrocytes. The transformed cells are blocked in differentiation at around the colony-forming unit-erythroid stage of development but can be induced to differentiate in vitro. Analysis of the expression and assembly of components of the membrane skeleton indicates that these cells simultaneously synthesize alpha-spectrin, beta-spectrin, ankyrin, and protein 4.1 at levels that are comparable to those of mature erythroblasts. However, they do not express any detectable amounts of anion transporter. The peripheral membrane skeleton components assemble transiently and are subsequently rapidly catabolized, resulting in 20-40-fold lower steady-state levels than are found in maturing erythrocytes. Upon spontaneous or chemically induced terminal differentiation of these cells expression of the anion transporter is initiated with a concommitant increase in the steady-state levels of the peripheral membrane-skeletal components. These results suggest that during erythropoiesis, expression of the peripheral components of the membrane skeleton is initiated earlier than that of the anion transporter. Furthermore, they point a key role for the anion transporter in conferring long-term stability to the assembled erythroid membrane skeleton during terminal differentiation.     

Sodium gradient-dependent L-glutamate transport is localized to the canalicular domain of liver plasma membranes. Studies in rat liver sinusoidal and canalicular membrane vesicles

The driving forces for L-glutamate transport were determined in purified canalicular (cLPM) and basolateral (i.e. sinusoidal and lateral; blLPM) rat liver plasma membrane vesicles. Initial rates of L-glutamate uptake in cLPM vesicles were stimulated by a Na+ gradient (Na+o greater than Na+i), but not by a K+ gradient. Stimulation of L-glutamate uptake was specific for Na+, temperature sensitive, and independent of nonspecific binding. Sodium-dependent L-glutamate uptake into cLPM vesicles exhibited saturation kinetics with an apparent Km of 24 microM, and a Vmax of 21 pmol/mg X min at an extravesicular sodium concentration of 100 mM. Specific anionic amino acids inhibited L-[3H]glutamate uptake and accelerated the exchange diffusion of L-[3H]glutamate. An outwardly directed K+ gradient (K+i greater than K+o) further increased the Na+ gradient (Na+o greater than Na+i)-dependent uptake of L-glutamate in cLPM vesicles, resulting in a transient accumulation of L-glutamate above equilibrium values (overshoot). The K+ effect had an absolute requirement for Na+. In contrast, in blLPM the initial rates of L-glutamate uptake were only minimally stimulated by a Na+ gradient, an effect that could be accounted for by contamination of the blLPM vesicles with cLPM vesicles. These results indicate that hepatic Na+ gradient-dependent transport of L-glutamate occurs at the canalicular domain of the plasma membrane, whereas transport of L-glutamate across sinusoidal membranes results mainly from passive diffusion. These findings provide an explanation for the apparent discrepancy between the ability of various in vitro liver preparations to transport glutamate and suggest that a canalicular glutamate transport system may serve to reabsorb this amino acid from bile.     

Restriction enzyme analysis of tomato chloroplast and chromoplast DNA

Plastid DNA was isolated from the chloroplasts of tomato (Lycopersicon esculentum var Traveler 76) leaves and the chromoplasts of ripe tomato fruit. Comparisons of the two DNAs were made by restriction endonuclease analysis using PvuII, HpaI, and Bg1I. No differences in the electrophoretic banding patterns of the restricted plastid DNAs were detected, indicating that no major rearrangements, losses, or gains of plastid DNA accompany the transition from chloroplast to chromoplast.     

Control of the rate of cell enlargement: Excision,wall relaxation,and growth-induced water potentials

A new guillotine thermocouple psychrometer was used to make continuous measurements of water potential before and after the excision of elongating and mature regions of darkgrown soybean (Glycine max L. Merr.) stems. Transpiration could not occur, but growth took place during the measurement if the tissue was intact. Tests showed that the instrument measured the average water potential of the sampled tissue and responded rapidly to changes in water potential. By measuring tissue osmotic potential ( _s ), turgor pressure ( _p ) could be calculated. In the intact plant, _s and _p were essentially constant for the entire 22 h measurement, but _s was lower and _p higher in the elongating region than in the mature region. This caused the water potential in the elongating region to be lower than in the mature region. The mature tissue equilibrated with the water potential of the xylem. Therefore, the difference in water potential between mature and elongating tissue represented a difference between the xylem and the elongating region, reflecting a water potential gradient from the xylem to the epidermis that was involved in supplying water for elongation. When mature tissue was excised with the guillotine, _s and _p did not change. However, when elongating tissue was excised, water was absorbed from the xylem, whose water potential decreased. This collapsed the gradient and prevented further water uptake. Tissue _p then decreased rapidly (5 min) by about 0.1 MPa in the elongating tissue. The _p decreased because the cell walls relaxed as extension, caused by _p , continued briefly without water uptake. The _p decreased until the minimum for wall extension (Y) was reached, whereupon elongation ceased. This was followed by a slow further decrease in Y but no additional elongation. In elongating tissue excised with mature tissue attached, there was almost no effect on water potential or _p for several hours. Nevertheless, growth was reduced immediately and continued at a decreasing rate. In this case, the mature tissue supplied water to the elongating tissue and the cell walls did not relax. Based on these measurements, a theory is presented for simultaneously evaluating the effects of water supply and water demand associated with growth. Because wall relaxation measured with the psychrometer provided a new method for determining Y and wall extensibility, all the factors required by the theory could be evaluated for the first time in a single sample. The analysis showed that water uptake and wall extension co-limited elongation in soybean stems under our conditions. This co-limitation explains why elongation responded immediately to a decrease in the water potential of the xylem and why excision with attached mature tissue caused an immediate decrease in growth rate without an immediate change in _p Abbreviations and symbols L tissue conductance for water - m wall extensibility - Y average yield threshold (MPa) - _o water potential of the xylem - _p turgor pressure - _s osmotic potential - _w water potential of the elon gating tissue     

ATP-gamma-S (adenosine 5'-0(3-thiotriphosphate)) blocks progesterone-induced maturation of the Xenopus oocyte

ATP-gamma-S microinjection into Xenopus oocyte prevents progesterone induced maturation. Inhibition is time and dose dependent; 50% inhibition occurs when 50 nl of 0.5 mM ATP-gamma-S solution are microinjected/oocyte 1 hr prior to the hormonal trigger. ATP-gamma-S inhibited oocytes can be induced to mature (100%) following microinjection of extracts containing maturation promoting factor (MPF). Our results suggest that the maturation protein(s) has been stabilized in ovo by ATP-gamma-S microinjection, in its phosphorylated inhibitory form.     

Antigenic probes locate the myosin subfragment 1 interaction site on the N-terminal part of actin

The interaction of two different anti-actin antibody populations with the myosin subfragment 1-F-actin rigor complex has been studied. In contrast with the 1–7 sequence, the 18–28 sequence appears to be strongly implicated in the contact area of the myosin head on the actin polypeptide chain.     

Induction by chemically modified actin derivatives of antibody specificity: A relation between modified sites and antibody interactions with monomeric and filamentous actins

A comparison of specific antibodies induced by unfolded actins modified either by oxidation or by arylation of lysine residues was reported. We have focused our work on binding properties with filamentous actin and located its preferential antigenic sites for the anti-arylated-actin antibodies in the C-part of the molecule. An interference of anti-oxidized actin antibodies upon actin polymerisation has also been reported.     

The incorporation of iron into apoferritin as mediated by ceruloplasmin

Ceruloplasmin, a copper ferroxidase, promotes the incorporation of Fe(III) into the iron storage protein, apoferritin. The product formed is identical to ferritin as judged by polyacrylamide electrophoresis and iron/protein measurements. Of several proteins examined, only apoferritin accumulates the Fe(III) produced by ceruloplasmin. When ceruloplasmin was replaced by tyrosinase, which we have shown to have ferroxidase activity, no iron incorporation into apoferritin was observed. It is proposed that Fe(III) is transferred directly and specifically to apoferritin. These data support a more specific role for ceruloplasmin in iron metabolism than has previously been proposed.