Cell-specific regulation of protein synthesis in the sea urchin gastrula: A two-dimensional electrophoretic study

Protein synthesis in the two cell types of echinoid early gastrulae was analyzed by two-dimensional polyacrylamide gel electrophoresis and fluorography. Epithelial cells and primary mesenchyme cells were isolated from early gastrulae as described by M. A. Harkey and A. H. Whiteley, 1980 (Wilhelm. Roux's. Arch.189, 111–112). Newly synthesized proteins were labeled with [³H]valine, extracted in SDS buffer, and analyzed electrophoretically. Of the 454 labeled proteins analyzed, 58 incorporated [³H]valine at markedly different relative rates in the two cell types, and 69 were labeled exclusively in one or the other cell type. The most rapidly synthesized proteins in gastrula cells constituted a class which exhibited a much higher degree of cell specificity than the total protein population. Several of these rapidly synthesized proteins were analyzed individually. Among those that were synthesized preferentially in primary mesenchyme cells, two low-molecular-weight, acidic proteins, designated PM28 and PM32, accounted for 9–14% of the total protein synthesis in primary mesenchyme cells but were barely detectable in epithelial cells. Those proteins that were synthesized preferentially in the epithelial cells included several low-molecular-weight species, probably histones, and the cytoskeletal proteins, actin and tubulin. These data indicate that the primary mesenchyme and epithelium of the early gastrula differ profoundly with respect to the synthesis of specific proteins.     

Phosphate transport in fertilized sea urchin eggs. II. Effects of metabolic inhibitors and studies on differentiation

Metabolic inhibitors were applied after the transport system was fully developed in concentrations sufficient to block cleavage. 0.5–1.0 × 10^?4 M cyanide and anaerobiosis caused from negligible to moderate (40%) inhibition of phosphate uptake. The inhibition occurred late in the breeding season, and the inhibitory action of cyanide on uptake was associated with irreversible developmental effects. Azide (3 × 10^?3 M) did not inhibit uptake when the chamber method was used, but the aliquot and Hopkins' tube methods gave considerable inhibition. Purified preparations of 2,4-dinitrophenol (1 × 10^?4 M) did not inhibit uptake. Sodium iodoacetate (up to 0.05 M) and phlorizin (0.005 M) exerted no effect. Calculations of the minimal work requirement for the transport process reveal that this amounts to only a small fraction (0.24% at an external phosphate concentration of 2 μM) of the total available metabolic energy. Exposure of eggs at five minutes after insemination (lag phase) to cyanide (5 × 10^?5 M), anaerobic conditions, or azide (3 × 10^?3 M) blocked the expected increase of phosphate uptake. Removal of the inhibitors led to resumption of development and the appearance of the phosphate transport system in an essentially normal pattern. Exposure of eggs to 1.4–2.0 × 10^?4 M p-chloromercuribenzoate (p-CMB) during the accumulation phase severely depressed phosphate uptake, but cleavage was not inhibited nor delayed; recovery from the inhibition was accelerated by 1 × 10^?3 M cysteine. Exposure to p-CMB during the lag phase blocked the appearance of the transport system; cleavage proceeded normally. After the removal of p-CMB little reversal occurred until the addtion of 1 × 10^?3 M cysteine, when the phosphate transport system developed in an essentially normal manner. Trypsin (0.001–0.01%) neither activates the transport system in unfertilized eggs, nor inactivates it in denuded fertilized eggs by removal of surface proteins. The data are consistent with the conclusion that (1) the phosphate transport system is newly synthesized at fertilization in energy dependent reactions, and (2) phosphate transport is a carrier mediated process not directly dependent on metabolic energy.     

Blood parasites of some waterfowl from Victoria, Australia.

A total of 316 anatids (5 species) from Serendip Wildlife Research Station, Lara, Victoria, were examined for blood parasites. Twenty-two of the ducks (all five species) harbored Haemoproteus nettionis and one also harbored Plasmodium relictum. None of 12 dusky moorhens (Gallinula tenebrosa) were infected. There was no significant difference in the prevalence of H. nettionis between species or age groups of ducks. No evidence of infection with Leucocytozoon, Trypanosoma or microfilaria was obtained.     

Purification and properties of alpha-ketoglutarate reductase from Micrococcus aerogenes

Micrococcus aerogenes grown in media containing glutamate has high levels of glutamate dehydrogenase and alpha-ketoglutarate reductase. The latter enzyme catalyzes the reversible reduction of alpha-ketoglutarate to alpha-hydroxyglutarate in the presence of reduced nicotinamide adenine dinucleotide (NADH). The enzyme has a high specificity for both substrates in either direction and displays Michaelis-Menten kinetics at moderate substrate concentrations. K(m) values of 0.12 to 0.17 mm alpha-ketoglutarate and 0.3 mm NADH for the forward reaction were calculated from data obtained at low substrate concentrations. At high concentrations, this reaction was inhibited by both substrates. The reverse reaction, which proceeded at 0.1 to 0.2 times the rate of the forward reactions, was inhibited by one of the products, alpha-ketoglutarate. K(m) values for the substrates of this reaction were 10 mm for alpha-hydroxyglutarate and 1 mm for nicotinamide adenine dinucleotide. alpha-Ketoglutarate reductase has a molecular weight of 7.5 x 10(4) to 8.2 x 10(4) and is composed of identical polypeptide chains with a molecular weight of 3.6 x 10(4) to 3.8 x 10(4).     

Bacillus subtilis bacteriophages SP82, SPO1, and phie: a comparison of DNAs and of peptides synthesized during infection.

The genomes of Bacillus subtilis phages phie, SPO1, and SP82 were compared by DNA-DNA hybridization, analysis of DNA fragments produced by digestion with restriction endonucleases, comparison of the arrays of peptides synthesized during infection, and phage neutralization. DNA-DNA hybridization experiments indicated that about 78% of the SP82 DNA was homologous with SPO1 DNA, whereas 40% of the phie DNA was homologous to either SPO1 or SP82 DNA. Agarose gel electrophoresis was used to compare the molecular weights of DNA fragments produced by cleavage of SP82, SPO1, and phie DNAs with the restriction endonucleases Hae III, Sal I, Hpa II, and Hha I. Digestion of the DNAs with Hae III and Sal I produced only a few fragments, whereas digestion with Hpa II and Hha I yielded 29 to 40 fragments, depending on the DNA and the enzyme. Comparing the Hpa II fragments, 51% of the SP82 fragments had mobilities which matched those of SPO1 fragments, 32% of the SP82 fragments matched the phie fragments, and 34% of the SPO1 fragments matched the phie fragments. Comparing the Hha I digestion products, 62% of the SP82 fragments had mobilities matching the SPO1 fragments, 24% of the SP82 fragments matched the phie fragments, and 22% of the SPO1 fragments matched the phie fragments. Analysis of peptides by electrophoresis on one-dimensional sodium dodecyl sulfate-polyacrylamide slab gels showed that approximately 70 phage-specific peptides were synthesized in the first 24 min of each infection. With mobility and the intervals of synthesis as criteria, 66% of the different SP82 peptides matched the SPO1 peptides, 34% of the SP82 peptides matched the phie peptides, and 37% of the SPO1 peptides matched the phie peptides. Phage neutralization assays using antiserum to SP82 yielded K values of 510 for SP82, 240 for SPO1, and 120 for phie.     

Elastic response of the roots of field crops

The elasticity of root tips was investigated in experiments of the static clamped-beam type. The response was non-linear. The results were interpreted in terms of a generalized elastic modulus M , and two parameters, α and β, which account for the non-linearity.
Seminal axes and primary lateral roots of 16 crop species grown in the laboratory show significant inter-species differences but insignificant inter-variety differences in the elastic properties. A smaller set of results for field-grown roots with two tillage treatments shows that, in nearly all cases, the elastic modulus is smaller and the diameter is larger than with laboratory-grown roots. Environmental factors such as water potential, and nutrition have significant influences on the diameters and elastic behaviour of roots.     

A fluorescent derivative of methotrexate as an intracellular marker for dihydrofolate reductase in L1210 cells

Fluorescein isothiocyanate coupled via a diaminopentyl-linking group to methotrexate (G.R. Gapski, J. M. Whiteley, J. I. Rader, P. L. Cramer, G. B. Henderson, V. Neef, and F. M. Huennekens, 1975, J. Med. Chem.18, 526–528) produces a fluorescent compound which is a strong inhibitor of dihydrofolate reductase (K_i = 60 nM) purified from L1210 murine leukemia cells. The fluorescent methotrexate derivative is preferentially taken up by methotrexate-resistant rather than wild-type L1210 cells grown in culture and acts as a visual marker for dihydrofolate reductase (K_D = 50 nM) during both purification and polyacrylamide electrophoresis. Uptake, which is proportional to the level of dihydrofolate reductase (often an indicator of the degree of acquired cellular methotrexate resistance), occurs slowly and via a route that is distinct from the carrier-mediated system utilized by these cells to transport methotrexate.     

The effect of the width of the ventilation-perfusion distribution on arterial blood oxygen content

We investigate the effect of the width of ventilation-perfusion distributions on arterial blood oxygen content. We assume that the perfusion within the alveolar volume is a continuous function of ventilation-perfusion ratio, known as the continuous ventilation-perfusion distribution, and then write down the conservation of mass equations in the lung incorporating the nonlinear relationship between oxygen concentration in the gas phase and blood oxygen content. We solve these equations for various unimodal and bimodal ventilation-perfusion distributions believed to occur in practice and calculate the arterial blood oxygen content in each case. When a subject has a unimodal ventilation-perfusion distribution we show that the fraction of cardiac output to that mode (i.e. the fraction of non-shunted blood) has a large effect on arterial oxygen blood content. However, the width of the distribution has only a negligible effect on arterial oxygen blood content. For a bimodal ventilation-perfusion distribution the location and fraction of cardiac output to each mode has a large effect on arterial oxygen blood content. Again, the width of each mode of the distribution has little effect on arterial oxygen blood content. As a result there is little point, from a clinical perspective, in developing techniques for investigating the width of modes of these distributions since all relevant clinical information is contained in the nature (i.e. unimodal or bimodal) and in the location of the modes.     

The deformation of soil by penetrometers and root tips ofPisum sativum

Channels were formed by seminal roots ofPisum sativum and a steel penetrometer of similar dimeter in blocks of remoulded and weathered soil. For both types of channels, the soil was equilibrated and maintained at –12kPa matric water potential during formation. Small samples of soil containing channels were then excavated and examined using a scanning electron microscope. Sections of root channels were found to contain a clearly differentiated zone of newly remoulded soil containing oriented clay. In contrast to channels created by the rigid steel probe, the newly remoulded zone surrounding root channels did not exhibit either a region of maximum soil compression at the channel surface or a radial pattern of shear failure and compression. This micromorphological evidence suggests that exudates may have an additional role to play in reducing the mechanical strength of soil in the proximity of the root tip. The mechanism is thought to operate through an accumulation of soil water related to solute potential and a resultant increase in matric potential.