Trehalose-6-phosphate synthetase activity in extracts of Dictyostelium discoideum.

Trehalose-6-phosphate (T-6-P) synthetase activity in extracts of Dictyostelium discoideum has been reexamined in an effort to resolve discrepancies between the results of previous studies (R. Roth and M. Sussman (1966). Biochim. Biophys. Acta, 122, 225; K. A. Killick and B. E. Wright (1972). J. Biol. Chem., 247, 2967). We find that T-6-P synthetase is not cold sensitive as reported by Killick and Wright (1972), is not present in bacterial-grown vegetative cells (though subject to some modulation by other nutritional conditions), and is not in our hands unmasked or activated by ammonium sulfate fractionation. We conclude that the pattern of T-6-P synthetase accumulation and disappearance during fruiting body construction in D. discoideum is as originally described by R. Roth and M. Sussman (1968). J. Biol. Chem., 243, 5081) and confirmed elsewhere (P. C. Newell et al. (1972). J. Mol. Biol., 63, 373; R. W. Brackenbury et al. (1974). J. Mol. Biol., 90, 529; B. D. Hames and J. M. Ashworth (1974). Biochem. J., 142, 301).     

Proteolysis in eucaryotic cells: Aminopeptidases and dipeptidyl aminopeptidases of yeast revisited

Using nine different l-aminoacyl-4-nitroanilides and four different dipeptidyl-4-nitroanilides, aminopeptidases and dipeptidyl aminopeptidases active at pH 7.5 and (or) pH 5.5 in logarithmically growing and stationary-phase cells of Saccharomyces cerevisiae were searched for. Ion-exchange chromatography was used to separate the proteins of the soluble cell extract. Besides the three already-characterized aminopeptidases—aminopeptidase I (P. Matile, A. Wiemken, and W. Guyer (1971) Planta (Berlin)96, 43–53; J. Frey and K. H. Röhm (1978) Biochim. Biophys. Acta527, 31–41), aminopeptidase II (J. Frey and K. H. Röhm (1978) Biochim. Biophys. Acta527, 31–41; J. Knüver (1982) Thesis, Fachbereich Chemie, Marburg, FRG), and aminopeptidase Co (T. Achstetter, C. Ehmann, and D. H. Wolf (1982) Biochem. Biophys. Res. Commun.109, 341–347)—12 additional aminopeptidase activities are found in soluble cell extracts eluting from the ion-exchange column. These activities differ from the characterized aminopeptidases in one or more of the parameters such as charge, size, substrate specificity, inhibition pattern, pH optimum for activity and regulation. Also, a particulate aminopeptidase, called aminopeptidase P, is found in the nonsoluble fraction of disintegrated cells. Besides the described particulate X-prolyl-dipeptidyl aminopeptidase (M. P. Suarez Rendueles, J. Schwencke, N. Garcia-Alvarez and S. Gascon (1981) FEBS Lett.131, 296–300), three additional dipeptidyl aminopeptidase activities of different substrate specificities are found in the soluble extract.     

Cell death during development of the topographical distributions of cutaneous sensory neurons in amphibian ganglia

During development of the topographical distribution of cutaneous sensory neurons in amphibian ganglia, neurons innervating back skin are eliminated from the ventral half of the ganglia (M. R. Bennett and K. Lai, 1981, Develop. Biol., 86, 212–223). The possibility that the emergence of the mature topographical distribution is due to cell death has been tested. Neurons with axons in the dorsal cutaneous nerve at stage 14 have been labeled with horseradish peroxidase (HRP), and the tadpoles allowed to survive till stage 22, when the distribution of HRP-labeled cells was determined. Labeled cells died throughout the ganglia, but in much greater numbers in ventral than dorsal ganglia; this cell death was sufficient to account for the elimination of ventral cells with dorsal projections described in Bennett and Lai (1981). The projections of sensory neurons may be specified according to their position in the ganglion; those with axons in the incorrect ramus may then be eliminated by cell death.     

Cotranslational cleavage of immunoglobulin light chain precursors by plasmacytoma microsomes.

Murine plasmacytoma endoplasmic reticulum which has been freed of ribosomes by EDTA treatment is capable of the cotranslational proteolytic processing of representative λ₁,λ₂, and k immunoglobulin light chain precursors. Messenger RNA fractions from the MOPC-104E, MOPC-315, and MOPC-46B tumor lines were used to direct the synthesis of the light chain precursors in a cell-free system derived from Krebs II ascites cells. The precursor cleavage activity of the plasmacytoma membranes is comparable in activity and in characteristics to that of two well-defined membrane preparations: Krebs II ascites intracellular membranes (E. Szczesna and I. Boime, 1976, Proc. Nat. Acad. Sci. USA73, 1179–1183) and EDTA-treated rough endoplasmic reticulum from canine pancreas (34., 35., J. Cell Biol.67, 852–862). The efficiency of the cleavage reaction appears to be dependent upon the precursor being utilized as a substrate. An assay suitable for a preliminary characterization of the plasmacytoma membrane preparations is described.     

Cell surface glycosyltransferase activities during normal and mutant (TT) mesenchyme migration

Migrating cells possess surface glycosyltransferase activity toward extracellular substrates, and the appearance of enzyme activity coincides with the onset of cellular migration (Shur, 1977a, Shur, 1977b, Develop. Biol.58, 23–39, 40–55; E. A. Turley and S. Roth, 1979, Cell17, 109–115). In this paper, surface glycosyltransferases were examined during normal and $\text{T}\text{T}$ mutant mesenchyme migration. Of six glycosyltransferases that were assayed, only galactosyltransferase was present at significant levels on the cell surface, despite the presence of a variety of intracellular glycosyltransferases. All controls have been performed to show clearly the enzyme activity was cell surface localized. In both normal and $\text{T}\text{T}$ embryos, surface galactosyltransferase activity was localized, by autoradiography, primarily to migrating mesenchymal cells, and to a lesser degree, to presumptive neural epithelium. During primitive streak formation, putative $\text{T}\text{T}$ embryos were devoid of surface galactosyltransferase activity. However, as development progressed, the $\text{T}\text{T}$ level of activity eventually exceeded wild-type levels by two- to sixfold and was evident in $\text{T}\text{T}$ tissues prior to the onset of microscopic pathology. Other surface enzymes assayed did not show any $\text{T}\text{T}\text{-}\text{dependent}$ increase in activity. The extracellular galactosyl acceptors were not chloroform:methanol soluble, and glycopeptides prepared by exhaustive Pronase digestion were excluded from Sephadex G-50. This large galactosylated glycoconjugate was readily digestable with endo-β-galactosidase, and, therefore, is similar to the poly-N-acetyllactosamine chains previously identified on early embryonic tissues (A. Kapadia, T. Feizi, and M. J. Evans, 1981, Exp. Cell. Res.131, 185–195; T. Muramatsu, G. Gachelin, M. Damonneville, C. Delarbre, and F. Jacob, 1979, Cell18, 183–191; A. Heifetz, W. J. Lennarz, B. Libbus, and Y. -C. Hsu, 1980, Develop. Biol.80, 398–408). These results support an involvement of surface galactosyltransferases in mesenchyme formation and during migration on poly-N-acetyllactosamine substrates.     

A note on the sampling theory for infinite alleles and infinite sites models

This note considers sampling theory for a selectively neutral locus where it is supposed that the data provide nucleotide sequences for the genes sampled. It thus anticipates that technical advances will soon provide data of this form in volume approaching that currently obtained from electrophoresis. The assumption made on the nature of the data will require us to use, in the terminology ofKimura (Theor. Pop. Biol.2, 174–208 (1971)), the “infinite sites” model of Karlin and McGregor (Proc. Fifth Berkeley Symp. Math. Statist. Prob.4, 415–438 (1967)) rather that the “infinite alleles” model of Kimura and Crow (Genetics49, 174–738 (1964)). We emphasize that these two models refer not to two different real-world circumstances, but rather to two different assumptions concerning our capacity to investigate the real world. We compare our results where appropriate with corresponding sampling theory of Ewens (Theor. Pop. Biol.3, 87–112 (1972)) for the “infinite alleles” model. Note finally that some of our results depend on an assumption of independence of behavior at individual sites; a parallel paper byWatterson (submitted for publication (1974)) assumes no recombination between sites. Real-world behavior will lie between these two assumptions, closer to the situation assumed by Watterson than in this note. Our analysis provides upper bounds for increased efficiency in using complete nucleotide sequences.     

An autoradiographic analysis of the time of appearance of neurons in the developing chick neural retina

The pattern of incorporation of [³H]thymidine into the chick neural retina has been used to establish the time and order in which different classes of neuroepithelial cells withdraw from the cell cycle and initiate migration and differentiation.The posterior pole of the retina is the first to form during development. In this region most neuroepithelial cells complete mitotic activity between the third and sixth day of incubation. Presumptive ganglion cells initiate the withdrawal process, and they are soon followed by the neuroepithelial precursors of amacrine, horizontal, and receptor cells. Bipolar cell precursors are the last to begin and the last to complete cell cycle activity. It is worthy of note, however, that, in any given region of the retina, neuroepithelial cells of all types cease mitosis in close, overlapping succession.These results are in reasonable agreement with those previously published on the chick retina by Fujita and Horii (1963), and other investigators on the mouse (Mus), killifish (Fundulus), and toad (Xenopus). The present data are also consistent with those proposals of Angevine (1970), Jacobson, 1968a, Jacobson, 1968b, Jacobson, 1970, and others that relate the cessation of mitotic activity of neuroepithelial cells to the determination of neuronal size, axon length, and the specification of neuronal connections.     

An approach to a physico-chemical model of olfactory stimulation in vertebrates by single compounds

During recent years, numerous attempts have been made to correlate both quantitative (Davies &; Taylor, 1959; Engen, 1962; Beck, 1964; Engen, Cain &; Rovee, 1968; Cain, 1969; Dravnieks &; Laffoit, 1970; Laffort, 1969a,b) and qualitative (Davies, 1965; Amoore &; Venstrom, 1965; Döving, 1966a,b; Wright &; Michels, 1964; Leveteau &; MacLeod, 1969) odorous properties of single compounds to their molecular properties. These attempts have been only partially successful.In the present paper we will try to explain the several odorous properties of single compounds on the basis of the non-specific properties of odorants involved in solubility.This model is a first approach, and although it gives statistically highly significant relations, it is not as accurate as those advanced with respect to the physical and sensory dimensions of stimuli in the fields of vision and audition.We will first give the present definitions of the most suitable physicochemical parameters, and then advance quantitative and qualitative models for single compounds. Quantitative odorous properties are: odour threshold, rate of change of odour intensity with odorant concentration in the suprathreshold region, and the somewhat controversial upper odour intensity. Qualitative properties refer to odour character.     

Quantitative dependence of mitochondrial oxidative phosphorylation on oxygen concentration: a mathematical model.

The model of Wilson and co-workers (2., 3., Arch. Biochem. Biophys. 182, 749–762) for the regulation of mitochondrial oxidative phosphorylation has been extended to include the dependence on oxygen tension. The derived rate expression correctly describes the observed dependence of cellular energy metabolism on oxygen tension, including the oxygen dependence at “normoxic” physiological values. Experimental evidence is presented that oxidative phosphorylation by suspensions of isolated rat liver mitochondria is also dependent on oxygen concentration up to values of at least 100 μM.     

The fine structure of Ameson pulvis (Microspora,Microsporida) and its implications regarding classification and chromosome cycle

Nosema pulvisPerez, 1905, Ameson pulvis (Perez) Sprague, 1977, in muscles of the crabs Carcinus maenas and C. mediterraneus from the coast of France, was observed with the electron microscope. It was found to be structurally similar to the type species A. michaelis (Sprague, 1970). Sprague, 1977, having moniliform sporogonial plasmodia, unikaryotic sporoblasts, and hirsute sporulation stages. It is treated as distinct from A. michaelis because it has slightly smaller spores (by comparison with syntype material of A. michaelis) and appears to have fewer coils in the polar filament. The results require the removal of the genus Ameson from the family Nosematidae Labbé, 1899, where Sprague (1977) had placed it under the erroneous supposition that its sporoblasts are diplokaryotic. Ameson is transferred to family Unikaryonidae Sprague, 1977. Ameson is distinguished from PereziaLéger and Duboscq, 1909, shown by Ormieres et al. to have a similar developmental pattern, by presence of appendages on its sporulation stage. A. nelsoni (Sprague, 1950), the third, and only other species of Ameson, lacks the appendages and is transferred to genus Perezia.     

Negative chemotaxis does not control quail neural crest cell dispersion

Negative chemotaxis has been proposed to direct dispersion of amphibian neural crest cells away from the neural tube (V. C. Twitty, 1949, Growth13(Suppl. 9), 133–161). We have reexamined this hypothesis using quail neural crest and do not find evidence for it. When pigmented or freshly isolated neural crest cells are covered by glass shards to prevent diffusion of a “putative” chemotactic agent away from the cells and into the medium, we find a decrease in density of cells beneath the coverslip as did Twitty and Niu (1948, J. Exp. Zool.108, 405–437). Unlike those investigators, however, we find the covered cells move slower than uncovered cells and that the decrease in density can be attributed to cessation of cell division and increased cell death in older cultures, rather than directed migration away from each other. In cell systems where negative chemotaxis has been demonstrated, a “no man's land” forms between two confronted explants (Oldfield, 1963, Exp. Cell Res.30, 125–138). No such cell-free space forms between confronted neural crest explants, even if the explants are closely covered to prevent diffusion of the negative chemotactic material. If crest cell aggregates are drawn into capillary tubes to allow accumulation of the putative material, the cells disperse farther, the wider the capillary tube bore. This is contrary to what would be expected if dispersion depended on accumulation of this material. Also, no difference in dispersion is noted between cells in the center of the tubes versus cells near the mouth of the tubes where the tube medium is freely exchanging with external fresh medium. Alternative hypotheses for directionality of crest migration in vivo are discussed.     

Inhibition and activation of fat cell adenylate cyclase by GTP is mediated by structures of different size

The adenylate cyclase system in the plasma membrane of fat cells contains regulatory components that either stimulate or inhibit activity in response to ligands acting at the cell surface. GTP is required for both the stimulation by various hormones (catecholamines and peptide hormones) and the inhibition by adenosine. We have analyzed the effects of high-energy radiation on the stimulatory and inhibitory processes and conclude that these processes are mediated by structures of different functional size. Moreover, the fat cell cyclase system, when analyzed under conditions in which the inhibitory action of GTP is minimally expressed, displays targets of the same size as those previously observed for those involved in the activation of the hepatic enzyme by glucagon and guanine nucleotides (W. Schlegel, E. S. Kempner, and M. Rodbell, 1979, J. Biol. Chem.254, 5168–5176). These findings extend our recent evidence for the nonidentity of the two GTP-mediated processes (D. M. F. Cooper, W. Schlegel, M. C. Lin, and M. Rodbell, 1979, J. Biol. Chem.254, 8927–8931).     

A model for propagation of action potentials in smooth muscle

A modified Hodgkin & Huxley (1952) model for axons was used to simulate smooth muscle action potentials. The modifications were such as to match our own experimental results and published data on the passive and active behavior of smooth muscle.A brief account of the modifications introduced to the HH model is as follows. The resting ionic conductances were obtained from the data of Casteels (1969). Chloride conductance was replaced by an ad hoc leakage conductance ($\text{g}\text{?}{}_{\mathrm{L}}$) in order to obtain a resting membrane resistance of about 11 kΩcm². The ionic equilibrium potentials were according to Kao & Nishiyama (1969). The rate constants m, n and h have similar form to those in axons, but their different numerical values produce action potentials that match the duration of the smooth muscle action potential (about 16 ms) at half its maximum amplitude. The effective membrane capacitance was taken as 2.5 μF/cm².The results obtained by implementing those smooth muscle parameters in the HH formulation include: (a) a membrane potential that matches the main characteristics of experimentally recorded action potentials in uterine smooth muscle and guinea-pig taenia-coli, and (b) a propagated action potential which, on a cable diameter of 5 μm (similar to the diameter of a single smooth muscle cell), has a speed of propagation within the range of the values experimentally recorded in smooth muscle. This observed velocity of propagation is not compatible with a large cable and it is concluded that “functional units” are not required to sustain propagation of action potentials in smooth muscle.     

Transport and metabolism of N-δ-chloroacetyl-l-ornithine by Saccharomyces cerevisiae

The general amino acid transport system of Saccharomyces cerevisiae functions in the uptake of neutral, basic, and acidic amino acids (M. Grenson, C. Hou, and M. Crabeel, 1970,J. Bacteriol. 103, 770–777; J. Rytka, 1975,J. Bacteriol.121, 562–570; C. Darte and M. Grenson, 1975,Biochem. Biophys. Res. Commun.67, 1028–1033). We have previously demonstrated that this transport system can be inhibited by the amino acid, N-δ-chloroacetyl-l-ornithine (NCAO) (F. S., Larimore and R.J. Roon, 1978,Biochemistry17, 431–436). In the present study radiolabeled NCAO was synthesized and its transport and metabolism studied. Under initial rate conditions: (a) NCAO was transported by the general amino acid transport system with a K_m of 52 μm, a V of 32 nmol/min/mg cells, and a pH optimum of 5.0; (b) the V for NCAO transport in gap mutants, which lack the general amino acid transport system, was approximately 1% of that observed with wild-type cells; (c) the V for NCAO in cells deprived of glucose was less than 5% of that observed when glucose was present. NCAO was transiently concentrated more than 1000-fold by yeast cells when glucose served as an energy source. The internal pool of NCAO was metabolized by the yeast cells and the products were excreted. When 100 μm [¹⁴C]NCAO was incubated with a yeast cell suspension for 8 h, more than 95% of the compound was converted into two ninhydrin-negative excretory products. The effect of NCAO on the growth of yeast cells was determined. Wild-type strains did not grow when 1 mm NCAO was present in the medium. The growth of gap mutants was not inhibited by 1 mm NCAO.     

Kinship and covariance

Price's (1970) covariance theorem can be used to derive an expression for gene frequency change in kin selection models in which the fitness effect of an act is independent of the genotype of the recipient. This expression defines a coefficient of relatedness which subsumes r(Wright, 1922), b(Hamilton, 1972), ρ (Orlove &; Wood, 1978), and R(Michod &; Hamilton, 1980). The new coefficient extends the domain of Hamilton's rule to models in which the average gene frequency of actors differs from that of recipients.     

Analysis of glial cell differentiation in peripheral nervous tissue: II. Neurons promote S100 synthesis by purified glial precursor cell populations

Isolated sensory neurons in vitro do not contain or synthesize S100, whereas glial cell precursor populations do. These precursor cells, when isolated from other cell types, produce low levels of S100 but never undergo the developmental transition to produce high levels of S100. When glial cell precursors are combined with isolated, live or paraformaldehyde-fixed sensory neurons, the precursor cells do undergo the second transition, and accumulate high levels of S100. Peroxidase-anti-peroxidase immunohistochemical staining for S100 confirms previous conclusions (B. Holton and J. A. Weston, 1982, Develop. Biol.89, 64–71) that only those glial cells which are closely apposed to neurons contain augmented levels of S100. This stimulation appears to be specific to neuronal/glial interactions since live or fixed fibroblasts, when cocultured with glial precursor cells, do not promote accumulation of S100 by the glial cells.     

Liberation of labile sulfur from ferredoxins by alkaline zinc reagent: an appraisal of the methylene blue method.

In disagreement with reported observation by Suhara and her colleagues (K. Suhara, S. Takemori, M. Katagiri, K. Wada, H. Kobayashi, and H. Matsubara, 1975, Anal. Biochem.68, 632–636) we found that more than 90% of labile sulfur was liberated from adrenodoxin within 5 min at 22°C. This rate was faster than those of spinach and clostridial ferredoxins, a result also at variance with Suhara's observation. At low temperature, the reaction was clearly biphasic, and spinach ferredoxin showed a similar profile. In the absence of zinc acetate, activation energies of the decomposition reaction of iron-sulfur center of OH^? were obtained as 39, 26, and 11 kcal/mol for adrenal, spinach, and clostridial ferredoxins, respectively. The adrenal reaction became faster as the dipole moment of the solvent increased. In the presence of 4 m urea and 1 m KCl, the rate was enhanced by approximately 26-fold, relative to the reaction without the addition of urea. In conclusion, the liberation reaction of adrenal labile sulfur with alkaline zinc reagent is fast at 22°C, indicating no need for modification of the original method (T. Kimura and K. Suzuki, 1967, J. Biol. Chem.242, 485–491; P. E. Brumly, R. W. Miller, and V. Massey, 1965, J. Biol. Chem.240, 2222–2228).     

Iron-ethylenediaminetetraacetic acid (EDTA)-catalyzed superoxide dismutation revisited: An explanation of why the dismutase activity of Fe-EDTA cannot be detected in the cytochrome c/xanthine oxidase assay system

The recent assertion of J. Diguiseppi and I. Fridovich (1980, Arch. Biochem. Biophys., 203, 145–150) that Fe-EDTA does not catalyze superoxide dismutation is disputed. By directly observing superoxide generated during pulse radiolysis, we have confirmed the results of a previous study (G. J. McClune, J. A. Fee, G. A. McClusky, and J. T. Groves, 1977, J. Amer. Chem. Soc., 99, 5220–5222) which concluded that Fe-EDTA catalyzed superoxide dismutation. We also demonstrate that the reaction of Fe(II)-EDTA, formed during catalyzed superoxide dismutation, with cytochrome c, the probe molecule in the cytochrome c/xanthine oxidase/xanthine assay system for superoxide dismutase activity, is sufficiently rapid (H. L. Hodges, R. A. Holwerda, and H. B. Gray, 1974, J. Amer. Chem. Soc., 96, 3132–3137) to obscure the weak catalysis of superoxide dismutation by Fe-EDTA.     

Crystalline aggregation of a proteolytic fragment of the major head protein of bacteriophage T4.

An analysis has been made of the composition and structure of the two types of sheets assembled from material from dissociated bacteriophage T2 (Poglazov &; Mesyhanzhinov, 1967) and T4 capsids. Serological techniques have been used to show that both types of sheet are assembled from proteolytic fragment of P23¹, the major capsid constituent. The two types of sheets have been found to interconvert depending on the concentration of Mg²⁺ ions in the buffer. Computer modelling experiments show that the “hexagonal” and “rectangular” morphologies observed in the negative stain are due to in-register and staggered associations, respectively, of a single basic hexagonal lattice. Analysis by polyacrylamide gel electrophoresis of samples of sheets and dissociated capsids, together with previous results from immune electron microscopy (Kistler et al., 1978), suggest that hexamers of the proteolytic fragment are derived conservatively from capsomers of the phage head.The value of this proteolytic P23 fragment has been twofold: (1) it has proved to be a useful peptide in the ongoing primary sequence determination of P23 and (2) antibodies raised against it have been employed to follow the fate of P23 antigenic sites during various steps of phage capsid maturation (Kistler et al., 1978).