Compartmental restrictions and blastema formation during pattern regulation in Drosophila imaginal leg discs

Clonal analysis was used to study the regulative behavior of cells in Drosophila imaginal leg discs. Such studies performed during normal development of the leg have revealed a clonal restriction known as the anterior-posterior compartment boundary (E. Steiner, 1976, Wilhelm Roux Arch. Entwicklungsmech. Organismen180, 9–30). When we tested a regulating fragment that contained cells from both anterior and posterior compartments this clonal restriction was maintained in the original fragment. However, in material produced by regulation, clones of cells induced at the time of fragmentation differentiated anterior and posterior structures. Clonal restriction was observed in the regulated material when clones were induced during in vivo culture. We deduced the position of the dividing cells that contributed to regulative growth from the positions of the clones within the differentiated disc tissue. We observed that initially the majority of dividing cells originated from and were localized to an area close to a region of the wound, resembling a blastema. As regulation proceeded, a localization of dividing cells was maintained. However, the blastema changed its position from the original material into the regulated part.     

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.     

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.     

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.     

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.     

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.     

Disruption of the tryptophan binding site in the human serum albumin dimer

Monomer and dimer fractions of human serum albumin (HSA) obtained from charcoal-treated Fraction V HSA have very similar fluorescence and circular dichroism spectra, but the dimer neither binds l-tryptophan nor reacts rapidly with p-nitrophenyl acetate. The latter reaction presumably occurs in a major binding site of the monomer as many strongly bound ligands including l-tryptophan, small fatty acid anions (e.g., S.-W. M. Koh and G. E. Means, 1979, Arch. Biochem. Biophys.192, 73–79), and several drugs (e.g, N. P. Sollenne and G. E. Means, 1979, Mol. Pharmacol.15, 754–757) all decrease rates of reaction in direct proportion to their concentration. Binding data for those ligands indicate the presence of less than one binding site per molecule of charcoal-treated Fraction V HSA, and thus appear to reflect only its content of albumin monomer. The absence of that binding site in the dimer may reflect its inclusion within the dimer interface.     

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).     

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.     

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.     

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.     

Is the most frequent allele the oldest?

Exact and approximate expressions are obtained for the probability that the most frequent allele is oldest, in neutral allele models in which all mutations produce new alleles. The higher the mutation rate, the less likely is it that the most frequent allele would be oldest. The results are in agreement with simulation studies by Ewens and Gillespie (1974) (Theor. Popul. Biol.6, 35–57), and limit the range of validity of a suggestion made by Crow (1972) (J. Hered.63, 306–316) with respect to the statistical testing of the neutral allele hypothesis.     

Refinements in the rapid isolation of glutamine synthetase from Escherichia coli

In an earlier paper, we described a procedure for the isolation of glutamine synthetase and the protein product of the groE gene (pgroE) by polyethyleneimine precipitation and affinity chromatography on a Blue Dextran column (Z. F. Burton and D. Eisenberg, 1980, Arch. Biochem. Biophys.205, 478–488). Subsequently we found that several of the steps can be omitted when isolating glutamine synthetase. Two procedures are described which are very rapid and quantitative for the recovery of glutamine synthetase activity and which are useful for handling quantities of cells at least up to 500 g.     

Development of the cation-induced stacking capacity during the biogenesis of higher plant thylakoids

We studied the capacity of the thylakoid membrane to form grana stacks in the presence of cations, monovalent or divalent, added to N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine “low-salt” disorganized plastids during their greening. Grana stacking was monitored by the yield of heavy subchloroplast fractions separated by differential centrifugation after digitonin disruption of plastids (J. H. Argyroudi-Akoyunoglou, 1976, Arch. Biochem. Biophys., 176, 267–274). Primary thylakoids of the agranal protochloroplasts formed in periodic light do not show the cation-induced stacking capacity of the mature green chloroplast thylakoids. Similarly, the cation effect saturates at lower cation concentrations in mature chloroplasts than in plastids of the early stages of greening. The capacity for cation-induced stacking and for saturation of the effect at low cation concentrations appears gradually after exposure to continuous light and parallel to the appearance of chlorophyll b and the polypeptides of the 25,000–30,000 molecular weight range of lipid-free thylakoids, probably derived from the chlorophyll b-rich chlorophyll protein Complex II. The thylakoid peripheral stroma proteins ribulosediphosphate carboxylase and the coupling factor protein are not involved in the cation-induced stacking, since their removal (H. Strotmann, H. Hesse, and K. Edelmann, 1973, Biochim. Biophys. Acta, 314, 202–210) does not affect the thylakoid aggregation.     

Pigeon liver malic enzyme: Involvement of an arginyl residue at the binding site for malate and its analogs

Treatment of malic enzyme with arginine-specific reagents phenylglyoxal or 2,3-butanedione results in pseudo-first-order loss of oxidative decarboxylase activity. Inactivation by phenylglyoxal is completely prevented by saturating concentrations of NADP⁺, Mn²⁺, and substrate analog hydroxymalonate. Double log plots of pseudo-first-order rate constant versus concentration yield straight lines with identical slopes of unity for both reagents, suggesting that reaction of one molecule of reagent per active site is associated with activity loss. In parallel experiments, complete inactivation is accompanied by the incorporation of four [¹⁴C]phenylglyoxal molecules, and the loss of two arginyl residues per enzyme subunit, as determined by the colorimetric method of Yamasaki et al (R. B. Yamasaki, D. A. Shimer, and R. E. Feeney (1981) Anal. Biochem., 14, 220–226). These results confirm a 2:1 ratio for the reaction between phenylglyoxal and arginine (K. Takahashi (1968) J. Biol. Chem., 243, 6171–6179) and yield a stoichiometry of two arginine residues reacted per subunit for complete inactivation, of which one is essential for enzyme activity as determined by the statistical method of Tsou (C. L. Tsou (1962) Acta Biochim. Biophys. Sinica, 2, 203–211) and the Ray and Koshland analysis (W. J. Ray and D. E. Koshland (1961) J. Biol. Chem., 236, 1973–1979). Amino acid analysis of butanedione-modified enzyme also shows loss of arginyl residues, without significant decrease in other amino acids. Modification by phenylglyoxal does not significantly affect the affinity of this enzyme for NADPH. Binding of l-malate and its dicarboxylic acid analogs oxalate and tartronate is abolished upon modification, as is binding of the monocarboxylic acid α-hydroxybutyrate. The latter result indicates binding of the C-1 carboxyl group of the substrate to an arginyl residue on the enzyme.     

Further characterization and thiophosphorylation of smooth muscle myosin

(i) Myosin from chicken gizzards was purified by a modification of an earlier procedure (M. N. Malik, 1978,Biochemistry17, 27–32). When this myosin, as well as that prepared by the method of A. Sobieszek and R. D. Bremel (1975,Eur. J. Biochem.55, 49–60), was analyzed by gradient slab gel using the discontinuous buffer system of Neville (1971,J. Biol. Chem.246, 6328–6334), a closely spaced doublet in the heavy chain and four light chains were observed as opposed to one heavy chain and two light chains with the method of Weber and Osborn (1969, J. Biol. Chem.244, 4406–4412). These findings raise the possibility of the existence of myosin isoenzymes in smooth muscle. (ii) The purified gizzard myosin was found to be free of kinase and phosphatase. Phosphorylation or thiophosphorylation of myosin was observed only by exogenously adding kinase. A maximum of 1.2 mol of ³²P/mol of myosin and 2.3 mol of ³⁵S/mol of myosin were obtained. The actin-activated ATPase activity depended upon the extent of thiophosphorylation of myosin; a four- to fivefold increase in the activity was observed when myosin was fully thiophosphorylated. Thiophosphorylated myosin was found to be more stable than phosphorylated myosin.     

Investigation of the pH dependence of proton uptake by porcine heart mitochondrial malate dehydrogenase upon binding of NADH

The pH dependence of proton uptake upon binding of NADH to porcine heart mitochondrial malate dehydrogenase (l-malate: NAD⁺ oxidoreductase, EC 1.1.1.37) has been investigated. The enzyme has been shown to exhibit a pH-dependent uptake of protons upon binding NADH at pH values from 6.0 to 8.5. Enzyme in which one histidine residue has been modified per subunit by the reagent iodoacetamide (E. M. Gregory, M. S. Rohrbach, and J. H. Harrison, 1971, Biochim. Biophys. Acta253, 489–497) was used to establish that this specific histidine residue was responsible for the uptake of a proton upon binding of NADH to the native enzyme. It has also been established that while there is no enhancement of the nucleotide fluorescence upon addition of NADH to the iodoacetamide-modified enzyme, NADH is nevertheless binding to the modified enzyme with the same stoichiometry as with native enzyme. The data are discussed in relation to the involvement of the essential histidine residue in the catalytic mechanism of “histidine dehydrogenases” recently proposed by Lodola et al. (A. Lodola, D. M. Parker, R. Jeck, and J. J. Holbrook, 1978, Biochem. J.173, 597–605) and the catalytic mechanism of “malate dehydrogenases” recently proposed by L. H. Bernstein and J. Everse (1978, J. Biol. Chem.253, 8702–8707).     

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.     

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).     

Human galactose-1-phosphate uridylyltrsferase: purification and comparison of the red blood cell and placental enzymes

Galactose-1-phosphate Uridylyltransferase (uridine diphosphoglucose: α-d-galactose-1-phosphate Uridylyltransferase, EC 2.7.7.12) has been purified from human red blood cells and placental tissue. The placental enzyme was obtained as a homogeneous protein with a specific activity of about 100 units/mg of protein by a combination of previously published methods (G. R. Helmer, Jr., and V. P. Williams, 1981,Arch. Biochem. Biophys.210, 573–580) and concanavalin A-Sepharose chromatography. The properties of the two enzyme forms have been examined with respect to subunit size, electrophoretic properties, isozyme distribution, kinetic patterns, and immunological properties.