Hemoglobin alpha-chain variation in macaques:primary structures of the alpha 1 and alpha II chains from the adult hemoglobins of Malaysian Macaca nemestrina.

The complete primary structures of each of the two α chains commonly found in the adult hemoglobins of Malaysian Macaca nemestrina (pig-tailed macaques) were obtained from the intact chains and five fragments produced by two nonenzymatic and three enzymatic cleavage reactions. The two chains differ at a single site, the α^I chains having a glutaminyl residue and the α^II chains having a histidinyl residue in position 78. Both chains differ from their human counterpart at five positions, the extent of divergence being similar to that observed for most of the α chains from other species of Macaca that have been analyzed to date. Elucidation of the structural difference between the α^I and α^II chains demonstrates that the high degree of heterogeneity observed among the hemoglobin phenotypes of M. nemestrina is a consequence of underlying genetic variability and not a result of postsynthetic modification of genetically identical proteins. Comparisons with the hemoglobin phenotypes found in other species of Macaca support the contention that chromosomes bearing linked α^I and α^II genes, as well as those bearing single α^I and α^II alleles, combine in zygotes to produce the phenotypic variation observed in members of Malaysian populations of M. nemestrina.     

Reconstitution of binding protein-dependent ribose transport in spheroplasts of Escherichia coli K-12.

Purified Escherichia coli K-12 ribose binding protein was used to reconstitute the high affinity ribose transport system in spheroplasts derived from ribose-induced cells. It was not possible to reconstitute ribose transport in spheroplasts derived from uninduced cells or from transport-negative mutant strains, suggesting that one or more additional inducible components are required for binding protein-dependent ribose transport. It was possible to reconstitute transport in a ribokinase-deficient mutant which constitutively transports but does not utilize ribose.     

Hemogloblin α-gene duplication in macaques: Individual Macaca nemestrina with three structurally different α chains

The diversity of hemoglobin phenotypes observed among Malaysian Macaca nemestrina (pig-tailed macaques) has been attributed, in part, to the presence of duplicated α-chain loci in some members of this species. To date, evidence in support of this view has been indirect, consisting of variation in proportions of α^I (Asp₇₁, Gln₇₈) and α^II (Asp₇₁, His₇₈) chains among presumptive heterozygotes. However, the discovery that erythrocytes from some M. nemestrina contain α^I and α^II chains in company with a third, or α^III, chain (Gly₇₁, Gln₇₈) provides direct evidence of duplicated α-chain loci.     

Binding of terbium(III) to yeast enolase

Several independent criteria indicate 2 mol of terbium(III) bind to yeast enolase in the absence of substrate-fluorescence titrations of enzyme and metal, effects on thermal stability and published ultrafiltration and inhibition experiments. These measurements also suggest the terbium binding sites are the same as those normally occupied by “conformational” magnesium. Terbium binds much more strongly than magnesium, however, and measurements of the kinetics of the absorbance change in the terbium-enzyme on adding excess EDTA suggest the terbium-enzyme dissociation constant is about $\text{1}\text{500}$ that of the magnesium-enzyme. Measurements of enzyme activity as a function of substrate concentration show that terbium permits no enzymatic activity. However, magnesium competes more effectively with the lanthanide if the substrate analogue 3-aminoenolpyruvate 2-phosphate (AEP) is present.The fluorescence of the lanthanide is not readily observed on exciting the terbium-enzyme at 280 nm, indicating the absence of tyrosines or tryptophans in the coordination sphere of the metal. Excitation of terbium using 488 nm radiation from an argon ion laser shows the fluorescence of the metal is enhanced by binding to the enzyme. EDTA and carbonate have similar effects. This suggests carboxyl groups are involved in binding metal at the conformational sites of yeast enolase. Measurements of lifetimes of enzyme-bound terbium in the presence and absence of D₂O indicated three moles of water remained on each of the bound metals, independently of the buffer used. If enzyme-bound terbium is assumed to be nine-coordinate, the metal must bind to six groups from the enzyme. The presence of substrate does not markedly affect the emission spectrum of the bound terbium or the number of water molecules remaining on the metal, but calorimetric measurements show that substrate binds to the terbium enzyme.     

Age-dependent changes in myogenic precursor cell compartment sizes. Evidence for the existence of a stem cell

Individual myogenic cells were isolated from the pectoralis muscles of chick embryos from days 8-14 of embryogenesis. When separately cloned, these cells produced three types of colonies in culture: (1) Positive: all cells in the clone were terminally differentiated muscle cells; (2) negative: no cells in the clone were terminally differentiated muscle; (3) mixed: some cells in the clone were terminally differentiated muscle. Positive clones from all ages tended to contain 2n cells (n = 0, 1, 2, 3, 4). Negative clones were found in all sizes and did not cluster around powers of 2 in cell number. Mixed clones were, by far, the most common type among those clones larger than 24 in cell number. Estimates of cell numbers in embryonic muscle tissue revealed that, while the numbers of cells in all myogenic compartments increased steadily with embryonic age, the number and percentage of precursor cells that produced large mixed clones increased dramatically. Subclones, prepared from populations of cells equivalent to large mixed clones, yielded both small positive and large mixed colonies. This indicated that the precursors to the large mixed clones were also precursors to the smaller positive clones. These observations suggest a model for the myogenic lineage in which there exists a stem cell that can generate, by a series of asymmetric divisions, cohorts of terminally differentiated muscle cells. The model can explain the asynchrony of production of terminally differentiated muscle cells both in vitro and in vivo.     

The evolutionary origin of proinsulin: Amino acid sequence homology with the trypsin-related serine proteases detected and evaluated by new statistical methods

Proinsulins and pancreatic serine proteases were analyzed for possible amino acid sequence similarity, using an adapted version of the nucleotide sequence alignment technique of Sankoff (1972). The technique allowed us to determine simultaneously the statistical significance of both the sequence alignment and the number of gaps necessary to achieve that alignment. In the course of this work, it was realized that a rigorous analysis required non-parametric statistics.For the B-chain (amino-terminal) of insulin a highly significant gap-free sequence alignment with the serine proteases was found. For the A-chain (carboxy-terminal) of insulin a sequence alignment of modest statistical significance with two gaps could be obtained, while the search for a corresponding alignment for the C-peptide remained unsuccessful. Presumably the rapid evolution of the C-peptide has obscured its origin. Reconstruction of ancestral sequences was of no help. In contrast to the amino acid sequences, three-dimensional structures of the two protein families are quite different.Considering current histophysiological understanding of ontogeny and phylogeny of exocrine and endocrine pancreas, the observed sequence similarity of proinsulins and serine proteases was interpreted to mean that the two protein families have diverged from a common genetic ancestor. Moreoever, from the organismic distribution of these proteins it was concluded that at least one serine protease existed first, and that proinsulin was generated after duplication of a serine protease gene and subsequent drastic modification, such as a large deletion. Thus proinsulin, basically an anabolic hormone, is derived from a serine protease, an enzyme involved in digestion. This constitutes a refinement of a similar proposal by Steiner et al. (1973).The emergence of proinsulin seems to have occurred after coelenterates diverged, and possibly before most other major animal phyla diverged from the line leading to vertebrates, i.e. 520 to 700 million years ago. The evolution of proinsulin seems to have paralleled the evolution of endocrine cells. Homology of the secreted products of endocrine and exocrine cells was most readily reconciled with a common embryological and phylogenetic origin of the two cell types, as considered by Pictet & Rutter (1972).     

Hormonal control of rates of metamorphic development in the tobacco hornworm Manduca sexta

The rate of metamorphosis in Manduca appears to be under continuous regulation by the circulating titer of the ecdysteroids. Ecdysteroids promote development during the first third of adult differentiation. We present here several lines of evidence indicating that the role of the ecdysteroids then changes to being inhibitory during the later stages of adult differentiation. Abdomen ligation, which precipitously reduces the levels of ecdysteroids in the abdomen, accelerates the rates of tissue development in this region. This acceleration can be counteracted by ecdysteroid injection or by implantation of prothoracic glands. Infusion of ecdysteroids into insects late in development results in a dose-dependent depression in the rate of subsequent development. The effectiveness of a given dosage of steroid is dependent on the developmental stage, with older animals being more affected. Last, the normal ecdysteroid titer declines in a stepwise fashion over the last 3 days of development and these steps are paralleled by a drop-off in the effectiveness of abdomen ligation over this same period. It is concluded that this effect of the ecdysteroids late in development provides a mechanism to ensure that the various tissues of the insect complete metamorphosis in a coordinated fashion.     

Effects of protease inhibitors on sperm-related events in sea urchin fertilization

Evidence for sperm-borne proteolytic enzymes exposed during the acrosome reaction in sea urchin sperm has been accumulating. To investigate the possible role(s) such enzymes have in fertilization, we studied the effects of several protease inhibitors on sperm-related events. Soybean trypsin inhibitor, Nα-p-tosyl-l-lysine, chloromethyl ketone, phenylmethylsulfonyl fluoride, and chymostatin neither reduced the number of acrosome reactions nor interfered with gamete binding. p-Nitrophenyl-p′-guanidinobenzoate caused sperm to fuse into irregular clumps, rendering them unable to fertilize eggs. However, l-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK), an inhibitor of chymotrypsin, prevented the acrosome reaction in Strongylocentrotus purpuratus, S. droebachiensis, and Lytechinus pictus. The effects of TPCK on sperm in subsequent steps of fertilization were also investigated. First, gamete binding assays were performed on fixed eggs. This precluded any effects TPCK might have had on egg-derived secretions (e.g., proteases). Binding of prereacted sperm occurred with both fixed and living eggs. However, fertilization of living eggs in the presence of TPCK was greatly reduced, even though sperm had been prereacted with egg jelly. Vitelline coats were then removed from eggs by trypsin treatment. Eggs in TPCK fertilized and developed normally after the above treatment. These observations are consistent with the hypothesis of a sperm protease participating in the acrosome reaction and the penetration of the egg vitelline coat in the sea urchin.     

Reversible inhibition of aggregation-related cohesivity in Dictyostelium discoideum by diffusible metabolites

Evidence presented elsewhere (G.B. Williams, E.M. Elder, and M. Sussman 1984, Dev. Biol. 105, 377-388) indicates that NH3 and certain carboxylic acids including propionate, succinate, and acetate modulate the cAMP relay in Dictyostelium discoideum. The former appears to act as a cAMP accumulation inhibitor, the latter as cAMP release inhibitors. The cohesive properties of aggregation competent cells have been assayed quantitatively in the presence of these modulators. The following results were obtained: (1) At pH 7.5, EDTA-resistant cohesivity was greatly inhibited by NH4C within the concentration range tested (30-3.8 mM). Even at the higher concentrations the effect was not immediate but required ca. 10 min for full expression. At the lower concentrations, the inhibitory level was only slightly reduced but the time for full expression progressively increased. At pH 6.5, the level of inhibition was marginal, indicating that NH3 is the active molecular species. By themselves, neither ambient pH nor ionic strength appeared to affect cohesive performance within the ranges employed. The inhibition was immediately and completely reversed upon removal of NH4Cl or a shift of ambient pH from 7.5 to 6.5. The presence of cycloheximide did not affect the recovery of cohesivity after NH4Cl removal. (2) The presence of 15 mM succinate, propionate, or acetate also reduced cell cohesivity. The timing and extent of the inhibition were identical at pH 7.5 and 6.5. The inhibition was expressed immediately and was reversible. Each of the acids acted synergistically with NH4Cl. The relative potencies of these metabolites acting singly or in combination as inhibitors of cohesivity corresponded roughly to their potencies as modulators of the cAMP relay (Williams et al., 1984). (3) The sensitivity to the metabolites was stage specific, being maximal during and shortly after aggregation and disappearing abruptly at 11-12 hr. This corresponds to the time at which this cohesive system, responsible for the end-to-end cell associations evident during aggregation (H. Beug, G. Gerisch, S. Kempff, V. Riedel, and G. Cremer, 1970, Exp. Cell. Res. 63, 147-158) is supplanted by a newly arisen, serologically and genetically distinct system which thereafter maintains the integrity of the aggregate (C. Steinemann and R.W. Parish, 1980, Nature (London) 286, 721-724; D.K. Wilcox and M. Sussman, 1981, Dev. Biol. 82, 102-112, and Proc. Natl. Acad. Sci. USA 78, 358-362; C.L. Saxe III and M. Sussman, 1982, Cell 29, 755-759). The activities of the metabolites, detailed above, are discussed in relation to their previously demonstrated activities as morphogens.     

Human thrombin: Partial primary structure

Human thrombin, prepared from an extrinsically activated prothrombin, was finally chromatographed on an affinity column of p-chlorobenzylamido-?-aminocaproyl agarose. After reduction and s-pyridylethylation, the A and B chains were separated. The A chain contains 36 residues and no carbohydrate and has a formula weight of 4093. Its sequence corresponds to 14–49 of the bovine A chain sequence with nine replacements. Human B chain contains 264 residues. The carbohydrate content is 3.5% neutral sugar, 2.6% glucosamine, and 1.5% sialic acid. High-speed sedimentation equilibrium in guanidine gave a minimum molecular weight of 33,500. Sequenator analysis yielded the first 50 amino-terminal residues and established the positions of three of the cyanogen bromide fragments. A fourth fragment lacked homoserine and was placed at the carboxyl-terminus. The four remaining fragments, comprising half of the B chain, were tentatively assigned positions by assuming homology with bovine thrombin and with pancreatic serine proteases. Within the B chain, the active site histidine and serine were both in highly conservative regions, and an Arg-Tyr bond has been identified as a biological degradation site. Sixty-nine percent of the primary structure of human thrombin was identified by sequenator analysis. In comparing this sequence with that reported for the bovine molecule, 26 replacements are present.     

Temperature-sensitive periods and autonomy of pleiotropic effects of l(1)Nts1, a conditional notch lethal in Drosophila.

Analysis of temperature-shift experiments using strains homo- and/or hemizygous for a temperature-sensitive (ts) mutation of the Notch locus, l(1)N^ts1, has permitted us to localize temperature-sensitive periods (TSPs) both for lethality and for adult ectodermal morphology defects. Discrete TSPs for lethality are localized to the first half of the embryonic period, to the second larval instar, to the third larval instar, and to a 15 hr period immediately after pupation. TSPs for adult morphology defects are localized to the second and third larval instars for eyeless-headless and duplicated antenna, to the third larval instar for small and rough (spl-like) eye, eye scar, fused leg segments, shortened tarsal leg segments, Notch wings, and extra macrochaetae, and to the early pupal period for extra and missing microchaetae, fa^g-like rough eye and thick wing vein defects. Within the third larval instar, distinct patterns of eye, wing, and leg defects are observed. There is a striking similarity between the adult morphology defects and TSPs of l(1)N^ts1 and those of the larval and adult locomotor mutant, shi^ts1 (C. A. Poodry, L. Hall, and D. T. Suzuki, 1973, Develop. Biol.32, 373–386). Expression of l(1)N^ts1 also has been studied in genetic mosaics, in which we find that the pleiotropic effects of l(1)N^ts1 are autonomously expressed.     

The role of ribose-binding protein in transport and chemotaxis in Escherichia coli K12.

The ribose-binding protein of Escherichia coli [Willis, R. C., and Furlong, C. E. (1974) J. Biol. Chem.249, 6926–6929] has been shown to be a required common receptor component for high-affinity ribose transport and for chemotaxis toward this attractant. Mutants devoid of the ribose-binding protein are missing high-affinity ribose transport and do not respond chemotactically to this sugar, whereas the response to other attractants is normal. Eight independently isolated ribose-positive revertant strains regained the binding protein, high-affinity ribose transport, and ribose chemotaxis. One revertant which grows slowly on ribose as a sole carbon source did not regain the binding protein, high-affinity transport, or ribose chemotaxis.     

The binding of copper ions to daunomycin and adriamycin

Using visible absorption, CD, ¹H nmr, and epr spectroscopy, the Cu(II) binging properties of daunomycin, adriamycin, and N-trifluoroacetyl daunomycin in water and ethanol have been explored. The drugs form two water soluble complexes having Cu-drug stoichiometries of 1:1 and 1:2, and with apparent pK_as of formation of 5.6 and 6.5, respectively. At pH values above ～8, the drugs form insoluble polymeric complexes with Cu(II). Similar species are also observed in ethanol. The structure of the compounds have been interpreted in terms of binding of the deprotonated hydroxyquinone portion of the drug to the copper ion. No evidence for the binding of the amino group on daunosamine was found.     

A spectroscopic and electrochemical study of the interaction between copper (II) glycylglycyl-L-histidine-N-methylamide and iron (III) tetra-p-sulfophenylporphine

A binuclear complex has been produced by the reaction of an iron porphyrin (sodium tetra-p-sulfophenylporphine iron (III)-FeTPPS) with a copper metallo-tripeptide (copper (II) glycylglycyl-L-histidine-N-methylamide-CuGGH) in aqueous solution. The system has been characterized by electron spin resonance (ESR) spectroscopy, optical absorption spectroscopy, and electrochemical methods. Room-temperature ESR spectra of the copper complex and low-temperature ESR spectra of the iron porphine provide evidence for the formation of a binuclear complex. These findings are supported by absorption spectroscopy and electrochemical studies, and lead to a value of ca. 2 X 10(-3) M-1 (at room temperature) for the equilibrium constant for complex formation. The relevance of this system to the enzymic active site of mammalian cytochrome c oxidase is discussed.     

The effect of cell density on the expression of cell adhesive properties in a cloned rat astrocytoma (C-6)

The cell-cell adhesion characteristic of C-6 astrocytoma cells changes as a function of cell density. Cell suspensions prepared from monolayers having a density lower than 1 × 10⁵ cells/cm² show maximal affinity for plasma membranes and cells obtained from monolayers at densities greater than 1 × 10⁶ cells/cm² shows minimal affinity for plasma membranes. The adhesive component retained on plasma membranes is present at essentially equal levels in membranes prepared from cells at different density. This modulation in cell surface affinity appears to be due to cell-cell contact and appears to represent a suitable model for the study of the modulation of cell-cell adhesion as a result of cell contact.     

Lymphotoxin and interferon production by rossette-separated human peripheral blood leukocytes.

When normal (CBA × DBA)F₁ spleen cells are cultured for 4 days in polyacrylamide vessels, clones of cytotoxic lymphocytes (CLs) are generated. The specificity of these apparently spontaneous CL clones has been investigated by assaying cells from individual clones against pairs of different target cells. CL clones were found to discriminate between the two parental strain splenic blasts, between splenic blasts and syngeneic tumour cells, and between two F₁ splenic blasts induced with different mitogens (LPS and PHA). The CL clones generated spontaneously in culture also discriminate between semisyngeneic targets [DBA blasts and (CBA × DBA)F₁ blasts]. Significant cross-reactivity however, was detected when CL clones were assayed against normal P815 targets and TNP-modified P815 targets.     

Group mating among Norway rats I. Sex differences in the pattern and neuroendocrine consequences of copulation

The copulatory pattern of groups of rats (Rattus norvegicus) was studied in the laboratory in a seminatural environment. In a given mating session, every oestrous female copulated with each male; likewise, every male copulated with each oestrous female. While individual males and females experienced similar amounts of copulation, there were dramatic sex differences in sequence and temporal pattern. Males mated in a multiple intromission pattern and had more ejaculatory series when several females were in oestrus. In contrast, females received intromissions and ejaculations in a random order, not in the sequence of a male ejaculatory series. Males copulated at shorter intervals than females did, a temporal sex difference that was determined by the pattern of female solicitations and male approaches. These sex differences are used to discuss the different units of analysis that are appropriate for male and female sexual behaviour in this species. Furthermore, the sex differences in the temporal pattern of copulation which emerged during group mating parallel the known sex differences in the temporal parameters of the neuroendocrine reflexes which mediate successful reproduction in the domestic strain.     

The binding of copper(II) and zinc(II) to oxidized glutathione

1H and 13C NMR studies of Zn(II) binding to oxidized glutathione (GSSG) in aqueous solution over the pH range 4-11 show that it forms a complex with a 1:1 Zn:GSSG stoichiometry. At pH values between 6 and 11 the metal ligands are the COO- and NH2 groups of the glutamate residues. Below pH 5 the glycine end of the molecule also binds to the metal ions. EPR and visible absorption spectra of Cu(II) GSSG solutions suggest that similar complexes are formed with Cu(II). The solid products obtained from these solutions are shown by analysis and EPR to be primarily binuclear with Cu2GSSG stoichiometry, although the structures depend on the pH and stoichiometry of the solution from which they were obtained.