Towards quantification of vocal imitation in the zebra finch

The transition from an amorphous subsong into mature song requires a series of vocal changes. By tracing song elements during development, we have shown that the imitation trajectory to the target could not be predicted based on monotonic progression of vocal changes, indicating an internal component that imposes constraints on song development. Here we further examine the nature of constraints on song imitation in the zebra finch. We first present techniques for identifying and tracing distinctive vocal changes, and then we examine how sequences of vocal change are expressed and coordinated. Examples suggest two types of constraints on song imitation, based on the nature of the temporal context. Developmentally diachronic constraints are imposed by sequential dependencies between vocal changes as a function of developmental time, whereas developmentally synchronic constraints are given by the acoustic context of notes within the song. Finally, we show that the tendency of birds to copy certain sounds in the song model before others might be related to such constraints. We suggest that documenting the full range of distinctive vocal changes and the coordination of their expression would be useful for testing mechanisms of vocal imitation.     

Purification and characterization of a hemagglutinin isolated from the leaves of Chenopodium (Chenopodium amaranticolor).

A hemagglutinin was isolated and purified from the leaves of Chenopodium (Chenopodium amaranticolor) using ion-exchange chromatography and affinity chromatography on fetuin-agarose matrix. It agglutinated rabbit erythrocytes. The hemagglutinin had a native molecular mass of 58 kDa, as estimated by gel filtration and showed a single band of molecular mass of 33 kDa on SDS-PAGE. It showed hemagglutination activity over the pH range 3-12 and was found to be stable up to 70 degrees C. On isoelectrofocussing, the pI of this hemagglutinin was estimated to be 5.25. However, it was found to contain seven charge variants when isoelectrofocussing was performed in presence of 6M urea.     

Structure of a biologically active neurotensin-related peptide obtained from pepsin-treated albumin(s)

Using a radioimmunoassay toward the COOH-terminal region of neurotensin, an immunoreactive and biologically active neurotensin-related peptide (NRP) has been isolated from pepsin-treated fractions of bovine, canine, human, and rat plasma. Bovine NRP was identified as H-Ile-Ala-Arg-Arg-His-Pro-Tyr-Phe-Leu-OH, which is similar in structure to both neurotensin and angiotensin I. Canine and human NRP also had the above amino acid composition, whereas that obtained from rat plasma had valine substituted for isoleucine. At their concentrations in pepsin-treated plasmas (2-6 microM) rat, human and canine NRP were shown to increase vascular permeability when injected intradermally into rats and to release histamine from rat mast cells in vitro. The pure peptides also cross-reacted very effectively at nanomolar concentrations in a radioreceptor assay for neurotensin. The protein(s) which liberated NRP upon pepsin treatment were purified about 7-fold and shown to behave like albumin during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isoelectric focusing, and high pressure liquid chromatography on muBondapak C4. In addition, the purified preparations were found to react with anti-albumin antisera during immunodiffusion. Although the amino acid sequence of NRP was not found in albumin, a partial sequence homology was noted for NRP and various segments of bovine albumin. Using V8 protease, glutamyl residues were shown to lie within 3-4 amino acids of each end of NRP, as also occurs for the related segments in albumin. These results suggest that a subset of albumin-related protein(s) could serve as precursor(s) to biologically active neurotensin-related peptide(s).     

Binding of thiocyanate to lactoperoxidase: 1H and 15N nuclear magnetic resonance studies

The binding of thiocyanate to lactoperoxidase (LPO) has been investigated by 1H and 15N NMR spectroscopy. 1H NMR of LPO shows that the major broad heme methyl proton resonance at about 61 ppm is shifted upfield by addition of the thiocyanate, indicating binding of the thiocyanate to the enzyme. The pH dependence of line width of 15N resonance of SC15N- in the presence of the enzyme has revealed that the binding of the thiocyanate to the enzyme is facilitated by protonation of an ionizable group (with pKa of 6.4), which is presumably distal histidine. Dissociation constants (KD) of SC15N-/LPO, SC15N-/LPO/I-, and SC15N-/LPO/CN- equilibria have been determined by 15N T1 measurements and found to be 90 +/- 5, 173 +/- 20, and 83 +/- 6 mM, respectively. On the basis of these values of KD, it is suggested that the iodide ion inhibits the binding of the thiocyanate but cyanide ion does not. The thiocyanate is shown to bind at the same site of LPO as iodide does, but the binding is considerably weaker and is away from the ferric ion. The distance of 15N of the bound thiocyanate ion from the iron is determined to be 7.2 +/- 0.2 A from the 15N T1 measurements.     

Correlation of polarized absorption spectroscopic and X-ray diffraction studies of crystalline cytosolic aspartate aminotransferase of pig hearts

Absorption spectra of large, well-formed crystals of cytosolic aspartate aminotransferase have been recorded using plane polarized light. Making use of measurements of crystal thickness we have calculated extinction coefficients with the electric vector of the light parallel to both the a and c axes of the crystals of the enzyme in space group P2(1)2(1)2(1). The spectra have been resolved into components with lognormal distribution curves and the resulting integrated intensities have been used to calculate the c/a polarization ratios for the absorption bands of the bound co-enzyme pyridoxal 5'-phosphate. From the polarization ratio and the co-ordinates of the co-enzyme ring atoms, provided by X-ray crystallography, we have assigned principal molecular directions of the transition dipole moment within the plane of the co-enzyme ring. Of two possible orientations, only one predicts the correct crystal extinction coefficients for the 436 nm band. In this orientation, when viewed from the B face of the ring (i.e. looking into the active site of the enzyme), the transition moment is related to the N-1-C-4 axis of the ring by counterclockwise rotation by 27 degrees. A tentative assignment of the principal molecular directions of the transition moment has also been made for the 368 nm band of the high pH form of the enzyme. In each case, the plane of the co-enzyme ring was located from the atomic co-ordinates of the ring atoms and of those atoms attached directly to the ring. The projection of the N-1 to C-4 axis on to this plane was used to evaluate the orientation of the transition moment, which was presumed to lie precisely within the plane of the ring. We have tilted this plane systematically to evaluate the error in transition moment direction resulting from uncertainties in the atomic co-ordinates. When 2-methylaspartate is diffused into the crystals if forms a Schiff base with the co-enzyme in which the ring has tilted about 32 degrees from its original position and the polarization ratio of the 436 nm band drops from 1.6 in the free enzyme to about 0.38. On the assumption that the orientation of the transition moment within the co-enzyme does not change during this rotation, this value of the polarization ratio is within experimental error of that predicted from X-ray structures on the two forms. The 2-methylaspartate binds only to subunit 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutations induced by ionizing radiation in a plasmid replicated in human cells. I. Similar, nonrandom distribution of mutations in unirradiated and X-irradiated DNA

The Escherichia coli supF gene encoding the suppressor tyrosine tRNA in a human shuttle plasmid, pZ189, was used as a target for molecular analysis of X-ray-induced mutations in human lymphoblastoid cells. Following replication of the in vitro-irradiated plasmid in human cells, the mutant supF-containing molecules were cloned by phenotypic screening in E. coli and the nature of the mutations was determined by direct sequencing of the tRNA gene. At 160 Gy the mutant frequency was 13 times (0.39%) that observed in unirradiated controls (0.031%). When control plasmid was replicated directly in E. coli, the mutant frequency was 16 times less than that of the plasmid passaged through the human cells. The distribution of mutations was highly nonrandom and remarkably similar in both irradiated and control DNAs. The majority of the mutations were transitions involving G.C pairs and occurred selectively at most 5'-TC (3'-AG) sequences. These mutations at C's were preferentially distributed in the nontranscribed strand. We propose that mutations in the control plasmid result from oxidative damages that occur during and/or after its incorporation into human cells and that these damages are similar to those induced by ionizing radiation. The hot spots for mutations suggest that the proximate nucleotide sequence and the overall conformation of the target DNA are important in the production and/or processing of these damages during repair and replication.     

Xenopsin-related peptide(s) are formed from xenopsin precursor by leukocyte protease(s) and cathepsin D

Lysates of isolated rat polymorphonuclear leukocytes and macrophages were found to generate xenopsin-related peptides when incubated with a liver extract used as a source of precursor. The lysosomal enzyme, cathepsin D, was also shown to display this property and to share with the lysate a similar pH dependence (optimum, approximately pH 3.5) and sensitivity to the acid protease inhibitor, pepstatin A (ID50: lysate, 10 nM; cathepsin D, 30 nM). When subjected to HPLC on mu-Bondapak C-18, the xenopsin-related peptides generated by the lysate eluted near to those formed by cathepsin D and when tested in a radioreceptor assay for neurotensin, they displayed similar cross-reactivities (peak 2, approximately 50%; peak 1, approximately 100%). These results indicate that cathepsin D from lysed granulocytes can process precursor protein(s) to form radioreceptor-active xenopsin-related peptides.     

Characterization of three-subunit chloroplast coupling factor

The delta- and epsilon-polypeptides were removed from chloroplast coupling factor 1 (CF1). The resulting enzyme, CF1(-delta, epsilon), is a stable active ATPase containing only alpha-, beta-, and gamma-polypeptides. The dependence of the steady-state kinetics of ATP hydrolysis catalyzed by CF1(-delta, epsilon) on the concentrations of ATP and ADP was found to be essentially the same as by activated CF1. Nucleotide binding studies with CF1(-delta, epsilon) revealed three binding sites: a nondissociable ADP site (site 1), a tight MgATP binding site (site 2), and a site that binds ADP and ATP with a dissociation constant in the micromolar range (site 3). Similar results have been obtained with CF1. For both CF1 and CF1(-delta, epsilon), the binding of MgATP at site 2 is tight only in the presence of Mg2+. Fluorescence resonance energy transfer was used to map distances between the gamma-sulfhydryl ("dark" site) and gamma-disulfide and between the gamma-sulfhydryl and the three nucleotide sites. These distances are within 5% of the corresponding distances on CF1. These results indicate that removal of the delta- and epsilon-polypeptides from CF1 does not cause significant changes in the structure, kinetics, and nucleotide binding sites of the enzyme.     

Influence of ventrolateral surface of medulla on reflex tracheal constriction

To assess the role of structures located superficially near the ventrolateral surface of the medulla on the reflex constriction of tracheal smooth muscle that occurs when airway and pulmonary receptors are stimulated mechanically or chemically, experiments were conducted in alpha-chloralose-anesthetized, paralyzed, and artificially ventilated cats. Pressure changes within a bypassed segment of the trachea were used as an index of alterations smooth muscle tone. The effects of focal cooling of the intermediate areas or topically applied lidocaine on the ventral surface of the medulla on the response of the trachea to mechanical and chemical stimulation of airway receptors were examined. Atropine abolished tracheal constriction induced by mechanical stimulation of the carina or aerosolized histamine, showing that the responses were mediated over vagal pathways. Moderate cooling of the intermediate area (20 degrees C) or local application of lidocaine significantly decreased the tracheal constrictive response to mechanical activation of airway receptors. Furthermore, when the trachea was constricted by histamine, cooling of the intermediate area significantly diminished the increased tracheal tone, whereas rewarming restored tracheal tone to the previous level. These findings suggest that under the conditions of the experiments the ventral surface of the medulla plays an important role in constriction of the trachea by inputs from intrapulmonary receptors and in the modulation of parasympathetic outflow to airway smooth muscle.     

Baseline frequency of sister-chromatid exchanges (SCE) in newborn lymphocytes and its relationship to in vivo aging in humans

Heparinised cord blood from newborns and peripheral venous blood from three other age groups of individuals (1-75 years) have been cultured in vitro to obtain baseline frequencies of SCE and to see if the frequency of baseline SCE in vitro varies as a function of aging in vivo. The results demonstrate an age-dependent variation in the frequency of SCEs. Although the SCE frequency was lowest (5.10/cell) in 1-5-year-old infants, a significantly higher (P less than 0.001) frequency (8.97/cell) was observed in the cord blood of newborns. In old age, the level of SCE also increased. The plausible reason(s) for such observations is discussed.