Synthesis and characterization of chimeric silkworm silk

A synthetic gene encoding a chimeric silklike protein was constructed that combined a polyalanine encoding region (Ala)(18), a sequence slightly longer than the (Ala)(12-13) found in the silk fibroin from the wild silkworm Samia cynthia ricini, and a sequence encoding GVGAGYGAGAGYGVGAGYGAGVGYGAGAGY, found in the silk fibroin from the silkworm Bombyx mori. A tetramer of the chimeric repeat sequence encoding a approximately 29 kDa protein was expressed as a fusion protein in Escherichia coli. In comparison to S. c. ricini silk, the chimeric protein demonstrated improved solubility because it could be dissolved in 8 M urea. The purified protein assumed an alpha-helical structure based on solid-state (13)C CP/MAS NMR and was less prone to conformational transition to a beta-sheet, unlike native silk proteins from S. c. ricini. Model peptides representing the crystalline region of S. c. ricini silk fibroin, (Ala)(12) and (Ala)(18), formed beta-sheet structures. Therefore, the solubility and structural transitions of the chimeric protein were significantly altered through the formation of this chimeric silk. This experimental strategy to the study of silk structure and function can be used to develop an improved understanding of the contributions of protein domains in repetitive silkworm and spider silk sequences to structure development and structural transitions.     

Antidiabetic effect of Lactobacillus GG in streptozotocin-induced diabetic rats

Neonatally streptozotocin-induced diabetic (n-STZ) rats were given food containing Lactobacillus GG cells (GG) or a control diet (control), from 9 to 18 weeks of age. The GG cells significantly lowered the blood hemoglobin A(1C) (HbA(1C)) level and improved glucose tolerance in n-STZ rats (p<0.05). In the GG group, the serum insulin level at 30 min after glucose loading was significantly higher than in the control group (p<0.05).     

Determination of the torsion angles of alanine and glycine residues of model compounds of spider silk (AGG)(10) using solid-state NMR methods

Spiders synthesize several kinds of silk fibers. In the primary structure of spider silk, one of the major ampullate (dragline, frame) silks, spidroin 1, and flagelliform silk (core fibers of adhesive spiral), there are common repeated X-Gly-Gly (X = Ala, Leu, Pro, Tyr, Glu, and Arg) sequences, which are considered to be related to the elastic character of these fibers. In this paper, two dimensional spin diffusion solid-state NMR under off magic angle spinning (OMAS), ¹³C chemical shift contour plots, and Rotational Echo DOuble Resonance (REDOR) were applied to determine the torsion angles of one Ala and two kinds of Gly residues in the Ala-Gly-Gly sequence of ¹³C=O isotope-labeled (Ala-Gly-Gly)₁₀. The torsion angles were determined to be (, ) = (–90°, 150° ) within an experimental error of ±10° for each residue. This conformation is characterized as 3₁ helix which is in agreement with the structure proposed from the X-ray powder diffraction pattern of poly(Ala-Gly-Gly). The 3₁ helix of (Ala-Gly-Gly)₁₀ does not change by formic acid treatment although (Ala-Gly)₁₅ easily changes from the silk I conformation (the structure of Bombyx mori silk fibroin before spinning in the solid state) to silk II conformation (the structure of the silk fiber after spinning) by such treatment. Thus, the 3₁ helix conformation of (Ala-Gly-Gly)₁₀ is considered very stable. Furthermore, the torsion angles of the 16th Leu residue of (Leu-Gly-Gly)₁₀ were also determined as (, ) = (–90°, 150° ) and this peptide is also considered to take 3₁ helix conformation.     

Synthesis and hybridization property of novel 2',5'-isoDNA mimic chiral peptide nucleic acids

2',5'-isoDNA mimic chiral peptide nucleic acid (isoPNA) monomers derived from D- and L-aspartic acids were synthesized. These novel monomers were incorporated in aminoethylglycine peptide nucleic acid (aegPNA) thymine dodecamers, and the hybridization properties to RNA and DNA were demonstrated by UV thermal denaturation.     

Nonlinear time-course of lumbar muscle fatigue using recurrence quantifications

 Isometric skeletal muscle fatigue is usually assumed to be a linear process based upon the monotonic decrease in spectral frequency of the EMG. Since spectral analysis by fast Fourier transform (FFT) constitutes a linear transformation of the data, the present study was designed to reevaluate the time-course of muscle fatigue with a nonlinear tool, recurrence quantification analysis (RQA). Surface EMG recordings were obtained from the multifidus muscle of 17 human subjects during isometric posture-holding of the upper torso. The process of muscle fatigue was found to be linear for 59% of the subjects by FFT criteria, but nonlinear for 76% by RQA criteria. As a demonstrative control, both slow and fast transients occurring within a nonlinear mathematical process could be accurately depicted by RQA, but not by FFT. It is concluded that assessment of EMG patterns by nonlinear techniques can give insight into the time-course of fatiguing muscles attributed to the summation of several nonlinear and competing processes. Received: 12 November 1998 / Accepted in revised form: 29 November 1999     

Effects of Neonatal Melatonin Administration on the Extra-Hypothalamic Regions in Rat Brains: Effects on the Serotonergic System

The effects of 100g melatonin injection at postnatal day 5 (PD 5) on the development of the central serotonergic systems in male and female rats were investigated. The contents of serotonin (5-HT) and 5-hydroxy-3-indolacetic acid (5-HIAA) were measured in several extra-hypothalamic regions at 3, 10 and 42 weeks of age. The neonatal melatonin administration increased both 5-HT and 5-HIAA levels in the striatum throughout the examined period. In the hippocampus, an increase in 5-HIAA contents by neonatal melatonin administration was found at 3 weeks but not 10 or 42 weeks of age. There were no significant differences in the effects of melatonin between male and female rats. These results indicated that exogenous melatonin administration during the early neonatal period influenced the development of the serotonergic systems in extrahypothalamic regions including the hippocampus and the striatum.     

Structure and dynamics of photosynthetic membrane-bound proteins in Rhodobacter Sphaeroides,studied with solid-state NMR spectroscopy

The photosynthetic purple bacteria such as Rb. sphaeroides possesses an intracytoplasmic membrane (ICM) and a variety of pigment-binding membrane proteins located in the ICM, acting as photoreceptor. Such photosynthetic apparatus is concentrated in the ICM. It is composed of three multimeric membrane-bound proteins; light-harvesting complexes (LH 1, LH 2), a reaction center (RC) and a cytochrome b/c1 complex. We have purified these membranes, which are called chromatophores, and characterized the structure and dynamics of the photosynthetic membrane-bound proteins by means of multi-nuclear solid state NMR. First, the isotropic chemical shift of carbonyl carbons in natural abundance and [1-¹³C] Phe labeled chromatophores indicates that the membrane-bound proteins take mainly the helical conformation. Second, the chemical shifts of side-chain resonances of uniformly ¹⁵N-labeled chromatophores indicate the side-chain histidine residue is mainly hydrogen bonded, whereas structural heterogeneity of arginine and lysine side-chains are probed by those wide distribution of ¹⁵N shifts. Thirdly, the [β-²H₃]Ala and [ε-²H₂]Tyr labeling of the chromatophores are performed and dynamics of the [β-²H]Ala and the [ε-²H₂]Tyr labeled chromatophores are studied by means of ²H solid state NMR. The dynamics of [β-²H₃]Ala is found to be a 10⁸Hz three-site jump motion with 10° liberation along the Cα-Cβ bond axis. The ²H-NMR powder pattern spectrum of [ε-²H₂] Tyr labeled chromatophores was interpreted with an averaged correlation time of 5×10⁵ Hz with 180° two-fold flips, the result of the averaging of two kinds of split spectra in terms of motional time scale. This revised version was published online in June 2006 with corrections to the Cover Date.     

ZAP-70 Association with T Cell Receptor ζ (TCRζ): Fluorescence Imaging of Dynamic Changes upon Cellular Stimulation

The nonreceptor protein tyrosine kinase ZAP-70 is a critical enzyme required for successful T lymphocyte activation. After antigenic stimulation, ZAP-70 rapidly associates with T cell receptor (TCR) subunits. The kinetics of its translocation to the cell surface, the properties of its specific interaction with the TCRζ chain expressed as a chimeric protein (TTζ and Tζζ), and its mobility in different intracellular compartments were studied in individual live HeLa cells, using ZAP-70 and Tζζ fused to green fluorescent protein (ZAP-70 GFP and Tζζ–GFP, respectively). Time-lapse imaging using confocal microscopy indicated that the activation-induced redistribution of ZAP-70 to the plasma membrane, after a delayed onset, is of long duration. The presence of the TCRζ chain is critical for the redistribution, which is enhanced when an active form of the protein tyrosine kinase Lck is coexpressed. Binding specificity to TTζ was indicated using mutant ZAP-70 GFPs and a truncated ζ chimera. Photobleaching techniques revealed that ZAP-70 GFP has decreased mobility at the plasma membrane, in contrast to its rapid mobility in the cytosol and nucleus. Tζζ– GFP is relatively immobile, while peripherally located ZAP-70 in stimulated cells is less mobile than cytosolic ZAP-70 in unstimulated cells, a phenotype confirmed by determining the respective diffusion constants. Examination of the specific molecular association of signaling proteins using these approaches has provided new insights into the TCRζ–ZAP-70 interaction and will be a powerful tool for continuing studies of lymphocyte activation.