Simple method for the determination of choline and acetylcholine by pyrolysis gas chromatography

An improved purification procedure is described for the simultaneous assay of endogenous choline and acetylcholine by pyrolysis gas chromatography, particularly for providing a simple and effective method for propionylation of choline in the presence of acetylcholine. The reaction was carried out in acetonitrile solution prepared by dissolving the evaporated residue of the supernatant of brain homogenate. Thus samples for propionylation were prepared without the use of ion-exchange chromatography.     

A new structural protein located in the Z lines of chicken skeletal muscle.

A new structural protein was purified from a prolonged 0.6 M KI extract of residues of chicken skeletal myofibrils, from which myosin, actin, and some other proteins had been removed. The protein had a chain weight of 55,000. The indirect immunofluorescence technique using antiserum against the 55,000 dalton protein revealed that the protein was exclusively located in the Z lines of a myofibril. The new protein formed lattice structures in vitro which were similar to those observed in the Z lines in situ.     

Equilibrium and kinetics of the thermal unfolding of yeast 5S ribosomal RNA.

The equilibrium and kinetics of thermal unfolding of yeast 5S ribosomal RNA have been studied by optical methods, in a low ionic strength environment without Mg2+, to follow the disruption of the secondary structure base pairs in the molecule. The equilibrium results demonstrated that all of the helical regions melted simultaneously, and the kinetics of the thermal unfolding were first order. These findings suggest the validity of the two-state approximation for the unfolding reaction under the present conditions. The total number of secondary structure base pairs estimated from our experiment was consistent with that contained in the conformational model based on the Raman spectrum rather than that in the one derived by the enzymic digestion method. Taking our results on the kinetic behavior of the thermal unfolding overall, we propose that the 5S RNA has a partly melted secondary structure under the solvent conditions used.     

Biochemical and topographical studies on Escherichia coli cell surface. IV. Giant spheroplast formation from a filamentous cell.

Long, nonseptate filamentous cells consisting of 5 to 40 single-cell unit lengths were formed from Escherichia coli surface mutant ONT-3 by treatment with a sublethal concentration of sodium dodecyl sylfate. As distinct from several other elongated cells (e.g., thymine-starved filaments), it was found here that stable giant spheroplasts, 5 to 10 micrometers in diameter, were produced by the action of lysozyme in the presence of bovine serum albumin via the gradual fusion of distinct spheroplasting bulbs.     

beta-actinin, a regulatory protein of muscle. Purification, characterization and function.

beta-Actinin, a minor regulatory protein of muscle, was purified from skeletal muscles of rabbit and chicken by DEAE-Sephadex chromatography. beta-Actinin consisted of two subunits, beta I and betaII, with chain weights of 37,000 and 34,000 daltons, respectively. The amino acid compositions were similar, though not identical. It appears that each of the two subunits is associated in solution. beta-Actinin had the following effects on actin: (1) inhibition of reassociation of F-actin fragments; (2) inhibition of network formation of F-actin; (3) inhibition of growth of F-actin fragments; (4) retardation of depolymerization of F-actin and (5) acceleration of polymerization of G-actin. All these actions of beta-actinin can be explained in terms of action as an "ending factor". Experimental evidence favored the view that beta-actinin is bound to one end of the F-actin filament, namely to the end opposite to the direction of polymerization. Fluorescence-labeled anti-beta-actinin stained the middle portion of the A band of myofibrils. Based on the finding that the stain was unchanged on removal of myosin, it is suggested that beta-actinin is located at the free ends of the I filaments of myofibrils. Thus is seems likely that beta-actinin functions as an ending factor for actin filaments.     

Carpesiolin from Carpesium abrotanoides

Carpesiolin and carabrone were obtained from Carpesium abrotanoides as antifungal and antibacterial components, respectively, and the structure of carpesiolin was elucidated.     

A transcriptional fusion of genes encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase in dinoflagellates

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase are enzymes essential for glycolysis and gluconeogenesis. Dinoflagellates possess several types of both GAPDH and enolase genes. Here, we identify a novel cytosolic GAPDH-enolase fusion protein in several dinoflagellate species. Phylogenetic analyses revealed that the GAPDH moiety of this fusion is weakly related to a cytosolic GAPDH previously reported in dinoflagellates, ciliates, and an apicomplexan. The enolase moiety has phylogenetic affinity with sequences from ciliates and apicomplexans, as expected for dinoflagellate genes. Furthermore, the enolase moiety has two insertions in a highly conserved region of the gene that are shared with ciliate and apicomplexan homologues, as well as with land plants, stramenopiles, haptophytes, and a chlorarachniophyte. Another glycolytic gene fusion in eukaryotes is the mitochondrion-targeted triose-phosphate isomerase (TPI) and GAPDH fusion in stramenopiles (i.e. diatoms and oomycetes). However, unlike the mitochondrial TPI-GAPDH fusion, the GAPDH-enolase fusion protein appears to exist in the same compartment as stand-alone homologues of each protein, and the metabolic reactions they catalyze in glycolysis and gluconeogenesis are not directly sequential. It is possible that the fusion is post-translationally processed to give separate GAPDH and enolase products, or that the fusion protein may function as a single bifunctional polypeptide in glycolysis, gluconeogenesis, or perhaps more likely in some previously unrecognized metabolic capacity.     

The effect of posture on respiratory activity of the abdominal muscles

The purpose of this study was to determine the influence of posture on the expiratory activity of the abdominal muscles. Fifteen young adult men participated in the study. Activities of the external oblique abdominis, internal oblique abdominis, and rectus abdominis muscles were measured electromyographically in various postures. We used a pressure threshold in order to activate the abdominal muscles as these muscles are silent at rest. A spirometer was used to measure the lung volume in various postures. Subjects were placed in the supine, standing, sitting, and sitting-with-elbow-on-the-knee (SEK) positions. Electromyographic activity and mouth pressure were measured during spontaneous breathing and maximal voluntary ventilation under the respiratory load. We observed that the lung volume changed with posture; however, the breathing pattern under respiratory load did not change. During maximal voluntary ventilation, internal oblique abdominis muscle expiratory activity was lower in the SEK position than in any other position, external oblique abdominis muscle inspiratory activity was lower in the supine position than in any other position, and internal oblique abdominis muscle activity was higher in the standing position than in any other position. During spontaneous breathing, external oblique abdominis muscle activity was higher during expiration and inspiration in the SEK position than in any other position. The internal oblique abdominis muscle activity was higher during both inspiration and expiration in the standing position than in any other position. The rectus abdominis muscle activity did not change with changes in posture during both inspiration and expiration. Increase in the external oblique abdominis activity in the SEK position was due to anatomical muscle arrangement that was consistent with the direction of lower rib movement. On the other hand, increase in the internal oblique abdominis activity in the standing position was due to stretching of the abdominal wall by the viscera. We concluded that differences in activity were due to differences in the anatomy of the abdominal muscles and the influence of gravity.     

Investigations of molecular recognition aspects related to the enantiomer separation of 2-methoxy-2-(1-naphthyl)propionic acid using quinine carbamate as chiral selector: An NMR and FT-IR spectroscopic as well as X-ray crystallographic study

The chiral recognition mechanism of a cinchona alkaloid-based chiral stationary phase (CSP) showing high enantiomer discrimination potential for 2-methoxy-2-(1-naphthyl)propionic acid (MalphaNP acid) was investigated. Conformational and structural analyses of the 1:1 complexes of 9-O-(tert-butylcarbamoyl) quinine selector (SO) and MalphaNP acid (selectand, SA) were carried out employing NMR spectroscopy in solution, Fourier-transform infrared (FT-IR) spectroscopy, and solid-state X-ray diffraction analysis. Intramolecular NOEs of a soluble analogue of the CSP afforded the conformational states of the free and complexed form of the selector. The (1)H-NMR spectra revealed that the free form of the SO constitutes anti-open as well as anti-closed and/or syn-closed conformers. Upon complexation with the (S)-MalphaNP acid enantiomer to form the more stable diastereomeric associate, a conformational transition of the selector takes place, resulting in the synthesis of the anti-open conformer nearly exclusively. FT-IR spectra reveal that, besides the primary ion-pairing interaction, stereoselective hydrogen bonding stabilizes the more stable complex via the amide hydrogen of the SO. X-ray diffraction analysis of 9-O-(tert-butylcarbamoyl)quinine and (S)-MalphaNP acid complex further revealed the occurrence of a bidentate H-bond-mediated ionic interaction between SO and SA as well as the lack of pi-pi interaction in the 1:1 complex, and corroborated the conclusions derived from spectroscopic and chromatographic studies.     

Structure and function of bacterial super-biosystem responsible for import and depolymerization of macromolecules

Generally, when microbes assimilate macromolecules, they incorporate low-molecular-weight products derived from macromolecules through the actions of extracellular degrading enzymes. However, a Gram-negative bacterium, Sphingomonas sp. A1, has a smart biosystem for the import and depolymerization of macromolecules. The bacterial cells directly incorporate a macromolecule, alginate, into the cytoplasm through a "superchannel", as we named it. The superchannel consists of a pit on the cell surface, alginate-binding proteins in the periplasm, and an ATP-binding cassette transporter in the inner membrane. Cytoplasmic polysaccharide lyases depolymerize alginate into the constituent monosaccharides. Other than the proteins characterized so far, novel proteins (e.g., flagellin homologs) have been found to be crucial for the import and depolymerization of alginate through genomics- and proteomics-based identification, thus indicating that the biosystem is precisely constructed and regulated by diverse proteins. In this review, we focus on the structure and function of the bacterial biosystem together with the evolution of related proteins.