Phosphorylation of AMP by inorganic phosphorylating agents

AMP was phosphorylated by inorganic phosphorylating agents: cyclo-triphosphate and diphosphonate, in aqueous solution (70-80 degrees C, pH 6-12). The molecular structures of phosphorylated products were established by use of phosphorus-31 NMR and high-performance liquid chromatography (HPLC). The OH groups on AMP were phosphorylated by both phosphorylating agents to form 2'- or 3'-phosphate but an OH group on dAMP was not phosphorylated. Phosphorylation of OH group proceeds in two steps: formation of hydrogen bond between OH group and phosphorylating agent; subsequent nucleophilic attack of OH group on a phosphorus atom. Phosphate group on AMP was phosphorylated by diphosphonate but not by cyclo-triphosphate. The difference in the reactivities is explained in terms of charge repulsion between AMP and agents.     

Enzymatic isomerization and epimerization of D-erythrose 4-phosphate and its quantitative analysis by gas chromatography/mass spectrometry

An enzyme preparation from beef liver catalyzed the isomerization and epimerization of D-erythrose 4-phosphate to D-erythrulose 4-phosphate and D-threose 4-phosphate. The presence of D-erythrulose 4-phosphate and D-threose 4-phosphate was demonstrated by several analytical methods. After dephosphorylation, the presence of D-erythrulose and D-threose was confirmed by thin-layer chromatography, gas-liquid chromatography and an enzymatic method depending upon D-erythrulose reductase. The enzymatic products were also identified and simultaneously quantitated by a new procedure using gas chromatography/mass spectrometry. Each of three tetroses was distinguished by the combination of the reduction with sodium borodeuteride and the determination of relative intensities of the ion pairs m/z 379 and 380 of sugar tetritol trifluoroacetate. By gas chromatography/mass spectrometry, we observed that D-threose 4-phosphate was also converted into D-erythrulose 4-phosphate and D-erythrose 4-phosphate. At the equilibrium, about 90% of the tetrose 4-phosphate existed in the form of D-erythrulose 4-phosphate. On the basis of gas chromatography/mass spectrometric evidence together with gas chromatographic and thin-layer chromatographic patterns, it is suggested that the single enzyme of the beef liver catalyzed both reactions of isomerization and epimerization of aldotetrose 4-phosphate.     

Evaluation of decay and termite resistance of wood treated with copper in combination with boron and N′-N-(1, 8-naphthalyl) hydroxylamine (NHA-Na)

This study evaluated the relative ability of various combinations of copper sulfate with either boric acid or calcium-precipitating agent, N′-N-(1, 8-naphthalyl) hydroxylamine (NHA-Na), to inhibit fungal degradation and attack by Formosan subterranean termites (Coptotermes formosanus Shiraki). Wood specimens were treated with either 1%, 0.5%, or 0.1% concentrations of copper sulfate, boric acid, NHA-Na, copper sulfate + boric acid, or copper sulfate + NHA-Na mixtures. Treated specimens were subjected to laboratory decay-resistance tests by using petri dishes inoculated with the Basidiomycetes fungi Tyromyces palustris and Trametes versicolor for 12 weeks. Treated wood specimens were also subjected to termite-resistance tests under laboratory conditions. Increased efficacy of copper sulfate against the brown-rot fungus T. palustris was observed when either boric acid or NHA-Na was added. The most effective treatments against the fungi tested were NHA-Na only treatments at 1% and 0.5% concentration levels. Boric acid treatments were not able to protect wood against decay after leaching because of excessive leaching of boron. Similar results were obtained in termite-resistance tests in comparison with decay-resistance tests. These results indicate that the efficacy of the treatments in preventing fungal and termite attack is a function of the type of preservative.     

A 5-bp Insertion in Mip Causes Recessive Congenital Cataract in KFRS4/Kyo Rats

We discovered a new cataract mutation, kfrs4, in the Kyoto Fancy Rat Stock (KFRS) background. Within 1 month of birth, all kfrs4/kfrs4 homozygotes developed cataracts, with severe opacity in the nuclei of the lens. In contrast, no opacity was observed in the kfrs4/+ heterozygotes. We continued to observe these rats until they reached 1 year of age and found that cataractogenesis did not occur in kfrs4/+ rats. To define the histological defects in the lenses of kfrs4 rats, sections of the eyes of these rats were prepared. Although the lenses of kfrs4/kfrs4 homozygotes showed severely disorganised fibres and vacuolation, the lenses of kfrs4/+ heterozygotes appeared normal and similar to those of wild-type rats. We used positional cloning to identify the kfrs4 mutation. The mutation was mapped to an approximately 9.7-Mb region on chromosome 7, which contains the Mip gene. This gene is responsible for a dominant form of cataract in humans and mice. Sequence analysis of the mutant-derived Mip gene identified a 5-bp insertion. This insertion is predicted to inactivate the MIP protein, as it produces a frameshift that results in the synthesis of 6 novel amino acid residues and a truncated protein that lacks 136 amino acids in the C-terminal region, and no MIP immunoreactivity was observed in the lens fibre cells of kfrs4/kfrs4 homozygous rats using an antibody that recognises the C- and N-terminus of MIP. In addition, the kfrs4/+ heterozygotes showed reduced expression of Mip mRNA and MIP protein and the kfrs4/kfrs4 homozygotes showed no expression in the lens. These results indicate that the kfrs4 mutation conveys a loss-of-function, which leads to functional inactivation though the degradation of Mip mRNA by an mRNA decay mechanism. Therefore, the kfrs4 rat represents the first characterised rat model with a recessive mutation in the Mip gene.     

Stimulation of in vitro mitochondrial protein synthesis by yeast cytoplasmic extracts is caused by guanyl nucleotides

Micromolar concentrations of GDP or GTP stimulate protein synthesis by isolated yeast mitochondria 3- to 10-fold even if alpha-ketoglutarate and an ATP-regenerating system are present. No stimulation is observed with GMP, UTP, CTP, TTP, and the nonhydrolyzable GTP analogues guanyl(beta, gamma-methylene) diphosphate and guanyl imidodiphosphate. This stimulatory effect of exogenously added guanyl nucleotides may answer the long standing question why protein synthesis by isolated mitochondria is so slow. It can also explain previous reports by two other laboratories that a high speed supernatant from yeast cells stimulates protein synthesis by isolated mitochondria. The supernatant contains nondialyzable GMP which is converted to GDP under the conditions used for assaying mitochondrial protein synthesis. The stimulatory effect of high speed supernatants is abolished by 5'-nucleotidase (which degrades GMP) or by trypsin (which destroys supernatant protein(s) necessary for converting GMP to GDP). No evidence was obtained that the stimulatory effect of high speed supernatants was caused by precursors to cytoplasmically made cytochrome c oxidase subunits.     

Unique distributions of natriuretic hormones in dog brain

We examined the regional distributions of atrial natriuretic polypeptide (ANP) and digoxin-like immunoreactivity (DLI) in dog brain, using specific radioimmunoassay. The molecular form of the dog brain ANP was similar to that of alpha-hANP, in gel filtration and reversed-phase HPLC. Distribution of ANP in dog brain differed from rat brain. A significant amount of ANP-like immunoreactivity (ANPLI) was observed in the periaqueductal grey, ventral thalamus and spinal cord, however, only a trace amount was seen in the hypothalamus. The DLI was widely distributed in the dog brain, especially with over 2 ng/g wet wt. of the immunoreactivity content in mammillary body, septum, striatal body, hypothalamus and periaqueductal grey. Different from the localizations of natriuretic hormones in rat brain, the periaqueductal grey matter in dog brain may be an important source of both natriuretic hormones.     

Nucleotide sequence of the PR-1 gene of Nicotiana tabacum

A gene encoding one of the pathogenesis-related proteins, PR1a, and two related pseudogenes were isolated from Nicotiana tabacum. The cloned PR1a gene (pPR-gamma) and one of the pseudogenes (pPR-alpha) were sequenced and found to have similar structures. The sequence of pPR-gamma was quite similar to that of the cDNA clone of PR1a. The plasmid pPR-gamma did not contain an intron and had a typical promoter sequence in the 5'-flanking region.     

ADP-ribosylation of a Mr 21,000 membrane protein by type D botulinum toxin

When crude membrane fraction from bovine adrenal gland was incubated with type D botulinum toxin in the presence of NAD, a membrane protein with a molecular weight of 21,000 was specifically ADP-ribosylated. This ADP-ribosylation occurred dependent on the dose of the toxin and was abolished by prior boiling ADP-ribose transfer to the membrane protein was significantly suppressed when agmatine and L-arginine methyl ester were included in the reaction mixture. Dithiothreitol stimulated this ADP-ribosylation about 3-fold. Incubation of membrane fractions from mouse brain and pancreas with this toxin also resulted in ADP-ribosylation of a protein of the same molecular weight. These results suggested that type D botulinum toxin catalyzed transfer of an ADP-ribose moiety of NAD to the specific membrane protein common to secretory cells.     

A simple and rapid assay for heme attachment to apocytochrome c

A method for assaying the covalent attachment of heme to apoprotein of cytochrome c was developed. 125I-labeled apoprotein was chemically prepared from 125I-labeled yeast cytochrome c (iso-1-cytochrome c). After incubation of 125I-apocytochrome c with yeast mitochondria, the product was extracted with Triton X-100, digested with trypsin in the presence or absence of a reducing agent, and then precipitated in trichloroacetic acid. The resulting precipitates were collected on nitrocellulose membranes and counted for radioactivity. The radioactivity correlated well with the appearance of a heme-containing peptide in the trypsin digested peptide fragments of cytochrome c. This procedure is simpler and faster than the previously reported methods.     

Infrageneric variation of the low molecular weight carbohydrate composition of the seeds of the genusVicia (Leguminosae)

The low molecular weight carbohydrate compositions of the seeds of 29 species ofVicia, namelyV. amoena, V. amurensis, V. bifolia, V. dumetorum, V. fauriei, V. japonica, V. nipponica, V. pisiformis, V. pseudo-orobus, V. sylvatica, V. unijuga, V. venosa, V. cassubica, V. orobus, V. cracca agg.,V. hirsuta, V. villosa agg.,V. tetrasperma,V. oroboides, V. sepium, V. cuspidata, V. grandiflora, V. lathyloides, V. sativa agg.,V. bithynica, V. faba, V. narbonensis, V. hybrida andV. lutea were determined by gas liquid chromatography. The carbohydrate compositions were found to be species-specific. Principal component analysis of the carbohydrate composition data showed that these species can be divided into three groups. Although, as far as the examined species were concerned, these groups were not correlated with the known subgenera, significant correlation between the groups and the known sections was detected in the subgenusVicia. The carbohydrate composition character would be important to clarify the relationships among closely related taxa of the genusVicia.