Molecular cloning of two tandemly arranged peroxidase genes from Populus kitakamiensis and their differential regulation in the stem

A genomic library was prepared from Populus kitakamiensis and screened with the cDNA for an anionic peroxidase from P. kitakamiensis. One genomic clone was isolated that contained two tandemly oriented genes for anionic peroxidases, prxA3a and prxA4a. Both genes consisted of four exons and three introns; the introns had consensus nucleotides, namely, GT and AG, at their 5 and 3 ends, respectively. The prxA3a and prxA4a genes encoded 347 and 343 amino acid residues, respectively, including putative signal sequences at the amino-termini. Putative promoters and polyadenylation signals were found in the flanking regions of both genes. The sequence of the coding region of prxA3a was completely identical to that of the cDNA clone pA3, whereas the sequence of the coding region of prxA4a was only 73% identical to that of the cDNA clone pA3. Northern blot analysis showed that the patterns of expression of the mRNAs that corresponded to prxA3a and prxA4a differed in stems of P. kitakamiensis.     

Phylogenetic position of Dictyostelium inferred from multiple protein data sets

The phylogenetic position of Dictyostelium inferred from 18S rRNA data contradicts that from protein data. Protein trees always show the close affinity of Dictyostelium with animals, fungi, and plants, whereas in 18S rRNA trees the branching of Dictyostelium is placed at a position before the massive radiation of protist groups including the divergence of the three kingdoms. To settle this controversial issue and to determine the correct position of Dictyostelium, we inferred the phylogenetic relationship among Dictyostelium and the three kingdoms Animalia, Fungi, and Plantae by a maximum-likelihood method using 19 different protein data sets. It was shown at the significance level of 1 SE that the branching of Dictyostelium antedates the divergence of Animalia and Fungi, and Plantae is an outgroup of the Animalia-Fungi-Dictyostelium clade.Correspondence to: T. Miyata     

Real time imaging of single fluorophores on moving actin with an epifluorescence microscope.

Relatively simple modifications of an ordinary epifluorescence microscope have greatly reduced its background luminescence, allowing continuous and real time imaging of single fluorophores in an aqueous medium. Main modifications were changing the excitation light path and setting an aperture stop so that stray light does not scatter inside the microscope. A simple and accurate method using actin filaments is presented to establish the singularity of the observed fluorophores. It was possible, at the video rate of 30 frames/s, to image individual tetramethylrhodamine fluorophores bound to actin filaments sliding over heavy meromyosin. The successful imaging of moving fluorophores demonstrates that conventional microscopes may become a routine tool for studying dynamic interactions among individual biomolecules in physiological environments.     

Spatiotemporal relationships among early events of fertilization in sea urchin eggs revealed by multiview microscopy.

Four early events of egg fertilization, changes in intracellular calcium concentration and intracellular pH, reorientation of the surface membrane, and the elevation of the fertilization envelope, were imaged in real time and in pairs in single sea urchin eggs. The paired imaging allowed the correlation of the four events spatially and temporally. Three of them propagated as waves starting at the sperm entry site. The earliest was the calcium wave, visualized with fluorescent indicator dyes. After a delay of 10 s there followed a large decrease in the fluorescence polarization of membrane-bound dyes, which we interpret as arising from membrane reorientation as a result of cortical granule exocytosis and microvillar elongation. With a further delay of 15 s the fertilization envelope was seen to rise in transmitted light. All three waves propagated with similar velocities of approximately 10 microns/s, supporting the view that calcium triggers the latter two events. The fluorescence polarization changed in two steps with a clear pause of 10-20 s in between. The second step, which also propagated as wave, reflects either further elongation of microvilli or straightening of irregular microvilli. This second step was abolished by cytochalasin B and was coincident with an increase in cytoplasmic pH, suggesting that pH-induced actin reorganization may play a role. The cytoplasmic alkalinization, imaged with a fluorescent probe, was quite different from the other events in that it took place homogeneously throughout the egg and slowly (over 100 s). Apparently, the alkalinization is not on a direct downstream pathway of calcium origin. An opposing possibility, that the alkalinization may in fact be triggered by the traveling calcium wave, is also discussed.     

An Estimate of Divergence Time of Parazoa and Eumetazoa and That of Cephalochordata and Vertebrata by Aldolase and Triose Phosphate Isomerase Clocks

Previously we suggested that four proteins including aldolase and triose phosphate isomerase (TPI) evolved with approximately constant rates over long periods covering the whole animal phyla. The constant rates of aldolase and TPI evolution were reexamined based on three different models for estimating evolutionary distances. It was shown that the evolutionary rates remain essentially unchanged in comparisons not only between different classes of vertebrates but also between vertebrates and arthropods and even between animals and plants, irrespective of the models used. Thus these enzymes might be useful molecular clocks for inferring divergence times of animal phyla. To know the divergence time of Parazoa and Eumetazoa and that of Cephalochordata and Vertebrata, the aldolase cDNAs from Ephydatia fluviatilis, a freshwater sponge, and the TPI cDNAs from Ephydatia fluviatilis and Branchiostoma belcheri, an amphioxus, have been cloned and sequenced. Comparisons of the deduced amino acid sequences of aldolase and TPI from the freshwater sponge with known sequences revealed that the Parazoa–Eumetazoa split occurred about 940 million years ago (Ma) as determined by the average of two proteins and three models. Similarly, the aldolase and TPI clocks suggest that vertebrates and amphioxus last shared a common ancestor around 700 Ma and they possibly diverged shortly after the divergence of deuterostomes and protostomes.     

Evolution and structure of two ADP-ribosylation enterotoxins, Escherichia coli heat-labile toxin and cholera toxin

Nucleotide sequence comparisons of the heat-labile enterotoxin (LTh) genes of E. coli pathogenic for humans with cholera toxin (CT) genes suggest that the two toxin genes have evolved from a common ancestry by a series of single base changes, while conserving the catalytic fragment A1 (ADP-ribose transferase). Based on the local hydrophilicity profiles of LTh and CT peptides, a transmembrane segment appears to be present in A1 in both toxins.     

On the antisymmetry of the amino acid code table

Antisymmetry of the amino acid code table in terms of codon degeneracy is pointed out, and it is related to a physico-chemical problem of codon-anticodon interaction energy. A strong negative correlation between molecular weight of an amino acid and its codon degeneracy is pointed out, and its implication to the origin of the amino acid code table is discussed. Finally, an earlier form of the amino acid code table is proposed.     

Stimulation of glycosaminoglycan sulfotransferase from chick embryo cartilage by basic proteins and polyamines

A soluble glycosaminoglycan sulfotransferase (3'-phosphoadenylylsulfate:chondroitin 4'-sulfotransferase, EC 2.8.2.5) from chick embryo cartilage has been prepared free from endogenous acceptor. The reaction with this enzyme preparation was stimulated by basic proteins and polyamines, the degree of stimulation being dependent on the chemical nature of both basic compounds and acceptor glycosaminoglycans. A maximum stimulation was obtained when protamine (basic compound) and chondroitin (acceptor) were involved in the reaction mixture at a molar ratio of protamine to repeating disaccharide units of chondroitin, 1:100. The stimulation of sulfotransferase activity by basic substances was much higher than that by Mn2+. However, increasing the Mn2+ concentration immediately reduced the stimulation by basic substances. The Km value for 3'-phosphoadenosine 5'-phosphosulfate of the sulfotransferase, when chondroitin was used as acceptor, was 1 . 10(-6) M in the presence of 25 microgram/ml protamine, compared to 2 . 10(-5) M in the absence of protamine. These observations indicate that the basic proteins and polyamines may interact with acceptor polysaccharide, thereby causing an increase in the affinity of the enzyme toward 3'-phosphoadenosine 5'-phosphosulfate.     

Effect of furosemide on urinary excretion of prostaglandin E in normal volunteers and pateints with essential hypertension

Urinary excretion of prostaglandin E was measured radioimmunologically in 19 healthy persons ( 15 men and 4 women ) and in 16 patients ( 10 men and 6 women ) with essential hypertension before and after the administration of furosemide. The excretion rates were increased from 26.3±3.0 to 64.5±11.3 ng/hr in the former and from 11.9±2.7 to 26.9±85 ng/hr in the latter. There was a significant difference between them, healthy subjects showing a greater increase than patients with essential hypertension.There was an obvious sexual difference in urinary excretion of prostaglandin. In men, greater increase in the excretion rates was found than in the women. Greater increases were also obtained in healthy men than in hypertensive men and in healthy women than in hypertensive women. The present results suggest that furosemide enhances urinary excretion of prostaglandin E by mechanisms which entails either an increase in prostaglandin synthesis or a decrease in renal metabolism.     

Identification of a cross-linked peptide of a covalent complex between adrenodoxin reductase and adrenodoxin.

A cross-linked complex between bovine NADPH-adrenodoxin reductase (AR) and adrenodoxin (AD) was prepared with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and purified, as described previously [Hara, T. & Kimura, T. (1989) J. Biochem. 105, 594-600]. The covalent complex was S-pyridylethylated and digested with lysylendopeptidase, and the resulting peptides were separated by reversed-phase HPLC to identify the cross-linked peptide. Comparison of the HPLC chromatograms of the peptides showed that (i) two tandem peptides (K-4 and K-5) from AD and a peptide (K-1) from AR were missing in the chromatogram of the peptides of the covalent complex and (ii) a single new peak was observed in the chromatogram of the peptides from the covalent complex. Amino acid composition and sequence analyses showed that the newly observed peptide was a covalently cross-linked peptide formed between a peptide K-4-K-5 (Ile-25-Lys-98) derived from AD and a peptide K-1 (Ser-1-Lys-27) derived from AR, in which an amide bond had been formed between the epsilon-amino group of Lys-66 in AD and the gamma-carboxyl group of Glu-4 in AR. These results indicate that the binding site of AR with AD is localized in the amino-terminal part of AR and that of AD with AR is localized around Lys-66 of AD. The six clustered basic amino acid residues (His-24, Lys-27, His-28, His-29, Arg-31, and His-33) present in the amino-terminal portion of AR and the eight clustered acidic amino acid residues (Glu-65, Glu-68, Asp-72, Glu-73, Glu-74, Asp-76, Asp-79, and Asp-86) present in the middle part of AD may play an important role in the complex formation.