Ribosomal Protein Genes S23 and L35 from Amphioxus Branchiostoma belcheri tsingtauense： Identification and Copy Number

The complete cDNA and deduced amino acid sequences of the ribosomal proteins S23 （AmphiS23） and L35 （AmphiL35） from amphioxus Branchiostoma belcheri tsingtauense were identified in this study. AmphiS23 cDNA is 546 bp long and encodes a protein of 143 amino acids. It has a predicted molecular mass of 15,851 Da and a pI of 10.7. AmphiL35 cDNA comprises 473 bp, and codes for a protein of 123 amino acids with a predicted molecular mass of 14,543 Da and a pI of 10.8. AmphiS23 shares more than 83% identity with its homologues in the vertebrates and more than 84% identity with those in the invertebrates. AmphiL35 is more than 63% identical to its counterparts in the vertebrates and more than 52% identical to those in the invertebrates. Southern blot analysis demonstrated the existence of 1-2 copies of the S23 gene and 2-3 copies of the L35 gene in the genome of amphioxus B. belcheri tsingtauense. This is in sharp contrast to the presence of 6-13 copies of the S23 gene and 15-17 copies of the L35 gene in therat genome. It is clear that the housekeeping genes like S23 and L35 underwent a large-scale duplication in the vertebrate lineage, reinforcing the gene/genome duplication hypothesis.     

Characterisation of AmphiAmR4, an amphioxus (Branchiostoma floridae) α2-adrenergic-like G-protein-coupled receptor

Little is known about the evolutionary relationship between vertebrate adrenergic receptors and invertebrate octopamine and tyramine receptors. The complexity of the adrenergic signalling system is believed to be an innovation of the vertebrate lineage but the presence of noradrenaline has been reported in some invertebrate species. The cephalochordate, amphioxus (Branchiostoma floridae), is an ideal model organism for studying the evolution of vertebrate GPCRs, given its unique position at the base of the chordate lineage. Here, we describe the pharmacological characterisation and second messenger coupling abilities of AmphiAmR4, which clusters with α₂-adrenergic receptors in a phylogenetic tree but also shares a high sequence similarity to invertebrate octopamine/tyramine receptors in both BLAST and Hidden Markov Model analyses. Thus, it was of particular interest to determine if AmphiAmR4 displayed similar functional properties to the vertebrate α₂-adrenergic receptors or to invertebrate octopamine or tyramine receptors. When stably expressed in Chinese hamster ovary (CHO) cells, noradrenaline couples the receptor to both the activation of adenylyl cyclase and to the activation of the MAPKinase pathway. Pharmacological studies with a wide range of agonists and antagonists suggest that AmphiAmR4 functions as an α₂-adrenergic-like receptor when expressed in CHO cells.     

Über den Feinbau der Chorda dorsalis von Branchiostoma lanceolatum

Zusammenfassung Die Chorda dorsalis von Branchiostoma lanceolatum, Pall. wurde elektronenmikroskopisch untersucht. Die Feinstruktur der Chordaplatten zeigt keine Ähnlichkeit mit der der bisher untersuchten Chordazellen der Wirbeltiere, sondern gleicht derjenigen der schräggestreiften Muskeln von Anneliden und Mollusken. Dicke und dünne Filamente sind in A-, H- und I-Bändern angeordnet. Im I-Band verläuft ein Z-Streifen. Peripher ist ein dem sarkoplasmatischen Retikulum entsprechendes Zisternensystem ausgebildet. In dicken Chordaplatten verläuft ein Tubulussystem. Lateral sind die Platten eng mit der Kollagenfaserhülle verzahnt. Wahrscheinlich bilden sich die endgültigen Chordaplatten durch Zerlegung einer Ausgangszelle in Scheiben. Die Müllerschen Zellen stehen in zytoplasmatischem Zusammenhang mit den Chordaplatten und bilden Kontaktstellen mit dem Neuralrohr aus.

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The fine structure of the notochord of Branchiostoma lanceolatum

Summary The notochord of the lancelet (Branchiostoma lanceolatum, Pall.) was investigated with the electron microscope. The ultrastructure of the notochordal plates does not show any similarity to the notochordal cells of the vertebrates investigated so far, but resembles closely the fine structure of the oblique-striated muscles of annelids and mollusks. Thick and thin filaments are arranged in A-, H- and I-bands. Through the I-band passes a Z-line. Along the periphery a system of cisterns is developed which corresponds to the peripheral sarcoplasmic reticulum. Within the thick plates a system of tubules can be found. Special formations connect the plates with the surrounding collagen sheath. Presumably, the definite formation of the notochordal plate occurs by the splitting of a parent plate into several discs. Müller's cells are found to be in cytoplasmic connection with the plates. Via the notochordal horns they come into contact with nerve cells of the spinal cord.

     

EF1α is a useful internal reference for studies of gene expression regulation in amphioxus Branchiostoma japonicum

Amphioxus is a well-known model organism widely used for interspecies comparative genome study, developmental homology analysis and comparative immunological investigation. However, no study has been performed so far to evaluate the internal reference for quantitative RT-PCR (qRT-PCR) studies of gene expression in this important species. In this study, two software applications (geNorm and NormFinder) were used to evaluate the expression stability of 4 housekeeping genes (ACTB, GAPDH, 18S rRNA and EF1α) in 8 different normal tissues (whole body, gut, gut-free body, hepatic caecum, gill, hind-gut, notochord and muscle) and 2 tissues (gut and gut-free body) challenged with LPS and LTA in amphioxus Branchiostoma japonicum. Our results showed that in the normal tissues, the expression of 18S rRNA was most abundant, whereas the expression levels of the other three genes were close to each other, with the expression of ACTB being most unstable. Following challenge with LPS and LTA, all the four genes exhibited varied degrees of expression changes in the different tissues and the expression stabilities of the genes were also affected by the different experimental conditions. Yet, the overall ranking results produced by the two algorithms consistently indicated that the expression of EF1α showed the most least variation in the different tissues, suggesting that EF1α is a suitable internal control for qRT-PCR studies in amphioxus B. japonicum.     

Synthesis and Cytotoxicity of О- and N-Acyl Derivatives of Azepanobetulin

Russian Journal of Bioorganic Chemistry - A five-stage synthesis of azepanobetulin from betulin with a total yield of 47% has been carried out. The acylation of azepanobetulin with anhydrides or...     

Cloning and sequencing of inulinase and β-fructofuranosidase genes of a deep-sea Microbulbifer species and properties of recombinant enzymes

An inulinase-producing Microbulbifer sp. strain, JAM-3301, was isolated from a deep-sea sediment. An inulin operon that contained three open reading frames was cloned and sequenced. Two of the three genes were expressed. One product was an endo-inulinase, and the other was a β-fructofuranosidase. Both enzymes worked together to effectively degrade inulin.     

Identification and Characterisation of the α and β Subunits of Succinyl CoA Ligase of Tomato

Despite the central importance of the TCA cycle in plant metabolism not all of the genes encoding its constituent enzymes have been functionally identified. In yeast, the heterodimeric protein succinyl CoA ligase is encoded for by two single-copy genes. Here we report the isolation of two tomato cDNAs coding for α- and one coding for the β-subunit of succinyl CoA ligase. These three cDNAs were used to complement the respective Saccharomyces cerevisiae mutants deficient in the α- and β-subunit, demonstrating that they encode functionally active polypeptides. The genes encoding for the subunits were expressed in all tissues, but most strongly in floral and leaf tissues, with equivalent expression of the two α-subunit genes being expressed to equivalent levels in all tissues. In all instances GFP fusion expression studies confirmed an expected mitochondrial location of the proteins encoded. Following the development of a novel assay to measure succinyl CoA ligase activity, in the direction of succinate formation, the evaluation of the maximal catalytic activities of the enzyme in a range of tissues revealed that these paralleled those of mRNA levels. We also utilized this assay to perform a preliminary characterisation of the regulatory properties of the enzyme suggesting allosteric control of this enzyme which may regulate flux through the TCA cycle in a manner consistent with its position therein.     

Preparation and Characterization of Oligonucleotides of D- and L-2’ Deoxyuridine

Abstract

The synthesis of oligonucleotides of 2'deoxyuridine containing both the natural D-2'deoxyribose and the unnatural L-2'deoxyribose is described. Units up to the 18-mer have been made via a modified triester procedure and characterized by HPLC.     

Rate of utilization of glucose and `compartmentation'' of α-oxoglutarate and glutamate in rat brain

1. The rate of incorporation of ¹⁴C into pyruvate, α-oxoglutarate, lactate and glucose of rat tissues was measured after the subcutaneous injection of uniformly labelled glucose. 2. In rat brain the specific radioactivities of lactate and glucose were similar to that of alanine. In liver the specific radioactivity of glucose was considerably higher than that of lactate or alanine. 3. The specific radioactivities of α-oxo acids of rat brain were lower than those of corresponding amino acids, alanine and glutamate. These findings have been explained in relation to metabolic compartments in vivo. 4. The approximate estimated rate of glucose utilization in rat brain in vivo is 0·96μmole/g. of brain/min.     

Summer-autumn ichthyoplankton of the Sea of Okhotsk and the Sea of Japan and special traits of feeding of fish larvae and fry in 2003–2004

In summer-autumn of 2003–2004, the ichthyoplankton of the Sea of Okhotsk comprised 35 species. In this period the most widely distributed and numerous were larvae of the lord Hemilepidotus gilberti, the Pacific stout sand lance Ammodytes hexapterus, and the Sakhalin dab Limanda sakhalinensis. The maximum catches of fish larvae were attributed to coastal waters off eastern Sakhalin and to the shelf of the northern part of the Sea of Okhotsk. In November of 2003, the ichthyoplankton of the Sea of Japan was represented by fish larvae belonging mainly to the boreal ichthyocomplex. The catches consisted predominantly of larvae of the arabesque greenling Pleurogrammus azonus, the ronquil Bathymaster derjugini, and the rockfish Sebastes owstoni. Fish larvae and fry in the northwestern part of the Sea of Japan were caught principally within 43°–45° N and 137°–139° E above the depth 1500–2000 m. The food spectrum of fish larvae in the Sea of Okhotsk and the Sea of Japan comprised over 20 plankters of various size belonging to seven taxa. Irrespective of fish species, the food items common of all fish were copepods Pseudocalanus minutus and Oithona similis. The daily rations were calculated for mass species (Hemilepidotus gilberti, Ammodytes hexapterus, Hexagrammos stelleri, Pleurogrammus azonus, Bathymaster derjugini, and Sebastes owstoni). The larvae of all considered species in the Sea of Japan and in the Sea of Okhotsk fed predominantly in the light period of the day.     

α-Lactalbumin binding and membrane integrity—effect of charge and degree of unsaturation of glycerophospholipids

Several studies have shown that the physical state of the phospholipid membrane has an important role in protein-membrane interactions, involving both electrostatic and hydrophobic forces. We have investigated the influence of the interaction of the calcium-depleted, (apo)-conformation of bovine α-lactalbumin (BLA) on the integrity of anionic glycerophospholipid vesicles by leakage experiments using fluorescence spectroscopy. The stability of the membranes was also studied by measuring surface tension/molecular area relationships with phospholipid monolayers. We show that the degree of unsaturation of the acyl chains and the proportion of charged phospholipid species in the membranes made of neutral and acidic glycerophospholipids are determinants for the association of BLA with liposomes and for the impermeability of the bilayer. Particularly, tighter packing counteracted interaction with BLA, while unsaturation—leading to looser packing—promoted interaction and leakage of contents. Equimolar mixtures of neutral and acidic glycerophospholipids were more permeable upon protein binding than pure acidic lipids. The effect of lipid structure on BLA-membrane interaction and bilayer integrity may throw new light on the membrane disrupting mechanism of a conformer of human α-lactalbumin (HAMLET) that induces death of tumour cells but not of normal cells.     

Hydrophilicity and antigenicity of proteins — A case study of myoglobin and hemoglobin

Hydrophilicity index is used to locate antigenic determinants on two related groups of proteins-myoglobin and hemoglobin. The data on 41 species (including 34 mammals) of myoglobin show that average hydrophilicity for the complete myoglobin molecules as well as the average hydrophilicity for all hydrophilic regions put together seem to remain constant; the variation in the size and location of the antigenic determinants in these species is very small indicating that the antigenic sites are not shifted during evolution. In the case of both the proteins there is a good agreement between the antigenic sites picked up by using hydrophilicity index and the experimentally determined antigenic sites. The data on 56 species of hemoglobin α-chains and 44 species of hemoglobinβ-chains showed that although there are few sites on hemoglobin which have remained invariant during evolution, there is a significant variation in other sites in terms of either a splitting of a site, or a drastic change in the hydrophilicity values and/or a length of the site. Comparison of the hydrophilicity data on these two groups of proteins suggests that hemoglobins which perform a variety of functions as compared to myoglobins are evolving faster than myoglobins supporting the contention of earlier workers.     

Analysis and modeling of time-variant amplitude–frequency couplings of and between oscillations of EEG bursts

Low-frequency (0.5-2.5 Hz) and individually defined high-frequency (7-11 or 8-12 Hz; 11-15 or 14-18 Hz) oscillatory components of the electroencephalogram (EEG) burst activity derived from thiopental-induced burst-suppression patterns (BSP) were investigated in seven sedated patients (17-26 years old) with severe head injury. The predominant high-frequency burst oscillations (>7 Hz) were detected for each patient by means of time-variant amplitude spectrum analysis. Thereafter, the instantaneous envelope (IE) and the instantaneous frequency (IF) were computed for these low- and high-frequency bands to quantify amplitude-frequency dependencies (envelope-envelope, envelope-frequency, and frequency-frequency correlations). Time-variant phase-locking, phase synchronization, and quadratic phase couplings are associated with the observed amplitude-frequency characteristics. Additionally, these time-variant analyses were carried out for modeled burst patterns. Coupled Duffing oscillators were adapted to each EEG burst and by means of these models data-based burst simulations were generated. Results are: (1) strong envelope-envelope correlations (IE courses) can be demonstrated; (2) it can be shown that a rise of the IE is associated with an increase of the IF (only for the frequency bands 0.5-2.5 and 7-11 or 8-12 Hz); (3) the rise characteristics of all individually averaged envelope-frequency courses (IE-IF) are strongly correlated; (4) for the 7-11 or 8-12 Hz oscillation these associations are weaker and the variation between the time courses of the patients is higher; (5) for both frequency ranges a quantitative amplitude-frequency dependency can be shown because higher IE peak maxima are accompanied by stronger IF changes; (6) the time range of significant phase-locking within the 7-11 or 8-12 Hz frequency bands and of the strongest quadratic phase couplings (between 0.5-2.5 and 7-11 or 8-12 Hz) is between 0 and 1,000 ms; (7) all phase coupling characteristics of the modeled bursts accord well with the corresponding characteristics of the measured EEG burst data. All amplitude-frequency dependencies and phase locking/coupling properties described here are known from and can be discussed using coupled Duffing oscillators which are characterized by autoresonance properties.     

Journal of Genetics and Genomics

(Started in 1974, Monthly)WWW.jgenetgenomics.org (Free electronic papers published in 1974-2005)Journal of Genetics and Genomics (JGG) is one of China's leading journals in the life science. JGG covers all areas of     

Rates of entry and oxidation of acetate,glucose, d(?)-β-hydroxybutyrate,palmitate, oleate and stearate,and rates of production and oxidation of propionate and butyrate in fed and starved sheep

1. Rates of entry and oxidation of a range of metabolites have been measured in tracheostomized sheep (diet, 800g. of lucerne chaff and 100g. of maize/day) by combining isotope-dilution techniques with the continuous measurement of total respiratory gas exchange, and ¹⁴CO₂ production during the intravenous or intraruminal infusion of ¹⁴C-labelled substrates. 2. Mean entry rates in fed and starved (24hr.) sheep respectively, expressed as mg./min./kg. body wt.^0·75, were: glucose, 5·0 (range 4·8–5·1, 2 observations) and 3·8 (3·2–4·2, 4); acetate, 10·8 (9·1–13·5, 4) and 5·8 (1); d(−)-β-hydroxybutyrate, 1·4 (1) and 1·5 (0·8–2·4, 4); palmitate, oleate and stearate (starved sheep only) 1·0 (0·6–1·9, 7), 0·9 (0·2–1·6, 10) and 0·9 (0·5–1·1, 11) respectively. 3. Production rates of propionate and butyrate in continuously feeding sheep were 6·4 (4·7–8·3, 4) and 4·3 (3·4–6·1, 4) mg./min./kg.^0·75 respectively, and in starved (24hr.) sheep were 2·5 (2·2–2·9, 2) and 1·0 (0·8–1·2, 2) mg./min./kg.^0·75 respectively. 4. Calculated terminal values for the specific radioactivity of respiratory ¹⁴CO₂ during measurements of entry rates and production rates were used to calculate the contributions of individual substrates to overall oxidative metabolism. Mean values for fed and starved sheep respectively were: glucose, 9·1 (8·6–9·6, 2) and 11·2 (5·9–15·1, 4)%; acetate, 31·6 (26·8–38·1, 4) and 22·1 (1)%; d(−)-β-hydroxybutyrate, 10·4 (1) and 4·8 (1·9–7·7, 4)%; propionate, 23·0 (13·8–29·9, 4) and 7·1 (6·8–7·4, 2)%; butyrate, 16·5 (13·7–20·5, 4) and 5·3 (5·2–5·3, 2)%; palmitate, oleate and stearate (starved sheep only), 4·7 (2·0–7·7, 7), 4·0 (1·2–6·6, 10) and 4·4 (3·8–5·8, 9)% respectively. The sum of these values for individual substrates in fed and starved sheep, excluding that of β-hydroxybutyrate and after correction of the glucose value for the known interrelations of this substrate with propionate, accounted for 76% and 58% respectively of total production of carbon dioxide. 5. Calculations based on the proportion of substrate entry directly oxidized indicated that the substrates studied accounted for 63% (fed sheep) and 43% (starved sheep) of total energy expenditure measured by oxygen uptake. The contribution of β-hydroxybutyrate was excluded, and corrections were made for glucose–propionate interrelations, and for the different rates of oxidation of the methyl and carboxyl fragments of acetate. 6. The present results have been combined with those obtained earlier in this Laboratory to examine the relationships between rates of substrate entry and oxidation, and concentrations of substrate in blood. Rates of entry of acetate, glucose, d(−)-β-hydroxybutyrate, palmitate and oleate (but not stearate) were well correlated with concentration in blood, and substrate contribution to production of carbon dioxide showed a similar correlation to blood concentration, except with glucose. 7. It was concluded that the general technique is of potential value in providing valid quantitative parameters of animal metabolism.     

A comparison of enzymatic phosphorylation and phosphatidylation of β-l- and β-d-nucleosides

Enzymatic 5′-monophosphorylation and 5′-phosphatidylation of a number of β-l- and β-d-nucleosides was investigated. The first reaction, catalyzed by nucleoside phosphotransferase (NPT) from Erwinia herbicola, consisted of the transfer of the phosphate residue from p-nitrophenylphosphate (p-NPP) to the 5′-hydroxyl group of nucleoside; the second was the phospholipase d (PLD)-catalyzed transphosphatidylation of l-α-lecithin with a series of β-l- and β-d-nucleosides as the phosphatidyl acceptor resulted in the formation of the respective phospholipid-nucleoside conjugates. Some β-l-nucleosides displayed similar or even higher substrate activity compared to the β-d-enantiomers.