Structural characterization and promoter activity analysis of the γ-kafirin gene from sorghum

A genomic clone encoding the γ-kafirin gene from sorghum was isolated and sequenced. A 2938 bp sequenced fragment includes an intronless open reading frame of 636 nucleotides encoding a putative polypeptide of 212 amino acids. Comparison of the deduced amino acid sequence of γ-kafirin with the published sequences of γ-prolamins of maize, and Coix revealed highly conserved domains. The N-terminal region of these proteins contains the conserved hexapeptide PPPVHL, which is repeated eight times in γ-zein, four times in γ-kafirin and three times in γ-coixin. The number of PPPVHL repeats accounts predominantly for the differences in the molecular weights of γ-prolamins. Several putative regulatory sequences common to the γ-kafirin and γ-zein genes were identified in both the 5′ and the 3′ flanking regions. Putative GCN4-like regulatory sequences were found at positions ?192 and ?476 in the 5′ flanking region of γ-kafirin. In the 3′ noncoding region, three putative polyadenylation signals, two AATAAT and one AATGAA, were found at positions + 658, + 716, and + 785, respectively. In order to investigate the role of the putative GCN4-like motifs and other possible cis-acting element(s) of the γ-kafirin promoter, a series of deleted and chimeric promoter constructs were introduced into maize, Coix and sorghum tissues by particle bombardment. Histochemical analysis of β-glucuronidase (GUS) activity in different tissues indicated that the element(s) responsible for tissue specificity is probably located in the 285-bp proximal region of the promoter, while the remaining promoter sequence seems to carry the element(s) responsible for the quantitative response.     

First record of ponyfishes (Perciformes: Leiognathidae) from the Oligocene of the Grube Unterfeld (“Frauenweiler”) clay pit

The first and presently only record of a ponyfish is described from the Lower Oligocene of the Grube Unterfeld (“Frauenweiler”) clay pit at Rauenberg (S Germany) and compared with other extant and fossil representatives of the Leiognathidae. It differs from all extant (and probably also fossil) members of this family in the coalescence of the ascending and articular processes of the premaxilla and the presence of a straight maxilla with a prominent dorsal crest. It furthermore differs from most extant and probably also fossil Leiognathidae in the combination of the following characters: the presence of multiple-rowed villiform teeth on the oral jaws; the presence of three supraneurals; third and fourth dorsal as well as third anal fin spines that are smooth at their anterior margins; delicate longitudinal surface striations on the neural and haemal spines of the vertebral column as well as on the dorsal and anal fin pterygiophores and on the dorsal, anal and pelvic fin spines; small but not plate-like distal expansions on the dorsal and haemal spines of the fourth preural centrum; the presence of autogenous hypurals; and a squamation that extends anteriorly onto the occipital and otic regions but not onto the cheeks. In terms of its general morphological traits, it resembles Leiognathoides altapinna, a species that has been reported from contemporary localities in Switzerland and Russia, although there are also some deviations that may be indicative of a different species. However, with the available materials, it is difficult to decide whether these differences are of taxonomic significance, so the new record is assigned to the genus Leiognathoides as cf. L. altapinna. The Oligocene specimens from France as well as the Lower Miocene specimens from the Czech Republic clearly belong to different taxa and probably not to the genus Leiognathoides at all. With regard to the palaeoecological and palaeogeographical framework, the new record indicates a tropical to subtropical climate and a waterway connection with the eastern Paratethys.     

Isolation and characterization of an antimicrobial peptide from bovine hemoglobin ��-subunit

In this study, a novel 18-residue linear antimicrobial peptide derived from the central part of the bovine hemoglobin ??-subunit was identified. The peptide was purified by a combination of cationic exchange and reversed-phase high-performance liquid chromatography. The sequence was determined to be VNFKLLSHSLLVTLASHL. The theoretical molecular weight of this peptide was calculated to be 1992.38 Da, which is the same as that determined (1992.401 Da) by matrix-assisted laser desorption ionization mass spectrometry. Sequence analysis showed that there is a high degree of homology in this peptide among hemoglobin ??-subunits of bovine, sheep, deer, porcine, and human. In a radial-diffusion plate assay, this purified peptide exhibited antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Candida albicans.     

Structural analysis in nonsymbiotic hemoglobins: What can we learn from inner cavities?

Plants contain three classes of hemoglobins which are not associated with nitrogen fixing bacteria, and have been accordingly termed nonsymbiotic hemoglobins. The function of nonsymbiotic hemoglobins is as yet mostly unknown. A NO dioxygenase activity has been proposed and demonstrated for some of them in vitro. In this context, a sound molecular mechanism that relates the structure with the biological activity is crucial to suggest a given physiological role. Insight into such a mechanism is now facilitated by recent progress made in both experimental and computational techniques. These studies have highlighted a number of key structural features implicated in the function of nonsymbiotic hemoglobins. The bis-histidyl hexacoordination of the heme in both its ferric and ferrous states provides a powerful and general tool to modulate reactivity, protein dynamics, and shape of the cavities. In addition, the specific arrangement of distal cavity residues provides effective protection against autoxidation. Inspection of the static crystal structures available for both liganded and unliganded states seems unsufficient to explain the function of these proteins. Function appears to be intimately linked with protein flexibility, which influences the dynamical behavior of inner cavities, capable of delivering apolar reactants to the reaction site, and removing charged reaction products. In this mini review, we demonstrate how the integration of information derived from experimental assays and computational studies is valuable and can shed light into the linkage between structural plasticity of nonsymbiotic hemoglobins and their biological role.     

Structural characterization of hemoglobin NSeattle:α2Aβ261Lys→Glu

Hemoglobin N_Seattle is an abnormal human hemoglobin found in a Seattle man of Negro ancestry. The structure of hemoglobin N_Seattle was found by chemical analysis to differ from the structure of normal hemoglobin by the substitution of a glutamic acid residue for the lysine residue in position 61 of the β chain. The structural formula is α₂β₂^{61 Glu}.     

Structural characterization of the interactions between palladin and α-actinin

The interaction between α-actinin and palladin, two actin-cross-linking proteins, is essential for proper bidirectional targeting of these proteins. As a first step toward understanding the role of this complex in organizing cytoskeletal actin, we have characterized binding interactions between the EF-hand domain of α-actinin (Act-EF34) and peptides derived from palladin and generated an NMR-derived structural model for the Act-EF34/palladin peptide complex. The critical binding site residues are similar to an α-actinin binding motif previously suggested for the complex between Act-EF34 and titin Z-repeats. The structure-based model of the Act-EF34/palladin peptide complex expands our understanding of binding specificity between the scaffold protein α-actinin and various ligands, which appears to require an α-helical motif containing four hydrophobic residues, common to many α-actinin ligands. We also provide evidence that the Family X mutation in palladin, associated with a highly penetrant form of pancreatic cancer, does not interfere with α-actinin binding.     

The purification,characterization and ligand-binding kinetics of hemoglobins from root nodules of the non-leguminous Casuarina glauca — Frankia symbiosis

The presence of a membrane-bound hemoglobin in aqueous extracts of nitrogen-fixing Casuarina root nodules (Davenport, H.E. (1960) Nature 186, 653–654) has been confirmed. By strictly anaerobic grinding and extraction under carbon monoxide, with inclusion of soluble polyvinylpyrrolidone and zwitterionic detergent in extraction buffer, soluble carboxyhemoglobin was obtained. This was purified by anaerobic ‘adsorption’ chromatography on Sephacryl S-200 (Pharmacia) followed by aerobic molecular exclusion chromatography on Sephadex G-75 (Pharmacia) to yield very stable oxyhemoglobin. By preparative-scale isoelectric focusing Casuarina oxyhemoglobin is separable into three major components comprising approx. 20% of applied protein, and very many minor components. Monomeric Casuarina hemoglobin is similar to other plant hemoglobins in respect of molecular weight (≈ 17 500), optical spectra, extremely rapid kinetics of binding to oxygen and carbon monoxide and high oxygen affinity (P₅₀ ≈ 0.074 torr). Hence, it is possible that this protein functions in the Casuarina symbiosis as does leghemoglobin in leguminous nitrogen-fixing symbioses. Western blot analysis showed close immunological relationships between the non-leguminous Casuarina and Parasponia hemoglobins and a weaker relationship between these two proteins and soybean leghemoglobin. It is proposed that these hemoglobins from widely separated plant orders have a common evolutionary origin.     

First partial cranium of Togocetus from Kpogamé (Togo) and the protocetid diversity in the Togolese phosphate basin

Earliest cetaceans (whales) originated from the early Eocene of Indo-Pakistan, but the group dispersed through most of the oceans of the planet by the late middle to late Eocene. This late Eocene global distribution indicates that important dispersal events took place during the middle Eocene (Lutetian), a globally undersampled time interval that is well documented in the Togolese phosphate series. We report here the first discovery of a partial cetacean cranium from middle Eocene deposits of Togo (West Africa). A 3D model of the cranium and teeth was reconstructed in order to reveal hidden anatomical features. The dental and cranial characteristics of the Togolese specimen recall those of protocetid taxa described in Africa, Asia, and North America, but also display significant differences. In particular, we show that the new specimen shares a number of morphological features with the Togolese taxon Togocetus. Such a hypothesis is further supported by a cladistic analysis including 45 taxa and 167 morphological characters, which recovers the new specimen close to Togocetus as the first offshoot of protocetids. Phylogenetic analysis including all the protocetids remains of Kpogamé confirms the singular diversity of the Togolese phosphate basin, and enables to examine potential connections with faunas from contemporaneous localities in Africa.     

Application of “spider-web” mode in discovery and identification of Q-markers from Xuefu Zhuyu capsule

BackgroundThe selection of quality control indicators in a complex system is a key scientific issue for the study of Chinese materia medica (CMM), which is directly related to its safety and efficacy. In order to scientifically understand and control the quality of CMM, quality marker (Q-marker) has been recently raised as a new concept, which provided a novel research idea for the quality control and evaluation of CMM.PurposeBy a new and integrated “spider-web” mode, Q-markers of Xuefu Zhuyu capsule (XZC) were comprehensively uncovered, conducing to great improvement of quality control of XZC.MethodsMainly established by three dimensions derived from six variables including content, stability and activity, “spider-web” mode was constructed to evaluate Q-marker property of candidate compounds by taking regression area of the tested compounds into account.ResultsThe candidate compounds with larger regression area were preferentially adopted as Q-markers, which should possess the satisfactorily integrated properties of content, stability and activity. Six compounds, naringin, isoliquiritin, paeoniflorin, protocatechuic acid, neohesperidin and ferulic acid, were identified and preferred as Q-markers of XZC.ConclusionBased on “spider-web” mode, Q-markers from Xuefu Zhuyu capsule were successfully screened, which would substantially perform quality control of XZC and prove the feasibility of “spider-web” mode in solving the selection of quality control indicators from compound formulae.     

A new Veturius (Veturius) of the “cephalotes” group from southern Colombia,Putumayo (Coleoptera: Passalidae)

A new species of Veturius (Veturius) Kaup from southern Colombia, Putumayo, lowland forests, is described and illustrated. V. paya n. sp. belongs to the South American “cephalotes” species group and is sister species of V. cephalotes (Le Peletier & Serville, 1825). Both species are distinguished by few characters of the head and thorax. They seem to be allopatric close to the western distributional limits of V. cephalotes. The endemism of V. paya n. sp. corresponds to a geographic vicariance in the axis of the Amazon Basin. An overview is given on the phylogeny and the chorology of the completed “cephalotes” species group.     

Artemia hemoglobins: Increase in net synthesis of the β-polypeptide (relative to the α-polypeptide) in hypoxia

Previous studies have shown that in the brine shrimp there are three dimeric hemoglobins with polypeptide composition α₂, αβ, β₂. Concentrations of the α- and β-polypeptides increase in hypoxia. We now report a two-dimensional electrophoretic method for assay of radiolabelled polypeptidesin each hemoglobin. Net synthesis (synthesis minus degradation) of the β-chain, relative to that of the α-chain, increases more than 3-fold (in male and female adults) within 3 days following a downshift in oxygen concentration from 0.2 to 0.1 mM in the culture medium. 3 days after downshift (2 days after in vivo incorporation of radiolabelled leucine), the β-homodimer contained 10–20% of the radiolabel in the three hemoglobins although β₂ was usually not detectable in the protein stain of an overloaded gel. The amount of radioactive leucine incorporated per unit amount of protein was more than 300-times greater in the β₂ homodimer than in the β-subunit of the heterodimer, suggesting that β₂ does not dissociate rapidly during electrophoresis on the first dimension non-denaturing gel. This evidence for stable association of the two β-monomers and the 5–8 heme-binding domains within each monomer (in vivo and during electrophoresis on non-denaturing gels) allows us to exclude one of two alternative interpretations of genetic data published previously. We present an independent line of evidence for the dimer model of the native hemoglobins (which states that each polypeptide has many heme-binding domains).     

Structural and enzymatic characterization of the isoamylase1 homo-oligomer and the isoamylase1–isoamylase2 hetero-oligomer from rice endosperm

The present study established that there are two distinct polymeric forms of isoamylase1 (ISA1) in rice endosperm: presumably a homo-pentamer of ISA1 and a hetero-hexamer composed of five ISA1 and one ISA2. The molecular sizes of the homo- and hetero-oligomers, which could be fractionated by hydrophobic chromatography, were approximately 420–480 and 510–550 kDa, respectively. The hetero-oligomer exhibited higher affinities for various branched polyglucans, especially for phytoglycogen, which had a K _m value that was approximately 12 times lower relative to that with the homo-oligomer, although no marked differences were found in chain preferences for debranching of amylopectin and phytoglycogen between these forms. The hetero-oligomer was active even when incubated at 50°C for 10 min, while the homo-multimer was completely inactivated at 40°C in 10 min. When the ISA1 homo-oligomer was incubated with the ISA2 protein expressed in Escherichia coli and applied onto a nondenature polyacrylamide gel, additional debranching activity bands which were specific for the purified ISA1–ISA2 preparation were also detected, indicating that ISA1 and ISA2 combine to form a hetero-oligomer. These results suggest that the hetero-oligomer plays a predominant role in the amylopectin biosynthesis in rice endosperm although the homo-oligomer can complement the function of the hetero-oligomer at least to some extent.Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Saponins and Phylogeny: Example of the “Gypsogenin group” Saponins

The chemical structure of triterpenoid saponins is quite complicated, especially the glucuronide oleanane-type triterpene carboxylic acid 3,28-bidesmosides (GOTCAB) saponins. Moreover, triterpenoid saponins are numerous as a result of this complicated structure. This review tries to explain this diversity in terms of plant classification and phylogeny. The study focuses on the three main successive steps of the biosynthetic pathway of the triterpenoid saponins: cyclisation, oxidation and glycosylation showing the relationship between triterpenols and sterols in terms of cell membrane evolution and the importance of which metabolic intermediates involved as aglycones of the triterpenoid saponins, represented progressively less and less in the advanced groups of the plant kingdom as they are more and more oxidised. This oxidation seems to reflect a better adaptation to new environmental conditions of some of these groups. By their enormous chemical diversity, the triterpenoid saponins seem to be good candidates to study the phylogeny of the flowering plants. A first attempt is given using recent advances in botanical classification for the orders and families by the Angiosperm Phylogenic Group (APG). This study was simplified in the first step focusing only on the “gypsogenin group” of the GOTCAB saponins. For example, as a result, these compounds are mainly concentrated in advanced groups such as Caryophyllideae, primitive Rosideae and Asterideae.     

The isolation and partial characterization of α2-macroglobulin from the serum of the ostrich (Struthio camelus)

• 1.1. α₂-Macroglobulin (α₂M) activity is present in the serum of the ostrich, Struthio camelus. The chromogenic synthetic peptide substrates BAPNA and ATNA were hydrolysed by trypsin and chymotrypsin, respectively, in the presence of ostrich serum and the α₂M in ostrich serum protected trypsin from being inhibited by soybean trypsin inhibitor. Ostrich α₂M proved to be a potent inhibitor of bovine pancreatic trypsin and chymotrypsin.
• 2.2. α₂M was purified to apparent homogeneity by PEG precipitation, DEAE-Toyopearl 650M, Bio-Gel A-5m and Zn²⁺-affinity chromatography.
• 3.3. Ostrich α₂M migrated as a single band (M_r 779,000) during non-denaturing gradient gel electrophoresis and showed increased mobility after reaction with trypsin. Denaturation dissociated ostrich α₂ M into half-molecules. Denaturation with reduction further dissociated the protein into quarter-subunits.
• 4.4. Isoelectric focusing revealed a pI of 5.3.
• 5.5. The amino acid composition of ostrich α₂M is typical of an α₂M, comparing favourably with those of other animal species. The carbohydrate composition of the purified protein, in percentage dry weight of the molecule, was galactose: mannose (1:1), 4.55; N-acetylglucosamine, 2.35; N-acetylneuraminic acid, 0.58; and fucose, 0.77.
• 6.6. α₂M was assessed immunologically by Ouchterlony double-diffusion and Western blot analysis with polyvalent antisera directed against ostrich α₂M.
• 7.7. Ostrich α₂M seems to show many physical, chemical and kinetic properties similar to those of other known α₂Ms, but is expected to differ from other αMs when considering the primary structure of the bait region, the area differing among α Ms from different species and determining its specificity.

     

Spectrophotometric, electron paramagnetic resonance and oxygen binding studies on the hemoglobin from the marine polychaete Perinereis aibuhitensis (Grübe): comparative physiology of hemoglobin

The physicochemical properties of giant hemoglobin (Hb) of the marine polychaete Perinereis aibuhitensis were extensively studied and the following results were obtained. (1) Light absorption spectra of the oxy, deoxy, CO, met, and cyanomet derivatives were similar to those for human Hb, except for a somewhat peculiar shape and pH-dependence of the met derivative, and high absorbance values around 277 nm for all these derivatives of Perinereis Hb. Abnormal pH dependence for the met derivative was confirmed by powder electron parmagnetic resonance (EPR) spectroscopy, which revealed that a water molecule does not coordinate to the heme iron as a sixth ligand. The high absorption around 277 nm is indicative of the existence of some non-heme polypeptide chains and/or a high content of aromatic residues in the molecule. (2) UV difference and derivative spectra revealed oxygenation-induced conformational changes in the protein moiety that are related to the degree of cooperativity. (3) The EPR spectrum for the nitrosyl derivative showed well-resolved triplet-triplet splittings due to 14N, indicating that the proximal residue is probably a histidine. (4) The oxygen affinity and cooperativity of this Hb were pH-dependent. Mg2+ markedly increased the oxygen affinity, the Bohr effect, and the cooperativity, which was maximal at physiological pH. CO2 and anions such as 2,3-diphosphoglycerate and inositol hexaphosphate had no effect on the oxygenation properties. Thus, different from vertebrate Hb, the oxygen-binding properties of this Hb are regulated by divalent cations which bind preferentially to the oxy form. The low temperature-dependence of oxygen affinity observed for this Hb is a sign of adaptation to the environment by this poikilothermic organism. (5) By using a graphic method, the minimal functional unit that preserves the full cooperativity (allosteric unit) was inferred to be the one containing 6 heme groups and its significance is discussed in connection with the structural hierarchy of the molecule.     

Untangling the Echiniscus Gordian knot: paraphyly of the “arctomys group” (Heterotardigrada: Echiniscidae)

The genus Echiniscus C.A.S. Schultze, 1840, one of the earliest established and speciose tardigrade genera, has been hypothesized to be polyphyletic. Moreover, the presence or absence of trunk appendages, the type of cuticular sculpturing and body colour have been argued to hold taxonomic significance at the genus level in Echiniscus-line taxa. Here, by combining morphological and genetic analyses, we demonstrate that the so-called “arctomys group”, i.e. Echiniscus spp. lacking trunk appendages, comprises numerous separate evolutionary lineages within the family Echiniscidae. As a result, we erect five new echiniscid genera: Barbaria gen. nov. , presumably of Neotropical (Gondwanan) origin, previously classified as the Echiniscus bigranulatus group; the pantropical and subtropical Kristenseniscus gen. nov. (the tessellatus group), characterized by a peculiar subdivision of dorsal plates; Claxtonia gen. nov. (the wendti group), with large and evident endocuticular pillars in the form of polygons; Nebularmis gen. nov. (the reticulatus group), with an elusive dorsal sculpturing; and Viridiscus gen. nov. (the viridis group), with body colour ranging from light green through brownish to even almost black. Additionally, we briefly address appendaged Echiniscus s.s. and divide the genus into several groups based on dorsal plate sculpturing and suggest that these could also represent separate supraspecific entities.