Morphology and Molecular Phylogeny of Two New Marine Euplotids,Pseudodiophrys nigricans n. g., n. sp., and Paradiophrys zhangi n. sp. (Ciliophora: Euplotida)

ABSTRACT. The morphology, infraciliature, and molecular phylogeny of Pseudodiophrys nigricans n. g., n. sp., and Paradiophrys zhangi n. sp., isolated from a sandy beach near Qingdao, China, were investigated. Pseudodiophrys is characterized by the Diophrys‐like ciliature pattern, but having only a single, reduced undulating membrane. Pseudodiophrys nigricans, the type species by monotypy, is described from live and silver‐impregnated specimens. Paradiophrys zhangi is similar to the type species Paradiophrys irmgard but can be recognized by its border body and the number (7 vs. 8–10) and arrangement (in rows vs. sparsely distributed) of the frontoventral cirri. Small subunit (SSU) rRNA gene sequence data support the validity of both species. Phylogenetic analyses based on the SSU rRNA gene sequence data currently available for uronychiids and other related taxa indicate that P. nigricans is most closely related to Diophrys scutum and Diophrys apoligothrix, while P. zhangi clusters most closely with Apodiophrys ovalis within a clade that also includes two other Paradiophrys species.     

Two New Diophrys‐Like Genera and Their Type Species,Apodiophrys ovalis n. g., n. sp. and Heterodiophrys zhui n. g., n. sp. (Ciliophora: Euplotida), with Notes on Their Molecular Phylogeny

ABSTRACT. Based on both morphological and molecular information, two new euplotid genera Apodiophrys n. g. and Heterodiophrys n. g. are described in the present paper. Apodiophrys n. g. is defined as sculptured Diophryinae with bipartite adoral zone; frontoventral cirri arranged in Diophrys‐pattern; marginal cirri located in two clearly separated groups. Heterodiophrys n. g. is recognizable by the combination of Diophrys‐like frontoventral cirri and the unique structure of several marginal cirri that are arranged in a long row. The type species for both new genera, Apodiophrys ovalis n. sp. and Heterodiophrys zhui n. sp., collected from southern China sea, are described. The small subunit rRNA (SSU rRNA) gene sequences for both new taxa are determined. Phylogenetic analyses based on these data indicate that Apodiophrys is most closely related to Paradiophrys, which then clusters with Uronychia species. Thus, Apodiophrys–Paradiophrys is separated from other typical Diophrys‐like genera in the SSU rRNA gene trees. The new genus Heterodiophrys is basal to the sister group of Diophrys–Diophryopsis, hence belongs to the “core”Diophrys‐complex.     

Microscopic investigation of three species of Diophrys (Ciliophora,Euplotida, Uronychiidae) from Japan,including Diophrys peculiaris nov. spec

Three species of Diophrys, D. peculiaris nov. spec., D. cf. scutum and D. oligothrix, isolated from the New Nagasaki Fishing Port, Nagasaki, Japan, were investigated using live observation and protargol impregnation. Diophrys peculiaris nov. spec. can be recognized by having two characteristic clusters of rod-like structures and two groups of dikinetids located on anterior dorsal portion of cell. Morphogenetic data show that this part of the life cycle basically proceeds as in congeners, except for the formation of dikinetids under the rod-like structures. In the opisthe, the origin of dikinetids under the rod-like structures is still unknown, but the old dikinetids under the rod-like structures may be retained by the proter. The Japanese population of Diophrys cf. scutum resembles other populations of D. scutum well except for moniliform macronuclear segments. Our populations of D. oligothrix correspond well with other populations in terms of general morphology and ciliary pattern, in particular the continuous dorsal kineties with loosely arranged cilia.     

The Cortical Morphogenetic Cycle Associated with Cell Division in Diophrys Dujardin, 1841 (Ciliophora,Hypotrichida)1

Morphogenesis of cell division was investigated in Diophrys scutum, D. oligothrix, and D. appendiculata utilizing both light microscopy of living and stained specimens and SEM of preserved specimens. The cortical morphogenetic pattern of Diophrys is similar to that of other members of the family Euplotidae. The opisthe oral primordium, which develops in a subsurface pouch, forms posterior to the parental buccal cavity. The proter inherits the parental adoral zone of membranelles (AZM) apparently unchanged. The endoral membrane forms to the right of the posterior end of the AZM in the proter, in association with the developing AZM in the opisthe. The paroral cirrus and membrane develop from a single streak that first appears along the right edge of the buccal cavity in the proter to the right of the developing buccal structures of the opisthe. Frontal and transverse cirri develop in both proter and opisthe from five separate cirral primordia that form to the right of the buccal cavity. Left marginal cirri do not develop in association with the corresponding parental structures. Kinetosomes formed within the opisthe oral primordium, or kinetosomes that were part of any parental ciliary structure, do not appear to become part of any developing paroral structures, frontal, transverse, or left marginal cirri. Speciation within the genus Diophrys and evolution of the family Euplotidae as they relate to the morphogenesis of cortical structure are discussed.     

A Bacterium Belonging to the Rickettsiaceae Family Inhabits the Cytoplasm of the Marine Ciliate Diophrys appendiculata (Ciliophora, Hypotrichia)

Bacteria of the family Rickettsiaceae (order Rickettsiales, α-Proteobacteria) are mainly known to be endosymbionts of arthropods with the capability to infect also vertebrate cells. Recently, they have also been found as leech endocytobionts. In the present paper, we report the first finding of a bacterium belonging to the family Rickettsiaceae in a natural population of a marine ciliate protozoan, namely Diophrys appendiculata, collected in the Baltic Sea. Bacteria were unambiguously identified through morphological characterization and the “full-cycle rRNA approach” (i.e., 16S rRNA gene characterization and use of specifically designed oligonucleotide probes for in situ detection). Symbionts are rod-shaped bacteria that grow freely in the cytoplasm of the host cell. They present two different morphotypes, similar in size, but different in cytoplasmic density. These are typical morphological features of members of the family Rickettsiaceae. 16S rRNA gene sequence showed that Diophrys symbionts share a high similarity value (>92%) with bacteria belonging to the genus Rickettsia. Phylogenetic analysis revealed that these new endosymbionts are clearly included in the clade of the family Rickettsiaceae, but they occupy an independent phylogenetic position with respect to members of the genus Rickettsia. This is the first report of a member of this family from a host protozoan and from a marine habitat. This result shows that this bacterial group is more diversified and widespread than supposed so far, and that its ecological relevance could until now have been underestimated. In light of these considerations, the two 16S rRNA oligonucleotide probes here presented, specific for members of the Rickettsiaceae, can represent useful tools for further researches on the presence and the spread of these microorganisms in the natural environment.     

Efficient Preparation of (R)‐2‐chloromandelic Acid Via a Recycle Process of Resolution

Efficient preparation of (R)‐2‐chloromandelic acid (R)-1 based on a recycle process of resolution is described. In the process, the desired (R)-1 was obtained by coordination‐mediated resolution with D‐O,O'‐di‐(p‐toluoyl)‐tartaric acid in the presence of Ca²⁺. Meanwhile, the undesired (S)-1 could be racemized in the presence of sodium hydroxide and the product was suitable for further resolution. A carbanion mechanism for the racemization of (S)-1 is proposed. Chirality 27:281–285, 2015. © 2015 Wiley Periodicals, Inc.     

Chitosan‐heparin polyelectrolyte multilayers on cortical bone: Periosteum‐mimetic,cytophilic, antibacterial coatings

Cortical bone allografts suffer from high rates of failure due to poor integration with host tissue, leading to non‐union, fracture, and infection following secondary procedures. Here, we report a method for modifying the surfaces of cortical bone with coatings that have biological functions that may help overcome these challenges. These chitosan‐heparin coatings promote mesenchymal stem cell attachment and have significant antibacterial activity against both S. aureus and E. coli. Furthermore, their chemistry is similar to coatings we have reported on previously, which effectively stabilize and deliver heparin‐binding growth factors. These coatings have potential as synthetic periosteum for improving bone allograft outcomes. Biotechnol. Bioeng. 2013; 110: 609–618. © 2012 Wiley Periodicals, Inc.     

The effect of deuterium oxide on the stability of the collagen model peptides H‐(Pro‐Pro‐Gly)10‐OH,H‐(Gly‐Pro‐4(R)Hyp)9‐OH,and Type I collagen

The collagen triple helix has a larger accessible surface area per molecular mass than globular proteins, and therefore potentially more water interaction sites. The effect of deuterium oxide on the stability of collagen model peptides and Type I collagen molecules was analyzed by circular dichroism and differential scanning calorimetry. The transition temperatures (T_m) of the protonated peptide (Pro‐Pro‐Gly)₁₀ were 25.4 and 28.7°C in H₂O and D₂O, respectively. The increase of the T_m of (Pro‐Pro‐Gly)₁₀ measured calorimetrically at 1.0°C min^?1 in a low pH solution from the protonated to the deuterated solvent was 5.1°C. The increases of the T_m for (Gly‐Pro‐4(R)Hyp)₉ and pepsin‐extracted Type I collagen were measured as 4.2 and 2.2°C, respectively. These results indicated that the increase in the T_m in the presence of D₂O is comparable to that of globular proteins, and much less than reported previously for collagen model peptides [Gough and Bhatnagar, J Biomol Struct Dyn 1999, 17, 481–491]. These experimental results suggest that the interaction of water molecules with collagen is similar to the interaction of water with globular proteins, when the ratio of collagen to water is very small and collagen is monomerically dispersed in the solvent. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 93–101, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Molecular characterization of gap region in 28S rRNA molecules in brine shrimp <Emphasis Type="Italic">Artemia parthenogenetica</Emphasis> and planarian <Emphasis Type="Italic">Dugesia japonica</Emphasis>

In most insects and some other protostomes, a small stretch of nucleotides can be removed from mature 28S rRNA molecules, which could create two 28S rRNA subunits (28Sα and 28Sβ). Thus, during electrophoresis, the rRNA profiles of these organisms may differ significantly from the standard benchmark since the two subunits co-migrate with the 18S rRNA. To understand the structure and mechanism of the atypical 28S rRNA molecule, partial fragments of 28Sα and 28Sβ in brine shrimp Artemia parthenogenetica and planarian Dugesia japonica were cloned using a modified technology based on terminal transferase. Alignment with the corresponding sequences of 28S rDNAs indicates that there are 41 nucleotides in A. parthenogenetica and 42 nucleotides in D. japonica absent from the mature rRNAs. The AU content of the gap sequences of D. japonica and A. parthenogenetica is high. Both the gaps may form stem-loop structure. In D. japonica a UAAU cleavage signal is identified in the loop, but it is absent in A. parthenogenetica. Thus, it is proposed that the gap processing of 28S rRNA was a late enzyme-dependent cleavage event in the rRNA maturational process based on the AU rich gap sequence and the formation of the stem-loop structure to expose the processing segment, while the deletion of the gap region would not affect the structure and function of the 28S rRNA molecule.     

Chemosensory behavior of semi‐restrained Caenorhabditis elegans

A new behavioral assay is described for studying chemosensation in the nematode Caenorhabditis elegans. This assay presents three main characteristics: (1) the worm is restrained by gluing, preserving correlates of identifiable behaviors; (2) the amplitude and time course of the stimulus are controlled by the experimenter; and (3) the behavior is recorded quantitatively. We show that restrained C. elegans display behaviors comparable to those of freely moving worms. Moreover, the chemosensory response of wild‐type glued animals to changes in salt concentration is similar to that of freely moving animals. This glued‐worm assay was used to reveal new chemosensory deficits of the potassium channel mutant egl‐2. We conclude that the glued worm assay can be used to study the chemosensory regulation of C. elegans behavior and how it is affected by neuronal or genetic manipulations. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Neosurugatoxin blocks an α-bungarotoxin-sensitive neuronal nicotinic acetylcholine receptor

Neosurugatoxin (NSTX), a neurotoxin isolated from the Japanese ivory mollusc Babylonia japonica, is a potent neuronal nicotinic acetylcholine receptor (nAChR) antagonist. Hitherto, NSTX has been found to block only neuronal nAChRs that are insensitive to α-Bgt. Here, we report for the first time that NSTX blocks an α-Bgt-sensitive nAChR on an identified insect motor neurone. Bath-applied NSTX at a concentration of 10 nM and above reversibly blocks the nicotine-induced depolarizations recorded from the cockroach (Periplaneta americana) fast coxal depressor motor neurone (D_f) and is without effect on GABA-induced responses detected on the same cell. NSTX is among the most potent blockers tested to date on nAChRs of motor neurone D_f. © 1993 Wiley-Liss, Inc.     

Histological development of the long‐snouted seahorse Hippocampus guttulatus during ontogeny

The objective of the present study was to describe histological development of the European long‐snouted seahorse Hippocampus guttulatus, to increase understanding of the biology and physiology of the species. Most vital organs were present in juveniles by the time of their release from the male's pouch. Digestive tract specialization occurred at 89 effective day‐degrees (D°_eff), corresponding to 15 days post partum (dpp), with development of the first intestinal loop and mucosal folding. At 118 D°_eff (20 dpp), lipids were being mobilized from the liver and oocytes attained the perinuclear stage. The fovea emerged at 177 D°_eff (30 dpp), contemporaneous with the shift from pelagic to benthic behaviour in juveniles. At this stage, the most interesting feature was the formation of the second intestinal loop. Male gonads were never observed during the study (from 0 to 354 D°_eff; 0–60 dpp), but the first oogonia were present at 30 D°_eff (5 dpp). In 354 D°_eff (60 dpp) juveniles, oocytes were observed in a cortical alveoli stage, indicating maturity. Low digestive efficiency was observed at early stages, which was due to a poorly developed gastrointestinal tract and an immature digestive tract prior to 89 D°_eff. The present study demonstrates that approximately 89 and 177 D°_eff represent two important transitional stages in the early development of H. guttulatus. At a temperature of approximately 19 ± 1°C and an age of 1 month (177 D°_eff), main organs were fully functional, suggesting that the adult phenotype was largely established by that age, with females becoming mature at the age of 2 months (354 D°_eff).     

How far bowalization affects phytodiversity,life forms and plant morphology in Sub‐humid tropic in West Africa

Bowal or ferricrete, the final of land degradation, occurred only in tropical region. This study aimed at assessing the effects of bowalization on phytodiversity, life forms and morphological response of plant species using Combretum nigricans Leprieur ex Guill. & Perr. as a case study. Morphological parameters (height, number of stems, number of branches, diameter at breast height and crown diameter) of C. nigricans were determined in the sub‐humid zone of Benin. Plant communities were determined according to Multi‐Response Permutation Procedures analysis. Plant communities were more diversified on sand‐clay and concretion soils (control) compared with those described on bowal. C. nigricans developed more stems (3.6 ± 1.4 stems vs. 1.3 ± 0.4 stems), more branches (5.9 ± 2.4 branches vs. 3.2 ± 0.6 branches) and large crown diameter (5 ± 1.48 m vs. 3.4 ± 1.2 m) on bowal than on sand‐clay soil. The best adapted life forms on bowal were therophytes. Bowalization induced loss of phytodiversity, changes in species life forms and provoked local adaptation of tree species.     

Resolution of 1‐n‐Butyl‐3‐Methyl‐3‐Phospholene 1‐Oxide With TADDOL Derivatives and Calcium Salts of O,O'‐Dibenzoyl‐(2R,3R)‐ or O,O'‐di‐p‐Toluoyl‐(2R,3R)‐tartaric Acid

The resolution methods applying (?)‐(4R,5R)‐4,5‐bis(diphenylhydroxymethyl)‐2,2‐dimethyldioxolane (“TADDOL”), (?)‐(2R,3R)‐α,α,α',α'‐tetraphenyl‐1,4‐dioxaspiro[4.5]decan‐2,3‐dimethanol (“spiro‐TADDOL”), as well as the acidic and neutral Ca²⁺ salts of (?)‐O,O'‐dibenzoyl‐ and (?)‐O,O'‐di‐p‐toluoyl‐(2R,3R)‐tartaric acid were extended for the preparation of 1‐n‐butyl‐3‐methyl‐3‐phospholene 1‐oxide in optically active form. In one case, the intermediate diastereomeric complex could be identified by single‐crystal X‐ray analysis. The absolute P‐configuration of the enantiomers of the phospholene oxide was also determined by comparing the experimentally obtained and calculated CD spectra. Chirality 26:174–182, 2014. © 2014 Wiley Periodicals, Inc.     

Redefinition of the Genera Diophrys and Paradiophrys and Establishment of the Genus Diophryopsis N. G. (Ciliophora, Hypotrichida): Implication for the Species Problem

ABSTRACT. Species belonging to the genus Diophrys Dujardin, 1841, are easily recognized due to possession of the usual complement of approximately seven frontoventral cirri, five transverse cirri, two left marginal cirri, and three large caudal cirri. Separation of these species has been based upon differences in cell length and width, the number and arrangement of cilia in dorsal kinetics, the configuration of the adoral zone of oral polykinetids, the number and distribution of cirri within cirral groups, and the number and arrangement of macronuclei. Jankowski used some of these characteristics to divide the genus into two genera, Diophrys and Paradiophrys, with several subgenera [Jankowski, A. W. 1978. Systematic revision of the class Polyhymenophora (Spirotricha), morphology, systematics and evolution. Tezisy Dokl. Zool. Inst. Akad. Nauk SSSR, 197839-40. (in Russian); Jankowski, A. W. 1979. Systematics and phylogeny of the order Hypotrichida Stein, 1859 (Protozoa, Ciliophora). Trudy. Zool. Inst. Akad. Nauk SSSR, 86 :48–85. (in Russian with English summary)]. Data obtained from light microscopic examination of stained (nigrosin-butanol, Chatton-Lwoff, and Protargol) cells in interphase or division supports and modifies the use of particular structural features of these ciliates for the purpose of taxonomic classification. The structural variability within and among populations of different species within the genera Diophrys (D. appendiculata, D. oligothrix, and D. scutum) and Paradiophrys (P. irmgard and P. multinucleata) is described. D. hystrix is redescribed as the type of the new genus Diophryopsis n. g. Comparative information on the cortical morphogenesis of division of selected species within each genus is reviewed. Two taxonomic classifications of these hypotrichs are discussed: 1) a listing of diagnoses and synonymies and 2) a binary key for identification of all species at the light microscope level. An alternative evolutionary explanation of variations among isolates is presented.     

Unexpected Loss of Stereoselectivity in Ring‐Opening Reaction of 2‐Alkoxy‐2‐Thio‐1,3,2‐Oxathiaphospholanes With a Pyrophosphate Anion

A reaction of DBU promoted ring opening in nucleoside‐3'‐O‐ and nucleoside‐5'‐O‐(2‐thio‐4,4‐pentamethylene‐1,3,2‐oxathiaphospholane) monomers with a pyrophosphate or a methylenediphosphonate anion proceeds with substantial loss of stereoselectivity. Depending on the absolute configuration of the phosphorus atom, so far widely accepted the stereoretentive mechanism of condensation is accompanied by a stereoinvertive one, most likely employing an intramolecular ligand–ligand exchange in an uncharged intermediate. Chirality 27:155–122, 2015. © 2014 Wiley Periodicals, Inc     

Label‐free determination of protein–ligand binding constants using mass spectrometry and validation using surface plasmon resonance and isothermal titration calorimetry

We performed a systematic comparison of three label‐free methods for quantitative assessment of binding strengths of proteins interacting with small molecule ligands. The performance of (1) nanoelectrospray ionization mass spectrometry (nESI‐MS), (2) surface plasmon resonance (SPR), and (3) isothermal titration calorimetry (ITC) was compared for the determination of dissociation constants (K_D). The model system studied for this purpose was the human carbonic anhydrase I (hCAI) with eight known and well characterized sulfonamide inhibitors (Krishnamurthy et al., Chem. Rev. 2008, 108: 946–1051). The binding affinities of the inhibitors chosen vary by more than four orders of magnitude e.g., the K_D value determined for ethoxzolamide by nESI‐MS was 5 ± 1 nM and the K_D value for sulfanilamide was 145.7 ± 10.0 µM. The agreement of the determined K_D values by the three methods investigated was excellent for ethoxzolamide and benzenesulfonamide (variation with experimental error), good for acetazolamide and 4‐carboxybenzenesulfonamide (variation by ～ one order of magnitude), but poor for others e.g., sulpiride. The accuracies of the K_D values are determined, and advantages and drawbacks of the individual methods are discussed. Moreover, we critically evaluate the three examined methods in terms of ease of the measurement, sample consumption, time requirement, and discuss their limitations. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect of head‐to‐tail cyclization on conformation of histatin‐5

Histatin‐5 (Hst‐5, DSHAKRHHGYKRKFHEKHHSHRGY) is a member of a histidine‐rich peptide family secreted by major salivary glands, exhibiting high fungicidal activity against Candida albicans. In the present work, we demonstrate the 3D structure of the head‐to‐tail cyclic variant of Hst‐5 in TFE solution determined using NMR spectroscopy and molecular dynamics simulations. The cyclic histatin‐5 reveals a helix‐loop‐helix motif with α‐helices at positions Ala⁴‐His⁷ and Lys¹¹‐Ser²⁰. Both helical segments are arranged relative to each other at an angle of ca. 142°. The head‐to‐tail cyclization increases amphipathicity of the peptide, this, however, does not affect its antimicrobial potency. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Argentina adulterina (Rosaceae‐Potentilleae), a new species from New Guinea

Argentina adulterina Soják sp. nova from New Guinea (Irian Jaya) is described and compared with allied species. Habitually it is similar to A. irianensis, but in leaflet dentation it agrees with A. biloba. Leaves and leaflets of these three species are illustrated and a depiction of calyces of A. adulterina and A. irianensis is presented (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)