Nuclease Digestion of Chromatin from the Eukaryotic Algae Olisthodiscus luteus,Peridinium balticum,and Crypthecodinium cohnii 1, 2

Chromatin from a uninucleate dinoflagellate, Crypthecodinium cohnii, a binucleate dinoflagellate, Peridinium balticum, and a chromophyte, Olisthodiscus luteus, was examined by nuclease digestion and the results were compared to those from vertebrates. Gel analysis of the products of staphylococcal (micrococcal) nuclease digestion revealed a DNA repeat unit of 220(±5) base pairs for O. luteus and 215(±5) for P. balticum. Limit digestion gave a core particle of 140 base pairs, revealing that these longer repeat sizes are due to longer linker regions. No repeating subunit structure was found upon electrophoresis of digests of C. cohnii nuclei. Examination of the DNA fragments produced by DNAse I digestion of nuclei isolated from P. balticum and O. luteus showed the same ladder of ten base multiples as seen in chromatin from other eukaryotes. Examination of the kinetics of digestion by DNAse II of Peridinium chromatin revealed less susceptibility when compared to DNAse I digestions while 70% of Olisthodiscus chromatin and 35% of C. cohnii chromatin was sensitive to DNAse II. These data, taken together with previous results from Euglena, indicate that while algal chromatin is similar to that of higher eukaryotes in regard to DNAse I and II action, it differs in that the linker DNA is longer. In addition, the Hl-like histone from O. luteus and P. balticum is located in the linker DNA as in higher eukaryotes.     

Two new species of <Emphasis Type="Italic">Anthobothrium</Emphasis> van Beneden, 1850 (Tetraphyllidea: Phyllobothriidae) from carcharhinid sharks,with a redescription of <Emphasis Type="Italic">Anthobothrium laciniatum</Emphasis> Linton, 1890

Anthobothrium laciniatum Linton, 1890 is redescribed based on specimens taken from the dusky shark Carcharhinus obscurus (Lesueur) collected from the Northwestern Atlantic Ocean, and a neotype is designated. A. laciniatum differs from A. cornucopia van Beneden, 1850, A. altavelae Euzet & Ben Hassine, 2002, A. lesteri Williams, Burt & Caira, 2004 and A. spinosum Subhapradha, 1955 in total length. It further differs from A. cornucopia, A. altavelae and A. spinosum in proglottid number, and differs from A. galeorhini Suriano, 2002, A. cornucopia, and A. spinosum in testis number. A. lyndoni n. sp. is described from the sandbar shark C. plumbeus (Nardo). This new species differs from A. laciniatum in ovarian width and from A. cornucopia, A. altavelae, A. galeorhini and A. spinosum in the total number of proglottids. It further differs from A. cornucopia, A. galeorhini, and A. spinosum in total length, and from A. cornucopia and A. galeorhini in the number of testes. A. lyndoni n. sp. differs from A. lesteri in bothridial muscle and ovarian morphology. Anthobothrium caseyi n. sp. is described from Prionace glauca (Linnaeus). This new species differs conspicuously from the other six species of Anthobothrium van Beneden, 1850 (sensu stricto) in the shape of its proglottid laciniations. The taxonomic status of 43 species that have been associated with Anthobothrium is addressed. Taxonomic actions regarding Anthobothrium during the past century have resulted in a polyphyletic taxon.     

Permeation through Phospholipid Bilayers,Skin‐Cell Penetration,Plasma Stability,and CD Spectra of α‐ and β‐Oligoproline Derivatives

After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell‐penetrating properties of polycationic proline‐containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002 , 124, 8876) is acknowledged, according to which fluorescein‐labeled tetradecaproline is slowly taken up by rat kidney cells (NRK‐49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004 , 1, 1111) observation that a hexa‐β³‐Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo‐L ‐ and oligo‐D ‐α‐prolines, as well as of oligo‐β²h‐ and oligo‐β³h‐prolines without and with fluorescence labels ( 1 – 8 ; Fig. 1). Permeation through protein‐free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2–4). This methodology is applied for the first time for quantitative determination of translocation rates of cell‐penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6–8, resp.). The stabilities of oligoprolines in heparin‐stabilized human plasma increase with decreasing chain lengths (Figs. 9–11). Time‐ and solvent‐dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption.     

<i>Trichoderma</i>-Induced Improvement in Growth,Photosynthetic Pigments,Proline, and Glutathione Levels in <i>Cucurbita pepo</i> Seedlings under Salt Stress

Salt stress is one of the major abiotic stress in plants. However, traditional approaches are not always efficient in conferring salt tolerance. Experiments were conducted to understand the role of Trichoderma spp. (T. harzianum and T. viride) in growth, chlorophyll (Chl) synthesis, and proline accumulation of C. pepo exposed to salinity stress. There were three salt stress (50, 100, and 150 mM NaCl) lavels and three different Trichoderma inoculation viz. T. harzianum, T. viride, and T. harzianum + T. viride. Salt stress significantly declined the growth in terms of the shoot and root lengths; however, it was improved by the inoculation of Trichoderma spp. C. pepo inoculated with Trichoderma exhibited increased synthesis of pigments like chl a, chl b, carotenoids, and anthocyanins under normal conditions. It was interesting to observe that such positive effects were maintained under salt-stressed conditions, as reflected by the amelioration of the salinity-mediated decline in growth, physiology and antioxidant defense. The inoculation of Trichoderma spp. enhanced the synthesis of proline, glutathione, proteins and increased the relative water content. In addition, Trichoderma inoculation increased membrane stability and reduced the generation of hydrogen peroxide. Therefore, Trichoderma spp. can be exploited either individually or in combination to enhance the growth and physiology of C. pepo under saline conditions.     

Ndst1, a heparan sulfate modification enzyme, regulates neuroectodermal patterning by enhancing Wnt signaling in Xenopus

Neural tissue is derived from three precursor regions: neural plate, neural crest, and preplacodal ectoderm. These regions are determined by morphogen-mediated signaling. Morphogen distribution is generally regulated by binding to an extracellular matrix component, heparan sulfate (HS) proteoglycan. HS is modified by many enzymes, such as N-deacetyl sulfotransferase 1 (Ndst1), which is highly expressed in early development. However, functions of HS modifications in ectodermal patterning are largely unknown. In this study, we analyzed the role of Ndst1 using Xenopus embryos. We found that ndst1 was expressed in anterior neural plate and the trigeminal region at the neurula stage. ndst1 overexpression expanded the neural crest (NC) region, whereas translational inhibition reduced not only the trigeminal region, but also the adjacent NC region, especially the anterior part. At a later stage, ndst1 knocked-down embryos showed defects in cranial ganglion formation. We also found that Ndst1 activates Wnt signaling pathway at the neurula stage. Taken together, our results suggest that N-sulfonated HS accumulates Wnt ligand and activates Wnt signaling in ndst1-expressing cells, but that it inhibits signaling in non-ndst1-expressing cells, leading to proper neuroectodermal patterning.     

Cloning,Expression, Purification,and Characterization of a Novel Esterase from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>

Lactobacillus plantarum is an important lactic acid bacterium, usually found as natural inhabitant of food, such as fermented vegetables and meat products. However, little information about lactic acid bacteria, especially concerning L. plantarum, as a source of useful enzymes has been reported. The aim of this study was to clone, express in Escherichia coli, purify, and characterize an esterase from L. plantarum ATCC 8014. The esterase gene (1014 bp) was amplified and cloned in pET14b expression vector to express a His₆-tagged protein in E. coli. Recombinant L. plantarum esterase was purified by Ni-NTA resin, presenting an apparent molecular mass of about 38 kDa. It presented highest activity at pH 6.0 and 40°C. Also, it presented preference for p-nitrophenyl butyrate, but hydrolyzed more efficiently p-nitrophenyl acetate. Besides, this study shows, for the first time, CD data about secondary structure of an esterase from L. plantarum.     

Validation of <Emphasis Type="Italic">Christianella</Emphasis> Guiart, 1931 (Cestoda: Trypanorhyncha) and its taxonomic relationship with <Emphasis Type="Italic">Grillotia</Emphasis> Guiart, 1927

Christianella Guiart, 1931 (Cestoda: Trypanorhyncha) is redefined as a subgenus of Grillotia Guiart, 1927 based on the type-species, G. (C.) minuta (van Beneden, 1849), from the elasmobranch Squatina squatina (Linnaeus). Grillotia smarisgora (Wagener, 1854) is treated as a synonym of G. (C.) minuta, as are G. angeli Dollfus, 1969 and G. bothridiopunctata Dollfus, 1969. Other species included in the subgenus are G. (C.) carvajalregorum Menoret & Ivanov, 2009 (formerly Progrillotia dollfusi Carvajal & Rego, 1983), G. (C.) australis Beveridge & Campbell, 2001, G. (C.) longispinis (Linton, 1890) n. comb. (formerly Rhynchobothrium longispine Linton, 1890) and G. (C.) yuniariae Palm, 2004. The subgenus is similar to Grillotia Guiart, 1927 (sensu stricto), having two bothria and an atypical heteroacanthous armature, but differs in having a single row of intercalary hooks, fewer, elongate segments with testes often in longitudinal columns, a distinctive basal armature, an internal seminal vesicle which extends beyond the cirrus or hermaphroditic sac and no uterine pore. The adults of three species are known, all parasitising members of Squatina Duméril.     

<Emphasis Type="Italic">Ambrosiella</Emphasis><Emphasis Type="Italic">beaveri,</Emphasis> sp. nov., Associated with an exotic ambrosia beetle, <Emphasis Type="Italic">Xylosandrus mutilatus</Emphasis> (Coleoptera: Curculionidae,Scolytinae), in Mississippi,USA

Xylosandrus mutilatus is an Asian ambrosia beetle that has recently established in Mississippi, Texas, Alabama, and possibly Florida, USA. We investigated the fungi associated with the mycangia (specialized fungus-transporting structures) of X. mutilatus in Mississippi. Mycangia consistently yielded an Ambrosiella sp. which was subsequently found to be closely related to, but distinct from, other Ambrosiella species affiliated with Ceratocystis. This Ambrosiella is described herein as Ambrosiella beaveri sp. nov. Also isolated were Geosmithia lavendula, G. obscura, and a yeast, Candida homelintoma. It is likely Ambrosiella beaveri was introduced along with the beetle into North America.     

Listeria monocytogenes exopolysaccharide: origin,structure, biosynthetic machinery and c‐di‐GMP‐dependent regulation

Elevated levels of the second messenger c‐di‐GMP activate biosynthesis of an unknown exopolysaccharide (EPS) in the food‐borne pathogen Listeria monocytogenes. This EPS strongly protects cells against disinfectants and desiccation, indicating its potential significance for listerial persistence in the environment and for food safety. We analyzed the potential phylogenetic origin of this EPS, determined its complete structure, characterized genes involved in its biosynthesis and hydrolysis and identified diguanylate cyclases activating its synthesis. Phylogenetic analysis of EPS biosynthesis proteins suggests that they have evolved within monoderms. Scanning electron microscopy revealed that L. monocytogenes EPS is cell surface‐bound. Secreted carbohydrates represent exclusively cell‐wall debris. Based on carbohydrate composition, linkage and NMR analysis, the structure of the purified EPS is identified as a β‐1,4‐linked N‐acetylmannosamine chain decorated with terminal α‐1,6‐linked galactose. All genes of the pssA‐E operon are required for EPS production and so is a separately located pssZ gene. We show that PssZ has an EPS‐specific glycosylhydrolase activity. Exogenously added PssZ prevents EPS‐mediated cell aggregation and disperses preformed aggregates, whereas an E72Q mutant in the presumed catalytic residue is much less active. The diguanylate cyclases DgcA and DgcB, whose genes are located next to pssZ, are primarily responsible for c‐di‐GMP‐dependent EPS production.     

Two new species of <Emphasis Type="Italic">Lonchocarpus</Emphasis> (Leguminosae,Papilionoideae, Millettieae) from Brazil

Two new species of Lonchocarpus from Brazil, assigned to sect. Laxiflori Benth., are described and illustrated and their affinities, geographical distribution and conservation status are discussed. Lonchocarpus laticiferus from arboreal caatinga vegetation of Bahia State, and deciduous forest of Minas Gerais State is related to L. montanus A. M. G. Azevedo ex M. J. Silva & A. M. G. Azevedo. Lonchocarpus graciliflorus, known only from the rain forest of Paraná, is morphologically similar to L. longiunguiculatus M. J. Silva & A. M. G. Azevedo.     

NdgR,an IclR-like regulator involved in amino-acid-dependent growth,quorum sensing,and antibiotic production in <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>

NdgR (regulator for nitrogen source-dependent growth and antibiotic production), an IclR-like regulator, has been initially identified as a binding protein to the promoters of doxorubicin biosynthetic genes in Streptomcyes peucetius by DNA affinity capture assay method. NdgR is well conserved throughout the Streptomcyes species and many other bacteria such as Mycobacteria and Corynebacteria. In Streptomcyes coelicolor, ndgR deletion mutant showed slow cell growth and defects in differentiation and enhances the production of actinorhodin (ACT) in minimal media containing certain amino acids where wild-type strain could not produce ACT. Although deletion mutant of ndgR showed different antibiotic production in minimal media containing Leu or Gln, it only showed reduced mRNA expression levels of the genes involved in leucine metabolism. Neither NdgR-dependent expression of glnA nor direct binding of NdgR protein to glnA, glnII, and glnR promoters was observed. However, ScbR, which is governed by NdgR shown by gel mobility shift assay, binds to promoter of glnR, suggesting indirect regulation of glutamine metabolism by NdgR. NdgR protein binds to intergenic region of ndgR–leuC, and scbR–scbA involved in γ-butyrolactone. Two-dimensional gel analysis has shown a global effect of ndgR deletion in protein expression, including up-regulated proteins involved in ACT synthesis and down-regulation of chaperones such as GroEL, GroES, and DnaK. These results suggest a global regulatory role for NdgR in amino acid metabolisms, quorum sensing, morphological changes, antibiotic production, and expression of chaperonines in S. coelicolor. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

On 3LEZ,a Deep‐Sea Halophilic Protein with in vitro Class‐A β‐Lactamase Activity: Molecular‐Dynamics,Docking, and Reactivity Simulations

This work shows that a deep‐sea protein, 3LEZ, with known in vitro β‐lactamase activity, proved stable, substantially in the conformation detected by X‐ray diffraction of the crystal, when subjected to molecular‐dynamics (MD) simulations under conditions compatible with shallow seas. Docking simulations showed that the β‐lactamase active site S85 of 3LEZ (S70 in Ambler numbering) is the preferential binding pocket for not only β‐lactam antibiotics and inhibitors, but, surprisingly, also for a wide variety of other biologically active compounds in various chemical classes, including marine metabolites. In line with the in vitro β‐lactamase activity, a) affinities on docking β‐lactam antibiotics and inhibitors onto 3LEZ were found to roughly parallel published K_m and K_i values, obtained from Michaelis? Menten kinetics under room conditions, and b) DFT calculations agreed with experiments that the irreversible reaction of the β‐lactamase inhibitor clavulanic acid with the whole S85 catalytic center of 3LEZ is spontaneous. These observations must be viewed in the light that a) the compounds in other chemical classes showed comparable affinities, and, in some cases, even higher than β‐lactams, for the S85 active site, b) K_m and K_i data are not available at the high hydrostatic pressure of the deep sea, where 3LEZ is believed to have evolved, c) an inverse order of affinities for the β‐lactams, with respect to both experimentation and simulations at room conditions, was observed from comparative docking simulations with 3LEZ derived from MD under high hydrostatic pressure. Although MD requires a general assessment for high hydrostatic pressure before c) above is given the same weight as all other observations, this work questions the conclusion that the in vitro determined β‐lactamase activity represents the ecological role of 3LEZ.     

Cloning and characterization of an allene oxide cyclase,PpAOC3, in <Emphasis Type="Italic">Physcomitrella patens</Emphasis>

Allene oxide cyclase (AOC) is a key enzyme in the octadecanoid pathway of flowering plants that synthesizes 12-oxo-phytodienoic acid (OPDA), which is a biosynthetic precursor of the signal molecule jasmonic acid (JA). A database search of the Physcomitrella patens genome revealed the presence of an AOC gene unique from the two previously reported AOC genes, PpAOC1 and PpAOC2. After cloning the identified AOC gene, designated PpAOC3, the obtained cDNA sequence (897 bp) was larger than the predicted AOC gene (765 bp) in the database because a speculated intron was not fully deleted. Although PpAOC3 did not display high similarity with AOC proteins from other species, recombinant PpAOC3 exhibited AOC activity and translocated to chloroplasts, as is observed for other AOC proteins. Notably, the expression profile of PpAOC3 differed from the other PpAOCs, as its expression in protonemata was higher than that in gametophores. Although the function of oxylipins such as OPDA and JA remains elusive in P. patens, further characterization of the enzymes in the octadecanoid pathway and the role of oxylipin will aid in the elucidation of physiological processes in this model bryophyte.     

A revision of the Haploporinae Nicoll, 1914 (Digenea: Haploporidae) from mullets (Mugilidae): <Emphasis Type="Italic">Haploporus</Emphasis> Looss, 1902 and <Emphasis Type="Italic">Lecithobotrys</Emphasis> Looss, 1902

The status of the nominal species of Haploporus Looss, 1902 and Lecithobotrys Looss, 1902 is re-assessed by means of a comparative morphological study based on newly collected specimens from the western Mediterranean, the re-examination of museum material and a critical evaluation of published data. H. benedeni (Stossich, 1887) (type-species) is described and H. lateralis Looss, 1902 is considered to be its junior synonym. Additional data are given for H. pseudoindicus Rekharani & Madhavi, 1985, H. spinosus Machida, 1996 and H. magnisaccus Machida, 1996. Species parasitising Valamugil spp. from the Indo-West Pacific region, H. indicus Rekharani & Madhavi, 1985, H. spinosus, H. magnisaccus, H. mugilis Liu & Yang, 2002 and H. muscolosaccus Machida, 2003, are considered incertae sedis with respect to their generic affiliation. H. pacificus (Manter, 1963) (syn. Neohaploporus pacificus Manter, 1963), H. pseudoindicus and H. musculosaccus are designated as species inquirendae and H. lossii Al-Bassel, 1990 is considered to be a nomen nudum. Lecithobotrys putrescens Looss, 1902 is described based on newly collected material from Liza spp. Pseudolecithobotrys n. g. is erected to accommodate Lecithobotrys stomachicola Machida, 1996, as P. stomachicola (Machida, 1996) n. comb., from the North Pacific. L. aegyptiacus Hassan, El-Aziz, Khidr & Abu Samak, 1990 is considered to be a synonym of Saccocoelium tensum Looss, 1902, and L. brisbanensis (Martin, 1974) (syn. Paralecithobotrys brisbanensis Martin, 1974), L. vitellosus Sharma & Gupta, 1970 and L. suezcanali Nisreen Ezz El-Dien, Abdel-Rahman, El-Gawady, Imam & Fahmy, 1990 are regarded as species inquirendae. New generic diagnoses are presented for both Haploporus and Lecithobotrys.

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Enhancing Optical,Electronic, Crystalline,and Morphological Properties of Cesium Lead Halide by Mn Substitution for High‐Stability All‐Inorganic Perovskite Solar Cells with Carbon Electrodes

In this work all‐inorganic perovskite CsPbIBr₂ are doped with Mn to compensate their shortcomings in band structure for the application of perovskite solar cells (PSCs). The novel Mn‐doped all‐inorganic perovskites, CsPb_1?xMn_xI_1+2xBr_2?2x, are prepared in ambient atmosphere. As the concentration of Mn²⁺ ions increases, the bandgaps of CsPb_1?xMn_xI_1+2xBr_2?2x decrease from 1.89 to 1.75 eV. Additionally, when the concentration of Mn dopants is appropriate, this novel Mn‐doped all‐inorganic perovskite film shows better crystallinity and morphology than its undoped counterpart. These advantages alleviate the energy loss in hole transfer and facilitate the charge‐transfer in perovskites, therefore, PSCs based on these novel CsPb_1?xMn_xI_1+2xBr_2?2x perovskite films display better photovoltaic performance than the undoped CsPbIBr₂ perovskite films. The reference CsPbIBr₂ cell reaches a power conversion efficiency (PCE) of 6.14%, comparable with the previous reports. The CsPb_1?xMn_xI_1+2xBr_2?2x cells reach the highest PCE of 7.36% (when x = 0.005), an increase of 19.9% in PCE. Furthermore, the encapsulated CsPb_0.995Mn_0.005I_1.01Br_1.99 cells exhibit good stability in ambient atmosphere. The storage stability measurements on the encapsulated PSCs reveal that PCE is dropped by only 8% of the initial value after >300 h in ambient. Such improved efficiency and stability are achieved using low‐cost carbon electrodes (without expensive hole transport materials and Au electrodes).     

<Emphasis Type="Italic">Legionella</Emphasis>, Protozoa,and Biofilms: Interactions Within Complex Microbial Systems

Currently, the investigation of Legionella ecology falls into two distinct areas of research activity: (1) that Legionella multiply within water sources by parasitizing amoebic or ciliate hosts or (2) that Legionella grows extracellularly within biofilms. Less focus has been given to the overlaps that may occur between these two areas or the likelihood that Legionella employs multiple survival strategies to persist in water sources. It is likely that Legionella interacts with protozoa, bacteria, algae, fungi, etc., and biofilm components in a more complex fashion than multiplication or death due to the presence or absence of single components of these complex microbial systems. This paper addresses gaps that exist in the understanding of Legionella ecology and serves to pinpoint areas of future research. To assume that only one other class of organism is important to Legionella ecology may limit our understanding of how this bacterium proliferates in heated water sources and also limit our strategies for its control in the built environment.     

Gene cloning,expression, and characterization of phenolic acid decarboxylase from <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> RM84

Phenolic acid decarboxylase (PAD) catalyzes the synthesis of vinyl phenols from hydroxycinnamic acids. The gene encoding PAD from Lactobacillus brevis was cloned and expressed as a fusion protein in Escherichia coli. The recombinant PAD enzyme is a heat-labile enzyme that functions optimally at 22°C and pH 6.0. The purified enzyme did not show thermostability at temperatures above 22°C. L. brevis PAD is able to decarboxylate exclusively the hydroxycinnamic acids, such as p-coumaric, caffeic, and ferulic acids, with K _m values of 0.98, 0.96, and 0.78 mM, respectively. The substrate specificity exhibited by L. brevis PAD is similar to the PAD isolated from Bacillus subtilis and B. pumilus, but different from that of L. plantarum and Pediococcus pentosaceus. As the C-terminal region may be involved in determining PAD substrate specificity and catalytic capacity, amino acid differences among these proteins could explain the differences observed. The substrate specificity shown by L. brevis PAD shows promise for the synthesis of high-added value products from plant wastes.