The Microstructure,Proteomics and Crystallization of the Limpet Teeth

Limpets are marine mollusks that use mineralized teeth, one of the hardest and strongest biomaterials, to feed on algae on intertidal rocks. However, most of studies only focus on the ultrastructure and chemical composition of the teeth while the molecular information is largely unknown, limiting our understanding of this unique and fundamental biomineralization process. The study investigates the microstructure, proteomics, and crystallization in the teeth of limpet Cellana toreuma. It is found that the limpets formed alternatively tricuspid teeth and unicuspid teeth. Small nanoneedles are densely packed at the tips or leading regions of the cusps. In contrast, big nanoneedles resembling chemically synthesized goethite are loosely packed in the trailing regions of the cusps. Proteins extracted from the whole radula, such as ferritin, peroxiredoxin, arginine kinase, GTPase‐Rabs, and clathrin, are identified by proteomics. A goethite‐binding experiment coupled with proteomics and RNA‐seq highlights six chitin‐binding proteins (CtCBPs). Furthermore, the extracted proteins from the cusps of radula or the framework chitin induce packing of crystals and possibly affect crystal polymorphs in vitro. This study provides insight into the unique biomineralization process in the limpet teeth at the molecular levels, which may guide biomimetic strategies aimed at designing hard materials at room temperature.     

Biomineralization of limpet teeth: a cryo-TEM study of the organic matrix and the onset of mineral deposition

The continuously growing limpet radula contains teeth at various stages of maturity and thus provides an excellent opportunity for studying the processes and mechanisms of their mineralization. We report here on our structural investigations of the pre-formed chitin matrix and the initial deposition and growth of goethite (α-FeOOH) crystals within the matrix. By using cryo-techniques, in which unstained sections of the teeth are examined in a frozen-hydrated state in a transmission electron microscope (TEM), we were able to characterize the process without introducing artifacts normally associated with the staining, dehydration, and embedding required for conventional TEM. The unmineralized matrix consists of relatively well ordered, densely packed arrays of chitin fibers, with only a few nanometers between adjacent fibers. There are clearly no pre-formed compartments that control goethite crystal size and shape; rather, crystals must push aside or engulf the fibers as they grow. By examining teeth nearly row-by-row around the onset of mineralization, we were able to image the first-formed mineral within the chitin matrix. These linear deposits of goethite appear to nucleate on the chitin fibers, which thus control the orientation of the crystals. Crystal growth, on the other hand, is apparently not influenced by the matrix, in contrast to many other biomineralization systems.     

Hemolymph ferritin and radula structure in the limpets Patelloida alticostata and Patella peronii (Mollusca: Gastropoda)

• 1.1. The mean concentration of total hemolymph iron was 3060μg/100 ml in Patella peronii and 2950μg/100 ml in Patelloida alticostata.
• 2.2. Ferritin was found to act as a major iron-binding protein in the hemolymph of both P. peronii and P. alticostata.
• 3.3. P. alticostata ferritin has a molecular weight of approximately 505,000, while that of P. peronii has a mol wt. of approximately 520,000.
• 4.4. The lateral radula teeth of both species are mineralized by deposits of silica (SiO₂) and iron in the form of goethite (α-FeOOH).
• 5.5. Hemolymph ferritin is suggested to act as a high capacity transport system to supply iron to the mineralizing front of the radula.

     

Action and immunoreactivity of neuropeptides in the buccal neuromuscular system of a prosobranch mollusc,Rapana thomasiana

Summary In a prosobranch mollusc, Rapana thomasiana, the catch-relaxing peptide H-Ala-Met-Pro-Met-Leu-Arg-Leu-NH₂ (CARP) was found to depress the contraction of the radula protractor and retractor elicited by electrical stimulations. The action of CARP was in contrast to that of other neuropeptides, H-Phe-Met-Arg-Phe-NH₂ (FMRFamide) and H-Phe-Leu-Arg-Phe-NH₂ (FLRFamide), which enhanced the contraction of the radula protractor and retractor, respectively. By immunohistochemical examinations, FMRFamide-like immunoreactive neurons were found on the rostral side of the right buccal ganglion and the caudal side of the left ganglion, where some CARP-like immunoreactive neurons were also distributed, indicating a possible coexistence of FMRFamide and CARP. FMRFamide- and CARP-like immunoreactivities were also detected in the neuropile of buccal ganglia, radula nerves arising from the ganglia, and nerve fibers in the radula muscles. The present results suggest that FMRFamide- and CARP-like peptides are involved in the regulation of the contraction of the radula muscles.     

Scanning electron microscope observations of the organic matrix in the otolith of the teleost fish Fundulus heteroclitus (Linnaeus) and Tilapia nilotica (Linnaeus)

Scanning electron microscope observations of sagitta otoliths of Fundulus heteroclitus (Linnaeus) and Tilapia nilotica (Linnaeus) have revealed that the “discontinuous zone” is a narrow band of organic matrix consisting of fibers ≈900 Å thick, that in turn are composed of thin fibers ≈200 Å thick. The “incremental zone” is the crystalline layer with crystals elongated perpendicular to the otolith periphery that are usually terminated at the discontinuous zones. The crystals are embedded in organic matrix fibers that appear similar to and continuous with the fibers of the discontinuous zones. Frequently, these fibers aggregate into matrix sheets. Based on these findings, a possible process of otolith formation is proposed.     

Age determination and estimation of larval period in field caught abalone (Haliotis discus hannai Ino 1953) larvae and newly metamorphosed post-larvae by counts of radular teeth rows

We developed an age determination method for larval and newly metamorphosed post-larval abalone Haliotis discus hannai in a laboratory experiment and determined the age of field caught individuals. Laboratory experiments showed that competent veliger larvae (4 days after fertilization) had a radula and regularly added rows of radular teeth with age in the absence of metamorphosis. Under environmentally relevant temperatures (17-22 °C), the number of rows of radular teeth increased linearly with age, but slopes of the regression lines were different among temperatures. Rows of radular teeth were added more slowly at lower temperatures. The effect of temperature on the development rate of the radula was quantified by the regression and the temperature coefficient, Q₁₀. The radular development of newly metamorphosed post-larvae, which had not acquired a peristomal shell (adult shell), was comparable with that of veliger larvae, although older post-larvae had a larger number of rows of radula than those of the same age of veliger larvae. From these results, an age determination method of veliger larvae and newly metamorphosed post-larvae was established, using the number of rows of radular teeth. The age of veliger larvae and newly metamorphosed post-larvae was determined by the age determination method for samples collected in August to October of 2003 and 2004 for which the thermal history of the coastal water of Miyagi Prefecture Japan was available. Only 9.1% of veliger larvae (n = 8) captured in the field had formed a radula and these were estimated to be 4-6 days old. The remaining 90.9% of larvae (n = 80) that had not formed a radula were classified as younger than 4 days old. All newly metamorphosed post-larvae (n = 24) that had metamorphosed on substrata were estimated to be 4-6 days old. Results of the field study indicate that these abalone metamorphosed within a few days after the acquisition of competence (4 days after fertilization) at this site, which has suitable crustose algal habitat.     

Insights into the composition,morphology, and formation of the calcareous shell of the serpulid Hydroides dianthus

To date, the calcareous tubes of serpulid marine worms have not been studied extensively in a biomineralization context. The structure and composition of the tube shell and adhesive cement of the marine tubeworm Hydroides dianthus were studied using a variety of characterization techniques, including powder XRD, FTIR, SEM, EDX, and AFM. The tube and cement were determined to be inorganic–organic composite materials, consisting of inorganic aragonite (CaCO₃) and Mg-calcite ((Ca_0.8Mg_0.2)CO₃) crystals, and both soluble and insoluble organic matrices (SOM and IOM). SEM imaging revealed a variety of crystal morphologies. AFM nanoindentation of the inorganic components yielded Young’s moduli of ～20 GPa in the wet state, and ～50 GPa in the dry state. Amino acid analysis of the SOM indicated substantial amounts of acidic and non-polar neutral amino acids. Part of the insoluble organic tube lining was identified as being composed of collagen-containing fibres aligned in a criss-crossed structure. The SOM and organic tube lining were found to contain carboxylated and sulphated polysaccharides. In an artificial seawater solution, the SOM and the organic tube lining mediated CaCO₃ mineralization in vitro.     

On the visibility of leading eigenvalues in compartmental models

The system matrix eigenvalue with the largest real part (leading eigenvalue) of any input-output (IO) connectable compartmental model is real and visible (appears explicitly) in the impulse response, and thus it governs the asymptotic response of the model. Its visible multiplicity is calculated here by decomposing the model into strongly connected components and applying the Perron-Frobenius theorem. Cascade models and fully visible eigenvalues are defined, and it is shown that for any cascade model the leading eigenvalue is fully visible in the impulse response. A necessary and sufficient condition is given for full visibility of the leading eigenvalue of any IO-connectable model. As a corollary, if an IO-connectable compartmental model has one or more traps, the leading eigenvalue λ₁ = 0 always has visible multiplicity one.     

Redox and Lewis acid–base activities through an electronegativity-hardness landscape diagram

Chemistry is the science of bond making and bond breaking which requires redistribution of electron density among the reactant partners. Accordingly acid–base and redox reactions form cardinal components in all branches of chemistry, e.g., inorganic, organic, physical or biochemistry. That is the reason it forms an integral part of the undergraduate curriculum all throughout the globe. In an electronegativity (χ)- hardness (η) landscape diagram the diagonal χ?=?η line separates reducing agents from oxidizing agents as well as Lewis acids from Lewis bases. While electronegativity is related to the degree of electron transfer between two reactants, hardness is related to the resistance to that process. Accordingly the electronegativities of oxidizing agents/Lewis acids are generally greater than the corresponding hardness values and the reverse is true for reducing agents/Lewis bases. Electrophiles and nucleophiles are also expected to follow similar trends.     

Ultrastructure of otolith increments and checks in the teleost fish Oreochromis niloticus

An otolith increment is composed of an incremental zone and a discontinous zone. Observation of the otoliths of Oreochromis niloticus with transmission and scanning electron microscopy reveals that organic fibers are concentrated in the discontinuous zone and are relatively scarce in the incremental zone. On the other hand, calcium carbonate crystals are chiefly packed in the incremental zone and are less dense in the discontinuous zone. Both fibers and crystals are oriented perpendicular to the growth increments. Otolith checks, or discontinuities, contain even denser fibers and fewer fine crystals than does the discontinuous zone. A higher proportion of individual fibers is prominently stained in the discontinuous zone and check than in the incremental zone. Other features of individual fibers appear to be the same among the three zones. The crystals on either side of a check or a discontinuous zone resemble each other both in size and orientation. Zonation of protein and mineral components of otoliths can be understood in terms of both incremental growth and the daily cycle of deposition.     

The role of water chemistry in the distribution of Austropotamobius pallipes (Crustacea Decapoda Astacidae) in Piedmont (Italy)

Over the last decades, the populations of Austropotamobius pallipes have decreased markedly all over Europe. If we evaluate the ecological factors that determine its presence, we will have information that could guide conservation decisions. This study aims to investigate the chemical-physical demands of A. pallipes in NW Italy. To this end, we investigated 98 sites. We performed Principal Component Analysis using chemical-physical parameters, collected in both presence and absence sites. We then used principal components with eigenvalue > 1 to run Discriminant Function Analysis and Logistic Regression. The statistics on the concentration of Ca²⁺, water hardness, pH and BOD₅ were significantly different in the presence and in the absence sites. pH and BOD₅ played the most important role in separating the presence from the absence locations. These findings are further evidence that we should reduce dissolved organic matter and fine particles in order to contribute to species management and conservation.     

A New Genus and Species of the Family Anulidentaliidae (Scaphopoda: Dentaliida) and its Systematic Implications

Shell, radula, and anatomy of Epirhabdoides ivanovi new genusand species are described from a sample of the Russian VitjazExpedition from the Japan Trench. It is distinguished from thesimilar Laevidentalium sominium by shell morphometrics and radulamorphology. The radula is almost identical with that of Anulidentaliumbambusa (Anulidentaliidae). The anatomy of the mantle margins,however, including dorsolateral slits at the anterior mantlemargin and a connective tissue bolster at the posterior mantleopening is that of the family Rhabdidae. In a parsimony analysisEpirhabdoides ivanovi takes an intermediate position betweena basal grade of Gadilinidae and the remaining Dentaliida implyingconvergent evolution of mantle characters. An alternative butless parsimonious tree with E. ivanovi as sister taxon to Rhabdusrequires convergences in radula characters. This is the firstdocumented case of convergent anatomical features among Scaphopodaand enhances the need of radula and soft part investigationof the conchologically little informative, smooth-shelled dentaliidgroups. (Received 16 March 1998; accepted 1 June 1998)     

Isolation and Characterization of a Genetically Tractable Photoautotrophic Fe(II)-Oxidizing Bacterium, Rhodopseudomonas palustris Strain TIE-1

We report the isolation and characterization of a phototrophic ferrous iron [Fe(II)]-oxidizing bacterium named TIE-1 that differs from other Fe(II)-oxidizing phototrophs in that it is genetically tractable. Under anaerobic conditions, TIE-1 grows photoautotrophically with Fe(II), H₂, or thiosulfate as the electron donor and photoheterotrophically with a variety of organic carbon sources. TIE-1 also grows chemoheterotrophically in the dark. This isolate appears to be a new strain of the purple nonsulfur bacterial species Rhodopseudomonas palustris, based on physiological and phylogenetic analysis. Fe(II) oxidation is optimal at pH 6.5 to 6.9. The mineral products of Fe(II) oxidation are pH dependent: below pH 7.0 goethite (α-FeOOH) forms, and above pH 7.2 magnetite (Fe₃O₄) forms. TIE-1 forms colonies on agar plates and is sensitive to a variety of antibiotics. A hyperactive mariner transposon is capable of random insertion into the chromosome with a transposition frequency of ~10⁻⁵. To identify components involved in phototrophic Fe(II) oxidation, mutants of TIE-1 were generated by transposon mutagenesis and screened for defects in Fe(II) oxidation in a cell suspension assay. Among approximately 12,000 mutants screened, 6 were identified that are specifically impaired in Fe(II) oxidation. Five of these mutants have independent disruptions in a gene that is predicted to encode an integral membrane protein that appears to be part of an ABC transport system; the sixth mutant has an insertion in a gene that is a homolog of CobS, an enzyme involved in cobalamin (vitamin B₁₂) biosynthesis.     

Sterols of the lichen Pseudevernia furfuracea

A mixture of C₂₇, C₂₈ and C₂₉ sterols was isolated from the lichen Pseudevernia furfuracea and characterized by means of GLC and MS. Mono-, di- and tri-unsaturated sterols were identified as well as a small amount of fully saturated sterols (stanols). Only a part of the total sterols present in the lichen tissue was easily extractable with organic solvents. Another portion was only obtained after saponification of the lichen residue remaining after extraction with organic solvents. The composition of these two fractions difrered considerably, the former contained predominantly 5a,8a-epidioxy-5a-ergosta-6,22-dien-3β-ol (ergosterol peroxide) and 24-ethylcholesta-5,22-dien-3β-ol while in the latter 24-ethylcholesta-5,22-dien- 3β-ol and C₂₈ triene sterols were the main components.     

Molecular topology in crystals and fibers of hemoglobin S

Several lines of evidence indicate a close correspondence between the linear double filaments in the crystal form of hemoglobin S grown from solutions containing polyethylene glycol and the seven pairs of helical filaments that occur in the 14-filament fibers of hemoglobin S. An analysis of the adjustments to the intermolecular contacts required to convert the double filaments from crystals to fibers is presented here. In addition, postulated contacts between the helical double filaments, which are distinct from any of the contacts of the crystals, are specified for the first time. The movements from crystals to fibers are described in terms of three rotation angles: α, the inclination of the filaments with respect to the fiber axis; δ, the tilt of successive molecules along the filaments; and ω, the rotation of successive molecules along the filaments. On the basis of the fiber structure determined by three-dimensional reconstruction of electron micrographs and the assignment of filament pairs from data on incomplete fibers, the various angles have been evaluated. For the filaments at various radii in the fibers, a varies from 3 ° to 12 °, δ varies from 1 ° to 4 ° and ω is constant at 9 °. The effects of the rotations on the contacts between molecules of hemoglobin S at various positions in the fibers are characterized using surface maps based on polar coordinates. For each residue on the surface of hemoglobin the centroid position of its side-chain is located by a longitude, a latitude and an altitude. Locations on the maps are assigned for the contacts within the helical double filaments, as well as 11 classes of new contacts describing the potential interaction sites between double filaments. The resulting maps (1) deduce roles for the various α mutants of hemoglobin known to influence fiber formation that have been identified by the Benesches; (2) distinguish effects for the α chain mutants on the same (cis) or opposite (trans) α₁β₁ dimer as the β6 Val in asymmetric tetramers; (3) propose new sites where effects of mutations on fiber formation may be found; and (4) suggest why some mutants may inhibit, while others enhance, fiber formation. Concerning the last point, the possibility of certain mutants “correcting” the effects of other mutants is proposed as a test of contact assignments.     

Seasonal aberrant radular formation in Thais bronni (Dunker) and T. clavigera (Küster) (Gastropoda : Muricidae)

Radulae of Thais bronni (Dunker) and T. clavigera (Küster) were examined at Mukaishima Island for a period of 2 yr, 1982 to 1984. Radulae of both species are similar in morphology, both having the basic pentacuspid rachidian plan. Sexual dimorphism of the radula was not observed, but rachidian tooth changes morphologically in different growth stages. Seasonal conditions affect the size and shape of the radula; in winter it is clearly malformed and strikingly thin. These aberrant parts of the radula comprised some dozens of rows, in which only several extremely thin rows exist. Results of experiments using T. clavigera under different water temperature conditions showed that the radula is rarely produced below 10 °C and that rate of radular production and replacement increases with increase in temperature. These results suggest that in the field the radula of these species is replaced entirely 2–2.5 times per year and 10–15 times during the life of the animal.     

Soil fungi rather than bacteria were modified by invasive plants,and that benefited invasive plant growth

Background and aims

Positive relationships between temperature and soil respiration rate are widely observed, but it remains unclear if the relationships are due to increases in soil organic matter mineralisation (R _om ), or in root and rhizosphere respiration (R _roots ), or increases in both. This study aims to determine the relative sensitivity of R _om and R _roots to temperature in soils with differing properties.

Methods

Taking advantage of the difference in stable carbon isotopic composition provided by C₃ and C₄ plants, we partitioned soil respiration into R _om and R _roots for two soils with contrasting clay mineralogy, pH and carbon content over a 24 °C temperature range (from 12 to 36 °C).

Results

The Chromosol (dominated by illite, with near neutral pH and low organic carbon content) showed an increase in the proportion of R _om with temperature, indicating an increase in the decomposition of soil organic carbon. In contrast, the Ferrosol (dominated by hematite and goethite, with acidic pH and high organic carbon) showed no change in the proportion of R _om with warming, and a negative priming effect at the highest temperature.

Conclusions

The observed positive priming effect for the Chromosol and a negative priming effect for the Ferrosol are consistent with contrasting mineralogy, reflecting the relatively weaker bond strength between soil carbon and illites in the Chromosol compared to the Ferrosol.     

The Structure of B-DNA in Oriented Fibers

Abstract

Native, general sequence B-form DNA in uniaxially oriented fibers is a ten-fold helix with identical antiparallel strands: this is to say the molecular symmetry is 2 2 10₁. The diffraction patterns indicate that local variations, however significant, must be modest. This is true also for the lithium salt of calf thymus DNA in fibers that are polycrystalline as well as oriented. The contents of its orthorhombic unit cells are arranged with P2₁2₁2₁ symmetry which permits the molecular symmetry to be merely two-fold. The molecular structure of DNA in such conditions resembles, conformationally and molecularly, that of B-type DNA in oligonucleotide single crystals and in oriented polycrystalline fibers of polyoligonucleotides, and therefore provides a basis for evaluating the variations that may be due to sequence effects in polyoligonucleotides in fibers and oligonucleotides in single crystals.     

The buccal apparatus with radula of a ceratitic ammonoid from the German Middle Triassic

Complete ammonoid mouth parts including both mandibles and the radula are rare. A newly prepared specimen of Ceratites penndorfi of the late Anisian from the Franconian Muschelkalk reveals one such anaptychus-type jaw apparatus including several more or less clearly recognisable structures such as the shapes and proportions of the inner and outer lamellae of both mandibles. The lower mandible has a short inner and a long outer lamella with an ovoid outline, while the upper mandible has a slightly arched rostrum and the inner lamella carries two wings. The radula, as far as it is preserved, appears to be homodont with oblique, simply conical, monocuspidate teeth. Some other structures are here illustrated, described and interpreted as both mandibles and oesophageal remains. Additional structures of organic origin are preserved such as the radula, but others are difficult to interpret. Some, if not all, of these structures also were body parts of the ceratite, such as perhaps the radular support and/or the oesophagus. All mouthparts, which are preserved in the specimen described herein, are carbonised except for the radula remains, which are phosphatic. The taphonomy of ceratite mouth parts in the Muschelkalk is shortly discussed.     

Differences in rhizome aeration of Phragmites australis in a constructed wetland

Waterlogged soils are a challenging environment for plants due to anoxic conditions and enrichment of phytotoxic substances in the rhizosphere. High contents of organic matter in the substrate as present in constructed wetlands (CW) may amplify these effects. The influence of substrate organic matter on the rhizome aeration of Phragmites australis (common reed) was investigated in a CW for sewage treatment on two sites with different organic concentrations: (a) inflow part of the bed with a high organic charge (high-organic site) and (b) outflow zone of the bed with a lower organic charge (low-organic site). Several diurnal oxygen (O₂) courses were recorded inside the rhizomes using micro-optodes. Maximum O₂ percentages in the rhizomes were generally the same at both sites, but minimum O₂ values were lower in the low-organic site than in the high-organic site, leading to higher amplitudes at the low-organic site. The results suggest that oxygen release from the roots is hampered under high-organic compared to low-organic conditions. This observation might be explained by changes in gas conductivity of the roots, but also by alterations in microbial oxygen demand, under different organic burden.