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How the turtle forms its shell: a paracrine hypothesis of carapace formation.

We propose a two-step model for the evolutionary origin of the turtle shell. We show here that the carapacial ridge (CR) is critical for the entry of the ribs into the dorsal dermis. Moreover, we demonstrate that the maintenance of the CR and its ability to attract the migrating rib precursor cells depend upon fibroblast growth factor (FGF) signaling. Inhibitors of FGF allow the CR to degenerate, with the consequent migration of ribs along the ventral body wall. Beads containing FGF10 can rearrange rib migration in the chick, suggesting that the CR FGF10 plays an important role in attracting the rib rudiments. The co-ordinated growth of the carapacial plate and the ribs may be a positive feedback loop (similar to that of the limbs) caused by the induction of Fgf8 in the distal tips of the ribs by the FGF10-secreting mesenchyme of the CR. Once in the dermis, the ribs undergo endochrondral ossification. We provide evidence that the ribs act as signaling centers for the dermal ossification and that this ossification is due to bone morphogenetic proteins secreted by the rib. Thus, once the ribs are within the dermis, the ossification of the dermis is not difficult to achieve. This relatively rapid means of carapace formation would allow for the appearance of turtles in the fossil record without obvious intermediates.

Animals↗

Microbial degradation of shrimp-shell waste.

A total of 40 strains of bacteria were isolated and tested for their potentiality to degrade chitin and utilize shrimp-shell waste for the production of chitinase. The activity ratio, percentage of weight loss and enzyme activity were determined for cultures exhibiting highest chitinolytic activities. The most active organisms were identified as Alcaligenes denitrificans, Bacillus amyloliquefaciens, B. megaterium and B. subtilis. The potentiality of the first two organisms to degrade chitin was reported for the first time.

Alcaligenes↗

Droplet and particle size relationship and shell thickness of inhalable lactose particles during spray drying.

To find means of controlling the size and density of particles intended for inhalation the relationship between droplet and particle size during spray drying was investigated. Lactose solutions were atomized with a two-fluid nozzle and dried in a laboratory spray drier. The effects of nozzle orifice diameter, atomization airflow and feed concentration on droplet and particle size were examined. Mass median diameter of both droplets and particles were analyzed with laser diffraction. In addition, scanning electron microscopy and transmission electron microscopy were used for studies of particle shape and morphology. It was demonstrated that nozzle orifice diameter and airflow, but not feed concentration controlled the droplet size during atomization. Increasing droplet size increased particle size but the effect was also influenced by feed concentration. Particles from solutions of a low concentration (1% w/w) were smaller than those from higher concentrations (5-20% w/w). This may be partly explained by lower yields at higher feed concentrations, but may also be related to differences in drying rate. Spray-dried lactose solutions formed hollow particles, and it was suggested that the shell thickness of the particles increased with increasing feed concentration.

Administration, Inhalation↗

Physicochemical characterization of papain entrapped in ionotropically cross-linked kappa-carrageenan gel beads for stability improvement using Doehlert shell design.

This work examines the influence of various process parameters on papain entrapped in cross-linked kappa-carrageenan beads for improvement of its stability. A Doehlert shell design (DSD) was employed to investigate the effect of three process variables, namely kappa-carrageenan concentration, KCl concentration, and hardening time, on the entrapment, time required for 50% enzyme release (T50), time required for 90% enzyme release (T90), and particle size. The beads were prepared by dropping the kappa-carrageenan containing papain into a magnetically stirred KCl solution. Topographical characterization was carried out by scanning electron microscopy and entrapment was confirmed by Fourier transform infrared spectroscopy and differential scanning calorimetry. Stability testing was carried out according to the International Conference on Harmonization (ICH) guidelines for zone III and IV. A polymeric matrix was prepared with kappa-carrageenan (3.5% w/v) and potassium chloride (0.5 M) using the ionotropic gelation method, with a hardening time of 20 min. Beads characterized by a spherical disc shape with a collapsed center, an absence of aggregates, an entrapment of 82.75%, a T90 value of 55.36 min, and a composite index of 88.55 were produced. The shelf-life of the enzyme-loaded beads was found to increase to 3.63 years compared with 1.01 years for the conventional formulation. It can be inferred that the proposed methodology can be used to prepare papain-loaded kappa-carrageenan beads for stability improvement.

Algorithms↗

Water migration from soft gelatin capsule shell to fill material and its effect on drug solubility.

The bioavailability of some poorly water-soluble drugs was reported to increase due to a change in dosage form from a tablet to a solution encapsulated in soft gelatin capsules. However, the objective of increasing the bioavailability may be defeated if the drug crystallizes from a solution inside the capsule. In this study, a water-insoluble drug [alpha-pentyl-3-(2-quinolinylmethoxy)benzenemethanol; REV 5901] was solubilized in both polyethylene glycol 400 (PEG 400) and a 6:1 mixture of Gelucire 44/14:PEG 400. The solutions were then encapsulated in soft elastic gelatin capsules with a fill weight of 700 mg (drug, 125 mg), and water migration from the capsule shell into the fill material and its effect on the solubility of the drug were investigated. Gelucire 44/14 is a mixture of hydrogenated fatty acid esters with a mp of 44 degrees C; PEG 400 was added to reduce the mp of solution to approximately 36 degrees C for easier encapsulation. After equilibration of capsules at ambient condition, the amount of water in the PEG 400 solution was 6.3%. This reduced the solubility of the drug by 45%, resulting in drug crystallization. The solubility decreased exponentially with the increase in water content. The water in the encapsulated Gelucire:PEG solution was only 1.1%, which did not affect the solubility significantly.

Capsules↗

The first solvation shell of magnesium ion in a model protein environment with formate, water, and X-NH3, H2S, imidazole, formaldehyde, and chloride as ligands: an Ab initio study.

The first coordination shell of an Mg(II) ion in a model protein environment is studied. Complexes containing a model carboxylate, an Mg(II) ion, various ligands (NH3, H2S, imidazole, and formaldehyde) and water of hydration about the divalent metal ion were geometry optimized. We find that for complexes with the same coordination number, the unidentate carboxylate-Mg(II) ion is greater than 10 kcal mol-1 more stable than the bidentate orientation. Imidazole was found to be the most stable ligand, followed in order by NH3, formaldehyde, H2O, and H2S.

Cations↗

Stable carbon and oxygen isotopic determination of sub-microgram quantities of CaCO3 to analyze individual foraminiferal shells.

We have developed an analytical system to determine stable isotopic compositions (delta13C and delta18O) of sub-microgram quantities of CaCO3 for the purpose of analyzing individual foraminiferal shells, using continuous-flow isotope ratio mass spectrometry (CF-IRMS). The system consists of a micro-volume CaCO3 decomposition tube, stainless steel CO2 purification vacuum line with a quantity-regulating unit, helium-purged CO2 purification line, gas chromatograph, and a CF-IRMS system. By using this system, we can determine stable carbon and oxygen isotopic compositions as low as 0.2 microg of CaCO3, with standard deviations of +/-0.10 per thousand for delta13C and +/-0.18 per thousand for delta18O within a 4-h reaction time and 30-min analysis period.

Animals↗

Violation of frozen shell approximation in dissociative electron capture by halogenated anthraquinones.

A series of halogenated anthraquinone (AQ) derivatives has been studied by means of electron capture negative ion (NI) mass spectrometry (ECNI-MS). 1Cl-AQ and 2Br-AQ display dramatically steep positive temperature dependencies of Hal(-) ion abundance in the low electron energy region. Molecular NI intensity decreases rapidly with increasing temperature in the case of 1I-AQ. In the case of 2Br-AQ, a metastable NI peak (m/z 22.9) corresponding to the process BrAQ(-) --> Br(-) + AQ(0) was recorded. This means that the characteristic dissociation lifetime of the molecular NI Br-AQ(-) is at least approximately 25 micros at the energy approximately 0.67 eV in the low-temperature spectrum (T approximately 80 degrees C), and at the energy approximately 0.13 eV in the hot spectrum (T approximately 290 degrees C). Together with the observed temperature dependence of the 2Br-AQ curves of effective yield (CEY), this proves that this anion dissociates according to Coulson's model. The same halogen anion behavior is observed in the case of 1Cl-AQ. There are three consecutive stages in the process of molecular NI dissociation of Cl- and Br-substituted AQ, namely, electron capture into the empty pi-orbital by means of the shape resonance mechanism, followed by a radiationless transition into the ground electronic pi-state of the anion, as predicted by Compton in the case of the parabenzoquinone molecule, and, finally, a fluctuative dissociation of the molecular NI accompanied by the transition from the pi-term into the sigma-term, so-called predissociation. Calculations show reasonable agreement with the experimental data. In the case of 1I-AQ, an effect of inversion of empty levels in the process of electron capture by the molecule takes place, a violation of the so-called frozen shell approximation. The phenomenon found may be of significance not only in the case of ECNI-MS, but also in other experimental investigations using low-energy electron-molecule and ion-molecule collisions.

Anthraquinones↗

Vesicular acetylcholine transporter in the rat nucleus accumbens shell: subcellular distribution and association with mu-opioid receptors.

Cholinergic interneurons in the nucleus accumbens shell (AcbSh) are implicated in the reinforcing behaviors that develop in response to opiates active at mu-opioid receptors (MOR). We examined the electron microscopic immunocytochemical localization of the vesicular acetylcholine transporter (VAChT) and MOR to determine the functional sites for storage and release of acetylcholine (ACh), and potential interactions involving MOR in this region of rat brain. VAChT was primarily localized to membranes of small synaptic vesicles in axon terminals. Less than 10% of the VAChT-labeled terminals were MOR-immunoreactive. In contrast, 35% of the cholinergic terminals formed symmetric or punctate synapses with dendrites showing an extrasynaptic plasmalemmal distribution of MOR. Membranes of tubulovesicles in other selective dendrites were also VAChT-labeled, and almost half of these dendrites displayed plasmalemmal MOR immunoreactivity. The VAChT-labeled dendritic tubulovesicles often apposed unlabeled axon terminals that formed symmetric synapses. Our results indicate that in the AcbSh MOR agonists can modulate the release of ACh from vesicular storage sites in axon terminals as well as in dendrites where the released ACh may serve an autoregulatory function involving inhibitory afferents. These results also suggest, however, that many of the dendrites of spiny projection neurons in the AcbSh are dually influenced by ACh and opiates active at MOR, thus providing a cellular substrate for ACh in the reinforcement of opiates.

Acetylcholine↗

The paraventricular nucleus of the thalamus as an interface between the orexin and CART peptides and the shell of the nucleus accumbens.

The paraventricular nucleus of the thalamus (PVT) receives afferents from the brainstem and has been thought to relay arousal related information to specific limbic forebrain areas, including the nucleus accumbens. More recent anatomical observations suggest that the PVT also receives afferents from various hypothalamic nuclei. The present anatomical experiments investigated the innervation of the PVT by fibers immunoreactive for orexin and cocaine and amphetamine related transcript (CART), two feeding-related peptides highly concentrated in the hypothalamus. Emphasis was placed on identifying the relationship between these neuropeptides and PVT neurons projecting to the shell of the nucleus accumbens (NacSh). Infusion of a retrograde tracer into the NacSh labeled numerous cells of the midline and intralaminar thalamus, most of which were restricted to the PVT. The retrograde tracer, orexin fibers, and CART fibers were immunopositive throughout the entire PVT whereas no overlap between signals was evident within adjacent thalamic regions. High-magnification light and confocal microscopy showed that both orexin and CART fibers made frequent contact with retrogradely labeled neurons throughout the anteroposterior PVT. Furthermore, single PVT cells retrogradely labeled from the NacSh were apposed by both orexin and CART fibers. The present experiments provide the first evidence suggesting a role for the PVT as a relay of hypothalamic activity to the nucleus accumbens. The PVT may function to link visceral arousal signals with limbic regions involved in behavioral responses.

Animals↗

Chronic treatment with Delta-9-tetrahydrocannabinol alters the structure of neurons in the nucleus accumbens shell and medial prefrontal cortex of rats.

The potential of repeated exposure to Delta(9)-tetrahydrocannabinol (Delta(9)-THC) to produce long-lasting changes in synaptic connections in a manner similar to other drugs of abuse was evaluated in Sprague-Dawley rats. For 12 days, rats received two i.p. injections per day (8 h apart) of vehicle, a low dose of Delta(9)-THC (0.5 mg/kg), or escalating doses of Delta(9)-THC (0.5-4.0 mg/kg). Thirty days later, they were evaluated for sensitized locomotor activity (during the night cycle) for 60 min on each of three trials. Using a within-groups design, rats were tested following an injection of vehicle, 0.5 mg/kg Delta(9)-THC or 2.0 mg/kg Delta(9)-THC. The rats showed no evidence of sensitized locomotor activity in any group. Twenty-four hours after the final sensitization test, their brains were removed and then processed for Golgi-Cox staining. Prior exposure to Delta(9)-THC (both the low dose and the escalating doses) increased the length of the dendrites as well as the number of dendritic branches in the shell of the nucleus accumbens and in the medial prefrontal cortex, but not in the hippocampus, striatum, orbital frontal cortex, parietal cortex, or occipital cortex. These results are similar to those evident in brains of rats sensitized to amphetamine, and support previous findings that cannabinoids promote DA activity in the mesolimbic DA system.

Animals↗

Molluscan shellfish biomarker study of the Quebec, Canada, Saguenay Fjord with the soft-shell clam, Mya arenaria.

A spatial and temporal survey of six sites in the Saguenay Fjord and of one adjacent site in the St. Lawrence River estuary (Quebec, Canada) was undertaken to study the possible effects of anthropogenic contaminant input on soft-shell clam (Mya arenaria) populations. Bivalve sampling sites were selected because they reflected a range of areas representative of either no known (or apparent) pollution sources or of areas potentially influenced by different gradients and types of contamination sources. The most upstream site selected in the Saguenay Fjord, nearest to a highly populated and industrialized sector, and the most downstream site, near its mouth with the St. Lawrence River estuary, spanned a distance of some 70 km and encompassed the entire intertidal area suitable for Mya arenaria habitat. To measure effects in collected animals, we used a comprehensive battery of biomarkers composed of metallothionein-like proteins (MT), 7-ethoxyresorufin O-deethylase activity (EROD), DNA damage (DD), lipid peroxidation (LPO), vitellinlike proteins (Vn), phagocytosis (PHAG), nonspecific esterase (NspE) activity, and condition factor (weight-to-length ratio of clams). Vn, PHAG, DD, and NspE biomarkers were assayed in hemolymph (or hemocytes), whereas others (MT, EROD, LPO) were determined in the digestive gland. Whole-tissue metal content was also quantified in clams collected in the spatial survey. The spatial survey conducted in June 1997 showed significant effects at all sites, and principal component analysis indicated in addition that the more important responses were linked to the MT, LPO, and NspE biomarkers. Clams collected from sites closest to the upstream reaches of the fjord generally displayed higher levels of tissue metals (cadmium, manganese), as well as greater responses of NspE activity, MT, LPO, and PHAG. Animals collected from sites influenced by municipal wastewaters had higher levels of Vn, suggesting the presence of environmental estrogens. The results of the temporal survey (six monthly samplings of clams at three sites from May through October, 1997) showed that the bivalve reproductive cycle (vitellogenesis and spawning) can modulate the expression of several biomarkers. Vn levels, for example, were positively correlated with DD and EROD and negatively correlated with MT, suggesting that reproduction can influence the susceptibility of clams to some contaminants. Discrimination analysis over the 6 months of sampling revealed that the mean value of the discriminant function changed significantly over time, suggesting important changes in the relative contribution of each biomarker. In short, this study has provided evidence that clam populations in the Saguenay Fjord are impacted by multiple sources of contamination whose effects can be modulated by reproduction.

Animals↗

Finding of a homarine-synthesizing enzyme in turban shell and some properties of the enzyme.

A homarine-synthesizing enzyme was found for the first time in cell-free extract from turban shell (Batillus cornutus) and the enzyme was purified 36.2-fold and characterized. Properties of the enzyme were as follows: substrates were picolinic acid (pyridine-2-carboxylic acid) and S-adenosyl-L-methionine; optimum temperature for the enzymic reaction was 25 degrees C; optimum pH for the enzymic reaction was 6.3; and the Km values for picolinic acid and S-adenosyl-L-methionine were calculated at 317 and 14.5 microM, respectively. Among pyridine carboxylic acids, only picolinic acid was methylated with S-adenosyl-L-methionine by this enzyme. The molecular weight of the enzyme was estimated to be 70,800. The enzyme activity was inhibited by heavy metal ions, S-adenosyl-L-homocysteine, adenosine, homocysteine, and sinefungin. Homarine, which is an osmotic pressure regulator, morphogen, etc.; is enzymatically synthesized by the methylation of picolinic acid with S-adenosyl-L-methionine and the enzyme activity may be controlled by S-adenosyl-L-homocysteine (reaction product) and its related compounds.

Adenosine↗

Adrenomedullary hormonal and glycemic responses to high ambient temperature in the soft-shelled turtle, Lissemys punctata punctata.

The aim of the investigation was to study the influence of high ambient temperature on adrenomedullary activity and blood glucose levels in adult female soft-shelled turtles (Lissemys punctata punctata). Experiments were carried out at 25 degrees, 35 degrees, and 38 degrees, and one group was exposed to 38 degrees for 15 days and then maintained at 25 degrees for another 15 days. Exposure to a low ambient temperature of 25 degrees had no clear effect on adrenomedullary function with respect to histology (nuclear diameter), epinephrine and norepinephrine concentrations, and blood glucose level of turtles, but higher temperatures of 35 degrees and 38 degrees stimulated adrenomedullary activity as well as blood glucose level in turtles compared with controls (30 degrees ). The extent of these changes was greater at 38 degrees than that at 35 degrees, and withdrawal from high ambient temperature reversed the effect in turtles.

Adrenal Medulla↗

Hierarchical Structure of Lattices in Film Formation of Core-Shell Type Polymer Microspheres

By adding polystyrene homopolymer in the solid state, super-lattice structures of poly(4-vinyl pyridine) core-polystyrene shell type polymer microspheres were hierarchically changed from a disordered state to a face-centered cubic via a body-centered cubic. The transition of the lattice structures was investigated from the view point of the excluded volume of the microsphere, which was controlled by a changing of the molecular weights and blend tarios of polystyrene homopolymers.

Journal Article↗

Fungal virus capsids, cytoplasmic compartments for the replication of double-stranded RNA, formed as icosahedral shells of asymmetric Gag dimers.

The primary functions of most virus capsids are to protect the viral genome in the extra-cellular milieu and deliver it to the host. In contrast, the capsids of fungal viruses, like the cores of all other known double stranded RNA viruses, are not involved in host recognition but do shield their genomes, and they also carry out transcription and replication. Nascent (+) strands are extruded from transcribing virions. The capsids of the yeast virus L-A are composed of Gag (capsid protein; 76 kDa), with a few molecules of Gag-Pol (170 kDa). Analysis of these 420 A diameter shells and those of the fungal P4 virus by cryo-electron microscopy and image reconstruction shows that they share the same novel icosahedral structure. Both capsids consist of 60 equivalent Gag dimers, whose two subunits occupy non-equivalent bonding environments. Stoichiometry data on other double-stranded RNA viruses indicate that the 120-subunit structure is widespread, implying that this molecular architecture has features that are particularly favorable to the design of a capsid that is also a biosynthetic compartment.

Capsid↗

Pressure-induced formation of inactive triple-shelled rotavirus particles is associated with changes in the spike protein Vp4.

Rotaviruses are non-enveloped, triple-shelled particles that cause enteritis in animals and humans. The interactions among the different viral proteins located in the three concentric layers make the rotavirus particle an excellent model for physico-chemical and biological studies of viral assemblage. SA11-4S rotaviruses subjected to high pressure were inactivated by more than five log units. After pressure treatment, the particles were recovered with slight structural changes when compared to the control. Electron microscopy suggested subtle changes in the viral outer layer in some pressurised particles. Fluorescence spectroscopy showed that much more dramatic changes were produced by urea denaturation than by pressure. Based on the fluorescence spectrum, the genome resistance to ribonuclease, and the absence of changes in hydrodynamic properties, there was little or no disruption of the capsid under pressure. On the other hand, hemagglutination assays indicated that the main component affected by pressure was the spike protein VP4, thus accounting for changes in interaction with host cells and greatly reduced infectivity. The changes leading to inactivation did not cause removal of VP4 from the outer capsid, as verified by size-exclusion chromatography. Antibodies raised against pressurised material were as effective as antibodies raised against the intact virus, based on their neutralisation titre in plaque reduction assays, enzyme-linked immunosorbent assays and direct interaction with the particle, as measured by gel-filtration chromatography. Therefore, the new conformation of the pressurised particle did not result in loss of immunogenicity. We propose that pressure alters the receptor-binding protein VP4 by triggering changes similar to those produced when the virus interacts with target cells. As the changes in VP4 conformation caused by pressure occur prior to virus exposure to target cells, it leads to non-infectious particles and may lead to the exposure of previously occult epitopes, important for vaccine development.

Capsid↗

A Study of Solvation-Shell Symmetry in Electrolyte Solutions Using Quadrupolar NMR Relaxation of the Nuclei of Monoatomic Ions

The spin-lattice relaxation rates of 7Li, 23Na, 27Al, 71Ga, and 139La nuclei in electrolyte solutions have been investigated as functions of the isotopic composition of the solvent. The results show that quadrupolar relaxation of the nuclei of monoatomic ions is extremely sensitive to the symmetry of the solvation shells. The data are compared to the results for the 2H and 17O nuclei of the solvent molecules and the 14N nucleus of the nitrate anion. A semiquantitative description of the observed effects is given.

Journal Article↗

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