Diurnal polyp expansion behavior in stony corals may enhance carbon availability for symbionts photosynthesis

Photosynthesis of zooxanthellate stony corals may be limited by inorganic carbon at high irradiances. We demonstrated that oxygen consumption of expanded corals is higher than that of contracted corals in both night-expanding and day-expanding corals. It is assumed that at the single-polyp level, the expansion of tentacles increases the surface area for solute exchange with the surrounding water, which may alleviate potential carbon limitation and excess oxygen levels in the tissue under high irradiance. We investigated this hypothesis using stable carbon isotope (613 C) analysis of coral species from the Red Sea exhibiting different morphologies. delta C-13 ratios in zooxanthellae of branched coral colonies with small polyp size that extend their tentacles during daytime (diurnal morphs) showed lower delta C-13 values in their zooxanthellae - 13.83 +/- 1.45 parts per thousand, compared to corals from the same depth with large polyps, which are usually massive and expand their tentacles only at night (nocturnal morphs). Their algae delta C-13 was significantly higher, averaging - 11.33 +/- 0.59 parts per thousand. Carbon isotope budget of the coral tissue suggests that branched corals are more autotrophic, i.e., that they depend on their symbionts for nutrition compared to massive species, which are more heterotrophic and depend on plankton predation. (c) 2005 Elsevier B.V. All rights reserved.

Meganema perideroedes gen. nov., sp nov., a filamentous alphaproteobacterium from activated sludge

An industrial wastewater treatment plant at Grindsted, Denmark, has suffered from bulking problems for several years caused by filamentous bacteria. Five strains were isolated from the sludge by micromanipulation, Phylogenetic analysis of the 16S rRNA gene sequences showed that the strains formed a monophyletic cluster in the Alphaproteobacteria, and they were phenotypically different from their closest relatives and from all hitherto known filamentous bacteria described (closest relative Brevundimonas vesicularis ATCC 11426(T), 89(.)8% sequence similarity). In pure culture, the cells (1(.)5-2(.)0 mu m) in filaments are Gram-negative and contain polyphosphate and polyhydroxyalkanoates. The optimum temperature for growth is 30 degrees C and the strains grow in 2 % NaCl and are oxidase- and catalase-positive. Ubiquinone 10 is the major quinone. The major fatty acid (C-18: 1 omega 7c) and smaller amounts of unsaturated fatty acids, 3-hydroxy fatty acids with a chain length of 16 and 18 carbon atoms and small amounts of 10-methyl-branched fatty acids with 18 carbon atoms (C-19: 0 10-methyl) affiliated the strains with the Methylobacterium/Xanthobacter group in the Alphaproteobacteria. The G + C content of the DNA is 42(.)9 mol% (for strain Gr1(T)). The two most dissimilar isolates by 16S rRNA gene comparison (Gr1(T) and Gr10; 97(.)7 % identical) showed 71(.)5 % DNA-DNA relatedness. Oligonucleotide probes specific for the pure cultures were designed for fluorescence in situ hybridization and demonstrated that two filamentous morphotypes were present in the Grindsted wastewater treatment plant. It is proposed that the isolates represent a new genus and species, Meganema perideroedes gen. nov., sp. nov. The type strain of Meganema perideroedes is strain Gr1(T) (=DSM 15528(T) =ATCC BAA-740(T)).

Mutations in the regulatory subunit of yeast acetohydroxyacid synthase affect its activation by MgATP

Isoleucine, leucine and valine are synthesized via a common pathway in which the first reaction is catalysed by AHAS (acetohydroxyacid synthase; EC 2.2.1.6). This heterotetrameric enzyme is composed of a larger subunit that contains the catalytic machinery and a smaller subunit that plays a regulatory role. The RSU (regulatory subunit) enhances the activity of the CSU (catalytic sub unit) and mediates end-product inhibition by one or more of the branched-chain amino acids, usually valine. Fungal AHAS differs front that in other organisms in that the inhibition by valine is reversed by MgATP. The fungal AHAS RSU also differs from that in other organisms in that it contains a sequence insert. We suggest that this insert may form the MgATP-binding site and we have tested this hypothesis by mutating ten highly conserved amino acid residues of the yeast AHAS RSU. The modified subunits were tested for their ability to activate the yeast AHAS CSU, to confer sensitivity to valine inhibition and to mediate reversal of the inhibition by MgATP. All but one of the mutations resulted in substantial changes in the properties of the RSU. Unexpectedly, four of them gave a protein that required mgATP in order for strong stimulation of the CSU and valine inhibition to be observed. A model to explain this result is proposed. Five of the mutations abolished MgATP activation and are suggested to constitute the binding site for this modulator.

Specializations of the granular prefrontal cortex of primates: Implications for cognitive processing

The biological underpinnings of human intelligence remain enigmatic. There remains the greatest confusion and controversy regarding mechanisms that enable humans to conceptualize, plan, and prioritize, and why they are set apart from other animals in their cognitive abilities. Here we demonstrate that the basic neuronal building block of the cerebral cortex, the pyramidal cell, is characterized by marked differences in structure among primate species. Moreover, comparison of the complexity of neuron structure with the size of the cortical area/region in which the cells are located revealed that trends in the granular prefrontal cortex (gPFC) were dramatically different to those in visual cortex. More specifically, pyramidal cells in the gPFC of humans had a disproportionately high number of spines. As neuron structure determines both its biophysical properties and connectivity, differences in the complexity in dendritic structure observed here endow neurons with different computational abilities. Furthermore, cortical circuits composed of neurons with distinguishable morphologies will likely be characterized by different functional capabilities. We propose that 1. circuitry in V1, V2, and gPFC within any given species differs in its functional capabilities and 2. there are dramatic differences in the functional capabilities of gPFC circuitry in different species, which are central to the different cognitive styles of primates. In particular, the highly branched, spinous neurons in the human gPFC may be a key component of human intelligence. (C) 2005 Wiley-Liss, Inc.

Visualisation of macropinosome maturation by the recruitment of sorting nexins

We report that phosphoinositol-binding sorting nexin 5 ( SNX5) associates with newly formed macropinosomes induced by EGF stimulation. We used the recruitment of GFP-SNX5 to macropinosomes to track their maturation. Initially, GFP-SNX5 is sequestered to discrete subdomains of the macropinosome; these subdomains are subsequently incorporated into highly dynamic, often branched, tubular structures. Time-lapse videomicroscopy revealed the highly dynamic extension of SNX5-labelled tubules and their departure from the macropinosome body to follow predefined paths towards the perinuclear region of the cell, before fusing with early endosomal acceptor membranes. The extension and departure of these tubular structures occurs rapidly over 5-10 minutes and is dependent upon intact microtubules. As the tubular structures depart from the macropinosome there is a reduction in the surface area and an increase in tension of the limiting membrane of the macropinosome. In addition to the recruitment of SNX5 to the macropinosome, Rab5, SNX1 and EEA1 are also recruited by newly formed macropinosomes, followed by the accumulation of Rab7. SNX5 forms heterodimers with SNX1 and this interaction is required for endosome association of SNX5. We propose that the departure of SNX5-positive tubules represents a rapid mechanism of recycling components from macropinosomes thereby promoting their maturation into Rab7-positive structures. Collectively these findings provide a detailed real-time characterisation of the maturation process of the macropinocytic endosome.

A windows program for the derivation of steady-state equations in enzyme systems

Despite the number of computer-assisted methods described for the derivation of steady-state equations of enzyme systems, most of them are focused on strict steady-state conditions or are not able to solve complex reaction mechanisms. Moreover, many of them are based on computer programs that are either not readily available or have limitations. We present here a computer program called WinStes, which derives equations for both strict steady-state systems and those with the assumption of rapid equilibrium, for branched or unbranched mechanisms, containing both reversible and irreversible conversion steps. It solves reaction mechanisms involving up to 255 enzyme species, connected by up to 255 conversion steps. The program provides all the advantages of the Windows programs, such as a user-friendly graphical interface, and has a short computation time. WinStes is available free of charge on request from the authors. (c) 2006 Elsevier Inc. All rights reserved.

Solution properties of star and linear poly(N-isopropylacrylamide)

The LCST transitions of novel N-isopropylacrylamide ( NIPAM) star polymers, prepared using the four-armed RAFT agent pentaerythritoltetrakis(3-(S-benzyltrithiocarbonyl) propionate) (PTBTP) and their hydrolyzed linear arms were studied using H-1 NMR, PFG-NMR, and DLS. The aim was to determine the effect of polymer architecture and the presence of end groups derived from RAFT agents on the LCST. The LCST transitions of star PNIPAM were significantly depressed by the presence of the hydrophobic star core and possibly the benzyl end groups. The effect was molecular weight dependent and diminished once the number of repeating units per arm >= 70. The linear PNIPAM exhibited an LCST of 35 degrees C, regardless of molecular weight; the presence of both hydrophilic and hydrophobic end groups after hydrolysis from the star core was suggested to cancel effects on the LCST. A significant decrease in R-H was observed below the LCST for star and linear PNIPAM and was attributed to the formation of n-clusters. Application of a scaling law to the linear PNIPAM data indicated the cluster size n = 6. Tethering to the hydrophobic star core appeared to inhibit n-cluster formation in the lowest molecular weight stars; this may be due to enhanced stretching of the polymer chains, or the presence of larger numbers of n-clusters at temperatures below those measured.

The motor neurite terminal determines where neuromuscular synapses form in the absence of agrin

Agrin is a proteoglycan secreted by motor neurite terminals that functions to initiate and maintain AChR clusters at the nerve terminal. This led to the theory that neurite terminals decide where neuromuscular synapses form by secreting agrin. However, initiation of AChR clustering occurs in the absence of the innervating motoneuron and in the absence of agrin. In this instance, the muscle, not the nerve, is deciding the location of neuromuscular synapses by drawing neurite terminals towards pre-existing AChR clusters. If this were true, one would expect the initial innervation patterns to be the same in agrin-deficient mice and wild-type mice. To test this we quantified the intramuscular axonal branching and synapse formation in the diaphragm at E14.5 in agrin-deficient mice and wild-type mice. Heterozygote mothers were anaesthetised with Nembutal (30 mg) and killed via cervical dislocation. In the diaphragm, the nerve trunk runs down the centre of the muscle and extends branches primarily toward the lateral side. In agrin-deficient mice however, we found significantly more branches exited the phrenic nerve trunk, branched in the periphery and extended further on the medial side. Moreover, we found that the percentage α-bungarotoxin/synaptophysin colocalisations, markers of pre- and postsynaptic differentiation, respectively, was the same in agrin-deficient mice and wild-type mice. These results show that initial innervation patterns are not the same in agrin-deficient mice and wild-type mice indicating neurite terminals, not muscle, decide the placement of neuromuscular synapses in the absence of agrin.

Migration of additives from thermoplastic polymers

A homologous series of ultra-violet stabilisers containing 2-hydroxybenzophenone (HBP) moiety as a uv absorbing chromophore with varying alkyl chain lengths and sizes were prepared by known chemical synthesis. The strong absorbance of the HBP chromophore was utilized to evaluate the concentration of these stabilisers in low density polyethylene films and concentration of these stabilisers in low density polyethylene films and in relevant solvents by ultra-violet/visible spectroscopy. Intrinsic diffusion coefficients, equilibrium solubilities, volatilities from LDPE films and volatility of pure stabilisers were studied over a temperature range of 5-100oC. The effects of structure, molecular weight and temperature on the above parameters were investigated and the results were analysed on the basis of theoretical models published in the literature. It has been found that an increase in alkyl chain lengths does not change the diffusion coefficients to a significant level, while attachment of polar or branched alkyl groups change their value considerably. An Arrhenius type of relationship for the temperature dependence of diffusion coefficients seems to be valid only for a narrow temperature range, and therefore extrapolation of data from one temperature to another leads to a considerable error. The evidence showed that increase in additive solubility in the polymer is favoured by lower heat of fusions and melting points of additives. This implies the validity of simple regular solution theory to provide an adequate basis for understanding the solubility of additives in polymers The volubility of stabilisers from low density polyethylene films showed that of an additive from a polymer can be expressed in terms of a first-order kinetic equation. In addition the rate of loss of stabilisers was discussed in relation to its diffusion, solubility and volatility and found that all these factors may contribute to the additive loss, although one may be a rate determining factor. Stabiliser migration from LDPE into various solvents and food simulants was studied at temperatures 5, 23, 40 and 70oC; from the plots of rate of migration versus square root time, characteristic diffusion coefficients were obtained by using the solution of Fick's diffusion equations. It was shown that the rate of migration depends primarily on partition coefficients between solvent and the polymer of the additive and also on the swelling action of the contracting media. Characteristic diffusion coefficients were found to approach to intrinsic values in non swelling solvents, whereas in the case of highly swollen polymer samples, the former may be orders of magnitude greater than the latter.

Synthetic polymers for ophthalmic applications

The contact lens represents a well-established important class of biomaterials. This thesis brings together the literature, mostly Japanese and American patents, concerned with an important group of polymers, `rigid gas permeable contact lens materials'. A comparison is made of similarities in the underlying chemical themes, centring on the use of variants of highly branched siloxy compounds with polymerizable methacrylate groups. There is a need for standard techniques to assess laboratory behaviour in relation to in vitro performance. A major part of the present work is dedicated to the establishment of such standardised techniques. It is apparent that property design requirements in this field (i.e. oxygen permeability, surface and mechanical properties) are to some extent conflicting. In principle, the structural approaches used to obtain high oxygen permeability lead to surface properties that are less than ideal in terms of compatibility with tears. PMMA is known to have uniquely good (but not perfect) surface properties in this respect; it has been used as a starting point in attempting to design new materials that possess a more acceptable compromise of transport and surface properties for ocular use. Initial examination of the oxygen permeabilities of relatively simple alkyl methacrylates, show that butyl methacrylate which has a permeability some fifty times greater than PMMA, represents an interesting and hitherto unexplored group of materials for ophthalmic applications. Consideration was similarly given to surface modification techniques that would produce materials having the ability to sustain coherent tear film in the eye without markedly impairing oxygen transport properties. Particular attention is paid to the use of oxygen plasma techniques in this respect. In conclusion, similar design considerations were applied to an extended wear hydrogel lens material in an attempt to overcome mechanical stability deficiencies which manifest themselves lq`in vivo' but not `in vitro'. A relatively simple structure modification, involving steric shielding of the amide substituent group, proved to be an effective solution to the problem.

Development of plasmid dna formulations for application as microspheric dna vaccines

The advent of DNA vaccines has heralded a new technology allowing the design and elicitation of immune responses more adequate for a wider range of pathogens. The formulation of these vaccines into the desired dosage forms extends their capability in terms of stability, routes of administration and efficacy. This thesis describes an investigation into the fabrication of plasmid DNA, the active principle of DNA vaccines, into microspheres, based on the tenet of an increased cellular uptake of microparticulate matter by phagocytic cells. The formulation of plasmid DNA into microspheres using two methods, is presented. Formulation of microspheric plasmid DNA using the double emulsion solvent evaporation method and a spray-drying method was explored. The former approach involves formation of a double emulsion, by homogenisation. This method produced microspheres of uniform size and smooth morphology, but had a detrimental effect on the formulated DNA. The spray-drying method resulted in microspheres with an improved preservation of DNA stability. The use of polyethylenimine (PEI) and stearylamine (SA) as agents in the microspheric formulation of plasmid DNA is a novel approach to DNA vaccine design. Using these molecules as model positively-charged agents, their influence on the characteristics of the microspheric formulations was investigated. PEI improved the entrapment efficiency of the plasmid DNA in microspheres, and has minimal effect on either the surface charge, morphology or size distribution of the formulations. Stearylamine effected an increase in the entrapment efficiency and stability of the plasmid DNA and its effect on the micropshere morphology was dependent on the method of preparation. The differences in the effects of the two molecules on microsphere formulations may be attributable to their dissimilar physico-chemical properties. PEI is water-soluble and highly-branched, while SA is hydrophobic and amphipathic. The positive charge of both molecules is imparted by amine functional groups. Preliminary data on the in vivo application of formulated DNA vaccine, using hepatitis B plasmid, showed superior humoral responses to the formulated antigen, compared with free (unformulated) antigen.

Tumour associated proteolysis and protein metabolism

The effect of cancer cachexia on protein metabolism has been studied in mice transplanted with the MAC16 adenocarcinoma. The progressive cachexia induced by the MAC16 tumour was characterised by a reduction in carcass nitrogen between 16-30% weight loss and a reciprocal increase in tumour nitrogen content. Carcass nitrogen loss was accompanied by a concomitant decrease in gastrocnemius muscle weight and nitrogen content and also by a decrease in liver nitrogen content. The loss of gastrocnemius muscle throughout the progression of cachexia was attributable to a 60% decrease in the rate of protein synthesis and a 240% increase in the rate of protein degradation. The loss of skeletal muscle protein that may be partially mediated by an increased rate of protein degradation has been correlated with a circulatory catabolic factor present only in cachectic tumour-bearing animals, that degrades host muscle in vitro. The proteolysis-inducing factor was found to be heat stable, not a serine protease and was inhibited by indomethacin and eicosapentaenoic acid (EPA) in a dose-related manner. The proteolytic factor induced prostaglandin E2 formation in the gastrocnemius muscle of non tumour-bearing animals and this effect was inhibited by indomethacin and EPA. In vivo studies show EPA (2.0g/kg-1 by gavage) to effectively reverse the decrease in body weight in animals bearing the MAC16 tumour with a concomitant reduction in tumour growth. Muscle from animals treated with EPA showed a decrease (60%) in protein degradation without an effect on protein synthesis. In vivo studies show branched chain amino acid treatment to be ineffective in moderating the cachectic effect of the MAC16 tumour. The action of the factor was largely mimicked by triarachidonin and trilinoleia. The increased serum levels of arachidonic acid in cachectic tumour-bearing animals may thus be responsible for increased protein degradation through prostanoid metabolism. The understanding of protein metabolism and catabolic factors in the cachectic animal may provide future avenues for the reversal of cachexia and the treatment of cancer.metabolism and catabolicmetabolism and cat

Metabolic induction and early responses of mouse blastocyst developmental programming following maternal low protein diet affecting life-long health

Previously, we have shown that a maternal low protein diet, fed exclusively during the preimplantation period of mouse development (Emb-LPD), is sufficient to induce by the blastocyst stage a compensatory growth phenotype in late gestation and postnatally, correlating with increased risk of adult onset cardiovascular disease and behavioural dysfunction. Here, we examine mechanisms of induction of maternal Emb-LPD programming and early compensatory responses by the embryo. Emb-LPD induced changes in maternal serum metabolites at the time of blastocyst formation (E3.5), notably reduced insulin and increased glucose, together with reduced levels of free amino acids (AAs) including branched chain AAs leucine, isoleucine and valine. Emb-LPD also caused reduction in the branched chain AAs within uterine fluid at the blastocyst stage. These maternal changes coincided with an altered content of blastocyst AAs and reduced mTORC1 signalling within blastocysts evident in reduced phosphorylation of effector S6 ribosomal protein and its ratio to total S6 protein but no change in effector 4E-BP1 phosphorylated and total pools. These changes were accompanied by increased proliferation of blastocyst trophectoderm and total cells and subsequent increased spreading of trophoblast cells in blastocyst outgrowths. We propose that induction of metabolic programming following Emb-LPD is achieved through mTORC1signalling which acts as a sensor for preimplantation embryos to detect maternal nutrient levels via branched chain AAs and/or insulin availability. Moreover, this induction step associates with changes in extra-embryonic trophectoderm behaviour occurring as early compensatory responses leading to later nutrient recovery. © 2012 Fleming et al.

Do little embryos make big decisions? How maternal dietary protein restriction can permanently change an embryo's potential, affecting adult health

Periconceptional environment may influence embryo development, ultimately affecting adult health. Here, we review the rodent model of maternal low-protein diet specifically during the preimplantation period (Emb-LPD) with normal nutrition during subsequent gestation and postnatally. This model, studied mainly in the mouse, leads to cardiovascular, metabolic and behavioural disease in adult offspring, with females more susceptible. We evaluate the sequence of events from diet administration that may lead to adult disease. Emb-LPD changes maternal serum and/or uterine fluid metabolite composition, notably with reduced insulin and branched-chain amino acids. This is sensed by blastocysts through reduced mammalian target of rapamycin complex 1 signalling. Embryos respond by permanently changing the pattern of development of their extra-embryonic lineages, trophectoderm and primitive endoderm, to enhance maternal nutrient retrieval during subsequent gestation. These compensatory changes include stimulation in proliferation, endocytosis and cellular motility, and epigenetic mechanisms underlying them are being identified. Collectively, these responses act to protect fetal growth and likely contribute to offspring competitive fitness. However, the resulting growth adversely affects long-term health because perinatal weight positively correlates with adult disease risk. We argue that periconception environmental responses reflect developmental plasticity and 'decisions' made by embryos to optimise their own development, but with lasting consequences.