Medium Chains Research Articles

Assessment of Aquatic Toxicity and Oxygen Depletion during Aerobic Biodegradation of Vegetable Oil: Effect of Oil Loading and Mixing Regime

The potential ecological impacts of aerobic biodegradation of vegetable oils on contaminated water columns was investigated in the laboratory at different oil loadings (100, 333, and 1,000 gal acre(-1)) and mixing regimes (fully, moderately, and nonmixed microcosms). The impacts were estimated by use of the Microtox assay and dissolved oxygen concentration measurements. The results of the Microtox assay showed no major toxicity at the 100 gal acre(-1) loading. Furthermore, oxygen was not completely depleted from the water column at this oil coverage. At higher oil loadings, oxygen was fully depleted from the mixed and nonmixed water columns. A transient toxicity in the aqueous phase was observed in the case of the moderately mixed microcosms at 333 gal acre(-1) and was maintained at moderate levels (EC(50) ∼ 30%) in the nonmixed microcosms. A substantial increase in toxicity (EC(50) ∼ 10%) was observed in both mixing conditions when the initial oil loading was increased to 1,000 gal acre(-1). At all oil loadings, significant toxicity (EC(50) < 2%) was found in the solid phase due to the strong partition of lipids to the biomass. Long and medium chains fatty acids associated with the measured toxicity were detected in both liquid and solid phases.

Effect of high temperature on color, chlorophyll fluorescence and volatile biosynthesis in green-ripe banana fruit

Banana (Musa AAA group) is one of the most consumed fruits in the world. It has been reported that chlorophyll breakdown and color formation in banana is inhibited by ripening temperatures above 24 °C. At this temperature thylakoid membranes are retained resulting in reduced chlorophyll degradation. In this study, green mature, untreated banana fruit were obtained from a local wholesale market and half of the fruit were subjected to ethylene treatment at 10 μL L −1 for 24 d. After ethylene treatment, both treated and untreated fruit were stored at 20 °C or 30 °C for 7 d. Fruit were sampled after 0, 1, 4 and 7 d of storage and evaluated for color, chlorophyll fluorescence, volatile production and expression of genes related to volatile biosynthesis. Storage at 30 °C reduced yellow color development in the peel, decreased chlorophyll fluorescence (Fv/Fm), but increased Fo, indicating possible heat stress of the fruit. A total of 19 volatile compounds were identified using SPME/GC/MS. Both ethylene treatment and high temperature enhanced volatile production. The increase of Fo and hexanoate and acetate esters are coincided with the stress of high temperature. Using real time PCR (qPCR), expression of genes related to volatile biosynthesis including branched-chain amino acid transaminase ( BCAT), lipoxygenase (LOX), hydroperoxide lyase ( HPL), pyruvate decarbolxylase ( PDC), alcohol dehydrogenases ( ADH, short and medium chains), and alcohol acetyl transferase ( AAT) were investigated in both peel and pulp tissues. Among the tested genes, BCAT, HPL, ADH and AAT in peel and pulp tissues increased significantly in response to ethylene and storage at 30 °C. PDC, ADH and BCAT (in pulp tissue) were induced by storage at 30 °C in ethylene-treated fruit. LOX decreased during ripening and storage at 30 °C in the peel, but increased in the pulp tissue of ethylene-treated fruit. This study demonstrates that both ethylene and high temperature influence volatile biosynthesis in banana fruit at the transcriptional level and confirms findings that high temperature causes stress in banana fruit during ripening.

Studies on Immobilization and Partial Characterization of Lipases from Wheat Seeds (Triticum aestivum)

The objective of this study was to provide some features on immobilization and partial characterization of lipases from wheat seeds. The optimum pH and temperature were found to be 5.5 and 32-37°C, respectively. The stability of the concentrated enzymatic extract to high temperatures (25, 35, 45, and 55°C) showed that the incubation of the extract at 55°C led to its complete inactivation. The concentrated enzymatic extract kept 90% of its hydrolytic and esterification activities until 70 and 40days of storage at 4°C, respectively. The extract presented higher hydrolytic specificity to substrates of medium and long chains and higher esterification affinity to fatty acids of short and medium chains and alcohols with two and three carbon atoms. After the immobilization process using activated coal and sodium alginate as supports, an enhancement of about threefold in lipase activity was observed. The development of the present work permitted us to point out some characteristics of lipases from wheat seeds necessary for the proposition of new future industrial applications for this important biocatalyst.

Prospective life cycle carbon abatement for pyrolysis biochar systems in the UK

Life cycle assessment (LCA) of slow pyrolysis biochar systems (PBS) in the UK for small, medium and large scale process chains and ten feedstocks was performed, assessing carbon abatement and electricity production. Pyrolysis biochar systems appear to offer greater carbon abatement than other bioenergy systems. Carbon abatement of 0.7–1.3 t CO 2 equivalent per oven dry tonne of feedstock processed was found. In terms of delivered energy, medium to large scale PBS abates 1.4–1.9 t CO 2e/MWh, which compares to average carbon emissions of 0.05–0.30 t CO 2e/MWh for other bioenergy systems. The largest contribution to PBS carbon abatement is from the feedstock carbon stabilised in biochar (40–50%), followed by the less certain indirect effects of biochar in the soil (25–40%)—mainly due to increase in soil organic carbon levels. Change in soil organic carbon levels was found to be a key sensitivity. Electricity production off-setting emissions from fossil fuels accounted for 10–25% of carbon abatement. The LCA suggests that provided 43% of the carbon in the biochar remains stable, PBS will out-perform direct combustion of biomass at 33% efficiency in terms of carbon abatement, even if there is no beneficial effect upon soil organic carbon levels from biochar application.

Amylolysis of wheat starches. I. Digestion kinetics of starches with varying functional properties

The susceptibility of wheat ( Triticum aestivum L.) starches to hydrolysis by pancreatic α-amylase in vitro was investigated using a series of 35 starches with slightly enriched amylose content within a narrow range (36–43%), but widely differing functional properties. After 2 h of incubation with α-amylase, native starch granules were digested to different extents, but there were no differences between any of the starches once they were gelatinized. Cooling the starch for 72 h at 4 °C after cooking reduced the susceptibility of all of the starches to enzymic digestion by a similar extent, whereas addition of monopalmitin decreased the digestibility of the starches that contained amylose, but did not affect the digestibility of waxy starches that were also included in the study. Amylopectin chain length distribution of partly digested starch granules displayed increased proportion of short and medium chains and decreased proportion of long chains in comparison to native granules. Separated large (A) and small (B) starch granules from three of the starches differed significantly in their susceptibility to in-vitro digestion. A predictive model of the susceptibility of starch in the different forms was developed from the physico-chemical and functional properties of the starches.

Lipolysis of natural long chain and synthetic medium chain galactolipids by pancreatic lipase-related protein 2

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the most abundant lipids in nature, mainly as important components of plant leaves and chloroplast membranes. Pancreatic lipase-related protein 2 (PLRP2) was previously found to express galactolipase activity, and it is assumed to be the main enzyme involved in the digestion of these common vegetable lipids in the gastrointestinal tract. Most of the previous in vitro studies were however performed with medium chain synthetic galactolipids as substrates. It was shown here that recombinant guinea pig (Cavia porcellus) as well as human PLRP2 hydrolyzed at high rates natural DGDG and MGDG extracted from spinach leaves. Their specific activities were estimated by combining the pH-stat technique, thin layer chromatography coupled to scanning densitometry and gas chromatography. The optimum assay conditions for hydrolysis of these natural long chain galactolipids were investigated and the optimum bile salt to substrate ratio was found to be different from that established with synthetic medium chains MGDG and DGDG. Nevertheless the length of acyl chains and the nature of the galactosyl polar head of the galactolipid did not have major effects on the specific activities of PLRP2, which were found to be very high on both medium chain [1786+/-100 to 5420+/-85U/mg] and long chain [1756+/-208 to 4167+/-167U/mg] galactolipids. Fatty acid composition analysis of natural MGDG, DGDG and their lipolysis products revealed that PLRP2 only hydrolyzed one ester bond at the sn-1 position of galactolipids. PLRP2 might be used to produce lipid and free fatty acid fractions enriched in either 16:3 n-3 or 18:3 n-3 fatty acids, both found at high levels in galactolipids.

Nanostructure, Morphology, and Properties of Fluorous Copolymers Bearing Ionic Grafts

In order to probe the effects of polymer microstructure on the properties of proton conducting polymer membranes, three series of fluorous−ionic graft copolymers, partially sulfonated poly([vinylidene difluoride-co-chlorotrifluoroethylene]-g-styrene) [P(VDF-co-CTFE)-g-SPS], comprising controlled graft lengths and degrees of sulfonation were synthesized. The parent building block was a poly(vinylidene difluoride-co-chlorotrifluoroethylene) [P(VDF-co-CTFE)] macroinitiator (Mn = 3.12 × 105 g/mol) synthesized to contain 1 chloro group per 17 repeat units, onto which polystyrene, having degrees of polymerization of 35, 88, and 154 units per graft, was grown by atom transfer radical polymerization (ATRP). These graft copolymers, termed short, medium, and long graft chains, were sulfonated to different extents to provide a series of polymers with varying ion exchange capacity (IEC). The resulting P(VDF-co-CTFE)-g-SPS copolymers were cast into proton exchange membranes, and their nanostructure, morphology, and pr...

Physicochemical properties and structure of starches from Chinese rice cultivars

Starches from 10 different non-waxy rice cultivars in China were isolated and investigated for their physicochemical properties including the swelling power, solubility, syneresis, thermal, rheological and textural properties, and structure. The range of amylose content in 10 rice starches was between 18.1 and 31.6%. The branch chain length distribution of amylopectin revealed that the proportions of short (DP6–11), medium (DP12–17), and longer chains (DP18–23) were 4.6–11.1%, 47.0–51.6% and 25.3–31.8%, respectively. The relationships between different properties of rice starches were determined using Pearson correlation analysis. Amylose content was negatively correlated to swelling power ( r = −0.632, p ≤ 0.05) and positively correlated to gel hardness ( r = 0.776, p ≤ 0.01). The percentage of short chains (DP6–11) was negatively related to the transition temperatures ( T o, T p and T c), T o G′ and T G ′max, while the percentage of medium chains (DP12–23) was positively related. Various branch chain length amylopectins played different roles in rheological behavior of starch dispersions. The percentage of short chains (DP6–11) was positively correlated to G′ and G″( r = 0.832 and r = 0.775, respectively, p ≤ 0.01), and negatively correlated to tan δ peak ( r = −0.717, p ≤ 0.05). However, the percentage of longer chains (DP18–23) showed a negative relation with G′ and G″ ( r = −0.794, p ≤ 0.01; r = −0.741, p ≤ 0.05), and a positive relation with tan δ peak ( r = 0.668, p ≤ 0.05).

Reação de cultivares de batata a Streptomyces scabies, agente causal da sarna comum profunda

Este trabalho visou caracterizar quanto a critérios morfológicos e fisiológicos isolados de Streptomyces causadores de sarna comum profunda em batata; avaliar o comportamento de cultivares em relação à doença e a variação na agressividade entre os isolados da bactéria. Os isolados de Streptomyces apresentaram coloração cinza em meio extrato de levedura e malte e cadeias de esporos espiraladas, produzidas sobre um micélio aéreo. Ocorreu produção de melanina em meio de tirosina-ágar e a utilização de oito fontes de carbono recomendadas, propriedades estas que correspondem às descritas para S. scabies. Batatas-semente sadias de seis cultivares foram plantadas em substrato infestado com seis isolados de S. scabies separadamente e as plantas cultivadas em vasos e ambiente aberto. A severidade da doença foi estimada com auxílio de uma escala diagramática e avaliou-se o rendimento de tubérculos (g/planta). As cvs. Mondial e Jaete Bintje foram as mais resistentes à sarna comum com severidade média nos dois experimentos de 10,5% e 14,0%, respectivamente, seguidas por Asterix (17,4%), Ágata (21,8%), Monalisa (23,0%) e Cupido (23,3%). A agressividade dos isolados variou quantitativamente, com severidade maior para o isolado M4 (34,7%) e menor para o isolado M1 (6,2%). Redução da produção de tubérculos foi verificada na cv. Jaete Bintje, no primeiro experimento, e quando as cultivares foram infectadas pelos isolados A1, M2 e M3, no primeiro experimento, e pelo isolado M4, no segundo experimento.

Study of chain branching in natural rubber using size-exclusion chromatography coupled with a multi-angle light scattering detector (SEC-MALS)

The different rheological behaviour of natural rubber (NR) compared to industrial synthetic poly( cis-1,4-isoprene) (SR) has been attributed to the gel phase and long-chain branching. Previous studies on branching in NR were carried out using the fractionation technique by precipitation to obtain narrow molar mass distribution. In this study, chain branching of poly( cis-1,4-isoprene) in NR was characterised by size-exclusion chromatography coupled with an online multi-angle light scattering detector (SEC-MALS). The nanoaggregates adsorbed on the column packing interfered with branching characterisation for short and medium chains ( M w < 1000 kg/mol). Using a master curve of linear standard poly( cis-1,4-isoprenes), SEC-MALS revealed no or very little branching in the higher chains (1000 < M w < 10,000 kg/mol) of natural rubber contrary to previous studies. This study showed that the soluble portion of NR samples was composed of almost linear poly( cis-1,4-isoprene) and nanoaggregates with rather compact structures.

Tryptophan fluorescence reveals induced folding of Vibrio harveyi acyl carrier protein upon interaction with partner enzymes

We have introduced tryptophan as a local fluorescent probe to monitor the conformation of Vibrio harveyi acyl carrier protein (ACP), a small flexible protein that is unfolded at neutral pH but must undergo reversible conformational change during the synthesis and delivery of bacterial fatty acids. Consistent with known 3D structures of ACP, steady-state fluorescence and quenching experiments indicated that Trp at positions 46, 50, and 72 are buried in the hydrophobic core upon Mg 2+-induced ACP folding, whereas residues 25 and 45 remain in a hydrophilic environment on the protein surface. Attachment of fatty acids to the phosphopantetheine prosthetic group progressively stabilized the folded conformation of all Trp-substituted ACPs, but longer chains (14:0) were less effective than medium chains (8:0) in shielding Trp from acrylamide quenching in the L46W protein. Interaction with ACP-dependent enzymes LpxA and holo-ACP synthase also caused folding of L46W; fluorescence quenching indicated proximity of Trp-45 in helix II of ACP in LpxA binding. Our results suggest that divalent cations and fatty acylation produce differing environments in the ACP core and also reveal enzyme partner-induced folding of ACP, a key feature of “natively unfolded” proteins.

Analysis of Factors Motivating the Managers’ Career

The present article analyzes what factors determine managers’ strive for career, making an assumption that a possibility for career has to be provided for the most initiative and qualified employees, a possibility which at the same time would be a motivating factor itself. Only motivated employees who have possibilities to realize their goals and expectations in organization can successfully strive for results in organization performance. Analysis of managers’ career expectations is relevant when considering today’s condition in Lithuania, for many qualified employees, possessing both knowledge and working experience but seeing no perspective in their current job or position, leave their workplaces and search for jobs abroad. Such analyses are useful in order to hold qualified professionals in the Lithuanian market, irrespective of the fact if they work in managing position or not, for if an organization lacks professional managers capable to recruit a united team, it will lack good employees and activities of such an organization will be doomed to failure. This article analyzes environmental factors determine managers’ motivation to seek for a career in Lithuanian organizations. Continuous changes taking place necessitate a new approach to the role of managers in an organization, and an assessment of their professional and career related expectations. In order to achieve successes in organization activity, involvement of employees and managers in their career planning must be evaluated anew, by integrating their personal and organizational interests. Growing requirements for managers suggest paying greater attention to implementation of their career possibilities in organization. For only motivated employees, having possibilities to implement their goals and expectation within the organization can develop activity of that organization effectively. Analysis of business enterprises has revealed that more than 30 % of surveyed managers are not satisfied with their current position. The most common reasons, next to hard work and small salaries, are the following: the job does not tally with their speciality, there is lack for professional improvement, and a job lacks creativity. When inducing managers to pursue career, a very important role is played by possibilities to improve one’s qualification. Irrespective of age, one of the obstacles to career is insufficient education: 42.79 % of participatits, mainly being managers of medium and lower chains, referred to this reason. Another reason of unwillingness for career is the fact that a person is satisfied with the current job or salary: 45.95 % of participatits, most of them having greater experience in the enterprise. 53.8% of the participants have the possibility to seek career, and it is more desirable for persons giving smaller working experience. All the age groups value the career possibilities and the reasons thereof in a quite similar way, the intentions to change jobs, however, differ greatly (χ2=38.078, p=0.003: such intentions are more common to persons with small working experience. Reasons of those intentions vary as well: managers of small enterprises most often named insufficient salary and wearisome working conditions, while one of the most relevant reasons in large enterprises is inadequate ranking of employees.

Mapping P2X and P2Y receptor proteins in striatum and substantia nigra: An immunohistological study

Our work aimed to provide a topographical analysis of all known ionotropic P2X1–7 and metabotropic P2Y1,2,4,6,11–14 receptors that are present in vivo at the protein level in the basal ganglia nuclei and particularly in rat brain slices from striatum and substantia nigra. By immunohistochemistry-confocal and Western blotting techniques, we show that, with the exception of P2Y11,13 receptors, all other subtypes are specifically expressed in these areas in different amounts, with ratings of low (P2X5,6 and P2Y1,6,14 in striatum), medium (P2X3 in striatum and substantia nigra, P2X6,7 and P2Y1 in substantia nigra) and high. Moreover, we describe that P2 receptors are localized on neurons (colocalizing with neurofilament light, medium and heavy chains) with features that are either dopaminergic (colocalizing with tyrosine hydroxylase) or GABAergic (colocalizing with parvalbumin and calbindin), and they are also present on astrocytes (P2Y2,4, colocalizing with glial fibrillary acidic protein). In addition, we aimed to investigate the expression of P2 receptors after dopamine denervation, obtained by using unilateral injection of 6-hydroxydopamine as an animal model of Parkinson’s disease. This generates a rearrangement of P2 proteins: most P2X and P2Y receptors are decreased on GABAergic and dopaminergic neurons, in the lesioned striatum and substantia nigra, respectively, as a consequence of dopaminergic denervation and/or neuronal degeneration. Conversely, P2X1,3,4,6 on GABAergic neurons and P2Y4 on astrocytes augment their expression exclusively in the lesioned substantia nigra reticulata, probably as a compensatory reaction to dopamine shortage. These results disclose the presence of P2 receptors in the normal and lesioned nigro-striatal circuit, and suggest their potential participation in the mechanisms of Parkinson’s disease.

Nano-emulsions and nanocapsules by the PIT method: An investigation on the role of the temperature cycling on the emulsion phase inversion

This paper focuses on the phenomenological understanding of temperature cycling process, applied to the phase inversion temperature (PIT) method. The role of this particular thermal treatment on emulsions phase inversion, as well as its ability to generate nano-emulsions have been investigated. In order to propose a general study, we have based our investigations on a given formulation of nano-emulsions classically proposed in the literature [Heurtault, B., Saulnier, P., Pech, B., Proust, J. E., Benoit, J.P., 2002. A novel phase inversion-based process for the preparation of lipid nanocarriers. Pharm. Res. 19, 875; Lamprecht, A., Bouligand, Y, Benoit, J.P., 2002. New lipid nanocapsules exhibit sustained release properties for amiodarone. J. Control. Release 84, 59–68], using a polyethoxylated model nonionic surfactant, a polyoxyehtylene-660-12-hydroxy stearate, stabilizing the emulsion composed of caprilic triglycerides (triglycerides medium chains), salt water (and also phospholipidic amphiphiles neutral for the formulation). Characterization of nano-emulsions was performed by dynamic light scattering (DLS) which provides the hydrodynamic diameter, but also the polydispersity index (PDI), as a fundamental criteria to judge the quality of the dispersion. Another aspect of the characterization was done following the emulsion inversion and structure by electrical conductivity through the temperature scan. Overall, the role such a temperature cycling process on the formulation of nano-emulsions appears to be relatively important, and globally enhanced as the surfactant concentration is lowered. Actually, both the hydrodynamic diameter and the PDI decrease as a function of the number and temperature cycles up to stabilize a steady state. Eventually, such a cycling process allows the generation of nano-emulsions in ranges of compositions largely expanded when compared with the classical PIT method. These general and interesting trends emerge from the results, are discussed and essentially explained by regarding the behavior of the nonionic surfactants towards the water/oil interface, linking partitioning coefficients, temperature variation, and surfactant water/oil interfacial concentration. In that way, this paper proposes new insights into the phenomena governing the PIT method, by originally investigating the temperature cycling process.

Studies on tuber starches. II. Molecular structure, composition and physicochemical properties of yam ( Dioscorea sp.) starches grown in Sri Lanka

Starch from varieties of Dioscorea esculenta (Lour.) Burkill (kukulala, java-ala, nattala) and Dioscorea alata L. (hingurala, raja-ala) tubers grown in Sri Lanka was isolated and its morphology, composition, structure and physicochemical properties were studied. The yield of starch was in the range 12.2–18.0% on a whole tuber basis. The shape and size of D. esculenta and D. alata starch granules were round to polygonal (3–10 μm) and spade shaped (30–45 μm), respectively. There was considerable variation in total amylose, phosphorous, bound lipid, amylose leaching, swelling factor, gelatinization parameters, extent of retrogradation, pasting characteristics, susceptibility towards enzyme and acid hydrolysis crystallinity and lipid complexed amylose chains between and among the starches belonging to the two species. The chain length distribution of debranched amylopectins of the starches showed that the proportion of short branched chains (dp 6–12), medium chains (dp 25–36) and average chain length of amylopectin were significantly ( P < 0.05) higher in the D. esculenta starches. However, variations in amylopectin structure among varieties of both species were not significant ( P < 0.05). D. esculenta starches exhibited a ‘B’-type powder X-ray diffraction pattern. However, in the D. alata starches, hingurala and raja-ala displayed ‘C’- and ‘B’-type patterns, respectively. The results showed that some of the starches from the above species could be used in foods subjected to thermal processing and frozen storage, respectively, with minimal physical or chemical modification.

Evidence for a Functional Quorum-Sensing Type AI-1 System in the Extremophilic Bacterium Acidithiobacillus ferrooxidans

Acidithiobacillus ferrooxidans is one of the main acidophilic chemolithotrophic bacteria involved in the bioleaching of metal sulfide ores. The bacterium-mineral interaction requires the development of biofilms, whose formation is regulated in many microorganisms by type AI-1 quorum sensing. Here, we report the existence and characterization of a functional type AI-1 quorum-sensing system in A. ferrooxidans. This microorganism produced mainly acyl-homoserine lactones (AHL) with medium and large acyl chains and different C-3 substitutions, including 3-hydroxy-C8-AHL, 3-hydroxy-C10-AHL, C12-AHL, 3-oxo-C12-AHL, 3-hydroxy-C12-AHL, C14-AHL, 3-oxo-C14-AHL, 3-hydroxy-C14-AHL, and 3-hydroxy-C16-AHL. A quorum-sensing genetic locus that includes two open reading frames, afeI and afeR, which have opposite orientations and code for proteins with high levels of similarity to members of the acyl synthase (I) and transcriptional regulator (R) protein families, respectively, was identified. Overexpression of AfeI in Escherichia coli and the associated synthesis of AHLs confirmed that AfeI is an AHL synthase. As determined by reverse transcription-PCR, the afeI and afeR genes were transcribed in A. ferrooxidans. The transcription levels of the afeI gene were higher in cells grown in sulfur and thiosulfate media than in iron-grown cells. Phosphate starvation induced an increase in the transcription levels of afeI which correlated with an increase in AHL levels. Two afe boxes which could correspond to the AfeR binding sites were identified upstream of the afeI gene. This is the first report of a functional type AI-1 quorum-sensing system in an acidophilic chemolithotrophic microorganism, and our results provide a very interesting opportunity to explore the control and regulation of biofilm formation during the bioleaching process.

Antisense downregulation of the barley limit dextrinase inhibitor modulates starch granule size distribution, starch composition and amylopectin structure.

The barley protein limit dextrinase inhibitor (LDI), structurally related to the alpha-amylase/trypsin inhibitor family, is an inhibitor of the starch debranching enzyme limit dextrinase (LD). In order to investigate the function of LDI, and the consequences for starch metabolism of reduced LDI activity, transgenic barley plants designed to downregulate LDI by antisense were generated. Homozygous antisense lines with reduced LDI protein level and activity were analysed and found to have enhanced free LD activity in both developing and germinating grains. In addition the antisense lines showed unpredicted pleiotropic effects on numerous enzyme activities, for example, alpha- and beta-amylases and starch synthases. Analysis of the starch showed much reduced numbers of the small B-type starch granules, as well as reduced amylose relative to amylopectin levels and reduced total starch. The chain length distribution of the amylopectin was modified with less of the longer chains (>25 units) and enhanced number of medium chains (10-15 units). These results suggest an important role for LDI and LD during starch synthesis as well as during starch breakdown.

Basolateral targeting by leucine-rich repeat domains in epithelial cells.

The asymmetric distribution of proteins to basolateral and apical membranes is an important feature of epithelial cell polarity. To investigate how basolateral LAP proteins (LRR (for leucine-rich repeats) and PDZ (for PSD-95/Discs-large/ZO-1), which play key roles in cell polarity, reach their target membrane, we carried out a structure-function study of three LAP proteins: Caenorhabditis elegans LET-413, human Erbin and human Scribble (hScrib). Deletion and point mutation analyses establish that their LRR domain is crucial for basolateral membrane targeting. This property is specific to the LRR domain of LAP proteins, as the non-LAP protein SUR-8 does not localize at the basolateral membrane of epithelial cells, despite having a closely related LRR domain. Importantly, functional studies of LET-413 in C. elegans show that although its PDZ domain is dispensable during embryogenesis, its LRR domain is essential. Our data establish a novel paradigm for protein localization by showing that a subset of LRR domains direct subcellular localization in polarized cells.

Functional dissection of a eukaryotic dicistronic gene: transgenic stonedB, but not stonedA, restores normal synaptic properties to Drosophila stoned mutants.

The dicistronic Drosophila stoned mRNA produces two proteins, stonedA and stonedB, that are localized at nerve terminals. While the stoned locus is required for synaptic-vesicle cycling in neurons, distinct or overlapping synaptic functions of stonedA and stonedB have not been clearly identified. Potential functions of stoned products in nonneuronal cells remain entirely unexplored in vivo. Transgene-based analyses presented here demonstrate that exclusively neuronal expression of a dicistronic stoned cDNA is sufficient for rescue of defects observed in lethal and viable stoned mutants. Significantly, expression of a monocistronic stonedB trangene is sufficient for rescuing various phenotypic deficits of stoned mutants, including those in organismal viability, evoked transmitter release, and synaptotagmin retrieval from the plasma membrane. In contrast, a stonedA transgene does not alleviate any stoned mutant phenotype. Novel phenotypic analyses demonstrate that, in addition to regulation of presynaptic function, stoned is required for regulating normal growth and morphology of the motor terminal; however, this developmental function is also provided by a stonedB transgene. Our data, although most consistent with a hypothesis in which stonedA is a dispensable protein, are limited by the absence of a true null allele for stoned due to partial restoration of presynaptic stonedA by transgenically provided stonedB. Careful analysis of the effects of the monocistronic transgenes together and in isolation clearly reveals that the presence of presynaptic stonedA is dependent on stonedB. Together, our findings improve understanding of the functional relationship between stonedA and stonedB and elaborate significantly on the in vivo functions of stonins, recently discovered phylogenetically conserved stonedB homologs that represent a new family of "orphan" medium (mu) chains of adaptor complexes involved in vesicle formation. Data presented here also provide new insight into potential mechanisms that underlie translation and evolution of the dicistronic stoned mRNA.

Synthesis of pyroglutamic acid fatty esters through lipase-catalyzed esterification with medium chains alcohols

The lipase-catalyzed synthesis of fatty alcohol esters of pyroglutamic acid (pGlu) was investigated. The results showed that, a strategy involving ethyl pyroglutamic ester intermediate was advantageous since this compound had a better solubility in the chosen solvent than the free pyroglutamic acid. The effects of the enzyme load, type of solvent, substrates molar ratio and fatty alcohol type were studied. It was shown that the most optimized reaction conditions were the ones using a 10% (w/w) quantities of Candida antarctica lipase with acetonitrile as solvent and a molar ratio of substrates corresponding to a 1:5 excess of fatty alcohols. Under such conditions, octyl, decyl and dodecyl esters of pyroglutamic acid were obtained in satisfactory yields between 65 and 70% with good reaction kinetics within 24 h.