Body Membrane Protein Research Articles

Phagolysosomes break down the membrane of a non-apoptotic corpse independent of macroautophagy.

Cell corpses must be cleared in an efficient manner to maintain tissue homeostasis and regulate immune responses. Ubiquitin-like Atg8/LC3 family proteins promote the degradation of membranes and internal cargo during both macroautophagy and corpse clearance, raising the question how macroautophagy contributes to corpse clearance. Studying the clearance of non-apoptotic dying polar bodies in Caenorhabditis elegans embryos, we show that the LC3 ortholog LGG-2 is enriched in the polar body phagolysosome independent of membrane association or autophagosome formation. We demonstrate that ATG-16.1 and ATG-16.2, which promote membrane association of lipidated Atg8/LC3 proteins, redundantly promote polar body membrane breakdown in phagolysosomes independent of their role in macroautophagy. We also show that the lipid scramblase ATG-9 is needed for autophagosome formation in early embryos but is dispensable for timely polar body membrane breakdown or protein cargo degradation. These findings demonstrate that macroautophagy is not required to promote polar body degradation, in contrast to recent findings with apoptotic corpse clearance in C. elegans embryos. Determining how membrane association of Atg8/LC3 promotes the breakdown of different types of cell corpses in distinct cell types or metabolic states is likely to give insights into the mechanisms of immunoregulation during normal development, physiology, and disease.

Momordica charantia seed proteins - Purification, biochemical characterization of a class II α-mannosidase isoenzyme and its interaction with the lectin and protein body membrane

Momordica charantia seeds contain a galactose specific lectin and mixture of glycosidases. These bind to lectin-affigel at pH 5.0 and are all eluted at pH 8.0. From the mixture, α-mannosidase was separated by gel filtration (purified enzyme Mr ∼ 238 kDa). In native PAGE (silver staining) it showed three bands that stained with methylumbelliferyl substrate (possible isoforms). Ion exchange chromatography separated two isoforms in 0.5 M eluates and one isoform in 1.0 M eluate. In SDS-PAGE it dissociated to Mr ∼70 and 45 kDa subunits, showing antigenic similarity to jack bean enzyme. MALDI analysis confirmed the 70 kDa band to be α-mannosidase with sequence identity to the genomic sequence of Momordica charantia enzyme (score 83, 29 % sequence coverage). The pH, temperature optima were 5.0 and 60o C respectively. Kinetic parameters KM and Vmax estimated with p-nitrophenyl α-mannopyranoside were 0.85 mM and 12.1 U/mg respectively. Swainsonine inhibits the enzyme activity (IC50 value was 50 nM). Secondary structural analysis at far UV (190–300 nm) showed 11.6 % α-helix and 36.5 % β-sheets. 2.197 mg of the enzyme was found to interact with 3.75 mg of protein body membrane at pH 5.0 and not at pH 8.0 suggesting a pH dependent interaction.

Isolation of Momordica charantia seed lectin and glycosidases from the protein bodies: Lectin-glycosidase (β-hexosaminidase) protein body membrane interaction reveals possible physiological function of the lectin

Momordica charantia seeds are known to contain a galactose specific lectin that has been well characterized. Seed extracts also contain glycosidases such as the β-hexosaminidase, α-mannosidase and α-galactosidase. In the present study, lectin was affinity purified from the seed extracts and protein bodies isolated by sucrose density gradient centrifugation. From the protein bodies, lectin was identified and β-hexosaminidase was isolated by lectin affinity chromatography and subsequently separated from other glycosidases by gel filtration. In the native PAGE, the purified β-hexosaminidase migrated as a single band with a molecular weight of ∼235 kDa and by zymogram analysis using 4-methylumbelliferyl N-acetyl-β-D-glucosaminide substrate it was confirmed as β-hexosaminidase. Under reducing conditions in SDS-PAGE, the purified enzyme dissociated into three bands (Mr 33, 20 and 15 kDa). The prominent bands (20 and 15 kDa) showed immunological cross-reactivity with the human Hexosaminidase B antibody in a western blot experiment. In gel digestion of the purified enzyme, followed by proteomic analysis using tandom MS/MS revealed sequence identity as compared to the genomic sequence of the Momordica charantia with a score of 57 (24% sequence coverage). Additionally, by CD analysis the purified β-hexosaminidase showed 39.1% of α-helix. Furthermore, secondary structure variations were observed in presence of substrate, lectin and at different pH values. Protein body membrane prepared from the isolated protein bodies showed a pH dependent interaction with the purified lectin and mixture of glycosidases.

Proteomic analysis of metabolic mechanisms associated with fatty acid biosynthesis during Styrax tonkinensis kernel development.

Styrax tonkinensis is a white-flowered tree with considerable potential as a feedstock source for biodiesel production from the oily seed contained within its nutlike drupes. Transcriptome changes during oil accumulation have been previously reported, but not concurrent changes in the proteome. Using proteomic analysis of samples collected at 50, 70, 100 and 130 days after flowering (DAF), we identified 1472 differentially expressed proteins (DEPs). Based on their expression patterns, we grouped the DEPs into nine clusters and analyzed the pathway enrichment. Proteins related to starch and sucrose metabolism were most abundant at 50 DAF. Proteins involved in fatty acid (FA) biosynthesis were mainly grouped into a cluster that peaked at 70 DAF. Proteins related to protein processing in endoplasmic reticulum had two major patterns, trending either upwards or downwards, while proteins involved in amino acid biosynthesis showed more complex relationships. We identified 42 key enzymes involved in lipid accumulation during kernel development, including the acetyl-CoA carboxylase complex (ACC) and the pyruvate dehydrogenase complex (PDC). One oil body membrane protein, oleosin, continuously increased during kernel development. A regulatory network of oil accumulation processes was built based on protein and available transcriptome expression data, which were in good temporal agreement. This analysis placed ACC and PDC in the center of the network, suggesting that the glycolytic provision of substrate plays a central regulatory role in FA biosynthesis and oil accumulation. © 2021 Society of Chemical Industry.

A plant Bro1 domain protein BRAF regulates multivesicular body biogenesis and membrane protein homeostasis

Plant development, defense, and many physiological processes rely on the endosomal sorting complex required for transport (ESCRT) machinery to control the homeostasis of membrane proteins by selective vacuolar degradation. Although ESCRT core components are conserved among higher eukaryotes, the regulators that control the function of the ESCRT machinery remain elusive. We recently identified a plant-specific ESCRT component, FREE1, that is essential for multivesicular body/prevacuolar compartment (MVB/PVC) biogenesis and vacuolar sorting of membrane proteins. Here we identify a plant-specific Bro1-domain protein BRAF, which regulates FREE1 recruitment to the MVB/PVC membrane by competitively binding to the ESCRT-I component Vps23. Altogether, we have successfully identified a role for BRAF, whose function as a unique evolutionary ESCRT regulator in orchestrating intraluminal vesicle formation in MVB/PVCs and the sorting of membrane proteins for degradation in plants makes it an important regulatory mechanism underlying the ESCRT machinery in higher eukaryotes.

Evaluation of Etanercept Treatment in Newborn Rat Model with Hyperoxic Lung Injury

ABSTRACTBackground: Many factors contribute to the development of BPD basically by increasing inflammation in preterm lungs. However, premature neonates have insufficient anti-inflammatory capacity. We aimed to evaluate the effect of etanercept, an anti-TNF agent, on BPD development in newborn rat model with hyperoxia-induced lung injury. Methods: Thirty-two newborn rats were divided into 3 groups as control group (Group 1, n = 11), hyperoxia + placebo group (Group 2, n = 10), and hyperoxia + etanercept group (Group 3, n = 11). Histopathological and biochemical analysis were performed in order to assess inflammation and oxidative stress. Superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activities, and malondialdehyde (MDA) levels were studied, histopathological scoring and radial alveolar count were applied in lung tissue. Lamellar body membrane protein, vascular endothelial growth factor (VEGF), nuclear factor-kappaB (NF-κB) gene expressions were studied in immunohistochemical evaluation of tissue samples. All three groups were compared with each other in terms of all parameters. Results: SOD and GSH-Px activities were significantly higher, whereas MDA levels were lower in group 3, compared to group 2 (p < 0.001). Histopathological scores were lower, lamellar body membrane protein expression and radial alveolar count were higher in group 3 (p < 0.05). NF-κB expression was higher in group 2, but lower in group 3 in comparison with group 1. Expression of VEGF was decreased in group 2 but came close to group 1 with etanercept treatment in group 3. Conclusions: We found etanercept treatment to be protective in newborn rats with hyperoxia-induced lung damage.

Proteomic analysis of oil body membrane proteins accompanying the onset of desiccation phase during sunflower seed development

A noteworthy metabolic signature accompanying oil body (OB) biogenesis during oilseed development is associated with the modulation of the oil body membranes proteins. Present work focuses on 2-dimensional polyacrylamide gel electrophoresis (2-D PAGE)-based analysis of the temporal changes in the OB membrane proteins analyzed by LC-MS/MS accompanying the onset of desiccation (20–30 d after anthesis; DAA) in the developing seeds of sunflower (Helianthus annuus L.). Protein spots unique to 20–30 DAA stages were picked up from 2-D gels for identification and the identified proteins were categorized into 7 functional classes. These include proteins involved in energy metabolism, reactive oxygen scavenging, proteolysis and protein turnover, signaling, oleosin and oil body biogenesis-associated proteins, desiccation and cytoskeleton. At 30 DAA stage, exclusive expressions of enzymes belonging to energy metabolism, desiccation and cytoskeleton were evident which indicated an increase in the metabolic and enzymatic activity in the cells at this stage of seed development (seed filling). Increased expression of cruciferina-like protein and dehydrin at 30 DAA stage marks the onset of desiccation. The data has been analyzed and discussed to highlight desiccation stage-associated metabolic events during oilseed development.

Protective Effects of Valproic Acid, a Histone Deacetylase Inhibitor, against Hyperoxic Lung Injury in a Neonatal Rat Model.

ObjectiveHistone acetylation and deacetylation may play a role in the pathogenesis of inflammatory lung diseases. We evaluated the preventive effect of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, on neonatal hyperoxic lung injury.MethodsForty newborn rat pups were randomized in normoxia, normoxia+VPA, hyperoxia and hyperoxia+VPA groups. Pups in the normoxia and normoxia+VPA groups were kept in room air and received daily saline and VPA (30 mg/kg) injections, respectively, while those in hyperoxia and hyperoxia+VPA groups were exposed to 95% O2 and received daily saline and VPA (30 mg/kg) injections for 10 days, respectively. Growth, histopathological, biochemical and molecular biological indicators of lung injury, apoptosis, inflammation, fibrosis and histone acetylation were evaluated.ResultsVPA treatment during hyperoxia significantly improved weight gain, histopathologic grade, radial alveolar count and lamellar body membrane protein expression, while it decreased number of TUNEL(+) cells and active Caspase-3 expression. Expressions of TGFβ3 and phospho-SMAD2 proteins and levels of tissue proinflammatory cytokines as well as lipid peroxidation biomarkers were reduced, while anti-oxidative enzyme activities were enhanced by VPA treatment. VPA administration also reduced HDAC activity while increasing acetylated H3 and H4 protein expressions.ConclusionsThe present study shows for the first time that VPA treatment ameliorates lung damage in a neonatal rat model of hyperoxic lung injury. The preventive effect of VPA involves HDAC inhibition.

Histamine and Histamine Receptor Involvement in Sensory‐Motor Integration of Gill Lateral Cell Cilia Activity in the Bivalve Crassostrea viginica

Gill lateral ciliated cells of Crassostrea virginica are innervated by serotonin and dopamine nerves. Motor aspects have been well studied, but the sensory side hasn't. Histamine (HIS) a neurotransmitter in a variety of invertebrates rarely has been reported in bivalves. We quantified HIS in C. virginica and found gill lateral cell cilia beating changed in response to mantle sensory inputs including HIS. The mechanism involves mantle rim sensory tentacles and visceral ganglia. HIS does not alter beating when applied to gill. We hypothesize HIS receptors are present in mantle and can be confirmed by Western Blot (WB). For WB, mantle body and rim lysates were prepared in NP-40 detergent buffer containing protease inhibitor followed by centrifuging to obtain supernatant with solubilized mantle body and rim membrane proteins. H2 receptor immunoreactivity was revealed by 1̊ antibody followed by HRP-conjugated 2̊ antibody. Mantle body and rim H2 receptor protein was resolved by colormetric development using CN/DAB substrate kit. We also did a dose response of HIS H1, H2 and H3 receptor agonists and antagonist at the mantle rim and observed cilia beating in gill. WB showed a 70 kD band corresponding to HIS H2 receptors in mantle body and rim, and H2 agonists and antagonists had the strongest effects on cilia beating. The study shows mantle body and rim have HIS H2 receptors and demonstrates a physiological role of HIS in sensory-motor integration of gill lateral cell cilia activity.

Cytidine 5′-diphosphocholine ameliorates hyperoxic lung injury in a neonatal rat model

Bronchopulmonary dysplasia (BPD) is an important cause of morbidity. The aim of this study was to evaluate the preventive effect of cytidine 5'-diphosphocholine (CDP-choline) treatment on hyperoxic lung injury in a neonatal rat model. A total of 30 newborn pups were divided into control, hyperoxia, and hyperoxia + CDP-choline groups. After birth, pups in the control group were kept in room air and received saline injections, whereas those in hyperoxia and hyperoxia + CDP-choline groups were exposed to 95% O₂ and received daily injections of saline and CDP-choline throughout postnatal day 10, respectively. Histopathological scoring, radial alveolar count, lamellar body membrane protein expression, fibrosis, proinflammatory cytokine levels, lung tissue and bronchoalveolar lavage (BAL) fluid phospholipid content, and apoptosis were evaluated. Hyperoxia-induced severe lung damage was reduced significantly by CDP-choline treatment. Radial alveolar count and lamellar body membrane protein expression were significantly recovered, and the number of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling-positive cells, active caspase-3 expression, and tissue proinflammatory cytokine levels were decreased by CDP-choline administration. Lung tissue and BAL phospholipid contents showed significant increases after CDP-choline administration. These data show that CDP-choline ameliorates hyperoxic lung injury in a neonatal rat model. It may therefore be suggested that CDP-choline may be a novel therapeutic option for the prevention of BPD.

A novel conserved targeting motif found in ABCA transporters mediates trafficking to early post-Golgi compartments

The ATP binding cassette, class A (ABCA) proteins are homologous polytopic transmembrane transporters that function as lipid pumps at distinct subcellular sites in a variety of cells. Located within the N terminus of these transporters, there exists a highly conserved xLxxKN motif of unknown function. To define its role, human ABCA3 was employed as a primary model representing ABCA transporters, while mouse ABCA1 was utilized to support major findings. Transfection studies showed colocalization of both transporters with surfactant protein C (SP-C), a marker peptide for successful protein targeting to lysosomal-like organelles. In contrast, alanine mutation of xLxxKN resulted in endoplasmic reticulum retention. As proof of principle, swapping xLxxKN for the known lysosomal targeting motif of SP-C resulted in post-Golgi targeting of the SP-C chimera. However, these products failed to reach their terminal processing compartments, suggesting that the xLxxKN motif only serves as a Golgi exit signal. We propose a model whereby an N-terminal signal sequence, xLxxKN, directs ABCA transporters to a post-Golgi vesicular sorting station where additional signals may be required for selective delivery of individual transporters to final subcellular destinations.

Sodium chloride stress induces nitric oxide accumulation in root tips and oil body surface accompanying slower oleosin degradation in sunflower seedlings

Present work highlights the involvement of endogenous nitric oxide (NO) in sodium chloride (NaCl)-induced biochemical regulation of seedling growth in sunflower (Helianthus annuus L., cv. Morden). The growth response is dependent on NaCl concentration to which seedlings are exposed, they being tolerant to 40 mM NaCl and showing a reduction in extension growth at 120 mM NaCl. NaCl sensitivity of sunflower seedlings accompanies a fourfold increase in Na(+) /K(+) ratio in roots (as compared to that in cotyledons) and rapid transport of Na(+) to the cotyledons, thereby enhancing Na(+) /K(+) ratio in cotyledons as well. A transient increase in endogenous NO content, primarily contributed by putative NOS activity in roots of 4-day-old seedlings subjected to NaCl stress and the relative reduction in Na(+) /K(+) ratio after 4 days, indicates that NO regulates Na(+) accumulation, probably by affecting the associated transporter proteins. Root tips exhibit an early and transient enhanced expression of 4,5-diaminofluorescein diacetate (DAF-2DA) positive NO signal in the presence of 120 mM NaCl. Oil bodies from 2-day-old seedling cotyledons exhibit enhanced localization of NO signal in response to 120 mM NaCl treatment, coinciding with a greater retention of the principal oil body membrane proteins, i.e. oleosins. Abolition of DAF positive fluorescence by the application of specific NO scavenger [2-phenyl-4,4,5,5-tetramethyllimidazoline-1-oxyl-3-oxide (PTIO)] authenticates the presence of endogenous NO. These novel findings provide evidence for a possible protective role of NO during proteolytic degradation of oleosins prior to/accompanying lipolysis.

A rapid and non‐destructive screenable marker, FAST, for identifying transformed seeds of Arabidopsis thaliana

The creation of transgenic plants has contributed extensively to the advancement of plant science. Establishing homozygous transgenic lines is time-consuming and laborious, and using antibiotics or herbicides to select transformed plants may adversely affect the growth of some transgenic plants. Here we describe a novel technology, which we have named FAST (fluorescence-accumulating seed technology), that overcomes these difficulties. Although this technology was designed for use in Arabidopsis thaliana, it may be adapted for use in other plants. The technology is based on the expression of a fluorescent co-dominant screenable marker FAST, under the control of a seed-specific promoter, on the oil body membrane. The FAST marker harbors a fusion gene encoding either GFP or RFP with an oil body membrane protein that is prominent in seeds. The marker protein was only expressed in a specific organ (i.e. in dry seeds) and at a specific time (i.e. during dormancy), which are desirable features of selectable and/or screenable markers. This technique provides an immediate and non-destructive method for identifying transformed dry seeds. It identified the heterozygous transformed seeds among the T(1) population and the homozygous seeds among the T(2) population with a false-discovery rate of <1%. The FAST marker reduces the length of time required to produce homozygous transgenic lines from 7.5 to 4 months. Furthermore, it does not require sterilization, clean-bench protocols or the handling of large numbers of plants. This technology should greatly facilitate the generation of transgenic Arabidopsis plants.

Temporal and spatial analysis of lipid accumulation, oleosin expression and fatty acid partitioning during seed development in sunflower (Helianthus annuus L.)

Biochemical and fluorescence microscopic imaging approach has been adopted to investigate the accumulation of oil bodies at specific stages of seed development in Helianthus annuus L. cv. Morden. Seed filling in sunflower is marked with a rapid accumulation of proteins and lipids upto 30 DAA, after which protein accumulation declines whereas lipids continue to accumulate. Earliest signs of lipid accumulation are evident as early as during globular stage of embryo development. Spatially, a developing seed exhibits enhanced lipid deposition in peripheral cells. Oil body biogenesis is observed as early as 10 DAA, as is evident from the fluorescence microscopic detection of Nile red-positive entities in the protoplasts. To begin with, expression of one of the oleosin (the principal oil body membrane proteins) isoforms (16 kDa), is slower than the other two (17.5 and 20 kDa). Fatty acid composition of oil body lipids is quite similar to that of total seed lipids. An enhanced accumulation of linoleic acid is evident during later stages of seed filling. The proportion of major saturated fatty acids, palmitic (16:0) and stearic (18:0), however, do not alter much during the later phases of seed development. Present findings provide new information on oil body development, lipid accumulation and fatty acid composition, for a better understanding of the phasing of physiological and biochemical events associated with oilseed development.

The Maize Floury1 Gene Encodes a Novel Endoplasmic Reticulum Protein Involved in Zein Protein Body Formation

The maize (Zea mays) floury1 (fl1) mutant was first reported almost 100 years ago, but its molecular identity has remained unknown. We report the cloning of Fl1, which encodes a novel zein protein body membrane protein with three predicted transmembrane domains and a C-terminal plant-specific domain of unknown function (DUF593). In wild-type endosperm, the FL1 protein accumulates at a high level during the period of zein synthesis and protein body development and declines to a low level at kernel maturity. Immunogold labeling showed that FL1 resides in the endoplasmic reticulum surrounding the protein body. Zein protein bodies in fl1 mutants are of normal size, shape, and abundance. However, mutant protein bodies ectopically accumulate 22-kD alpha-zeins in the gamma-zein-rich periphery and center of the core, rather than their normal discrete location in a ring at outer edge of the core. The 19-kD alpha-zein is uniformly distributed throughout the core in wild-type protein bodies, and this distribution is unaffected in fl1 mutants. Pairwise yeast two-hybrid experiments showed that FL1 DUF593 interacts with the 22-kD alpha-zein. Results of these studies suggest that FL1 participates in protein body formation by facilitating the localization of 22-kD alpha-zein and that this is essential for the formation of vitreous endosperm.

α 1G T‐type Ca 2+ channel controls plasma membrane P‐selectin expression in pulmonary microvascular endothelial cells

The objective of this study was to determine the in vivo relevance of our previous in vitro observation, that CaV3.1 (α1G) T-type Ca2+ channel controls the thrombin-stimulated exocytosis of Weibel-Palade bodies in pulmonary microvascular endothelial cells. We utilized confocal fluorescence microscopy and isolated perfused lung to characterize the surface expression of P-selectin, a Weibel-Palade body membrane protein, at the apical plasma membrane of lung endothelial cells following thrombin stimulation in α1G knockout mice and their wild-type littermate mice. We found that in wild-type mice, thrombin stimulation induced membrane P-selectin expression in lung endothelial cells in both septal capillaries and extra-alveolar vessels. Pharmacological T-type Ca2+ channel blockade using mibefradil prevented thrombin stimulated-membrane P-selectin expression in septal but not extra-alveolar endothelia. In α1G knockout mice, thrombin stimulated-membrane P-selectin expression was attenuated in septal capillary endothelial cells, but not extra-alveolar vessel endothelial cells, compared to those of wild-type mice. Taken together, these results suggest that α1G T-type Ca2+ channel importantly contributes to agonist-induced lung microvascular endothelial cell procoagulant phenotype. Supported by HL-66299.

CEACAM6: A HORMONALLY REGULATED PROTEIN IN DIFFERENTIATING HUMAN FETAL LUNG TYPE II CELLS.

Ceacam6 (carcinoembryonic antigen cell adhesion molecule) is a GPI-anchored membrane glycoprotein recently identified in human fetal lung undergoing in vitro hormone-induced differentiation of type II cells. The protein is a tumor biomarker in adult lung and selected other tissues, where it has antimicrobial activity; however, expression and function in lung have not been characterized. Undifferentiated epithelial cells were isolated by collagenase/trypsin digestion from human fetal lung (16-21 wk gestation) and cultured 1-5 d in the absence (control) or presence of DCI (dexamethasone, 10 nM; 8-Br-cAMP, 0.1 mM; isobutylmethylxanthine, 0.1 mM). Some cells in control media were treated with adenovirus Ad12A2 (0.5-6 pfu/cell) to overexpress thyroid transcription factor-1 (TTF-1) or were transfected by electroporation (AMAXA nucleofection technology) with siRNAs (200 nM) for TTF-1 or Ceacam6 to knockdown expression in the presence of DCI. CEACAM6 mRNA and protein were up-regulated ≈10-fold by exposure of fetal lung epithelial cells to either DCI or to a dose of Ad12A2 that produced a level of recombinant TTF-1 comparable with endogenous TTF-1 in DCI-treated cells. By confocal immunofluorescence, CEACAM6 localized to both intracellular vesicles, costaining with SP-B (ie, lamellar bodies), and to the plasma membrane of type II cells. Knockdown of TTF-1 (46 ± 4% of control) in DCI-treated cells reduced CEACAM6 mRNA and protein by 80% (n = 4). Knockdown of CEACAM6 expression by siRNA (88 ± 3%, n = 3) did not alter expression or staining patterns of TTF-1, SP-B or DC-LAMP (a lamellar body membrane protein). Secretion rates for CEACAM6 were similar for both DCI- and Ad12A2-treated cells, with no response to treatment with secretagogues that stimulate surfactant secretion. Treatment of cells with mannosamine, an inhibitor of GPI anchor synthesis, blocked ≈50% of CEACAM6 secretion, consistent with GPI-anchor involvement in the secretory mechanism. We conclude that CEACAM6 expression in primary fetal lung epithelial cells is regulated by glucocorticoid and cAMP and is dependent on TTF-1. CEACAM6 is associated with intracellular surfactant but is secreted primarily by a constitutive pathway independent of lamellar bodies. CEACAM6 may contribute to the innate defense mechanisms of the mature type II cell.

Preferential phospholipase A 2 activity on the oil bodies in cotyledons during seed germination in Helianthus annuus L. cv. Morden

Oil body mobilization during seed germination in oilseed plants is expected to involve controlled degradation of oil body membrane proteins prior to or accompanying the mobilization of triacylglycerols (TAGs). Present findings demonstrate that phosphatidylcholine (PC), a constituent of the oil body membranes, starts getting depleted with a concomitant accumulation of its degradation product—lysophosphatidylcholine (LPC), during seedling development in dark-grown sunflower ( Helianthus annuus L. cv. Morden) seedling cotyledons. An increase in the LPC content was observed between 2 and 5 d of seedling development. A simultaneous enhancement of phospholipase A 2 (PLA 2) activity was also observed in the total soluble protein fraction, indicating its possible role in the degradation of PC, leading to the formation of LPC. Evidence obtained both from in vitro and in vivo experiments have indicated enhanced PLA 2 activity in association with oil bodies in seedling cotyledons. Enzyme activity was further localized by in vivo assay using a PLA 2-specific, fluorogenic substrate: PED6. Intracellular PED6-binding sites fluoresced bright green upon excitation at 505 nm (em 507 nm). Oil body surface begins to exhibit preferential PLA2 activity in protoplasts from 2 d old seedling cotyledons onwards. The enzyme activity enhanced further on the surface of oil bodies in 4–5 d old seedling cotyledons, coinciding with the phase of maximal lipolysis. These observations suggest the involvement of PLA 2 in causing changes in the oil body membrane phospholipids, thereby making them susceptible to subsequent lipolytic action during seed germination.

Langmuir and Langmuir–Blodgett films of proline-rich N-terminal domain peptide of γ-zein

The proline-rich N-Terminal domain peptides of γ-zein (VHLPPP)n with n=1 and 3 (peptides I and II) are shown to form stable Langmuir films at air/water interface and the films have been characterized using surface pressure–molecular area (π–A), surface potential–molecular area (ΔV–A) isotherms, respectively. The longer peptide sequence does not show dramatic increase in surface or interfacial properties suggesting that the minimum length of n=1 is sufficient to achieve the necessary surface properties. Brewster angle micrographs also agreed with these results. The high surface-active nature of the peptide suggests a fairly non-polar character at air/water interface and at solid/air interface when coated expresses a high surface energy.Additives such as isopropyl alcohol (IPA) and polyvinyl alcohol (PVA) with the peptides showed more homogenous films at the air/water interface and also improved mechanical and tensile properties. The organized assembly of peptide I at the air/water and solid/air interface suggests that even thin layer of the peptide could play an important role in coating the inner surface of protein body membrane in storage proteins. Composite films of such short peptides with biocompatible polymers may find applications as surface coatings and in biomaterials.

Exploring the interaction of the surfactant N-terminal domain of gamma-Zein with soybean phosphatidylcholine liposomes.

Zeins are maize storage proteins that accumulate inside large vesicles called protein bodies. gamma-Zein lines the inner surface of the protein body membrane, and its N-terminal, proline-rich, repetitive domain with the sequence (VHLPPP)(8) appears to be necessary for the accumulation of the protein within the organelle. Synthetic (VHLPPP)(8) adopts an amphipathic polyproline II conformation and forms cylindrical micelles in aqueous solution. Here we explore the interaction of (VHLPPP)(8) with soybean phosphatidylcholine unilamellar lipid vesicles and examine its effect on the stability and permeability of the liposome membrane. The amphipathic N-terminal domain of gamma-zein interacts with the membrane and assembles to form extended domains over the phospholipid membrane. The interaction between the peptide and the membrane increases the stability and permeability of the liposome membrane. The spontaneous amphipathic aggregation of (VHLPPP)(8) on the membrane suggests a mechanism of gamma-zein deposition inside maize protein bodies.