Exocrine glands of the ant Myrmoteras iriodum

This paper describes the morphological characteristics of nine major exocrine glands in workers of the formicine ant Myrmoteras iriodum. The elongate mandibles reveal along their entire length a conspicuous intramandibular gland, which contains both class‐1 and class‐3 secretory cells. The secretory cells of the mandibular glands show a peculiar appearance, with a branched end apparatus, which is unusual for ants. The other major glands (pro‐ and postpharyngeal gland, infrabuccal cavity gland, labial gland, metapleural gland, venom gland and Dufour gland) show common features for formicine ants. The precise function of the glands could not yet be experimentally demonstrated, and to clarify this will depend on the availability of live material of these enigmatic ants in future.     

The expression analysis of silk gland-enriched intermediate-size non-coding RNAs in silkworm Bombyx mori

Small non-protein coding RNAs （ncRNAs） play important roles in development, stress response and other cellular processes. Silkworm is an important model for studies on insect genetics and control of Lepidopterous pests. We have previously identified 189 novel intermediate-size ncRNAs in silkworm Bombyx mori, including 40 ncRNAs that showed altered expression in different developmental stages. Here we characterized the functions of these 40 ncRNAs by measuring their expressions in six tissues of the fifth instar larvae using Northern blot and real-time polymerase chain reaction assays. We identified nine ncRNAs （four small nucleolar RNAs and five unclassified ncRNAs） that were enriched in silk gland, including four ncRNAs that showed silk gland-specific expression. We further showed that three of nine silk gland-enriched ncRNAs were predominantly expressed in the anterior silk gland, whereas another three ncRNAs were highly accumulated in the posterior silk gland, suggesting that they may play different roles in fibroin synthesis. Furthermore, an unclassified ncRNA, Bm- 152, exhibited converse expression pattem with its antisense host gene gartenzwerg in diverse tissues, and might regulate the expression of gartenzwerg through RNA-protein complex. In addition, two silk gland-enriched ncRNAs Bm-102 and Bm-159 can be found in histone modification complex, which indicated that they might play roles through epigenetic modifications. Taken together, we provided the first expression and preliminary functional analysis of silk gland-enriched ncRNAs, which will help understand the molecular mechanism of silk gland-development and fibroin synthesis.     

Ablation of ceramide synthase 2 exacerbates dextran sodium sulphate‐induced colitis in mice due to increased intestinal permeability

Ceramides mediate crucial cellular processes including cell death and inflammation and have recently been implicated in inflammatory bowel disease. Ceramides consist of a sphingoid long‐chain base to which fatty acids of various length can be attached. We now investigate the effect of alerting the ceramide acyl chain length on a mouse model of colitis. Ceramide synthase (CerS) 2 null mice, which lack very‐long acyl chain ceramides with concomitant increase of long chain bases and C16‐ceramides, were more susceptible to dextran sodium sulphate‐induced colitis, and their survival rate was markedly decreased compared with that of wild‐type littermates. Using mixed bone‐marrow chimeric mice, we showed that the host environment is primarily responsible for intestinal barrier dysfunction and increased intestinal permeability. In the colon of CerS2 null mice, the expression of junctional adhesion molecule‐A was markedly decreased and the phosphorylation of myosin light chain 2 was increased. In vitro experiments using Caco‐2 cells also confirmed an important role of CerS2 in maintaining epithelial barrier function; CerS2‐knockdown via CRISPR‐Cas9 technology impaired barrier function. In vivo myriocin administration, which normalized long‐chain bases and C16‐ceramides of the colon of CerS2 null mice, increased intestinal permeability as measured by serum FITC‐dextran levels, indicating that altered SLs including deficiency of very‐long‐chain ceramides are critical for epithelial barrier function. In conclusion, deficiency of CerS2 influences intestinal barrier function and the severity of experimental colitis and may represent a potential mechanism for inflammatory bowel disease pathogenesis.     

Crystal structures of ligand‐bound octaprenyl pyrophosphate synthase from Escherichia coli reveal the catalytic and chain‐length determining mechanisms

Octaprenyl pyrophosphate synthase (OPPs) catalyzes consecutive condensation reactions of one allylic substrate farnesyl pyrophosphate (FPP) and five homoallylic substrate isopentenyl pyrophosphate (IPP) molecules to form a C₄₀ long‐chain product OPP, which serves as a side chain of ubiquinone and menaquinone. OPPs belongs to the trans‐prenyltransferase class of proteins. The structures of OPPs from Escherichia coli were solved in the apo‐form as well as in complexes with IPP and a FPP thio‐analog, FsPP, at resolutions of 2.2–2.6 Å, and revealed the detailed interactions between the ligands and enzyme. At the bottom of the active‐site tunnel, M123 and M135 act in concert to form a wall which determines the final chain length. These results represent the first ligand‐bound crystal structures of a long‐chain trans‐prenyltransferase and provide new information on the mechanisms of catalysis and product chain elongation. Proteins 2015; 83:37–45. © 2014 Wiley Periodicals, Inc.     

Long‐Chain O‐Ascarosyl‐alkanediols Are Constitutive Components of Caenorhabditis elegans but Do Not Induce Dauer Larva Formation

Two long‐chain ascarosides, O‐ascarosylnonacosane‐2,28‐diol ( 1 ) and O‐ascarosyluntriacontane‐2,30‐diol ( 2 ), were isolated from Caenorhabditis elegans and detected in all developmental stages of the worm. The long‐chain ascarosides were shown to be minor lipid components, and it was also shown that they do not induce dauer larva formation.     

Covalently attached fatty acyl chains alter the aggregation behavior of an amyloidogenic peptide derived from human β2‐microglobulin

Aggregation of a polypeptide chain into highly ordered amyloid aggregates is a complex process. Various factors, both extrinsic and intrinsic to the polypeptide chain, have been shown to perturb this process, leading to a drastic change in the amyloidogenic behavior, which is reflected in the polymorphism of amyloid aggregates at various levels of self‐assembly. In this paper, we have investigated the ability of covalently linked long‐chain fatty acids in modulating the self‐assembly of an aromatic amino acid‐rich highly amyloidogenic sequence derived from the amino acid region 59–71 of human β₂‐microglobulin by thioflavin T (ThT) fluorescence microscopy, circular dichroism, and fluorescence spectroscopy. Our results indicate that under identical conditions of dissolution and concentration, each peptide enhances the fluorescence of ThT. However, the aggregates are morphologically distinct. For the same peptide, the aggregate morphologies are dependent on peptide concentration. Further, an optimum concentration, which varies with solution ionic strength, is required for the formation of fibrillar aggregates. We show that covalent modification of this amyloidogenic sequence, with long‐chain fatty acids, affects the way the higher order amyloid structures assemble from the cross‐β units, in fatty acyl chain‐dependent and position‐dependent manner. Our data indicate that noncovalent interactions leading to amyloid fibril formation can be modulated by the hydrophobicity of covalently attached long‐chain fatty acids resulting in self‐assembly of the peptide chain to form nonfibrillar aggregates. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

New ultrastructural data from the larva of Paragordius varius (Nematomorpha)

Larvae of Paragordius varius (Leidy, 1851) were investigated using transmission electron microscopy, providing new observations concerning the cuticular structures of the preseptum, the postseptal gland and the pseudointestine. The cuticle is three‐layered but the basal layer is variable in structure. The hooks possess long, intracuticular roots and the stylets have apical teeth. Both postseptal gland and pseudointestine are secretory in function. The postseptal gland is composed of eight cells, which are all connected by a duct leading through the septum into the preseptum. Inside the postseptal gland, the duct wall has numerous pores. The pseudointestine is composed of four cells surrounding a cavity with an amorphous content. A flattened duct within a syncytium with five nuclei leads to a ventral pore close to the posterior end.     

Diethyl‐Ether Flower Washings of Dianthus cruentus Griseb. (Caryophyllaceae): Derivatization Reactions Leading to the Identification of New Wax Constituents

Some carnation species (Dianthus spp., Caryophyllaceae) exhibit a strong resistance to drought stress that is connected with the increased surface wax formation. Wax composition is unknown for the majority of Dianthus spp. Herein, mass spectral and gas chromatographic data, in combination with synthesis and chemical transformations (transesterification and synthesis of dimethyl disulfide adducts), enabled the identification of 151 constituents of diethyl‐ether washings of fresh flowers of Dianthus cruentus Griseb . from Serbia. The flower wax contained, along with the dominant ubiquitous long‐chain n‐alkanes, homologous series of n‐ and branched (iso‐ and anteiso‐) long‐chain hexyl alkanoates/alkenoates and alkyl/alkenyl benzoates. The branching position in the mentioned hexyl esters was probed by synthesis of esters of three isomeric hexanols that were spectrally characterized (¹H‐ and ¹³C‐NMR, IR, MS). The washings also contained long‐chain (Z)‐ and (E)‐alkenes (C₂₃?C₃₅) with several different double bond regiochemistries. Fifty‐five of these constituents (eight hexyl esters, two benzoates, and forty‐five alkenes) were detected for the first time in Plantae, while ten of these represent completely new compounds. The rare occurrence of these wax constituents makes them possible chemotaxonomic markers of this particular Dianthus sp.     

The biosynthesis of Dufour's gland constituents in queens of the honeybee (Apis mellifera)

Chemical analysis of queen Dufour's gland by GC/MS revealed the presence of long chain esters and hydrocarbons. To evaluate whether the biosynthesis of Dufour's gland components is modulated by physiological factors, we studied thede novo biosynthesis of esters and hydrocarbons in virgin and mated queen glandsin vivo andin vitro. Using [1-¹⁴C] sodium acetate as a precursor, it was shown thatin vivo, esters and hydrocarbons comprised the majorde novo products. The relative proportion of the hydrocarbon fraction was significantly higher in mated queens than in virgins, while in esters the situation was reversed. Comparision between reproductive and non reproductive virgin queens revealed that mating and not egg laying induced changes in glandular activity. Glands incubatedin vitro, on the other hand, synthesized mainly esters and alcohols but not hydrocarbons. These results suggest that either hydrocarbons are produced elsewhere and subsequently sequestered by Dufour's gland, or that the gland biosynthetic expressionin vivo is modulated. Differences between the gland activityin vivo andin vitro further support the hypothesis of a physiological mechanism in regulation of glandular performance.     

Crystal structure and enhanced activity of a cutinase‐like enzyme from Cryptococcus sp. strain S‐2

The structural and enzymatic characteristics of a cutinase‐like enzyme (CLE) from Cryptococcus sp. strain S‐2, which exhibits remote homology to a lipolytic enzyme and a cutinase from the fungus Fusarium solani (FS cutinase), were compared to investigate the unique substrate specificity of CLE. The crystal structure of CLE was solved to a 1.05 Å resolution. Moreover, hydrolysis assays demonstrated the broad specificity of CLE for short and long‐chain substrates, as well as the preferred specificity of FS cutinase for short‐chain substrates. In addition, site‐directed mutagenesis was performed to increase the hydrolysis activity on long‐chain substrates, indicating that the hydrophobic aromatic residues are important for the specificity to the long‐chain substrate. These results indicate that hydrophobic residues, especially the aromatic ones exposed to solvent, are important for retaining lipase activity. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Metabolic engineering of oilseed crops to produce high levels of novel acetyl glyceride oils with reduced viscosity,freezing point and calorific value

Seed oils have proved recalcitrant to modification for the production of industrially useful lipids. Here, we demonstrate the successful metabolic engineering and subsequent field production of an oilseed crop with the highest accumulation of unusual oil achieved so far in transgenic plants. Previously, expression of the Euonymus alatus diacylglycerol acetyltransferase (EaDAcT) gene in wild‐type Arabidopsis seeds resulted in the accumulation of 45 mol% of unusual 3‐acetyl‐1,2‐diacyl‐sn‐glycerols (acetyl‐TAGs) in the seed oil (Durrett et al., 2010 PNAS 107:9464). Expression of EaDAcT in dgat1 mutants compromised in their ability to synthesize regular triacylglycerols increased acetyl‐TAGs to 65 mol%. Camelina and soybean transformed with the EaDAcT gene accumulate acetyl‐triacylglycerols (acetyl‐TAGs) at up to 70 mol% of seed oil. A similar strategy of coexpression of EaDAcT together with RNAi suppression of DGAT1 increased acetyl‐TAG levels to up to 85 mol% in field‐grown transgenic Camelina. Additionally, total moles of triacylglycerol (TAG) per seed increased 20%. Analysis of the acetyl‐TAG fraction revealed a twofold reduction in very long chain fatty acids (VLCFA), consistent with their displacement from the sn‐3 position by acetate. Seed germination remained high, and seedlings were able to metabolize the stored acetyl‐TAGs as rapidly as regular triacylglycerols. Viscosity, freezing point and caloric content of the Camelina acetyl‐TAG oils were reduced, enabling use of this oil in several nonfood and food applications.     

Stylar polymorphism on the edge: unusual flower traits in Moroccan Narcissus broussonetii (Amaryllidaceae)

Heterostyly and related polymorphisms (e.g. stigma‐height dimorphism) have been used as model systems for studying the origin and maintenance of plant population variability. Stigma‐height dimorphism frequently occurs in Narcissus and is associated with a particular flower shape. In the present study, we describe a new, peculiar case of stigma‐height dimorphism in Narcissus broussonetii, a species on the margin of the geographical distribution of the genus. We determined the stylar condition of N. broussonetii and its variation across populations, analyzed perianth morphology and its relationship with stylar variation, and compared this species with other stylar dimorphic species of the genus. We also studied the incompatibility system and pollination ecology of the species. Narcissus broussonetii is a style‐dimorphic species, as suggested in early studies that were subsequently neglected, and displays unusual flower morphology, with a long floral tube and a virtual absence of a corona. The species shows a late‐ (ovarian) acting incompatibility system and crosses within and between morphs are fertile. We observed short‐tongued diurnal and long‐tongued nocturnal pollinators. Our findings confirm that the presence of the observed dimorphism across populations is most probably the result of the joint action of a nonheteromorphic incompatibility system, extremely long and narrow floral tubes, and a combined role of short‐ and long‐tongued pollinators. © 2015 The Linnean Society of London, Botanical Journal of the Linnean Society, 2015, 177 , 644–656.     

Microanatomy of the male and female reproductive tracts in the long‐tailed butterfly ray Gymnura poecilura,an elasmobranch with unusual characteristics

The anatomy of the male and female reproductive systems was investigated in the long‐tailed butterfly ray Gymnura poecilura using gross observation and light microscopy. The testes are highly asymmetrical, to the extent that only the left testis is functional and the right testis is completely absent. Both of the male genital ducts are present and symmetrical, although spermatozoa only occur in the left duct. The genital ducts are straight and unconvoluted, with regular incomplete internal partitions throughout. Females do not possess a right ovary, nor do the oviducal glands exhibit distinct club and papillary zones, and the baffle zone lacks baffle plates. In all sections of the gland, the tubules display different secretory activities depending on the proximity to the gland lumen. The gland produces a thin egg membrane that encases each egg individually, while the endometrium is formed into trophonemata.     

Medium chain acyl-thioester hydrolase activity in goat and rabbit mammary gland fatty acid synthetase complexes

The goat mammary gland fatty acid synthetase hydrolysed both medium (C_8:0, C_10:0) and long (C_16:0, C_18:0) chain length acyl CoA esters, whereas the enzyme from rabbit mammary gland only hydrolysed long chain length acyl CoA esters. The medium chain acyl-thioester hydrolase activity of goat mammary gland fatty acid synthetase was much less sensitive to inhibition by phenylmethanesulfonyl-fluorid than the long chain acylthioester hydrolase activity. These results indicate the presence of either two acyl-thioester hydrolases with different specificity or one acyl-thioester hydrolase containing two different active sites.     

The Arabidopsis cer26 mutant,like the cer2 mutant,is specifically affected in the very long chain fatty acid elongation process

Plant aerial organs are covered by cuticular waxes, which form a hydrophobic crystal layer that mainly serves as a waterproof barrier. Cuticular wax is a complex mixture of very long chain lipids deriving from fatty acids, predominantly of chain lengths from 26 to 34 carbons, which result from acyl‐CoA elongase activity. The biochemical mechanism of elongation is well characterized; however, little is known about the specific proteins involved in the elongation of compounds with more than 26 carbons available as precursors of wax synthesis. In this context, we characterized the three Arabidopsis genes of the CER2‐like family: CER2, CER26 and CER26‐like . Expression pattern analysis showed that the three genes are differentially expressed in an organ‐ and tissue‐specific manner. Using individual T–DNA insertion mutants, together with a cer2 cer26 double mutant, we characterized the specific impact of the inactivation of the different genes on cuticular waxes. In particular, whereas the cer2 mutation impaired the production of wax components longer than 28 carbons, the cer26 mutant was found to be affected in the production of wax components longer than 30 carbons. The analysis of the acyl‐CoA pool in the respective transgenic lines confirmed that inactivation of both genes specifically affects the fatty acid elongation process beyond 26 carbons. Furthermore, ectopic expression of CER26 in transgenic plants demonstrates that CER26 facilitates the elongation of the very long chain fatty acids of 30 carbons or more, with high tissular and substrate specificity.     

CYP116B5: a new class VII catalytically self‐sufficient cytochrome P450 from Acinetobacter radioresistens that enables growth on alkanes

A gene coding for a class VII cytochrome P450 monooxygenase (CYP116B5) was identified from Acinetobacter radioresistens S13 growing on media with medium (C14, C16) and long (C24, C36) chain alkanes as the sole energy source. Phylogenetic analysis of its N‐ and C‐terminal domains suggests an evolutionary model involving a plasmid‐mediated horizontal gene transfer from the donor Rhodococcus jostii RHA1 to the receiving A. radioresistens S13. This event was followed by fusion and integration of the new gene in A. radioresistens chromosome. Heterologous expression of CYP116B5 in Escherichia coli BL21, together with the A. radioresistens Baeyer–Villiger monooxygenase, allowed the recombinant bacteria to grow on long‐ and medium‐chain alkanes, showing that CYP116B5 is involved in the first step of terminal oxidation of medium‐chain alkanes overlapping AlkB and in the first step of sub‐terminal oxidation of long‐chain alkanes. It was also demonstrated that CYP116B5 is a self‐sufficient cytochrome P450 consisting of a heme domain (aa 1–392) involved in the oxidation step of n‐alkanes degradation, and its reductase domain (aa 444–758) comprising the NADPH‐, FMN‐ and [2Fe2S]‐binding sites. To our knowledge, CYP116B5 is the first member of this class to have its natural substrate and function identified.