Humoral factor activating the Sarcophaga lectin gene in cultured fat body

On culture of the fat body of Sarcophaga peregrina (flesh fly) larvae in modified Grace's medium, the Sarcophaga lectin gene was activated only when the medium was supplemented with larval hemolymph. The expressions of the genes for the storage protein and the sarcocystatin A were not affected by supplementing the medium with the hemolymph. Thus the hemolymph contained a factor that specifically activated the Sarcophaga lectin gene. This factor seemed to be a heat-stable, low molecular weight compound that was probably not a peptide, and to activate genes in the fat body for defense proteins that are known to be expressed in response to injury of Sarcophaga larvae.     

Differential expression of sex-related fat body proteins during the larval–pupal developmental stages of the silkworm (Bombyx mori)

The silkworm fat body is the site of many intermediary metabolic processes, and a source of sustenance for growth throughout the life cycle. Fat body proteins are responsible for storing nutrients, providing energy, and regulating hormones, and they have been identified using proteomic approaches. However, detailed differential expression of sex-related fat body proteins has not previously been evaluated. In the present study, we characterized the differential expression of sex-related fat body proteins, by using 2-dimensional gel electrophoresis (2-DE) followed by mass spectrometry identification and bioinformatics methods. We extracted the fat body proteins from 5-day-old fifth instar larvae (L5), 10-day-old fifth instar larvae (corresponding to the end of spinning [LE]), and 0-day-old pupae (P0) of the multivoltine silkworm variety “Da Zao”. We confirmed the presence of 11 important sex-specific expression proteins and 14 stage-specific expression proteins. We accurately identified 13 of these specific expression proteins, including actin, calponin-like protein, 75 kDa subunit NADH, receptor for activated protein kinase C from Bombyx mori (BmRACK), IMP (inosine monophosphate) cyclohydrolase, tropomyosin 1, β-tubulin, hypothetical protein, antichymotrypsin precursor, and 30 K protein precursor. We showed that BmRACK was differentially expressed between male and female silkworms. We discuss the biological roles of the specific expression proteins during the larval–pupal developmental stages.     

Characterizing the normal proteome of human ciliary body

Background

The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body.

Results

In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis.

Conclusions

More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia.     

Mining the Giardia genome and proteome for conserved and unique basal body proteins

Giardia lamblia is a flagellated protozoan parasite and a major cause of diarrhoea in humans. Its microtubular cytoskeleton mediates trophozoite motility, attachment and cytokinesis, and is characterised by an attachment disk and eight flagella that are each nucleated in a basal body. To date, only 10 giardial basal body proteins have been identified, including universal signalling proteins that are important for regulating mitosis or differentiation. In this study, we have exploited bioinformatics and proteomic approaches to identify new Giardia basal body proteins and confocal microscopy to confirm their localisation in interphase trophozoites. This approach identified 75 homologs of conserved basal body proteins in the genome including 65 not previously known to be associated with Giardia basal bodies. Thirteen proteins were confirmed to co-localise with centrin to the Giardia basal bodies. We also demonstrate that most basal body proteins localise to additional cytoskeletal structures in interphase trophozoites. This might help to explain the roles of the four pairs of flagella and Giardia-specific organelles in motility and differentiation. A deeper understanding of the composition of the Giardia basal bodies will contribute insights into the complex signalling pathways that regulate its unique cytoskeleton and the biological divergence of these conserved organelles.     

Synthesis and release of proteins from cultured larval fat body of the southwestern corn borer,Diatraea grandiosella

The larval fat body of the southwestern corn borer, Diatraea grandiosella, was cultured in vitro to examine the relationship between proteins present in the fat body, those released into the medium, and those present in the haemolymph. While the incorporation of [³H]leucine into fat body proteins was high in last instar pre-diapausing and non-diapausing larvae, it fell in early diapausing larvae to about 11% of that found in prediapausing larvae. Incorporation of [³H]leucine into the diapause-associated protein of the fat body increased gradually in pre-diapausing larvae and reached a maximum in newly-diapaused larvae at a time when the incorporation of [³H]leucine into other proteins of the fat body had declined. The proteins released from the cultured fat body showed identical electrophoretic properties and close immunochemical relationships to most of those present in the haemolymph. Small amounts of the diapause-associated protein were released in vitro from the fat body of larvae of different ages in diapause. Lipophorin was also released in vitro from the fat body of non-diapausing and diapausing larvae, and shown to be immunochemically identical to the lipophorin present in the haemolymph.     

Use of primary fat body cultures for the study of baculovirus replication

Fat body cultures of Trichoplusia ni and Estigmene acrea were established for use in the study of the two baculoviruses Autographa californica nuclear polyhedrosis virus (AcNPV) and Estigmene acrea granulosis virus (EaGV), respectively. Multiplication of AcNPV observed by phase and electron microscopy was correlated with an increase in viral specific proteins as determined by indirect enzyme-linked immunosorbent assay (ELISA). Although EaGV morphogenesis was not observed in fat body cultures, an increase in specific proteins of this virus could be detected with the ELISA.     

Protein synthesis by the milk gland and fat body of the tsetse fly,Glossina pallidipes

The patterns of protein synthesis by the milk gland and the fat body of female Glossinapallidipes during the pregnancy cycle were studied by incubation with [³⁵S]methionine both in vivo and in vitro. The pattern of protein synthesis by the milk gland changed with the stage of the larva in the uterus. Very little synthesis occurred in the milk gland until the first instar larva hatched. Then four proteins (13, 16, 24 and 72 kDa) were prominently synthesized. As the larva matured, the synthesis of 19, 38, 40 and 72 kDa proteins increased, whereas that of the 13 and 24 kDa proteins decreased. Just before larviposition, only the 16 and 72 kDa proteins were still being synthesized. The milk gland secreted into the medium primarily the 13, 16, 19 and 72 kDa proteins, all of which were found in the larval gut after a 5 hr pulse of labeled methionine in vivo. During most of the pregnancy cycle protein synthesis in the fat body was low compared to that of the milk gland and only small amounts of several low molecular weight proteins (less than or equal to 16 kDa) were released into the medium. But when a large third instar larva was present in the uterus, the fat body synthesized and secreted a 72 kDa and a 15–17 kDa complex of proteins.     

SLC7 amino acid transporters of the yellow fever mosquito Aedes aegypti and their role in fat body TOR signaling and reproduction

BackgroundAn important function of the fat body in adult female mosquitoes is the conversion of blood meal derived amino acids (AA) into massive amounts of yolk protein precursors. A highly efficient transport mechanism for AAs across the plasma membrane of the fat body trophocytes is essential in order to deliver building blocks for the rapid synthesis of large amounts of these proteins. This mechanism consists in part of AA transporter proteins from the solute carrier family. These transporters have dual function; they function as transporters and participate in the nutrient signal transduction pathway that is activated in the fat body after a blood meal. In this study we focused on the solute carrier 7 family (SLC7), a family of AA transporters present in all metazoans that includes members with strong substrate specificity for cationic AAs.Methodology/principal findingsWe identified 11 putative SLC7 transporters in the genome sequence of Aedes aegypti. Phylogenetic analysis puts five of these in the cationic AA transporter subfamily (CAT) and six in the heterodimeric AA transporter (HAT) subfamily. All 11 A. aegypti SLC7 genes are expressed in adult females. Expression profiles are dynamic after a blood meal. We knocked down six fat body-expressed SLC7 transporters using RNAi and found that these ‘knockdowns’ reduced AA-induced TOR signaling. We also determined the effect these knockdowns had on the number of eggs deposited following a blood meal.Conclusions/significanceOur analysis stresses the importance of SLC7 transporters in TOR signaling pathway and mosquito reproduction.     

Protein release from the larval fat body of the southwestern corn borer, Diatraea grandiosella: An in vitro study

The release of protein from the perivisceral fat body of non-diapausing, pre-diapausing and diapausing larvae of the southwestern corn borer, Diatraea grandiosella, was examined in vitro. Time course studies showed a selective release of proteins into macromolecule-free Grace's medium. The rate of release of individual proteins differed. The release of some proteins was partially inhibited by the incorporation of potassium cyanide (10^?2 M) and ouabain (5 × 10^?3 M) into the medium. During a 5 min incubation a single major high molecular weight protein fraction was released at a high rate from the fat body of both non-diapausing and diapausing larvae. A low molecular weight protein (the diapause-associated protein) was also released readily from the fat body of diapausing larvae. Although most proteins released from the fat body in vitro appeared to be present in the haemolymph in vivo, one notable exception was the absence of the diapause-associated protein from the haemolymph. The method holds promise for facilitating further studies of protein release from insect fat body.     

Proteins of the fat body of non-diapausing and diapausing larvae of the southwestern corn borer, Diatraea grandiosella: Effect of juvenile hormone

The proteins of the fat body of non-diapausing, pre-diapausing, and newly-diapaused larvae of the southwestern corn borer, Diatraea grandiosella, were examined. Since a low titre of juvenile hormone (JH) is present in the haemolymph throughout the final instar of non-diapausing larvae, the hormone does not appear to stimulate the pre-metamorphic synthesis of proteins. In contrast, the high titre of JH in the haemolymph during the final instar of pre-diapausing larvae appears to stimulate the synthesis of selected proteins. For example, pre-diapausing larvae store in their fat body a low molecular weight protein which has been named the ‘diapause-associated protein’. When non-diapausing larvae were treated topically with C₁₇-JH or a JH mimic, from 50 to 70% entered a diapause-like state as fully grown larvae. These hormone-treated larvae accumulated the diapause-associated protein and a high molecular weight protein in their fat bodies. Both of these proteins were shown to be released from the fat body of newly-diapaused larvae in vitro, and may function in the haemolymph during diapause. The high molecular weight protein, isolated from the haemolymph, was shown to contain neutral and polar lipids, including biochromes. Its storage in the fat body and release into the haemolymph may be essential for the transport of lipids during diapause. The fat body proteins of newly-diapaused larvae of the southern cornstalk borer, Diatraea crambidiodes, were also examined electrophoretically. They were found to contain a similar protein pattern to that of D. grandiosella, including the presence of a diapause-associated protein.     

A larval serum protein of the silkworm,Bombyx mori: cDNA sequence and developmental specificity of the transcript

The Bombyx mori larval serum protein (BmLSP) is a major component of larval hemolymph proteins until early in the last instar. The cDNA for BmLSP was cloned from a library constructed from fat body RNA of penultimate instar larvae, and the complete nucleotide sequence of the 909 base pair cDNA insert was determined. The deduced 262 amino acid polypeptide included a 16 amino acid residue signal peptide and a 15 amino acid sequence prosegment. A homology search showed that BmLSP has significant similarity with microvitellogenin of Manduca sexta and the 30K proteins of B. mori. Tissue distribution and developmental profile of BmLSP mRNA were analyzed by northern hybridization. BmLSP mRNA was abundant in fat body but not detected in midgut and silk gland. BmLSP mRNA was present during the feeding periods of the fourth and fifth instar larvae, but absent during the larval molt and after the onset of cocoon spinning.     

Metabolic shift from lipogenesis to glycogenesis in the last instar larval fat body of the silkworm,Bombyx mori

When [¹⁴C]glucose was injected into the last instar larvae of the silkworm, Bombyx mori, the label was incorporated into various tissues at varying degrees depending on the developmental stages. Fat body exhibited high incorporation rates throughout the feeding periods. Silk glands became active in incorporation but midgut decreased toward larval maturation. The pulse labeling experiment clearly demonstrated that the metabolic shift from lipogenesis to glycogenesis occurred in fat body at the middle of the last instar; a predominant incorporation was found in lipids when [¹⁴C]glucose was injected at the early stage, while at the late stage glycogen synthesis became most active. Incorporation into fat body proteins was not a major factor throughout the instar. Extirpation of silk glands enhanced incorporation into glycogen and proteins at the late stage but did not affect lipid synthesis. Long-term chase showed that fat body lipids and proteins synthesized at the early stage were totally carried over into the pupal fat body, while much glycogen produced at the late stage was used during the larval-pupal transformation with the remainder carried over into the pupa.From these results the metabolic shift from lipogenesis to glycogenesis in fat body is discussed in relation to the storage function of the fat body for pupal metamorphosis.     

Heat shock protein 9 mRNA expression increases during fruiting body differentiation in Grifola frondosa and other edible mushrooms

We isolated a homolog of Schizosaccharomyces pombe HSP9 from Grifola frondosa and designated it Gf.HSP9. The Gf.HSP9 gene consisted of four exons and three introns and encoded 84 amino acid residues. We have also identified related HSP9 genes from Pleurotus eryngii (Pe.HSP9), Hypsizygus marmoreus (Hm.HSP9), and Lentinula edodes (Le.HSP9). The predicted tertiary structures of these HSP9s were the same. In addition, the expression pattern of their mRNA increased during fruiting body in a differentiation-dependent manner. These results suggested that HSP9 was associated with the fruiting body differentiation in these four edible mushrooms.     

The heterogeneous nuclear ribonucleoprotein (hnRNP) glorund functions in the Drosophila fat body to regulate lipid storage and transport

The availability of excess nutrients in Western diets has led to the overaccumulation of these nutrients as triglycerides, a condition known as obesity. The full complement of genes important for regulating triglyceride storage is not completely understood. Genome-wide RNAi screens in Drosophila cells have identified genes involved in mRNA splicing as important lipid storage regulators. Our lab has shown that a group of splicing factors called heterogeneous nuclear ribonucleoproteins (hnRNPs) regulate lipid metabolism in the fly fat body; however, the identities of all the hnRNPs that function to control triglyceride storage are not known. Here, we used the GAL4/UAS system to induce RNAi to the hnRNP glorund (glo) in the Drosophila fat body to assess whether this hnRNP has any metabolic functions. Decreasing glo levels resulted in less triglycerides being stored throughout the fly. Interestingly, decreasing fat body glo expression resulted in increased triglyceride storage in the fat body, but blunted triglyceride storage in non-fat body tissues, suggesting a defect in lipid transport. Consistent with this hypothesis, the expression of apolipophorin (apolpp), microsomal triglyceride transfer protein (mtp), and apolipoprotein lipid transfer particle (apoltp), apolipoprotein genes important for lipid transport through the fly hemolymph, was decreased in glo-RNAi flies, suggesting that glo regulates the transport of lipids from the fly fat body to surrounding tissues. Together, these results indicate that glorund plays a role in controlling lipid transport and storage and provide additional evidence of the link between gene expression and the regulation of lipid metabolism.     

Heterogeneity in thylakoid membrane proteome of Synechocystis 6803

Cell-free extracts of Synechocystis 6803 were fractionated by successive ultracentrifugation at 40,000 × g, 90,000 × g and 150,000 × g to obtain the three thylakoid fractions designated as 40 k, 90 k and 150 k fractions respectively. These fractions showed differences in absorption and emission spectra. Nano-LC-ESI-Q-TOF MS analysis identified 123 proteins belonging to membrane as well as cytosolic fraction. Out of these proteins, there were 22 proteins with transmembrane helices and 12 proteins with signal peptide. There were 77 proteins common across all the three fractions. Most of these proteins were subunits of photosynthetic complexes, CF₀–CF₁ ATP synthase or ribosomal proteins. Among the rest of the proteins, 8 were exclusive to 40 k fraction, 3 were exclusive to 90 k fraction and 13 were exclusive to 150 k fraction. There were 10 proteins common between 40 k and 90 k fractions and 12 proteins common between 90 k and 150 k fractions. There were no common proteins detected between 40 k and 150 fractions. The results suggested existence of heterogeneity in thylakoids of Synechocystis 6803, which may lead to micro-compartmentation and functional heterogeneity in the thylakoids of this organism as seen previously.     

Escape of the cercarial body of Gorgoderina vitelliloba from its anterior tail chamber

Mitchell J. B. and Mason A. R. 1978. Escape of the cercarial body of Gorgoderina vitelliloba from its anterior tail chamber. International Journal for Parasitology8: 193–198. Before the cercarial body of Gorgoderina vitelliloba can encyst in the body of its second intermediate host it must escape from the anterior tail chamber where it has been confined throughout the free living cercarial phase. Escape from the tail chamber is influenced by pH, enzymes, bile salts and by the physical nature of its environment. The cercarial body effects its escape after first becoming activated and then breaking its connection with the tail.