Adult Fat Body Research Articles

Presence of the storage seed protein vicilin in internal organs of larval Callosobruchus maculatus (Coleoptera: Bruchidae)

Variant vicilins (7S storage globulins) of cowpea seeds ( Vigna unguiculata) are considered as the main resistance factor present in some African genotypes against the bruchid Callosobruchus maculatus. It has been suggested that the toxic properties of vicilins may be related to their recognition and interaction with glycoproteins and other membrane constituents along the digestive tract of the insect. However, the possibility of a systemic effect has not yet been investigated. The objective of this work was to study the fate of 7S storage globulins of V. unguiculata in several organs of larvae of the cowpea weevil C. maculatus. Results demonstrated binding of vicilins to brush border membrane vesicles, suggesting the existence of specific receptors. Vicilins were detected in the haemolymph, in the midgut, and in internal organs, such as fat body and malpighian tubules. There is evidence of accumulation of vicilins in the fat body of both larvae and adults. The absorption of vicilins and their presence in insect tissues parallels classical sequestration of secondary compounds.

Lipophorin and its Receptor in Lepidoptera

ABSTRACTLipophorin (Lp) has an approximate native molecular weight of 730 kDa for Bombyx mori and consists of ApoLp‐I and ApoLp‐II with molecular weights of 250 kDa and 90 kDa for B. mori and 230 kDa and 80 kDa for Hyphantria cunea and 230 kDa and 49 kDa for Lymantria dispar, respectively. Lipid in Lp was mostly composed of neutral lipid. Lp of B. mori maintains constant level during larval and pupal stages but greatly increases during adult stage in both male and female. Lp of H. cunea appeared in great amounts in protein yolk bodies of ovary when vitellogenesis is actively taking place and was present in testicular fluid but not in the peritoneal sheath and cysts of testis. ApoLp‐III of B. mori has a molecular weight of 17 kDa and similar amino acid composition as those of other species Lp. H. cunea apoLp‐III has a molecular weight of 18 kDa and was present in all stages and in the protein body of ovary and in the cyst of testis. ApoLp‐III is synthesized in larval and adult fat body. cDNA sequence of Spodoptera litura apoLp‐III encodes a 188 amino acid polypeptide including a 22 amino acid leader peptide. Galleria mellonella Lp receptor has an approximate molecular weight of 97 kDa and 110 kDa under non‐reducing and reducing conditions, respectively and bound HDLp specifically. Lp receptor cDNA of G. mellonella showed th pattern of the VLDL receptor belonging to the LDL receptor family. The variant Lp receptors were expressed in the fat body of G. mellonella; one is a Lp receptor which lacks 84 bp of O linked sugar domain and the other is a full length form of the Lp receptor. The Lp receptor from the fat body of G. mellonella was differently expressed depending on the tissue and the developmental stages with specific abundance in prepupal stage.

Increased expression of unusual EP repeat-containing proteins in the midgut of the tsetse fly ( Glossina) after bacterial challenge

Proteins containing a glutamic acid–proline (EP) repeat epitope were immunologically detected in midguts from eight species of Glossina (tsetse flies). The molecular masses of the tsetse EP proteins differed among species groups. The amino acid sequence of one of these proteins, from Glossina palpalis palpalis, was determined and compared to the sequence of a homologue, the tsetse midgut EP protein of Glossina m. morsitans. The extended EP repeat domains comprised between 36% ( G. m. morsitans) and 46% ( G. p. palpalis) of the amino acid residues, but otherwise the two polypeptide chains shared most of their sequences and predicted functional domains. The levels of expression of tsetse EP protein in adult teneral midguts were markedly higher than in midguts from larvae. The EP protein was detected by immunoblotting in the fat body, proventriculus and midgut, the known major immune tissues of tsetse and is likely secreted as it was also detected in hemolymph. The EP protein was not produced by the bacterial symbionts of tsetse midguts as determined by genome analysis of Wigglesworthia glossinidia and immunoblot analysis of Sodalis glossinidius. Bacterial challenge of G. m. morsitans, by injection of live E. coli, induced augmented expression of the tsetse EP protein. The presence of EP proteins in a wide variety of tsetse, their constitutive expression in adult fat body and midguts and their upregulation after immunogen challenge suggest they play an important role as a component of the immune system in tsetse.

Response to Comment on "Long-Lived Drosophila with Overexpressed dFOXO in Adult Fat Body"

Reduced insulin/insulin-like growth factor signaling (IIS) has been shown to extend life span in Caenorhabditis elegans, Drosophila , and mice ([ 1 ][1]–[ 3 ][2]), and adipose tissue has been implicated in determination of life span extension by reduced IIS in these model organisms ([ 3 ][2]–[ 6

Comment on "Long-Lived Drosophila with Overexpressed dFOXO in Adult Fat Body"

Aging can be retarded when insulin-like signals are reduced in a limited set of tissues in several animal models ([ 1 ][1]). Current research aims to determine which tissues are necessary and sufficient for mediating effects on life span ([ 2 ][2]–[ 4 ][3]). Giannakou et al . ([ 5 ][4]) assert

Ornithine decarboxylase activity during the development of Anastrepha fraterculus (Diptera, Tephritidae)

Ornithine decarboxylase (ODC) (EC 4.1.1.17) is very important for polyamine biosynthesis, which is required for main biological events. In the present study, ODC activity was measured in samples of Anastrepha fraterculus's egg, larva, pupa body and abdomen, adult body, ovaries, and fat body of young females, and in ovaries of mature flies. The kinetic parameters (Km app and Vmax) for ODC activity were determined for pupa, larva, and young ovary. ODC activity showed fluctuations during A. fraterculus's life development. In its earlier stages, prior to emergence, the egg has high ODC-specific activity probably due to embryogenesis, which is characterized by a high rate of cell division. This enzyme activity is also significantly high in the ovary and fat body of young females possibly related to the increased oogenesis and vitellogenesis. The kinetic parameters (Km app and Vmax) had great variation. Our results using GTP showed that the great variation in kinetic parameters can be accounted for by post-translational modifications.

Long-lived Drosophila with overexpressed dFOXO in adult fat body.

Reduced activity of the insulin/insulin-like growth factor signaling (IIS) pathway increases life-span in diverse organisms. We investigated the timing of the effect of reduced IIS on life-span and the role of a potential target tissue, the fat body. We overexpressed dFOXO, a downstream effector of IIS, in the adult Drosophila fat body, which increased life-span and reduced fecundity of females but had no effect on male life-span. The role of FOXO transcription factors and the adipose tissue are therefore evolutionarily conserved in the regulation of aging, and reduction of IIS in the adult is sufficient to mediate its effects on life-span and fecundity.

Drosophila dFOXO controls lifespan and regulates insulin signalling in brain and fat body.

In Drosophila melanogaster, ageing is slowed when insulin-like signalling is reduced: life expectancy is extended by more than 50% when the insulin-like receptor (InR) or its receptor substrate (chico) are mutated, or when insulin-producing cells are ablated. But we have yet to resolve when insulin affects ageing, or whether insulin signals regulate ageing directly or indirectly through secondary hormones. Caenorhabditis elegans lifespan is also extended when insulin signalling is inhibited in certain tissues, or when repressed in adult worms, and this requires the forkhead transcription factor (FOXO) encoded by daf-16 (ref. 6). The D. melanogaster insulin-like receptor mediates phosphorylation of dFOXO, the equivalent of nematode daf-16 and mammalian FOXO3a. We demonstrate here that dFOXO regulates D. melanogaster ageing when activated in the adult pericerebral fat body. We further show that this limited activation of dFOXO reduces expression of the Drosophila insulin-like peptide dilp-2 synthesized in neurons, and represses endogenous insulin-dependent signalling in peripheral fat body. These findings suggest that autonomous and non-autonomous roles of insulin signalling combine to control ageing.

Lipid transfer particle mediates the delivery of diacylglycerol from lipophorin to fat body in larval Manduca sexta

This work analyzed the process of lipid storage in fat body of larval Manduca sexta, focusing on the role of lipid transfer particle (LTP). Incubation of fat bodies with [(3)H]diacylglycerol-labeled lipophorin resulted in a significant accumulation of diacylglycerol (DAG) and triacylglycerol (TAG) in the tissue. Transfer of DAG to fat body and its storage as TAG was significantly inhibited (60%) by preincubating the tissue with anti-LTP antibody. Lipid transfer was restored to control values by adding LTP to fat body. Incubation of fat body with dual-labeled DAG lipophorin or its treatment with ammonium chloride showed that neither a membrane-bound lipoprotein lipase nor lipophorin endocytosis is a relevant pathway to transfer or to storage lipids into fat body, respectively. Treatment of fat body with suramin caused a 50% inhibition in [(3)H]DAG transfer from lipophorin. Treatment of [(3)H]DAG-labeled fat body with lipase significantly reduced the amount of [(3)H]DAG associated with the tissue, suggesting that the lipid is still on the external surface of the membrane. Whether this lipid represents irreversibly adsorbed lipophorin or a DAG lipase-sensitive pool is unknown. Nevertheless, these results indicate that the main pathway for DAG transfer from lipophorin to fat body is via LTP and receptor-mediated processes.

Primary characterization and basal promoter activity of two hexamerin genes of Musca domestica

Hexamerins are high molecular-weight proteins found in the hemolymph of insects and have been proposed to function as storage proteins. In previous studies, two Musca domestica hexamerins, designated Hex-L and Hex-F were characterized. Hex-L is synthesized exclusively by the larval fat bodies, is secreted into the hemolymph and likely provides a source of amino acids and energy during metamorphosis. Hex-F synthesis is induced by a proteinaceous meal and occurs only in the adult insect fat bodies. Hex-F also is secreted into the hemolymph and it has been suggested that in females it may be an amino acid reservoir to be used during the final stages of egg formation. Genomic clones containing full-length copies of the genes MdHexL1 and MdHexF1, encoding subunits of the larval and the adult female hexamerin, respectively, were isolated. Complete nucleotide sequences, including the 5′-end untranscribed regions, were determined and analyzed for each of the genes. Comparisons of the conceptual translation products of the cloned genes indicated that MdHexL1 and MdHexF1 are related to the larval serum proteins (LSP) 1 and 2 of Calliphora vicina and Drosophila melanogaster. DNA fragments containing the putative promoters of the two hexamerin genes were compared and cloned into a plasmid vector so as to drive the expression of the GFP reporter gene. The constructs were assayed in vitro in transfected S2 Drosophila melanogaster cells demonstrating that the cloned M. domestica DNA fragments exhibit promoter activity.Hex L, Hex F Larval and female hexamerins respectively LSP Larval serum protein MdHexL1, MdHexF1 hexamerins of Musca domestica

Primary characterization and basal promoter activity of two hexamerin genes of Musca domestica

Hexamerins are high molecular-weight proteins found in the hemolymph of insects and have been proposed to function as storage proteins. In previous studies, two Musca domestica hexamerins, designated Hex-L and Hex-F were characterized. Hex-L is synthesized exclusively by the larval fat bodies, is secreted into the hemolymph and likely provides a source of amino acids and energy during metamorphosis. Hex-F synthesis is induced by a proteinaceous meal and occurs only in the adult insect fat bodies. Hex-F also is secreted into the hemolymph and it has been suggested that in females it may be an amino acid reservoir to be used during the final stages of egg formation. Genomic clones containing full-length copies of the genes MdHexL1 and MdHexF1, encoding subunits of the larval and the adult female hexamerin, respectively, were isolated. Complete nucleotide sequences, including the 5'-end untranscribed regions, were determined and analyzed for each of the genes. Comparisons of the conceptual translation products of the cloned genes indicated that MdHexL1 and MdHexF1 are related to the larval serum proteins (LSP) 1 and 2 of Calliphora vicina and Drosophila melanogaster. DNA fragments containing the putative promoters of the two hexamerin genes were compared and cloned into a plasmid vector so as to drive the expression of the GFP reporter gene. The constructs were assayed in vitro in transfected S2 Drosophila melanogaster cells demonstrating that the cloned M. domestica DNA fragments exhibit promoter activity.

Molecular cloning and characterization of the cathepsin B‐like proteinase from the cotton boll worm, Helicoverpa armigera

An enzyme purified from the ovaries of Helicoverpa armigera, as an active form with molecular mass of 30 kDa on SDS-PAGE, was identified as a cysteine proteinase because it could be inhibited by E-64, a specific inhibitor of cysteine proteinase, and required reducing conditions for activity. This enzyme was further identified as a cathepsin B-like cysteine proteinase by partial amino acid sequencing. A cDNA encoding this proteinase was cloned from H. armigera, using degenerate primers and RACE techniques. Results of Northern blots indicated that the mRNA encoding the proteinase was transcribed in the ovaries, the fat bodies of female and male adults, pupae and in the larvae. No mRNA was detected from the larval epidermis or from the midgut. Hence, transcription of the cathepsin B-like cysteine proteinase from H. armigera was tissue-specific, but not gender- or developmental stage-specific. However, proteolytic activities were only detected from ovaries, and adult female and male fat bodies. No activity was observed from pupal and larval fat bodies, from the larval epidermis or from the midgut. Only one form of mRNA of approximately 1100 bases was detected, and in situ hybridization showed that the transcripts were distributed in the adult female fat bodies, follicular cells and the oocytes. Since the proteinase expressed in ovaries was able to degrade vitellin in vitro, it may be involved in the degradation of vitellin during embryonic development.

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Acyl-CoA Δ9-desaturases play essential roles in fatty acid metabolism and the regulation of cell membrane fluidity. In this research, a cDNA sequence was obtained from Trichoplusia ni adult fat body mRNA by using RT–PCR with degenerate primers based on other characterized Δ9-desaturase sequences. The remainder of the sequence was amplified using 3′- and 5′-RACE. A 1439 bp cDNA reconstructed from three overlapping PCR products contains an ORF encoding a 353-amino acids (aa) protein that shows clear homology (greater than 50% aa identity and greater than 65% aa similarity to characterized insect and vertebrate desaturases). The ORF of this cDNA was subcloned into an expression vector, which relieved the unsaturated fatty acid (UFA) auxotrophy of a desaturase-deficient yeast strain following genetic transformation. The newly characterized desaturase from T. ni produced fatty acids Δ9-16 and Δ9-18 in a 1:6 ratio, compared to a 5:1 ratio, respectively, with the yeast Δ9 desaturase. A Northern blot hybridization and a RT–PCR experiment showed that temporal and tissue-specific patterns of expression of the corresponding mRNA are distinct from those of the Δ11-desaturase mRNA present in the pheromone glands of adult females. Based on its homology to other desaturases, the widespread distribution of its corresponding mRNA in various tissues, and its functional assay, we conclude that this cDNA encodes the apoprotein corresponding to the desaturase component of the metabolic Δ9-desaturase complex of T. ni.

Functional characterization and immunolocalization of the transporter encoded by the life-extending gene Indy.

Caloric restriction extends life span in a variety of species, highlighting the importance of energy balance in aging. A new longevity gene, Indy (for I'm not dead yet), which doubles the average life span of flies without a loss of fertility or physical activity, was postulated to extend life by affecting intermediary metabolism. We report that functional studies in Xenopus oocytes show INDY is a metabolite transporter that mediates the high-affinity, disulfonic stilbene-sensitive flux of dicarboxylates and citrate across the plasma membrane by a mechanism that is not coupled to Na(+), K(+), or Cl(-). Immunocytochemical studies localize INDY to the plasma membrane with most prominent expression in adult fat body, oenocytes, and the basolateral region of midgut cells and show that life-extending mutations in Indy reduce INDY expression. We conclude that INDY functions as a novel sodium-independent mechanism for transporting Krebs and citric acid cycle intermediates through the epithelium of the gut and across the plasma membranes of organs involved in intermediary metabolism and storage. The life-extending effect of mutations in Indy is likely caused by an alteration in energy balance caused by a decrease in INDY transport function.

CDNA cloning of two different serine protease inhibitor precursors in the migratory locust, Locusta migratoria

Recently, a novel serine protease-inhibiting peptide family, designated as the 'pacifastin family', has been described in locusts and crayfish. All members of this family possess a characteristic cysteine-rich domain. The present study describes the cDNA cloning, sequencing and transcript distribution of two novel pacifastin-related peptide precursors in the migratory locust, Locusta migratoria. Only one of the encoded peptides (HI) was identified previously, whereas six others represent new members of the pacifastin family. Northern blot analysis showed that both precursor transcripts are present in adult locust fat body. These could not be detected in the midgut. Interestingly, an in silico data mining approach of the expressed sequence tags (EST) database revealed the existence of Manduca sexta and Bombyx mori cDNAs that display pronounced sequence similarities with these locust pacifastin-related transcripts.

Cloning and functional expression of a fat body-specific chitinase cDNA from the tsetse fly, Glossina morsitans morsitans

A chitinase cDNA, GChit1 was isolated from Glossina morsitans morsitans and shown to be specifically expressed in fat body tissue. GChit1 is encoded by a 1.6 kb mRNA with a putative open reading frame (ORF) of 460 amino acids (predicted pI=7.5, m.w.= 51 kDa ) that contains a signal peptide domain and two potential N-linked glycosylation sites. The ORF exhibits homology to various chitinases characterized from insects. It has the conserved catalytic site residues and the cysteine-rich 3′-end domain associated with chitin binding although the serine/threonine rich domain is apparently missing. Southern blot data indicate that GChit1 is present as a single-copy locus in the Glossina genome. Northern analysis indicates that transcripts for GChit1 can be detected only from the fat body of adult flies. Similarly, chitinase activity could be detected in fat body but not in the gut or salivary gland tissues. The full-length cDNA was expressed in vitro in Drosophila S2 cells and the molecule was produced in a soluble form. Polyclonal antibodies raised against recGChit1 could recognize a protein of about 50 kDa in adult fat body extracts. In addition to fat body, chitinase protein was detected by Western analysis from the milk gland tissue of pregnant females as well as from the intrauterine larval and pupal developmental stages. No chitinase specific mRNA transcripts could be observed, however from larvae and pupae. The intrauterine larva of tsetse may receive the protein from its mother via the milk gland route. The molecular characteristics of GChit and its product and the potential role of this chitinase in tsetse biology are discussed.

Physiological and biochemical aspects of flight metabolism in cocoon-enclosed adults of the fruit beetle, Pachnoda sinuata

We studied several aspects of flight metabolism in cocoon-enclosed adults of the fruit beetle Pachnoda to investigate their flight capability. The majority of adults which were forcefully removed from their pupal cocoon flew off within 5 min of exposure to bright sunlight. Most of the beetles which did not fly voluntarily were, however, capable of flight. Compared with 2–4 week old adults of the same species, cocoon-enclosed adults have higher reserves of glycogen in flight muscles and fat body, whereas the level of total carbohydrates in the haemolymph and the concentration of proline in haemolymph, flight muscles and fat body were similar.Enzymes involved in carbohydrate breakdown (MDH, GAPDH) were more active in flight muscles and fat body of cocoon-enclosed adults compared with adults, while enzymes of proline metabolism in the flight muscles (AlaT, NAD-ME) and fat body (AlaT, NADP-ME) had similar activities in cocoon-enclosed adults and adults. An enzyme of the β-oxidation of fatty acids (HOAD) had similar activities in flight muscles and fat body of cocoon-enclosed adults and adults.Mitochondria isolated from flight muscles of adults removed prematurely from their cocoon favour the oxidation of proline and pyruvate. Pyruvate, however, is oxidized at higher rates than by mitochondria isolated from flight muscles of adults.During a short lift-generating flight, cocoon-enclosed adults proved that their flight muscles are capable of strong flight performance. During these flights, cocoon-enclosed adults consume proline and carbohydrates at a similar rate to that of adults.The endogenous AKH peptide, Mem-CC, has hyperprolinaemic and hypertrehalosaemic activity in cocoon-enclosed adults. The hypertrehalosaemic effect, however, is stronger in cocoon-enclosed adults than in adults.The content of Mem-CC in corpora cardiaca of larvae (3rd instar), cocoon-enclosed adults and 1 day-old adults is similar at 5–6 pmol per pair of corpora cardiaca, whereas it is higher in 10 day-old adults and 20 day-old adults (37 and 15 pmol per pair corpora cardiaca, respectively).From these results we conclude that cocoon-enclosed adults comply with all the prerequisites for flight performance before they leave their pupal cocoon. Furthermore, cocoon-enclosed adults have a more pronounced carbohydrate-based metabolism before they leave their cocoon compared with adults, which suggests that carbohydrate breakdown is mainly involved in such activities as leaving the cocoon and burrowing activity thereafter.

Ecdysteroids, juvenile hormone and vitellogenesis in the cockroach Leucophaea maderae.

Topical application of 400 micro g of the juvenile hormone analog, methoprene, to females of the penultimate instar of Leucophaea maderae failed to induce vitellogenin synthesis. However, last instar females showed an increasing response level in making vitellogenin as they aged during the first half of the instar. In the second half of the last instar the response to methoprene declined to nearly zero when the prothoracic glands have become highly active. Then, a few days before the metamorphic molt the responsiveness reached maximal levels, i.e., comparable to adult females. These data suggest that the fat body develops competency to produce vitellogenin during the last nymphal instar, but increasing titers of ecdysone then interfere with the action of methoprene and consequently production of vitellogenin is curtailed. When prothoracic glands from the second half of the last instar were implanted into adult females, the normal activation of the corpora allata, or their accelerated activation induced by mating, did not occur. Likewise, an activation of the corpora allata due to the severance of the NCCI was not observed when prothoracic glands had been implanted prior to such operations. Thus, ecdysone released by the prothoracic glands appeared to directly inhibit the isolated corpora allata in vivo i.e. without the mediation by the brain. Methoprene applied to allatectomized adult females induced vitellogenin synthesis in a dose dependent manner. This induction was, however, quantitatively reduced by implanted active prothoracic glands, particularly when low doses of methoprene had been applied. Methoprene higher than 5 micro g overcame the inhibitory potency of the implanted prothoracic glands. The effect of the prothoracic glands, i.e. ecdysone, appears to signal an interference with the action of methoprene at the target tissues, the fat body. The exposure of the fat body to a given juvenile hormone/ecdysone ratio dictates the apparent effectiveness of ecdysone. The precise mode of the interaction of juvenile hormone and ecdysone on the adult fat body is not known. These data show that ecdysone inhibits vitellogenesis by two independent mechanisms: 1) inhibition of the corpora allata resulting in the inhibition of juvenile hormone production and 2) inhibition of vitellogenin synthesis by the fat body. Both of these mechanisms appear to be operative in immature and mature animals. However, the action of ecdysones on the fat body is only seen after it had acquired competency to make vitellogenin during the last nymphal instar.

A novel GATA factor transcriptionally represses yolk protein precursor genes in the mosquito Aedes aegypti via interaction with the CtBP corepressor.

In anautogenous mosquitoes, vitellogenesis, the key event in egg maturation, requires a blood meal. Consequently, mosquitoes are vectors of many devastating human diseases. An important adaptation for anautogenicity is the previtellogenic arrest (the state of arrest) preventing the activation of the yolk protein precursor (YPP) genes Vg and VCP prior to blood feeding. A novel GATA factor (AaGATAr) that recognizes GATA binding motifs (WGATAR) in the upstream region of the YPP genes serves as a transcriptional repressor at the state of arrest. Importantly, AaGATAr can override the 20-hydroxyecdysone transactivation of YPP genes, and its transcriptional repression involves the recruitment of CtBP, one of the universal corepressors. AaGATAr transcript is present only in the adult female fat body. Furthermore, in nuclear extracts of previtellogenic fat bodies with transcriptionally repressed YPP genes, there is a GATA binding protein forming a band with mobility similar to that of AaGATAr. The specific repression of YPP genes by AaGATAr in the fat body of the female mosquito during the state of arrest represents an important molecular adaptation for anautogenicity.

Molecular cloning of a female-specific cDNA with unique repeat sequences from the fat body of the adult locust, Locusta migratoria

A cDNA clone encoding a 25-kDa protein (25K) was isolated from a cDNA library made from RNA isolated from the adult fat body and ovaries of the locust, Locusta migratoria. The longest open reading frame of this cDNA clone encodes a 225-amino acid polypeptide, the N-terminal end of which was similar to the 21-kDa and 19-kDa juvenile hormone induced proteins identified in the locust hemolymph, but the C-terminal end was different. The C-terminal end of the 25K cDNA contained seven unique repeat elements of 10 amino acids each, most of which are polar residues. Expression of the 25K mRNA was tissue-, development- and sex-specific. A 1.2-kb mRNA was detected using the 25K cDNA as a probe only in the fat body of adult females. The mRNA started to appear at day 4 after the insect molted to the adult and rapidly increased by day 6. The mRNA was absent in the ovarian follicle cells and fat body of adult males. In vitro transcription and translation of the 25K cDNA produced a protein that migrated around 32 kDa on sodium dodecyl sulfate polyacrylamide gels. The 25K cDNA was expressed in a baculovirus expression system and the protein produced also migrated around 32 kDa.