Preparation of two recombinant crustacean hyperglycemic hormones from the giant freshwater prawn, Macrobrachium rosenbergii, and their hyperglycemic activities

Crustacean hyperglycemic hormone (CHH) is released from the X-organ/sinus gland complex located in the eyestalks, and regulates glucose levels in the hemolymph. In the giant freshwater prawn (Macrobrachium rosenbergii), two cDNAs encoding different CHH molecules were previously cloned by other workers. One of these (Mar-CHH-2) was expressed only in the eyestalks, whereas the other (Mar-CHH-L) was expressed in the heart, gills, antennal gland, and thoracic ganglion, but not in the eyestalks. However, their biological activities had not yet been characterized. Therefore, in this study, recombinant Mar-CHH-2 (rMar-CHH-2) and Mar-CHH-L (rMar-CHH-L) were produced using an E. coli expression system, by expression in bacterial cells and recovery in the insoluble fraction. Thereafter, rMar-CHH-2 and rMar-CHH-L were subjected to refolding and were subsequently purified by reversed-phase HPLC. The rMar-CHH-2 and rMar-CHH-L thus obtained exhibited the same disulfide bond arrangements as those of other CHHs reported previously, indicative of natural conformation. In in vivo bioassay, rMar-CHH-2 showed significant hyperglycemic activity, whereas rMar-CHH-L had no effect. These results indicate that Mar-CHH-L does not function as a CHH, but may have some other, unknown function.     

Immunochemical analysis and immunocytochemical localization of crustacean hyperglycemic hormone from the eyestalk of Macrobrachium rosenbergii

Heptapeptide (YANAVQV-NH2 = T-) and octapeptide (YANAVQTV-NH2 = T+), the putative C-terminus of crustacean hyperglycemic hormone (CHH) from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii, was synthesized by solid phase peptide synthesis and conjugated to bovine serum albumin, then used for immunization in swiss mice. Specificity of the antisera against both peptides was determined by indirect immunoperoxidase ELISA. The best response of antiserum against each peptide was used to determine the presence of the natural CHH in the eyestalk extract after separation by one step of RP-HPLC using dot-ELISA. The peptide immunoreactive substances were found in fraction 30 using anti-T- antiserum and in fraction 38 using anti-T+ antiserum. However, the CHH activity was found only in fractions 37-39. Immunocytochemical localization of peptide immunoreactive substances in the eyestalk of M. rosenbergii using the anti-T- antiserum did not show any specific staining. In contrast, the anti-T+ antiserum revealed specific staining on a group of 24 +/- 5 neurons in medulla terminalis ganglionic x-organ and their processes through the sinus gland. Similar results were also obtained using the eyestalk of another species, the giant tiger prawn Penaeus monodon, in which 34 +/- 4 neuronal cells were recognized. These results strongly indicate that the anti-T+ antibody can bind to the natural CHH while the anti-T- antibody can not; therefore, this isoform of CHH in M. rosenbergii should consist of 72 residues and threonine is predicted to be present at position 71.     

Identification and characterization of a hyperglycemic hormone from freshwater giant prawn, Macrobrachium rosenbergii

Crustacean hyperglycemic hormone (CHH), a physiologically important neurohormone stored in the sinus gland of eyestalks, primarily regulates carbohydrate metabolism and also plays significant roles in reproduction, molting and other physiological processes. In the freshwater giant prawn, Macrobrachium rosenbergii, an injection of X-organ sinus gland (XOSG) extract evoked a hyperglycemic response, peaked in 1 h. The hyperglycemic effect of the eyestalk extract was maximal at the dose of 0.5 eyestalk equivalent. CHH fractionated by RP-HPLC, in M. rosenbergii was identified by its hyperglycemic activity and partial amino acid sequence, and the molecular weight of 8534 was determined by matrix-assisted laser desorption ionization mass spectrometry--time of flight analysis (MALDI-TOF). The amino acid sequence of the first 25 residues of CHH showed 72% homology with the first 25 residues of CHH A and CHH B of the American lobster Homarus americanus.     

Differential expression of CMG peptide and crustacean hyperglycemic hormones (CHHs) in the eyestalk of the giant tiger prawn Penaeus monodon

Mouse antiserum against C-terminal amide of Pem-CMG (a peptide in the family of CHH/MIH/GIH) penta-deca peptide (RPRQRNQYRAALQRLamide=CMG-15) was generated and used for localization of the peptide in tissue and extract of the eyestalk of Penaeus monodon by means of immunohistochemistry and dot-ELISA in comparison with anti-T+ antiserum (T+=YANAVQTVamide : the putative C-terminal amide of crustacean hyperglycemic hormone (CHH) of Macrobrachium rosenbergii). The anti-CMG-15 antiserum did not show cross-reactivity to T+ peptide by dot-ELISA and vice versa for anti-T+ antiserum. In dot-ELISA of eyestalk extract of P. monodon after one step separation by RP-HPLC, anti-CMG-15 antiserum recognized different peptide fractions (F38-39) from those recognized by anti-T+ antiserum (F19, 40-41 and 47-51). Most of the T+ immunoreactive fractions (except F19) show higher hyperglycemic activity than the CMG immunoreactive fractions. In immunohistochemical localization, anti-CMG antiserum recognized only 2-3 neurons in medulla terminalis X-organ complex (MTXO) with long processes terminated in the sinus gland. The CMG-immunoreactive neurons were clearly distinct from CHH containing neurons situated in the same area. This evidence confirms the existing of CMG peptide which may play distinct roles from CHHs in hormonal regulation in P. monodon.     

Quantitation of peptide hormone in single cultured secretory neurons of the crab,Cardisoma carnifex

The content of crustacean hyperglycemic hormone (CHH) in single cultured neurons of the crab Cardisoma carnifex was determined by a sensitive enzyme-linked immunosorbent assay (ELISA), using purified CHH (1–50 pg) of the crab Carcinus maenas as standard. The somata were dissociated from the group of 150 peptidergic neurons that form the X-organ—sinus gland neuroendocrine system. As previously reported, the neurons show immediate regenerative outgrowth in defined culture conditions, and develop, generally, into one of two morphological types: cells that produce broad, lamelliform growth cones (veils), and others that are characterized by branching of neurites. In this study, all but one of 64 veiling cells taken after various times in culture up to 12 days contained CHH. They could be readily categorized as having high (>33 pg; mean 86±5, S.E., n=47) or low (33pg; mean 22±2.5; n=17) Carcinus CHH equivalents. Thus, CHH is associated with neurons showing veiling outgrowth, but veiling neurons with low CHH form a distinct, but not morphologically distinguishable group. They may contain an isoform of CHH with limited cross-reactivity. In 24 branching neurons assayed, Carcinus CHH equivalents averaged 7.2±2 pg. This figure includes 14 neurons in which CHH was undetectable, and one that had 40 pg of Carcinus CHH equivalents. There was no significant change of the hormone content in cells of either type during 6 days of culturing.     

Purification and partial characterization of seven glutathione S-transferase isoforms from the clam Ruditapes decussatus.

This paper deals with the purification and the partial characterization of glutathione S-transferase (GST) isoforms from the clam Ruditapes decussatus. For the first step of purification, two affinity columns, reduced glutathione (GSH)-agarose and S-hexyl GSH-agarose, were mounted in series. Four affinity fractions were thus recovered. Further purification was performed using anion exchange chromatography. Seven fractions, which present a GST activity with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate, were collected and analyzed by RP-HPLC. Seven distinct GST isoforms were purified, six of them were homodimers, the last one was a heterodimer consisting of the subunits 3 and 6. Kinetic parameters were studied. Results showed that isoforms have distinct affinity and Vmax for GSH and CDNB as substrates. The catalytic activity of the heterodimer isoform appeared to be a combination of the ability of each subunit. The immunological properties of each purified isoform were investigated using three antisera anti-pi, anti-mu and anti-alpha mammalian GST classes. Three isoforms (3-3, 6-6 and 3-6) seem to be closely related to the pi-class GST. Both isoforms 1-1 and 2-2 cross-reacted with antisera to pi and alpha classes and the isoform 5-5 cross-reacted with the antisera to mu and pi classes. Subunit 4 was recognized by the three antisera used, and its N-terminal amino acid analysis showed high identity (53%) with a conserved sequence of an alpha/m micro /pi GST from Fasciola hepatica.     

Cloning and sequence analysis of cDNA for precursor of a crustacean hyperglycemic hormone

Crustacean hyperglycemic hormone (CHH) from Carcinus maenas, a 72 amino acid neuropeptide, originates in neurosecretory perikarya in the eyestalk ganglia. Poly (A)RNA was isolated from these perikarya and a cDNA library was prepared. Screening of 20,000 clones with a 26-mer oligonucleotide, corresponding to a partial sequence of CHH, yielded one positive clone with an insert of approximately 2,000 bp, which contained the complete coding sequence for a pre-pro CHH. This precursor consists of a putative 26 amino acid signal sequence, a 38 amino acid peptide of unknown function (Peptide C), and the CHH sequence at the carboxyl end. The CHH-sequence is flanked N-terminally by a Lys-Arg cleavage site and C-terminally by the tetrapeptide Gly-Arg-Lys-Lys which is followed by the stop codon.     

Seven novel FMRFamide-like neuropeptide sequences from the eyestalk of the giant tiger prawn Penaeus monodon

FMRFamide-like immunoreactivity (FLI) was localized in the eyestalk of Penaeus monodon by immunohistochemistry using a combination of three anti-FMRFamide-like peptide (FLPs) monoclonal antibodies. Approximately 3000 small neuronal cell bodies in the lamina ganglionalis; 100 medium to large size at the ganglion between the medulla interna and the medulla terminalis; and 250 medium size around the medulla terminalis were stained intensely. The neuronal processes in neuropils of the medulla externa, medulla interna, medulla terminalis, sinus gland and some nerve fibers in the optic nerve were also recognized. The small cell bodies, approximately 1500 cells, anterior to the medulla externa were stained inconsistently and the neuronal processes were not observed from these cells. Isolation of FLPs from 9000 eyestalks was performed using methanol/acetic/water (90:1:9) extraction. After the extract was partially purified using C18 cartridges, it was further purified by five to seven steps of RP-HPLC using three kinds of columns: C18; C8; and cyano, and three solvent systems: acetonitrile/trifluoro acetic acid; aceonitrile/heptafluoro butyric acid; and acetonitrile/triethyl ammonium acetate. Dot-ELISA using the combination of the same antibodies was used to monitor FLPs in the fractions during purification processes. Seven new sequences of FLPs were identified which can be divided into four subgroups according to the primary structure of the C-terminus: (1) GDRNFLRFamide; (2) AYSNLNYLRFamide; (3) AQPSMRLRFamide, SQPSMRLRFamide, SMPSLRLRFamide and DGRTPALRLRFamide; and (4) GYRKPPFNGSIFamide. These data indicate the high complexity of this peptide family in which multiple forms are usually exist.     

Two type I crustacean hyperglycemic hormone (CHH) genes in Morotoge shrimp (Pandalopsis japonica): cloning and expression of eyestalk and pericardial organ isoforms produced by alternative splicing and a novel type I CHH with predicted structure shared with type II CHH peptides

Crustacean hyperglycemic hormone (CHH) peptide family members play critical roles in growth and reproduction in decapods. Three cDNAs encoding CHH family members (Pj-CHH1ES, Pj-CHH1PO, and Pj-CHH2) were isolated by a combination of bioinformatic analysis and conventional cloning strategies. Pj-CHH1ES and Pj-CHH1PO were products of the same gene that were generated by alternative mRNA splicing, whereas Pj-CHH2 was the product of a second gene. The Pj-CHH1 and Pj-CHH2 genes had four exons and three introns, suggesting the two genes arose from gene duplication. The three cDNAs were classified in the type I CHH subfamily, as the deduced amino acid sequences had a CHH precursor-related peptide sequence positioned between the N-terminal signal sequence and C-terminal mature peptide sequence. The Pj-CHH1ES isoform was expressed at a higher level in the eyestalk X-organ/sinus gland (XO/SG) complex and at a lower level in the gill. The Pj-CHH1PO isoform was expressed at higher levels in the XO/SG complex, brain, abdominal ganglion, and thoracic ganglion and at a lower level in the epidermis. Pj-CHH2 was expressed at a higher level in the thoracic ganglion and at a lower level in the gill. Real-time polymerase chain reaction was used to quantify the effects of eyestalk ablation on the mRNA levels of the three Pj-CHHs in the brain, thoracic ganglion, and gill. Eyestalk ablation reduced expression of Pj-CHH1ES in the brain and Pj-CHH1PO and Pj-CHH2 in the thoracic ganglion. Sequence alignment of the Pj-CHHs with CHHs from other species indicated that Pj-CHH2 had an additional alanine at position #9 of the mature peptide. Molecular modeling showed that the Pj-CHH2 mature peptide had a short alpha helix (α1) in the N-terminal region, which is characteristic of type II CHHs. This suggests that Pj-CHH2 differs in function from other type I CHHs.     

Laccase production by a monokaryotic strain of Pycnoporus cinnabarinus derived from a dikaryotic strain

Monokaryotic Pycnoporus cinnabarinus strains were obtained from the dikaryotic strain I-938. One of these, designated MK18, consistently produced high laccase activity. In cultures of MK18 and I-938 where ferulic acid was added as laccase inducer, laccase activity was enhanced about 2.5-fold reaching 3400 U/l for the MK18 strain. Laccase was purified to homogeneity and under the selected growth conditions, only one isoform of the enzyme was produced. The N-terminal sequence was similar to the amino terminal sequence of laccase II from Trametes versicolor. The enzyme was stable at 60 C for more than 1 h.     

Development of high oleic and low linoleic acid transgenics in a zero erucic acid Brassica juncea L. (Indian mustard) line by antisense suppression of the fad2 gene

A zero erucic acid (C22:1) line of Brassica juncea (VH486), adapted to the agronomic conditions of Northern India, has been modified for its fatty acid composition in the seed oil with antisense constructs using the sequence of fad2 gene of B. rapa. The full-length B. rapa fad2 cDNA sequence was determined by 5 and 3 RACE of a partial sequence available in the EST database. Construct pASfad2.1 contained 315 to 1251 bp and construct pASfad2.2 contained 1 to 1251 bp fragment of the fad2 gene, both in antisense orientation, driven by a truncated napin promoter. Analysis of the levels of linoleic acid (C18:2) in the BC1 seeds of single-copy transgenics showed that the construct pASfad2.2 gave better suppression of the fad2 gene as compared to the construct pASfad2.1. The BC1 transgenic seeds containing the pASfad2.2 construct segregated into two distinct classes of C18:2>20% (putative null homozygotes) and C18:2<20% (putative heterozygotes) in a 1:1 ratio, while the T1 seeds segregated into three classes, C18:2>20%, C18:2 between 12% and 20%) and C18:2<12% (putative homozygotes) in a 1:2:1 ratio. Putative homozygous T1 seeds (C18:2<12% analyzed by the half-seed method) of four of the transgenic lines were grown to establish T2 homozygous lines. These had ca. 73% C18:1 and 8 to 9% each of C18:2 and C18:3 (-linolenic acid) fractions in comparison to ca. 53% C18:1, 24% C18:2 and 16% C18:3 in the parental line VH486.     

Characterization of a low molecular mass autophosphorylating protein in cultured sugarcane cells and its identification as a nucleoside diphosphate kinase.

A low molecular mass (18 kD) phosphoprotein (pp18) was characterized and purified from cultured sugarcane (Saccharum officinarum L.) cell line H50-7209. Autophosphorylation assays were used to detect pp18 after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Only pp18 was detected by a brief in situ phosphorylation method, whereas additional putative protein kinases were detected by an extended method. pp18 was present in both microsomal membrane and soluble fractions and exhibited anomalous turnover of 32P label during in vitro phosphorylation experiments with highest levels present at shorter incubation times. Two major isoforms of the protein were identified in two-dimensional isoelectric focusing/SDS-PAGE of crude extracts and microsomal fractions. The levels of pp18 were enhanced approximately 4-fold by heat shock at 36 degrees C and the elevated pp18 decayed after heat shock was discontinued. pp18 was purified to apparent homogeneity, could be phosphorylated on serine residues, and also exhibited kinase-like activity toward histone H1. The amino acid sequence obtained from a cyanogen bromide digest was greater than 80% identical to nucleoside diphosphate (NDP) kinases from a variety of organisms. Biochemical analysis of the purified protein confirmed the identity as NDP kinase. Thus, NDP kinase appears to be modulated by heat shock in plants.     

Determination of alpha-naphthylisothiocyanate and metabolites alpha-naphthylamine and alpha-naphthylisocyanate in rat plasma and urine by high-performance liquid chromatography

A rapid and sensitive high-performance liquid chromatographic (HPLC) assay for the determination of alpha-naphthylisothiocyanate (1-NITC) and two metabolites alpha-naphthylamine (1-NA) and alpha-naphthylisocyanate (1-NIC) in rat plasma and urine has been developed. The chromatographic analysis was carried out using reversed-phase isocratic elution with a Partisphere C(18) 5-microm column, a mobile phase of acetonitrile-water (ACN-H(2)O 70:30, v/v), and detection by ultraviolet (UV) absorption at 305 nm. The lower limits of quantitation (LLQ) in rat plasma, urine, and ACN were 10, 30, and 10 ng/ml for 1-NITC; 30, 100, and 30 ng/ml for 1-NA; and 30 ng/ml in ACN for 1-NIC. At low (10 ng/ml), medium (500 ng/ml), and high (5000 ng/ml) concentrations of quality control samples (QCs), the range of within-day and between-day accuracies were 95-106 and 97-103% for 1-NITC in plasma, respectively. Stability studies showed that 1-NITC was stable at all tested temperatures in ACN, and at -20 and -80 degrees C in plasma, urine, and ACN precipitated plasma and urine, but degraded at room temperature and 4 degrees C. 1-NA was stable in all of the tested matrices at all temperatures. 1-NIC was unstable in plasma, urine, and ACN precipitated plasma and urine, but stable in ACN. The degradation product of 1-NITC and 1-NIC in universal buffer was confirmed to be 1-NA. 1-NITC and 1-NA were detected and quantified in rat plasma and urine, following the administration of a 25 mg/kg i.v. dose of 1-NITC to a female Sprague-Dawley rat.     

Purification and cDNA cloning of chloroplastic monodehydroascorbate reductase from spinach

The chloroplastic isoform of monodehydroascorbate (MDA) radical reductase was purified from spinach chloroplasts and leaves. The cDNA of chloroplastic MDA reductase was cloned, and its deduced amino acid sequence, consisting of 497 residues, showed high homology with those of putative organellar MDA reductases deduced from cDNAs of several plants. The amino acid sequence of the amino terminal of the purified enzyme suggested that the chloroplastic enzyme has a transit peptide consisting of 53 residues. A southern blot analysis suggested the occurrence of a gene encoding another isoform homologous to the chloroplastic isoform in spinach. The recombinant enzyme was highly expressed in Eschericia coli using the cDNA, and purified to a homogeneous state with high specific activity. The enzyme properties of the chloroplastic isoform are presented in comparison with those of the cytosolic form.     

Two genetic variants of the crustacean hyperglycemic hormone (CHH) from the Australian crayfish, Cherax destructor: detection of chiral isoforms due to posttranslational modification

From sinus glands of the Australian crayfish Cherax destructor, two genetic variants of the crustacean hyperglycemic hormone (CHH) were isolated by HPLC and fully characterized by mass spectrometry and Edman sequencing. Both CHH A (8350.38 Da) and CHH B (8370.34 Da) consist of 72 amino acid residues, with pyroGlu as N-terminus and an amidated (Val-NH2) C-terminus. They differ in 14 residues (81% identity). Both sequences are significantly different from those of the hitherto known three CHHs of Astacoidea species (Northern hemisphere crayfish), which among themselves are extremely conserved. This may reflect the long, separate evolution of the Astacoidea lineage and the Parastacoidea (Southern hemisphere crayfish) lineage, to which Cherax belongs. CHH A and CHH B genes are expressed at comparable levels, as indicated by the similar amounts of mature peptides in the sinus gland. In addition to each of the major peptides, which share the identical N-terminal tripeptide pyroGlu-Val-L-Phe, one chiral isoform containing pyroGlu-Val-D-Phe was identified. Compared to the main peptides, the amounts of the D-isoforms are lower, but significant, amounting to 30-40% of L-isoforms. These results demonstrate that two genes can give rise to a total of four different peptides in the secretory terminals of the sinus gland. All peptides gave a highly significant hyperglycemic in vivo response in C. destructor.     

Three more novel FMRFamide-like neuropeptide sequences from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii

In addition to five FMRFamide-like peptides (FLPs) previously isolated from the eyestalk of the giant freshwater prawn Macrobrachium rosenbergii (16), three more new FLPs (Mar-FLP6-8) were identified from minor immunoreactive fractions of 5,000 eyestalk extracted in methanol/acetic acid/water: DGGRNFLRFamide, GYGDRNFLRFamide and VSHNNFLRFamide. These three peptides share 5-6 common residues at the C-terminus with Mar-FLP1,2 and 3. This evidence reveals that the structural diversity and complexity of the FLP family in M. rosenbergii are similar to that found in other invertebrate species.     

Crustacean hyperglycemic and vitellogenesis-inhibiting hormones in the lobster Homarus gammarus

Crustacean hyperglycemic hormone (CHH) and vitellogenesis-inhibiting hormone (VIH), produced by the X organ-sinus gland neurosecretory complex, belong to a peptide group referred to as the CHH family, which is widely distributed in arthropods. In this study, genetic variants and post-translationally modified isoforms of CHH and VIH were characterized in the European lobster Homarus gammarus. With the use of RP-HPLC and ELISA with specific antibodies that discriminate between stereoisomers of CHH and VIH, two groups of CHH-immunoreactive peaks were characterized from HPLC fractions of sinus gland extract (CHH A and CHH B); each group contained two variants (CHH and D-Phe3CHH). In the same way, two VIH-immunoreactive peaks (VIH and D-Trp4VIH) were demonstrated in HPLC fractions from sinus gland extract. The masses of these different neuropeptides were determined by FT-ICR MS: CHH A and CHH B spectra exhibited monoisotopic ions at 8557.05 Da and 8527.04 Da, respectively, and both VIH isomers displayed an m/z value of 9129.19 Da. Two full-length cDNAs encoding preprohomones of CHH A and CHH B and only one cDNA for VIH precursor were cloned and sequenced from X organ RNA. Comparison of CHH sequences between European lobster and other Astacoidea suggests that the most hydrophobic form appeared first during crustacean evolution.     

Fatty acid composition of chlorenchyma membrane fractions from three desert succulents grown at moderate and high temperatures

To help understand the tolerances of desert succulents to extremely high temperatures (above 60°C), the effect of growth temperature on fatty acid composition of various membrane fractions from three species was investigated. When maintained at day/night air temperatures of 30°C/20°C, their chlorenchyma fatty acid compositions were similar to one another and to those of mesophytic leaves, except that desert succulents had appreciably less linolenic acid (18:3) and more oleic acid (18:1) and hence greater fatty acid saturation. The differences were observed in the chloroplast, mitochondrial and microsomal fractions and were more apparent in the nonpolar lipids than the total lipids. For all membrane fractions of Ferocactus acanthodes, a shift to 50°C/40°C resulted in a decrease in 18:3 and an increase in 18:1 and hence an increase in fatty acid saturation level. For Agave deserti and Carnegiea gigantea, however, increasing the day/night air temperatures did not result in increased fatty acid saturation, although their high-temperature tolerances increase about as much as that of F. acanthodes as the air temperature is increased. Thus, acquisition of high-temperature tolerance need not be accompanied by marked changes in fatty acid saturation or composition.