Biological activity and identification of neuropeptides in the neurosecretory complexes of the cabbage pest insect, Mamestra brassicae (Noctuidae; Lepidoptera).

The need for more environmentally sound strategies of plant protection has become a driving force in physiological entomology to combat insect pests more efficiently. Since neuropeptides regulate key biological processes, these "special agents" or their synthetic analogues, mimetics, agonists or antagonists may be useful tools. We examined brain-suboesophageal ganglia and corpora cardiaca-corpora allata complexes of the cabbage moth, Mamestra brassicae, in order to obtain clues about possible peptide candidates which may be appropriate for the biological control of this pest. With the aid of bioassays, reversed phase high performance liquid chromatography, and mass spectrometry, five neuropeptides were unequivocally identified and the presence of a further three were inferred solely by comparing mass spectra with known peptides. Only one neuropeptide with adipokinetic capability was identified in M. brassicae. Data from the established homologous bioassay indicated that the cabbage moths rely on a lipid-based metabolism which is aided by an adipokinetic hormone (viz. Manse-AKH) that had previously been isolated in many different lepidopterans. Other groups of neuropeptides identified in this study are: FLRFamides, corazonin, allatostatin and pheromonotropic peptide.

Cyclic AMP mediates the elevation of proline by AKH peptides in the cetoniid beetle, Pachnoda sinuata.

The role of cyclic nucleotides in the transduction of the hyperprolinaemic and hypertrehalosaemic signal of the endogenous neuropeptide Mem-CC was investigated in the cetoniid beetle Pachnoda sinuata. Flight and injection of Mem-CC into the haemocoel of the beetle induce an increase of cAMP levels in the fat body of the beetle. This increase is tissue-specific and does not occur in brain and flight muscles. An elevation of cAMP levels was also found when in vitro preparations of fat body tissue were subjected to Mem-CC. Elevation of the cAMP concentration after injection of Mem-CC is time- and dose-dependent: the maximum response is measured after 1 min, and a dose of 25 pmol Mem-CC is needed. Injection of cpt-cAMP, a cAMP analogue which penetrates the cell membrane, causes a stimulation of proline synthesis but no mobilisation of carbohydrate reserves. The same is measured when IBMX, an inhibitor of phosphodiesterase, is injected. cGMP seems not to be involved in synthesis of proline nor carbohydrate release, because injection of cpt-cGMP has no influence on the levels of proline, alanine and carbohydrates in the haemolymph. Although glycogen phosphorylase of the fat body is activated by Mem-CC in a time- and dose-dependent manner, it cannot be stimulated by cpt-cAMP. The combined data suggest that cAMP is involved in regulation of proline levels by Mem-CC but not in regulation of carbohydrates. Octopamine has no effect on metabolites in the haemolymph and is not capable of activating glycogen phosphorylase, indicating that it is not involved in the regulation of substrates in this beetle. Furthermore, the requirements of the receptor of Mem-CC are different for eliciting a hypertrehalosaemic and a hyperprolinaemic effect, respectively, suggesting that differentiation in signal transduction begins at the receptor level.

The role of calcium in the activation of glycogen phosphorylase in the fat body of the fruit beetle, Pachnoda sinuata, by hypertrehalosaemic hormone.

The role of calcium in the mediation of the hypertrehalosaemic signal of the endogenous neuropeptide Mem-CC was investigated in vitro and in vivo in the cetoniid beetle Pachnoda sinuata. The presence of Mem-CC increases the influx of extracellular 45Ca(2+) into the fat body as well as the efflux of 45Ca(2+) from pre-loaded fat body into the incubation medium. Extracellular calcium is essential to exert maximal activation of the fat body glycogen phosphorylase by saturating doses of Mem-CC (0.3 nM). This effect of extracellular Ca(2+) is dose-dependent: maximal activation of glycogen phosphorylase by Mem-CC is achieved at calcium concentrations of approximately 1.2 mM and the ED(50) was calculated to be 0.6 mM. Both, thimerosal and thapsigargin caused a stimulation of carbohydrate metabolism in the fat body, suggesting that a release of calcium from the endoplasmic reticulum is involved in this process. However, neither entry of extracellular calcium nor the release from the endoplasmic reticulum are sufficient alone for a full activation of the phosphorylase. The results of the present study suggest that calcium from extracellular as well as from intracellular sources is part of the second messenger system for the transduction of the hypertrehalosaemic signal of Mem-CC in the fat body of P. sinuata.

Hormonal stimulation of proline synthesis in the fat body of the fruit beetle, Pachnoda sinuata, is calcium dependent.

The role of calcium in the transduction of the hyperprolinaemic signal of the endogenous neuropeptide Mem-CC was investigated in the cetoniid beetle Pachnoda sinuata using in vivo and in vitro methods to measure changes in the concentration of proline and its precursor alanine. Extracellular calcium is necessary for maximal stimulation of proline synthesis at saturating doses of Mem-CC (0.3 nM) in vitro. This effect depends on the dose of Ca(2+): maximal proline synthesis of 2.1 micromol mg(-1) protein h(-1) was stimulated by Mem-CC at calcium levels of 0.5 mM, and the EC(50) was 0.16 mM. Using the ionophore A 23187 in vivo and in vitro, we demonstrated that the extracellular calcium acts, via an influx into the cell, on the stimulation of proline production and alanine consumption. The release of calcium from intracellular sources is part of the signalling process: the agent thapsigargin, which inhibits the Ca(2+)-ATPase, is able to stimulate proline synthesis in vivo and in vitro. Thimerosal, however, which triggers the release of calcium from IP3-sensitive stores in the endoplasmic reticulum, had no influence on proline production nor alanine consumption, indicating that inositolphosphates are not part of the transduction of the hyperprolinaemic signal of Mem-CC. Both substances, thapsigargin and thimerosal, stimulate calcium entry in vitro from the medium (similar to Mem-CC), which indicates that a capacitative calcium entry takes place. Neither the entry of extracellular calcium nor the release from the endoplasmic reticulum, however, are alone sufficient for a full stimulation of proline synthesis in vitro. The results of the present study suggest that calcium from extra- as well as from intracellular sources is part of the second messenger system for the transduction of the hyperprolinaemic signal of Mem-CC in the fat body of P. sinuata. Calcium acts most likely via the elevation of cAMP levels: the concentration of this cyclic nucleotide in the fat body during in vitro incubation was elevated by 487% by Mem-CC in the presence of calcium, while the increase was only 122% when calcium was absent.

A third active AKH is present in the pyrgomorphid grasshoppers Phymateus morbillosus and Dictyophorus spumans.

The corpora cardiaca of the African pyrgomorphid grasshoppers Phymateus morbillosus and Dictyophorus spumans contain three adipokinetic hormones (AKHs): besides two already known AKHs, Phm-AKH-I and Scg-AKH-II (Gäde et al., 1996 [Gäde, G., Kellner, R., Rinehart, K.L., 1996. Pyrgomorphid grasshoppers of the genus Phymateus contain species-specific decapeptides of the AKH/RPCH family regulating lipid-mobilisation during flight. Physiol. Entomol. 21, 193-202]), a new AKH-III, denoted Phm-AKH-III, pGlu-Ile-Asn-Phe-Thr-Pro-Trp-Trp-NH(2), has been characterised. This is only the second AKH-III identified so far, thus, only three insect species - all of them grasshoppers - contain three active AKHs. Phm-AKH-III differs from Lom-AKH-III from the migratory locust, Locusta migratoria, only in position 2: isoleucine is present instead of leucine. The structure of the Phm-AKH-III was confirmed by synthesis, subsequent mass determination and reversed-phase high-performance liquid chromatography. The synthetic peptide also induced hyperlipaemia in D. spumans and L. migratoria.

Identification of multiple peptides homologous to cockroach and cricket allatostatins in the stick insect Carausius morosus.

Eighteen peptides were isolated from brain extracts of the stick insect Carausius morosus. The peptides were purified in four steps by high-performance liquid chromatography, monitored by their ability to inhibit juvenile hormone biosynthesis by corpora allata of the cricket Gryllus bimaculatus in vitro, and chemically characterised by Edman degradation and mass spectrometry. We obtained complete primary-structure information for nine peptides, four of which belong to the peptide family characterised by a common C-terminal pentapeptide sequence -YXFGLamide. The remaining five belong to the W(2)W(9)amide peptide family, nonapeptides characterised by having the amino acid tryptophan in positions 2 and 9. The amino-acid sequence of two other peptides could not be completely resolved by means of Edman degradation; however, these peptides could be allocated to the -YXFGLamide and the W(2)W(9)amide family, respectively, by comparison of retention times, co-elution and mass spectrometry. Both classes of neuropeptides strongly inhibit juvenile hormone biosynthesis in crickets but show no inhibiting effect on the corpora allata of the stick insect.

Mathematical modelling of insect neuropeptide potencies. Are quantitatively predictive models possible?

The potencies of natural adipokinetic hormones and synthetic variants have been determined in Locusta migratoria using the lipid mobilisation assay in vivo, and/or the acetate uptake assay in vitro. These data are combinations of previously published and unpublished data (a total of sixty-nine analogues), and form data sets for the construction of mathematical models of the hormone potencies. The sequence variations of amino acids in both natural and artificial adipokinetic hormone analogues were described using continuous descriptor scales z(1)', z(2)', and z(3)', each previously published scale being derived from various properties of the amino acids. By means of these z'-scales and partial least squares regression we attempted to model the potencies in Locusta migratoria of adipokinetic hormones in the two assays. Correlations (r(2) values) between predicted and actual potencies of the different peptides were up to 0.73. We discuss the potential of the partial least squares method for formulating quantitative relationships between different hormone structures and their potencies, and describe how the procedure might be used in structure-activity prediction with the construction of an optimised peptide data set.

Differential distribution of pyrokinin-isoforms in cerebral and abdominal neurohemal organs of the American cockroach.

Different pyrokinin isoforms were identified from major neurohemal organs of the American cockroach. During their isolation they were recognized by bioassay using a hyperneural muscle preparation that is sensitive to pyrokinins. All structures were elucidated by sequence analysis and mass spectrometry. The primary structures of the novel peptides isolated from the retrocerebral complex are LVPFRPRL-NH2 (designated Pea-PK-3) and DHLPHDVYSPRL-NH2 (designated Pea-PK-4). A pyrokinin, labeled Pea-PK-5, was isolated from abdominal perisympathetic organs. Structural analysis of this peptide yielded the sequence GGGGSGETSGMWFGPRL-NH2. The threshold concentrations of the identified pyrokinins for an eliciting effect on contractions of the hyperneural muscle preparations differed dramatically. This indicates that the different distribution of pyrokinin-isoform observed in neurohemal organs may be associated with different functions. This is the first report of a differential distribution of peptide-isoforms in the neurohemal organs of insects.

Using a mailed survey to predict hospital admission among patients older than 80.

OBJECTIVE: To determine if the results of a questionnaire mailed to older patients can help identify those patients at greatest risk of hospital admission. DESIGN: A longitudinal cohort study. SETTING: A prepaid managed care plan in the Denver metropolitan area. PARTICIPANTS: Of the 4414 eligible patients at least 81 years old, 3745 (84.8%) responded. MEASUREMENTS: We studied the predictive power of self-reported demographic, health status, medical history, health habits, functional status (including Katz' activities of daily living and OARS instrumental activities of daily living), and socioeconomic status data to identify those older adults at greatest risk of hospitalization within 4.5 months of completing the survey. We derived our predictive model on one-half the subjects and tested its validity it on the other half. RESULTS: Univariate analysis revealed 25 variables significantly associated with hospital admission. In a logistic regression model, four significant variables successfully stratified the patients by risk of admission. These four variables are: the presence of heart disease, the presence of diabetes, need for help preparing meals, and limited physical independence (requiring the help of a person or mechanical aid to get around). In addition, there was an antagonistic interaction between the presence of heart disease and limited physical independence. The model stratified patients from low risk (4.5% chance of admission) to high risk (39% chance of admission). As measured by the Hosmer-Lemeshow statistic and the area under a receiver-operator characteristic (ROC) curve, this model fit both the derivation and validation subjects well. CONCLUSIONS: A mailed questionnaire achieved a high response rate, and the information collected produced an effective model predictive of hospitalization in the short term. Four easily ascertained pieces of information identify those patients older than age 81 at increased risk.

A randomized trial of group outpatient visits for chronically ill older HMO members: the Cooperative Health Care Clinic.

OBJECTIVE: To compare the impact of group outpatient visits to traditional "physician-patient dyad" care among older chronically ill HMO members on health services utilization and cost, self-reported health status, and patient and physician satisfaction. DESIGN: A 1-year randomized trial. SETTING: A group model HMO in the Denver Metropolitan area. PARTICIPANTS: Three hundred twenty-one members aged 65 and older, randomized to a group visit intervention (n = 160) or to usual care (n = 161). INTERVENTION: Patients with high health services utilization and one or more chronic conditions had monthly group visits with their primary care physician and nurse. Visits included health education, prevention measures, opportunities for socialization, mutual support, and for one-to-one consultations with their physician, where necessary. MEASUREMENTS: Health services utilization and associated cost, health status, and patient and physician satisfaction. RESULTS: Outcome measures obtained after a 1-year follow-up period showed that group participants had fewer emergency room visits (P = .009), visits to subspecialists (P = .028), and repeat hospital admissions per patient (P = .051). Group participants made more visits (P = .021) and calls (P = .038) to nurses than control group patients and fewer calls to physicians (P = .019). In addition, a greater percentage of group participants received influenza and pneumonia vaccinations (P < .001). Group participants had greater overall satisfaction with care (P = .019), and participating physicians reported higher levels of satisfaction with the groups than with individual care. No differences were observed between groups on self-reported health and functional status. Cost of care per member per month was $14.79 less for the group participants. CONCLUSIONS: Group visits for chronically ill patients reduce repeat hospital admissions and emergency care use, reduce cost of care, deliver certain preventive services more effectively, and increase patient and physician satisfaction.

The metabolic neuropeptides of the corpus cardiacum from the potato beetle and the American cockroach are identical.

Two neuropeptides with adipokinetic activity in Locusta migratoria and hypertrehalosaemic activity in Periplaneta americana were purified by high performance liquid chromatography from the corpus cardiacum of the Colorado potato beetle, Leptinotarsa decemlineata. The sequences of both peptides, designated Led-CC-I and Led-CC-II, were determined by pulsed-liquid phase sequencing employing Edman degradation after deblocking enzymatically the N-terminal pyroglutamate residue. The C-terminal of both peptides were blocked and neither molecule was cleaved by carboxypeptidase. Both peptides were found to be octapeptides; Led-CC-I has the primary structure pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH2, and Led-CC-II has the primary sequence pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH2. These structures are identical to the two hypertrehalosaemic hormones from the American cockroach. Preliminary experiments show that the synthetic peptides are apparently involved in the control of amino acid metabolism during flight of the potato beetle.

The primary structure of the hypertrehalosemic neuropeptide from tenebrionid beetles: a novel member of the AKH/RPCH family.

A hypertrehalosemic neuropeptide from the corpora cardiac of the two tenebrionid beetle species, Tenebrio molitor and Zophobas rugipes, was purified by high performance liquid chromatography, and its sequence determined by pulsed-liquid phase sequencing employing Edman degradation after deblocking enzymatically the N-terminal pyroglutamate residue. Additionally, the C-terminus of the peptide was blocked as shown by the lack of breakdown using carboxypeptidase. In both species an identical octapeptide, designated Tem-HrTH, with the following amino acid sequence, was found: pGlu-Leu-Asn-Phe-Ser-Pro-Asn-Trp-NH2. This primary sequence has an 88% homology with the hypertrehalosemic hormone I (Pea-CAH-I) from the American cockroach as well as with the red pigment-concentrating hormone (RPCH) of prawns. Injection of the synthetic peptide into larvae or young adults of T. molitor or adult Z. rugipes increases the hemolymph carbohydrate levels in a dose-dependent manner. Thin layer chromatography identified the elevated sugar component of the hemolymph as the disaccharide trehalose. Carbohydrate release from larval fat body in vitro was also shown upon administration of a low concentration of synthetic Tem-HrTH.

Structure-activity relationships for Periplaneta americana hypertrehalosemic hormone. I: The importance of side chains and termini.

Single amino acid replacement analogues for the native hypertrehalosemic hormone I of the American cockroach, Periplaneta americana (Pea-CAH-I: pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH2), have been prepared by solid-phase peptide synthesis, and complete dose-response curves have been measured in P. americana monitoring the carbohydrate-mobilizing activity in vivo. All analogues that elicited hypertrehalosemia showed similar time-response courses, indicating that transport and degradation rates were comparable. Comparison of the potency and efficacy parameters of the analogues under study in the dose-response curves revealed four activity groups: 1) analogues that had the aromatic amino acids at positions 4 (phenylalanine) or 8 (tryptophan) replaced by alanine and glycine, respectively, had trace activity; 2) analogues with alanine at positions 1 or 2 had low potencies and an apparent biphasic dose-response relationship without much observable loss of efficacy; 3) analogues with glycine at positions 6 and 7 had potencies and efficacies most similar to Pea-CAH-I; and 4) analogues that had either an alanine instead of asparagine residue at position 3, or had a substitution of the carboxylamide function at the C-terminus by a carboxyl function reached apparent saturation, but only achieved 50-57% of the maximum activity of the native peptide. The potency profile for the analogue set is consistent with the importance of the N-terminal pentapeptide and the C-terminal tryptophan interacting with receptor(s) more closely than the side chains at positions 6 and 7, which are predicted to be the corner residues of a beta-turn. Finally, the biphasic dose-response curves observed for more than one analogue suggest the potential that receptors for Pea-CAH-I exist in more than one form.

Isolation and primary structures of neuropeptides of the AKH/RPCH family from various termite species.

We have isolated neuropeptides of the AKH/RPCH family from extracts of whole heads of four termite species (Mastotermes darwiniensis, Microhodotermes viator, Hodotermes mossambicus, and Trinervitermes trinervoides) using the effect of mobilizing lipids in Locusta migratoria for bioassay. Isolation was essentially achieved by two steps of reversed-phase chromatography (on phenyl-support followed by C-18). The peptides were identified by Edman degradation after deblocking with oxoprolyl peptidase. Each termite species contained only one AKH/RPCH family member. The primary structure in M. darwiniensis and T. trinervoides is pGlu-Val-Asn-Phe-Ser-Pro-Asn-Trp-NH2, a peptide previously found mainly in cockroaches and code named Pea-CAH-I. The peptide from M. viator has the primary sequence pGlu-Ile-Asn-Phe-Thr-Pro-Asn-Trp-NH2; it is a novel member of the family and is code-named Miv-CC (Microhodotermes viator corpus cardiacum peptide). Phylogenetic relations between the known cockroach and mantid AKH/RPCH octapeptides and the termite peptides from this study could be revealed employing the parsimony method. Based on a computer analysis, using PAUP 3.1.1., we concluded that termites are plesiomorphic with regard to cockroaches, and mantids are the sister taxon to the termite/cockroach group.

A novel peptide in the AKH/RPCH family isolated from the corpora cardiaca of the Emperor dragonfly, Anax imperator.

Using a heterologous (in locusts and cockroaches) and a homologous bioassay, we have isolated the neuropeptide pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp-NH2 from extracts of corpora cardiaca of the Emperor dragonfly, Anax imperator. The sequence elucidation was achieved by Edman degradation of the deblocked peptide and by electrospray mass spectrometry. Low concentrations of the synthetic peptide injected into the Emperor dragonfly increased the hemolymph lipid concentration, suggesting a possible role of the peptide in lipid homeostasis during flight. Therefore, it is named Ani-AKH, Anax imperator adipokinetic hormone.

Characterization and sequence elucidation of a novel peptide with molt-inhibiting activity from the South African spiny lobster, Jasus lalandii.

We have isolated a peptide from extracts of sinus glands from a South African spiny lobster species, Jasus lalandii, by high-performance liquid chromatography (HPLC) and identified it as a putative molt-inhibiting hormone (MIH) by (i) an in vitro assay with J. lalandii Y-organs to measure the inhibition of ecdysteroid synthesis and (ii) an immunoassay using antiserum raised against MIH of the edible crab. The MIH of J. lalandii has 74 amino acid residues, a molecular mass of 9006 Da, a free N-terminus and an amidated C-terminus. The full primary sequence has been obtained from sequencing various digest fragments (tryptic, endoproteinase Asp-N, cyanogen bromide) of the unreduced (native) peptide: RFTFDCPGMMGQRYLYEQVEQVCDDCYNLYREEKIAVNCRENCFLNSWFTVCLQATMREHETPRFDIWR SIILKA-NH(2). Structural comparisons with other peptides show that the J. lalandii MIH belongs to the peptide family which includes the crustacean hyperglycemic hormone, molt-inhibiting hormone and vitellogenesis-inhibiting hormone (cHH/MIH/VIH). This novel peptide has 36-43% sequence identity to putative MIHs from other decapod crustaceans and 32-34% identity to the two cHH peptides previously identified in this spiny lobster species. This is the first report of a peptide with MIH activity in the Palinuridae infraorder.

Sequence analyses of two neuropeptides of the AKH/RPCH-family from the lubber grasshopper, Romalea microptera.

Two neuropeptides with adipokinetic activity in Locusta migratoria and hypertrehalosaemic activity in Periplaneta americana were purified by high-performance liquid chromatography from the corpus cardiacum of the lubber grasshopper, Romalea microptera. The sequences of both peptides, designated Ro I and Ro II, were determined by gas-phase sequencing employing Edman degradation after the N-terminal pyroglutamate residue was enzymatically deblocked, as well as by fast atom bombardment mass spectrometry. Ro I was found to be a decapeptide with the primary structure: pGlu-Val-Asn-Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2, whereas Ro II is an octapeptide with the structure: pGlu-Val-Asn-Phe-Ser-Thr-Gly-Trp-NH2. Ro II is identical with AKH-G isolated from the cricket Gryllus bimaculatus. Synthetic materials having the assigned structures were found to be chromatographically, mass spectrometrically, and biologically indistinguishable from the natural peptides, confirming the sequences and establishing the Romalea peptides as members of the AKH/RPCH-family of peptides.

Isolation and structural elucidation of two pyrokinins from the retrocerebral complex of the American cockroach.

By monitoring the contractile activity of the hyperneural muscle of the American cockroach in vitro two peptides were isolated from the retrocerebral complex of the American cockroach. Three purification steps using reversed-phase high performance liquid chromatography on C-18 columns containing trifluoroacetic acid or heptafluorobutyric acid as organic modifiers were sufficient to achieve homogeneous peptide preparations. The structures of both peptides were elucidated by a combination of Edman degradation and mass spectrometry which yielded the following structures: His-Thr-Ala-Gly Phe-Ile-Pro-Arg-Leu-NH2 (Pea-PK-1) and Ser-Pro-Pro-Phe-Ala-Pro-Arg-Leu-NH2 (Pea-PK-2). The C-terminal sequence Phe-X-Pro-Arg-Leu-NH2 characterized the peptides as members of the insect pyrokinin family. The synthetic peptides were shown to have the same retention times as the natural peptides. The occurrence of both peptides in the retrocerebral complex suggests a physiological role as neurohormones. The effects of the synthetic pyrokinis were clearly distinguishable in their actions on the hyperneural muscle. Regarding the threshold concentrations, Pea-PK-2 was only 0.3% as active as Pea-PK-1.

Primary structures of a second hyperglycemic peptide and of two truncated forms in the spiny lobster, Jasus lalandii.

We have isolated a 72-amino acid peptide from extracts of sinus glands of the South African rock lobster, Jasus lalandii, and identified it, functionally and immunologically, as a hyperglycemic hormone. This is the second peptide with hyperglycemic activity found in this palinurid species and, because it occurs in smaller quantities (approximately 3 pmol/sinus gland) than the previously identified hyperglycemic hormone [14], this minor isoform is designated Jala cHH-II. The complete elucidation of the primary structure of cHH-II, as determined by automated Edman degradation of the N-terminus enzymatic digests of the non-reduced peptide, chemical cleavage and mass spectrometry, is presented here. Jala cHH-II (molecular mass of 8357 Da) is more hydrophobic than Jala cHH-I (8380 Da). The two cHHs have a free N-terminus a blocked C-terminus; and share 90% sequence homology. We also present structural data of a further two peptides isolated from sinus gland extracts that were immunopositive to cHH antisera. These peptides, with masses of 7665 and 7612 Da, structurally represent C-terminally truncated forms of the major and the minor Jala cHH peptides, respectively, but do not have any hyperglycemic activity in vivo. We demonstrate that the prevalence of these truncated forms can be reduced by the addition of proteases to the homogenization buffer during preparation of the tissues.

Molecular cloning and localization of a cDNA encoding a crustacean hyperglycemic hormone from the South African spiny lobster, Jasus lalandii.

A cDNA, encoding a crustacean hyperglycemic hormone (cHH) of the South African spiny lobster, Jasus lalandii has been cloned. The cDNA consists of 1773 bp with an open reading frame of 399 bp that encodes a preprohormone of 133 amino acid residues. The preprohormone consists of a 25 amino acid hydrophobic signal peptide, a 32 amino acid cHH precursor-related peptide (CPRP) and the cHH sequence of 72 amino acid residues. The cHH sequence is flanked N-terminally by a Lys-Arg cleavage site and C-terminally by Gly-Lys, where Gly serves as an amidation site. The deduced amino acid sequence of the CPRP is in complete agreement with a peptide previously elucidated from sinus glands of J. lalandii, code-named CPRP 2 and the sequence of the cHH peptide matches that of the minor cHH isoform of J. lalandii, i.e. crustacean hyperglycemic hormone-II (cHH-II), which was also previously obtained by peptide sequencing. In situ hybridization on eyestalks revealed strong cHH-II mRNA expression in a subset of neurosecretory cells of the X-organ.