Distribution and characterization of Corazonin in the central nervous system of Triatoma infestans (Insecta: Heteroptera)

The distribution of corazonin in the central nervous system of the heteropteran insect Triatoma infestans was studied by immunohistochemistry. The presence of corazonin isoforms was investigated using MALDI-TOF mass spectrometry in samples containing the brain, the subesophageal ganglion, the corpora cardiaca-corpus allatum complex and the anterior part of the aorta. Several groups of immunopositive perikarya were detected in the brain, the subesophageal ganglion and the thoracic ganglia. Regarding the brain, three clusters were observed in the protocerebrum. One of these clusters was formed by somata located near the entrance of the ocellar nerves whose fibers supplied the aorta and the corpora cardiaca. The remaining groups of the protocerebrum were located in the lateral soma cortex and at the boundary of the protocerebrum with the optic lobe. The optic lobe housed immunoreactive somata in the medial soma layer of the lobula and at the level of the first optic chiasma. The neuropils of the deutocerebrum and the tritocerebrum were immunostained, but no immunoreactive perikarya were detected. In the subesophageal ganglion, immunostained somata were found in the soma layers of the mandibular and labial neuromeres, whereas in the mesothoracic ganglionic mass, they were observed in the mesothoracic, metathoracic and abdominal neuromeres. Immunostained neurites were also found in the esophageal wall. The distribution pattern of corazonin like immunoreactivity in the central nervous system of this species suggests that corazonin may act as a neurohormone. Mass spectrometric analysis revealed that [Arg⁷]-corazonin was the only isoform of the neuropeptide present in T. infestans tissue samples.     

Characterization of a diuretic hormone receptor from the tobacco hornworm,Manduca sexta

We have characterized a diuretic hormone receptor from the tobacco hornworm, Manduca sexta. A single high affinity binding site for the 41 amino acid M. sexta diuretic hormone was found in membranes prepared from Malpighian tubules of fifth stadium larvae. The site has a Kd = 79 pM and Bmax = 3.1 pmol/mg protein. The dissociation rate constant was determined to be 0.11 min^?1 with a corresponding half-life of 6.4 min. Receptor binding of the hormone is inhibited by Ca²⁺ and Mg2⁺, while Na⁺ and K⁺ inhibit binding to a lesser extent. Truncated diuretic hormone analogs in which up to 20 amino acids were removed from the N-terminus maintain high affinity for the receptor. A diuretic hormone from Locusta migratoria which has 43% sequence identity with the M. sexta diuretic hormone also possesses a high affinity for the receptor. Conformational analysis of the M. sexta diuretic hormone indicates the core region of the peptide assumes a helical conformation, which may have implications in the binding of the peptide to the receptor. © 1993 Wiley-Liss. Inc.     

Effects of lignoids on a hematophagous bug, Rhodnius prolixus: feeding, ecdysis and diuresis

The effects of lignoids on feeding, ecdysis and diuresis in fourth-instar larvae of Rhodnius prolixus (Hemiptera) were investigated. Up to 100 microg/ml burchellin, podophyllotoxin, pinoresinol, sesamin, licarin A, or nordihydroguaiaretic acid (NDGA) in the diet did not induce antifeedant effects. Pinoresinol and NDGA significantly inhibited ecdysis. In experiments in vivo, burchellin and podophyllotoxin reduced the production of urine after feeding. 5-Hydroxytryptamine (5-HT), a diuretic hormone, partially counteracted this effect of burchellin. In experiments in vitro, using isolated Malpighian tubules, (i) burchellin reduced diuretic hormone levels in the hemolymph but not the amount of diuretic hormone stored in the thoracic ganglionic masses (including axons), (ii) burchellin decreased the volume of urine secreted by isolated Malpighian tubules, and (iii) 5-HT could not overcome the effect of burchellin upon the Malpighian tubules. We conclude that burchellin interfered with the release, but not with the production of diuretic hormone by the thoracic ganglionic mass or induced an antidiuretic hormone and directly affected the Malpighian tubules.     

Binding of heparin to antithrombin III: The use of dansyl and rhodamine labels

Low molecular weight heparin of low-anticoagulant activity and high molecular weight heparin of correspondingly high activity were prepared by chromatography on protamine-Sepharose; preparations subjected to limited N-desulfation (5–10% free amino groups) by solvolysis were labeled with 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) or rhodamine B isothiocyanate (RITC). The fluorescent heparins retained approximately 50% of the original anticoagulant activities. Dansyl-heparin on binding to antithrombin III (ATIII) exhibited a 2.5-fold enhancement of dansyl fluorescence intensity. This effect could be prevented by excess unlabeled heparin. A 7900 molecular weight dansyl-heparin preparation bound to ATIII with a stoichiometry of close to 2:1 and with an apparent association constant for binding (K_a) of 4.9 × 10⁵, m^?1, whereas a 21,600 molecular weight fraction bound at 0.7:1 with the protein and with an apparent K_a = 7.9 × 10⁵, m^?1. When ATIII reacted with a mixture of low molecular weight dansyl-heparin and low molecular weight RITC-heparin, there was enhancement of RITC fluorescence emission when excited at the dansyl excitation maximum; this effect was not observed when either of the labeled heparin species was prepared from high molecular weight material. The results are consistent with the proposal that a single molecule of high molecular weight, high-activity heparin occupies two sites when it binds to ATIII, whereas low molecular weight, low-activity heparin binds to the two sites separately.     

The synthesis of human placental lactogen hormone (hPL) in a cell-free wheat germ system

Total polysomes from human term placenta were incubated in a wheat germ cell-free system during 1 hr at 25° C. Human placental lactogen hormone was identified among the proteins synthesized in vitro by immune precipitation and subsequent sodium dodecylsulphate electrophoresis of the immunoprecipitate. The zone containing the radioactivity from the immunoprecipitate [³H]-labelled lactogen hormone comigrated exactly with the radioactivity zone from added [¹⁴C]-labelled marker hormone. This result indicates that the molecular weight of the synthesized product must be equal or very similar to that of the native protein hormone.     

Serotonin has kinin-like activity in stimulating secretion by Malpighian tubules of the house cricket Acheta domesticus

Serotonin stimulates secretion by Malpighian tubules (MT) of a number of insects, and functions as a diuretic hormone in Rhodnius prolixus and in larval Aedes aegypti. Serotonin is here shown to be a potent stimulant of secretion by MT of the house cricket, Acheta domesticus, with an apparent EC₅₀ of 9.4 nmol L⁻¹, although its diuretic activity is just 25% of the maximum achievable with either the native CRF-related peptide, Achdo-DH, or a crude extract of the corpora cardiaca. In this respect, the diuretic activity of serotonin is similar to that of the cricket kinin Achdo-KI, and when tested together their actions are not additive, which suggests they target the same transport process. Consistent with this suggestion, the activity of serotonin is chloride-dependent and is associated with a non-selective stimulation of NaCl and KCl transport. In common with Achdo-KI, serotonin has no effect on cAMP production by isolated MT, and both act synergistically with exogenous 8bromo-cAMP in stimulating fluid secretion, most likely by promoting the release of Ca²⁺ from intracellular stores. A number of serotonin agonists and antagonists were tested to determine the pharmacological profile of receptors on cricket MT. The results are consistent with the diuretic activity of serotonin being mediated through a 5-HT₂-like receptor.     

Purification and molecular properties of a soluble ferredoxin from Rhodopseudomonas capsulata

A soluble ferredoxin was purified from the photosynthetic bacterium Rhodopseudomonas capsulata and characterized. Unlike Rhodospirillum rubrum, where two soluble ferredoxins have been found, only a single species was found in Rps. capsulata. The amino acid composition, ultraviolet-visible spectral properties, molecular weight (12000) and biological activity were determined. The ultraviolet-visible spectrum is similar to that of other bacterial ferredoxins, with a maximum when oxidized at 380 nm (? = 26.1 · 10³ M^-1 · cm^-1). The possible roles of this ferredoxin in the cellular metabolism are discussed.     

Action patterns of immobilized dextranase

Dextranase, isolated from Penicillium funiculosum and P. lilacinum, was immobilized on porous, silanized-silica beads and a phenol-formaldehyde resin. A commercial dextran of relatively low molecular weight (～2 × 10⁶) was degraded by immobilized dextranase, with the formation of reducing sugars, but with little decrease in viscosity. In contrast, soluble dextranase caused rapid loss of viscosity, but only a slight increase in reducing sugar. Native dextran of high molecular weight, from Leuconostoc mesenteroides NRRL B-512 (F), was attacked very slowly by immobilized dextranase, with the release of oligosaccharides of low molecular weight.     

Immunoreactive Parathyroid Hormone in Circulation of Man

WE have reported that parathyroid hormone (PTH) is secreted from the parathyroid in vivo as a polypeptide of eighty-four amino-acids, identical to the hormone stored in the glands (molecular weight of 9,500), but that the hormonal polypeptide is cleaved after it enters the general circulation¹. A large hormonal fragment from this cleavage, with a molecular weight of approximately 7,500, has been identified in the circulation. The fragment differs immunologically from the hormone secreted and extracted from the glands¹. To analyse the biological significance of the metabolism of the hormone and the chemical nature and hormonal activity of the large circulating fragment, we have developed radioimmunoassays that specifically measure the amino-terminal (N-assay) and carboxyl-terminal (C-assay) regions of the hormonal molecule. We now report that much higher concentrations of immunoreactive hormone are found in the general circulation by the C-assay than by the N-assay. The studies with the N-assay indicate that the large fragment has lost a portion of the amino-terminal sequence required for biological activity⁹. Since the fragment is present in much higher concentration than native uncleaved hormone, we must conclude that much of the immunoreactive PTH detected in the circulation is biologically inactive.     

In vitro oxidation of indoleacetic Acid by soluble auxin-oxidases and peroxidases from maize roots

Soluble auxin-oxidases were extracted from Zea mays L. cv LG11 apical root segments and partially separated from peroxidases (EC 1.11.1.7) by size-exclusion chromatography. Auxin-oxidases were resolved into one main peak corresponding to a molecular mass of 32.5 kilodaltons and a minor peak at 54.5 kilodaltons. Peroxidases were separated into at least four peaks, with molecular masses from 32.5 to 78 kilodaltons. In vitro activity of indoleacetic acid-oxidases was dependent on the presence of MnCl₂ and p-coumaric acid. Compound(s) present in the crude extract and several synthetic auxin transport inhibitors (including 2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid) inhibited auxin-oxidase activity, but had no effect on peroxidases. The products resulting from the in vitro enzymatic oxidation of [³H] indoleacetic acid were separated by HPLC and the major metabolite was found to cochromatograph with indol-3yl-methanol.     

Biochemical and structural studies of actomyosin-like proteins from non-muscle cells. Isolation and characterization of myosin from amoebae of Dictyostelium discoideum

A myosin-like protein was purified from amoebae of the cellular slime mold Dictyostelium discoideum. The purification utilized newly discovered solubility properties of actomyosin in sucrose. The amoebae were extracted with a 30% sucrose solution containing 0.1 m-KCl, and actomyosin was selectively precipitated from this crude extract by removal of the sucrose. The myosin and actin were then solubilized in a buffer containing KI and separated by gel filtration.The purified Dictyostelium myosin bears a very close resemblance to muscle myosin. The amoeba protein contains two heavy chains, about 210,000 molecular weight each, and two classes of light chains, 16,000 and 18,000 molecular weight. Dictyostelium myosin is insoluble at low ionic strength and forms bipolar thick filaments. The myosin possesses ATPase activity that is activated by Ca²⁺ but not EDTA, and is inhibited by Mg²⁺; under optimal conditions the specific activity of the enzyme is 0.09 μmol P₁/min per mg myosin.Dictyostelium myosin interacts with Dictyostelium actin or muscle actin, as shown by electron microscopy and by measurements of enzymatic activity. The ATPase activity of Dictyostelium myosin, in the presence of Mg²⁺ at low ionic strength, exhibits an average ninefold activation when actin is added.     

Isolation of the diapause hormone from the silkworm,Bombyx mori

The diapause hormone (DH) secreted from the suboesophageal ganglion is responsible for the embryonic diapause in the silkworm, Bombyx mori. It was extracted from two million mated male adult heads. At least two species of DH were separated from the extracts (a complex lipid fraction) by gel permeation chromatography with Sephadex LH-20 by organic eluants. One of them was further purified by repeating gel permeation column chromatography with Merckogel^® Type OR 6000 in the cold and dark to give finally a single peak with high DH activity. This preparation seems to be chemically pure, and its molecular weight is chromatographically estimated to be between 2000 and 4000. Other characteristics of DH are also presented. Biological activity of the final DH preparation is discussed with reference to other insect hormones such as juvenile hormone and α-ecdysone.     

The effect of greening of sorghum leaves on the molecular weight of a complex containing 4-hydroxycinnamic Acid hydroxylase activity

During the greening of leaves of Sorghum bicolor var. Wheatland milo, the activity of 4-hydroxycinnamic (p-coumaric) acid hydroxylase in pH 6 buffered extracts was shifted from a relatively low to a high molecular weight fraction. Differences between these forms found in etiolated and green leaves were based on differential centrifugation, ammonium sulfate precipitation, and on elution patterns from Agarose A-15m. Both molecular weight forms were precipitated by protamine sulfate at pH 6, and approximately 40 to 80% of the activity of each form was associated with a 500 to 37,000g pellet when tissues were ground at pH 8 in media of either high or low osmotic concentration. Although no fraction with hydroxylase activity was ever found without any chlorogenic acid oxidase activity, the two activities frequently varied independently, and could be partially separated from each other, using the above techniques. Comparisons were made with the very small molecular weight form of 4-hydroxycinnamic acid hydroxylase characteristic of tissues of first internodes. The significance of these results in terms of possible multienzyme complexes capable of converting phenylalanine and tyrosine to cinnamic acid derivatives is discussed.     

Comparative immunochemistry of phytochrome

Partially purified high molecular weight preparations of phytochrome, estimated to be close to 440,000 molecular weight based upon chromatography through a calibrated Bio-Gel P-300 column, were obtained from Garry and Newton oats (Avena Sativa L., cv. Garry and cv. Newton), rye (Secale cereale L., cv. Balbo), barley (Horedum vulgare L., cv. Harrison), and pea (Pisum sativum L., cv. Alaska) by a sequence of three chromatographic steps: brushite, diethylaminoethyl cellulose, and Bio-Gel P-300. No significant differences were observed between these preparations during purification or subsequent handling. In addition, a low molecular weight form of phytochrome was purified from Garry oats. Two specific antisera against a low molecular weight form of phytochrome (60,000 molecular weight) obtained from etiolated Garry oat seedlings are characterized and used to compare the phytochrome preparations. Double diffusion assays indicated antigenic identity between all preparations except that pea phytochrome yielded a spur when compared to oat phytochrome. Micro complement fixation assays yielded complete identity between Garry and Newton oat phytochrome, reduced activity with rye and barley phytochrome, and a complete lack of activity with pea phytochrome at the serum dilutions assayed. Immunoelectrophoretic assays indicated that all high molecular weight phytochrome preparations were homogeneous by this criterion and that there were only slight differences between the preparations in electrophoretic mobility. Large and small forms of phytochrome isolated from Garry oats were found to be very similar antigens when tested with the anti-small phytochrome sera, although the small form was observed to electrophorese at a much slower rate than the large.     

Vitellogenin synthesis in female larvae of the gypsy moth,Lymantria dispar (L.): suppression by juvenile hormone

• 1.1. Fat body from feeding-phase, last instar gypsy moth females incorporates l-[³⁵S]methionine in vitro into two vitellogenins with the same molecular masses (165 and 180 kDa) as the apo-vitellogenins found in teh hemolymph and the apo-vitellins in teh eggs.
• 2.2. Both apo-vitellogenins are observed in the medium of fat body cultures, but only the 180 kDa apo-vitellogenin is observed in extracts of cultured tissue.
• 3.3. Synthesis and accumulation of the apo-vitellogenins are suppressed in a dose-dependent manner by topical treatment with the juvenile hormone analog, methoprene, prior to day 4.
• 4.4. This suppression suggests that a declining juvenile hormone titre is involved in the initiation of vitellogenin synthesis.

     

Heterogeneity of mitochondrial DNA from Saccharomyces carlsbergensis: renaturation and sedimentation studies

The renaturation kinetics of mitochondrial DNA from the yeast Saccharomyces carlsbergensis have been studied at different temperatures and molecular weights. At renaturation temperatures 25 deg. C below the mean denaturation temperature (T_m) in 1 M-sodium chloride the renaturation rate constant is found to decrease with increasing molecular weight of the reacting strands. This unusual molecular weight dependency gradually disappears with an increase in the renaturation temperature. At a temperature 10 deg. C below the melting point, the rate constant shows the normally expected increase with the square root of the molecular weight. From the renaturation data at this temperature, the molecular weight of the mitochondrial genome is estimated to be about 5·0 × 10⁷. The same size of genome was found from renaturation at low molecular weight and 25 deg. C below the T_m.The sedimentation properties of denatured mitochondrial DNA at pH values 7·0 to 12·5 were used to study the conformation of this DNA in 1 M-sodium chloride. The results obtained support the conclusion from the renaturation studies: that the pieces of denatured mitochondrial DNA with a molecular weight above 2 × 10⁵ to 3 × 10⁵, in 1 M-sodium chloride at 25 deg. C below the mean denaturation temperature are not fully extended random coils. Presumably, interaction between adenine and thymine-rich sequences, which are clustered at certain distances within the molecules, is the molecular basis for these observations.     

Stable Radical Content and Anti-Radical Activity of Roasted Arabica Coffee: From In-Tact Bean to Coffee Brew

The roasting of coffee beans generates stable radicals within melanoidins produced by non-enzymatic browning. Roasting coffee beans has further been suggested to increase the antioxidant (AO) capacity of coffee brews. Herein, we have characterized the radical content and AO capacity of brews prepared from Coffea arabica beans sourced directly from an industrial roasting plant. In-tact beans exhibited electron paramagnetic resonance signals arising from Fe³⁺, Mn²⁺ and at least three distinct stable radicals as a function of roasting time, whose intensity changed upon grinding and ageing. In coffee brews, the roasting-induced radicals were harboured within the high molecular weight (> 3 kD) melanoidin-containing fraction at a concentration of 15 nM and was associated with aromatic groups within the melanoidins. The low molecular weight (< 3 kD) fraction exhibited the highest AO capacity using DPPH as an oxidant. The AO activity was not mediated by the stable radicals or by metal complexes within the brew. While other non-AO functions of the roasting-induced radical and metal complexes may be possible in vivo, we confirm that the in vitro antiradical activity of brewed coffee is dominated by low molecular weight phenolic compounds.     

Isolation and immunological studies of high and low molecular weight cysteine proteinase inhibitors in bovine serum

Two kinds of cysteine proteinase inhibitor (M_r 145 000 and M_r 15 500) were purified from bovine serum. These purified inhibitors showed a single band on SDS-polyacrylamide gel electrophoresis, respectively. The isoelectric point of the high molecular weight inhibitor was found to be 4.4 and that of the low molecular weight inhibitor was 8.6. The high molecular weight inhibitor inhibited papain and cathepsin H, but had little activity against cathepsin B. While the low molecular weight inhibitor was a strong inhibitor of papain and cathepsin H and showed a weak inhibition of cathepsin B. These two inhibitors showed different immunological reactivities.     

Organization of the glycolytic enzymes in Escherichia coli

Differential centrifugation of osmotically lysed lysozyme-EDTA spheroplasts from Escherichia coli sedimented 50–70% of the glycolytic activities examined in a low speed pellet; the remaining activity, occurring in a high speed supernatant, contained the soluble enzymes of the cell. The distribution pattern of the enzymes could be altered by extrusion of the spheroplasts through the French Press or by lysis at different pH values. Electron micrographs of the pellet fraction revealed lysed spheroplasts mostly devoid of cellular constituents but consisting of cytoplasmic membranes surrounded by partially degraded cell wall fragments. Washing of the pellet showed that the enzymes were not all bound to the same degree to the membrane fraction. Throughput activity of the glycolytic pathway was demonstrated for the membrane fraction, but none was observed for the soluble fraction of the cell (i.e. for enzymes present in the supernatants) unless these were first concentrated by ultrafiltration. The supernatant from the lysed spheroplasts, together with a further supernatant obtained by washing the membrane pellet, was concentrated by ultrafiltration and chromatographed on a Bio-Gel column. The eluate contained glycolytic activities both in fractions corresponding to relatively high and relatively low molecular weight material The high molecular weight species, containing a proportion of all the enzymes studied, had a molecular weight of at least 1.2 × 10⁶. A multienzyme aggregate containing one each of the glycolytic enzymes would have a molecular weight of ～ 1.3 × 10⁶. The specific rate of pyruvate formation from glucose by the high molecular weight species was similar to that obtained from a preparation in which the fractions containing all the low molecular weight material enzyme activities were pooled and concentrated by ultrafiltration. Using the high molecular weight material, studies were made of the ability of added unlabelled glycolytic intermediates to compete for catalytic sites with intermediates produced endogenously from [¹⁴C₆] glucose. The relatively weak competition observed indicated a high degree of protection afforded the labelled intermediates derived from [¹⁴C₆] glucose.     

Auditory interneurones in the mesothoracic ganglion of crickets

Auditory interneurone responses in the mesothoracic ganglion of the cricket Gryllus bimaculatus were investigated with special regard to temporal features of the calling song. Units representing five response types were found. One type codes verse syllables and intensity. The second codes syllables of highfrequency verses. The third responds as a pulse marker. The fourth shows adaptation and the response pattern depends on the verse frequency. The fifth fires a burst at verse onset.Responses of mesothoracic units recorded in two other cricket species do not differ markedly from those of Gryllus bimaculatus. Particularly, no tuning is found to species-specific differences in their calling songs.The stimulus direction can affect the threshold in different ways: dependence at all frequencies, dependence only between 3 and 6 kHz, and independence are found. The dependence is mainly expressed by a higher threshold for contralateral sounds.The mesothoracic branching of a few neurones was demonstrated by extracellular CoS-staining. These cells pass through the ganglion as connective fibres giving off small branches into the ventro-medial and dorso-medial neuropiles.