Release from the brain and acquisition by the corpus cardiacum of a neurohormone in vitro

Hindgut stimulating neurohormone (HSN) was synthesized and stored by cultured brains of the cockroach, Leucophaea maderae. The presence of either HSN or cultured corpora cardiaca in the medium caused the brains to release their accumulated HSN. The corpora cardiaca were able to sequester HSN from the medium when the concentration was above a threshold level. Thus, the corpus cardiacum may provide an homeostatic mechanism for maintaining physiological levels of neurohormone.     

In vitro synthesis and release of proteins by fat body and ovarian tissue of Leucophaea maderae during the sexual cycle

Fat body, ovaries without their surrounding connective tissue, and ovarian connective tissue of the ovoviviparous cockroach Leucophaea maderae were incubated in vitro. Incorporation of ¹⁴C-labelled amino acids into proteins by all three tissues was investigated quantitatively and qualitatively during oöcyte maturation (corpora allata active), immediately after ovulation, and during the gestation period (corpora allata inactive). Proteins in the tissues and in the incubation media were processed separately. The qualitative analysis involving disk electrophoretic separation of Ringer soluble tissue proteins or of proteins in the incubation media and subsequent autoradiography of the dried gels showed that all three tissues synthesize the same 26 proteins in all stages of the sexual cycle. An additional fraction is produced by the ovarian connective tissue only. All three tissues synthesize proteins at a higher rate during oöcyte maturation than during gestation.     

Isolation,primary structure and synthesis of leucomyosuppressin,an insect neuropeptide that inhibits spontaneous contractions of the cockroach hindgut

1. A neuropeptide that inhibits spontaneous contractions of the isolated cockroach hindgut was purified from head extracts of the cockroach, Leucophaea maderae.2. The inability of aminopeptidase M to degrade the peptide and the presence of glutamic acid in the hydrolysate suggested N-terminal blocking by pyroglutamic acid. The N-terminal pGlu was removed enzymatically and the unblocked fragment was sequenced with an automated peptide sequencer.3. The structure determined (pGlu-Asp-Val-Asp-His-Val-Phe-Leu-Arg-Phe-NH₂) was synthesized and shown to be both chemically and biologically identical with the natural product.4. Leucomyosuppressin is the first inhibitory neuropeptide isolated and structurally identified from an insect source.     

Analysis of cockroach oothecae and exuviae by solid-state 13C-NMR spectroscopy

Sclerotized oothecae from four species of cockroaches, Periplaneta americana, P. fuliginosa, Blatta orientalis and Blattella germanica, were examined by solid-state ¹³C-nuclear magnetic resonance and chemical analyses. The oothecae were composed of protein, water, calcium oxalate, diphenolic compounds, lipid, and uric acid. Calcium oxalate was the major soluble component in egg cases of P. americana, P. fuliginosa, and B. orientalis. Oothecae of B. germanica had approx. 10-fold less calcium oxalate and extractable diphenols than the other species. The major diphenolic compound extracted in cold dilute perchloric acid was 3,4-dihydroxybenzoic acid. Exuviae from P. americana, B. germanica, Gromphadorhina portentosa, Blaberus craniifer, and Leucophaea maderae also were examined by solid-state ¹³C-NMR. They contained protein, diphenols, and lipid, as well as chitin, which accounted for 30–42% of the organic content, depending upon the species.     

Enzymatic and immunological characterization of the conditioning factor for epidermal outgrowth in the cockroach Leucophaea maderae

A partial characterization of a haemocyte derived conditioning factor which permits rapid in vitro outgrowth of insect epidermis (Leucophaea maderae) was attempted. The factor resisted denaturating agents such as heat (100°C), ethanol, acetone, formol, and acetic acid. It was not extractable by hot pyridine or detergent, nor destroyed by lipase, RNase, DNase, or glycolytic enzymes. The factor was, however, completely inactivated by all four proteolytic enzymes tested. Rabbit antisera were produced against a haemocyte homogenate and tested in vitro. One of the antisera contained antibodies against the conditioning factor. The nature and origin of the conditioning factor are discussed in some detail.     

Immunocytochemical localization of leucomyosuppressin-like peptides in the CNS of the cockroach,Leucophaea maderae

Immunocytochemistry was used to determine sites of synthesis and pathways for the transport of the neuropeptide, Leucomyosuppressin (pQDVDHVFLRFamide) in the cockroach,Leucophaea maderae. This study led to identification of neurons in the brain and thoracic ganglia reactive to polyclonal antibodies raised against this peptide. No immunoreactive cells were found in the subsophageal or abdominal ganglia. Although the corpus cardiacum contained no intrinsic cells immunoreactive to LMS antibodies, the periphery of this organ and that of the nervi corporis allati contain an abundance of LMS-reactive terminals.     

Isolation,primary structure,and synthesis of leucokinins V and VI: Myotropic peptides of Lleucophaea maderae

1. Two additional octapeptides which stimulate the contractile activity of the cockroach hindgut have been isolated from head extracts of the cockroach, Leucophaea maderae.2. A series of four high performance liquid-chromatographic (HPLC) fractionations yielded sufficient quantities of pure peptides for micro amino acid analysis and primary sequence determinations. The sequence determined for two peptides were: Gly-Ser-Gly-Phe-Ser-Ser-Trp-Gly-NH₂ and pGlu-Ser-Ser-Phe-His-Ser-Trp-Gly-NH₂. These octapeptides were designated leucokinins V and VI (L-V, L-VI), respectively.3. Minimum concentrations of natural L—V and L-VI required to produce a response from the isolated cockroach hindgut were 4.5 × and 4.9 × 10⁻¹¹ M, respectively. These concentrations were quite similar to the threshold concentrations observed for the synthetic products.     

Synthesis, transport, and release of a neurohormone by cultured neuroendocrine glands from the cockroach, Leucophaea maderae

A hindgut-stimulating neurohormone synthesized in vitro by the neurosecretory cells of cultured brains of Leucophaea maderae passes through the nervi corporis cardiaci I into the corpora cardiaca and is released into the culture medium. As much as 90 per cent of the hormone breaks down in the medium during a 3-day incubation period, and the amount recovered represents only a small fraction of the amount actually released.     

Differential adhesion of the haemocytes of Leucophaea maderae (Blattaria) to a glass surface

A system for the study of insect haemocytes in vitro is described. The system was used to analyse the adhesive properties of the haemocytes of the cockroach, Leucophaea maderae. The two main types of haemocytes, plasmatocytes and granulocytes, showed considerable differences in adhesive properties, which allowed the production of nearly homogeneous monolayers consisting of either plasmatocytes or granulocytes. The much stronger adhesion of the plasmatocytes is discussed in relation to their role in phagocytosis and encapsulation.     

Density-gradient centrifugation isolation of hormone-containing neurosecretory granules from the cockroach,Leucophaea maderae

Neurosecretory granules (NSG) containing hindgut-stimulating neurohormone (HSN) from Leucophaea maderae were isolated by densitygradient centrifugation of cockroach brain homogenates.High concentrations of HSN were consistently found in isolates containing large numbers of NSG. HSN was measured by bioassay and the NSG were identified by electron microscopy.     

Adenylate cyclase in the hindgut of the cockroach,Leucophaea maderae (F.)

An adenylate cyclase from the hindgut of the cockroach, Leucophaea maderae (F.), has been investigated. Several properties of the enzyme, including subcellular distribution, pH optimum, stimulation by NaF, effect of ATP and Mg²⁺, effect of divalent cations and effect of hindgut-stimulating neurohormone (HSN) were determined.Studies with isolated hindguts revealed that cyclic AMP potentiated the effect of HSN fourfold. However, HSN had no effect on basal- or fluoride-stimulated activities of adenylate cyclase in vitro. These observations are discussed with reference to the mode of action of HSN.     

Comparative actions of the neuropeptides leucopyrokinin and periplanetin CCI on the visceral muscle systems of the cockroaches Leucophaea maderae and Periplaneta americana

1. The effects of two structurally-related peptides, leucopyrokinin (LPK) and periplanetin CC-I (CCI), on contractile activities of visceral muscle systems were compared in the two cockroaches from which these peptides were originally isolated.2. LPK elicited consistent proctolin-like responses on the hindgut, foregut, oviduct and heart of the Madeira cockroach, Leucophaea maderae, with increases in both amplitude and frequency of contraction. CCI, on the other hand, elicited a mostly tonic response on these tissues.3. For the American cockroach, Periplaneta americana, the responses elicited by LPK and CCI were tonic in nature.4. With the exception of the response of the L. maderae hindgut and heart to LPK, threshold levels for either LPK or CCI on all other tissues of both roaches were considerably higher (10–100 times greater) than those for proctolin on the same tissues.5. The maximum response to any concentration of LPK or CCI on the foregut and oviduct of L. maderae and that on the foregut and hindgut of P. americana never reached more than 60% of the maximum contraction achieved with proctolin.     

The Leucophaea maderae hindgut preparation: A rapid and sensitive bioassay tool for the isolation of insect myotropins of other insect species

The isolated hindgut preparation of the cockroach, Leucophaea maderae has provided an effective bioassay tool for the isolation of certain structural types of insect myotropic peptides. Initially, the preparation was used to monitor excitatory and inhibitory activities of numerous HPLC fractions in a study that resulted in the structural characterization of 12 Leucophaea neuropeptides. Subsequently, the preparation was used as the bioassay for the isolation and structural characterization of myotropic neuropeptides of the house cricket, Acheta domesticus, and the locust, Locusta migratoria. Five novel myotropic peptides from the cricket were structurally characterized, and 32 separate myotropic compounds were isolated from nervous tissue of the locust. At present, 8 of the locust peptides have been structurally characterized. Isolation studies using this bioassay have been responsible for the discovery of 25 unique neuropeptides, 4 new peptide families, and the initial demonstration of the natural analog phenomenon in insects.     

Regulation of adenylate cyclase from Leucophaea maderae by calcium,calmodulin and forskolin

1. Adenylate cyclase was assayed in homogenates ofhindgut tissue from Leucophaea maderae (L.). The 10,000 g supernatant enzyme was stimulated by calmodulin.2. Trifluoperazine inhibited calmodulin-stimulated adenylate cyclase activity.3. Elevated calcium levels (> 100 μM) inhibited natural and calmodulin-stimulated enzymic activity.4. Forskolin (1 mM) stimulated adenylate cyclase activity by approximately 10-fold.     

Isolation and properties of a preparation of arterial collagen solubilized by pretreatment with alkali (author's transl)]

The acid-soluble, highly cross-linked aorta collagen, of which about 30% can be converted into a soluble form by alkali treatment, followed by extraction with aetic acid, was obtained predominantly in the form of monomeric, helical molecules, as indicated by the value for the intrinsic viscosity and its behaviour in sodium dodecylsulphate disc electrophoresis. Apart from decreased values for tyrosine (0.26%), arginine (4.4%) and aspartic acid (3.9%), the amino acid composition of the aorta collagen fraction was similar to that of the acid-soluble calf skin collagen. This finding, together with the cyanogen bromide peptide pattern, shows that the collagen extracted from the artery is predominantly type I. Treatment with alkali probably shortens the alpha1-CB6-peptide by about 45 amino acids. The collagen extracted from artery was compared with acid soluble skin collagen by sodium dodecylsulphate polyacrylamide electrophoresis. The arterial collagen showed a marked increase in the rations alpha1 to alpha2 (4:1), alpha to beta (3:1) and beta11 to beta12 (2.5:1). Compared with acid soluble skin collagen, the aorta collagen contained twice as much galactose and glucose (13.5 and 9.6 nmol/mg protein respectively), which are bound to hydroxylysine. 50% of the hydroxylysine residues are unsubstituted, 15% are present as galactosyl hydroxylysine, and 35% as glucosyl-galactosyl hydroxylysine. On the basis of its reported properties, arterial collagen obtained by the method of Fujii appears to be a suitable substrate for the study of the enzymic synthesis and enzymic degradation of hydroxylysine glycosides of native arterial collagen.     

Effects of food and water availability,and of NCA-1 section,upon juvenile hormone biosynthesis and oöcyte development in adult female Periplaneta americana

The combined stimuli from feeding, drinking, mating and crowding are required for the highest rates of oöcyte development in maturing adult female Periplaneta americana. A graded series of “sexually suppressed” females can be produced by withholding one or more of these stimuli, and this stepwise retardation of ovarian development appears to be achieved by a progressive increase in corpus allatum restrain. It seems that all of these environmental cues are centrally integrated such that juvenile hormone-dependent processes can proceed at an appropriate pace. Water availability is evidently the most important factor. Water-deprived females are sexually unreceptive, and are found to have very low rates of juvenile hormone biosynthesis and ovarian development. This holds true even when they are provided with food. In contrast, 75% of starved females are sexually receptive if allowed free access to drinking water. At the same time they have enhanced corpus allatum activity, and show significant oöcyte growth.The mode of regulation of corpus allatum function in adult female P. americana appears to be significantly different to the model proposed for the cockroaches Leucophaea maderae and Diploptera punctata. Allatotropic signals may be more important than inhibitory signals in the former species. The glands continue to be moderately active in fed, mated female P. americana after NCA-1 section (although a major peak of corpus allatum activity is not obvious), and the rate of oöcyte development is not greatly reduced. However, NCA-1 mediated inhibition of juvenile hormone biosynthesis is less readily demonstrated. We could observe no enhancement of corpus allatum activity nor stimulation of oöcyte growth after unilateral NCA-1 section when the operation was performed on starved virgins, and the same result was found after bilateral NCA-1 section when starvation or virginity were separately enforced. A slight stimulation of juvenile hormone biosynthesis, together with a small increase in oöcyte development, could only be demonstrated after both NCA-1 were cut in starved virgins.We conclude that neurally mediated corpus allatum inhibition in has yet to be adequately verified, and that the available evidence does not contradict the theory that juvenile hormone biosynthesis in adult females could be regulated predominantly by chemicals released into the haemolymph.     

Synthesis of vitellogenin after long-term ovariectomy in a cockroach

Synthesis of vitellogenin and its release into the haemolymph of vitellogenic and ovariectomized females of Leucophaea maderae were monitored. Six-and-a-half months after ovariectomy, release of vitellogenin from the fat bodies had declined to about 12% of that in normal females. At this time, vitellogenin made up over 70% of the total haemolymph proteins in ovariectomized females, whereas in normal vitellogenic females it represented only 5–6%. Synthesis of vitellogenin on ergastoplasmic membranes of the fat bodies continued in ovariectomized females at nearly the same level as in unoperated ones. Most of this vitellogenin was stored in the fat bodies. From these data it is concluded that a high vitellogenin titre in the haemolymph inhibits its further release from the fat bodies, yet has no or very little effect on vitellogenin production. In this species the ovaries do not exert any control over vitellogenin synthesis.     

Isolation and partial characterization of a second myotropic peptide from the hindgut of the cockroach,Leucophaea maderae

• 1.1. Proctolin and a second myotropic peptide were extracted from the hindgut of the cockroach Leucophaea maderae with methanol-water-acetic acid (90:9:1). The two peptides were easily separated by HPLC on a μ-Bondapak-phenyl column.
• 2.2. Like proctolin, the second peptide was heat stable and was inactivated by the exopeptidases aminopeptidase M and carboxypeptidase Y.
• 3.3. The response of the isolated hindgut to the new peptide was distinguishable from the response to proctolin by the following features: (a) a longer interval following application (1–4 min) to reach a maximum contraction, and (b) a much larger amplitude for single phasic contractions. Like proctolin, the new peptide could cause a protracted stimulation of the hindgut for more than 2 hr.

     

Structure,haemolymph titre,and regulatory effects of juvenile homone during ovarian maturation in Leucophaea maderae

Juvenile hormone (JH) synthesized and secreted in vitro by the corpora allata of mated adult Leucophaea maderae females was determined to be JH III (methyl-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate).The haemolymph titre of JH was determined during maturation of the terminal oöcytes in the first reproductive cycle of L. maderae. In virgin females, JH is not detectable in the haemolymph during the first eight days following adult emergence; however, by 10 days after emergence, trace quantities of JH are apparent. Mating stimuli induce a dramatic increase in the concentration of haemolymph JH, with a peak occurring approximately 12 days after mating; thereafter, the JH concentration declines until it has reached an undetectable level 19 days after mating, at the time of chorion deposition.During ovarian maturation, changes in the rates of synthesis of vitellogenin by the fat body and DNA by the ovary correlate closely with the haemolymph titre of JH. However, no such correlation exists between the JH titre and the extensive ovarian protein synthesis that occurs in L. maderae coincident with chorion formation.The effects of JH I and JH III on both vitellogenin synthesis and ovarain DNA synthesis are statistically similar.