Nachweis von häutungsaktiven Stoffen in isolierten Prothorakaldrüsen und Oenocyten beiBombyx mori während des 5. Larvenstadiums

Summary The content of moulting hormones has been determined in homogenates of isolated prothoracic glands and oenocytes during the 5th instar of the silkworm,Bombyx mori by means of the Calliphora bioassay.Prothoracic glands show variable activity in the production of moulting hormones, reaching a maximum near the end of the larval period. Comparable activities, but at higher levels, could be demonstrated in oenocytes. Controls with doubled quantities of tissue produced in a proportionate reaction in the bioassay. Fat bodies were inactive.Prothoracic glands and oenocytes incubated together resulted in a slower pupation index than would be expected from the sum of single determinations of oenocytes and prothoracic glands. This is explained by the ability of prothoracic glands to build conjugates of ecdysones.Die Arbeit wurde mit Unterstützung der Deutschen Forschungsgemeinschaft durchgeführt     

Synchrony of juvenile hormone-sensitive periods for internal and external development in last-instar larvae of Oncopeltus fasciatus

Breakdown of the moulting glands in Oncopeltus can be completely inhibited by topical application of a juvenile hormone analogue prior to day 2 of the fifth instar, and partially inhibited by application prior to day 4. The analogue-sensitive period for the inhibition of external metamorphosis is very similar to that for the inhibition of cell death in the moulting glands. A decline in response to the analogue between days 2 and 4 is correlated with rising ecdysteroid levels in the haemolymph. That this rise in ecdysteroids may be responsible for termination of the juvenile hormone-sensitive period is suggested by premature loss of sensitivity to the analogue by the moulting glands following premature exposure to 20-hydroxyecdysone.     

In vitro stimulation of cell death in the moulting glands of Oncopeltus fasciatus by 20-hydroxyecdysone

The moulting glands of the milkweed bug, Oncopeltus fasciatus, normally degenerate just before the time of ecdysis to an adult (day 7 of the fifth instar). Morphologically normal cell death can be prematurely stimulated in vitro by 20-hydroxyecdysone. Breakdown is triggered by a 24-hr period of exposure to 20-hydroxyecdysone, but an additional incubation period is required before clear signs of degeneration are manifested. Glands removed after the onset of endogenous ecdysteroid secretion degenerate in vitro in the absence of added hormones. Thus, in the moulting glands of Oncopeltus, ecdysteroids appear to act as an important trigger for metamorphic cell death.     

Prothoracotropic action of ecdysone analogues in Bombyx mori

The prothoracotropic action of ecdysone analogues was examined, using the brainless, diapausing pupae of Bombyx mori with or without inclusion of prothoracic glands. The most effective of those hormones to stimulate prothoracic glands to secrete the moulting hormone was found to be cyasterone. The other analogues such as ponasterone A, ecdysterone, and inokosterone showed a lower activity with regard to prothoracotropic action. The female prothoracic glands were found to be more sensitive to the ecdysones than the male ones. The time lag from hormone injection to emergence indicated the dual actions of the injected ecdysones, directly on the target organs and indirectly on the prothoracic glands subsequent to secreting the moulting hormone.     

Effectiveness of imaging modalities for screening IgG4-related dacryoadenitis and sialadenitis (Mikulicz’s disease) and for differentiating it from Sj?gren’s syndrome (SS), with an emphasis on sonography

IntroductionThe aim of this study was to clarify the effectiveness of various imaging modalities and characteristic imaging features in the screening of IgG4-related dacryoadenitis and sialadenitis (IgG4-DS), and to show the differences in the imaging features between IgG4-DS and Sjögren’s syndrome (SS).MethodsThirty-nine patients with IgG4-DS, 51 with SS and 36 with normal salivary glands were enrolled. Images of the parotid and submandibular glands obtained using sonography, 2-[¹⁸F]-fluoro-2-deoxy-D-glucose positron emission tomography/computed tomography (FDG-PET/CT), computed tomography (CT) and magnetic resonance imaging (MRI) were retrospectively analyzed. Six oral and maxillofacial radiologists randomly reviewed the arranged image sets under blinded conditions. Each observer scored the confidence rating regarding the presence of the characteristic imaging findings using a 5-grade rating system. After scoring various findings, diagnosis was made as normal, IgG4-DS or SS, considering all findings for each case.ResultsOn sonography, multiple hypoechoic areas and hyperechoic lines and/or spots in the parotid glands and obscuration of submandibular gland configuration were detected mainly in patients with SS (median scores 4, 4 and 3, respectively). Reticular and nodal patterns were observed primarily in patients with IgG4-DS (median score 5). FDG-PET/CT revealed a tendency for abnormal ¹⁸F-FDG accumulation and swelling of both the parotid and submandibular glands in patients with IgG4-DS, particularly in the submandibular glands. On MRI, SS had a high score regarding the findings of a salt-and-pepper appearance and/or multiple cystic areas in the parotid glands (median score 4.5). Sonography showed the highest values among the four imaging modalities for sensitivity, specificity and accuracy. There were significant differences between sonography and CT (p = 0.0001) and between sonography and FDG-PET/CT (p = 0.0058) concerning accuracy.ConclusionsChanges in the submandibular glands affected by IgG4-DS could be easily detected using sonography (characteristic bilateral nodal/reticular change) and FDG-PET/CT (abnormal ¹⁸F-FDG accumulation). Even inexperienced observers could detect these findings. In addition, sonography could also differentiate SS. Consequently, we recommend sonography as a modality for the screening of IgG4-DS, because it is easy to use, involves no radiation exposure and is an effective imaging modality.     

Production of cyclic AMP and adenosine by the brain and prothoracic glands of Manduca sexta

Relatively large amounts of cyclic AMP are produced by the prothoracic glands (source of the insect moulting hormone or moulting hormone percursor) of the tobacco hornworm, Manduca sexta. Pharate pupal glands produce more cyclic AMP than early fifth instar larval glands, and the addition of aminophylline enhances cyclic AMP accumulation. The much lower cyclic AMP level in the absence of aminophylline indicates the presence of potent cyclic AMP phosphodiesterase activity. Brains (sources of the prothoracicotropic hormone) also produce cyclic AMP but at a lower rate. Brains efficiently produce adenosine from ATP while β-ecdysone inhibits adenosine formation in early fifth instar larval brains. β-Ecdysone stimulates adenyl cyclase in brains of both stages when aminophylline and fluoride are present but has no effect on cyclic AMP accumulation in prothoracic glands. The absence of fluoride greatly reduces the amount of cyclic AMP produced by prothoracic glands when aminophylline is present. No cyclic AMP is accumulated in prothoracic glands when both fluoride and aminophylline are absent or in brains when fluoride is absent, notwithstanding the presence of aminophylline. Other insect tissues were also analysed for cyclic AMP production and none showed levels nearly as high as the prothoracic glands, suggesting a close relationship between cyclic AMP production and the function of the gland.     

Prothoracic gland activity and blood titres of ecdysone and ecdysterone during the last larval instar of Locusta migratoria L.

The kinetics of secretion of ecdysone by the prothoracic glands of Locusta migratoria were studied during the last larval instar. Three stages of intense production of ecdysone (α-ecdysone) were monitored during this developmental period: they correspond to three peaks of moulting hormone concentration in the blood, which indicates that the main regulation of the moulting hormone titre is achieved through variations in prothoracic gland activity. In the haemolymph the ratio of ecdysone to ecdysterone (20-hydroxy-ecdysone) is in favour of ecdysone during the two first moulting hormone peaks ecdysterone being by far predominant over ecdysone at the time of the third (major) peak; these results support previous studies on the metabolic fate of injected labelled ecdysone during the same developmental period in Locusta migratoria.     

Actin exposure upon tissue injury is a targetable wound site-specific protein marker

BackgroundIdentification of wound-specific markers would represent an important step toward damaged tissue detection and targeted delivery of biologically important materials to injured sites. Such delivery could minimize the amount of therapeutic materials that must be administered and limit potential collateral damage on nearby normal tissues. Yet, biological markers that are specific for injured tissue sites remain elusive.MethodsIn this study, we have developed an immunohistological approach for identification of protein epitopes specifically exposed in wounded tissue sites.ResultsUsing ex-vivo tissue samples in combination with fluorescently-labeled antibodies we show that actin, an intracellular cytoskeletal protein, is specifically exposed upon injury. The targetability of actin in injured sites has been demonstrated in vivo through the specific delivery of anti-actin conjugated particles to the wounded tissue in a lethal rat model of grade IV liver injury.ConclusionsThese results illustrate that identification of injury-specific protein markers and their targetability for specific delivery is feasible.General significanceIdentification of wound-specific targets has important medical applications as it could enable specific delivery of various products, such as expression vectors, therapeutic drugs, hemostatic materials, tissue healing, or scar prevention agents, to internal sites of penetrating or surgical wounds regardless of origin, geometry or location.     

Moulting cycles in European quail (Coturnix coturnix coturnix) under constant photoperiodic conditions

Abstract

Male and female European quails (Coturnix coturnix coturnix) were studied whilst being maintained in LD 12:12, at 20° C, for four years from birth. The birds exhibited the following phases: end of the first phase of the post‐juvenile moult, fattening and a period of nocturnal activity. Then sexual development is observed up to complete maturation at the age of four months. The second part of post juvenile moulting occurs towards the age of 5 to 6 months. During the following 6 months an important phase of reproduction is observed, thus completing their first year of life. The same sequence is repeated during the following years: a moult which lasts for 6 months in two (sometimes three) partial phases separated by episodes of reproduction; then an important period of reproduction (6 months) without moulting. The birds are capable of reproducing at all times except during moulting. The sequence of moulting begins each year in the same months as those for natural French populations. It thus seems that the annual periodicity and the organisation itself of the phenomenon of moulting is controlled in European quail by an endogenous circannual rhythm. In contrast, the natural cyclic expression of sexuality appears to be dependent on abiotic environmental and social factors.     

Fine structure of the cuticle and structural changes occuring during moulting in the mite Tetranychus urticae. II. Moulting process (Chelicerata,Acarina)

Summary Processes occurring during moulting in Tetranychus urticae (Acari, Tetranychidae) are described by means of electron microscopy.Moulting is characterized by a pre-ecdysial phase which is initiated by the detachment of cuticle and epidermis. Epicuticular material is deposited as plaques but fuses to form a continuous layer. The epidermis folds up and ridges become determined. Procuticular material is synthesized inside the epidermis and packed into granules which accumulate below the epicuticular portions already deposited. Prior to ecdysis, portions of the old cuticle are dissolved. Ecdysis is achieved by moulting glands which effect bursting of the old cuticle. During the post-ecdysial phase, the endocuticle is synthesized during which a lamellation becomes obvious.Processes occuring during moulting are compared to published information on the tick cuticle.     

Changes in ionic conductances and in sensitivity to amiloride during the natural moulting cycle of toad skin (Bufo viridis,L.)

Summary The resistance of the apical membranes of toad skin (Bufo viridis) was measured during its natural moulting cycle using a fast flow technique. The skin behaved in all periods of the moulting cycle as a nearly perfect sodium electrode. In the presence of amiloride (10^–4 M), the total resistance of the same skin was identical with solutions which contained either sodium or potassium. The resistance of the skin with potassium was sensitive to amiloride in the period just after moulting. The resistance of skins which were made shunted by treating them with urea on the outside was insensitive to amiloride in solutions containing potassium; a small effect was still observed with sodium. It is suggested that the transient sensitivity to amiloride, with potassium, is the result of differentiation of the sodium specific sites at the apical membranes of the skin.     

Ultrastructural location of calcium and magnesium during mineralisation of the cuticle of the shore crab,as determined by the K-pyroantimonate method and X-ray microanalysis

We have investigated the distribution of Ca²⁺ and Mg²⁺ in the new cuticle of moulting shore crabs (Carcinus maenas), using the K-pyroantimonate method in combination with X-ray microanalysis in order to identify antimony precipitates. During the premoult period, Ca²⁺ and Mg²⁺ accumulate in well-defined sites of the new pigmented layer. After moulting, mineralisation appears to begin preferntially at these sites. These form a honeycomb-like structure that quickly increases the rigidity of the new cuticle, with a small recruitment of material from extraneous sources. Mineralisation of the principal layer, on the other hand, immediately follows deposition of the organic matrix. Our experiments also provide evidence that the epidermal cell extensions associated with the pore canals are the means by which Ca²⁺ and Mg²⁺ are transferred from the epidermis into the mineralising cuticular layers. The plasma membrane of these cell extensions appears densely lined by particles of antimony precipitate that probably mark the location of the transporting sites. Shortly after moulting, the distribution of mineral deposits is such that the cell extensions cross the mineralised lamellae of the principal layer and constitute preferential access routes to the pigmented layer, where mineralisation is still in progress.     

Role of the Y organ and the effect of ecdysterone during the regeneration of an appendage in the oniscoid Porcellio dilatatus

The rôle of the moulting hormone in regeneration was studied in Porcellio dilatatus by destroying the Y-organs (moulting glands) and by injecting synthetic ecdysterone. The destruction of the Y-organs prevents the formation of the regeneration bud of the limb. Several successive injections of a very weak solution of ecdysterone allow the formation of a regeneration bud in animals without Y-organs. A single injection of ecdysterone at a dose which induces apolysis blocks regeneration by making all the epidermal cells secrete cuticle, including those of the regeneration bud.     

Moulting diving ducks and their food supply

Numbers of moulting diving ducks at Myvatn, north Iceland, were monitored over a period of 25 years; aquatic insects (Chironomidae and Simuliidae), a major food resource, were monitored with window traps for 23 years. The response of the duck populations to changes in the food situation during the moulting period varied between species. Histrionicus histrionicus and Melanitta nigra invariably go to the sea to moult. Three species (Aythya fuligula, Clangula hyemalis and Mergus serrator) have shown long term variation in moult migration habits. Aythya marila and Bucephala islandica stay for moulting, their numbers supplemented by birds from elsewhere. A. marila is a generalist feeder and therefore at low risk of starving, the strategy of Bucephala islandica is to move within the water system where the food situation tends to alternate between the lake and the river. Numbers of Bucephala islandica males moulting on Lake Myvatn were strongly and positively correlated with chironomid numbers and those moulting on the river Laxá with Simulium vittatum, the main food resource there. We did not find convincing evidence that numbers of moulting A. marila and C. hyemalis responded to variation in the food supply during the study period. This applied also to A. fuligula in the South Basin of Myvatn, but numbers in the North Basin were positively associated with chironomid numbers. M. serrator moulting on Myvatn showed negative correlations with the chironomids, perhaps reflecting a negative association between chironomids and its main food, Gasterosteus aculeatus. Apart from safety considerations for a flightless bird, the choice of a moulting site is apparently influenced by the local food conditions on one hand and by the opportunities and risks involved in migrating to distant moulting sites with an unknown food situation on the other hand.     

Somatotrophs and lactotrophs: an immunohistochemical study of Gallus domesticus pituitary gland at different stages of induced moult

The objective of this study was to determine the distribution of somatotrophs and lactotrophs and conduct a morphometrical analysis of immunoreactive somatotrophs and lactotrophs in the pituitary glands of White Leghorn Hens (Gallus domesticus) during the period of induced moult. We divided the periods of induced moulting into three phases viz. 7, 14 and 21 days. The labeled alkalinephsphatase method with anti-GH (growth hormone) and anti-PRL (prolactin) as a primary antibody was used to detect somatotrophs and lactotrophs, in the midsagital sections of chicken adenohypophysis. Immunohistochemistry showed that somatotrophs are not only confined to the cephalo-caudal axis but can also be found in the caudal lobe; while lactotrophs were distributed in both lobes of the anterior pituitary gland at all stages of moulting (7, 14 and 21 days). Lactotrophs were of different shapes but somatotrophs were oval to round in morphology. At the given stages of induced moulting, some hypertrophied lactotrophs were also present after 7 days of induced moult in the anterior pituitary gland. However, there were moulting-related changes: from 7 to 21 days of induced moulting the immunoreactive-PRL cell population decreased, while the mean lactotroph size was more than that of somatotrophs. Basic quantitative and morphological information relating to somatotrophs and lactotrophs during the period of induced moult in laying hens is reported here and the changes brought about by induced moulting are restricted to PRL positive cells rather than GH positive cells.Key words: Moult, pituitary gland, somatotrophs, lactotrophs, chicken.     

Forelimb regeneration in thyroidectomized adult newts following organ culture and autografting of the thyroid glands

Summary Thyroidectomy and organ culture of adult newt thyroid glands three days prior to forelimb amputation was followed by autografting the glands subcutaneously into the animal's lower jaw region 9, 18 or 25 days postamputation (GC^{9, 18, 25} day series). This was an attempt, utilizing 515 animals, to elucidate further the role of the thyroids in regeneration. Amputated limbs of the thyroidectomized (Thx) and autografted muscle explant (MC = sham) cases underwent stumping or were significantly delayed in their regeneration rate and displayed abnormal morphogenesis compared with control regenerates. In the GC⁹ series newts, regenerates were identical to controls 45 days postamputation. However, regenerates of the GC¹⁸ series cases exhibited delayed and abnormal development at 45 days; but they were not as delayed and had fewer abnormalities than those cases in the Thx and MC groups. Results of the GC²⁵ series newts were similar to those of the Thx group. Within 5 days of autografting the thyroids, epidermal moulting resumed and long-term survival ensued. We conclude that normal limb regeneration in the adult newt is thyroid hormone(s) dependent, specifically the later stages of growth, differentiation and morphogenesis.Supported by grant A-1208 from the Natural Sciences and Engineering Research Council of Canada to R.A.L.     

Prothoracicotropic hormone bioassay: Bombyx larva assay

Summary

An assay for the prothoracicotropic hormone (PTTH) has been established using in situ activation of the prothoracic glands (PG) of Bombyx mori in its larva-to-larva development. The timing of PTTH release was estimated by examining developmental response of 4th instar larvae to brain removal and neck ligation, and changes in the haemolymph ecdysteroid titer and ecdysone-releasing activity of PG in vitro during the development. Injection of Bombyx brain extracts into 4th instar larvae neck-ligated shortly before full activation of PG elicited larval moulting rather than precocious pupation in headless larvae. This developmental shift was regarded as due to the action of PTTH, and the PTTH unit has been defined from a linear log dose-response relationship. Materials chromatographically fractionated from Bombyx brain extracts were examined for the presence of stage- and species-specific PTTH molecules by using this Bombyx larva assay and Bombyx and Samia pupa assays previously developed. The same fractions were active when assayed by Bombyx larva and pupa assays.     

Identification and Characterization of a Specific Receptor for Cholecystokinin on Isolated Fundic Glands from Guinea Pig Gastric Mucosa Using a Biologically Active 125I-CCK-8 Probe

Abstract

Specific binding sites for cholecystokinin (CCK) have been identified and characterized in fundic glands isolated by collagenase treatment from guinea pig gastric mucosa using a biologically active ¹²⁵I-labeled derivative of the C-terminal octa-peptide of CCK (¹²⁵IIE-CCK-8). The time course of binding to these glands was rapid, temperature dependent and saturable. At 24, 30 and 37° C, half-maximal binding was reached at 5 min and full binding at 30 min. The addition of a large excess of CCK-8 after 15 and 30 min of binding at 24° C caused a prompt and rapid decline in radioligand bound showing that the interaction was reversible. There was a progressive decline in the amount of ¹²⁵IIE-CCK-8 bound to fundic glands with increasing concentrations of CCK-8 and other structurally related peptides. Gastrin II displaced 50% of the radioligand at 1.6nM, CCK-8 at 3.2nM, gastrin I at 16nM, and desulfated-CCK-8 and pentagastrin at 59nM. Secretin did not displace the radioligand from fundic glands at 1.0uM. The binding was also tissue specific as glands isolated from the antral mucosa did not contain specific binding sites for ¹²⁵IIE-CCK-8. This data provides evidence for specific receptors for CCK on gastric fundic glands that may be involved in the control of acid and pepsinogen secretion.     

Feedback inhibition of ecdysone production by 20-hydroxyecdysone in Pieris brassicae pupae

The effects of exogenous moulting hormones, ecdysone and 20-hydroxyecdysone on ecdysteroid production were studied in vivo in Pieris brassicae pupae. Both hormones inhibit ecdysteroid production; however, 20-hydroxyecdysone is much more efficient than ecdysone, and it is likely that the ecdysone effect is due to its partial conversion into 20-hydroxyecdysone. These results suggest that 20-hydroxyecdysone acts on ecdysteroid production as a negative-feedback regulator. Furthermore, since 20-hydroxyecdysone elicits inhibition in headless pupae, it is suggested that 20-hydroxyecdysone acts directly upon the prothoracic glands.