Studies on ribonucleic acid synthesis in the venom glands of Vipera palaestinae (Ophidia, Reptilia)

RNA metabolism in the venom glands of Vipera palaestinae was studied at different stages after manual extraction of the venom (milking). The rate of (32)P incorporation into gland RNA was found to be maximal at 1-4 days after milking in correlation with the height of the secretory epithelium. Venom production attained a maximum only after 8-16 days, in parallel with the accumulation of stable species of cellular RNA.     

Localization of some enzymes in the main venom glands of viperid snakes

Several secretory and nonsecretory enzymes were localized histochemically in the main venom gland of 13 viperid snakes. All secretory cells show the intracellular oxidative enzymes succinate dehydrogenase and monoamine oxidase. The granular reactions obtained for both enzymes resemble mitochondria in distribution. Distinctive cells with a very high succinate dehydrogenase activity are dispersed among the secretory cells of all species except Atractaspis. Nonspecific acid phosphatase activity is found in the supranuclear region of the secretory cells in species that do not secrete this enzyme and throughout the cytoplasm in snakes that secrete the enzyme. Nonspecific alkaline phosphatase activity occurs in the secretory cells of those snakes whose venom shows this activity. Leucine amino peptidase (aryl amidase) activity is found in the venom and in the secretory cells of all the species. In Vipera palaestinae both the venom and the secretory cells of the main venom gland contain nonspecific esterase, L-amino acid oxidase and phosphodiesterase activities. The localization of phosphodiesterase and L-amino acid oxidase do not show major differences between glands at different intervals from an initial milking. Adenosine-monophosphate phosphatase activity is localized in the supranuclear region of the secretory cells in the glands of Vipera palaestinae and Aspis cerastes. Its activity is found in the venom of Aspis only.     

Asynchrony in the synthesis of secretory proteins in the venom gland of the snake Vipera palaestinae.

1. Venom of Vipera palastinae was subjected to isoelectrofocusing on polyacrylamide gel. The protein separation profiles were similar for different venom samples; more than 25 protein bands with a wide range of pI values could be demonstrated by this technique. 2. Labelled venom was obtained 8h after an intracardial injection of [3H]leucine. The relative radioactivities of four out of 12 main protein bands were significantly different in the venom synthesized during the 2nd day of the venom regeneration cycle as compared with the venom of the 4th day. The comparison was made in venom samples obtained from the two glands of the same snake at two different secretory stages. 3. It is concluded that the asynchronous synthesis of exportable proteins after the initiation of a new venom regeneration cycle is responsible for the non-parallel secretion of some venom proteins by the venom gland of Vipera palaestinae during the first few days after milking.     

Cost of venom regeneration in Parabuthus transvaalicus (Arachnida: Buthidae)

Scorpion venom has many components, but is mainly made up of water, salts, small molecules, peptides, and proteins. One can reasonably assume that the production and storage of this complex secretion is an expensive metabolic investment. However, to date, no study has addressed the costs associated with the regeneration of venom by scorpions. Using a closed-system respirometer, we examined the difference in oxygen consumption between milked and unmilked scorpions to determine the metabolic costs associated with the first 72 h of subsequent venom synthesis. During this time period, milked scorpions had a significantly higher (39%) metabolic rate than unmilked scorpions. The regenerated venom from a second milking had significantly lower (74%) protein concentration, suggesting that venom regeneration was incomplete after 72 h. The protein content in the regenerated venom was not correlated with oxygen consumption. The significant increase in oxygen consumption after milking supports existing hypotheses about the metabolic cost associated with venom regeneration and provides further insight on why scorpions appear to be judicious in their stinger use.     

Differential proteomic analysis of laser-microdissected penetration glands of avian schistosome cercariae with a focus on proteins involved in host invasion

Schistosome invasive stages, cercariae, leave intermediate snail hosts, penetrate the skin of definitive hosts, and transform to schistosomula which migrate to the final location. During invasion, cercariae employ histolytic and other bioactive products of specialized holocrine secretory cells – postacetabular (PA) and circumacetabular (CA) penetration glands. Although several studies attempted to characterize protein composition of the in vitro-induced gland secretions in Schistosoma mansoni and Schistosoma japonicum, the results were somewhat inconsistent and dependent on the method of sample collection and processing. Products of both gland types mixed during their secretion did not allow localization of identified proteins to a particular gland. Here we compared proteomes of separately isolated cercarial gland cells of the avian schistosome Trichobilharzia szidati, employing laser-assisted microdissection and shotgun LC-MS/MS, thus obtaining the largest dataset so far of the representation and localization of cercarial penetration gland proteins. We optimized the methods of sample processing with cercarial bodies (heads) first. Alizarin-pre-stained, chemically non-fixed samples provided optimal results of MS analyses, and enabled us to distinguish PA and CA glands for microdissection. Using 7.5 × 10⁶ μm³ sample volume per gland replicate, we identified 3347 peptides assigned to 792 proteins, from which 461 occurred in at least two of three replicates in either gland type (PA = 455, 40 exclusive; CA = 421, six exclusive; 60 proteins differed significantly in their abundance between the glands). Peptidases of five catalytic types accounted for ca. 8% and 6% of reliably identified proteins in PA and CA glands, respectively. Invadolysin, nardilysin, cathepsins B2 and L3, and elastase 2b orthologs were the major gland endopeptidases. Two cystatins and a serpin were highly abundant peptidase inhibitors in the glands. While PA glands generally had rich enzymatic equipment, CA glands were conspicuously abundant in venom allergen-like proteins.     

A medium for the maintenance of Chironomus tentans salivary glands in vitro

A synthetic medium based upon the chemical composition of fourth instar Chironomus haemolymph was formulated for the in vitro culture of Chironomus tentans salivary glands.Salivary glands maintained in the medium for up to 4 days appeared morphologically normal. Secretion-free glands, obtained from pilocarpine-treated larvae, accumulated proteinaceous material in the gland lumen and exhibited a 46% increase in total gland protein after 24 hr in the medium. Cycloheximide almost totally inhibited the accumulation of secretion material and the increase in total gland protein by cultured glands.Glands cultured for up to 4 days continued to incorporate ¹⁴C-leucine into acid-insoluble total protein and ³H-uridine into total RNA, but at reduced levels. The incorporation of both isotopes was almost completely inhibited by cycloheximide.Autoradiographic squash preparations of glands pulse-labelled with ³H-thymidine after 3 days in culture revealed a normal pattern of asynchronous chromosomal DNA replication. Glands cultured for up to 4 days exhibited ³H-uridine incorporation into nucleoli and into distinct chromosomal regions which corresponded with sites of cytochemically demonstrable acidic protein.The chromosomes of cultured glands appeared morphologically and cytochemically normal, except for some regression of the Balbiani rings. Addition of ecdysterone to media containing glands previously cultured for 3 days resulted in puff induction at the IV-2-B chromosomal locus.     

Karyometric analysis of the supraoptic nucleus and the paraventricular nucleus before and after milking the cow

With mathematical-statistical methods the reaction of the Nucleus supraopticus (N.S.O.) and Nucleus paraventricularis (N.P.V.) were investigated before and after the milking of the cows. The frequency-distributions of the tests of the nuclear volumes in the N.S.O. and N.P.V. show an increase of the mean values of the nuclear volumes from the unmilked cows to those studied 15 minutes after the milking. The mean values of the nuclei volumes of the cows studied 60 minutes after the milking fall off, but they are higher than the values of the unmilked cows. The results of the histological-histochemical investigations mainly coincided with the results obtained by caryometry. It is discussed, that the changes in the nuclear bolumes show an increased protein synthesis and that -- combined with the release of oxytocin -- the milk stimulus caused a secretory synthesis in the neurosecretory cells, mainly in the N.P.V.     

Development of secretagogue response in rat pancreatic acinar cells

Two to 3 days prior to birth, acinar cells of the rat pancreas acquire morphologic and biochemical characteristics of the adult gland. To determine if differentiation of the secretory apparatus coincides temporally with the capacity of the cell to respond to secretory stimuli, lobules of embryonic, neonatal, and adult rat pancreas were compared for their ability to respond to secretagogues presumed to act directly via hormone receptors [caerulein and carbamylcholine (carbachol)] or indirectly (cyclic nucleotide analogs and the Ca²⁺ ionophore A23187). Of all agents tested, only dibutyryl cAMP elicited discharge of secretory proteins at day 20 in utero and preceded hormone stimulation by 1 day. A23187 elicited discharge by Day 21 in utero; its action was near adult levels in contrast to hormonal stimuli whose effect was maximal only at birth. All secretagogues required Ca²⁺ and energy to induce discharge. Pulse-chase autoradiography of lobules from Day 20 embryonic glands indicated that the acinar cells were capable of transporting [³H]leucine-labeled proteins to zymogen granules at rates roughly equivalent to those in adult glands. SDS gel electrophoretograms confirmed that the bulk of ¹⁴C-amino acid incorporation into proteins at a given age was primarily into exportable proteins. The results indicate that acinar cells synthesize and package secretory proteins into zymogen granules about 2 days before they are capable of responding to hormonal stimuli and to intracellular effectors.     

Venom and Dufour's glands of the emerald cockroach wasp Ampulex compressa (Insecta,Hymenoptera, Sphecidae): Structural and biochemical aspects

The digger wasp species Ampulex compressa produces its venom in two branched gland tubules. They terminate in a short common duct, which is bifurcated at its proximal end. One leg is linked with the venom reservoir, the other one extends to the ductus venatus. Each venom gland tubule possesses, over its entire length, a cuticle-lined central duct. Around this duct densely packed class 3 gland units each composed of a secretory cell and a canal cell are arranged. The position of their nuclei was demonstrated by DAPI staining. The brush border of the secretory cells surrounds the coiled end-apparatus. Venom is stored in a bladder like reservoir, which is surrounded by a thin reticulated layer of muscle fibres. The reservoir as a whole is lined with class 3 gland units. The tubiform Dufour's gland has a length of about 350 μm (∅ 125 μm) only and is surrounded by a network of pronounced striated muscle fibres. The glandular epithelium is mono-layered belonging to the class 1 type of insect epidermal glands. The gland cells are characterized by conspicuous lipid vesicles. Secretion of material via the gland cuticle into the gland lumen is apparent. Analysis of the polypeptide composition demonstrated that the free gland tubules and the venom reservoir contain numerous proteins ranging from 3.4 to 200 kDa. The polypeptide composition of the Dufour's gland is completely different and contains no lectin-binding glycoproteins, whereas a dominant component of the venom droplets is a glycoprotein of about 80 kDa. Comparison of the venom reservoir contents with the polypeptide pattern of venom droplets revealed that all of the major proteinaceous constituents are secreted. The secreted venom contains exclusively proteins present in the soluble contents of the venom gland. The most abundant compound class in the Dufour's gland consisted of n-alkanes followed by monomethyl-branched alkanes and alkadienes. Heptacosane was the most abundant n-alkane. Furthermore, a single volatile compound, 2-methylpentan-3-one, was identified in various concentrations in the lipid extract of the Dufour's gland.     

SECRETORY PROTEIN SYNTHESIS IN THE STIMULATED RAT PAROTID GLAND : Temporal Dissociation of the Maximal Response from Secretion

Administration of the β-adrenergic drug, isoproterenol (IPR), affects the release of 98% of stored amylase from rat parotid gland acinar cells. A period of 6 h elapses from the onset of secretion to the maximum [¹⁴C]phenylalanine (Phe) incorporation into total protein and amylase. 10 h after IPR administration the rate of [¹⁴C]Phe incorporation into total protein was no longer elevated above that of control. Incorporation into amylase, however, remained elevated above the control by 2.3 times. This latent period may reflect: (a) reduced amounts of available ATP which occurs as a result of the process of secretion as well as (b) the time required for reorganization of cellular organelles and membranes after secretion. The latent period after IPR-induced secretion appears similar to the latent period which has recently been reported to occur after physiologic release of amylase from the parotid gland during the diurnal feeding cycle of the rat. These observations support the existence of a positive feedback system operant in the parotid acinar cell linking the release of secretory proteins with their synthesis. The period of greatest protein synthesis is, however, temporally dissociated from the secretory process.     

Ultrastructural and histochemical study of collagen fibres types I and III in the venom gland of Bothrops jararaca during secretory cycle

Fragments of snake (Bothrops jararaca) venom gland were analysed by light and transmission electron microscopy in order to characterize the changes in collagen fibres types I and III in the intertubular gland septa during the secretory cycle. The snakes were sacrificed at 45 days (unmilked group), 6 h, 4 and 8 days after manual extraction of the venom. The fragments were fixed, processed according to standard histologic technique for embedding in paraffin, and stained with haematoxylin-eosin and Gomori's trichrome and submitted to Gomori's silver impregnation technique and picrosirius-polarization method. For transmission electron microscopy the fragments were fixed and processed for embedding in Spurr's medium. At the 45th day (the gland at rest), when the secretory activity was at a minimum, the septa were narrow and filled with densely packed collagen fibrils. At 6 h, the septa were enlarged and exhibited wide spaces filled with finely granular Alcian Blue-positive material. Until the 8th day, the septa were narrower and the histologic aspect resembled that of the gland at rest. The results demonstrated structural modifications in the glandular septa according to the different periods of the secretory cycle. These modifications can be associated with the transformation in the secretory epithelium during the venom synthesis cycle.     

Biochemical differentiation of lone star tick,Amblyomma americanum (L), salivary glands: Effects of attachment,feeding and mating

Salivary glands of female Amblyomma americanum (L.) are stimulated to differentiate by attachment to a host, subsequent feeding and mating. Incorporation of [³H]uridine into ribosomal and transfer RNAs as well as the synthesis of poly(A⁺)mRNA and protein parallel the pattern of increasing enzymatic activity and secretory ability of the glands. Unfed ticks contained 3.5 ± 0.47 ng poly(A⁺)mRNA/gland pr. By the second day of feeding this had increased more than 5-fold. The greatest amount of poly(A⁺)mRNA found in rapid-feeding phase females (body wt > 100 mg) was 370 ± 80 ng/gland pr. Poly(A⁺)mRNA mass doubles on the final day of feeding, just as the ticks exceeded 100 mg in wt. Ticks attached 1 to 10 days had increasingly greater amounts of salivary monosomes, 60 and 40S ribosomal subunits, and polysomes. Polysomal mass/gland pr also attained its maximum above 100 mg tick wt at the slow/rapid-feeding phase boundary; exceeding by 20 times that of unfed ticks. Degenerating glands from replete ticks continued to synthesize protein. In vitro incorporation of [³H]leucine was greatest within 24 h of attachment. Fluorographs of [³H]leucine labeled protein showed that mating caused a drop in incorporation after the 4th day of feeding. Glands from unmated females attached the same number of days continued to incorporate [³H]leucine at higher levels than those from mated females.     

Morphology and ultrastructure of the venom glands of the northern pacific rattlesnake Crotalus viridis oreganus

The venom gland of Crotalus viridis oreganus is composed of two discrete secretory regions: a small anterior portion, the accessory gland, and a much larger main gland. These two glands are joined by a short primary duct consisting of simple columnar secretory cells and basal horizontal cells. The main gland has at least four morphologically distinct cell types: secretory cells, the dominant cell of the gland, mitochondria-rich cells, horizontal cells, and “dark” cells. Scanning electron microscopy shows that the mitochondria-rich cells are recessed into pits of varying depth; these cells do not secrete. Horizontal cells may serve as secretory stem cells, and “dark” cells may be myoepithelial cells. The accessory gland contains at least six distinct cell types: mucosecretory cells with large mucous granules, mitochondria-rich cells with apical vesicles, mitochondria-rich cells with electron-dense secretory granules, mitochondria-rich cells with numerous cilia, horizontal cells, and “dark” cells. Mitochondria-rich cells with apical vesicles or cilia cover much of the apical surface of mucosecretory cells and these three cell types are found in the anterior distal tubules of the accessory gland. The posterior regions of the accessory gland lack mucosecretory cells and do not appear to secrete. Ciliated cells have not been noted previously in snake venom glands. Release of secretory products (venom) into the lumen of the main gland is by exocytosis of granules and by release of intact membrane-bound vesicles. Following venom extraction, main gland secretory and mitochondria-rich cells increase in height, and protein synthesis (as suggested by rough endoplasmic reticulum proliferation) increases dramatically. No new cell types or alterations in morphology were noted among glands taken from either adult or juvenile snakes, even though the venom of each is quite distinct. In general, the glands of C. v. oreganus share structural similarities with those of crotalids and viperids previously described.     

Comparing the secretory pathway in honeybee venom and hypopharyngeal glands

We provide insights into the secretory pathway of arthropod gland systems by comparing the royal jelly-producing hypopharyngeal glands and the venom-producing glands of the honeybee, Apis mellifera. These glands have different functions and different product release characteristics, but both belong to the class 3 types of insect glands, each being composed of two cells, a secretory cell and a microduct-forming cell. The hypopharyngeal secretory cells possess an extremely elongate tubular invagination that is filled with a cuticular structure, the end-apparatus, anchored against the cell membrane by a conspicuous series of actin rings. In contrast, venom glands have no actin rings, but instead have an actin-rich brush border surrounding the comparatively short and narrow end-apparatus. We relate these cytoskeletal differences to the production system and utilisation of secretions; venom is stored in a reservoir whereas royal jelly and enzymes are produced on demand. Fluorescence-based characterisation of the actin cytoskeleton combined with scanning electron microscopy of the end-apparatus allows for detailed characterisation of the point of secretion release in insect class 3 glands.     

Ultrastructure of the venom gland of the scorpion,Centruroides sculpturatus (Ewing)

The venom apparatus of the scorpion, C. sculpturatus (Ewing) was studied with light and electron microscopy. Each of the paired glands is lined by secretory epithelium made up of a single layer of columnar cells. Extensive folding in the epithelial layer creates a primitive acinar gland. The secretory products are either membrane-bound or unbound vesicles with discrete morphologies and are observed in the extruded venom, within the lumen of the gland, and within single secretory cells. The venom apparatus, including connective tissues, nerve cells, and muscle tunic is described and correlations are made with observations in other Athropods.