The toxin mimic FS48 from the salivary gland of Xenopsylla cheopis functions as a Kv1.3 channel-blocking immunomodulator of T cell activation.

The Kv1.3 channel has been widely demonstrated to play crucial roles in the activation and proliferation of T cells, which suggests that selective blockers could serve as potential therapeutics for autoimmune diseases mediated by T cells. We previously described that the toxin mimic FS48 from salivary gland of Xenopsylla cheopis downregulates the secretion of proinflammatory factors by Raw 264.7 cells by blocking the Kv1.3 channel and the subsequent inactivation of the proinflammatory MAPK/NF-κB pathways. However, the effects of FS48 on human T cells and autoimmune diseases are unclear. Here, we described its immunomodulatory effects on human T cells derived from suppression of Kv1.3 channel. Kv1.3 currents in Jurkat T cells were recorded by whole-cell patch-clamp, and Ca2+ influx, cell proliferation, and TNF-α and IL-2 secretion were measured using Fluo-4, CCK-8, and ELISA assays, respectively. The in vivo immunosuppressive activity of FS48 was evaluated with a rat DTH model. We found that FS48 reduced Kv1.3 currents in Jurkat T cells in a concentration-dependent manner with an IC50 value of about 1.42 µM. FS48 also significantly suppressed Kv1.3 protein expression, Ca2+ influx, MAPK/NF-κB/NFATc1 pathway activation, and TNF-α and IL-2 production in activated Jurkat T cells. Finally, we show that FS48 relieved the DTH response in rats. We therefore conclude that FS48 can block the Kv1.3 channel and inhibit human T cell activation, which most likely contributes to its immunomodulatory actions and highlights the great potential of this evolutionary-guided peptide as a drug template in future studies.

Stingless Bee-Collected Pollen (Bee Bread): Chemical and Microbiology Properties and Health Benefits.

Stingless bee-collected pollen (bee bread) is a mixture of bee pollen, bee salivary enzymes, and regurgitated honey, fermented by indigenous microbes during storage in the cerumen pot. Current literature data for bee bread is overshadowed by bee pollen, particularly of honeybee Apis. In regions such as South America, Australia, and Southeast Asia, information on stingless bee bee bread is mainly sought to promote the meliponiculture industry for socioeconomic development. This review aims to highlight the physicochemical properties and health benefits of bee bread from the stingless bee. In addition, it describes the current progress on identification of beneficial microbes associated with bee bread and its relation to the bee gut. This review provides the basis for promoting research on stingless bee bee bread, its nutrients, and microbes for application in the food and pharmaceutical industries.

Study of the pharmacokinetics of eriodictyol-6-C-ß-d-glucoside, a flavonoid of rooibos (Aspalathus linearis) extract, after its oral administration in mice.

Systemic dry syndrome affects quality of life, and various effective methods are being developed for its treatment. We recently found that rooibos (Aspalathus linearis) extract activates muscarinic M3 receptor and improves dryness in mice and humans. We identified eriodictyol-6-C-ß-D-glucoside (E6CG) as the active component affecting the secretory functions of exocrine glands; however, the pharmacokinetics and distribution of E6CG in exocrine glands have not been elucidated in mice receiving rooibos extract. We have developed a quantification method using LC-MS/MS to detect E6CG without an internal standard. Experiments on C57BL/6 mice administered rooibos extract showed that E6CG was transferred into blood plasma, with its concentration levels peaking 19.3 min after treatment. Substantial levels of E6CG were detected in the submandibular, sublingual, parotid, and lacrimal glands and in the sweat glands in palm skin. This study reports that rooibos extracts containing E6CG can be used as functional foods for improving systemic dryness.

A novel protease-activated receptor 1 inhibitor from the leech Whitmania pigra.

Whitmania pigra has been used as a traditional Chinese medicine (TCM) for promoting blood circulation, alleviating blood coagulation, activating meridians and relieving stasis for several hundred years. However, the therapeutic components of this species, especially proteins and peptides were poorly exploited. Until now only a few of them were obtained by using chromatographic isolation and purification. In recent decade, transcriptome techniques were rapidly developed, and have been used to fully reveal the functional components of many animal venoms. In the present study, the cDNA of the salivary gland of Whitmania pigra was sequenced by illumina and the transcriptome was assembled by using Trinity. The proteome were analysed by LC-MS/MS. Based on the data of the transcriptome and the proteome, a potential antiplatelet protein named pigrin was found. Pigrin was cloned and expressed using P. pastoris GS115. The antiplatelet andantithrombotic bioactivities of pigrin were tested by using aggregometer and the rat arterio-venous shunt thrombosis model, respectively. Thebleeding time of pigrin was measured by a mice tail cutting method. The docking of pigrin and protease-activated receptor 1 (PAR1) or collagen were conducted using the ZDOCK Server. Pigrin was able to selectively inhibit platelet aggregation stimulated by PAR1 agonist and collagen. Pigrin attenuated thrombotic formation in vivo in rat, while did not prolong bleeding time at its effective dosage. There are significant differences in the key residues participating in binding of Pigrin-Collagen complex from Pigrin-PAR1 complex. In conclusion,a novel PAR1 inhibitor pigrin was found from the leech Whitmania pigra. This study helped to elucidate the mechanism of the leech for the treatment of cardiovascular disorder.

[Effect of ingestion on antithrombin activity in different tissues of Whitmania pigra].

To study the effects of ingestion on antithrombin activities in different tissues of Whitmania pigra, the salivary glands, ingluvies, intestine and muscle of adult leeches were weighted on the 1st d, 3rd d, 5th d, 7th d and 11th d after feeding, respectively, and meanwhile antithrombin activity was measured by antithrombin titration method. The results showed that the antithrombin activity of salivary glands in different stages was significantly higher than that in other tissues (P<0.05). The activity of antithrombin in muscle tissue increased initially and then decreased with the prolongation of the time after feeding, and the peak was observed on the 5th day after feeding (P<0.05). The activity of antithrombin in the salivary glands, gluvies and intestine were found the highest on the 1st day after feeding(P<0.05), and then gradually decreased with the prolongation of the time of stopping the diet. The total amount of antithrombin activity on the 5th day was increased by 49.5%, 73.5%, 69.1% and 126.0% comparing with the 1st, 3rd, 7th and 11th day after feeding, respectively (P<0.05). In summary, both the feeding behavior and the food can induce the secretion of anticoagulant substances in the salivary glands and the digestive tract. The total amount of antithrombin activity was the highest on the 5th day after feeding and the 5th day after feeding was suggested as harvesting time.

Identification and characterization of a novel neuropeptide (neuropeptide Y-HS) from leech salivary gland of Haemadipsa sylvestris.

The present study was designed to identify immunomodulatory components from the leech salivary gland of Haemadipsa sylvestris. The Sephadex G-50, Resource(TM) S column chromatography and reverse-phase high performance liquid chromatography (RP-HPLC) were used to isolate and purify the salivary gland extracts (SGE). Structural analysis of isolated compounds was based on Edman degradation and matrix assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS). The cDNA encoding the precursor of the compound was cloned from the cDNA library of the salivary gland of H. sylvestris. The levels of inflammatory mediators, including tumor necrosis factor-α (TNF-α), interferon γ (IFN-γ), interleukin-6 (IL-6), and monocyte chemotactic protein-1 (MCP-1) were assayed using an enzyme-linked immunosorbent assay (ELISA). The effects on cell proliferation and cell viability were observed using MTT assay. A novel neuropeptide Y (Neuropeptide Y-HS) from the leech salivary gland of H. sylvestris was purified and characterized. It was composed of 36 amino acid residues and the amino acid sequence was determined to be FLEPPERPAVFTSVEQMKSYIKALNDYYLLLGRPRF-NH2, containing an amidated C-terminus. It showed significant inhibitory effects on the production of inflammatory cytokines including TNF-α, IFN-γ, IL-6, and MCP-1. Neuropeptide Y was identified from leeches for the first time. The presence of neuropeptide Y-HS in leech salivary gland may help get blood meal from hosts and inhibit inflammation.

The Postpharyngeal Gland: Specialized Organ for Lipid Nutrition in Leaf-Cutting Ants.

There are several hypotheses about the possible functions of the postpharyngeal gland (PPG) in ants. The proposed functions include roles as cephalic or gastric caeca and diverticulum of the digestive tract, mixing of hydrocarbons, nestmate recognition, feeding larvae, and the accumulation of lipids inside this gland, whose origin is contradictory. The current study aimed to investigate the functions of these glands by examining the protein expression profile of the PPGs of Atta sexdens rubropilosa (Hymenoptera, Formicidae). Mated females received lipid supplementation and their glands were extracted and analyzed using a proteomic approach. The protocol used combined two-dimensional electrophoresis and shotgun strategies, followed by mass spectrometry. We also detected lipid ß-oxidation by immunofluorescent marking of acyl-CoA dehydrogenase. Supplying ants with lipids elicited responses in the glandular cells of the PPG; these included increased expression of proteins related to defense mechanisms and signal transduction and reorganization of the cytoskeleton due to cell expansion. In addition, some proteins in PPG were overexpressed, especially those involved in lipid and energy metabolism. Part of the lipids may be reduced, used for the synthesis of fatty alcohol, transported to the hemolymph, or may be used as substrate for the synthesis of acetyl-CoA, which is oxidized to form molecules that drive oxidative phosphorylation and produce energy for cellular metabolic processes. These findings suggest that this organ is specialized for lipid nutrition of adult leaf-cutting ants and characterized like a of diverticulum foregut, with the ability to absorb, store, metabolize, and mobilize lipids to the hemolymph. However, we do not rule out that the PPG may have other functions in other species of ants.

Functional characterisation of vizottin, the first factor Xa inhibitor purified from the leech Haementeria vizottoi.

The strategic position of factor Xa (FXa) in blood coagulation makes it a compelling target for the development of new anticoagulants. Blood-sucking animals have in their salivary glands mixtures of anticoagulants, which could be used for designing novel antithrombotic compounds. Herein, we describe Vizottin, the first FXa inhibitor from the salivary complex of the leech Haementeria vizottoi . Vizottin was purified by gel filtration and reverse-phase chromatography, and shown to have anticoagulant effects in human plasma, prolonging the recalcification time in a dose-dependent manner (IC50 40 nM). Vizottin induced blood incoagulability in FX-deficient plasma, whereas in normal and reconstituted plasma, Vizottin doubled the prothrombin time at 160 nM. This peptide competitively inhibited human FXa (K(i) 2 nM) like FXa inhibitors from other leeches, albeit via a distinct mechanism of action. At high concentrations, vizottin inhibited the amidolytic activity of factor VIIa/tissue factor (IC50 96.4 nM). Vizottin inhibited FXa in the prothrombinase complex and Gla-domainless FXa. Moreover, vizottin did not interfere with FX activation induced by RVV-X, a known enzyme that requires the Gla-domain of FX for activation. Competition experiments in the presence of FXa and GGACK-FXa (active site blocked) demonstrated that the inhibition of FXa by vizottin is through binding to the active site rather than an exosite. This novel inhibitor appears to exert its inhibitory effects through direct binding to the active site of FXa in a time-dependent manner, but not involving a tight-binding model. In this context, vizottin is a promising model for designing novel anticoagulants for the treatment of thrombotic diseases.

[Steroids, histamine and serotonin in medicinal leech salivary gland secretions].

Lipids represent 20% of the total weight of dried pool of medicinal leech salivary gland secretion (SGS) received from about 50 individual animals. There were detected steroids, but not pospholipids in SGS lipid fraction. Immunochemiluminescent analysis identified free steroid hormones in SGS: cortisol, progesterone, testosterone, estradiol and dehydroepiandrosterone. Microchromatografic-mass-spectrometric analysis of SGS and of low-molecular weight fraction (LMW) (molecular masses ranged from 220 to 850 Da) has shown the multicomponent nature of the LMW fraction. Using standart preparations as the reference steroid hormones (cortisol, dehydroepiandrosterone, androstenedione and testosterone) and histamine and serotonine have been identified in SGS.

Metal distribution and metallothionein induction after cadmium exposure in the terrestrial snail Helix aspersa (Gastropoda, Pulmonata).

The aim of the present work was to study the effect of Cd2+ exposure on metallothionein (MT) induction and on the distribution of metals (Cd, Cu, and Zn) in the terrestrial pulmonate Helix aspersa. In particular, the soluble and nonsoluble pools of the accumulated metals and their tissue distribution in uncontaminated and contaminated edible snails were investigated after a two-week exposure to Cd2+. In the soluble cytosolic pool of the midgut gland of H. aspersa, three metal-specific putative MT isoforms were separated following a fractionation protocol with diethylaminoethyl cellulose, size-exclusion chromatography, ultrafiltration, and reversed-phase high-performance liquid chromatography (RP-HPLC). Interestingly, one of the above isoforms seems to bind both Cd and Cu, which may in addition mobilize, after induction by Cd2+, some of the intracellular Cu and, thus, perhaps increase the Cu pool in the cytosolic fraction. The cDNA and its translated amino acid sequence of a Cd2+-binding MT isoform from the snail midgut gland was characterized and attributed to one of the putative MT isoforms obtained by RP-HPLC. The amino acid sequence of this Cd-MT isoform of H. aspersa differed from similar sequences described in other terrestrial pulmonates, such as Helix pomatia or Arianta arbustorum, by only a few amino acids (n = 4 and 8, respectively). That the identified Cd-MT from H. aspersa is inducible by Cd2+ also was shown, chromatographic evidence aside, by a specific polymerase chain reaction protocol on a cDNA basis, which included a noninducible housekeeping gene as a control.

Molecular analysis of calreticulin expressed in salivary glands of Rhipicephalus (Boophilus) microplus indigenous to Thailand.

The tropical cattle tick, Rhipicephalus (Boophilus) microplus, is an important ectoparasite of livestock in Thailand that causes economic losses due to the direct effects of tick feeding and by the pathogens they transmit. Intensive acaricide use has several drawbacks, which spurred efforts to develop anti-tick vaccines. Vaccines targeting concealed antigens localized in the tick midgut result in reduced tick fecundity, but molecules localized in the tick salivary glands, which could play a role in pathogen transmission, remain largely unexplored for R. microplus. Calreticulin (CRT) is a protein found in tick salivary glands and saliva, and CRT might facilitate tick feeding and pathogen transmission through anti-thrombotic and complement-inhibition activities. This then suggests that CRT should be evaluated as a vaccine candidate antigen to control cattle ticks in Thailand. The objective of this work was to clone, sequence, and analyze cDNA encoding CRT from salivary glands of R. microplus indigenous to Thailand. Nucleotide sequence analysis showed an open reading frame of 1233 bp. Comparison of the amino acid sequence showed 65-99% identities to other known CRTs from Oryctolagus cuniculus, Rattus norvegicus, Homo sapiens, Bos taurus, R. sanguineus, and R. microplus. The N- and P-domains of CRT were the most conserved, whereas the C-domain was high acid and more variable. CRT primary sequences were most conserved among mammals. Further investigations are warranted to determine whether immunization with Thai B. microplus CRT can affect tick performances and experimental pathogen transmission.

The neurite-stimulating influence of components of medicinal leech salivary gland secretions in organotypic culture of spinal ganglia.

The effects of components from medicinal leech (Hirudo medicinalis) salivary gland secretions and the therapeutic agent Piyavit on the growth of chick embryo neurites in organotypic culture were studied. Native destabilase and bdellin A at concentrations of 0.01, 0.02, 0.05, and 0.1 ng/ml, bdellin B at a concentration of 0.05 ng/ml, and eglin at a concentration of 0.1 ng/ml had neurite-stimulating activity, evident on the third day of organotypic culture of spinal ganglia. The stimulatory activity of destabilase was lost after revere-phase chromatography. The neurite-stimulating activity of the extract of the therapeutic agent Piyavit (200 ng/ml) in organotypic ganglion culture appeared to result from the neurite-stimulating salivary gland components within this agent, suggesting that Piyavit could be used for the treatment of neurodegenerative disorders.

Purification, cloning, expression, and mechanism of action of a novel platelet aggregation inhibitor from the salivary gland of the blood-sucking bug, Rhodnius prolixus.

Rhodnius prolixus aggregation inhibitor 1 (RPAI-1), a 19-kDa protein isolated from the salivary gland of R. prolixus, was purified by strong cation exchange and reverse-phase high performance liquid chromatographies. Based on 49 amino-terminal amino acid sequences of RPAI-1, primers were produced to generate probes to screen an R. prolixus salivary gland cDNA library. A phage containing the full-length clone of RPAI-1 codes for a mature protein of 155 amino acids. RPAI-1 shows sequence homology to triabin and pallidipin, lipocalins from Triatoma pallidipennis. The cDNA sequence was cloned in Pet17B Escherichia coli expression vector, producing an active peptide. RPAI-1 inhibits human platelet-rich plasma aggregation triggered by low concentrations of ADP, collagen, arachidonic acid, thromboxane A(2) mimetics (U46619), and very low doses of thrombin and convulxin. Here we show that ADP is the target of RPAI-1 since (i) RPAI-1 inhibits ADP-dependent large aggregation formation and secretion triggered by U46619, without affecting Ca(2+) increase and shape change; (ii) ADP restored the inhibition of U46619-induced platelet aggregation by RPAI-1, (iii) PGE(1)-induced increase of cAMP (which is antagonized by U46619 in an ADP-dependent manner) was restored by RPAI-1, (iv) RPAI-1 inhibits low concentrations of ADP-mediated responses of indomethacin-treated platelets, and (v) RPAI-1 binds to ADP, as assessed by large zone chromatography. RPAI-1 affects neither integrin alpha(2)beta(1)- nor glycoprotein VI-mediated platelet responses. We conclude that RPAI-1 is the first lipocalin described that inhibits platelet aggregation by a novel mechanism, binding to ADP.

Analysis of human sodium iodide symporter gene expression in extrathyroidal tissues and cloning of its complementary deoxyribonucleic acids from salivary gland, mammary gland, and gastric mucosa.

The ability to concentrate iodide is a fundamental property of normally functioning thyroid tissue and represents the first step in the production of thyroid hormones. Iodide uptake has been demonstrated in various extrathyroidal tissues, including salivary gland, gastric mucosa, and lactating mammary gland. Recently, cloning and molecular characterization of the human sodium iodide symporter (hNIS) have been reported; however, the patterns of hNIS gene expression in human tissues have remained unidentified. To examine the profiles of human hNIS gene expression in various normal human tissues, we performed high-stringency Northern blot analysis using a 32P-labeled hNIS-specific complementary DNA (cDNA) probe (nucleotides 1184-1667). To detect rare hNIS transcripts in small tissue samples, RT-PCR was performed with a pair of hNIS-specific oligonucleotide primers designed to amplify a portion (nucleotides 1184-1667) of the hNIS gene. hNIS-specific transcripts were confirmed by Southern hybridization using a digoxigenin-labeled internal hNIS-specific oligonucleotide probe (nucleotides 1460-1477). To monitor cDNA integrity and quantity, and to rule out DNA contamination and illegitimate transcription, all samples were coamplified with two pairs of intron-spanning primers designed to amplify fragments of the human beta-actin and thyroglobulin genes, respectively. Using Northern blot analysis, hNIS transcripts of approximately 4 kb were detected in thyroid gland and parotid gland but not in a broad range of endocrine and nonendocrine tissues. RT-PCR and Southern hybridization revealed hNIS gene expression in thyroid gland, salivary gland, parotid gland, submandibular gland, pituitary gland, pancreas, testis, mammary gland, gastric mucosa, prostate and ovary, adrenal gland, heart, thymus, and lung. By contrast, hNIS transcripts were not detected in normal orbital fibroblasts, colon, and nasopharyngeal mucosa. To further analyze hNIS gene sequences in parotid gland, mammary gland, and gastric mucosa, the EXPAND High Fidelity PCR System and three sets of overlapping NIS oligonucleotide primers were used for amplification and cloning. The resulting PCR products were subcloned into pBluescript-SKII(-)vector, and at least two independent cDNA clones derived from each tissue were subjected to automated sequencing. The nucleotide sequences of hNIS cDNA derived from parotid gland, mammary gland, and gastric mucosa revealed full identity with the recently published human thyroid-derived NIS cDNA sequence. In conclusion, our results demonstrate markedly variable levels of hNIS gene expression in several extrathyroidal tissues. Although the physiological role of hNIS in these tissues awaits further study, our results suggest that the capacity to actively transport iodine may be a feature common to several secretory and endocrine tissues. The diminished capacity to transport and concentrate iodide in extrathyroidal tissues (such as parotid gland, mammary gland, and gastric mucosa), compared with thyroid gland, does not seem to be caused by an altered primary structure of the hNIS cDNA. Variability of NIS gene expression levels in normal extrathyroidal tissues may rather be caused by differences in NIS gene transcriptional activity. Further studies will address this hypothesis and examine the mechanisms of tissue-specific regulation of NIS gene expression.

Urinary excretion of the TRH-like peptide pyroglutamyl-glutamyl-prolineamide in rats.

TRH-like immunoreactivity (TRH-LI) was estimated in methanolic extracts of rat tissues and blood by RIA using antiserum 4319, which binds most peptides with the structure pGlu-X-ProNH2, or antiserum 8880, which is specific for TRH (pGlu-His-ProNH2). TRH-LI (determined with antiserum 4319) and TRH (determined with antiserum 8880) contents were 8 and 8 ng/g in brain, 216 and 222 ng/g in hypothalamus, 6.5 and 6 ng/g in pancreas, 163 and 116 ng/g in male pituitary, 105 and 77 ng/g in female pituitary, 1 and 0.1 ng/g in salivary gland, 61 and 42 ng/g in thyroid, 12 and 3 ng/g in adrenal, 3 and 0.3 ng/g in prostate, and 11 and 0.8 ng/g in ovary respectively. Blood TRH-LI (antiserum 4319) and TRH (antiserum 8880) levels were 31 and 18 pg/ml in male rats, and 23 and 10 pg/ml in female rats respectively. Unextracted serum obtained from blood kept for at least 1 h at room temperature no longer contained authentic TRH but still contained TRH-LI (males 20.3 +/- 3.1, females 15.9 +/- 3.0 pg/ml; means +/- S.E.M.). Isocratic reverse-phase HPLC showed that TRH-LI in serum is largely pGlu-Glu-ProNH2 (< EEP-NH2), a peptide previously found in prostate and anterior pituitary. In urine, TRH-LI (antiserum 4319) and TRH (antiserum 8880) levels were 3.21 +/- 0.35 and 0.32 +/- 0.04 ng/ml in male rats and 3.75 +/- 0.22 and 0.37 +/- 0.04 ng/ml in female rats respectively (means +/- S.E.M.). Anion-exchange chromatography on QAE-Sephadex showed that urine of normally fed rats contains both basic/neutral TRH-LI (b/n TRH-LI) and acidic TRH-LI (aTRH-LI) in a ratio of approximately 40:60, and further analysis by HPLC indicated that aTRH-LI represents < EEP-NH2. Analysis of food extracts and urine from fasted rats demonstrated that b/n TRH-LI is derived from food particles spilled by the rats during urine collection, while aTRH-LI is endogenously produced. While urinary aTRH-LI levels were higher in female than in male rats (2.99 +/- 0.41 vs 2.04 +/- 0.20 ng/ml), the daily urinary excretion was similar in both sexes (females 15.6 +/- 1.4, males 19.5 +/- 2.0 ng/day). Intravenously injected < EEP-NH2 disappeared from serum with a half-life of approximately 1 h, and was recovered unchanged and quantitatively in urine. In contrast, when < EEP-NH2 was administered with food, only approximately 0.5% was recovered in urine. The urinary clearance rate of serum TRH-LI amounted to 0.52 +/- 0.10 ml/min in males and 0.34 +/- 0.05 ml/min in females. In view of the presence of < EEP-NH2 in the anterior pituitary gland, and the regulation of its content in parallel with gonadotrophins, we examined the possibility that serum < EEP-NH2 is of pituitary origin and correlates with gonadotrophin secretion. However, treatments that alter pituitary < EEP-NH2 content and gonadotrophin release had no effect on serum TRH-LI or urinary aTRH-LI. In conclusion, the TRH-like peptide < EEP-NH2 is present in rat serum and is excreted into the urine. Moreover, < EEP-NH2 in serum and urine is not derived from rat food and is probably not of pituitary origin.

The effect of essential fatty acid deficiency on the fatty acid composition of different salivary glands and saliva in rats.

The main purpose was to compare the changes in fatty acid composition of lipids induced by essential fatty acid (EFA) deficiency in the rat submandibular, parotid and sublingual glands. Three groups of rats were fed for 28 weeks (1 week gestation, 3 weeks lactation and 24 weeks thereafter) diets containing 7% hydrogenated coconut oil (HCO) (EFA-deficient in both n-6 and n-3), 7% soybean oil (SBO) (control) and 7% safflower oil (SFO) (deficient in n-3). Rats were killed and salivary glands were dissected out. Lipids were extracted and the fatty acid composition of total lipids and phospholipids was determined by gas chromatography-mass spectrometry. The fatty-acid compositional changes indicative of an EFA deficiency, such as decreases in the levels of 18:2 n-6, along with an accumulation of 20:3 n-9, were generally observed in all the salivary glands of rats fed 7% HCO diet. In the submandibular glands, the proportions of 16:1, 18:1 n-9 and 18:1 n-7 were also higher in the HCO-fed group than in the other two groups. There were some differences in the fatty acid composition of the three glands. Total lipids of parotid gland had higher levels of 12:0 and 18:1 n-9 as compared to the other two glands. The levels of 18:0, 20:3 n-9, 20:3 n-6 and 20:4 n-6 were, however, lower in the parotid gland as compared with the other glands. In total phospholipids of rats fed SBO- and SFO-containing diets, the sublingual gland had lower levels of 18:2 n-6 and higher levels of 20:4 n-6 than the parotid or the submandibular. These differences in fatty acid composition may be related to possible differences in chain elongation/desaturation. The changes in fatty acid composition were also reflected in total lipids of plasma, liver and whole saliva of rats fed the various diets. A number of fatty acids were identified in saliva by gas chromatography-mass spectrometry.

Osmium ammine-B and electron spectroscopic imaging of ribonucleoproteins: correlation of stain and phosphorus.

Ultra-thin sections of Chironomus salivary glands were stained in a non-Feulgen procedure with osmium ammine-B and imaged at several electron energy-loss windows. For two types of RNP-containing structures (ie Balbiani ring granules and endoplasmic reticulum), a significant spatial correlation was observed between stain distribution and net phosphorus distribution. Non-Feulgen osmium ammine-B staining does not require the use of ultra-thin sections and can approximate the distribution of nucleic acid phosphorus.

Differential expression of type 2 and type 3 inositol 1,4,5-trisphosphate receptor mRNAs in various mouse tissues: in situ hybridization study.

The inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ release channel responsible for mobilizing stored Ca2+. Three different receptor types have been molecularly cloned, and their genes have been classified into a family. The gene for the type 1 receptor (IP3R1) is predominantly expressed in cerebellar Purkinje neurons, but its gene product is localized widely in a variety of tissues; however, there is little information on what types of cells express the other two receptor types, type 2 and type 3 (IP3R2 and IP3R3, respectively). We studied the expression of the IP3R gene family in various mouse tissues by in situ hybridization histochemistry. Compared with IP3R1, the levels of expression of IP3R2 and IP3R3 mRNAs were low in all of the tissues tested. IP3R2 mRNA was localized in the intralobular duct cells of the submandibular gland, the urinary tubule cells of the kidney, the epithelial cells of epididymal ducts and the follicular granulosa cells of the ovary, while the IP3R3 mRNA was distributed in gastric cells, salivary and pancreatic acinar cells and the epithelium of the small intestine. All of these cells which express either IP3R2 or IP3R3 mRNA are known to have a secretory function in which IP3/Ca2+ signalling has been shown to be involved, and thus either IP3R2 or IP3R3 may be a prerequisite to secretion in these cells.

A review of electron probe X-ray microanalysis studies of salivary gland cells.

Electron probe X-ray microanalysis (EPXMA) has now been successfully applied to several salivary gland preparations. This paper briefly reviews the principles underlying this technique and the specific sample preparation procedures which permit accurate measurement of elemental concentrations in the various intracellular spaces. Findings from salivary gland studies indicate that cytoplasmic and nuclear spaces of nonstimulated acinar cells have high concentrations of K and P, and low concentrations of Mg, Ca, and S; and that mature secretory granules have high concentrations of Ca and S, and relatively low concentrations of K and P. No consistent differences have been found between the elemental concentrations of mucous and serous secretory granules. In vivo and in vitro EPXMA studies of the elemental changes associated with secretory granule maturation indicate there are at least two stages in this process: an early stage during which granule S concentration increases in parallel with mass density as condensing vacuoles mature into secretory granules, and a late stage during which granule mass density and protein content increase with no further elemental concentration changes. Findings from other in vivo and in vitro studies indicate that secretory granule membranes are permeable to Na, K, and Cl ions because the granular concentrations of these elements are altered by electrochemical gradients. Recent EPXMA results indicate that cells stimulated with parasympathomimetic agonists have decreased K and Cl concentrations, and increased Na concentrations. Furthermore, the magnitude of these changes are quantitatively consistent with changes measured using radio-isotope equilibration and other techniques. In contrast, cells stimulated with the beta-adrenergic agonist, isoproterenol, have increased concentrations of Na and Cl, but unchanged K concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of clonidine on the calcium content and morphology of rat salivary glands.

These effects were examined with and without pretreatment of animals with reserpine and the adrenergic antagonists prazosin (alpha 1), yohimbine (alpha 2) and propranolol (beta). The effects of clonidine on glandular concentrations of norepinephrine and dopamine also were examined. These effects were compared with those of xylazine, a presynaptic alpha 2-adrenergic agonist. A single, high dose of clonidine followed by an overnight fast caused marked increases in calcium content and acinar secretory granules in the submandibular and sublingual glands, similar to those caused by reserpine. However, the calcium content of the parotid gland was not altered by clonidine, although there seemed to be a modest increase in acinar secretory granules. The clonidine-induced increase in submandibular calcium content could not be attributed to any adrenergic receptor activity since it was not blocked by either alpha- or beta-adrenergic antagonists. Unlike reserpine, clonidine did not affect catecholamine concentrations in the parotid and submandibular glands. Pretreatment with reserpine did not significantly alter the clonidine-induced increase in submandibular calcium content. It is likely that the greater accumulation of acinar secretory granules is related to the increased calcium stores of the glands in clonidine- and/or reserpine-treated rats. The large differences in calcium content among the three glands might be attributable, in part, to differences in the calcium-binding capacity of their secretory granules. Possible mechanisms for the clonidine effects on salivary-gland calcium include disturbances in membrane-associated pools or gating mechanisms for calcium, which need further study.