The activity of platelet activating factor-acetyl hydrolase (PAF-AH) in the salivary glands of Rhodnius prolixus

In this work, we investigated the activity of the platelet activating factor acetyl hydrolase (PAF-AH) in the salivary gland homogenates and saliva of Rhodnius prolixus. PAF-AH activity in the salivary gland homogenates was lower than in the saliva. Preliminary characterization of the enzyme demonstrated that it hydrolyzed the substrate 2-thio-PAF, was detectable just in 1 pair of salivary gland homogenates in 0.5 ml buffer, and was stable under different conditions. PMSF, TPCK, TLCK, pepstatin A and p-BPB all inhibited the PAF-AH activity. Enzyme specific activity in salivary gland homogenates diminished immediately after feeding of 5th-instar larvae, and increased before feeding by adult insects. 2-Thio-PAF induced platelet-aggregation that was inhibited by previous incubation of the substrate with salivary gland homogenates or saliva. The relevance of PAF-AH for providing Rhodnius with a feeding mechanism for facilitating the sucking of a high volume of blood meal in a short period is discussed.     

Composition and variability of the saliva of coreids in relation to phytoxicoses and other aspects of the salivary physiology of phytophagous Heteroptera

In the saciform, principal salivary glands ofMictis profana (Fabr.) (Coreidae: Heteroptera, Pentatomorpha), the contents of all lobes other than the posterior form gels consistent with their contributing to the solidifying saliva (stylet sheath); the posterior lobe secretes most if not all of a sucrose-hydrolysing enzyme that occurs in the nongelling (watery) saliva. Evidence for the occurrence of such an enzyme in the saliva of other coreids is presented. That inM. profana has a pH optimum near neutral and a substrate specificity consistent with sucrase (sucrose α-D-glucohydrolase, EC 3.2.1.48) as distinct from plant invertase (β-D-fruccofuranosidase, EC 3.2.1.26). Apart from some maltose-hydrolysing activity in the salivary glands, also consistent with sucrase, no other carbohydrases and neither proteinase nor lipase were detected. Phosphatases were found in gland extracts but not in secreted saliva. The saliva contains catechol oxidase (EC 1.10.3.1) from the accessory gland and ducts. Topical application of pilocarpine caused individualM. profana to secrete up to 58 μl watery saliva which showed continuous and independent variation of sucrase activity (up to ca 0.012 Units/μl) and pH (6–8), although high sucrase content tended to coincide with high pH. Total protein varied up to 10 μg/μl, and free amino acids up to 1.8 μg/μl leucine eq. Of the many proteins and/or protein subunits separable by electrophoresis of gland contents and saliva, four had sucrase activity, the most mobile with MW ca 66 000. TLC indicatedinter alia phenyl alanine and tyrosine, but no DOPA nor other diphenolic substrates of the catechol oxidase in the watery saliva. The soluble components of the saliva, which also has marked surfactant properties, are discussed in relation to the feeding process of coreids and the characteristic lesions they produce in their food plants.     

Molecular cloning of a novel calcium-binding protein in the secreted saliva of the green rice leafhopper Nephotettix cincticeps

Green rice leafhoppers (Nephotettix cincticeps) secrete watery and coagulable saliva in the feeding process. In our study, the watery salivary secretion was concentrated by ultrafiltration from “fed diet” and subjected to SDS-PAGE. The N-terminal amino acid sequence of the most predominant band at 84 kDa (designated NcSP84) was analyzed by Edman degradation. This sequence was completely consistent with the most abundant protein in the salivary gland extracts, which was separated by two-dimensional gel electrophoresis. Based on the N-terminal amino acid sequence, the complete cDNA of this protein was cloned by 5′- and 3′-RACE using degenerate primers. The deduced NcSP84 contained an open reading frame of 2061 bp encoding a putative 687 amino acids with a putative signal sequence composed of 19 amino acids. The nucleotide and amino acid sequences of NcSP84 did not share statistically significant homology with any sequences in public databases. Motif search predicted that this protein had EF-hands, the most common motif found in Ca²⁺ -binding proteins. As predicted, NcSP84 exhibited Ca²⁺-binding activity. The SDS-PAGE mobility of purified NcSP84 bound to Ca²⁺ tended to decline discretely, depending on the concentration of CaCl₂ with which it was mixed for 1 h before adding SDS buffer. In situ hybridization and immunohistochemistry showed that the NcSP84 gene and gene product were expressed and stored in type III cells, which are the largest lobes in the primary salivary glands. The NcSP84 protein was detected in the phloem sap of rice exposed to leafhoppers, verifying that the NcSP84 protein was injected into the sieve tubes. These results suggest that NcSP84 could be secreted into the sieve tubes during feeding, which might bind Ca²⁺ ions that flow into sieve tubes in response to stylet puncturing. This might suppress sieve-element clogging and facilitate continuous ingestion from sieve tubes.     

Preliminary characterisation of digestive proteases of the green mirid, Creontiades dilutus (Hemiptera: Miridae)

Protease activities in the secreted saliva, salivary glands and midgut of the green mirid, Creontiades dilutus, were investigated. The saliva and salivary glands had more protease activity than the midgut, but no differences in protease activity levels were detected between male and female mirids, adult mirids and third instar nymphs, or between fed and starved mirids.In the salivary glands, chymotrypsin-like serine proteases predominated, as characterised by inhibitor specificity, basic pH optima, and hydrolysis of N-benzoyl-L-tyrosine p-nitroanilide and N-succinyl-ala-ala-pro-leu p-nitroanilide. The pH optimum of midgut extracts was acidic (pH 4), implying that acidic proteases predominate. However, protease activity was inhibited substantially by both aprotinin and E-64, suggesting the presence of both serine and cysteine proteases in the midgut of the green mirid.     

Phenoloxidase of peach (Prunus persica) endocarp: Its relationship with peroxidases and lignification

A preliminary characterization of a phenoloxidase from extracts of soluble and ionically-bound cell wall proteins of peach ( Prunus persica L. Batsch, cv. Redhaven) endocarp is described in the present study to establish differences with peroxidases from the same plant tissue. The phenoloxidase activity was detected mainly in the first stage of peach fruit growth, while peroxidase activity and lignin content increased along the second stage of growth. There were clear differences between the two enzymes. The phenoloxidase had a pI value of 5.6, different from those of peroxidases isoenzymes with various pIs ranging from 3.6 to 9.6. The oxidase molecular mass was 112 kDa, similar to other phenoloxidases described in the literature, while all peroxidase isoenzymes showed a molecular mass of around 40 kDa. The specific activities of phenoloxidase against different substrates and its inhibition by various effectors suggest that the endocarp oxidase described here is probably a metal-dependent polyphenol oxidase, displaying attributes of both catechol oxidase (EC 1.10.3.1) and laccase (EC 1.10.3.2).     

Identification and characterization of tyrosylprotein sulfotransferase from human saliva

Tyrosylprotein sulfotransferase (TPST), the enzyme responsible for the sulfation of tyrosine residues, has been identified and characterized in submandibular salivary glands previously (William et al. Arch Biochem Biophys 338: 90-96). Tyrosylprotein sulfotransferase catalyses the sulfation of a variety of secretory and membrane proteins and is believed to be present only in the cell. In the present study, this enzyme was identified for the first time in human saliva. Analysis of human saliva and parotid saliva for the presence of tyrosylprotein sulfotransferase revealed tyrosine sulfating activity displayed by both whole saliva and parotid saliva at pH optimum of 6.8. In contrast to tyrosylprotein sulfotransferase isolated from submandibular salivary glands, salivary enzyme does not require the presence of Triton X-100, NaF and 5'AMP for maximal activity. Similar to the submandibular TPST, the enzyme from saliva also required MnCl2 for its activity. Maximum TPST activity was observed at 20 mM MnCl2. The enzyme from saliva was immunoprecipitated and purified by immunoaffinity column using anti-TPST antibody. Affinity purified salivary TPST showed a single band of 50-54 kDa. This study is the first report characterizing a tyrosylprotein sulfotransferase in a secretory fluid.     

Purification of a novel extracellular laccase from solid-state culture of the edible mushroom <Emphasis Type="Italic">Lentinula edodes</Emphasis>

The laccases (EC 1.10.3.2) secreted into solid-state culture by Lentinula edodes were analyzed. The fungus secreted at least two laccases in the solid-state culture. One laccase was purified to a homogeneous preparation using anion-exchange, hydrophobic, and size-exclusion chromatography. SDS-PAGE analysis showed that the purified laccase, Lcc6, was a monomeric protein of 58.5 kDa. The optimum pH for enzyme activity was about 3.5, and the laccase was most active at 40°C. The N-terminal amino acid sequence of Lcc6 did not correspond to the sequence of Lcc1, which was previously purified from L. edodes. Lcc6 had decolorization activity to some chemical dyes.     

A salivary sheath protein essential for the interaction of the brown planthopper with rice plants

Salivary secretions, including gel saliva and watery saliva, play crucial roles in the interaction between the insect and plant during feeding. In this study, we identified a salivary gland-specific gene encoding a salivary sheath protein (NlShp) in Nilaparvata lugens. NlShp has two alternative splicing variants; both are expressed at high levels during the nymph and adult stages. Immunohistochemical staining showed that the NlShp were synthesized in the principal gland cells of the salivary gland. LC-MS/MS and western blot analysis confirmed that NlShp was one of the components of the salivary sheath. Simultaneously knocking down the two NlShp variants by RNA interference inhibited both salivary flange and salivary sheath formation and resulted in a lethal phenotype within four days for the brown planthopper (BPH) feeding on rice plants, indicating that the salivary sheath and salivary flanges were essential for plant-associated feeding. Despite the salivary sheath deficiency, no obvious phenotype was observed in the NlShp-knockdown BPHs fed on artificial diet. The electrical penetration graph (EPG) results showed that salivary sheath-deficient BPHs exhibited a prolonged nonpenetration period, scarce sap period, and increased stylet movement on rice plants and eventually starved to death. Our results provided evidence that the interaction between the salivary sheath and host plant might be a critical step in successful BPH feeding. According to present research, we propose a salivary sheath required feeding model for piercing-sucking insects and provide a potential target for rice planthopper management.     

Purification,characterization, and identification of a novel bifunctional catalase-phenol oxidase from <Emphasis Type="Italic">Scytalidium thermophilum</Emphasis>

A novel bifunctional catalase with an additional phenol oxidase activity was isolated from a thermophilic fungus, Scytalidium thermophilum. This extracellular enzyme was purified ca. 10-fold with 46% yield and was biochemically characterized. The enzyme contains heme and has a molecular weight of 320 kDa with four 80 kDa subunits and an isoelectric point of 5.0. Catalase and phenol oxidase activities were most stable at pH 7.0. The activation energies of catalase and phenol oxidase activities of the enzyme were found to be 2.7 +/- 0.2 and 10.1 +/- 0.4 kcal/mol, respectively. The pure enzyme can oxidize o-diphenols such as catechol, caffeic acid, and L-DOPA in the absence of hydrogen peroxide and the highest oxidase activity is observed against catechol. No activity is detected against tyrosine and common laccase substrates such as ABTS and syringaldazine with the exception of weak activity with p-hydroquinone. Common catechol oxidase inhibitors, salicylhydroxamic acid and p-coumaric acid, inhibit the oxidase activity. Catechol oxidation activity was also detected in three other catalases tested, from Aspergillus niger, human erythrocyte, and bovine liver, suggesting that this dual catalase-phenol oxidase activity may be a common feature of catalases.     

Salivary signals of European corn borer induce indirect defenses in tomato

Plants can recognize the insect elicitors and activate its defense mechanisms. European Corn Borer (ECB; Ostrinia nubilalis) saliva, produced from the labial salivary glands and released through the spinneret, is responsible for inducing direct defenses in host plants. Glucose oxidase (GOX) present in the ECB saliva induced direct defenses in tomato. By contrast, GOX activity in ECB saliva was insufficient to trigger defenses in maize, suggesting that host-specific salivary elicitors are responsible for inducing direct defenses in host plants. Our current study further examined whether ECB saliva can trigger indirect defenses in tomato. Relative expression levels of TERPENE SYNTHASE5 (TPS5) and HYDROPEROXIDE LYASE (HPL), marker for indirect defenses in host plants, were monitored. Quantitative real-time PCR analysis revealed that ECB saliva can induce the expression of TPS5 and HPL, suggesting that salivary signals can induce indirect defenses in addition to the direct defenses. Further experiments are required to identify different ECB elicitors that are responsible for inducing direct and indirect defenses in host plants.     

A tick B-cell inhibitory protein from salivary glands of the hard tick, Hyalomma asiaticum asiaticum

Some studies done to date suggest that B-cell inhibitory factor occurred in tick saliva. In this study, a novel protein having B-cell inhibitory activity was purified and characterized from the salivary glands of the hard tick, Hyalomma asiaticum asiaticum. This protein was named B-cell inhibitory factor (BIF). The cDNA encoding BIF was cloned by cDNA library screening. The predicted protein from the cDNA sequence is composed of 138 amino acids including the mature BIF. No similarity was found by Blast search. The lipopolysaccharide-induced B-cell proliferation was inhibited by BIF. This is the first report of the identification and characterization of B-cell inhibitory protein from tick. The current study facilitates the study of identifying the interaction among tick, Borrelia burgdorferi, the causative agent of Lyme disease, and host.     

Aphids secrete watery saliva into plant tissues from the onset of stylet penetration

Aphid feeding requires the secretion of two types of saliva: gelling saliva (from the principal gland) that forms an intercellular sheath for the penetrating stylet, and watery saliva [from accessory salivary glands (ASGs)] that facilitates intracellular penetration and phloem feeding. Plant viruses can be used as salivary markers to investigate key steps in aphid feeding, and penetration can be monitored electrically using the electrical penetration graph (EPG) approach. We conducted a series of EPG‐controlled transmission experiments using Cucurbit aphid‐borne yellows virus [CABYV; Polerovirus spec. (Luteoviridae)], which is retained in the ASGs, as a marker for watery saliva secretions. The melon aphid, Aphis gossypii Glover (Hemiptera: Aphididae), was used as a vector and melon seedlings, Cucumis melo L. (Cucurbitaceae), as host plants. Viruliferous aphids were interrupted at various stages during stylet penetration, i.e., during intercellular penetration prior to intracellular puncture and following a potential drop within the first probe. Viruliferous aphids and leaf disc samples obtained from the stylet penetration site were used to detect CABYV by quantitative real‐time RT‐PCR. Approximately half of the inoculated leaf discs were found to be infected with CABYV after very brief (12.9 ± 1.9 s) intercellular stylet probes and before intracellular stylet puncture. The number of virus particles ejected during such probes was similar to the number ejected by aphids during longer probes including a single intracellular puncture. Our results therefore suggest that watery saliva is secreted by aphids from the onset of stylet penetration.     

Boophilus microplus: its saliva contains microphilin, a small thrombin inhibitor

Saliva of the cattle tick Boophilus microplus contains two thrombin inhibitors, BmAP and microphilin. This work presents the purification and characterization of microphilin. It was purified from the saliva by gel filtration, ultrafiltration through a 3 kDa cut-off membrane and affinity chromatography in a thrombin-Sepharose column. Analysis by mass spectrometry showed a molecular mass of 1770 Da. Microphilin is the smallest salivary thrombin inhibitor peptide known to date. It inhibits fibrinocoagulation and thrombin-induced platelet aggregation with an IC(50) of 5.5 microM, is temperature resistant and its inhibitory activity was abolished by protease K treatment. Microphilin did not inhibit the amidolytic activity of the enzyme upon a small chromogenic substrate, but inhibited the hydrolysis of a substrate that binds both catalytic site and exosite I. Therefore, we propose that microphilin blocks thrombin at exosite I.     

棉铃虫幼虫唾液腺cDNA文库的构建及EST分析

棉铃虫Helicoverpa armigera (Hübner)幼虫唾液中的各种酶类及各种生化组分在棉铃虫与植物相互作用及协同进化中起到重要作用; 唾液腺是棉铃虫唾液成分的合成器官。本研究通过构建棉铃虫幼虫唾液腺全长cDNA文库, 测序得到1 502条EST序列, 聚类分析后获得821个unigenes, 为筛选棉铃虫与寄主互作信号因子提供基因信息资源。使用Blast2 GO软件对821个unigenes进行了比对和功能注释, 初步获得棉铃虫幼虫唾液腺中mRNA的构成特征。结果显示, 在棉铃虫唾液腺ESTs文库中, 鉴定得到脂类相关消化酶基因17个, 糖类相关消化酶基因5个, 半胱氨酸蛋白酶基因1个, 丝氨酸蛋白酶基因20个（其中16个为新发现）, 提示唾液腺的主要功能是分泌消化酶进行预消化; 还发现在棉铃虫幼虫唾液腺中存在表皮蛋白、 气味结合蛋白和化学感受蛋白基因。结果为研究棉铃虫预消化系统打下基础。     

Laccase activity in melanin-producing strains of Sinorhizobium meliloti

Laccase-like activity was detected in melanin-producing strains of Sinorhizobium meliloti mainly in cells at the stationary growth phase when copper was added to the medium. The laccase showed both syringaldazine and ABTS (2,2'-azino-bis-ethylbenzthiazoline-6-sulfonic acid) oxidase activities and was activated by the addition of 1.7 mM sodium dodecyl sulfate. Activity was totally inhibited by the addition of 1.0 mM EDTA, suggesting that the enzyme is a metal-dependent one. The enzyme was found to be cytosolic having an optimum pH of 5.0, an estimated molecular mass of 95 kDa and a K(m) of 4 microM for syringaldazine. Both laccase and tyrosinase activities were detected in melanin-producing S. meliloti strains. Plant growth-promoting (PGP) effect in rice by a laccase-producing S. meliloti strain when co-inoculated with Azospirillum brasilense Cd was observed. PGP effect by co-inoculation significantly increased plant yield compared to A. brasilense by itself. To the best of our knowledge this is the first report on laccase production in rhizobia and cooperation between Azospirillum and Sinorhizobium in rice.     

Purification of β-glucosidase from the salivary glands of the green rice leafhopper, Nephotettix cincticeps (Uhler) (Hemiptera: Cicadellidae), and its detection in the salivary sheath

The green rice leafhopper, Nephotettix cincticeps (Uhler), is an insect pest of rice and discharges β-glucosidase (EC 3.2.1.21) from its salivary glands during feeding. To investigate the biological function of this enzyme, we purified it from the heads of 18,000 adult females by acetone precipitation and a series of chromatography steps: gel filtration, cation-exchange chromatography, metal-affinity chromatography and hydrophobic interaction chromatography. During cation-exchange chromatography, β-glucosidases were eluted in three peaks (isozymes). These β-glucosidases were monomeric proteins of 58 kDa as estimated by SDS-PAGE and 62 kDa based on gel filtration. All of the purified β-glucosidase isozymes exhibited maximum activity for p-nitrophenyl β-glucoside (NPGlc) and p-nitrophenyl β-galactopyranoside (NPGal) at pH 5.5 and 5.0, respectively. There was no significant difference in substrate specificity among the three isozymes. The Km values were estimated to be 0.13 μM for NPGlc and 0.9 μM for NPGal. Among the oligosaccharide substrates examined, laminaribiose (Glc β1-3 Glc) was the most extensively hydrolyzed, sophorose (Glc β1-2 Glc) and cellobiose (Glc β1-4 Glc) were comparatively well hydrolyzed, and gentiobiose (Glc β1-6 Glc), lactose (Gal β1-4 Glc), laminaritriose, cellotriose and cellotetraose were poorly hydrolyzed. Among the glycoside substrates examined, salicin was considerably well hydrolyzed. β-Glucosidase was detected in the salivary sheaths by activity staining with a fluorescent substrate. The salivary β-glucosidase of N. cincticeps may be involved in the hydrolysis of a phenol glucoside present in the saliva, which is a step in the solidification of gelling saliva to form salivary sheaths.     

Catechol 1,2-dioxygenase from the new aromatic compounds - Degrading Pseudomonas putida strain N6

This study aimed to characterization of catechol 1,2-dioxygenase from a Gram-negative bacterium, being able to utilize a wide spectrum of aromatic substrates as a sole carbon and energy source. Strain designated as N6, was isolated from the activated sludge samples of a sewage treatment plant at Bentwood Furniture Factory Jasienica, Poland. Morphology, physio-biochemical characteristics and phylogenetic analysis based on 16S rDNA sequence indicate that strain belongs to Pseudomonas putida. When cells of strain N6 grown on protocatechuate or 4-hydroxybenzoic acid mainly protocatechuate 3,4-dioxygenase was induced. The activity of catechol 1,2-dioxygenase was rather small. The cells grown on benzoic acid, catechol or phenol showed high activity of only catechol 1,2-dioxygenase. This enzyme was optimally active at 35 °C and pH 7.4. Kinetic studies showed that the value of K_m and V_max was 85.19 ??M and 14.54 ??M min⁻¹ respectively. Nucleotide sequence of gene encoding catechol 1,2-dioxygenase in strain N6 has 100% identity with catA genes from two P. putida strains. The deduced 301-residue sequence of enzyme corresponds to a protein of molecular mass 33.1 kDa. The deduced molecular structure of the catechol 1,2-dioxygenase from P. putida N6 was very similar and characteristic for the other intradiol dioxygenases.