Preliminary characterisation of esterase and platelet-activating factor (PAF)-acetylhydrolase activities from cat flea (Ctenocephalides felis) salivary glands

Naphthyl esterase and platelet-activating factor (PAF)-acetylhydrolase activities were detected in the salivary glands of the cat flea, Ctenocephalides felis. Salivary naphthyl esterase activity is disgorged during exploratory probing. Whole extracts of salivary glands contain esterase activity against the short-chain naphthyl esters alpha-naphthyl acetate (approximately 210pmol/min/gland pair; 10.0micromol/min/mg specific activity; K(m) approximately 59microM) and beta-naphthyl acetate (approximately 110pmol/min/gland pair; 5.2micromol/min/mg specific activity; K(m) approximately 132microM). Salivary gland extracts have PAF-acetylhydrolase activity (approximately 5pmol/min/gland pair; 0.24micromol/min/mg specific activity) but do not have detectable acetylcholinesterase activity. Native-PAGE and IEF resolve three and six salivary gland naphthyl esterase bands, respectively, and both patterns are different from carcass esterases. Salivary gland naphthyl esterase activity binds reversibly to Concanavalin A, and enzymatic deglycosylation with glycopeptidase F produced a new, fast-migrating salivary gland naphthyl esterase band on Native-PAGE. Renaturation of esterase activity after SDS-PAGE gave approximately 56kDa, approximately 57kDa and approximately 58kDa naphthyl-esterase-positive bands. On gel filtration naphthyl esterase and PAF-acetylhydrolase activities co-elute as a single peak with an apparent molecular weight of approximately 59kDa. This partially purified pool of enzyme had esterase activity against a series of short-chain alpha- and beta-naphthyl esters. The heterogeneity of salivary gland esterases, their relationship to PAF-acetylhydrolase, and the possible physiological functions of salivary gland PAF-acetylhydrolase activity are discussed.     

Comparative analysis of the malate dehydrogenases isolated from the cytosolic fraction of several tissues of guinea-pig Cavia porcellus

The specific activities of the malate dehydrogenase and lactate dehydrogenase present in the soluble fraction of several guinea-pig tissues are reported. The electrophoretic patterns showed always two forms (A and B) with malate dehydrogenase activity and the five isoenzymes of lactate dehydrogenase. Chromatography of the different soluble fractions through 5' AMP-Sepharose allowed both molecular forms of malate dehydrogenase to be separated and obtained free from lactate dehydrogenase. Comparative studies of the two forms of malate dehydrogenase evidenced that the A and B forms exhibited cytosolic and mitochondrial characteristics, respectively.     

Patterns and variability in electrophoretic polypeptide profiles of human saliva in a healthy population

Electrophoretic polypeptide profiles of normal human saliva differ markedly between different reports. Since both methodological variations and polymorphism may explain these differences, in this study we aimed to establish whether or not the salivary electrophoretic polypeptide profiles of subjects from a healthy population share discrete molecular features. To this end, parotid, submandibular/sublingual and whole salivas were collected separately from each of 40 young and 34 elderly clinically healthy adults and processed for SDS-polyacrylamide gel electrophoresis and Coomassie blue staining. Each type of glandular saliva displayed a different group of invariant (i.e. present in every subject) electrophoretic polypeptide bands while whole saliva showed a profile that reflected mostly the combined contribution of the major salivary glands. Some minor variant (i.e. absent in some subjects) bands were identified in each type of saliva. Regarding those interindividual variations, no age- or sex-dependence was appreciated. Altogether, these results demonstrate the occurrence of distinctive electrophoretic polypeptide patterns, in addition to some minor variations, for each type of normal saliva, thus providing a background for further populational studies on salivary polypeptide profiles.     

Morphology and electrophoretic protein profiles of female salivary glands in four Oriental black fly species (Diptera: Simuliidae).

Saliva of female flies is responsible for localized hypersensitivity reactions and life-threatening systemic hemorrhagic syndromes in humans and animals. In this study, morphology and electrophoretic protein profiles of female salivary glands of Oriental black flies in the subgenus Simulium Latreille s. str., Simulium (Simulium) nigrogilvum, S. (S.) rufibasis, S. (S.) nodosum, and subgenus Gomphostilbia Enderlein, S. (Gomphostilbia) asakoae were analyzed. The paired female salivary glands of the four simuliid species were morphologically similar and situated on either side of the esophagus. Each gland is composed of two main parts, a secretory arm and a reservoir. In each species, the size of the gland correlated with salivary gland protein contents. SDS-PAGE analysis revealed differences of electrophoretic protein profiles and specifically major protein bands of the female salivary glands in each species, suggesting that protein profiles might be useful for construction of an additional tool to distinguish these black fly species. The information obtained from this study is an initial step for further research on salivary proteins that are involved in vertebrate hemostatic response.     

雌雄文昌鱼同工酶的表型差异

本文应用聚丙烯酰胺凝胶电泳结合生化染色方法分析了雌雄文昌鱼中苹果酸酶、苹果酸脱氢酶、酸性磷酸酶和酯酶四种同工酶的酶谱。首次发现苹果酸酶、苹果酸脱氢酶和酸性磷酸酶表型在文昌鱼雌性和雄性个体之间存在差异 ,而在同一性别不同个体之间无差异。酯酶表型较复杂 ,不但在不同性别个体之间而且在同一性别不同个体之间都出现一定差异     

Variability in esterases of Metarhizium anisopliae

The variability in esterases of the entomogenous fungus Metarhizium anisopliae was determined electrophoretically on 8.5% polyacrylamide gel. Ten isolates from diverse taxonomic groups of insects were analyzed. The electrophoretic analysis showed differences and similarities between these isolates and it was possible to distinguish six different patterns. The results obtained show a great polymorphism for the esterase system of M. anisopliae.     

Comparative esterase electrophoretic polymorphism of Escherichia coli isolates obtained from animal and human sources

To determine whether enzyme electrophoretic polymorphism in Escherichia coli populations was influenced by environmental background, the mobilities of four electrophoretically variable esterases (A, B, C and I) were examined. The distinction between isolates was established by significant differences in the electrophoretic distribution and the genetic diversity coefficient of individual esterases. Principal components analysis on each population and on all strains revealed three groups of allozymes. The first, characterized by slow electrophoretic mobilities of esterase B, was frequently observed in strains obtained from human extra-intestinal infections and rarely in commensal organisms. The second, characterized by fast mobilities of esterases A and B, was frequently found in animal isolates. The third, characterized by prominence of the most common mobilities of esterases B and A, was recovered in all populations. These results were confirmed by discriminant analysis. Among the 610 strains investigated, 316 electrophoretic types (distinctive combinations of allozymes of the four varieties of esterases) were distinguished, illustrating high esterase polymorphism.     

CELLULAR AND SECRETORY PROTEINS OF THE SALIVARY GLANDS OF SCIARA COPROPHILA DURING THE LARVAL-PUPAL TRANSFORMATION

The cellular and secretory proteins of the salivary gland of Sciara coprophila during the stages of the larval-pupal transformation were examined by electrophoresis in 0.6 mm sheets of polyacrylamide gel with both SDS-continuous and discontinuous buffer systems. After SDS-electrophoresis, all electrophoretograms of both reduced and nonreduced proteins from single glands stained with Coomassie brilliant blue revealed a pattern containing the same 25 bands during the stages of the larval-pupal transformation. With the staining procedures used in this study, qualitative increases and decreases were detected in existing proteins and enzymes. There was no evidence, however, for the appearance of new protein species that could be correlated with the onset of either pupation or gland histolysis. Electrophoretograms of reduced samples of anterior versus posterior gland parts indicated that no protein in the basic pattern of 25 bands was unique to either the anterior or posterior gland part. Electrophoretograms of reduced samples of secretion collected from either actively feeding or "cocoon"-building animals showed an electrophoretic pattern containing up to six of the 25 protein fractions detected in salivary gland samples, with varied amounts of these same six proteins in electrophoretograms of secretion samples from a given stage. Zymograms of non-specific esterases in salivary gland samples revealed a progressive increase in the amount of esterase reaction produce in one major band and some decrease in the second major band during later stages of the larval-pupal transformation.     

Zymographic demonstration of lactate and malate dehydrogenases isoenzymes in the rodent salivary glands

Summary Analysis of lactate and malate dehydrogenase zymograms of rodent salivary glands showed species and organ specific patterns.Lactate dehydrogenase isoenzyme patterns occupied the middle positions in relation to those of skeletal and heart muscle. Activities of the major salivary glands were in the order submaxillary gland>parotid>sublingual gland. Zymogram of the mouse and rat showed LDH₄ and LDH₅ high activity patterns, while that of the rabbit was the fast moving active one. Hamster salivary gland exhibited a neutral type of the former and the latter.Malate dehydrogenase isoenzyme exhibited very similar patterns for the mouse, rat and hamster. Malate dehydrogenase zymogram of rabbit showed 3 active bands, which was different from the other rodents.     

Comparative electrophoretic polymorphism of esterases and other enzymes in Escherichia coli

The electrophoretic polymorphism of esterases was compared with that of other enzymes in Escherichia coli populations by investigating allozyme distribution of four esterases (A, B, C and I) within both the subspecific groups I, II and III and the new groups A, B1, B2, C, D and E, which have been distinguished by electrophoretic analysis of 11 and 35 enzymes respectively in the 72 reference strains of the ECOR collection. Electrophoretic distribution of esterases was distinct for each of the three subspecific groups as indicated by distributions of allozymes and electrophoretic types (distinctive combination of allozyme for the four esterases). Esterase polymorphisms of the three subspecific groups appeared to have similar features to those of three previously studied natural populations of strains obtained from human and animal gastro-intestinal tracts and extra-intestinal infections in humans. Multiple correspondence analyses using data obtained from the four esterases and the 11 other enzymes also distinguished the groups A, B1, B2, C, D and E. All strains of group B2 showed the B2 electrophoretic pattern of esterase B, which appeared to be a marker of a distinct cluster of strains frequently implicated in extra-intestinal infections.     

Individual variations in the content of giant secretory polypeptides in salivary glands of Chironomus

Salivary glands in aquatic larvae of Chironomus are responsible for formation of a fiber that larvae use to construct feeding tubes. Major constituents of this fiber include a family (the sp-I family) of high M _r (1 × 10⁶) secretory polypeptides. Because of our interest in the polypeptide composition and polymerization of the salivary fiber we conducted a survey of the electrophoretic pattern of sp-I components found in salivary glands obtained from individual larvae. The survey encompassed ten strains of Chironomus tentans, three strains of Chironomus pallidivittatus and four strains of Chironomus thummi. Salivary glands from C. tentans and C. pallidivittatus contained at least four sp-I components (sp-Ia, sp-Ib, sp-Ic and sp-Id) that behave identically with regard to their electrophoretic mobility and detectability when larvae were exposed to galactose or glycerol. Sp-I components in C. thummi were generally fewer and not directly comparable by electrophoretic mobility to sp-I components in the other two species. During this survey two important alterations were observed in the electrophoretic pattern of sp-I components obtained from C. tentans and C. pallidivittatus. First, all four sp-I components exhibited, with a low frequency, double bands that appeared as slow-versus-fast electrophoretic variants of a particular component. Secondly, the relative steady-state level of each sp-I component fluctuated in comparison to other sp-I components in the same extract. This fluctuation varied such that any one sp-I component might appear as a single prominent component. Sp-I components are encoded by a multigene family located in Balbiani rings (BRs). Results presented here, in conjunction with known nucleotide sequence data from BR genes, led us to the following conclusions. The slow and fast electrophoretic variants observed for each sp-I component suggest that each corresponding BR gene may be able to expand and/or contract in size. The observed degree of independent fluctuation in the steady-state level of each sp-I component suggests that each BR gene may be able to regulate its expression independently from the others. Finally, the observation that salivary glands sometimes contained only one prominent sp-I component led us to hypothesize that, whereas salivary fibers might typically be heteropolymers comprised of two or more types of sp-I components, homopolymers comprised of only one sp-I component may also exist.     

家蚕蛹-成虫变态期中肠和涎腺超微结构变化与功能

为进一步明确家蚕Bombyx mori变态期间消化系统的生理功能以及溶茧酶的来源器官,通过透射电镜观察和酶活性检测,对家蚕蛹 成虫变态期中肠和涎腺的超微结构、水解酶活力以及中肠内容物的变化进行了观察和分析。结果表明: 蛹第7日到羽化前1日家蚕中肠细胞和刚羽化成虫的涎腺细胞中,均可观察到大量的分泌泡、分泌颗粒、微绒毛等分泌细胞的结构特征以及活跃的分泌现象。潜成虫的中肠和涎腺中都存在活性较高的水解酶活力,其中每毫克中肠组织中蛋白酶活力、酯酶活力和纤溶酶活力分别为726 U、751 U和263 U,每毫克涎腺组织中上述3种酶的活力分别为603 U、523 U和147 U,说明中肠和涎腺可能都具有分泌溶茧酶的功能。家蚕蛹期中肠内容物的主要成分是蛋白质、脂质和糖,三者占内含物总量的95%以上,其中蛋白质含量占78.8%～80.2%。中肠内容物的重量在刚化蛹时为20.1～21.9 mg/头,化蛹后7日内无明显变化,化蛹第9日内容物重量减少63.01%～66.17%,到成虫羽化时已所剩无几,可能是因内容物被消化吸收所致。据此推测,在家蚕变态期中肠还具有贮存和释放营养物质的功能,而溶茧酶的另一个功能可能是分解消化中肠内容物。     

Kallikrein localization in rodent salivary glands and kidney with the immunoglobulin-enzyme bridge technique.

Kallikrein has been localized in rodent kidney and salivary glands by means of an immunoglobulin-enzyme bridge technique. In sections of kidney, anti-kallikrein antibodies bound to the apical region of certain distal tubule segments in the cortex, to reabsorption droplets of proximal convoluted tubules, and to certain duct segments in the papilla. In salivary glands of both male and female rats and mice, and apical rim of most striated duct cells of submandibular, parotid and sublingual glands and granular tubules of submandibular glands exhibited immunoreactivity. Granular intercalated duct cells in female submandibular glands also displayed immunostaining for kallikrein. Phenylephrine administration resulted in loss of immunoreactive granules from the granular convoluted tubule cells of male mouse submandibular gland. This response was paralleled by a biochemically demonstrable decrease in kallikrein-like tosylarginine methyl ester (TAME) esterase activity.     

Cytoskeletal F-actin patterns in whole-mounted larval and adult salivary glands of the fleshfly, Sarcophaga bullata.

The patterns of filamentous actin were analysed in different larval, pupal and adult stages in the salivary glands of the fleshfly Sarcophaga bullata. Using the rhodamine labelled phalloidin staining method in combination with detergent extraction specific actin filament distribution was detected. The salivary glands which are histolysed during the process of metamorphosis show distinct cellular morphology and actin filament patterns in larvae and adults. The large third instar larval salivary gland cells contain a well developed apicolateral microvillar zone. In third instar larvae this microvillar zone invaginates and expands in the basal part of the lateral membranes. Larval salivary gland cells also contain numerous parallel basal actin bundles. The larval glands are histolysed during metamorphosis and adult glands are formed out of the imaginal cell group. At the onset of metamorphosis these basal actin bundles form a network of crossing bundles. The filamentous actin patterns of the proximal part of adult gland cells is confined to the apicolateral microvillar membranes. The cells in the distal, tubular part of the adult salivary glands show intense staining of their folded lateral membranes.     

Characterization of Enterobacter cloacae and E. sakazakii by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases

Acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases of 34 strains of Enterobacter cloacae and 22 strains of Enterobacter sakazakii were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gel. The two species could be separated on the basis of distinct electrophoretic patterns of all enzymes analysed. Glutamate dehydrogenase and acid phosphatase were detected exclusively in E. cloacae, whereas esterase bands were more intensively stained in E. sakazakii. For each species, two zymotypes could be distinguished, on the basis of electrophoretic mobilities of malate dehydrogenase and banding patterns of esterase for E. cloacae, and by both isoelectric point and electrophoretic mobilities of an esterase and of lactate and malate dehydrogenases for E. sakazakii. The high degree of enzyme polymorphism within the two species permitted precise identification of strains. The variations in electrophoretic patterns might therefore provide useful epidemiological markers.     

Proteins of microdissected polytenic cells

Proteins extracted from unfixed and ethanol-acetic acid fixed salivary glands were compared by SDS-polyacrylamide gel electrophoresis. The electropherograms were nearly identical when care was taken to prevent degradation in the unfixed glands. Steady state proteins from microdissected nuclei and cytoplasms showed approximately the same number of species but displayed fundamentally different electrophoretic distributions. It was calculated that the maximum number of copies for individual polypeptide species ranged from 3.8×10⁸ to 1.8×10¹⁰ in large polytene nuclei. A comparison of electropherograms from steady state nuclear proteins and nuclear proteins labeled with ³H-leucine after various periods of in vitro incubation of the glands, may suggest different turnover rates for individual protein species. An unexpected effect of incubation of the explanted salivary glands in a synthetic medium was observed. There are differences, on a quantitative level, between certain labeled proteins which accumulate in the nucleus at the beginning and at the end of a relatively long period of incubation.     

Electron microscopic immunogold localization of salivary mucins MG1 and MG2 in human submandibular and sublingual glands.

The human salivary mucins MG1 and MG2 are well characterized biochemically and functionally. However, there is disagreement regarding their cellular and glandular sources. The aim of this study was to define the localization and distribution of these two mucins in human salivary glands using a postembedding immunogold labeling method. Normal salivary glands obtained at surgery were fixed in 3% paraformaldehyde-0.1% glutaraldehyde and embedded in Lowicryl K4M or LR Gold resin. Thin sections were labeled with rabbit antibodies to MG1 or to an N-terminal synthetic peptide of MG2, followed by gold-labeled goat anti-rabbit IgG. The granules of all mucous cells of the submandibular and sublingual glands were intensely reactive with anti-MG1. No reaction was detected in serous cells. With anti-MG2, the granules of both mucous and serous cells showed reactivity. The labeling was variable in both cell types, with mucous cells exhibiting a stronger reaction in some glands and serous cells in others. In serous granules, the electron-lucent regions were more reactive than the dense cores. Intercalated duct cells near the acini displayed both MG1 and MG2 reactivity in their apical granules. In addition, the basal and lateral membranes of intercalated duct cells were labeled with anti-MG2. These results confirm those of earlier studies on MG1 localization in mucous cells and suggest that MG2 is produced by both mucous and serous cells. They also indicate differences in protein expression patterns among salivary serous cells.