Vitellin of the sweet potato whitefly,Bemisia tabaci: Biochemical characterization and titer changes in the adult

SDS-PAGE of the sweet potato whitefly (Bemisia tabaci) egg extract showed one major band (approximately 190 kDa) and two minor bands (approximately 75 kDa and 67 kDa). A distinct 190 kDa band was also present in male extract. On SDS gels the vitellin band of the greenhouse whitefly (Trialeurodes vaporarium) was larger, about 220 kDa. The native molecular mass of sweet potato whitefly vitellin was estimated to be 375 kDa using 4–20% native pore-limiting gel electrophoresis. Its isoelectric point was estimated to be 7.3 using isoelectric focusing. Two-dimensional gel electrophoresis and densitometry were used to estimate vitellin subunit composition; the data suggest that the sweet potato whitefly vitellin is likely to be a 380 kDa native molecule formed by two 190 kDa subunits. The two minor bands (75 kDa and 67 kDa) may be breakdown products of the native vitellin. This conclusion was supported by a Western blot of an SDS-PAGE gel of partially degraded female and egg extracts, which showed that polyclonal antiserum raised against the 190 kDa polypeptide recognized the 75 kDa and 67 kDa bands. Seven hybridoma cell lines secreting monoclonal antibodies against the 190 kDa band were screened, and one of them (S1A2G9H2) was mass produced. The antibody recognized the 190 kDa band in a Western blot. All the screened monoclonal antibodies were female and egg-specific by ELISA and/or Western blot, suggesting that the 190 kDa band in male extract was not a vitellin. A sensitive ELISA was established that could detect as little as 1/40 of an egg equivalent of vitellin using the monoclonal antibody from S1A2G9H2. Profiles of female sweet potato whitefly reproductive activities (egg laying, amount of vitellin in the female, and total vitellin produced by a female) within 2 days after eclosion were determined. Arch. Insect Biochem. Physiol. 34:223–237, 1997. © 1997 Wiley-Liss, Inc.     

Plasma membrane-associated tumor necrosis factor. A non-integral membrane protein possibly bound to its own receptor

Purified plasma membranes from LPS-activated human blood monocytes produced significant lysis and growth inhibition of the TNF-sensitive L929 murine fibroblast cell line. Unactivated human monocyte plasma membranes did not display either activity. Anti-TNF serum specifically inhibited the anti-tumor activity of activated monocyte membranes whereas anti-IL-1 serum or non-specific rabbit serum decreased neither the lysis nor growth inhibition of L929 cells. Membrane-associated TNF did not behave as an integral protein as it could be eluted from the plasma membranes by either high salt or low pH treatment. Plasma membranes cleared of membrane-associated TNF by high salt treatment were able to bind TNF, and this binding was specifically inhibited by preincubation of rTNF with specific anti-TNF serum. Western blot analysis of plasma membranes showed a membrane-associated TNF with a m. w. of approximately 17 kDa present only in the activated monocytes. When the plasma membranes were preincubated with the cross-linker agent dissuccinimidyl suberate, Western blot analysis revealed the presence of a TNF-binding protein with a Mr of approximately 102 kDa. These studies indicate that unlike IL-1, membrane-associated TNF is not an integral membrane protein and that TNF may be associated with the monocyte membrane by occupying the TNF R.     

Differential hydrolysis of homocysteine thiolactone by purified human serum (192)Q and (192)R PON1 isoenzymes

Human serum paraoxonase 1 (PON1) is a HDL-associated enzyme that catalyzes the hydrolysis of a variety of aromatic carboxylic acid esters and several organophosphates. Recently it has been suggested that a physiological substrate of serum PON1 is homocysteine thiolactone which is a putative risk factor in atherosclerosis. In this study, human (192)Q and (192)R PON1 isoenzymes were purified from the respective phenotype human serum, using a protocol consisting of ammonium sulfate precipitation and four chromatography steps: gel filtration, ion-exchange, non-specific affinity, and a second ion-exchange. Using paraoxon as substrate, overall purification fold was found as 742 for (192)R PON1 and 590 for (192)Q PON1. The final purified enzymes were shown as single protein bands close to 45kDa on SDS-PAGE and confirmed by Western blot. Substrate kinetics were studied with phenyl acetate, paraoxon and homocysteine thiolactone. Both PON1 isoenzymes showed mixed type inhibition with phenyl acetate. K(m) values of (192)Q and (192)R PON1 for homocysteine thiolactone were 23.5mM and 22.6mM respectively. For (192)R PON1, the V(max) was 2.5-fold and k(cat)/K(m) was 2.6-fold higher than those for (192)Q PON1 when homocysteine thiolactone is used as substrate. The present data suggest that defining (192)Q and (192)R PON1 isoforms could be a good predictor and prognostic marker in the cardiovascular risk assessment.     

Immunolocalization of betaine aldehyde dehydrogenase in porcine kidney.

Polyclonal anti-BADH serum was raised in rabbits against native BADH purified from porcine kidney. The antiserum cross-reacted strongly with BADH purified from kidney, Amaranthus palmierii, and Pseudomona aeuroginosa (1:1000), and weakly with Amaranthus hypochondriacus L (1:100). Antibodies bound to purified native kidney BADH in immunoblots showed a major band of an apparent molecular mass of 340 kDa and a subunit with an apparent molecular mass of 52 kDa. Data on activity assays showed higher activity in cortex sections (81.3 nmol/min/mg protein) than in medulla sections (21.3 nmol/min/mg protein). Immunolocalization of BADH in kidney tissue sections showed that BADH is found in cortex and medulla. In inner medulla, the enzyme was mainly localized in cells surrounding the tubules. Western blot analysis on extracts from the cortex and medulla sections showed higher concentration of BADH protein in cortex than in medulla. These results were in accordance with immunolocalization and activity analysis.     

A study of the heterogenous structure of guinea pig lysosomal beta-mannosidase using a polyclonal antibody

Lysosomal beta-mannosidase (EC 3.2.11.25) has a functional size of 120-150 kDa, but the enzyme purified from guinea pig liver (GPL) reportedly gave a single band corresponding to a molecular mass of 110 kDa. In order to investigate the subunit structure and tissue-specific expression of beta-mannosidase, we prepared a polyclonal antibody against GPL beta-mannosidase in rabbits which immunoprecipitated beta-mannosidase activity, free from other lysosomal hydrolase activity. Following storage at -20 degrees C and SDS polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol, a sample of purified GPL beta-mannosidase gave a major Coomassie blue staining band at 97 kDa. This was confirmed by Western blot analysis, which also revealed a faster moving 37 kDa protein. In contrast, Western blot analysis of fresh GPL homogenate prepared in the presence of proteinase inhibitors showed a major band at 150 kDa. Upon freezing and thawing, we observed immunoreactive bands at 120 and 20 kDa and finally, immunoreactive bands at 97, 37 and 20 kDa. The formation of the 97, 37 and 20 kDa forms from the 150 kDa species was accelerated by an n-butanol/ether extraction of the associated lipids, suggesting some tight hydrophobic association of these subunits. In contrast to liver, both fresh and freeze-thawed preparations of guinea pig kidney (GPK) yielded only the 97, 37 and 20 kDa subunit forms confirming that these are the major beta-mannosidase subunits. Endo-F treatment converted both the liver and kidney 97 kDa into a 91 kDa form and the 37 kDa form into a 35 kDa form, whereas the 20 kDa form was unaffected. Total beta-mannosidase activity, as measured with the synthetic substrate 4MU-beta-mannoside was unaffected by dissociation of the 150 form into the 97, 37 and 20 kDa subunits, suggesting that these are the functional forms of the enzyme rather than proteolytic degradation products.     

Purification and characterization of myrosinase from horseradish (Armoracia rusticana) roots.

Myrosinase (beta-thioglucoside glucohydrolase; EC 3.2.3.147) from horseradish (Armoracia rusticana) roots was purified to homogeneity by ammonium sulfate fractionation, Q-sepharose, and concanavalin A sepharose affinity chromatography. The purified protein migrated as a single band with a mass of about 65 kDa on SDS-polyacrylamide gel electrophoresis. Using LC-MS/MS, this band was identified as myrosinase. Western blot analysis, using the anti-myrosinase monoclonal antibody 3D7, showed a single band of about 65 kDa for horseradish crude extract and for the purified myrosinase. The native molecular mass of the purified myrosinase was estimated, using gel filtration, to be about 130 kDa. Based on these data, it appeared that myrosinase from horseradish root consists of two subunits of similar molecular mass of about 65 kDa. The enzyme exhibited high activity at broad pH (pH 5.0-8.0) and temperature (37 and 45 degrees C). The purified enzyme remained stable at 4 degrees C for more than 1 year. Using sinigrin as a substrate, the Km and Vmax values for the purified enzyme were estimated to be 0.128 mM and 0.624 micromol min(-1), respectively. The enzyme was strongly activated by 0.5 mM ascorbic acid and was able to breakdown intact glucosinolates in a crude extract of broccoli.     

Iron-ascorbic acid-induced oxidant stress and its quenching by paraoxonase 1 in HDL and the liver: comparison between humans and rats

Paraoxonase 1 (PON1) is a serum enzyme closely associated with high-density lipoprotein (HDL), which may protect against atherosclerosis by hydrolyzing lipid peroxides and several organophosphorus compounds. The purpose of the present study was to test the hypothesis that lipid peroxidation modifies the activity and protein mass of PON1 in humans and rats. Our findings revealed that the bulk of the activity monitored by the hydrolysis of paraoxon and phenyl acetate was confined to liver intracellular endoplasmic reticulum-derived microsomes and was mostly recovered in circulating HDL3. Confirmation was obtained by the determination of PON1 expression by Western blot. It is noteworthy that PON1 levels were consistently decreased in human sera, HDL, and liver microsomes compared with rat counterparts. Concomitant with iron-ascorbate-mediated lipid peroxidation, there was a decline in PON1 activity and protein in both HDL3 and microsomes, which was attenuated by butylated hydroxytoluene antioxidant treatment. The current data indicate that PON1 localization in microsomes and HDL3 could represent a selective cellular and lipoprotein response to oxidative stress. This was tested by the iron-ascorbate oxygen-radical generating system. It is also proposed that the increased PON1 level may have a function related to the well-known atherosclerosis resistance of rats.     

Purification and immunological characterization of acid β-galactosidase from dog liver

• 1.1. Dog liver acid β-galactosidase was isolated in high yield and purified to homogeneity using a series of chromatographies on Con A-Sepharose, decyl-agarose, anion-exchange HPLC and gel-filtration HPLC.
• 2.2. Non-denaturing gel filtration by HPLC gave a single homogeneous peak corresponding to molecular mass of 180–190 kDa. During SDS-PAGE analysis, the single peak dissociated into a major band corresponding to molecular mass of 32 kDa with minor bands at 18 and 13 kDa.
• 3.3. Polyclonal antibodies raised against the purified enzyme immunoprecipitated β-galactosidase activity specifically from dog liver extracts and recognized a single 32 kDa band in Western blot analysis of dog tissue homogenates. This antibody did not crossreact with any protein band in tissue homogenates from other species examined except cat.
• 4.4. Western blot analysis of tissue extracts from dogs affected with G_MI-gangliosidosis showed the presence of a 32 kDa band similar to that of controls.

     

Mouse serum paraoxonase-1 lactonase activity is specific for medium-chain length fatty acid lactones

Recent studies suggest that paraoxonase-1 (PON1), complexed with high-density lipoproteins, is the major lactonase in the circulation. Using 5-hydroxy eicosatetraenoate δ-lactone (5-HETEL) as the substrate, we observed lactonase activity in serum from Pon1-/- mice. However, 6-12 carbon fatty acid γ- and δ-lactones were not hydrolyzed in serum from Pon1-/- mice. Serum from both wild-type and Pon1-/- mice contained a lactonase activity towards 5-HETEL and 3-oxo-dodecanoyl-homoserine lactone that was resistant to inactivation by EDTA. This lactonase activity was sensitive to the serine esterase inhibitor phenyl methyl sulfonyl fluoride and co-eluted with carboxylesterase activity by size-exclusion chromatography. Analysis of serum from the Es1e mouse strain, which has a deficiency in the carboxylesterase, ES-1, proved that this activity was due to ES-1. PON1 activity predominated at early time points (30 s), whereas both PON1 and ES-1 contributed equally at later time points (15 min). When both PON1 and ES-1 were inhibited, 5-HETEL was stable in mouse serum. Thus, while long-chain fatty acid lactones are substrates for PON1, they can be hydrolyzed by ES-1 at neutral pH. In contrast, medium-chain length fatty acid lactones are stable in mouse serum in the absence of PON1, suggesting that PON1 plays a specific role in the metabolism of these compounds.     

Increased Expression of Complement Component C3 in the Plasma of Obese Zucker fa and LA/N fef Rats Compared with Their Lean Counterparts

Objectives : The objectives of this study were to determine whether there are differences in the electrophoretic profiles of plasma proteins from lean and obese rats and to identify a protein that was found to be more abundant in the plasma of obese rats. Research Methods and Procedures : Plasma proteins from lean and obese Zucker fa and LA/N/fa^frats were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The identity of a band that was differentially expressed was determined by amino acid sequencing and Western blot analysis. Results : A band migrating approximately the same distance as the 116 kDa molecular weight marker was more prominent in plasma from obese rats than in plasma of lean rats. Partial sequencing of the peptide revealed that 17 of the first 18 amino acids at the amino terminus were identical with the corresponding residues in the α-chain of complement component C3. Western blot analysis confirmed the identity of the peptide as complement component C3. Complement C3 activity was measured using a hemolytic assay to determine whether there was a corresponding increase in the biological activity of this component in the serum of obese rats. Serum from obese rats was found to have 1.8 times as much complement component C3 activity as serum from lean rats. Discussion : Elevated levels of complement C3 in genetically obese rats may be relevant because increased amounts of C3 could serve as a reservoir from which increased amounts of acylation stimulating protein, a cleavage product of complement C3, could be produced.     

Identification of immunodominant Trypanosoma musculi antigens recognized by monoclonal antibody and curative immunoglobulin G2a antibody.

Trypanosoma musculi obtained from normal or irradiated (900 rad) hosts or from in vitro cultures were lysed and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Similar protein banding patterns with a molecular weight (mol. wt) range from 34 to 68 kDa were observed between the two bloodstream forms. In comparison, lysates of cultured parasites showed a unique banding pattern of antigens within the same mol. wt range. Western blot of bloodstream form lysates, probed with immune plasma (IP), revealed a wide range of parasite proteins. However, when probed with the IgG2a-enriched fraction of IP, a major band of approximately 66 kDa was detected on the blot. Several bands of higher mol. wt were also observed. When anti-T. musculi monoclonal antibodies were used to probe the blot, the 66 kDa protein was again recognized. Using indirect fluorescence, live bloodstream form parasites were analysed by flow cytometry and the p66 protein was determined to be a surface molecule. Finally, lysates of 35S-methionine-labelled trypanosomes were immunoprecipitated with Sepharose linked anti-T. musculi monoclonal antibodies and the eluted ligand analysed by SDS-PAGE and autoradiographed. The 66 kDa band was identified, therefore confirming that this protein was of parasite origin.     

Species- and substrate-specific stimulation of human plasma paraoxonase 1 (PON1) activity by high chloride concentration

Paraoxonase 1 (PON1), contained in plasma high-density lipoproteins, plays an important role in the protection of plasma lipoproteins and cell membranes from oxidative damage. Previous studies indicate that human PON1 is stimulated by high NaCl concentrations. The aim of this study was to characterize in more detail the effect of salts on serum PON1. Paraoxon-hydrolyzing activity of human serum was stimulated by 81.6% following the addition of 1 M NaCl. The effect of NaCl was dose-dependent between 0.5 and 2 M. PON1 activity toward phenyl acetate was reduced by 1 M NaCl by 55.2%. Both the paraoxon- and phenyl acetate-hydrolysing activity was slightly lower in heparinized plasma than in serum, but NaCl had similar stimulatory and inhibitory effects on these activities, respectively. In rat, rabbit, and mouse, NaCl reduced PON1 activity. KCl had a similar effect on human PON1 as NaCl. Sodium nitrite also stimulated human PON1 but much less effectively than chloride salts. In contrast, sucrose, sodium acetate and sodium lactate had no significant effect. NaBr was a less effective PON1 activator than NaCl, whereas the effect of NaJ was non-significant. The activity of human PON1 toward homogentisic acid lactone and gamma-decanolactone was unaltered by NaCl. These data indicate that: 1) high concentrations of chlorides stimulate human PON1 activity toward paraoxon but not other substrates, 2) PON1 is inhibited by Cl(-) in other mammalian species, 3) the potency of human PON1 activation by halogene salts increases with decreasing atomic mass of the halide anion.     

A homogeneous cell-based assay for measurement of endogenous paraoxonase 1 activity

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme that plays an important role in organophosphate detoxification and prevention of atherosclerosis. Thus, there is significant interest in identifying nutritional and pharmacological enhancers of PON1 activity. To identify such compounds, we developed a rapid homogeneous assay to detect endogenous cell-associated PON1 activity. PON1 activity was measured by the simple addition of fluorigenic PON1 substrate DEPFMU to live Huh7 cells in medium and monitoring change in fluorescence. A specific PON1 inhibitor, 2-hydroxyquinoline, was used to confirm that the observed activity was due to PON1. The assay was optimized and characterized with regard to time course, substrate and sodium chloride concentration, number of cells, and tolerance to dimethyl sulfoxide and serum. Aspirin, quercetin, and simvastatin are compounds reported to increase PON1 expression. Consistent with the literature and Western blot data, these compounds enhanced PON1 activity in this assay with comparable efficacies and potencies. A known toxic compound did not increase assay signal. This assay method also detected PON1 activity in normal hepatocytes. Thus, a novel homogeneous assay for detection of endogenous PON1 expression has been developed and is amenable to high-throughput screening for the identification of small molecules that enhance PON1 expression.     

Novel purification strategy for human PON1 and inhibition of the activity by cephalosporin and aminoglikozide derived antibiotics

Human serum paraoxonase (PON1, EC 3.1.8.1.) is a high-density lipid (HDL)-associated, calcium-dependent enzyme; its physiological substrates are not known. In this study, a new purification strategy for human PON1 enzyme was developed using two-step procedures, namely ammonium sulfate precipitation and sepharose-4B-l-tyrosine-1-napthylamine hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent MW of 43 kDa. Overall purification rate of our method was found 227-fold. The V(max) and K(m) of the purified enzyme were determined 227.27 EU and 4.16 mM, respectively. The in vitro effects of commonly used antibiotics, namely gentamycin sulfate and cefazolin sodium was also investigated on the purified human serum PON1 enzyme and human liver PON1 enzyme from human hepatoma cell (HepG2). Gentamycin sulfate and cefazolin sodium caused a dose- and time-dependent decrease on PON1 activity in HepG2 cells. Moreover, gentamycin sulfate and cefazolin sodium were effective inhibitors on purified human serum PON1 activity with IC(50) of 0.887 and 0.0084 values, respectively. The kinetics of interaction of gentamycin sulfate and cefazolin sodium with the purified human serum PON1 indicated a different inhibition pattern. Cefazolin sodium showed a competitive inhibition with K(i) of 0.012+/-0.00065 mM. However, Gentamycin sulfate was inhibited in non-competitive manner with K(i) of 0.026+/-0.015. In order to determine the inhibition statue of these drugs on a living system, the effects of same antibiotics on PON1 enzyme activity of mouse serum PON1 and liver PON1 were investigated in vivo. Gentamycin sulfate (3.2 mg/kg) and cefazolin sodium (106.25 mg/kg) leads to the significant decrease in mouse serum PON1 after 2, 4, 6 h and 2, 4 h drug administration, respectively. Cefazolin sodium did not exhibit any inhibition effect for the liver PON1, in vivo.     

Cryogenic changes in proteases and antiprotease activities of buffalo (Bubalus bubalis) and cattle (Bos taurus) semen

The postthaw motility and fertility of buffalo and cattle semen is reduced when they are cryopreserved for artificial insemination. In the present study, an attempt was made to characterize the cryogenic changes in proteases and antiprotease activities (APA) of buffalo and cattle semen because these proteolysis regulators have been reported to be associated with sperm motility and fertility. Buffalo sperm demonstrated at least two major proteases of 45 and 42 kDa and three minor proteases of 95, 52, and 33 kDa. Similarly, cattle sperm demonstrated three major proteases of 62, 45, and 42 kDa and two minor proteases of 85 and 78 kDa. Buffalo seminal plasma demonstrated at least three major proteases of 78, 68, and 62 kDa and one minor protease of 98 kDa and cattle seminal plasma demonstrated one major protease of 68 kDa and two minor proteases of 78 and 75 kDa. Except for the 45 kDa protease, most of the previously mentioned proteases were found to be metalloproteinases. Compared with fresh sperm, cryopreserved buffalo and cattle sperm demonstrated a major protease band of 52/49 kDa and the activity of this protease reduced progressively with the duration of cryopreservation. On the contrary, compared with the fresh seminal plasma, cryopreserved buffalo and cattle semen extenders displayed the presence of a new protease band of 45 kDa and demonstrated that this protease activity was leaked from buffalo and cattle cryopreserved spermatozoa. Buffalo and cattle seminal plasmas displayed at least two major APA of 86 and 26 kDa. Compared with buffalo, cattle seminal plasma demonstrated significantly greater APA. Thus, the present study demonstrated the presence of an array of proteases and APA in buffalo and cattle semen and the activities of which changed during cryopreservation. The leakage of the specific protease activity and changes in the proteases and APA might be attributed to reduced motility and fertility of cryopreserved semen in these species.     

Posttranslationally processed forms of the human chemokine HCC-1

HCC-1 is the only CC-chemokine known so far which circulates in nanomolar concentrations in human plasma. Its physiological function is not well defined. Posttranslational processing of HCC-1 was shown to modulate its biological properties. In this study several different processed forms of HCC-1 were isolated. Western blot analysis of human plasma extracts revealed a HCC-1 immunoreactive double band at 8-10 kDa indicating the presence of two distinct HCC-1 peptides. These peptides were isolated from a peptide library of human blood filtrate and represent predominantly HCC-1 (1-74) and glycosylated HCC-1 (1-74). Glycosylated HCC-1 exhibits a molecular mass of 9621 Da due to O-glycosylation at position 7 (Ser-7) with two N-acetylneuraminic acids and the disaccharide N-acetylgalactosamine galactose. Furthermore N-terminally truncated HCC-1 (3-74) and HCC-1 (4-74) were identified in the peptide library. In hemofiltrate approximately 3% of total HCC-1 represents HCC-1 (3-74) and approximately 1% represents HCC-1 (4-74) whereas the major products are nonglycosylated HCC-1 (1-74) and glycosylated HCC-1 (1-74). Our data imply that HCC-1 (1-74), HCC-1 (3-74), HCC-1 (4-74) and glycosylated HCC-1 (1-74) circulate in human blood. The N-terminal processing and modification of HCC-1 might be of importance in displaying its full biological activity.     

Isolation of a galactose-free 20-kDa fragment exhibiting butyrylcholine esterase and aryl acylamidase activity from human serum butyrylcholine esterase by limited alpha-chymotrypsin digestion

Purified human serum butyrylcholine esterase (approximately 90-kDa subunit), which also exhibits aryl acylamidase activity, was subjected to limited alpha-chymotrypsin digestion. Three major protein fragments of approximately 50 kDa, approximately 21 kDa and approximately 20 kDa were found to be produced, as observed by SDS-gel electrophoresis of the chymotryptic digest. The purified butyrylcholine esterase could fully bind to a Ricinus-communis-agglutinin-Sepharose column but after chymotryptic digestion about 15-20% of the enzyme activity remained unbound and was recovered in the run-through fractions. Sephadex G-75 chromatography of the chymotryptic digest showed an enzymatically active fragment eluted at an approximate molecular mass of 20 kDa, apart from the undigested butyrylcholine esterase eluted at the void volume. The butyrylcholine esterase fragment that did not bind to Ricinus communis agglutinin also was eluted at an approximate molecular mass of 20 kDa from a Sephadex G-75 column. This enzymatically active low-molecular-mass fragment from Sephadex G-75 chromatography showed a single protein band of approximately 20 kDa on SDS-gel electrophoresis. Neutral sugar analysis of the approximately 20 kDa fragment showed the presence of mannose only, whereas the undigested butyrylcholine esterase showed the presence of both mannose and galactose. Amino-terminal-sequence analysis of the approximately 20 kDa fragment showed the sequence Arg-Val-Gly-Ala-Leu, which agrees with amino acid residues 147-151 reported for human serum butyrylcholine esterase [Lockridge et al. (1987) J. Biol. Chem. 262, 549-557]. Both cholinesterase and aryl acylamidase activities were co-eluted in all chromatographic procedures. The results suggested that limited alpha-chymotrypsin digestion of human serum butyrylcholine esterase resulted in the formation of a approximately 20-kDa enzymatically active fragment with Arg147 as its N-terminal residue and which was devoid of galactose.     

Production and characterization of an antiserum which recognizes the native receptor for thyrotropin-releasing hormone.

Despite attempts in several laboratories, it has been difficult to prepare antiserum to the thyrotropin-releasing hormone receptor (TRHR). We have prepared a polyclonal anti-rat TRHR antiserum by immunization of rabbits with a synthetic peptide corresponding to the C-terminus of the TRHR. The specificity of the antiserum was assessed by enzyme-linked immunosorbent assay. The affinity-purified antibody recognized a major broad band at 50-60 kDa and a minor broad band at 100-120 kDa in Western blot analysis of membrane proteins from TRHR-transfected, but not control, HEK293t cells. Binding to both bands was abolished by preincubation with the immunizing peptide but not control peptide. The approach was repeated with rat pituitary F4C1 cells, which lack endogenous TRHRs; membranes from F4C1 cells transfected with TRHR cDNA, but not control cells, showed specific binding by Western blot. Using laser confocal microscopy, the TRHR was visualized on the plasma membrane of transfected, but not control, F4C1 cells. Similar confocal findings were observed in TRHR-transfected HEK293t cells. Within 5 min after TRH addition, the TRHR signal translocated from the plasma membrane to the cytoplasm of F4C1 cells transfected with TRHR cDNA. Ten minutes after TRH addition, the TRHR signal formed aggregates in the cytoplasm. Thirty minutes after TRH treatment, both cytoplasmic and plasma membrane localizations were observed, suggesting recycling of some TRHRs back to the plasma membrane. These observations are consistent with our previous findings using an epitope-tagged TRHR. In conclusion, we have prepared an antiserum that recognizes the native TRHR by Western blot analysis and confocal microscopy.     

Altered fractalkine cleavage potentially promotes local inflammation in NOD salivary gland

Introduction

In the nonobese diabetic (NOD) mouse model of Sjögren's syndrome, lymphocytic infiltration is preceded by an accumulation of dendritic cells in the submandibular glands (SMGs). NOD mice also exhibit an increased frequency of mature, fractalkine receptor (CX3C chemokine receptor [CX3CR]1) expressing monocytes, which are considered to be precursors for tissue dendritic cells. To unravel further the role played by fractalkine-CX3CR1 interactions in the salivary gland inflammation, we studied the expression of fractalkine in NOD SMGs.

Methods

We studied protein expression using Western blot analysis of whole tissue lysates. Protease activity was measured in salivary gland tissue lysates using fluorimetric substrates. Digestive capacity of enzymes was determined by in vitro incubation of recombinant enzyme and fractalkine, followed by protein staining and Western blot.

Results

Fractalkine was detected in salivary glands of both NOD and control mice at all ages. Western blot analysis showed fractalkine cleavage with increasing age, which was more pronounced in NOD mice. This cleavage resulted in a decrease in the 31 kDa form of the protein, and the generation of an approximately 19 kDa band. Furthermore, in NOD animals older than 15 weeks, we noted the presence of a unique approximately 17 kDa fragment. This cleavage was organ specific, because it did not occur in brain or pancreas. Increased gelatinase and α-secretase activity were detected in NOD SMG and contributed to cleavage of the 31 kDa protein. Because aberrant cleavage products may induce autoimmunity, we studied the presence of autoantibodies against fractalkine. Indeed, NOD mice exhibited significantly more antibodies against fractalkine than did control animals.

Conclusion

These data indicate that aberrant proteolytic activity in the NOD SMG results in increased fractalkine cleavage and generation of a unique fractalkine fragment. This specific cleavage may contribute to autoimmunity.