Lentil Lectin Affinity Research Articles

Isolation and characterization of the first phosphodiesterase (Bj-PDE) from the venom of Bothrops jararacussu snake

Phosphodiesterases are exonucleases that sequentially hydrolyse phosphodiester bonds of polynucleotides from the 3′-end and release 5-mononucleotides. After more than one decade without any advance in the study of Bothropic phosphodiesterases, we described here the isolation of the first phosphodiesterase from Bothrops jararacussu, which we named Bj-PDE. A five-step column chromatography procedure (size exclusion, hydrophobic interaction, cation exchange, lentil lectin affinity, and blue sepharose affinity) enabled isolation of Bj-PDE with preserved and stable enzymatic activity (bis(p-nitrophenyl) phosphate substrate), Km = 6.9 mM (± 0.7 mM), kcat/Km = 1.7 × 104 M−1 s−1 (± 0.2 × 104 M−1 s−1), MW = 116 kDa (SDS-PAGE), optimum activity around 45 °C at pH 8.0, and stability for 81 days at different storage temperatures (8, −20, and − 80 °C). Ca2+ and Mg2+ ions positively influenced Bj-PDE activity, while EDTA had the opposite action. Zn2+ restored >50 % of enzyme activity after its inhibition by EDTA. The Bj-PDE partial sequence identified by mass spectrometry was very similar to the sequence of BATXPDE1 from Bothrops atrox, which was evolutionarily close to this new PDE. Therefore, our study represents an important progress on the isolation of this minor toxin and sheds new lights on the properties and bioprospection of bothropic phosphodiesterases.

Evaluation of two Taenia solium cysticercal antigenic preparations (vesicular fluid and a glycoprotein fraction with affinity for lentil lectin) for the immunodiagnosis of neurocysticercosis by enzyme-linked immunosorbent assay (ELISA)

To evaluate the performance of two antigenic preparations (vesicular fluid - VF and a glycoprotein fraction, LLa-Gp fraction, purified from a whole parasite extract by lentil lectin affinity chromatography) from Taenia solium cysticerci for the immunodiagnosis of neurocysticercosis. Fifty-six cerebrospinal fluid (CSF) samples (22 from patients with neurocysticercosis and 34 from patients with other neurological disorders) and 57 serum samples (22 from patients with neurocysticercosis, 18 from patients with other infections and 17 from presumably healthy persons) were assayed for anticysticercal IgG antibodies with an enzyme-linked immunosorbent assay (ELISA). The VF ELISA showed 100% sensitivity and specificity in CSF and serum samples, whereas the sensitivity and specificity of the LLa-Gp ELISA were, respectively, 90.9% and 97.1%, with the CSF samples and 95.5% and 100% with serum samples. There was no significant difference in the sensitivity and specificity of the two antigenic preparations used to screen CSF and serum samples. Considering the complexity and high cost of obtaining the LLa-Gp fraction, VF could be more suitable for screening specific antibodies by ELISA in CSF and serum samples from patients with neurocysticercosis.

Dual expression of the HA protein of H5N2 avian influenza virus in a baculovirus system

Baculovirus/insect cell system is used widely for recombinant protein production. The hemagglutinin (HA) gene of H5N2 avian influenza virus (AIV) 1209 strain and the enhanced green fluorescent protein (EGFP) gene were cloned into pFastBac DUAL vector that has two promoters and cloning sites, allowing simultaneous expression of these two genes. The HA protein of AIV was fused with a hexahistidine (His 6) tag for purification. The coexpression of EGFP allowed identification of the recombinant baculoviruses in Sf-9 insect cells, eliminating cumbersome and time-consuming assays. A recombinant baculovirus, Bac-HA, was generated by transfecting pBac-HA to bacmid inside DH10B AC Escherichia coli by site-specific transposition, followed by transfection into the Sf-9 cells. Fluorescence in the insect cells was observed from 3 days post-infection. The expressed HA protein was confirmed by Western blot using an anti-HA monoclonal antibody. Also, different detergents and incubation times on ice were tested. The two-stage extraction with Triton X-100 or Tween 20 and incubation on ice for 2 h exhibited high efficiency. Since purification of HA with ConA resin resulted in low protein recovery, lentil lectin affinity column was used and was useful for HA purification.

The Drosophila Acetylcholine Receptor Subunit Dα5 Is Part of an α-Bungarotoxin Binding Acetylcholine Receptor

The central nervous system of Drosophila melanogaster contains an alpha-bungarotoxin-binding protein with the properties expected of a nicotinic acetylcholine receptor. This protein was purified 5800-fold from membranes prepared from Drosophila heads. The protein was solubilized with 1% Triton X-100 and 0.5 M sodium chloride and then purified using an alpha-cobratoxin column followed by a lentil lectin affinity column. The purified protein had a specific activity of 3.9 micromol of 125I-alpha-bungarotoxin binding sites/g of protein. The subunit composition of the purified receptor was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This subunit profile was identical with that revealed by in situ labeling of the membrane-bound protein using the photolyzable methyl-4-azidobenzoimidate derivative of 125I-alpha-bungarotoxin. The purified receptor reveals two different protein bands with molecular masses of 42 and 57 kDa. From sedimentation analysis of the purified protein complex in H2O and D2O and gel filtration, a mass of 270 kDa was calculated. The receptor has a s(20,w) of 9.4 and a Stoke's radius of 7.4 nm. The frictional coefficient was calculated to be 1.7 indicating a highly asymmetric protein complex compatible with a transmembrane protein forming an ion channel. The sequence of a peptide obtained after tryptic digestion of the 42-kDa protein allowed the specific identification of the Drosophila D alpha5 subunit by sequence comparison. A peptide-specific antibody raised against the D alpha5 subunit provides further evidence that this subunit is a component of an alpha-bungarotoxin binding nicotinic acetylcholine receptor from the central nervous system of Drosophila.

Schistosoma mansoni gene GP22 encodes the tegumental antigen sm25: (1) antibodies to a predicted B-cell epitope of Sm25 cross-react with other candidate vaccine worm antigens; (2) characterization of a recombinant product containing tandem-repeats of this peptide as a vaccine.

Monospecific antibodies against two putative epitopes of schistosome protein encoded by gene GP22 (182 codons, no introns) were used to probe worm extracts fractionated by lentil-lectin affinity chromatography or by electrophoresis. Anti-peptide-alpha (codons 70-84) exclusively identifies the N-glycanase-sensitive, 25 kDa tegumental glycoprotein Sm25 in the lectin-bound fraction of detergent-solubilized adult worm extract S3. In contrast, antipeptide-delta (codons 151-162) does not react with Sm25 but cross-reacts with other schistosome proteins, including candidate vaccine antigens paramyosin (Sm97) and glutathione-S-transferases (Sm26, Sm28, Sj26). Recombinant protein r4 x 47, constructed to express multiple copies of codon sequence 117-163 (containing delta), reacts with anti-delta and is uniquely recognized by protective Fischer twice-infected (F-2x) rat antiserum. Immunization with r4 x 47 induces antibodies with cross reactivities similar to anti-delta, but which also recognize Sm25. Despite these cross-reactivities with protective antigens, rodents vaccinated with r4 x 47 were not protected against cercarial infection. On the basis of these data, two hypotheses are proposed: (1) antigenic epitopes other than delta are present within the r4 x 47 sequence which induce antibodies reactive with Sm25 and/or (2) peptide-delta assumes alternative antigenic conformations, dependent upon the context of neighbouring sequences, some of which mimic epitopes of proteins encoded by other schistosome genes. These mimotopes are not targets of protective antibodies.

Molecular and immunological characterization of soluble aggregated A/Victoria/3/75 (H3N2) influenza haemagglutinin expressed in insect cells.

A/Victoria/3/75 (H3N2)-derived cDNA coding for a secreted haemagglutinin (HA0s) was cloned into the polyhedrin promoter-based pVL1392 transfer vector, and a recombinant baculovirus was isolated. 5 to 10 micrograms/ml of secreted HA were obtained following infection of Spodoptera frugiperda-9 cells. Gel filtration revealed the presence in the cell supernatant of immunoreactive HA molecules with varying M(r). The high M(r) fraction (aHA0s) could be purified by Matrex Cellufine Sulphate and Lentil-lectin affinity chromatography, followed by Sephacryl S300 HR gel filtration. aHA0s consisted of aggregated, non-covalently linked subunits which were not cleaved into HA1 and HA2 polypeptides; aHA0s was highly susceptible to trypsin treatment and reacted with two low pH-specific monoclonal antibodies, suggesting that a HA0s consists of monomeric subunits. When the expression medium was adjusted to pH 8.5, no aHA0s was observed, suggesting that aggregation occurred in the cells due to a low intracellular pH. Balb/c mice immunized with purified aHA0s developed high, aHA0s-specific antibody titres. Despite these high titres, almost no binding to trimeric viral HA was observed, and immunized mice were not protected against a challenge with homologous mouse-adapted X47 virus. However, when virus was subjected to low pH, resulting in a profound conformational rearrangement, strong binding was observed. Moreover, binding to the low pH-treated HA of different drift variants, isolated between 1968 and 1989, occurred.

Utility of lentil lectin affinity of α-fetoprotein in the diagnosis of hepatocellular carcinoma

Aims/Methods: Frozen sera obtained from 70 patients (35 with hepatocellular carcinoma and 35 with benign chronic liver disease) with serum α-fetoprotein > 20 ng/ml were studied to evaluate the diagnostic indices of lentil lectin affinity of α-fetoprotein in detecting hepatocellular carcinoma. Results: The proportion of α-fetoprotein-L3 was significantly higher in patients with hepatocellular carcinoma than in those with benign chronic liver disease (41.0±33.6% vs. 16.4±15.3%, p<0.001). This difference led to a sensitivity, specificity, positive predictive value, negative predictive value and accuracy of 57, 89, 83, 67 and 73%, respectively, in detecting hepatocellular carcinoma using the proportion of α-fetoprotein-L3 > 35% as a parameter. Within a 1-year period, 1500 high-risk person were collaborating, leading to 22 cases with serum total α-fetoprotein > 20 ng/ml. These 22 cases included six pregnant women. The parameter, α-fetoprotein-L3 > 35% was used along with sonography to detect hepatocellular carcinoma for the remaining 16 cases. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy were 75, 83, 60, 91 and 81%, respectively, by the porportion of α-fetoprotein-L3 > 35%; and 100, 92, 80, 100 and 94%, respectively, by sonography. Conclusions: Lentil lectin affinity of α-fetoprotein provides a moderately high sensitivity and a high specificity in the detection of hepatocellular carcinoma for persons with high α-fetoprotein levels. It may be a useful adjuvant tool of sonography and total α-fetoprotein level in a mass survey of hepatocellular carcinoma for a high-risk population.

UDP galactose:ceramide galactosyltransferase and glutamate/aspartate transporter. Copurification, separation and characterization of the two glycoproteins.

The oligodendrocyte-specific UDP-galactose:ceramide galactosyltransferase (CGT) is the key enzyme involved in the biosynthesis of the oligodendrocyte- and myelin-specific cerebrosides. The galactosyltransferase was isolated and purified to homogeneity from Triton-X-100-solubilized rat brain microsomes by ion exchange, dye ligand and lectin affinity chromatography as a 64-kDa protein homogenous in SDS/PAGE. It copurified with the brain-specific Na(+)-dependent high-affinity L-glutamate/aspartate neurotransmitter transporter (GLAST-1) of the central nervous system. Differential lentil lectin affinity chromatography led to the separation of two glycoproteins with very similar physical properties. CGT was identified as a high-mannose glycoprotein and GLAST-1 as a hybrid glycoprotein, both with a molecular mass of 64 kDa. Deglycosylation reduced the molecular mass of the two proteins to 59 kDa. A 70-kDa isoform of GLAST-1 was isolated from whole brain by wheat germ lectin affinity chromatography. Deglycosylation again reduced the molecular mass to 59 kDa. Therefore the 70-kDa isoform differs only in the degree of glycosylation from the 64-kDa GLAST-1 isoform. The two isoproteins form homodimers of 130 and 140 kDa, respectively. They were isolated and characterized with protein-chemical and immunological methods. Oligonucleotides derived from respective peptide sequences of CGT and GLAST-1 were successfully applied to the cloning of CGT and the first high-affinity glutamate neurotransmitter transporter (GLAST-1) in glia of the central nervous system as well.

Enzymo-rocket electrophoretic assay and crossed affinity enzymoimmunoelectrophoresis and its ap-lication in diagnosis of plimary liver cancer

A sensitive and accurate quantitative method of alphafetoprotein (AFP), enzymo-rocket dectrophoretie assay (EREA), was developed by introducing horseradish peroxidase-labeled anti-btmmn AFP antibody into rocket electrophoresis. The lower limit of quantitation by this method is about 10 ng/ml of AFP. The dose-nesponse curve covers a broader range of concentrations of AFP (10--4000 ng/ml) than RIA (0---400 ng/ml). 3fine accm-acy and precision is comparable to that of RIA (r=0.986). Sermn AFP measured in 100 patients with primary liver cancer by this method, 88% had levels over 25 ng/ml. The crossed affinity enzymoimmtmoelectrophoresis is a combination of lentil lectin (LCA) affinity electrophoresis and enzymo-rocket electrophoresis, it has been possible to separate the AFP into two variants, LCA-reactive (LCA-R) and LCA-nonreactive (LCA-N) fractions. The advantages of this method are high sensitivity, rapid (6--7 h), and can be effectively used to differentiate the primary liver cancer and benign liver disease.

Characterization of the ATPase activity of P-glycoprotein from multidrug-resistant Chinese hamster ovary cells

P-Glycoprotein (Pgp) was isolated from CHRC5 membranes by selective detergent extraction and further purified by lentil lectin affinity chromatography. The purified product displayed a very high basal ATPase activity (1.65 mumol/min per mg protein in the absence of added drugs or lipids) with an apparent Km for ATP of 0.4 mM. There was no evidence of cooperativity, suggesting that the two ATP sites operate independently of each other. Pgp ATPase activity was stimulated by verapamil, trifluoperazine and colchicine, and inhibited by daunomycin and vinblastine. All drugs and chemosensitizers acted as mixed activators or inhibitors, producing changes in both the Vmax of the ATPase and the Km for ATP. ADP competitively inhibited Pgp ATPase, with a Ki of 0.2 mM. The macrolide antibiotics bafilomycin A1, concanamycin A and concanamycin B, inhibited Pgp ATPase at concentrations of 0.1-10 microM, and at an inhibitor:protein stoichiometry of 0.65-1.0 mumol/mg protein, which is at the low end of the range characteristic of P-type ATPases. Pgp ATPase was relatively selective for adenine nucleotides. Several phospholipids stimulated Pgp ATPase activity in a dose-dependent manner, whereas others produced inhibition. Metabolic labelling showed that the endogenous phospholipids associated with purified Pgp consisted largely of phosphatidylethanolamine and phosphatidylserine, with only a small amount of phosphatidylcholine. 32P-Labelling studies indicated that purified Pgp was partially phosphorylated. It can be concluded that Pgp is a constitutively active, adenine nucleotide-specific ATPase whose catalytic activity can be modulated by both drugs and phospholipids.

Serum thyrotropin (TSH) heterogeneity in euthyroid subjects and patients with subclinical hypothyroidism: the core fucose content of TSH-releasing hormone-released TSH is altered, but not the net charge of TSH

The aims of the present study were to determine the influence of brief subclinical hypothyroidism on the isoforms of serum thyrotropin (TSH) and to examine the net charge of TSH in different metabolic states. Sera were obtained from euthyroid subjects (n = 7) and from patients with subclinical hypothyroidism (n = 8) before and 30 min after the intravenous administration of 200 micrograms thyrotropin-releasing hormone (TRH). The TSH from human pituitary extracts (IRP 68/38), basal and TRH-stimulated serum TSH was immunoconcentrated and further analysed by isoelectric focusing (IEF) and lentil lectin affinity chromatography. TSH immunoreactivity was determined in each specimen or fraction with an automated highly sensitive chemiluminometric TSH assay. We found that basal TSH in subclinical hypothyroidism, and TRH-released TSH in euthyroidism and in subclinical hypothyroidism is distributed in a similar neutral to acidic pattern, which significantly differs from the more alkaline to neutral isoform pattern of intrapituitary TSH (P < 0.05). IEF analysis of pituitary standard TSH revealed 3 major peaks (pI values 7.5; 6.6; 5.8) whereas in most euthyroid or subclinically hypothyroid subjects 5 peaks were found. Lentil lectin affinity chromatography revealed that TRH-released TSH in euthyroid subjects has more core fucose residues than TSH from patients with subclinical hypothyroidism (64.6 +/- 6.7 vs 12.5 +/- 2.7%, P < 0.0001). Thus pituitary standard TSH seems to be less mature material than circulating TSH. Perhaps no alteration in the IEF pattern of TSH was detected during early hypothyroidism because sialylation of TSH was increasing as sulfatation was decreasing. Nevertheless, a change in the core fucose content of TSH was detectable by lentil analysis.

Loss of biological activity of arginine vasopressin during its degradation by vasopressinase from pregnancy serum

Degradation of AVP by placental vasopressinase may precipitate gestational diabetes insipidus, which in some cases is accompanied by pre-eclampsia. Abnormally elevated vasopressinase has also been reported in pre-eclampsia without diabetes insipidus. This association between excessive vasopressinase production and pre-eclampsia might be explained if the products of AVP degradation by vasopressinase retained pressor activity even after anti-diuretic activity had been destroyed. Recent evidence indicates that such products may raise blood pressure in rats. The objective of this study was, therefore, to purify vasopressinase and investigate its action on both the V1 and V2 receptor-stimulating activity of AVP. Vasopressinase was purified from pooled pregnancy serum by ammonium sulphate precipitation, followed by sequential ion exchange, lentil lectin affinity and gel filtration chromatography. Purified enzyme was then used to degrade AVP and the loss of both immunoreactivity and biological activity monitored. Loss of V1 receptor-stimulating activity and V2 receptor-stimulating activity was compared by two-way ANOVA. Blood was obtained from healthy women between week 34 and the end of pregnancy. Pooled serum from 20-30 patients was used as starting material for the purification of vasopressinase. AVP immunoreactivity was measured by radioimmunoassay, V1 receptor-stimulating activity by a platelet aggregation bioassay, and V2 receptor-stimulating activity by adenylate cyclase stimulation in LLC-PK1 target cells. Purified vasopressinase was a dimeric protein of molecular weight 330 kDa, which cleaved the synthetic substrate S-benzyl-L-cysteine-4-nitroanilide with a Km of 0.33 mM. Incubation of AVP (0.1 mM) with vasopressinase (0.66 milligrams) at 37 degrees C led to a parallel loss of both AVP immunoreactivity and biological activity. The rates of loss of V1 and V2 receptor mediated activities were not significantly different. We report the first direct comparison between the loss of V1 and V2 receptor mediated activities of vasopressinase degraded AVP. There was no significant retention of V1, relative to V2, receptor mediated activity. AVP degradation products are unlikely to be pathogenic in hypertensive pregnancy.

A protective "concealed" antigen from Boophilus microplus. Purification, localization, and possible function.

A membrane protein that can be used successfully to vaccinate cattle against the tick Boophilus microplus has been purified and characterized. The mature protein, which is referred to as Bm91, has an apparent m.w. of 86,000, an isoelectric point of 4.8 to 5.2, and is glycosylated, with an affinity for lentil lectin. Bm91 is of relatively low abundance, with approximately 300 to 400 micrograms being recovered from 1 kg of semiengorged adult female ticks. The protein is located largely in the salivary gland and gut of these ticks. Partial amino acid sequence data for the protein show striking similarities to that of mammalian angiotensin-converting enzyme, suggesting that the Ag may have an enzymatic function. The protein seems not to be recognized by sera from cattle with extensive exposure to ticks under natural conditions. The immunity induced by vaccination, therefore, represents another example of vaccination against a hematophagous parasite with "concealed" Ags.

Different forms of 130 kD connective tissue protein are specific for boundaries in the nervous system and basement membrane of muscle cells in leech

The nervous system and muscle tissue of the leech express two different organ-specific forms of connective tissue protein. The nervous system-specific form appears in regional boundaries separating cell bodies, axonal tracts and areas of the neuropile during late embryogenesis. In contrast, the muscle-specific form appears earlier during development in the basement membrane of muscle cells. In extraction experiments both forms behave like extracellular matrix proteins and because of their molecular weight, are considered members of a group of cell type-specific 130 kD proteins (leech gp130s). However, the two forms differ in their posttranslational modification. As determined by Con A and lentil lectin affinity chromatography, only the nervous system-specific, but not the muscle-specific form, has fucosylated and high mannose N-linked carbohydrates. These differences in the developmental onset and glycosylation suggest that nervous system-specific and muscle-specific connective tissue proteins are regulated differently and participate in different molecular interactions.

Synthesis of 1-N-glycyl beta-oligosaccharide derivatives. Reactivity of Lens culinaris lectin with a fluorescent labeled streptavidin pseudoglycoprotein and immobilized neoglycolipid.

The lectin from Lens culinaris (lentil) has a binding specificity for glycopeptides bearing 6-O-linked fucose on the reducing terminus on complex-type N-linked oligosaccharides. Lentil lectin therefore provides an excellent example of a carbohydrate binding protein in which high-affinity interactions are dependent on the integrity of the oligosaccharide core structure. We report here the synthesis of the 1-N-glycyl beta-derivative of Gal beta 4GlcNAc beta 2Man alpha 6(Gal beta 4GlcNAc beta 2Man alpha 3)Man beta 4GlcNAc beta 4(Fuc alpha 6)-GlcNAc (Gal-2F) and its subsequent biotinylation and palmitoylation. The biotin derivative when bound to a streptavidin-fluorescein isothiocyanate (FITC) conjugate was able to bind to both concanavalin A (ConA) and lentil lectin affinity columns. In contrast, synthesis of the biotin derivative of the glycamine derivative of Gal-2F and subsequent binding to streptavidin-FITC afforded reactivity to a ConA affinity column but not to a lentil lectin affinity column. Lentil lectin also bound to plastic microtiter plates containing the adsorbed palmitoyl-1-N-glycyl beta-derivative. No binding occurred when the homologous glycamine neoglycolipid was used. These results suggest the 1-N-glycyl beta-derivative of oligosaccharides may have general utility as an intermediate in the synthesis of novel glycoconjugate probes.

Ricin and lentil lectin-affinity chromatography reveals oligosaccharide heterogeneity of thyrotropin secreted by 12 human pituitary tumors

Some patients with thyrotropin (TSH)-producing pituitary tumors are more hyperthyroid than others despite similar TSH levels in serum, suggesting that qualitatively different TSH molecules with differing bioactivities may be secreted by different tumors. We used ricin and lentil lectin-affinity chromatography to test whether the TSH oligosaccharides varied among 12 patients with TSH-producing tumors. We found that each tumor secreted heterogeneous isoforms of TSH that differed in their extents of exposed galactose (Gal) residues, and their degrees of sialylation and core fucosylation. These biochemical parameters also varied markedly for TSH secreted by different tumors. Isoforms appeared to reflect poor sialyltransferase activity in two tumors and efficient sialyltransferase in the remainder. TSH secreted by tumors was more fucosylated than TSH secreted by control euthyroid persons. There was an inverse relationship between the sialylation and fucosylation of tumor TSH. No simple relationship between TSH oligosaccharide structures and bioactivity was evident, although mixtures of isoforms having the least and most sialylated TSH seemed to be the most bioactive clinically. In three patients from whom serum and medium TSH were both available, TSH in serum was more sialylated than TSH secreted by the tumor in vitro, perhaps reflecting slow clearance of sialylated isoforms form the circulation. Core fucosylation of serum TSH was less than that of medium TSH. These data prove that human tumors secrete TSH with heterogeneous oligosaccharide structures.

Fucosylation of glycoproteins begins in the rough endoplasmic reticulum of mouse active thyrotrophs.

Our aim was to determine whether fucosylation of glycoproteins begins in the rough endoplasmic reticulum (RER) of active thyrotrophs. This would contrast with most cells studied, in which fucosylation generally is associated with the Golgi apparatus. Mouse thyrotropic tumor tissue was incubated with [35S]methionine for 2, 5, 7, 10, 30, and 90 minutes. TSH and free alpha-subunits were immunoprecipitated from cell lysates, and they displayed a time-dependent increase in affinity for lentil lectin (which binds oligosaccharides having core fucose), even at short times. Since no 20-30 minute lag in onset of TSH- and free alpha-subunit-lentil binding was appreciated, as might have been expected had fucosylation begun only in the Golgi, it appeared that fucosylation was beginning in the RER of thyrotrophs. Pituitary tissue from euthyroid and hypothyroid mice was incubated with [3H]fucose, then subjected to electron microscopic autoradiography. The pituitaries of hypothyroid mice had numerous "thyroidectomy cells," which had 40% of silver grains over dilated cisternae of RER. "Nonthyroidectomy" cells had few silver grains over RER; most were over secretory granules and Golgi areas. Thus, active mouse thyrotrophs appear to shift the subcellular site of fucosylation partially from Golgi to RER, and this phenomenon may represent one cellular mechanism whereby the endocrine regulation of the structure of TSH oligosaccharides is accomplished.

Binding of thyrotropin to lentil lectin is unchanged by thyrotropin-releasing hormone administration in three patients with thyrotropin-producing pituitary adenomas.

Glycoproteins have increased affinity for lentil lectin when fucose residues are bound to N-acetylglucosamine in the "core region" of their asparagine-linked oligosaccharides. In three patients with thyrotropin (TSH)-producing pituitary tumors, the proportion of serum TSH isoforms that bound to lentil (70.8% +/- 15%) was higher than that seen for TSH from normal persons (32.5 +/- 8%). Unlike normal subjects, the concentration of TSH circulating in the tumor patients after acute administration of TSH-releasing hormone (TRH) did not rise, and the TSH did not exhibit increased binding to lentil compared to basal TSH. The TSH binding to lentil in one tumor patient decreased after metoclopramide, but TSH binding to lentil generally remained unchanged after metoclopramide or L-dopa administration. We conclude that human thyrotropic tumor tissue, unlike normal thyrotrophs, generally fails to release more highly fucosylated isoforms of TSH after pharmacologic stimulation, perhaps because the tumor tissue is less readily modulated by endocrine stimuli, or because the TSH is already relatively highly fucosylated.

Cross-Neutralizing Antibodies in Rabbits Immunized with HIV-1 gp160 Purified from Simian Cells Infected with a Recombinant Vaccinia Virus

A recombinant vaccinia virus in which the transcription of the human immunodeficiency virus type 1 (BRU isolate) env gene is driven by the 11K late vaccinia promoter yields about 10-fold higher amounts of gp160 env protein upon infection of monkey cells than does a recombinant in which gp160 is expressed using the 7.5K early-late promoter. The gp160 was purified from detergent lysates of infected cells by lentil lectin affinity chromatography followed by immunoaffinity chromatography, and was obtained in yields of 1-2 mg/10(9) cells of material estimated to be about 70% pure. Pairs of rabbits were immunized with purified gp160 using either one of five different adjuvants or an immunostimulating complex. In all cases a substantial humoral immune response was obtained after boosting, including an activity that neutralized the homologous (BRU) isolate of HIV-1. In some cases, this activity also neutralized two distantly related isolates, SF2 and MN.

Isolation and characterization of a thermolabile β-2 macroglycoprotein (‘thermolabile substance’ or ‘Hakata antigen’) detected by precipitating (auto) antibody in sera of patients with systemic lupus erythematosus

A novel thermolabile beta-2 macroglycoprotein ('thermolabile substance' (TLS) or 'Hakata antigen' (HA], which was detected by the precipitating (auto) antibodies of patients with systemic lupus erythematosus, was isolated and characterized. The purification procedure entailed the following steps: isoelectric precipitation in the range between pH 5.2-6.1, hydroxyapatite absorption chromatography, 35% saturated ammonium sulfate precipitation, Sephadex G-200 gel filtration, Pevikon block electrophoresis, lentil lectin affinity chromatography and immobilized rabbit anti-human whole serum IgG column chromatography. Utilizing these procedures, 0.1 mg of HA was purified from 3 1 of pooled human serum. The molecular mass of HA was determined as 650 kDa by Sepharose 4B gel filtration. On SDS-PAGE analysis, HA showed a single band at 35 kDa under reduced conditions and numerous ladder bands between 35 kDa to more than 300 kDa under nonreduced conditions. On analytical ultracentrifugation, HA gave a molecular mass of 520 kDa with a single meniscus and a sedimentation constant of 12.0. The amino acid and carbohydrate analysis of reduced and S-pyridylethylated HA revealed that it contained five residues of hydroxyproline and an N-linked type sugar chain.