Binding of aflatoxin B1, G1 and M to plasma albumin

The aflatoxins B₁, G₁ and their metabolites exist in the systemic blood as protein conjugate. This conjugation is specific to plasma albumin and proceeds enzymatically by liver and kidney cells. The aflatoxin-albumin conjugate is permanent and the conjugation is an irreversible one. This may interpret the acute liver damage of animal ingested a single dose of aflatoxin (3, 4). The existence of bound aflatoxin-albumin in the systematic blood could be considered as one factor of low excretions of aflatoxins and their metabolites in urine (5, 6, 7).     

Advanced procedures for labeling of antibodies with quantum dots

Semiconductor quantum dots (QDs) are proved to be unique fluorescent labels providing excellent possibilities for high-throughput detection and diagnostics. To explore in full QDs’ advantages in brightness, photostability, large Stokes shift, and tunability by size fluorescence emission, they should be rendered stable in biological fluids and tagged with the target-specific capture molecules. Ideal QD-based nanoprobes should not exceed 15 nm in diameter and should contain on their surface multiple copies of homogeneously oriented highly active affinity molecules, for example, antibodies (Abs). Direct conjugation of QDs with the Abs through cross-linking of QDs’ amines with the sulfhydryl groups issued from the reduced Abs’ disulfide bonds is the common technique. However, this procedure often generates conjugates in which the number of functionally active Abs on the surface of QDs does not always conform to expectations and is often low. Here we have developed an advanced procedure with the optimized critical steps of Ab reduction, affinity purification, and QD–Ab conjugation. We succeeded in reducing the Abs in such a way that the reduction reaction yields highly functional, partially cleaved, 75-kDa heavy–light Ab fragments. Affinity purification of these Ab fragments followed by their tagging with the QDs generates QD–Ab conjugates with largely improved functionality compared with those produced according to the standard procedures. The developed approach can be extended to conjugation of any type of Ab with different semiconductor, noble metal, or magnetic nanocrystals.     

Parasporin-2Ab,a Newly Isolated Cytotoxic Crystal Protein from <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

A novel crystal protein that exhibited potent cytotoxicity against human leukemic T-cells was cloned from the Bacillus thuringiensis TK-E6 strain. The protein, designated as parasporin-2Ab (PS2Ab), was a polypeptide of 304 amino acid residues with a predicted molecular weight of 33,017. The deduced amino acid sequence of PS2Ab showed significant homology (84% identitiy) to parasporin-2Aa (PS2Aa) from the B. thuringiensis A1547 strain. Upon processing of PS2Ab with proteinase K, the active form of 29 kDa was produced. The activated PS2Ab showed potent cytotoxicity against MOLT-4 and Jurkat cells and the EC₅₀ values were estimated as 0.545 and 0.745 ng/mL, respectively. The cytotoxicity of PS2Ab was significantly higher than that of PS2Aa reported elsewhere. Although both cytotoxins were structurally related, it was thought that the minor differences found were responsible for the different cytotoxicities of PS2Ab and PS2Aa.     

Crystal structure of the complex between the F(ab)' fragment of the cross-neutralizing anti-HIV-1 antibody 2F5 and the F(ab) fragment of its anti-idiotypic antibody 3H6

The monoclonal antibody 2F5 neutralizes a broad range of human immunodeficiency virus-1 isolates via a conserved epitope on the viral glycoprotein gp41. The conformation of the principal epitope is a type I β-turn centered on gp41 residues ⁶⁶⁴DKW⁶⁶⁶; in addition, binding studies indicate that residues N- and C-terminal to this core as well as structurally more distant parts of gp41 also contribute to the interaction. Ab2/3H6 is an anti-idiotypic antibody that inhibits the interaction between 2F5 and gp41 and as such, Ab2/3H6 may, in principle, possess a paratope that mimics the gp41 epitope. To establish the potential of Ab2/3H6 to serve as a guide for the design of vaccine components against human immunodeficiency virus, we investigated the crystal structure of the heterodimeric complex of Ab2/3H6 F_ab and 2F5 F_ab′. Ab2/3H6 F_ab binds to 2F5 F_ab′ via a helix-like protrusion formed by residues ^58(H)RYSPSLNTRL^67(H) of the 2F5 F_ab′ variable domain and proximal to but not overlapping with the gp41 ⁶⁶⁴DKW⁶⁶⁶ epitope-binding pocket. This defines Ab2/3H6 as an anti-idiotypic antibody of the Ab2γ class, i.e., an antigen-inhibitable idiotype that does not carry the internal image of the linear primary gp41 ⁶⁶²ELDKWAS⁶⁶⁸ epitope.     

ICAM-1 Targeted Nanogels Loaded with Dexamethasone Alleviate Pulmonary Inflammation

Lysozyme dextran nanogels (NG) have great potential in vitro as a drug delivery platform, combining simple chemistry with rapid uptake and cargo release in target cells with “stealth” properties and low toxicity. In this work, we study for the first time the potential of targeted NG as a drug delivery platform in vivo to alleviate acute pulmonary inflammation in animal model of LPS-induced lung injury. NG are targeted to the endothelium via conjugation with an antibody (Ab) directed to Intercellular Adhesion Molecule-1(ICAM-NG), whereas IgG conjugated NG (IgG-NG) are used for control formulations. The amount of Ab conjugated to the NG and distribution in the body after intravenous (IV) injection have been quantitatively analyzed using a tracer isotope-labeled [¹²⁵I]IgG. As a proof of concept, Ab-NG are loaded with dexamethasone, an anti-inflammatory therapeutic, and the drug uptake and release kinetics are measured by HPLC. In vivo studies in mice showed that: i) ICAM-NG accumulates in mouse lungs (∼120% ID/g vs ∼15% ID/g of IgG-NG); and, ii) DEX encapsulated in ICAM-NG, but not in IgG-NG practically blocks LPS-induced overexpression of pro-inflammatory cell adhesion molecules including ICAM-1 in the pulmonary inflammation.     

Direct Determination of Copper,Lead and Cadmium in Tea Infusions by Flameless Atomic Absorption Spectrometry

Two kinds of “group A saponin,” Aa and Ab, are present as the main constituent in soybean seed. The saponin composition and content in F₁ and F₂ seeds derived from the cross parents of Aa and Ab types were analyzed. The “group A saponin” was of Aa–Ab type in all the F₁ seeds, and the ratio of Aa type : Aa–Ab type : Ab type was 1:2:1 in the F₂ seeds. From these results, it appears that Aa and Ab were controlled by codominant allelic alternatives at a single locus. An investigation of the saponin composition of the seed hypocotyls of 18 wild lines revealed some lines in which “group A saponin” was absent.     

Baseline susceptibility of Asian corn borer (Ostrinia furnacalis (Guenée)) populations in Vietnam to Cry1Ab insecticidal protein

Susceptibility of Asian corn borer (ACB), Ostrinia furnacalis (Guenée) to Bacillus thuringiensis (Bt) Cry1Ab protein was studied between 2015 and 2016 with 11 ACB populations, collected from various geographical regions in Vietnam. A concentration range of Cry1Ab from 0.20 to 26.10 ng/cm² of diet was evaluated against F₁ ACB neonates using diet surface-overlay bioassays. Mortality data was recorded daily until seven days after infestation. Growth inhibition was recorded at the end of seven days. The median lethal concentration (LC₅₀) varied ≈3-fold among the different populations, ranging from 0.58 to 1.83 ng/cm² of diet with an overall mean of 0.86 ng/cm² of diet. Even the lowest concentration of 0.20 ng/cm² caused 73.53% growth inhibition. >90% growth inhibition was achieved at 0.82 ng/cm² or higher concentrations. The results reflect natural variation in Bt susceptibility among ACB populations rather than variation caused by prior exposure to selection pressures. LC₉₉ value (17.26 ng/cm²) was generated by pooling mortality data across different populations. The upper fiducial limit of LC₉₉ (24.38 ng/cm²) could be a potential diagnostic dose for future resistance monitoring programs. The findings from this study suggest that ACB populations in Vietnam are highly susceptible to Cry1Ab protein. This is the first report of Cry1Ab susceptibility of different ACB populations in Vietnam and will serve as a baseline for future resistance monitoring work.     

Mathematical theory of immunoradiometric (labeled antibody) assays

Immunoradiometric assays (IRMA) are distinguished from conventional radioimmunoassays (RIA) by use of purified radioactively labeled antibody (Ab) instead of labeled antigen (Ag). Immunoadsorbent (ImAd) is used to precipitate free (unbound) Ab, leaving the bound Ab in the supernatant fraction, which is counted. The Ag-Ab reaction is treated as reversible, and as obeying second-order kinetics. The differential equations describing this system have been formulated, solved, and used for computer simulation studies. The sensitivity of the assay is primarily determined by the affinity constant, K. There is an optimal duration of the ImAd reaction (t_max), to maximize the slope of the dose-response curve. As the amount of ImAd increases, t_max decreases, and the maximal slope increases. For any arbitrary ImAd concentration, there is an optimal Ab concentration. Divalency of the Ab results in decreased specific activity, and a decrease in the sensitivity of the assay. The sensitivity for IRMAs is comparable to that for RIAs. Factors affecting the choice between IRMA and RIA are discussed.     

Relationships between Cell Cycle and Conjugation in 3 Hypotrichs*

SYNOPSIS. Relationships between the cell cycle and the beginning of conjugation were analyzed for 3 hypotrichs: Diophrys scutum, Oxytricha bifaria, and Euplotes crassus. The first 2 species enter conjugation with micronuclei in G₁; the latter species with a micronucleus in G₂. The 1st micronuclear division of conjugating E. crassus is mitotic. Thus meiotic DNA replication occurs when the cells of each species have already entered the mating process. Cells from asynchronous populations start conjugation with their macronuclei primarily in G₁ or more rarely at the beginning of the S stage in a percentage significantly different from that expected on the basis of random mating among all cells in the population. Also, macronuclear replication, when already begun, was blocked in cells undergoing conjugation. Therefore only the G₁ or the very early S stages of the cell cycle are compatible with conjugation in the 3 analyzed species.     

Susceptibility of northern corn rootworm Diabrotica barberi (Coleoptera: Chrysomelidae) to mCry3A and eCry3.1Ab Bacillus thuringiensis proteins

The susceptibility of the northern corn rootworm Diabrotica barberi (Smith & Lawrence) to mCry3A and eCry3.1Ab proteins derived from Bacillus thuringiensis (Bt) was determined using a diet bioassay. Northern corn rootworm neonates were exposed to different concentrations of mCry3A and eCry3.1Ab, incorporated into artificial diet. Larval mortality was evaluated after 7 d. The mCry3A and eCry3.1Ab proteins were found to be toxic to the northern corn rootworm larvae. The LC₅₀ and LC₉₉ values for mCry3A were 5.13 and 2482.31 μg/mL, respectively. For eCry3.1Ab, the LC₅₀ and LC₉₉ values were 0.49 and 213.01 μg/mL. Based on the estimated lethal concentrations, eCry3.1Ab protein was more efficacious to northern corn rootworm larvae than mCry3A. These lethal concentration values will be used as diagnostic doses for routine annual monitoring for change in susceptibility of field collected northern corn rootworm to mCry3A, and eCry3.1Ab toxins.     

Effect of ethylene on [C]indole-3-acetic Acid metabolism in leaf tissues of woody plants

The effect of ethylene on [¹⁴C]indole-3-acetic acid (IAA) metabolism was investigated in defoliation sensitive leaf tissues of citrus (Citrus sinensis) and resistant leaf tissues of eucalyptus (Eucalyptus camaldulensis). IAA metabolites were fractionated into 80% ethanol-soluble, H₂O-soluble, NaOH-soluble, and insoluble components. In citrus, pretreatment with 25 microliters per liter ethylene for 24 hours significantly increased the amount of ethanol- and H₂O-extractable conjugates during the first hour of incubation in [¹⁴C]IAA and increased 3- to 4-fold the formation of NaOH-extractable conjugates during the entire 6-hour incubation period. However, induction of the IAA-aspartate conjugation system was inhibited by ethylene. In eucalyptus, ethylene pretreatment only slightly stimulated the formation of IAA metabolites. Increased formation of ethanol-extractable conjugates in ethylene-pretreated eucalyptus tissues was observed only after 6 hours of incubation. Chromatographic analysis indicated that the ethanol and H₂O extracts of both species contained various low molecular weight conjugates, whereas in citrus leaf tissues high molecular weight conjugates accounted for most of the greater radioactivity detected in the NaOH extracts as a result of ethylene-pretreatment. It is suggested that ethylene may reduce the level of endogenous IAA in citrus leaf tissues by stimulating IAA conjugation.     

Vaccination of Heifers with Anaflatoxin Improves the Reduction of Aflatoxin B1 Carry Over in Milk of Lactating Dairy Cows

It was previously reported that injection of anaflatoxin B₁ (AnAFB₁) conjugated to keyhole limpet hemocyanin (KLH), together with Freund''s adjuvant, was effective in inducing in cows a long lasting titer of anti-aflatoxin B₁ (AFB₁) antibodies (Abs), cross-reacting with other aflatoxins, which were able to hinder, proportionally to their titer, the secretion of aflatoxin M₁ (AFM₁) into the milk of cows continuously fed with AFB₁. According to anti-AFB₁ Ab titer, 50% of the vaccinated cows were recognized as high responder animals. In an attempt to prepare a more effective formulation for vaccination of cows, it was compared the immunogenicity, in Holstein Friesian heifers, of AnAFB₁ covalently conjugated to KLH or to recombinant diphtheria toxin (CRM₁₉₇) molecules, and injected together with various adjuvants. This study demonstrated that injection of AnAFB₁ conjugated to KLH and mixed with complete (priming) and incomplete Freund''s adjuvant (boosters), as in the previous schedule of immunization, was the most effective regimen for inducing Ab responses against AFB₁, although pre-calving administration could increase the effectiveness of vaccination, resulting in 100% high responder animals. After one booster dose at the beginning of the milk production cycle, anti-AFB₁ Ab titers were comparable to those recorded at the end of the immunization schedule, and proved to be effective in reducing significantly AFB₁ carry over, as AFM₁, from feed to milk. Pre-calving vaccination of dairy heifers with conjugated AnAFB₁, adjuvated with complete and incomplete Freund''s adjuvant, may represent the most effective tool for preventing the public health hazard constituted by milk and cheese contaminated with aflatoxins.     

Determination of baseline susceptibility to Cry1Ab protein for Asian corn borer (Lep., Crambidae)

Abstract: Although transgenic Bacillus thuringiensis (Bt) corn can provide a new tool for control of the Asian corn borer (ACB), Ostrinia furnacalis (Guenée), concern has been raised regarding the possibility of the target insect evolving resistance to the Bt protein under intensive selection pressure from Bt corn. Therefore, it is necessary to establish baseline data to enable detection of changes in susceptibility in field populations after prolonged exposure to Bt corn. Susceptibility to purified Cry1Ab protein from Bt was determined for 10 populations of ACB from the major corn‐growing regions of China, ranging geographically from Heilongjiang Province in the northeast to Shaanxi Province in the east‐central part. Neonate ACB were exposed to semi‐artificial diet incorporated with increasing Cry1Ab protein concentrations, and mortality and growth inhibition were evaluated after 7 days. The range of LC₅₀ (50% lethal concentration) among the populations was 0.10 to 0.81 μg/g (Cry1Ab protein/diet). Differences (P < 0.05) in susceptibility among the populations were significant. LC₅₀s generated from the Huanghuaihai Summer Corn Region were higher than those from the Spring Corn Regions. Bt was one of the significant natural biomortality factors of overwintering generation ACB. There was a significant correlation between percentage of the larvae infected with Bt and their LC₅₀ values to Cry1Ab protein in geographic distinct populations (r = 0.7350*, d.f. = 8, _r0.05 = 0.632). Based on the background of Bt formulations used for corn insect pests control in these areas, these differences were not caused by prior exposure to Bt insecticides. Instead, the small differences likely reflect natural Bt selection pressure. Because the variation in susceptibility to Cry1Ab was small (<10‐fold), the ACB apparently is susceptible to Cry1Ab across its range within China.     

Different rates of metabolism of two chloroacetanilide herbicides in pioneer 3320 corn

The in vivo rates of uptake and detoxification of alachlor and metolachlor were determined using Pioneer corn 3320 seedlings. Equal amounts of the radiolabeled herbicides were applied to etiolated coleoptiles and, at various intervals after treatment, the unabsorbed radioactivity was removed and quantified. Analysis of 80% methanol extracts by reverse phase liquid chromatography showed no significant differences in the rate of uptake of metolachlor and alachlor. However, the rate of glutathione conjugation of alachlor in vivo was two- to threefold greater than the rate for metolachlor at 2 and 4 hours after herbicide application. Since the initial step in detoxification is conjugation of the chloroacetanilide to glutathione, the activities of the enzymes responsible for conjugation, the glutathione-S-transferases (GST) were also analyzed in vitro, using crude extracts and the purified GST enzymes. The specific activities of the extracts were consistent with the results in vivo. Using alachlor as a substrate, the specific activity for glutathione conjugation was almost threefold higher than that for metolachlor. Kinetic analysis of purified GST III indicates that the enzyme has a higher affinity for alachlor (K_mapp = 1.69 millimolar) than for metolachlor (K_mapp = 8.9 millimolar).     

Tracking the source of Bacillus thuringiensis Cry1Ab endotoxin in the environment

The application of Bacillus thuringiensis (Bt) and the growing of genetically-modified crops are currently practised to control infestations of crop-eating insects. The increasing use of these biopesticides could lead to an increase in Cry1Ab endotoxin in both terrestrial and aquatic environments. The aim of this study was to quantify levels of Cry1Ab endotoxin and locate its source in the environment. Agricultural soils and surface waters were spiked with crystals (biopesticide-Dipel^®) or with pure Bt-corn endotoxin. Cry1Ab concentrations were then determined with immunoassays. Additionally, surface water, soils and sediments were sampled in an area sprayed with Bt kurstaki and at a site where genetically-modified corn expressing Cry1Ab is grown. Isotopic analysis was performed on the endotoxin from Bt and Bt corn to characterize the proportions of ¹³C/¹²C and ¹⁵N/¹⁴N. The results showed that Bt-corn endotoxin is degraded more rapidly in water than in soils (t_1/2: 4 and 9 days, respectively), while crystals appeared to be more resilient, as expected. The isotopic patterns of ¹³C and ¹⁵N in Bt-corn endotoxin differed markedly from Bt, making it possible to track the source of Cry1Ab in the environment. Preliminary field surveys indicate that Cry1Ab is fairly uncommon in aquatic environments, being found only at trace concentrations when it is detected.     

Stereoselective sulfate conjugation of isoproterenol in humans: Comparison of hepatic,intestinal, and platelet activity

The stereochemistry of sulfate conjugation of isoproterenol (ISO) was examined with human liver, intestine, and platelets as the phenolsulfotransferase (PST) enzyme source and PAP³⁵S as the cosubstrate. With the hepatic cytosol, two distinct sulfation reactions were identified, a high affinity reaction (K_m 5 to 50 μM) and a low affinity reaction (K_m 360 to 2,900 μM). The efficiency of sulfation (V_max/K_m) for both reactions was 5-fold higher for (+)- than for (?)-ISO. When the hepatic PSTs were resolved by ionexchange chromatography, it could be shown that the high affinity reaction was catalyzed by the monoamine (M) form and the low affinity reaction by the phenol (P) form of PST. Only the high affinity (M form) sulfation was detected in the jejunal cytosol with a V_max/K_m value 6.1-fold higher for (+)- than for (?)-ISO. Finally the platelet, as a potentially useful model tissue, also demonstrated only the high affinity M form reaction with a V_max/K_m value 5.7-fold higher for (+)- than for (?)-ISO. In summary, this study has shown that sulfation of ISO by PSTs in various human tissues is stereoselective and favors the inactive (+)-enantiomer over the active (?)-enantiomer by about 5-fold, a finding which should be considered in the therapeutic use of chiral drugs cleared by sulfate conjugation. © 1993 Wiley-Liss, Inc.     

Enhancement of cytotoxic and proliferative responses of lymphocytes from melanoma patients by incubation with monoclonal antibodies against ganglioside GD3

Summary Previous studies have shown that monoclonal antibodies (M.Ab) to the ganglioside GD₃ may induce partial remissions in tumour growth in patients with melanoma. In vitro studies demonstrated that M.Abs to GD₃ may also enhance lymphocyte responses to phytohemagglutinin and interleukin 2 (IL2). The present study extended these findings by showing that the IL2-dependent proliferative and cytotoxic response of T cell clones derived from a melanoma patient and a patient with the Vogt-Koyanagi-Harada syndrome were enhanced by pre-incubation of T cells with M.Ab to GD₃. The degree of enhancement increased with the duration of pre-incubation from 2 to 24 h and applied to both T4⁺ and T8⁺ clones. The potentiation of these responses was not specific for M.Abs to GD₃ but was also seen with M.Abs to GD₂ and the T10 structure on T cells but not with M.Abs to the transferrin receptor or an isotype control M.Ab. Incubation of lymphocytes from a melanoma patient with M.Ab to GD₃ during culture with autologous melanoma cells enhanced the proliferative response to the tumour. The expression of IL2 receptors (Tac epitope) on the T cells showed variable enhancement after incubation with M.Ab to GD₃ but the significance of these findings in relation to the mechanism of the enhanced responses is not known. These results suggest that certain M.Abs may stimulate cell-mediated immune responses against tumour cells and that this may provide an additional mode of action of M.Abs against tumours in vivo     

The metabolism of estriol in the guinea-pig

Labeled estriol (E₃) was injected i.v. into intact guinea-pigs and animals with biliary fistulas. It was shown that biliary excretion constitutes a major route for the conjugates of E₃, probably followed by a significant enterohepatic circulation. The conjugation pattern of E₃ revealed the preponderant amount of the steroid to be excreted as a 3-glucosiduronate, both in the urine and bile. In contrast, the 3,16α-disulfate of E₃ was found in significant amount only in the bile. Unconjugated E₃ and its sulfate constituted a minor percentage of the radioactivity found both in bile and urine. The E₃ nucleus remained intact during the conjugation processes in the guinea-pig. The kidney was shown to glucoronidate E₃in vitro to some extent, but to a much lower degree than observed with kidneys of other species; sulfation of E₃ by the guinea-pig kidney was of an even much lower magnitude. Thus, we interpret the results to indicate that in guinea-pigs, the liver plays a paramount role in the conjugation of E₃ followed by biliary excretion of most of the conjugates and subsequent entero-hepatic circulation. The role of the kidney in the conjugation of E₃ in the guinea pig appears to be secondary to that of the liver.