Risks of ingestion of aflatoxin-contaminated groundnuts in Benin: scale measurements, beliefs, and socioeconomic factors.

This study evaluates farmers' beliefs and perceived risks of aflatoxin (AF) on the consumption, production, and marketing of groundnuts. A survey was conducted with 181 farmers in Benin to assess their beliefs of AF effects on the marketing of groundnuts, and finally human and animal health. Awareness and action factors were also evaluated. Relationships of the belief and action factors with socioeconomic variables were evaluated using multiple indicators and multiple causes (MIMIC) models within a socioeconomic framework using a health belief model (HBM). The results indicate that the scale of the various constructs is reliable and the validity conforms to expectations. The unifactorial models developed in this study provide a satisfactory fit with NFl, CFI, and GFI exceeding 0.90. The results reveal that gender, age, and years of experience in farming significantly impact farmers' action regarding the reduction of AF in groundnut production and marketing. Male farmers are more likely to be aware of AF problems in groundnuts and feel more susceptible to the problems than their female counterparts. Gender and education seem to be dominating factors in the perception of barriers to mitigating the effects of AF, and male and older farmers are more likely to perceive the benefits of producing and marketing good quality groundnuts.

Evidence for IRF-1-dependent gene expression deficiency in interferon unresponsive HepG2 cells.

Induction of the antiproliferative and antiviral state by IFNs (type I and II) is dramatically impaired in HepG2 cells. We show here that RNase L, IDO, GBP-2 and iNOS genes normally expressed as a secondary response to IFN are no longer inducible in HepG2 cells, while induction of primary response genes (IRF-1, PKR, p48-ISGF3gamma, 2-5AS, 6-16 and p56-(trp)tRNA) are unaffected. On the basis of previous data implicating transcription factor IRF-1 in the induction of some IFN-induced genes, we tested the effects of transfecting an IRF-1 oligonucleotide antisense in HeLa cells and found specifically impaired IFN induction of secondary response genes (RNase L, IDO and GBP-2). This raised the possibility that IRF-1 was defective in HepG2 cells. However, some molecular and biochemical analyses reveal that IRF-1 is induced normally by IFNs and retains its normal size, cellular location, phosphorylation status and ability to bind the IDO promoter in vitro. Therefore, we conclude that although the primary response pathway is fully functional, some aspects of the secondary pathway involving IRF-1 (but not IRF-1 itself) are defective in HepG2 cells. It may be possible that the promoter region of these deficient HepG2-genes requires an unidentified transcription factor in addition to de novo IRF-1, which could be elicited by a cooperative activator.

Lack of 2',5'-oligoadenylate-dependent RNase expression in the human hepatoma cell line HepG2.

2',5'-adenylate oligonucleotide (2-5A)-dependent RNase and 2-5A-synthetase are two enzymes of the 2-5A system strongly implicated in the basal control of RNA decay of both interferon-treated and untreated cells. RNase is activated by a 2-5A produced by 2-5A-synthetase, both enzymes being overexpressed by type I-interferon (alpha/beta). We described here for the first time a cell line completely deficient in RNase and its mRNA, while p69 2-5A-synthetase was normally interferon alpha/beta-induced. The complete absence of this RNase in human hepatoma cells (HepG2) was shown using three different methods based on the binding of a [32P]-labeled 2-5A probe of high specific activity to its binding site. Negative Western blotting assay with a specific monoclonal antibody correlated the previous findings. RNase-specific mRNA was not detectable even after treatment of cells with 1000 units/ml of interferon alpha/beta. This is not due to a mutation of the gene because an intronless genomic DNA sequence encoding 2-5A-binding site was cloned and expressed. It is likely that the expression of 2-5A-dependent RNase was impaired at the transcriptional level while having the known IFN alpha/beta-transcriptional regulatory factors as revealed by induction of p69 2-5A-synthetase gene. This may account for a differential activation of 2-5A-dependent RNase and 2-5A-synthetase genes by type I-interferon, and suggests that other members of regulatory transcription factors, different from IRF-1 and STAT proteins, may participate in two different interferon alpha/beta signaling pathways.

Microheterogeneity of rat, mouse and human alpha1-fetoprotein as revealed by polyacrylamide gel electrophoresis and by crossed immuno-affino-electrophoresis with different lectins.

Polyacrylamide gel electrophoresis and crossed immuno-affino-electrophoresis with several free lectins have been used to characterize and to compare the molecular heterogeneity of rat, mouse and human alpha1-fetoproteins. Each alpha1-fetoprotein contains a variable number of electrophoretic variants depending on the gel porosity. In SDS electrophoresis, two molecular size populations are present in rat alpha1-fetoprotein (Mr = 74 000 and 72 000) and in mouse alpha1-fetoprotein (Mr = 73 000 and 72 000) but only one is observed in human alpha1-fetoprotein (Mr = 70 000). The crossed immuno-affino-electrophoresis patterns square with affinity chromatography results and reveal a marked and characteristic heterogeneity for the three alpha1-fetoprotein species with Concanavalin A, Ricinus communis and Lens culinaris lectins. No lectin-alpha-fetoprotein interaction is apparent with Ulex, Lotus and wheat germ lectins. Since similar patterns are obtained whether with purified alpha1-fetoprotein or with unfractionated fresh fetal sera, it is likely that this heterogeneity is not a consequence of artefactual molecular modifications arising during the purification procedure.

Oestrone binding properties of nine molecular variants of rat alpha-foetoprotein.

Rat alpha-foetoprotein was separated into nine molecular variants by electrophoresis and affinity chromatography on Ricinus communis agglutinin and concanavalin-A. The nine variants are able to bind oestrone with the same capacity of one binding site per alpha-foetoprotein molecule. The association constants seem to vary with the sialic acid composition of the iso-alpha-foetoprotein.

Rat alpha-fetoprotein heterogeneity. Comparative chemical study of the two electrophoretic variants and their Ricinus lectin-binding properties.

Two electrophoretic forms of rat alpha-fetoprotein were purified using immunosorbent chromatography and preparative electrophoresis on polyacrylamide gel slabs. Some of their respective chemical properties and their affinity for the Ricinus communis lectin (RCAI) were compared. Electrophoresis on polyacrylamide gradient gel in the presence of sodium dodecyl sulfate indicated a slight difference in molecular 74 000 for the slow alpha-fetoprotein (AFPA) and 72000 for the fat alpha-fetoprotein (AFPB). no significant difference in amino acid composition between AFPA and AFPB was found. A residue of valine was identified at the C-germinal end of both alpha-fetoproteins. The analysis of the CNRr-cleavage products reveals slight differences between AFPS and AFPB. The slow moving alpha-fetoprotein could be further fractionated on RCAI-sepharose column in two components, AFPA1 and AFPA2 differing by their sialic acid content.

Localization of a molecular form of interferon-regulated RNase L in the cytoskeleton.

RNase L (also termed 2-5A-dependent RNase) is a crucial enzyme involved in the molecular mechanism of interferon (IFN) action. Activated by 2',5'-oligoadenylate oligomers (2-5A), this enzyme controls the regulation of RNA stability in IFN-treated or virus-infected mammalian cells. Knowledge of RNase location within cells may provide additional information about its function. Previous work located RNase as a detergent-soluble molecule in nuclei and cytoplasm. In this study, we demonstrate that this enzyme was also present in a detergent-insoluble fraction associated with proteins of the cytoskeleton. A cellular fractionation procedure was used to prepare the cytoskeleton, which was shown to contain 2-5A binding activity not due to cytoplasmic contaminants. In contrast to the cytoplasmic fraction, which contained RNase L with a 2-5A-accessible site, the insoluble RNase molecular form of the cytoskeleton could not be assayed by the classic radiobinding method or the covalent UV cross-linking procedure, which only detects the 2-5A binding site in an open position, that is, free of 2-5A or with an unmasked 2-5A site. The 2-5A binding site present in the cytoskeleton was completely masked and not directly accessible to its 2-5A activator. This particular molecular form of RNase can be detected after a specific denaturing-renaturing treatment of the cytoskeleton, which separates the RNase from cytoskeletal proteins, unmasking the 2-5A site. The cytoskeletal RNase was no longer present at this site when cells were stimulated for a short time with 12-O-tetradecanoylphorbol-13-acetate (TPA). Our data suggest the existence of a pathway that targets the RNase to another subcellular location. To explore the issue further, we examined in vitro the ability of calcium and phospholipid-dependent protein kinase C (PKC) to catalyze significant phosphorylation of the RNase.

Upregulation of a myristylated 74-kDa protein by interferon treatment of Daudi cells.

Labeling of unstimulated human Daudi B lymphoblastoid cells with exogenously added [3H]myristate resulted in acylation of a broad spectrum of different proteins, most of which are currently unknown. Among this array of labeled proteins, a unique 74-kDa acylated protein was induced in interferon (IFN)-treated cells. In the present study, we defined the myristylation kinetics of this protein and examined the subcellular distribution before and after activation with IFN-alpha/beta. This acylated protein was detected only at a very low level in the membrane fraction of untreated cells, and its level increased 3-4-fold by treatment with IFN. This induction occurred over a short period of time and was IFN-alpha/beta dose-dependent. No significant induction was observed with IFN-gamma. Incorporation of [3H]myristate was completely abolished by cycloheximide. The fatty acid associated with this protein was probably linked to a nascent chain through an amide linkage, as it was not released by alkaline hydroxylamine treatment and was identified as myristic acid by HPLC after its release from the polypeptide chain by acid methanolysis. In contrast to other IFN-induced proteins, whose synthesis started at 10 h and was maintained for 20 h, this protein was present in the plasma membrane for a short period of time, between 4 and 6 h after IFN-alpha/beta treatment, and was no longer present in this cellular compartment. This event appears to be transient and suggests that a degradation or a negative regulation of transcription starts from 6-7 h after continuous IFN treatment. As many other myristylated proteins are implicated in cellular regulation, it is possible that this 74-kDa protein may have a regulatory role in cell proliferation and the inhibition of viral replication.

Analytical and comparative chromatographic maps of oligosaccharides produced by acetolysis and partial acid hydrolysis of glycoprotids.

The authors describe a chromatographic mapping procedure of oligosaccharides present in acetolysates and partial acid hydrolysates of glycopeptides or glycoproteins. Oligosaccharides are first fractionated on charcoal-Celite columns and then identified by paper chromatography. This procedure is sensitive and reproducible and allows to compare the structure of N-glycans and of glycoproteins from various sources.

Interferons and oncogenes in the control of cell growth and differentiation: working hypothesis and experimental facts.

This short review summarizes available evidence for (i) growth regulatory properties of exogenous as well as recently described autocrine IFNs, (ii) down-regulation of cellular oncogene expression with emphasis on c-myc and (iii) the possible involvement of the IFN-regulated 2-5A pathway at these levels. Initially described as a part of the IFN-induced antiviral mechanism, this double-stranded RNA-activated pathway leads to the preferential degradation of viral mRNAs in IFN-treated virus-infected cells probably through localized activation at the site of virus replication. Such mechanisms could be involved in the regulation of the stability of rapidly turning over mRNAs as for instance c-myc mRNA in IFN-treated cells. Whatever the elegance of the concept, however, experimental evidence is essentially circumstantial; tools developed in our group to strengthen the demonstration are briefly described.

Cardiac and plasma atrial natriuretic peptide after 9-day hindlimb suspension in rats.

To determine atrial natriuretic peptide (ANP) adaptation to simulated weightlessness, immunoreactive plasma (ir-NH2- and ir-COOH-terminals) and atrial (ir-COOH-terminal) ANP levels, atrial mRNA expression, immunoreactive cardiocyte ANP levels (ir-NH2- and ir-COOH-terminals), and ultrastructural observations of granules in atrial cardiocytes were assessed in male Wistar rats after a 9-day hindlimb suspension. Plasma ir-NH2- and ir-COOH-terminal ANP concentrations decreased by 17 (P < 0.05) and 37% (P < 0.05), respectively, in suspended rats. A concomitant ir-COOH-terminal ANP content reduction was also observed in left (31%; P < 0.01) and right atria (25%; P < 0.05). Atrial ANP mRNA expression was severely depleted in the right atrium and less so in the left atrium after 9 days of hindlimb suspension. Immunocytochemistry observations demonstrated lowered NH2- and COOH-terminal ANP immunoreactivities in left and right atria from suspended rats. A reduced number of storage granules (dense granules) in both atria was also noted on ultrastructural analysis. It was concluded that ANP biosynthesis, storage, and release were decreased after a 9-day hindlimb suspension.

Direct analysis of the lectin reactivity of alpha-fetoprotein in maternal serum by crossed affinity radio-immunoelectrophoresis.

Affinity experiments with the lentil (Lens culinaris) lectin have revealed the existence of two distinct molecular populations of alpha-fetoprotein: lectin reactive and lectin non-reactive. Using a combination of crossed lectin immunoelectrophoresis and radio-immunoelectrophoresis, it has been possible to obtain directly the lentil lectin affinity patterns of alpha-fetoprotein present in maternal sera. The lentil lectin reactivity of maternal alpha-fetoprotein decreases almost linearly with the gestational age from week 15 to 35.

Cardiac atrial natriuretic peptide (ANP) in rat dams and fetuses developed in space (NIH-R1 and NIH-R2 experiments).

NIH-R1 and R2 missions, conducted by NASA, allowed us to study the effects of the microgravitational environment 1) on cardiac ANP in pregnant rats, spaceflown for 11 days and dissected after a 2-day readaptation to Earth's gravity, after natural delivery, and 2) on maturation of cardiac ANP system in rat fetuses developed for 11 days in space and dissected on the day of landing, 2 days before birth. Immunocytochemical and electron microscopy analyses showed a typical formation of ANP-containing granules in atrial myocytes, in both dams and fetuses. Using competitive RT-PCR and radioimmunoassays, we observed that, after 2 days of readaptation to Earth's gravity, cardiac ANP biosynthesis of rat dams flown in space was increased by about twice, when compared to Synchronous and Vivarium Control rats. More obviously, rat fetuses developed in space and dissected on the day of landing displayed an altered maturation of cardiac ANP, evidenced by an increased mRNA biosynthesis (by about 6 fold, p<0.05), whereas the cardiac ANP storage was slightly reduced (by about twice, p<0.05) in both Flight and Synchronous Control groups, in comparison with Vivarium Control rats. These last results suggest that ANP metabolism during development is impacted by the microgravitational environment, but also by the housing conditions designed for space flight.

Hydrazinolysis and nitrous acid deamination of the carbohydrate moiety of alpha1-acid glycoprotein.

Hydrazinolysis followed by nitrous acid deamination of alpha1-acid glycoprotein gave acidic and neutral mono- and oligo-saccharides that contain 2,5-anhydro-D-mannose as reducing residue: alpha-D-Manp-(1 leads to 3)-[alpha-D-Manp-(1 leads to 6)]-beta-D-Manp-(1 leads to 4)-2,5-anhydro D-mannose (1), beta D-Galp-(1 leads to 4)-2,5-anhydro-D-mannose (3), 2,5-anhydro-D-mannose, and two N-acetylneuraminic acid-containing oligosaccharides having the common partial sequence: NeuNAc-(2 leads to ?)-[BETA-D-Galp-(1 leads to 4)-2,5-anhydro-D-mannose] (5). This specific cleavage of 2-amino-s-deoxy-D-glucosyl linkages released almost quantitatively a very limited number of saccharides. Reduction with sodium borotritide of the products of cleavage allowed the precise determination of the molar proportion of 1, 3, and free 2,5-anhydro-D-mannose.