The maize locus, Opaque‐2, controls the expression in developing endosperm of structural genes encoding a family of storage proteins, the 22 kd zeins, and an abundant albumin, termed b‐32. It is shown that the promoter of the b‐32 gene is activated in vivo in the presence of the O2 gene product and that the information necessary for this activation resides in a 440 bp DNA fragment containing five O2 binding sites (GATGAPyPuTGPu). Two of these sites are embedded in copies of the ‘endosperm box’, a motif thought to be involved in endosperm‐specific expression, which is also represented in 22 kd zein promoters. The O2 protein is also shown to be capable of binding in vitro and activating in vivo, its own promoter.

The structure of the zein regulatory gene Opaque 2 of Zea mays has been determined by sequence analysis of genomic and cDNA clones. The size of 02 mRNA is 1751 bp [poly(A) tail not included] containing a major open reading frame (ORF) of 1380 bp preceded by three short ORFs of 3, 21 and 20 amino acid residues. The main ORF comprises 1362 bp and is composed of six exons ranging in size from 465 to 61 bp and five introns of 678 bp to 83 bp. A putative protein 454 amino acids long was derived by the theoretical translation of the genomic sequences corresponding to exons. The opaque 2 protein contains a domain similar to the leucine zipper motif identified in DNA binding proteins of animal protooncogenes such as fos, jun and myc, and in the transcriptional activators GCN4 and C/EBP. The region of 30 amino acid residues next to the leucine repeats towards the N terminus is rich in basic amino acids and is also homologous to a domain present in fos, jun and GCN4.Moreover, in the carboxy terminal region an amino acid motif closely resembling a metal binding domain is present.

A/B toxins, produced by bacteria and plants, are among the deadliest molecules known. The B chain binds the cell, whereas the A chain exerts the toxic effect. Both anti-A chain and anti-B chain Abs can neutralize toxins in vivo and in vitro. B chain Abs block binding of the toxin to the cell. It is not known how anti-A chain Abs function. Working with ricin toxin, we demonstrate that immunization with A chain induces greater protection than immunization with B chain. A panel of mAbs, binding to A chain, B chain, or both chains, has been produced and characterized. Immunologic characteristics evaluated include isotype, relative avidity, and epitope specificity. The ability to inhibit ricin enzymatic or cell binding activity was studied, as was the ability to block ricin-mediated cellular cytotoxicity on human and murine cell lines. Finally, the in vivo protective efficacy of the Abs in mice was studied. The Ab providing the greatest in vivo protective efficacy was directed against the A chain. It had the greatest relative avidity and the greatest ability to block enzymatic function and neutralize cytotoxicity. Interestingly, we also obtained an anti-A chain Ab that bound with high avidity, blocked enzymatic activity, did not neutralize cytotoxicity, and actually enhanced the in vivo toxicity of ricin. Anti-A chain Abs with moderate avidity had no in vivo effect, nor did any anti-B chain Abs.

IL-35 is induced during atherosclerosis development and inhibits mitochondrial reactive oxygen species-H3K14 acetylation-AP-1-mediated EC activation.

Background: Interleukin-35 is a novel inhibitory cytokine. Results: Interleukin-35 inhibits vascular endothelial cell activation by suppressing MAPK-AP1-mediated VACM-1 expression in LPS-induced acute inflammation. Conclusion: Interleukin-35 suppresses acute vascular endothelium response. Significance: Interleukin-35 may be an attractive reagent for anti-inflammatory therapy.

Mucosal tolerance induction generally requires multiple or large Ag doses. Because microfold (M) cells have been implicated as being important for mucosal tolerance induction and because reovirus attachment protein σ1 (pσ1) is capable of binding M cells, we postulated that targeting a model Ag to M cells via pσ1 could induce a state of unresponsiveness. Accordingly, a genetic fusion between OVA and the M cell ligand, reovirus pσ1, termed OVA-pσ1, was developed to enhance tolerogen uptake. When applied nasally, not parenterally, as little as a single dose of OVA-pσ1 failed to induce OVA-specific Abs even in the presence of adjuvant. Moreover, the mice remained unresponsive to peripheral OVA challenge, unlike mice given multiple nasal OVA doses that rendered them responsive to OVA. The observed unresponsiveness to OVA-pσ1 could be adoptively transferred using cervical lymph node CD4+ T cells, which failed to undergo proliferative or delayed-type hypersensitivity responses in recipients. To discern the cytokines responsible as a mechanism for this unresponsiveness, restimulation assays revealed increased production of regulatory cytokines, IL-4, IL-10, and TGF-β1, with greatly reduced IL-17 and IFN-γ. The induced IL-10 was derived predominantly from FoxP3+CD25+CD4+ T cells. No FoxP3+CD25+CD4+ T cells were induced in OVA-pσ1-dosed IL-10-deficient (IL-10−/−) mice, and despite showing increased TGF-β1 synthesis, these mice were responsive to OVA. These data demonstrate the feasibility of using pσ1 as a mucosal delivery platform specifically for low-dose tolerance induction.

The Glossy2 (Gl2) locus of maize is required for the formation of the epicuticular wax layer of young plants. gl2 mutant seedlings can be visually identified because of their glossy leaf surface which is different from the dull surface of wild-type seedlings. The Gl2 locus was isolated by transposon tagging. Seven unstable mutations, gl2-m2 to gl2-m8, were induced in a parental strain carrying an active transposable Activator (Ac) element in the unstable wx-m7 allele. Genetic tests on the gl2-m2 allele indicated that it was not caused by the Ac element but by the insertion of the transposable element Enhancer/Suppressor-Mutator (En/Spm). A Sa/l restriction fragment segregating with the mutant phenotype was identified, by Southern analysis, using sequences from the En/Spm element as a probe. Part of the fragment was cloned and was shown to carry part of the unstable gl2-m2 allele. These gl2 sequences were used to identify a genomic fragment carrying the wild-type allele and to isolate its corresponding cDNA sequence. The predicted Glossy2 protein consists of 426 amino acids. No similar amino acid sequence was found in protein data banks and the biochemical function of the Gl2 gene product is still unknown. The wild-type Gl2 transcript is found predominantly in juvenile leaves. The transcript level in the leaves of seedlings homozygous for a stable recessive gl2-ref allele is hardly detectable.

The maize Opaque-2 (O2) protein is a transcription factor of the basic/leucine-zipper class, involved in the regulation of endosperm proteins including the 22kDa alpha-zein storage proteins and b32 protein. In this study we have focussed our attention on the relationship between O2 and the cyPPDK1 gene, which encodes a cytoplasmic pyruvate orthophosphate dikinase (PPDK) isoform. The results of this study showed that PPDK activity is detectable in wild-type maize endosperms, while in o2 mutant endosperms, the levels of PPDK protein, mRNA and enzymatic activity are reduced, indicating that O2 is involved in the regulation of cyPPDK1 in this tissue. By employing transient expression experiments in tobacco mesophyll protoplasts, we have demonstrated that the O2 protein can activate expression of a chloramphenicol acetyl transferase reporter gene placed under the control of the cyPPDK1 promoter. An in vitro binding assay and DNaseI footprint analysis demonstrated that a specific sequence in the cyPPDK1 promoter can be recognized and protected by maize O2 protein. The regulation by the O2 locus of cyPPDK1 reported here, and control of alpha-zein synthesis by O2 suggest that the O2 protein may play a more general role in maize endosperm development than previously thought.