Guard cells shrink and close stomatal pores when air humidity decreases (i.e. when the difference between the vapor pressures of leaf and atmosphere [VPD] increases). The role of abscisic acid (ABA) in VPD-induced stomatal closure has been studied using ABA-related mutants that respond to VPD in some studies and not in others. The importance of ABA biosynthesis in guard cells versus vasculature for whole-plant stomatal regulation is unclear as well. Here, we show that Arabidopsis (Arabidopsis thaliana) lines carrying mutations in different steps of ABA biosynthesis as well as pea (Pisum sativum) wilty and tomato (Solanum lycopersicum) flacca ABA-deficient mutants had higher stomatal conductance compared with wild-type plants. To characterize the role of ABA production in different cells, we generated transgenic plants where ABA biosynthesis was rescued in guard cells or phloem companion cells of an ABA-deficient mutant. In both cases, the whole-plant stomatal conductance, stunted growth phenotype, and leaf ABA level were restored to wild-type values, pointing to the redundancy of ABA sources and to the effectiveness of leaf ABA transport. All ABA-deficient lines closed their stomata rapidly and extensively in response to high VPD, whereas plants with mutated protein kinase OST1 showed stunted VPD-induced responses. Another strongly ABAinsensitive mutant, defective in the six ABA PYR/RCAR receptors, responded to changes in VPD in both directions strongly and symmetrically, indicating that its VPD-induced closure could be passive hydraulic. We discuss that both the VPD-induced passive hydraulic stomatal closure and the stomatal VPD regulation of ABA-deficient mutants may be conditional on the initial pretreatment stomatal conductance.

Immunoprecipitation of mRNA-protein complexes is a method that can be used to study RNA binding protein (RBP)-RNA interactions. In this protocol, an antibody targeting an RBP of interest is used to immunoprecipitate the RBP and any interacting molecules from a cell lysate. Reverse transcription followed by PCR is then used to identify individual mRNAs isolated with the RBP. This method focuses on examining an association between a specific RBP-mRNA complex, and it is best suited for a small scale screening of known or putative binding partners. It can also be used as a second, independent method to verify RBP-mRNA interactions discovered through more universal screening techniques. We describe the immunoprecipitation protocol in practical detail and discuss variations of the method as well as issues associated with it. The procedure takes three days to complete.

Tumor-associated macrophages (TAMs) expressing the multi-ligand endocytic receptor mannose receptor (CD206/MRC1) contribute to tumor immunosuppression, angiogenesis, metastasis, and relapse. Here, we describe a peptide that selectively targets MRC1-expressing TAMs (MEMs). We performed in vivo peptide phage display screens in mice bearing 4T1 metastatic breast tumors to identify peptides that target peritoneal macrophages. Deep sequencing of the peptide-encoding inserts in the selected phage pool revealed enrichment of the peptide CSPGAKVRC (codenamed “UNO”). Intravenously injected FAM-labeled UNO (FAM-UNO) homed to tumor and sentinel lymph node MEMs in different cancer models: 4T1 and MCF-7 breast carcinoma, B16F10 melanoma, WT-GBM glioma and MKN45-P gastric carcinoma. Fluorescence anisotropy assay showed that FAM-UNO interacts with recombinant CD206 when subjected to reducing conditions. Interestingly, the GSPGAK motif is present in all CD206-binding collagens. FAM-UNO was able to transport drug-loaded nanoparticles into MEMs, whereas particles without the peptide were not taken up by MEMs. In ex vivo organ imaging, FAM-UNO showed significantly higher accumulation in sentinel lymph nodes than a control peptide. This study suggests applications for UNO peptide in diagnostic imaging and therapeutic targeting of MEMs in solid tumors.

Galanin is a neuropeptide with a wide variety of biological functions, including that of a strong endogenous anticonvulsant. No nonpeptide ligands, capable of activating galanin receptors, are available today. Based on known pharmacophores of galanin, a combinatorial library was designed, synthesized, and screened at the rat hippocampal galanin receptor. A low molecular weight galanin receptor agonist, 7-((9-fluorenylmethoxycarbonyl)cyclohexylalanyllysyl)amino-4-methylcoumarin (galnon) was found to displace 125 I-galanin with micromolar affinity at Bowes cellular and rat hippocampal membranes. Autoradiographic binding assay on rat spinal cord sections confirmed the ability of galnon to displace 125 I-galanin from its binding sites. Galnon inhibited adenylate cyclase activity, suggesting an agonist action at galanin receptors. When injected i.p. galnon reduced the severity and increased the latency of pentylenetetrazole-induced seizures in mice and reversed the proconvulsant effects of the galanin receptor antagonist M35, injected into a lateral ventricle. Intrahippocampal injection of galnon also shortened the duration of self-sustaining status epilepticus in rats, confirming its agonist properties in vivo. Pretreatment of rats with antisense peptide nucleic acid targeted to galanin receptor type 1 mRNA abolished the effect of galnon, suggesting mediation of its anticonvulsant properties through this receptor subtype. These findings introduce a systemically active nonpeptide galanin agonist anticonvulsant.