Dissection of Nodule Development by Supplementation of \u3cem\u3eRhizobium leguminosarum\u3c/em\u3e biovar \u3cem\u3ephaseoli\u3c/em\u3e Purine Auxotrophs with 4-Aminoimidazole-5-Carboxamide Riboside

Purine auxotrophs of Rhizobium leguminosarum biovar phaseoli CFN42 elicit uninfected pseudonodules on bean (Phaseolus vulgaris L.). Addition of 4-aminoimidazole-5-carboxamide (AICA) riboside to the root medium during incubation of the plant with these mutants leads to enhanced nodule development, although nitrogenase activity is not detected. Nodules elicited in this manner had infection threads and anatomical features characteristic of normal nodules, such as peripheral vasculature rather than the central vasculature of the pseudonodules that were elicited without AICA riboside supplementation. Although 105 to 106 bacteria could be recovered from these nodules after full development, bacteria were not observed in the interior nodule cells. Instead, large cells with extensive internal membranes were present. Approximately 5% of the normal amount of leghemoglobin and 10% of the normal amount of uricase were detected in these nodules. To promote the development of true nodules rather than pseudonodules, AICA riboside was required no later than the second day through no more than the sixth day following inoculation. After this period, removal of AICA riboside from the root medium did not prevent the formation of true nodules. This observation suggests that there is a critical stage of infection, reached before nodule emergence, at which development becomes committed to forming a true nodule rather than a pseudonodule

Infection of Soybean and Pea Nodules by \u3cem\u3eRhizobium\u3c/em\u3e spp. Purine Auxotrophs in the Presence of 5-aminoimidazole-4-Carboxamide Riboside

Purine auxotrophs of various Rhizobium species are symbiotically defective, usually unable to initiate or complete the infection process. Earlier studies demonstrated that, in the Rhizobium etli-bean symbiosis, infection by purine auxotrophs is partially restored by supplementation of the plant medium with 5-amino-imidazole-4-carboxamide (AICA) riboside, the unphosphorylated form of the purine biosynthetic intermediate AICAR. The addition of purine to the root environment does not have this effect. In this study, purine auxotrophs of Rhizobium fredii HH303 and Rhizobium leguminosarum 128C56 (bv. viciae) were examined. Nutritional and genetic characterization indicated that each mutant was blocked in purine biosynthesis prior to the production of AICAR. R. fredii HH303 and R. leguminosarum 128C56 appeared to be deficient in AICA riboside transport and/or conversion into AICAR, and the auxotrophs derived from them grew very poorly with AICA riboside as a purine source. All of the auxotrophs elicited poorly developed, uninfected nodules on their appropriate hosts. On peas, addition of AICA riboside or purine to the root environment led to enhanced nodulation; however, infection threads were observed only in the presence of AICA riboside. On soybeans, only AICA riboside was effective in enhancing nodulation and promoting infection. Although AICA riboside supplementation of the auxotrophs led to infection thread development on both hosts, the numbers of bacteria recovered from the nodules were still 2 or more orders of magnitude lower than in fully developed nodules populated by wild-type bacteria. The ability to AICA riboside to promote infection by purine auxotrophs, despite serving as a very poor purine source for these strains, supports the hypothesis that AICAR plays a role in infection other than merely promoting bacterial growth

Porcine vas deferens luminal pH is acutely increased by systemic xylazine administration

Data are accumulating to demonstrate that pH regulation in the male reproductive tract has a vital role in modulating sperm cell fertilizing capacity and, therefore, male fertility. Bicarbonate uptake by sperm cells is required for the achievement of motility levels required for fertilization. Vas deferens epithelial cells can carry out measurable bicarbonate secretion, but the available literature to date reports that the vas deferens luminal content is typically acidic. This study aimed to determine pH in the boar vas deferens lumen and whether modulatory mechanisms exist for regulation of pH in this compartment of the male reproductive tract. A fiber-optic pH probe was used to assess pH in the vas deferens of anesthetized adult boars. The mean pH, derived from multiple measurements at variable positions along the vas deferens lumen, was 7.39 ± 0.09. Furthermore, administration of xylazine, an alpha-2 adrenergic receptor agonist, rapidly (< 10 min) alkalinized the vas deferens lumen in most cases. Since the duct was transected proximal to the site of measurements, the observations rule out the possibility that alkalinization resulted from secretion in more proximal portions of the duct. These results indicate that the boar vas deferens lumen can be alkaline, and suggest that porcine vas deferens epithelia increase net bicarbonate secretion in vivo, following systemic alpha-2 adrenergic stimulation. This secretory response greatly changes the luminal environment to which sperm cells are exposed, which will initiate or enhance motility, and is expected to modulate male fertility

Channel Replacement Therapy for Cystic Fibrosis

No abstract available

Effect of diaminopropionic acid (Dap) on the biophysical properties of a modified synthetic channel-forming peptide

Citation: Bukovnik, U., Sala-Rabanal, M., Francis, S., Frazier, S. J., Schultz, B. D., Nichols, C. G., & Tomich, J. M. (2013). Effect of diaminopropionic acid (Dap) on the biophysical properties of a modified synthetic achannel-forming peptide. Molecular Pharmaceutics, 10(10), 3959-3966.Channel replacement therapy, based on synthetic channel-forming peptides (CFPs) with the ability to supersede defective endogenous ion channels, is a novel treatment modality that may augment existing interventions against multiple diseases. Previously, we derived CFPs from the second transmembrane segment of the α-subunit of the glycine receptor, M2GlyR, which forms chloride-selective channels in its native form. The best candidate, NK₄-M2GlyR T19R, S22W (p22-T19R, S22W), was water-soluble, incorporated into cell membranes and was nonimmunogenic, but lacked the structural properties for high conductance and anion selectivity when assembled into a pore. Further studies suggested that the threonine residues at positions 13, 17, and 20 line the pore of assembled p22-T19R, S22W, and here we used 2,3-diaminopropionic acid (Dap) substitutions to introduce positive charges to the pore-lining interface of the predicted p22-T19R, S22W channel. Dap-substituted p22-T19R, S22W peptides retained the α-helical secondary structure characteristic of their parent peptide, and induced short-circuit transepithelial currents when exposed to the apical membrane of Madin-Darby canine kidney (MDCK) cells; the sequences containing multiple Dap-substituted residues induced larger currents than the peptides with single or no Dap substitutions. To gain further insights into the effects of Dap residues on the properties of the putative pore, we performed two-electrode voltage clamp electrophysiology on Xenopus oocytes exposed to p22-T19R, S22W or its Dap-modified analogues. We observed that Dap-substituted peptides also induced significantly larger voltage-dependent currents than the parent compound, but there was no apparent change in reversal potential upon replacement of external Na⁺, Cl⁻ or K⁺, indicating that these currents remained nonselective. These results suggest that the introduction of positively charged side chains in predicted pore-lining residues does not improve anion-to-cation selectivity, but results in higher conductance, perhaps due to higher oligomerization numbers

A new treatment for neonatal scours

Scours account for significant losses to the US swine industry every year. A common treatment for scours is the administration of broad-spectrum antibiotics, a practice with increasing unpopularity in the eyes of consumers. Currently, no treatment is available to reduce or eliminate the fluid losses associated with scours that is both inexpensive and easy to use. In the present study, a variety of prospective drugs were used to determine if a single compound might inhibit the effects of bacterial toxins in a laboratory setting. The results indicate that a new class of drugs, which we call DASUs, likely will prove useful for the treatment of watery diarrhea. Additional studies are underway to validate this conclusion.; Swine Day, Manhattan, KS, November 18, 199

Structural and biophysical properties of a synthetic channel-forming peptide: designing a clinically relevant anion selective pore

The design, synthesis, modeling and in vitro testing of channel-forming peptides derived from the cys-loop superfamily of ligand-gated ion channels are part of an ongoing research focus. Over 300 different sequences have been prepared based on the M2 transmembrane segment of the spinal cord glycine receptor α-subunit. A number of these sequences are water-soluble monomers that readily insert into biological membranes where they undergo supramolecular assembly, yielding channels with a range of selectivities and conductances. Selection of a sequence for further modifications to yield an optimal lead compound came down to a few key biophysical properties: low solution concentrations that yield channel activity, greater ensemble conductance, and enhanced ion selectivity. The sequence NK[subscript]4-M2GlyR T19R, S22W (KKKKPARVGLGITTVLTMRTQW) addressed these criteria. The structure of this peptide has been analyzed by solution NMR as a monomer in detergent micelles, simulated as five-helix bundles in a membrane environment, modified by cysteine-scanning and studied for insertion efficiency in liposomes of selected lipid compositions. Taken together, these results define the structural and key biophysical properties of this sequence in a membrane. This model provides an initial scaffold from which rational substitutions can be proposed and tested to modulate anion selectivity. This article is part of a Special Issue entitled: Protein Folding in Membranes

NC-1059: A Channel-Forming Peptide That Modulates Drug Delivery across In Vitro Corneal Epithelium

PURPOSE. The goal of this study was to determine whether a synthetic peptide, NC-1059, can modulate the corneal epithelium to increase the permeation of therapeutic agents across this barrier. METHODS. An in vitro system employing transformed human corneal epithelial (THCE) cells was optimized for this study. Culture conditions were identified to promote formation of a confluent monolayer that rapidly develops a substantial transepithelial electrical resistance. Electrical parameters were measured with a modified Ussing flux chamber, and solute flux was quantified with fluorescently labeled compounds. RESULTS. NC-1059 causes a concentration-dependent increase in short-circuit current and an increase in transepithelial electrical conductance when assessed in modified Ussing chambers. The effect of NC-1059 on transepithelial electrical resistance was reversible. To test for paracellular permeability and size exclusion, FITC-labeled dextran ranging in size from 10 to 70 kDa was used. Dextran permeated the corneal cell monolayer in the presence, but not the absence, of NC-1059. Fluorescein sodium and carboxyfluorescein were then used as low molecular weight markers with similar NC-1059 -modulated kinetics being observed. Maximum permeation for the fluorescein derivatives occurred 30 to 90 minutes after a 5-minute NC-1059 exposure. A prototypical drug, methotrexate, also exhibited increased permeation in the presence of NC-1059. CONCLUSIONS. NC-1059 enhances drug permeation across cultured corneal epithelial cell monolayers by transiently affecting the paracellular pathway. Thus, NC-1059 is a lead compound for development of cotherapeutic agents to enhance access and effectiveness of ophthalmic compounds. (Invest Ophthalmol Vis Sci. 2009;50:3337-3345) DOI:10.1167/iovs.08-3053 D rug delivery of ocular therapeutics presents several challenges. There are many difficulties in attaining and sustaining adequate therapeutic levels in the eye while avoiding systemic toxicity. Although there are several routes of administration available for the eye (e.g., topical, subconjunctival, retrobulbar, intracameral, and systemic), topical application of therapeutic agents to the eye offers several advantages including localized drug effects with limited entry into systemic circulation, better accessibility than can be achieved by systemic delivery, avoidance of first-pass hepatic metabolism, convenience, and simplicity. Despite the benefits of topical application, many factors limit the bioavailability of therapeutic agents. For instance, the maximum volume that can be contained within the conjunctival cul-de-sac and precorneal tear film is approximately 30 L, and the solutes are eliminated rapidly from the precorneal area by lacrimal secretions. The cornea is the outermost transparent portion of the eye and provides the primary barrier through which ocular absorption must occur

<i>De Novo</i> and Rare Variants at Multiple Loci Support the Oligogenic Origins of Atrioventricular Septal Heart Defects

Congenital heart disease (CHD) has a complex genetic etiology, and recent studies suggest that high penetrance de novo mutations may account for only a small fraction of disease. In a multi-institutional cohort surveyed by exome sequencing, combining analysis of 987 individuals (discovery cohort of 59 affected trios and 59 control trios, and a replication cohort of 100 affected singletons and 533 unaffected singletons) we observe variation at novel and known loci related to a specific cardiac malformation the atrioventricular septal defect (AVSD). In a primary analysis, by combining developmental coexpression networks with inheritance modeling, we identify a de novo mutation in the DNA binding domain of NR1D2 (p.R175W). We show that p.R175W changes the transcriptional activity of Nr1d2 using an in vitro transactivation model in HUVEC cells. Finally, we demonstrate previously unrecognized cardiovascular malformations in the Nr1d2tm1-Dgen knockout mouse. In secondary analyses we map genetic variation to protein-interaction networks suggesting a role for two collagen genes in AVSD, which we corroborate by burden testing in a second replication cohort of 100 AVSDs and 533 controls (p = 8.37e-08). Finally, we apply a rare-disease inheritance model to identify variation in genes previously associated with CHD (ZFPM2, NSD1, NOTCH1, VCAN, and MYH6), cardiac malformations in mouse models (ADAM17, CHRD, IFT140, PTPRJ, RYR1 and ATE1), and hypomorphic alleles of genes causing syndromic CHD (EHMT1, SRCAP, BBS2, NOTCH2, and KMT2D) in 14 of 59 trios, greatly exceeding variation in control trios without CHD (p = 9.60e-06). In total, 32% of trios carried at least one putatively disease-associated variant across 19 loci,suggesting that inherited and de novo variation across a heterogeneous group of loci may contribute to disease risk

Hippocampus Leads Ventral Striatum in Replay of Place-Reward Information

While the brain is asleep, the hippocampus plays first fiddle in the orchestra of memory; spatial information is reactivated in the hippocampus shortly in advance of emotional memory traces in the vental striatum