Genetic regulation of gibberellin deactivation in Pisum

The regulation of gibberellin (GA) deactivation was examined using the sin (slender) mutation in the garden pea (Pisum sativum L.). This mutation blocks the deactivation of GA₂₀, the precursor of the bioactive GA₁. Firstly, crosses were made to combine sin with the GA biosynthesis mutations na, lhⁱ and le-3. The combination sin na produced a novel phenotype, with long (‘slender’) basal internodes and extremely short (‘nana’) upper internodes. In contrast, the double mutant sin lhⁱ was phenotypically dwarf. The mutation sin causes an accumulation of GA₂₀ in maturing seeds, and this was unaffected by na, since the na mutation is not expressed in seeds. In contrast, lhⁱ seeds did not accumulate GA₂₀, since lhⁱ imposes an early block on GA biosynthesis. Secondly, the effects of sin on several steps in GA deactivation were investigated. In maturing seeds, the mutation sin blocks two steps in GA₂₀ metabolism, namely, GA₂₀ to GA₂₉, and GA₂₉ to GA₂₉-catabolite. In the vegetative plant, on the other hand, sin blocked the step GA₂₀ to GA₂₉, but not GA₂₉ to GA₂₉-catabolite; the steps GA₂₀ to GA₈₁ and GA₂₀ to GA₁ were also not impaired in this mutant. It is clear that the effects of sin, like those of na, are strongly organ-specific. The presence of separate enzymes for the steps GA₂₀ to GA₂₉ and GA₂₉ to GA₂₉-catabolite was suggested by the observation that GA₈ inhibited the latter step, but not the former, and by the inability of GA₂₀ and GA₂₉ to inhibit each other's metabolism. It is suggested that the Sin gene may be a regulatory gene controlling the expression of two structural genes involved in GA deactivation.     

Molecular genetic analysis of the response of three soil microbial communities to the application of 2, 4-D

The responses of three different soil microbial communities to the experimental application of 2, 4-dichlorophenoxyacetic acid (2, 4-D) were evaluated with a variety of molecular genetic techniques. Two of the three soil communities had histories of prior direct exposure to 2, 4-D, and one had no prior direct application of any herbicide. Dominant 2, 4-D degrading strains isolated from these soils the previous year were screened for hybridization with three catabolic genes (tfdA, tfdAII, and tfdB) cloned from the well-studied 2, 4-D degradative plasmid, pJP4, revealing varying degrees of similarity with the three genes. Hybridization of total community DNA from the three soils with the tfd gene probes also indicated that pJP4-like tfd genes were not harboured by a significant percentage of the community. Community level response was evaluated by the comparison of different treatments by Random Amplified Polymorphic DNA (RAPD) fingerprints and by community DNA cross-hybridization. No differences between treatments within the same soil were detected in any of the RAPD fingerprints generated with 17 primers. Community DNA cross-hybridization also indicated that the application of 2, 4-D at the applied rates did not quantitatively affect the structure of the soil microbial communities present in the three soils during the time-frame studied.     

Comparison of amide proton exchange in reduced and oxidizedRhodobacter capsulatus cytochrome c2: A1H−15N NMR study

Summary The hydrogen-deuterium exchange rates of the reduced and oxidized forms ofRhodobacter' capsulatus cytochrome c₂ were studied by¹H–¹⁵N homonuclear multiple quantum correlation spectroscopy. Minimal differences were observed for the N- and C-terminal helices on changing redox state suggesting that although these helices are structurally important they do not affect the relative stability of the two redox states and hence may not be important in determining the redox potential differences observed amongst the class I c-type cytochromes. However, significant differences were observed for other regions of the protein. For example, all slow exchanging protons of the helix spanning Phe⁸² to Asp⁸⁷ are similarly affected on reduction indicating that the unfolding equilibrium of this helix is altered between the two redox states. Other regions are not as simple to interpret; however, the difference in NH exchange rates between the redox states for a number of residues including His¹⁷, Leu³⁷, Arg⁴³, Ala⁴⁵, Gly⁴⁶, Ile⁵⁷, Val⁵⁸, Leu⁶⁰, Gly⁶¹ and Leu¹⁰⁰ suggest that interactions affecting the causes of these differences may be important factors in determining redox potential.Abbreviations NMR nuclear magnetic resonance - HMQC homonuclear multiple quantum correlation - NOESY nuclear Overhauser effect spectroscopy     

Pro----Ala-35 Rhodobacter capsulatus cytochrome c2 shows dynamic not structural differences. A 1H and 15N NMR study

Comparative analysis of nuclear Overhauser effects show that the time average conformation of the wild-type and mutant Pro----Ala-35 Rhodobacter capsulatus cytochrome c2 are indistinguishable. The ring resonances of Phe-51 and Tyr-53 show that their ring flip rates increase in P35A. NH proton exchange studies show that the exchange rates of the NH of Gly-34 and the NpiH of His-17 increase by approximately 10(2) in P35A suggesting that their respective hydrogen bonds are destabilized in this protein. However, 3JchiNH 1H and 15N chemical shift data argue that these bonds are intact. These data are compatible if the replacement of a Pro with an Ala residue forms a cavity or increases local flexibility thus reducing steric hinderance and increasing solvent accessibility.     

The XbaI-BlnI-CeuI genomic cleavage map of Salmonella enteritidis shows an inversion relative to Salmonella typhimurium LT2

We have established the genomic cleavage map of Salmonella enteritidis strain SSU7998 using pulsed-field gel electrophoresis. The chromosome of 4600kb was analysed by XbaI (16 fragments), I-CeuI (7 fragments) and BlnI (12 fragments); the genome also contains a plasmid of 60 kb. Cleavage sites of I-CeuI, in the large subunit ribosomal RNA gene, are conserved from Salmonella typhimurium and Escherichia coli K-12, and the XbaI and BinI sites in glt-tRNA are also conserved, but other sites are less conserved. Transposon Tn10, located at 60 different positions in the chromosome of S. typhimurium, was transduced by bacteriophage P22 into S. enteritidis and the insertion mapped using the XbaI and BlnI sites on Tn10. Gene order in S. enteritidis is identical to S. typhimurium LT2 and similar to E. coli K-12 except for an inversion of 815 kb, which covers the terminus region including T1 and T2. Endpoints are in the NDZs, or non-divisible zones, in which inversion endpoints were not detected in experiments in E. coli K-12 and S. typhimurium LT2. This inversion resembles the inversion between S. typhimurium and E. coli, but is longer at both ends.     

Macroevolutionary insights into sedges (Carex: Cyperaceae): The effects of rapid chromosome number evolution on lineage diversification

Changes in holocentric chromosome number due to fission and fusion have direct and immediate effects on genome structure and recombination rates. These, in turn, may influence ecology and evolutionary trajectories profoundly. Sedges of the genus Carex (Cyperaceae) comprise ca. 2000 species with holocentric chromosomes. The genus exhibits a phenomenal range in the chromosome number (2n = 10 − 132) with almost not polyploidy. In this study, we integrated the most comprehensive cytogenetic and phylogenetic data for sedges with associated climatic and morphological data to investigate the hypothesis that high recombination rates are selected when evolutionary innovation is required, using chromosome number evolution as a proxy for recombination rate. We evaluated Ornstein–Uhlenbeck models to infer shifts in chromosome number equilibrium and selective regime. We also tested the relationship between chromosome number and diversification rates. Our analyses demonstrate significant correlations between morphology and climatic niche and chromosome number in Carex. Nevertheless, the amount of chromosomal variation that we are able to explain is very small. We recognized a large number of shifts in mean chromosome number, but a significantly lower number in climatic niche and morphology. We also detected a peak in diversification rates near intermediate recombination rates. In combination, these analyses point toward the importance of chromosome evolution to the evolutionary history of Carex. Our work suggests that the effect of chromosome evolution on recombination rates, not just on reproductive isolation, may be central to the evolutionary history of sedges.     

Geographical vs. ecological diversification in Carex section Phacocystis (Cyperaceae): Patterns hidden behind a twisted taxonomy

Carex section Phacocystis (Cyperaceae) is one of the most diverse and taxonomically complex groups of sedges (between 116 and 147 species), with a worldwide distribution in a wide array of biomes. It has a very complicated taxonomic history, with numerous disagreements among different treatments. We studied the biogeography and niche evolution in a phylogenetic framework to unveil the relative contribution of geographical and ecological drivers to diversification of the group. We used a large species sampling of the section (82% of extant species) to build a phylogeny based on four DNA regions, constrained with a phylogenomic HybSeq tree and dated with six fossil calibrations. Our phylogenetic results recovered section Phacocystis s.s. (core Phacocystis) as sister to section Praelongae. Ancestral area reconstruction points toward the N Pacific as the cradle for the crown diversification of section Phacocystis during the Middle Miocene. Wide distributions were recurrently inferred across deep nodes. Large Northern Hemisphere lineages with geographical congruence were retrieved, pointing toward the importance of allopatric divergence at deep phylogenetic levels, whereas within-area speciation emerges as the predominant pattern at shallow phylogenetic level. The Southern Hemisphere (Neotropics, SW Pacific) was colonized several times from the Northern Hemisphere. The global expansion of Carex section Phacocystis did not entail major ecological changes along the inner branches of the phylogeny. Nevertheless, ecological differentiation seems to gain importance toward recent times.     

Identification and characterization of bi-thiazole-2,2′-diamines as kinase inhibitory scaffolds

Based on bioinformatics interrogation of the genome, > 500 mammalian protein kinases can be clustered within seven different groups. Of these kinases, the mitogen-activated protein kinase (MAPK) family forms part of the CMGC group of serine/threonine kinases that includes extracellular signal regulated kinases (ERKs), cJun N-terminal kinases (JNKs), and p38 MAPKs. With the JNKs considered attractive targets in the treatment of pathologies including diabetes and stroke, efforts have been directed to the discovery of new JNK inhibitory molecules that can be further developed as new therapeutics. Capitalizing on our biochemical understanding of JNK, we performed in silico screens of commercially available chemical databases to identify JNK1-interacting compounds and tested their in vitro JNK inhibitory activity. With in vitro and cell culture studies, we showed that the compound, 4′-methyl-N²-3-pyridinyl-4,5′-bi-1,3-thiazole-2,2′-diamine (JNK Docking (JD) compound 123, but not the related compound (4′-methyl-N ~ 2 ~ -(6-methyl-2-pyridinyl)-4,5′-bi-1,3-thiazole-2,2′-diamine (JD124), inhibited JNK1 activity towards a range of substrates. Molecular docking, saturation transfer difference NMR experiments and enzyme kinetic analyses revealed both ATP- and substrate-competitive inhibition of JNK by JD123. In characterizing JD123 further, we noted its ATP-competitive inhibition of the related p38-γ MAPK, but not ERK1, ERK2, or p38-α, p38-β or p38-δ. Further screening of a broad panel of kinases using 10 μM JD123, identified inhibition of kinases including protein kinase Bβ (PKBβ/Aktβ). Appropriately modified thiazole diamines, as typified by JD123, thus provide a new chemical scaffold for development of inhibitors for the JNK and p38-γ MAPKs as well as other kinases that are also potential therapeutic targets such as PKBβ/Aktβ.     

The Relaxin Receptor (RXFP1) Utilizes Hydrophobic Moieties on a Signaling Surface of Its N-terminal Low Density Lipoprotein Class A Module to Mediate Receptor Activation

The peptide hormone relaxin is showing potential as a treatment for acute heart failure. Although it is known that relaxin mediates its actions through the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1), little is known about the molecular mechanisms by which relaxin binding results in receptor activation. Previous studies have highlighted that the unique N-terminal low density lipoprotein class A (LDLa) module of RXFP1 is essential for receptor activation, and it has been hypothesized that this module is the true “ligand” of the receptor that directs the conformational changes necessary for G protein coupling. In this study, we confirmed that an RXFP1 receptor lacking the LDLa module binds ligand normally but cannot signal through any characterized G protein-coupled receptor signaling pathway. Furthermore, we comprehensively examined the contributions of amino acids in the LDLa module to RXFP1 activity using both gain-of-function and loss-of-function mutational analysis together with NMR structural analysis of recombinant LDLa modules. Gain-of-function studies with an inactive RXFP1 chimera containing the LDLa module of the human LDL receptor (LB2) demonstrated two key N-terminal regions of the module that were able to rescue receptor signaling. Loss-of-function mutations of residues in these regions demonstrated that Leu-7, Tyr-9, and Lys-17 all contributed to the ability of the LDLa module to drive receptor activation, and judicious amino acid substitutions suggested this involves hydrophobic interactions. Our results demonstrate that these key residues contribute to interactions driving the active receptor conformation, providing further evidence of a unique mode of G protein-coupled receptor activation.     

Proliferation-Linked Apoptosis of Adoptively Transferred T Cells after IL-15 Administration in Macaques

The adoptive transfer of antigen-specific effector T cells is being used to treat human infections and malignancy. T cell persistence is a prerequisite for therapeutic efficacy, but reliably establishing a high-level and durable T cell response by transferring cultured CD8⁺ T cells remains challenging. Thus, strategies that promote a transferred high-level T cell response may improve the efficacy of T cell therapy. Lymphodepletion enhances persistence of transferred T cells in mice in part by reducing competition for IL-15, a common γ-chain cytokine that promotes T cell memory, but lymphodepleting regimens have toxicity. IL-15 can be safely administered and has minimal effects on CD4⁺ regulatory T cells at low doses, making it an attractive adjunct in adoptive T cell therapy. Here, we show in lymphoreplete macaca nemestrina, that proliferation of adoptively transferred central memory-derived CD8⁺ effector T (T_CM/E) cells is enhanced in vivo by administering IL-15. T_CM/E cells migrated to memory niches, persisted, and acquired both central memory and effector memory phenotypes regardless of the cytokine treatment. Unexpectedly, despite maintaining T cell proliferation, IL-15 did not augment the magnitude of the transferred T cell response in blood, bone marrow, or lymph nodes. T cells induced to proliferate by IL-15 displayed increased apoptosis demonstrating that enhanced cycling was balanced by cell death. These results suggest that homeostatic mechanisms that regulate T cell numbers may interfere with strategies to augment a high-level T cell response by adoptive transfer of CD8⁺ T_CM/E cells in lymphoreplete hosts.