The zebrafish <Emphasis Type="Italic">lysozyme C</Emphasis> promoter drives myeloid-specific expression in transgenic fish

Background  

How different immune cell compartments contribute to a successful immune response is central to fully understanding the mechanisms behind normal processes such as tissue repair and the pathology of inflammatory diseases. However, the ability to observe and characterize such interactions, in real-time, within a living vertebrate has proved elusive. Recently, the zebrafish has been exploited to model aspects of human disease and to study specific immune cell compartments using fluorescent reporter transgenic lines. A number of blood-specific lines have provided a means to exploit the exquisite optical clarity that this vertebrate system offers and provide a level of insight into dynamic inflammatory processes previously unavailable.     

Characterization of the specific DNA-binding properties of Tnp26, the transposase of insertion sequence IS26

The bacterial insertion sequence (IS) IS26 mobilizes and disseminates antibiotic resistance genes. It differs from bacterial IS that have been studied to date as it exclusively forms cointegrates via either a copy-in (replicative) or a recently discovered targeted conservative mode. To investigate how the Tnp26 transposase recognizes the 14-bp terminal inverted repeats (TIRs) that bound the IS, amino acids in two domains in the N-terminal (amino acids M1–P56) region were replaced. These changes substantially reduced cointegration in both modes. Tnp26 was purified as a maltose-binding fusion protein and shown to bind specifically to dsDNA fragments that included an IS26 TIR. However, Tnp26 with an R49A or a W50A substitution in helix 3 of a predicted trihelical helix–turn–helix domain (amino acids I13–R53) or an F4A or F9A substitution replacing the conserved amino acids in a unique disordered N-terminal domain (amino acids M1–D12) did not bind. The N-terminal M1–P56 fragment also bound to the TIR but only at substantially higher concentrations, indicating that other parts of Tnp26 enhance the binding affinity. The binding site was confined to the internal part of the TIR, and a G to T nucleotide substitution in the TGT at positions 6 to 8 of the TIR that is conserved in most IS26 family members abolished binding of both Tnp26 (M1–M234) and Tnp26 M1–P56 fragment. These findings indicate that the helix–turn–helix and disordered domains of Tnp26 play a role in Tnp26–TIR complex formation. Both domains are conserved in all members of the IS26 family.     

Regulation of mitochondrial fission by GIPC-mediated Drp1 retrograde transport

Dynamin-related protein 1 (Drp1) is a key regulator of mitochondrial fission, a large cytoplasmic GTPase recruited to the mitochondrial surface via transmembrane adaptors to initiate scission. While Brownian motion likely accounts for the local interactions between Drp1 and the mitochondrial adaptors, how this essential enzyme is targeted from more distal regions like the cell periphery remains unknown. Based on proteomic interactome screening and cell-based studies, we report that GAIP/RGS19-interacting protein (GIPC) mediates the actin-based retrograde transport of Drp1 toward the perinuclear mitochondria to enhance fission. Drp1 interacts with GIPC through its atypical C-terminal PDZ-binding motif. Loss of this interaction abrogates Drp1 retrograde transport resulting in cytoplasmic mislocalization and reduced fission despite retaining normal intrinsic GTPase activity. Functionally, we demonstrate that GIPC potentiates the Drp1-driven proliferative and migratory capacity in cancer cells. Together, these findings establish a direct molecular link between altered GIPC expression and Drp1 function in cancer progression and metabolic disorders.     

Camera traps are an effective tool for monitoring insect–plant interactions

Insect and pollinator populations are vitally important to the health of ecosystems, food production, and economic stability, but are declining worldwide. New, cheap, and simple monitoring methods are necessary to inform management actions and should be available to researchers around the world. Here, we evaluate the efficacy of a commercially available, close‐focus automated camera trap to monitor insect–plant interactions and insect behavior. We compared two video settings—scheduled and motion‐activated—to a traditional human observation method. Our results show that camera traps with scheduled video settings detected more insects overall than humans, but relative performance varied by insect order. Scheduled cameras significantly outperformed motion‐activated cameras, detecting more insects of all orders and size classes. We conclude that scheduled camera traps are an effective and relatively inexpensive tool for monitoring interactions between plants and insects of all size classes, and their ease of accessibility and set‐up allows for the potential of widespread use. The digital format of video also offers the benefits of recording, sharing, and verifying observations.     

Increase in Alfalfa Nodulation, Nitrogen Fixation, and Plant Growth by Specific DNA Amplification in Sinorhizobium meliloti

To improve symbiotic nitrogen fixation on alfalfa plants, Sinorhizobium meliloti strains containing different average copy numbers of a symbiotic DNA region were constructed by specific DNA amplification (SDA). A DNA fragment containing a regulatory gene (nodD1), the common nodulation genes (nodABC), and an operon essential for nitrogen fixation (nifN) from the nod regulon region of the symbiotic plasmid pSyma of S. meliloti was cloned into a plasmid unable to replicate in this organism. The plasmid then was integrated into the homologous DNA region of S. meliloti strains 41 and 1021, which resulted in a duplication of the symbiotic region. Sinorhizobium derivatives carrying further amplification were selected by growing the bacteria in increased concentrations of an antibiotic marker present in the integrated vector. Derivatives of strain 41 containing averages of 3 and 6 copies and a derivative of strain 1021 containing an average of 2.5 copies of the symbiotic region were obtained. In addition, the same region was introduced into both strains as a multicopy plasmid, yielding derivatives with an average of seven copies per cell. Nodulation, nitrogenase activity, plant nitrogen content, and plant growth were analyzed in alfalfa plants inoculated with the different strains. The copy number of the symbiotic region was critical in determining the plant phenotype. In the case of the strains with a moderate increase in copy number, symbiotic properties were improved significantly. The inoculation of alfalfa with these strains resulted in an enhancement of plant growth.     

Diacylglycerol kinase inhibition prevents IL-2-induced G1 to S transition through a phosphatidylinositol-3 kinase-independent mechanism.

Stimulation via IL-2R ligation causes T lymphocytes to transit through the cell cycle. Previous experiments by our group have demonstrated that, in human T cells, IL-2 binding induces phosphatidic acid production through activation of the alpha isoform of diacylglycerol kinase. In this study, using the IL-2-dependent mouse T cell line CTLL-2, we demonstrate that pharmacological inhibition of IL-2-induced diacylglycerol kinase activation is found to block IL-2-induced late G1 to S transition without affecting cell viability. Herein, we demonstrate that diacylglycerol kinase inhibition has a profound effect on the induction of the protooncogenes c-myc, c-fos, and c-raf by IL-2, whereas expression of bcl-2 and bcl-xL are not affected. When the IL-2-regulated cell cycle control checkpoints are examined in detail, we demonstrate that inhibition of diacylglycerol kinase activation prevents IL-2 induction of cyclin D3 without affecting p27 down-regulation. The strict control of cell proliferation exerted by phosphatidic acid through activation of diacylglycerol kinase is independent of other well-characterized IL-2R-derived signals, such as the phosphatidylinositol-3 kinase/Akt pathway, indicating the existence of a different and important mechanism to control cell division.