Yellow Fluorescent Protein Expression Research Articles

Identification of Intestinal Lamina Propria Plasma Cells by Surface Transmembrane Activator and CAML Interactor Expression.

Plasma cells secrete an abundance of Abs and are a crucial component of our immune system. The intestinal lamina propria harbors the largest population of plasma cells, most of which produce IgA. These Abs can bind to beneficial gut bacteria to reinforce intestinal homeostasis and provide protection against enteric pathogens. Plasma cells downregulate many cell-surface proteins commonly used to identify B cells. In mice, expression of the surface marker CD138 has been widely used to identify plasma cells in lymph nodes, bone marrow, and spleen. Intestinal plasma cells require liberation via extensive tissue processing involving treatment with collagenase. We report that detection of CD138 surface expression is reduced following collagenase treatment. Using a mouse in which yellow fluorescent protein expression is controlled by the plasma cell requisite transcription factor Blimp-1, we show that surface detection of transmembrane activator and CAML interactor captures a significant proportion of Ab-secreting plasma cells in the intestinal lamina propria and gut-draining mesenteric lymph nodes. Additionally, we describe a flow cytometry panel based on the detection of surface markers to identify murine B cell subsets in the intestinal lamina propria and, as a proof of concept, combine it with a cutting-edge fate-tracking system to characterize the fate of germinal center B cells activated in early life. By identifying plasma cells and other key intestinal B subsets in a manner compatible with several downstream applications, including sorting and culturing and invitro manipulations, this efficient and powerful approach can enhance studies of mucosal immunity.

Identification and characterization of sugar-regulated promoters in Chaetomium thermophilum

The thermophilic fungus Chaetomium thermophilum has been used extensively for biochemical and high-resolution structural studies of protein complexes. However, subsequent functional analyses of these assemblies have been hindered owing to the lack of genetic tools compatible with this thermophile, which are typically suited to other mesophilic eukaryotic model organisms, in particular the yeast Saccharomyces cerevisiae. Hence, we aimed to find genes from C. thermophilum that are expressed under the control of different sugars and examine their associated 5’ untranslated regions as promoters responsible for sugar-regulated gene expression. To identify sugar-regulated promoters in C. thermophilum, we performed comparative xylose- versus glucose-dependent gene expression studies, which uncovered a number of enzymes with induced expression in the presence of xylose but repressed expression in glucose-supplemented media. Subsequently, we cloned the promoters of the two most stringently regulated genes, the xylosidase-like gene (XYL) and xylitol dehydrogenase (XDH), obtained from this genome-wide analysis in front of a thermostable yellow fluorescent protein (YFP) reporter. With this, we demonstrated xylose-dependent YFP expression by both Western blotting and live-cell imaging fluorescence microscopy. Prompted by these results, we expressed the C. thermophilum orthologue of a well-characterized dominant-negative ribosome assembly factor mutant, under the control of the XDH promoter, which allowed us to induce a nuclear export defect on the pre-60S subunit when C. thermophilum cells were grown in xylose- but not glucose-containing medium. Altogether, our study identified xylose-regulatable promoters in C. thermophilum, which might facilitate functional studies of genes of interest in this thermophilic eukaryotic model organism.

Characterization of orexin input to dopamine neurons of the ventral tegmental area projecting to the medial prefrontal cortex and shell of nucleus accumbens

Orexin neurons are involved in homeostatic regulatory processes, including arousal and feeding, and provide a major input from the hypothalamus to the ventral tegmental area (VTA) of the midbrain. VTA neurons are a central hub processing reward and motivation and target the medial prefrontal cortex (mPFC) and the shell part of nucleus accumbens (NAcs). We investigated whether subpopulations of dopamine (DA) neurons in the VTA projecting either to the mPFC or the medial division of shell part of nucleus accumbens (mNAcs) receive differential input from orexin neurons and whether orexin exerts differential electrophysiological effects upon these cells. VTA neurons projecting to the mPFC or the mNAcs were traced retrogradely by Cav2-Cre virus and identified by expression of yellow fluorescent protein (YFP). Immunocytochemical analysis showed that a higher proportion of all orexin-innervated DA neurons projected to the mNAcs (34.5%) than to the mPFC (5.2%). Of all sampled VTA neurons projecting either to the mPFC or mNAcs, the dopaminergic (68.3 vs. 79.6%) and orexin-innervated DA neurons (68.9 vs. 64.4%) represented the major phenotype. Whole-cell current clamp recordings were obtained from fluorescently labeled neurons in slices during baseline periods and bath application of orexin A. Orexin similarly increased the firing rate of VTA dopamine neurons projecting to mNAcs (1.99 ± 0.61 Hz to 2.53 ± 0.72 Hz) and mPFC (0.40 ± 0.22 Hz to 1.45 ± 0.56 Hz). Thus, the hypothalamic orexin system targets mNAcs and to a lesser extent mPFC-projecting dopaminergic neurons of the VTA and exerts facilitatory effects on both clusters of dopamine neurons.

Novel cis-regulatory elements as synthetic promoters to drive recombinant protein expression from the Chlamydomonas reinhardtii nuclear genome

Eukaryotic green microalgae represent a sustainable, photosynthetic biotechnology platform for generating high-value products. The model green alga Chlamydomonas reinhardtii has already been used to generate high value bioproducts such as recombinant proteins and terpenoids. However, low, unstable, and variable nuclear transgene expression has limited the ease and speed of metabolic engineering and recombinant protein expression in this system. Here, novel genetic devices for transgene expression in C. reinhardtii have been developed by identifying cis-regulatory DNA elements capable of driving high transgene expression in C. reinhardtii promoters using de novo motif discovery informatics approaches. Thirteen putative motifs were synthesized as concatemers, linked to a common minimal basal promoter, and assayed for their activity to drive expression of a yellow fluorescent protein reporter gene. Following transformation of the vectors into C. reinhardtii by electroporation, in vivo measurements of yellow fluorescent protein expression by flow cytometry revealed that five of the DNA motifs analyzed displayed significantly higher reporter expression compared to the basal promoter control. Two of the concatemerized motifs, despite being much smaller minimal cis-regulatory elements, drove reporter expression at levels approaching that of the conventionally-used AR1 promoter. This analysis provides insight into C. reinhardtii promoter structure and gene regulation, and provides a new toolbox of cis-regulatory elements that can be used to drive transgene expression at a variety of expression levels.

A novel distal convoluted tubule-specific Cre-recombinase driven by the NaCl cotransporter gene.

Cre-lox technology has revolutionized research in renal physiology by allowing site-specific genetic recombination in individual nephron segments. The distal convoluted tubule (DCT), consisting of distinct early (DCT1) and late (DCT2) segments, plays a central role in Na+ and K+ homeostasis. The only established Cre line targeting the DCT is Pvalb-Cre, which is limited by noninducibility, activity along DCT1 only, and activity in neurons. Here, we report the characterization of the first Cre line specific to the entire DCT. CRISPR/Cas9 targeting was used to introduce a tamoxifen-inducible IRES-Cre-ERT2 cassette downstream of the coding region of the Slc12a3 gene encoding the NaCl cotransporter (NCC). The resulting Slc12a3-Cre-ERT2 mice were crossed with R26R-YFP reporter mice, which revealed minimal leakiness with 6.3% of NCC-positive cells expressing yellow fluorescent protein (YFP) in the absence of tamoxifen. After tamoxifen injection, YFP expression was observed in 91.2% of NCC-positive cells and only in NCC-positive cells, revealing high recombination efficiency and DCT specificity. Crossing to R26R-TdTomato mice revealed higher leakiness (64.5%), suggesting differential sensitivity of the floxed site. Western blot analysis revealed no differences in abundances of total NCC or the active phosphorylated form of NCC in Slc12a3-Cre-ERT2 mice of either sex compared with controls. Plasma K+ and Mg2+ concentrations and thiazide-sensitive Na+ and K+ excretion did not differ in Slc12a3-Cre-ERT2 mice compared with controls when sex matched. These data suggest genetic modification had no obvious effect on NCC function. Slc12a3-Cre-ERT2 mice are the first line generated demonstrating inducible Cre recombinase activity along the entire DCT and will be a useful tool to study DCT function.

Abstract WP135: Engineered Exosomes With Elevated Mir-27a Improve Neurological Outcome in Ischemic Mice

Background: Augmentation of axonal growth enhances improvement of neurological function after stroke. Our previous in vitro study demonstrated that miR-27a promotes axonal growth of embryonic neurons. The present in vivo study investigated whether exosomes carrying elevated miR-27a (27a-exos) enhance axonal growth and neurological outcome after stroke. Methods and Results: The 27a-exos were isolated from medium of mesenchymal stromal cells (MSCs) transfected with lenti-miR-27 vector. In order to investigate axonal growth and underlying mechanisms, Emx1-Thy1-YFP mice with selective expression of yellow fluorescent protein (YFP) in pyramidal neurons were subjected to the permanent middle cerebral artery occlusion (MCAO). Quantitative RT-PCR analysis showed that compared with exosomes derived from empty-vector transfected MSCs (empty-exos), 27a-exos had 3.7 fold increased miR-27a. 27a-exos (3x10 11 particles) were injected intravenously into mice at 24h after MCAO. Compared with control mice, the empty-exos (n=7) significantly (p<0.05) reduced the foot-fault (5±0.1 vs 11±0.5 in control) and the times to remove adhesive paper (seconds, 47±3 vs 70±4). However, compared with empty-exos, 27a-exos (n=7) further significantly (p<0.05) reduced foot-fault (3.0±0.2) and times to remove the paper (38±1.7). Histopathological and in situ hybridization analysis showed that 27a-exos localized to neurons and significantly (p<0.05) increased miR-27a in Emx1-YFP+ neurons (4±0.7 vs 1 in empty-exos), and augmented (p<0.05) the density of YFP+/pNFH+ axons (7±2 vs 1 in empty-exos). Moreover, 27a-exos substantially decreased the levels of the inhibitory protein Sema6a in the YFP+ axons in peri-infarct areas. Conclusion: Our data suggest that tailored 27a-exos augment the therapeutic effect of exosomes on stroke recovery and that suppression of axonal inhibitory proteins such as Sema6a may contribute to the 27a-exo-enhanced axonal outgrowth to promote neurological recovery post stroke.

Transition of Macrophages to Fibroblast-Like Cells in Healing Myocardial Infarction

BackgroundMacrophages and fibroblasts are 2 major cell types involved in healing after myocardial infarction (MI), contributing to myocardial remodeling and fibrosis. Post-MI fibrosis progression is characterized by a decrease in cardiac macrophage content. ObjectivesThis study explores the potential of macrophages to express fibroblast genes and the direct role of these cells in post-MI cardiac fibrosis. MethodsProlonged in vitro culture of human macrophages was used to evaluate the capacity to express fibroblast markers. Infiltrating cardiac macrophages was tracked in vivo after experimental MI of LysM(Cre/+);ROSA26(EYFP/+) transgenic mice. The expression of Yellow Fluorescent Protein (YFP) in these animals is restricted to myeloid lineage allowing the identification of macrophage-derived fibroblasts. The expression in YFP-positive cells of fibroblast markers was determined in myocardial tissue sections of hearts from these mice after MI. ResultsExpression of the fibroblast markers type I collagen, prolyl-4-hydroxylase, fibroblast specific protein-1, and fibroblast activation protein was evidenced in YFP-positive cells in the heart after MI. The presence of fibroblasts after MI was evaluated in the hearts of animals after depletion of macrophages with clodronate liposomes. This macrophage depletion significantly reduced the number of Mac3+Col1A1+ cells in the heart after MI. ConclusionsThe data provide both in vitro and in vivo evidence for the ability of macrophages to transition and adopt a fibroblast-like phenotype. Therapeutic manipulation of this macrophage-fibroblast transition may hold promise for favorably modulating the fibrotic response after MI and after other cardiovascular pathological processes.

TMIC-08. IDENTIFICATION OF TUMOR SPECIFIC LYMPHOCYTES IN A MURINE GLIOMA MODEL

Abstract INTRODUCTION One major challenge in the use of immunotherapy for glioblastoma is the identification of tumor specific lymphocytes within the tumor microenvironment. Previous studies in non-glioma cancer models have identified CD39 and PD1 as lymphocyte markers of tumor specificity. In this study, we demonstrate the existence of this specific T cell phenotype and show correlation with anti-tumor activity. METHODS We implanted KR158B murine glioma cells into right caudate nucleus into yellow fluorescent protein (YFP) interferon-gamma reporter mice for a total of 104 cells using stereotaxis. At 4 weeks, mice were sacrificed followed by organ harvest. Samples taken included tumor, cervical draining lymph nodes, spleen, and peripheral blood. These were processed into single cell suspensions and then stained for CD3, CD8, CD39, and PD1. Flow cytometry was performed on a BD FACSCanto II. Gating analysis was performed with FlowJo while data analysis was performed with GraphPad Prism. RESULTS Gating for CD3+ CD8+ on tumor lymphocytes reveals a significant population of CD39 and PD1 double positive (DP) cells (61.4%) not found in the peripheral organs (< 1.0%). Evaluation of these cells for YFP expression demonstrated significantly greater median fluorescence intensity (MFI) in the DP group as opposed to either of the single positive (SP) or double negative (DN) groups (DP = 225.4, SP-CD39 = 40.54, SP-PD1 = 82.7, DN = 29.5, p < 0.0001). CONCLUSION Here we demonstrate the existence of a unique phenotype of CD39+ and PD1+ expression on lymphocytes within the tumor microenvironment. These DP cells also had high levels of interferon gamma production compared SP and DN groups. This phenotype is not observed in peripheral circulating lymphocytes. Enrichment for this subpopulation will allow for delivery of purified lymphocyte-based immunotherapy in this murine glioma model.

Migratory and Dancing Macrophage Subsets in Atherosclerotic Lesions.

Macrophages are essential regulators of atherosclerosis. They secrete cytokines, process lipoproteins and cholesterol, and take up apoptotic cells. Multiple subsets of plaque macrophages exist and their differential roles are emerging. Here, we explore macrophage heterogeneity in atherosclerosis plaques using transgenic fluorescent mice in which subsets of macrophages are labeled by GFP (green fluorescent protein), YFP (yellow fluorescent protein), neither, or both. The objective was to define migration patterns of the visible subsets and relate them to their phenotypes and transcriptomes. Apoe-/-Cx3cr1GFPCd11cYFP mice have 4 groups of macrophages in their aortas. The 3 visible subsets show varying movement characteristics. GFP and GFP+YFP+ macrophages extend and retract dendritic processes, dancing on the spot with little net movement while YFP macrophages have a more rounded shape and migrate along the arteries. RNA sequencing of sorted cells revealed significant differences in the gene expression patterns of the 4 subsets defined by GFP and YFP expression, especially concerning chemokine and cytokine expression, matrix remodeling, and cell shape dynamics. Gene set enrichment analysis showed that GFP+ cells have similar transcriptomes to cells found in arteries with tertiary lymphoid organs and regressing plaques while YFP+ cells were associated with progressing and stable plaques. The combination of quantitative intravital imaging with deep transcriptomes identified 4 subsets of vascular macrophages in atherosclerosis that have unique transcriptomic profiles. Our data link vascular macrophage transcriptomes to their in vivo migratory function. Future work on the functional significance of the change in gene expression and motility characteristics will be needed to fully understand how these subsets contribute to disease progression.

Identification of Tumor Specific Lymphocytes Cells in a Murine Glioma Model

Abstract INTRODUCTION One major challenge in the use of immunotherapy for glioblastoma is the identification of tumor specific lymphocytes within the tumor microenvironment. Previous studies in nonglioma cancer models have identified CD39 and PD1 as lymphocyte markers of tumor specificity. In this study, we demonstrate the existence of this specific T cell phenotype and show correlation with antitumor activity. METHODS We implanted KR158B murine glioma cells into right caudate nucleus into yellow fluorescent protein (YFP) interferon-gamma reporter mice for a total of 104 cells using stereotaxis. At 4 wk, mice were sacrificed followed by organ harvest. Samples taken included tumor, cervical draining lymph nodes, spleen, and peripheral blood. These were processed into single cell suspensions and then stained for CD3, CD8, CD39, and PD1. Flow cytometry was performed on a BD Canto II. Gating analysis was performed with FlowJo while data analysis was performed with GraphPad Prism. RESULTS Gating for CD3+ CD8+ on tumor lymphocytes reveals a significant population of CD39 and PD1 double positive (DP) cells (61.4%) not found in the peripheral organs (<1.0%). Evaluation of these cells for YFP expression demonstrated significantly greater median fluorescence intensity (MFI) in the DP group as opposed to either of the single positive (SP) or double negative (DN) groups (DP = 225.4, SP-CD39 = 40.54, SP-PD1 = 82.7, DN = 29.5, P < .001). CONCLUSION Here we demonstrate the existence of a unique phenotype of CD39+ and PD1+ expression on lymphocytes within the tumor microenvironment. These DP cells also had high levels of interferon gamma production compared SP and DN groups. This phenotype is not observed in peripheral circulating lymphocytes. Enrichment for this subpopulation will allow for delivery of purified lymphocyte-based immunotherapy in this murine glioma model.

The NT11, a novel fusion tag for enhancing protein expression in Escherichia coli.

The Escherichia coli (E. coli) expression system has been widely used to produce recombinant proteins. However, in some heterologous expressions, there are still difficulties in large-scale production. The use of fusion partners is one of the strategies for improving the expression levels of proteins in E. coli host. Here, we demonstrate a novel fusion element, the NT11-tag, which enhances protein expression. The NT11-tag was derived from the first 11 amino acid residues within the N-terminal N-half domain of a duplicated carbonic anhydrase (dCA) from Dunaliella species. Previously, we have found that the tag improves expression of the C-half domain of dCA when linked to its N-terminus. To verify its use as a protein production enhancer tag, two kinds of CAs derived from Hahella chejuensis (Hc-CA) and Thermovibrio ammonifican (Ta-CA) and the yellow fluorescent protein (YFP) were used as model proteins to measure their increased expression upon fusion with the NT11-tag. The NT11-tag amplified protein expression in E. coli by 6.9- and 7.6-fold for Ta-CA and YFP, respectively. Moreover, the tag also enhanced the soluble expression of Hc-CA, Ta-CA, and YFP by 1.7-, 5.0-, and 3.2-fold, respectively. Furthermore, protein yield was increased without inhibiting protein function. These results indicate that the use of the NT11-tag is a promising method for improving protein production in E. coli.

Direct comparison of the abilities of bone marrow mesenchymal versus hematopoietic stem cells to reverse hyperglycemia in diabetic NOD.SCID mice

ABSTRACTBoth bone marrow-derived hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC) improve glycemic control in diabetic mice, but their kinetics and associated changes in pancreatic morphology have not been directly compared. Our goal was to examine the time course of improvements in glucose tolerance and associated changes in β-cell mass and proliferation following transplantation of equivalent numbers of HSC or MSC from the same bone marrow into diabetic non-obese diabetic severe combined immune deficiency (NOD.SCID) mice. We used transgenic mice with a targeted expression of yellow fluorescent protein (YFP) driven by the Vav1 gene promoter to genetically tag HSC and progeny. HSC were separated from bone marrow by fluorescence-activated cell sorting and MSC following cell culture. Equivalent numbers of isolated HSC or MSC were transplanted directly into the pancreas of NOD.SCID mice previously made diabetic with streptozotocin. Glucose tolerance, serum insulin, β-cell mass and β-cell proliferation were examined up to 28 days following transplant. Transplantation with MSC improved glucose tolerance within 7 days and serum insulin levels increased, but with no increase in β-cell mass. Mice transplanted with HSC showed improved glucose tolerance only after 3 weeks associated with increased β-cell proliferation and mass. We conclude that single injections of either MSC or HSC transiently improved glycemic control in diabetic NOD.SCID mice, but with different time courses. However, only HSC infiltrated the islets and were associated with an expanded β-cell mass. This suggests that MSC and HSC have differing mechanisms of action.

Ischemic stroke activates the VE-cadherin promoter and increases VE-cadherin expression in adult mice.

Endothelial cells (ECs) are a key component of the blood-brain barrier (BBB). Healthy ECs in the BBB form inter-endothelial junctions, including adherens junctions (AJs). Under pathological conditions, such as after ischemic stroke, the BBB may be functionally compromised. However, gene and protein expression patterns involving endothelial AJs have not been well studied. Because expression levels of endothelial AJs are considered to be related to BBB functionality, we investigated the expression pattern of a representative endothelial AJ marker, VE-cadherin, in healthy and diseased mice. We first examined the expression of VE-cadherin in developing mouse brains. In addition, to a mouse model of cerebral infarction, we investigated the expression pattern of VE-cadherin in pathologic brains. Furthermore, using the Cre-LoxP system, we established a strain of mice expressing yellow fluorescent protein (YFP) under the control of the VE-cadherin promoter and investigated the expression pattern of YFP-expressing ECs in developing and pathologic murine brains. VE-cadherin protein and YFP expression driven by the VE-cadherin promoter both showed that VE-cadherin expression was weak during embryonic stages, followed by a steady increase postnatally, which then decreased during adulthood. However, following ischemic stroke, imunohistochemistry of VE-cadherin demonstrated an upregulation in ECs within ischemic regions, concomitant with YFP upregulation. These findings reveal that ischemic stroke activates the VE-cadherin promoter and increases VE-cadherin protein expression, which suggests that endothelial VE-cadherin is involved in the reconstruction of the BBB following ischemic stroke.

Vitamin K3 Induces the Expression of the Stenotrophomonas maltophilia SmeVWX Multidrug Efflux Pump.

Stenotrophomonas maltophilia is an opportunistic pathogen with increasing prevalence, which is able to cause infections in immunocompromised patients or in those with a previous pathology. The treatment of the infections caused by this bacterium is often complicated due to the several intrinsic antibiotic resistance mechanisms that it presents. Multidrug efflux pumps are among the best-studied mechanisms of S. maltophilia antibiotic resistance. Some of these efflux pumps have a basal expression level but, in general, their expression is often low and only reaches high levels when the local regulator is mutated or bacteria are in the presence of an effector. In the current work, we have developed a yellow fluorescent protein (YFP)-based sensor with the aim to identify effectors able to trigger the expression of SmeVWX, an efflux pump that confers resistance to quinolones, chloramphenicol, and tetracycline when it is expressed at high levels. With this purpose in mind, we tested a variety of different compounds and analyzed the fluorescence signal given by the expression of YFP under the control of the smeVWX promoter. Among the tested compounds, vitamin K3, which is a compound belonging to the 2-methyl-1,4-naphthoquinone family, is produced by plants in defense against infection, and has increasing importance in human therapy, was able to induce the expression of the SmeVWX efflux pump. In addition, a decrease in the susceptibility of S. maltophilia to ofloxacin and chloramphenicol was observed in the presence of vitamin K3, in both wild-type and smeW-deficient strains.

Zn2+-Inducible Expression Platform for Synechococcus sp. Strain PCC 7002 Based on the smtA Promoter/Operator and smtB Repressor.

This is the first metal-responsive expression system in cyanobacteria, to our knowledge, that does not exhibit low sensitivity for induction, which is one of the major hurdles for utilizing this class of genetic tools. In addition, high levels of expression can be generated that approximate those of established constitutive systems, with the added advantage of titratable control. Together, these properties establish this Zn2+-inducible system, which is based on the smtA7942 operator/promoter and smtB7942 repressor, as a versatile gene expression platform that expands the genetic toolbox of Synechococcus sp. strain PCC 7002.

Identification and Regulation of Genes for Cobalamin Transport in the Cyanobacterium Synechococcus sp. Strain PCC 7002.

The cyanobacterium Synechococcus sp. strain PCC 7002 is a cobalamin auxotroph and utilizes this coenzyme solely for the synthesis of l-methionine by methionine synthase (MetH). Synechococcus sp. strain PCC 7002 is unable to synthesize cobalamin de novo, and because of the large size of this tetrapyrrole, an active-transport system must exist for cobalamin uptake. Surprisingly, no cobalamin transport system was identified in the initial annotation of the genome of this organism. With more sophisticated in silico prediction tools, a btuB-cpdA-btuC-btuF operon encoding components putatively required for a B12 uptake (btu) system was identified. The expression of these genes was predicted to be controlled by a cobalamin riboswitch. Global transcriptional profiling by high-throughput RNA sequencing of a cobalamin-independent form of Synechococcus sp. strain PCC 7002 grown in the absence or presence of cobalamin confirmed regulation of the btu operon by cobalamin. Pérez et al. (A. A. Pérez, Z. Liu, D. A. Rodionov, Z. Li, and D. A. Bryant, J Bacteriol 198:2743-2752, 2016, http://dx.doi.org/10.1128/JB.00475-16) developed a cobalamin-dependent yellow fluorescent protein reporter system in a Synechococcus sp. strain PCC 7002 variant that had been genetically modified to allow cobalamin-independent growth. This reporter system was exploited to validate components of the btu uptake system by assessing the ability of targeted mutants to transport cobalamin. The btuB promoter and a variant counterpart mutated in an essential element of the predicted cobalamin riboswitch were fused to a yfp reporter. The combined data indicate that the btuB-cpdA-btuF-btuC operon in this cyanobacterium is transcriptionally regulated by a cobalamin riboswitch. With a cobalamin-regulated reporter system for expression of yellow fluorescent protein, genes previously misidentified as encoding subunits of a siderophore transporter were shown to encode components of cobalamin uptake in the cyanobacterium Synechococcus sp. strain PCC 7002. This study demonstrates the importance of experimental validation of in silico predictions and provides a general scheme for in vivo verification of similar cobalamin transport systems. A putative cobalamin riboswitch was identified in Synechococcus sp. strain PCC 7002. This riboswitch acts as a potential transcriptional attenuator of the btu operon that encodes the components of the cobalamin active-transport system.

A proton current associated with sour taste: distribution and functional properties.

Sour taste is detected by taste receptor cells that respond to acids through yet poorly understood mechanisms. The cells that detect sour express the protein PKD2L1, which is not the sour receptor but nonetheless serves as a useful marker for sour cells. By use of mice in which the PKD2L1 promoter drives expression of yellow fluorescent protein, we previously reported that sour taste cells from circumvallate papillae in the posterior tongue express a proton current. To establish a correlation between this current and sour transduction, we examined its distribution by patch-clamp recording. We find that the current is present in PKD2L1-expressing taste cells from mouse circumvallate, foliate, and fungiform papillae but not in a variety of other cells, including spinal cord neurons that express PKD2L1. We describe biophysical properties of the current, including pH-dependent Zn(2+) inhibition, lack of voltage-dependent gating, and activation at modest pH values (6.5) that elicit action potentials in isolated cells. Consistent with a channel that is constitutively open, the cytosol of sour taste cells is acidified. These data define a functional signature for the taste cell proton current and indicate that its expression is mostly restricted to the subset of taste cells that detect sour.

Thy1.2 YFP-16 transgenic mouse labels a subset of large-diameter sensory neurons that lack TRPV1 expression.

The Thy1.2 YFP-16 mouse expresses yellow fluorescent protein (YFP) in specific subsets of peripheral and central neurons. The original characterization of this model suggested that YFP was expressed in all sensory neurons, and this model has been subsequently used to study sensory nerve structure and function. Here, we have characterized the expression of YFP in the sensory ganglia (DRG, trigeminal and vagal) of the Thy1.2 YFP-16 mouse, using biochemical, functional and anatomical analyses. Despite previous reports, we found that YFP was only expressed in approximately half of DRG and trigeminal neurons and less than 10% of vagal neurons. YFP-expression was only found in medium and large-diameter neurons that expressed neurofilament but not TRPV1. YFP-expressing neurons failed to respond to selective agonists for TRPV1, P2X2/3 and TRPM8 channels in Ca2+ imaging assays. Confocal analysis of glabrous skin, hairy skin of the back and ear and skeletal muscle indicated that YFP was expressed in some peripheral terminals with structures consistent with their presumed non-nociceptive nature. In summary, the Thy1.2 YFP-16 mouse expresses robust YFP expression in only a subset of sensory neurons. But this mouse model is not suitable for the study of nociceptive nerves or the function of such nerves in pain and neuropathies.

Enteric plexuses of two choline-acetyltransferase transgenic mouse lines: chemical neuroanatomy of the fluorescent protein-expressing nerve cells.

We studied cholinergic circuit elements in the enteric nervous system (ENS) of two distinct transgenic mouse lines in which fluorescent protein expression was driven by the choline-acetyltransferase (ChAT) promoter. In the first mouse line, green fluorescent protein was fused to the tau gene. This construct allowed the visualization of the fiber tracts and ganglia, however the nerve cells were poorly resolved. In the second mouse line (ChATcre-YFP), CRE/loxP recombination yielded cytosolic expression of yellow fluorescent protein (YFP). In these preparations the morphology of enteric neurons could be well studied. We also determined the neurochemical identity of ENS neurons in muscular and submucous layers using antibodies against YFP, calretinin (CALR), calbindin (CALB), and vasoactive intestinal peptide (VIP). Confocal microscopic imaging was used to visualize fluorescently-conjugated secondary antibodies. In ChATcre-YFP preparations, YFP was readily apparent in somatodendritic regions of ENS neurons. In the myenteric plexus, YFP/CALR/VIP staining revealed that 34% of cholinergic cells co-labeled with CALR. Few single-stained CR-positive cells were observed. Neither YFP nor CALR co-localized with VIP. In GFP/CALB/CALR staining, all co-localization combinations were represented. In the submucosal plexus, YFP/CALR/VIP staining revealed discrete neuronal populations. However, in separate preparations, double labeling was observed for YFP/CALR and CALR/VIP. In YFP/CALR/CALB staining, all combinations of double staining and triple labeling were verified. In conclusion, the neurochemical coding of ENS neurons in these mouse lines is consistent with many observations in non-transgenic animals. Thus, they provide useful tools for physiological and pharmacological studies on distinct neurochemical subtypes of ENS neurons.

Traumatic brain injury-induced axonal phenotypes react differently to treatment.

Injured axons with distinct morphologies have been found following mild traumatic brain injury (mTBI), although it is currently unclear whether they reflect varied responses to the injury or represent different stages of progressing pathology. This complicates evaluation of therapeutic interventions targeting axonal injury. To address this issue, we assessed axonal injury over time within a well-defined axonal population, while also evaluating mitochondrial permeability transition as a therapeutic target. We utilized mice expressing yellow fluorescent protein (YFP) in cortical neurons which were crossed with mice which lacked Cyclophilin D (CypD), a positive regulator of mitochondrial permeability transition pore opening. Their offspring were subjected to mTBI and the ensuing axonal injury was assessed using YFP expression and amyloid precursor protein (APP) immunohistochemistry, visualized by confocal and electron microscopy. YFP(+) axons initially developed a single, APP(+), focal swelling (proximal bulb) which progressed to axotomy. Disconnected axonal segments developed either a single bulb (distal bulb) or multiple bulbs (varicosities), which were APP(-) and whose ultrastructure was consistent with ongoing Wallerian degeneration. CypD knock-out failed to reduce proximal bulb formation but decreased the number of distal bulbs and varicosities, as well as a population of small, APP(+), callosal bulbs not associated with YFP(+) axons. The observation that YFP(+) axons contain several pathological morphologies points to the complexity of traumatic axonal injury. The fact that CypD knock-out reduced some, but not all, subtypes highlights the need to appropriately characterize injured axons when evaluating potential neuroprotective strategies.