Targeted Disruption of the Intracellular Domain of Receptor FgfrL1 in Mice

FgfrL1 is the fifth member of the fibroblast growth factor receptor (Fgfr) family. Studies with FgfrL1 deficient mice have demonstrated that the gene plays an important role during embryonic development. FgfrL1 knock-out mice die at birth as they have a malformed diaphragm and lack metanephric kidneys. Similar to the classical Fgfrs, the FgfrL1 protein contains an extracellular part composed of three Ig-like domains that interact with Fgf ligands and heparin. However, the intracellular part of FgfrL1 is not related to the classical receptors and does not possess any tyrosine kinase activity. Curiously enough, the amino acid sequence of this domain is barely conserved among different species, with the exception of three motifs, namely a dileucine peptide, a tandem tyrosine-based motif YXXΦ and a histidine-rich sequence. To investigate the function of the intracellular domain of FgfrL1, we have prepared genetically modified mice that lack the three conserved sequence motifs, but instead contain a GFP cassette (FgfrL1ΔC-GFP). To our surprise, homozygous FgfrL1ΔC-GFP knock-in mice are viable, fertile and phenotypically normal. They do not exhibit any alterations in the diaphragm or the kidney, except for a slight reduction in the number of glomeruli that does not appear to affect life expectancy. In addition, the pancreas of both FgfrL1ΔC-GFP knock-in and FgfrL1 knock-out mice do not show any disturbances in the production of insulin, in contrast to what has been suggested by recent studies. Thus, the conserved motifs of the intracellular FgfrL1 domain are dispensable for organogenesis and normal life. We conclude that the extracellular domain of the protein must conduct the vital functions of FgfrL1.     

In Vivo Regulation of Erythropoiesis by Chemically Inducible Dimerization of the Erythropoietin Receptor Intracellular Domain

Erythropoietin (Epo) and its receptor (EpoR) are required for the regulation of erythropoiesis. Epo binds to the EpoR homodimer on the surface of erythroid progenitors and erythroblasts, and positions the intracellular domains of the homodimer to be in close proximity with each other. This conformational change is sufficient for the initiation of Epo-EpoR signal transduction. Here, we established a system of chemically regulated erythropoiesis in transgenic mice expressing a modified EpoR intracellular domain (amino acids 247–406) in which dimerization is induced using a specific compound (chemical inducer of dimerization, CID). Erythropoiesis is reversibly induced by oral administration of the CID to the transgenic mice. Because transgene expression is limited to hematopoietic cells by the Gata1 gene regulatory region, the effect of the CID is limited to erythropoiesis without adverse effects. Additionally, we show that the 160 amino acid sequence is the minimal essential domain of EpoR for intracellular signaling of chemically inducible erythropoiesis in vivo. We propose that the CID-dependent dimerization system combined with the EpoR intracellular domain and the Gata1 gene regulatory region generates a novel peroral strategy for the treatment of anemia.     

The Extracellular Domain of Lrp5/6 Inhibits Noncanonical Wnt Signaling In Vivo

Lrp5/6 are crucial coreceptors for Wnt/β-catenin signaling, a pathway biochemically distinct from noncanonical Wnt signaling pathways. Here, we examined the possible participation of Lrp5/6 in noncanonical Wnt signaling. We found that Lrp6 physically interacts with Wnt5a, but that this does not lead to phosphorylation of Lrp6 or activation of the Wnt/β-catenin pathway. Overexpression of Lrp6 blocks activation of the Wnt5a downstream target Rac1, and this effect is dependent on intact Lrp6 extracellular domains. These results suggested that the extracellular domain of Lrp6 inhibits noncanonical Wnt signaling in vitro. In vivo, Lrp6−/− mice exhibited exencephaly and a heart phenotype. Surprisingly, these defects were rescued by deletion of Wnt5a, indicating that the phenotypes resulted from noncanonical Wnt gain-of-function. Similarly, Lrp5 and Lrp6 antisense morpholino-treated Xenopus embryos exhibited convergent extension and heart phenotypes that were rescued by knockdown of noncanonical XWnt5a and XWnt11. Thus, we provide evidence that the extracellular domains of Lrp5/6 behave as physiologically relevant inhibitors of noncanonical Wnt signaling during Xenopus and mouse development in vivo.     

S/T Phosphorylation of DLL1 Is Required for Full Ligand Activity In Vitro but Dispensable for DLL1 Function In Vivo during Embryonic Patterning and Marginal Zone B Cell Development

Interaction of Notch receptors with Delta- and Serrate-type ligands is an evolutionarily conserved mechanism that mediates direct communication between adjacent cells and thereby regulates multiple developmental processes. Posttranslational modifications of both receptors and ligands are pivotal for normal Notch pathway function. We have identified by mass spectrometric analysis two serine and one threonine phosphorylation sites in the intracellular domain of the mouse Notch ligand DLL1. Phosphorylation requires cell membrane association of DLL1 and occurs sequentially at the two serine residues. Phosphorylation of one serine residue most likely by protein kinase B primes phosphorylation of the other serine. A DLL1 variant, in which all three identified phosphorylated serine/threonine residues are mutated to alanine and valine, was more stable than wild-type DLL1 but had reduced relative levels on the cell surface and was more effectively cleaved in the extracellular domain. In addition, the mutant variant activated Notch1 significantly less efficient than wild-type DLL1 in a coculture assay in vitro. Mice, however, whose endogenous DLL1 was replaced with the phosphorylation-deficient triple mutant developed normally, suggesting compensatory mechanisms under physiological conditions in vivo.     

APP Intracellular Domain Impairs Adult Neurogenesis in Transgenic Mice by Inducing Neuroinflammation

Background

A devastating aspect of Alzheimer''s disease (AD) is the progressive deterioration of memory due to neuronal loss. Amyloid precursor protein (APP) occupies a central position in AD and APP-derived amyloid-β (Aβ) peptides are thought to play a pivotal role in disease pathogenesis. Nonetheless, it is becoming clear that AD etiology is highly complex and that factors other than Aβ also contribute to AD pathogenesis. APP intracellular domain (AICD) is generated together with Aβ and we recently showed that AICD transgenic mice recapitulate pathological features of AD such as tau hyperphosphorylation, memory deficits and neurodegeneration without increasing the Aβ levels. Since impaired adult neurogenesis is shown to augment memory deficits in AD mouse models, here we examined the status of adult neurogenesis in AICD transgenic mice.

Methodology/Principal Finding

We previously generated transgenic mice co-expressing 59-residue long AICD fragment and its binding partner Fe65. Hippocampal progenitor cell proliferation was determined by BrdU incorporation at 1.5, 3 and 12 months of age. Only male transgenic and their respective wilt type littermate control mice were used. We find age-dependent decrease in BrdU incorporation and doublecortin-positive cells in the dentate gyrus of AICD transgenic mice suggesting impaired adult neurogenesis. This deficit resulted from decreased proliferation and survival, whereas neuronal differentiation remained unaffected. Importantly, this impairment was independent of Aβ since APP-KO mice expressing AICD also exhibit reduced neurogenesis. The defects in adult neurogenesis are prevented by long-term treatment with the non-steroidal anti-inflammatory agents ibuprofen or naproxen suggesting that neuroinflammation is critically involved in impaired adult neurogenesis in AICD transgenic mice.

Conclusion/Significance

Since adult neurogenesis is crucial for spatial memory, which is particularly vulnerable in AD, these findings suggest that AICD can exacerbate memory defects in AD by impairing adult neurogenesis. Our findings further establish that AICD, in addition to Aβ, contributes to AD pathology and that neuroinflammation plays a much broader role in AD pathogenesis than previously thought.     

In Vivo and In Vitro Activity by Diverse Chelators against Trypanosoma brucei brucei1

A system of prescreens and screen has been developed to select chelators as potential drugs against Trypanosoma brucei brucei EATRO 110. The chelators tested were nearly all commercially available, low molecular, and having moderate to high affinity for Fe(III). We prescreened 70 compounds showing heme-sparing or inhibitory activity in a Crithidia fasciculata growth system having excess Fe and minimal hemin. Of these, 45 were highly trypanocidal for suspensions of bloodstream T. b. brucei; criteria of activity here were immobilization, lysis, and loss of infectivity. Eighteen of the chelators highly active in the suspension prescreen were tried in T. b. brucei-infected mice. Thirteen of these chelators were curative in mice with 24-h infections, that is, they allowed survival >30 days beyond the untreated controls. 3,4-Dihydroxycinnamic acid (caffeic acid). 2,9-dimethyl-1, 10 phenanthroline (neocuproine), and 2-pyridinecarboxaldehyde-2-pyridyl-hydrazone cured five out of five mice after an i.v. dose of 100 mg/kg. Salicylaldehyde thiosemicarbazone cured five out of five mice at an i.p. dose of 500 mg/kg. Lesser activity was shown by several other chelators.     

Comparison of Monkeypox Viruses Pathogenesis in Mice by In Vivo Imaging

Monkeypox viruses (MPXV) cause human monkeypox, a zoonotic smallpox-like disease endemic to Africa, and are of worldwide public health and biodefense concern. Using viruses from the Congo (MPXV-2003-Congo-358) and West African (MPXV-2003-USA-044) clades, we constructed recombinant viruses that express the luciferase gene (MPXV-Congo/Luc+and MPXV-USA-Luc+) and compared their viral infection in mice by biophotonic imaging. BALB/c mice became infected by both MPXV clades, but they recovered and cleared the infection within 10 days post-infection (PI). However, infection in severe combined immune deficient (SCID) BALB/c mice resulted in 100% lethality. Intraperitoneal (IP) injection of both MPXV-Congo and MPXV-Congo/Luc+resulted in a systemic clinical disease and the same mean time-to-death at 9 (±0) days post-infection. Likewise, IP injection of SCID-BALB/c mice with MPXV-USA or the MPXV-USA-Luc+, resulted in similar disease but longer (P<0.05) mean time-to-death (11±0 days) for both viruses compared to the Congo strains. Imaging studies in SCID mice showed luminescence in the abdomen within 24 hours PI with subsequent spread elsewhere. Animals infected with the MPXV-USA/Luc+had less intense luminescence in tissues than those inoculated with MPXV-Congo/Luc+, and systemic spread of the MPXV-USA/Luc+virus occurred approximately two days later than the MPXV-Congo/Luc+. The ovary was an important target for viral replication as evidenced by the high viral titers and immunohistochemistry. These studies demonstrate the suitability of a mouse model and biophotonic imaging to compare the disease progression and tissue tropism of MPX viruses.     

Maintenance of Bone Homeostasis by DLL1‐Mediated Notch Signaling

Adult bone mass is maintained through a balance of the activities of osteoblasts and osteoclasts. Although Notch signaling has been shown to maintain bone homeostasis by controlling the commitment, differentiation, and function of cells in both the osteoblast and osteoclast lineages, the precise mechanisms by which Notch performs such diverse and complex roles in bone physiology remain unclear. By using a transgenic approach that modified the expression of delta‐like 1 (DLL1) or Jagged1 (JAG1) in an osteoblast‐specific manner, we investigated the ligand‐specific effects of Notch signaling in bone homeostasis. This study demonstrated for the first time that the proper regulation of DLL1 expression, but not JAG1 expression, in osteoblasts is essential for the maintenance of bone remodeling. DLL1‐induced Notch signaling was responsible for the expansion of the bone‐forming cell pool by promoting the proliferation of committed but immature osteoblasts. However, DLL1‐Notch signaling inhibited further differentiation of the expanded osteoblasts to become fully matured functional osteoblasts, thereby substantially decreasing bone formation. Osteoblast‐specific expression of DLL1 did not alter the intrinsic differentiation ability of cells of the osteoclast lineage. However, maturational arrest of osteoblasts caused by the DLL1 transgene impaired the maturation and function of osteoclasts due to a failed osteoblast‐osteoclast coupling, resulting in severe suppression of bone metabolic turnover. Taken together, DLL1‐mediated Notch signaling is critical for proper bone remodeling as it regulates the differentiation and function of both osteoblasts and osteoclasts. Our study elucidates the importance of ligand‐specific activation of Notch signaling in the maintenance of bone homeostasis. J. Cell. Physiol. 232: 2569–2580, 2017. © 2016 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals Inc.     

Identification of an Effective Early Signaling Signature during Neo-Vasculogenesis In Vivo by Ex Vivo Proteomic Profiling

Therapeutic neo-vasculogenesis in vivo can be achieved by the co-transplantation of human endothelial colony-forming progenitor cells (ECFCs) with mesenchymal stem/progenitor cells (MSPCs). The underlying mechanism is not completely understood thus hampering the development of novel stem cell therapies. We hypothesized that proteomic profiling could be used to retrieve the in vivo signaling signature during the initial phase of human neo-vasculogenesis. ECFCs and MSPCs were therefore either transplanted alone or co-transplanted subcutaneously into immune deficient mice. Early cell signaling, occurring within the first 24 hours in vivo, was analyzed using antibody microarray proteomic profiling. Vessel formation and persistence were verified in parallel transplants for up to 24 weeks. Proteomic analysis revealed significant alteration of regulatory components including caspases, calcium/calmodulin-dependent protein kinase, DNA protein kinase, human ErbB2 receptor-tyrosine kinase as well as mitogen-activated protein kinases. Caspase-4 was selected from array results as one therapeutic candidate for targeting vascular network formation in vitro as well as modulating therapeutic vasculogenesis in vivo. As a proof-of-principle, caspase-4 and general caspase-blocking led to diminished endothelial network formation in vitro and significantly decreased vasculogenesis in vivo. Proteomic profiling ex vivo thus unraveled a signaling signature which can be used for target selection to modulate neo-vasculogenesis in vivo.     

Establishment of HSV1 Latency in Immunodeficient Mice Facilitates Efficient In Vivo Reactivation

The establishment of latent infections in sensory neurons is a remarkably effective immune evasion strategy that accounts for the widespread dissemination of life long Herpes Simplex Virus type 1 (HSV1) infections in humans. Periodic reactivation of latent virus results in asymptomatic shedding and transmission of HSV1 or recurrent disease that is usually mild but can be severe. An in-depth understanding of the mechanisms regulating the maintenance of latency and reactivation are essential for developing new approaches to block reactivation. However, the lack of a reliable mouse model that supports efficient in vivo reactivation (IVR) resulting in production of infectious HSV1 and/or disease has hampered progress. Since HSV1 reactivation is enhanced in immunosuppressed hosts, we exploited the antiviral and immunomodulatory activities of IVIG (intravenous immunoglobulins) to promote survival of latently infected immunodeficient Rag mice. Latently infected Rag mice derived by high dose (HD), but not low dose (LD), HSV1 inoculation exhibited spontaneous reactivation. Following hyperthermia stress (HS), the majority of HD inoculated mice developed HSV1 encephalitis (HSE) rapidly and synchronously, whereas for LD inoculated mice reactivated HSV1 persisted only transiently in trigeminal ganglia (Tg). T cells, but not B cells, were required to suppress spontaneous reactivation in HD inoculated latently infected mice. Transfer of HSV1 memory but not OVA specific or naïve T cells prior to HS blocked IVR, revealing the utility of this powerful Rag latency model for studying immune mechanisms involved in control of reactivation. Crossing Rag mice to various knockout strains and infecting them with wild type or mutant HSV1 strains is expected to provide novel insights into the role of specific cellular and viral genes in reactivation, thereby facilitating identification of new targets with the potential to block reactivation.     

Identification of a Cardiac Specific Protein Transduction Domain by In Vivo Biopanning Using a M13 Phage Peptide Display Library in Mice

Background

A peptide able to transduce cardiac tissue specifically, delivering cargoes to the heart, would be of significant therapeutic potential for delivery of small molecules, proteins and nucleic acids. In order to identify peptide(s) able to transduce heart tissue, biopanning was performed in cell culture and in vivo with a M13 phage peptide display library.

Methods and Results

A cardiomyoblast cell line, H9C2, was incubated with a M13 phage 12 amino acid peptide display library. Internalized phage was recovered, amplified and then subjected to a total of three rounds of in vivo biopanning where infectious phage was isolated from cardiac tissue following intravenous injection. After the third round, 60% of sequenced plaques carried the peptide sequence APWHLSSQYSRT, termed cardiac targeting peptide (CTP). We demonstrate that CTP was able to transduce cardiomyocytes functionally in culture in a concentration and cell-type dependent manner. Mice injected with CTP showed significant transduction of heart tissue with minimal uptake by lung and kidney capillaries, and no uptake in liver, skeletal muscle, spleen or brain. The level of heart transduction by CTP also was greater than with a cationic transduction domain.

Conclusions

Biopanning using a peptide phage display library identified a peptide able to transduce heart tissue in vivo efficiently and specifically. CTP could be used to deliver therapeutic peptides, proteins and nucleic acid specifically to the heart.     

The Establishment of an In Vivo HIV-1 Infection Model in Humanized B-NSG Mice

Suitable animal models for human immunodeficiency virus type 1(HIV-1) infection are important for elucidating viral pathogenesis and evaluating antiviral strategies in vivo. The B-NSG(NOD-PrkdcscidIl2rgtm1/Bcge) mice that have severe immune defect phenotype are examined for the suitability of such a model in this study. Human peripheral blood mononuclear cells(PBMCs) were engrafted into B-NSG mice via mouse tail vein injection, and the repopulated human T-lymphocytes were observed at as early as 3-weeks post-transplantation in mouse peripheral blood and several tissues.The humanized mice could be infected by HIV-1, and the infection recapitulated features of T-lymphocyte dynamic observed in HIV-1 infected humans, meanwhile the administration of combination antiretroviral therapy(cART) suppressed viral replication and restored T lymphocyte abnormalities. The establishment of HIV-1 infected humanized B-NSG mice not only provides a model to study virus and T cell interplays, but also can be a useful tool to evaluate antiviral strategies.     

Context-Dependent Functional Divergence of the Notch Ligands DLL1 and DLL4 In Vivo

Notch signalling is a fundamental pathway that shapes the developing embryo and sustains adult tissues by direct communication between ligand and receptor molecules on adjacent cells. Among the ligands are two Delta paralogues, DLL1 and DLL4, that are conserved in mammals and share a similar structure and sequence. They activate the Notch receptor partly in overlapping expression domains where they fulfil redundant functions in some processes (e.g. maintenance of the crypt cell progenitor pool). In other processes, however, they appear to act differently (e.g. maintenance of foetal arterial identity) raising the questions of how similar DLL1 and DLL4 really are and which mechanism causes the apparent context-dependent divergence. By analysing mice that conditionally overexpress DLL1 or DLL4 from the same genomic locus (Hprt) and mice that express DLL4 instead of DLL1 from the endogenous Dll1 locus (Dll1^Dll4ki), we found functional differences that are tissue-specific: while DLL1 and DLL4 act redundantly during the maintenance of retinal progenitors, their function varies in the presomitic mesoderm (PSM) where somites form in a Notch-dependent process. In the anterior PSM, every cell expresses both Notch receptors and ligands, and DLL1 is the only activator of Notch while DLL4 is not endogenously expressed. Transgenic DLL4 cannot replace DLL1 during somitogenesis and in heterozygous Dll1^Dll4ki/+ mice, the Dll1^Dll4ki allele causes a dominant segmentation phenotype. Testing several aspects of the complex Notch signalling system in vitro, we found that both ligands have a similar trans-activation potential but that only DLL4 is an efficient cis-inhibitor of Notch signalling, causing a reduced net activation of Notch. These differential cis-inhibitory properties are likely to contribute to the functional divergence of DLL1 and DLL4.     

In Vivo Bioluminescence Imaging for the Study of Intestinal Colonization by Escherichia coli in Mice

Bioluminescence imaging (BLI) is emerging as a powerful tool for real-time monitoring of infections in living animals. However, since luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. Strains of Escherichia coli harboring the genes for either the bacterial luciferase from Photorhabdus luminescens or the PpyRE-TS and PpyGR-TS firefly luciferase mutants of Photinus pyralis (red and green thermostable P. pyralis luciferase mutants, respectively) have been engineered and used to monitor intestinal colonization in the streptomycin-treated mouse model. There was excellent correlation between the bioluminescence signal measured in the feces (R² = 0.98) or transcutaneously in the abdominal region of whole animals (R² = 0.99) and the CFU counts in the feces of bacteria harboring the luxABCDE operon. Stability in vivo of the bioluminescence signal was achieved by constructing plasmid pAT881(pGB2ΩP_amiluxABCDE), which allowed long-term monitoring of intestinal colonization without the need for antibiotic selection for plasmid maintenance. Levels of intestinal colonization by various strains of E. coli could be compared directly by simple recording of the bioluminescence signal in living animals. The difference in spectra of light emission of the PpyRE-TS and PpyGR-TS firefly luciferase mutants and dual bioluminescence detection allowed direct in vitro and in vivo quantification of two bacterial populations by measurement of red and green emitted signals and thus monitoring of the two populations simultaneously. This system offers a simple and direct method to study in vitro and in vivo competition between mutants and the parental strain. BLI is a useful tool to study intestinal colonization.Among the wide variety of bacteria that colonize the gastrointestinal tracts of mammals, Escherichia coli is the most abundant facultative anaerobe of the human intestinal microflora. Aside from being part of the normal flora, E. coli is also a versatile organism capable of causing a variety of intestinal and extraintestinal diseases (18). The mechanisms that allow commensal E. coli to colonize the intestine and survive successfully in this niche remain poorly characterized. Conventional mice display natural resistance to colonization by commensal E. coli, but oral administration of streptomycin, which alters the intestinal microflora, allows colonization of the mouse large intestine by this species (25). The streptomycin-treated mouse model has been used extensively to study the factors of gram-negative bacteria implicated in the intestinal colonization process. However, this model is limited to the viable plate counts of bacteria in the feces and misses some critical information, such as the kinetics of colonization, the fate of the bacterial cells across the digestive tract, and the site of colonization. A better understanding of colonization would be facilitated by direct in vivo follow-up of this process.Bioluminescence imaging (BLI) technology is emerging as a powerful tool for the study of a wide range of biological processes in live animals, including real-time monitoring of infections (16). Bioluminescence systems emit visible light due to the luciferase-mediated oxidation of a luciferin substrate. A variety of luciferin-luciferase systems with different peak emissions have been identified in nature from numerous species (14). The luciferase of the soil bacterium Photorhabdus luminescens has been expressed successfully in gram-negative and gram-positive bacteria. This system emits blue-green light, with an emission maximum of approximately 490 nm, and does not require the addition of an exogenous substrate since the luciferase operon contains the genes required for synthesis of the substrate. Therefore, this luciferase has been used extensively to monitor bacterial infections in the living mouse. One of the first investigations with Salmonella enterica serovar Typhimurium transformed with the lux operon of P. luminescens evaluated the tissue distribution and the virulence of various S. Typhimurium strains (9). Subsequent modification of the lux operon led to the generation of highly bioluminescent Staphylococcus aureus and allowed the monitoring of infections due to this species in living mice (11). The modified lux operon was engineered into a lux-kan transposon cassette for chromosomal integration in gram-positive bacteria, such as S. aureus, Streptococcus pneumoniae, group A Streptococcus, and Listeria monocytogenes (16). Replication of L. monocytogenes in the lumen of the gall bladder was demonstrated for the first time by BLI (13).Bioluminescent E. coli was used in the neutropenic mouse thigh model of infection to evaluate the in vivo activity of antimicrobial agents (29). Bioluminescence was as indicative of therapeutic efficacy as CFU counts but, in addition, allowed real-time monitoring of the infection and of treatment efficacy in the same animal; however, only short-term monitoring (12 h) could be performed.Because luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. After oral administration of bioluminescent Salmonella to susceptible mice, the bioluminescent signal recorded in the abdominal region was greatly enhanced after air exposure (9). It was therefore assumed that direct bioluminescence imaging of intestine-colonizing microorganisms would not be optimal unless oxygen was provided exogenously or as the result of the close interaction between cells and the bacteria (9). However, the bacterial luciferase was used to trace in real time the colonization dynamics by Citrobacter rodentium of the gastrointestinal tracts of living animals, demonstrating that the gut represents a semianaerobic environment that allows the study of bacterial colonization by BLI (33).Factors essential for colonization are best studied in cocolonization experiments (7, 17). There are several luciferases with distinct emission spectra (34) that could be used in competition experiments to trace simultaneously two bacterial populations in the same living animal. However, in order not to impose additional and different metabolic burdens on the bacteria under study, the exogenous luciferases ideally have to be similar to allow comparison between strains. The thermostable luciferase variants PpyRE-TS and PpyGR-TS, derived from wild-type luciferase from the North American firefly Photinus pyralis, emit red (612 nm) and green (552 nm) light, respectively, at 37°C and are encoded by single genes of 1,650 bp, differing by only 9 bp (4). Bioluminescence color is determined by the Ser₂₈₄Thr (PpyRE-TS) and Val₂₄₁Ile, Gly₂₄₆Ala, and Phe₂₅₀Ser (PpyGR-TS) amino acid changes (5, 34). By use of optical filters, the emission spectra are readily distinguishable (4, 5). Five additional mutations provide enhanced thermostability at 37°C (4), improving the compatibility of the enzymes with bacterial culture conditions and BLI in animal models.While the luciferase mutants and all firefly luciferases use as substrates firefly luciferin and ATP to produce light, in vivo imaging is commonly performed with endogenous ATP and requires only exogenous administration of the luciferase substrate.The aim of this study was to develop a dynamic mouse model using in vivo bioluminescence imaging systems to monitor bacterial colonization in situ and in real time in whole living animals. Various strains of E. coli harboring the genes for the bacterial luciferase from P. luminescens or the firefly luciferase mutants (PpyRE-TS and PpyGR-TS) from P. pyralis have been engineered and used to follow bacterial intestinal colonization in mice. BLI was found to be well adapted to compare the intestine-colonizing capacities of various E. coli strains and to monitor cocolonization in vivo by use of dual bioluminescence emission.     

In Vivo and In Vitro Manifestations of Adrenergic Blockade in Bordetella pertussis-vaccinated Mice

The in vivo metabolic events which follow the administration of epinephrine, norepinephrine, or isoproterenol were examined in normal, Bordetella pertussis-vaccinated, and alpha and beta adrenergically blocked mice. The normal hyperglycemic response to epinephrine was suppressed in all experimental groups. The pertussis-sensitized and beta-blocked animals produced similar split patterns of altered response not duplicated by the alpha-blocking compounds. Those catecholamines that normally increase free fatty acids and lactic acid in the circulation failed to do so in the pertussis-sensitized and beta-blocked animals; the inhibition of free fatty acid mobilization was also demonstrated with adipose tissue incubated in vitro. An extract of the pertussis organism added to incubation media prevented the catecholamine-induced free fatty acid response. The epinephrine-stabilizing effect of bovine serum albumin (Cohn-fraction V) was observed. The results of these studies further emphasize a correlation between pertussis-sensitized and beta-adrenergically blocked mice.     

Intracellular Localization of the HCS2 Gene Products in Identified Snail Neurons In Vivo and In Vitro

SUMMARY 1. The HCS2 (Helix command specific 2) gene expressed in giant command neurons for withdrawal behavior of the terrestrial snail Helix lucorum encodes a unique hybrid precursor protein that contains a Ca-binding (EF-hand motif) protein and four small peptides (CNP1-CNP4) with similar Tyr-Pro-Arg-X aminoacid sequence at the C terminus. Previous studies suggest that under conditions of increased intracellular Ca²⁺ concentration the HCS2 peptide precursor may be cleaved, and small physiologically active peptides transported to the release sites. In the present paper, intracellular localization of putative peptide products of the HCS2-encoded precursor was studied immunocytochemically by means of light and electron microscopy.2. Polyclonal antibodies against the CNP3 neuropeptide and a Ca-binding domain of the precursor protein were used for gold labeling of ultrathin sections of identified isolated neurons maintained in culture for several days, and in same identified neurons freshly isolated from the central nervous system.3. In freshly isolated neurons, the gold particles were mainly localized over the cytoplasmic secretory granules, with the density of labeling for the CNP3 neuropeptide being two-fold higher than for the calcium-binding domain. In cultured neurons, both antibodies mostly labeled clusters of secretory granules in growth cones and neurites of the neuron. The density of labeling for cultured neurons was the same for both antibodies, and was two-fold higher than for the freshly isolated from the central nervous system neurons.4. The immunogold particles were practically absent in the bodies of cultured neurons.5. The data obtained conform to the suggestion that the HCS2 gene products are transported from the cell body to the regions of growth or release sites.     

Enhancement of Osteogenic Induction by LL37 Modified with a Collagen-Binding Domain In Vitro and In Vivo

International Journal of Peptide Research and Therapeutics - Bone defect diseases, particularly those induced by inflammation, pose a challenge for the design of ideal drug-loading scaffolds that...