Literature context: 00; Life Technologies; A-11055; RRID:AB_142672) secondary antibodies for 1 hou
The glycoprotein FSH, a product of pituitary gonadotrope cells, regulates ovarian follicle development in females and spermatogenesis in males. FSH is a heterodimer of the common α gonadotropin subunit and the hormone-specific FSHβ subunit (a product of the Fshb gene). Using a conditional knockout approach (Cre-lox), we previously demonstrated that Fshb expression in mice depends on the transcription factors forkhead box L2 (FOXL2) and SMAD4. Deletion of Foxl2 or Smad4 alone led to FSH deficiency, female subfertility, and oligozoospermia in males. Simultaneous deletion of the two genes yielded a greater suppression of FSH and female sterility. The Cre-driver used previously was first active during embryonic development. Therefore, it is unclear whether FOXL2 and SMAD4 play important roles in the development or adult function of gonadotropes, or both. To address this question, we developed a tamoxifen-inducible Cre-driver line, which enabled Foxl2 and Smad4 gene deletions in gonadotropes of adult mice. After tamoxifen treatment, females with previously demonstrated fertility exhibited profound reductions in FSH levels, arrested ovarian follicle development, and sterility. FSH levels were comparably reduced in males 1 or 2 months after treatment; however, spermatogenesis was unaffected. These data indicate that (1) FOXL2 and SMAD4 are necessary to maintain FSH synthesis in gonadotrope cells of adult mice, (2) FSH is essential for female reproduction but appears to be unnecessary for the maintenance of spermatogenesis in adult male mice, and (3) the inducible Cre-driver line developed here provides a powerful tool to interrogate gene function in gonadotrope cells of adult mice.
Literature context: (H+L) Molecular Probes, A11055, RRID:AB_142672, donkey, polyclonal 1:200 PFA o
Vocalization is a behavioral feature that is shared among multiple vertebrate lineages, including fish. The temporal patterning of vocal communication signals is set, in part, by central pattern generators (CPGs). Toadfishes are well-established models for CPG coding of vocalization at the hindbrain level. The vocal CPG comprises three topographically separate nuclei: pre-pacemaker, pacemaker, motor. While the connectivity between these nuclei is well understood, their neurochemical profile remains largely unexplored. The highly vocal Gulf toadfish, Opsanus beta, has been the subject of previous behavioral, neuroanatomical and neurophysiological studies. Combining transneuronal neurobiotin-labeling with immunohistochemistry, we map the distribution of inhibitory neurotransmitters and neuromodulators along with gap junctions in the vocal CPG of this species. Dense GABAergic and glycinergic label is found throughout the CPG, with labeled somata immediately adjacent to or within CPG nuclei, including a distinct subset of pacemaker neurons co-labeled with neurobiotin and glycine. Neurobiotin-labeled motor and pacemaker neurons are densely co-labeled with the gap junction protein connexin 35/36, supporting the hypothesis that transneuronal neurobiotin-labeling occurs, at least in part, via gap junction coupling. Serotonergic and catecholaminergic label is also robust within the entire vocal CPG, with additional cholinergic label in pacemaker and prepacemaker nuclei. Likely sources of these putative modulatory inputs are neurons within or immediately adjacent to vocal CPG neurons. Together with prior neurophysiological investigations, the results reveal potential mechanisms for generating multiple classes of social context-dependent vocalizations with widely divergent temporal and spectral properties.
Literature context: y anti-goat IgG (H+L) (RRID:AB_142672 and RRID:AB_141788), and Alexa
LIM-domain containing transcription factors (LIM-TFs) are conserved factors important for embryogenesis. The specificity of these factors in transcriptional regulation is conferred by the complexes that they form with other proteins such as LIM-domain-binding (Ldb) proteins and LIM-domain only (LMO) proteins. Unlike LIM-TFs, these proteins do not bind DNA directly. LMO proteins are negative regulators of LIM-TFs and function by competing with LIM-TFs for binding to Ldb's. Although the LIM-TF Lmx1a is expressed in the developing mouse hindbrain, which provides many of the extrinsic signals for inner ear formation, conditional knock-out embryos of both sexes show that the inner ear source of Lmx1a is the major contributor of ear patterning. In addition, we have found that the reciprocal interaction between Lmx1a and Lmo4 (a LMO protein within the inner ear) mediates the formation of both vestibular and auditory structures. Lmo4 negatively regulates Lmx1a to form the three sensory cristae, the anterior semicircular canal, and the shape of the utricle in the vestibule. Furthermore, this negative regulation blocks ectopic sensory formation in the cochlea. In contrast, Lmx1a negatively regulates Lmo4 in mediating epithelial resorption of the canal pouch, which gives rise to the anterior and posterior semicircular canals. We also found that Lmx1a is independently required for the formation of the endolymphatic duct and hair cells in the basal cochlear region.SIGNIFICANCE STATEMENT The mammalian inner ear is a structurally complex organ responsible for detecting sound and maintaining balance. Failure to form the intricate 3D structure of this organ properly during development most likely will result in sensory deficits on some level. Here, we provide genetic evidence that a transcription factor, Lmx1a, interacts with its negative regulator, Lmo4, to pattern various vestibular and auditory components of the mammalian inner ear. Identifying these key molecules that mediate formation of this important sensory organ will be helpful for designing strategies and therapeutics to alleviate hearing loss and balance disorders.
Literature context: 42; catalog #A-11055, RRID:AB_142672). Finally, all sections were st
The nucleus accumbens (NAc) plays a central role in reinforcement and motivation. Around 95% of the NAc neurons are medium spiny neurons (MSNs), divided into those expressing dopamine receptor D1 (D1R) or dopamine receptor D2 (D2R). Optogenetic activation of D2-MSNs increased motivation, whereas inhibition of these neurons produced the opposite effect. Yet, it is still unclear how activation of D2-MSNs affects other local neurons/interneurons or input terminals and how this contributes for motivation enhancement. To answer this question, in this work we combined optogenetic modulation of D2-MSNs with in loco pharmacological delivery of specific neurotransmitter antagonists in rats. First, we showed that optogenetic activation of D2-MSNs increases motivation in a progressive ratio (PR) task. We demonstrated that this behavioral effect relies on cholinergic-dependent modulation of dopaminergic signalling of ventral tegmental area (VTA) terminals, which requires D1R and D2R signalling in the NAc. D2-MSN optogenetic activation decreased ventral pallidum (VP) activity, reducing the inhibitory tone to VTA, leading to increased dopaminergic activity. Importantly, optogenetic activation of D2-MSN terminals in the VP was sufficient to recapitulate the motivation enhancement. In summary, our data suggests that optogenetic stimulation of NAc D2-MSNs indirectly modulates VTA dopaminergic activity, contributing for increased motivation. Moreover, both types of dopamine receptors signalling in the NAc are required in order to produce the positive behavioral effects.
Literature context: at IgG Invitrogen A11055 RRID:AB_142672 Alexa Fluoro 594 Donkey anti-go
Cells demonstrate plasticity following injury, but the extent of this phenomenon and the cellular mechanisms involved remain underexplored. Using single-cell RNA sequencing (scRNA-seq) and lineage tracing, we uncover that myoepithelial cells (MECs) of the submucosal glands (SMGs) proliferate and migrate to repopulate the airway surface epithelium (SE) in multiple injury models. Specifically, SMG-derived cells display multipotency and contribute to basal and luminal cell types of the SMGs and SE. Ex vivo expanded MECs have the potential to repopulate and differentiate into SE cells when grafted onto denuded airway scaffolds. Significantly, we find that SMG-like cells appear on the SE of both extra- and intra-lobular airways of large animal lungs following severe injury. We find that the transcription factor SOX9 is necessary for MEC plasticity in airway regeneration. Because SMGs are abundant and present deep within airways, they may serve as a reserve cell source for enhancing human airway regeneration.
Literature context: A-11055 also A11055 lot number RRID:AB_142672). Finally, slices were washed w
Pain is often described as a "biopsychosocial" process, yet social influences on pain and underlying neural mechanisms are only now receiving significant experimental attention. Expression of pain by one individual can be communicated to nearby individuals by auditory, visual, and olfactory cues. Conversely, the perception of another's pain can lead to physiological and behavioral changes in the observer, which can include induction of hyperalgesia in "bystanders" exposed to "primary" conspecifics in which hyperalgesia has been induced directly. The current studies were designed to investigate the neural mechanisms responsible for the social transfer of hyperalgesia in bystander mice housed and tested with primary mice in which hyperalgesia was induced using withdrawal (WD) from voluntary alcohol consumption. Male C57BL/6J mice undergoing WD from a two-bottle choice voluntary alcohol-drinking procedure served as the primary mice. Mice housed in the same room served as bystanders. Naïve, water-drinking controls were housed in a separate room. Immunohistochemical mapping identified significantly enhanced Fos immunoreactivity (Fos-ir) in the anterior cingulate cortex (ACC) and insula (INS) of bystander mice compared to naïve controls, and in the dorsal medial hypothalamus (DMH) of primary mice. Chemogenetic inactivation of the ACC but not primary somatosensory cortex reversed the expression of hyperalgesia in both primary and bystander mice. These studies point to an overlapping neural substrate for expression of socially transferred hyperalgesia and that expressed during alcohol WD.
Literature context: Molecular Probes, Cat# A-11055, RRID:AB_142672) and an anti-rabbit secondary a
The nicotinic acetylcholine receptor alpha2 subunit (Chrna2) is a specific marker for oriens lacunosum-moleculare (OLM) interneurons in the dorsal CA1 region of the hippocampus. It was recently shown using a Chrna2-cre mice line that OLM interneurons can modulate entorhinal cortex and CA3 inputs and may therefore have an important role in gating, encoding, and recall of memory. In this study, we have used a combination of electrophysiology and optogenetics using Chrna2-cre mice to determine the role of Chrna2 interneurons in the subiculum area, the main output region of the hippocampus. We aimed to assess the similarities between Chrna2 subiculum and CA1 neurons in terms of the expression of interneuron markers, their membrane properties, and their inhibitory input to pyramidal neurons. We found that subiculum and CA1 dorsal Chrna2 cells similarly expressed the marker somatostatin and had comparable membrane and firing properties. The somas of Chrna2 cells in both regions were found in the deepest layer with axons projecting superficially. However, subiculum Chrna2 cells displayed more extensive projections with dendrites which occupied a significantly larger area than in CA1. The post-synaptic responses elicited by Chrna2 cells in pyramidal cells of both regions revealed comparable inhibitory responses elicited by GABAA receptors and, interestingly, GABAB receptor mediated components. This study provides the first in-depth characterization of Chrna2 cells in the subiculum, and suggests that subiculum and CA1 Chrna2 cells are generally similar and may play comparable roles in both sub-regions.
Literature context: +L) Molecular Probes: A-11055; RRID:AB_142672 Donkey 1:500
The outer mitochondrial membrane translocator protein (TSPO) binds cholesterol with high affinity and is involved in mediating its delivery into mitochondria, the rate-limiting step in hormone-induced steroidogenesis. Specific ligand binding to TSPO has been shown to initiate steroid formation. However, recent studies of the genetic deletion of Tspo have provided conflicting results. Here, we address and extend previous studies by examining the effects of Tspo-specific mutations on steroid formation in hormone- and cyclic adenosine monophosphate (cAMP)-responsive MA-10 cells, using the CRISPR/Cas9 system. Two mutant subcell lines, nG1 and G2G, each carrying a Tspo exon2-specific genome modification, and two control subcell lines, G1 and HH, each carrying a wild-type Tspo, were produced. In response to dibutyryl cAMP, the nG1 and G2G cells produced progesterone at levels significantly lower than those produced by the corresponding control cells G1 and HH. Neutral lipid homeostasis, which provides free cholesterol for steroid biosynthesis, was altered significantly in the Tspo mutant cells. Interestingly, the mitochondrial membrane potential (ΔΨm) of the Tspo mutant cells was significantly reduced compared with that of the control cells, likely because of TSPO interactions with the voltage-dependent anion channel and tubulin at the outer mitochondrial membrane. Steroidogenic acute regulatory protein (STAR) expression was induced in nG1 cells, suggesting that reduced TSPO affected STAR synthesis and/or processing. Taken together, these results provide further evidence for the critical role of TSPO in steroid biosynthesis and suggest that it may function at least in part via its regulation of ΔΨm and effects on STAR.
Literature context: (secondary antibody) Invitrogen RRID:AB_142672, RRID:AB_141788
Luminal fluid reabsorption plays a fundamental role in male fertility. We demonstrated that the ubiquitous GPCR signaling proteins Gq and β-arrestin-1 are essential for fluid reabsorption because they mediate coupling between an orphan receptor ADGRG2 (GPR64) and the ion channel CFTR. A reduction in protein level or deficiency of ADGRG2, Gq or β-arrestin-1 in a mouse model led to an imbalance in pH homeostasis in the efferent ductules due to decreased constitutive CFTR currents. Efferent ductule dysfunction was rescued by the specific activation of another GPCR, AGTR2. Further mechanistic analysis revealed that β-arrestin-1 acts as a scaffold for ADGRG2/CFTR complex formation in apical membranes, whereas specific residues of ADGRG2 confer coupling specificity for different G protein subtypes, this specificity is critical for male fertility. Therefore, manipulation of the signaling components of the ADGRG2-Gq/β-arrestin-1/CFTR complex by small molecules may be an effective therapeutic strategy for male infertility.
Literature context: ermo Fisher Scientific A-11055, RRID:AB_142672 Donkey polyclonal anti-mouse Ig
Genetic lineage tracing has revealed that Lgr5+ murine colon stem cells (CoSCs) rapidly proliferate at the crypt bottom. However, the spatiotemporal dynamics of human CoSCs in vivo have remained experimentally intractable. Here we established an orthotopic xenograft system for normal human colon organoids, enabling stable reconstruction of the human colon epithelium in vivo. Xenografted organoids were prone to displacement by the remaining murine crypts, and this could be overcome by complete removal of the mouse epithelium. Xenografted organoids formed crypt structures distinctively different from surrounding mouse crypts, reflecting their human origin. Lineage tracing using CRISPR-Cas9 to engineer an LGR5-CreER knockin allele demonstrated self-renewal and multipotency of LGR5+ CoSCs. In contrast to the rapidly cycling properties of mouse Lgr5+ CoSCs, human LGR5+ CoSCs were slow-cycling in vivo. This organoid-based orthotopic xenograft model enables investigation of the functional behaviors of human CoSCs in vivo, with potential therapeutic applications in regenerative medicine.
Literature context: Fisher Scientific Cat# A-11055, RRID:AB_142672 Bacterial and Virus Strains
Human autoantibodies to contactin-associated protein-like 2 (CASPR2) are often associated with neuropathic pain, and CASPR2 mutations have been linked to autism spectrum disorders, in which sensory dysfunction is increasingly recognized. Human CASPR2 autoantibodies, when injected into mice, were peripherally restricted and resulted in mechanical pain-related hypersensitivity in the absence of neural injury. We therefore investigated the mechanism by which CASPR2 modulates nociceptive function. Mice lacking CASPR2 (Cntnap2-/-) demonstrated enhanced pain-related hypersensitivity to noxious mechanical stimuli, heat, and algogens. Both primary afferent excitability and subsequent nociceptive transmission within the dorsal horn were increased in Cntnap2-/- mice. Either immune or genetic-mediated ablation of CASPR2 enhanced the excitability of DRG neurons in a cell-autonomous fashion through regulation of Kv1 channel expression at the soma membrane. This is the first example of passive transfer of an autoimmune peripheral neuropathic pain disorder and demonstrates that CASPR2 has a key role in regulating cell-intrinsic dorsal root ganglion (DRG) neuron excitability.
Literature context: hermoFisher Scientific, A11055, RRID:AB_142672), anti-goat 568 (ThermoFisher S
While cholinergic neuromodulation is important for locomotor circuit operation, the specific neuronal mechanisms that acetylcholine employs to regulate and fine-tune the speed of locomotion are largely unknown. Here, we show that cholinergic interneurons are present in the zebrafish spinal cord and differentially control the excitability of distinct classes of motoneurons (slow, intermediate and fast) in a muscarinic dependent manner. Moreover, we reveal that m2-type muscarinic acetylcholine receptors (mAChRs) are present in fast and intermediate motoneurons, but not in the slow motoneurons, and that their activation decreases neuronal firing. We also reveal a strong correlation between the muscarinic receptor configuration on motoneurons and the ability of the animals to locomote at different speeds, which might serve as a plasticity mechanism to alter the operational range of the locomotor networks. These unexpected findings provide new insights into the functional flexibility of motoneurons and how they execute locomotion at different speeds.
Literature context: sher scientific Cat #: A-11055, RRID:AB_142672 Anti-Mouse EpCAM APC (G8.8) eBi
Tissue regeneration requires dynamic cellular adaptation to the wound environment. It is currently unclear how this is orchestrated at the cellular level and how cell fate is affected by severe tissue damage. Here we dissect cell fate transitions during colonic regeneration in a mouse dextran sulfate sodium (DSS) colitis model, and we demonstrate that the epithelium is transiently reprogrammed into a primitive state. This is characterized by de novo expression of fetal markers as well as suppression of markers for adult stem and differentiated cells. The fate change is orchestrated by remodeling the extracellular matrix (ECM), increased FAK/Src signaling, and ultimately YAP/TAZ activation. In a defined cell culture system recapitulating the extracellular matrix remodeling observed in vivo, we show that a collagen 3D matrix supplemented with Wnt ligands is sufficient to sustain endogenous YAP/TAZ and induce conversion of cell fate. This provides a simple model for tissue regeneration, implicating cellular reprogramming as an essential element.
Literature context: 1055, RRID:AB_142672:1/100; Molecular Probes, Cat# A-1
There is increasing evidence indicating that aquaporins (AQPs) exert an influence in cell signaling by the interplay with the TRPV4 Ca2+ channel. Ca2+ release from intracellular stores and plasma membrane hyperpolarization due to opening of Ca2+ -activated potassium channels (KCa) are events that have been proposed to take place downstream of TRPV4 activation. A major mechanism for Ca2+ entry, activated after depletion of intracellular Ca2+ stores and driven by electrochemical forces, is the store-operated Ca2+ entry (SOCE). The consequences of the interplay between TRPV4 and AQPs on SOCE have not been yet investigated. The aim of our study was to test the hypothesis that AQP2 can modulate SOCE by facilitating the interaction of TRPV4 with KCa channels in renal cells. Using fluorescent probe techniques, we studied intracellular Ca2+ concentration and membrane potential in response to activation of TRPV4 in two rat cortical collecting duct cell lines (RCCD1 ), one not expressing AQPs (WT-RCCD1 ) and the other transfected with AQP2 (AQP2-RCCD1 ). We found that AQP2 co-immunoprecipitates with TRPV4 and with the small-conductance potassium channel (SK3). We also showed that AQP2 is crucial for the activation of SK3 by TRPV4, leading to hyperpolarization of the plasma membrane. This seems to be relevant to modulate the magnitude of SOCE and is accompanied by TRPV4 translocation to the plasma membrane only in AQP2 expressing cells. These findings open the perspective to further investigate whether the interplay between different AQPs with TRPV4 and KCa channels can be an important mechanism to modulate SOCE with physiological relevance.
Literature context: Fisher Scientific Cat# A-11055; RRID:AB_142672 Mouse anti-human PCNA antibody
Despite expression of oncogenic KRAS, premalignant pancreatic intraepithelial neoplasia 1 (PanIN1) lesions rarely become fully malignant pancreatic ductal adenocarcinoma (PDAC). The molecular mechanisms through which established risk factors, such as chronic pancreatitis, acinar cell damage, and/or defective autophagy increase the likelihood of PDAC development are poorly understood. We show that accumulation of the autophagy substrate p62/SQSTM1 in stressed KrasG12D acinar cells is associated with PDAC development and maintenance of malignancy in human cells and mice. p62 accumulation promotes neoplastic progression by controlling the NRF2-mediated induction of MDM2, which acts through p53-dependent and -independent mechanisms to abrogate checkpoints that prevent conversion of differentiated acinar cells to proliferative ductal progenitors. MDM2 targeting may be useful for preventing PDAC development in high-risk individuals.
Literature context: 1:250 (IF) Invitrogen A-11055, RRID:AB_142672 Anti-rabbit IgG AlexaFluor 488
The present study explores tissue and cellular distribution of ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and the gene and protein expression in rat spinal cord during the course of experimental autoimmune encephalomyelitis (EAE). Given that NTPDase2 hydrolyzes ATP with a transient accumulation of ADP, the expression of ADP-sensitive P2 purinoceptors was analyzed as well. The autoimmune disease was actively induced in Dark Agouti female rats and the changes were analyzed 10, 15 and 29 days after the induction. These selected time points correspond to the onset ( Eo ), peak ( Ep ) and recovery ( Er ) from EAE. In control animals, NTPDase2 was confined in the white matter, in most of the glial fibrillary acidic protein (GFAP)-immunoreactive (ir) astrocytes and in a considerable number of nestin-ir cells, while the other cell types were immunonegative. Immunoreactivity corresponding to NTPDase2 decreased significantly at Eo and Ep and then returned to the baseline levels at Er . The preservation of the proportion of GFAP single-labeled and GFAP/NTPDase2 double-labeled elements along the course of EAE indicated that changes in NTPDase2-ir occurred at fibrous astrocytes that typically express NTPDase2 in normal conditions. Significant downregulation of P2Y1 and P2Y12 receptor proteins at Eo and several-fold induction of P2Y12 and P2Y13 receptor proteins at Ep and/or Er were observed implying that the pathophysiological process in EAE may be linked to ADP signaling. Cell-surface expression of NTPDase2, NTPDase1/CD39 and ecto-5'-nucleotidase (eN/CD73) was analyzed in CD4+ T cells of a draining lymph node by fluorescence-activated cell sorting. The induction of EAE was associated with a transient decrease in a number of CD4+ NTPDase2+ T cells in a draining lymph node, whereas the recovery was characterized by an increase in NTPDase2+ cells in both CD4+ and CD4- cell populations. The opposite was found for NTPDase1/CD39+ and eN/CD73+ cells, which slightly increased in number with progression of the disease, particularly in CD4- cells, and then decreased in the recovery. Finally, CD4+ NTPDase2+ cells were never observed in the spinal cord parenchyma. Taken together, our results suggest that the process of neuroinflammation in EAE may be associated with altered ADP signaling.
Literature context: Thermo Scientific; Cat# A11055, RRID:AB_142672). The sections were mounted on
Acupuncture, a traditional medical procedure practised for over 2000 years in Asia, stimulates specific but poorly defined sites called acupoints. To date, no unique anatomical acupoint structures have been found. However, noxious sensory signals from visceral organs produce hypersensitive spots on the skin (neurogenic spots), caused by cutaneous neurogenic inflammation, in the dermatome that overlaps with visceral afferent innervations. Here, we show that an acupoint is one form of neurogenic inflammation on the skin. Various studies have demonstrated that acupoints show mechanical hypersensitivity and have high electrical conductance. Stimulation of acupoints produces needling sensations caused by the activation of small diameter afferent nerve fibres and therapeutic effects on the associated visceral organs, which is likely due to the release of endogenous opioids. The present study provides experimental evidence that neurogenic spots exhibit all the characteristics of the acupoints listed above. In addition, the stimulation of neurogenic spots by electrical, mechanical, or chemical means alleviated pathological conditions in rat colitis and hypertension models via the endogenous opioid system. Our results suggest that acupoints associated with internal organs may be identical to neurogenic inflammatory spots on the skin, which are produced by activation of somatic afferents in abnormal conditions of visceral organs.
Literature context: gG Molecular Probes Cat#A11055, RRID:AB_142672 Alexa 488-conjugated goat anti-
Intestinal organoids hold great promise as a valuable tool for studying and treating intestinal diseases. The currently available sources of human intestinal organoids, tissue fragments or pluripotent stem cells, involve invasive procedures or complex differentiation protocols, respectively. Here, we show that a set of four transcription factors, Hnf4α, Foxa3, Gata6, and Cdx2, can directly reprogram mouse fibroblasts to acquire the identity of fetal intestine-derived progenitor cells (FIPCs). These induced FIPCs (iFIPCs) form spherical organoids that develop into adult-type budding organoids containing cells with intestinal stem cell properties. The resulting stem cells produce all intestinal epithelial cell lineages and undergo self-renewing cell divisions. After transplantation, the induced spherical and budding organoids can reconstitute colonic and intestinal epithelia, respectively. The same combination of four defined transcription factors can also induce human iFIPCs. This alternative approach for producing intestinal organoids may well facilitate application for disease analysis and therapy development.
Literature context: # A11055; RRID:AB_142672 Mouse Anti
Targeting the tumor vasculature with antibody-drug conjugates (ADCs) is a promising anti-cancer strategy that in order to be realized must overcome several obstacles, including identification of suitable targets and optimal warheads. Here, we demonstrate that the cell-surface protein CD276/B7-H3 is broadly overexpressed by multiple tumor types on both cancer cells and tumor-infiltrating blood vessels, making it a potentially ideal dual-compartment therapeutic target. In preclinical studies CD276 ADCs armed with a conventional MMAE warhead destroyed CD276-positive cancer cells, but were ineffective against tumor vasculature. In contrast, pyrrolobenzodiazepine-conjugated CD276 ADCs killed both cancer cells and tumor vasculature, eradicating large established tumors and metastases, and improving long-term overall survival. CD276-targeted dual-compartment ablation could aid in the development of highly selective broad-acting anti-cancer therapies.
Literature context: . A11055, RRID:AB_142672; or AlexaF
This study refines the characterization of the rat parietal cortical domain in terms of cyto- and chemoarchitecture as well as thalamic connectivity. We recognize three subdivisions of the posterior parietal cortex (PPC), which are architectonically distinct from the neighboring somatosensory and visual cortices. Furthermore, we show that the different parietal areas are differently connected with thalamic nuclei. The medial portion of PPC (mPPC) is connected primarily with the medial portion of the lateral posterior nucleus (LP), whereas the lateral portion (lPPC) connects with the posterior complex (Po). The more caudolateral part of PPC (PtP) also projects to Po but can be distinguished from lPPC based on architectonic criteria. The primary somatic and visual cortices, neighboring PPC, are preferentially connected with the primary ventral posterior and dorsolateral geniculate nuclei, respectively, and less with the associational Po and LP. Particularly the border between the secondary visual cortex and the PPC has been a matter of controversy, but here we show that, although PPC subareas are connected with Po and medial LP, the medial and lateral secondary visual cortices are connected with lateral LP and a portion of medial LP different from that connected with PPC. The resulting delineations and specifications of connectivity with thalamic nuclei together with upcoming studies of cortical connectivity will facilitate detailed studies on the role of the subdivisions of PPC in the rat as diverse, higher order associative cortical areas, comparable to those described in the primate.for J. Comp. Neurol. 524:3774-3809, 2016. © 2016 Wiley Periodicals, Inc.
Literature context: ","term_text":"A21244"}}A21244 [RRID: AB_142672]1:1000Donkey anti-goat (AlexaFl
Ischemic stroke is a leading cause of adult disability, including cognitive impairment. Our laboratory has previously shown that treatment with function-blocking antibodies against the neurite growth inhibitory protein Nogo-A promotes functional recovery after stroke in adult and aged rats, including enhancing spatial memory performance, for which the hippocampus is critically important. Since spatial memory has been linked to hippocampal neurogenesis, we investigated whether anti-Nogo-A treatment increases hippocampal neurogenesis after stroke. Adult rats were subject to permanent middle cerebral artery occlusion followed 1 week later by 2 weeks of antibody treatment. Cellular proliferation in the dentate gyrus was quantified at the end of treatment, and the number of newborn neurons was determined at 8 weeks post-stroke. Treatment with both anti-Nogo-A and control antibodies stimulated the accumulation of new microglia/macrophages in the dentate granule cell layer, but neither treatment increased cellular proliferation or the number of newborn neurons above stroke-only levels. These results suggest that anti-Nogo-A immunotherapy does not increase post-stroke hippocampal neurogenesis.
Literature context: # A11055; RRID:AB_142672 AlexaFluor
Two complementary approaches were used in search of the intracellular targets of the toxic PR poly-dipeptide encoded by the repeat sequences expanded in the C9orf72 form of amyotrophic lateral sclerosis. The top categories of PRn-bound proteins include constituents of non-membrane invested cellular organelles and intermediate filaments. PRn targets are enriched for the inclusion of low complexity (LC) sequences. Evidence is presented indicating that LC sequences represent the direct target of PRn binding and that interaction between the PRn poly-dipeptide and LC domains is polymer-dependent. These studies indicate that PRn-mediated toxicity may result from broad impediments to the dynamics of cell structure and information flow from gene to message to protein.
GPR88 is a neuronal cerebral orphan G-protein-coupled receptor (GPCR) that has been linked to various psychiatric disorders. However, no extensive description of its localization has been provided so far. Here, we investigate the spatiotemporal expression of the GPR88 in prenatal and postnatal rat tissues by using in situ hybridization and immunohistochemistry. GPR88 protein was initially detected at embryonic day 16 (E16) in the striatal primordium. From E16-E20 to adulthood, the highest expression levels of both protein and mRNA were observed in striatum, olfactory tubercle, nucleus accumbens, amygdala, and neocortex, whereas in spinal cord, pons, and medulla GPR88 expression remains discrete. We observed an intracellular redistribution of GPR88 during cortical lamination. In the cortical plate of the developing cortex, GPR88 presents a classical GPCR plasma membrane/cytoplasmic localization that shifts, on the day of birth, to nuclei of neurons progressively settling in layers V to II. This intranuclear localization remains throughout adulthood and was also detected in monkey and human cortex as well as in the amygdala and hypothalamus of rats. Apart from the central nervous system, GPR88 was transiently expressed at high levels in peripheral tissues, including adrenal cortex (E16-E21) and cochlear ganglia (E19-P3), and also at moderate levels in retina (E18-E19) and spleen (E21-P7). The description of the GPR88 anatomical expression pattern may provide precious functional insights into this novel receptor. Furthermore, the GRP88 nuclear localization suggests nonclassical GPCR modes of action of the protein that could be relevant for cortical development and psychiatric disorders. J. Comp. Neurol. 524:2776-2802, 2016. © 2016 Wiley Periodicals, Inc.
Literature context: at 1:400 (RRID:AB_142672) (Table 2)
Functional imaging studies have revealed that certain brainstem areas are activated during migraine attacks. The neuropeptide calcitonin gene-related peptide (CGRP) is associated with activation of the trigeminovascular system and transmission of nociceptive information and plays a key role in migraine pathophysiology. Therefore, to elucidate the role of CGRP, it is critical to identify the regions within the brainstem that process CGRP signaling. In situ hybridization and immunofluorescence were performed to detect mRNA expression and define cellular localization of calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1), respectively. To define CGRP receptor binding sites, in vitro autoradiography was performed with [(3)H]MK-3207 (a CGRP receptor antagonist). CLR and RAMP1 mRNA and protein expression were detected in the pineal gland, medial mammillary nucleus, median eminence, infundibular stem, periaqueductal gray, area postrema, pontine raphe nucleus, gracile nucleus, spinal trigeminal nucleus, and spinal cord. RAMP1 mRNA expression was also detected in the posterior hypothalamic area, trochlear nucleus, dorsal raphe nucleus, medial lemniscus, pontine nuclei, vagus nerve, inferior olive, abducens nucleus, and motor trigeminal nucleus; protein coexpression of CLR and RAMP1 was observed in these areas via immunofluorescence. [(3)H]MK-3207 showed high binding densities concordant with mRNA and protein expression. The present study suggests that several regions in the brainstem may be involved in CGRP signaling. Interestingly, we found receptor expression and antagonist binding in some areas that are not protected by the blood-brain barrier, which suggests that drugs inhibiting CGRP signaling may not be able to penetrate the central nervous system to antagonize receptors in these brain regions.
In the stomach, somatostatin (SST) acts as a general paracrine negative regulator of exocrine secretion of gastric acid and pepsinogen and endocrine secretion of gastrin, ghrelin, and histamine. Using reporter mice expressing red fluorescent protein (RFP) under control of the SST promotor, we have characterized the G protein-coupled receptors expressed in gastric Sst-RFP-positive cells and probed their effects on SST secretion in primary cell cultures. Surprisingly, besides SST, amylin and PYY were also highly enriched in the SST cells. Several receptors found to regulate SST secretion were highly expressed and/or enriched. 1) The metabolite receptors calcium-sensing receptor and free fatty acid receptor 4 (GPR120) functioned as positive and negative regulators, respectively. 2) Among the neurotransmitter receptors, adrenergic receptors α1a, α2a, α2b, and β1 were all highly expressed, with norepinephrine and isoproterenol acting as positive regulators. The muscarinic receptor M3 acted as a positive regulator, whereas M4 was conceivably a negative regulator. 3) Of the hormone receptors, the GLP-1 and GIP receptors, CCKb (stimulated by both CCK and gastrin) and surprisingly the melanocortin MC1 receptor were all positive regulators. 4) The neuropeptide receptors for calcitonin gene-related peptide, adrenomedullin, and vasoactive intestinal peptide acted as positive regulators, no effect was observed using galanin and nociceptin although transcripts for the corresponding receptors appeared highly expressed. 5) The SST receptors 1 and 2 functioned in an autocrine negative feedback loop. Thus, the article provides a comprehensive map of receptors through which SST secretion is regulated by hormones, neurotransmitters, neuropeptides and metabolites that act directly on the SST cells in the gastric mucosa.
Literature context: 8 or 546, RRID:AB_142672 and AB_142
The insular cortex is involved in the perception of interoceptive signals, coding of emotional and affective states, and processing information from gustatory, olfactory, auditory, somatosensory, and nociceptive modalities. This information represents an important component of episodic memory, mediated by the parahippocampal-hippocampal region. A comprehensive description of insular projections to the latter region is lacking. Previous studies reported that insular projections do not target any of the subdivisions in the hippocampal formation (the dentate gyus, the cornu ammonis [CA] fields 1, 2, and 3 and the subiculum), but, in contrast, target the parahippocampal region (perirhinal, postrhinal, lateral and medial entorhinal cortices, and pre- and parasubiculum). The present study examined the topographical and laminar organization of insular projections to the parahippocampal region in the rat with the use of anterograde tracing. Notably, our results corroborated the absence of hippocampal projections. We further showed that the perirhinal and the lateral entorhinal cortices received extensive projections from the insular cortex, primarily from its agranular areas. With the exception of a weak projection to the postrhinal cortex, projections to the remaining parahippocampal areas were either absent or very sparse. The projections to the lateral entorhinal cortex displayed a preference for the deep layers of its most lateral subdivisions, known also to receive hippocampal inputs. Projections to the perirhinal cortex primarily targeted the superficial layers with a preference for its ventral subdivision, referred to as area 35. Our findings indicate that only processed information, reflecting emotional and affective states, but not primary gustatory and viscerosensory information, has direct access to the parahippocampal-hippocampal system.
Formation of migratory extravillous trophoblasts (EVTs) is critical for human placentation and hence embryonic development. However, key regulatory growth factors, hormones, and nuclear proteins controlling the particular differentiation process remain poorly understood. Here, the role of the Wingless (Wnt)-dependent transcription factor T-cell factor-4 (TCF-4) in proliferation and motility was investigated using different trophoblast cell models. Immunofluorescence of first-trimester placental tissues revealed induction of TCF-4 and nuclear recruitment of its coactivator β-catenin in nonproliferating EVTs, whereas membrane-associated β-catenin decreased upon differentiation. In addition, EVTs expressed the TCF-4/β-catenin coactivator Pygopus 2 as well as repressors of the Groucho/transducin-like enhancer of split family. Western blotting revealed Pygopus 2 expression and up-regulation of integrin α1 and nuclear TCF-4 in purified first-trimester cytotrophoblasts (CTBs) differentiating on fibronectin. Concomitantly, elevated TCF-4 mRNA, quantitated by real-time PCR, and increased TCF-dependent luciferase reporter activity were noticed in EVTs of villous explant cultures and differentiated primary CTBs. Gene silencing using specific small interfering RNA decreased TCF-4 transcript and protein levels, TCF-dependent reporter activity as well as basal and Wnt3a-stimulated migration of trophoblastic SGHPL-5 cells and primary CTBs through fibronectin-coated transwells. In contrast, proliferation of SGHPL-5 cells and primary cells, measured by cumulative cell numbers and 5-bromo-2'-deoxy-uridine labeling, respectively, was not affected. Moreover, siRNA-mediated down-regulation of TCF-4 in primary CTBs diminished markers of the differentiated EVT, such as integrin α1 and α5, Snail1, and Notch2. In summary, the data suggest that Wnt/TCF-4-dependent signaling could play a role in EVT differentiation promoting motility and expression of promigratory genes.
We have previously demonstrated that rat islets express a high density of angiotensin type 2 receptors and that activation of this receptor evokes insulinotropic effect. In this study, we evaluated the protective effects of Compound 21 (C21), a nonpeptide angiotensin type 2 receptor agonist, on islets in streptozotocin (STZ)-induced diabetes. Rats were assigned to five groups: normal, STZ, and STZ plus C21 (0.24, 0.48, and 0.96 mg/kg·d). C21 was continually infused by a sc implanted osmotic minipump for 14 days, and STZ was bolus injected on day 7. Body weight, water intake, urine excretion, and blood glucose were monitored daily. On the last day, the rats received an oral glucose tolerance test, and the pancreata were saved to examine islet morphology and biochemical parameters of oxidative stress and apoptosis. We found that, compared with control STZ rats, C21-treated STZ rats displayed less water intake and urine excretion, lower blood glucose, higher serum insulin concentration, and improved glucose tolerance. These rats had more islets, larger islet mass, and up-regulated insulin protein and proinsulin 2 mRNA expressions in the pancreas. Their islets displayed lower superoxide, decreased gp91 expression, and increased superoxide dismutase 1 expression as well as less apoptosis and down-regulated caspase-3 expression. In the epididymal adipose tissue of these rats, we found a decreased adipocyte size and up-regulated adipocyte protein 2 expression. The protective effects of C21 on β-cells against the toxic effects of STZ were also confirmed in cultured INS-1E cells. These data suggest that C21 ameliorates STZ-induced diabetes by protecting pancreatic islets via antioxidative and antiapoptotic effects.
Because oxidative stress promotes insulin resistance in obesity and type 2 diabetes, it is crucial to find effective antioxidant for the purpose of decreasing this threat. In this study, we explored the effect of astaxanthin, a carotenoid antioxidant, on insulin signaling and investigated whether astaxanthin improves cytokine- and free fatty acid-induced insulin resistance in vitro. We examined the effect of astaxanthin on insulin-stimulated glucose transporter 4 (GLUT4) translocation, glucose uptake, and insulin signaling in cultured rat L6 muscle cells using plasma membrane lawn assay, 2-deoxyglucose uptake, and Western blot analysis. Next, we examined the effect of astaxanthin on TNFα- and palmitate-induced insulin resistance. The amount of reactive oxygen species generated by TNFα or palmitate with or without astaxanthin was evaluated by dichlorofluorescein staining. We also compared the effect of astaxanthin on insulin signaling with that of other antioxidants, α-lipoic acid and α-tocopherol. We observed astaxanthin enhanced insulin-stimulated GLUT4 translocation and glucose uptake, which was associated with an increase in insulin receptor substrate-1 tyrosine and Akt phosphorylation and a decrease in c-Jun N-terminal kinase (JNK) and insulin receptor substrate-1 serine 307 phosphorylation. Furthermore, astaxanthin restored TNFα- and palmitate-induced decreases in insulin-stimulated GLUT4 translocation or glucose uptake with a concomitant decrease in reactive oxygen species generation. α-Lipoic acid enhanced Akt phosphorylation and decreased ERK and JNK phosphorylation, whereas α-tocopherol enhanced ERK and JNK phosphorylation but had little effect on Akt phosphorylation. Collectively these findings indicate astaxanthin is a very effective antioxidant for ameliorating insulin resistance by protecting cells from oxidative stress generated by various stimuli including TNFα and palmitate.