ABSTRACT
Diverse molecular species of sulfatide with differences in FA lengths, unsaturation degrees, and hydroxylation statuses are expressed in the kidneys. However, the physiological functions of specific sulfatide species in the kidneys are unclear. Here, we evaluated the distribution of specific sulfatide species in the kidneys and their physiological functions. Electron microscopic analysis of kidneys of Cst-deficient mice lacking sulfatide showed vacuolar accumulation in the cytoplasm of intercalated cells in the collecting duct, whereas the proximal and distal tubules were unchanged. Immunohistochemical analysis revealed that vacuolar H+-ATPase-positive vesicles were accumulated in intercalated cells in sulfatide-deficient kidneys. Seventeen sulfatide species were detected in the murine kidney by iMScope MALDI-MS analysis. The distribution of the specific sulfatide species was classified into four patterns. Although most sulfatide species were highly expressed in the outer medullary layer, two unique sulfatide species of m/z 896.6 (predicted ceramide structure: t18:0-C22:0h) and m/z 924.6 (predicted ceramide structure: t18:0-C24:0h) were dispersed along the collecting duct, implying expression in intercalated cells. In addition, the intercalated cell-enriched fraction was purified by fluorescence-activated cell sorting using the anti-vacuolar H+-ATPase subunit 6V0A4, which predominantly contained sulfatide species (m/z 896.6 and 924.6). The Degs2 and Fa2h genes, which are responsible for ceramide hydroxylation, were expressed in the purified intercalated cells. These results suggested that sulfatide molecular species with ceramide composed of phytosphingosine (t18:0) and 2-hydroxy FAs, which were characteristically expressed in intercalated cells, were involved in the excretion of NH3 and protons into the urine.
Subject(s)
Sulfoglycosphingolipids , Vacuolar Proton-Translocating ATPases , Animals , Ceramides , Kidney/metabolism , Mice , Sphingosine/analogs & derivatives , Vacuolar Proton-Translocating ATPases/metabolismABSTRACT
OBJECTIVE: Glioma stem cells (GSCs) are responsible for tumor initiation, therapeutic resistance, and recurrence. CD146 is mainly expressed in dividing GSCs and regulates cell cycle progression. However, the evaluation of the efficacy of targeted therapy against CD146 in vivo remains to be investigated. In this study, the authors aimed to develop gene therapy targeting GSCs using chitosan oligosaccharide lactate (COL) nanoparticles (NPs) conjugated with folic acid-polyethylene glycol (FA-PEG-COL NPs) for in vitro and in vivo delivery of CD146 small-interfering RNA (siCD146) and to determine the effect of CD146 knockdown on tumor growth. METHODS: To examine the uptake of NPs by tumor cells, immunofluorescence staining, flow cytometry, and in vivo imaging were performed. The knockdown effect of siCD146 was measured by western blot and water-soluble tetrazolium salt-8 assay in mouse glioma cells. The efficacy of siRNA therapy-targeted GSCs was evaluated by monitoring tumor growth through in vivo imaging and histological analysis. RESULTS: In vivo accumulation of the FA-PEG-COL NPs in subcutaneous and intracranial gliomas following NP administration via a mouse tail vein was observed. Additionally, in vitro delivery of siCD146 ionically cross-linked NPs, reduced CD146 levels, and suppressed growth in the glioma tumor sphere. Evaluation of the in vivo therapeutic effects of siCD146-cross-linked NPs in a mouse glioma model revealed significant suppression of intracranial tumor growth, with complete removal of the tumor observed in some mice on histological examination. Furthermore, delivery of siCD146 significantly reduced the Ki-67 index in residual tumor tissues relative to that in control mice. CONCLUSIONS: CD146 is a potential therapeutic target, and folic acid-conjugated NPs delivering siRNA may facilitate gene therapy in malignant gliomas.
Subject(s)
Brain Neoplasms/therapy , Folic Acid/administration & dosage , Glioma/therapy , Nanoparticles/administration & dosage , RNA, Small Interfering/administration & dosage , Animals , Brain Neoplasms/genetics , Brain Neoplasms/pathology , CD146 Antigen/antagonists & inhibitors , CD146 Antigen/genetics , Cell Line, Tumor , Chickens , Gene Targeting/methods , Genetic Therapy/methods , Glioma/genetics , Glioma/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Nude , RNA, Small Interfering/geneticsABSTRACT
Regenerative medicine using umbilical cord blood (UCB) cells shows promise for the treatment of cerebral palsy. Although the efficacy of this therapy has been seen in the clinic, the mechanisms by which UCB cells interact and aid in the improvement of symptoms are not clear. We explored the chemokine expression profile in damaged brain tissue in the neonatal mouse ischemia-reperfusion (IR) brain injury model that was infused with human UCB (hUCB) cells. IR brain injury was induced in 9-day-old NOD/SCID mice. hUCB cells were administered 3 weeks post brain injury. Chemokine expression profiles in the brain extract were determined at various time points. Inflammatory chemokines such as CCL1, CCL17, and CXCL12 were transiently upregulated by 24 hours post brain injury. Upregulation of other chemokines, including CCL5, CCL9, and CXCL1 were prolonged up to 3 weeks post brain injury, but most chemokines dissipated over time. There were marked increases in levels of CCL2, CCL12, CCL20, and CX3CL1 in response to hUCB cell treatment, which might be related to the new recruitment and differentiation of neural stem cells, leading to the induction of tissue regeneration. We propose that the chemokine expression profile in the brain shifted from responding to tissue damage to inducing tissue regeneration. hUCB cell administration further enhanced the production of chemokines, and chemokine networks may play an active role in tissue regeneration in neonatal hypoxic-ischemic brain injury.
Subject(s)
Brain Injuries/etiology , Brain Injuries/pathology , Chemokines/genetics , Cord Blood Stem Cell Transplantation , Fetal Blood/cytology , Reperfusion Injury/etiology , Reperfusion Injury/pathology , Animals , Biomarkers , Brain Injuries/therapy , Chemokines/metabolism , Cord Blood Stem Cell Transplantation/methods , Cytokines/metabolism , Disease Models, Animal , Humans , Mice , Mice, Inbred NOD , Mice, SCID , Reperfusion Injury/therapyABSTRACT
BACKGROUND: Neonatal hypoxia-ischemia induces massive brain damage during the perinatal period, resulting in long-term consequences to central nervous system structural and functional maturation. Although neural progenitor cells (NPCs) migrate through the parenchyma and home in to injury sites in the rodent brain, the molecular mechanisms are unknown. We examined the role of chemokines in mediating NPC migration after neonatal hypoxic-ischemic brain injury. METHODS: Nine-day-old mice were exposed to a 120-minute hypoxia following unilateral carotid occlusion. Chemokine levels were quantified in mouse brain extract. Migration and proliferation assays were performed using embryonic and infant mouse NPCs. RESULTS: The neonatal hypoxic-ischemic brain injury resulted in an ipsilateral lesion, which was extended to the cortical and striatal areas. NPCs migrated toward an injured area, where a marked increase of CC chemokines was detected. In vitro studies showed that incubation of NPCs with recombinant mouse CCL11 promoted migration and proliferation. These effects were partly inhibited by a CCR3 antagonist, SB297006. CONCLUSIONS: Our data implicate an important effect of CCL11 for mouse NPCs. The effective activation of NPCs may offer a promising strategy for neuroregeneration in neonatal hypoxic-ischemic brain injury.
Subject(s)
Brain Injuries/pathology , Brain/pathology , Chemokine CCL11/pharmacology , Hypoxia-Ischemia, Brain/pathology , Neural Stem Cells/drug effects , Animals , Animals, Newborn , Benzamides/pharmacology , Brain/metabolism , Brain Injuries/metabolism , Carotid Arteries/surgery , Cell Movement/drug effects , Cell Proliferation/drug effects , Cerebrovascular Disorders/surgery , Chemokine CCL11/metabolism , Chemokines, CC/metabolism , Disease Models, Animal , Hypoxia-Ischemia, Brain/metabolism , Mice , Mice, Inbred NOD , Mice, SCID , Neural Stem Cells/cytology , Neural Stem Cells/metabolism , Phenylalanine/analogs & derivatives , Phenylalanine/pharmacology , Primary Cell Culture , Receptors, CCR3/antagonists & inhibitors , Receptors, CCR3/genetics , Receptors, CCR3/metabolismABSTRACT
OBJECTIVE: Neonatal hypoxic-ischemic encephalopathy (HIE) is caused by brain injury that occurs in a developing fetus or infant. Stem cell transplantation can reportedly induce functional recovery in animal models of HIE. Murine neonatal splenocytes are enriched with immature blood stem cells and are used for the investigation of murine models of syngeneic transplantation. The aim of this study was to investigate the therapeutic potential of newborn splenocytes in a murine model of neonatal ischemia-reperfusion brain injury. METHODS: C57BL/6N mice (postnatal day 7) underwent right common carotid artery occlusion with an aneurysm clip. Following hypoxic exposure, reperfusion was achieved by unclamping the artery. Newborn splenocytes were transplanted intravenously at 3 weeks after injury. RESULTS: The splenocytes transplanted group tended to show an improvement in behavioral tests, but it was not significantly different compared with the control groups. The transplanted cells were localized in various organs including injured brain tissue over 3 weeks. In the penumbra region of the brain, vascular endothelial growth factor (VEGF) expression was upregulated after transplantation. CONCLUSIONS: These results showed that syngeneic transplantation of newborn splenocytes achieved the long-term survival of the grafts and exerted influence the microenvironment in the injured brains of mice.
Subject(s)
Hypoxia-Ischemia, Brain/therapy , Reperfusion Injury/therapy , Spleen/cytology , Stem Cell Transplantation , Animals , Blotting, Western , Flow Cytometry , Hypoxia-Ischemia, Brain/diagnosis , Immunohistochemistry , Magnetic Resonance Imaging , Mice , Mice, Inbred C57BL , Reperfusion Injury/diagnosis , Transplantation, Isogeneic , Treatment OutcomeABSTRACT
Human umbilical cord blood (HUCB) cells are rich source of immature stem cells, which have the potential to repair lost tissue. Intractable central nervous system (CNS) disorders are important targets for regenerative medicine, and the application of HUCB cells is being investigated in animal models of CNS disorders. Transplantation of HUCB has induced functional improvements in these animal models due to multiple therapeutic effects including neuroprotection, anti-inflammation, angiogenesis, and neurogenesis. HUCB cells are easily available and safer than other stem cells used in transplantation therapy. In this review, we focus on HUCB transplantation as an encouraging therapeutic approach for animal models of neonatal hypoxic-ischemic brain injury and ischemic stroke.
Subject(s)
Cord Blood Stem Cell Transplantation/methods , Hypoxia-Ischemia, Brain/surgery , Stroke/surgery , Animals , Disease Models, Animal , Humans , Infant, NewbornABSTRACT
Ureaplasma spp. is detected in the urogenital tract, including the vagina, cervix, chorioamnion, and placenta. Their colonization is associated with histologic chorioamnionitis (CAM), often observed in placentas from preterm delivery. We isolated Ureaplasma spp. from 63 preterm placentas among 151 specimens, which were delivered at <32 wk of gestation. Of the 63 placentas, 52 (83%) revealed CAM in cultures positive for Ureaplasma spp., however, CAM was observed only in 30% (26/88) of cultures negative for Ureaplasma spp. (p < 0.01). Colonization by Ureaplasma spp. was an independent risk factor for CAM (OR, 11.27; 95% CI, 5.09-24.98). Characteristic neutrophil infiltration was observed in the amnion and subchorion (bistratified pattern) in cultures positive for Ureaplasma spp. FISH analysis of CAM placenta with male infant pregnancy indicated that bistratified infiltrated neutrophils showed the XX karyotype and umbilical vein infiltrated neutrophils showed XY karyotype. The distribution of sulfoglycolipid, the receptor of Ureaplasma spp., was mainly detected in the amnion. Ureaplasmal urease D protein and ureB gene were both detected in the amnion, indicating direct colonization by Ureaplasma spp.
Subject(s)
Chorioamnionitis/microbiology , Placenta/microbiology , Premature Birth/microbiology , Ureaplasma Infections/microbiology , Ureaplasma/isolation & purification , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Case-Control Studies , Chorioamnionitis/genetics , Chorioamnionitis/immunology , Chromosomes, Human, X , Chromosomes, Human, Y , Female , Gestational Age , Glycolipids/metabolism , Humans , Immunohistochemistry , In Situ Hybridization, Fluorescence , Logistic Models , Male , Neutrophil Infiltration , Odds Ratio , Placenta/immunology , Polymerase Chain Reaction , Pregnancy , Premature Birth/genetics , Premature Birth/immunology , Risk Assessment , Risk Factors , Ureaplasma/genetics , Ureaplasma/metabolism , Ureaplasma Infections/complications , Ureaplasma Infections/genetics , Ureaplasma Infections/immunology , Urease/genetics , Urease/metabolismABSTRACT
Certain glycosphingolipids play important roles as cellular receptor for bacterial toxins with high specificity and strong affinity. In particular AB(5) toxins exhibit typical modes of cell attachment with B5 and invasion and biological effects in cells with A subunit. Subtilase cytotoxin (SubAB) is the prototype of a recently discovered AB(5) cytotoxin family produced by certain strains of Shiga toxigenic Escherichia coli, and shows highly specific serine protease activity toward endoplasmic reticulum chaperone Bip. Since this toxin bound to a mimic of ganglioside GM2, GM2 has been considered to be possible receptor for SubAB. Using six kinds of glycosylation-defective knockout mice lacking certain group of glycosphingolipids, sensitivity to SubAB in vivo was analyzed. Consequently, all mutant mice died at around 70h after intraperitoneal injection of 10 microg (or 7.5 microg) of SubAB as well as wild type mice. These results indicated none of glycolipids are not pivotal receptor for SubAB in the body.
Subject(s)
Escherichia coli Proteins/toxicity , G(M2) Ganglioside/metabolism , Molecular Mimicry , Subtilisins/toxicity , Animals , Escherichia coli Proteins/metabolism , G(M2) Ganglioside/genetics , Glycosylation , Mice , Mice, Knockout , Subtilisins/metabolismABSTRACT
Bifidobacterium bifidum is a useful probiotic agent exhibiting health-promoting properties and contains d-aspartate as an essential component of the cross-linker moiety in the peptidoglycan. To help understand D-aspartate biosynthesis in B. bifidum NBRC 14252, aspartate racemase, which catalyzes the racemization of D- and L-aspartate, was purified to homogeneity and characterized. The enzyme was a monomer with a molecular mass of 27 kDa. This is the first report showing the presence of a monomeric aspartate racemase. Its enzymologic properties, such as its lack of cofactor requirement and susceptibility to thiol-modifying reagents in catalysis, were similar to those of the dimeric aspartate racemase from Streptococcus thermophilus. The monomeric enzyme, however, showed a novel characteristic, namely, that its thermal stability significantly increased in the presence of aspartate, especially the D-enantiomer. The gene encoding the monomeric aspartate racemase was cloned and overexpressed in Escherichia coli cells. The nucleotide sequence of the aspartate racemase gene encoded a peptide containing 241 amino acids with a calculated molecular mass of 26 784 Da. The recombinant enzyme was purified to homogeneity and its properties were almost the same as those of the B. bifidum enzyme.