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1.
Immunol Lett ; 242: 1-7, 2022 02.
Article in English | MEDLINE | ID: mdl-35007661

ABSTRACT

SARS-CoV-2 harbors a unique S1/S2 furin cleavage site within its spike protein, which can be cleaved by furin and other proprotein convertases. Proteolytic activation of SARS-CoV-2 spike protein at the S1/S2 boundary facilitates interaction with host ACE2 receptor for cell entry. To address this, high titer antibody was generated against the SARS-CoV-2-specific furin motif. Using a series of innovative ELISA-based assays, this furin site blocking antibody displayed high sensitivity and specificity for the S1/S2 furin cleavage site, including with a P681R mutation, and demonstrated effective blockage of both enzyme-mediated cleavage and spike-ACE2 interaction. The results suggest that immunological blocking of the furin cleavage site may afford a suitable approach to stem proteolytic activation of SARS-CoV-2 spike protein and curtail viral infectivity.


Subject(s)
Angiotensin-Converting Enzyme 2/metabolism , Antibodies, Viral/immunology , Furin/metabolism , Spike Glycoprotein, Coronavirus/immunology , Amino Acid Motifs/genetics , Amino Acid Motifs/immunology , Antibodies, Viral/pharmacology , Humans , Mutation , Nose/enzymology , Proprotein Convertases/metabolism , Protein Binding/drug effects , Proteolysis/drug effects , SARS-CoV-2/drug effects , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism
2.
Am J Physiol Endocrinol Metab ; 314(5): E512-E521, 2018 05 01.
Article in English | MEDLINE | ID: mdl-29351476

ABSTRACT

Three-dimensional (3D) pseudoislets (PIs) can be used for the study of insulin-producing ß-cells in free-floating islet-like structures similar to that of primary islets. Previously, we demonstrated the ability of islet-derived endothelial cells (iECs) to induce PIs using murine insulinomas, where PI formation enhanced insulin production and glucose responsiveness. In this report, we examined the ability of iECs to spontaneously induce the formation of free-floating 3D PIs using the EndoC-ßH1 human ß-cell line murine MS1 iEC. Within 14 days, the coculturing of both cell types produced fully humanized EndoC-ßH1 PIs with little to no contaminating murine iECs. The size and shape of these PIs were similar to primary human islets. iEC-induced PIs demonstrated reduced dysregulated insulin release under low glucose levels and higher insulin secretion in response to high glucose and exendin-4 [a glucagon-like peptide-1 (GLP-1) analog] compared with monolayer cells cultured alone. Interestingly, iEC-PIs were also better at glucose sensing in the presence of extendin-4 compared with PIs generated on a low-adhesion surface plate in the absence of iECs and showed an overall improvement in cell viability. iEC-induced PIs exhibited increased expression of key genes involved in glucose transport, glucose sensing, ß-cell differentiation, and insulin processing, with a concomitant decrease in glucagon mRNA expression. The enhanced responsiveness to exendin-4 was associated with increased protein expression of GLP-1 receptor and phosphokinase A. This rapid coculture system provides an unlimited number of human PIs with improved insulin secretion and GLP-1 responsiveness for the study of ß-cell biology.


Subject(s)
Endothelial Cells/metabolism , Glucagon-Like Peptide 1/metabolism , Glucose/metabolism , Insulin-Secreting Cells/metabolism , Islets of Langerhans/cytology , Islets of Langerhans/metabolism , Cells, Cultured , Coculture Techniques/methods , Endothelial Cells/cytology , Endothelial Cells/drug effects , Glucagon-Like Peptide 1/pharmacology , Glucose Intolerance/metabolism , Glucose Intolerance/pathology , Humans , Insulin/metabolism , Insulin Resistance/physiology , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/drug effects , Signal Transduction/drug effects
3.
Diabetol Metab Syndr ; 8(1): 60, 2016.
Article in English | MEDLINE | ID: mdl-27563355

ABSTRACT

BACKGROUND: Gestational diabetes mellitus (GDM) affects approximately 7-17 % of all pregnancies and has been recognized as a significant risk factor to neonatal and maternal health. Postpartum, GDM significantly increases the likelihood of developing type 2 diabetes (T2D). While it is well established that insulin resistance and impaired ß-cell function contribute to GDM development, the role of active ß-cell loss remains unknown. Differentially methylated circulating free DNA (cfDNA) is a minimally invasive biomarker of ß-cell loss in type 1 diabetes mellitus. Here we use cfDNA to examine the levels of ß-cell death in women with GDM. METHODS: Second to third-trimester pregnant women with GDM were compared with women with normal pregnancy (PRG), women at postpartum (PP), and non-pregnant (NP) women. Fasting glucose levels, insulin, and C-peptide levels were measured. Serum samples were collected and cfDNA purified and bisulfite treated. Methylation-sensitive probes capable of differentiating between ß-cell-derived DNA (demethylated) and non-ß-cell-derived DNA (methylated) were used to measure the presence of ß-cell loss in the blood. RESULTS: GDM was associated with elevated fasting glucose levels (GDM = 185.9 ± 5.0 mg/dL) and reduced fasting insulin and c-peptide levels when compared with NP group. Interestingly, ß-cell derived insulin DNA levels were significantly lower in women with GDM when compared with PRG, NP, and PP groups (demethylation index: PRG = 7.74 × 10(-3) ± 3.09 × 10(-3), GDM = 1.01 × 10(-3) ± 5.86 × 10(-4), p < 0.04; NP = 4.53 × 10(-3) ± 1.62 × 10(-3), PP = 3.24 × 10(-3) ± 1.78 × 10(-3)). CONCLUSIONS: These results demonstrate that ß-cell death is reduced in women with GDM. This reduction is associated with impaired insulin production and hyperglycemia, suggesting that ß-cell death does not contribute to GDM during the 2nd and 3rd trimester of pregnancy.

4.
EBioMedicine ; 10: 227-35, 2016 Aug.
Article in English | MEDLINE | ID: mdl-27381476

ABSTRACT

Multiple sclerosis (MS) is a neurodegenerative disease of the central nervous system (CNS). Minimally invasive biomarkers of MS are required for disease diagnosis and treatment. Differentially methylated circulating-free DNA (cfDNA) is a useful biomarker for disease diagnosis and prognosis, and may offer to be a viable approach for understanding MS. Here, methylation-specific primers and quantitative real-time PCR were used to study methylation patterns of the myelin oligodendrocyte glycoprotein (MOG) gene, which is expressed primarily in myelin-producing oligodendrocytes (ODCs). MOG-DNA was demethylated in O4(+) ODCs in mice and in DNA from human oligodendrocyte precursor cells (OPCs) when compared with other cell types. In the cuprizone-fed mouse model of demyelination, ODC derived demethylated MOG cfDNA was increased in serum and was associated with tissue-wide demyelination, demonstrating the utility of demethylated MOG cfDNA as a biomarker of ODC death. Collected sera from patients with active (symptomatic) relapsing-remitting MS (RRMS) demonstrated a higher signature of demethylated MOG cfDNA when compared with patients with inactive disease and healthy controls. Taken together, these results offer a minimally invasive approach to measuring ODC death in the blood of MS patients that may be used to monitor disease progression.


Subject(s)
Biomarkers , Multiple Sclerosis/blood , Multiple Sclerosis/diagnosis , Oligodendroglia/pathology , Adult , Animals , Cell Line , Central Nervous System/metabolism , Central Nervous System/pathology , DNA Methylation , Female , Humans , Mice , Multiple Sclerosis/genetics , Multiple Sclerosis, Relapsing-Remitting/blood , Multiple Sclerosis, Relapsing-Remitting/diagnosis , Multiple Sclerosis, Relapsing-Remitting/genetics , Myelin-Oligodendrocyte Glycoprotein/genetics , Oligodendroglia/metabolism , Schwann Cells/metabolism
5.
PLoS One ; 11(4): e0152662, 2016.
Article in English | MEDLINE | ID: mdl-27111653

ABSTRACT

In type 1 diabetes (T1D), ß-cell loss is silent during disease progression. Methylation-sensitive quantitative real-time PCR (qPCR) of ß-cell-derived DNA in the blood can serve as a biomarker of ß-cell death in T1D. Amylin is highly expressed by ß-cells in the islet. Here we examined whether demethylated circulating free amylin DNA (cfDNA) may serve as a biomarker of ß-cell death in T1D. ß cells showed unique methylation patterns within the amylin coding region that were not observed with other tissues. The design and use of methylation-specific primers yielded a strong signal for demethylated amylin in purified DNA from murine islets when compared with other tissues. Similarly, methylation-specific primers detected high levels of demethylated amylin DNA in human islets and enriched human ß-cells. In vivo testing of the primers revealed an increase in demethylated amylin cfDNA in sera of non-obese diabetic (NOD) mice during T1D progression and following the development of hyperglycemia. This increase in amylin cfDNA did not mirror the increase in insulin cfDNA, suggesting that amylin cfDNA may detect ß-cell loss in serum samples where insulin cfDNA is undetected. Finally, purified cfDNA from recent onset T1D patients yielded a high signal for demethylated amylin cfDNA when compared with matched healthy controls. These findings support the use of demethylated amylin cfDNA for detection of ß-cell-derived DNA. When utilized in conjunction with insulin, this latest assay provides a comprehensive multi-gene approach for the detection of ß-cell loss.


Subject(s)
B-Lymphocytes/pathology , Biomarkers/metabolism , DNA Methylation , Diabetes Mellitus, Type 1/pathology , Islet Amyloid Polypeptide/genetics , Adolescent , Animals , Child , Female , Humans , Male , Mice
6.
J Biol Chem ; 290(24): 15250-9, 2015 Jun 12.
Article in English | MEDLINE | ID: mdl-25911095

ABSTRACT

The co-culturing of insulinoma and islet-derived endothelial cell (iEC) lines results in the spontaneous formation of free-floating pseudoislets (PIs). We previously showed that iEC-induced PIs display improved insulin expression and secretion in response to glucose stimulation. This improvement was associated with a de novo deposition of extracellular matrix (ECM) proteins by iECs in and around the PIs. Here, iEC-induced PIs were used to study the expression and posttranslational modification of the ECM receptor integrin ß1. A wide array of integrin ß subunits was detected in ßTC3 and NIT-1 insulinomas as well as in primary islets, with integrin ß1 mRNA and protein detected in all three cell types. Interestingly, the formation of iEC-induced PIs altered the glycosylation patterns of integrin ß1, resulting in a higher molecular weight form of the receptor. This form was found in native pancreas but was completely absent in monolayer ß-cells. Fluorescence-activated cell sorting analysis of monolayers and PIs revealed a higher expression of integrin ß1 in PIs. Antibody-mediated blocking of integrin ß1 led to alterations in ß-cell morphology, reduced insulin gene expression, and enhanced glucose secretion under baseline conditions. These results suggest that iEC-induced PI formation may alter integrin ß1 expression and posttranslational modification by enhancing glycosylation, thereby providing a more physiological culture system for studying integrin-ECM interactions in ß cells.


Subject(s)
Integrin beta1/metabolism , Islets of Langerhans/cytology , Islets of Langerhans/metabolism , Animals , Base Sequence , Cell Line, Tumor , Cell Membrane/metabolism , DNA Primers , Endothelium/cytology , Endothelium/metabolism , Glycosylation , Humans , Male , Mice , Mice, Inbred C57BL , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction
7.
PLoS One ; 8(8): e72260, 2013.
Article in English | MEDLINE | ID: mdl-24015227

ABSTRACT

ß cell pseudoislets (PIs) are used for the in vitro study of ß-cells in a three-dimensional (3-D) configuration. Current methods of PI induction require unique culture conditions and extensive mechanical manipulations. Here we report a novel co-culture system consisting of high passage ß-cells and islet-derived endothelial cells (iECs) that results in a rapid and spontaneous formation of free-floating PIs. PI structures were formed as early as 72 h following co-culture setup and were preserved for more than 14 d. These PIs, composed solely of ß-cells, were similar in size to that of native islets and showed an increased percentage of proinsulin-positive cells, increased insulin gene expression in response to glucose stimulation, and restored glucose-stimulated insulin secretion when compared to ß-cells cultured as monolayers. Key extracellular matrix proteins that were absent in ß-cells cultured alone were deposited by iECs on PIs and were found in and around the PIs. iEC-induced PIs are a readily available tool for examining ß cell function in a native 3-D configuration and can be used for examining ß-cell/iEC interactions in vitro.


Subject(s)
Cell Differentiation , Endothelial Cells/physiology , Insulin-Secreting Cells/physiology , Animals , Cell Culture Techniques , Cell Line, Tumor , Collagen Type IV/metabolism , Islets of Langerhans/cytology , Laminin/metabolism , Mice
8.
Biochemistry ; 49(27): 5753-9, 2010 Jul 13.
Article in English | MEDLINE | ID: mdl-20515078

ABSTRACT

The cationic lipid 1,3-dimyristoylamidopropane-2-[bis(2-dimethylaminoethane)] carbamate (1,3lb2) was applied as a delivery system for small interfering RNA (siRNA) to inhibit the production of vascular endothelial growth factor (VEGF) in vitro in human prostate carcinoma cell line PC-3. VEGF protein silencing peaked at 94% when cationic lipid-nucleic acid complexes (lipoplexes) were formulated at a nitrogen:phosphorothioate ratio (N:P) of 2 with a dose concentration of 53.7 nM, and the performance of these lipoplexes was not impeded by serum. Knockdown efficiency was maintained for at least 72 h, and an IC(50) of 12 nM lasted for 48 h. Only 20% of the total siRNA became cell-associated at this N:P, at a rate of 25 ng/h. Lipoplexes of the optimal formulation were relatively monodisperse, having an average diameter of 634 nm and a zeta potential of -21.3 mV. Formation of the 1,3lb2-siRNA complex reached 94% at an N:P of 2 and was positively cooperative; the binding constant was calculated in the range of 10(5) M(-1), and a Hill coefficient of 3 was determined. 1,3lb2 was found to be a nontoxic and potent carrier of siRNA that binds to the nucleic acid effectively and whose lipoplexes promote long-lasting inhibition, have high biological activity at low N:Ps, and are functional in the presence of serum.


Subject(s)
Gene Silencing , Gene Transfer Techniques , Lipids/chemistry , RNA Interference , RNA, Small Interfering/chemistry , RNA, Small Interfering/metabolism , Carbamates/pharmacology , Cations/chemistry , Cations/metabolism , Cations/pharmacology , Cell Line , Chemistry, Pharmaceutical , DNA/pharmacology , Genes , Genetic Therapy , Humans , Lipids/pharmacology , Male , Nucleic Acids/pharmacology , Plasmids , RNA, Small Interfering/pharmacology , Transfection , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor A/pharmacology
9.
FEBS J ; 275(1): 148-62, 2008 Jan.
Article in English | MEDLINE | ID: mdl-18067582

ABSTRACT

Novel N,N'-diacyl-1,3-diaminopropyl-2-carbamoyl bivalent cationic lipids were synthesized and their physicochemical properties in lamellar assemblies with and without plasmid DNA were evaluated to elucidate the structural requirements of these double-chained pH-sensitive surfactants for potent non-viral gene delivery and expression. The highest in vitro transfection efficacies were induced at +/-4:1 by the dimyristoyl, dipalmitoyl and dioleoyl derivatives 1,3lb2, 1,3lb3 and 1,3lb5, respectively, without inclusion of helper lipids. Transfection activities were reduced in the presence of either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine alone or in combination with cholesterol for all derivatives except 1,3lb5, which maintained reporter gene expression levels at +/-4:1 and yielded increased lipofection activity at a lower charge ratio of +/-2:1. Ethidium bromide displacement indicated efficient plasmid DNA binding and compaction by the transfection-competent analogs. Dynamic light-scattering and electrophoretic mobility studies revealed lipoplexes of the active lipids with large particle sizes (mean diameter>or=500 nm) and zeta potentials with positive values (low ionic strength) or below neutrality (high ionic strength). Langmuir film balance studies showed high in-plane elasticity of these derivatives in isolation. In agreement with the monolayer experiments, fluorescence polarization studies verified the fluid nature of the highly transfection-efficient amphiphiles, with gel-to-liquid crystalline phase transitions below physiological temperature. The active compounds also interacted with endosome-mimicking vesicles to a greater extent than the poorly active derivative 1,3lb4, as revealed by fluorescence resonance energy transfer experiments. Taken together, the results suggest that well-hydrated and highly elastic cationic lipids with increased acyl chain fluidity and minimal cytotoxicity elicit high transfection activity.


Subject(s)
Fatty Acids/chemistry , Transfection , Cations/chemistry , Dimethylamines/chemical synthesis , Dimethylamines/chemistry , Fatty Acids/chemical synthesis , Kinetics , Particle Size , Plasmids/chemistry , Propanolamines/chemistry , Surface-Active Agents/chemistry , Temperature
10.
Biophys Chem ; 129(2-3): 137-47, 2007 Sep.
Article in English | MEDLINE | ID: mdl-17573180

ABSTRACT

The transfection activity and physicochemical properties of the dimyristoyl derivatives from three novel series of double-chained tertiary cationic lipids were compared. Two of the derivatives were constructed as isomers with different linkages of the same bis-(2-dimethylaminoethane) polar headgroup and hydrophobic chains to the diaminopropanol backbone, while the third was designed with a hydrophilic region containing only a single ionizable amine group. Such systematic molecular changes offer a great opportunity to delineate factors critical for transfection activity, which in this work include the intramolecular distance between the hydrophobic chains and pH-expandability of the polar headgroup. The physical studies comprised a variety of techniques, including pKa determination, Langmuir monolayer studies, fluorescence anisotropy, gel electrophoresis mobility shift assay, ethidium bromide displacement assay, particle size distribution, and zeta potential. These studies are crucial in the development of lipid-based gene delivery systems with improved efficacy. Physicochemical characterization revealed that a symmetric bivalent pH-expandable polar headgroup in combination with greater intramolecular space between the hydrophobic chains provide for high transfection activity through efficient binding and compaction of pDNA, increased acyl chain fluidity, and high molecular elasticity.


Subject(s)
Gene Transfer Techniques , Lipids/chemistry , Transfection/methods , Animals , Cations/chemistry , DNA/chemistry , DNA/metabolism , Electrophoretic Mobility Shift Assay , Hydrogen-Ion Concentration , Melanoma, Experimental/metabolism , Particle Size , Quaternary Ammonium Compounds/chemistry , Transition Temperature
11.
Colloids Surf B Biointerfaces ; 57(1): 108-17, 2007 May 15.
Article in English | MEDLINE | ID: mdl-17336044

ABSTRACT

The in vitro transfection activity of a novel series of N,N'-diacyl-1,2-diaminopropyl-3-carbamoyl-(aminoethane) derivatives was evaluated against a mouse melanoma cell line at different +/- charge ratios, in the presence and absence of helper lipids. Only the unsaturated derivative N,N'-dioleoyl-1,2-diaminopropyl-3-carbamoyl-(aminoethane), (1,2lmp[5]) mediated significant increase in the reporter gene level which was significantly boosted in the presence of DOPE peaking at +/- charge ratio of 2. The electrostatic interactions between the cationic liposomes and plasmid DNA were investigated by gel electrophoresis, fluorescence spectroscopy, dynamic light scattering and electrophoretic mobility techniques. In agreement with the transfection results, 1,2lmp[5]/DOPE formulation was most efficient in associating with and retarding DNA migration. The improved association between the dioleoyl derivative and DNA was further confirmed by ethidium bromide displacement assay and particle size distribution analysis of the lipoplexes. Differential scanning calorimetry studies showed that 1,2lmp[5] was the only lipid that exhibited a main phase transition below 37 degrees C. Likewise, 1,2lmp[5] was the only lipid found to form all liquid expanded monolayers at 23 degrees C. In conclusion, the current findings suggest that high in vitro transfection activity is mediated by cationic lipids characterized by increased acyl chain fluidity and high interfacial elasticity.


Subject(s)
DNA/administration & dosage , Lipids/chemistry , Propane/analogs & derivatives , Propane/chemistry , Transfection/methods , Animals , Calorimetry, Differential Scanning , Cations , Cell Line, Tumor , Cell Survival , Chemical Phenomena , Chemistry, Physical , Electrophoresis, Agar Gel , Ethidium , Indicators and Reagents , Light , Lipids/chemical synthesis , Lipids/toxicity , Liposomes , Melanoma, Experimental/genetics , Membranes, Artificial , Mice , Particle Size , Phosphatidylethanolamines/chemistry , Plasmids/genetics , Propane/chemical synthesis , Propane/toxicity , Scattering, Radiation
12.
Bioconjug Chem ; 16(6): 1411-22, 2005.
Article in English | MEDLINE | ID: mdl-16287237

ABSTRACT

Novel N,N'-diacyl-1,2-diaminopropyl-3-carbamoyl[bis-(2-dimethylaminoethane)] bivalent cationic lipids were synthesized and evaluated for in vitro transfection activity against a murine melanoma cell line. In the absence of the helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), only the dioleoyl derivative 22 (1,2lb5) elicited transfection activity. The transfection activity of this lipid was reduced when formulated with DOPE. Contrary to that, the dimyristoyl derivative 19 (1,2lb2) mediated no activity when used alone but induced the highest levels of marker gene expression in the presence of DOPE. In an effort to correlate the transfection activity with cationic lipid structures, the physicochemical properties of cationic lipids in isolation and of lipoplexes were studied with surface tensiometry, photon correlation spectroscopy, gel electrophoresis mobility shift assay, and fluorescence techniques. In regard to the lipoplex properties, gel electrophoresis mobility shift assay and EtBr exclusion fluorescence assay revealed that the 1,2lb5 was the only lipid to associate and condense plasmid DNA, respectively. Photon correlation spectroscopy analysis found that 1,2lb5/DNA complexes were of relatively small size compared to all other lipoplexes. With respect to the properties of isolated lipids, Langmuir monolayer studies and fluorescence anisotropy on cationic lipid dispersions verified high two-plane elasticity and increased fluidity of the transfection competent dioleoyl derivative 1,2lb5, respectively. The results indicate that high transfection activity is mediated by cationic lipids characterized by an expanded mean molecular area, high molecular elasticity, and increased fluidity.


Subject(s)
Amines/chemical synthesis , Lipids/chemistry , Transfection/methods , Animals , Cations , Cell Line, Tumor , Elasticity , Membrane Fluidity , Mice , Phosphatidylethanolamines , Plasmids/administration & dosage , Surface-Active Agents/chemical synthesis
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