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2.
Viruses ; 12(3)2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32121564

RESUMO

While antiretroviral therapy increases the longevity of people living with HIV (PLWH), about 30% of this population suffers from three or more concurrent comorbidities, whose mechanisms are not well understood. Chronic activation and dysfunction of the immune system could be one potential cause of these comorbidities. We recently demonstrated reduced macrophage infiltration and delayed resolution of inflammation in the lungs of HIV-transgenic mice. Additionally, trans-endothelial migration of HIV-positive macrophages was reduced in vitro. Here, we analyze macrophages' response to LPS challenge in the kidney and peritoneum of HIV-Tg mice. In contrast to the lung infiltration, renal and peritoneal macrophage infiltrations were similar in WT and HIV-Tg mice. Higher levels of HIV-1 gene expression were detected in lung macrophages compared to peritoneal macrophages. In peritoneal macrophages, HIV-1 gene expression was increased when they were cultured at 21% O2 compared to 5% O2, inversely correlating with reduced trans-endothelial migration at higher oxygen levels in vitro. The resolution of macrophage infiltration was reduced in both the lung and the peritoneal cavity of HIV-Tg mice. Taken together, our study described the organ-specific alteration of macrophage dynamics in HIV-Tg mice. The delayed resolution of macrophage infiltration might constitute a risk factor for the development of multiple comorbidities in PLWH.

3.
Front Microbiol ; 10: 2145, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31572348

RESUMO

Ebola virus (EBOV) is a non-segmented negative-sense RNA virus that causes a severe human disease. The ongoing EBOV outbreak in the Eastern part of Democratic Republic of the Congo has resulted to date in over 2500 confirmed cases including over 1500 deaths. Difficulties with vaccine administration indicate the necessity for development of new general drugs and therapeutic strategies against EBOV. Host Ser/Thr protein phosphatases, particularly PP1 and PP2A, facilitate EBOV transcription by dephosphorylating the EBOV VP30 protein and switching activity of the polymerase complex toward replication. Previously, we developed small molecule 1E7-03 that targeted host protein phosphatase-1 (PP1) and induces phosphorylation of EBOV VP30 protein thus shifting transcription-replication balance and inhibiting EBOV replication. Here, we developed a new EBOV inhibitor, 1E7-07, that potently inhibits EBOV replication and displays significantly improved metabolic stability when compared to previously described 1E7-03. Proteome analysis of VP30 shows that 1E7-07 increases its phosphorylation on Thr-119 and Ser-124 over 3-fold with p < 0.001, which likely contributes to EBOV inhibition. We analyzed 1E7-07 binding to PP1 using a mass spectrometry-based protein painting approach. Combined with computational docking, protein painting shows that 1E7-07 binds to several PP1 sites including the RVxF site, C-terminal groove and NIPP1-helix binding pocket. Further analysis using surface plasmon resonance and a split NanoBiT system demonstrates that 1E7-07 binds primarily to the RVxF site. Together, detailed analysis of 1E7-07 binding to PP1 and identification of the RVxF site as the main binding site opens up an opportunity for future development of PP1-targeting EBOV inhibitors.

4.
Anim Biotechnol ; : 1-7, 2019 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-30773109

RESUMO

Lactoferrin (Lf) is a multifunctional bi-lobate iron-binding glycoprotein belonging to transferrin family with a mass of approximately 80 kD. Being ubiquitously present in almost all biological secretions, it performs important biological functions. One of the earliest and very well-documented functions of Lf is the antibacterial effect against broad spectrum Gram-negative and Gram-positive bacteria. In this study, buffalo Lf N-lobe cDNA was amplified, cloned and expressed as a fusion protein in Escherichia coli cells using pQE30 expression vector. After post-induction confirmation of expressed protein by SDS-PAGE, purification of recombinant protein using Ni-NTA was attempted and the yield of recombinant buffalo N-lobe Lf was estimated to be 1 mg/ml. Antibacterial activity of recombinant buffalo Lf N-lobe was assessed on pathogenic E. coli and Staphylococcus aureus strains. Peptic digest of recombinant N-lobe buffalo Lf showed antibacterial activity comparable to commercially available bovine Lf. The successful expression and characterization of functional recombinant N-lobe of buffalo Lf expressed in E. coli opens new vistas for developing alternate therapeutics, particularly against the diseases caused by Gram-negative microbes such as septicemia and diarrhea in newborn calves and mastitis in dairy animals.

5.
Theriogenology ; 125: 152-156, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30447494

RESUMO

Crossbreds of low-producing indigenous cattle and high-producing exotic dairy bulls (Holstein Friesian and Jersey) have contributed in ensuring that India continues to be the world's top milk-producing country. However, subfertility observed in crossbred male progenies has been a major obstacle in exploitation of heterosis due to crossbreeding. There is sufficient scientific evidence in support of genetic and epigenetic regulation of key physiological processes including spermatogenesis. Bovine Vasa Homology (Bvh) is considered a molecular marker for the study of gametogenesis. Significant negative correlation between DNA methylation and gene expression has been reported in cattle-yaks hybrids and their parents. The present study analyzed promoter methylation status and expression profile of Bvh gene in spermatozoa from exotic Holstein Friesian cattle, indigenous Sahiwal cattle and their crossbreds with varying semen motility parameters. The degree of methylation of the Bvh promoter region was significantly higher in poor motility crossbred bulls (13.3%) as compared to good motility crossbreds (5.3%), Sahiwal (3%) and Holstein Friesian bulls (1%) (P < 0.05). Gene expression analysis revealed significantly higher mRNA abundance of Bvh in purebreds (Holstein Friesian and Sahiwal) as compared to crossbred counterparts (P < 0.001). Inverse correlation observed in this study between promoter methylation and gene expression of Bvh gene in spermatozoa from crossbred bulls with poor motility phenotype as compared to purebred parents provides an important insight into understanding the graded fertility of crossbred bulls.


Assuntos
Bovinos/genética , Infertilidade Masculina/veterinária , Sêmen , Motilidade Espermática/genética , Espermatozoides/fisiologia , Animais , Regulação da Expressão Gênica , Marcadores Genéticos , Infertilidade Masculina/genética , Masculino , Motilidade Espermática/fisiologia , Espermatogênese/genética
6.
Environ Microbiol ; 21(2): 814-826, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30585380

RESUMO

The well-known role of antibiotics in killing sensitive organisms has been challenged by the effects they exert at subinhibitory concentrations. Unfortunately, there are very few published reports on the advantages these molecules may confer to their producers. This study describes the construction of a genetically verified deletion mutant of Streptomyces flaviscleroticus unable to synthesize chromomycin. This mutant was characterized by a rapid loss of viability in stationary phase that was correlated with high oxidative stress and altered antioxidant defences. Altered levels of key metabolites in the mutant signalled a redistribution of the glycolytic flux toward the PPP to generate NADPH to fight oxidative stress as well as reduction of ATP-phosphofructokinase and Krebs cycle enzymes activities. These changes were correlated with a shift in the preference for carbon utilization from glucose to amino acids. Remarkably, chromomycin at subinhibitory concentration increased longevity of the non-producer and restored most of the phenotypic features' characteristic of the wild type strain. Altogether these observations suggest that chromomycin may have antioxidant properties that would explain, at least in part, some of the phenotypes of the mutant. Our observations warrant reconsideration of the secondary metabolite definition and raise the possibility of crucial roles for their producers.

7.
J Infect Dis ; 218(suppl_5): S627-S635, 2018 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-30169869

RESUMO

Background: Ebola virus (EBOV) infection causes severe hemorrhagic fever. EBOV transcription is controlled by host protein phosphatase 1 (PP1), which dephosphorylates VP30 protein. We previously developed 1E7-03, a compound targeting a noncatalytic site of PP1 that induced VP30 phosphorylation and inhibited EBOV transcription. Here, we attempted to further improve 1E7-03, which was not stable in murine serum. Results: High-throughput screening with EBOV-green fluorescent protein was conducted on 72 1E7-03 analogs and identified 6 best inhibitory and the least toxic compounds. A parallel in silico screening of compounds from the ZINC database by docking to PP1 identified the best-binding compound C31, which was also present among the top 6 compounds found in the viral screen. C31 showed the best EBOV inhibitory activity among the top 6 compounds and also inhibited EBOV minigenome. C31 bound to the PP1 C-terminal groove in vitro and increased VP30 phosphorylation in cultured cells. C31 demonstrated improved stability in mouse plasma and cell permeability, compared with 1E7-03. It was also detected for 24 hours after injection in mice. Conclusion: C31 represents a novel PP1-targeting EBOV inhibitor with improved pharmacological properties that can be further evaluated for future antifiloviral therapy.


Assuntos
Antivirais/farmacologia , Ebolavirus/efeitos dos fármacos , Proteína Fosfatase 1/metabolismo , Replicação Viral/efeitos dos fármacos , Animais , Domínio Catalítico , Estabilidade de Medicamentos , Ebolavirus/fisiologia , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Fosforilação , Proteína Fosfatase 1/química , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo
8.
Retrovirology ; 15(1): 39, 2018 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-29792216

RESUMO

BACKGROUND: HIV-1 transcription activator protein Tat is phosphorylated in vitro by CDK2 and DNA-PK on Ser-16 residue and by PKR on Tat Ser-46 residue. Here we analyzed Tat phosphorylation in cultured cells and its functionality. RESULTS: Mass spectrometry analysis showed primarily Tat Ser-16 phosphorylation in cultured cells. In vitro, CDK2/cyclin E predominantly phosphorylated Tat Ser-16 and PKR-Tat Ser-46. Alanine mutations of either Ser-16 or Ser-46 decreased overall Tat phosphorylation. Phosphorylation of Tat Ser-16 was reduced in cultured cells treated by a small molecule inhibitor of CDK2 and, to a lesser extent, an inhibitor of DNA-PK. Conditional knock-downs of CDK2 and PKR inhibited and induced one round HIV-1 replication respectively. HIV-1 proviral transcription was inhibited by Tat alanine mutants and partially restored by S16E mutation. Pseudotyped HIV-1 with Tat S16E mutation replicated well, and HIV-1 Tat S46E-poorly, but no live viruses were obtained with Tat S16A or Tat S46A mutations. TAR RNA binding was affected by Tat Ser-16 alanine mutation. Binding to cyclin T1 showed decreased binding of all Ser-16 and Ser-46 Tat mutants with S16D and Tat S46D mutationts showing the strongest effect. Molecular modelling and molecular dynamic analysis revealed significant structural changes in Tat/CDK9/cyclin T1 complex with phosphorylated Ser-16 residue, but not with phosphorylated Ser-46 residue. CONCLUSION: Phosphorylation of Tat Ser-16 induces HIV-1 transcription, facilitates binding to TAR RNA and rearranges CDK9/cyclin T1/Tat complex. Thus, phosphorylation of Tat Ser-16 regulates HIV-1 transcription and may serve as target for HIV-1 therapeutics.


Assuntos
Regulação Viral da Expressão Gênica , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Serina/metabolismo , Transcrição Genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Células Cultivadas , Ciclina T/química , Ciclina T/genética , Ciclina T/metabolismo , Quinase 2 Dependente de Ciclina/química , Quinase 2 Dependente de Ciclina/genética , Quinase 2 Dependente de Ciclina/metabolismo , Quinase 9 Dependente de Ciclina/química , Quinase 9 Dependente de Ciclina/metabolismo , Técnicas de Silenciamento de Genes , Infecções por HIV/genética , Interações Hospedeiro-Patógeno , Humanos , Modelos Biológicos , Modelos Moleculares , Mutação , Fosforilação , Ligação Proteica , Conformação Proteica , RNA Viral , Ubiquitinação , Replicação Viral , eIF-2 Quinase/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética
9.
Haematologica ; 103(5): 787-798, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29519868

RESUMO

Sickle cell disease patients are at increased risk of developing a chronic kidney disease. Endothelial dysfunction and inflammation associated with hemolysis lead to vasculopathy and contribute to the development of renal disease. Here we used a Townes sickle cell disease mouse model to examine renal endothelial injury. Renal disease in Townes mice was associated with glomerular hypertrophy, capillary dilation and congestion, and significant endothelial injury. We also detected substantial renal macrophage infiltration, and accumulation of macrophage stimulating protein 1 in glomerular capillary. Treatment of human cultured macrophages with hemin or red blood cell lysates significantly increased expression of macrophage membrane-associated protease that might cleave and activate circulating macrophage stimulating protein 1 precursor. Macrophage stimulating protein 1 binds to and activates RON kinase, a cell surface receptor tyrosine kinase. In cultured human renal glomerular endothelial cells, macrophage stimulating protein 1 induced RON downstream signaling, resulting in increased phosphorylation of ERK and AKT kinases, expression of Von Willebrand factor, increased cell motility, and re-organization of F-actin. Specificity of macrophage stimulating protein 1 function was confirmed by treatment with RON kinase inhibitor BMS-777607 that significantly reduced downstream signaling. Moreover, treatment of sickle cell mice with BMS-777607 significantly reduced glomerular hypertrophy, capillary dilation and congestion, and endothelial injury. Taken together, our findings demonstrated that RON kinase is involved in the induction of renal endothelial injury in sickle cell mice. Inhibition of RON kinase activation may provide a novel approach for prevention of the development of renal disease in sickle cell disease.


Assuntos
Aminopiridinas/farmacologia , Anemia Falciforme/fisiopatologia , Endotélio Vascular/efeitos dos fármacos , Rim/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Piridonas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Animais , Células Cultivadas , Endotélio Vascular/lesões , Endotélio Vascular/patologia , Humanos , Rim/lesões , Rim/patologia , Macrófagos/patologia , Camundongos
11.
Oncotarget ; 8(44): 76749-76769, 2017 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-29100346

RESUMO

We recently identified the protein phosphatase-1 - targeting compound, 1E7-03 which inhibited HIV-1 in vitro. Here, we investigated the effect of 1E7-03 on HIV-1 infection in vivo by analyzing its metabolic stability and antiviral activity of 1E7-03 and its metabolites in HIV-1 infected NSG-humanized mice. 1E7-03 was degraded in serum and formed two major degradation products, DP1 and DP3, which bound protein phosphatase-1 in vitro. However, their anti-viral activities were significantly reduced due to inefficient cell permeability. In cultured cells, 1E7-03 reduced expression of several protein phosphatase-1 regulatory subunits including Sds22 as determined by a label free quantitative proteomics analysis. In HIV-1-infected humanized mice, 1E7-03 significantly reduced plasma HIV-1 RNA levels, similar to the previously described HIV-1 transcription inhibitor F07#13. We synthesized a DP1 analog, DP1-07 with a truncated side chain, which showed improved cell permeability and longer pharmacokinetic retention in mice. But DP1-07 was less efficient than 1E7-03 as a HIV-1 inhibitor both in vitro and in vivo, indicating that the full side chain of 1E7-03 was essential for its anti-HIV activity. Analysis of 1E7-03 stability in plasma and liver microsomes showed that the compound was stable in human, primate and ferret plasma but not in rodent plasma. However, 1E7-03 was not stable in human liver microsomes. Our findings suggest that 1E7-03 is a good candidate for future development of HIV-1 transcription inhibitors. Further structural modification and advanced formulations are needed to improve its metabolic stability and enhance its antiviral activity in non-human primate animals and humans.

12.
Indian J Ophthalmol ; 65(9): 853-858, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-28905830

RESUMO

PURPOSE: Resections and plications tighten recti although the latter are less traumatic, potentially reversible, quicker, and vascularity preserving. To compare inflammation, scarring, and alignment in horizontal strabismus, operated uniocularly by either resections or plications (with recessions): recession and resection (R&R) or recession and plication (R&P) groups. This was a prospective, patient and assessor blind, randomized trial. METHODS: All consenting strabismus patients qualifying for the first-time uniocular horizontal rectus surgeries underwent detailed ocular examination and were randomized into standard R&R or R&P groups. For the latter, we folded the tendon-muscle strap the desired amount using 6-0 polyglactin, suturing it to its insertion, entailing no disinsertion. We compared the groups for inflammatory grades (individually for congestion, chemosis, discharge, foreign-body sensation, and drop intolerance and aggregated to a total inflammatory score (TIS), scar visibility (SV) at 1 m, and successful alignment (≤10 prism diopter of orthotropia). We used Mann-Whitney and Fisher's exact tests, with significance at P ≤ 0.05. RESULTS: We randomized 40 patients: 22 to R&R and 18 to R&P. The groups were comparable in age, strabismus onset and duration, and strabismus amount. The inflammatory scores, both individual and TIS, were comparable at all time-points: all P > 0.05. SV proportions were not significantly different: 16/22 in R&R versus 9/18 in R&P; P = 0.19. There were no significant differences in success rates: 14/22 versus 10/18, P = 0.74. CONCLUSION: Our study shows that plication is similarly effective as resection, when combined with recession in horizontal strabismus, and should be resorted to more frequently.


Assuntos
Movimentos Oculares , Músculos Oculomotores/cirurgia , Estrabismo/cirurgia , Visão Binocular/fisiologia , Adolescente , Criança , Pré-Escolar , Feminino , Humanos , Masculino , Músculos Oculomotores/fisiopatologia , Procedimentos Cirúrgicos Oftalmológicos , Estudos Prospectivos , Estrabismo/fisiopatologia , Resultado do Tratamento
13.
Oman J Ophthalmol ; 10(2): 76-80, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28757690

RESUMO

CONTEXT: Surgery for horizontal strabismus reportedly has a success rate of 60%-80%. However, which preoperative factors are predictive of this success is not clear. AIMS: To identify prognostic factors those are predictive of successful outcome in horizontal strabismus surgery. SETTINGS AND DESIGN: Observational analytical study using multiple logistic regression (MLR). SUBJECTS AND METHODS: We assessed the medical records of patients who had undergone first-time horizontal muscle strabismus surgery between 2002 and 2013, where complete follow-up data were available for ≥6 weeks, and also, we collected data prospectively on patients operated between January 2014 and September 2015. Successful outcome was defined as a postoperative angle of deviation within 10 prism diopter of orthophoria at ≥6 weeks postoperatively. Independent variables considered were age at onset, age at surgery, duration, gender, deviation - type and amount, logMAR visual acuity (VA) - mean and of the poorer eye, mean refractive error, amount of anisometropia, and presence of dense amblyopia. Only those with P < 0.2 on univariate analyses (UAs) were included in the MLR, with significance set at P ≤ 0.05. STATISTICAL ANALYSES: UA (Chi-square for categorical variables and t-tests for continuous variables), followed by logistic regression analysis. RESULTS: Of 113 patients, on UA, type of deviation (P = 0.01), age at surgery (P = 0.16), absence of dense amblyopia (P = 0.002), and logMAR VA of the poorer eye (P = 0.005) qualified for the inclusion in MLR. On MLR, esotropia (ET) (odds ratio [OR]: 4.46) and absence of dense amblyopia (OR: 5.90) were associated with success. CONCLUSIONS: With an overall success rate of 83%, ET and absence of dense amblyopia were significantly predictive of surgical success.

14.
Curr Pharm Des ; 23(28): 4122-4132, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28677499

RESUMO

BACKGROUND: Despite efficient suppression of HIV-1 replication, current antiviral drugs are not able to eradicate HIV-1 infection. Permanent HIV-1 suppression or complete eradication requires novel biological approaches and therapeutic strategies. Our previous studies showed that HIV-1 transcription is regulated by host cell protein phosphatase-1. We also showed that HIV-1 transcription is sensitive to the reduction of intracellular iron that affects cell cycle-dependent kinase 2. We developed protein phosphatase 1-targeting small molecules that inhibited HIV-1 transcription. We also found an additional class of protein phosphatase-1-targeting molecules that activated HIV-1 transcription and reported HIV-1 inhibitory iron chelators and novel curcumin analogs that inhibit HIV-1. Here, we review HIV-1 transcription and replication with focus on its regulation by protein phosphatase 1 and cell cycle dependent kinase 2 and describe novel small molecules that can serve as future leads for anti-HIV drug development. RESULTS: Our review describes in a non-exhaustive manner studies in which HIV-1 transcription and replication are targeted with small molecules. Previously, published studies show that HIV-1 can be inhibited with protein phosphatase-1-targeting and iron chelating compounds and curcumin analogs. These results are significant in light of the current efforts to eradicate HIV-1 through permanent inhibition. Also, HIV-1 activating compounds can be useful for "kick and kill" therapy in which the virus is reactivated prior to its inhibition by the combination antiretroviral therapy. CONCLUSION: The studies described in our review point to protein phosphatase-1 as a new drug target, intracellular iron as subject for iron chelation and novel curcumin analogs that can be developed for novel HIV-1 transcription- targeting therapeutics.


Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/tratamento farmacológico , Proteína Fosfatase 1/metabolismo , Animais , Curcumina/análogos & derivados , Curcumina/farmacologia , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Desenho de Fármacos , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/isolamento & purificação , Humanos , Quelantes de Ferro/farmacologia , Transcrição Genética/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
15.
J Food Sci Technol ; 53(11): 4007-4013, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28035156

RESUMO

The present study embarked on the objective of optimizing improved sous-vide processing condition for development of ready-to-cook Pangasius steaks with extended shelf-life using response surface methodology. For the development of improved sous-vide cooked product, Pangasius steaks were treated with additional hurdles in various combinations for optimization. Based on the study, suitable combination of chitosan and spices was selected which enhanced antimicrobial and oxidative stability of the product. The Box-Behnken experimental design with 15 trials per model was adopted for designing the experiment to know the effect of independent variables, namely chitosan concentration (X1), cooking time (X2) and cooking temperature (X3) on dependent variable i.e. TBARS value (Y1). From RSM generated model, the optimum condition for sous-vide processing of Pangasius steaks were 1.08% chitosan concentration, 70.93 °C of cooking temperature and 16.48 min for cooking time and predicted minimum value of multiple response optimal condition was Y = 0.855 mg MDA/Kg of fish. The high correlation coefficient (R2 = 0.975) between the model and the experimental data showed that the model was able to efficiently predict processing condition for development of sous-vide processed Pangasius steaks. This research may help the processing industries and Pangasius fish farmer as it provides an alternative low cost technology for the proper utilization of Pangasius.

16.
Biology (Basel) ; 5(4)2016 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-27918433

RESUMO

Protein phosphatase 1 (PP1), a cellular serine/threonine phosphatase, is targeted to cellular promoters by its major regulatory subunits, PP1 nuclear targeting subunit, nuclear inhibitor of PP1 (NIPP1) and RepoMan. PP1 is also targeted to RNA polymerase II (RNAPII) by NIPP1 where it can dephosphorylate RNAPII and cycle-dependent kinase 9 (CDK9). Here, we show that treatment of cells with a small molecule activator of PP1 increases the abundance of a neuregulin-1 (NRG-1)-derived peptide. NRG-1 mRNA and protein levels were increased in the cells stably or transiently expressing mutant NIPP1 (mNIPP1) that does not bind PP1, but not in the cells expressing NIPP1. Expression of mNIPP1 also activated the NRG-1 promoter in an NF-κB-dependent manner. Analysis of extracts from mNIPP1 expressing cells by glycerol gradient centrifugation showed a redistribution of PP1 and CDK9 between large and small molecular weight complexes, and increased CDK9 Thr-186 phosphorylation. This correlated with the increased CDK9 activity. Further, RNAPII co-precipitated with mNIPP1, and phosphorylation of RNAPII C-terminal domain (CTD) Ser-2 residues was greater in cells expressing mNIPP1. In mNIPP1 expressing cells, okadaic acid, a cell-permeable inhibitor of PP1, did not increase Ser-2 CTD phosphorylation inhibited by flavopiridol, in contrast to the NIPP1 expressing cells, suggesting that PP1 was no longer involved in RNAPII dephosphorylation. Finally, media conditioned with mNIPP1 cells induced the proliferation of wild type 84-31 cells, consistent with a role of neuregulin-1 as a growth promoting factor. Our study indicates that deregulation of PP1/NIPP1 holoenzyme activates NRG-1 expression through RNAPII and CDK9 phosphorylation in a NF-κB dependent manner.

17.
Indian J Ophthalmol ; 64(4): 323-5, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27221688

RESUMO

We report a case of a 20-year-old female having systemic hypertension who presented with right-sided proptosis, chemosis, and diminished vision, preceded by an acute episode of unilateral throbbing headache. Imaging studies revealed a right-sided direct, spontaneous carotid-cavernous fistula (CCF), aneurysm of internal carotid artery, bleed in the parieto-frontal lobe, and swelling of extraocular muscles. Abdominal ultrasound revealed a small contracted right kidney measuring 64 mm × 27 mm. A direct spontaneous CCF can occur spontaneously following rupture of intracranial aneurysm without any history of trauma or connective tissue disorder. Prompt diagnosis of intravascular malformations at initial presentation can prevent neurological complications and vision loss. A team approach including emergency physicians, neurosurgeons, and ophthalmologists is needed for the proper management of such patients.


Assuntos
Fístula Carotidocavernosa/diagnóstico , Nefropatias/congênito , Rim/anormalidades , Fístula Carotidocavernosa/complicações , Fístula Carotidocavernosa/terapia , Angiografia Cerebral , Embolização Terapêutica/métodos , Exoftalmia/diagnóstico , Exoftalmia/etiologia , Feminino , Humanos , Rim/diagnóstico por imagem , Nefropatias/complicações , Nefropatias/diagnóstico , Imagem por Ressonância Magnética , Oftalmoscopia , Doenças Raras , Tomografia Computadorizada por Raios X , Adulto Jovem
18.
Chembiochem ; 17(13): 1211-5, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27124688

RESUMO

Inorganic pyrophosphate (PPi) is produced from nucleoside triphosphates in important biosynthetic reactions and is considered a diagnostic marker for various diseases, such as cancer, crystal deposition disease, and arthritis. Traditional methods for biological PPi detection rely on off-line analytics after sample destruction. Molecular probes for imaging this biologically important analyte with temporal and spatial control in living cells are currently in demand. Herein, we report an Fe(III) -salen complex as the first small reaction-based probe for endogenous mitochondrial PPi following a disassembly approach. Significantly, we successfully applied this complex for the detection of increased cellular PPi levels, and its performance was not affected by the presence of mitochondrial ATP in living cells.


Assuntos
Complexos de Coordenação/farmacologia , Difosfatos/análise , Compostos Férricos/farmacologia , Corantes Fluorescentes/farmacologia , Mitocôndrias/química , Trifosfato de Adenosina , Aldeídos/química , Permeabilidade da Membrana Celular , Complexos de Coordenação/química , Compostos Férricos/química , Compostos Férricos/toxicidade , Corantes Fluorescentes/química , Corantes Fluorescentes/toxicidade , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Estrutura Molecular , Proteínas de Transporte de Fosfato/antagonistas & inibidores , Probenecid/farmacologia
19.
Infect Dis (Auckl) ; 9: 21-32, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27013886

RESUMO

Highly active antiretroviral (ARV) therapy (HAART) for chronic suppression of HIV replication has revolutionized the treatment of HIV/AIDS. HAART is no panacea; treatments must be maintained for life. Although great progress has been made in ARV therapy, HIV continues to replicate in anatomical and intracellular sites where ARV drugs have restricted access. Nanotechnology has been considered a platform to circumvent some of the challenges in HIV/AIDS treatment. Dispersion polymerization was used to fabricate two types (PMM and ECA) of polymeric nanoparticles, and each was successfully loaded with four ARV drugs (zidovudine, lamivudine, nevirapine, and raltegravir), followed by physicochemical characterization: scanning electron microscope, particle size, zeta potential, drug loading, and in vitro availability. These nanoparticles efficiently inhibited HIV-1 infection in CEM T cells and peripheral blood mononuclear cells; they hold promise for the treatment of HIV/AIDS. The ARV-loaded nanoparticles with polyethylene glycol on the corona may facilitate tethering ligands for targeting specific receptors expressed on the cells of HIV reservoirs.

20.
Blood Adv ; 1(3): 170-183, 2016 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-28203649

RESUMO

The low incidence of HIV-1 infection in patients with sickle cell disease (SCD) and inhibition of HIV-1 replication in vitro under the conditions of low intracellular iron or heme treatment suggests a potential restriction of HIV-1 infection in SCD. We investigated HIV-1 ex vivo infection of SCD peripheral blood mononuclear cells (PBMCs) and found that HIV-1 replication was inhibited at the level of reverse transcription (RT) and transcription. We observed increased expression of heme and iron-regulated genes, previously shown to inhibit HIV-1, including ferroportin, IKBα, HO-1, p21, and SAM domain and HD domain-containing protein 1 (SAMHD1). HIV-1 inhibition was less pronounced in hepcidin-treated SCD PBMCs and more pronounced in the iron or iron chelators treated, suggesting a key role of iron metabolism. In SCD PBMCs, labile iron levels were reduced and protein levels of ferroportin, HIF-1α, IKBα, and HO-1 were increased. Hemin treatment induced ferroportin expression and inhibited HIV-1 in THP-1 cells, mimicking the HIV-1 inhibition in SCD PBMCs, especially as hepcidin similarly prevented HIV-1 inhibition. In THP-1 cells with knocked down ferroportin, IKBα, or HO-1 genes but not HIF-1α or p21, HIV-1 was not inhibited by hemin. Activity of SAMHD1-regulatory CDK2 was decreased, and SAMHD1 phosphorylation was reduced in SCD PBMCs and hemin-treated THP-1 cells, suggesting SAMHD1-mediated HIV-1 restriction in SCD. Our findings point to ferroportin as a trigger of HIV-1 restriction in SCD settings, linking reduced intracellular iron levels to the inhibition of CDK2 activity, reduction of SAMHD1 phosphorylation, increased IKBα expression, and inhibition of HIV-1 RT and transcription.

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