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Tuberculosis (T.B.) remains a prominent global cause of health challenges and death, exacerbated by drug-resistant strains such as multidrug-resistant tuberculosis MDR-TB and extensively drug-resistant tuberculosis XDR-TB. For an effective disease management strategy, it is crucial to understand the dynamics of T.B. infection and the impacts of treatment. In the present article, we employ AI-based machine learning techniques to investigate the immunity impact of medications. SEIPR epidemiological model is incorporated with MDR-TB for compartments susceptible to disease, exposed to risk, infected ones, preventive or resistant to initial treatment, and recovered or healed population. These masses' natural trends, effects, and interactions are formulated and described in the present study. Computations and stability analysis are conducted upon endemic and disease-free equilibria in the present model for their global scenario. Both numerical and AI-based nonlinear autoregressive exogenous NARX analyses are presented with incorporating immediate treatment and delay in treatment. This study shows that the active patients and MDR-TB, both strains, exist because of the absence of permanent immunity to T.B. Furthermore, patients who have recovered from tuberculosis may become susceptible again by losing their immunity and contributing to transmission again. This article aims to identify patterns and predictors of treatment success. The findings from this research can contribute to developing more effective tuberculosis interventions.
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INTRODUCTION: Total tau (t-tau) and phosphorylated tau (p-tau) are abnormally elevated in the brain and cerebrospinal fluid of individuals with Alzheimer's disease (AD). Tau is also present in the salivary gland tissue and saliva, and salivary measures might produce an accurate, accessible, and inexpensive biomarker. METHODS: Using unstimulated saliva and Western blot analysis, we quantified the p-tau/t-tau ratio at different phosphorylation sites. RESULTS: We found that for one phosphorylation site, S396, p-tau/t-tau ratio was significantly elevated in patients with AD compared with normal elderly control subjects. The elevation in saliva, however, did not correlate with cerebrospinal fluid tau or with brain measures such as hippocampal volume. DISCUSSION: There is significant elevation of p-tau/t-tau ratio for the S396 phosphorylation site. Large variation in the AD salivary tau levels, however, limits the utility of this test as a clinical biomarker.
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BACKGROUND: Insulin resistance is positively correlated with body iron. It is unclear whether iron is a cause or an outcome of insulin resistance. Insulin resistance precedes type 2 diabetes mellitus. Offspring of type 2 diabetics are insulin resistant as compared to those of the non-diabetics. The present study was designed to compare and correlate insulin resistance with iron parameters (including serum ferritin, transferrin saturation and blood haemoglobin) in non-diabetic offspring of type 2 diabetics and non-diabetic offspring of non-diabetics. METHODS: It was a cross-sectional study, conducted on one hundred and twenty male subjects 20-40 years of age. They were divided into two groups, each group having 60 subjects. Group A included non-diabetic offspring of type 2 diabetics, while Group B included non-diabetic offspring of non-diabetics. Fasting blood sample was taken and examined for glucose, haemoglobin, insulin, iron, TIBC and ferritin. Data was analysed by SPSS-17. RESULTS: Insulin resistance and iron parameters were significantly higher (p<0.05) in non-diabetic offspring of type 2 diabetics as compared to those of the non-diabetics. There was significant positive correlation (p=0.027) between insulin resistance and serum iron in non-diabetic offspring of type 2 diabetics. There was also significant positive correlation between insulin resistance and serum iron, transferrin saturation and haemoglobin in non-diabetic offspring of non-diabetics. CONCLUSION: Non-diabetic offspring of type 2 diabetics have iron load and insulin resistance, that predispose them to the development of type 2 diabetes.
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Diabetes Mellitus Tipo 2/sangre , Resistencia a la Insulina , Insulina/sangre , Hierro/sangre , Adulto , Estudios Transversales , Diabetes Mellitus Tipo 2/epidemiología , Diabetes Mellitus Tipo 2/genética , Ferritinas/sangre , Estudios de Seguimiento , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Pakistán/epidemiología , Factores de RiesgoRESUMEN
BACKGROUND: Nigella sativa or "Kalonji" is a naturally occurring plant in Pakistan and other countries which possesses a wide range of medicinal properties, the anti-inflammatory property being one of these. Diclofenac sodium is a commonly used anti-inflammatory drug. The purpose of this study was to compare the anti-inflammatory effect of ethanolic extract of Nigella sativa seeds with that of diclofenac sodium in albino rats. METHODS: This laboratory randomized controlled trial (RCT) was conducted in the Physiology Department, Services Institute of Medical Sciences (SIMS), Lahore. The study was carried out on 90 male albino rats. Five percent formalin in a dose of 50 µl was injected into sub-plantar surface of right hind paw of each rat to produce inflammation. The rats were randomly divided into three groups of thirty each. Group A was given normal saline (control); group B was given Nigella sativa seed extract; and group C received diclofenac sodium, as a reference drug. Increase in paw diameter, and total and differential leukocyte counts were measured as markers of inflammation. RESULTS: Nigella sativa seeds extract caused significant (p<0.05) reduction in the paw inflammatory response in albino rats. The effect was longer in duration than the effect caused by diclofenac sodium; however, the extract was comparatively less potent than diclofenac sodium. The extract had no significant effect (p>0.05) on the total or differential leukocyte counts. CONCLUSION: Our results suggest that ethanolic extract of Nigella sativa seeds possesses potent anti-inflammatory effect, in albino rats however, this effect is comparatively less but prolonged than that produced by diclofenac sodium.
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Diclofenaco/farmacología , Inflamación/tratamiento farmacológico , Nigella sativa , Extractos Vegetales/farmacología , Animales , Antiinflamatorios no Esteroideos/farmacología , Modelos Animales de Enfermedad , Masculino , Ratas , SemillasRESUMEN
BACKGROUND: Cardiovascular disease (CVD) is a leading cause of mortality and morbidity in diabetes mellitus (DM). Studies indicate that atherosclerosis has slowly altered from a model of chronic degenerative disease affecting patients with advanced ages to a model of subclinical chronic inflammatory disease present in childhood. DM is a risk factor for atherosclerosis and asymptomatic low grade inflammation occurs prior to unconcealed vascular lesions in these patients. A low grade inflammation can be determined by serum C-reactive protein (CRP). The aim of this study was to evaluate serum CRP levels in recently diagnosed type-1 diabetic children to predict early cardiovascular complications. METHODS: In this cross-sectional study, serum CRP levels were determined in 39 diabetic children and 40 healthy children as control. CRP concentrations were determined by ELISA by an automated ELISA analyzer. The values were expressed as mean+standard deviation and data from patients and controls was compared by t-test. RESULTS: Serum CRP levels were significantly elevated in diabetic children as compared to controls (p<0.001). CONCLUSION: Serum CRP can be used as a potent biochemical markers in addition to other traditional risk factors like dyslipidemia, hypertension, obesity and smoking to detect hieh risk Datients.
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Aterosclerosis/sangre , Proteína C-Reactiva/metabolismo , Diabetes Mellitus Tipo 1/sangre , Adolescente , Aterosclerosis/etiología , Biomarcadores/sangre , Niño , Diabetes Mellitus Tipo 1/complicaciones , Femenino , Humanos , Inflamación/sangre , Inflamación/complicaciones , Masculino , Medición de RiesgoRESUMEN
b-Amyloid peptide accumulation, tau hyperphosphorylation, and synapse loss are characteristic neuropathological symptoms of Alzheimer's disease (AD). Tau hyperphosphorylation is suggested to inhibit the association of tau with microtubules, making microtubules unstable and causing neurodegeneration. The mechanism of tau phosphorylation in AD brain, therefore, is of considerable significance. Although PHF-tau is phosphorylated at over 40 Ser/Thr sites, Ser(262) phosphorylation was shown to mediate b-amyloid neurotoxicity and formation of toxic tau lesions in the brain. In vitro, PKA is one of the kinases that phosphorylates tau at Ser(262), but the mechanism by which it phosphorylates tau in AD brain is not very clear. 14-3-3z is associated with neurofibrillary tangles and is upregulated in AD brain. In this study, we show that 14-3-3z promotes tau phosphorylation at Ser(262) by PKA in differentiating neurons. When overexpressed in rat hippocampal primary neurons, 14-3-3z causes an increase in Ser(262) phosphorylation, a decrease in the amount of microtubule-bound tau, a reduction in the amount of polymerized microtubules, as well as microtubule instability. More importantly, the level of pre-synaptic protein synaptophysin was significantly reduced. Downregulation of synaptophysin in 14-3-3z overexpressing neurons was mitigated by inhibiting the proteosome, indicating that 14-3-3z promotes proteosomal degradation of synaptophysin. When 14-3-3z overexpressing neurons were treated with the microtubule stabilizing drug taxol, tau Ser(262) phosphorylation decreased and synaptophysin level was restored. Our data demonstrate that overexpression of 14-3-3z accelerates proteosomal turnover of synaptophysin by promoting the destabilization of microtubules. Synaptophysin is involved in synapse formation and neurotransmitter release. Our results suggest that 14-3-3z may cause synaptic pathology by reducing synaptophysin levels in the brains of patients suffering from AD.
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Proteínas 14-3-3/metabolismo , Enfermedad de Alzheimer/metabolismo , Regulación de la Expresión Génica/fisiología , Hipocampo/citología , Chaperonas Moleculares/metabolismo , Neuronas/metabolismo , Sinaptofisina/metabolismo , Proteínas tau/metabolismo , Análisis de Varianza , Animales , Clonación Molecular , Cartilla de ADN/genética , Regulación de la Expresión Génica/efectos de los fármacos , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Microscopía Fluorescente , Microtúbulos/efectos de los fármacos , Paclitaxel/farmacología , Fosforilación/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , RatasRESUMEN
Alzheimer's disease (AD) is characterized by the presence of abnormal, straight filaments and paired helical filaments (PHFs) that are coated with amorphous aggregates. When PHFs are treated with alkali, they untwist and form filaments with a ribbonlike morphology. Tau protein is the major component of all of these ultrastructures. 14-3-3ζ is present in NFTs and is significantly upregulated in AD brain. The molecular basis of the association of 14-3-3ζ within NFTs and the pathological significance of its association are not known. In this study, we have found that 14-3-3ζ is copurified and co-immunoprecipitates with tau from NFTs of AD brain extract. In vitro, tau binds to both phosphorylated and nonphosphorylated tau. When incubated with 14-3-3ζ, tau forms amorphous aggregates, single-stranded, straight filaments, ribbonlike filaments, and PHF-like filaments, all of which resemble the corresponding ultrastructures found in AD brain. Immuno-electron microscopy determined that both tau and 14-3-3ζ are present in these ultrastructures and that they are formed in an incubation time-dependent manner. Amorphous aggregates are formed first. As the incubation time increases, the size of amorphous aggregates increases and they are incorporated into single-stranded filaments. Single-stranded filaments laterally associate to form double-stranded, ribbonlike, and PHF-like filaments. Both tau and phosphorylated tau aggregate in a similar manner when they are incubated with 14-3-3ζ. Our data suggest that 14-3-3ζ has a role in the fibrillization of tau in AD brain, and that tau phosphorylation does not affect 14-3-3ζ-induced tau aggregation.
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Proteínas 14-3-3/metabolismo , Enfermedad de Alzheimer/patología , Ovillos Neurofibrilares/patología , Proteínas tau/metabolismo , Humanos , Ovillos Neurofibrilares/ultraestructura , FosforilaciónRESUMEN
In the normal brain, tau protein is phosphorylated at a number of proline- and non-proline directed sites, which reduce tau microtubule binding and thus regulate microtubule dynamics. In Alzheimer disease (AD), tau is abnormally hyperphosphorylated, leading to neurofibrillary tangle formation and microtubule disruption, suggesting a loss of regulatory mechanisms controlling tau phosphorylation. Early growth response 1 (Egr-1) is a transcription factor that is significantly up-regulated in AD brain. The pathological significance of this up-regulation is not known. In this study, we found that lentivirus-mediated overexpression of Egr-1 in rat brain hippocampus and primary neurons in culture activates proline-directed kinase Cdk5, inactivates PP1, promotes tau phosphorylation at both proline-directed Ser(396/404) and non-proline-directed Ser(262) sites, and destabilizes microtubules. Furthermore, in Egr-1(-/-) mouse brain, Cdk5 activity was decreased, PP1 activity was increased, and tau phosphorylation was reduced at both proline-directed and non-proline-directed sites. By using nerve growth factor-exposed PC12 cells, we determined that Egr-1 activates Cdk5 to promote phosphorylation of tau and inactivates PP1 via phosphorylation. When Cdk5 was inhibited, tau phosphorylation at both proline- and non-proline directed sites and PP1 phosphorylation were blocked, indicating that Egr-1 acts through Cdk5. By using an in vitro kinase assay and HEK-293 cells transfected with tau, PP1, and Cdk5, we found that Cdk5 phosphorylates Ser(396/404) directly. In addition, by phosphorylating and inactivating PP1, Cdk5 promotes tau phosphorylation at Ser(262) indirectly. Our results indicate that Egr-1 is an in vivo regulator of tau phosphorylation and suggest that in AD brain increased levels of Egr-1 aberrantly activate an Egr-1/Cdk5/PP1 pathway, leading to accumulation of hyperphosphorylated tau, thus destabilizing the microtubule cytoskeleton.
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Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Hipocampo/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Animales , Quinasa 5 Dependiente de la Ciclina/genética , Quinasa 5 Dependiente de la Ciclina/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Activación Enzimática/genética , Femenino , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Microtúbulos/genética , Microtúbulos/metabolismo , Células PC12 , Fosforilación/genética , Ratas , Ratas Long-Evans , Receptores de Neuropéptido Y , Proteínas tau/genéticaRESUMEN
In Parkinson disease (PD) brain, a progressive loss of dopaminergic neurons leads to dopamine depletion in the striatum and reduced motor function. Lewy bodies, the characteristic neuropathological lesions found in the brain of PD patients, are composed mainly of α-synuclein protein. Three point mutations in the α-synuclein gene are associated with familial PD. In addition, genome-wide association studies indicate that α-synuclein and Tau protein synergistically increase disease susceptibility in the human population. To determine the mechanism by which α-synuclein and Tau act together, we have used PD-causing neurotoxin MPTP and pathogenic α-synuclein mutants A30P, E46K, and A53T as models. We found that exposure of human neuroblastoma M17 cells to MPTP enhances the intracellular α-synuclein protein level, stimulates Tau protein phosphorylation at Ser(262), and induces apoptosis. In mouse brain, ablation of α-synuclein function significantly suppresses Tau phosphorylation at Ser(262). In vitro, α-synuclein binds to phosphorylated Ser(214) of Tau and stimulates PKA-catalyzed Tau phosphorylation at Ser(262). PD-associated α-synuclein mutations increase α-synuclein binding to Tau and stimulate Tau phosphorylation at Ser(262). In HEK-293 cells, α-synuclein and its all PD-associated mutants destabilize the microtubule cytoskeleton in a similar extent. In contrast, when co-expressed with Tau, these PD-associated mutants destabilize microtubules with significantly higher potency than WT. Our results demonstrate that α-synuclein is an in vivo regulator of Tau protein phosphorylation at Ser(262) and suggest that PD-associated risk factors such as environmental toxins and α-synuclein mutations promote Tau phosphorylation at Ser(262), causing microtubule instability, which leads to loss of dopaminergic neurons in PD brain.
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1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Microtúbulos/metabolismo , Mutación Missense , Neurotoxinas/farmacología , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Sustitución de Aminoácidos , Línea Celular Tumoral , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Estudio de Asociación del Genoma Completo , Células HEK293 , Humanos , Microtúbulos/genética , Fosforilación/efectos de los fármacos , Fosforilación/genética , alfa-Sinucleína/genética , Proteínas tau/genéticaRESUMEN
OBJECTIVE: To determine the analgesic effect of ethanolic extract of Nigella sativa seeds on experimentally-induced pain in albino mice. STUDY DESIGN: Randomized controlled trial (RCT). PLACE AND DURATION OF STUDY: Physiology Department, Services Institute of Medical Sciences (SIMS), Lahore, from May to September, 2009. METHODOLOGY: The study was carried out in 90 male albino mice using acetic acid induced writhing test as a chemical model of nociception. The mice were divided in three groups of 30 each. Group A was given normal saline (control); group B was given Nigella sativa seed extract in a dose of 50 mg/kg; and group C received diclofenac sodium, as a reference drug. Number of writhings in treated and control groups were compared. RESULTS: The ethanolic extract of Nigella sativa seeds given intraperitoneally caused significant (p < 0.05) analgesic effect on nociceptive response initiated by 0.6% acetic acid; although this analgesic effect was less than that produced by diclofenac sodium. CONCLUSION: Ethanolic extract of Nigella sativa possessed significant analgesic effect in mice.
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Analgesia/métodos , Nigella sativa , Dolor/tratamiento farmacológico , Fitoterapia , Extractos Vegetales/uso terapéutico , Semillas , Animales , Masculino , RatonesRESUMEN
BACKGROUND: Few studies have studied the role of homocysteine in migraineurs and have produced conflicting results. The MTHFR C677T genotype has been associated with increased risk of migraine in selected clinical samples. We assessed the association of the MTHFR C677T variant with migraine, the corresponding homocysteine levels and their correlation. METHOD: We studied 27 random adult migraineurs with aura (MWA), migraine without aura (MWOA), and 32 non-migraineurs (controls) from Lahore, Pakistan in this pilot study which is still under progress. RESULTS: We found significant differences in homocysteine levels between various diagnostic groups (K-W test: p=0.005). One-way ANOVA, post-hoc tests revealed significant differences in homocysteine levels between Non-migraineurs, MWA (p=0.002, CI: 1.93 - 9.19) and MWoA (p=0.002, CI: -9.19 - -1.9). We found a significant association between the migraine group and C677T-MTHFR variant mutant allele (C/T) (p=0.039). We did not find a significant association between C677T-MTHFR variant and homocysteine levels. CONCLUSION: In this pilot study, we found plasma homocysteine levels to be significantly associated with MWOA. Additionally, plasma homocysteine levels were lower in MWA than in MWOA. Furthermore, we did not find a relationship between homocysteine levels and the MTHFR variant (SNP rs1801133). Lastly, there may be a relationship between the MTHFR variant (SNP rs1801133) and migraine in this population.
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Homocisteína/sangre , Metilenotetrahidrofolato Reductasa (NADPH2)/genética , Migraña sin Aura/sangre , Migraña sin Aura/genética , Adulto , Estudios de Casos y Controles , Estudios de Cohortes , Femenino , Humanos , MasculinoRESUMEN
BACKGROUND: In postmenopausal women, the two major causes of bone loss are oestrogen deficiency after menopause and age related processes. Bone turnover increases to high levels and oestrogen deficiency may induce calcium loss by indirect effects on extra skeletal calcium homeostasis. Objective of this study was to evaluate calcium status in pre-menopausal and postmenopausal women. METHODS: This cross sectional study was carried out in 34 premenopausal women and 33 postmenopausal women, in Department of Physiology, Services Institute of Medical Sciences, Lahore. Height and weight of each woman were taken to find out the body mass index (BMI). Serum calcium, parathyroid hormone and calcitonin levels of each subject were determined. RESULTS: Premenopausal women were obese (BMI>30 Kg/m2) while postmenopausal women were overweight (BMI>25 Kg/m2). Serum calcium levels were significantly lower in postmenopausal women than in pre-menopausal women, while serum parathyroid hormone levels were significantly higher in postmenopausal woman. Serum calcitonin level was not significantly different in the two groups. CONCLUSION: Postmenopausal women are calcium deficient and have increased bone turnover as indicated by increased serum parathyroid hormone levels.
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Calcio/metabolismo , Posmenopausia/metabolismo , Premenopausia/metabolismo , Adulto , Factores de Edad , Anciano , Estudios Transversales , Femenino , Humanos , Persona de Mediana Edad , Estado Nutricional , PakistánRESUMEN
Proteosomal degradation of proteins is one of the major mechanisms of intracellular protein turnover. Failure of the proteosome to degrade misfolded protein is implicated in the accumulation of alpha-synuclein in Parkinson's disease (PD). Heme oxygenase-1 (HO-1), an enzyme that converts heme to free iron, carbon monoxide (CO) and biliverdin (bilirubin precursor) is expressed in response to various stressors. HO-1 is up-regulated in PD- and Alzheimer's disease-affected neural tissues. In this study, we found that HO-1 over-expression engenders dose-dependent decreases in alpha-synuclein protein levels in human neuroblastoma M17 cells. When over-expression of HO-1 was silenced in HO-1 transfected cells, level of alpha-synuclein was restored. Likewise, treatment of HO-1 over-expressing cells with the HO-1 inhibitor, tin mesoporphyrin, the iron chelator deferoxamine or antagonist of CO-dependent cGMP activation, methylene blue, mitigated the HO-1-induced reduction in alpha-synuclein levels. Furthermore, when HO-1 over-expressing cells were treated with the proteosome inhibitors, lactacystin and MG132, level of alpha-synuclein was almost completely restored. In contrast to the effect on alpha-synuclein [wild-type (WT)] levels, HO-1 over-expression did not significantly impact PD-associated alpha-synuclein (A30P) levels in these cells. HO-1 also significantly reduced aggregation of alpha-synuclein (WT) but not that of A30P. Our results suggest that HO-1, which is expressed when neurons are exposed to toxic stimuli capable of inducing protein misfolding, triggers proteosomal degradation of proteins and prevents intracellular accumulation of protein aggregates and inclusions. Resistance to HO-1 induced proteosomal degradation may render the familial PD-associated A30P mutation prone to toxic intracellular aggregation.
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Regulación Enzimológica de la Expresión Génica/genética , Hemo-Oxigenasa 1/genética , Neuroblastoma/metabolismo , Enfermedad de Parkinson/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , alfa-Sinucleína/metabolismo , Animales , Silenciador del Gen , Hemo-Oxigenasa 1/biosíntesis , Hemo-Oxigenasa 1/deficiencia , Humanos , Mutación , Neuroblastoma/enzimología , Neuroblastoma/genética , Enfermedad de Parkinson/genética , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/fisiología , Desnaturalización Proteica/genética , Estabilidad Proteica , Ratas , Células Tumorales Cultivadas , alfa-Sinucleína/antagonistas & inhibidores , alfa-Sinucleína/genéticaRESUMEN
In Alzheimer disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) and other tauopathies, tau accumulates and forms paired helical filaments (PHFs) in the brain. Tau isolated from PHFs is phosphorylated at a number of sites, migrates as approximately 60-, 64-, and 68-kDa bands on SDS-gel, and does not promote microtubule assembly. Upon dephosphorylation, the PHF-tau migrates as approximately 50-60-kDa bands on SDS-gels in a manner similar to tau that is isolated from normal brain and promotes microtubule assembly. The site(s) that inhibits microtubule assembly-promoting activity when phosphorylated in the diseased brain is not known. In this study, when tau was phosphorylated by Cdk5 in vitro, its mobility shifted from approximately 60-kDa bands to approximately 64- and 68-kDa bands in a time-dependent manner. This mobility shift correlated with phosphorylation at Ser(202), and Ser(202) phosphorylation inhibited tau microtubule-assembly promoting activity. When several tau point mutants were analyzed, G272V, P301L, V337M, and R406W mutations associated with FTDP-17, but not nonspecific mutations S214A and S262A, promoted Ser(202) phosphorylation and mobility shift to a approximately 68-kDa band. Furthermore, Ser(202) phosphorylation inhibited the microtubule assembly-promoting activity of FTDP-17 mutants more than of WT. Our data indicate that FTDP-17 missense mutations, by promoting phosphorylation at Ser(202), inhibit the microtubule assembly-promoting activity of tau in vitro, suggesting that Ser(202) phosphorylation plays a major role in the development of NFT pathology in AD and related tauopathies.
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Quinasa 5 Dependiente de la Ciclina/metabolismo , Mutación Missense , Proteínas tau/metabolismo , Animales , Bovinos , Cromosomas Humanos Par 17/química , Cromosomas Humanos Par 17/genética , Cromosomas Humanos Par 17/metabolismo , Quinasa 5 Dependiente de la Ciclina/química , Quinasa 5 Dependiente de la Ciclina/genética , Humanos , Microtúbulos/genética , Fosforilación , Tauopatías/genética , Tauopatías/metabolismo , Proteínas tau/química , Proteínas tau/genéticaRESUMEN
Extracellular matrix (ECM)-rich cartilage-derived chondrocytes are difficult to transfect with DNA/RNA. We modified the classical calcium phosphate transfection method by detaching adherent chondrocytes with trypsin and resuspending in CaPo4-nucleic acid precipitate followed by readherence for 24h. Due to the absence of ECM, chondrocytes could be transfected with 80% efficiency. Potent gene silencing with several antisense oligonucleotides and small interfering RNAs and strong promoter-luciferase activity could be achieved. This approach is applicable to any adherent or suspended cells and has utility in gene knockdown, ectopic overexpression, promoter regulation studies, and gene delivery in tissue engineering and gene therapy applications.
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Fosfatos de Calcio/química , Condrocitos/metabolismo , ADN/metabolismo , ARN/metabolismo , Transfección/métodos , Fosfatos de Calcio/metabolismo , Condrocitos/citología , Matriz Extracelular/metabolismo , Técnicas de Transferencia de Gen , HumanosRESUMEN
Transforming growth factor beta (TGF-beta1) promotes cartilage matrix synthesis and induces tissue inhibitor of metalloproteinases-3 (TIMP-3), which inhibits matrix metalloproteinases, aggrecanases and TNF-alpha-converting enzyme implicated in articular cartilage degradation and joint inflammation. TGF-beta1 activates Akt, ERK and Smad2 pathways in chondrocytes. Here we investigated previously unexplored roles of specific Smads in TGF-beta1 induction of TIMP-3 gene by pharmacological and genetic knockdown approaches. TGF-beta1-induced Smad2 phosphorylation and TIMP-3 protein expression could be inhibited by the Smad2/3 phosphorylation inhibitors, PD169316 and SB203580 and by Smad2-specific siRNA. Specific inhibitor of Smad3 (SIS3) and Smad3 siRNA abolished TGF-beta induction of TIMP-3. Smad2/3 siRNAs also down regulated TIMP-3 promoter-driven luciferase activities, suggesting transcriptional regulation. SiRNA-driven co-Smad4 knockdown abrogated TIMP-3 augmentation by TGF-beta. TIMP-3 promoter deletion analysis revealed that -828 deletion retains the original promoter activity while -333 and -167 deletions display somewhat reduced activity suggesting that most of the TGF-beta-responsive, cis-acting elements are found in the -333 fragment. Chromatin Immunoprecipitation (ChIP) analysis confirmed binding of Smad2 and Smad4 with the -940 and -333 promoter sequences. These results suggest that receptor-activated Smad2 and Smad3 and co-Smad4 critically mediate TGF-beta-stimulated TIMP-3 expression in human chondrocytes and TIMP-3 gene is a target of Smad signaling pathway.
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Condrocitos/metabolismo , Proteínas Smad/metabolismo , Inhibidor Tisular de Metaloproteinasa-3/genética , Factor de Crecimiento Transformador beta/farmacología , Células Cultivadas , Condrocitos/efectos de los fármacos , Inmunoprecipitación de Cromatina , Humanos , Isoquinolinas/farmacología , Regiones Promotoras Genéticas , Piridinas/farmacología , Pirroles/farmacología , Interferencia de ARN , Transducción de Señal , Proteína Smad2/antagonistas & inhibidores , Proteína Smad2/genética , Proteína Smad2/metabolismo , Proteína smad3/antagonistas & inhibidores , Proteína smad3/genética , Proteína Smad4/antagonistas & inhibidores , Proteína Smad4/genética , Inhibidor Tisular de Metaloproteinasa-3/biosíntesis , Factor de Crecimiento Transformador beta/antagonistas & inhibidoresRESUMEN
Oncostatin M (OSM) stimulates cartilage degradation in rheumatoid arthritis (RA) by inducing matrix metalloproteinases (MMPs) and aggrecanases (ADAMTS; a disintegrin and metalloproteinase with thrombospondin motif). Transforming growth factor beta (TGF-beta1) induces cartilage repair in joints but in excessive amounts, promotes inflammation. OSM and TGF-beta1 also induce tissue inhibitor of metalloproteinase-3 (TIMP-3), an important natural inhibitor of MMPs, aggrecanases, and tumor necrosis factor alpha converting enzyme (TACE), the principal proteases involved in arthritic inflammation and cartilage degradation. We studied cartilage protective mechanisms of the antiinflammatory cytokine, interleukin-4 (IL-4). IL-4 strongly (MMP-13 and TIMP-3) or minimally (ADAMTS-4) suppressed OSM-induced gene expression in chondrocytes. IL-4 did not affect OSM-stimulated phosphorylation of extracellular signal-regulated kinases (ERKs), protein 38 (p38), c-Jun N-terminal kinase (JNK) and Stat1. Lack of additional suppression with their inhibitors suggested that MMP-13, ADAMTS-4, and TIMP-3 inhibition was independent of these mediators. IL-4 also downregulated TGF-beta1-induced TIMP-3 gene expression, Smad2, and JNK phosphorylation. Additional suppression of TIMP-3 RNA by JNK inhibitor suggests JNK implication. The cartilage protective effects of IL-4 in animal models of arthritis may be due to its inhibition of MMPs and ADAMTS-4 expression. However, suppression of TIMP-3 suggests caution for using IL-4 as a cartilage protective therapy.
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Cartílago Articular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-4/farmacología , Oncostatina M/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/antagonistas & inhibidores , Proteínas ADAM/biosíntesis , Proteínas ADAM/genética , Proteína ADAMTS4 , Animales , Cartílago Articular/citología , Bovinos , Células Cultivadas/efectos de los fármacos , Condrocitos/metabolismo , Regulación hacia Abajo , Metaloproteinasa 13 de la Matriz/biosíntesis , Metaloproteinasa 13 de la Matriz/genética , Proteínas Quinasas Activadas por Mitógenos/biosíntesis , Proteínas Quinasas Activadas por Mitógenos/genética , Fosforilación/efectos de los fármacos , Procolágeno N-Endopeptidasa/biosíntesis , Procolágeno N-Endopeptidasa/genética , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , ARN Mensajero/biosíntesis , Proteínas Recombinantes/antagonistas & inhibidores , Factor de Transcripción STAT1/antagonistas & inhibidores , Factor de Transcripción STAT1/biosíntesis , Factor de Transcripción STAT1/genética , Transducción de Señal/efectos de los fármacos , Proteína Smad2/biosíntesis , Proteína Smad2/genética , Inhibidor Tisular de Metaloproteinasa-3/biosíntesis , Inhibidor Tisular de Metaloproteinasa-3/genéticaRESUMEN
Transforming growth factor beta (TGF-beta1) induces cartilage extracellular matrix synthesis and tissue inhibitor of metalloproteinases-3 (TIMP-3), an important natural inhibitor of matrix metalloproteinases, aggrecanases and TNF-alpha-converting enzyme, which are implicated in cartilage degradation and joint inflammation. This study tested the hypothesis that Akt/protein kinase B signaling pathway could mediate TGF-beta1 induction of TIMP-3 in human articular chondrocytes. TGF-beta activated phosphorylation of Akt in a delayed and sustained fashion that correlated with TIMP-3 mRNA induction. Phosphatidylinositol kinase (PI3K) inhibitors, Wortmannin and LY294002 and Akt inhibitor (NL-71-101) significantly inhibited TGF-beta-induced Akt phosphorylation, TIMP-3 expression, TIMP-3 promoter (-940 to +376)-driven luciferase activity and Sp1 transcription factor binding. PI3K p85, Akt and Sp1 small interfering RNA (siRNA)-driven knockdown of the respective gene products significantly suppressed TGF-beta-induced TIMP-3 gene expression. TGF-beta-stimulated phosphorylation of p70S6 Kinase and TIMP-3 protein induction was inhibited by rapamycin. Thus TGF-beta induces TIMP-3 gene expression in human chondrocytes partly through PI3K/Akt pathway and Sp1 transcription factor and by translational mechanisms via mammalian target of rapamycin (mTOR) signaling. TGF-beta induction of pro-survival Akt cascade and TIMP-3 may be related to strengthening of cartilage extracellular matrix, increased chondrocyte viability and maintenance of joint tissue integrity.
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Condrocitos/metabolismo , Proteína Oncogénica v-akt/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Transducción de Señal , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Androstadienos/farmacología , Cartílago Articular/citología , Células Cultivadas , Condrocitos/efectos de los fármacos , Cromonas/farmacología , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Humanos , Modelos Biológicos , Morfolinas/farmacología , Fosforilación , Factor de Crecimiento Transformador beta/farmacología , WortmaninaRESUMEN
In the preceding paper, we showed that GSK3beta phosphorylates tau at S(202), T(231), S(396), and S(400) in vivo. Phosphorylation of S(202) occurs without priming. Phosphorylation of T(231), on the other hand, requires priming phosphorylation of S(235). Similarly, priming phosphorylation of S(404) is essential for the sequential phosphorylation of S(400) and S(396) by GSK3beta. The priming kinase that phosphorylates tau at S(235) and S(404) in the brain is not known. In this study, we find that in HEK-293 cells cotransfected with tau, GSK3beta, and Cdk5, Cdk5 phosphorylates tau at S(202), S(235), and S(404). S(235) phosphorylation enhances GSK3beta-catalyzed T(231) phosphorylation. Similarly, Cdk5 by phosphorylating S(404) stimulates phosphorylation of S(400) and S(396) by GSK3beta. These data indicate that Cdk5 primes tau for GSK3beta in intact cells. To evaluate if Cdk5 primes tau for GSK3beta in mammalian brain, we examined localizations of Cdk5, tau, and GSK3beta in rat brain. We also analyzed the interaction of Cdk5 with tau and GSK3beta in brain microtubules. We found that Cdk5, GSK3beta, and tau are virtually colocalized in rat brain cortex. When bovine brain microtubules are analyzed by FPLC gel filtration, Cdk5, GSK3beta, and tau coelute within an approximately 450 kDa complex. From the fractions containing the approximately 450 kDa complex, tau, Cdk5, and GSK3beta co-immunoprecipitate with each other. In HEK-293 cells transfected with tau, Cdk5, and GSK3beta in different combinations, tau binds to Cdk5 in a manner independent of GSK3beta and to GSK3beta in a manner independent of Cdk5. However, Cdk5 and GSK3beta bind to each other only in the presence of tau, suggesting that tau connects Cdk5 and GSK3beta. Our results suggest that in the brain, tau, Cdk5, and GSK3beta are components of an approximately 450 kDa complex. Within the complex, Cdk5 phosphorylates tau at S(235) and primes it for phosphorylation of T(231) by GSK3beta. Similarly, Cdk5 by phosphorylating tau at S(404) primes tau for a sequential phosphorylation of S(400) and S(396) by GSK3beta.
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Quinasa 5 Dependiente de la Ciclina/farmacología , Glucógeno Sintasa Quinasa 3/metabolismo , Proteínas tau/metabolismo , Animales , Sitios de Unión , Encéfalo/metabolismo , Bovinos , Células Cultivadas , Quinasa 5 Dependiente de la Ciclina/metabolismo , Técnica del Anticuerpo Fluorescente , Glucógeno Sintasa Quinasa 3 beta , Humanos , Masculino , Fosforilación , Unión Proteica , Ratas , Ratas Sprague-Dawley , Serina/metabolismo , Transfección , Proteínas tau/farmacologíaRESUMEN
BACKGROUND: Pakistan Medical and Dental Council has recently replaced 'composite scheme' of first Professional MBBS examination with 'split scheme'. In composite scheme First Professional MBBS examination is offered after 2 years in medical college while in split scheme the same examination is split into two parts, Part-I after first year and Part-II after completion of 2nd year in medical college. METHODS: This study analyzed the results of two batches of students successively passing the 1st professional MBBS examination at Nishtar Medical College, Multan under two different evaluation systems. One batch of students (N-49) passed under composite scheme in 2001 while the other batch (N-50) passed under the split scheme of 1st professional MBBS examination in 2002. RESULTS: Results showed a significant (P<0.05) increase in the pass percentage of students under the split scheme (74.65%) as compared with the composite scheme (64.03%). Students secured more total marks (615.36 +/- 35.99) under the split system as compared with the total marks obtained (587.22 +/- 33.85) under the composite system. More students (65.35%) obtained first division (60% or more marks) under the split scheme as compared with the composite scheme (36.29%). CONCLUSION: Students' performance seems to be better under the split scheme as compared with the composite scheme of 1st professional MBBS examination.