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1.
PLoS One ; 16(7): e0254921, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34293021

RESUMO

AIM: Diabetic foot ulcer (DFU) is a major concern in diabetes and its control requires in-depth molecular investigation. The present study aimed to screen the expression of microRNA-210 (miR-210) and its association in hypoxic pathway in DFU patients. METHODS: The study consists of 3 groups of circulation samples (50 in each group of: healthy volunteers, T2DM and T2DM with DFU) and 2 groups of tissue samples (10 in each group of: control and T2DM with DFU). Expression of miR-210 and hypoxia inducible factor-1 alpha (HIF-1α), and its responsive genes such as VEGF, TNF-α, IL-6, BCl2, Bax and Caspase 3 were analyzed by RT-PCR, Western blot and ELISA analyses. RESULTS: The HIF-1α expression decreased in DFU patients with increased miR-210 expression in both circulation and tissue biopsies. The circulatory IL-6 and inflammatory gene TNF-α expression was increased in DFU compared to healthy controls and T2DM subjects. Further, we found there was no alteration in the angiogenic marker, VEGF expression. In comparison, anti-apoptotic BCl2 was decreased and Bax and Caspase 3 was increased in DFU tissues relative to control. CONCLUSIONS: The study showed that there was an inverse relationship between miR-210 and HIF-1α expression in patients with DFU, indicating that miR-210 may regulate the expression of the hypoxic gene.


Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Pé Diabético/metabolismo , Regulação da Expressão Gênica , MicroRNAs/biossíntese , Cicatrização , Adulto , Proteínas Reguladoras de Apoptose/biossíntese , Proteínas Reguladoras de Apoptose/genética , Citocinas/biossíntese , Citocinas/genética , Diabetes Mellitus Tipo 2/genética , Pé Diabético/genética , Feminino , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade
2.
Nitric Oxide ; 82: 48-58, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30439561

RESUMO

Angiogenesis is the formation of new blood vessels that occurs by two distinct processes following sprouting angiogenesis (SA) and intussusceptive angiogenesis (IA). Nitric oxide (NO) is known for its pro-angiogenic functions. However, no clear mechanisms are delineated on its role in promoting angiogenesis in reparative wound healing. We propose that NO regulates SA to IA transition and vice versa in wound milieu. We have used three models which include a new chick embryo extra-vasculature (CEV) burn wound model, adult Tie2-GFP transgenic Zebrafish caudal fin regeneration model and Zebrafish skin wound model to study the mechanisms underlying behind the role of NO in wound healing. Wounds created in CEV were treated with NO donor (Spermine NONOate (SPNO)), NOS inhibitor (L-nitro-l-arginine-methyl ester (l-NAME)), NaNO2, NaNO3, and beetroot juice, a nitrite-rich juice respectively and the pattern of wound healing was assessed. Morphological and histological techniques tracked the wound healing at the cellular level, and the molecular changes were investigated by using real-time RT-PCR gene expression analysis. The result concludes that NO donor promotes wound healing by activating SA at an early phase of healing while NOS inhibitor induces wound healing via IA. At the later phase of wound healing NO donor followed IA while NOS inhibitor failed to promote wound repair. The current work underpinned a differential regulation of NO on angiogenesis in wound milieu and this study would provide new insights in designing therapeutics for promoting wound repair.


Assuntos
Neovascularização Patológica/tratamento farmacológico , Óxido Nítrico/farmacologia , Cicatrização/efeitos dos fármacos , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Embrião de Galinha , Modelos Animais de Doenças , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Óxido Nítrico/metabolismo , Peixe-Zebra/metabolismo
3.
Int J Biol Macromol ; 119: 833-837, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30086330

RESUMO

Hypoxia inducible factor 1 alpha (HIF-1α) is a key regulator of the genes involved in the cellular response to hypoxia. This study aims to determine the HIF-1α gene polymorphism and its association with protein expression in diabetic subjects with and without diabetic foot ulcers (DFU). A total of 529 patients with T2DM (N = 185), DFU (N = 199) and Control (N = 145) were accounted for the study. PCR-RFLP experiment was carried out in order to find the allelic and genotypic comparison of HIF-1α gene in various groups of patients. There was a highly increased frequency of GA, RR value of 3.533(2.099-5.950) with p-value of 0.0001 on DFU patients when compared to that of control subjects with risk allele of GA, RR value of 1.756 (1.294-2.384) with p-value of 0.00001. Thus, we found that there was a significant association of HIF-1α polymorphism in exon 12 among DFU patients when compared to control groups. The circulatory HIF-1α protein expression study indicated a decreased expression in DFU levels when compared to T2DM and control. Overall, the study showed that there is an association of HIF-1α polymorphism (G1970A) in diabetes and DFU patients when compared to the healthy group.


Assuntos
Diabetes Mellitus Tipo 2/genética , Pé Diabético/genética , Éxons , Estudos de Associação Genética , Predisposição Genética para Doença , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Mutação , Adulto , Alelos , Biomarcadores , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Feminino , Expressão Gênica , Frequência do Gene , Genótipo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/sangue , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Polimorfismo de Nucleotídeo Único
4.
Int J Biol Macromol ; 103: 242-247, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28522400

RESUMO

Lysyl oxidase (LOX) is an extra-cellular matrix-modifying enzyme that has been linked to cell proliferation, metastasis, angiogenesis and wound healing. This study was designed to examine the association of LOX gene polymorphism G473A, G>A, (rs1800449) located in exon 1 of the LOX gene in diabetic subjects with and without diabetic foot ulcers (DFU) and its impact of expression on DFU. Genotypic analysis of 906 samples showed a significant increase in the presence of 'A' allele in type 2 diabetes mellitus (T2DM) and DFU when compared to that of control subjects. Allele wise analysis showed a higher frequency of 'A' allele in the T2DM (36.23%, OR 1.069, P value 0.29) and DFU (41.69%, OR 1.195, P value 0.003) when compared to that of control subjects (33.17%). Interestingly, real time RT-PCR results showed significant increased transcript level of the LOX gene on the AA genotype of DFU when compared to that of the AA genotype of T2DM and control subjects. Our finding predicts that there is an association of LOX gene polymorphism (G473A) on diabetes and DFU patients when compared to that of healthy controls. Thus, this study merits further evaluation on a mechanistic approach of this gene.


Assuntos
Pé Diabético/enzimologia , Pé Diabético/genética , Polimorfismo de Nucleotídeo Único , Proteína-Lisina 6-Oxidase/genética , Estudos de Casos e Controles , Feminino , Genótipo , Humanos , Masculino
5.
Biomark Med ; 11(4): 377-388, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28326825

RESUMO

Diabetic foot ulcer (DFU) is one of the major complications of diabetes and about 1% of people with diabetes have to go for lower limb amputation. With better understanding of the pathological basis of DFU, number of biomarkers like atrial natriuretic peptides, galectin-3, and cardiac troponins for diabetic cardiomyopathy, cystatin C for diabetics nephropathy and C-reactive protein for infection and procalcitonin could aid in early and noninvasive diagnosis especially when clinical signs are misleading. Predictive role of novel biomarkers in primary prevention however, requires additional studies considering sex, age and multiple complications in DFU. The current review provides an insight about the novel and emerging biomarkers of diabetes and its complications with special reference to DFUs.


Assuntos
Biomarcadores/metabolismo , Pé Diabético/diagnóstico , Proteína C-Reativa/análise , Calcitonina/análise , Cistatina C/análise , Complicações do Diabetes , Pé Diabético/etiologia , Pé Diabético/patologia , Humanos , MicroRNAs/metabolismo , Óxido Nítrico/metabolismo , Peptídeo Hidrolases/metabolismo
6.
Diabetes Res Clin Pract ; 109(3): 533-40, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26113285

RESUMO

AIM: Adaptation to low oxygen tension (hypoxia) in cells and tissues leads to the transcriptional induction of series of genes and the primary factor mediating this response is the hypoxia-inducible factor-1α. This study was designed in order to examine the HIF-1α gene polymorphism, p582s (rs11549465) in Exon-12 of HIF-1α gene in diabetic subjects with and without foot ulcers (DFU) and to find its expression under these pathological conditions. METHODS: A total of 224 subjects from our tertiary care centre were included, which consists of healthy controls (N=66), type 2 diabetes mellitus (T2DM) (N=79) and T2DM with foot ulcers (DFU) (N=79). Allelic and genotypic comparisons between the different groups were evaluated by PCR-RFLP. The gene expression studies on selected samples (N=15 of each group) were done by Semi-quantitative real time PCR. RESULTS AND DISCUSSIONS: Genotype analysis showed a significant increase in presence of 'T' allele in T2DM & DFU when compared to that of control subjects. Allele wise analysis showed a higher frequency of 'T' allele in the T2DM (62.03%) when compared to that of control subjects (53.79%). Interestingly, semi-quantitative RT-PCR results showed decreased expression of HIF-1α gene on DFU when compared to that of T2DM and control subjects. CONCLUSION: Our findings predict that there is an association of HIF-1α gene polymorphism on foot ulcer patients when compare to that of healthy controls. Semi-quantitative real time studies showed decreased HIF-1α gene expression on foot ulcer patients suggesting its possible role on the pathogenesis.


Assuntos
Pé Diabético/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Polimorfismo de Nucleotídeo Único , Adulto , Idoso , Substituição de Aminoácidos/genética , Estudos de Casos e Controles , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/genética , Pé Diabético/epidemiologia , Feminino , Frequência do Gene , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Fragmento de Restrição , Prolina/genética , Serina/genética
7.
Toxicon ; 77: 32-9, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24184517

RESUMO

The saliva of ticks is critical to their survival as parasites and hematophagous animals. In this study, we have purified an enzyme with trypsin-like activity from the saliva of the tick vector of Lyme Disease, Ixodes scapularis. This enzyme, named as IXOSP (I. scapularis salivary serine protease), is a 29.9 kDa molecule with N-terminus FPxMVxLRIKxR. A BLAST search identified IXOSP as a secreted serine protease (AAY66740) with a conserved catalytic triad His, Asp, and Ser. In vitro studies demonstrated that IXOSP cleaves chromogenic substrates with arginine in the P1 position, by a mechanism inhibited by PMSF or aprotinin. Gene expression studies revealed that IXOSP is expressed at different tick developmental stages, including eggs, and unfed or fed adult tick salivary glands, but not in nymphs or in the midgut. While the physiological substrate for IXOSP remains to be identified, we demonstrated that I. scapularis saliva activate protein C (PC) resulting in the production of activated PC, a potent anticoagulant that also regulates a myriad of inflammatory responses through protease activated receptors. In contrast, the salivary glands of Anopheles gambiae, Anopheles stephensi, Anopheles albimanus, Aedes aegypti, Lutzomyia longipalpis, and Phlebotomus ariasi did not activate protein C. These discoveries are discussed in the context of blood coagulation, inflammation and vector-host interactions.


Assuntos
Hemostasia/efeitos dos fármacos , Ixodes/enzimologia , Oligopeptídeos/isolamento & purificação , Saliva/enzimologia , Serina Proteases/isolamento & purificação , Sequência de Aminoácidos , Animais , Biologia Computacional , Primers do DNA/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Espectrometria de Massas , Dados de Sequência Molecular , Oligopeptídeos/genética , Oligopeptídeos/toxicidade , Rhode Island , Análise de Sequência de Proteína , Serina Proteases/genética , Serina Proteases/toxicidade , Especificidade da Espécie
8.
PLoS One ; 7(11): e48567, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23152782

RESUMO

Transitional cell carcinoma (TCC) of the urinary bladder is the most common cancer of the urinary tract. Most of the TCC cases are of the superficial type and are treated with transurethral resection (TUR). However, the recurrence rate is high and the current treatments have the drawback of inducing strong systemic toxicity or cause painful cystitis. Therefore, it would be of therapeutic value to develop novel concepts and identify novel drugs for the treatment of bladder cancer. Ki-67 is a large nucleolar phosphoprotein whose expression is tightly linked to cell proliferation, and curcumin, a phytochemical derived from the rhizome Curcuma longa, has been shown to possess powerful anticancer properties. In this study, we evaluated the combined efficacy of curcumin and a siRNA against Ki-67 mRNA (Ki-67-7) in rat (AY-27) and human (T-24) bladder cancer cells. The anticancer effects were assessed by the determination of cell viability, apoptosis and cell cycle analysis. Ki-67-7 (10 nM) and curcumin (10 µM), when treated independently, were moderately effective. However, in their combined presence, proliferation of bladder cancer cells was profoundly (>85%) inhibited; the rate of apoptosis in the combined presence of curcumin and Ki-67-7 (36%) was greater than that due to Ki-67-7 (14%) or curcumin (13%) alone. A similar synergy between curcumin and Ki-67-7 in inducing cell cycle arrest was also observed. Western blot analysis suggested that pretreatment with Ki-67-7 sensitized bladder cancer cells to curcumin-mediated apoptosis and cell cycle arrest by p53- and p21-independent mechanisms. These data suggest that a combination of anti-Ki-67 siRNA and curcumin could be a viable treatment against the proliferation of bladder cancer cells.


Assuntos
Antineoplásicos/farmacologia , Curcumina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Antígeno Ki-67/genética , RNA Interferente Pequeno/genética , Ribonuclease III/metabolismo , Neoplasias da Bexiga Urinária/genética , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Carcinoma de Células de Transição/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Humanos , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Neoplasias da Bexiga Urinária/tratamento farmacológico
9.
Pharmacol Res ; 65(1): 48-55, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21933712

RESUMO

Tumor necrosis factor alpha (TNF-α) plays a major role in the pathogenesis of many inflammatory diseases. Neutralizing TNF-α by antibodies or antisense oligodeoxynucleotides, alleviate disease symptoms. In this study, we introduce the new generation of gene-silencing molecules, namely the small interfering RNAs (siRNAs) to reduce TNF-α. Although siRNAs of 19-21bp are commonly used, it is reported that longer siRNAs have much higher efficacies. Here, we report the identification of a 27-mer Dicer-substrate siRNA (DsiRNA) against TNF-α mRNA. Primary cells of rat Kupffer cells were transfected with five 27-mer siRNA constructs (si27-1, si27-2 si27-3, si27-4 and si27-5) for 24h, following which, TNF-α secretion was induced by exposure to LPS (0.1µg/ml) for 2h. TNF-α released to the medium was measured by ELISA. Of the five si27 constructs, si27-3 had the highest inhibitory effect on TNF-α secretion. At 10nM, si27-3 inhibited TNF-α secretion by 80% compared to a 60% inhibition by a 21-mer (SSL3). Following encapsulation in anionic liposomes, si27-3 at 100µg/kg body weight, on two successive days by intravenous administration, inhibited the secretion of TNF-α by 50%. These data demonstrate the identification of a highly efficacious siRNA formulation, which can be used in the treatment of TNF-α mediated diseases.


Assuntos
Terapia Genética/métodos , Células de Kupffer/enzimologia , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ribonuclease III/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Células Cultivadas , Regulação para Baixo , Injeções Intravenosas , Células de Kupffer/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Lipossomos , Masculino , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/administração & dosagem , Ratos , Ratos Sprague-Dawley , Especificidade por Substrato , Fatores de Tempo , Transfecção , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/genética
10.
BMC Biochem ; 12: 32, 2011 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-21708020

RESUMO

BACKGROUND: Ixodes scapularis, commonly known as the blacklegged or deer tick, is the main vector of Lyme disease in the United States. Recent progress in transcriptome research has uncovered hundreds of different proteins expressed in the salivary glands of hard ticks, the majority of which have no known function, and include many novel protein families. We recently identified transcripts coding for two putative cytosolic sulfotransferases in these ticks which recognized phenolic monoamines as their substrates. In this current study, we characterize the genetic expression of these two cytosolic sulfotransferases throughout the tick life cycle as well as the enzymatic properties of the corresponding recombinant proteins. Interestingly, the resultant recombinant proteins showed sulfotransferase activity against both neurotransmitters dopamine and octopamine. RESULTS: The two sulfotransferase genes were coded as Ixosc SULT 1 & 2 and corresponding proteins were referred as Ixosc Sult 1 and 2. Using gene-specific primers, the sulfotransferase transcripts were detected throughout the blacklegged tick life cycle, including eggs, larvae, nymphs, adult salivary glands and adult midgut. Notably, the mRNA and protein levels were altered upon feeding during both the larval and nymphal life stages. Quantitative PCR results confirm that Ixosc SULT1 was statistically increased upon blood feeding while Ixosc SULT 2 was decreased. This altered expression led us to further characterize the function of these proteins in the Ixodid tick. The sulfotransferase genes were cloned and expressed in a bacterial expression system, and purified recombinant proteins Ixosc Sult 1(R) and 2(R) showed sulfotransferase activity against neurotransmitters dopamine and octopamine as well as the common sulfotransferase substrate p-nitrophenol. Thus, dopamine- or octopamine-sulfonation may be involved in altering the biological signal for salivary secretion in I. scapularis. CONCLUSIONS: Collectively, these results suggest that a function of Ixosc Sult 1 and Sult 2 in Ixodid tick salivary glands may include inactivation of the salivation signal via sulfonation of dopamine or octopamine.


Assuntos
Ixodes/enzimologia , Sulfotransferases/genética , Sulfotransferases/metabolismo , Animais , Sangue , Clonagem Molecular , Dopamina/metabolismo , Comportamento Alimentar , Regulação Enzimológica da Expressão Gênica , Ixodes/genética , Ixodes/crescimento & desenvolvimento , Estágios do Ciclo de Vida , Neurotransmissores/metabolismo , Nitrofenóis/metabolismo , Ninfa/enzimologia , Octopamina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Glândulas Salivares/enzimologia , Homologia de Sequência de Aminoácidos , Transdução de Sinais
11.
ACS Chem Biol ; 6(2): 176-84, 2011 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-21043483

RESUMO

Blacklegged ticks (Ixodes scapularis) transmit the causative agent of Lyme disease in the Northeastern United States. Current research focuses on elucidating biochemical pathways that may be disrupted to prevent pathogen transmission, thereby preventing disease. Genome screening reported transcripts coding for two putative sulfotransferases in whole tick extracts of the nymphal and larval stages. Sulfotransferases are known to sulfonate phenolic and alcoholic receptor agonists such as 17ß-estradiol, thereby inactivating the receptor ligands. We used bioinformatic approaches to predict substrates for Ixosc Sult 1 and Ixosc Sult 2 and tested the predictions with biochemical assays. Homology models of 3D protein structure were prepared, and visualization of the electrostatic surface of the ligand binding cavities showed regions of negative electrostatic charge. Molecular docking identified potential substrates including dopamine, R-octopamine and S-octopamine, which docked into Ixosc Sult 1 with favorable binding affinity and correct conformation for sulfonation. Dopamine, but not R- or S-octopamine, also docked into Ixosc Sult 2 in catalytic binding mode. The predictions were confirmed using cytosolic fractions of whole tick extracts. Dopamine was a good substrate (K(m) = 0.1-0.4 µM) for the native Ixodes scapularis sulfotransferases from larval and nymphal stages regardless of their fed/unfed status. Octopamine sulfonation was detected only after feeding when gene expression data suggests that Ixosc Sult 1 is present. Because dopamine is known to stimulate salivation in ticks through receptor stimulation, these results imply that the function(s) of Ixosc Sult 1 or 2 may include inactivation of the salivation signal via sulfonation of dopamine and/or octopamine.


Assuntos
Monoaminas Biogênicas/metabolismo , Biologia Computacional/métodos , Doença de Lyme/metabolismo , Neurotransmissores/metabolismo , Sulfotransferases/metabolismo , Carrapatos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Monoaminas Biogênicas/química , Bioensaio , Dopamina/química , Dopamina/metabolismo , Cinética , Doença de Lyme/enzimologia , Doença de Lyme/transmissão , Dados de Sequência Molecular , Neurotransmissores/química , Octopamina/química , Octopamina/metabolismo , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , Sulfotransferases/química , Sulfotransferases/genética , Carrapatos/enzimologia
12.
Biochem Biophys Res Commun ; 390(3): 511-5, 2009 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-19852941

RESUMO

A novel antimicrobial peptide was isolated from the saliva of the Lyme disease tick vector, Ixodes scapularis, henceforth designated as ISAMP (I. scapularis Antimicrobial Peptide). ISAMP was purified using a sequential method including ultra filtration, gel filtration and reverse-phase high performance liquid chromatography. The purified peak had a molecular weight of 5.3kDa by MALDI/TOF-MS and its amino acid sequence, determined by Edman degradation was PDxGxPxxVKAGRxPxxSI. A BLASTP search revealed that the protein is a putative 5.3kDa secreted protein (AAM93656) from I. scapularis. The predicted protein is composed of 69 amino acids with no conserved domain motifs. Purified ISAMP was found to have antimicrobial activities against bacteria. Gene expression studies were carried out to observe ISAMP expression in different tick tissues. RT-PCR results indicated that the gene was expressed in hemocytes, fat body and salivary gland but virtually no expression was observed in the midgut. ISAMP is only similar to other Ixodid tick proteins, thus it is a member of a unique family.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Ixodes/metabolismo , Proteínas e Peptídeos Salivares/química , Proteínas e Peptídeos Salivares/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/isolamento & purificação , Peptídeos Catiônicos Antimicrobianos/metabolismo , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Escherichia coli/efeitos dos fármacos , Hemolíticos/química , Hemolíticos/isolamento & purificação , Hemolíticos/farmacologia , Proteínas e Peptídeos Salivares/isolamento & purificação , Proteínas e Peptídeos Salivares/metabolismo , Ultrafiltração
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