RESUMO
Small heat shock protein beta-1 (HSPB-1) plays an essential role in the protection of cells against environmental stress.Elucidation of its molecular, structural, and biological characteristics in a naturally wild-type model is essential. Although the sequence information of the HSPB-1 gene is available for many mammalian species, the HSPB-1 gene of Arabian camel (Arabian camel HSPB-1) has not yet been structurally characterized. We cloned and functionally characterized a full-length of Arabian camel HSPB-1 cDNA. It is 791 bp long, with a 5'-untranslated region (UTR) of 34 bp, a 3'-UTR of 151 bp with a poly(A) tail, and an open reading frame (ORF) of 606 bp encoding a protein of 201 amino acids (accession number: MF278354). The tissue-specific expression analysis of Arabian camel HSPB-1 mRNA was examined using quantitative real-time PCR (qRT-PCR); which suggested that Arabian camel HSPB-1 mRNA was constitutionally expressed in all examined tissues of Arabian camel, with the predominately level in the esophagus tissue. Peptide mass fingerprint-mass spectrometry (PMF-MS) analysis of the purified Arabian camel HSPB-1 protein confirmed the identity of this protein. Phylogenetic analysis showed that the HSPB-1 protein of Arabian camel is grouped together with those of Bactrian camel and Alpaca. Comparing the modelled 3D structure of Arabian camel HSPB-1 protein with the available protein 3D structure of HSPB-1 from human confirmed the presence of α-crystallin domain, and high similarities were noted between the two structures by using super secondary structure prediction.
Assuntos
Camelus/genética , Biologia Computacional , Proteínas de Choque Térmico/genética , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Clonagem Molecular , DNA Complementar/genética , Expressão Gênica , Proteínas de Choque Térmico/química , Modelos Moleculares , Filogenia , Estrutura Secundária de Proteína , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: Rhazya stricta Decne. is a medicinal plant that is widespread in Saudi Arabia and desert areas of the Arabian Peninsula. Its extract contains alkaloids, tannins, and flavonoids that are involved in different biological activities. The study aim was to evaluate the effects of Rhazya stricta plant extracts on the proliferation and differentiation of NTERA-2 (NT2) pluripotent embryonal carcinoma cells. METHODS: Soxhlet extraction was carried out using different solvents to extract stems, leaves and fruit parts of this plant. Cytotoxicity was evaluated by an MTS cell viability assay. The ability of the plant extract to induce cell differentiation was examined phenotypically using an inverted light microscope. The expression of pluripotency markers was investigated by reverse transcriptase polymerase chain reaction (RT-PCR) and immunocytochemistry. Phytochemical screening of chloroform stem extracts was carried out and a chromatographic fingerprint was generated using gas chromatography - mass spectrometry (GC-MS). RESULTS: Chloroform stem extract induced differentiation of NT2 cells at 5 µg/ml, and the differentiated cells exhibited neurite formation. Following induction of differentiation, there was significant down-regulation of the pluripotency marker genes Oct4 and Sox2. In addition, the surface antigen pluripotency marker, TRA-1-60, was strongly down-regulated. Phytochemical analysis of the extract showed the presence of alkaloids and saponins. The chromatogram revealed the presence of fifteen compounds with different retention times. CONCLUSION: Our results demonstrate for the first time that chloroform stem extract of R. stricta can induce neuronal differentiation of stem cells at an early stage and may contain potential therapeutic agent that can be used in neurodegenerative diseases.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apocynaceae/química , Carcinoma Embrionário/fisiopatologia , Diferenciação Celular/efeitos dos fármacos , Extratos Vegetais/farmacologia , Antineoplásicos Fitogênicos/isolamento & purificação , Carcinoma Embrionário/tratamento farmacológico , Carcinoma Embrionário/genética , Carcinoma Embrionário/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismoRESUMO
Krüppel-like factor 4 (KLF4) is a pluripotency transcription factor that helps in generating induced pluripotent stem cells (iPSCs). We sequenced for the first time the full coding sequence of Camelus dromedarius KLF4 (cKLF4), which is also known as the Arabian camel. Bioinformatics analysis revealed the molecular weight and the isoelectric point of cKLF4 protein to be 53.043 kDa and 8.74, respectively. The predicted cKLF4 protein sequence shows high identity with some other species as follows: 98% with Bactrian camel and 89% with alpaca KLF4 proteins. A three-dimensional (3D) structure was built based on the available crystal structure of the Mus musculus KLF4 (mKLF4) of 82 residues (PDB: 2 WBS) and by predicting 400 residues using bioinformatics software. The comparison confirms the presence of the zinc finger domains in cKLF4 protein. Phylogenetic analysis showed that KLF4 from the Arabian camel is grouped with the Bactrian camel, alpaca, cattle, and pig. This study will help in the annotation of KLF4 protein and in generating camel-induced pluripotent stem cells (CiPSCs).
