Deficiency of Vitamin B12, Folate and Methionine During Preconception Period Causes Altered Immune Responses, Obesity, Insulin Resistance and Hypertension in Adult Progeny

Munculnya penyakit degeneratif pada orang dewasa bisa berhubungan dengan fase perkembangan sewaktu di dalam rahim. Asupan nutrisi selama tahap perkembangan inidapat mempengaruhi gen yang meregulasi epigenetik. Telah banyak penelitian yang menemukan bahwa asupan nutrisi selama kehamilan akan mempengaruhi status kesehatan anak yang akan dilahirkan. Akan tetapi, baru sedikit yang meneliti efek dari nutrisi tertentu pada saat sebelum konsepsi dan sebelum kehamilan. Fase ini penting, sebab fase ini adalah waktunya oosit tumbuh dan berkembang. Secara umum, setelah masa perkembangan oosit , DNA metilasi dalam jaringan akan menetap. Riset menunjukkan adanya efek pembatasan asupan nutrisi tertentu, yaitu vitamin B12, vitamin B9 (asam folat) dan metionin, pada masa prekonsepsi dalam diet ibu terhadap terjadinya obesitas, diabetes mellitus, hipertensi dan berkurangnya respon imun pada anak setelah dewasa. Kekurangan ketiga nutrisi tersebut ternyata dapat menyebabkan terjadinya epigenetik melalui DNA hipometilasi pada pulaupulau CpG. Hipometilasi pada DNA tersebut berhubungan dengan fenotipe beberapa parameter klinis yang berupa obesitas, diabetes mellitus, hipertensi dan berkurangnya respon imun. Penemuan-penemuan mengenai hal ini berguna sebagai sumber referensi mengenai nutrisi bagi perencanaan kehamilan untuk mengurangi faktor resiko penyakit degeneratif pada keturuna

Uji Mutagenik Ames untuk Melengkapi Data Keamanan Ekstrak Gambir (Uncaria gambir Roxb.)

The main compound of Uncaria gambir Roxb. (gambir), catechin and it’s derivates have been believed to be potential as antiviral. Epigallocatechin gallate (EGCG) and epicatechin are catechin derivates which are found to be potential as antiviral against Human Immunodeficiency Virus (HIV). However, gambir extract also contains quercetin that has possibility to be mutagenic. Therefore, a preliminary study towards safety of those compounds within gambir extract, mutagenicity assay using Ames Method has been performed.Sample (gambir extract) was obtained from West Sumatera, Indonesia. The extract was characterized according to Farmakope Herbal Indonesia and WHO methods. Mutagenicity test by Ames method utilized a colorimetric microplate in 6 various concentration (125 mg/mL; 62.5 mg/mL; 31.25 mg/mL; 15.625 mg/mL; 7.81 mg/mL dan 3.91 mg/mL) against mutant bacteria Salmonella typhimurium TA 98, Salmonella typhimurium TA 100 and Escherichia coli WP2 uvrA with and without the addition of S-9 enzyme. Extract of gambir in this study contains 86.60% of catechin, 12.92% moisture content, 22.49% water-soluble extract content, 80.63% ethanol-soluble extract content, 0.81% total ash, 0.32% acid insoluble ash content and 10.38% in dryness level. From the mutagenicity test and calculation, fold increase (over baseline) of the sample in 6 various concentration with and without adding S-9 enzyme are lower than 2. Gambir extract from West Sumatra with catechin contains 86.6% hasn’t showed mutagenic effect due to the fold increase (over baseline) of mutagenicity test lower than 2

Purifikasi Katekin dari Ekstrak Gambir (Uncaria gambir Roxb.)

Abstract Uncaria gambir Roxb., one of native plants of Indonesia contains high levels of catechins. Catechins are very potential to be used for medicinal raw materials because theirs effects are proven to be antibacterial, antiviral, and antidyslipidemic. Derivatization of catechin can be developed to produce drug compounds that are effective as antiviral agents for HIV. Derivatization process needs pure catechin isolate as a starting material in order to obtain maximum result so that the isolation process is one of the important stages. In this study, isolation and purification of catechin isolate from Uncaria gambir Roxb. extract was carried out, which will then be the starting material in the derivatization of catechin. Gambir extract used for catechin isolation was a quality 1 gambir extract obtained from Padang, West Sumatra. Gambier extract is characterized according to the standard method stated in Farmakope Herbal Indonesia. Isolation of catechin from gambir extract was done by percolation method using ethyl acetate solvents. Pure catechin isolate was obtained using a vacuum liquid chromatograpy method with a series of solvent hexane and ethyl acetate gradients. Catechin purification was monitored using Thin Layer Chromatography (TLC) method with eluent chloroform:ethyl acetate:formic acid (5:4:1, then identified using High Performance Liquid Chromatography (HPLC), Nuclear Magnetic Resonance (NMR) Spectroscopy, and Liquid Cromatography- Mass Spectroscopy (LC-MS). The purity of catechin isolate obtained was 99.80%± 0.132. Abstrak Uncaria gambir Roxb., salah satu tanaman asli Indonesia yang mengandung katekin dengan kadar yang tinggi. Katekin sangat potensial digunakan untuk bahan baku obat karena efeknya terbukti sebagai antibakteri, antivirus, dan antidislipidemia. Derivatisasi katekin dapat dikembangkan untuk menghasilkan senyawa obat yang efektif sebagai antivirus untuk HIV. Untuk derivatisasi ini diperlukan isolat katekin murni sebagai starting material agar diperoleh hasil yang maksimal sehingga proses purifikasi isolat merupakan salah satu tahap yang penting. Dalam penelitian ini dilakukan isolasi dan pemurnian isolat katekin dari ekstrak Uncaria gambir Roxb. yang selanjutnya akan menjadi bahan awal dalam derivatisasi katekin. Ekstrak gambir yang digunakan untuk isolasi katekin adalah ekstrak gambir kualitas 1 yang diperoleh dari Padang, Sumatera Barat. Ekstrak gambir dikarakterisasi sesuai dengan metode standar yang tertera dalam Farmakope Herbal Indonesia. Isolasi katekin dari ekstrak gambir dilakukan dengan metode perkolasi menggunakan pelarut etil asetat. Isolat katekin murni diperoleh menggunakan metode kromatografi cair vakum (KCV) dengan serangkaian gradien pelarut heksana dan etil asetat. Pemurnian katekin dimonitor menggunakan metode kromatografi lapis tipis (KLT) dengan eluen kloroform : etil asetat : asam format (5:4:1), kemudian diidentifikasi menggunakan High Performance Liquid Chromatography (HPLC), spektroskopi Nuclear Magnetic Resonance (NMR), dan Liquid Cromatography-Mass Spectroscopy (LC- MS). Kemurnian isolat katekin yang didapatkan 99,80%± 0,132

Uji Mutagenik Ames Untuk Melengkapi Data Keamanan Ekstrak Gambir (Uncaria Gambir Roxb.)

The main compound of Uncaria gambir Roxb. (gambir), catechin and it\u27s derivates have been believed to be potential as antiviral. Epigallocatechin gallate (EGCG) and epicatechin are catechin derivates which are found to be potential as antiviral against Human Immunodeficiency Virus (HIV). However, gambir extract also contains quercetin that has possibility to be mutagenic. Therefore, a preliminary study towards safety of those compounds within gambir extract, mutagenicity assay using Ames Method has been performed.Sample (gambir extract) was obtained from West Sumatera, Indonesia. The extract was characterized according to Farmakope Herbal Indonesia and WHO methods. Mutagenicity test by Ames method utilized a colorimetric microplate in 6 various concentration (125 mg/mL; 62.5 mg/mL; 31.25 mg/mL; 15.625 mg/mL; 7.81 mg/mL dan 3.91 mg/mL) against mutant bacteria Salmonella typhimurium TA 98, Salmonella typhimurium TA 100 and Escherichia coli WP2 uvrA with and without the addition of S-9 enzyme. Extract of gambir in this study contains 86.60% of catechin, 12.92% moisture content, 22.49% water-soluble extract content, 80.63% ethanol-soluble extract content, 0.81% total ash, 0.32% acid insoluble ash content and 10.38% in dryness level. From the mutagenicity test and calculation, fold increase (over baseline) of the sample in 6 various concentration with and without adding S-9 enzyme are lower than 2. Gambir extract from West Sumatra with catechin contains 86.6% hasn\u27t showed mutagenic effect due to the fold increase (over baseline) of mutagenicity test lower than 2

A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1

BACKGROUND: Previously we described a transdominant negative mutant of the HIV-1 Tat protein, termed Nullbasic, that downregulated the steady state levels of unspliced and singly spliced viral mRNA, an activity caused by inhibition of HIV-1 Rev activity. Nullbasic also altered the subcellular localizations of Rev and other cellular proteins, including CRM1, B23 and C23 in a Rev-dependent manner, suggesting that Nullbasic may disrupt Rev function and trafficking by intervening with an unidentified component of the Rev nucleocytoplasmic transport complex. RESULTS: To seek a possible mechanism that could explain how Nullbasic inhibits Rev activity, we used a proteomics approach to identify host cellular proteins that interact with Nullbasic. Forty-six Nullbasic-binding proteins were identified by mass spectrometry including the DEAD-box RNA helicase, DDX1. To determine the effect of DDX1 on Nullbasic-mediated Rev activity, we performed cell-based immunoprecipitation assays, Rev reporter assays and bio-layer interferometry (BLI) assays. Interaction between DDX1 and Nullbasic was observed by co-immunoprecipitation of Nullbasic with endogenous DDX1 from cell lysates. BLI assays showed a direct interaction between Nullbasic and DDX1. Nullbasic affected DDX1 subcellular distribution in a Rev-independent manner. Interestingly overexpression of DDX1 in cells not only restored Rev-dependent mRNA export and gene expression in a Rev reporter assay but also partly reversed Nullbasic-induced Rev subcellular mislocalization. Moreover, HIV-1 wild type Tat co-immunoprecipitated with DDX1 and overexpression of Tat could rescue the unspliced viral mRNA levels inhibited by Nullbasic in HIV-1 expressing cells. CONCLUSIONS: Nullbasic was used to further define the complex mechanisms involved in the Rev-dependent nuclear export of the 9 kb and 4 kb viral RNAs. All together, these data indicate that DDX1 can be sequestered by Nullbasic leading to destabilization of the Rev nucleocytoplasmic transport complex and decreased levels of Rev-dependent viral transcripts. The outcomes support a role for DDX1 in maintenance of a Rev nuclear complex that transports viral RRE-containing mRNA to the cytoplasm. To our knowledge Nullbasic is the first anti-HIV protein that specifically targets the cellular protein DDX1 to block Rev’s activity. Furthermore, our research raises the possibility that wild type Tat may play a previously unrecognized but very important role in Rev function. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12977-014-0121-9) contains supplementary material, which is available to authorized users

A HIV-1 Tat mutant protein disrupts HIV-1 Rev function by targeting the DEAD-box RNA helicase DDX1

BACKGROUND: Previously we described a transdominant negative mutant of the HIV-1 Tat protein, termed Nullbasic, that downregulated the steady state levels of unspliced and singly spliced viral mRNA, an activity caused by inhibition of HIV-1 Rev activity. Nullbasic also altered the subcellular localizations of Rev and other cellular proteins, including CRM1, B23 and C23 in a Rev-dependent manner, suggesting that Nullbasic may disrupt Rev function and trafficking by intervening with an unidentified component of the Rev nucleocytoplasmic transport complex. RESULTS: To seek a possible mechanism that could explain how Nullbasic inhibits Rev activity, we used a proteomics approach to identify host cellular proteins that interact with Nullbasic. Forty-six Nullbasic-binding proteins were identified by mass spectrometry including the DEAD-box RNA helicase, DDX1. To determine the effect of DDX1 on Nullbasic-mediated Rev activity, we performed cell-based immunoprecipitation assays, Rev reporter assays and bio-layer interferometry (BLI) assays. Interaction between DDX1 and Nullbasic was observed by co-immunoprecipitation of Nullbasic with endogenous DDX1 from cell lysates. BLI assays showed a direct interaction between Nullbasic and DDX1. Nullbasic affected DDX1 subcellular distribution in a Rev-independent manner. Interestingly overexpression of DDX1 in cells not only restored Rev-dependent mRNA export and gene expression in a Rev reporter assay but also partly reversed Nullbasic-induced Rev subcellular mislocalization. Moreover, HIV-1 wild type Tat co-immunoprecipitated with DDX1 and overexpression of Tat could rescue the unspliced viral mRNA levels inhibited by Nullbasic in HIV-1 expressing cells. CONCLUSIONS: Nullbasic was used to further define the complex mechanisms involved in the Rev-dependent nuclear export of the 9 kb and 4 kb viral RNAs. All together, these data indicate that DDX1 can be sequestered by Nullbasic leading to destabilization of the Rev nucleocytoplasmic transport complex and decreased levels of Rev-dependent viral transcripts. The outcomes support a role for DDX1 in maintenance of a Rev nuclear complex that transports viral RRE-containing mRNA to the cytoplasm. To our knowledge Nullbasic is the first anti-HIV protein that specifically targets the cellular protein DDX1 to block Rev’s activity. Furthermore, our research raises the possibility that wild type Tat may play a previously unrecognized but very important role in Rev function. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12977-014-0121-9) contains supplementary material, which is available to authorized users