Izolovanje funkcionalne ukupne RNK iz lišća i polena lipe (Tilia cordata)

The conditions required for the isolation of high quality total RNA from European linden (Tilia cordata) leaves and pollen were determined. Pure total RNA was isolated from linden leaves utilizing a Qiagen plant mini kit, while the total RNA isolated from linden pollen using this method was degraded. Successful isolation of total RNA from both linden pollen and leaves, however, was achieved following TRIzol (TM) preparation of the total RNA. The total RNA isolated using TRIzol (TM) was contaminated with genomic DNA but treatment with the enzyme DNase, in solution or on-column, efficiently removed the genomic DNA. Furthermore, the conditions for the elimination of genomic DNA contamination on-column and isolation of pure total RNA from leaves were optimized. The isolated total RNA from both leaves and pollen was used successfully in first-and second-strand cDNA synthesis reactions and in a reverse transcription polymerase chain reaction (RT-PCR), demonstrating that the total RNA isolated using this method was functional. In conclusion, pure and functional total RNA from T. cordata leaves and pollen (27.8 +/- 7.9 mu g g(-1) leaves; 25.7 +/- 1.1 mu g g(-1) pollen) could be obtained and was suitable for application in further molecular biology studies.Uspostavljeni su uslovi za izolovanje ukupne RNK iz lišća i polena evropske lipe (Tilia cordata). Korišćenjem komercijalno dostupnog pribora za izolovanje RNK iz biljaka izolovana je čista ukupna RNK iz lišća lipe, dok je korišćenjem iste metode dobijena degradirana RNK iz polena lipe. Uspešno izolovanje RNK iz lišća i polena je dobijeno korišćenjem TRIzol reagensa. RNK izolovana ovim metodom je kontaminirana genomskom DNK, koja je uspešno eliminisana korišćenjem enzima DNaze. Dalje su optimizovani i uslovi uklanjanja genomske DNK pomoću DNaze. Izolovana ukupna RNK iz oba izvora je dalje uspešno iskorišćena za sintezu prvog i drugog lanca klonske DNK, kao i u reverzno-transkriptivnoj PCR reakciji, dokazujući time da je korišćenjem ovog metoda izolovana funkcionalna ukupna RNK. U zaključku, dobijena je čista i funkcionalna RNK iz lišća i polena T. cordata (27,8±7,9 μg g-1 lišća; 25,7±1,1 μg g-1 polena) koja se može koristiti u daljim molekularno-biološkim istraživanjima

Isolation of functional total RNA from Tilia cordata leaves and pollen

The conditions required for the isolation of high quality total RNA from European linden (Tilia cordata) leaves and pollen were determined. Pure total RNA was isolated from linden leaves utilizing a Qiagen plant mini kit, while\ud the total RNA isolated from linden pollen using this method was degraded. Successful isolation of total RNA from both linden pollen and leaves, however, was achieved following TRIzol™ preparation of the total RNA. The total RNA isolated using TRIzol™ was contaminated with genomic DNA but treatment with the enzyme DNase, in solution or on-column, efficiently removed the genomic DNA. Furthermore, the conditions for the elimination of genomic DNA contamination on-column and isolation of pure total RNA from leaves were optimized. The isolated total RNA from both leaves and pollen was used successfully in first- and second-strand cDNA synthesis reactions and in a reverse transcription polymerase chain reaction (RT-PCR), demonstrating that the total RNA isolated using this method was functional. In conclusion, pure and functional total RNA from T. cordata leaves and pollen (27.8±7.9 μg g-1 leaves; 25.7±1.1 μg g-1 pollen) could be obtained and was suitable for application in further molecular biology studies

Studies on the role(s) of post-translational modification(s) of human apolipoprotein B during very low density lipoprotein assembly and secretion.

The role of two post-translational modifications, N-linked glycosylation and palmitoylation, of human apolipoprotein B (apoB) in the biosynthesis of hepatic apoB-containing very low density lipoproteins (VLDL) was investigated. Working with tunicamycin-treated rat hepatoma McA-RH7777 cells stably expressing human apoB variants, we found that inhibition of N-linked glycosylation decreased the secretion of apoB variants, without affecting translation. Detailed biochemical analysis was performed following site-specific mutagenesis at consensus N-linked glycosylation sites (using asparagine-to-glutamine substitution) within recombinant human apoB variants (i.e. apoB17, -B37, -B48, and -B50). Four notable features associated with the requirement of N-linked oligosaccharides during apoB biosynthesis were observed: (a) N-linked oligosaccharides were required for efficient secretion of the apoB polypeptide. (b) N-linked oligosaccharides were required for the assembly and secretion of the apoB-containing VLDL. (c) Removal of N-linked oligosaccharides was associated with accumulation of total intracellular apoB without specific apoB retention in the ER, and (d) Expression of mutant apoB lacking N-glycans was associated with changes in mass or activity of microsomal triglyceride transfer protein (MTP). Similar biochemical analysis was performed following site-specific mutagenesis at potential palmitoylation sites (using cysteine-to-serine substitution) within human apoB48. The cysteine-to-serine substitution had no effect on the secretion of apoB48-containing lipoproteins or apoB intracellular distribution. Thus, while N-linked glycosylation at the amino terminus of apoB represents an important requisite for proper biogenesis of apoB-containing VLDL, palmitoylation is not required for apoB-containing VLDL assembly and secretion

Insights into proteolytic processing of the major peanut allergen Ara h 2 by endogenous peanut proteases

BACKGROUND: The major peanut allergens are Ara h 1, Ara h 2 and Ara h 6. Proteolytic processing has been shown to be required for the maturation process of Ara h 6. The aim of this study was to examine whether Ara h 2 undergoes proteolytic processing and, if so, whether proteolytic processing influences its ability to bind human immunoglobulin E (IgE). RESULTS: Ara h 2 isolated from peanut extract under conditions of protease inhibition revealed a single additional peak for its two known isoforms (Ara h 2.01 and Ara h 2.02), corresponding to a C-terminally truncated form lacking a dipeptide (RY). Ara h 2 isolated in the absence of protease inhibition, however, yielded two additional peaks, identified as C-terminally truncated forms lacking either a dipeptide (RY) or a single tyrosine residue. The IgE-binding capacity of the Ara h 2 truncated forms was not altered. CONCLUSION: Ara h 2 undergoes proteolytic processing by peanut proteases that involves C-terminal removal of a dipeptide. Hence Ara h 2 isolated from peanut extract is a complex mixture of two isoforms expressed by different genes, Ara h 2.01 and Ara h 2.02, as well as truncated forms generated by the proteolytic processing of these isoforms

Isolation of functional total RNA from Tilia cordata leaves and pollen

Conditions for isolation of high quality total RNA from European linden (Tilia cordata) leaves and pollen were determined. Pure total RNA was isolated from linden leaves utilizing Qiagen plant mini kit while total RNA isolated from linden pollen using this method was degraded. Successful isolation of total RNA from both linden pollen and leaves, however, was achieved following TRIZOL™ preparation of total RNA. Total RNA isolated using TRIZOL™ was contaminated with genomic DNA but treatment with enzyme DNase, in solution or on-column, efficiently removed the genomic DNA. Furthermore, conditions for elimination of genomic DNA contamination on-column and isolation of pure total RNA from leaves were optimized. Isolated total RNA from both leaves and pollen was used successfully in firstand second-strand cDNA synthesis reactions, as well as, in RT-PCR, demonstrating that the total RNA isolated using this method is functional. In conclusion, pure and functional total RNA from Tilia cordata leaves and pollen (27.8 ± 7.9μg/g leaves; 25.7 ± 1.1μg/g pollen) can be obtained and applicable for further molecular biology studies