A convenient procedure for the isolation of intact translatable mRNA by potassium iodide gradient centrifugation

High concentrations of KI were found to efficiently protect RNA against degradation by RNases. When a sufficient amount of solid KI was added to cell lysates or subcellular fractions (9 g per 10 ml), the solutions could be stored at room temperature for several days without measurable degradation of mRNA. Ribonucleic acids were selectively sedimented when these KI-containing solutions were centrifuged at 72,000 x g for 24 h. The RNA pellets were found to be readily dissolved in bidistilled water and the redissolved RNA could be immediately submitted to oligo(dT)-cellulose chromatography to isolate the poly(A)-containing RNA. However, extraction with phenol/chloroform was found to be necessary, if total RNA or poly(A)-minus RNA was to be analysed. This procedure was found to be superior to other methods currently in use - especially with respect to the isolation of intact, translatable high-molecular-weight mRNA.     

A convenient procedure for the synthesis of ceramides

A procedure for the preparation of ceramides by direct coupling of long-chain bases and fatty acids in the presence of a mixed carbodiimide is described. This method has been used to prepare ceramides containing sphing-4-enine or sphinganine and various saturated and unsaturated fatty acids as well as saturated 2-hydroxy acids. Ceramides containing 4-hydroxy sphinganine and saturated nonhydroxy acids have also been prepared. The yields were 60-75%. The characterization of these compounds by gas-liquid chromatography-mass spectrometry as trimethylsilyl derivatives has been previously reported. Some of the ceramides are further characterized in this report by infrared spectroscopy and one compound, in addition, by elementary analysis. Use of racemic constituents for 2-hydroxy acid ceramide syntheses leads to the formation of diastereoisomers which separate by thin-layer chromatography. These were characterized by gas-liquid chromatography-mass spectrometry as the trimethylsilyl derivatives and by infrared spectroscopy. Their configurations were established by syntheses with optically active constituents.     

A simple procedure for isolation of eukaryotic mRNA polyadenylation factors.

We have devised a simple chromatographic procedure which isolates five polyadenylation factors that are required for polyadenylation of eukaryotic mRNA. The factors were separated from each other by fractionation of HeLa cell nuclear extract in two consecutive chromatographic steps. RNA cleavage at the L3 polyadenylation site of human adenovirus 2 required at least four factors. Addition of adenosine residues required only two of these factors. The fractionation procedure separates two components that are both likely to be poly(A) polymerases. The candidate poly(A) polymerases were interchangeable and participated during both RNA cleavage and adenosine addition. They were discriminated from each other by chromatographic properties, heat sensitivity and divalent cation requirement. We have compared our data with published information and have been able to correlate the activities that we have isolated to previously identified polyadenylation factors. However, we have not been able to assign one of the candidate poly(A) polymerases to a previously identified poly(A) polymerase. This simple fractionation procedure can be used for generating an in vitro reconstituted system for polyadenylation within a short period of time.     

A novel approach to the synthesis of oligodeoxyribonucleotide boranophosphates.

Deoxyribonucleoside 3'-boranophosphate derivatives including adenine, cytosine, guanine, and thymine bases were synthesized in good yields by the use of a new boranophosphorylation reaction. The reaction was found to be effective for the formation of internucleotidic boranophosphate linkages.     

A one-step procedure for isolation and resolution of the Phaseolus vulgaris isolectins by affinity chromatography

A new fast-working one-step method for the resolution of the Phaseolus vulgaris isolectins is described which requires inexpensive materials.     

A cystatin-based affinity procedure for the isolation and analysis of papain-like cysteine proteinases from tissue extracts

Cysteine-proteinases (CP) of the papain family can be affinity-adsorbed by egg white cystatin C coupled to Sepharose 4B, thus allowing their selective isolation from either tissue or cultured cell extracts as well as biolological fluids and culture media. CP complexed by immobilized cystatin are further analyzed by means of SDS-PAGE and Western blot followed by serial or parallel immunological detection. The single-step affinity adsorption of papain-like enzymes has the advantage, over immunoprecipitation techniques, of yielding the simultaneous and comprehensive picture of most CP, as both precursor and mature forms, in a given sample. Moreover, cell extraction in the presence of immobilized cystatin ensures a fast complexation of CP, avoiding artifacts, due to conversion, degradation, and, eventually, subtraction of constitutive enzymes from the sample because of their interactions with endogenous inhibitors. This will provide a pattern that might reflect more closely the real CP levels in intact cells. The method may be useful in the field of biochemistry, cell biology, and, possibly, clinical chemistry to perform rapid analyses of papain-like enzymes and to monitor changes in both cellular and extracellular CP profiles along with different physiopathological conditions.     

A simple procedure for the isolation of protein disulphide-isomerase.

A two-step procedure is described for the purification of protein disulphide-isomerase (PDI). This procedure is based on the previous finding that the beta-subunit of the prolyl 4-hydroxylase tetramer (alpha 2 beta 2) is identical with PDI [Koivu, Myllylä, Helaakoski, Pihlajaniemi, Tasanen & Kivirikko (1987) J. Biol. Chem. 262, 6447-6449; Pihlajaniemi, Helaakoski, Tasanen, Myllylä, Huhtala, Koivu & Kivirikko (1987) EMBO J. 6, 643-649]. The procedure involves purification of the prolyl 4-hydroxylase tetramer by a simple affinity chromatography and subsequent isolation of the beta-subunit from the dissociated tetramer by ion-exchange chromatography.     

A convenient procedure for the synthesis of nonsymmetrical bivalent selective serotonin reuptake inhibitors using polymer-supported reagents

A convenient synthesis of nonsymmetrical bivalent inhibitors of the serotonin transporter is described. The synthesis utilizes polymer-supported reagents that allow for rapid access to novel bivalent ligands without the need for isolation or purification of synthetic intermediates.     

A simple and convenient method for acyclonucleoside synthesis.

Introduction of acyclic chain for synthesis of acyclonucleoside derivatives was achieved in a simple and convenient way. Silylated pyrimidine or purine bases were treated with 1,3-dioxolane, trimethyl chlorosilane and metal iodide, such as KI and NaI, all together at room temperature. By this method, 2-thiopyrimidine derivatives were also obtained in good yield, using 2 molecular equivalents of 1,3-dioxolane.     

A convenient microtitre tray procedure for yeast identification

Media for yeast identification tests were incorporated into the wells of a microtitre tray. The tests included fermentation and assimilation of carbohydrates, assimilation of nitrogen compounds, growth in vitamin-free medium, resistance to cycloheximide, and observations for cell morphology and sporulation. Results of tests conducted in the trays showed very good agreement with those obtained by conventional methods. Eighteen reference yeasts were correctly identified from tests conducted in the trays. The trays of media could be stored, and provided a convenient system for yeast identification.     

A convenient microtitre tray procedure for yeast identification

Media for yeast identification tests were incorporated into the wells of a microtitre tray. The tests included fermentation and assimilation of carbohydrates, assimilation of nitrogen compounds, growth in vitamin-free medium, resistance to cycloheximide, and observations for cell morphology and sporulation. Results of tests conducted in the trays showed very good agreement with those obtained by conventional methods. Eighteen reference yeasts were correctly identified from tests conducted in the trays. The trays of media could be stored, and provided a convenient system for yeast identification.     

Facile synthesis of benzyl beta-D-galactofuranoside. A convenient intermediate for the synthesis of D-galactofuranose-containing molecules

Benzyl beta-D-galactofuranoside was efficiently obtained from 1,2,3,5,6-penta-O-benzoyl-alpha,beta-D-galactofuranose, via benzyl 2,3,5,6-tetra-O-benzoyl-beta-D-galactofuranoside. Conditions for the O-debenzylation were investigated in order to evaluate the synthetic application of the benzyl group as an anomeric protector of a galactofuranose moiety in synthetic strategies involving galactofuranose.     

A convenient method for saponin isolation in tumour therapy

Saponinum album (Merck), which is a crude mixture of saponins from Gypsophila paniculata L., was shown to improve the anti cancer therapy when used in vivo in combination with saporin-based targeted toxins. Unfortunately saponinum album cannot be used for further development since Merck has ceased its production in the 1990s. As pure saponins are mandatory for use in medical purposes we developed a convenient method for saponin isolation directly from the roots of Gypsophila paniculata L. The developed method is rapid, cheap and scaling up is also possible. By combining dialysis and HPLC three saponins were isolated in a one-step procedure. Chemical structures of the purified saponins were characterized by extensive one and two-dimensional NMR-spectroscopy and by using ESI-TOF-MS. The biological activities of the purified saponins were also investigated. The method presented herein enabled a rapid and cheap isolation of saponins for tumour therapy.