A simple and efficient procedure for the synthesis of 5''-aminoalkyl oligodeoxynucleotides.

Synthetic deoxyoligonucleotides have been 5'-aminoalkylated at the end of step-wise synthesis on the polymer support. This was achieved through the activation of the 5'-hydroxyl group as its 5'-imidazolyl derivative using carbonyldiimidazole, which was subsequently displaced with hexamethylene diamine to yield the title compound. The alkyl carbamate linkage thus generated withstands the deprotection conditions used in oligonucleotide synthesis. Purification by gel electrophoresis and further derivatization at the 5'-amino group with N-hydroxysuccinimidobiotin is described.     

A simple enzymatic procedure for the synthesis of a hydroxylated polyester from glycerol and adipic acid

Lipase B from Candida antarctica catalyzed regioselectively the polyesterification of glycerol and adipic acid. UV-MALDI-TOF-MS analysis of the polymers shows low molecular weight polyesters (1314-1716) with very narrow polydispersities (1.0–1.2).     

A simple and efficient procedure for the rapid homogenization of cultured animal cells grown in monolayer

Cell swelling takes place rapidly when animal cells in monolayer culture are treated with hypotonic buffer in situ; scraping of the swollen cells causes virtually 100% cell lysis. Because this procedure avoids the use of scraping and centrifugation to collect the cells and the use of Dounce homogenizers for cell disruption, recovery of the cell extract is very high. 3-Hydroxy-3-methylglutaryl coenzyme A reductase activities of cell extracts prepared by this method are virtually identical to those prepared by the conventional procedure involving Dounce homogenization.     

A simple procedure for the efficient estimation of potato cyst nematode larvae in lactophenol-treated roots

Counts of potato cyst nematode larvae in whole roots of potatoes may be readily made by liberating the larvae from lactophenol treated roots into water using a blender, removing root debris by filtration and counting larvae directly in water. The method obviates the health hazard associated with high levels of phenolic fumes when counting intact root systems or pieces directly in lactophenol and is also considerably faster and more efficient.     

A simple and efficient procedure for cryopreservation of embryogenic cells of aromatic Indica rice varieties

Embryogenic suspension cells of two commercially cultivated aromatic Indica rice varieties, Basmati 385 and Pusa Basmati 1, were cryopreserved using a simple one-step freezing procedure that does not require a controlled-rate freezer. The procedure involves osmotic pre-conditioning of cells with mannitol, addition of a cryoprotectant solution consisting of sucrose, dimethyl sulfoxide, glycerol, proline, and modified R₂ medium, cooling to –25°C for 2 h in a freezer, and then storage in liquid nitrogen. After rapid thawing at 45°C, these cultures showed post-thaw cell viability of 5.6 to 10.5% and formed actively dividing, readyto-use cell suspensions in 20–35 d when cultured directly into liquid medium. Plants were regenerated from cell clumps as well as from colonies formed by protoplasts that were isolated from suspension cells re-established from cryopreserved cells, with frequencies higher (54–98%) than, or comparable to, those obtained from three to four-month-old original non-frozen cell cultures. Cell viability and regeneration frequencies of post-thawed Pusa Basmati 1 cultures were similar to those obtained from the suspension cells cryopreserved using the conventional slow-freezing procedure which involves pre-freezing cells to –40°C at the rate of –0.2°C per min prior to immersion in liquid nitrogen. In Basmati 385, however, cells frozen at ––25°C showed lower post-thaw cell viability than those preserved using the slow-freezing procedure, but these cells produced cell suspensions that had greater shoot morphogenetic potential. The study indicates the beneficial effect of this simple freezing procedure, not only for preserving desirable cultured cells but also for an enrichment of embryogenic cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DMSO dimethylsulfoxide - LN liquid nitrogen - MS Murashige and Skoog (1962) medium - NAA -napthaleneacetic acid - pcv packed cell volume - TTC 2,3,5-triphenyltetrazolium chloride     

A simple and efficient procedure for the isolation of high-quality phage lambda DNA using a DEAE-cellulose column

A simple and rapid procedure for purifying large quantities of bacteriophage lambda particles and DNA is described. The procedure involves DEAE-cellulose column chromatography of the phage particles and elution of the phage particles from the column with a low-ionic-strength buffer. The resulting phage were well separated from RNA, DNA, and proteins derived from Escherichia coli host cells. The lambda DNA was prepared from the purified phage particles by the conventional method of phenol extraction and ethanol precipitation. This procedure did not use nucleases, proteases, detergents, or CsCl density gradient centrifugation. The lambda DNA obtained by this method was equivalent in purity to the material prepared by CsCl density gradient centrifugation and amenable to restriction enzyme digestion, ligation, radiolabeling, and double-stranded DNA sequencing. A detailed protocol is described for obtaining 0.5 to 1.0 mg DNA from a 1-liter liquid lysate in less than 5 h. This procedure is simple, inexpensive, and timesaving, and is particularly suitable for large-scale isolation of lambda DNA.     

Gene cloning and characterization of a deaminase from the 4-amino-3-hydroxybenzoate-assimilating Bordetella sp. strain 10d

The 4-amino-3-hydroxybenzoate-assimilating Bordetella sp. strain 10d produces a deaminase that catalyzes the deamination of 2-amino-5-carboxymuconic 6-semialdehyde. A gene encoding the deaminase, ahdB , was cloned and expressed in Escherichia coli; ahdB is located downstream from the previously reported genes encoding 4-amino-3-hydroxybenzoate 2,3-dioxygenase ( ahdA ) and a LysR-type regulator. The deduced amino acid sequence of ahdB shows 30–33% identity to those of previously reported 2-aminomuconate deaminases. We identified a region (RAGDFLXVSG) conserved in AhdB and three other deaminases. The recombinant enzyme AhdB was purified to homogeneity. After a coupled enzyme assay with purified AhdA, AhdB, and the substrate 4-amino-3-hydroxybenzoate, the final product, formed by the action of AhdA, AhdB, and by nonenzymatic decarboxylation, was identified by HPLC, MS, and ¹H-nuclear magnetic resonance analyses as 2-hydroxymuconic 6-semialdehyde.     

6-Methylpurine derived sugar modified nucleosides: Synthesis and evaluation of their substrate activity with purine nucleoside phosphorylases

6-Methylpurine (MeP) is cytotoxic adenine analog that does not exhibit selectivity when administered systemically, and could be very useful in a gene therapy approach to cancer treatment involving Escherichia coli PNP. The prototype MeP releasing prodrug, 9-(β-d-ribofuranosyl)-6-methylpurine, MeP-dR has demonstrated good activity against tumors expressing E. coli PNP, but its antitumor activity is limited due to toxicity resulting from the generation of MeP from gut bacteria. Therefore, we have embarked on a medicinal chemistry program to identify non-toxic MeP prodrugs that could be used in conjunction with E. coli PNP. In this work, we report on the synthesis of 9-(6-deoxy-β-d-allofuranosyl)-6-methylpurine (3) and 9-(6-deoxy-5-C-methyl-β-d-ribo-hexofuranosyl)-6-methylpurine (4), and the evaluation of their substrate activity with several phosphorylases. The glycosyl donors; 1,2-di-O-acetyl-3,5-di-O-benzyl-α-d-allofuranose (10) and 1-O-acetyl-3-O-benzyl-2,5-di-O-benzoyl-6-deoxy-5-C-methyl-β-d-ribohexofuran-ose (15) were prepared from 1,2:5,6-di-O-isopropylidine-α-d-glucofuranose in 9 and 11 steps, respectively. Coupling of 10 and 15 with silylated 6-methylpurine under Vorbrüggen glycosylation conditions followed conventional deprotection of the hydroxyl groups furnished 5′-C-methylated-6-methylpurine nucleosides 3 and 4, respectively. Unlike 9-(6-deoxy-α-l-talo-furanosyl)-6-methylpurine, which showed good substrate activity with E. coli PNP mutant (M64V), the β-d-allo-furanosyl derivative 3 and the 5′-di-C-methyl derivative 4 were poor substrates for all tested glycosidic bond cleavage enzymes.     

A simple procedure for the determination of the trapped volume of liposomes

A new method is described for determining the volume of the aqueous compartment of liposomes. Liposomes are prepared in a solution of the fluorescent dye, calcein. The fraction of the total volume that is within the liposomes is obtained as the fraction of the fluorescence that remains after adding cobalt(II) ions which, when chelated by calcein, quench its fluorescence. The method is rapid, simple and accurate. Separation of the liposomes from the medium is not required. The procedure is equally well suited to the assay of permeability characteristics of liposomal membranes.     

A simple procedure for the purification of rat alpha-fetoprotein

A simple purification procedure for obtaining a high yield of electrophoretically and immunologically pure rat α-fetoprotein from amniotic fluid is described. Rat amniotic fluid is passed through an anti-rat albumin immunoabsorbent column to remove albumin. The albumin-free eluate is then chromatographed on DEAE-Sephacel to separate α-fetoprotein from transferrin and other minor protein contaminants. This two-step purification procedure results in a recovery of approximately 70% of the rat α-fetoprotein originally present in the amniotic fluid.     

A simple and rapid procedure to establish embryogenic cell suspensions as a source of protoplasts for efficient plant regeneration from two Chinese commercial rice cultivars

An efficient and rapid procedure has been developed to establish embryogenic cell suspension cultures of two Japonica Chinese commercial rice cultivars, Zhonghua 8 and Eryi 105. Embryogenic cell suspensions of both varieties were established from 0.5–1.0 g fresh weight of embryogenic callus in AA medium within 2.5 months of the initiation of callus from sterilised seeds. The previously reported subculture of callus on semi-solid medium for 4–8 weeks prior to transfer into liquid medium was unnecessary and caused delay in the establishment of embryogenic cell suspensions. Protoplasts were isolated reproducibly from cell suspensions up to 18 months after their initiation, with protoplast plating efficiencies attaining 0.15–0.37%. Reproducible plant regeneration from 14–26% of the protoplast-derived tissues was achieved without the requirement for nurse cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

A novel procedure for simple and efficient genotyping of single nucleotide polymorphisms by using the Zn2+-cyclen complex

The analysis of single nucleotide polymorphisms (SNPs) is increasingly utilized in the study of various genetic determinants. Here, we introduce a simple, rapid, low-cost and accurate procedure for the detection of SNPs by polyacrylamide gel electrophoresis (PAGE) with a novel additive, the Zn2+- cyclen complex (cyclen = 1,4,7,10-tetraazacyclododecane). The method is based on the difference in mobility of mutant DNA (in the same length) in PAGE, which is due to Zn2+-cyclen binding to thymine bases accompanying a total charge decrease and a local conformation change of target DNA. Various nucleotide substitutions (e.g. AT to GC) in DNA fragments (up to 150 bp) can be visualized with ethidium bromide staining. Furthermore, heteroduplex and homoduplex DNAs are clearly separated as different bands in the gel. We demonstrate the analysis of single- and multiple-nucleotide substitutions in a voltage-dependent sodium channel gene by using this novel procedure (Zn2+-cyclen-PAGE).     

A simple procedure for the separation of insoluble collagen and elastin

It is possible to solubilize selectively collagen from a collagen-elastin mixture by the use of a phenol:acetic acid:water solvent. This technique can be successfully applied to vascular tissue which has been salt-extracted and delipidated. As well as separating both of the major insoluble structural proteins from the one tissue sample, the method is very simple and inexpensive and requires minimal attention.     

A simple and efficient method for the preparation of GDP-fucose.

A crude extract from hog submaxillary gland was found to synthesize guanosine diphosphate (GDP)-fucose, when incubated with fucose, ATP and GTP, the two enzymatic steps (fucose

fucose-1-phosphate

GDP-fucose) proceeding without noticeable side reactions. Column chromatography on Dowex 1 (HCO₃^?), gel filtration on Sephadex G-15 and preparative paper chromatography gave pure GDP-fucose in an overall yield of 81%. The sugar nucleotide was shown to be active as a glycosyl donor in fucose-incorporating systems.     

A simple and efficient procedure for isolating plant chromatin which is suitable for studies of DNase I-sensitive domains and hypersensitive sites

Summary A simple and rapid procedure has been developed for the isolation of chromatin from plant leaves. The molecular weight of the DNA extracted from these chromatin preparations is comparable to that of DNA isolated by a conventional purification procedure (CTAB-CsCl-method). These results suggest that almost no degradation occurs during the isolation procedure. The effect of DNase I on three different groups of genes was studied; one of them, encoding the NADPH-protochlorophyllide oxidoreductase (PCR), represents a gene which is actively transcribed in etiolated leaf tissue. The other genes examined encode the hordein seed storage protein and 26S ribosomal RNA. The hordein genes are known to be inactive in leaves.The hordein and rDNA genes were found to be resistant to low levels of DNase I, while the gene for the PCR was highly sensitive to DNase I. During the course of digestion of the PCR gene, discrete cleavage products are generated. These indicate the presence of DNase I hypersensitive sites in the vicinity of the PCR gene in etiolated leaves. As a control naked DNA has been digested with DNase I. No differences in sensitivity between the PCR gene and the hordein genes can be detected.     

A simple procedure for the manual isolation and identification of plant heterokaryons

A simple procedure is described for the aseptic manual isolation of individual heterokaryons. Heterokaryons were identified with bright field illumination using an inverted microscope and isolated by means of a micro-manipulator and capillary pipette coupled to a specially constructed syringe. When cell suspension protoplasts were labelled with fluorescein isothiocyanate and fused with mesophyll protoplasts, the heterokaryons exhibited an apple green cytoplasmic fluorescence (from cell suspension protoplasts) and a red chloroplast fluorescence (from mesophyll protoplasts). By this double fluorescence procedure, these manually isolated heterokaryons, identified initially using brightfield could be confirmed as heterokaryons.