Abnormal tubulovesicular particles in brains of hamsters with scrapie

Abnormal tubulovesicular particles of an average diameter of 23 nm have been observed in brains of mice with scrapie as well as in other animals with spongiform encephalopathies, but they were thought to be absent from the brains of hamsters with scrapie in which the highest known concentrations of the infectious agent occur. We observed in neuronal processes of hamsters as well as mice clusters of those tubulovesicular structures, most often in postsynaptic terminals. Such particles have now been seen regularly in both experimental and natural scrapie in all species examined as well as in other spongiform encephalopathies.     

A simple method to enrich mRNA from total prokaryotic RNA

Isolation of prokaryotic mRNA by the poly(dT) method has been difficult, primarily due to the great instability of the poly(A) sequence in its mRNA. We developed a simple method to remove rRNA from total RNA ofStaphylococcus aureus by cloning a PCR-amplifiedS. aureus rRNA gene fragment into a plasmid, and then synthesizing biotin-labeled antisense rRNA to subtract rRNA. By using this method,S. aureus rRNA is significantly reduced and mRNA is enriched. This method may be used to prepare prokaryotic mRNA for many molecular biology applications.     

A rapid, efficient method for isolating DNA from yeast

A method is described for the purification of chromosomal and plasmid DNA from the yeast Saccharomyces cerevisiae. This method is rapid, gives 75% of theoretical yield, and produces DNA that can be cut with restriction endonucleases. Yeast cells are treated with zymolyase, and the resulting spheroplasts are lysed in the presence of the chaotropic agent guanidine hydrochloride. After a brief ethanol precipitation, protein is removed by treatment with proteinase K followed by phenol-chloroform extraction. After ethanol precipitation, the DNA is sufficiently pure for restriction analysis or for the transformation of Escherichia coli.     

A rapid and efficient method for the purification of tyrosinase from Neurospora.

A short and efficient procedure consisting of two chromatographic steps is described for the isolation of tyrosinase from Neurospora. The first step, Celite-column chromatography, resulted in the isolation of four copper-containing proteins from the crude mycelial extract. Anion-exchange chromatography on DEAE-Sephadex of these proteins resulted in the isolation of electrophoretically and serologically pure tyrosinase. More than 70% of the initial tyrosinase activity was recovered in the final enzyme preparation, which had a specific activity of 450 units/mg.     

A rapid and efficient PCR-based mutagenesis method applicable to cell physiology study

PCR-based mutagenesis is a cornerstone of molecular biology and protein engineering studies. Herein we describe a rapid and highly efficient mutagenesis method using type IIs restriction enzymes. A template gene is amplified into two separate PCR fragments using two pairs of anchor and mutagenic primers. Mutated sequences are located near the recognition site of a type IIs restriction enzyme. After digestion of two fragments with a type IIs enzyme, exposed cohesive ends that are complementary to each other are then ligated together to generate a mutated gene. We applied this method to introduce multiple site-directed mutations in EGFP and Bcl-2 family genes and observed perfect mutagenesis efficiency at the desired sites. This efficient and cost-effective mutagenesis method can be applied to a wide variety of structural and functional studies in cell physiology. Type IIs restriction enzyme; enhanced green fluorescent protein; Bcl-2     

Purification of scrapie agent from infected animal brains and raising of antibodies to the purified fraction

The fraction (P4) containing scrapie infectivity was obtained by treatment of scrapie-infected mouse brains with the detergent sarcosyl, differential centrifugation, and proteolytic enzyme digestion. Scrapie infectivity in the P4 fraction was purified 239-2,390 times with respect to protein. Similar fractions were also prepared from the brain of a sheep naturally infected with scrapie. Morphological observation of the P4 fractions revealed that the main components were unique rods of 3-5 X 60-200 nm, which resembled scrapie-associated fibrils (SAF) or prion rods. The P4 fractions formed three major broad bands of polypeptides with molecular weights (MWs) of about 24.5K, 21K, and 17K dalton (Kd) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and some low MW polypeptides were also present in the fraction. Rabbits immunized with this fraction prepared from mouse brains raised antibodies against the three major polypeptides.     

A hybridization and enzyme blocking method to enrich minor cDNA sequences

A method is presented which allows for the enrichment of low frequency cDNA sequences. The crucial step in the procedure is the hybridization of a pool of cDNA to homologous or heterologous RNA to a Rot value which will leave minor sequences in a single strand cDNA form while the major sequences form cDNA:RNA hybrids. This allows subsequent enzymatic differentiation between major and minor sequences resulting ultimately in the degradation of the major sequences. The procedure is general and simple as it requires no column chromatography step. The method is designed to integrate into a widely used cDNA cloning protocol and results either in double-stranded cDNA which can be used for molecular cloning or as a source of probes for hybridization.     

A rapid and efficient method to remove labelled molecular probes from nucleic acids immobilized on solid supports

A rapid and efficient method is described for the removal of radio-active molecular probes from nucleic acids immobilized on nylon membranes. This method involves boiling in distilled water in a microwave oven. This procedure can be completed within ten minutes, does not require the use of any buffers or reagents, and produces results comparable with conventional buffer-wash procedures recommended by the suppliers of the transfer membranes.     

Immunoglobulins in urine of hamsters with scrapie

In the prion diseases, a prolonged, asymptomatic incubation period precedes the onset of neurologic dysfunction. At present, a noninvasive test is not available for the presymptomatic diagnosis of prion disease, and thus the report of a test for prions using urine has been of great interest (Shaked, G. M., Shaked, Y., Kariv-Inbal, Z., Halimi, M., Avraham, I., and Gabizon, R. (2001) J. Biol. Chem. 276, 31479-31482). Using Western immunoblots with the anti-prion protein (PrP) 3F4 monoclonal antibody and an anti-mouse IgG secondary antibody, a protease-resistant PrP was reported in the urine of Syrian hamsters and humans with prion disease. Here we have demonstrated that this purportedly "protease-resistant PrP" band in the urine of diseased hamsters is detectable using the anti-mouse IgG secondary antibody in the absence of the 3F4 monoclonal antibody. Mass spectrometric analysis identified an immunoglobulin light chain in the band but found no PrP peptides. No similar band was found in the urine of uninfected hamsters or in brain homogenates from normal or prion-infected hamsters. Moreover, the band in the urine of infected hamsters was not detected using two chimeric human-mouse recombinant anti-PrP antibody fragments followed by an anti-human IgG secondary antibody. Our results indicate that the band detected under previously published conditions is due to the cross-reactivity of the anti-mouse IgG antibody with IgG light chains and possibly heavy chain fragments in urine, but not with PrP.     

A rapid and highly efficient method for transformation of sugarcane callus

Modern sugarcane cultivars have complex genetic characteristics and low fertility that render their genetic improvement through traditional breeding difficult. Genetic engineering methodology to introduce foreign genes provides new opportunities for the genetic improvement of sugarcane cultivars. One of prerequisites for successful insertion of a gene cassette into the plant genome is the availability of an efficient transformation protocol. An improved protocol for Agrobacterium-mediated transformation of sugarcane is described. Between 85 and 100% of calli transformed using this procedure produced new calli, and 100% of them were positive for the inserted gene. The whole procedure permitted the production of transgenic calli in a short time (1.5 mo). The transformed calli can be cultured further for the production of the inserted gene-encoded enzyme by using cell culture, or they can be regenerated into transgenic plants. This protocol may be implemented also for the generation of transgenic plants from other species.     

A rapid and efficient route to benzazole heterocycles

This protocol describes a rapid, high-yielding, microwave-mediated route that affords benzazole heterocycles in high crude purity and represents a significant advancement toward an environmentally friendly reaction. The reaction of aryl isothiocyanates with o-nucleophilic anilines produces thiourea intermediates that, in the presence of a carbodiimide-functionalized resin, cyclize to benzazoles with the safe removal of one equivalent of hydrogen sulfide. This procedure takes ～ 8.5 h to complete: 1-3 h for setup, 4.5 h for benzazole formation and 2 h for workup and purification.     

Ultrastructural studies of scrapie-associated fibrils and prion protein from hamster brains infected with scrapie

We report here about the purification of prion protein 27-30 (PrP 27-30) and scrapie-associated fibrils (SAF) from hamsters infected with the 263K strain of scrapie. Ultrastructural analysis of fractions from scrapie-infected brains revealed numerous fibrils measuring approximately 20 nm in diameter and 100-200 nm in length. The substructure of these fibrils consisted of protofilaments which were usually straight and rarely helically arranged. We conclude that the electron microscopic appearance of SAF depends much on the purification scheme.     

A rapid and efficient method for the isolation of restrictable total DNA from plants of the genusAbelmoschus

A rapid, simple and efficient protocol is given for the extraction of restrictable total DNA from plants of the genusAbelmoschus, for which the main obstacle is the stickiness of the solution, after grinding of green leaves. This problem is resolved using cotyledons of dark-grown seedlings.     

A rapid and efficient assay for extracting DNA from fungi

AIMS: A method for the rapid extraction of fungal DNA from small quantities of tissue in a batch-processing format was investigated. METHODS AND RESULTS: Tissue (< 3.0 mg) was scraped from freshly-grown fungal isolates. The tissue was suspended in buffer AP1 and subjected to seven rounds of freeze/thaw using a crushed dry ice/ethanol bath and a boiling water bath. After a 30 min boiling step, the tissue was quickly ground against the wall of the microfuge tube using a sterile pipette tip. The Qiagen DNeasy Plant Tissue Kit protocol was then used to purify the DNA for PCR/sequencing applications. CONCLUSIONS: The method allowed batch DNA extraction from multiple fungal isolates using a simple yet rapid and reliable assay. SIGNIFICANCE AND IMPACT OF THE STUDY: Use of this assay will allow researchers to obtain DNA from fungi quickly for use in molecular assays that previously required specialized instrumentation, was time-consuming or was not conducive to batch processing.     

A rapid and efficient protocol to purify biologically active recombinant proteins from mammalian cells

Here, we describe a simple and efficient method for the expression and purification of active recombinant proteins in mammalian cells. This method uses the expression of T7 epitope-tagged proteins in transiently transfected 293T cells grown in monolayer, followed by anti-T7-agarose affinity chromatography. This procedure yields approximately between 75 and 100 microg of biologically active protein/150 cm(2) flask that can be used for biochemical studies. We have tested this protocol for the expression of the prototype SR protein, SF2/ASF, which is a member of the SR protein family with a role in constitutive and alternative splicing. We show that SF2/ASF purified using this protocol is able to complement an S100 HeLa extract, demonstrating that is biologically active. Moreover, expression of a novel SR-related protein that it is required for the second step of pre-mRNA splicing also rendered an active protein. In summary, we present a protocol based on transient transfection of mammalian cells that results in easy purification of significant amounts of biologically active proteins.     

A rapid and efficient method for primary culture of human adipose-derived stem cells

Adipose tissue contains some populations, adipose-derived stem cells (ADSCs) which can differentiate into adipogenic, chondrogenic, osteogenic, myogenic, and endothelial cells. Furthermore, adipose tissue can be easily obtained in large quantities through a simple liposuction. ADSCs are thought to be an alternate source of autologous adult stem cells for cell-based therapy. However, it is time-consuming and inefficient to harvest ADSCs by using a traditional collagenase-digestion method. To meet the demand of large quantities of ADSCs in the basic and applied research of regenerative medicine, we developed a rapid and efficient method for isolation and culture of primary ADSCs. The results indicated that the ADSCs obtained with our method possessed strong abilities of proliferation and colony formation in vitro, and could keep low level of cell senescence with stable population doubling during long-term culture in vitro. Furthermore, these harvested ADSCs were capable to differentiate into osteogenic and adipogenic lineages in the specific induction medium. In addition, the results of flow cytometry analysis indicated that these ADSCs could positively express multiple CD markers, such as CD44, CD105, CD29, CD90, and CD13, and hardly expressed CD31, CD34, CD45, and CD106, which was homologous to the mesenchymal stem cells. Therefore, the ADSCs isolated with our method are consistent with previously reported characteristics of the ADSCs. This new method that we established in this study is an efficient tool to isolate and culture the stem cells from adipose tissue.