Determination of base specificity of multiple ribonucleases from crude samples

Ribonucleases are widely found in the tissues of living organisms, but the functions of individual ribonucleases are not clear. To facilitate characterization of individual ribonucleases, I have developed a rapid method to separate and identify each ribonuclease from a crude sample by gel electrophoresis instead of by time-consuming purification steps. The ribonucleases in a crude sample are first separated by RNA-cast SDS-polyacrylamide gel electrophoresis and then eluted from the gel after ethidium bromide staining. To determine the base specificity of each ribonuclease, a 5 labelled oligonucleotide with known sequence is added to the enzyme eluate and the digested products are analyzed by denaturing gel electrophoresis. The base specificity of bovine pancreatic ribonuclease (RNase A), bullfrog oocyte-specific ribonuclease (RC-RNase), human serum ribonucleases and sweet potato leaf ribonucleases were determined by this method. Other properties of individual ribonucleases, e.g. substrate preference, may also be determined from crude samples by this method without further purification steps.Abbreviations RNase ribonuclease - SDS sodium dodecyl sulfate     

Diffusivity of Cu2+ in calcium alginate gel beads

The diffusivity of Cu(2+) in calcium alginate beads calculated by the shrinking core model (SCM) was reevaluated in this work. The results obtained in this work were significantly different than those by the original authors. There were excellent agreements between the results obtained by the SCM in this work and those by the more rigorous linear absorption model (LAM) by the original authors. (c) 1994 John Wiley & Sons, Inc.     

The use of hollow fiber cross-flow microfiltration in bioaccumulation and continuous removal of heavy metals from solution by Saccharomyces cerevisiae

Cross-flow microfiltration was shown to retain Saccharomyces cerevisiae biomass utilized for heavy metal bioaccumulation. The passage of metal-laden influent through a series of sequential bioaccumulation systems allowed for further reductions in the levels of copper, cadmium, and cobalt in the final effluent than that afforded by a single bioaccumulation process. Serial bioaccumulation systems also allowed for partial separation of metals from dual metal influents. More than one elemental metal cation could be accumulated simultaneously and in greater quantities than when a single metal was present in the effluent (Cu(2+) 0.43 mmol, Cu(2+) + Cd(2+) 0.67 mmol, and Cu(2+) + Co(2+) 0.83 mmol/g yeast dry mass when the initial concentration of each of the metal species was 0.2 mmol.L(-1)). Co-accumulation of two different metal cations allowed higher total levels of bioaccumulation than found with a single metal. The flux rate was 2.9 x 10(2) L.h(-2)mum(-2) using a polypropylene microfiltration membrane (0.1 mum pore size) at 25 degrees C. (c) 1994 John Wiley & Sons, Inc.     

Isolation of high-molecular-weight plant DNA for DNA damage quantitation: relative effects of solar 297 nm UVB and 365 nm radiation

Quantitation of UV-induced DNA damages in nanogram quantities of non-radiactive DNA from irradiated plants by gel electrophoresis requires a prompt, efficient, high-yield method of isolating DNA yielding high-molecular-weight, enzymatically digestible DNA. To meet these criteria we devised a high-yield method for isolating from plant tissue, DNA whose single-strand molecular length is greater than about 170 kb. Leaf tissue is embedded in agarose plugs, digested with Proteinase K in the presence of detergent, and treated with phenylmethylsulfonyl fluoride (PMSF). The agarose plugs are then soaked with buffer appropriate to the desired enzyme treatment. Evaluation of the DNA on neutral and alkaline gels indicates its high molecular length and low frequency of single-strand breaks. The DNA can be digested with damage-specific and other endonucleases. The method is especially suitable for DNA damage quantitation, as tissue processing is carried out immediately after harvesting (allowing DNA lesion measurement at precisely known times after irradiation), and many samples can be easily handled at once. It should also be useful for molecular analysis of large numbers of plant samples available only in small quantities. We here use this method to quantitate DNA damage induced by 297 and 365 nm radiation, and calculate the relative damaging effects of these wavebands in today's solar spectrum.     

Cell Lysis Due to Ultrasound Gel In Fine Needle Aspirates; an Important New Artefact In Cytology

An important new artefact in cytopathology is described. the initial observation of the artefact followed contamination of a breast fine needle aspiration (FNA) sample by ultrasound gel which was used to localize the lesion. the changes proved reproducible ex vivo. the changes varied depending on the conditions and degree of contamination, and ranged from cell swelling to leakage of nuclear chromatin and cell lysis. This artefact is discussed in the context of other major sources of cytology artefact. Pathologists and radiologists should beware of the detrimental effects of ultrasound gel on cytology specimens.     

The anaerobic filtration of dairy waste: Results of a pilot trial

Results are presented from a pilot scale (4·3 m³) upflow anaerobic filter for the treatment of the wastewater from ice-cream manufacture. The reactor was completely mixed by gas production but the solids or sludge held within the reactor were shown to be affected by the liquid velocities. The reactor was subject to a number of organic and hydraulic shocks and this reduced the consistency of COD removal. Daily loading rates varied from 0 to 18 kg COD/m³/day but with an average load of 5·5 kg/m³/day the mean COD removal was 70%. This was compared with previous work and shown to be a typical performance for an anaerobic filter. Alkalinity and carboxylic acid data are also presented and were within the normal, stable, operating range. Previous research on the anaerobic treatment of industrial effluents has shown alkalinity to be the most important factor controlling reliability.     

Mitochondrial DNA control region polymorphisms: genetic markers for ecological studies of marine turtles

We describe a rapid and sensitive method for the detection of population-specific genetic markers in mitochondrial DNA (mtDNA) and the use of such markers to analyse population structure of marine turtles. A series of oligonucleotide primers specific for the amplification of the mtDNA control region in Cheloniid turtles were designed from preliminary sequence data. Using two of these primers, a 384–385-bp sequence was amplified from the 5′ portion of the mtDNA control region of 15 green turtles Chelonia mydas from 12 different Indo-Pacific rookeries. Fourteen of the 15 individuals, including some with identical whole-genome restriction fragment patterns, had sequences that differed by one or more base substitutions. Analysis of sequence variation among individuals identified a total of 41 nucleotide substitutions and a 1-bp insertion/deletion. Comparison with evidence from whole-genome restriction enzyme analysis of the same individuals indicated that this portion of the control region is evolving approximately eight times faster than the average rate and that the sequence analysis detected approximately one fifth of the total variation present in the genome. Restriction enzyme analysis of amplified products from an additional 256 individuals revealed significant geographic structuring in the distribution of mtDNA genotypes among five of the 10 rookeries surveyed extensively. Additional geographic structuring of genotypes was identified through denaturing gradient gel electrophoresis (DGGE) of amplified products. Only two of the 10 rookeries surveyed could not be differentiated, indicating that the Indo-Pacific C. mydas include a number of genetically differentiated populations, with minimal female-mediated gene flow among them. Important applications for genetic markers in the conservation and management of marine turtles include the identification of appropriate demographic units for research and management (i.e. genetically discrete populations) and assessment of the composition of feeding and harvested populations.     

Genome size and restriction fragment length polymorphism analysis of Vibrio cholerae strains belonging to different serovars and biotypes

Abstract The genome size of Vibrio cholerae has been determined by pulsed field gel electrophoresis following digestion of chromosomal DNA with endonucleases. The genome size of all the classical strains examined was about 3000 kb and that of El Tor biotype was 2500 kb. The Not I and S fi I digestion patterns of the genomes of several V. cholerae straimns belonging to different serovars and biotypes showed distinct restriction fragment length polymorphism (RFLP). RFLP analysis together with the genome size can be used to differentiate strains of different serovars and biotypes of V. cholerae .     

Pulsed Field Gel Electrophoresis Analysis of Borrelia burgdorferi Sensu Lato Isolated in Japan and Taxonomic Implications with Lyme Disease Spirochetes

Genomic DNAs of Borrelia burgdorferi sensu lato isolates obtained in Japan sharing different rRNA gene ribotypes were digested with rare-cutter restriction endonucleases and the fragments obtained were separated by pulsed field gel electrophoresis (PFGE). The sizes of large restriction cleavage bands with MluI endonuclease were quite similar among isolates in each ribotype group. On the other hand, the PFGE profiles obtained with the other enzymes (NruI, Sal I or SplI) were rather divergent, and Japanese isolates were distinguishable from the United States and European isolates. The Japanese isolates classified as ribotypes group II (Borrelia garinii) and III (B. afzelii) showed different PFGE patterns from that of European isolates. The isolates grouped into ribotype IV revealed distinctively different PFGE profiles. These results indicate that the Japanese isolates may be genetically divergent and distinct from the United States and European isolates.