Mitochondrial data are not suitable for resolving placental mammal phylogeny

Mitochondrial data have traditionally been used in reconstructing a variety of species phylogenies. The low rates of recombination and thorough characterization of mitochondrial data across vertebrate species make it a particularly attractive phylogenetic marker. The relatively low number of fully sequenced mammal genomes and the lack of extensive sampling within Superorders have posed a serious problem for reaching agreement on the placement mammal species. The use of mitochondrial data sequences from large numbers of mammals could serve to circumvent the taxon-sampling deficit. Here we assess the suitability of mitochondrial data as a phylogenetic marker in mammal phylogenetics. MtDNA datasets of mammal origin have been filtered as follows: (i) we have sampled sparsely across the phylogenetic tree, (ii) we have constrained our sampling to genes with high taxon coverage, (iii) we have categorised rates across sites in a phylogeny independent manner and have removed fast evolving sites, and (iv), we have sampled from very shallow divergence times to reduce phylogenetic conflict. However, topologies obtained using these filters are not consistent with previous studies and are discordant across different genes. Individual mitochondrial genes, and indeed all mitochondrial genes analysed as a supermatrix, resulted in poor resolution of the species phylogeny. Overall, our study highlights the limitations of mitochondrial data, not only for resolving deep divergences and but also for shallow divergences in the mammal phylogeny.     

A silver impregnation method for nervous tissue suitable for routine use with mounted sections.

A simple, reliable silver impregnation method for nervous tissue is described for tissues fixed in various fixatives including formalin, Bouin, and Susa. Sections are impregnated in a solution containing 1 g Protargol, 2 ml of a 1% Cu(NO3)2 solution, 2 ml of a 1% AgNO3 solution, and 2-4 drops 30% H2O2 in 100 ml distilled water. Sections are impregnated 2-5 days at 37 C and thereafter reduced in a hydroquinone-formalin solution. This is followed by gold toning and subsequent reduction, dehydration and mounting. This method has been found to be very reliable and selective.     

Antioxidants suitable for use with chemiluminescence to identify oxyradical species

From a panel of 24 alleged antioxidants the most suitable antioxidants (AO) for use with chemiluminescence (CL) experiments were determined. Superoxide dismutase (SOD), using luminol as the chemiluminescence probe (Lum-CL), was inhibitory only towards O2*- and not HO* or (1)O2. SOD was thus a suitable antioxidant for O2*-, as was tiron. Tiron had advantages, however, since SOD acted as a pro-oxidant in the presence of H2O2 or H2O2/HO* generators. The two most suitable antioxidants for (1)O2 were diphenylisobenzofuran (DBF) and tryptophan, for both Lum and Lucigenin-CL (Luc-CL). Desferrioxamine, with both Lum and Luc-CL, was a very effective scavenger for HO*, but appeared to be an even more effective scavenger for (1)O2. Cysteamine showed the best discrimination between IC50s when the two (1)O2 generators NaOCl/H2O2 and NDPO2 were compared. Cysteamine was, therefore, the only scavenger that was appropriate for studies with hypochlorite. Melatonin, with Lum-CL, was found to be the most suitable scavenger for HO*. Mannitol, the classical AO for HO*, was not suitable when used with CL since it acted as a pro-oxidant. Some of the AOs revealed either calyx- or bell-shaped CL inhibition profiles and presumably, therefore, may act as both pro- or antioxidants at different concentrations. Antioxidants showing these kinds of dual activities should be used with caution in CL studies.     

A rapid procedure for the isolation of yeast mitochondrial DNA suitable for restriction fragment analysis

A method for the rapid isolation of mitochondrial DNA from the yeast Saccharomyces cerevisiae is described. Cells are first disrupted by vortexing with glass beads and the mitochondiral DNA is then extracted directly from the cell lysate by poly-l-lysine-kieselguhr-exchange chromatography. The method is unique from most other published procedures in that there is no requirement for the isolation of either a crude or purified mitochondrial preparation. Mitochondrial DNA isolated by this procedure is shown to yield restriction endonuclease fragment patterns identical to those obtained from DNA isolated by other previously reported procedures.     

A suitable and efficient procedure for the removal of decontaminating antibiotics from tissue allografts

The presence of residual antibiotics in tissue allografts after decontamination with antibiotic cocktails may result in widely documented adverse effects in predisposed subjects. Moreover, antibiotic residues may mask contaminating microorganisms, resulting in falsely negative sterility tests, with potential risk of post-surgical infections. The objective of the present study was to define a rinsing procedure capable of eliminating antibiotic residues from cardiovascular, bone and skin tissues after decontamination with BASE.128. Different washing patterns, employing BASE medium, were applied. The presence of antibiotic residues in tissue homogenates was assessed by agar diffusion test at different stages of tissue processing. To test whether antibiotic residues can result in falsely negative microbiological analysis, we induced a superficial tissue contamination with known inoculum concentration. By employing four different porcine tissues, we here report direct evidence that the presence of even limited amounts of antibiotics in decontaminated tissues interferes with sterility testing. This has implications in terms of increased risk of infections in allograft recipients. To minimize this risk, we developed a procedure for extensive removal of antibiotics from allografts, allowing for subsequent detection of microbial contaminations that may occur during transportation, storage or processing prior to allograft transplantation. Our study emphasizes the importance of validating all processes and analytical methods in tissue banking, in order to warrant tissue safety. This will minimize the risks of post-surgical infections as well as antibiotic-induced anaphylaxis in predisposed patients.     

The use of a microcomputer in a biomedical research project

The use of an inexpensive microcomputer system in an NIH-supported biomedical research project is explained. Computer programs which store and analyze air pollution data; records from hospitals, doctors' offices, pharmacies, and schools; and data from a community public health survey are described. The analysis consists primarily of calculations of various temporal and geographical correlation coefficients in order to determine whether particulate air pollution is affecting public health in southern Puerto Rico.     

A procedure for purifying jack bean urease for clinical use

Urease with a purity meeting the requirements of analytical use was purified from jack bean meal through steps consisting of 20% acetone extraction, heat treatment, acid precipitation, and lyophilization. For extraction of urease, one part of bean meal was mixed with 5 parts of 20% acetone containing 1 mM EDTA and 1 mM 2-mercaptoethanol, and stirred at 20 degrees C for 5 min. Milky substances in the extract were removed by heat treatment. Urease in the clear yellow supernatant was precipitated by adjusting the pH of the solution to 5.4 with citric acid. The acid precipitated urease was neutralized by dissolving in 0.015 M phosphate buffer, pH 8.5 (final pH 6.8 to 7.0) and then lyophilized. By this procedure, the purity of the enzyme was increase 14.7 fold, the recovery of activity was 63%, and the yield was 6.75 g from 1 kg of bean seeds. The specific activity of the preparation was 411 units/mg protein (240 units/mg solid), and the free ammonia content was less than 0.01 microgram per unit. Some other proteins were present in the urease preparation as examined by gel filtration and gradient polyacrylamide gel electrophoresis. The molecular weight of the enzyme estimated by gel filtration was 480,000. However, two urease activity bands with molecular weight of 230,000 and 480,000 were observed in the polyacrylamide gel electrophoregram. From the result of determination of blood urea nitrogen (BUN), this simple purification procedure could be used for practical preparation of urease from jack bean meal for clinical analysis.     

A queueing model for a two-stage stochastic manufacturing system with overlapping operations

This paper presents analytical expressions for estimating average process batch flow times through a stochastic manufacturing system with overlapping operations. It is shown that the traditional queueing methodology cannot be directly applied to this setting, as the use of the overlapping operations principle causes the arrival process of sublots at the second stage to be a non-renewal process. An embedded queueing model is then proposed, which provides a tool to estimate the flow time reductions caused by the use of overlapping operations. Moreover, we provide expressions to estimate the production disruptions occurring at the second stage. The results of our research confirm the general intuition that the overlapping operations principle leads to less congestion, and hence a smoother flow of work through the system. On the other hand however, lot splitting inevitably requires more material handling on the shop floor. The expressions provided in this paper allow the quantification of the trade-off between these two effects, e.g., by gauging them within the scope of a cost model.     

A simple reliable procedure for obtaining metaphases from human leukemic bone-marrow aspirates suitable for Giemsa banding

Summary Short incubation of heparinized human leukemic bone-marrow cells in phosphate buffered saline containing colcemid and overnight chilling of fixed cells yields metaphases with elongated and well-spread chromosomes. This technique enables us to do trypsin-Giemsa banding of chromosomes obtained from leukemic marrow cells otherwise difficult to band.     

The use of a microcomputer to collect activity data

ABSTRACT. Use of small microcomputers to collect and record from the outputs of activity detectors is described in simple terms for the uninitiated. Provided simple external latches are used, the microcomputer may be programmed in a high level language, such as BASIC, and the system can be used to control light or temperature cycles. Data are acquired and control effected through an 8-bit parallel 'user' port, as fitted to most microcomputers, and details are given of the installation of such a port.     

Synthesis of an unsaturated mixed-acid phosphatidylinositol of natural configuration. A new procedure for resolving racemic alcohols

DL-1,2,4,5,6-Penta-O-acetyl-myo-inositol was resolved into antipodes through the salts of its acid oxalate with quinidine and (?)-α-phenethylamine. The optically active pentaacetates were regenerated from the oxalates by lead tetraacetate oxidation. The optical purity of these pentaacetates was confirmed by their transformation into optically pure 1D- and 1L-myo-inositol 1-phosphates. The 1D-isomer of the pentaacetate was used as starting compound for the synthesis of a mixed acid phosphatidylinositol having the natural configuration at all the asymmetric centres and the normally predominating 1-saturated 2-unsaturated distribution of the fatty acid residues. From the 1L-pentaacetate an isomer of phosphatidylinositol with the unnatural 1L-configuration of the myo-inositol residue has been obtained.     

A gel electrophoresis system for resolving over 500 nucleotides with a single sample loading

A procedure for the resolution of over 500 nucleotides in a single loading on a sequencing gel is described. The method uses exponential wedge spacers to achieve compression of the band spacing in the lower portion of the gel. The method is simple and reliable and reduces the cost of electrophoresis and autoradiography supplies by at least a factor of two.