A simple and efficient method for DNA extraction from grapevine cultivars andVitis species

A quick, simple, and reliable method for the extraction of DNA from grapevine species, hybrids, andAmpelopsis brevipedunculata (Vitaceae) has been developed. This method, based on that of Doyle and Doyle (1990), is a CTBA-based extraction procedure modified by the use of NaCl to remove polysaccharides and PVP to eliminate polyphenols during DNA purification. The method has also been used successfully for extraction of total DNA from other fruit species such as apple (Malus domestica), apricot (Prunus armeniaca), cherry (Prunus avium), peach (Prunus persica), plum (Prunus domestica), and raspberry (Rubus idaeus). DNA yield from this procedure is high (up to 1 mg/g of leaf tissue). DNA is completely digestible with restriction endonucleases and amplifiable in the polymerase chain reaction (PCR), indicating freedom from common contaminating compounds.     

Voltage-Gated Potassium Channel Genes Are Clustered in Paralogous Regions of the Mouse Genome

Cloning of the Drosophila Shaker gene established that a neurological phenotype including locomotor dysfunction can be caused by a mutation in a voltage-gated potassium (K) channel gene. Shaker sequences have been used to isolate a large family of related K channel genes from both flies and mammals. Toward elucidating the evolutionary relationship between loci and the potential causal connection that K channels may have to mammalian genetic disorders, we report here the genetic mapping of 12-16 different murine, voltage-gated K channel genes. We find that multiple genes, in some cases from distantly related K channel subfamilies, occur in clusters in the mouse genome. These mapping results suggest that the K channel gene subfamilies arose through ancient localized gene duplication events, followed by chromosomal duplications and rearrangements as well as further gene duplication. We also note that several neurologic disorders of both mouse and human are associated with the chromosomal regions containing K channel genes.     

HPLC and NMR methods for the quantitation of the (R)-enantiomer in (−)-(S)-timolol maleate drug raw materials

HPLC and ¹H-NMR methods for the quantitation of the (R)-enantiomer in (?)-(S)-timolol maleate were developed and validated. The HPLC method requires a 25 cm × 4.6 mm 5 μm Chiracel OD-H (cellulose tris-3,5-dimethylphenylcarbamate) column, a mobile phase of 0.2% (v/v) diethylamine and 4% (v/v) isopropanol in hexane at a flow rate of 1 ml/min and UV detection at 297 nm. A system suitability test was devised to verify the separation of the (R)- and (S)-enantiomers of timolol from other drug-related impurities. The NMR method requires the use of a high-field NMR spectrometer (>360 MHz) and a chiral solvating agent, (?)-(R)-2,2,2-trifluoro-1-(9-anthrylethanol) (R-TFAE). The limits of quantitation were 0.05% and 0.2% (m/m) for HPLC and NMR, respectively. The methods were applied to the determination of the (R)-enantiomer in eight lots of raw material. The results for the two methods were in very good agreement, with results ranging from 0.1 to 4.1% (m/m) by HPLC and none detected to 4.3% (m/m) by NMR. The USP method for specific rotation was found to be unsuitable for detecting the presence of low levels of the (R)-enantiomer in (?)-(S)-timolol maleate. © 1994 Wiley-Liss, Inc.     

PrP in pathology and pathogenesis in scrapie-infected mice

PrP accumulation in the brains of mice infected with scrapie takes several different forms: amyloid plaques, widespread accumulation in neuropile, and perineuronal deposits. PrP is also sometimes detected within microglia and in or around astrocytes. There are dramatic and reproducible differences between scrapie strains in the relative prominence of these changes and their distribution in the brain. Depending on the scrapie strain, PrP pathology is targeted precisely to particular brain areas, often showing a clear association with identifiable groups of neurons. These results suggest that PrP changes are primarily associated with neurons, and that different scrapie strains recognize and selectively replicate in different populations of neurons. Immunostaining at the ultrastructural level demonstrates an association of PrP with neurite plasmalemma, around amyloid plaques, and in areas of widespread neuropile and perineuronal accumulation. It is probable that PrP is encoded by theSinc gene, which controls the incubation period of scrapie in mice. Studies using the intraocular infection route show that theSinc gene controls the onset rather than the rate of replication, suggesting that PrP may be involved in cell-to-cell spread of infection. The accumulation of PrP at the surface of neurons is consistent with such a role.     

NMR View: A computer program for the visualization and analysis of NMR data

Summary NMR View is a computer program designed for the visualization and analysis of NMR data. It allows the user to interact with a practically unlimited number of 2D, 3D and 4D NMR data files. Any number of spectral windows can be displayed on the screen in any size and location. Automatic peak picking and facilitated peak analysis features are included to aid in the assignment of complex NMR spectra. NMR View provides structure analysis features and data transfer to and from structure generation programs, allowing for a tight coupling between spectral analysis and structure generation. Visual correlation between structures and spectra can be done with the Molecular Data Viewer, a molecular graphics program with bidirectional communication to NMR View. The user interface can be customized and a command language is provided to allow for the automation of various tasks.Inquiries concerning the availability of NMR View and the Molecular Data Viewer should be sent via email to johnsonb@merck.com or to Bruce A. Johnson, Merck Research Laboratories, RY80Y-103, P.O. Box 2000, Rahway, NJ 07065, U.S.A.     

The radiosensitivity of spermatogonial stem cells in C3H/101 F1 hybrid mice

The radiosensitivity of spermatogonial stem cells of C3H/HeH × 101/H F₁ hybrid mice was determined by counting undifferentiated spermatogonia at 10 days after X-irradiation. During the spermatogenic cycle, differences in radiosensitivity were found, which were correlated with the proliferative activity of the spermatogonial stem cells. In stage VIII_irr, during quiescence, the spermatogonial stem cells were most radiosensitive with a D₀ of 1.4 Gy. In stages XI_irr−V_irr, when the cells were proliferatively active, the D₀ was about 2.6 Gy. Based on the D₀ values for sensitive and resistant spermatogonia and on the D₀ for the total population, a ratio of 45:55% of sensitive to resistant spermatogonial stem cells was estimated for cell killing.

When the present data were compared with data on translocation induction obtained in mice of the same genotype, a close fit was obtained when the translocation yield (Y; in % abnormal cells) after a radiation dose D was described by Y = e^τD, with τ = 1 for the sensitive and τ = 0.1 for the resistant spermatogonial stem cells, with a maximal e^τD of 100.     

Effects of the parasitic botfly Philornis carinatus on nestling house wrens,Troglodytes aedon,in Costa Rica

I studied the life cycle of a botfly (Diptera: Muscidae: Philornis carinatus) and examined the effects of botfly ectoparasitism on nestling house wrens (Passeriformes: Troglodytidae: Troglodytes aedon) during three years in Costa Rica. At three study sites, I found that nestlings were relatively unaffected by botflies, in contrast to all other studies of birds infected with philornid botflies. At Monteverde, the main study site, infected chicks grew slightly slower and had slightly shorter tarsi and wing chords than uninfected chicks, but both groups fledged at similar weights. Since weight at fledging is the only growth character associated with post-fledging survivorship, botfly infections likely cost wrens little in terms of fitness. At all sites, fledging success did not differ between infected and uninfected nests. Botfly infections were more prevalent at two lower elevation sites than at the high elevation Monteverde side. Infection prevalence increased during the nesting season at all study sites, which suggests a botfly life cycle in which adult population levels increase during the wren breeding season and then decline during a dormant period when wrens are not nesting. Finally, botflies may attack chicks throughout the period before fledging, but there is no indication they locate nests before hatching. In sum, botfly parasitism on wrens appears to be benign, perhaps because the study sites are at the edge of the botfly's range or because wrens are not a preferred host.     

The sequence of electron carriers in the reaction of cytochromec oxidase with oxygen

Kinetic studies of the electron transfer processes performed by cytochrome oxidase have assigned rates of electron transfer between the metal centers involved in the oxidation of ferrocytochromec by molecular oxygen. Transient-state studies of the reaction with oxygen have led to the proposal of a sequence of carriers from cytochromec, to Cu_A, to cytochromea, and then to the binuclear (i.e., cytochromea ₃-Cu_B) center. Electron exchange rates between these centers agree with relative center-to-center distances as follows; cytochromec to Cu_A 5–7 Å, cytochromec to cytochromea 20–25 Å, Cu_A to cytochromea 14–16 Å and cytochromea to cytochrome a₃-Cu_B 8–10 Å. It is proposed that the step from cytochromea to the binuclear center is the key control point in the reaction and that this step is one of the major points of energy transduction in the reaction cycle.