Methods to improve the yield and quality of DNA from dried and processed figs

We describe here a molecular method that can be used to detect genome traits of a given horticultural item at each stage from the farm to the market. We developed a procedure to extract and amplify by PCR DNA obtained from complex matrixes, such as dried figs and fig jam. Few fragmented DNA molecules can be recovered from food products. However, we were able to increase the yield of PCR reactions by successfully applying an enzymatic repair protocol to retrieved DNA.     

Gating in iodate-modified single cardiac Na+ channels

Summary Elementary Na⁺ currents were recorded at 19°C during 220-msec lasting step depolarizations in cell-attached and inside-out patches from cultured neonatal rat cardiocytes in order to study the modifying influence of iodate, bromate and glutaraldehyde on single cardiac Na⁺ channels.Iodate (10 mmol/liter) removed Na⁺ inactivation and caused repetitive, burst-like channel activity after treating the cytoplasmic channel surface. In contrast to normal Na⁺ channels under control conditions, iodate-modified Na⁺ channels attain two conducting states, a short-lasting one with a voltage-independent lifetime close to 1 msec and, likewise tested between –50 and +10 mV, a long-lasting one being apparently exponentially dependent on voltage. Channel modification by bromate (10 mmol/liter) and glutaraldehyde (0.5 mmol/liter) also included the occurrence of two open states. Also, burst duration depended apparently exponentially on voltage and increased when shifting the membrane in the positive direction, but there was no evidence for two bursting states. Chemically modified Na⁺ channels retain an apparently normal unitary conductance (12.8±0.5 pS). Of the two substates observed, one of them is remarkable in that it is mostly attained from full-state openings and is very short living in nature; the voltage-independent lifetime was close to 2 msec. Despite removal of inactivation, open probability progressively declined during membrane depolarization. The underlying deactivation process is strongly voltage sensitive but, in contrast to slow Na⁺ inactivation, responds to a voltage shift in the positive direction with a retardation in kinetics. Chemically modified Na⁺ channels exhibit a characteristic bursting state much shorter than in DPI-modified Na⁺ channels, a difference not consistent with the hypothesis of common kinetic properties in noninactivating Na⁺ channels.     

Purkyn?'s description of pressure phosphenes and modern neurophysiological studies on the generation of phosphenes by eyeball deformation

(a) When a subject indents one of his eyeballs in total darkness, he immediately perceives light extending slowly across the whole visual field of the indented eye. The appearance and the time course of these pressure or deformation phosphenes are described. (b) With simultaneous binocular indentation of the eyeballs a flickering patterned phosphene is observed. (c) A short history of the research on pressure phosphenes and its consequences for the theories of vision is presented. (d) Purkyn?'s observations of monocular deformation phosphenes are described. He repeatedly noted patterned light structures, which most observers only perceive with simultaneous binocular eyeball deformation. It is suggested that Purkyn?'s deviating observations were caused by amblyopia of one eye. (e) The neurophysiological basis of the monocular pressure phosphenes was investigated by means of microelectrode recordings from single optic tract fibers. The activity of single retinal ganglion cells (on-center, off-center neurons, latency class I [Y-neurons] or latency class II [X-neurons]), was recorded in anaesthetized cats. Eyeball deformation in total darkness led to an activation of the on-center ganglion cells, while the off-center ganglion cells were inhibited. The latency and strength of this activation or inhibition varied considerably between different neurons, but were fairly constant in the same neuron when the eyeball indentation was repeated after a pause of 1-3 min. The latency and strength of neuronal activation or inhibition seemed to be dependent mainly upon the neuron location relative to the point of eyeball indentation. Some on-center neurons also exhibited a short activation at "deformation off". (f) The antagonistic response type of on-center and off-center ganglion cells was also observed when the eyeball was deformed as a hydrostatic open system and the intraocular pressure was kept at 25 mm Hg basic pressure. (g) Dark adaptation up to 45 min affected the deformation responses of retinal neurons only to a small degree, if at all. This corresponds to the observation that deformation phosphenes in a human observer changed little during the course of dark adaptation. (h) We assume that the activation of on-center and inhibition of off-center ganglion cells by eyeball deformation are caused by retinal stretching, which also leads to horizontal cell stretch. Stretching the horizontal cell membrane probably generates an increase in membrane sodium conductivity and a depolarization of the membrane potential. This depolarization of the horizontal cell membrane potential is transmitted either directly or indirectly (via receptor synapses) from the horizontal to the bipolar cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

FMRFamide-like immunoreactivity in the brain and pituitary of the teleost. Eigenmannia lineata (Gymnotiformes)

The distribution of cells immunoreactive for the molluscan tetrapeptide FMRFamide in the brain and the pituitary of Eigenmannia was investigated immunohistochemically by the use of the peroxidase-antiperoxidase (PAP) technique and unlabelled antibodies. FMRFi neurons were located in the ganglion of the nervus terminalis at the rostroventral side of the bulbus olfactorius. FMRFi perikarya were also found in a dorsomedial diencephalic nucleus, in the nucleus ventromedialis, in some liquor-contacting neurons of the nucleus lateralis tuberis and of the nucleus recessus lateralis and posterior. The perikarya of the midbrain pre-pacemaker nucleus were only weakly immunoreactive for FMRFamide while large FMRFi neurons (T-cells) occurred in lamina VI of the torus semicircularis, in the brain stem, in dorsal and medial layers of the lobus lineae lateralis posterior (LLLp) and in the medullary electric organ pacemaker nucleus (pm). FMRFi fibers and nerve endings were found in the bulbus olfactorius, in medial areas of the telencephalon, and rather densely in the rostral diencephalon. Ventrocaudally to most of the hypothalamic nuclei the occurrence of immunoreactive fibres increased; many coursed to the pituitary through the pituitary stalk. FMRFi fibres also appeared in the deep layers of the tectum opticum, in the torus semicircularis, in the medial and lateral medulla and below the pacemaker nucleus. Wherever FMRFamide-immunoreactivity occurred fibres and nerve endings could be found in close contact with blood vessels.     

A single mutation in 16S rRNA that affects mRNA binding and translation-termination.

A single base change in 16S rRNA (C726 to G) has previously been shown to have a dramatic effect on protein synthesis in E. coli (1). This paper more specifically details the effects of the mutation on mRNA binding and translation-termination. The in vitro technique of toeprinting (2) was used to demonstrate that 30S subunits containing the mutation 726G had an altered binding affinity for mRNA by comparison to the wild type. In addition, expression of the mutant ribosomes in vivo resulted in exclusive suppression of the UGA nonsense codon. This effect was supported by in vitro studies that showed the mutant ribosomes to have an altered binding affinity for Release Factor-2.     

Inhibitors of sterol synthesis. Chemical syntheses and spectral properties of 26-oxygenated derivatives of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one and their effects on 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells.

26-Oxygenated derivatives of delta 8(14)-15-ketosterols have been synthesized from (25R)-3 beta,26-diacetoxy-5 alpha-cholest-8(14)-en-15-one (IX) as part of a program to prepare potential metabolites and analogs of 3 beta-hydroxy-5 alpha-cholest-8(14)-en-15-one (I), a potent regulator of cholesterol metabolism. Partial hydrolysis of IX gave a mixture, from which the 3 beta,26-diol II and the 26-acetate (XI) and 3 beta-acetate (X) monoesters were isolated. Mitsunobu reaction of XI followed by hydrolysis gave (25R)-3 alpha,26-dihydroxy-5 alpha-cholest-8(14)-en-15-one (VI). Oxidation of XI with pyridinium chlorochromate followed by hydrolysis of the acetate gave (25R)-26-hydroxy-5 alpha-cholest-8(14)-ene-3,15-dione (VII). Oxidation of X with Jones reagent followed by hydrolysis of the acetate gave (25R)-3 beta-hydroxy-15-keto-5 alpha-cholest-8(14)-en-26-oic acid (IVa). Jones oxidation of II gave (25R)-3,15-diketo-5 alpha-cholest-8(14)-en-26-oic acid (VII). 1H and 13C nuclear magnetic resonance assignments and analyses of mass spectral fragmentation data are presented for each of the new compounds and their derivatives. The 3,15-diketone VII was found to be highly active in lowering the levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in CHO-K1 cells, with a potency comparable to that of I. In contrast, 3 alpha,26-diol VI was less potent than I or VII. The two carboxylic acid analogs IVa and VIII were considerably less potent than VI in lowering the levels of HMG-CoA reductase activity.     

Characterization of crystals of genetically engineered human manganese superoxide dismutase

The genetically engineered human manganese superoxide dismutase crystallizes in space group P2(1)2(1)2 with a = 75.51 A, b = 79.00 A, c = 67.95 A. At room temperature the crystals are not stable against radiation, so we cooled them to 90 K and collected a data set to 3 A resolution at this temperature.     

Assignment of the human gene for muscle-type phosphofructokinase (PFKM) to chromosome 1 (region cen leads to q32) using somatic cell hybrids and monoclonal anti-M antibody

Human phosphofructokinase (PFK; EC 2.7.1.11) is under the control of three structural loci which encode muscle-type (M), live-type (L), and platelet-type (P) subunits; human diploid fibroblasts and leukocytes express all three loci. In order to assign human PFKM locus to a specific chromosome we have analyzed human x Chinese hamster somatic cell hybrids for the expression of human M subunits, using an anti-human M subunit-specific mouse monoclonal antibody. In 18 of 19 hybrids studied, the expression of the PFKM locus segregated concordantly with the presence of chromosome 1 (discordance rate 0.05) as indicated by chromosome and isozyme marker analysis. The discordance rates for all the other chromosomes were 0.32 or greater, indicating that the PFKM locus is on chromosome 1. For the regional mapping of PFKM, eight hybrids were studied that contained one of five distinct regions of chromosome 1. These results further localize the human PFKM locus to region cen leads to q32 chromosome 1.     

High temporal resolution of ion fluxes in semi-natural ecosystems – gain of information or waste of resources?

Monitoring programs of ion concentrations and fluxes in semi-natural ecosystems are confronted with the task to gain as much information as possible with simultaneously minimizing costs and efforts. The aim of this study was (i) to assess how much of the heterogeneity of solution concentrations is lost because of temporal integration of measurements and (ii) to estimate the error in ion fluxes due to temporal integration. High resolution measurements (daily interval) of ion concentrations (sulfate, nitrate, chloride, pH and EC) in throughfall, soil solutions and runoff at the catchment Lehstenbach (Fichtelgebirge, Northeast Bavaria, Germany) were compared over a two year period with the reference monitoring program (biweekly measurement interval). Evaluation of the maximum temporal heterogeneity of ion concentrations in throughfall, soil solution and runoff (expressed as minimum, maximum, median and 25–75% percentile) did not result in an overall higher heterogeneity of the high resolution measurements compared to the reference program. The calculation of runoff fluxes from the reference data (biweekly concentration) resulted in significant errors of up to 25% for time periods < 1 year (high resolution data was considered the "true" value and set as 100%). However, errors became minor (< 10%) if longer time periods were considered. The suitability of different interpolation methods to up-scale biweekly concentration data for the calculation of runoff fluxes was evaluated in this study. We concluded for the monitoring programs at the Lehstenbach catchment that a biweekly measurement interval seemed to be suitable to capture the heterogeneity of ion concentrations and fluxes (and thus temporal trends). In comparison, high resolution measurements with a daily measurement interval were higher in cost, work and time resources and had a relatively low information gain. While the introduced methods are applicable in all monitoring programs, conclusions on temporal resolution of measurements are most likely not valid for systems where ion concentrations have a low autocorrelation length (e.g., agricultural or urban systems with nitrate or pesticide treatment; tropical systems with extreme temperature or hydrological events).