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PubMed1995

Carbon isotope variation in Douglas-fir foliage: improving the delta(13)C-climate relationship.

The natural abundance of stable carbon isotopes in the annual rings of forest trees is used as a tracer of environmental changes such as climate and atmospheric pollution. Although tree-ring delta(13)C varies by about 2 per thousand from year to year, variability within the foliage can be as high as 6 per thousand. Recent studies have shown that branch length affects stomatal response, which influences the integrated foliar delta(13)C signal. To improve the ability of delta(13)C to predict climate differences, we examined the relationship between branch length and foliar delta(13)C in Pseudotsuga menziesii (Mirb.) Franco from four sites across a steep climate gradient in Oregon. The transect spanned the boundary between the ranges of the coastal variety, P. menziesii var. menziesii (three sites), and the Rocky Mountain variety, P. menziesii var. glauca (one site). At the most maritime site, branch length explained 76% of within-site variation of 5 per thousand, whereas at the harshest site, branch length accounted for only 15% of this variation. We considered the possibility that cavitation in the water-conducting xylem obscures the branch length effect in the harsher climates. Cavitation, as measured by dye perfusion, was most extensive at sites where the branch length effect in the coastal variety was weakest. Trees at the site with the most substantial cavitation displayed seasonal xylem refilling. Branch length standardization significantly improved the relationship between delta(13)C and climate. With standardization to constant length, delta(13)C values were significantly related to the degree that climatic variables, as modeled with a forest growth simulation model, constrain transpiration (R(2) = 0.69, P < 0.0001). Without standardization, the R(2) was 0.27. We conclude that sampling standard length branches or tree rings from trees of similar shape and size is desirable when seeking correlations between isotopic composition and climate.

Journal Article↗
PubMed1997

The genome of Moloney murine leukemia virus can be integrated by the integrase of human immunodeficiency virus type 1 expressed alone in vivo.

An in vivo integration assay using the expressed human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein and plasmids carrying a copy of the infectious Moloney murine leukemia virus (MuLV) provirus genome as substrates is presented. The HIV-1 IN gene was taken from vector pINSD and cloned into vector pXT1 to give pXT1-IN. Two and three nucleotides from the circle junction on one pair of U3 and U5 attachment (att) sequences on an infectious MuLV provirus vector pMLV-K were changed by means of site-directed mutagenesis to that of the corresponding HIV-1 att sequences to generate vector pMLV*(U3U5). The MuLV IN sequence was partially deleted for vectors pMLV-K and pMLV*(U3U5) to generate vectors pMLV delta IN and pMLV*(U3U5) delta IN. Integration of these wild type and MuLV IN partially deleted or att mutated MuLV provirus vectors in the transfected cells by the expressed HIV-1 IN was monitored by means of a non-radioactive reverse transcriptase (RT) assay for released and collected virions. No RT activity was detected for the NIH/3T3 cell singly transfected with vector pMLV delta IN. However some RT activities were observed for the HIV-1 IN expressing cell transfected either with vectors pMLV delta IN or pMLV*(U3U5) delta IN. This indicated that in the absence of other HIV-1 proteins expressed the MuLV provirus genome was integrated by the expressed HIV-1 IN protein. The integration of these MuLV provirus genomes was further confirmed by polymerase chain reaction analysis on the genomic DNA extracted from the transfected cells using the MuLV IN sequence remained from partial deletion as a target.

Cell Line↗
PubMed1999

Factors and processes involved in membrane potential build-up in yeast: diS-C3(3) assay.

No methods are currently available for fully reliable monitoring of membrane potential changes in suspensions of walled cells such as yeast. Our method using the Nernstian cyanine probe diS-C3(3) monitors even relatively fast changes in membrane potential delta psi by recording the shifts of probe fluorescence maximum lambda max consequent on delta psi-dependent probe uptake into, or exit from, the cells. Both increased [K+]out and decreased pHout, but not external NaCl or choline chloride depolarise the membrane. The major ion species contributing to the diS-C3(3)-reported membrane potential in S. cerevisiae are thus K+ and H+, whereas Na+ and Cl- do not perceptibly contribute to measured delta psi. The strongly pHout-dependent depolarisation caused by the protonophores CCCP and FCCP, lack of effect of the respiratory chain inhibitors rotenone and HQNO on the delta psi, as well as results obtained with a respiration-deficient rho- mutant show that the major component of the diS-C3(3)-reported membrane potential is the delta psi formed on the plasma membrane while mitochondrial potential forms a minor part of the delta psi. Its role may be reflected in the slight depolarisation caused by the F1F0-ATPase inhibitor azide in both rho- mutant and wildtype cells. Blocking the plasma membrane H(+)-ATPase with the DMM-11 inhibitor showed that the enzyme participates in delta psi build-up both in the absence and in the presence of added glucose. Pore-forming agents such as nystatin cause a fast probe entry into the cells signifying membrane damage and extensive binding of the probe to cell constituents reflecting obviously disruption of ionic balance in permeabilised cells. In damaged cells the probe therefore no longer reports on membrane potential but on loss of membrane integrity. The delta psi-independent probe entry signalling membrane damage can be distinguished from the potential-dependent diS-C3(3) uptake into intact cells by being insensitive to the depolarising action of CCCP.

Carbonyl Cyanide m-Chlorophenyl Hydrazone↗
PubMed2026

Simplifying multiplex genome engineering in Saccharomyces cerevisiae with intron-mediated Random Assembly and INtegration (RAIN).

Engineering of multistep enzymatic pathways often involves extensive optimization of heterologous gene expression levels and requires cloning of promoter and open reading frames (ORFs) to generate expression cassettes. We present work on a nascent method for multiplex genome engineering in&#xa0;Saccharomyces cerevisiae that negates the requirement for cloning of expression cassettes. Our system, Random Assembly and INtegration (RAIN), uses intron-mediated homologous recombination (HR) for random&#xa0;in vivo assembly of exogenous promoter and ORF libraries, which are combined and cotransformed in a one-pot method. The libraries include consensus homology arms which target long terminal repeat regions of the Ty1 retrotransposon, providing over a hundred possible integration loci. In this way, our developmental system aims to negate the need for&#xa0;in vitro combinatorial cloning of promoters and ORFs to generate expression cassettes, simplifying in vitro DNA preparation before multiplex genome engineering. This paper presents findings from a series of experiments to demonstrate a proof of concept for the RAIN system. These include: the first reported use of intron-mediated assembly of promoters and ORFs for expression of a functional gene product; up to three markerless genomic integrations; and up to five integrations with antibiotic selection. We also present a number of innovations to improve integration efficiency during multiplex engineering in S. cerevisiae including: SGS1 gene knockout; disruption of heteroduplex rejection; modified Cas9 expression architecture; and overexpression of HR genes RAD52, MRE11, and RAD59. To demonstrate how our system can be used for single transformation phenotype engineering of multiple strains, we also transformed a library of methylotrophy associated genes to generate four new strains that were able to grow on a solid minimal medium with methanol as the sole additional carbon source. Our findings contribute to the ongoing efforts to improve multiplex genome engineering tools in S. cerevisiae, and provide the foundations for further development of a novel toolbox for generating useful genetic diversity for metabolic pathway engineering.

Saccharomyces cerevisiae↗
PubMed1997

Bronchial temperature as a key to the interior pulmonary capillary bed of anaesthetized dogs.

The terminal airways are separated from the surrounding pulmonary capillaries by a tissue layer of a few micrometers in thickness only. Therefore, it should be possible to gain information about in vivo transcapillary heat transport of the interior pulmonary vascular bed by recording the terminal bronchial temperature. For this purpose, we studied temperature-time curves in the pulmonary artery, bronchial system and the aorta of six anaesthetized dogs permanently instrumented for measuring pulmonary blood flow. Thermistors recorded temperature changes at the three locations after injection of 5 ml cold solution into the right atrium. From the observed temperature-time curves mean transit times between the three recording sites were calculated for various pulmonary blood flows (integral of delta Ttdt/integral of delta Tdt) (range 1.1-3.5 1/ min). We found that the temperature-time curves of the bronchial system resemble typical "dilution" curves and are interspaced between those in the pulmonary artery and those in the aorta. Regardless of pulmonary blood flow, mean transit times from the pulmonary artery to the distal bronchial system and from there to the aorta were about equal. We conclude that transcapillary heat transfer generates bronchial temperature-time curves which permit an estimation of the relation of precapillary to postcapillary mean transit times in the interior of the lung.

Animals↗
PubMed2001

delta(15)N as an integrator of the nitrogen cycle.

Natural abundances of the rare stable isotope of nitrogen, 15N, are now being used widely in research on N cycling in organisms and ecosystems. 15N natural abundances are used in fundamentally different ways from traditional 15N tracers by integrating N cycle processes via N isotope fractionations and the mixing of various N-containing pools. This approach of using 15N natural abundances still requires certain technical and conceptual advances before it can be used routinely in ecological research. Here, the basis of the natural abundance 15N approach and opportunities for applying it in ecology are reviewed, and recent progress towards overcoming some of the key technical problems and in revealing large-scale patterns in N cycle processes is discussed.

Journal Article↗
PubMed2005

Efficient synthesis of an imidazole-substituted delta-amino acid by the integration of chiral technologies.

Two methods to produce (2S)-5-amino-2-(1-n-propyl-1H-imidazol-4-ylmethyl)-pentanoic acid were investigated. Diastereoisomeric salt resolution, using the quinidine salt, gave the desired intermediate in 98% ee and 33% yield. Asymmetric hydrogenation of various substrates gave high conversions, with up to 83% ee. Integration of these two approaches via asymmetric hydrogenation of a quinidine salt substrate followed by crystallization provided the desired intermediate in 94% ee and 76% yield.

Amino Acids↗
PubMed2003

Simulation of calcium sparks in cut skeletal muscle fibers of the frog.

Spark mass, the volume integral of Delta F/F, was investigated theoretically and with simulations. These studies show that the amount of Ca2+ bound to fluo-3 is proportional to mass times the total concentration of fluo-3 ([fluo-3T]); the proportionality constant depends on resting Ca2+ concentration ([Ca2+]R). In the simulation of a Ca2+ spark in an intact frog fiber with [fluo-3T] = 100 microM, fluo-3 captures approximately one-fourth of the Ca2+ released from the sarcoplasmic reticulum (SR). Since mass in cut fibers is several times that in intact fibers, both with similar values of [fluo-3T] and [Ca2+]R, it seems likely that SR Ca2+ release is larger in cut fiber sparks or that fluo-3 is able to capture a larger fraction of the released Ca2+ in cut fibers, perhaps because of reduced intrinsic Ca2+ buffering. Computer simulations were used to identify these and other factors that may underlie the differences in mass and other properties of sparks in intact and cut fibers. Our spark model, which successfully simulates calcium sparks in intact fibers, was modified to reflect the conditions of cut fiber measurements. The results show that, if the protein Ca2+-buffering power of myoplasm is the same as that in intact fibers, the Ca2+ source flux underlying a spark in cut fibers is 5-10 times that in intact fibers. Smaller source fluxes are required for less buffer. In the extreme case in which Ca2+ binding to troponin is zero, the source flux needs to be 3-5 times that in intact fibers. An increased Ca2+ source flux could arise from an increase in Ca2+ flux through one ryanodine receptor (RYR) or an increase in the number of active RYRs per spark, or both. These results indicate that the gating of RYRs, or their apparent single channel Ca2+ flux, is different in frog cut fibers--and, perhaps, in other disrupted preparations--than in intact fibers.

Animals↗
PubMed1985

Effects of body heating during sleep interruption.

This study assessed the effects that elevating body temperature had on sleep structure in the third and fourth sleep cycles, cycles typically characterized by a high propensity for REM sleep and diminished levels of delta amplitude and incidence. The sleep of eight women and two men was interrupted for 30 min on each of 3 consecutive nights following an undisturbed adaptation night. The subjects were awakened each night following the end of the second REM sleep period. On 2 nights, subjects were immersed to midthorax in water at either 34 degrees C (TW condition) or 41 degrees C (HW condition) for 20 min. A third interruption without immersion (NW condition) was performed to provide a second type of baseline condition. The HW condition induced a mean tympanic temperature rise of 2.5 degrees C, that returned to baseline levels in approximately 60 min. Analysis of sleep patterns focused on the two sleep cycles following interruption. The mean of the two baseline conditions (TW + NW/2) was compared with the HW condition. Sleep onset latency, REM latency, REM duration, and eye movement activity in REM were unaffected by heating. Heating evoked increases in both total NREM and slow wave sleep, though these increases were delayed until the second cycle following sleep onset (i.e., appearing in the fourth, but not the third, NREM period). These were paralleled by increases in two objective measures of delta activity: integrated slow-wave amplitude (33% increase) and slow-wave density (10% increase).

Adult↗
PubMed2006

Stoichiometry and valence measurements of niobium oxides using electron energy-loss spectroscopy.

Qualitative and quantitative electron energy-loss spectroscopy analyses have been performed on niobium and stable niobium oxides (NbO, NbO(2) and Nb(2)O(5)). At integration windows (Delta) greater than 75 eV, k-factor analysis can be used to distinguish between the stoichiometry of the three oxides within 5.7% error. As seen in other metal oxides, with increasing oxidation state the metal ionization edges shift to higher energies relative to the O-K edge. Normalized M(2,3) white-line intensities show a strong correlation with 4d occupancy for each compound. The data are in correspondence with that observed in the literature for 4d transition metals using normalized L(2,3) white lines. Lastly, a distinctive energy-loss near-edge, structure of the O-K edge was observed for each oxide, which could be used as a fingerprint for analysis of unknowns.

Journal Article↗
PubMed1989

Chloroplast ATP synthase contains one single copy of subunit delta that is indispensable for photophosphorylation.

F0F1 ATP synthases synthesize ATP in their F1 portion at the expense of free energy supplied by proton flow which enters the enzyme through their channel portion F0. The smaller subunits of F1, especially subunit delta, may act as energy transducers between these rather distant functional units. We have previously shown that chloroplast delta, when added to thylakoids partially depleted of the coupling factor CF1, can reconstitute photophosphorylation by inhibiting proton leakage through exposed coupling factor CF0. In view of controversies in the literature, we reinvestigated two further aspects related to subunit delta, namely (a) its stoichiometry in CF0CF1 and (b) whether or not delta is required for photophosphorylation. By rocket immunoelectrophoresis of thylakoid membranes and calibration against purified delta, we confirmed a stoichiometry of one delta per CF0CF1. In CF1-depleted thylakoids photophosphorylation could be reconstituted not only by adding CF1 and subunit delta but, surprisingly, also by CF1 (-delta). We found that the latter was attributable to a contamination of CF1 (-delta) preparations with integral CF1. To lesser extent CF1 (-delta) acted by complementary rebinding to CF0 channels that were closed because they contained delta [CF0(+delta)]. This added catalytic capacity to proton-tight thylakoid vesicles. The ability of subunit delta to control proton flow through CF0 and the absolute requirement for delta in restoration of photophosphorylation suggest an essential role of this small subunit at the interface between the large portions of ATP synthase: delta may be part of the coupling site between electrochemical, conformational and chemical events in this enzyme.

Amino Acids↗
PubMed1998

Adjacent asparagines in the NR2-subunit of the NMDA receptor channel control the voltage-dependent block by extracellular Mg2+.

1. The voltage-dependent block of N-methyl-D-aspartate (NMDA) receptor channels by extracellular Mg2+ is a critical determinant of its contribution to CNS synaptic physiology. The function of the narrow constriction of the channel in determining the block was investigated by analysing the effects of a set different amino acid substitutions at exposed residues positioned at or near this region. NMDA receptor channels, composed of wild-type and mutant NR1- and NR2A-subunits, were expressed in Xenopus oocytes or human embryonic kidney (HEK) 293 cells. 2. In wild-type channels, the voltage dependence (delta) of the block Mg2+ was concentration dependent with values of delta of integral of 0.82 in 0.07 mM and higher concentrations. Under bionic conditions with high extracellular Mg2+ and K+ as the reference ion, Mg2+ weakly permeated the channel. Over intermediate potentials (approximately -60 to -10 mV), this weak permeability had no apparent effect on the block but at potentials negative to approximately -60mV, it attenuated the extent and voltage dependence of the block. 3. Substitutions of glycine, serine, glutamine or aspartate for the N-site asparagine in the NR1-subunit enhanced the extent of block over intermediate potentials but left the voltage dependence of the block unchanged indicating that structural determinants of the block remained. These same substitutions either attenuated or left unchanged the apparent Mg2+ permeability. 4. In channels containing substitutions of glycine, serine or glutamine for the N-site asparagine in the NR2A-subunit, the block Mg2+ was reduced at negative potentials. Over intermediate potentials, the block was not strongly attenuated except for the glutamine substitution which reduced the voltage dependence of the block to integral of 0.57 in 0.7 mM Mg2+. 5. Equivalent substitutions for the N + 1 site asparagine in the NR2A-subunit strongly attenuated the block over the entire voltage range. In 0.7 mM Mg2+, the voltage dependence of the block was reduced to 0.50 (glycine), 0.53 (serine) and 0.46 (glutamine). 6. Channels containing substitutions of the N-site or N + 1 site asparagines in the NR2A-subunit showed an increased Mg2+ permeability suggesting that these adjacent asparagines form a barrier for inward Mg2+ flux. Changes in this barrier contribute, at least in part, to the mechanism underlying disruption of the block following substitution of these residues. 7. The adjacent NR2A-subunit asparagines are positioned at or near the narrow constriction of the channel. Pore size, however, did not determine how effectively Mg2+ blocks mutant channels. 8. It is concluded that, at the narrow constriction in the NMDA receptor channel, the adjacent NR2A-subunit asparagines, the N-site and N + 1 site, but not the N-site asparagine of the NR1-subunit, form a critical blocking site for extracellular Mg2+. The contribution to the blocking site, in contrast to the prevailing view, is stronger for the N + 1 site than for the N-site asparagine. The block may involve binding of Mg2+ to these residues.

Amino Acid Sequence↗
PubMed2002

Engineering of polyploid Saccharomyces cerevisiae for secretion of large amounts of fungal glucoamylase.

We engineered Saccharomyces cerevisiae cells that produce large amounts of fungal glucoamylase (GAI) from Aspergillus awamori var. kawachi. To do this, we used the delta-sequence-mediated integration vector system and the heat-induced endomitotic diploidization method. delta-Sequence-mediated integration is known to occur mainly in a particular chromosome, and the copy number of the integration is variable. In order to construct transformants carrying the GAI gene on several chromosomes, haploid cells carrying the GAI gene on different chromosomes were crossed with each other. The cells were then allowed to form spores, which was followed by dissection. Haploid cells containing GAI genes on multiple chromosomes were obtained in this way. One such haploid cell contained the GAI gene on five chromosomes and exhibited the highest GAI activity (5.93 U/ml), which was about sixfold higher than the activity of a cell containing one gene on a single chromosome. Furthermore, we performed heat-induced endomitotic diploidization for haploid transformants to obtain polyploid mater cells carrying multiple GAI genes. The copy number of the GAI gene increased in proportion to the ploidy level, and larger amounts of GAI were secreted.

Chromosomes, Fungal↗
PubMed1987

Early feeding of a high-cholesterol diet enhances intestinal permeability to lipids in rabbits.

This study was undertaken in young and growing rabbits to establish the of feeding a high- (2%) cholesterol diet (HC) on the intestinal in vitro uptake of a homologous series of saturated fatty acids, dodecanol, cholesterol, and bile acids. In the jejunum there was an increase in the uptake of myristic acid and dodecanol in HC as compared with animals fed Purina Chow, whereas in the ileum the uptake of stearic acid, cholesterol, and taurocholic acid was reduced. There was increased jejunal uptake of medium-chain length fatty acids, increased incremental change in free energy (integral of delta Fw----l), increased uptake of palmitic, taurocholic acid, and glycocholic acid and reduced uptake of myristic acid and dodecanol in animals fed the high-cholesterol diet for 10 days followed by Purina Chow for 18 days (HC-chow), as compared with HC. There was also increased ileal uptake of palmitic acid in HC-chow as compared with HC but reduced ileal uptake of stearic acid, cholesterol, taurocholic acid, and glycocholic acid. In the colon, there was increased uptake of stearic acid and dodecanol in HC-C as compared with HC, but reduced uptake of cholesterol, taurocholic acid and glycocholic acid. These changes in lipid uptake were not explained by alterations in the mucosal surface area or in the animals' body weight gain. It is proposed that early feeding experiences with a high-cholesterol diet may alter the normal development of intestinal transport of lipids with increased jejunal permeability to medium-chain fatty acids and increased effective resistance of the intestinal unstirred water layer.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

[The genetic aspects of the neuropsychology of verbal memory in schizophrenia].

Genetic study methods were used while examining the families with schizophrenic patients to study the relationships between computerized tomographic and resting ECG parameters with memory for information, which differed in degrees and ways of organization. In patients, memory performance was affected both by genotype-controlled high-frequency alpha-rhythm subranges and by integral delta-rhythm values determined by environmental factors. A significant role of the right hemisphere was found for all forms of remembering. In a group of relatives, predictors of different memory forms differed in frequency and topographic characteristics to a greater degree. These predictors mainly included neuromorphological parameters and power values of alpha-rhythm ranges. Most predictors were under considerable genetic determination.

Adolescent↗
PubMed1986

Evaluation of a filter/integrator system for quantifying delta activity in the sleep electroencephalogram.

The integrated output of a delta bandpass filter was evaluated as a means of quantifying delta activity in the sleep electroencephalogram (EEG). Total integrated slow wave amplitude (ISWA) and a density measure (ISWA/20-s epoch) were determined from four complete NREM sleep periods (NREMPs) on each of 2 baseline nights from 15 subjects. Total ISWA displays a linear decline across NREMPs, whereas ISWA/20-s epoch shows a curvilinear decline. Both measures show highly significant correlations with manually staged slow wave sleep and a high degree of intra-individual consistency between nights, for both whole-night values and those of individual NREMPs. The slow-wave density index (ISWA/20-s epoch) demonstrated consistently higher between-night correlations across NREMPs than did the total ISWA index, particularly in the first NREMP, suggesting it may better characterize the nightly distribution of delta activity in an individual's sleep EEG.

Delta Rhythm↗
PubMed2005

Rotational line-integrated photoabsorption cross sections corresponding to the delta(0,0) band of NO: a molecular quantum-defect orbital procedure.

The rotational line-integrated photoabsorption cross sections corresponding to the delta(0,0) band of the nitric oxide (NO) molecule at 295 K, calculated with the molecular quantum-defect orbital methodology, are in rather good accord with the experimental measurements available in the literature. The achieved results are of straightforward use in atmospheric chemistry, such as in the assessment of the NO photodissociation rate constant, which is of great relevance for atmospheric modeling.

Journal Article↗
PubMed1994

Prostaglandins in the treatment of cancer.

Prostaglandins (PGs) with antiproliferative activity against tumor cells consist of the cyclopentenone PGs and the alkylidene cyclopentenone PGs. Such PGs are PGD2, PGJ2, delta 12-PGJ2, PGA1, delta 7-PGA1, and PGA2. Both PGJ2 and delta 12-PGJ2 are ultimate metabolites of PGD2 and have potent antiproliferative activity on tumor cells. delta 12-PGJ2 was identified in human urine, whereas delta 7-PGA1 has not been found in the human body. One important characteristic of both delta 7-PGA1 and delta 12-PGJ2 is that they have little cross resistance with cisplatin and adriamycin in vitro and in vivo. delta 7-PGA1 has 5-fold greater antitumor activity than delta 12-PGJ2. Methyl ester-delta 7PGA1 (methyl-delta 7-PGA1) is stable chemically and can be easily synthesized in large amounts. All four isomers of methyl-delta 7-PGA1 showed the same antiproliferative activities on ovarian carcinoma cells. In addition, methyl-delta 7-PGA1 integrated in lipid microspheres (lipo-methyl-delta 7-PGA1) is more soluble in water than methyl-delta 7-PGA1 alone. Hence, lipo-methyl-delta 7-PGA1 was selected for extensive preclinical studies. Intravenous administration of lipo-methyl-delta 7-PGA1 could inhibit the growth of both HeLa S3 and Lovo colon cancer cells transplanted subcutaneously in nude mice. Lipo-methyl-delta 7-PGA1 by intraperitoneal administration could prolong the survival of scid mice bearing 2008C/13* cells resistant to cisplatin. The combined administration of cisplatin and lipo-methyl-delta 7-PGA1 prolonged the survival of nude mice bearing HRA cells compared with each single agent alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗