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PubMed1987

Power spectrum analysis in the study of ocular mechanisms.

The use of power-spectrum analysis in the study of ocular dynamics is becoming increasingly widespread. This paper summarizes the key points of signal acquisition and power-spectrum analysis, emphasizing aspects which we have found to be of relevance when the technique is applied to experiments in binocular accommodation. The considerations and conclusions are relevant to other areas of investigation into ocular dynamics and similar biological systems.

Accommodation, Ocular↗
PubMed2006

Cloning and sequence analysis of maize FAD2 gene.

Delta-12 desaturases are involved in the conversion of oleic acid to linoleic acid in plant. Based on the conserved oligo amino acid residues of the published delta-12 desaturase genes from other higher plant species, a cDNA fragment was amplified by RT-PCR (reverse transcriptase-polymerase chain reaction) from the total RNA of immature maize embryos. According to bioinformation analysis of the cDNA sequence, a specific fragment of FAD2 gene was isolated by RT-PCR from immature maize embryos, and DNA of the same length was amplified from maize genome. Results of sequence analysis indicate that they are all 1 164 bp long, and have just an open reading frame (ORF) coding for 387 amino acids, and there is no intron in the FAD2 ORF (GenBank accession DQ496227). The deduced amino acid sequence of the cloned FAD2 showed high identity to those of other plant delta-12 fatty acid desaturases. It contains three histidine motifs and two long stretches of hydrophobic residues, indicative of an integral membrane protein spanning membrane four times. Analysis by semi-quantitive RT-PCR showed that FAD2 was strongly expressed in maize immature embryos than in leaves, stems and roots.

Amino Acid Sequence↗
PubMed1978

Energy-related pollutants in the environment: use of short-term tests for mutagenicity in the isolation and identification of biohazards.

In an effort to gather information on the potential genetic hazards of existing or proposed energy-generating or -conversion systems, we have begun a correlated analytical and genetic analysis of a number of technologies. The work is divided into two phases: one deals with known compounds expected to occur in the environment through energy production, conversion, or use; the other deals with actual samples from existing or experimental processes. To approach the problems of coping with and testing large numbers of compounds, we set up a form of the "tier system." Operating units utilizing Salmonella, Escherichia coli, yeast, human leukocytes, mammalian cells, and Drosophila have been initiated. Various liquid-liquid extraction methods and column chromatographic separations have been applied to crude products and effluents from oil-shale, coal-liquefaction, and coal-gasification processes. Mutagenicity of the various fractions is assayed by means of reversion of histidine-requiring auxotrophs of Salmonella typhimurium; comparative studies are carried out with the other genetic systems. In order to incorporate metabolic activation of these fractions and compounds, rat liver homogenates (S-9) are used in the various assays. Results implicate chemicals occurring in the basic (ether-soluble) and the neutral fractions as potential genetic hazards. Chemical constituents of these fractions (identified or predicted) were tested individually for their mutagenic activity.

Animals↗
PubMed2005

The residue E351 is essential for the activity of human 21-hydroxylase: evidence from a naturally occurring novel point mutation compared with artificial mutants generated by single amino acid substitutions.

Congenital adrenal hyperplasia (CAH) [OMIM 201910] is a group of autosomal recessive disorders, caused in 90-95% of cases by a deficiency of steroid 21-hydroxylase due to mutations in the CYP21A2 gene. The functional and structural effects of a novel rare missense mutation (E351K) in CYP21A2 found in a male patient with simple virilizing CAH were studied. The novel E351K point mutation is located in the ERR triad of the 21-hydroxylase. The ERR triad is a glutamine-arginine-arginine motif conserved in all cytochrome P450 sequences. The glutamate and first arginine residue are invariant in all P450 cytochrome enzymes, whereas the second arginine residue is present as arginine, histidine, or asparagine. Although the ERR triad is involved in some way to heme binding by the cytochrome P450 monooxygenases, the E351K mutation leads to severe but not complete loss of CYP21 enzyme activity. The functional analysis in COS-7 cells revealed a reduced conversion of 17-hydroxyprogesterone to 11-deoxycortisol of 1.1+/-0.5% (SD) and of progesterone to 11-deoxycorticosterone of 1.2+/-0.3% of wild-type activity. Analyzing the artificial mutants (E351D, E351I) of the E351 residue did not show a restoration of the in vitro 21-hydroxylase activity. These effects could be readily explained by structural changes induced by the mutations, which were rationalized by a three-dimensional-model structure of the CYP21 protein. The combination of in vitro enzyme function and computerized protein analysis of the E351 residue of the CYP21 protein provides experimental evidence for the ERR triad being a fundamental structural element of cytochrome P450 enzymes.

Amino Acid Substitution↗
PubMed1986

Arachidonic acid metabolism by canine tracheal epithelial cells. Product formation and relationship to chloride secretion.

Canine tracheal epithelial cells freshly isolated from mongrel dog trachea were used to study relationships between arachidonic acid metabolism and chloride ion movement. High performance liquid chromatography (HPLC) analysis of the cell incubation media after the addition of A23187 showed the presence of prostaglandin H synthase and lipoxygenase-derived metabolites. The major prostaglandin H synthase metabolite identified by HPLC, gas chromatography, and mass spectrometry was prostaglandin (PG) D2. The major lipoxygenase metabolites were leukotriene (LT) C4 and LTB4. LTB4 was identified by HPLC, UV spectroscopy, and gas chromatography. Straight phase HPLC of the methyl esters indicated only a minor formation of LTB4 isomers. LTC4 was identified by HPLC, UV spectroscopy, and conversion to LTD4 by gamma-glutamyl transpeptidase. Analysis by radioimmunoassays indicated approximately 1-2 ng of LTB4 and peptide LT formed by 10(6) cells after A23187 stimulation. The addition of ionophore A23187 caused a rapid release of arachidonic acid metabolites which was completed within 5 min of stimulation. Cl- secretion was measured in parallel studies of excised tracheas in Ussing chambers. Cl- secretion occurred at 2-3 min after the addition of ionophore, and the most rapid change occurred with the highest PGD2 concentrations. Indomethacin produced a concentration-dependent inhibition of PGD2 formation and Cl- movement. The addition of PGE2, PGD2, and PGH2 effectively stimulated Cl- secretion. LTC4 also stimulated Cl- secretion, but the stimulation was inhibited by indomethacin. These results indicate that canine tracheal epithelial cells metabolize arachidonic acid via both prostaglandin H synthase and lipoxygenase enzymes. It appears that endogenous PGD2 formation is the important variable controlling the Cl- ion movement in canine trachea.

Animals↗
PubMed2005

Mechanism of the Schiff base forming fructose-1,6-bisphosphate aldolase: structural analysis of reaction intermediates.

The glycolytic enzyme fructose-1,6-bisphosphate aldolase (FBPA) catalyzes the reversible cleavage of fructose 1,6-bisphosphate to glyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Catalysis of Schiff base forming class I FBPA relies on a number of intermediates covalently bound to the catalytic lysine. Using active site mutants of FBPA I from Thermoproteus tenax, we have solved the crystal structures of the enzyme covalently bound to the carbinolamine of the substrate fructose 1,6-bisphosphate and noncovalently bound to the cyclic form of the substrate. The structures, determined at a resolution of 1.9 A and refined to crystallographic R factors of 0.148 and 0.149, respectively, represent the first view of any FBPA I in these two stages of the reaction pathway and allow detailed analysis of the roles of active site residues in catalysis. The active site geometry of the Tyr146Phe FBPA variant with the carbinolamine intermediate supports the notion that in the archaeal FBPA I Tyr146 is the proton donor catalyzing the conversion between the carbinolamine and Schiff base. Our structural analysis furthermore indicates that Glu187 is the proton donor in the eukaryotic FBPA I, whereas an aspartic acid, conserved in all FBPA I enzymes, is in a perfect position to be the general base facilitating carbon-carbon cleavage. The crystal structure of the Trp144Glu, Tyr146Phe double-mutant substrate complex represents the first example where the cyclic form of beta-fructose 1,6-bisphosphate is noncovalently bound to FBPA I. The structure thus allows for the first time the catalytic mechanism of ring opening to be unraveled.

Archaeal Proteins↗
PubMed2002

Detecting gene-gene interactions using affected sib pair analysis with covariates.

Interest has recently focussed on allowing for interactions between loci as a way to increase power to detect linkage. In this paper, a simplified logistic regression method was used to perform affected sib pair analyses allowing for the inclusion of data from other loci. A systematic search of two-locus disease models was carried out to determine the situations in which this was advantageous. If IBD information is available (e.g. from a genome scan), it is unlikely that allowing for interactions will give a large lod score in the absence of linkage evidence from sinlge-locus analysis. Furthermore, allowing for interactions rarely gave a significant increase in power to detect linkage over a single-locus analysis, except for heterogeneity models with low K(P). Conversely, the availability of disease-associated genotypes may greatly increase the power both to detect linkage to a second locus and interaction between the loci. These results indicate that when only IBD information is available, two-locus analysis of genome scan data should be restricted to regions giving peaks under single-locus analysis. If disease-associated genotypes are available, it may be worth re-analysing the whole genome.

Genetic Linkage↗
PubMed2006

MRI of hippocampus and entorhinal cortex in mild cognitive impairment: a follow-up study.

The concept of mild cognitive impairment (MCI) has been proposed to represent a transitional stage between normal aging and dementia. We studied the predictive value of the MRI-derived volumes of medial temporal lobe (MTL) structures, white matter lesions (WML), neuropsychological tests, and Apolipoprotein E (APOE) genotype on conversion of MCI to dementia and AD. The study included 60 subjects with MCI identified from population cohorts. During the mean follow-up period of 34 months, 13 patients had progressed to dementia (9 to Alzheimer's disease (AD)). In Cox regression analysis the baseline volumes of the right hippocampus, the right entorhinal cortex and CDR sum of boxes predicted the progression of MCI to dementia during the follow-up. In a bivariate analysis, only the baseline volumes of entorhinal cortex predicted conversion of MCI to AD. The Mini-Mental State Examination (MMSE) score at baseline, WML load, or APOE genotype were not significant predictors of progression. The MTL volumetry helps in identifying among the MCI subjects a group, which is at high risk for developing AD.

Aged↗
PubMed2004

Spectral and correlation analyses of the verapamil-induced conversion of ventricular fibrillation to tachycardia in isolated rat hearts.

Ventricular tachycardia (VT) is considered to be the most common precursor of ventricular fibrillation (VF). However, the mechanisms underlying the transition from VT to VF remain unclear. Here, we investigated whether and how perfusion of the heart with verapamil, a blocker of L-type calcium channels, changed the macro-dynamics of the heart between VT and VF. The experiments were performed with Langendorff perfused isolated rat hearts, in which left ventricular pressure and left ventricular cardiomyogram were measured. Sustained VT or VF was induced by burst pacing of the left ventricular muscles. During sustained VF, verapamil perfusion resulted in the conversion of VF to VT. A cross-correlation analysis between left ventricular cardiomyogram and left ventricular pressure revealed that the correlation coefficient was small during VF, but became larger during VT. This study showed that inactivation of L-type Ca(2+) channels occurred during verapamil-induced conversion of pacing-induced sustained VF to VT, and characterized the changes in macro-dynamics of the heart associated with the transition.

Animals↗
PubMed1990

Comparative evaluation of verapamil, flecainide and propafenone for the acute conversion of atrial fibrillation to sinus rhythm.

In 69 consecutive patients with recent-onset atrial fibrillation, we compared the usefulness of verapamil, flecainide and propafenone for the acute conversion to sinus rhythm within 1 hour of the start of intravenous infusion. The patients were randomized to receive verapamil, 10 mg in 5 minutes (n = 29), flecainide, 2 mg/kg in 10 minutes (n = 20) or propafenone, 2 mg/kg in 10 minutes (n = 20). The 3 treatment groups did not differ significantly regarding patient baseline characteristics. Conversion rate was 14% for verapamil, 50% for flecainide and 25% for propafenone. A significant difference (p = 0.02) was found only between the verapamil and flecainide results. Mean time to conversion did not differ significantly. Stepwise multiple regression analysis revealed that the only variables correlating with the final result were the type of treatment (p = 0.001) and the duration of arrhythmia (p = 0.005). Mean duration of arrhythmia in converted patients was significantly shorter than in non-converted patients (9.7 [6.6] vs 34.0 [46.1] hours, p = 0.03). Serious side effects were seen only during propafenone infusion (2 patients developed severe hypotension necessitating discontinuation of therapy and administration of intravenous fluids). We conclude that intravenous flecainide appears to be more effective than the other 2 drugs for the acute conversion of recent-onset atrial fibrillation to sinus rhythm.

Adult↗
PubMed1984

Neonatal denervation inhibits the normal postnatal decrease in endplate channel open time.

The apparent mean channel open time (tau) of acetylcholine receptors (AChR) at skeletal muscle endplates decreases greater than 3-fold during development. In rat soleus muscles, the change occurs between postnatal days 8 and 18 as channels with long apparent open times (tau = 4.5 msec) disappear while channels with short apparent open times (tau = 1.5 msec) increase in number. We studied the role of innervation in this process by denervating neonatal soleus muscles prior to channel conversion. Tau at the denervated endplates was assayed at various times between days 8 and 18 by using fluctuation analysis. We found that early denervation blocked, or at least delayed, channel conversion. Unexpectedly, there was enhanced extrajunctional ACh sensitivity in the innervated muscles contralateral to the denervated ones. This observation allowed us to compare the apparent open times of junctional AChRs with those of extrajunctional AChRs 200 micron distant in the same innervated fibers. In developing muscles, tau at the extrajunctional sites decreased in parallel with tau at the endplates. Thus, neural regulation of AChR channel gating extends well beyond the endplate boundaries.

Acetylcholinesterase↗
PubMed1992

R339H and P453S: CYP21 mutations associated with nonclassic steroid 21-hydroxylase deficiency that are not apparent gene conversions.

Steroid 21-hydroxylase deficiency is the most common enzymatic defect causing congenital adrenal hyperplasia, an inherited disorder of cortisol biosynthesis. All mutations thus far characterized that cause this disorder appear to result from recombinations between the gene encoding the enzyme, CYP21B (CYP21), and the adjacent pseudogene, CYP21A (CYP21P). These are either deletions caused by unequal crossing-over during meiosis or apparent transfers of deleterious sequences from CYP21A to CYP21B, a phenomenon termed gene conversion. However, a small percentage of alleles do not carry such a mutation. We analyzed DNA from a patient with the mild, nonclassic form of 21-hydroxylase deficiency, who carried one allele that had no gene conversions detectable by hybridization with oligonucleotide probes. Sequence analysis revealed that this allele carried two missense mutations, R339H and P453S, neither of which has been previously observed in CYP21A or CYP21B. Each of these mutations was introduced into CYP21 cDNA which was then expressed in COS1 cells using a vaccinia virus system. Each mutation reduced the ability of the enzyme to 21-hydroxylate 17-hydroxyprogesterone to 50% of normal and the ability to metabolize progesterone to 20% of normal. Thus, each of these mutations represents a potential nonclassic 21-hydroxylase deficiency allele that is not the result of an apparent gene conversion.

Adrenal Hyperplasia, Congenital↗
PubMed2000

Further characterization of Delta(8)-sphingolipid desaturases from higher plants.

A previously cloned cDNA from Helianthus annuus codes for a fusion protein composed of an N-terminal cytochrome b(5) and a C-terminal desaturase domain. For a functional identification, this cDNA was expressed in Saccharomyces cerevisiae and the structures of sphingolipid long-chain bases were analysed. The expression of this sunflower enzyme resulted in the formation of new Delta(8)-trans/cis-phytosphingenine from C(18)- and C(20)-phytosphinganine present in wild-type yeast cells. To elucidate the substrate specificity, the recently cloned Delta(8)-sphingolipid desaturases from Arabidopsis thaliana and Brassica napus were expressed in the yeast mutant sur2Delta that lacked the sphinganine C(4)-hydroxylase and was thus unable to form phytosphinganine. Long-chain base analysis of the transformed mutant cells did not show any conversion of C(18)- or C(20)-sphinganine into Delta(8)-sphingenine, whereas exogenously added C(18)-phytosphinganine was desaturated to Delta(8)-trans/cis-phytosphingenine. Furthermore, GLC-MS analysis did not reveal the presence of any Delta(9)-regioisomers as reported before. These results show that the sunflower gene codes for a Delta(8)-sphingolipid desaturase which accepts C(18)- and C(20)-phytosphinganine. The absence of Delta(8)-sphingenine as desaturation product in the transformed mutant suggests that C(4)-hydroxylation of sphinganine precedes Delta(8)-desaturation. Therefore, in yeast, the substrate for the plant Delta(8)-sphingolipid desaturase seems to be the phytosphinganine residue.

Arabidopsis↗
PubMed1971

Mutational and physiological enhancement of photosynthetic energy conversion in Rhodopseudomonas capsulata.

Photosynthetic growth of Rhodopseudomonas capsulata, a nonsulfur purple bacterium, is severely inhibited by inorganic arsenate when this anion is present equimolar to orthophosphate in the culture medium. Studies on an arsenate-resistant mutant indicate that its resistance can be explained by a significantly increased photophosphorylation capacity of the energy-converting machinery, which can compensate for the decrease in adenosine triphosphate regeneration rate caused by esterification of arsenate in place of phosphate. Repeated subculture of the mutant in the presence of arsenate, under certain conditions, yields cell populations that have strikingly elevated contents of membrane-bound reaction-center bacteriochlorophyll, cytochromes, and photo-phosphorylation coupling factor. Membranes from such cells show unusually high photophosphorylation activity, and can be expected to provide a useful experimental material for more refined analysis of the mechanism of the energy-conversion process.

Adenosine Triphosphate↗
PubMed1997

The effect of curing light variations on bulk curing and wall-to-wall quality of two types and various shades of resin composites.

OBJECTIVES: This study evaluated the influence of light intensity and irradiation time variations on the curing efficacy of two types and various shades of resin composites and the effect of reduced light intensity on the preservation of wall-to-wall continuity. MATERIALS AND METHODS: Three microfilled composites (in three different shades) and one hybrid composite were used in this study. Polymerization shrinkage, and the hardness and adaptation of adhesive restorations in dentin cavities were determined at light intensities of 175 and 700 mW/cm2 and irradiation times of 10 and 60 s. Data were compared using in a general linear model analysis. RESULTS: Shrinkage measurements were the indication of conversion and conversion rate. Reduced intensity slowed down the rate of polymerization but did not reduce the conversion as long as an irradiation time of 60 s was employed. High-energy irradiation caused increased separation of the composite from the tooth structure. On the basis of obtaining optimal conversion and adaption, it was demonstrated that the irradiation time to be more effective than irradiation energy. SIGNIFICANCE: Light-cured composites require an understanding of their structure, pigmentation and irradiation parameters to obtain optimal performance. High intensity light-curing does not necessarily lead to optimal quality.

Analysis of Variance↗
PubMed1978

Pyrimidine metabolism in microplasmodia of Physarum polycephalum.

If microplasmodia of Physarum polycephalum are exposed to 14C-labelled pyrimidine nucleosides or bases, an unusual pattern of metabolism is found. Only the nucleosides are taken up. Analysis of the distribution of the radioactivity in the cells revealed that ribonucleosides and deoxyribonucleosides are incorporated into nucleotides; however, a substantial catabolism takes place. Thus incubation with [2-14C]pyrimidine nucleosides readily gives rise to [14C]O2, particularly in the case of [2-14C]thymidine. Due to this a significant part of the trichloroacetic-acid-insoluble radioactivity from exogenously supplied [2-14C]thymidine is not associated with DNA. The pattern of labelling of nucleoside triphosphates from exogenously supplied nucleosides indicated that the de novo synthesis of nucleotides was only partly repressed. An unusual conversion of deoxycytidine into cytidine was noted. Enzyme analysis on cell-free extracts revealed that pyrimidine nucleosides can be salvaged by kinases and that their initial catabolism is initiated by hydrolases. Incubation of microplasmodia with pyrimidine analogues showed that only nucleoside analogues are toxic. The experimental results have led us to propose a scheme for the metabolism of pyrimidine nucleosides and bases in Physarum polycephalum.

Biological Transport↗
PubMed2002

Quantitative assessment of solid waste treatment systems in the industrial ecology perspective by exergy analysis.

Solid waste treatment options (recycling, incineration, and landfilling; the two latter processes both with co-generation of heat and electricity) have been studied for cardboard, newspaper, polyethylene, poly(ethylene terephthalate), polypropylene, polystyrene, and poly(vinyl chloride) waste. The conversion processes have been analyzed in terms of the second law of thermodynamics. The analysis allows calculating the exergy (useful energy) embodied in conversion products that can be obtained from the required inputs for the treatment processes. Taking into account the waste materials and the resources to convert them, it proved that recycling is the most efficient option for polyethylene with an efficiency of 62.5% versus 43.6% for incineration and 0.9% for landfilling. Next, waste treatment has been put into the broader perspective of industrial ecology. Exergetic efficiencies of industrial metabolic options have been calculated. Here resources for manufacturing and converting solid products have been considered. Furthermore, selection of one type of conversion excludes the generation of other potential conversion products. Therefore, it has to be taken into account that these latter products still have to be produced starting from virgin resources. Recycling proved to be the most efficient strategy: the ratio eta between exergy embodied in all delivered products on one hand, and all exergy withdrawn from the ecosphere or from waste materials on the other hand, is the highest. For polyethylene, eta proved to be 0.568, whereas eta is 0.503 and 0.329 for incineration and landfilling, respectively. On the other hand, if R the ratio between exergy of delivered products on one hand and exergy of virgin materials on the other hand is calculated, the differences between the industrial metabolic options are larger. Recycling polyethylene showed a ratio R of 0.936, whereas ratios of 0.772 and 0.531 were found for incineration and landfilling, respectively. It has been shown that the exergy concept allows a quantitative comparison of different industrial metabolic options, contributing to a better assessment of sustainability of technology with respect to resource management.

Conservation of Natural Resources↗
PubMed1987

Identification of a mammalian glutaminyl cyclase converting glutaminyl into pyroglutamyl peptides.

Extracts from bovine pituitary were found to contain an activity catalyzing the conversion of glutaminyl peptides such as [Gln1]gonadotropin-releasing hormone, [Gln1, Gly4]thyrotropin-releasing hormone (H-Gln-His-Pro-Gly-OH), and H-Gln-Tyr-Ala-OH to the respective pyroglutamyl peptides. The TRH precursor fragment H-Lys-Arg-Gln-His-Pro-Gly-Lys-Arg-OH and the D-glutaminyl stereoisomer of H-Gln-Tyr-Ala-OH did not react under the same conditions. The conversion products were identified by Edman degradation, amino acid analysis, and reversed-phase HPLC. That this activity was exhibited by an enzyme, glutaminyl cyclase, was concluded from the protein character of the activity (revealed by its abolition with trypsin and heat), the Michaelis-Menten relationship between substrate concentration and conversion rate, and the substrate specificity. It was determined that glutaminyl cyclase had a molecular weight of 43,000-50,000, a pH optimum at pH 8, and Km and Vmax values in the range of 60-130 microM and 390-690 pmol/microgram per hr, respectively. Glutaminyl cyclase was not observed to require ATP and could be inhibited with 1.0 M ammonium chloride, which increased the Km and decreased the Vmax value. The subcellular distribution of glutaminyl cyclase corresponded to the one of peptidylglycine alpha-amidating monooxygenase believed to catalyze C-terminal amidations during posttranslational precursor processing. It was also observed that the formation of pyroglutamyl from glutaminyl peptides occurred nonenzymatically; however, the enzymatic reaction carried out with crude extract was found to be approximately 70 times faster than the nonenzymatic reaction enhanced by phosphate. It is speculated that glutaminyl cyclase may participate in the posttranslational processing of hormonal precursors to pyroglutamyl peptides.

Acyltransferases↗