White blood cell telomerase activity and incident respiratory illness among community-dwelling elderly vaccinated against seasonal influenza.

Immune cell telomerase activity may impact vaccine response in the elderly. Fifty persons aged 60-100 years were tested for post-influenza vaccination telomerase RNA expression (TERT) in peripheral blood mononuclear cells to assess for an association with influenza antibody levels and influenza-like illness or incident respiratory infection (IRI) in the year following vaccination. High rates of seroprotective influenza antibody (> or = 1:40 titers) were observed post-vaccination (86-92% to vaccine viral strains), with no association to TERT. No IRI occurred among persons in the top quartile of TERT expression, whereas the IRI rate was 33% in the lower three quartiles (Kaplan-Meier P=0.028). TERT expression was also IRI significantly higher in those who did not experience IRI than those who did in the follow-up period (0.845 vs. 0.301, P=0.024). These data suggest that telomerase expression may correlate with immune capacity for vaccine response in the elderly and could represent a target for recognizing risk for vaccine failure.

Oxygen radical absorbance capacity of the phenolic compounds in plant extracts fractionated by high-performance liquid chromatography.

The oxygen radical absorbance capacity (ORAC) assay has been used to quantify the antioxidative properties of phytonutrients in fruit and vegetable extracts. Using aqueous methanol extracts of tea and spinach as a model systems, separation of the components in the extracts by HPLC followed by semiautomatic ORAC analysis of the column fractions permitted the determination of peroxyl-radical-scavenging profiles, demonstrating the relative abilities of the individual extract components to scavenge peroxyl radicals. ORAC values for up to 80 HPLC fractions were measured, confirming the major contribution of epigallocatechin gallate in the peroxyl radical scavenging of green tea extracts. Although the flavonoids in spinach extracts provided resistance to peroxyl radicals, components that did not bind to the HPLC column and simple phenolic compounds may also be important contributors to the total ORAC activity of spinach leaf extracts. Application of these procedures to plants believed to provide certain human health benefits by reducing free radicals may allow the identification and characterization of the specific components responsible for the free-radical-scavenging activities.

A device for the semiautomatic determination of oxygen-radical absorbance capacity.

The oxygen-radical absorbance capacity (ORAC) assay has become a standard method to quantify the antioxidative properties of phytonutrients in fruit and vegetable extracts. However, it is usually not possible to determine directly the contribution of specific phytonutrients to the total ORAC value. Separation of the components in the plant extracts by HPLC followed by ORAC analyses of the column fractions might permit the determination of free radical-scavenging profiles. The accurate determination of ORAC values may require 1 to 2 h/sample. Considering the number of samples that would be generated by a single HPLC separation, a device was constructed which permits up to 45 simultaneous ORAC analyses. Varying degrees of automation were included in the design. Furthermore, since the assay has a Q(10) for peroxyl radical-scavenging of about 3, elevation of the assay temperature from the standard 37 to 47 degrees C significantly reduced the assay times. Relatively simple modifications would allow the apparatus to be used in a variety of time-dependent fluorescence and absorbance assays.

Identification of two cytosolic ascorbate peroxidase cDNAs from soybean leaves and characterization of their products by functional expression in E. coli.

Screening of a cDNA library from soybean (Glycine max (L.) Merr. cv. Century) with probes based upon cytosolic ascorbate peroxidase (APx; EC 1.11.1.11) genes identified two full-length clones (SOYAPx1, SOYAPx2) apparently encoding for different soybean leaf cytosolic APxs. The deduced amino acid sequences of the two APx cDNA products differed in 13 of the 250 amino acids. The SOYAPx1 cDNA was identical to the cytosolic APx cDNA previously found in soybean root nodules. Escherichia coli expression systems were developed using both soybean APx cDNAs. Recombinant SOYAPx1 and SOYAPx2 were then utilized to characterize the enzymatic properties of the two APx cDNA products.

Pediatric mortality probability estimated from pre-ICU severity of illness.

OBJECTIVE: The Pediatric Risk of Mortality (PRISM) score is a measure of illness severity based on abnormalities observed in the bedside examination and laboratory assessment. PRISM scores obtained after pediatric intensive care unit (PICU) admission predict mortality probability, but no previous efforts to evaluate mortality risk before PICU admission have been reported. Our study was performed on patients admitted to PICUs at four pediatric tertiary care centers to derive a quantitative estimate of hospital mortality probability as a function of PRISM scores obtained at referring hospitals before PICU transfer. Performance of the model was tested by evaluating accuracy of mortality predictions obtained from pre-ICU PRISM scores in a separate validation set of patients. METHODS: Patients were randomized to the derivation or validation sets. Data were recorded prospectively from observations made at hospitals referring to the study PICUs. Patients included 780 infants and children with medical and surgical emergencies and trauma. Electively admitted patients were excluded from analysis. RESULTS: The relationship between mortality probability (P) and the pre-ICU PRISM score is expressed by the equation: P = er/(1 + er). In this equation, r is an empirical function of the pre-ICU PRISM score: r = .197 x PRISM - 4.705. The mortality probability rises from near 0 at low scores, approaching 1 (certainty) above a PRISM score of 40. Mortality probability exceeds 10% at a score of 13 and exceeds 50% at a score of 24. Performance of predictions in the validation set of patients was evaluated for five categories of mortality probability. The observed number of deaths corresponded to predicted mortality across the range of illness severity. When compared for each tertiary institution, observed mortality rates were similar to predictions for three of four institutions. For data obtained at institution D, the observed mortality of 17% significantly exceeded the 7% predicted rate. In infants younger than 1 year, as well as children 1 year and older, observed mortality rates were similar to predicted. CONCLUSIONS: The pre-ICU PRISM score as a measure of illness severity provides an estimate of hospital mortality probability. Further investigation is required to determine the use of pre-ICU mortality estimates in making clinical decisions.

Purification of Mitochondrial Glutamate Dehydrogenase from Dark-Grown Soybean Seedlings.

Proteins in extracts from cotyledons, hypocotyls, and roots of 5-d-old, dark-grown soybean (Glycine max L. Merr. cv Williams) seedlings were separated by polyacrylamide gel electrophoresis. Three isoforms of glutamate dehydrogenase (GDH) were resolved and visualized in gels stained for GDH activity. Two isoforms with high electrophoretic mobility, GDH1 and GDH2, were in protein extracts from cotyledons and a third isoform with the lowest electrophoretic mobility, GDH3, was identified in protein extracts from root and hypocotyls. Subcellular fractionation of dark-grown soybean tissues demonstrated that GDH3 was associated with intact mitochondria. GDH3 was purified to homogeneity, as determined by native and sodium dodecyl sulfate-polyacrylamide gels. The isoenzyme was composed of a single 42-kD subunit. The pH optima for the reductive amination and the oxidative deamination reactions were 8.0 and 9.3, respectively. At any given pH, GDH activity was 12- to 50-fold higher in the direction of reductive amination than in the direction of the oxidative deamination reaction. GDH3 had a cofactor preference for NAD(H) over NADP(H). The apparent Michaelis constant values for [alpha]-ketoglutarate, ammonium, and NADH at pH 8.0 were 3.6, 35.5, and 0.07 mM, respectively. The apparent Michaelis constant values for glutamate and NAD were 15.8 and 0.10 mM at pH 9.3, respectively. To our knowledge, this is the first biochemical and physical characterization of a purified mitochondrial NAD(H)-dependent GDH isoenzyme from soybean.

Modification of the Cellular Heat Sensitivity of Cucumber by Growth under Supplemental Ultraviolet-B Radiation.

The effect of ultraviolet B (UV-B) radiation on the thermal sensitivity of cucumber (Cucumis sativus L.) was studied using UV-B-sensitive cv Poinsett 76 and UV-B-resistant cv Ashley grown under control and elevated (300 mW m-2) UV-B radiation levels. Using both cotyledon and leaf discs, the ability of the tissue to reduce triphenyl tetrazolium chloride (TTC) was determined after treatment at 50[deg]C for various times. Semilogarithmic plots of TTC reduction as a function of time at 50[deg]C were curvilinear. They were monophasic for the control cucumber and biphasic for cucumber grown in the presence of elevated UV-B. Treatment of cucumber plants at 37[deg]C for 24 h or of tissue discs at acute UV-B levels for 1 h further modified their response to elevated temperature. These results suggest that growth of cucumber under enhanced UV-B radiation levels increased its ability to withstand elevated temperatures.

Estimation and Analysis of Cucumber (Cucumis sativus L.) Leaf Cellular Heat Sensitivity.

Triphenyl tetrazolium chloride (TTC) reduction by cucumber (Cucumis sativus L. cv Poinsett 76 and cv Ashley) leaf discs was used as a viability assay to examine the effect of temperature pretreatment on the tissue response to acute hyperthermia. Semi-logarithmic plots of TTC reduction as a function of incubation time at different temperatures from 40 to 60[deg]C resembled the heat survival curves of animal cells. Heat inactivation rates were obtained and subjected to "quasi" Arrhenius analyses by analytical methods derived from the animal studies. The Arrhenius plots of TTC reduction rates for cv Ashley leaf discs preincubated at 25 or 37[deg]C and for cv Poinsett 76 preincubated at 37[deg]C were linear with the same activation energy (Ea) of about 80 kcal mol-1. The Arrhenius plot of cv Poinsett 76 preincubated at 25[deg]C was nonlinear with an Ea of about 80 kcal mol-1 at temperatures below 46[deg]C and an Ea of about 27.5 kcal mol-1 at temperatures above 47[deg]C. The significance of these differences is discussed in terms of the role of protein denaturation in the thermal sensitivity of cucumber disc reduction of TTC and the applicability of these methods to the analysis of plant cellular heat sensitivity.

Ultraviolet-Induced Photodegradation of Cucumber (Cucumis sativus L.) Microsomal and Soluble Protein Tryptophanyl Residues in Vitro.

The in vitro effects of ultraviolet B (280-320 nm) radiation on microsomal membrane proteins and partially purified ribulose bisphosphate carboxylase (Rubisco) from cucumber (Cucumis sativus L.) was investigated by measuring the direct photolytic reduction of tryptophan fluorescence and the formation of fluorescent photooxidation products. Exposure of microsomes and Rubisco to monochromatic 300-nm radiation resulted in the loss of intrinsic tryptophan fluorescence and the production of blue-emitting fluorophores. The major product of tryptophan photolysis was tentatively identified as N-formylkynurenine (N-FK). Even though the rates of tryptophan photodegradation and N-FK formation were similar, the amount of blue fluorescence produced was significantly higher in the microsomes relative to Rubisco. Studies with various free radical scavengers and other modifiers indicated that tryptophan photodegradation requires oxygen and that the subsequent formation of N-FK may involve reactive oxygen species. The optimum wavelengths for loss of typtophan fluorescence were 290 nm for the microsomes and 280 nm for Rubisco. The temperature dependence of tryptophan fluorescence and rate of tryptophan photodegradation indicated an alteration in the cucumber microsomal membranes at about 24[deg]C, which influenced protein structure and tryptophan photosensitivity.

Temperature-Induced Protein Conformational Changes in Barley Root Plasma Membrane-Enriched Microsomes: III. Effect of Temperature and Cations on Protein Sulfhydryl Reactivity.

Temperature and cations modified the reaction of barley (Hordeum vulgare L. cv Conguest) root plasma membrane protein sulfhydryl groups with N-4-(7-diethylamino-4-methylcoumarin-3-yl)-phenyl maleimide (CPM). The pseudo-first-order rate constants for the formation of fluorescent CPM-protein adducts increased as the temperature was raised above 30 degrees C, suggesting changes in protein conformation. Monovalent [K(I), Na(I), L(I)] and certain divalent cations [Ba(II), Mg(II)] increased the reaction rates. Other divalent cations [Ca(II), Mn(II), Ni(II), Co(II), Sr(II), Cd(II), Hg(II)] decreased the rate of fluorescent adduct formation. Na(I) promoted and Ca(II) delayed the onset of the temperature-dependent increases in reaction rates. The results are discussed in terms of lipid-mediated, temperature-dependent changes in membrane protein conformation and ion-protein interactions.

Analysis of aluminum and divalent cation binding to wheat root plasma membrane proteins using terbium phosphorescence.

A phosphorescent trivalent cation, terbium [Tb(III)], has been used to study the binding of different polyvalent cations to the proteins of wheat (Triticum aestivum L.) root plasma membranes. The phosphorescence emission intensity of Tb(III) was enhanced after Tb(III) binding to wheat root plasma membranes as a result of nonradiative resonance energy transfer from the membrane protein tyrosine and phenylalanine residues. Complex, saturable Tb(III) binding was observed, suggesting multiple binding sites. Bound Tb(III) could be displaced by divalent cations in the general order: Mn(II) > Ca(II) > Mg(II). Al(III) was very effective in reducing the protein-enhanced Tb(III) phosphorescence at pH values below 5. Al(III) also altered the Tb(III) phosphorescence lifetime, suggesting Al(III)-induced changes in membrane protein conformation. The more Al(III)-sensitive wheat cultivar (Anza) bound Al(III) with higher affinity than the more tolerant cultivar (BH 1146). At pH 5.5 where Al(III) did not displace bound Tb(III), low levels of Al(III) reduced the ability of Mn(II) to decrease Tb(III) phosphorescence. The significance of these results is discussed with respect to the mechanisms of Al(III) tolerance in wheat and the potential beneficial effects of Al(III) in reducing Mn(II) phytotoxicity.

Temperature-induced protein conformational changes in barley root plasma membrane-enriched microsomes: I. Effect of temperature on membrane protein and lipid mobility.

A protein spin label and lipid spin probes were used to study the temperature-dependent motion of protein and lipid, respectively, in barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes. Using membranes from seedlings grown at 20 degrees C, the temperature-dependence of the relative motion of membrane surface spin probes and a spin label covalently attached to membrane proteins suggested abrupt changes in the lipid and protein mobilities at about 12 degrees C. Spin probe spin-spin exchange broadening and fluorescent probe eximer formation indicated apparent temperature-induced alterations in probe lateral diffusion within the membrane at about 12 to 14 degrees C. The results suggest the presence of temperature-induced quasicrystalline lipid clusters which may influence the activity of membrane-bound enzymes.

Temperature-Induced Protein Conformational Changes in Barley Root Plasma Membrane-Enriched Microsomes: II. Intrinsic Protein Fluorescence.

The membrane-bound proteins of barley (Hordeum vulgare L. cv Conquest) root plasma membrane-enriched microsomes displayed fluorescence typical of protein-associated trytophan residues. The protein fluorescence intensity was sensitive to variations in sample temperature. The temperature-induced decline in protein fluorescence intensity was nonlinear with slope discontinuities at about 12 and 32 degrees C. Detergents at levels above their critical micelle concentration enhanced protein fluorescence. Glutaraldehyde reduced protein fluorescence. Protein fluorescence polarization increased at temperatures above 30 degrees C. Both the rate of tryptophan photoionization and the fluorescence intensity of the photoionization products suggested alterations in membrane protein conformation between 12 and 32 degrees C. The quenching of the intrinsic protein fluorescence by acrylamide and potassium iodide indicated changes in accessibility of the extrinsic agents to the protein tryptophan residues beginning at about 14 degrees C. The results indicate thermally induced changes in the dynamics of the membrane proteins over the temperature range of 12 to 32 degrees C which could account for the complex temperature dependence of the barley root plasma membrane ATPase.

Orthodontic and surgical intervention to arrest tooth loss secondary to subgingival elastic.

Elastic bonds used for orthodontic tooth movement without benefit of attached mechanisms have been shown to migrate apically, causing severe periodontal damage and eventual tooth mobility and tooth loss. The literature indicates high mortality rates for such involved teeth. A case report is presented, illustrating initial successful retention of teeth following surgical removal of a displaced elastic band. Cooperation between oral surgeon, periodontist, and orthodontist, in conjunction with high standards of oral hygiene by the patient, may lead to successful retention of otherwise condemned teeth. It is also suggested that elastic band therapy be carefully supervised when used to move teeth without controlling attachment mechanisms.