Functional Significance of Calcium Binding to Tissue-Nonspecific Alkaline Phosphatase

The conserved active site of alkaline phosphatases (AP) contains catalytically important Zn²⁺ (M1 and M2) and Mg²⁺-sites (M3) and a fourth peripheral Ca²⁺ site (M4) of unknown significance. We have studied Ca²⁺ binding to M1-4 of tissue-nonspecific AP (TNAP), an enzyme crucial for skeletal mineralization, using recombinant TNAP and a series of M4 mutants. Ca²⁺ could substitute for Mg²⁺ at M3, with maximal activity for Ca²⁺/Zn²⁺-TNAP around 40% that of Mg²⁺/Zn²⁺-TNAP at pH 9.8 and 7.4. At pH 7.4, allosteric TNAP-activation at M3 by Ca²⁺ occurred faster than by Mg²⁺. Several TNAP M4 mutations eradicated TNAP activity, while others mildly influenced the affinity of Ca²⁺ and Mg²⁺ for M3 similarly, excluding a catalytic role for Ca²⁺ in the TNAP M4 site. At pH 9.8, Ca²⁺ competed with soluble Zn²⁺ for binding to M1 and M2 up to 1 mM and at higher concentrations, it even displaced M1- and M2-bound Zn²⁺, forming Ca²⁺/Ca²⁺-TNAP with a catalytic activity only 4–6% that of Mg²⁺/Zn²⁺-TNAP. At pH 7.4, competition with Zn²⁺ and its displacement from M1 and M2 required >10-fold higher Ca²⁺ concentrations, to generate weakly active Ca²⁺/Ca²⁺-TNAP. Thus, in a Ca²⁺-rich environment, such as during skeletal mineralization at pH 7.4, Ca²⁺ adequately activates Zn²⁺-TNAP at M3, but very high Ca²⁺ concentrations compete with available Zn²⁺ for binding to M1 and M2 and ultimately displace Zn²⁺ from the active site, virtually inactivating TNAP. Those ALPL mutations that substitute critical TNAP amino acids involved in coordinating Ca²⁺ to M4 cause hypophosphatasia because of their 3D-structural impact, but M4-bound Ca²⁺ is catalytically inactive. In conclusion, during skeletal mineralization, the building Ca²⁺ gradient first activates TNAP, but gradually inactivates it at high Ca²⁺ concentrations, toward completion of mineralization.     

Bradykinin-induced biphasic response in the rat isolated stomach fundus: Functional evidence for a novel bradykinin receptor

The responses of the rat isolated stomach fundus to bradykinin (BK) and des-Arg⁹-BK (DA-BK) have been examined. In rat isolated stomach fundus pre-contracted with BaCl₂ (0.5-1 mM), BK caused concentration-dependent biphasic responses characterized by relaxation followed by contraction. DA-BK also caused marked relaxations, but, unlike BK, induced only small contractions. Removal of the mucosal layer initially abolished the relaxant responses to BK and both responses to DA-BK without affecting BK-induced contractions, but repeated challenges with BK or DA-BK revealed a time-depeendent reappearance of the relaxant responses, suggesting “de novo” synthesis of BK receptors. Pretreatment of rat stomach fundus with tetrodotoxin (1 μM), atropine (1 μM), captopril (3 μM), prazosin (1 μM) or glibenclamide (1 μM) did not significantly modify the biphasic responses to BK (300 nM). The biphasic responses to DA-BK were antagonized selectivley by the B₁ receptor antagonist des-Arg⁹-[Leu ⁸]-BK (DAL- BK) (1 μM). In contrast, the biphasic responses to BK were unaffected by DAL-BK or by several selective peptide antagonists of B₂ receptors including NPC 431 (Thi^5,8, D-Phe⁷)-BK, NPC 349 (D-Arg Hyp³, Thi^5,8, D-Phe⁷)-BK, NPC 567 (D-Arg -Hyp³, D-Phe⁷)-BK and NPC 361 (D-Phe⁷)-BK (3 to 10 μM). These results are consistent with the view that the biphasic responses of the rat isolated stomach fundus to BK appear to be mediated by a novel BK receptor which is insensitive to blockade by B₁ and B₂ selective BK receptor antagonists.     

Effect of dexamethasone and cytochrome P 450 inhibitors on the formation of 7α-hydroxydehydroepiandrosterone by human adipose stromal cells

7α-Hydroxydehydroepiandrosterone (7α-OHDHA) is a major metabolite of dehydroepiandrosterone (DHA) using adipose stromal cells. To gain a better understanding of the factors regulating DHA metabolism, we examined the effect of dexamethasone and cytochrome P 450 inhibitors on the formation of 7α-OHDHA. Dexamethasone (10⁻⁹ to 10⁻⁷ M) stimulated 7α-OHDHA formation in a dose-dependent manner with a 2- to 5-fold stimulation at 10⁻⁷ M. The dexamethasone stimulated 7α-OHDHA formation was inhibited by RU486 in a dose-dependent manner with suppression to basal levels at 10⁻⁶ M. Progesterone (10⁻⁷ M) had no effect on 7α-OHDHA formation suggesting that the dexamethasone stimulation was acting through the glucocorticoid receptor. Conversion of DHA to 7α-OHDHA was inhibited by ketoconazole and metyrapone. An inhibition of 70–80% was obtained with ketoconazole and 25–60% with metyrapone at concentrations of 10⁻⁵ M. Aminoglutethimide phosphate was less effective than either ketoconazole or metyrapone in inhibiting 7α-OHDHA formation with <30% inhibition at 10⁻⁵ M. These studies indicate that 7-hydroxylation provides an alternative pathway for the metabolism of DHA in peripheral tissues. This pathway, which is regulated by glucocorticoids, may influence the amount of DHA available for conversion to androstenedione and its subsequent aromatization to estrone. The biological role of the 7-oxygenated metabolites and their effects on other steroidogenic pathways have not been established.     

Influence of plant growth regulators, polyamines and glycerol interaction on growth and morphogenesis of carposporelings of Grateloupia cultured in vitro

The influence of the plant growth regulators 2,4-D, GA₃, BA and kinetin, and the polyamines putrescine, spermidine and spermine were tested on axenic in vitro cultures of carposporelings of Grateloupia doryphora. The auxin 2,4-D (10^-3 M) and the polyamine spermine (10^-6 M and 10^-3 M) induced a callus (disorganised cell mass that arose from the organised tissue of the carposporeling, as demonstrated by microscopic monitoring of the tissue). Putrescine and spermidine (10^-3 M) transformed the carposporelings into cell masses that produced shoots. BA (10^-3 M) and kinetin (10^-6 M and 10^-3 M) were inhibitory. In 10^-1 M glycerol-containing culture medium, which is known to induce the formation of morphogenic cell masses, the addition of GA₃ M) resulted in the inhibition of the morphogenesis (i.e. shoot emission) in the cell mass. The kinetin at 10^-6 M inhibited morphogenesis, whilst at 10^-3 M inhibited even the formation of the cell masses. The combination of glycerol (10^-1 M) and the auxin 2,4-D (10^-6 and 10^-3 M) or the polyamines putrescine, spermidine and spermine (10^-6 and 10^-3 M) resulted in a bigger size of the cell masses that led to a higher amount of shoots per cell mass than in glycerol alone. This revised version was published online in June 2006 with corrections to the Cover Date.     

A lesson learned from the H3.3K27M mutation found in pediatric glioma

Glioblastoma (GBM) is the most aggressive primary brain tumor in human. Recent studies on high-grade pediatric GBM have identified two recurrent mutations (K27M and G34R/V) in genes encoding histone H3 (H3F3A for H3.3 and HIST1H3B for H3.1).^1,2 The two histone H3 mutations are mutually exclusive and give rise to tumors in different brain compartments.³ Recently, we⁴ and others⁵ have shown that the histone H3 K27M mutation specifically altered the di- and tri-methylation of endogenous histone H3 at Lys27. Genome-wide studies using ChIP-seq on H3.3K27M patient samples indicate a global reduction of H3K27me3 on chromatin. Remarkably, we also found a dramatic enrichment of H3K27me3 and EZH2 (the catalytic subunit H3K27 methyltransferase) at hundreds of gene loci in H3.3K27M patient cells. Here, we discuss potential mechanisms whereby H3K27me3 is enriched at chromatin loci in cells expressing the H3.3K27M mutation and report effects of Lys-to-Met mutations of other well-studied lysine residues of histone H3.1/H3.3 and H4 on the corresponding endogenous lysine methylation. We suggest that mutation(s) on histones may be found in a variety of human diseases, and the expression of mutant histones may help to address the function of histone lysine methylation and possibly other modifications in mammalian cells.     

Mometasone and desloratadine additive effect on eosinophil survival and cytokine secretion from epithelial cells

Background

Although antihistamines and topical corticosteroids are used in combination to treat allergic rhinitis, their additive effect has not been yet demonstrated. The aim was investigate the antiinflammatory additive effect of mometasone and desloratadine on cytokine and sICAM-1 secretion by epithelial cells, and on eosinophil survival stimulated by human epithelial cells secretions from nasal mucosa and polyps.

Methods

Epithelial cells obtained from nasal mucosa or polyps were stimulated with 10% fetal bovine serum in presence of mometasone (10^-11M-10^-5M) with/without desloratadine (10^-5M). Cytokine and sICAM-1 concentrations in supernatants were measured by ELISA. Peripheral blood eosinophils were incubated during 4 days with epithelial cell secretions with (10^-11M-10^-5M) and/or desloratadine (10^-5M) and survival assessed by Trypan blue. Results are expressed as percentage (mean ± SEM) compared to control.

Results

Fetal bovine serum stimulated IL-6, IL-8, GM-CSF and sICAM-1 secretion. In mucosa and polyp epithelial cells, mometasone inhibited this induced secretion while desloratadine inhibited IL-6 and IL-8. The combination of 10^-5M desloratadine and 10^-9M mometasone reduced IL-6 secretion (48 ± 11%, p < 0.05) greater extent than mometasone alone (68 ± 10%) compared to control (100%). Epithelial cell secretions induced eosinophil survival from day 1 to 4, this effect being inhibited by mometasone. At day 4, the combination of mometasone (10^-11M) and desloratadine (10^-5M) provoked an increased inhibition of eosinophil survival induced by cell secretions (27 ± 5%, p < 0.01) than mometasone (44 ± 7%) or desloratadine (46 ± 7%) alone.

Conclusions

These results suggest that the combination of desloratadine and mometasone furoate have a greater antinflammatory effect in an in vitro model of eosinophil inflammation than those drugs administered alone.     

Fucosyl-GM1 — A ganglioside associated with small cell lung carcinomas

Characterization of monosialogangliosides of a small cell lung carcinoma showed a unique composition. The tumour contained G_M2 and Fucosyl-G_M1 (Fuc-G_M1) with 2-hydroxy fatty acids as major ganglioside components. Three out of four other small cell carcinomas analysed contained also Fuc-G_M1 as a characteristic ganglioside. Fuc-G_M1 is suggested to be a small-cell lung carcinoma associated ganglioside antigen.Nomenclature: The gangliosides have been designated according to Svennerholm [25] G_M3 II³NeuAc-Lac-Cer - G_D3 II³(NeuAc)₂-LacCer - G_M2 II³NeuAc-GgOse₃Cer - G_M1 II³NeuAc-GgOse₄Cer - Fuc-G_M1 FuclV²Neu-AcII³-GgOse₄Cer - 3-L_M1 IV³NeuAc-nLcOse₄Cer - 6-L_M1 IV⁶NeuAc-nLcOse₄Cer     

Organogenesis in callus derived from stem and leaf tissues of apple and cherry rootstocks

Callus formation from stem internodes of the apple rootstocks M.9, M.25, M.26, M.27 and the cherry rootstock Colt, and from pith of Nicotiana tabacum cv. Wisconsin 38 was initiated on 4 -naphthaleneacetic acid (NAA)-based media (2.0–10.0 mg1^-1). Transfer of callus to corresponding media lacking NAA allowed regeneration of shoots from callus of M.25, M.27, Colt and tobacco but not of M.9 and M.26. With M.25 phloroglucinol (PG) depressed regeneration from 30 to 10% and no regeneration was observed in cultures grown in the presence of casein hydrolysate (CH) and glutathione (GSH).Organogenesis was also obtained from leaf discs of M.27 employing 6-benzyl-aminopurine (BAP) at 5.0mg 1^-1 and 2,4-dichlorophenoxy acetic acid (2,4-D) at 0.1 mg1^-1. The regenerated shoots have been multiplied and rooted.Organogenesis also occurred in M.26 from small (1–2mm), green, compact embryoid-like structures derived from stem and leaf surfaces of excised axillary shoots. These structures differentiated into shoots at a low frequency (< 1%) on media containing BAP (1.0mg1^-1) and indole-3-butyric acid (IBA) (0.1 mg1^-1) and could also be micropropagated by subsequent axillary shoot proliferation.     

IgE-mediated 14C-serotonin release from rat mast cells modulated by morphine and endorphins

The formaldehyde method was used to examine the interaction of PGE₁ with morphine, β-endorphin and Met-enkephalin on rat mast cells by their effects on IgE-mediated ¹⁴C-serotonin release. PGE₁ (2×10^?8?2×10^?5 M) caused a dose-related inhibition of the mediator release 1 min after an antigen challenge, and morphine (3×10^?7?3×10^?5 M) reversed this PGE₁ effect dose-dependently and stereospecifically; naloxone (2×10^?4 M) antagonized this action of morphine. β-Endorphin (3×10^?7?10^?5 M) and Met-enkephalin (3×10^?6?10^?4 M) mimicked this morphine action dose-dependently and were antagonized by naloxone (2×10^?4 M). These results suggest that morphine and endorphins modulate immunological mediator release from rat mast cells through opioid receptors.     

Stimulatory effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells: Activation of aminoacyl-tRNA synthetase

The effect of genistein and daidzein on protein synthesis in osteoblastic MC3T3-E1 cells in vitro was investigated to determine a cellular mechanism by which the isoflavones stimulate bone formation. Cells were cultured for 48 h in -minimal essential medium containing either vehicle, genistein (10^–7–10^–5 M) or daidzein (10^–7–10^–5 M). The 5,500 g supernatant of cell homogenate was used for assay of protein synthesis with [³H]leucine incorporation in vitro. The culture with genistein or daidzein caused a significant elevation of protein synthesis in the cell homogenate. The effect of genistein (10^–5 M) or daidzein (10^–5 M) in elevating protein synthesis was significantly prevented, when cells were cultured for 48 h in a medium containing either actinomycin D (10^–7 M) or cycloheximide (10^–6 M) in the absence or presence of isoflavones. Moreover, when genistein (10^–7–10^–5 M) or daidzein (10^–6 and 10^–5 M) was added to the reaction mixture containing the cell homogenate obtained from osteoblastic cells cultured without isoflavone, protein synthesis was significantly raised. This increase was markedly blocked by the addition of cycloheximide (10^–7 M). In addition, [³H]leucyl-tRNA synthetase activity in the cytosol of osteoblastic cells was significantly increased by the addition of genistein (10^–6 and 10^–5 M) or daidzein (10^–5 M) into the enzyme reaction mixture. The present study demonstrates that genistein or daidzein can stimulate protein synthesis in osteoblastic MC3T3-E1 cells. The isoflavones may have a stimulatory effect on osteoblastic bone formation due to increasing protein synthesis.     

A serotonin sensitive guanylate cyclase associated with specific neurotransmitter binding sites on isolated synaptic membranes from mature rat brain.

Results from this study indicate that adult rat brain posesses guanylate cyclase activity sensitive to serotonin (5-HT) and localized in the synaptic plasma membrane. The enzyme appears to have multiple activation sites for 5-HT with specific activity maxima at the 5-HT concentrations of 5 × 10^?10M and 7 × 10^?8M respectively. The rates of guanosine-3′:5′-monophosphate (cyclic GMP) formation at these concentrations of 5-HT are, respectively, 170% and 307% above the endogenous or basal production rate of 2.7±0.3picomoles/minute/milligram of synaptosomal membrane protein. We have also been able to identify $\text{four}$ distinct types (Type #1, #2, #3, and #4) of high affinity, specific binding sites for 5-HT on isolated synaptosomal membranes from rat brain. Dissociation constants of 2.6 × 10^?10M, 2.5 × 10^?9M, 7.0 × 10^?9M, and 4.6 × 10^?8M, characterize the binding of 5-HT to our sites of Type #1 through Type #4 respectively. The specific, high affinity binding was saturated at 5-HT concentrations of 5 × 10^?10M for the Type #1 sites, 5 × 10^?9M for our Type #2 sites, 1 × 10^?8M for our Type #3 sites, and 7 × 10^?8M for our Type #4 sites. The 5-HT concentrations producing saturation of our specific binding sites of Type #1 and Type #4 are virtually identical to those that elicit the two maxima of 5-HT stimulated cyclic GMP production, indicating that a membrane-bound guanylase cyclase may be closely associated with certain 5-HT receptors and/or re-uptake sites.     

Binding of 1α,25‐dihydroxyvitamin D3 to annexin II: Effect of vitamin D metabolites and calcium

We have recently reported that annexin II serves as a membrane receptor for 1α,25‐(OH)₂D₃ and mediates the rapid effect of the hormone on intracellular calcium. The purpose of these studies was to characterize the binding of the hormone to annexin II, determine the specificity of binding, and assess the effect of calcium on binding. The binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to purified annexin II was inhibited by 1α,25‐(OH)₂D₃ in a concentration‐dependent manner. Binding of the radiolabeled ligand to annexin II was markedly diminished by 1α,25‐(OH)₂D₃ at 24 μM, 18 μM, and 12 μM and blunted by 6 μM and 3 μM. At a concentration of 12 μM, 1β,25‐(OH)₂D₃ also diminished the binding of [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate to annexin II, but cholecalciferol, 25‐(OH)D₃, and 24,25‐(OH)₂D₃ did not. Saturation analyses of the binding of [³H]‐1α,25‐(OH)₂D₃ to purified annexin II showed a K_D of 5.5 × 10⁻⁹ M, whereas [³H]‐1β,25‐(OH)₂D₃ exhibited a K_D of 6.0 × 10⁻⁹ M. Calcium, which binds to the carboxy terminal domain of annexin II, had a concentration‐dependent effect on [¹⁴C]‐1α,25‐(OH)₂D₃ bromoacetate binding to annexin II, with 600 nM calcium being able to inhibit binding of the radiolabeled analog. The inhibitory effect of calcium was prevented by EDTA. Homocysteine, which binds to the amino terminal domain of annexin II, had no effect on the binding of the bromoacetate analog to the protein. The data indicate that 1α,25‐(OH)₂D₃ binding to annexin II is specific and suggest that the binding site may be located on the carboxy terminal domain of the protein. The ability of 1β,25‐(OH)₂D₃ to inhibit the binding of [¹⁴C]‐1α,25(OH)₂D₃ bromoacetate to annexin II provides a biochemical explanation for the ability of the 1β‐epimer to inhibit the rapid actions of the hormone in vitro. J. Cell. Biochem. 80:259–265, 2000. © 2000 Wiley‐Liss, Inc.     

Yolk polypeptide gene expression in Minute Drosophila females

Minutes have been considered for some time to be mutant at the sites of synthesis of some components of the protein synthetic apparatus. To study the hypothetical relationship between Minutes and suboptimal translation, a group of abundant proteins, the yolk polypeptides, was assayed in outcrossed females bearing M(3)w, M(3)h ^y , or M(1)n mutations. Recently emerged Minute females contained a lower amount of yolk polypeptides, in both ovarian and nonovarian tissues, than their non-Minute sisters. This low level correlated with the lower abundance of cytoplasmic RNA in Minutes compared to control females. By 1 week of age, both M(3)w and their non-Minute sibs contained the same amount of yolk polypeptides and the corresponding mRNA. The double heterozygote, ap ⁴/+;M(3)w/+, did not differ in yolk polypeptide content from control flies. M(3)w females demonstrated reduced fecundity during the period of low yolk polypeptide content but gradually increased egg deposition as yolk polypeptide levels rose. These results suggest that the low protein levels are due to the slower maturation of M(3)w, and not to less efficient translation machinery.This work was supported by the NSERC (Canada) and a Queen's University ARC grant.     

Tris(pyrazol-1-yl)borate and tris(pyrazol-1-yl)methane: A DFT study of their different binding capability toward Ag(I) and Cu(I) cations

Density functional theory has been used to study the electronic structure of [M(tp)] and [M(tpm)]⁺ conformers (M = Cu, Ag; tp = tris(pyrazol-1-yl)borate anion, tpm = tris(pyrazol-1-yl)methane) and the energetics of their interconversions. Results for the free tp ligand are similar to those of tpm [M. Casarin, D. Forrer, F. Garau, L. Pandolfo, C. Pettinari, A. Vittadini, J. Phys. Chem. A 112 (2008) 6723], indicating an intrinsic instability of the tripodal conformation (κ³-like). This points out that, though frequently observed, the κ³-coordinative mode is unlikely to be directly achieved through the interaction of M(I) with the κ³-like tp/tpm conformer. Analogously to the [M(tpm)]⁺ molecular ions, the energy barrier for the κ²-[M(tp)] → κ³-[M(tp)] conversion is computed to be negligible. Though κⁿ-[M(tp)] and κⁿ-[M(tpm)]⁺ (n = 1, 2, 3) have similar metal-ligand covalent interactions, the negative charge associated to the tp ligand makes the M-tp bonding stronger.     

Determination of glucose,urea and penicillin using enzyme-pH-electrodes

Conventional hydrogen ion glas electrodes have been used for the preparation of enzyme-pH-electrodes by either entrapping the enzymes within polyacrylamide gels around the electrode or as liquid layer trapped within a cellophane membrane. The enzymes were glucose oxidase, urase and penicillinase.The pH response to glucose concentration was about linear within 10^?1–10^?3 M glucose and for urea linear within 5·10^t—–5·10^?5M. The pH response to penicillin was about linear in the range from 10^?3–10^?2 M resulting in a pH shift of 1.4 units; reproduceable pH response was obtained down to concentrations of 3·10^?5 M.Studies as to the effect of buffer using an urease–pH-electrode showed a buffer concentration of 10^?2 M a substantial shift of about one pH-unit in the range of 10^?4 to 10^?2 M urea. Both urease- and penicillinase–pH-electrodes were tested as to stability showing no decrease in pH response except at high substrate concentration (1·10^?2 M) over a period of 2–3 weeks kept at room temperature.     

The effects of prostaglandins E2 and F2α on synaptosomal accumulation and release of H-norepinephrine

Prostaglandins (PG) of both the E and F series may serve as modulators of norepinephrine (NE) release from peripheral sympathetic neurons. We have studied the effects of PGE₂ and PGF_2α on the accumulation and release of ³H-NE in the CNS using synaptosomes isolated from rat hypothalami.The release of ³H-NE from synaptosomes superfused with Krebs-Ringer bicarbonate buffer was multiphasic with an initial fast release phase followed by a slower release. Raising KC1 concentration of the superfusion medium to 56mM during the slow release phase is known to stimulate ³H-NE release. PGE₂ (1 × 10⁻⁶M) attenuated ³H-NE release during the fast phase and reduced the amount of ³H-NE released due to KC1 stimulation. At lower concentrations of PGE₂ there was no change in the release profile. PGF_2α was without effect on ³H-NE release at all concentrations tested.The accumulation of ³H-NE was significantly diminished by PGE₂ at a concentration of 1 × 10⁻⁶M, while a lower concentration (1 × 10⁻⁷M) was ineffective. PGF_2α had no effect on ³H-NE accumulation at all concentrations investigated.     

Thyroid hormone: Aldosterone antagonism in cultured epithelial cells

Thyroid hormone (T₃) has been demonstrated to inhibit the action of aldosterone on sodium transport in toad urinary bladder and rat kidney. We have exammined the effect of T₃ on aldosterone action and specific nuclear binding in cultured epithelial cells derived from toad urinary bladder. In cell line TB6-C, addition of 5·10⁻⁸ M T₃ to culture media for up to 3 days results in no change in short-circuit current or transepithelial resistance. This concentration of T₃ completely inhibits the maximal increase in short-circuit current in response to 1·10⁻⁷ M aldosterone. The inhibition can be demonstrated with 18 h preincubation or with simultaneous addition of T₃ and aldosterone. The half-maximal concentration for the inhibition of the aldosterone effect is approx. 5·10⁻⁹ M T₃. T₃ has no effect on cyclic AMP-stimulated short-circuit current in these cells. The effect of T₃ on nuclear binding of [³H]aldosterone was examined using a filtration assay with data analysis by at least-squares curve-fitting program. Best fit was obtained with a model for two binding sites. The dissociation constants for the binding were K′_d1 = (0.82 ± 0.36)·10⁻¹⁰ M and K′_d2 = (3.2±0.60)·10⁻⁸ M.The half-maximal concentration for aldosterone-stimulated sodium transport in these cells is approx. 1·10⁻⁸ M. Analysis of nuclear aldosterone binding in cells preincubated for 18 h with 5·10⁻⁸ M T₃ showed a K′_d1 = (0.15 ± 0.10)·10⁻¹⁰ M and K′_d2 = (3.5 ± 0.10)·10⁻⁸ M. We conclude that T₃ i action of aldosterone on sodium transport at a site after receptor binding in the nucleus.     

Plasmid-controlled variation in the content of methylated bases in single-stranded DNA bacteriophages M13 and fd

The N⁶-methyladenine and 5-methylcytosine contents in the DNA of bacteriophages M13 and fd have been analyzed. The results are summarized as follows. (1) After growth in bacteria harboring the N-3ft^? drug resistance-factor, fd and M13 are observed to contain approximately 1 to 2 more 5-methylcytosine residues per DNA molecule than after growth in the parental drug-sensitive host; no effect on the N⁶-methyladenine content is produced by the plasmid. (2) After growth in bacteria harboring P1 prophage, fd and M13 are observed to contain approximately 2 to 3 more N⁶-methyladenine residues per DNA molecule than after growth in the parental P1-sensitive host; no apparent effect on the 5-methylcytosine content was produced by the P1 plasmid. (3) In agreement with others, fd carrying B-host specificity (fd·B) is observed to contain 2 more N⁶-methyladenine residues/DNA molecule than fd·K.     

A T Cell-Inducing Influenza Vaccine for the Elderly: Safety and Immunogenicity of MVA-NP+M1 in Adults Aged over 50 Years

Background

Current influenza vaccines have reduced immunogenicity and are of uncertain efficacy in older adults. We assessed the safety and immunogenicity of MVA-NP+M1, a viral-vectored influenza vaccine designed to boost memory T cell responses, in a group of older adults.

Methods

Thirty volunteers (aged 50–85) received a single intramuscular injection of MVA-NP+M1 at a dose of 1·5×10⁸ plaque forming units (pfu). Safety and immunogenicity were assessed over a period of one year. The frequency of T cells specific for nucleoprotein (NP) and matrix protein 1 (M1) was determined by interferon-gamma (IFN-γ) ELISpot, and their phenotypic and functional properties were characterized by polychromatic flow cytometry. In a subset of M1-specific CD8⁺ T cells, T cell receptor (TCR) gene expression was evaluated using an unbiased molecular approach.

Results

Vaccination with MVA-NP+M1 was well tolerated. ELISpot responses were boosted significantly above baseline following vaccination. Increases were detected in both CD4⁺ and CD8⁺ T cell subsets. Clonality studies indicated that MVA-NP+M1 expanded pre-existing memory CD8⁺ T cells, which displayed a predominant CD27⁺CD45RO⁺CD57⁻CCR7⁻ phenotype both before and after vaccination.

Conclusions

MVA-NP+M1 is safe and immunogenic in older adults. Unlike seasonal influenza vaccination, the immune responses generated by MVA-NP+M1 are similar between younger and older individuals. A T cell-inducing vaccine such as MVA-NP+M1 may therefore provide a way to circumvent the immunosenescence that impairs routine influenza vaccination.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00942071","term_id":"NCT00942071"}}NCT00942071