IL-2, -7, and -15, but not thymic stromal lymphopoeitin, redundantly govern CD4+Foxp3+ regulatory T cell development

Common gamma chain (gammac)-receptor dependent cytokines are required for regulatory T cell (Treg) development as gammac(-/-) mice lack Tregs. However, it is unclear which gammac-dependent cytokines are involved in this process. Furthermore, thymic stromal lymphopoietin (TSLP) has also been suggested to play a role in Treg development. In this study, we demonstrate that developing CD4(+)Foxp3(+) Tregs in the thymus express the IL-2Rbeta, IL-4Ralpha, IL-7Ralpha, IL-15Ralpha, and IL-21Ralpha chains, but not the IL9Ralpha or TSLPRalpha chains. Moreover, only IL-2, and to a much lesser degree IL-7 and IL-15, were capable of transducing signals in CD4(+)Foxp3(+) Tregs as determined by monitoring STAT5 phosphorylation. Likewise, IL-2, IL-7, and IL-15, but not TSLP, were capable of inducing the conversion of CD4(+)CD25(+)Foxp3(-) thymic Treg progenitors into CD4(+)Foxp3(+) mature Tregs in vitro. To examine this issue in more detail, we generated IL-2Rbeta(-/-) x IL-7Ralpha(-/-) and IL-2Rbeta(-/-) x IL-4Ralpha(-/-) mice. We found that IL-2Rbeta(-/-) x IL-7Ralpha(-/-) mice were devoid of Tregs thereby recapitulating the phenotype observed in gammac(-/-) mice; in contrast, the phenotype observed in IL-2Rbeta(-/-) x IL-4Ralpha(-/-) mice was comparable to that seen in IL-2Rbeta(-/-) mice. Finally, we observed that Tregs from both IL-2(-/-) and IL-2Rbeta(-/-) mice show elevated expression of IL-7Ralpha and IL-15Ralpha chains. Addition of IL-2 to Tregs from IL-2(-/-) mice led to rapid down-regulation of these receptors. Taken together, our results demonstrate that IL-2 plays the predominant role in Treg development, but that in its absence the IL-7Ralpha and IL-15Ralpha chains are up-regulated and allow for IL-7 and IL-15 to partially compensate for loss of IL-2.     

B7-2 regulates survival, phenotype, and function of APCs

The absence of B7-2-mediated costimulation protects NOD mice from the development of diabetes. Although the effects of B7-2 on T cell priming are well known, its impact on the function of APCs is not fully elucidated. We tested APC function and survival in mice lacking B7-2. A significant reduction in the phagocytic ability was observed in both splenic and pancreatic lymph node-associated dendritic cells (DCs) in B7-2 knockout (KO) mice. DCs from B7-2KO mice exhibited enhanced susceptibility to death, which was reflected by their reduced total cell numbers. Phenotypic analysis of APCs in B7-2KO mice revealed a significantly decreased proportion of CD8alpha+CD205+ DCs. Interestingly, an enhanced proportion of B7-H1+ and B7-DC+ DCs were observed in B7-2KO mice. Lastly, we found that B7-2 deficiency significantly diminished the PKC-epsilon response in APCs upon CD28-Ig stimulation. In conclusion our data suggests that B7-2 promotes the generation of a mature APC repertoire and promotes APC function and survival.     

Dysregulation of the inflammatory response to the parasite, Toxoplasma gondii, in P2X7 receptor-deficient mice

The P2X(7) receptor (P2X(7)R) is a two transmembrane receptor that is highly expressed on the surface of immune cells. Loss of function polymorphisms in this receptor have been linked to increased susceptibility to intracellular pathogens. P2X(7)R gene knockout (P2X(7)R(-/-); on a C57Bl/6J background), C57Bl/6J and BALB/c mice were infected with the avirulent ME49 strain of the intracellular parasite, Toxoplasma gondii, and susceptibility determined by monitoring weight loss. P2X(7)R(-/-) mice lost significantly more weight than C57Bl/6J mice from day 8p.i. C57Bl/6J, in turn, lost significantly more weight than BALB/c mice. Thus, by day 10p.i., P2X(7)R(-/-) mice had lost 5.7 ± 0.7% of their weight versus 2.4 ± 0.6% for C57Bl/6J mice, whereas BALB/c mice had gained 1.9 ± 0.5%; by day 12p.i., P2X(7)R(-/-) mice had lost 15.1±0.6%, C57Bl/6J had lost 10.1±0.8% and BALB/c had lost 4.8 ± 0.8% of their weight. Neither parasite burden nor liver pathology was greater in the P2X(7)R(-/-) mice than in C57Bl/6J mice but BALB/c mice had significantly smaller numbers of parasites and less pathology in their livers than these strains. Absence of the P2X(7) receptor did not affect IFN-γ, IL-12, IL-1β, monocyte chemoattractant protein-1 (MCP-1) or TNF production. However, both P2X(7)R(-/-) and C57Bl/6J mice produced more IL-1β and TNF than BALB/c mice. There was one important point of differentiation between the P2X(7)R(-/-) and C57Bl/6J mice, namely the significantly enhanced and prolonged production of nitric oxide, accompanied by delayed production of IL-10 in the P2X(7)R-deficient mice.     

Synthesis and preliminary biological evaluation of (2S,1'R,2'S)- and (2S,1'S,2'R)-2-(2'-phosphonocyclopropyl)glycines, two novel conformationally constrained l-AP4 analogues

Two novel constrained l-AP4 analogues, (2S,1'R,2'S)- and (2S,1'S,2'R)-2-(2'-phosphonocyclopropyl)glycines (7) and (8), were synthesized and evaluated as mGluR ligands. Compound 7 showed to be a group III mGluRs agonist with micromolar activity.     

Synthesis of (4R,6S,7R)-7-hydroxy-4,6-dimethyl-3-nonanone and (3R,5S,6R)- 6-hydroxy-3,5-dimethyl-2-octanone,the pheromone components of the bostrychid beetle,Dinoderus bifoveolatus

(4R,6S,7R)-7-Hydroxy-4,6-dimethyl-3-nonanone and (3R,5S,6R)-6-hydroxy-3,5-dimethyl-2-octanone, the pheromone components of the bostrychid beetle, Dinoderus bifoveolatus, as well as their (4R,6S,7S)- and (3R,5S,6S)-isomers were synthesized from (2R,4S,5R)- and (2R,4S,5S)-2,4-dimethyl-5-heptanolide, respectively.     

Determination of 7-methylguanine, N2,N2-dimethylguanosine, and pseudouridine in ultrafiltrated serum of healthy adults by high-performance liquid chromatography

7-Methylguanine (m7Gua), N2,N2-dimethylguanosine (m2(2)Guo), and pseudouridine (psi) are degradation products from RNA turnover and can be used as markers for the whole-body turnover of mRNA-cap, tRNA, and rRNA (in healthy individuals, urinary excretion of these catabolites follows a regular pattern; the relative molar ratio of psi:m7Gua:m2(2)Guo is approximately 100:19:6). HPLC methods were developed to measure serum concentrations of these RNA catabolites after deproteinization of the samples by ultrafiltration through microcollodion bags with a nominal exclusion Mr of 12,400. For healthy adults the following values (mean +/- SD) were found: psi, 2760 +/- 460 nmol/liter (n = 10); m7Gua, 129.7 +/- 24.0 nmol/liter (n = 13); m2(2)Guo, 31.0 +/- 3.7 nmol/liter (n = 9). The relative molar ratio of these substances in serum derived from our data is approximately 100:4.7:1.1. 7-Methylguanosine (m7Guo) added to serum is to a large extent converted to the corresponding free base, m7Gua, the form which is excreted in urine.     

Biochemical and immunological demonstration of prostaglandin D2, E2, and F2 alpha formation from prostaglandin H2 by various rat glutathione S-transferase isozymes

Glutathione S-transferase isozymes purified from normal rat liver (1-1, 1-2, 2-2, 3-3, 3-4, and 4-4), liver with hyperplastic nodules (7-7), brain (Yn1Yn1), and testis (Yn1Yn2) all had prostaglandin H2-converting activity. The prostaglandin H2 E-isomerase activity was high in 1-1 (1400 nmol/min/mg protein), 1-2 (1170), and 2-2 (420), moderate in 3-3, 3-4, 4-4, Yn1Yn1, and Yn1Yn2 (52-100), and weak but significant in 7-7 (33). The prostaglandin H2 D-isomerase activity was relatively high in 1-1 (170) and 1-2 (200), moderate in 2-2 (60) and Yn1Yn2 (43), and weak but marked in 3-3 (16), 4-4 (16), and 7-7 (14). The prostaglandin H2 F-reductase activity was remarkable in 1-1 (1250), 1-2 (920), and 2-2 (390), and weakly detected in 3-3 (24), 4-4 (28), and 7-7 (14). Glutathione was absolutely required for these prostaglandin H2-converting reactions, and its stoichiometric consumption was associated with F-reductase activity but not E- and D-isomerase activities. The Km values for glutathione and prostaglandin H2 were about 200 and 10-40 microM, respectively. By immunoabsorption analyses with various antibodies specific for each isozyme, we examined its contribution to the formation of prostaglandins D2, E2, and F2 alpha from prostaglandin H2 in 100,000g supernatants of rat liver, kidney, and testis. In the liver, about 90% of the F-reductase activity (9.8 nmol/min/mg protein) was shown to be catalyzed by the 1-2 group of isozymes. The E-isomerase activity (16.5) was catalyzed about 60 and 40% by the 1-2 and 3-4 groups, respectively; and the D-isomerase activity (3.7) was catalyzed by the 1-2 group (50%) and the 3-4 group and Yn1Yn2 (15-25%). In the kidney, the E-isomerase activity (9.4) was catalyzed by 1-1, 1-2 (40%), 2-2, 3-4 group, and 7-7 (10-20%). The F-reductase activity (3.3) was mostly catalyzed by the 1-2 group (75%). In the testis, the E-isomerase activity (3.9) was catalyzed by the 1-2 group (20-30%), the 3-4 group, and Yn1Yn2 (30-60%).     

Expression of B7 molecules in recipient, not donor, mice determines the survival of cardiac allografts.

Blockade of the CD28/CTLA4/B7 costimulatory pathway using CTLA4-Ig has great therapeutic potential, and has been shown to prolong allograft survival in a variety of animal models. To gain further insight into the mechanism by which costimulatory blockade prevents allograft rejection, we studied cardiac allograft survival in the complete absence of B7 costimulation using mice lacking B7-1 and B7-2 (B7-1/B7-2-/- mice). To determine the role of B7 on donor vs recipient cells, we used B7-1/B7-2-/- mice as either donors or recipients of allografts. Wild-type (WT) recipients acutely reject fully allogeneic hearts from both WT and B7-1/B7-2-/- mice. In contrast, B7-1/B7-2-/- recipients allow long-term survival of grafts from both WT and B7-1/B7-2-/- mice, with minimal histologic evidence of either acute or chronic rejection in grafts harvested after 90 days. The B7-1/B7-2-/- mice acutely reject B7-1/B7-2-/- allografts if CD28 stimulation is restored by the administration of Ab to CD28 and can mount an alloresponse in mixed lymphocyte reactions. Therefore, B7-1/B7-2-/- mice are capable of generating alloresponses both in vivo and in vitro. Our results demonstrate that in the alloresponse to mouse heterotopic cardiac transplantation, B7 molecules on recipient cells rather than donor cells provide the critical costimulatory signals. The indefinite survival of allografts into B7-1/B7-2-/- recipients further shows that the absence of B7 costimulation alone is sufficient to prevent rejection.     

Memory Th2 effector cells can develop in the absence of B7-1/B7-2, CD28 interactions,and effector Th cells after priming with an intestinal nematode parasite

B7-1/B7-2 interactions are required for many Th2-cell mediated primary immune responses including the response that follows infection with the intestinal nematode parasite, Heligmosomoides polygyrus. However, few studies have examined the role of B7-1/B7-2/CD28 interactions in the development of a Th2 memory immune response. We examined the development of the memory Th2 response to H. polygyrus in BALB/c mice deficient in both B7-1 and B7-2 (B7-1/B7-2(-/-)) and in BALB/c mice deficient in CD28 (CD28(-/-)). Following primary inoculation with H. polygyrus, adult worms in the gut were cleared with an anti-helminthic drug and mice were subsequently challenge-inoculated with H. polygyrus larvae. The memory Th2 response is readily distinguished by its inhibitory effect on adult worm maturation, resulting in marked reductions in adult worm egg production that are not observed during the primary immune response. Following H. polygyrus challenge inoculation, comparable decreases in egg production and similar increases in mesenteric lymph node cell IL-4 production were observed in B7-1/B7-2(-/-) and B7-1/B7-2(+/+) mice. However, elevations in total serum IgG1 and IgE were reduced, while increases in serum Ag-specific IgG1 and IgE and germinal center formation were blocked in H. polygyrus-challenged B7-1/B7-2(-/-) mice. In contrast, in H. polygyrus-challenged CD28(-/-) mice, marked elevations in Ag-specific IgG1 and IgE and increased germinal center formation were observed. The results of these studies demonstrate that effector Th2 memory cells that produce IL-4 and mediate host defense can develop when B7-1/B7-2 interactions, and associated effector Th2 cell development, are blocked during priming. However, humoral immunity is impaired and differentially affected in B7-1/B7-2(-/-) mice and CD28(-/-) mice following H. polygyrus challenge.     

Synthesis,characterization, anticancer,antimicrobial and carbonic anhydrase inhibition profiles of novel (3aR,4S,7R,7aS)-2-(4-((E)-3-(3-aryl)acryloyl) phenyl)-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoindole-1,3(2H)-dione derivatives

In the present study, a series of new hybrid compounds containing chalcone and methanoisoindole units 7a-n ((3aR,4S,7R,7aS)-2-(4-((E)-3-(3-aryl)acryloyl) phenyl)-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoindole-1,3(2H)-dione) were synthesized, characterized and investigated for their anticancer activity against C6 gliocarcinoma cell in rats, and antimicrobial activity against some human pathogen microorganisms. The compounds 7e, 7h, 7j, 7k, 7L and 7n showed very high anticancer activity with the inhibition range of 80.51–97.02% compared to 5-FU. Some of the compounds exhibited anti-microbial activity. Also, they evaluated for inhibition effects against human carbonic anhydrase I, and II isoenzymes (hCA I and II) with Ki values in the range of 405.26–635.68 pM for hCA I, and 245.40–489.60 pM for hCA II, respectively. These results demonstrated that 3aR,4S,7R,7aS)-2-(4-((E)-3-(3-aryl)acryloyl)phenyl)-3a,4,7,7a-tetrahydro-1H-4,7-methanoisoindole-1,3(2H)-dione derivatives could be used in different biomedical applications.     

Interactions between estrogen, tamoxifen, octylphenol, and two polychlorinated biphenyls in murine splenocytes.

Prior exposure of cultured murine splenocytes to 17beta-estradiol (E) protects them from the membrane disrupting effects of the xenoestrogen 4-tert-octylphenol (OP). Using splenocytes isolated from male Balb/c mice, we tested whether (a) the xenoestrogen, 2', 3', 4', 5'-tetrachloro-4-biphenylol (PCB-OH), or the polychlorinated biphenyl, 3, 3', 4, 4'-tetrachlorobiphenyl (PCB 77), which displays both estrogenic and anti-estrogenic actions, would compromise the membrane integrity of the cells and (b) E or tamoxifen (TX), another ligand for the E receptor, would protect the membranes of cells exposed to the agents. We also examined possible interactions between OP, PCB-OH, and PCB 77 on the cells. Splenocytes were cultured for 24 hr. Concentrations of OP (10(-5)-10(-9) M), PCB-OH (10(-6)-10(-16) M), or PCB 77 (10(-8)-10(-12) M) significantly compromised the membrane integrity of the cultured splenocytes in a dose response manner. Concentrations of E as high as 10(-5) M or TX as high as 10(-7) M were without effect. Incubation of splenocytes in medium containing E or TX at 10(-7) M for 2 hr prior to the subsequent addition of either OP, PCB-OH or PCB 77 (final concentrations of 10(-7), 10(-7), or 10(-8) M, respectively) blocked the membrane disrupting effects. Incubation of splenocytes in medium containing 10(-7) M E starting 2 hr after the addition of OP or PCB 77 or incubation of splenocytes in medium containing 10(-7) M TX starting 2 hr after the addition of OP or PCB-OH did not block the damaging effects of OP, PCB 77, or PCB-OH on the cell membranes. No interactions were observed when various combinations of OP, PCB-OH, or PCB 77 were used. These data suggest that: (a) TX acts like E in this system, (b) a prior response of splenocytes to E or TX can protect them from the potential cytotoxic effects of OP, PCB-OH, or PCB 77; and, (c) OP, PCB-OH, and PCB 77 were not additive in their actions.     

Structure-based design, synthesis, and biological evaluation of novel 1,4-diazepines as HDM2 antagonists

Crystallographic analysis of ligands bound to HDM2 suggested that 7-substituted 1,4-diazepine-2,5-diones could mimic the alpha-helix of p53 peptide and may represent a promising scaffold to develop HDM2-p53 antagonists. To verify this hypothesis, we synthesized and biologically evaluated 5-[(3S)-3-(4-chlorophenyl)-4-[(R)-1-(4-chlorophenyl)ethyl]-2,5-dioxo-7-phenyl-1,4-diazepin-1-yl]valeric acid (10) and 5-[(3S)-7-(2-bromophenyl)-3-(4-chlorophenyl)-4-[(R)-1-(4-chlorophenyl)ethyl]-2,5-dioxo-1,4-diazepin-1-yl]valeric acid (11). Preliminary in vitro testing shows that 10 and 11 substantially antagonize the binding between HDM2 and p53 with an IC(50) of 13 and 3.6 microM, respectively, validating the modeling predictions. Taken together with the high cell permeability of diazepine 11 determined in CACO-2 cells, these results suggest that 1,4-diazepine-2,5-diones may be useful in the treatment of certain cancers.     

Design,synthesis, and biological evaluation of a series of beta-lactam-based prodrugs

By use of pro-dual-drug concept the synthesis of 6-beta-[(R)-2-(clavaminio-9-N-yl)-2-(4-hydroxyphenylacetamido)]penicillanic acid (10), 6-beta-[(R)-2-(amino)-2-(4-(clavulano-9-O-yl)phenylacetamido)]penicillanic acid (13), (Z)-4-[2-(amoxycillin-4-O-yl)ethylidene]-2-(clavulano-9-O-yl)-3-methoxy-Delta(alpha,beta)-butenolide (19), and 3-[(amoxicillin-4-O-yl)methyl]-7-(phenoxyacetamido)-(1-oxo)-3-cephem-4-carboxylic acid (23) was accomplished. Unlike penicillin G, ampicillin, or amoxicillin, these four heretofore undescribed compounds 10, 13, 19, and 23 showed notable activity against beta-lactamase (betaL) producing microorganisms, Staphylococcus aureus A9606, S. aureus A15091, S. aureus A20309, S. aureus 95, Escherichia coli A9675, E. coli A21223, E. coli 27C7, Pseudomonas aeruginosa 18S-H, and Klebsiella pneumoniae A20634 TEM. In comparison with amoxicillin (9), alpha-amino-substituted compound 10 and butenolide derivative 19 showed a broadened spectrum of antibacterial activity; yet they were found to be less active than 13 and 23. Like clavulanic acid (7) or cephalosporin-1-oxide (21), the newly synthesized compounds 10, 13, 15, 16, 19, or 23 functioned as potent inhibitors of various bacterial betaLs.     

Aldose reductase inhibitors: flavonoids, alkaloids, acetophenones, benzophenones, and spirohydantoins of chroman

The inhibitory activity of various compounds, including 12 flavonoids, 10 alkaloids, 15 benzophenones, 5 acetophenones, and 7 spirohydantoins of chroman, was tested on rabbit lens aldose reductase, an enzyme involved in complications of diabetes. Almost all compounds tested were found to inhibit the enzyme at low concentrations (10(-5) M). The most potent inhibitor was 2R,4S-6-chloro-2-methylspiro(chroman-4,4'-imidazo-lidine+ ++)-2',5'-dione with an I50 value of 4.7 x 10(-8) M; other spirohydantoins showed similar potency. Polyhydroxybenzophenones were also potent inhibitors with an I50 value of about 10(-7) M. The possible structure-inhibitory activity relationships of the compounds tested are discussed.     

Energy flux, more so than energy balance, protein intake, or fitness level, influences insulin-like growth factor-I system responses during 7 days of increased physical activity.

The purpose of this study was to determine the impact of dietary factors and exercise-associated factors on the response of IGF-I and its binding proteins (IGFBPs) during a period of increased physical activity. Twenty-nine men completed a 4-day (days 1-4) baseline period of a controlled energy balanced diet while maintaining their normal physical activity level followed by 7 days (days 5-11) of a 1,000 kcal/day increase in physical activity above their normal activity levels. Two subject groups, one sedentary (Sed, mean Vo(2peak): 39 mlxkg(-1)xmin(-1), n = 7) and one fit (FIT1, mean Vo(2peak): 56 ml.kg(-1)xmin(-1), n = 8) increased energy intake to maintain energy balance throughout the 7-day intervention. In two other fit subject groups (FIT2, n = 7 and FIT3, n = 7), energy intake remained at baseline resulting in a 1,000 kcal/day exercise-induced energy deficit. Of these, FIT2 received an adequate protein diet (0.9 g/kg), and FIT3 received a high-protein diet (1.8 g/kg). For all four groups, IGF-I, IGFBP-3, and the acid labile subunit (ALS) were significantly decreased by day 11 (27 +/- 4%, 10 +/- 2%, and 19 +/- 4%, respectively) and IGFBP-2 significantly increased by 49 +/- 21% following day 3. IGFBP-1 significantly increased only in the two negative energy balance groups, FIT2 (38 +/- 6%) and FIT3 (46 +/- 8%). Differences in initial fitness level and dietary protein intake did not alter the IGF-I system response to an acute increase in physical activity. Decreases in IGF-I were observed during a moderate increase in physical activity despite maintaining energy balance, suggesting that currently unexplained exercise-associated mechanisms, such as increased energy flux, regulate IGF-I independent of energy deficit.     

Synthesis, radiolabeling, and preliminary biological evaluation of [3H]-1-[(S)-N,O-bis-(isoquinolinesulfonyl)-N-methyl-tyrosyl]-4-(o-tolyl)-piperazine, a potent antagonist radioligand for the P2X7 receptor

The design, synthesis, and preliminary biological evaluation of the first potent radioligand antagonist for the P2X(7) receptor, named [(3)H]-1-[(S)-N,O-bis-(isoquinolinesulfonyl)-N-methyl-tyrosyl]-4-(o-tolyl)-piperazine (compound 13), are reported. This compound bound to human P2X(7) receptors expressed in HEK transfected cells with K(D) and B(max) value of 3.46+/-0.1 nM and 727+/-73 fmol/mg of protein, respectively. The high affinity and facile labeling makes it a promising radioligand for a further characterization of P2X(7) receptor subtype.     

Modification of DNA with octadiynyl side chains: synthesis, base pairing, and formation of fluorescent coumarin dye conjugates of four nucleobases by the alkyne--azide "click" reaction

Oligonucleotides incorporating 5-(octa-1,7-diynyl)-2'-deoxycytidine 1a, 5-(octa-1,7-diynyl)-2'-deoxyuridine 2a and 7-deaza-7-(octa-1,7-diynyl)-2'-deoxyguanosine 3a, 7-deaza-7-(octa-1,7-diynyl)-2'-deoxyadenosine 4a were prepared. For this, the phosphoramidites 7, 10, and 13 were synthesized and employed in solid-phase oligonucleotide synthesis. The octa-1,7-diynyl nucleosides 1a- 4a were obtained from their corresponding iodo derivatives using the palladium-assisted Sonogashira cross-coupling reaction. The Tm values demonstrated that DNA duplexes containing octa-1,7-diynyl nucleosides show a positive influence on the DNA duplex stability when they are introduced at the 5-position of pyrimidines or at the 7-position of 7-deazapurines. The terminal alkyne residue of oligonucleotides were selectively conjugated to the azide residue of the nonfluorescent 3-azido-7-hydroxycoumarin ( 38) using the protocol of copper(I)-catalyzed [3 + 2] Huisgen--Sharpless--Meldal cycloaddition "click chemistry" resulting in the formation of strongly fluorescent 1,2,3-triazole conjugates. The fluorescence properties of oligonucleotides with covalently linked coumarin--nucleobase assemblies were investigated. Among the four modified bases, the 7-deazapurines show stronger fluorescence quenching than that of pyrimidines.     

Aging, opioid-receptor agonists and antagonists, and the vestibulosympathetic reflex in humans.

Animal studies indicate that opioids inhibit the firing rate of vestibular neurons, which are important in mediating the vestibulosympathetic reflex. Furthermore, this inhibition appears to be greater in more mature rats. In the present study, we tested the hypotheses that opioids inhibit the vestibulosympathetic reflex in humans and that endogenous opioids contribute to the age-related impairment of the vestibulosympathetic reflex. These hypotheses were tested by measuring muscle sympathetic nerve activity (MSNA), arterial blood pressure, and heart rate responses to otolith organ engagement during head-down rotation (HDR) in young (24 +/- 2 yr old) and older (63 +/- 2 yr) subjects before and after administration of either an opioid-receptor antagonist (16 mg naloxone in 9 young and 8 older subjects) or an opioid-receptor agonist (60 mg codeine in 7 young and 7 older subjects). Naloxone did not augment the reflex increase in MSNA during HDR in young (Delta7 +/- 2 vs. Delta4 +/- 2 bursts/min and Delta81 +/- 23 vs. Delta60 +/- 24% change in burst frequency and total MSNA before and after naloxone, respectively) or older subjects (Delta2 +/- 2 vs. Delta1 +/- 2 burst/min and Delta8 +/- 7 vs. Delta8 +/- 9% before and after naloxone). Similarly, codeine did not attenuate the increase in MSNA during HDR in young (Delta8 +/- 1 vs. Delta7 +/- 2 bursts/min and Delta53 +/- 4 vs. Delta64 +/- 16% before and after codeine) or older subjects (Delta6 +/- 4 vs. Delta3 +/- 3 bursts/min and Delta38 +/- 21 vs. Delta33 +/- 20%). Mean arterial blood pressure and heart rate responses to HDR were not altered by either naloxone or codeine. These data do not provide experimental support for the concept that opioids modulate the vestibulosympathetic reflex in humans. Moreover, endogenous opioids do not appear to contribute the age-associated impairment of the vestibulosympathetic reflex.     

Specificity and stability of a new PTH1 receptor antagonist,mouse TIP(7-39)

Parathyroid hormone 1 (PTH1) receptor antagonists might be of benefit in hypercalcemia of malignancy (HHM) and hyperparathyroidism. We previously identified bovine tuberoinfundibular peptide (7-39) (bTIP(7-39)) as a high-affinity PTH1 receptor antagonist. Mouse TIP(7-39) is an antagonist (rPTH1 K(B)=44 nM, rPTH2=940 nM) that is more potent than other known PTH1 receptor antagonists: bTIP(7-39) (210 nM), PTH-related protein (PTHrP)(7-34) (640 nM), and bPTH(7-34) (>3000 nM). Plasma proteases slowly (t(1/2)=81 min) inactivated [125I] mTIP(7-39). Intravenous plasma [125I]mTIP(7-39) was bi-phasically cleared (radioactivity t(1/2)=2.9 min (70%) and 120 min (30%), binding activity t(1/2)=3.6 min (92%), and t(1/2)=21 min (8%)). Loss of unlabeled mTIP(7-39) (250 microg/kg i.v.) receptor binding was similar. mTIP(7-39)'s high-affinity should facilitate animal evaluation of effects of PTH1 receptor antagonism.     

Strong, specific, monodentate G-C base pair recognition by N7-inosine derivatives in the pyrimidine.purine-pyrimidine triple-helical binding motif.

The nucleoside analogs 7-(2'-deoxy-alpha-D-ribofuranosyl)hypoxanthine (alpha7H,1), 7-(2'-deoxy-beta-D-ribofuranosyl)hypoxanthine (beta7H,2) and 7-7-(2'-O-methyl-beta-D- ribofuranosyl)hypoxanthine (beta7HOMe,3) were prepared and incorporated into triplex forming oligodeoxynucleotides, designed to bind to DNA in the parallel (pyrimidine.purine-pyrimidine) motif. By DNase I footprinting techniques and UV-melting curve analysis it was found that, at pH 7. 0, the 15mer oligonucleotides d(TTTTTMeCTXTMeCTMeCTMeCT) (MeC = 5-methyl-deoxycytidine, X =beta7H,beta7HOMe) bind to a DNA target duplex forming a H.G-C base triple with equal to slightly increased (10-fold) stability compared to a control oligodeoxynucleotide in which the hypoxanthine residue is replaced by MeC. Remarkably, triple-helix formation is specific to G-C base pairs and up to 40 microM third strand concentration, no stable triplex exhibiting H.A-T, H.T-A or H.C-G base arrangements could be found (target duplex concentration approximately 0.1 nM). Multiply substituted sequences containing beta7H residues either in an isolated [d(TTTTTbeta7HTbeta7HTbeta7HTbeta7HTbeta7HT)] or in a contiguous [d(TTTbeta7Hbeta7Hbeta7Hbeta7HTTTTbeta7HTTT)] manner still form triplexes with their targets of comparable stability as the control (MeC-containing) sequences at pH 7.0 and high salt or spermine containing buffers. General considerations lead to a structural model in which the recognition of the G-C base pair by hypoxanthine takes place via only one H-bond of the N-H of hypoxanthine to N7 of guanine. This model is supported by a molecular dynamics simulation. A general comparison of the triplex forming properties of oligonucleotides containing beta7H with those containing MeC or N7-2'-deoxyguanosine (N7G) reveals that monodentate recognition in the former case can energetically compete with bidentate recognition in the latter two cases.