CD28-independent, TRAF2-dependent costimulation of resting T cells by 4-1BB ligand

4-1BB ligand (4-1BBL) is a member of the tumor necrosis factor (TNF) family expressed on activated antigen-presenting cells. Its receptor, 4-1BB, is a member of the TNF receptor family expressed on activated CD4 and CD8 T cells. We have produced a soluble form of 4-1BBL using the baculovirus expression system. When coimmobilized on plastic with anti-CD3, soluble 4-1BBL induces interleukin (IL)-2 production by resting CD28+ or CD28- T cells, indicating that 4-1BBL can function independently of other cell surface molecules, including CD28, in costimulation of resting T cell activation. At low concentrations of anti-CD3, 4-1BBL is inferior to anti-CD28 in T cell activation. However, when 4-1BB ligand is provided together with strong TCR signals, then 4-1BBL and anti-CD28 are equally potent in stimulation of IL-2 production by resting T cells. We find that TNF receptor-associated factor (TRAF)1 or TRAF2 associate with a glutathione S-transferase-4-1BB cytoplasmic domain fusion protein in vitro. In T cells, we find that association of TRAF1 and TRAF2 with 4-1BB requires 4-1BB cross-linking. In support of a functional role for TRAF2 in 4-1BB signaling, we find that resting T cells isolated from TRAF2-deficient mice or from mice expressing a dominant negative form of TRAF2 fail to augment IL-2 production in response to soluble 4-1BBL. Thus 4-1BB, via the TRAF2 molecule, can provide CD28-independent costimulatory signals to resting T cells.     

Synergic antistaphylococcal properties of lactoferrin and lysozyme

Staphylococcus epidermidis colonises a wide range of implanted prosthetic devices, but rarely contact lenses -- despite a similarity in material composition. A conceivable explanation for this anomaly is the action of the tear defences, including the constitutive proteins lactoferrin and lysozyme. Therefore this study investigated the effect of lactoferrin, lysozyme and serum on the growth of S. epidermidis isolates in artificial tear fluid. Whether supplemented with serum alone or serum with either apolactoferrin or lysozyme, this medium induced a similar, strain-variable effect. However, simultaneous addition of these proteins induced a greater bactericidal or bacteristatic effect. Of those strains killed by the concerted action of apolactoferrin and lysozyme, the absence of serum led to a further increase in the bactericidal effect, whereas strains displaying bacteriostasis were unaffected by serum. Iron saturation of lactoferrin reversed the antimicrobial synergy of apolactoferrin and lysozyme. These results show synergy between lactoferrin and lysozyme which is dependent on the iron limitation of lactoferrin. As a bactericidal mechanism, this synergy is augmented by serum, but bacteriostasis remains unaffected by serum supplemention. Thus, the combination of lysozyme and lactoferrin may partly explain the low level of contact lens colonisation by S. epidermidis in vivo.     

11Beta-hydroxysteroid dehydrogenase type 2 in human pregnancy and reduced expression in intrauterine growth restriction

The evolutionary theory of aging predicts that the equilibrium gene frequency for deleterious mutations should increase with age at onset of mutation action because of weaker (postponed) selection against later-acting mutations. According to this mutation accumulation hypothesis, one would expect the genetic variability for survival (additive genetic variance) to increase with age. The ratio of additive genetic variance to the observed phenotypic variance (the heritability of longevity) can be estimated most reliably as the doubled slope of the regression line for offspring life span on paternal age at death. Thus, if longevity is indeed determined by late-acting deleterious mutations, one would expect this slope to become steeper at higher paternal ages. To test this prediction of evolutionary theory of aging, we computerized and analyzed the most reliable and accurate genealogical data on longevity in European royal and noble families. Offspring longevity for each sex (8409 records for males and 3741 records for females) was considered as a dependent variable in the multiple regression model and as a function of three independent predictors: paternal age at death (for estimation of heritability of life span), paternal age at reproduction (control for parental age effects), and cohort life expectancy (control for cohort and secular trends and fluctuations). We found that the regression slope for offspring longevity as a function of paternal longevity increases with paternal longevity, as predicted by the evolutionary theory of aging and by the mutation accumulation hypothesis in particular.     

Sex differences in the relative contributions of nitric oxide and EDHF to agonist-stimulated endothelium-dependent relaxations in the rat isolated mesenteric arterial bed

1. We have used the isolated, buffer-perfused, superior mesenteric arterial bed of male and female rats to assess the relative contributions of nitric oxide (NO) and the endothelium-derived hyperpolarizing factor (EDHF) to endothelium-dependent relaxations to carbachol. 2. Carbachol caused dose-related relaxations of methoxamine-induced tone in mesenteric vascular beds from male rats described by an ED50(M) of 0.43+/-0.15 nmol and a maximum relaxation (Rmax(M) of 89.6+/-1.2% (n=28) which were not significantly different from those observed in mesenteries from female rats (ED50(F)=0.72+/-0.19 nmol and Rax(F)=90.7+/-0.9%; n=22). 3. In the males, the addition of 100 microM NG-nitro-L-arginine methyl ester (L-NAME) caused the dose-response curve to carbachol to be significantly (P<0.001) shifted to the right 15 fold (ED50(M)=6.45+/-3.53 nmol) and significantly (P<0.01) reduced Rmax(M) (79.7+/-2.8%, n=13). By contrast, L-NAME had no effect on vasorelaxation to carbachol in mesenteries from female rats (ED50(f)= 0.89+/-0.19 nmol, Rmax(F)=86.9+/-2.3%, n=9). 4. Raising tone with 60 mM KCl significantly reduced the maximum relaxation to carbachol in mesenteries from male rats 2 fold (Rmax(M)=40.3+/-9.2%, n=4; P<0.001) and female rats by 1.5 fold (Rmax(F)=55.3+/-3.3%, n=6; P<0.001), compared with methoxamine-induced tone. The potency of carbachol was also significantly reduced 1.2 fold in preparations from males (ED50(M)=0.87+/-0.26 nmol; P<0.01) but not the females (ED50(F)=4.04+/-1.46 nmol). In the presence of both 60 mM KCl and L-NAME, the vasorelaxation to carbachol was completely abolished in mesenteries from both groups. 5. The cannabinoid receptor antagonist SR141716A (1 microM), which is also a putative EDHF antagonist, had no significant effect on the responses to carbachol in mesenteries from males or females (ED50(M)=1.41+/-0.74 nmol, Rmax(M)=89.4+/-2.5%, n=7; ED50(F)=2.17+/-0.95 nmol, Rmax(F)=89.9+/-1.8%, n=9). In mesenteries from male rats, in the presence of 100 microM L-NAME, SR141716A significantly (P<0.05) shifted the dose-response curve to carbachol 8 fold further to the right than that seen in the presence of L-NAME alone (ED50(M)= 53.8+/-36.8 nmol) without affecting Rmax(M) (72.4+/-4.8%, n=10). In mesenteries from female rats, the combined presence of L-NAME and SR141716A, significantly (P < 0.01) shifted the dose-response curve to carbachol 7.5 fold, (ED50(F)=6.66+/-2.46 nmol), as compared to L-NAME alone and significantly (P<0.001) decreased Rmax(F) (70.1+/-5.5%, n=8). 6. Vasorelaxations to the nitric oxide donor sodium nitroprusside (SNP), to the endogenous cannabinoid, anandamide (a putative EDHF) and to the ATP-sensitive potassium channel activator, levcromakalim, did not differ significantly between male and female mesenteric vascular beds. 7. The continuous presence of sodium nitroprusside (SNP; 20-60 nM) had no effect on vasorelaxation to carbachol in mesenteries from either males or females. In the presence of L-NAME, SNP significantly (P<0.05) reduced the potency of carbachol 6 fold, without affecting the maximal relaxation in mesenteries from male rats (ED50(M)=40.9+/-19.6 nmol, Rmax(M)=79.4+/-2.5%, n=11). Similarly in mesenteries from female rats, the ED50(F) was also significantly (P<0.01) increased 7 fold (6.24+/-2.02 nmol), while the Rmax(F) was unaffected (81.9+/-11.0%; n=4). 8 The results of the present investigation demonstrate that the relative contributions of agonist-stimulated NO and EDHF to endothelium-dependent relaxations in the rat isolated mesenteric arterial bed, differ between males and females. Specifically, although both NO and EDHF appear to contribute towards endothelium-dependent relaxations in males and females, blockade of NO synthesis alone has no effect in the female. This suggests that EDHF is functionally more important in females; one possible explanation for this is that in the absence of NO, the recently identified ability of EDHF to compensate for the loss of NO, is functio     

Homeless youth and their exposure to and involvement in violence while living on the streets

Sick building syndrome is the term given to a heterogeneous constellation of symptoms that affects workers in modern mechanically ventilated office buildings. Although the cause is unknown, there is evidence that the local environment of the work station is an important determinant of symptoms. In this study, investigators examined the effect of a new, individually controlled ventilation system on workers' symptoms. Investigators studied two groups of workers in one mechanically ventilated office building: (1) a control group at whose worksite no intervention was made and (2) an intervention group. The intervention consisted of installation of a device that allowed each worker control over the ventilation supplied to his or her worksite. Just before, and 4 and 16 mo after installation of this device, workers completed self-administered questionnaires regarding occurrence of symptoms. The new ventilation system resulted in higher air velocities, more variable temperatures, and higher concentrations of airborne dust and fungal spores. Four months after installation, workers with the new ventilation system reported fewer symptoms that were (a) work-related (p < .05) and that were work-related and frequent (p < .05); in addition, they reported fewer symptoms that reduced their capacity to work (p < .01). Sixteen months after installation, workers with the new device reported fewer symptoms than at baseline (although not as significantly), and they indicated that the indoor air quality improved their productivity by 11%, compared with a 4% reduction of productivity among the control group of workers (p < .001). Investigators concluded that the new ventilation system, which provided the workers with individual control over ventilation, was associated with important and sustained reduction in symptoms.     

Sites of alcohol and volatile anaesthetic action on GABA(A) and glycine receptors

Volatile anaesthetics have historically been considered to act in a nonspecific manner on the central nervous system. More recent studies, however, have revealed that the receptors for inhibitory neurotransmitters such as gamma-aminobutyric acid (GABA) and glycine are sensitive to clinically relevant concentrations of inhaled anaesthetics. The function of GABA(A) and glycine receptors is enhanced by a number of anaesthetics and alcohols, whereas activity of the related GABA rho1 receptor is reduced. We have used this difference in pharmacology to investigate the molecular basis for modulation of these receptors by anaesthetics and alcohols. By using chimaeric receptor constructs, we have identified a region of 45 amino-acid residues that is both necessary and sufficient for the enhancement of receptor function. Within this region, two specific amino-acid residues in transmembrane domains 2 and 3 are critical for allosteric modulation of both GABA(A) and glycine receptors by alcohols and two volatile anaesthetics. These observations support the idea that anaesthetics exert a specific effect on these ion-channel proteins, and allow for the future testing of specific hypotheses of the action of anaesthetics.     

Flexible and Conductive 3D Printable Polyvinylidene Fluoride and Poly(N,N‐dimethylacrylamide) Based Gel Polymer Electrolytes

In this work, 3D printable gel polymer electrolytes (GPEs) based on N,N‐dimethylacrylamide (DMAAm) and polyvinylidene fluoride (PVDF) in lithium chloride containing ethylene glycol solution are synthesized and their physicochemical properties are investigated. 3D printing is carried out with a customized stereolithography type 3D gel printer named “Soft and Wet Intelligent Matter‐Easy Realizer” and free forming GPE samples having variable shapes and sizes are obtained. Printed PVDF/PDMAAm‐based GPEs exhibit tunable mechanical properties and favorable thermal stability. Electrochemical proprieties of the printed GPEs are carried out via impedance spectroscopy in the temperature range of 25–90 °C by varying PVDF content. Ionic conductivity as high as 6.5 × 10^?4 S cm^?1 is achieved at room temperature for GPE containing low PVDF content (5 wt%) and conductivity of the GPEs is increased as temperature rises.     

Potent,Metabolically Stable 2‐Alkyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐adenines as Adenosine A2A Receptor Ligands

Inhibition of adenosine A_2A receptors has been shown to elicit a therapeutic response in preclinical animal models of Parkinson’s disease (PD). We previously identified the triazolo‐9H‐purine, ST1535, as a potent A_2AR antagonist. Studies revealed that ST1535 is extensively hydroxylated at the ω‐1 position of the butyl side chain. Here, we describe the synthesis and evaluation of derivatives in which the ω‐1 position has been substituted (F, Me, OH) in order to block metabolism. The stability of the compounds was evaluated in human liver microsomes (HLM), and the affinity for A_2AR was determined. Two compounds, (2‐(3,3‐dimethylbutyl)‐9‐methyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐purin‐6‐amine ( 3 b ) and 4‐(6‐amino‐9‐methyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐purin‐2‐yl)‐2‐methylbutan‐2‐ol ( 3 c ), exhibited good affinity against A_2AR (K_i=0.4 nM and 2 nM , respectively) and high in vitro metabolic stability (89.5 % and 95.3 % recovery, respectively, after incubation with HLM for two hours).     

Purification and crystallization of complexes modeling the active state of the fragile histidine triad protein

Fragile histidine triad protein (Fhit) is a diadenosine triphosphate (ApppA) hydrolase encoded at the human chromosome 3 fragile site which is frequently disrupted in tumors. Reintroduction of FHIT coding sequences to cancer cell lines with FHIT deletions suppressed the ability of these cell lines to form tumors in nude mice even when the reintroduced FHIT gene had been mutated to allow ApppA binding but not hydrolysis. Because this suggested that the tumor suppressor activity of Fhit protein depends on substrate-dependent signaling rather than ApppA catabolism, we prepared two crystalline forms of Fhit protein that are expected to model its biologically active, substrate-bound state. Wild-type and the His96Asn forms of Fhit were overexpressed in Escherichia coli, purified to homogeneity and crystallized in the presence and absence of ApppA and an ApppA analog. Single crystals obtained by vapor diffusion against ammonium sulfate diffracted X-rays to beyond 2.75 A resolution. High quality native synchrotron X-ray data were collected for an orthorhombic and a hexagonal crystal form.