Genetic variability in the tumor necrosis factor-lymphotoxin region influences susceptibility to rheumatoid arthritis.

The major histocompatibility complex class III tumor necrosis factor-lymphotoxin (TNF-LT) region (6p21.3) was investigated as a possible susceptibility locus for rheumatoid arthritis (RA). Inheritance of five TNF microsatellite markers was determined in 50 multiplex families. Overall, 47 different haplotypes were observed. One of these, the TNF a6, b5, c1, d3, e3 (H1) haplotype, was present in 35.3% of affected, but in only 20.5% of unaffected, individuals (P < .005). This haplotype accounted for 21.5% of the parental haplotypes transmitted to affected offspring and only 7.3% not transmitted to affected offspring (P = .0003). The TNF a6 and TNF c1 alleles were individually associated with RA (P = .0005 and .0008, respectively), as were the HLA-DRB1 "shared epitope" (SE) (P = .0001) and HLA-DRB1*0401 (P = .0018). Both univariate and bivariate conditional logistic regression analysis showed significant effects of TNF c1 and SE in increasing risk to RA (P < .001). Stratification by the presence of SE indicated an independent effect of the TNFc1 allele (P = .0003) and the HLA A1, B8, DR3 extended haplotype (always TNFa2, b3, c1, d1, e3) (P = .0027) in SE heterozygotes, while the H1 haplotype was associated with RA in SE homozygotes (P = .0018). The TNF-LT region appears to influence susceptibility to RA, distinct from HLA-DR.     

Mast cell heterogeneity

Increasing evidence for the existence of inter- and intra-species mast cell heterogeneity has expanded the potential biological role of this cell. Early studies suggesting that mast cells at mucosal sites differ morphologically and histochemically from connective tissue mast cells have been confirmed using isolated intestinal mucosal mast cells in the rat and more recently in man. These studies also established that mucosal mast cells are functionally distinct from connective tissue mast cells. Thus, mucosal and connective tissue mast cells differ in their responsiveness to a variety of mast cell secretagogues and antiallergic agents. Speculation about the therapeutic use of antiallergic drugs in disorders involving intestinal mast cells cannot, therefore, be based on extrapolation from studies of their effects on mast cells from other sites. Regulatory mechanisms for mast cell secretion may also be heterogeneous since mucosal mast cells differ from connective tissue mast cells in their response to a variety of physiologically occurring regulatory peptides. The development of techniques to purify isolated mast cell subpopulations will facilitate future analysis of the biochemical basis of the functional heterogeneity of mast cells.     

Cyclic 3'', 5''-AMP relay in dictyostelium discoideum. IV. Recovery of the cAMP signaling response after adaptation to cAMP

In dictyoselium discoideum, an increase in extracellular cAMP activates adenylate cyclase, leading to an increase in intracellular cAMP and the rate of cAMP secretion. Cells adapt to any constant cAMP stimulus after several minutes, but still respond to an increase in the concentration of the stimulus. We have now characterized the decay of adaptation (deadaptation) after the removal of cAMP stimuli. Levels of adaptation were established by the perfusion of [(3)H]adenosine-labeled amoebae with a defined cAMP stimulus. After a variable recovery period, the magnitude of the signaling response to a second stimulus was measured; its attenuation was taken as a measure of residual adaption to the first stimulus. The level of adaptation established by the first stimulus depended on both its magnitude and duration. Deadaptation began as soon as the first stimulus was removed. The magnitude of the response to the second stimulus increased with the recovery time in a first-order fashion, with a t(1/2)=3-4 min for stimuli of 10(-8) M to 10(-5) M cAMP. Responses to test stimuli, although reduced in magnitude, had an accelerated time-course when they closely followed a prior response that had not completely subsided. This effect is called priming; we believe it reveals a reversible, rate-limiting step that modulates the onset and termination of the signaling responses of amoebae that have not recently responded to a cAMP stimulus. We have suggested that the cAMP signaling response is controlled by two antagonistic cellular processes, excitation and adaptation. The data reported here imply that both the rate of rise in the adaptation process and the final level reached depend on the occupancy of cAMP surface receptors and that the decay of adaptation when external cAMP is removed proceeds with first-order kinetics.     

Biotinylation of a bombesin/gastrin-releasing peptide analogue for use as a receptor probe.

The development of a biotinylated bombesin/gastrin-releasing peptide (GRP) for use as a receptor probe is reported. The lysine13 of a GRP-27 was substituted by arginine and lysine was added to the amino terminus. Biotinylation of the N-terminal lysine was performed. The biotinylated peptide was purified by HPLC and characterized by mass spectral analysis. Binding studies with murine Swiss 3T3 fibroblasts, cells known to express bombesin/GRP receptors, yielded a dissociation curve for the biotinylated GRP-27 analogue (biotin-Lysyl[Asp12,Arg13]GRP-27) which was nearly identical to that of native GRP. Using studies of gastrin release from isolated canine G cells, equipotent functional activity of the biotinylated probe and unmodified GRP was demonstrated. Measurements of retained 125I-avidin confirmed that the biotin/avidin interaction could occur once the biotin-peptide complex was bound. Applicability of the probe was demonstrated with fluorescent microscopy using avidin-FITC on Swiss 3T3 fibroblasts. In conclusion, a novel biotinylated bombesin/GRP analogue has been developed which retains the functional characteristics of the native peptide and is a useful probe for receptor studies.     

Photolabeling of glucose-sensitive cytochalasin B binding proteins in erythrocyte, fibroblast and adipocyte membranes

(³H)Cytochalasin B has been photoincorporated into membrane fractions of the human erythrocyte, Rous sarcoma virus-transformed chicken embryo fibroblast and rat adipocyte. Identification of D-glucose sensitive cytochalasin B binding sites was achieved by photolyzing membranes with radioligand in the presence of 0.5–0.7M D- or L-glucose. In the erythrocyte the major labeled bands on SDS-polyacrylamide gels were at 55,000 and 46,000 daltons. In the virus-transformed fibroblasts a major labeled band was at 55,000 daltons, and in adipocyte microsomal membranes, peaks at 50,000 and 45,000 daltons were observed. Binding characteristics of these polypeptides suggest that they are the putative glucose transport proteins in these three cell types.     

Resistance of the intact and reconstituted adipocyte hexose transport system to irreversible inhibition by sulfhydryl and amino reagents

Sensitivity of the adipocyte D-glucose transport system in intact plasma membranes or following solubilization and reconstitution into phospholipid vesicles to several protein-modifying reagents was investigated. When intact plasma membranes were incubated with N-ethylmaleimide (20 mM) or fluorodinitrobenzene (4 mM), D-glucose transport activity was virtually abolished. However, washing the membranes free of unreacted reagents restored transport activity, indicating that covalent interaction with the membranes did not mediate the transport inhibition. Reaction of [³H] N-ethylmaleimide with plasma membranes under similar conditions resulted in extensive labeling of all protein fractions resolved on dodecyl sulfate gels. Similarly, addition of N-ethyl-maleimide to cholate-solubilized membrane protein had no effect on transport activity in artifical phospholipid vesicles reconstituted under conditions where the membrane protein was free of unreacted N-ethylmaleimide. Transport activity in plasma membranes was also inhibited by both reduced and oxidized dithiothreitol or glutathione (15 mM) in a readily reversible manner, consistent with a noncovalent mode of inhibition. Thus, the insulin-responsive adipocyte D-glucose transport system differs from the red cell hexose transport system in its remarkable insensitivity to modulation by covalent blockade of sulfhydryal or amino groups by the reagents studied.     

<Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis> aggravates vein graft intimal hyperplasia in a rat model

Background  

Along with angioplasty, autologus vein grafts are commonly used for artery bypass grafting in patients with advanced arterial stenosis and drug-resistant angina pectoris. Although initially a successful procedure, long-term functionality is limited due to proliferation and migration of smooth muscle cells. Like in atherosclerosis, common chronic infections caused by viruses and bacteria may contribute to this process of vein graft failure. Here we investigated the possible role of Chlamydia pneumoniae (Cpn) in the pathogenesis of venous graft failure in an experimental animal model. In 2 groups (n = 10 rats/group), an epigastric vein-to-common femoral artery interposition graft was placed. Immediately thereafter, rats were infected with Cpn (5*10⁸ IFU) or injected with control solutions. Rats were sacrificed three weeks after surgery and the grafts were harvested for morphometrical and immunohistochemical analysis.     

Nonentrained circadian rhythms of melatonin in submariners scheduled to an 18-hour day.

The human circadian timing system has previously been shown to free run with a period slightly longer than 24 h in subjects living in the laboratory under conditions of forced desynchrony. In forced desynchrony, subjects are shielded from bright light and periodic time cues and are required to live on a day length outside the range of circadian entrainment. The work schedule used for most personnel aboard American submarines is 6 h on duty alternating with 12 h off duty. This imposed 18-h cycle is too short for human circadian synchronization, especially given that there is no bright-light exposure aboard submarines. However, crew members are exposed to 24-h stimuli that could mediate synchronization, such as clocks and social contacts with personnel who are living on a 24-h schedule. The authors investigated circadian rhythms of salivary melatonin in 20 crew members during a prolonged voyage on a Trident nuclear submarine. The authors found that in crew members living on the 18-h duty cycle, the endogenous rhythm of melatonin showed an average period of 24.35 h (n = 12, SD = 0.18 h). These data indicate that social contacts and knowledge of clock time are insufficient for entrainment to a 24-h period in personnel living by an 18-h rest-activity cycle aboard a submarine.     

Opportunity or Orientation? Who Uses Urban Parks and Why

There is growing recognition that interactions with nature provide many desirable human well-being outcomes, yet increasing urbanization is degrading the quality and quantity of nature experiences. Thus, it has become increasingly important to understand how and why urban dwellers interact with nature. Studies of urban green space use have largely focused on the availability and ease of access to green space, suggesting that greater opportunities to experience such space will lead to increased use. However, a growing literature emphasizes the potential for an individual''s nature orientation to affect their interaction with green space. Here we measure the importance of both opportunity and orientation factors in explaining urban park use. An urban lifestyle survey was deployed across Brisbane, Australia in November 2012 to assess patterns of green space use. Participants (n = 1479) were asked to provide information on demographics, private yard use, park visitations in the past week, and their orientation toward nature. About 60% of those surveyed had visited a park in the past week, and while this park user population had significantly greater nearby park coverage (within a 250 m radius; p = 0.006), a much stronger determinant of visitation was their higher nature orientation (p<0.00001), suggesting that while both opportunity and orientation are important drivers for park visitation, nature orientation is the primary effect. Park users also spent significantly more time in their yards than non-park users (p<0.00001), suggesting that yard use does not necessarily compensate for lower park use. Park users with stronger nature orientation (i) spent more time in their yard, (ii) traveled further to green spaces, and (iii) made longer visits than park visitors with weaker nature orientation. Overall, our results suggest that measures to increase people''s connection to nature could be more important than measures to increase urban green space availability if we want to encourage park visitation.