Heat shock protein-27 attenuates foam cell formation and atherogenesis by down-regulating scavenger receptor-A expression via NF-κB signaling

Previously, we showed an inverse correlation between HSP27 serum levels and experimental atherogenesis in ApoE^?/? mice that over-express HSP27 and speculated that the apparent binding of HSP27 to scavenger receptor-A (SR-A) was of mechanistic importance in attenuating foam cell formation. However, the nature and importance of the interplay between HSP27 and SR-A in atheroprotection remained unclear. Treatment of THP-1 macrophages with recombinant HSP27 (rHSP27) inhibited acLDL binding (? 34%; p < 0.005) and uptake (? 38%, p < 0.05). rHSP27 reduced SR-A mRNA (? 39%, p = 0.02), total protein (? 56%, p = 0.01) and cell surface (? 53%, p < 0.001) expression. The reduction in SR-A expression by rHSP27 was associated with a 4-fold increase in nuclear factor-kappa B (NF-κB) signaling (p < 0.001 versus control), while an inhibitor of NF-κB signaling, BAY11-7082, attenuated the negative effects of rHSP27 on both SR-A expression and lipid uptake. To determine if SR-A is required for HSP27 mediated atheroprotection in vivo, ApoE^?/? and ApoE^?/? SR-A^?/? mice fed with a high fat diet were treated for 3 weeks with rHSP25. Compared to controls, rHSP25 therapy reduced aortic en face and aortic sinus atherosclerotic lesion size in ApoE^?/? mice by 39% and 36% (p < 0.05), respectively, but not in ApoE^?/?SR-A^?/? mice. In conclusion, rHSP27 diminishes SR-A expression, resulting in attenuated foam cell formation in vitro. Regulation of SR-A by HSP27 may involve the participation of NF-κB signaling. Lastly, SR-A is required for HSP27-mediated atheroprotection in vivo.     

A simple colorimetric determination of the manganese content in photosynthetic membranes

The functional Mn content of intact photosystem II membrane fragments was measured as 4.06 ± 0.13 Mn/reaction center when determined using a simple, sensitive colorimetric assay that will also work with thylakoids and core complexes. This procedure requires minimal sample material, does not need expensive assay equipment, requires four simple steps, and only takes 20–30 min to perform. These include (a) removal of the adventitious Mn ions by CaCl₂ treatment of the membranes, (b) extraction of the Mn from the O₂-evolving complex with hydrochloric acid, (c) purification of the extract by centrifugation followed by filtration of the supernatant through an Acrodisc syringe filter (0.2 μm nylon membrane), and (d) colorimetric determination of Mn in the extract using the reaction of the chromogenic agent, 3,3′,5,5′-tetramethylbenzidine, with previously oxidized Mn(II) cations carried out at high pH. The colorimetric assay itself has been used previously by Serrat (Mikrochim Acta 129:77–80, 1998) for assaying Mn concentrations in sea water and drinking water.     

Toward a framework for sulfoproteomics: Synthesis and characterization of sulfotyrosine-containing peptides

Tyrosine sulfation is one of the most common post-translational modifications in secreted and transmembrane proteins and a key modulator of extracellular protein-protein interactions. Several proteins known to be tyrosine sulfated play important roles in physiological processes, and in some cases a direct link between protein function and tyrosine sulfation has been established. In blood coagulation, tyrosine sulfation of factor VIII is required for efficient binding of von Willebrand factor; in leukocyte adhesion, tyrosine sulfation of the P-selectin glycoprotein ligand-1 mediates high-affinity binding to P-selectin; and in leukocyte chemotaxis, tyrosine sulfation of chemokine receptors is required for optimal interaction with chemokine ligands. Furthermore, tyrosine sulfation has been implicated in several infectious diseases. In particular, tyrosine sulfation of the HIV-1 co-receptor CCR5 is required for viral entry into host cells and tyrosine sulfation of the Duffy antigen/receptor for chemokines is crucial for erythrocyte invasion by the malaria parasite plasmodium vivax. Despite increasing interest in tyrosine sulfation in recent years, the sulfoproteome still remains largely unexplored. To date, only a relatively small number of sulfotyrosine-containing peptides and proteins have been identified, and a specific role for tyrosine sulfation has not been established for most of these. Here, we provide an overview of the biology and enzymology of tyrosine sulfation and discuss recent developments in preparative and analytical methods that are central to sulfoproteome research.     

Arg64β3‐Adrenoceptor Variant and the Components of Energy Expenditure

Objective: To determine Trp64Arg β₃‐adrenoceptor genotype‐specific differences in the components of energy expenditure. Hypothesis: We hypothesized that resting metabolic rate (RMR) and physical activity levels would be lower and that thermic effect of feeding (TEF) would be higher in those with the Arg64 allele. Research Methods and Procedures: RMR and TEF were measured by indirect calorimetry, physical activity by questionnaire, and total energy expenditure by the doubly labeled water method. Genotype‐specific measures were compared using ANOVA and analysis of covariance (ANCOVA). Results: RMR in Arg64 homozygotes was significantly lower than in Trp64 homozygotes [Arg64, 1373 ± 259 kcal/d (n = 15) vs. Trp64Arg, 1538 ± 238 kcal/d (n = 25) vs. Trp64, 1607 ± 290 kcal/d (n = 22); p < 0.01]. TEF was significantly higher in Arg64 homozygotes compared with Trp64 homozygotes (Arg64, 359 ± 28 kcal/d; Trp64Arg, 322 ± 22 kcal/d; and Trp64, 279 ± 23 kcal/d; p < 0.05). No differences were identified between genotypes in physical activity or in total energy expenditure. Discussion: Our results suggest that the Arg64 β₃‐adrenoceptor allele contributes significantly to the genetic variability in both RMR and TEF.     

Plant Species Numbers Predicted by a Topography-based Groundwater Flow Index

The lack of a clear understanding of the factors governing the often-great variation of species numbers over entire landscapes confounds attempts to manage biodiversity. We hypothesized that in a topographically variable boreal forest landscape the availability of shallow groundwater is a major determinant of plant species numbers. We then developed a topographically derived hydrologic index based on multidirectional flow algorithms to account for the variation in availability of such groundwater in the landscape. We found a positive correlation between species numbers of vascular plants in plots ranging from 0.01 to 200 m² and the hydrologic index. Generally, the landscape was relatively dry and species-poor, but interspersed patches with shallow groundwater had high species numbers and high proportions of regionally uncommon plant species. The index explained 30% of the variation in vascular plant number and correlated quite well (r_s = 0.50) with groundwater level, but not as well with a community H⁺ concentration value (instead of community pH, r_s = −0.31), based on species composition. In addition, we found a very strong correlation between species number and the community H⁺ concentration value (r_s −0.84). The hydrologic index is a useful tool for the identification of spatial of species number patterns across entire landscapes. This is an important step in identifying the areas most in need of protection or restoration, designing survey techniques, and understanding the fundamental processes that control the spatial distribution of species.     

Influence of phosphorylation by protein kinase A on CFTR at the cell surface and endoplasmic reticulum

Most, if not all, of the catalytic activity of the tandem catalytic domain-containing receptor-like protein tyrosine phosphatases (PTPs) resides in the membrane proximal domains (D1), with little to no activity associated with the membrane distal domains (D2). Two point mutations in the D2 domain of PTPalpha, which restore invariant amino acids found in the KNRY motif and WPD loop of all active D1 domains, synergistically confer D1-equivalent kinetic properties towards the phosphotyrosine analogue pNPP, and activate PTPalpha-D2 catalysed phosphopeptide hydrolysis (Lim et al., J. Biol. Chem. 273 (1998) 28986-28993; Buist et al., Biochemistry 38 (1999) 914-922). As all D2 domains lack these two D1-invariant amino acids, we have investigated whether other D2 domains are activated by such point mutations. Mutant PTPepsilon-D2, closely related to PTPalpha-D2 and belonging to a subgroup of D2 domains with minimal and conservative substitutions of D1-invariant amino acids, exhibits synergistic activation towards pNPP but not towards a phosphopeptide substrate. CD45-D2, belonging to another subgroup of D2 domains with considerable substitutions in D1-invariant amino acids, is not activated by these mutations, even in the context of a third mutation which restores the minimal essential active site sequence C(X(5))R, indicating that additional defects are sufficient to preclude catalysis. The ability of the KNRY and WPD replacements to activate PTPepsilon-D2 and PTPalpha-D2, but not CD45-D2, in conjunction with the extent and nature of their wild-type amino acid substitutions, suggests that these D2 domains are representative of two functionally distinct groups of D2 domain.     

The isolated Photosystem II reaction center: first attempts to directly measure the kinetics of primary charge separation

Direct measurements of the intrinsic rate of primary charge separation in the isolated Photosystem II (PS II) reaction center complex had to await the availability of suitable, stabilized reaction center materials as well as sophisticated femtosecond transient absorption spectroscopic techniques. The events that led to the first direct measurements of the primary charge separation act in PS II and discussions of the results thereafter are chronicled in this brief historical review. This revised version was published online in August 2006 with corrections to the Cover Date.