Molecular organization of the tear fluid lipid layer

The tear fluid protects the corneal epithelium from drying out as well as from invasion by pathogens. It also provides cell nutrients. Similarly to lung surfactant, it is composed of an aqueous phase covered by a lipid layer. Here we describe the molecular organization of the anterior lipid layer of the tear film. Artificial tear fluid lipid layers (ATFLLs) composed of egg yolk phosphatidylcholine (60 mol %), free fatty acids (20 mol %), cholesteryl oleate (10 mol %), and triglycerides (10 mol %) were deposited on the air-water interface and their physico-chemical behavior was compared to egg-yolk phosphatidylcholine monolayers by using Langmuir-film balance techniques, x-ray diffraction, and imaging techniques as well as in silico molecular level simulations. At low surface pressures, ATFLLs were organized at the air-water interface as heterogeneous monomolecular films. Upon compression the ATFLLs collapsed toward the air phase and formed hemispherelike lipid aggregates. This transition was reversible upon relaxation. These results were confirmed by molecular-level simulations of ATFLL, which further provided molecular-scale insight into the molecular distributions inside and dynamics of the tear film. Similar type of behavior is observed in lung surfactant but the folding takes place toward the aqueous phase. The results provide novel information of the function of lipids in the tear fluid.     

Cytotoxic T lymphocytes induce caspase-dependent and -independent cell death in neuroblastomas in a MHC-nonrestricted fashion

The MHC class I- restricted processing and presentation pathway is frequently nonfunctional in tumor cells; therefore, the direct targeting of tumor cells by CTLs may be difficult, if at all possible, to achieve. We used neuroblastoma (NB), which represents a striking example of a tumor with an impaired MHC class I pathway, as a model to study bystander effects of activated T lymphocytes on tumor cells. We found that NB cell lines are susceptible to killing by differentiated CD8(+) CTL clones in a MHC class I-nonrestricted manner that involves two programs of cell death distinguished on the basis of different kinetics, sensitivities to caspase inhibitors, and cytokine-blocking reagents. The "early" death exhibited characteristic features of apoptosis, whereas the "delayed" caspase-independent death exhibited features associated with necrosis and was partially inhibited by TNF-alpha-blocking and prevented by overexpression of Bcl-2 or Bcl-x(L). Our data reveal a previously unappreciated complexity of death pathways induced in tumor cells by immune activation and suggest that redirecting nonspecific effector CTLs to even a small proportion of NB cells or activating CTLs in a tumor's proximity may have therapeutic effects in patients with NB.     

Sensitization of IL-2 signaling through TLR-7 enhances B lymphoma cell immunogenicity

The innate ability of B lymphoma cells to escape control by tumor-reactive T cells must be overcome to develop effective immunotherapies for these diseases. Because signals from both the innate and adaptive immune systems direct the acquisition of strong immunogenicity by professional APCs, the effects of IL-2 and the TLR-7 agonist, S28690, on the immunogenic properties of chronic lymphocytic leukemia (CLL) B cells were studied. IL-2 with S28690 caused CLL cells to proliferate and increased their expression of B7-family members, production of TNF-alpha and IL-10, and levels of tyrosine-phosphorylated STAT-1 and STAT-3 proteins. S28690 increased CD25 expression on CLL cells and sensitized them to IL-2 signaling. However, IL-2 did not change TLR-7 expression or signaling in CLL cells. The ability to stimulate T cell proliferation required additional activation of protein kinase C, which inhibited tumor cell proliferation, "switched off" IL-10 production, and caused essentially all CLL cells (regardless of clinical stage) to acquire a CD83(high)CD80(high)CD86(high)CD54(high) surface phenotype marked by the activation of STAT-1 without STAT-3. These findings suggest that TLR-7 "licenses" human B cells to respond to cytokines of the adaptive immune system (such as IL-2) and provide a strategy to increase the immunogenicity of lymphoma cells for therapeutic purposes.     

Biological relevance of a stable biochemical interaction between the tombusvirus-encoded P19 and short interfering RNAs

The Tomato bushy stunt virus (TBSV)-encoded p19 protein (P19) is widely used as a robust tool to suppress RNA interference (RNAi) in various model organisms. P19 dimers appropriate 21-nucleotide (nt) duplex short interfering RNAs (siRNAs) generated by Dicer presumably to prevent programming of the RNA-induced silencing complex (RISC). In the context of virus infection, this model predicts that P19 mutants compromised for siRNA binding cannot prevent RISC-mediated degradation of TBSV RNA and thus reduce viral pathogenicity. To test this, we used P19/43 (R-->W), which is less pathogenic than wild-type P19 (wtP19), and P19/75-78 (RR-->GG), with pathogenicity properties (i.e., viral spread and symptom induction) comparable to those of a P19-null mutant. We demonstrate that P19/43 still suppresses RNAi-mediated viral RNA degradation in infected Nicotiana benthamiana, while P19/75-78 is unable to prevent this clearance of viral RNA, leading to an irreversible recovery phenotype. Gel filtration and immunoprecipitation assays show that at the onset of the infection, wtP19, P19/43, and P19/75-78 readily accumulate, and they form dimers. The wtP19 is stably associated with duplex approximately 21-nt TBSV siRNAs, while P19/75-78 does not bind these molecules, and the electrostatic interaction of P19/43 with siRNAs is perturbed for approximately 21-nt duplexes but not for longer siRNAs. This is the first clear demonstration of a direct correlation between a novel structurally orchestrated siRNA binding of an RNAi suppressor and its roles in viral pathogenesis. The findings should be particularly valuable for the RNAi field in general because the P19 mutants enable precise determination of siRNA appropriation effects.     

Activation of macrophage adenylate cyclase by stimulants of the oxidative burst and by arachidonic acid--two distinct mechanisms

The effect of agents stimulating the oxidative burst (OB) in oil-elicited guinea pig peritoneal macrophages (MPs) on cyclic adenosine 3′,5′-monophosphate (cAMP) levels was examined. We found that: (i) Phorbol myristate acetate (PMA), the Ca²⁺ ionophore A23187, concanavalin A (Con A), wheat germ agglutinin (WGA), N-formyl-l-methionyl-l-leucyl-l-phenylalanine (FMLP) and opsonized zymosan, elevated cAMP levels two- to fivefold; (ii) the biologically inactive PMA analog, 4-O-methyl-PMA, was proportionally less effective than PMA in stimulating cAMP accumulation; (iii) increased levels of cAMP were evident after 10 min of incubation with the stimulants, in the presence of the phosphodiesterase inhibitor 3-isobutyl methylxanthine (IBX); (iv) basal cAMP levels in MPs increased proportionally with the extracellular Ca²⁺ concentration; (v) the cAMP-elevating effect of all stimulants (with the exception of A23187) was more pronounced in low Ca²⁺ media, associated with lower basal cAMP levels. A23187 did not elevate cAMP levels in the absence of extracellular Ca²⁺; (vi) short-term incubation of MPs with arachidonic acid and with the arachidonic acid precursor, linoleic acid, induced an increase in the level of cAMP; (vii) the elevations in cAMP levels induced by OB stimulants were enhanced, not blocked, by mepacrine, 5,8,11,14-eicosatetraynoic acid (ETYA), indomethacin or aspirin, demonstrating that prostaglandin (PG) synthesis was not involved; (viii) the cAMP-elevating effect of arachidonic and linoleic acids was blocked by ETYA and indomethacin, indicating that it was mediated by PGs. The mechanism by which OB stimulants elevate cAMP levels could not be determined but changes in the cellular level of Ca²⁺ seem to play a pivotal role.     

The influence of fatty acids on determination of human serum albumin thiol group

During investigation of the changes of the Cys34 thiol group of human serum albumin (HSA) (isolated by affinity chromatography with Cibacron Blue (CB)) in diabetes, we found that the HSA-SH content was higher (11–33%) than the total serum thiol content. The influence of fatty acids (FA) binding to HSA on this discrepancy was investigated in vitro (using fluorescence and CD spectroscopy and GC) and with HSA samples from diabetic (n=20) and control groups (n=17). HSA-bound FA determine the selection of HSA molecules by CB and enhance reactivity and/or accessibility of the SH group. A high content of polyunsaturated FA (35.6%) leads to weaker binding of HSA molecules to CB. Rate constants of DTNB reaction with the SH group of HSA applied to a CB column, bound-HSA and unbound-HSA fractions, were 4.8×10^-3, 21.6×10^-3, and 11.2×10^-3 s^-1, respectively. The HSA-SH group of diabetics is more reactive compared with control individuals (rate constants 20.9×10^-3±4.4×10^-3 vs 12.9×10^-3±2.6×10^-3 s^-1, P<0.05). Recovery values of the SH group obtained after chromatography of HSA with bound stearic acid ranged from 110 to 140%, while those for defatted HSA were from 98.5 to 101.7%. Thus, HSA-bound FA leads to an increase of HSA-SH content and a contribution to total serum thiols, which make the determination of the thiol group unreliable.     

Molecular Dynamics as a Mathematical Mapping. III. Efficient Evaluation of the Differentiable Force Functions and Their Derivatives

Abstract

The fully continuous and differentiable framework for performing molecular dynamics calculations introduced in parts I and II of this paper [1,2] requires the evaluation of rather complex force functions and their spatial partial derivatives. This paper presents an efficient interpolation scheme for the evaluation of these quantities over a finite spatial domain.

The modified force function is approximated by a linear combination of Hermite cubic basis functions such that both the interpolant of the force and its spatial derivatives are continuous across the grid boundaries. In order to achieve better accuracy for a given grid size, a nonuniform rectilinear grid is constructed via iterative refinement procedure. The latter guarantees the accuracy of the force computed by interpolation within any specified tolerance > ε O.

For many potential functions of practical interest, it is possible for polynomial interpolants to be constructed for parts of the force functions which are independent of the potential parameters and system density (the so-called “separable force functions”). In such cases, a single interpolation grid which is applicable for a wide range of potential parameters and system densities can be constructed a priori.     

Leptin Receptor Gene Variation Predicts Weight Change in Subjects with Impaired Glucose Tolerance

The leptin receptor (OB‐R) gene is a promising candidate gene for type 2 diabetes, because leptin and its receptor play an important role in insulin secretion and the development of obesity. Therefore, we studied whether the pentanucleotide insertion polymorphism of the 3′‐untranslated region (3′UTR) of the OB‐R gene has an influence on the conversion from impaired glucose tolerance (IGT) to type 2 diabetes in the STOP‐Noninsulin‐Dependent Diabetes Mellitus trial. The STOP trial was a longitudinal, double‐blind, placebo‐controlled randomized trial that included 1429 subjects with IGT from high‐risk populations. Using the restriction fragment length polymorphism method, we genotyped 770 subjects whose DNA was available for the insertion/deletion polymorphism of the 3′UTR of the OB‐R gene. We did not find a relationship between the OB‐R polymorphism and the conversion from IGT to type 2 diabetes (p = 0.747). However, the insertion allele was associated with a significant reduction in weight (p = 0.016), BMI (p = 0.009), and waist circumference (p = 0.006) in all subjects. Women carrying the I allele had a larger waist circumference change (p = 0.036), whereas men lost more weight and had a greater decrease in BMI. The pentanucleotide insertion/deletion polymorphism in the 3′UTR of the OB‐R gene did not influence the conversion to type 2 diabetes in obese patients with IGT. However, this polymorphism was associated with a significant weight change, suggesting that it may potentially modulate the risk for type 2 diabetes.