Leukocyte function-associated antigen 1-mediated adhesion stability is dynamically regulated through affinity and valency during bond formation with intercellular adhesion molecule-1

Neutrophil rolling and transition to arrest on inflamed endothelium are dynamically regulated by the affinity of the beta(2) integrin CD11a/CD18 (leukocyte function associated antigen 1 (LFA-1)) for binding intercellular adhesion molecule (ICAM)-1. Conformational shifts are thought to regulate molecular affinity and adhesion stability. Also critical to adhesion efficiency is membrane redistribution of active LFA-1 into dense submicron clusters where multimeric interactions occur. We examined the influences of affinity and dimerization of LFA-1 on LFA-1/ICAM-1 binding by engineering a cell-free model in which two recombinant LFA-1 heterodimers are bound to respective Fab domains of an antibody attached to latex microspheres. Binding of monomeric and dimeric ICAM-1 to dimeric LFA-1 was measured in real time by fluorescence flow cytometry. ICAM-1 dissociation kinetics were measured while LFA-1 affinity was dynamically shifted by the addition of allosteric small molecules. High affinity LFA-1 dissociated 10-fold faster when bound to monomeric compared with dimeric ICAM-1, corresponding to bond lifetimes of 25 and 330 s, respectively. Downshifting LFA-1 into an intermediate affinity state with the small molecule I domain allosteric inhibitor IC487475 decreased the difference in dissociation rates between monomeric and dimeric ICAM-1 to 4-fold. When LFA-1 was shifted into the low affinity state by lovastatin, both monomeric and dimeric ICAM-1 dissociated in less than 1 s, and the dissociation rates were within 50% of each other. These data reveal the respective importance of LFA-1 affinity and proximity in tuning bond lifetime with ICAM-1 and demonstrate a nonlinear increase in the bond lifetime of the dimer versus the monomer at higher affinity.     

Momordicatin purified from fruits of Momordica charantia is effective to act as a potent antileishmania agent

Aqueous extract of the green fruits of the Indian plant Momordica charantia and purified Momordicatin structurally established as 4-(o-carboethoxyphenyl) butanol were evaluated in vitro and in vivo against kala-azar caused by Leishmania donovani. 50% inhibitory concentration (IC₅₀) against Leishmania promastigotes in vitro for the crude extract and momordicatin were 0.6 mg/L and 0.02 mg/L, respectively. When administered in the hamster model of visceral leishmaniasis, 100% parasite clearance was achieved at a dose of 300 mg/kg body weight of crude extract and 10 mg/kg body weight of Momordicatin. Fe containing parasite superoxide dismutase (SOD) was totally inhibited when treated with 0.72 mg/L crude extract and 0.20 mg/L Momordicatin, respectively, whereas Cu–Zn containing SOD present in host remained unaffected. Results reveal that the mode of action of these newly found antileishmanial agents is mediated through inhibiting parasite SOD which is one of the key enzymes of the oxidative burst. It may be proposed from the present study that both crude extract of Momordica charantia and Momordicatin obtained from the fruits of the said plant may be considered as potential candidates towards developing new chemotherapeutics against leishmaniasis.     

E4F and ATF, two transcription factors that recognize the same site, can be distinguished both physically and functionally: a role for E4F in E1A trans activation.

Previous experiments have identified an element in the adenovirus E4 promoter that is critical for E1A-dependent trans activation and that can confer inducibility to a heterologous promoter. This DNA element is a recognition site for multiple nuclear factors, including ATF, which is likely a family of DNA-binding factors with similar DNA recognition properties. However, ATF activity was found not to be altered in any demonstrable way as a result of adenovirus infection. In contrast, another factor that recognizes this element, termed E4F, was found at only very low levels in uninfected cells but was increased markedly upon adenovirus infection, as measured in DNA-binding assays. Although both the ATF activity and the E4F activity recognized and bound to the same two sites in the E4 promoter, they differed in their sequence recognition of these sites. Furthermore, E4F bound only to a small subset of the ATF recognition sites; for instance, E4F did not recognize the ATF sites in the E2 or E3 promoters. Various E4F and ATF binding sites were inserted into an expression vector and tested by cotransfection assays for responsiveness to E1A. We found that a sequence capable of binding E4F could confer E1A inducibility. In contrast, a sequence that could bind ATF but not E4F did not confer E1A inducibility. We also found that E4F formed a stable complex with the E4 promoter, whereas the ATF DNA complex was unstable and rapidly dissociated. We conclude that the DNA-binding specificity of E4F as well as the alterations in DNA-binding activity of E4F closely correlates with E1A stimulation of the E4 promoter.     

Fine Mapping Seronegative and Seropositive Rheumatoid Arthritis to Shared and Distinct HLA Alleles by Adjusting for the Effects of Heterogeneity

Despite progress in defining human leukocyte antigen (HLA) alleles for anti-citrullinated-protein-autoantibody-positive (ACPA⁺) rheumatoid arthritis (RA), identifying HLA alleles for ACPA-negative (ACPA⁻) RA has been challenging because of clinical heterogeneity within clinical cohorts. We imputed 8,961 classical HLA alleles, amino acids, and SNPs from Immunochip data in a discovery set of 2,406 ACPA⁻ RA case and 13,930 control individuals. We developed a statistical approach to identify and adjust for clinical heterogeneity within ACPA⁻ RA and observed independent associations for serine and leucine at position 11 in HLA-DRβ1 (p = 1.4 × 10⁻¹³, odds ratio [OR] = 1.30) and for aspartate at position 9 in HLA-B (p = 2.7 × 10⁻¹², OR = 1.39) within the peptide binding grooves. These amino acid positions induced associations at HLA-DRB1^∗03 (encoding serine at 11) and HLA-B^∗08 (encoding aspartate at 9). We validated these findings in an independent set of 427 ACPA⁻ case subjects, carefully phenotyped with a highly sensitive ACPA assay, and 1,691 control subjects (HLA-DRβ1 Ser11+Leu11: p = 5.8 × 10⁻⁴, OR = 1.28; HLA-B Asp9: p = 2.6 × 10⁻³, OR = 1.34). Although both amino acid sites drove risk of ACPA⁺ and ACPA⁻ disease, the effects of individual residues at HLA-DRβ1 position 11 were distinct (p < 2.9 × 10⁻¹⁰⁷). We also identified an association with ACPA⁺ RA at HLA-A position 77 (p = 2.7 × 10⁻⁸, OR = 0.85) in 7,279 ACPA⁺ RA case and 15,870 control subjects. These results contribute to mounting evidence that ACPA⁺ and ACPA⁻ RA are genetically distinct and potentially have separate autoantigens contributing to pathogenesis. We expect that our approach might have broad applications in analyzing clinical conditions with heterogeneity at both major histocompatibility complex (MHC) and non-MHC regions.     

Fine Mapping Major Histocompatibility Complex Associations in Psoriasis and Its Clinical Subtypes

Psoriasis vulgaris (PsV) risk is strongly associated with variation within the major histocompatibility complex (MHC) region, but its genetic architecture has yet to be fully elucidated. Here, we conducted a large-scale fine-mapping study of PsV risk in the MHC region in 9,247 PsV-affected individuals and 13,589 controls of European descent by imputing class I and II human leukocyte antigen (HLA) genes from SNP genotype data. In addition, we imputed sequence variants for MICA, an MHC HLA-like gene that has been associated with PsV, to evaluate association at that locus as well. We observed that HLA-C^∗06:02 demonstrated the lowest p value for overall PsV risk (p = 1.7 × 10⁻³⁶⁴). Stepwise analysis revealed multiple HLA-C^∗06:02-independent risk variants in both class I and class II HLA genes for PsV susceptibility (HLA-C^∗12:03, HLA-B amino acid positions 67 and 9, HLA-A amino acid position 95, and HLA-DQα1 amino acid position 53; p < 5.0 × 10⁻⁸), but no apparent risk conferred by MICA. We further evaluated risk of two major clinical subtypes of PsV, psoriatic arthritis (PsA; n = 3,038) and cutaneous psoriasis (PsC; n = 3,098). We found that risk heterogeneity between PsA and PsC might be driven by HLA-B amino acid position 45 (p_omnibus = 2.2 × 10⁻¹¹), indicating that different genetic factors underlie the overall risk of PsV and the risk of specific PsV subphenotypes. Our study illustrates the value of high-resolution HLA and MICA imputation for fine mapping causal variants in the MHC.     

Effect of polyamines on in vitro somatic embryogenesis in <Emphasis Type="Italic">Momordica</Emphasis><Emphasis Type="Italic">charantia</Emphasis> L.

The effects of exogenous polyamines (PAs) on enhancement of somatic embryogenic calli was investigated in Momordica charantia L. in vitro. Induction of somatic embryogenesis (SE) in leaf explants of M. charantia after 21 days of culture in Murashige and Skoog (MS) medium was determined using scanning electron microscopy. During induction of SE there were high titers of Putrescine (Put) as compared to Spermidine (Spd) and Spermine (Spm), a prerequisite for cell division. Addition of PAs to the embryogenic media resulted in an increase in fresh weights and number of somatic embryos of 21-day old embryogenic calli. Put at a concentration of 1 mM showed maximum increase in fresh weights of embryogenic calli (5 fold) and number of somatic embryos produced per 0.2 g of callus (2.5 fold). Moreover addition of PAs to the embryogenic media resulted in lowering of endogenous free PA level of 21-day old embryogenic calli. Thus, when the media was supplemented with exogenous PAs a positive correlation was found to exist between Somatic Embryogenesis enhancement and decrease in endogenous free PA levels.