VERNAL CONJUNCTIVITIS AS AN ATOPIC DISEASE

In a study of 30 cases of vernal conjunctivitis, antibodies to grass pollen were demonstrated in 16 of 29 patients tested by direct skin tests, in 11 of 30 tested by the Prausnitz-Kustner method and in 22 of 30 by the bis-diazotized benzidine hemagglutination method.A personal history of major atopic disease was found in 13 of 27 patients, and a family history of atopic disease in 16 of 26 patients questioned.Conjunctival eosinophilia was found in all cases. Results of the study indicated that vernal conjunctivitis is an atopic disease.     

Regulation of the Minichromosome Maintenance Protein 3 (MCM3) Chromatin Binding by the Prolyl Isomerase Pin1

Human Pin1 is a peptidyl prolyl cis/trans isomerase with a unique preference for phosphorylated Ser/Thr-Pro substrate motifs.Here we report that MCM3 (minichromosome maintenance complex component 3) is a novel target of Pin1. MCM3 interacts directly with the WW domain of Pin1. Proline-directed phosphorylation of MCM3 at S112 and T722 are crucial for the interaction with Pin1. MCM3 as a subunit of the minichromosome maintenance heterocomplex MCM2–7 is part of the pre-replication complex responsible for replication licensing and is implicated in the formation of the replicative helicase during progression of replication. Our data suggest that Pin1 coordinates phosphorylation-dependently MCM3 loading onto chromatin and its unloading from chromatin, thereby mediating S phase control.     

Suppression of EGFR autophosphorylation by FKBP12

FK506 binding proteins (FKBPs) represent a subfamily of peptidyl prolyl cis/trans isomerases that can control receptor-mediated intracellular signaling. The prototypic PPIase FKBP12 functionally interacts with EGFR. FKBP12 was shown to inhibit EGF-induced EGFR autophosphorylation with all internal phosphorylation sites equally affected. The inhibition of EGFR catalytic activity is conducted by targeting the EGFR kinase domain. The change of intracellular FKBP12 levels resulted in a change of EGFR autophosphorylation level. Collectively, our results demonstrate that FKBP12 forms an endogenous inhibitor of EGFR phosphorylation directly involved in the control of cellular EGFR activity.     

Hormonal influence on the secretory immune system of the eye: androgen modulation of IgA levels in tears of rats

The objective of the present studies was to determine whether hormones influence the level of IgA in tears of rats. Our results demonstrated that IgA concentrations in tears of male rats were significantly higher than those of females. This difference appeared to be due to an effect of androgens. Castration of male rats led to a significant decline in the content of tear IgA. Administration of testosterone offset this response and stimulated a time-dependent accumulation of IgA in tears of orchiectomized rats. This hormone action was only elicited by androgens, and not by other classes of steroid hormones. The testosterone-induced increase in tear IgA levels may have involved enhanced local production of IgA, and was found to occur in parallel with an elevation in tear secretory component concentrations. These findings indicate that androgens may regulate the ocular secretory immune system of the rat.     

Characterization of a localized basophil hypersensitivity lesion in guinea pig conjunctiva

Basophils accumulate in response to antigen challenge in cutaneous basophil hypersensitivity (CBH) reactions. Two ocular diseases, vernal conjunctivitis and contact-lens-associated conjunctivitis, are also characterized by this histopathology. We have refined a model previously developed in guinea pig conjunctiva by precisely defining the site of antigen injection and correlating the site with the clinical and histologic changes. Guinea pigs were primed by an intradermal injection of keyhole limpet hemocyanin (KLH) in the flank and challenged (Day 6) by injection of a small bolus of KLH just under the conjunctival epithelium. Twenty-four hours later histologic examination showed a perivascular infiltrate of inflammatory cells containing large numbers of basophils. Eosinophils, neutrophils, macrophages, and mast cells were also seen. Serial sections of the reaction site showed discrete boundaries. At all sites examined in antigen-challenged tissues, there were significantly more basophils than in control-injected conjunctiva. Insertion of a sterile needle or injection of PBS or KLH into normal conjunctiva induced a significant increase in neutrophils and some macrophages. Injection of graded doses of antigen into the conjunctiva of primed animals, resulted in a dose-dependent increase in basophils up to 50 micrograms KLH (optimal dose).     

Sfp-Type 4′-Phosphopantetheinyl Transferase Is Indispensable for Fungal Pathogenicity

In filamentous fungi, Sfp-type 4′-phosphopantetheinyl transferases (PPTases) activate enzymes involved in primary (α-aminoadipate reductase [AAR]) and secondary (polyketide synthases and nonribosomal peptide synthetases) metabolism. We cloned the PPTase gene PPT1 of the maize anthracnose fungus Colletotrichum graminicola and generated PPTase-deficient mutants (Δppt1). Δppt1 strains were auxotrophic for Lys, unable to synthesize siderophores, hypersensitive to reactive oxygen species, and unable to synthesize polyketides (PKs). A differential analysis of secondary metabolites produced by wild-type and Δppt1 strains led to the identification of six novel PKs. Infection-related morphogenesis was affected in Δppt1 strains. Rarely formed appressoria of Δppt1 strains were nonmelanized and ruptured on intact plant. The hyphae of Δppt1 strains colonized wounded maize (Zea mays) leaves but failed to generate necrotic anthracnose disease symptoms and were defective in asexual sporulation. To analyze the pleiotropic pathogenicity phenotype, we generated AAR-deficient mutants (Δaar1) and employed a melanin-deficient mutant (M1.502). Results indicated that PPT1 activates enzymes required at defined stages of infection. Melanization is required for cell wall rigidity and appressorium function, and Lys supplied by the AAR1 pathway is essential for necrotrophic development. As PPTase-deficient mutants of Magnaporthe oryzea were also nonpathogenic, we conclude that PPTases represent a novel fungal pathogenicity factor.     

LRRK2 dynamics analysis identifies allosteric control of the crosstalk between its catalytic domains

The 2 major molecular switches in biology, kinases and GTPases, are both contained in the Parkinson disease–related leucine-rich repeat kinase 2 (LRRK2). Using hydrogen–deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations, we generated a comprehensive dynamic allosteric portrait of the C-terminal domains of LRRK2 (LRRK2_RCKW). We identified 2 helices that shield the kinase domain and regulate LRRK2 conformation and function. One helix in COR-B (COR-B Helix) tethers the COR-B domain to the αC helix of the kinase domain and faces its activation loop, while the C-terminal helix (Ct-Helix) extends from the WD40 domain and interacts with both kinase lobes. The Ct-Helix and the N-terminus of the COR-B Helix create a “cap” that regulates the N-lobe of the kinase domain. Our analyses reveal allosteric sites for pharmacological intervention and confirm the kinase domain as the central hub for conformational control.

The Parkinson’s disease-related protein LRRK2 contains the two major molecular switches in biology; a kinase and a GTPase. This study uses hydrogen-deuterium exchange mass-spectrometry and molecular dynamics simulations to explore the conformational space of the four C-terminal domains of LRRK2, highlighting two essential regulatory helices that control LRRK2 dynamics.