Small molecule inhibitors of protein interaction with glycosaminoglycans (SMIGs), a novel class of bioactive agents with anti-inflammatory properties

Background

Small molecule inhibitors of biologically important protein–glycosaminoglycan (GAG) interactions have yet to be identified.

Methods

Compound libraries were screened in an assay of L-selectin–IgG binding to heparin (a species of heparan sulfate [HS-GAG]). Hits were validated, IC-50s established and direct binding of hits to HS-GAGs was investigated by incubating compounds alone with heparin. Selectivity of inhibitors was assessed in 11 different protein-GAG binding assays. Anti-inflammatory activity of selected compounds was evaluated in animal models.

Results

Screening identified a number of structurally-diverse planar aromatic cationic amines. Scaffolds similar to known GAG binders, chloroquine and tilorone, were also identified. Inhibitors displayed activity also against bovine kidney heparan sulfate. Direct binding of compounds to GAGs was verified by incubating compounds with heparin alone. Selectivity of inhibitors was demonstrated in a panel of 11 heparin binding proteins, including selectins, chemokines (IL-8, IP-10), Beta Amyloid and cytokines (VEGF, IL-6). A number of selected lead compounds showed dose-dependent efficacy in peritonitis, paw edema and delayed type hypersensitivity.

Conclusions

A new class of compounds, SMIGs, inhibits protein–GAG interaction by direct binding to GAGs. Although their IC-50s were in the low micro-molar range, SMIGs binding to HS-GAGs appeared to be stable in physiological conditions, indicating high avidity binding. SMIGs may interfere with major checkpoints for inflammatory and autoimmune events.

General significance

SMIGs are a class of structurally-diverse planar aromatic cationic amines that have an unusual mode of action — inhibiting protein–GAG interactions via direct and stable binding to GAGs. SMIGs may have therapeutic potential in inflammatory and autoimmune disorders.     

A comparative study of PKH67, DiI,and BrdU labeling techniques for tracing rat mesenchymal stem cells

Mesenchymal stem cells (MSCs) have generated a great deal of promise as a potential source of cells for cell-based therapies. Various labeling techniques have been developed to trace MSC survival, migration, and behavior in vitro or in vivo. In the present study, we labeled MSCs derived from rat bone marrow (rMSCs) with florescent membrane dyes PKH67 and DiI, and with nuclear labeling using 5 μM BrdU and 10 μM BrdU. The cells were then cultured for 6 d or passaged (1–3 passages). The viability of rMSCs, efficacy of fluorescent expression, and transfer of the dyes were assessed. Intense fluorescence in rMSCs was found immediately after membrane labeling (99.3?±?1.6% PKH67+ and 98.4?±?1.7% DiI+) or after 2 d when tracing of nuclei was applied (91.2?±?4.6% 10 μM BrdU+ and 77.6?±?4.6% 5 μM BrdU+), which remained high for 6 d. Viability of labeled cells was 91?±?3.8% PKH67+, 90?±?1.5% DiI+, 91?±?0.8% 5 μM BrdU+, and 76.9?±?0.9% 10 μM BrdU+. The number of labeled rMSCs gradually decreased during the passages, with almost no BrdU+ nuclei left at final passage 3. Direct cocultures of labeled rMSCs (PKH67+ or DiI+) with unlabeled rMSCs revealed almost no dye transfer from donor to unlabeled recipient cells. Our results confirm that labeling of rMSCs with PKH67 or DiI represents a non-toxic, highly stable, and efficient method suitable for steady tracing of cells, while BrdU tracing is more appropriate for temporary labeling due to decreasing signal over time.     

Composition of Bacterial Assemblages in Different Components of Reed Warbler Nests and a Possible Role of Egg Incubation in Pathogen Regulation

Bacteria play a central role in animal health. Yet, little is known about the acquisition of bacteria and the extent to which bacteria are acquired from different environmental sources. For example, bird nests host diverse bacteria associated with the eggs, nestlings and nesting material, but previous research has typically focussed on only a limited number of nest components at a time. It therefore remains unknown to what extent bacteria are transmitted between these components. Using both molecular and culture techniques, we characterised nest-associated bacterial assemblages throughout the entire nesting cycle of reed warblers by sampling bacteria on eggs before and during incubation, within nestling faeces, and on the nesting material of post-breeding nests. We found that bacterial assemblages clustered by nest component. Yet some overlap existed between nest components, suggesting that bacterial transmission across components is likely to occur. Eggs and nestlings from the same nest harboured more similar bacteria than expected by chance, suggesting an influence of environment or genetics on bacterial assemblages. Bacterial loads were not lower on incubated eggs. Instead, incubation was associated with a change in the structure of assemblages, including a decrease in potentially-harmful Gram-negative bacteria. In addition we show for the first time, that incubation is associated with the complete extinction of harmful haemolytic bacteria. Overall, our study appears to be the first to demonstrate differences in bacterial assemblages between bird nest components. In addition, we highlight the complexity of nest bacterial assemblages and provide new insights into the benefits of incubation.     

ATP analog-sensitive Pat1 protein kinase for synchronous fission yeast meiosis at physiological temperature

To study meiosis, synchronous cultures are often indispensable, especially for physical analyses of DNA and proteins. A temperature-sensitive allele of the Pat1 protein kinase (pat1-114) has been widely used to induce synchronous meiosis in the fission yeast Schizosaccharomyces pombe, but pat1-114-induced meiosis differs from wild-type meiosis, and some of these abnormalities might be due to higher temperature needed to inactivate the Pat1 kinase. Here, we report an ATP analog-sensitive allele of Pat1 [Pat1(L95A), designated pat1-as2] that can be used to generate synchronous meiotic cultures at physiological temperature. In pat1-as2 meiosis, chromosomes segregate with higher fidelity, and spore viability is higher than in pat1-114 meiosis, although recombination is lower by a factor of 2–3 in these mutants than in starvation-induced pat1⁺ meiosis. Addition of the mat-Pc gene improved chromosome segregation and spore viability to nearly the level of starvation-induced meiosis. We conclude that pat1-as2 mat-Pc cells offer synchronous meiosis with most tested properties similar to those of wild-type meiosis.     

Identification and functional characterisation of novel glucokinase mutations causing maturity-onset diabetes of the young in Slovakia

Heterozygous glucokinase (GCK) mutations cause a subtype of maturity-onset diabetes of the young (GCK-MODY). Over 600 GCK mutations have been reported of which ～65% are missense. In many cases co-segregation has not been established and despite the importance of functional studies in ascribing pathogenicity for missense variants these have only been performed for <10% of mutations. The aim of this study was to determine the minimum prevalence of GCK-MODY amongst diabetic subjects in Slovakia by sequencing GCK in 100 Slovakian probands with a phenotype consistent with GCK-MODY and to explore the pathogenicity of identified variants through family and functional studies. Twenty-two mutations were identified in 36 families (17 missense) of which 7 (I110N, V200A, N204D, G258R, F419S, c.580-2A>C, c.1113-1114delGC) were novel. Parental DNA was available for 22 probands (covering 14/22 mutations) and co-segregation established in all cases. Bioinformatic analysis predicted all missense mutations to be damaging. Nine (I110N, V200A, N204D, G223S, G258R, F419S, V244G, L315H, I436N) mutations were functionally evaluated. Basic kinetic analysis explained pathogenicity for 7 mutants which showed reduced glucokinase activity with relative activity indices (RAI) between 0.6 to <0.001 compared to wild-type GCK (1.0). For the remaining 2 mutants additional molecular mechanisms were investigated. Differences in glucokinase regulatory protein (GKRP) -mediated-inhibition of GCK were observed for both L315H & I436N when compared to wild type (IC(50) 14.6±0.1 mM & 20.3±1.6 mM vs.13.3±0.1 mM respectively [p<0.03]). Protein instability as assessed by thermal lability studies demonstrated that both L315H and I436N show marked thermal instability compared to wild-type GCK (RAI at 55°C 8.8±0.8% & 3.1±0.4% vs. 42.5±3.9% respectively [p<0.001]). The minimum prevalence of GCK-MODY amongst Slovakian patients with diabetes was 0.03%. In conclusion, we have identified 22 GCK mutations in 36 Slovakian probands and demonstrate that combining family, bioinformatic and functional studies can aid the interpretation of variants identified by molecular diagnostic screening.     

ER disruption and GFP degradation during non-regenerable transformation of flax with Agrobacterium tumefaciens

Flax is considered as plant species susceptible to Agrobacterium-mediated genetic transformation. In this study, stability of flax transformation by Agrobacterium rhizogenes versus Agrobacterium tumefaciens was tested by using combined selection for antibiotic resistance and visual selection of green fluorescent protein (GFP)-fusion reporter targeted to the endoplasmic reticulum (ER). Transformation with A. rhizogenes was stable for over 2 years, whereas transformation by A. tumefaciens resulted in non-regenerable stable transformation which was restricted solely to transgenic callus and lasted only 6–8 weeks. However, shoots regenerated from this callus appeared to be non-transgenic. Importantly, callus and root cells stably transformed with A. rhizogenes showed typical regular organization and dynamics of ER as visualized by GFP-ER marker. On the other hand, callus cells transformed with A. tumefaciens showed disintegrated ER structure and impaired dynamics which was accompanied with developmental degradation of GFP. Consequently, shoots which regenerated from such callus were all non-transgenic. Possible reasons for this non-regenerable flax transformation by A. tumefaciens are discussed.     

MS/MS library facilitated MRM quantification of native peptides prepared by denaturing ultrafiltration

ABSTRACT: Naturally occurring native peptides provide important information about physiological states of an organism and its changes in disease conditions but protocols and methods for assessing their abundance are not well-developed. In this paper, we describe a simple procedure for the quantification of non-tryptic peptides in body fluids. The workflow includes an enrichment step followed by two-dimensional fractionation of native peptides and MS/MS data management facilitating the design and validation of LC- MRM MS assays. The added value of the workflow is demonstrated in the development of a triplex LC-MRM MS assay used for quantification of peptides potentially associated with the progression of liver disease to hepatocellular carcinoma.