Immunity and autoimmunity to dsDNA and chromatin – the role of immunogenic DNA-binding proteins and nuclease deficiencies

Loss of immunological tolerance results in autoimmunity and may finally end in autoimmune disorders. For an autoimmune response against chromatin, autologous chromatin (nucleosomes) is assumed to activate both chromatin-specific B and T cells with a resulting anti-chromatin antibody response. As only fragmental elements of this process have been described, we do not have the full insight to justify this model in vivo. Early experimental immunization with methylated bovine serum albumin-DNA complexes elicited antibodies to various forms of synthetic ssDNA/dsDNA, but notably not to mammalian dsDNA. Thus, for a long time with intense research, the general result was that all forms of ssDNA and dsDNA, but mammalian B helical DNA, had an immunogenic potential. Summarizing these results, a preliminary conclusion was settled, saying that mammalian dsDNA was not immunogenic while other forms of DNA were really immunogenic in the situation where they were in complex with proteins. Recent studies have focused on nuclease deficiencies as a condition where chromatin may be presented to the immune system in an immunogenic form. However, although such deficiencies may provide information as to how chromatin may be exposed and targeted by relevant antibodies, data demonstrate that nuclease deficiencies is not in general correlated with autoimmunity to components of chromatin. This review discusses these topics, and provides information that may explain processes that account for anti-dsDNA antibody responses in vivo.     

Allelic exclusion of immunoglobulin genes: models and mechanisms

Summary: The allelic exclusion of immunoglobulin (Ig) genes is one of the most evolutionarily conserved features of the adaptive immune system and underlies the monospecificity of B cells. While much has been learned about how Ig allelic exclusion is established during B-cell development, the relevance of monospecificity to B-cell function remains enigmatic. Here, we review the theoretical models that have been proposed to explain the establishment of Ig allelic exclusion and focus on the molecular mechanisms utilized by developing B cells to ensure the monoallelic expression of Igκ and Igλ light chain genes. We also discuss the physiological consequences of Ig allelic exclusion and speculate on the importance of monospecificity of B cells for immune recognition.     

Silent information regulator 3: the Goldilocks of the silencing complex

A recent explosion of work surrounds the interactions between Sir3p (Silent Information Regulator 3) and chromatin. We review here the Sir3p functions related to its role in silencing in Saccharomyces cerevisiae. This unusual protein, which is absolutely required for silencing, is distantly related to the highly conserved replication initiator Orc1p, but is itself phylogenetically limited to “post-genome-duplicated” budding yeasts. Several recent studies revise earlier models for Sir3p action. Specifically, the N-terminal bromo-adjacent homology (BAH) domain plays a now well-defined role in silencing, and a picture is emerging in which both termini of Sir3p bind two locations on the nucleosome: (1) the loss of ribosomal DNA silencing (LRS) surface in the nucleosome core, and (2) the N-terminal histone tails for effective silencing at telomeres. We relate Sir3p structure and function, and summarize recent molecular studies of Sir3p/chromatin binding, Sir3p/Dot1p competition, and the possible role of O-Acetyl ADP ribose (O-AADPR) in Sir3p/chromatin binding. We emphasize recent genetic data that provide important new insights and settle controversies created by in vitro work. Finally, we synthesize these ideas to revise the model for how Sir3p mediates silent chromatin formation in yeast, in part through its affinity for the LRS region of the nucleosome, which must be “just right.”     

Biomimetic DNA nanoballs for oligonucleotide delivery

Here, we designed biomimetic DNA nanoballs for delivery of multiple antisense oligonucleotides (ASOs). DNA templates with ASOs-complementary sequences were amplified by rolling circle amplification (RCA). RCA products were loaded with two types of ASOs by hybridization, condensed using adenovirus-derived Mu peptide, and coated with hyaluronic acid (HA) for delivery into CD44-overexpressing tumor cells. HA-coated, Mu peptide-condensed, dual ASO-loaded DNA nanoballs (HMA nanoballs) showed considerable cellular entry of Cy5-incorporated RCA product DNA and fluorescent ASOs, whereas Mu peptide-condensed, dual ASO-loaded DNA nanoballs (MA nanoballs) revealed limited uptake. Dual ASOs, Dz13 and OGX-427, delivered by HMA nanoballs could reduce the levels of protein targets and exert anticancer effects. Enhanced tumor distribution was observed for fluorescent HMA nanoballs than the corresponding MA nanoballs. Upon intravenous co-administration with doxorubicin, HMA nanoballs exerted the greatest anti-tumor effects among the groups. These results suggest HMA nanoballs as a nanoplatform for sequence-specific delivery of multiple ASOs and other functional oligonucleotides.     

Regulation of Mec1 kinase activity by the SWI/SNF chromatin remodeling complex

ATP-dependent chromatin remodeling complexes alter chromatin structure through interactions with chromatin substrates such as DNA, histones, and nucleosomes. However, whether chromatin remodeling complexes have the ability to regulate nonchromatin substrates remains unclear. Saccharomyces cerevisiae checkpoint kinase Mec1 (ATR in mammals) is an essential master regulator of genomic integrity. Here we found that the SWI/SNF chromatin remodeling complex is capable of regulating Mec1 kinase activity. In vivo, Mec1 activity is reduced by the deletion of Snf2, the core ATPase subunit of the SWI/SNF complex. SWI/SNF interacts with Mec1, and cross-linking studies revealed that the Snf2 ATPase is the main interaction partner for Mec1. In vitro, SWI/SNF can activate Mec1 kinase activity in the absence of chromatin or known activators such as Dpb11. The subunit requirement of SWI/SNF-mediated Mec1 regulation differs from that of SWI/SNF-mediated chromatin remodeling. Functionally, SWI/SNF-mediated Mec1 regulation specifically occurs in S phase of the cell cycle. Together, these findings identify a novel regulator of Mec1 kinase activity and suggest that ATP-dependent chromatin remodeling complexes can regulate nonchromatin substrates such as a checkpoint kinase.     

Synthesis of 4‐[2‐(3,4‐dimethoxybenzyl)cyclopentyl]‐1,2‐dimethoxybenzene Derivatives and Evaluations of Their Carbonic Anhydrase Isoenzymes Inhibitory Effects

Rearrangement of 1,6‐bis(3,4‐dimethoxyphenyl)hexane‐1,6‐dione ( 8 ) gave two isomeric products having cyclopentene moiety. Starting from the major product (3,4‐dimethoxyphenyl)[2‐(3,4‐dimethoxyphenyl)cyclopent‐1‐en‐1‐yl]methanone ( 11 ), eight new compounds ( 16–23) were obtained by the reactions such as reduction (by catalytic hydrogenation and NaBH₄), nitration, 1,4‐addition, bromination, and esterification reactions. Carbonic anhydrases (CA, E.C.4.2.1.1) are ubiquitous metalloenzymes present in almost all living organism that catalyze a simple reaction, the conversion of carbon dioxide (CO₂) and water (H₂O) to bicarbonate ion (HCO₃^?) and a proton (H⁺). CA isoenzymes I and II (hCA I and II) inhibition effects of synthesized eleven new and four known compounds ( 8–13 and 15–23 ) were investigated. Inhibition studies of the hCA I and II with 4‐[2‐(3,4‐dimethoxybenzyl)cyclopentyl]‐1,2‐dimethoxybenzene derivatives revealed that they possess effective inhibitory potency. Cytosolic hCA I and II isoenzymes were potently inhibited by new synthesized 4‐[2‐(3,4‐dimethoxybenzyl)cyclopentyl]‐1,2‐dimethoxybenzene derivatives with K_is in the range of 313.16–1537.00 nm against hCA I and in the range of 228.31–1927.31 nm against hCA II, respectively.     

Comparison of volumetric analysis of lateral compaction & three different vertically compacted thermoplasticized obturation techniques using spiral CT – An in vitro study

The objective of endodontic obturation is to provide a complete seal along the length of root canal system. This study was conducted to compare volumetrically, the laterally compressed gutta percha with three different vertically compacted thermoplasticized obturating systems using spiral CT, an in vitro study. Materials and methods included preparation of access cavities in 20 single rooted mandibular first premolars, followed by cleaning and shaping. Obturation was done with different thermoplasticized obturation systems: Group A – cold lateral; Group B – Calamus dual; Group C – E & Q Plus; Group D – Obtura II. Volume analysis was done using spiral computed tomography (CT). The percentage difference was calculated & statistically analyzed using one-way ANOVA and multiple comparisons between groups using Tukey's HSD test. The results of study has shown the statistical significance between all the groups with the p-value <0.0001. Multiple comparisons between different groups show statistical significance in all groups except Group C and Group D. With this significance in results, it can be concluded that Calamus dual showed the highest volume of obturation, followed by Obtura II and E & Q Plus. The least volume of obturation was observed in cold lateral condensation technique.