An IRF5 Decoy Peptide Reduces Myocardial Inflammation and Fibrosis and Improves Endothelial Cell Function in Tight-Skin Mice

Interferon regulatory factor 5 (IRF5) has been called a “master switch” for its ability to determine whether cells mount proinflammatory or anti-inflammatory responses. Accordingly, IRF5 should be an attractive target for therapeutic drug development. Here we report on the development of a novel decoy peptide inhibitor of IRF5 that decreases myocardial inflammation and improves vascular endothelial cell (EC) function in tight-skin (Tsk/+) mice. Biolayer interferometry studies showed the Kd of IRF5D for recombinant IRF5 to be 3.72 ± 0.74x10^-6M. Increasing concentrations of IRF5D (0–100 μg/mL, 24h) had no significant effect on EC proliferation or apoptosis. Treatment of Tsk/+ mice with IRF5D (1mg/kg/d subcutaneously, 21d) reduced IRF5 and ICAM-1 expression and monocyte/macrophage and neutrophil counts in Tsk/+ hearts compared to expression in hearts from PBS-treated Tsk/+ mice (p<0.05). EC-dependent vasodilatation of facialis arteries isolated from PBS-treated Tsk/+ mice was reduced (~15%). IRF5D treatments (1mg/kg/d, 21d) improved vasodilatation in arteries isolated from Tsk/+ mice nearly 3-fold (~45%, p<0.05), representing nearly 83% of the vasodilatation in arteries isolated from C57Bl/6J mice (~55%). IRF5D (50μg/mL, 24h) reduced nuclear translocation of IRF5 in myocytes cultured on both Tsk/+ cardiac matrix and C57Bl/6J cardiac matrix (p<0.05). These data suggest that IRF5 plays a causal role in inflammation, fibrosis and impaired vascular EC function in Tsk/+ mice and that treatment with IRF5D effectively counters IRF5-dependent mechanisms of inflammation and fibrosis in the myocardium in these mice.     

Divergence in calls but not songs in the orchard oriole complex: Icterus spurius and I. fuertesi

Birdsong has important functions in attracting and competing for mates, and song characteristics are thought to diverge rapidly during the process of speciation. In contrast, other avian vocalizations that may have non‐reproductive functions, such as calls, are thought to be more evolutionarily conserved and may diverge more slowly among taxa. This study examines differences in both male song and an acoustically simpler vocalization, the ‘jeet’ call, between two closely related taxa, Icterus spurius and I. fuertesi. A previous study comparing song syllable type sharing within and between I. spurius and I. fuertesi indicated that their songs do not differ discernibly. Here we measured 18 acoustic characteristics of their songs and found strong evidence supporting this prior finding. In contrast, we measured 17 acoustic characteristics of jeet calls and found evidence of significant divergence between the two taxa in many of these characteristics. Calls in I. fuertesi have a longer duration, a larger frequency bandwidth, a lower minimum frequency, a lower beginning frequency, and greater levels of both frequency and amplitude modulation in comparison to the calls of I. spurius. In addition, I. fuertesi calls contain two distinct parts, while the calls of I. spurius have only one part. Thus, we find evidence of divergence in the calls of the two taxa but not their songs challenging the widespread assumption that complex bird song evolves more rapidly than other types of vocalizations. Understanding divergence in multiple vocalization types as well as other behavioral, morphological, and molecular traits is important to understanding the earliest stages of speciation.     

DNA-Protein Complex in Circular DNA from Phage ϕ29

THE DNA of the B. subtilis phage ?29 has been described as unpermuted linear duplex molecules¹ of molecular weight 11 × 10⁶, but the formation of circular molecules has also been indicated, suggesting the existence of cohesive ends^1,2.     

The chromatin structure of an actively expressed, single copy yeast gene.

When the yeast galactokinase gene is not active (repressed, not expressed, quiescent), there is an exceptionally regular nucleosome array on coding sequence galactokinase chromatin, as shown by both denaturing and non-denaturing gel analysis of staphylococcal nuclease digests. Expression of the gene results in a limited smearing of the nucleosome repeat peaks and an increase in interpeak DNA, appearing as a regular ladder of DNA bands on denaturing gels. On non-denaturing gels the pattern is more complex and molecular weight dependent. These data suggest an increase in intracore particle DNA accessibility, allowing staphylococcal nuclease to digest throughout the nucleosome in expressed chromatin. Comparison to bulk chromatin and to an operationally inactive gene (35S rDNA) show that the alteration is specific to expressed chromatin. In contrast, DNase I shows no differences in the digestion of the gene specific chromatin in expressed or inactive states.     

A giant type I polyketide synthase participates in zygospore maturation in Chlamydomonas reinhardtii

Polyketide synthases (PKSs) occur in many bacteria, fungi and plants. They are highly versatile enzymes involved in the biosynthesis of a large variety of compounds including antimicrobial agents, polymers associated with bacterial cell walls and plant pigments. While harmful algae are known to produce polyketide toxins, sequences of the genomes of non‐toxic algae, including those of many green algal species, have surprisingly revealed the presence of genes encoding type I PKSs. The genome of the model alga Chlamydomonas reinhardtii (Chlorophyta) contains a single type I PKS gene, designated PKS1 (Cre10.g449750), which encodes a giant PKS with a predicted mass of 2.3 MDa. Here, we show that PKS1 is induced in 2‐day‐old zygotes and is required for their development into zygospores, the dormant stage of the zygote. Wild‐type zygospores contain knob‐like structures (~50 nm diameter) that form at the cell surface and develop a central cell wall layer; both of these structures are absent from homozygous pks1 mutants. Additionally, in contrast to wild‐type zygotes, chlorophyll degradation is delayed in homozygous pks1 mutant zygotes, indicating a disruption in zygospore development. In agreement with the role of the PKS in the formation of the highly resistant zygospore wall, mutant zygotes have lost the formidable desiccation tolerance of wild‐type zygotes. Together, our results represent functional analyses of a PKS mutant in a photosynthetic eukaryotic microorganism, revealing a central function for polyketides in the sexual cycle and survival under stressful environmental conditions.     

On the occurrence of nucleosome phasing in chromatin.

We have found that DNAase I digestion of yeast, HeLa and chicken erythrocyte nuclei produces a pattern of DNA fragments spaced 10 bases apart and extending to at least 300 bases. This "extended ladder" of DNA fragments is most clearly seen with yeast, and least clearly with chicken erythrocytes. The appearance of regular and discrete bands at sizes much larger than the repeat size shows that the core particles (140 bp of DNA + H2A, H2B, H3 H4) in at least some fraction of chromatin are spaced in a particular fashion, by discrete lengths of spacer DNA, and not randomly. Based on the abundance of small repeats in yeast and from experiments with nucleosome oligomers, we conclude that the extended ladder and nucleosomal phasing probably arise mainly from regions in the chromatin in which nucleosome cores are closely packed or closely spaced (140-160 bp X n). Contributions from less closely packed but still accurately phased nucleosomes, however, cannot be entirely excluded.     

Quantitative analysis of the digestion of yeast chromatin by staphylococcal nuclease.

The DNA in intranuclear yeast chromatin is protected from rapid staphylococcal nuclease degradation so as to yield an oligomeric series of DNA sizes. The course of production and disappearance of the various oligomers agrees quantitatively with a theory of random cleavage by the enzyme at uniformly susceptible sites. The sizes of the oligomers are integral repeats of a basic size, about 160 base pairs, and 80-90% of the yeast genome is involved in this repeating structure. Within this repeat there exists a 140 base pair core of more nuclease-resistant DNA. During the course of digestion, the sizes of the oligomers decrease continuously. The widths of the distribution of DNA sizes increase in order: monomer (1 X repeat size, half width = 5-7 base pairs) less than dimer (2 X repeat size, half width = 30 base pairs) less than trimer (3 X repeat size, half width = 40-45 base pairs). The yeast genome thus seems to have variable spacing of the nucleaseresistant cores, to produce the average repeat size of about 160 base pairs. Also, the presence of more than one species of monomer and dimer at certain times of digestion suggests a possible heterogeneity in the subunit structure.     

Lipid dependence of diadinoxanthin solubilization and de-epoxidation in artificial membrane systems resembling the lipid composition of the natural thylakoid membrane

In the present study, the solubility and enzymatic de-epoxidation of diadinoxanthin (Ddx) was investigated in three different artificial membrane systems: (1) Unilamellar liposomes composed of different concentrations of the bilayer forming lipid phosphatidylcholine (PC) and the inverted hexagonal phase (H_II phase) forming lipid monogalactosyldiacylglycerol (MGDG), (2) liposomes composed of PC and the H_II phase forming lipid phosphatidylethanolamine (PE), and (3) an artificial membrane system composed of digalactosyldiacylglycerol (DGDG) and MGDG, which resembles the lipid composition of the natural thylakoid membrane. Our results show that Ddx de-epoxidation strongly depends on the concentration of the inverted hexagonal phase forming lipids MGDG or PE in the liposomes composed of PC or DGDG, thus indicating that the presence of inverted hexagonal structures is essential for Ddx de-epoxidation. The difference observed for the solubilization of Ddx in H_II phase forming lipids compared with bilayer forming lipids indicates that Ddx is not equally distributed in the liposomes composed of different concentrations of bilayer versus non-bilayer lipids. In artificial membranes with a high percentage of bilayer lipids, a large part of Ddx is located in the membrane bilayer. In membranes composed of equal proportions of bilayer and H_II phase forming lipids, the majority of the Ddx molecules is located in the inverted hexagonal structures. The significance of the pigment distribution and the three-dimensional structure of the H_II phase for the de-epoxidation reaction is discussed, and a possible scenario for the lipid dependence of Ddx (and violaxanthin) de-epoxidation in the native thylakoid membrane is proposed.     

Developmental distribution of CaM kinase II in the antennal lobe of the sphinx moth <Emphasis Type="Italic">Manduca sexta</Emphasis>

The antennal lobe (primary olfactory center of insects) is completely reorganized during metamorphosis. This reorganization is accompanied by changing patterns of calcium signaling in neurons and glial cells. In the present study, we investigated the developmental distribution of a major calcium-dependent protein, viz., calcium/calmodulin-dependent protein kinase II (CaM kinase II), in the antennal lobe of the sphinx moth Manduca sexta by using a monoclonal antibody. During synaptogenesis (developmental stages 6–10), we found a redistribution of CaM kinase II immunoreactivity, from a homogeneous distribution in the immature neuropil to an accumulation in the neuropil of the glomeruli. CaM kinase II immunoreactivity was less intense in olfactory receptor axons of the antennal nerve and antennal lobe glial cells. Western blot analysis revealed a growing content of CaM kinase II in antennal lobe tissue throughout metamorphosis. Injection of the CaM kinase inhibitor KN-93 into pupae resulted in a reduced number of antennal lobe glial cells migrating into the neuropil to form borders around glomeruli. The results suggest that CaM kinase II is involved in glial cell migration.This work was supported by the DFG LO779/2.