Direct activation of mitochondrial apoptosis machinery by c-Jun N-terminal kinase in adult cardiac myocytes.

Although oxidative stress causes activation of c-Jun N-terminal kinase (JNK) and apoptosis in many cell types, how the JNK pathway is connected to the apoptosis pathway is unclear. The molecular mechanism of JNK-mediated apoptosis was investigated in adult rat cardiac myocytes in culture as a model system that is sensitive to oxidative stress. Oxidative stress caused JNK activation, cytochrome c release, and apoptosis without new protein synthesis. Oxidative stress-induced apoptosis was abrogated by dominant negative stress-activated protein kinase/extracellular signal-regulated kinase kinase-1 (SEK1)-mediated inhibition of the JNK pathway, whereas activation of the JNK pathway by constitutively active SEK1 was sufficient to cause apoptosis. Inhibition of caspase-9, an apical caspase in the mitochondrial apoptosis pathway, suppressed oxidative stress-induced apoptosis, whereas inhibition of caspase-8 had no effect, indicating that both the JNK pathway and the mitochondrial apoptosis machinery are central to oxidative stress-induced apoptosis. Both JNK and SEK1 localized on mitochondria where JNK was activated by oxidative stress. Furthermore, active JNK caused the release of apoptogenic factors such as cytochrome c from isolated mitochondria in a cell-free assay. These findings indicate that the JNK pathway is a direct activator of mitochondrial death machinery without other cellular components and provide a molecular linkage from oxidative stress to the mitochondrial apoptosis machinery.     

A simulation study on the activation of cardiac CaMKII delta-isoform and its regulation by phosphatases

Although the highly conserved Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) is known to play an essential role in cardiac myocytes, its involvement in the frequency-dependent acceleration of relaxation is still controversial. To investigate the functional significance of CaMKII autophosphorylation and its regulation by protein phosphatases (PPs) in heart, we developed a new mathematical model for the CaMKIIδ isoform. Due to better availability of experimental data, the model was first adjusted to the kinetics of the neuronal CaMKIIα isoform and then converted to a CaMKIIδ model by fitting to kinetic data of the δ isoform. Both models satisfactorily reproduced experimental data of the CaMKII-calmodulin interaction, the autophosphorylation rate, and the frequency dependence of activation. The level of autophosphorylated CaMKII cumulatively increased upon starting the Ca²⁺ stimulation at 3 Hz in the δ model. Variations in PP concentration remarkably affected the frequency-dependent activation of CaMKIIδ, suggesting that cellular PP activity plays a key role in adjusting CaMKII activation in heart. The inhibitory effect of PP was stronger for CaMKIIα compared to CaMKIIδ. Simulation results revealed a potential involvement of CaMKIIδ autophosphorylation in the frequency-dependent acceleration of relaxation at physiological heart rates and PP concentrations.     

African Programme for Onchocerciasis Control 1995–2015: Model-Estimated Health Impact and Cost

Background

Onchocerciasis causes a considerable disease burden in Africa, mainly through skin and eye disease. Since 1995, the African Programme for Onchocerciasis Control (APOC) has coordinated annual mass treatment with ivermectin in 16 countries. In this study, we estimate the health impact of APOC and the associated costs from a program perspective up to 2010 and provide expected trends up to 2015.

Methods and Findings

With data on pre-control prevalence of infection and population coverage of mass treatment, we simulated trends in infection, blindness, visual impairment, and severe itch using the micro-simulation model ONCHOSIM, and estimated disability-adjusted life years (DALYs) lost due to onchocerciasis. We assessed financial costs for APOC, beneficiary governments, and non-governmental development organizations, excluding cost of donated drugs. We estimated that between 1995 and 2010, mass treatment with ivermectin averted 8.2 million DALYs due to onchocerciasis in APOC areas, at a nominal cost of about US$257 million. We expect that APOC will avert another 9.2 million DALYs between 2011 and 2015, at a nominal cost of US$221 million.

Conclusions

Our simulations suggest that APOC has had a remarkable impact on population health in Africa between 1995 and 2010. This health impact is predicted to double during the subsequent five years of the program, through to 2015. APOC is a highly cost-effective public health program. Given the anticipated elimination of onchocerciasis from some APOC areas, we expect even more health gains and a more favorable cost-effectiveness of mass treatment with ivermectin in the near future.     

S-peptide as a potent peptidyl linker for protein cross-linking by microbial transglutaminase from Streptomyces mobaraensis

We have found that ribonuclease S-peptide can work as a novel peptidyl substrate in protein cross-linking reactions catalyzed by microbial transglutaminase (MTG) from Streptomyces mobaraensis. Enhanced green fluorescent protein tethered to S-peptide at its N-terminus (S-tag-EGFP) appeared to be efficiently cross-linked by MTG. As wild-type EGFP was not susceptible to cross-linking, the S-peptide moiety is likely to be responsible for the cross-linking. A site-directed mutation study assigned Gln15 in the S-peptide sequence as the sole acyl donor. Mass spectrometric analysis showed that two Lys residues (Lys5 and Lys11) in the S-peptide sequence functioned as acyl acceptors. We also succeeded in direct monitoring of the cross-linking process by virtue of fluorescence resonance energy transfer (FRET) between S-tag-EGFP and its blue fluorescent color variant (S-tag-EBFP). The protein cross-linking was tunable by either engineering S-peptide sequence or capping the S-peptide moiety with S-protein, the partner protein of S-peptide for the formation of ribonuclease A. The latter indicates that S-protein can be used as a specific inhibitor of S-peptide-directed protein cross-linking by MTG. The controllable protein cross-linking of S-peptide as a potent substrate of MTG will shed new light on biomolecule conjugation.     

CRISPR/Cas9-based heritable targeted mutagenesis in Thermobia domestica: A genetic tool in an apterygote development model of wing evolution

Despite previous developmental studies on basally branching wingless insects and crustaceans, the evolutionary origin of insect wings remains controversial. Knowledge regarding genetic regulation of tissues hypothesized to have given rise to wings would help to elucidate how ancestral development changed to allow the evolution of true wings. However, genetic tools available for basally branching wingless species are limited. The firebrat Thermobia domestica is an apterygote species, phylogenetically related to winged insects. T. domestica presents a suitable morphology to investigate the origin of wings, as it forms the tergal paranotum, from which wings are hypothesized to have originated. Here we report the first successful CRISPR/Cas9-based germline genome editing in T. domestica. We provide a technological platform to understand the development of tissues hypothesized to have given rise to wings in an insect with a pre-wing evolution body plan.     

Mimicry of erythropoietin and interleukin-6 signalling by an antibody/cytokine receptor chimera in murine myeloid 32D cells

We have previously designed antibody-cytokine receptor chimeras that could respond to a cognate antigen. While these chimeric receptors were functional, it has not been investigated exactly how they mimic signal transduction through corresponding wild-type receptors. In this study, we compared the growth properties and the phosphorylation status of intracellular signal transducers between the erythropoietin receptor (EpoR)- or gp130-based chimeric receptors and wild-type EpoR or EpoR-gp130 chimera, respectively. Expression plasmids, encoding V(H) or V(L) region of anti-hen egg lysozyme (HEL) antibody HyHEL-10 tethered to a pair of extracellular D2 domain of EpoR and transmembrane/cytoplasmic domains of either EpoR or gp130, were constructed, and pairs of chimeric receptor combinations (V(H)-EpoR and V(L)-EpoR, V(H)-gp130 and V(L)-gp130, V(H)-EpoR and V(L)-gp130, V(H)-gp130 and V(L)-EpoR) were expressed in an IL-3-dependent myeloid cell line, 32D. Growth assay revealed that the transfectants all grew in a HEL-dependent manner. As for phosphorylation of Stat3, Stat5, ERK and Akt, the chimeric receptors showed similar activation pattern of signalling molecules with wild-type receptors, although the chimeric receptors showed ligand-independency and a little lower maximal phosphorylation than the corresponding wild-type receptors. The results demonstrate that antibody-receptor chimeras could substantially mimic wild-type receptors.     

A new class of LINEs (ATLN-L) from Arabidopsis thaliana with extraordinary structural features.

The Arabidopsis thaliana genome has about 250 copies of LINEs (here called ATLNs). Of these, some, called ATLN-Ls, have an extra sequence of about 2 kb in the region downstream of two consecutive open reading frames, orf1 and orf2. Interestingly, the extra sequences in these ATLN-L members have another open reading frame, designated as orf3. Each member is flanked by direct repeats of a target site sequence, showing that ATLN-L members with the three open reading frames have retrotransposed as a unit. The ATLN-L members are also distinct from other ATLN members: orf1 terminates with TAA (or TAG) and is located in the same frame as orf2, and the ATG initiation codon of orf2 is not present in the proximal region. A sequence that may form a pseudoknot structure in ATLN-L mRNA was present in the proximal region of orf2, therefore the TAA (or TAG) termination codon of orf1 is assumed to be suppressed to produce an Orf1-Orf2 transframe protein during the translation of the ATLN-L mRNA. The region between orf2 and orf3 is several hundred bp long, suggesting that orf3 expression is independent of orfl-orf2. The amino acid sequences of the proteins Orf1 and Orf3 are highly homologous in their N-terminal half regions that have a retroviral zinc-finger motif for RNA binding. Orf3, however, has a leucine-zipper motif in addition to the zinc-finger motif. The C-terminal regions of the Orf1 and Orf3 proteins have poor homology, but seem to have nuclear localization signals, suggesting that these proteins are involved in the transfer of ATLN-L mRNA to nuclei. A phylogenetic tree shows that Orf3 proteins form a branch distinct from the branches of the Orf1 proteins encoded by ATLN-L members. This indicates that an ancestor element of ATLN-Ls has incorporated the orf1 frame carried by another ATLN member into its distal region to orf1-orf2 during evolution.     

Three-dimensional CT examination of the mastication system in the giant anteater

The gross anatomy of the mastication system of the giant anteater (Myrmecophaga tridactyla) was examined by means of three-dimensional image analysis. The anteater rotates the mandibles medially and laterally to control its tongue when it is elongated and to house it when it is relaxed. Three-dimensional CT image analysis demonstrated that the shape and size of the oral cavity changes drastically when the mandibles are rotated. The oral cavity expands bilaterally when the dorsal part of the mandibles bend medially. Macroscopic observations and muscle-weight data supported the observation that the superficial temporal and medial pterygoid muscles act as the main medial and lateral rotators of the mandible, respectively. The low height of the mandibular ramus and the incomplete zygomatic arch in this species represent adaptations for the rotational movement of the mandibles, since they both contribute to the medially oriented transmission of force from the temporal muscles and to preventing collision between the mandibles and the cranium during the rotational movement.     

The alkaloid contents of sixty Nicotiana species

The alkaloid content (nicotine, nornicotine, anabasine, and anatabine) in leaves and roots of 60 Nicotiana species were analyzed by GC. All species contained alkaloids, the amounts varying with the species. There was no clearcut correlation between alkaloid amounts and subgeneric or sectional classification. The alkaloid content in the floral parts and immature and mature fruits of Nicotiana tabacum were also analyzed.