Dynamic Alterations to α-Actinin Accompanying Sarcomere Disassembly and Reassembly during Cardiomyocyte Mitosis

Although mammals are thought to lose their capacity to regenerate heart muscle shortly after birth, embryonic and neonatal cardiomyocytes in mammals are hyperplastic. During proliferation these cells need to selectively disassemble their myofibrils for successful cytokinesis. The mechanism of sarcomere disassembly is, however, not understood. To study this, we performed a series of immunofluorescence studies of multiple sarcomeric proteins in proliferating neonatal rat ventricular myocytes and correlated these observations with biochemical changes at different cell cycle stages. During myocyte mitosis, α-actinin and titin were disassembled as early as prometaphase. α-actinin (representing the sarcomeric Z-disk) disassembly precedes that of titin (M-line), suggesting that titin disassembly occurs secondary to the collapse of the Z-disk. Sarcomere disassembly was concurrent with the dissolution of the nuclear envelope. Inhibitors of several intracellular proteases could not block the disassembly of α-actinin or titin. There was a dramatic increase in both cytosolic (soluble) and sarcomeric α-actinin during mitosis, and cytosolic α-actinin exhibited decreased phosphorylation compared to sarcomeric α-actinin. Inhibition of cyclin-dependent kinase 1 (CDK1) induced the quick reassembly of the sarcomere. Sarcomere dis- and re-assembly in cardiomyocyte mitosis is CDK1-dependent and features dynamic differential post-translational modifications of sarcomeric and cytosolic α-actinin.     

Comprehensive Analysis to Improve the Validation Rate for Single Nucleotide Variants Detected by Next-Generation Sequencing

Next-generation sequencing (NGS) has enabled the high-throughput discovery of germline and somatic mutations. However, NGS-based variant detection is still prone to errors, resulting in inaccurate variant calls. Here, we categorized the variants detected by NGS according to total read depth (TD) and SNP quality (SNPQ), and performed Sanger sequencing with 348 selected non-synonymous single nucleotide variants (SNVs) for validation. Using the SAMtools and GATK algorithms, the validation rate was positively correlated with SNPQ but showed no correlation with TD. In addition, common variants called by both programs had a higher validation rate than caller-specific variants. We further examined several parameters to improve the validation rate, and found that strand bias (SB) was a key parameter. SB in NGS data showed a strong difference between the variants passing validation and those that failed validation, showing a validation rate of more than 92% (filtering cutoff value: alternate allele forward [AF]≥20 and AF<80 in SAMtools, SB<–10 in GATK). Moreover, the validation rate increased significantly (up to 97–99%) when the variant was filtered together with the suggested values of mapping quality (MQ), SNPQ and SB. This detailed and systematic study provides comprehensive recommendations for improving validation rates, saving time and lowering cost in NGS analyses.     

Interactive responses of a thalamic neuron to formalin induced lasting pain in behaving mice

Thalamocortical (TC) neurons are known to relay incoming sensory information to the cortex via firing in tonic or burst mode. However, it is still unclear how respective firing modes of a single thalamic relay neuron contribute to pain perception under consciousness. Some studies report that bursting could increase pain in hyperalgesic conditions while others suggest the contrary. However, since previous studies were done under either neuropathic pain conditions or often under anesthesia, the mechanism of thalamic pain modulation under awake conditions is not well understood. We therefore characterized the thalamic firing patterns of behaving mice in response to nociceptive pain induced by inflammation. Our results demonstrated that nociceptive pain responses were positively correlated with tonic firing and negatively correlated with burst firing of individual TC neurons. Furthermore, burst properties such as intra-burst-interval (IntraBI) also turned out to be reliably correlated with the changes of nociceptive pain responses. In addition, brain stimulation experiments revealed that only bursts with specific bursting patterns could significantly abolish behavioral nociceptive responses. The results indicate that specific patterns of bursting activity in thalamocortical relay neurons play a critical role in controlling long-lasting inflammatory pain in awake and behaving mice.     

Tolerance of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> K35 to lignocellulose-derived inhibitory compounds

The hydrolysis which converts polysaccharides to the fermentable sugars for yeast’s lingocellulosic ethanol production also generates byproducts which inhibit the ethanol production. To investigate the extent to which inhibitory compounds affect yeast’s growth and ethanol production, fermentations by Saccharomyces cerevisiae K35 were investigated in various concentrations of acetic acid, furfural, 5-hydroxymethylfurfural (5-HMF), syringaldehyde, and coumaric acid. Fermentation in hydrolysates from yellow poplar and waste wood was also studied. After 24 h, S. cerevisiae K35 produced close to theoretically predicted ethanol yields in all the concentrations of acetic acid tested (1 ∼ 10 g/L). Both furans and phenolics inhibited cell growth and ethanol production. Ethanol yield, however, was unaffected, even at high concentrations, except in the cases of 5 g/L of syringaldehyde and coumaric acid. Although hydrolysates contain various toxic compounds, in their presence, S. Cerevisiae K35 consumed close to all the available glucose and yielded more ethanol than theoretically predicted. S. Cerevisiae K35 was demonstrated to have high tolerance to inhibitory compounds and not to need any detoxification for ethanol production from hydrolysates.     

Cocoa procyanidins protect PC12 cells from hydrogen-peroxide-induced apoptosis by inhibiting activation of p38 MAPK and JNK

Oxidative stress induced by reactive oxygen species has been strongly associated with the pathogenesis of neurodegenerative disorders, including Alzheimer's disease. In this study, we investigated the possible protective effects of a cocoa procyanidin fraction (CPF) and procyanidin B2 (epicatechin-(4β-8)-epicatechin) – a major polyphenol in cocoa – against apoptosis of PC12 rat pheochromocytoma (PC12) cells induced by hydrogen peroxide (H₂O₂). CPF (1 and 5 μg/ml) and procyanidin B2 (1 and 5 μM) reduced PC12 cell death caused by H₂O₂, as determined by MTT and trypan blue exclusion assays. CPF and procyanidin B2 attenuated the H₂O₂-induced fragmentation of nucleus and DNA in PC12 cells. Western blot data demonstrated that H₂O₂ induced cleavage of poly(ADP-ribose)polymerase (PARP), downregulated Bcl-X_L and Bcl-2 in PC12 cells. Pretreatment with CPF or procyanidin B2 before H₂O₂ treatment diminished PARP cleavage and increased Bcl-X_L and Bcl-2 expression compared with those only treated with H₂O₂. Activation of caspase-3 by H₂O₂ was inhibited by pretreatment with CPF or procyanidin B2. Furthermore, H₂O₂-induced rapid and significant phosphorylation of c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and both of these effects were attenuated by CPF or procyanidin B2 treatment. These results suggest that the protective effects of CPF and procyanidin B2 against H₂O₂-induced apoptosis involve inhibiting the downregulation of Bcl-X_L and Bcl-2 expression through blocking the activation of JNK and p38 MAPK.     

Formylchromone derivatives as irreversible and selective inhibitors of human protein tyrosine phosphatase 1B. Kinetic and modeling studies

A series of formylchromone derivatives were synthesized as PTP1B inhibitors and some of them were potent against PTP1B with IC50 values as low as 1.0 microM. They exhibited remarkable selectivity for PTP1B over other human PTPases. Kinetic studies revealed that formylchromone derivatives are irreversible and active site-directed inhibitors. Molecular modeling study identified the orientation of the inhibitor bound at the active site of PTP1B.     

Structural chemoproteomics and drug discovery

Our laboratories have developed several technologies to accelerate drug discovery process on the basis of structural chemoproteomics. They include SPS technology for the efficient determination of protein structures, SCP technology for the rapid lead generation and SDF technology for the productive lead optimization. Using these technologies, we could determine many 3D structures of target proteins bound with biologically active chemicals including the structure of phosphodiesterase 5/Viagra complex and obtain highly potent compounds in animal models of obesity, diabetes, cancer and inflammation. In this paper, we will discuss concepts and applications of structural chemoproteomics for drug discovery.