Skeletal muscle type comparison of pyruvate dehydrogenase phosphatase activity and isoform expression: effects of obesity and endurance training

Pyruvate dehydrogenase (PDH) plays an important role in regulating carbohydrate metabolism in skeletal muscle. PDH is activated by PDH phosphatase (PDP) and deactivated by PDH kinase (PDK). Obesity has a large negative impact on skeletal muscle carbohydrate metabolism, whereas endurance training has been shown to improve regulatory control of skeletal muscle carbohydrate metabolism, more so when coupled with obesity. A majority of this literature has focused on PDK, with little information available on PDP. To determine the relative role of PDP in regulating skeletal muscle PDH activity with obesity and endurance training, obese and lean Zucker rats remained sedentary or were endurance trained (1 h/day, 5 days/wk) for a period of 8 wk. Soleus, red gastrocnemius, (RG), and white gastrocnemius (WG) muscles were sampled after the training period. The main findings were 1) obesity resulted in a 46% decrease in PDP activity expressed per milligram extracted mitochondrial protein only in RG, while PDP isoform content was unchanged; 2) 8 wk of endurance training led to a significant 1.4-2.2-fold increase in PDP activity of all muscle examined from obese rats, and the concomitant increase in PDP1 protein was only seen in soleus and RG; 3) 8 wk of endurance training led to a trending 1.4-2.2-fold increase in PDP activity of all muscle examined from obese rats, and the concomitant increase in PDP1 protein was only seen in soleus and RG; and 4) PDP2 protein content was not affected by obesity or training. These results suggest that decreased PDP activity in oxidative skeletal muscles may play a role in the impairment of carbohydrate metabolism in obese rats, which is reversible with endurance training.     

Phage display of ScFv peptides recognizing the thymidine(6–4)thymidine photoproduct

Solar ultraviolet (UV) radiation induces DNA photoproducts in skin cells and is the predominant cause of human skin cancers. To understand human susceptibility to skin cancer and to facilitate the development of prevention measures, highly specific reagents to detect and quantitate UV-induced DNA adducts in human skin will be needed. One approach towards this end is the use of monoclonal antibody-based molecular dosimetry methods. To facilitate the development of photoproduct-specific antibody reagents we have: (i) cloned and sequenced a single chain variable fragment (ScFv) gene coding for one such high affinity monoclonal antibody, αUVssDNA-1 (mAb C3B6), recognizing the thymidine(6–4)thymidine photoproduct; (ii) expressed and displayed the cloned ScFv gene on the surface of phage; (iii) selected functional recombinant phage by panning; (iv) purified the ScFv peptide; (v) shown that the purified ScFv peptide binds to UV-irradiated polythymidylic acid but not unirradiated polythymidylic acid. This is the first demonstration of the use of phage display to select a ScFv recognizing DNA damage. In addition, this is the initial step towards immortalizing the antibody gene for genetic manipulation, structure–function studies and application to human investigations.     

Closure of rRNA related gaps in the Chromobacterium violaceum genome with the PCR-assisted contig extension (PACE) protocol

In the finishing phase of the Chromobacterium violaceum genome project, the shotgun sequences were assembled into 57 contigs that were then organized into 19 scaffolds, using the information from shotgun and cosmid clones. Among the 38 ends resulting from the 19 scaffolds, 10 ended with sequences corresponding to rRNA genes (seven ended with the 5S rRNA gene and three ended with the 16S rRNA gene). The 28 non-ribosomal ends were extended using the PCR-assisted contig extension (PACE) methodology, which immediately closed 15 real gaps. We then applied PACE to the 16S rRNA gene containing ends, resulting in eight different sequences that were correctly assembled within the C. violaceum genome by combinatory PCR strategy, with primers derived from the non-repetitive genomic region flanking the 16S and 5S rRNA gene. An oriented combinatory PCR was used to correctly position the two versions (copy A and copy B, which differ by the presence or absence of a 100-bp insert); it revealed six copies corresponding to copy A, and two to copy B. We estimate that the use of PACE, followed by combinatory PCR, accelerated the finishing phase of the C. violaceum genome project by at least 40%.     

Photoacoustic and photothermal cytometry using photoswitchable proteins and nanoparticles with ultrasharp resonances

In the article by E. I. Galanzha et al. (doi: http://dx.doi.org/10.1002/jbio.201300140 ), published in J. Biophotonics 8, 81–93 (2015), the Conflict of Interest statement is missing. This erratum is published to correct this.