Protein kinase Cδ but not PKCα is involved in insulin-induced glucose metabolism in hepatocytes

The liver is a major insulin‐responsive tissue responsible for glucose regulation. One important mechanism in this phenomenon is insulin‐induced glycogen synthesis. Studies in our laboratory have shown that protein kinase Cs delta (PKCδ) and alpha (α) have important roles in insulin‐induced glucose transport in skeletal muscle, and that their expression and activity are regulated by insulin. Their importance in glucose regulation in liver cells is unclear. In this study we investigated the possibility that these isoforms are involved in the mediation of insulin‐induced glycogen synthesis in hepatocytes. Studies were done on rat hepatocytes in primary culture and on the AML‐12 (alpha mouse liver) cell line. Insulin increased activity and tyrosine phosphorylation of PKCδ within 5 min. In contrast, activity and tyrosine phosphorylation of PKCα were not increased by insulin. PKCδ was constitutively associated with IR, and this was increased by insulin stimulation. Suppression of PKCδ expression by transfection with RNAi, or overexpression of kinase dead (dominant negative) PKCδ reduced both the insulin‐induced activation of PKB/Akt and the phosphorylation of glycogen synthase kinase 3 (GSK3) and reduced significantly insulin‐induced glucose uptake. In addition, treatment of primary rat hepatocytes with rottlerin abrogated insulin‐induced increase in glycogen synthesis. Neither overexpression nor inhibition of PKCα appeared to alter activation of PKB, phosphorylation of GSK3 or glucose uptake in response to insulin. We conclude that PKCδ, but not PKCα, plays an essential role in insulin‐induced glucose uptake and glycogenesis in hepatocytes. J. Cell. Biochem. 113: 2064–2076, 2012. © 2012 Wiley Periodicals, Inc.     

A hierarchical approach to all-atom protein loop prediction

The application of all-atom force fields (and explicit or implicit solvent models) to protein homology-modeling tasks such as side-chain and loop prediction remains challenging both because of the expense of the individual energy calculations and because of the difficulty of sampling the rugged all-atom energy surface. Here we address this challenge for the problem of loop prediction through the development of numerous new algorithms, with an emphasis on multiscale and hierarchical techniques. As a first step in evaluating the performance of our loop prediction algorithm, we have applied it to the problem of reconstructing loops in native structures; we also explicitly include crystal packing to provide a fair comparison with crystal structures. In brief, large numbers of loops are generated by using a dihedral angle-based buildup procedure followed by iterative cycles of clustering, side-chain optimization, and complete energy minimization of selected loop structures. We evaluate this method by using the largest test set yet used for validation of a loop prediction method, with a total of 833 loops ranging from 4 to 12 residues in length. Average/median backbone root-mean-square deviations (RMSDs) to the native structures (superimposing the body of the protein, not the loop itself) are 0.42/0.24 A for 5 residue loops, 1.00/0.44 A for 8 residue loops, and 2.47/1.83 A for 11 residue loops. Median RMSDs are substantially lower than the averages because of a small number of outliers; the causes of these failures are examined in some detail, and many can be attributed to errors in assignment of protonation states of titratable residues, omission of ligands from the simulation, and, in a few cases, probable errors in the experimentally determined structures. When these obvious problems in the data sets are filtered out, average RMSDs to the native structures improve to 0.43 A for 5 residue loops, 0.84 A for 8 residue loops, and 1.63 A for 11 residue loops. In the vast majority of cases, the method locates energy minima that are lower than or equal to that of the minimized native loop, thus indicating that sampling rarely limits prediction accuracy. The overall results are, to our knowledge, the best reported to date, and we attribute this success to the combination of an accurate all-atom energy function, efficient methods for loop buildup and side-chain optimization, and, especially for the longer loops, the hierarchical refinement protocol.     

High-fidelity KKH variant of Staphylococcus aureus Cas9 nucleases with improved base mismatch discrimination

The Cas9 nuclease from Staphylococcus aureus (SaCas9) holds great potential for use in gene therapy, and variants with increased fidelity have been engineered. However, we find that existing variants have not reached the greatest accuracy to discriminate base mismatches and exhibited much reduced activity when their mutations were grafted onto the KKH mutant of SaCas9 for editing an expanded set of DNA targets. We performed structure-guided combinatorial mutagenesis to re-engineer KKH-SaCas9 with enhanced accuracy. We uncover that introducing a Y239H mutation on KKH-SaCas9’s REC domain substantially reduces off-target edits while retaining high on-target activity when added to a set of mutations on REC and RuvC domains that lessen its interactions with the target DNA strand. The Y239H mutation is modelled to have removed an interaction from the REC domain with the guide RNA backbone in the guide RNA-DNA heteroduplex structure. We further confirmed the greatly improved genome-wide editing accuracy and single-base mismatch discrimination of our engineered variants, named KKH-SaCas9-SAV1 and SAV2, in human cells. In addition to generating broadly useful KKH-SaCas9 variants with unprecedented accuracy, our findings demonstrate the feasibility for multi-domain combinatorial mutagenesis on SaCas9’s DNA- and guide RNA- interacting residues to optimize its editing fidelity.     

Roles of ferritin and iron in ischemic preconditioning of the heart

Iron and copper play major roles in biological systems, catalyzing free radical production and consequently causing damage. The relatively high levels of these metals, which are mobilized into the coronary flow following prolonged ischemia, have been incriminated as key players in reperfusion injury to the heart. In the present communication we investigated other roles of iron - providing protection to the ischemic heart via preconditioning (PC). PC was accomplished by subjecting isolated rat hearts to three episodes of 2 min ischemia separated by 3 min of reperfusion. Prolonged ischemia followed the PC phase. PC hearts (group I) were compared to hearts subjected to normal perfusion (group II, no ischemia) and to ischemia without PC (group III). Group I showed a marked improvement in the recovery of hemodynamic function vs. group III. Biochemical parameters further substantiated the PC protection provided to group I against prolonged ischemia. Correspondingly, group I presented markedly lower re-distribution and mobilization of iron and copper into the coronary flow, following prolonged ischemia, as evinced from the decrease in total levels, and in the 'free' fraction of iron and copper. During the PC phase no loss of cardiac function was observed. A small wave of re-distribution and mobilization of iron (typically less than 4-8% of the value of 35 min ischemia) was recorded. The cellular content of ferritin (Ft) measured in the heart was significantly higher in group I than in group III (0.90 and 0.54 microg/mg, respectively). Also, iron-saturation of Ft was significantly lower for PC hearts, compared to both groups II and III (0.22 vs. 0.32 and 0.31 microg/mg, for 35 min ischemia, respectively). These findings are in accord with the proposal that intracellular re-distribution and mobilization of small levels of iron, during PC, cause rapid accumulation of ferritin - the major iron-storage protein. It is proposed that iron play a dual role: (i) It serves as a signaling pathway for the accumulation of Ft following the PC phase. This iron is not involved in cardiac injury, but rather prepares the heart against future high levels of 'free' iron, thus reducing the degree of myocardial damage after prolonged ischemia. (ii) High levels of iron (and copper) are mobilized following prolonged ischemia and cause tissue damage.     

Sclerotome-derived Slit1 drives directional migration and differentiation of Robo2-expressing pioneer myoblasts

Pioneer myoblasts generate the first myotomal fibers and act as a scaffold to pattern further myotome development. From their origin in the medial epithelial somite, they dissociate and migrate towards the rostral edge of each somite, from which differentiation proceeds in both rostral-to-caudal and medial-to-lateral directions. The mechanisms underlying formation of this unique wave of pioneer myofibers remain unknown. We show that rostrocaudal or mediolateral somite inversions in avian embryos do not alter the original directions of pioneer myoblast migration and differentiation into fibers, demonstrating that regulation of pioneer patterning is somite-intrinsic. Furthermore, pioneer myoblasts express Robo2 downstream of MyoD and Myf5, whereas the dermomyotome and caudal sclerotome express Slit1. Loss of Robo2 or of sclerotome-derived Slit1 function perturbed both directional cell migration and fiber formation, and their effects were mediated through RhoA. Although myoblast specification was not affected, expression of the intermediate filament desmin was reduced. Hence, Slit1 and Robo2, via RhoA, act to pattern formation of the pioneer myotome through the regulation of cytoskeletal assembly.     

Direct evidence for the adaptive role of copy number variation on antifolate susceptibility in Plasmodium falciparum

Resistance to antimalarials targeting the folate pathway is widespread. GTP‐cyclohydrolase (gch1), the first enzyme in this pathway, exhibits extensive copy number variation (CN) in parasite isolates from areas with a history of longstanding antifolate use. Increased CN of gch1 is associated with a greater number of point mutations in enzymes targeted by the antifolates, pyrimethamine and sulphadoxine. While these observations suggest that increases in gch1 CN are an adaptation to drug pressure, changes in CN have not been experimentally demonstrated to directly alter drug susceptibility. To determine if changes in gch1 expression alone modify pyrimethamine sensitivity, we manipulated gch1 CN in several parasite lines to test the effect on drug sensitivity. We report that increases in gch1 CN alter pyrimethamine resistance in most parasites lines. However we find evidence of a detrimental effect of very high levels of gch1 overexpression in parasite lines with high endogenous levels of gch1 expression, revealing the importance of maintaining balance in the folate pathway and implicating changes in gch1 expression in preserving proper metabolic flux. This work expands our understanding of parasite adaptation to drug pressure and provides a possible mechanism for how specific mutations become fixed within parasite populations.     

Characterization of T-lymphocyte subpopulations infiltrating primary breast cancer

Summary Characterization of T-lymphocyte subpopulations adjacent to and infiltrating the primary tumor of breast cancer was carried out using a direct immunofluorescence procedure with the antibodies anti-(Leu-2a) for suppressor/cytotoxic (CD8⁺) and anti-(Leu-3a) for helper/inducer (CD4⁺) T-lymphocytes. Fifty-six primary malignant tumors with lymphoid infiltration were studied. The majority (58.9%) were infiltrating duct carcinoma. There were metastases to axillary lymph nodes in 6.67% of the patients. Massive lymphoid infiltration (>40 lymphocytes per ×400 microscopic field) was found in 19.6% of the tumors and moderate infiltration (20–40 lymphocytes per field) in 51.8%. In all the tumors studied there was a reversed CD4⁺/CD8⁺ ratio as compared to that found in normal peripheral blood. In 66.1% the CD4⁺/CD8⁺ ratio (helper/suppressor) was less than 1.0. The reversed ratio was due to a significant decrease in the number of helper cells (P<0.0005). The most significant drop was in the stroma area (P<0.0001) as well as in the tumor tissue (P=0.001). Of particular interest was the significant positive correlation between the age of the patients and an increased number of CD4⁺lymphocytes in the stroma (P=0.02). Significant negative correlations were found between a reduced number of CD4⁺ lymphocytes or CD4⁺/CD8⁺ ratio and several histological parameters: tumor diameter, pleomorphism, nucleus/cytoplasm ratio. There was also a significant positive correlation between the total number of CD8⁺ lymphocytes infiltrating the tumor tissue and the number of axillary lymph nodes with metastatic disease (P=0.03). It is suggested that the reversed ratio of CD4⁺/CD8⁺ lymphocytes may significantly affect the host/tumor immune surveillance.     

Calnexin overexpression sensitizes recombinant CHO cells to apoptosis induced by sodium butyrate treatment

Sodium butyrate (NaBu) can enhance the expression of foreign genes in recombinant Chinese hamster ovary (rCHO) cells, but it can also inhibit cell growth and induce cellular apoptosis. In this study, the potential role of calnexin (Cnx) expression in rCHO cells treated with 5 mM NaBu was investigated for rCHO cells producing tumor necrosis factor receptor FC. To regulate the Cnx expression level, a tetracycline-inducible system was used. Clones with different Cnx expression levels were selected and investigated. With regard to productivity per cell (q_p), NaBu enhanced the q_p by over twofold. Under NaBu treatment, Cnx overexpression further enhanced the q_p by about 1.7-fold. However, under NaBu stress, the cells overexpressing Cnx showed a poorer viability profile with a consistent difference of over 25% in the viability when compared to the Cnx-repressed condition. This drop in the viability was attributed to increased apoptosis seen in these cells as evidenced by enhanced poly (ADP-ribose) polymerase cleavage and cytochrome C release. Ca²⁺ localization staining and subsequent confocal imaging revealed elevated cytosolic Ca²⁺ ([Ca²⁺]_c) in the Cnx-overexpressing cells when compared to the Cnx-repressed condition, thus endorsing the increased apoptosis observed in these cells. Taken together, Cnx overexpression not only improved the q_p of cells treated with NaBu, but it also sensitized cells to apoptosis.     

LGN-dependent orientation of cell divisions in the dermomyotome controls lineage segregation into muscle and dermis

The plane of cell divisions is pivotal for differential fate acquisition. Dermomyotome development provides an excellent system with which to investigate the link between these processes. In the central sheet of the early dermomyotome, single epithelial cells divide with a planar orientation. Here, we report that in the avian embryo, in addition to self-renewing, a subset of progenitors translocates into the myotome where they generate differentiated myocytes. By contrast, in the late epithelium, individual progenitors divide perpendicularly to produce both mitotic myoblasts and dermis. To examine whether spindle orientations influence fate segregation, early planar divisions were randomized and/or shifted to a perpendicular orientation by interfering with LGN function or by overexpressing inscuteable. Clones derived from single transfected cells exhibited an enhanced proportion of mixed dermomyotome/myotome progeny at the expense of `like' daughter cells in either domain. Loss of LGN or Gαi1 function in the late epithelium randomized otherwise perpendicular mitoses and favored muscle development at the expense of dermis. Hence, LGN-dependent early planar divisions are required for the proper allocation of progenitors into either dermomyotome or myotome, whereas late perpendicular divisions are necessary for the normal balance between muscle and dermis production.