Haemodipsus brachylagi n. sp. (Phthiraptera: Anoplura: Polyplacidae), a new sucking louse from the pygmy rabbit in Nevada

The male and female of Haemodipsis brachylagi n. sp. (Phthiraptera: Anoplura) are described from specimens collected from a pygmy rabbit, Brachylagus idahoensis (Merriam) (Lagomorpha: Leporidae), from Nevada. Morphological features that differentiate the new species from other known species of Haemodipsus are elucidated, and an identification key to both sexes of the 3 species now known from this genus in North America is included. Geographical distributions of the other 4 species of Haemodipsus known from other parts of the world are highlighted.     

p66Shc mediates anoikis through RhoA

Detachment of parenchymal cells from a solid matrix switches contextual cues from survival to death during anoikis. Marked shape changes accompany detachment and are thought to trigger cell death, although a working model to explain the coordination of attachment sensation, shape change, and cell fate is elusive. The constitutive form of the adapter Shc, p52Shc, confers survival properties, whereas the longer p66Shc signals death through association with cytochrome c. We find that cells that lack p66Shc display poorly formed focal adhesions and escape anoikis. However, reexpression of p66Shc restores anoikis through a mechanism requiring focal adhesion targeting and RhoA activation but not an intact cytochrome c-binding motif. This pathway stimulates the formation of focal adhesions and stress fibers in attached cells and tension-dependent cell death upon detachment. p66Shc may thus report attachment status to the cell by imposing a tension test across candidate anchorage points, with load failure indicating detachment.     

Hypothermia-induced cardioprotection using extended ischemia and early reperfusion cooling

Optimal timing of therapeutic hypothermia for cardiac ischemia is unknown. Our prior work suggests that ischemia with rapid reperfusion (I/R) in cardiomyocytes can be more damaging than prolonged ischemia alone. Also, these cardiomyocytes demonstrate protein kinase C (PKC) activation and nitric oxide (NO) signaling that confer protection against I/R injury. Thus we hypothesized that hypothermia will protect most using extended ischemia and early reperfusion cooling and is mediated via PKC and NO synthase (NOS). Chick cardiomyocytes were exposed to an established model of 1-h ischemia/3-h reperfusion, and the same field of initially contracting cells was monitored for viability and NO generation. Normothermic I/R resulted in 49.7 +/- 3.4% cell death. Hypothermia induction to 25 degrees C was most protective (14.3 +/- 0.6% death, P < 0.001 vs. I/R control) when instituted during extended ischemia and early reperfusion, compared with induction after reperfusion (22.4 +/- 2.9% death). Protection was completely lost if onset of cooling was delayed by 15 min of reperfusion (45.0 +/- 8.2% death). Extended ischemia/early reperfusion cooling was associated with increased and sustained NO generation at reperfusion and decreased caspase-3 activation. The NOS inhibitor N(omega)-nitro-L-arginine methyl ester (200 microM) reversed these changes and abrogated hypothermia protection. In addition, the PKCepsilon inhibitor myr-PKCepsilon v1-2 (5 microM) also reversed NO production and hypothermia protection. In conclusion, therapeutic hypothermia initiated during extended ischemia/early reperfusion optimally protects cardiomyocytes from I/R injury. Such protection appears to be mediated by increased NO generation via activation of protein kinase Cepsilon; nitric oxide synthase.     

Elevation of the post-translational modification of proteins by O-linked N-acetylglucosamine leads to deterioration of the glucose-stimulated insulin secretion in the pancreas of diabetic Goto-Kakizaki rats

Many nuclear and cytoplasmic proteins are O-glycosylated on serine or threonine residues with the monosaccharide beta-N-acetylglucosamine, which is then termed O-linked N-acetylglucosamine (O-GlcNAc). It has been shown that abnormal O-GlcNAc modification (O-GlcNAcylation) of proteins is one of the causes of insulin resistance and diabetic complications. In this study, in order to examine the relationship between O-GlcNAcylation of proteins and glucose-stimulated insulin secretion in noninsulin-dependent type (type 2) diabetes, we investigated the level of O-GlcNAcylation of proteins, especially that of PDX-1, and the expression of O-GlcNAc transferase in Goto-Kakizaki (GK) rats, which are an animal model of type-2 diabetes. By immunoblot and immunohistochemical analyses, the expression of O-GlcNAc transferase protein and O-GlcNAc-modified proteins in whole pancreas and islets of Langerhans of 15-week-old diabetic GK rats and nondiabetic Wistar rats was examined. The expression of O-GlcNAc transferase at the protein level and O-GlcNAc transferase activity were increased significantly in the diabetic pancreas and islets. The diabetic pancreas and islets also showed an increase in total cellular O-GlcNAc-modified proteins. O-GlcNAcylation of PDX-1 was also increased. In the diabetic GK rats, significant increases in the immunoreactivities of both O-GlcNAc and O-GlcNAc transferase were observed. PUGNAc, an inhibitor of O-GlcNAcase, induced an elevation of O-GlcNAc level and a decrease of glucose-stimulated insulin secretion in isolated islets. These results indicate that elevation of the O-GlcNAcylation of proteins leads to deterioration of insulin secretion in the pancreas of diabetic GK rats, further providing evidence for the role of O-GlcNAc in the insulin secretion.     

Ubiquitin-independent degradation of cell-cycle inhibitors by the REGgamma proteasome

The cell-cycle regulator p21(Cip1) is degraded by proteasomes independently of ubiquitination. We now show that degradation of p21 in vivo does not require the 19S proteasome lid, which contains the ubiquitin-binding subunit. Instead, the major proteasomal pathway for p21 degradation involves an alternative proteasome lid, the REGgamma complex. REGgamma binds to p21 in vivo, and deletion of p21's REGgamma-binding site greatly extends its half-life. Knockdown of REGgamma by RNA interference stabilizes p21, p21 has a significantly extended half-life in REGgamma(-/-) murine embryonic fibroblasts, and the p21 abundance is elevated in REGgamma(-/-) mice. The role of REGgamma in cell-cycle regulation may extend beyond p21 regulation, because p16(INK4A) and p19(Arf) also bind to REGgamma and are stabilized in REGgamma-deficient cells.     

Cotranslational protein folding--fact or fiction?

MOTIVATION: Experimentalists have amassed extensive evidence over the past four decades that proteins appear to fold during production by the ribosome. Protein structure prediction methods, however, do not incorporate this property of folding. A thorough study to find the fingerprint of such sequential folding is the first step towards using it in folding algorithms, so assisting structure prediction. RESULTS: We explore computationally the existence of evidence for cotranslational folding, based on large sets of experimentally determined structures in the PDB. Our perspective is that cotranslational folding is the norm, but that the effect is masked in most classes. We show that it is most evident in alpha/beta proteins, confirming recent findings. We also find mild evidence that older proteins may fold cotranslationally. A tool is provided for determining, within a protein, where cotranslation is most evident.     

Structural characterization of AtmS13, a putative sugar aminotransferase involved in indolocarbazole AT2433 aminopentose biosynthesis

AT2433 from Actinomadura melliaura is an indolocarbazole antitumor antibiotic structurally distinguished by its unique aminodideoxypentose‐containing disaccharide moiety. The corresponding sugar nucleotide‐based biosynthetic pathway for this unusual sugar derives from comparative genomics where AtmS13 has been suggested as the contributing sugar aminotransferase (SAT). Determination of the AtmS13 X‐ray structure at 1.50‐Å resolution reveals it as a member of the aspartate aminotransferase fold type I (AAT‐I). Structural comparisons of AtmS13 with homologous SATs that act upon similar substrates implicate potential active site residues that contribute to distinctions in sugar C5 (hexose vs. pentose) and/or sugar C2 (deoxy vs. hydroxyl) substrate specificity. Proteins 2015; 83:1547–1554. © 2015 Wiley Periodicals, Inc.     

Structural Characterization of CalS8, a TDP-α-d-Glucose Dehydrogenase Involved in Calicheamicin Aminodideoxypentose Biosynthesis

Classical UDP-glucose 6-dehydrogenases (UGDHs; EC 1.1.1.22) catalyze the conversion of UDP-α-d-glucose (UDP-Glc) to the key metabolic precursor UDP-α-d-glucuronic acid (UDP-GlcA) and display specificity for UDP-Glc. The fundamental biochemical and structural study of the UGDH homolog CalS8 encoded by the calicheamicin biosynthetic gene is reported and represents one of the first studies of a UGDH homolog involved in secondary metabolism. The corresponding biochemical characterization of CalS8 reveals CalS8 as one of the first characterized base-permissive UGDH homologs with a >15-fold preference for TDP-Glc over UDP-Glc. The corresponding structure elucidations of apo-CalS8 and the CalS8·substrate·cofactor ternary complex (at 2.47 and 1.95 Å resolution, respectively) highlight a notably high degree of conservation between CalS8 and classical UGDHs where structural divergence within the intersubunit loop structure likely contributes to the CalS8 base permissivity. As such, this study begins to provide a putative blueprint for base specificity among sugar nucleotide-dependent dehydrogenases and, in conjunction with prior studies on the base specificity of the calicheamicin aminopentosyltransferase CalG4, provides growing support for the calicheamicin aminopentose pathway as a TDP-sugar-dependent process.