Structural basis of ligand recognition by PABC, a highly specific peptide-binding domain found in poly(A)-binding protein and a HECT ubiquitin ligase

The C-terminal domain of poly(A)-binding protein (PABC) is a peptide-binding domain found in poly(A)-binding proteins (PABPs) and a HECT (homologous to E6-AP C-terminus) family E3 ubiquitin ligase. In protein synthesis, the PABC domain of PABP functions to recruit several translation factors possessing the PABP-interacting motif 2 (PAM2) to the mRNA poly(A) tail. We have determined the solution structure of the human PABC domain in complex with two peptides from PABP-interacting protein-1 (Paip1) and Paip2. The structures show a novel mode of peptide recognition, in which the peptide binds as a pair of beta-turns with extensive hydrophobic, electrostatic and aromatic stacking interactions. Mutagenesis of PABC and peptide residues was used to identify key protein-peptide interactions and quantified by isothermal calorimetry, surface plasmon resonance and GST pull-down assays. The results provide insight into the specificity of PABC in mediating PABP-protein interactions.     

ANG II type 2 receptor regulates smooth muscle growth and force generation in late fetal mouse development

Although evidence from culture studies implicates the angiotensin II (ANG II) type 2 receptor (AT(2)R) in the regulation of growth and differentiation of arterial smooth muscle (SM) cells (SMC), the lack of its expression in adult arteries has precluded direct investigation of its role in vivo. The goal of the present study was to determine the role of AT(2)R in the control of fetal SMC growth, contractility, and differentiation during vascular development. Determination of isometric tension in fetal aortas showed potentiated ANG II-induced contraction by treatment with the selective AT(2)R antagonist PD-123319, demonstrating the presence of functional AT(2)Rs that mediate reduced force development in vascular SMC. In direct contrast to numerous cell culture studies, proliferation indexes were decreased rather than increased in aortic SMC of fetal homozygous AT(2)R knockout compared with wild-type or heterozygous knockout mice. Experiments using SMC tissues from heterozygous female AT(2)R knockout mice, which are naturally occurring chimeras for AT(2)R expression, showed that AT(2)R mRNA expression was exactly 50% of that of wild type. This indicated that loss of AT(2)R expression did not confer a selective advantage or disadvantage for SMC lineage determination and expansion. Real time RT-PCR analyses showed no significant difference in expression of SM-alpha-actin, SM myosin heavy chain, and myocardin in various SM tissues from all three genotypes, suggesting that knockout of AT(2)R had no effect on subsequent SMC differentiation. Taken together, results indicate that functional AT(2)R are expressed in fetal aorta and mediate reduced force development but do not significantly contribute to regulation of SMC differentiation.     

Methylenetetrahydrofolate reductase (MTHFR) polymorphisms and promoter methylation in cervical oncogenic lesions and cancer

The aim of this study was to investigate the role of methylenetetrahydrofolate reductase (MTHFR) polymorphisms and MTHFR methylation pattern in cervical lesions development among women from Romania, a country with high prevalence of human papillomavirus (HPV) cervical infections. To achieve this goal, blood samples and cervical cytology specimens (n = 77)/tumour tissue specimens (n = 23) were investigated. As control, blood and negative cytological smears (n = 50) were used. A statistically significant association was found between T allele of C677T polymorphism and cervical lesions, heterozygote women presenting a threefold increased risk (normal/cervical lesions and tumours: wild homozygote 34/41 (0.68/0.41), heterozygote 14/51 (0.28/0.51), mutant homozygote 2/8 (0.04/0.08); OR = 3.081, P = 0.0035). Using χ square test for the control group, the HPV‐negative and HPV‐positive patients with cervix lesions, a significant correlation between viral infection and T allele of C677T polymorphism (P = 0.0287) was found. The MTHFR promoter was methylated in all HGSIL and tumour samples, significant differences being noted between HPV‐positive samples, control group and cases of cervical dysplastic lesions without HPV DNA (P < 0. 0001) and between samples from patients with high‐risk (hr)HPV versus low‐risk (lr)HPV (P = 0.0026). No correlations between polymorphisms and methylation were observed. In Romania, individuals carrying T allele are susceptible for cervical lesions. MTHFR promoter methylation is associated with cervical severity lesions and with hrHPV.     

Size‐Dependent Charge Transfer in Blends of PbS Quantum Dots with a Low‐Gap Silicon‐Bridged Copolymer

The photophysics of bulk heterojunctions of a high‐performance, low‐gap silicon‐bridged dithiophene polymer with oleic acid capped PbS quantum dots (QDs) are studied to assess the material potential for light harvesting in the visible‐ and IR‐light ranges. By employing a wide range of nanocrystal sizes, systematic dependences of electron and hole transfer on quantum‐dot size are established for the first time on a low‐gap polymer–dot system. The studied system exhibits type II band offsets for dot sizes up to ca. 4 nm, whch allow fast hole transfer from the quantum dots to the polymer that competes favorably with the intrinsic QD recombination. Electron transfer from the polymer is also observed although it is less competitive with the fast polymer exciton recombination for most QD sizes studied. The incorporation of a fullerene derivative provides efficient electron‐quenching sites that improve interfacial polymer‐exciton dissociation in ternary polymer–fullerene–QD blends. The study indicates that programmable band offsets that allow both electron and hole extraction can be produced for efficient light harvesting based on this low‐gap polymer‐PbS QD composite.     

The identification and analysis of phosphorylation sites on the Atg1 protein kinase

Autophagy is a conserved, degradative process that has been implicated in a number of human diseases and is a potential target for therapeutic intervention. It is therefore important that we develop a thorough understanding of the mechanisms regulating this trafficking pathway. The Atg1 protein kinase is a key element of this control as a number of signaling pathways target this enzyme and its associated protein partners. These studies have established that Atg1 activities are controlled, at least in part, by protein phosphorylation. To further this understanding, we used a combined mass spectrometry and molecular biology approach to identify and characterize additional sites of phosphorylation in the Saccharomyces cerevisiae Atg1. Fifteen candidate sites of phosphorylation were identified, including nine that had not been noted previously. Interestingly, our data suggest that the phosphorylation at one of these sites, Ser-34, is inhibitory for both Atg1 kinase activity and autophagy. This site is located within a glycine-rich loop that is highly conserved in protein kinases. Phosphorylation at this position in several cyclin-dependent kinases has also been shown to result in diminished enzymatic activity. In addition, these studies identified Ser-390 as the site of autophosphorylation responsible for the anomalous migration exhibited by Atg1 on SDS-polyacrylamide gels. Finally, a mutational analysis suggested that a number of the sites identified here are important for full autophagy activity in vivo. In all, these studies identified a number of potential sites of regulation within Atg1 and will serve as a framework for future work with this enzyme.     

An interval timer controls the production of sexual morphs in Myzus persicae (Homoptera: Aphididae)

Abstract The interval timer mechanism was examined in two clones of Myzus persicae (Sulzer) originating from peach in Naousa, North Greece. Overwintering eggs were collected in April 1997 and after egg hatch the morphs of progeny were recorded at LD 10 : 14 h and 17 °C until the appearance of sexuals. In one clone, observations were continued until the 21st generation after egg hatch. In addition, the production of sexual morphs was examined in two other clones originating from Lehonia, Central Greece, when apterae (wingless females) were transferred from long to short days and 17 °C in the second and eighth generation of parthenogenetic rearing after their collection from peach trees. In clones from Naousa, the first gynoparae appear and comprise 10% of alate (winged) females in the second half of the third generation after egg hatch. The percentage of gynoparae increased in each consecutive generation, reaching 100% in fifth and seventh generations in clones Nb and Na, respectively. First males appeared later than gynoparae, in fifth and sixth generation in clones Nb and Na, respectively. In clone Nb, the number of males, after their appearance in fifth generation, was relatively constant from the sixth to the ninth generation (10.8–12.3). Then a significant increase was observed in the 10th generation, thereafter remaining relatively constant until the 21st generation (18.1–19.8). A similar trend in the number of males produced was observed in clones deriving from Lehonia.     

The occurrence of the Philadelphia chromosome in essential thrombocytosis

Summary The Philadelphia chromosome, assessed with banding techniques, was detected in 98.3% of bone marrow cells of a 46-year-old black female presenting with essential thrombocytosis. The patient has been followed for the past two years with no signs of chronic myelocytic leukemia. Her platelet counts remain elevated, and she shows no other hematologic changes. Comparisons with the 21q- marker associated with thrombocytosis are made. The role of the Ph' chromosome in myeloid malignant changes and the implications of the present findings in thrombocytosis are discussed.     

Increased abdominal-to-peripheral fat distribution contributes to altered autonomic-circulatory control with human aging

Autonomic nervous system (ANS) control of the circulation is altered with aging in adult humans. Similar changes are observed in obesity, particularly abdominal obesity. To determine whether age-associated differences in ANS-circulatory function can be partially explained by increased body fatness, we examined ANS function and three expressions of adiposity (total body fat, abdominal body fat, and abdominal-to-peripheral body fat distribution; dual-energy X-ray absorptiometry) in 43 healthy men: 27 young (25 +/- 1 yr) and 16 older (65 +/- 1). ANS functions assessed included 1) autonomic support of arterial blood pressure (BP; radial artery catheter), i.e., the reduction in BP during versus before acute ganglionic blockade (GB; intravenous trimethaphan); 2) baroreflex buffering, i.e., the increase in systolic BP with continuous incremental and bolus infusions of phenylephrine during versus before GB; 3) cardiovagal baroreflex sensitivity (Oxford technique); and 4) heart rate variability (time- and frequency-domain analyses). Covarying for abdominal-to-peripheral fat distribution reduced or abolished age-related differences in ANS support of BP, cardiovagal baroreflex sensitivity, and heart rate variability but did not affect age-related differences in baroreflex buffering. Covarying for abdominal and total fat had small selective or no effects on age-associated differences in autonomic-circulatory control. Abdominal-to-peripheral fat distribution explains a significant portion of the variance in a number of autonomic-circulatory functions attributable to aging. Therefore, the development of this fat pattern may contribute to several changes in ANS-cardiovascular function observed with aging. These results may help explain how changes in body fat distribution with advancing age are linked to impairments in circulatory control.