Structural studies of ß-turn-containing peptide catalysts for atroposelective quinazolinone bromination.

We describe herein a crystallographic and NMR study of the secondary structural attributes of a ß-turn-containing tetra-peptide, Boc-Dmaa-D-Pro-Acpc-Leu-NMe2, which was recently reported as a highly effective catalyst in the atroposelective bromination of 3-arylquinazolin-4(3H)-ones. Inquiries pertaining to the functional consequences of residue substitutions led to the discovery of a more selective catalyst, Boc-Dmaa-D-Pro-Acpc-Leu-OMe, the structure of which was also explored. This new lead catalyst was found to exhibit a type I'ß-turn secondary structure both in the solid state and in solution, a structure that was shown to be an accessible conformation of the previously reported catalyst, as well.

Dietary fibre linked to decreased inflammation in overweight minority youth.

OBJECTIVE: The objective of this study was to examine the relationship between diet and inflammation, and adiposity in minority youth. DESIGN AND METHODS: The study was designed as a cross-sectional analysis of 142 overweight (≥85th body mass index percentile) Hispanic and African-American adolescents (14-18 years) with the following measures: anthropometrics, adiposity via magnetic resonance imaging, dietary intake via 24-h dietary recalls, and inflammation markers from fasting blood draws utilizing a multiplex panel. Partial correlations were estimated and analysis of covariance (ancova) models fit to examine the relationship among dietary variables, inflammation markers and adiposity measures with the following a priori covariates: Tanner stage, ethnicity, sex, total energy intake, total body fat and total lean mass. RESULTS: Inference based on ancova models showed that the highest tertile of fibre intake (mean intake of 21.3 ± 6.1 g d(-1) ) vs. the lowest tertile of fibre intake (mean intake of 7.4 ± 1.8 g d(-1) ) was associated with 36% lower plasminogen activator inhibitor-1 (P = 0.02) and 43% lower resistin (P = 0.02), independent of covariates. Similar results were seen for insoluble fibre. No other dietary variables included in this study were associated with inflammation markers. CONCLUSIONS: These results suggest that increases in dietary fibre could play an important role in lowering inflammation and therefore metabolic disease risk in high-risk minority youth.

Association of gestational diabetes and breastfeeding on obesity prevalence in predominately Hispanic low-income youth.

OBJECTIVE: The goal of this study was to examine if breastfeeding duration by gestational diabetes mellitus status impacted the prevalence of obesity in offspring. METHODS: Data were obtained from a 2011 phone survey with caregivers of low-income children (2-4 years) participating in the Women, Infants and Children programme in Los Angeles County. The final sample included 2295 children, 84% Hispanic and 48% female. Chi-square and binary logistic regression were used to assess gestational diabetes status and breastfeeding duration on the prevalence of obesity, with the following a priori covariates: child's ethnicity, birth weight, age in months and sex. RESULTS: Breastfeeding and gestational diabetes were significantly associated with obesity prevalence (P < 0.01). Using gestational diabetes mellitus and no breastfeeding as the referent category, gestational diabetes mellitus offspring who were breastfed ≥12 months had a 72% decrease in obesity prevalence (adjusted odds ratio = 0.28, confidence interval 0.89-0.03, P = 0.05). CONCLUSIONS: These findings suggest that > 12 months of breastfeeding duration in the gestational diabetes mellitus group and any duration of breastfeeding in the non-gestational diabetes mellitus mothers is needed to reduce obesity levels in a primarily Hispanic population.

Increased eating frequency linked to decreased obesity and improved metabolic outcomes.

BACKGROUND: We previously reported that more frequent eating in overweight minority youth was linked to lower visceral adiposity and circulating triglycerides. The aim of this study was to examine this issue in more detail by assessing the relationship between eating frequency and adiposity and metabolic disease risk in a cohort of exclusively overweight Hispanic youth. METHODS: This analysis included 191 overweight (⩾ 85th percentile body mass index (BMI)) Hispanic youth (8-18 years) with the following cross-sectional measures: height, weight, BMI, dietary intake via multiple 24 h recalls, body composition via dual-energy X-ray absorptiometry, lipids and insulin action (insulin sensitivity, acute insulin response (AIR) and disposition index, a measure of ß-cell function) via a frequently sampled intravenous glucose tolerance test. Each eating occasion (EO) was defined as ⩾ 50 calories and ⩾ 15 min from any prior EO. Infrequent eaters (IEs) were classified as any subject who ate <3 EOs on any dietary recall (n = 32), whereas frequent eaters (FEs) always consumed ⩾ 3 EOs (n = 159). RESULTS: Using analyses of covariance, FEs compared with IEs consumed 23% more calories per day (P ⩽ 0.01), ate 40% more often and consumed 19% less calories per EO (P ⩽ 0.01). FEs also exhibited 9% lower BMI Z-scores (P ⩽ 0.01), 9% lower waist circumferences (P ⩽ 0.01), 29% lower fasting insulin (P = 0.02), 31% lower HOMA-IR (Homeostatic Model Assessment: Insulin Resistance) values (P = 0.02) and 19% lower triglycerides (P ⩽ 0.01), as well as an 11% higher AIR (P = 0.02) and 31% higher disposition index (P=0.01). The following a priori covariates were included: Tanner, sex, body fat and reported energy intake. CONCLUSION: These findings suggest that increased eating frequency is related to decreased obesity and metabolic disease risk in overweight Hispanic youth, despite increases in energy intake.

MAF mediates crosstalk between Ras-MAPK and mTOR signaling in NF1.

Mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene are common in cancer and can cause resistance to therapy. Using transcriptome analysis we identified MAF as an NF1- regulated transcription factor and verified MAF regulation through RAS/MAPK/AP-1 signaling in malignant peripheral nerve sheath tumor (MPNST) cell lines. MAF was also downregulated in human MPNST. Acute re-expression of MAF promoted expression of glial differentiation markers in MPNST cells in vitro, decreased self-renewal of embryonic precursors and transiently affected tumor cell phenotypes in vitro by increasing MPNST cell death and reducing metabolic activity and anchorage-independent growth. Paradoxically, chronic MAF overexpression enhanced MPNST cell tumor growth in vivo, correlating with elevated pS6 in vitro and in vivo. RAD001 blocked MAF-mediated tumor growth, and MAF regulated the mTOR pathway through DEPTOR. MAPK inhibition with NF1 loss of function is predicted to show limited efficacy due to reactivation of mTOR signaling via MAF.

Nuclear PTEN controls DNA repair and sensitivity to genotoxic stress.

Loss of function of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor gene is associated with many human cancers. In the cytoplasm, PTEN antagonizes the phosphatidylinositol 3-kinase (PI3K) signaling pathway. PTEN also accumulates in the nucleus, where its function remains poorly understood. We demonstrate that SUMOylation (SUMO, small ubiquitin-like modifier) of PTEN controls its nuclear localization. In cells exposed to genotoxic stress, SUMO-PTEN was rapidly excluded from the nucleus dependent on the protein kinase ataxia telangiectasia mutated (ATM). Cells lacking nuclear PTEN were hypersensitive to DNA damage, whereas PTEN-deficient cells were susceptible to killing by a combination of genotoxic stress and a small-molecule PI3K inhibitor both in vitro and in vivo. Our findings may have implications for individualized therapy for patients with PTEN-deficient tumors.

Inhibition of Eyes Absent Homolog 4 expression induces malignant peripheral nerve sheath tumor necrosis.

Malignant peripheral nerve sheath tumors (MPNSTs) are aggressive sarcomas without effective therapeutics. Bioinformatics was used to identify potential therapeutic targets. Paired Box (PAX), Eyes Absent (EYA), Dachsund (DACH) and Sine Oculis (SIX) genes, which form a regulatory interactive network in Drosophila, were found to be dysregulated in human MPNST cell lines and solid tumors. We identified a decrease in DACH1 expression, and increases in the expressions of PAX6, EYA1, EYA2, EYA4, and SIX1-4 genes. Consistent with the observation that half of MPNSTs develop in neurofibromatosis type 1 (NF1) patients, subsequent to NF1 mutation, we found that exogenous expression of the NF1-GTPase activating protein-related domain normalized DACH1 expression. EYA4 mRNA was elevated more than 100-fold as estimated by quantitative real-time PCR in most MPNST cell lines. In vitro, suppression of EYA4 expression using short hairpin RNA reduced cell adhesion and migration and caused cellular necrosis without affecting cell proliferation or apoptotic cell death. MPNST cells expressing shEYA4 either failed to form tumors in nude mice or formed very small tumors, with extensive necrosis but similar levels of proliferation and apoptosis as control cells. Our findings identify a role of EYA4 and possibly interacting SIX and DACH proteins in MPNSTs and suggest the EYA4 pathway as a rational therapeutic target.

Transfer of nitric oxide by blood from upstream to downstream resistance vessels causes microvascular dilation.

The discovery that hemoglobin, albumin, and glutathione carry and release nitric oxide (NO) may have consequences for movement of NO by blood within microvessels. We hypothesize that NO in plasma or bound to proteins likely survives to downstream locations. To confirm this hypothesis, there must be a finite NO concentration ([NO]) in arteriolar blood, and upstream resistance vessels must be able to increase the vessel wall [NO] of downstream arterioles. Arteriolar blood NO was measured with NO-sensitive microelectrodes, and vessel wall [NO] was consistently 25-40% higher than blood [NO]. Localized suppression of NO production in large arterioles over 500-1,000 microm with L-nitroarginine reduced the [NO] approximately 40%, indicating as much as 60% of the wall NO was from blood transfer. Flow in mesenteric arteries was elevated by occlusion of adjacent arteries to induce a flow-mediated increase in arterial NO production. Both arterial wall and downstream arteriolar [NO] increased and the arterioles dilated as the blood [NO] was increased. To study receptor-mediated NO generation, bradykinin was locally applied to upstream large arterioles and NO measured there and in downstream arterioles. At both sites, [NO] increased and both sets of vessels dilated. When isoproterenol was applied to the upstream vessels, they dilated, but neither the [NO] or diameter downstream arterioles increased. These observations indicate that NO can move in blood from upstream to downstream resistance vessels. This mechanism allows larger vessels that generate large [NO] to influence vascular tone in downstream vessels in response to both flow and receptor stimuli.

Differential protein expression during aging in ventricular myocardium of Fischer 344 x Brown Norway hybrid rats.

The aging heart undergoes well characterized structural changes associated with functional decline, though the underlying mechanisms are not understood. The aim of this study was to determine to what extent ventricular myocardial protein expression was altered with age and which proteins underwent protein nitration. Fischer 344 x Brown Norway F1 hybrid (FBN) rats of four age groups were used, 4, 12, 24, and 34 months. Differential protein expression was determined by 2-DE and proteins were identified by peptide mass fingerprinting. Altered protein nitration with age was assessed by immunoblotting. Over 1000 protein spots per sample were detected, and 255 were found to be differentially expressed when all aged groups were compared to young rats (4 months) (p0.05). A strong positive correlation between differential protein expression and increasing age (p=0.03, R(2)=0.997) indicated a progressive, rather than abrupt, change with age. Of 46 differentially expressed proteins identified, seventeen have roles in apoptosis, ten in hypertrophy, seven in fibrosis, and three in diastolic dysfunction, aging-associated processes previously reported in both human and FBN rat heart. Protein expression alterations detected here could have beneficial effects on cardiac function; thus, our data indicate a largely adaptive change in protein expression during aging. In contrast, differential protein nitration increased abruptly, rather than progressively, at 24 months of age. Altogether, the results suggest that differential myocardial protein expression occurs in a progressive manner during aging, and that a proteomic-based approach is an effective method for the identification of potential therapeutic targets to mitigate aging-related myocardial dysfunction.

Immediate and late outcomes of transarterial coil occlusion of patent ductus arteriosus in dogs.

Records from dogs (n = 125) that underwent attempted transarterial coil occlusion of patent ductus arteriosus (PDA) at the University of California, Davis, between 1998 and 2003, were reviewed, and a subset of these dogs (n = 31) in which the procedure was performed at least 12 months earlier were reexamined to determine long-term outcome. Coil implantation was achieved in 108 dogs (86%). Despite immediate complete ductal closure in only 34% of dogs, the procedure was hemodynamically successful as evidenced by a reduction in indexed left ventricular internal diameter in diastole (LVIDd; P < .0001), fractional shortening (P < .0001), and left atrial to aortic ratio (LA: Ao; P = .022) within 24 hours. Complete ductal closure was documented in 61% of dogs examined 12 to 63 months after coil occlusion. Long-standing residual ductal flow in the other 39% of dogs was not associated with increased indexed LVIDd or LA: Ao and was not hemodynamically relevant. Repeat intervention was deemed advisable in only 4 dogs with persistent (n = 1) or recurrent (n = 3) ductal flow. Complications included aberrant embolization (n = 27), death (n = 3), ductal reopening (n = 3), transient hemoglobinuria (n = 2), hemorrhage (n = 1), aberrant coil placement (n = 1), pulmonary hypertension (n = 1), and skin abscessation (n = 1). Serious infectious complications did not occur despite antibiotic administration to only 40% of these dogs. Transarterial coil occlusion was not possible in 14 dogs (11%) because of coil instability in the PDA and was associated with increased indexed minimum ductal diameter (P = .03), LVIDd (P = .0002), LVIDs (P = 0.001), and congestive left heart failure (P = .03) reflecting a relatively large shunt volume.

Epigenomic changes during leukemia cell differentiation: analysis of histone acetylation and cytosine methylation using CpG island microarrays.

Dysregulation of epigenetic control is an important participant in carcinogenesis. The PML/RAR alpha translocation in acute promyelocytic leukemia (APL) is an example where the resultant fusion protein recruits histone deacetylase complexes to target genes resulting in their inappropriate transcriptional repression. All-trans-retinoic acid (ATRA) acts as a ligand that relieves this repression and produces an epigenetic transcriptional reprogramming of the cancer cell. CpG island microarrays were used to analyze the DNA methylation and histone acetylation state of the human APL cell line NB4 before and after differentiation with ATRA as well as normal peripheral blood mononuclear cells (PBMC). Over 70 CpG islands within 1 kb of transcription start of a known gene are aberrantly methylated in NB4 cells compared with PBMC; however, no changes in cytosine methylation were detected following ATRA-induced differentiation. With respect to histone H4 acetylation, over 100 single-copy CpG islands within 1 kb of transcription start of a known human gene became hyperacetylated following ATRA-induced differentiation. One CpG island was aberrantly methylated in NB4 cells, but became hyperacetylated and was induced following ATRA treatment and was associated with the HoxA1 gene, suggesting it may be a target gene of ATRA in APL. In addition to single-copy sequences, a selective increase in acetylation was detected in satellite DNA when compared with other high-copy sequences, such as Alu or rDNA. In summary, ATRA stimulates complex epigenomic changes during leukemic cell differentiation, and monitoring these changes may help to identify new targets of epigenetic dysfunction.

Combining isometric knee extension exercises with hip adduction or abduction does not increase quadriceps EMG activity.

OBJECTIVE: To determine if the combined isometric contractions of knee extension/hip adduction and knee extension/hip abduction will elicit a different quadriceps and gluteus medius electromyographic (EMG) pattern as compared to isometric contraction of a uniplanar knee extension exercise. METHODS: Eight healthy young adult volunteers without history of knee or quadriceps injury participated. Surface EMG data were collected from the vastus medialis oblique (VMO), vastus lateralis (VL), and gluteus medius (Gmed) muscles of the dominant leg of each subject during three single leg, weight bearing, isometric exercises (uniplanar knee extension, knee extension/hip adduction, knee extension/hip abduction). All exercises were performed at a position of 60 degrees knee flexion. Three trials lasting 5 s each were performed for each of the three exercises. EMG data from each muscle were integrated and the maximum root mean square activity over a 0.5 s window for each trial was averaged. Analyses of variance were performed with exercise (straight extension, extension/adduction, extension/abduction) as the independent variable and VMO, VL, and Gmed activity and VMO:VL ratio as dependent variables. RESULTS: A significant main effect for exercise was found for the VMO (p = 0.006) and VL (p = 0.02), but not the Gmed (p = 0.25) or the VMO:VL ratio (p = 0.13). For the VMO and VL, the uniplanar knee extension task produced significantly more EMG activity than the extension/adduction or extension/abduction tasks. CONCLUSIONS: Uniplanar knee extension exercises may be more appropriate than combining isometric knee extension exercises with hip adduction or abduction when eliciting maximal VMO and VL contractions.

The debate regarding continuous use of oral contraceptives.

OBJECTIVE: To determine whether oral contraceptives (OCs) can be used safely in a continuous manner to prevent monthly withdrawal bleeding. DATA SOURCES: Search of MEDLINE (1966-2000), International Pharmaceutical Abstracts (1970-2000), and the Internet. DATA SYNTHESIS: Several prospective studies have assessed the efficacy of continuous cycle OCs in preventing monthly withdrawal bleeding. Monophasic OCs are useful for this purpose. In studies using traditional OC regimens as a control, patients receiving continuously administered OCs experienced more breakthrough bleeding and spotting. Continuous use of OCs decreased headache and other menstruation-associated symptoms. No long-term trials have assessed the impact of continuous use of OCs on risk of cancer, thromboembolic disease, or fertility, concems raised by opponents of the method. CONCLUSIONS: From the data available, continuous use of OCs is an effective method for delaying withdrawal bleeding. Long-term safety data are not available.

Fractured Whiteside Ortholoc II knee components.

A comprehensive failure analysis was performed on 6 femoral components and 1 tibial component that fractured in service. All were Whiteside Ortholoc II total knee arthroplasty components, manufactured from cast cobalt-chromium-molybdenum alloy and porous coated. Fracture surface analysis revealed fatigue-induced failure in all cases. Most fractures occurred at regions of high stress concentration, such as sharp corners, sintered beads, and thin sections. Metallurgical examination showed significant variation in grain size, interdendritic carbides, and hardness between samples. In some cases, continuous carbide networks and voids were prominent at the bead-substrate interface. Patient weight and surgical placement were identified as contributory factors in component failure. Limitations of cast cobalt-chromium-molybdenum alloy in weight-bearing applications must be emphasized, particularly when important determinants, such as design, metallurgy, and specific clinical factors, are less than optimal.

Inhibition of histone deacetylase activity causes cell type-specific induction of the PDGF-B promoter only in the absence of activation by its enhancer.

There is a strong correlation between the acetylation status of nucleosomal histones and transcriptional activity. Here we show that the histone deacetylase inhibitor trichostatin A (TSA) activates reporter gene constructs driven by the human platelet-derived growth factor B (PDGF-B) gene promoter. This activation showed an inverse correlation with the cell type-specific transcriptional activities of the promoter. The TSA response was minimal in three tumor cell lines that exhibit high-level promoter activity. In JEG-3 choriocarcinoma cells, however, where the basal promoter activity is considerably lower, there was a strong response to TSA. This was in contrast to constructs that included a PDGF-B enhancer, which were refractory to TSA effects, indicating a possible function of the enhancer in modulating acetylation status. Analysis of PDGF-B promoter mutants with respect to TSA induction revealed no specific TSA-responsive element, but suggested that association of nonacetylated histones to the PDGF-B promoter may be a default process in the absence of enhancer activation. TSA treatment of JEG-3 cells, either alone or in combination with the demethylating agent 5-azacytidine, failed to activate the silenced endogenous PDGF-B transcript, however, which appears to be repressed by additional mechanisms.

Enantioselective synthesis of a mitosane core assisted by diversity-based catalyst discovery.

[reaction: see text]. Synthesis of mitosane 1 in optically pure form is reported. A retrosynthetic plan that proceeds through racemic allylic alcohol 3 was carried out. This intermediate served as a test substrate for a rapid screen of a small library (152 members) of peptide-based kinetic resolution catalysts. Peptide 9 was found to effect kinetic resolution with k(rel) = 27. Alcohol (-)-3 was then converted to optically pure (-)-1 in eight steps.