A platform was developed to analyze MS/MS spectra from large peptides with low part-per-million mass accuracy, including a commercial-grade software suite. Termed Middle Down Proteomics, this ...platform identified 7454 peptides from 2−20 kDa (1472 unique) from 555 proteins after 23 LC-MS/MS injections of Lys-C digests of HeLa-S3 nuclear proteins. Along with greatly increased confidence for both peptide identification (expectation values from 10−89 to 10−4) and characterization (up to 18% of peptides were modified in some LC-MS/MS runs), fragmentation data with <2 ppm accuracy enabled error tolerant and routine multiplexed database searching–all clearly demonstrated in this study.
For automated production of tandem mass spectrometric data for proteins and peptides >3 kDa at >50 000 resolution, a dual online−offline approach is presented here that improves upon standard liquid ...chromatography−tandem mass spectrometry (LC−MS/MS) strategies. An integrated hardware and software infrastructure analyzes online LC−MS data and intelligently determines which targets to interrogate offline using a posteriori knowledge such as prior observation, identification, and degree of characterization. This platform represents a way to implement accurate mass inclusion and exclusion lists in the context of a proteome project, automating collection of high-resolution MS/MS data that cannot currently be acquired on a chromatographic time scale at equivalent spectral quality. For intact proteins from an acid extract of human nuclei fractionated by reversed-phase liquid chromatography (RPLC), the automated offline system generated 57 successful identifications of protein forms arising from 30 distinct genes, a substantial improvement over online LC−MS/MS using the same 12 T LTQ FT Ultra instrument. Analysis of human nuclei subjected to a shotgun Lys-C digest using the same RPLC/automated offline sampling identified 147 unique peptides containing 29 co- and post-translational modifications. Expectation values ranged from 10−5 to 10−99, allowing routine multiplexed identifications.
Fetal IGF-I is a determinant of birth weight, but whether maternal IGF-I plays a significant role is controversial. We sought to examine the relationships among maternal IGF-I, IGF-binding protein ...(IGFBP)-1, and IGFBP-2, with maternal and newborn anthropometry, in a cohort of 325 nondiabetic pregnant women of African origin. Blood was collected for IGF-I, IGFBP-1, and IGFBP-2 at 9, 25, and 35 wk gestation and in cord blood at delivery.
In the second and third trimesters, maternal IGF-I was significantly correlated (P < 0.005) with maternal body mass index and triceps skinfold thickness. Maternal IGFBP-1 and -2 had an inverse correlation (P < 0.0001), with maternal anthropometry. Maternal IGF-I at 35 wk, and fetal IGF-I by cord blood were significantly correlated with birth weight (P = 0.001 and 0.048, respectively). IGFBP-1 in the third trimester and cord blood were negatively correlated with birth weight (P = 0.012 and 0.002). In multiple regression analyses, maternal IGF-I at 35 wk, fetal IGF-I, maternal weight at the first antenatal visit, gender, and gestational age were significant independent factors in the determination of birth weight. In conclusion, maternal IGF-I levels, especially during late pregnancy, positively influence birth weight.
Ketosis-prone diabetes has been an enigmatic entity ever since it was first described in 1955 by Hugh-Jones. In that series, it compromised 6% of diabetic Jamaicans and was typified by periodic ...insulinopenia, insulin resistance, lean body habitus, and no fatty liver. Since then, it has been described in many locales, especially in India and Africa, among minorities in the United States, and not necessarily in lean individuals. It has a variety of other names, including the following: J-type diabetes, Flatbush diabetes, atypical type 2 diabetes, phasic insulin-dependent diabetes, and type 3 diabetes. It most closely resembles type 1B diabetes in the WHO classification and is more commonly seen in nonwhite populations with marginal nutritional status or chronic malnutrition. Thus, it is possible that these individuals may have â-cell dysfunction due to either reduced glucose-stimulated insulin secretion or reduced â-cell mass from early life, similar to that seen in adult survivors of marasmus.
An earlier onset of puberty is associated with increased cardiometabolic risk. We investigated whether this relation was independent of faster childhood growth or current size in an Afro-Caribbean ...birth cohort (n=259). Anthropometry was measured at birth and then 6-monthly. Tanner staging started at age 8 years. Cardiometabolic risk factors were measured at mean age 11.5 years. In boys, pubarchal stage and testicular size were associated with lower high-density lipoprotein cholesterol, higher systolic blood pressure, and higher homeostasis model assessment of insulin resistance score, but not after adjusting for current body mass index (BMI) or rate of growth (up to age 8 years). In girls, earlier menarche and greater breast development were associated with higher fasting glucose even after adjusting for current BMI or prior growth. Pubarchal stage was associated with systolic blood pressure, even after adjusting for current BMI and prior growth. We concluded that earlier puberty is independently associated with cardiometabolic risk in girls but not in boys.
1. Topical anesthesia with benzocaine or lidocaine occasionally causes methemoglobinemia, an uncommon but potentially fatal disorder where the blood has a reduced ability to transport oxygen. ...Previous in vitro studies using human whole blood have shown that benzocaine causes more methemoglobin (MetHb) formation than lidocaine, and that both compounds require metabolic transformation to form the MetHb producing species. In the current investigation, the active species of benzocaine forming the MetHb was investigated.
2. HPLC analysis of benzocaine samples incubated with human hepatic S9 showed the formation of a peak with the same UV spectrum and retention time as benzocaine hydroxylamine (BenzNOH). To confirm the activity of BenzNOH, MetHb production following exposure to the compound was determined in whole human blood using an Avoximeter 4000 CO-oximeter.
3. BenzNOH produced MetHb in a concentration dependent manner without the need for metabolic activation. Benzocaine in the presence of metabolic activation required a concentration of 500 μM to produce a similar degree of MetHb formation as 20 μM BenzNOH without activation. Previous work suggested that two metabolites of lidocaine may also form MetHb; N-hydroxyxylidine and 4-hydroxyxylidine. Of these two metabolites 4-hydroxyxylidine produced the most MetHb in whole blood in vitro in the absence of metabolic activation, however BenzNOH produced up to 14.2 times more MetHb than 4-hydroxyxylidine at a similar concentration.
4. These results suggest that the ability of benzocaine to form MetHb is likely to be mediated through its hydroxylamine metabolite and that this metabolite is inherently more active than the potentially MetHb-forming metabolites of lidocaine.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Severe acute malnutrition (SAM) in infants may present as one of two distinct syndromic forms: non-edematous (marasmus), with severe wasting and no nutritional edema; or edematous (kwashiorkor) with ...moderately severe wasting. These differences may be related to developmental changes prior to the exposure to SAM and phenotypic changes appear to persist into adulthood with differences between the two groups. We examined whether the different response to SAM and subsequent trajectories may be explained by developmentally-induced epigenetic differences.
We extracted genomic DNA from muscle biopsies obtained from adult survivors of kwashiorkor (n=21) or marasmus (n=23) and compared epigenetic profiles (CpG methylation) between the two groups using the Infinium® 450K BeadChip array.
We found significant differences in methylation of CpG sites from 63 genes in skeletal muscle DNA. Gene ontology studies showed significant differential methylation of genes in immune, body composition, metabolic, musculoskeletal growth, neuronal function and cardiovascular pathways, pathways compatible with the differences in the pathophysiology of adult survivors of SAM.
These findings suggest persistent developmental influences on adult physiology in survivors of SAM. Since children who develop marasmus have lower birth weights and after rehabilitation have different intermediary metabolism, these studies provide further support for persistent developmentally-induced phenomena mediated by epigenetic processes affecting both the infant response to acute malnutrition and later life consequences.
Supported by a Grant from the Bill and Melinda Gates Foundation (Global Health OPP1066846), Grand Challenge “Discover New Ways to Achieve Healthy Growth.”
Previous research has shown that infants who develop either kwashiorkor or marasmus in response to SAM differ in birth weight and subsequently have different metabolic patterns in both infancy and adulthood.
This study demonstrates epigenetic differences in the skeletal muscle of adult survivors of marasmus versus kwashiorkor and these differences are in genes that may underlie the longer-term consequences.
These data are compatible with the different clinical responses to SAM arising from developmentally-induced epigenetic changes laid down largely before birth and provide evidence for the predictive adaptive response model operating in human development.
•Infants who develop either kwashiorkor or marasmus differ in birth weight and subsequently have different metabolic patterns in later life.•We found epigenetic differences in the skeletal muscle of adult survivors of marasmus versus kwashiorkor which may underlie the longer-term consequences.•Different clinical responses to malnutrition may arise from developmentally-induced epigenetic changes resulting in predictive adaptive responses.
The biosynthesis of the coronamic acid fragment of the pseudomonal phytotoxin coronatine involves construction of the cyclopropane ring from a γ-chloro-l-allo-Ile intermediate while covalently ...tethered as a phosphopantetheinyl thioester to the carrier protein CmaD. The cyclopropane-forming catalyst is CmaC, catalyzing an intramolecular displacement of the γ-Cl group by the α carbon. CmaC can be isolated as a Zn2+ protein with about 10-fold higher activity over the apo form. CmaC will not cyclize free γ-chloro amino acids or their S-N-acetylcysteamine (NAC) thioester derivatives but will recognize some other carrier protein scaffolds. Turnover numbers of 5 min-1 are observed for Zn−CmaC, acting on γ-chloro-l-aminobutyryl-S-CmaD, generating 1-aminocyclopropane-1-carbonyl (ACC)-S-CmaD. Products were detected either while still tethered to the phosphopantetheinyl prosthetic arm by mass spectrometry or after thioesterase-mediated release and derivatization of the free amino acid. In D2O, CmaC catalyzed exchange of one deuterium into the aminobutyryl moiety of the γ-Cl-aminoacyl-S-CmaD, whereas the product ACC-S-CmaD lacked the deuterium, consistent with a competition for a γ-Cl-aminobutyryl α-carbanion between reprotonation and cyclization. CmaC-mediated cyclization yielded solely ACC, resulting from C−C bond formation and no azetidine carboxylate from an alternate N−C cyclization. CmaC could cyclize γ,γ-dichloroaminobutyryl to the Cl-ACC product but did not cyclize δ- or ε-chloroaminoacyl-S-CmaD substrates.
Nonobese nonalcoholic fatty liver disease is reported in several populations. However, because persons of African origin display unique fat accumulation, insulin resistance, and lipid profiles, we ...investigated fatty liver in nonobese persons of African origin.
We recruited 78 urban Jamaican volunteers. CT was used to estimate liver and abdominal fat and dual-energy X-ray absorptiometry to measure body composition. Fasting blood was collected for lipids, alanine aminotransferase (ALT), adiponectin, and fetuin-A. Homeostatic model assessment of insulin resistance (HOMA-IR), whole-body insulin sensitivity index (WBISI), insulinogenic index (IGI), and oral disposition index (oDI) were calculated after a 75-g oral glucose tolerance test.
Fifty-two percent of participants were male; mean (±SD) age was 28.5 ± 7.8 years, and body mass index was 22.4 ± 3.0 kg/m
. Mean liver attenuation (MLA) and liver/spleen (LS) ratio, both inversely correlated to liver fat, were 62.8 ± 4.3 HU and 1.2 ± 0.1, respectively; 3.8% of participants had liver fat >30% (LS ratio < 1). In age, sex, and BMI-adjusted correlations, MLA was negatively associated with weight (
= -0.30;
= 0.009) and height (
= -0.28;
= 0.017) and was associated with fasting glucose (
= 0.23;
= 0.05), fasting insulin (
= 0.42;
≤ 0.001) and HOMA-IR (
= 0.35;
= 0.004). Serum lipids, ALT, adiponectin, fetuin-A, WBISI, IGI, and oDI were not associated with liver fat.
In nonobese Afro-Caribbean participants, greater liver fat was associated with weight and height and lower fasting insulin and hyperinsulinemia appears to be influential in the reduction of NAFLD. These findings may be influenced by ethnicity, body size, and method of estimating liver fat.
Abstract We longitudinally explored the relationship of body size and adiponectin levels in 393 community-dwelling Afro-Jamaicans. Adiponectin levels were greater in women, increased with age and ...declined with abdominal adiposity. Multivariate regression analyses suggest that subcutaneous fat in women may contribute significantly to the variance in their adiponectin levels.
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