In search for non-steroidogenic functions of the prothoracic glands of the desert locust, Schistocerca gregaria: a peptidomic and proteomic approach

The only well established function of the prothoracic glands (PGs) of insects is the production of ecdysteroids. In gregarious locusts, like in most insect species, the PGs degenerate soon after the adult molt. In this way they resemble the thymus of mammals, a gland with an important role in the build up of the immune system in young animals. In adult solitarious locusts the PGs persist much longer, however without producing substantial amounts of ecdysteroids. In the literature the existence of a well developed rough endoplasmic reticulum and Golgi complex system has been repeatedly reported, suggesting an active role in peptide or/and protein synthesis and release. The nature of the secreted products remained unknown. Our pepdidomic analysis of an acidified methanolic extract of PGs of last instar gregarious nymphs did not yield any indication for the presence of known locust or other peptides. The peptide release assay was also negative. For our proteomic analysis, we developed an EST-based identification strategy. We successfully identified 50 protein spots on a two dimensional map. In addition to typical protein synthesis-related proteins, a number of proteins with a role in detoxification processes were found, suggesting some role of the PGs in the defense system.     

Transportomics: screening for substrates of ABC transporters in body fluids using vesicular transport assays

The ATP-binding cassette (ABC) genes encode the largest family of transmembrane proteins. ABC transporters translocate a wide variety of substrates across membranes, but their physiological function is often incompletely understood. We describe a new method to study the substrate spectrum of ABC transporters: We incubate extracts of mouse urine with membrane vesicles prepared from Spodoptera frugiperda Sf9 insect cells overproducing an ABC transporter and determine the compounds transported into the vesicles by LC/MS-based metabolomics. We illustrate the power of this simple "transportomics" approach using ABCC2, a protein present at sites of uptake and elimination. We identified many new substrates of ABCC2 in urine. These included glucuronides of plant-derived xenobiotics, a class of compounds to which humans are exposed on a daily basis. Moreover, we show that the excretion of these compounds in vivo depends on ABCC2: compared to wild-type mice, the urinary excretion of several glucuronides was increased up to 20-fold in Abcc2(-/-) mice. Transportomics has broad applicability, as it is not restricted to urine and can be applied to other ATP-dependent transport proteins as well.     

Evaluation of passively transferred, nonneutralizing antibody-dependent cellular cytotoxicity-mediating IgG in protection of neonatal rhesus macaques against oral SIVmac251 challenge

Previously, Ab-dependent cellular cytotoxicity (ADCC) was significantly correlated with reduced acute viremia upon intrarectal SIVmac251 challenge of immunized rhesus macaques. To directly assess ADCC protective efficacy, six neonatal macaques were infused s.c. with immune IgG (220 mg/kg) purified from the immunized animals and positive for ADCC and Ab-dependent cell-mediated viral inhibition (ADCVI) activities. Six neonates received control IgG. The neonates were challenged twice orally with 10(5) 50% inhibiting tissue culture-infective dose of SIVmac251 2 days post-IgG infusion. At challenge, plasma of neonates that received immune IgG did not neutralize SIVmac251 but had geometric mean ADCC titers of 48,130 and 232,850 against SIVmac251 -infected and gp120-coated targets, respectively. Peak ADCVI activity varied from 62 to 81%. ADCC activity declined with the 2-wk IgG half-life but was boosted at wk 4, together with de novo ADCC-mediating Abs in controls, by postchallenge viremia. ADCVI activity was similarly induced. No protection, assessed by viral burdens, CD4 counts, and time to euthanasia was observed. Possible factors contributing to the discrepancy between the previous correlation and lack of protection here include: the high oral challenge dose compared with the 400-fold lower intrarectal dose; the challenge route with regard to viral dissemination and distribution of infused IgG; insufficient NK effector activity and/or poor functionality in newborns; insufficient immune IgG; and the possibility that the previous correlation of ADCC with protection was augmented by cellular immune responses also present at challenge. Future studies should explore additional challenge routes in juvenile macaques using higher amounts of potent IgG preparations.     

Functional role of the conserved active site proline of triosephosphate isomerase

The importance of the fully conserved active site proline, Pro168, for the reaction mechanism of triosephosphate isomerase (TIM) has been investigated by studying the enzymatic and crystallographic properties of the P168A variant of trypanosomal TIM. In TIM, Pro168 follows the key catalytic residue Glu167, situated at the beginning of the flexible active site loop (loop 6). Turnover numbers of the P168A variant for its substrates are reduced approximately 50-fold, whereas the Km values are approximately 2 times lower. The affinity of the P168A variant for the transition state analogue 2-phosphoglycolate (2PG) is reduced 5-fold. The crystal structures of unliganded and liganded (2PG) P168A show that the phosphate moiety of 2PG is bound similarly as in wild-type TIM, whereas the interactions of the carboxylic acid moiety with the side chain of the catalytic Glu167 differ. The unique properties of the proline side chain at position 168 are required to transmit ligand binding to the conformational change of Glu167: the side chain of Glu167 flips from the inactive swung-out to the active swung-in conformation on ligand binding in wild-type TIM, whereas in the mutant this conformational change does not occur. Further structural comparisons show that in the wild-type enzyme the concerted movement of loop 6 and loop 7 from unliganded-open to liganded-closed appears to be facilitated by the interactions of the phosphate moiety with loop 7. Apparently, the rotation of 90 degrees of the Gly211-Gly212 peptide plane of loop 7 plays a key role in this concerted movement.     

The diastolic flow velocity-pressure gradient relation and dpv50 to assess the hemodynamic significance of coronary stenoses

To evaluate the hemodynamic impact of coronary stenoses, the fractional (FFR) or coronary flow velocity reserve (CFVR) usually is measured. The combined measurement of instantaneous flow velocity and pressure gradient (v-dp relation) is rarely used in humans. We derived from the v-dp relation a new index, dp(v50) (pressure gradient at flow velocity of 50 cm/s), and compared the diagnostic performance of dp(v50), CFVR, and FFR. Before coronary angiography was performed, patients underwent noninvasive stress testing. In all coronary vessels with an intermediate or severe stenosis, the flow velocity, aortic, and distal coronary pressure were measured simultaneously with a Doppler and pressure guidewire after induction of hyperemia. After regression analysis of all middiastolic flow velocity and pressure gradient data, the dp(v50) was calculated. With the use of the results of noninvasive stress testing, the dp(v50) cutoff value was established at 22.4 mmHg. In 77 patients, 124 coronary vessels with a mean 39% (SD 19) diameter stenosis were analyzed. In 43 stenoses, ischemia was detected. We found a sensitivity, specificity, and accuracy of 56%, 86%, and 76% for CFVR; 77%, 99%, and 91% for FFR; and 95%, 95%, and 95% for dp(v50). To establish that dp(v50) is not dependent on maximal hyperemia, dp(v50) was recalculated after omission of the highest quartile of flow velocity data, showing a difference of 3%. We found that dp(v50) provided the highest sensitivity and accuracy compared with FFR and CFVR in the assessment of coronary stenoses. In contrast to CFVR and FFR, assessment of dp(v50) is not dependent on maximal hyperemia.     

Beyond diversity: functional microbiomics of the human colon

Molecular tools have revealed wide microbial diversity in the human alimentary tract. Most intestinal microorganisms have not been cultured and the in situ functions of distinct groups of the intestinal microbiota are largely unknown but pivotal to understanding the role of these microorganisms in health and disease. Promising strategies to gain more insight into the functionality of the complex microbial communities in the human alimentary tract, including fermentation processes in the colon, are discussed. These research approaches could provide a basis for the definition of a healthy gut based on key properties of microbial functionality. This will also enable the development of direct nutritional strategies for intestinal disease prevention and health promotion.     

Sauropod dinosaurs evolved moderately sized genomes unrelated to body size

Sauropodomorph dinosaurs include the largest land animals to have ever lived, some reaching up to 10 times the mass of an African elephant. Despite their status defining the upper range for body size in land animals, it remains unknown whether sauropodomorphs evolved larger-sized genomes than non-avian theropods, their sister taxon, or whether a relationship exists between genome size and body size in dinosaurs, two questions critical for understanding broad patterns of genome evolution in dinosaurs. Here we report inferences of genome size for 10 sauropodomorph taxa. The estimates are derived from a Bayesian phylogenetic generalized least squares approach that generates posterior distributions of regression models relating genome size to osteocyte lacunae volume in extant tetrapods. We estimate that the average genome size of sauropodomorphs was 2.02 pg (range of species means: 1.77–2.21 pg), a value in the upper range of extant birds (mean = 1.42 pg, range: 0.97–2.16 pg) and near the average for extant non-avian reptiles (mean = 2.24 pg, range: 1.05–5.44 pg). The results suggest that the variation in size and architecture of genomes in extinct dinosaurs was lower than the variation found in mammals. A substantial difference in genome size separates the two major clades within dinosaurs, Ornithischia (large genomes) and Saurischia (moderate to small genomes). We find no relationship between body size and estimated genome size in extinct dinosaurs, which suggests that neutral forces did not dominate the evolution of genome size in this group.     

Non-Helicobacter pylori helicobacters detected in the stomach of humans comprise several naturally occurring Helicobacter species in animals

Besides the well-known gastric pathogen Helicobacter pylori , other Helicobacter species with a spiral morphology have been detected in a minority of human patients who have undergone gastroscopy. The very fastidious nature of these non- Helicobacter pylori helicobacters (NHPH) makes their in vitro isolation difficult. These organisms have been designated ' Helicobacter heilmannii '. However, sequencing of several genes detected in NHPH-infected tissues has shown that the ' H. heilmannii ' group comprises at least five different Helicobacter species, all of them known to colonize the stomach of animals. Recent investigations have indicated that Helicobacter suis is the most prevalent NHPH species in human. This species has only recently been isolated in vitro from porcine stomach mucosa. Other NHPH that colonize the human stomach are Helicobacter felis, Helicobacter bizzozeronii, Helicobacter salomonis and ' Candidatus Helicobacter heilmannii'. In numerous case reports of human gastric NHPH infections, no substantial information is available about the species status of the infecting strain, making it difficult to link the species with certain pathologies. This review aims to clarify the complex nomenclature of NHPH species associated with human gastric disease and their possible animal origin. It is proposed to use the term 'gastric NHPH' to designate gastric spirals that are morphologically different from H. pylori when no identification is available at the species level. Species designations should be reserved for those situations in which the species is defined.