Mutations within a human IgG2 antibody form distinct and homogeneous disulfide isomers but do not affect Fc gamma receptor or C1q binding

Human IgG2 antibodies may exist in at least three distinct structural isomers due to disulfide shuffling within the upper hinge region. Antibody interactions with Fc gamma receptors and the complement component C1q contribute to immune effector functions. These interactions could be impacted by the accessibility and structure of the hinge region. To examine the role structural isomers may have on effector functions, a series of cysteine to serine mutations were made on a human IgG2 backbone. We observed structural homogeneity with these mutants and mapped the locations of their disulfide bonds. Importantly, there was no observed difference in binding to any of the Fc gamma receptors or C1q between the mutants and the wild‐type IgG2. However, differences were seen in the apparent binding affinity of these antibodies that were dependent on the selection of the secondary detection antibody used.     

Consistent Association of Type 2 Diabetes Risk Variants Found in Europeans in Diverse Racial and Ethnic Groups

It has been recently hypothesized that many of the signals detected in genome-wide association studies (GWAS) to T2D and other diseases, despite being observed to common variants, might in fact result from causal mutations that are rare. One prediction of this hypothesis is that the allelic associations should be population-specific, as the causal mutations arose after the migrations that established different populations around the world. We selected 19 common variants found to be reproducibly associated to T2D risk in European populations and studied them in a large multiethnic case-control study (6,142 cases and 7,403 controls) among men and women from 5 racial/ethnic groups (European Americans, African Americans, Latinos, Japanese Americans, and Native Hawaiians). In analysis pooled across ethnic groups, the allelic associations were in the same direction as the original report for all 19 variants, and 14 of the 19 were significantly associated with risk. In summing the number of risk alleles for each individual, the per-allele associations were highly statistically significant (P<10⁻⁴) and similar in all populations (odds ratios 1.09–1.12) except in Japanese Americans the estimated effect per allele was larger than in the other populations (1.20; P_het = 3.8×10⁻⁴). We did not observe ethnic differences in the distribution of risk that would explain the increased prevalence of type 2 diabetes in these groups as compared to European Americans. The consistency of allelic associations in diverse racial/ethnic groups is not predicted under the hypothesis of Goldstein regarding “synthetic associations” of rare mutations in T2D.     

Effects of time and rainfall on PCR success using DNA extracted from deer fecal pellets

Non-invasive wildlife research using DNA from feces has become increasingly popular. Recent studies have attempted to solve problems associated with recovering DNA from feces by investigating the influence of factors such as season, diet, collection method, preservation method, extraction protocol, and time. To our knowledge, studies of this nature have not addressed DNA degradation over time in wet environments, and have not been performed on fecal pellets of ungulates. Therefore, our objective was to determine the length of time a fecal pellet from a Sitka black-tailed deer (Odocoileus hemionus sitkensis) could remain in the field in a temperate rainforest environment before the DNA became too degraded for individual identification. Pellets were extracted from the rectum of recently killed deer and placed in an environment protected from rainfall and in an environment exposed to rainfall. Pellets from each treatment group were sampled at intervals of 2, 7, 14, 21, and 28 days after deer harvest. DNA was extracted from sampled pellets and individual samples were genotyped using microsatellite markers. Amplification failure and errors (dropout and false alleles) were recorded to determine extent of DNA degradation. Eighty percent of samples in the protected environment and 22% of samples in the exposed environment were successfully genotyped during the 28-day experiment. With no samples being successfully genotyped in the exposed environment after 7 days, our study showed that rainfall significantly increases degradation rates of DNA from ungulate pellets.     

Is caseinomacropeptide from milk proteins,an inhibitor of gastric secretion?

The aim of this work was to study, in vivo, the effect of the ingestion of not glycosylated caseinomacropeptide (CMP) on gastric secretion. In Experiments #1 and #2, 7 calves fitted with a gastric pouch received either a diet without CMP (C diet) or C diet in which CMP was introduced (equal to and 5 folds that of CMP quantity contained in cow milk, diets CMP1 and CMP5, respectively). In Experiment #3, 2 calves (with gastric pouch) were fed C diet followed by an “iv perfusion” of CMP. In Experiment #4, 25 calves fed either C, CMP1 or CMP5 diets were fitted with a blood catheter for sample collections. The quantities of daily gastric secretions seemed few modified by CMP ingestion but the profile of these secretions was changed along the day. The most important result is that CMP can inhibit gastric secretions (mainly hydrochloric acid) stimulated by the meal, but there was no dose-dependent response. No similar observations were obtained after perfusion of CMP in jugular vein. CMP was not detected in blood. Results obtained in our experiments are not in favor of its significant intestinal absorption. Gastrin, somatostatin and VIP could be implicated in the mechanisms of regulation.     

Soil carbon and nitrogen mineralization following deposition of insect frass and greenfall from forests under elevated CO2 and O3

Elevated CO₂ and O₃ alter tree quality and the quality of herbivore inputs, such as frass, to forest soil. Altered quality or quantity of herbivore inputs to the forest floor can have large impacts on belowground processes. We collected green leaves and frass from whitemarked tussock moth caterpillars from aspen-birch stands at the Aspen Free Air CO₂ Enrichment (FACE) site near Rhinelander, WI, USA. Small or large quantities of frass, greenfall, or a 1:1 ratio of frass and greenfall were added to microcosms for each FACE treatment (control, +CO₂, +O₃, +CO₂+O₃). We measured initial frass and greenfall quality, and recorded microbial respiration, and nitrate leaching over 40 days. Elevated carbon dioxide (eCO₂) and tropospheric ozone (eO₃) significantly altered the carbon, nitrogen, and condensed tannin content of insect frass and green leaves. Although FACE treatments affected input quality, they had minimal effect on microbial respiration and no effect on nitrogen leaching. In contrast, input quantity substantially influenced microbial respiration and nitrate leaching. Respiratory carbon loss and nitrate immobilization were nearly double in microcosms receiving large amounts of herbivore inputs than those receiving no herbivore inputs. Small amounts of herbivore inputs, however, did not significantly alter microbial respiration or immobilization, suggesting that effects of herbivore inputs on soil processes will be detected only at moderate to high herbivory/input levels. These results suggest that subtle changes in frass and greenfall quality may not affect soil nutrient cycling. In contrast, environmental change induced increases in insect population size or frass and greenfall inputs to the soil may substantially impact nutrient cycling.     

Validation of QTLs for Orobanche crenata resistance in faba bean (Vicia faba L.) across environments and generations

Broomrape (Orobanche crenata Forsk.) is a major root–parasite of faba bean (Vicia faba L.), that seriously limits crop cultivation in the whole Mediterranean area. This parasitic weed is difficult to control, difficult to evaluate and the resistance identified so far is of polygenic nature. This study was conducted to identify genetic regions associated with broomrape resistance in recombinant inbred lines (RILs) and to validate their previous location in the original F₂ population derived from the cross between lines Vf6 and Vf136. A progeny consisting of 165 F₆ RILs was evaluated in three environments across two locations in 2003 and 2004. Two hundred seventy seven molecular markers were assigned to 21 linkage groups (9 of them assigned to specific chromosomes) that covered 2,856.7 cM of the V. faba genome. The composite interval mapping on the F₆ map detected more quantitative trait loci (QTL) than in the F₂ analysis. In this sense, four QTLs controlling O. crenata resistance (Oc2–Oc5) were identified in the RI segregant population in three different environments. Only Oc1, previously reported in the F₂ population, was not significant in the advanced lines. Oc2 and Oc3 were found to be associated with O. crenata resistance in at least two of the three environments, while the remaining two, Oc4 and Oc5, were only detected in Córdoba-04 and Mengíbar-04 and seemed to be environment dependent.     

Strategies for organ level tissue engineering

The field of tissue engineering has made considerable strides since it was first described in the late 1980s. The advent and subsequent boom in stem cell biology, emergence of novel technologies for biomaterial development and further understanding of developmental biology have contributed to this accelerated progress. However, continued efforts to translate tissue-engineering strategies into clinical therapies have been hampered by the problems associated with scaling up laboratory methods to produce large, complex tissues. The significant challenges faced by tissue engineers include the production of an intact vasculature within a tissue-engineered construct and recapitulation of the size and complexity of a whole organ. Here we review the basic components necessary for bioengineering organs-biomaterials, cells and bioactive molecules-and discuss various approaches for augmenting these principles to achieve organ level tissue engineering. Ultimately, the successful translation of tissue-engineered constructs into everyday clinical practice will depend upon the ability of the tissue engineer to "scale up" every aspect of the research and development process.     

Seasonal ultrasonic acoustic emissions of <Emphasis Type="Italic">Quercus ilex</Emphasis> L. trees in a Mediterranean forest

Ultrasonic acoustic emissions were measured in Quercus ilex trees of a Mediterranean forest in Catalonia (NE Spain) each season from summer of 2004 to autumn of 2005. Acoustic emissions were maximum during hot and dry summer periods. Acoustic emissions started below 17% soil moisture, 0.85 RWC, and 2.5 MPa leaf water potential. They were negatively correlated with soil moisture and leaf water potential. The relationship between acoustic emissions and leaf water potential was the strongest, indicating that xylem tension is the most important factor inducing both cavitation (acoustic emissions) and a decrease in leaf water potential. Future increase of xylem cavitation derived from climate change may result in growth and survival limitations for this species in the drier southern limits of its current distribution.     

Metabolism of glucagon-like peptide-2 in pigs: role of dipeptidyl peptidase IV

Little is known about the metabolism of the intestinotropic factor glucagon-like peptide-2 (GLP-2); except that it is a substrate for dipeptidyl peptidase IV (DPP-IV) and that it appears to be eliminated by the kidneys. We, therefore, investigated GLP-2 metabolism in six multicatheterized pigs receiving intravenous GLP-2 infusions (2 pmol/kg/min) before and after administration of valine-pyrrolidide (300 mumol/kg; a well characterized DPP-IV inhibitor). Plasma samples were analyzed by radioimmunoassays allowing determination of intact, biologically active GLP-2 and the DPP-IV metabolite GLP-2 (3-33). During infusion of GLP-2 alone, 30.9+/-1.7% of the infused peptide was degraded to GLP-2 (3-33). After valine-pyrrolidide, there was no significant formation of the metabolite. Significant extraction of intact GLP-2 was observed across the kidneys, the extremities (represented by a leg), and the splanchnic bed, resulting in a metabolic clearance rate (MCR) of 6.80+/-0.47 ml/kg/min and a plasma half-life of 6.8+/-0.8 min. Hepatic extraction was not detected. Valine-pyrrolidide addition did not affect extraction ratios significantly, but decreased (p=0.003) MCR to 4.18+/-0.27 ml/kg/min and increased (p=0.052) plasma half-life to 9.9+/-0.8 min. The metabolite was eliminated with a half-life of 22.1+/-2.6 min and a clearance of 2.07+/-0.11 ml/kg/min. In conclusion, intact GLP-2 is eliminated in the peripheral tissues, the splanchnic bed and the kidneys, but not in the liver, by mechanisms unrelated to DPP-IV. However, DPP-IV is involved in the overall GLP-2 metabolism and seems to be the sole enzyme responsible for N-terminal degradation of GLP-2.