Probing the equilibrium unfolding of ketosteroid isomerase through xenon-perturbed 1H–15N multidimensional NMR spectroscopy

We used xenon-perturbed ¹H–¹⁵N multidimensional NMR to investigate the structural changes in the urea-induced equilibrium unfolding of the dimeric ketosteroid isomerase (KSI) from Pseudomonas putida biotype B. Three limited regions located on the β3-, β5- and β6-strands of dimeric interface were significantly perturbed by urea in the early stage of KSI unfolding, which could lead to dissociation of the dimer into structured monomers at higher denaturant concentration as the interactions in these regions are weakened. The results indicate that the use of xenon as an indirect probe for multidimensional NMR can be a useful method for the equilibrium unfolding study of protein at residue level.     

Plasma Adiponectin Levels and Metabolic Factors in Nondiabetic Adolescents

Objectives: The relationship of plasma adiponectin levels with various anthropometric and metabolic factors has been surveyed extensively in adults. However, how plasma adiponectin levels are related to various anthropometric indices and cardiovascular risk factors in adolescents is not as vigorously studied. In this study, we investigated this among healthy nondiabetic adolescents. Research Methods and Procedures: Two hundred thirty nondiabetic subjects (125 boys and 105 girls, ～10 to 19 years old) were included. The plasma adiponectin, fasting plasma glucose, insulin, lipids and anthropometric indices including body height, weight, waist circumference, and hip circumference were examined. Body fat mass (FM) and percentage were obtained from DXA scan. The homeostasis model assessment was applied to estimate the degree of insulin resistance. Results: The plasma adiponectin levels were significantly higher in girls (30.79 ± 14.48 μg/mL) than boys (22.87 ± 11.41 μg/mL). The plasma adiponectin levels were negatively related to BMI, FM, FM percentage, waist circumference, waist‐to‐hip ratio, insulin resistance, plasma insulin, triglycerides, and uric acid levels, but positively with high‐density lipoprotein cholesterol (HDL‐C) with the adjustment for age and gender. Using different multivariate linear regression models, only age and HDL‐C were consistently related to the plasma adiponectin levels after adjustment for the other variables. Discussion: The relationship between plasma adiponectin and various anthropometric indices and metabolic factors, especially HDL‐C, previously reported in adults was present in the healthy nondiabetic adolescents. Whether variation of plasma adiponectin levels in healthy nondiabetic adolescents may influence their future coronary artery disease risk warrants further investigation.     

Elevated Soluble CD163 in Gestational Diabetes Mellitus: Secretion from Human Placenta and Adipose Tissue

Recently soluble CD163 (sCD163), a cleaved form of the macrophage receptor CD163, was identified as a macrophage-specific risk-predictor for developing Type 2 Diabetes. Here, we investigate circulating levels of sCD163 in gestational diabetes mellitus (GDM). Furthermore, given the role of the placenta in the pathogenesis of GDM, we assessed placental contribution to sCD163 secretion. Paired maternal (venous) and umbilical vein blood samples from GDM (n = 18) and Body Mass Index (BMI) matched control women (n = 20) delivered by caesarean section at 39–40 week gestation were assessed for circulating levels of sCD163, Tumour necrosis factor alpha (TNF-α) and Interleukin 6 (IL-6). Media from explant culture of maternal subcutaneous fat and corresponding placental tissues were assayed for these same molecules. CD163 positive cell numbers were determined in placental and adipose tissues of GDM and control women. We found significantly elevated circulating sCD163 levels in GDM mothers (688.4±46.9 ng/ml vs. 505.6±38.6 ng/ml) and their offspring (418.2±26.6 ng/ml vs. 336.3±24.4 ng/ml [p<0.05 for both]) as compared to controls, together with elevated circulating TNF-α and IL-6 levels. Moreover, both GDM placentae (268.1±10.8 ng/ml/mg vs. 187.6±20.6 ng/ml/mg) and adipose explants (41.1±2.7 ng/ml/mg vs. 26.6±2.4 ng/ml/mg) released significantly more sCD163 than controls. Lastly, significantly more CD163 positive cells were observed in GDM placentae (25.7±1.1 vs. 22.1±1.2) and adipose tissue (19.1±1.1 vs 12.7±0.9) compared to controls. We describe elevated sCD163 levels in GDM and identify human placenta as a novel source of sCD163 suggesting that placental tissues might contribute to the increased levels of circulating sCD163 in GDM pregnancies.     

Individual Variation in Advertisement Calls of Territorial Male Green Frogs, Rana clamitans: Implications for Individual Discrimination

Individuals of many territorial species discriminate between familiar territorial neighbors and unfamiliar strangers based on individual differences in acoustic signals. Many anuran amphibians are territorial and use primarily acoustic communication during social interactions, but evidence for acoustically mediated individual discrimination is available only for one species. As a first step in research designed to investigate individual discrimination in a second species of territorial frog, we examined patterns of within-male and among-male variability in 198 advertisement calls of 20 male green frogs, Rana clamitans . The aim was to determine which acoustic properties could be used by males to recognize their territorial neighbors and to estimate limits of reliability afforded by these properties for identifying different neighbors. All of the call properties that we examined exhibited significant inter-individual variation. Discriminant function analyses assigned between 52% and 100% of calls to the correct individual, depending on sample size and the call properties included in the model. This suggests that there is sufficient among-male variability to statistically identify individuals by their advertisement calls. The call properties of fundamental frequency and dominant frequency contributed the most towards discrimination among individuals. Based on their natural history and behavior and the results reported here, we suggest that male green frogs likely discriminate between strangers and adjacently territorial neighbors based on individual variation in advertisement calls.     

Factors affecting flow karyotype resolution.

BACKGROUND: One of the major factors which influences the chromosome purity achievable particularly during high speed sorting is the analytical resolution of individual chromosome peaks in the flow karyotype, as well as the amount of debris and fragmented chromosomes. We have investigated the factors involved in the preparation of chromosome suspensions that influence karyotype resolution. METHODS: Chromosomes were isolated from various human and animal cell types using a series of polyamine buffer isolation protocols modified with respect to pH, salt concentration, and chromosome staining time. Each preparation was analyzed on a MoFlo sorter (DAKO) configured for high speed sorting and the resolution of the flow karyotypes compared. RESULTS: High resolution flow cytometric data was obtained with chromosomes optimally isolated using hypotonic solution buffered at pH 8.0 and polyamine isolation buffer (with NaCl excluded) between pH 7.50 and 8.0. Extending staining time to more than 8 h with chromosome suspensions isolated from cell lines subjected to sufficient metaphase arrest times gave the best result with the lowest percentage of debris generated, tighter chromosome peaks with overall lower coefficients of variation, and a 1- to 5-fold increase in the yield of isolated chromosomes. CONCLUSIONS: Optimization of buffer pH and the length of staining improved karyotype resolution particularly for larger chromosomes and reduced the presence of chromosome fragments (debris). However, the most interesting and surprising finding was that the exclusion of NaCl in PAB buffer improved the yield and resolution of larger chromosomes.     

Detection of Coronavirus 229E Antibody by Indirect Hemagglutination

Tannic-acid treated sheep erythrocytes (fresh or glutaraldehyde preserved) were sensitized with 229E antigens from human embryonic lung (RU-1) cell cultures. Indirect hemagglutination (IHA) antigen titers in 229E-infected cell cultures paralleled virus infectivity and complement fixation (CF) antigen titers. The identity of the IHA antigen was confirmed by testing extracts from inoculated and control cell cultures for ability to inhibit IHA. Also, significant increases in IHA antibody were demonstrated with acute and convalescent serum pairs from patients with proven 229E infections. A comparison of IHA, neutralization and CF titers for 229E antibodies was made on human sera drawn from different populations. The IHA and neutralization results were in agreement on 93% of the 129 sera found to be positive by at least one of three tests. The number of antibody titers detected by the CF test was insufficient to permit comparison. Hyperimmune sera from animals immunized with OC 43 did not react with 229E by IHA. Also no increase in IHA antibody was demonstrated with acute and convalescent serum pairs from patients with seroconversions to OC 43. These findings suggest that the IHA test provides (i) a rapid and sensitive method for serodiagnosis of 229E infections and (ii) a simple and inexpensive method for seroepidemiological studies.     

15N NMR relaxation studies of Y14F mutant of ketosteroid isomerase: the influence of mutation on backbone mobility

The backbone dynamics of Y14F mutant of Delta(5)-3-ketosteroid isomerase (KSI) from Comamonas testosteroni has been studied in free enzyme and its complex with a steroid analogue, 19-nortestosterone hemisuccinate (19-NTHS), by (15)N NMR relaxation measurements. Model-free analysis of the relaxation data showed that the single-point mutation induced a substantial decrease in the order parameters (S(2)) in free Y14F KSI, indicating that the backbone structures of Y14F KSI became significantly mobile by mutation, while the chemical shift analysis indicated that the structural perturbations of Y14F KSI were more profound than those of wild-type (WT) KSI upon 19-NTHS binding. In the 19-NTHS complexed Y14F KSI, however, the key active site residues including Tyr14, Asp38 and Asp99 or the regions around them remained flexible with significantly reduced S(2) values, whereas the S(2) values for many of the residues in Y14F KSI became even greater than those of WT KSI upon 19-NTHS binding. The results thus suggest that the hydrogen bond network in the active site might be disrupted by the Y14F mutation, resulting in a loss of the direct interactions between the catalytic residues and 19-NTHS.