Peak Fractions Research Articles

SGTA Recognizes a Noncanonical Ubiquitin-like Domain in the Bag6-Ubl4A-Trc35 Complex to Promote Endoplasmic Reticulum-Associated Degradation

Elimination of aberrantly folded polypeptides from the endoplasmic reticulum (ER) by the ER-associated degradation (ERAD) system promotes cell survival under stress conditions. This quality control mechanism requires movement of misfolded proteins across the ER membrane for targeting to the cytosolic proteasome, a process facilitated by a "holdase" complex, consisting of Bag6 and the cofactors Ubl4A and Trc35. This multiprotein complex also participates in several other protein quality control processes. Here, we report SGTA as a component of the Bag6 system, which cooperates with Bag6 to channel dislocated ERAD substrates that are prone to aggregation. Using nuclear magnetic resonance spectroscopy and biochemical assays, we demonstrate that SGTA contains a noncanonical ubiquitin-like-binding domain that interacts specifically with an unconventional ubiquitin-like protein/domain in Ubl4A at least in part via electrostatics. This interaction helps recruit SGTA to Bag6, enhances substrate loading to Bag6, and thus prevents the formation of nondegradable protein aggregates in ERAD.

Characterization of the Molecular Architecture of Human Caveolin-3 and Interaction with the Skeletal Muscle Ryanodine Receptor

Caveolin-3 facilitates both caveolae formation and a range of cell signaling pathways, including Ca(2+) homeostasis. Caveolin-3 forms a disc-shaped nonamer that binds the Ca(2+)-release channel, RyR1. Multiple caveolin-3 nonamers bind to a single RyR1 homotetramer. First three-dimensional structural insights into caveolin-3 assembly, interactions with RyR1 suggest a novel role in muscle contraction and/or for channel localization within the membrane. Caveolin-3 (cav-3), an integral membrane protein, is a building block of caveolae as well as a regulator of a number of physiological processes by facilitating the formation of multiprotein signaling complexes. We report that the expression of cav-3 in insect (Sf9) cells induces caveola formation, comparable in size with those observed in native tissue. We have also purified the recombinant cav-3 determining that it forms an oligomer of ∼220 kDa. We present the first three-dimensional structure for cav-3 (using transmission electron microscopy and single particle analysis methods) and show that nine cav-3 monomers assemble to form a complex that is toroidal in shape, ~16.5 nm in diameter and ~ 5.5 nm in height. Labeling experiments and reconstitution of the purified cav-3 into liposomes have allowed a proposal for the orientation of the protein with respect to the membrane. We have identified multiple caveolin-binding motifs within the ryanodine receptor (RyR1) sequence employing a bioinformatic analysis. We have then shown experimentally that there is a direct interaction between recombinant cav-3 nonamers and purified RyR1 homotetramers that would imply that at least one of the predicted cav-3-binding sites is exposed within the fully assembled RyR1 structure. The cav-3 three-dimensional model provides new insights as to how a cav-3 oligomer can bind multiple partners in close proximity to form signaling complexes. Furthermore, a direct interaction with RyR1 suggests a possible role for cav-3 as a modifier of muscle excitation-contraction coupling and/or for localization of the receptor to regions of the sarcoplasmic reticulum.

Phenomenological Description of Tropical Clouds Using CloudSat Cloud Classification

Abstract Two years of tropical oceanic cloud observations are analyzed using the operational CloudSat cloud classification product and Cloud–Aerosol Lidar and Infrared Pathfinder Satellite Observations (CALIPSO) lidar. Relationships are examined between cloud types, sea surface temperature (SST), and location during the CloudSat early morning and afternoon overpasses. Based on CloudSat and combined lidar–radar products, the maximum and minimum cloud fractions occur at SSTs near 303 and 299 K, respectively, corresponding to deep convective/detrained cloud populations and the transition from shallow to deep convection. For SSTs below approximately 301 K, low clouds (stratiform and stratocumulus) are dominant (cloud fraction between 0.15 and 0.37) whereas high clouds are dominant for SSTs greater than about 301 K (cloud fraction between 0.18 and 0.28). Consistent with previous studies, most tropical low clouds are associated with lower SSTs, with a strong inverse linear relationship between low cloud frequency and SST. For all cloud types except nimbostratus, stratus, and stratocumulus, a sharp increase in frequency of occurrence is observed for SSTs between 299 and 300.5 K, deduced as the onset of deeper convection. Peak fractions of high, deep convective, altostratus, and altocumulus clouds occur at SSTs close to 303 K, while cumulus clouds, which have lower tops, show a smooth cloud fractional peak about 2° cooler. Deep convective and other high cloud types decrease sharply above SSTs of 303 K, in accordance with previous work suggesting a narrow window of tropical deep convection. Finally, significant cloud frequency differences exist between CloudSat early morning and afternoon overpasses, suggesting a diurnal cycle of some cloud types, particularly stratocumulus, high, and deep convective clouds.

Separation of Two Cytotoxic Saponins from the Roots of Adenophora triphylla var. japonica by High‐speed Counter‐current Chromatography

The roots of Adenophorae species have been reported to exhibit anti-obese, anti-oxidant, anti-cancer, and anti-bacterial activities. However, there has been no single report regarding the preparative isolation and biological activities of the chemical components from Adenophora triphylla. To develop an efficient method for the determination of the active fraction from the methanol extract from the roots of Adenophora triphylla and for the preparative isolation and purification of target compounds having cytotoxicity on carcinoma cells from the active fraction by high-speed counter-current chromatography (HSCCC). The Plant (5 kg, dry weight) was extracted with methanol. Three hundred grams of the dried methanol extract (885 g) were fractionated by open-column chromatography with a stepwise gradient of water-methanol. Preparative isolation of bioactive components was performed by HSCCC with a two-phase solvent system composed of ethyl acetate-n-butanol-0.2% trifluoroacetic acid in water (5:5:10, v/v). The cytotoxicity of column fractions and isolated compounds was evaluated by 2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide assay. The 70% MeOH column fraction showed inhibitory effects against three human carcinoma cells A549, AGS and HepG2. Two saponins were separated from 400 mg of the active fraction by HSCCC. After further purification with solid phase extraction column, 25 mg of peak fraction 1 and 20 mg of peak fraction 2 were obtained. Their structures were identified by ¹H-NMR, ¹³C-NMR, Fourier transform infrared, fast atom bombardment-MS and electrospray ionisation-MS/MS. They exhibited strong cytotoxic effects against three cancer cells. Two cytotoxic saponins were isolated for the first time from the roots of Adenophora triphylla by HSCCC.

Saccharomyces cerevisiae Dmc1 and Rad51 Proteins Preferentially Function with Tid1 and Rad54 Proteins, Respectively, to Promote DNA Strand Invasion during Genetic Recombination

The Saccharomyces cerevisiae Dmc1 and Tid1 proteins are required for the pairing of homologous chromosomes during meiotic recombination. This pairing is the precursor to the formation of crossovers between homologs, an event that is necessary for the accurate segregation of chromosomes. Failure to form crossovers can have serious consequences and may lead to chromosomal imbalance. Dmc1, a meiosis-specific paralog of Rad51, mediates the pairing of homologous chromosomes. Tid1, a Rad54 paralog, although not meiosis-specific, interacts with Dmc1 and promotes crossover formation between homologs. In this study, we show that purified Dmc1 and Tid1 interact physically and functionally. Dmc1 forms stable nucleoprotein filaments that can mediate DNA strand invasion. Tid1 stimulates Dmc1-mediated formation of joint molecules. Under conditions optimal for Dmc1 reactions, Rad51 is specifically stimulated by Rad54, establishing that Dmc1-Tid1 and Rad51-Rad54 function as specific pairs. Physical interaction studies show that specificity in function is not dictated by direct interactions between the proteins. Our data are consistent with the hypothesis that Rad51-Rad54 function together to promote intersister DNA strand exchange, whereas Dmc1-Tid1 tilt the bias toward interhomolog DNA strand exchange.

MR Cine DENSE Dyssynchrony Parameters for the Evaluation of Heart Failure: Comparison With Myocardial Tissue Tagging

We sought to assess the effectiveness of automated mechanical dyssynchrony (MD) parameters based on regional heterogeneity of strain (circumferential [CURE], longitudinal [LURE], and radial uniformity ratio estimates) relative to parameters based on regional time to peak contraction with cardiac magnetic resonance (CMR) cine DENSE (Displacement Encoding with Stimulated Echoes) validated with myocardial tissue tagging (MTT) strain data. Dyssynchrony measures based on the Fourier transformation (FT) of regional strain, such as CURE (previously evaluated in cardiac resynchronization therapy candidates), directly assess MD and yield straightforward global dyssynchrony indexes; however, performance relative to the 12-segment standard deviation of time to peak strain (SD12) or maximal regional delay in time to peak strain is unknown. Cine DENSE and MTT were obtained with CMR (1.5-T Siemens Avanto, Siemens, Erlangen, Germany) in 13 canines: 3 normal control subjects, 5 with tachycardia pacing-induced heart failure (HF) and left bundle branch ablation (LBBB-HF), and 5 with HF and narrow QRS (NQRS-HF). Strain and dyssynchrony parameters were determined with both CMR methods. Both HF groups had reduced peak strains and left ventricular ejection fraction compared with normal cases. There was strong agreement between cine DENSE and MTT on the basis of intraclass correlation coefficients (CURE: 0.99, 95% CI: 0.96 to 1.00; LURE: 0.92, 95% CI: 0.77 to 0.98; circumferential strain [E(CC)]: 0.95, 95% CI: 0.72 to 0.99; longitudinal strain [E(LL)]: 0.82, 95% CI: 0.42 to 0.97). The FT-based metrics (scale 0 to 1), in particular CURE, discriminated highly between LBBB-HF and NQRS-HF groups (median difference): CURE: 0.60, 95% CI: 0.43 to 0.76; LURE: 0.39, 95% CI: 0.19 to 0.58; radial uniformity ratio estimate: 0.22, 95% CI: 0.04 to 0.40). In contrast, relative confidence intervals for group differences in time-to-peak parameters were wide, indicating less consistent discrimination (median difference): SD12-E(CC): 52.5, 95% CI: -4.0 to 109.2; SD12-E(LL): 40.9, 95% CI: -5.3 to 87.1; SD12-radial strain: 42.0, 95% CI: 0.4 to 83.6). Correlations between FT-based and time-to-peak parameters were significant (CURE/SD12-E(CC): r = -0.62, p = 0.03; LURE/SD12-E(LL): r = -0.76, p = 0.005) but not as tight as correlations between time-to-peak parameters. Automated FT-based circumferential, radial, and longitudinal dyssynchrony measures compare favorably with time-to-peak parameters. Cine DENSE was effective for this application and validated with MTT. Further clinical evaluation in cardiac resynchronization therapy candidates with CMR or other imaging modalities is warranted.

Characterization of a Novel Activated Ran GTPase Mutant and Its Ability to Induce Cellular Transformation

Ran (Ras-related nuclear) protein, a member of the Ras superfamily of GTPases, is best known for its roles in nucleocytoplasmic transport, mitotic spindle fiber assembly, and nuclear envelope formation. Recently, we have shown that the overexpression of Ran in fibroblasts induces cellular transformation and tumor formation in mice (Ly, T. K., Wang, J., Pereira, R., Rojas, K. S., Peng, X., Feng, Q., Cerione, R. A., and Wilson, K. F. (2010) J. Biol. Chem. 285, 5815-5826). Here, we describe a novel activated Ran mutant, Ran(K152A), which is capable of an increased rate of GDP-GTP exchange and an accelerated GTP binding/GTP hydrolytic cycle compared with wild-type Ran. We show that its expression in NIH-3T3 fibroblasts induces anchorage-independent growth and stimulates cell invasion, as well as activates signaling pathways that lead to extracellular regulated kinase (ERK) activity. Furthermore, Ran(K152A) expression in the human mammary SKBR3 adenocarcinoma cell line gives rise to an enhanced transformed phenotype and causes a robust stimulation of both ERK and the N-terminal c-Jun kinase (JNK). Microarray analysis reveals that the expression of the gene encoding SMOC-2 (secreted modular calcium-binding protein-2), which has been shown to synergize with different growth factors, is increased by at least 50-fold in cells stably expressing Ran(K152A) compared with cells expressing control vector. Knocking down SMOC-2 expression greatly reduces the ability of Ran(K152A) to stimulate anchorage-independent growth in NIH-3T3 cells and in SKBR3 cells and also inhibits cell invasion in fibroblasts. Collectively, our findings highlight a novel connection between the hyper-activation of the small GTPase Ran and the matricellular protein SMOC-2 that has important consequences for oncogenic transformation.

Rapid calculation of the ion energy distribution on a plasma electrode

A model was developed to rapidly calculate the ion energy distribution (IED) on an electrode immersed in plasma, for a given voltage waveform applied to the electrode through a blocking capacitor. The model combined an equivalent circuit representation of the system, with an equation for a damped potential to which ions respond, during their transit through the sheath. Predicted IEDs on both conducting and insulating surfaces for a variety of applied voltage waveforms (spike, staircase, square wave, etc.) agreed with published experimental data. For these comparisons with experiments, peak broadening due to the resolution of the ion energy analyzer was also taken into account. Using “tailored” waveforms of the applied voltage, desired IEDs may be obtained in terms of peak energies and fraction of ions under each peak.

Expression of a small (p)ppGpp synthetase, YwaC, in the (p)ppGpp0 mutant of Bacillus subtilis triggers YvyD‐dependent dimerization of ribosome

To elucidate the biological functions of small (p)ppGpp synthetases YjbM and YwaC of Bacillus subtilis, we constructed RIK1059 and RIK1066 strains carrying isopropyl-β-D-thiogalactopyranoside (IPTG) inducible yjbM and ywaC genes, respectively, in the ΔrelA ΔyjbM ΔywaC triple mutant background. While the uninduced and IPTG-induced RIK1059 cells grew similarly in LB medium, the growth of RIK1066 cells was arrested following the addition of IPTG during the early exponential growth phase. Induction of YwaC expression by IPTG also severely decreased the intracellular GTP level and drastically altered the transcriptional profile in RIK1066 cells. Sucrose density gradient centrifugation analysis of the ribosomal fractions prepared from the IPTG-induced RIK1066 cells revealed three peaks corresponding to 30S, 50S, and 70S ribosome particles, and also an extra peak. Electron microscope studies revealed that the extra peak fraction contained dimers of 70S ribosomes, which were similar to the Escherichia coli 100S ribosomes. Proteomic analysis revealed that the 70S dimer contained an extra protein, YvyD, in addition to those found in the 70S ribosome. Accordingly, strain resulting from the disruption of the yvyD gene in the RIK1066 cells was unable to form 70S dimers following IPTG induction, indicating that YvyD is required for the formation of these dimers in B. subtilis.

Inhibiting Alternative Pathway Complement Activation by Targeting the Factor D Exosite

By virtue of its amplifying property, the alternative complement pathway has been implicated in a number of inflammatory diseases and constitutes an attractive therapeutic target. An anti-factor D Fab fragment (AFD) was generated to inhibit the alternative complement pathway in advanced dry age-related macular degeneration. AFD potently prevented factor D (FD)-mediated proteolytic activation of its macromolecular substrate C3bB, but not proteolysis of a small synthetic substrate, indicating that AFD did not block access of the substrate to the catalytic site. The crystal structures of AFD in complex with human and cynomolgus FD (at 2.4 and 2.3 Å, respectively) revealed the molecular details of the inhibitory mechanism. The structures show that the AFD-binding site includes surface loops of FD that form part of the FD exosite. Thus, AFD inhibits FD proteolytic function by interfering with macromolecular substrate access rather than by inhibiting FD catalysis, providing the molecular basis of AFD-mediated inhibition of a rate-limiting step in the alternative complement pathway.

Development of a Whole‐body Model of Vitamin A Metabolism in Neonatal Rats Treated with Oil or Vitamin A and Retinoic Acid (VARA) Based on Compartmental Analysis of Retinol Tracer Kinetics

Compartmental modeling has contributed greatly to our understanding of vitamin A (VA) metabolism in adult animals and humans. However, VA metabolism in neonates is virtually uncharacterized. Our objectives were to compare VARA supplementation vs. placebo oil on whole‐body retinol kinetics and to develop a whole‐body model of VA metabolism in neonatal rats, taking into account the inherent metabolic non‐steady state of neonates. On post‐natal day 4, rat pups (n=3/time point) received an oral dose of 11,12‐[3H]retinol admixed with oil or VARA. In pups treated with oil, radioactivity in plasma retinol rose quickly between 1 and 6 hours, with the absorption peak at 6 hr. Then there was a rapid disappearance of tracer, followed by a leveling off which reflects the recycling of retinol between plasma and extravascular tissues and a flat slope from 2 d to 8 d. Then radioactivity further declined into a terminal slope, which represents the initiation of retinol degradation. In pups treated with VARA, peak fraction of the oral dose in plasma was lower. VARA was also associated with a more rapid accumulation of radioactivity in liver and an increased fraction of the oral dose in lung. A compartmental model as viewed from the “plasma space” indicated extensive recycling of retinol between plasma and tissues in neonates, as in adults. Also, VARA stimulated the turnover of retinol between plasma and extravascular tissues.NIH HD‐66982Grant Funding Source : NIH

Macro-Thyrotropin: A Case Report and Review of Literature

Isolated elevation of TSH in the absence of thyroid symptoms can be very rarely caused by a macromolecule formed between TSH and Ig (macro-TSH), confounding the interpretation of thyroid function test results. We described the use of several commonly available laboratory-based approaches to investigate an isolated TSH elevation [232 mIU/liter; free T(4), 10 pmol/liter (reference interval, 10.0-23.0 pmol/liter), Vitros platform] in a clinically euthyroid elderly gentleman, which led to the diagnosis of macro-TSH. TSH concentration of the patient was significantly lower (122 mIU/liter) when measured on the Advia Centaur platform. Serial dilution of the patient's sample showed a nonlinear increase in TSH recovery at increasing dilution (nonlinearity). Polyethylene glycol precipitation and mixing the patient's sample with a hypothyroid patient sample showed reduced TSH recovery, suggesting the presence of a high molecular weight interfering substance and excess TSH binding capacity, respectively. Heterophile blocking tube studies and rheumatoid factors were negative. Gel filtration chromatography demonstrated a TSH peak fraction that approximated the molecular size of IgG; together with the excess TSH binding capacity, this indicated the presence of TSH-IgG macro-TSH. A review of 12 macro-TSH case reports showed that samples with macro-TSH produce over-recovery with dilution, return negative results on anti-animal and anti-heterophile blocking studies, and commonly have recovery of less than 20% when subjected to polyethylene glycol precipitation. Macro-TSH is an underrecognized laboratory interference. Routine laboratory techniques described above can help diagnose this rare entity. A close dialogue between the physician and the laboratory is important in approaching such cases.

Detection of Endolichenic Fungi Producing Antifungal Compound

To isolate a novel antifungal compound, we obtained 100 kinds of endolichenic fungi from Korean Lichen & Allied Bioresources Center and examined their antifungal capability. Three fungi Usnea rigidula (2326), Parmotrema pseudotinctorum (2202) and Myelochroa sp. (2292) showed high antifungal activity against Candida albicans when they grew in both liquid and solid media. We extracted the culture supernatants of these three fungi with chloroform and then with ethyl acetate. Chloroform fraction exhibited the highest antifungal activities when those fractions were examined for the growth inhibition of Candida albicans with disc diffusion method. The chloroform faction was on further analysis with column chromatography to see whether the inhibitors are already known or not. Two peak fractions were collected from 4-day culture extract for Usnea rigidula and from 6-day culture extract for Parmotrema pseudotinctorum on the HPLC. A peak fraction from chloroform extracts of 4-day culture filtrate of Parmotrema pseudotinctorum showed higher antifungal activities against C. albicans and C. glabrata than another peak fraction. It appears that the antifungal materials are relatively nonpolar as usnic acid often found in lichenic fungi.

Assembly, Purification, and Pre-steady-state Kinetic Analysis of Active RNA-dependent RNA Polymerase Elongation Complex

NS5B is the RNA-dependent RNA polymerase responsible for replicating hepatitis C virus (HCV) genomic RNA. Despite more than a decade of work, the formation of a highly active NS5B polymerase·RNA complex suitable for mechanistic and structural studies has remained elusive. Here, we report that through a novel way of optimizing initiation conditions, we were able to generate a productive NS5B·primer·template elongation complex stalled after formation of a 9-nucleotide primer. In contrast to previous reports of very low proportions of active NS5B, we observed that under optimized conditions up to 65% of NS5B could be converted into active elongation complexes. The elongation complex was extremely stable, allowing purification away from excess nucleotide and abortive initiation products so that the purified complex was suitable for pre-steady-state kinetic analyses of polymerase activity. Single turnover kinetic studies showed that CTP is incorporated with apparent K(d) and k(pol) values of 39 ± 3 μM and 16 ± 1 s(-1), respectively, giving a specificity constant of k(pol)/K(d) of 0.41 μM(-1) s(-1). The kinetics of multiple nucleotide incorporation during processive elongation also were determined. This work establishes a novel way to generate a highly active elongation complex of the medically important NS5B polymerase for structural and functional studies.

Massive monoclonal expansion of CD4 T-cells specific for aMycobacterium tuberculosisESAT-6 peptide

T-cell responses towards tuberculin (purified protein derivative; PPD) or the Mycobacterium tuberculosis-specific antigens early secretory antigenic target (ESAT)-6 and culture filtrate protein-10 are indicative of prior contact with mycobacterial antigens. In this study, we investigated the exceptional case of a 75-yr-old patient who devoted more than one-third of his CD4 T-cells against PPD and ESAT-6. Antigen-specific T-cells were characterised using flow cytometric intracellular cytokine staining, ELISPOT assay, proliferation assays, and T-cell receptor spectratyping. T-cell frequencies were far above those found in age-matched controls (median 0.33%, range 0.05-6.32%) and remained at high levels for >2 yrs. The patient initially presented with haemoptysis, but active tuberculosis was ruled out by repeated analysis of sputum and bronchoalveolar lavage fluid. Skin testing was negative and haemoptyses did not have a M. tuberculosis-related aetiology. Phenotypical and functional properties of specific T-cells were consistent with a terminally differentiated effector-memory phenotype with capacity to produce interferon-γ, interleukin-2 and tumour necrosis factor-α. Epitope mapping showed that the CD4 T-cells were directed against a single peptide from ESAT-6 (amino acid 5-20) that was presented in context of HLA-DR. T-cell receptor Vβ-spectratyping and sequencing of specific CD4 T-cells revealed a prominent peak fraction of monoclonal origin. In conclusion, similar to monoclonal gammopathies of undetermined significance, this may represent the first T-cell counterpart with known specificity against M. tuberculosis.

The Relationship between Nano Crystallite Structure and Internal Stress in Ni Coatings Electrodeposited by Watts Bath Electrolyte Mixed with Supercritical CO2

In this study, the internal stress of Ni coating electroplated in the supercritical-CO2 (Sc-CO2) mixed Watts bath was investigated with a focus on the effects of plating parameters and additives. Both the internal stresses of the Ni coatings and their corresponding microstructures were examined. A relationship is found that the internal stress of the Ni coating was inversely proportional to its grain size and the Ni{200}/Ni{111} peak fraction in the measured X-ray diffraction pattern. The smaller grain size, accompanied with higher Ni{111} intensity, resulted in the higher internal stress of the coating, and vice versa. A proposed theoretical model, which took both the grain size and preferred crystalline orientation into account, correlated closely with experimental results in the internal stress. As a result, the presence of surfactant in the Sc-CO2 electrolyte increased the internal stress of Ni coating. However, a reduction of internal stress is made possible by varying four factors: the raising of current density, the increasing of plating temperature, the lowering of plating pressure, and the addition of saccharin into electrolyte. A comparison to the internal stress of Ni coatings plated by the conventional method was also discussed in this study.

Neurally Adjusted Ventilatory Assist in Neonates Weighing

To report our experience using neurally adjusted ventilatory assist (NAVA), which allows a patient to synchronize spontaneous respiratory effort with mechanical ventilation, in the neonatal intensive care unit in neonates weighing <1500 g. This was a retrospective review performed between May 2008 and May 2009. A total of 52 neonates on conventional ventilation were converted to NAVA. We compared ventilatory parameters and blood gas values during conventional ventilation and then at various time intervals during NAVA and evaluated for complications. Statistical analyses were performed using the 2-tailed Student t-test and the Z-test for proportions for demographic data and Hotelling's T(2) test to compare repeated measures (P < .05). Peak inspiratory pressure and fraction of inspired oxygen decreased, and pH and partial pressure of carbon dioxide improved during use of NAVA. These changes were sustained for 24 hours. Compared with standard conventional ventilation, in preterm neonates NAVA appears to provide better blood gas regulation with lower peak inspiratory pressure and oxygen requirements.

Multidimensional profiling of plasma lipoproteins by size exclusion chromatography followed by reverse-phase protein arrays

The composition of lipoproteins and the association of proteins with various particles are of much interest in the context of cardiovascular disease. Here, we describe a technique for the multidimensional analysis of lipoproteins and their associated apolipoproteins. Plasma is separated by size exclusion chromatography (SEC), and fractions are analyzed by reverse-phase arrays. SEC fractions are spotted on nitrocellulose slides and incubated with different antibodies against individual apolipoproteins or antibodies against various apolipoproteins. In this way, tens of analytes can be measured simultaneously in 100 μl of plasma from a single SEC separation. This methodology is particularly suited to simultaneous analysis of multiple proteins that may change their distribution to lipoproteins or alter their conformation, depending on factors that influence circulating lipoprotein size or composition. We observed changes in the distribution of exchangeable apolipoproteins following addition of recombinant apolipoproteins or interaction with exogenous compounds. While the cholesteryl ester transfer protein (CETP)-dependent formation of pre-β-HDL was inhibited by the CETP inhibitors torcetrapib and anacetrapib, it was not reduced by the CETP modulator dalcetrapib. This finding was elucidated using this technique.

Total serum bilirubin level in umbilical cord blood and respiratory distress syndrome in very low birth weight infants

To explore the association between total serum bilirubin (TSB) level in umbilical cord blood (UCB) and the occurrence and/or severity of respiratory distress syndrome (RDS) in very low birth weight (VLBW) infants. We performed a prospective study of 579 VLBW infants. The mean TSB level in UCB (1.8 mg/dL) was used to divide the cohort into two groups: neonates with levels <1.8 mg/dL and those with levels >1.8 mg/dL. Demographic and clinical characteristics of the 2 groups, including diagnosis of RDS, need for ventilation, peak fraction of inspired oxygen (FiO2), oxygenation index (OI), and duration of respiratory support, were compared. Three hundred and twenty-four infants were included in the low-TSB group and 255 infants were included in the high-TSB group. RDS was less frequent in the high TSB group than in the low one (46.3% vs. 56.6%, P=0.01). A negative association between TSB level in UCB and the occurrence of RDS [odds ratio (OR)=0.620; 95% confidence interval (CI) 0.440-0.873, P=0.006] was observed. However, lower TSB in UCB was not associated with the maximum FiO2, OI, or duration of mechanical ventilation. VLBW infants with low TSB levels in UCB were more likely to develop RDS. However, in conjunction with surfactant replacement therapy, no correlation was found between TSB levels in UCB and the severity of RDS. The negative association between high TSB in UCB and RDS warrants further investigation.

Application of Denaturing High-Performance Liquid Chromatography (DHPLC) for the Identification of Fish: A New Way To Determine the Composition of Processed Food Containing Multiple Species

The identification of fish species in transformed food products is difficult because the existing methods are not adapted to heat-processed products containing more than one species. Using a common to all vertebrates region of the cytochrome b gene, we have developed a denaturing high-performance liquid chromatography (DHPLC) fingerprinting method, which allowed us to identify most of the species in commercial crab sticks. Whole fish and fillets were used for the creation of a library of referent DHPLC profiles. Crab sticks generated complex DHPLC profiles in which the number of contained fish species can be estimated by the number of major fluorescence peaks. The identity of some of the species was predicted by comparison of the peaks with the referent profiles, and others were identified after collection of the peak fractions, reamplification, and sequencing. DHPLC appears to be a quick and efficient method to analyze the species composition of complex heat-processed fish products.