Urinary cortisol sampling: a non‐invasive technique for examining cortisol concentrations in the Weddell seal,Leptonychotes weddellii

This study investigated the feasibility and validity of using non‐invasively collected ice urine samples to measure cortisol concentrations in Weddell seals. Radio‐immunoassays were used to determine urinary cortisol, and spectrophotometric assay was used to determine creatinine concentrations. This allowed for urinary cortisol/creatinine ratios (UCCR) to be compared between pure urine and urine collected from the ice. Urinary cortisol/creatinine ratios values of ice urine proved an effective method of studying cortisol concentrations in Weddell seals as there was no difference between pure urine and ice urine UCCR values. There were no inter‐sexual or age‐related differences in UCCR values in either pure or ice urine. Zoo Biol 0:1–8, 2006. © 2006 Wiley‐Liss, Inc.     

The eps15 homology (EH) domain-based interaction between eps15 and hrb connects the molecular machinery of endocytosis to that of nucleocytosolic transport

The Eps15 homology (EH) module is a protein-protein interaction domain that establishes a network of connections involved in various aspects of endocytosis and sorting. The finding that EH-containing proteins bind to Hrb (a cellular cofactor of the Rev protein) and to the related protein Hrbl raised the possibility that the EH network might also influence the so-called Rev export pathway, which mediates nucleocytoplasmic transfer of proteins and RNAs. In this study, we demonstrate that Eps15 and Eps15R, two EH-containing proteins, synergize with Hrb and Hrbl to enhance the function of Rev in the export pathway. In addition, the EH-mediated association between Eps15 and Hrb is required for the synergistic effect. The interaction between Eps15 and Hrb occurs in the cytoplasm, thus pointing to an unexpected site of action of Hrb, and to a possible role of the Eps15-Hrb complex in regulating the stability of Rev.     

Multiparameter metabolic analysis reveals a close link between attenuated mitochondrial bioenergetic function and enhanced glycolysis dependency in human tumor cells

Increased conversion of glucose to lactic acid associated with decreased mitochondrial respiration is a unique feature of tumors first described by Otto Warburg in the 1920s. Recent evidence suggests that the Warburg effect is caused by oncogenes and is an underlying mechanism of malignant transformation. Using a novel approach to measure cellular metabolic rates in vitro, the bioenergetic basis of this increased glycolysis and reduced mitochondrial respiration was investigated in two human cancer cell lines, H460 and A549. The bioenergetic phenotype was analyzed by measuring cellular respiration, glycolysis rate, and ATP turnover of the cells in response to various pharmacological modulators. H460 and A549 cells displayed a dependency on glycolysis and an ability to significantly upregulate this pathway when their respiration was inhibited. The converse, however, was not true. The cell lines were attenuated in oxidative phosphorylation (OXPHOS) capacity and were unable to sufficiently upregulate mitochondrial OXPHOS when glycolysis was disabled. This observed mitochondrial impairment was intimately linked to the increased dependency on glycolysis. Furthermore, it was demonstrated that H460 cells were more glycolytic, having a greater impairment of mitochondrial respiration, compared with A549 cells. Finally, the upregulation of glycolysis in response to mitochondrial ATP synthesis inhibition was dependent on AMP-activated protein kinase activity. In summary, our results demonstrate a bioenergetic phenotype of these two cancer cell lines characterized by increased rate of glycolysis and a linked attenuation in their OXPHOS capacity. These metabolic alterations provide a mechanistic explanation for the growth advantage and apoptotic resistance of tumor cells. oxygen consumption; oxidative phosphorylation; Warburg effect; real time     

Bone formation rather than inflammation reflects Ankylosing Spondylitis activity on PET-CT: a pilot study

Introduction

Positron Emission Tomography - Computer Tomography (PET-CT) is an interesting imaging technique to visualize Ankylosing Spondylitis (AS) activity using specific PET tracers. Previous studies have shown that the PET tracers [¹⁸F]FDG and [¹¹C](R)PK11195 can target inflammation (synovitis) in rheumatoid arthritis (RA) and may therefore be useful in AS. Another interesting tracer for AS is [¹⁸F]Fluoride, which targets bone formation. In a pilot setting, the potential of PET-CT in imaging AS activity was tested using different tracers, with Magnetic Resonance Imaging (MRI) and conventional radiographs as reference.

Methods

In a stepwise approach different PET tracers were investigated. First, whole body [¹⁸F]FDG and [¹¹C](R)PK11195 PET-CT scans were obtained of ten AS patients fulfilling the modified New York criteria. According to the BASDAI five of these patients had low and five had high disease activity. Secondly, an extra PET-CT scan using [¹⁸F]Fluoride was made of two additional AS patients with high disease activity. MRI scans of the total spine and sacroiliac joints were performed, and conventional radiographs of the total spine and sacroiliac joints were available for all patients. Scans and radiographs were visually scored by two observers blinded for clinical data.

Results

No increased [¹⁸F]FDG and [¹¹C](R)PK11195 uptake was noticed on PET-CT scans of the first 10 patients. In contrast, MRI demonstrated a total of five bone edema lesions in three out of 10 patients. In the two additional AS patients scanned with [¹⁸F]Fluoride PET-CT, [¹⁸F]Fluoride depicted 17 regions with increased uptake in both vertebral column and sacroiliac joints. In contrast, [¹⁸F]FDG depicted only three lesions, with an uptake of five times lower compared to [¹⁸F]Fluoride, and again no [¹¹C](R)PK11195 positive lesions were found. In these two patients, MRI detected nine lesions and six out of nine matched with the anatomical position of [¹⁸F]Fluoride uptake. Conventional radiographs showed structural bony changes in 11 out of 17 [¹⁸F]Fluoride PET positive lesions.

Conclusions

Our PET-CT data suggest that AS activity is reflected by bone activity (formation) rather than inflammation. The results also show the potential value of PET-CT for imaging AS activity using the bone tracer [¹⁸F]Fluoride. In contrast to active RA, inflammation tracers [¹⁸F]FDG and [¹¹C](R)PK11195 appeared to be less useful for AS imaging.     

Evidence for segment-nonspecific packaging of the influenza a virus genome

The influenza A virus genome is composed of eight negative-sense RNA segments (called vRNAs), all of which must be packaged to produce an infectious virion. It is not clear whether individual vRNAs are packaged specifically or at random, however, and the total vRNA capacity of the virion is unknown. We have created modified forms of the viral nucleoprotein (NP), neuraminidase (NA), and nonstructural (NS) vRNAs that encode green or yellow fluorescent proteins and studied the efficiency with which these are packaged by using a plasmid-based influenza A virus assembly system. Packaging was assessed precisely and quantitatively by scoring transduction of the fluorescent markers in a single-round infectivity assay with a flow cytometer. We found that, under conditions in which virions are limiting, pairs of alternatively tagged vRNAs compete for packaging but do so in a nonspecific manner. Reporters representing different vRNAs were not packaged additively, as would be expected under specific packaging, but instead appeared to compete for a common niche in the virion. Moreover, 3 to 5% of transduction-competent viruses were found to incorporate two alternative reporters, regardless of whether those reporters represented the same or different vRNAs - a finding compatible with random, but not with specific, packaging. Probabilistic estimates suggest that in order to achieve this level of dual transduction by chance alone, each influenza A virus virion must package an average of 9 to 11 vRNAs.     

Inhibitory action of cyclobutyrol on the secretion of biliary cholesterol and phospholipids.

A number of organic anions are known to decrease biliary secretion of cholesterol and phospholipid without affecting bile acid secretion. Cyclobutyrol (CB) is a choleretic agent which also inhibits biliary lipid secretion. Using isolated perfused rat liver we have studied this inhibition in relation to possible mechanisms suggested for other anions. Shortly after its administration to the isolated perfused liver, CB decreases biliary outputs of cholesterol and phospholipid, without changes in bile acid secretion, at low (450 nmol/min), high (1350 nmol/min) and nil taurocholate infusion rates. The absolute inhibition does not appear to be decreased by elevated bile acid secretion. There is a differential effect on secretion of cholesterol and phospholipid, more marked at low bile acid secretion rates. Biliary outputs of the canalicular membrane enzymes 5'-nucleotidase and alkaline phosphodiesterase I are also depressed by CB administration, but the anion does not affect the biliary output of bovine serum albumin or the output of rat serum albumin into the perfusion fluid. Since CB does not inhibit intracellular vesicular transport or apparently inhibit intracanalicular events, its effect is different from the effect of several other anions. From these studies it appears that the most likely effect of CB is exerted at the level of the canalicular membrane.     

Genetic stability of the D1Z2 region: implications for DNA genotyping and paternity testing.

The aim of the present study was to examine a single locus variable number tandem repeat for the purpose of DNA genotyping ("fingerprinting"). DNAs of 175 individuals from five ethnic groups (Black, Chinese, Japanese, Caucasian, and Melanesian) were analyzed. Restriction fragment length polymorphic analysis of random individuals revealed individual specific DNA patterns in all but one group. Among 20 Melanesian inhabitants of the Vanuatu islands in the southwest Pacific, three individuals were found to share a common pattern. This island population represents a "genetic isolate" and illustrates the importance of carrying out population studies on individual ethnic groups of interest. The complexity and the genetic stability of the D1Z2 region as revealed by the probe hMF1 make it an excellent candidate for DNA genotyping in paternity testing as 101 Caucasian individuals each had unique patterns for PstI and SinI digests.