A Streptococcus uberis transposon mutant screen reveals a negative role for LiaR homologue in biofilm formation.

AIMS: The environmental pathogen Streptococcus uberis causes intramammary infections in dairy cows. Because biofilm growth might contribute to Strep. uberis mastitis, we conducted a biological screen to identify genes potentially involved in the regulation of biofilm growth. METHODS AND RESULTS: By screening a transposon mutant library of Strep. uberis, we determined that the disruption of 13 genes (including hasA, coaC, clpP, miaA, nox and uidA) led to increased biofilm formation. One of the genes (SUB1382) encoded a homologue of the LiaR response regulator (RR) of the Bacillus subtilis two-component signalling system (TCS). Electrophoretic mobility shift assays revealed that DNA binding by LiaR was greatly enhanced by phosphorylation. Two-dimensional differential in-gel electrophoresis analyses of the liaR mutant and the parental Strep. uberis strain revealed five differentially produced proteins with at least a 1·5-fold change in relative abundance (P < 0·05). CONCLUSIONS: The DNA-binding protein LiaR is a potential regulator of biofilm formation by Strep. uberis. SIGNIFICANCE AND IMPACT OF THE STUDY: Several molecular primary and downstream targets involved in biofilm formation by Strep. uberis were identified. This provides a solid foundation for further studies on the regulation of biofilm formation in this important pathogen.

Novel microsatellite DNA markers indicate strict parthenogenesis and few genotypes in the invasive willow sawfly Nematus oligospilus.

Invasive organisms can have major impacts on the environment. Some invasive organisms are parthenogenetic in their invasive range and, therefore, exist as a number of asexual lineages (=clones). Determining the reproductive mode of invasive species has important implications for understanding the evolutionary genetics of such species, more especially, for management-relevant traits. The willow sawfly Nematus oligospilus Förster (Hymenoptera: Tenthredinidae) has been introduced unintentionally into several countries in the Southern Hemisphere where it has subsequently become invasive. To assess the population expansion, reproductive mode and host-plant relationships of this insect, microsatellite markers were developed and applied to natural populations sampled from the native and expanded range, along with sequencing of the cytochrome-oxidase I mitochondrial DNA (mtDNA) region. Other tenthredinids across a spectrum of taxonomic similarity to N. oligospilus and having a range of life strategies were also tested. Strict parthenogenesis was apparent within invasive N. oligospilus populations throughout the Southern Hemisphere, which comprised only a small number of genotypes. Sequences of mtDNA were identical for all individuals tested in the invasive range. The microsatellite markers were used successfully in several sawfly species, especially Nematus spp. and other genera of the Nematini tribe, with the degree of success inversely related to genetic divergence as estimated from COI sequences. The confirmation of parthenogenetic reproduction in N. oligospilus and the fact that it has a very limited pool of genotypes have important implications for understanding and managing this species and its biology, including in terms of phenotypic diversity, host relationships, implications for spread and future adaptive change. It would appear to be an excellent model study system for understanding evolution of invasive parthenogens that diverge without sexual reproduction and genetic recombination.

From aviation to automotive - a study on material selection and its implication on cost and weight efficient structural composite and sandwich designs.

The design of a composite material structure is often challenging as it is driven by the trade-off between lightweight performance and production costs. In this paper, the boundaries of this design trade-off and its implications on material selection, geometrical design and manufacturability are analysed for a number of design strategies and composite material systems. The analysis is founded on a methodology that couples weight-optimization and technical cost modelling through an application-bound design cost. Each design strategy is evaluated for three levels of bending and torsional stiffness. The resulting stiffness-versus cost-range together constructs the design envelope and provides guidelines on the suitability and improvement potential of each case. Design strategies researched include monolithic, u-beam-, sandwich-insert- and sandwich-stiffened plates. Considered material systems include carbon-, glass, recycled carbon-, lignin- and hemp-fibre reinforced composites. Optimized sandwich designs are shown to have lowest design cost. Glass-, recycled carbon-, lignin- and hemp-fibre reinforced composite materials are all shown to reduce costs but at lower stiffness performance. Ultimately, the case study demonstrates the importance of early structural design trade-off studies and material selection and justifies introducing novel fibre systems in low-cost applications of moderate stiffness levels.

Physical workload, low back pain and neck-shoulder pain: a Swedish twin study.

OBJECTIVES: To investigate if high physical workload is associated with low back pain (LBP) and/or neck-shoulder pain (NSP) when taking into account the influence of genetic and shared environmental factors. Further, the study aims to explore the potential influence of genetic and shared environmental factors in the associations between high physical workload and the three disorder subgroups: solely LBP, solely NSP, and concurrent LBP and NSP. METHODS: Data on 16,107 monozygotic and dizygotic twins, born during 1959-1985, were obtained from a cross-sectional study, performed in 2005-2006 by the Swedish Twin Registry. Odds ratios (ORs) calculated in cohort analyses and co-twin control analyses were used to assess the associations between high physical workload and LBP and NSP when controlling for genetic and shared environmental factors. RESULTS: In the cohort analysis, the association between high physical workload and the group with any one symptom (LBP and/or NSP) was OR 1.47 (95% CI 1.37 to 1.57). The co-twin control analyses indicated that the association was not confounded by genetic and shared environmental factors with OR 1.34 (95% CI 1.02 to 1.75) for dizygotic twins and OR 1.44 (95% CI 1.06 to 1.95) for monozygotic twins. In the cohort analyses the association with high physical workload was higher for concurrent LBP and NSP (OR 1.80 (95% CI 1.62 to 1.99)) than for solely LBP (OR 1.41 (95% CI 1.27 to 1.57)) and solely NSP (OR 1.31 (95% CI 1.20 to 1.43)). Concurrent LBP and NSP was the only group that showed a stepwise decrease of the point estimates between the cohort analysis and the co-twin control analyses, OR 1.71 (95% CI 1.00 to 2.94) for dizygotic twins, and OR 1.29 (95% CI 0.64 to 2.59) for monozygotic twins indicating confounding by genetic and shared environmental factors. CONCLUSIONS: High physical workload was associated with LBP and/or NSP even after adjusting for genetic or shared environmental factors. Only for concurrent LBP and NSP, genetic and shared environmental factors seemed to have an influence on the association with high physical workload.

Insulin-like growth factor binding proteins in human breast cancer tissue.

Breast tumor cell lines have been shown to secrete at least five distinct insulin-like growth factor (IGF) binding proteins (IGFBP), the secretion being related to the estrogen receptor (ER) content. In this study we investigated IGFBP mRNA expression and IGFBP content in relation to ER content in human breast tumors. Tissue specimens from 47 breast cancers were studied. In five cases the adjacent histologically normal tissue was also analyzed. IGFBP content in tissue homogenates was studied by Western ligand blot analysis, using [125I] IGF-I as a label, and IGFBP mRNA expression by reverse transcriptase polymerase chain reaction. The results show that human breast tumors express mRNAs encoding IGFBP-1, IGFBP-2, IGFBP-3, IGFBP-4 and IGFBP-5. The pattern of IGFBPs in different tumors varies. No correlation exists between ER content and IGFBPs with molecular weights 24,000 Mr, 28,000 Mr, 34,000 Mr or 43,000 Mr, whereas the 49,000 Mr IGFBP was more abundant in ER negative tumors (P less than 0.05). The IGFBP content was significantly (P less than 0.05) higher in five tumors than in their adjacent normal tissues suggesting that increased content of IGFBPs is a feature typically associated with the malignant transformation of breast tissue.

Geometrical distortions in two-dimensional gels: applicable correction methods.

Two-dimensional electrophoresis (2-DE) provides a rapid means for separating thousands of proteins from cell and tissue samples in one run. Although this powerful research tool has been enthusiastically applied in many fields of biomedical research, accurate analysis and interpretation of the data have provided many challenges. Several analysis steps are needed to convert the large amount of noisy data obtained with 2-DE into reliable and interpretable biological information. The goals of such analysis steps include accurate protein detection and quantification, as well as the identification of differentially expressed proteins between samples run on different gels. To achieve these goals, systematic errors such as geometric distortions between the gels must be corrected by using computer-assisted methods. A wide range of computer software has been developed, but no general consensus exists as standard for 2-DE data analysis protocol. The choice of analysis approach is an important element depending both on the data and on the goals of the experiment. Therefore, basic understanding of the algorithms behind the software is required for optimal results. This review highlights some of the common themes in 2-DE data analysis, including protein spot detection and geometric image warping using both spot- and pixel-based approaches. Several computational strategies are overviewed and their relative merits and potential pitfalls discussed. Finally, we offer our own personal view of future trends and developments in large-scale proteome research.

Sleep as a predictive factor for the onset and resolution of multi-site pain: a 5-year prospective study.

BACKGROUND: Disturbed sleep and pain often co-exist and the relationship between the two conditions is complex and likely reciprocal. This 5-year prospective study examines whether disturbed sleep can predict the onset of multi-site pain, and whether non-disturbed sleep can predict the resolution of multi-site pain. METHODS: The cohort (n = 1599) was stratified by the number of self-reported pain sites: no pain, pain from 1-2 sites and multi-site pain (≥3 pain sites). Sleep was categorized by self-reported sleep disturbance: sleep A (best sleep), sleep B and sleep C (worst sleep). In the no-pain and pain-from-1-2 sites strata, the association between sleep (A, B and C) and multi-site pain 5 years later was analysed. Further, the prognostic value of sleep for the resolution of multi-site pain at follow-up was calculated for the stratum with multi-site pain at baseline. In the analyses, gender, age, body mass index, smoking, physical activity and work-related exposures were treated as potential confounders. RESULTS: For individuals with no pain at baseline, a significantly higher odds ratio for multi-site pain 5 years later was seen for the tertile reporting worst sleep [odds ratio (OR) 4.55; 95% confidence interval (CI) 1.28-16.12]. Non-disturbed (or less disturbed) sleep had a significant effect when predicting the resolution of multi-site pain (to no pain) (OR 3.96; 95% CI 1.69-9.31). CONCLUSION: In conclusion, sleep could be relevant for predicting both the onset and the resolution of multi-site pain. It seems to be a significant factor to include in research on multi-site pain and when conducting or evaluating intervention programmes for pain.

Intrauterine progestin induces continuous insulin-like growth factor-binding protein-1 production in the human endometrium.

The effect of local intrauterine progestin on endometrial insulin-like growth factor-binding protein-1 (IGFBP-1) production was studied in 60 women using a levonorgestrel-releasing intrauterine device (IUD). Intrauterine progestin was a potent stimulator of stromal cell IGFBP-1 production, with 97% of endometrial specimens showing positive staining by immunohistological methods. After 5 yr or more of intrauterine progestin exposure, 100% (n = 20) of the tissues remained strongly positive for IGFBP-1. The IGFBP-1 content in endometrial tissue homogenates reached values as high as 10 micrograms/mg protein when measured by immunoradiometric assay. In contrast to the continuous endometrial IGFBP-1 production induced by local progestin, no such effect could be found in endometria from subjects with sc progestin-releasing implants or copper IUDs. Although the levonorgestrel-releasing IUD had a striking effect on local endometrial IGFBP-1 production, it had no effect on serum IGFBP-1 levels. By Western ligand blot analysis, the domainating IGF-binding species in endometria exposed to intrauterine progestin was of 28K mol wt, corresponding to IGFBP-1, whereas no IGFBP species of 31-43K, corresponding to IGFBP-2 or IGFBP-3, were detected.

Evolution of gall morphology and host-plant relationships in willow-feeding sawflies (Hymenoptera: Tenthredinidae).

There are over 200 species of nematine sawflies that induce galls on willows (Salix spp.). Most of the species are mono- or oligophagous, and they can be separated into seven or eight different groups based on the type of gall that they induce. We studied the evolution of different gall types and host plant associations by reconstructing the phylogeny of five outgroup and 31 ingroup species using DNA sequence data from the mitochondrial cytochrome b gene. Maximum-parsimony and maximum-likelihood analyses resulted in essentially the same phylogeny with high support for important branches. The results show that: (1) the galling species probably form a monophyletic group; (2) true closed galls evolved only once, via leaf folders; (3) with the possible exception of leaf rollers, all gall type groups are mono- or paraphyletic; (4) similar gall types are closer on the phylogeny than would be expected by a random process; (5) there is an apparent evolutionary trend in galling site from the leaf edge towards the more central parts of the host plant; and (6) many willow species have been colonized several times, which excludes the possibility of parallel cladogenesis between willows and the gallers; however, there are signs of restrictions in the evolution of host use. Many of the patterns in the evolutionary history of nematine gallers have also been observed in earlier studies on other insect gallers, indicating convergent evolution between the independent radiations.

The interleukin-1 system in gestational tissues at term: effect of labour.

Previous studies suggest that the interleukin-1 (IL-1) system is involved in preterm labour, at least in cases associated with intrauterine infection. To investigate the effect of term labour without infection on the IL-1 system, the messenger ribonucleic acid (mRNA) expression of IL-1 beta, IL-1 receptor type I (IL-1R tI), IL-1 receptor antagonist (IL-1ra), and IL-1 beta converting enzyme (ICE) were examined by Northern blot analysis and by reverse transcriptase-polymerase chain reaction (RT-PCR) in paired samples of decidua and placenta obtained from women having spontaneous vaginal delivery (group 1) or elective caesarean section (group 2) at term. In addition, concentrations of IL-1 beta and IL-1ra proteins were measured by ELISA in paired decidual and placental cytosols. In all decidual samples, IL-1 beta mRNA was expressed strongly, and the IL-1 beta concentration was 40- to 50-fold higher than in paired placental samples, in which the signal for IL-1 beta mRNA could be detected by RT-PCR only, and the amount of IL-1 beta protein was undetectable or very low. A comparison between the study groups revealed that the decidual IL-1 beta mRNA level tended to be higher in group 1, and the median IL-1 beta concentration in decidual cytosols was significantly higher in group 1 than in group 2 (P < 0.05). The IL-1R tI mRNA transcript was stronger in decidual than in paired placental samples in both groups. The mRNAs encoding ICE and IL-1ra were detected by RT-PCR in decidual and placental samples from both groups. The IL-1ra concentration tended to be higher in decidual cytosols than in paired placental cytosols, but there was no difference between the study groups. The IL-1ra/IL-1 beta ratio was significantly lower in decidual samples in women with spontaneous labour than in women without labour (P < 0.05). The results of this study confirm that decidua is the major site of IL-1 beta production and action in term gestational tissues. Furthermore, the results show that the major change in decidual/placental IL-1 system during parturition is the increase in decidual IL-1 beta production. Whether the increased IL-1 beta production precedes or is a consequence of labour, remains still unclear.

Human endometrial adenocarcinoma cell lines HEC 1B and KLE secrete insulin-like growth factor binding protein-1 and contain IGF-I receptors.

The production of insulin-like growth factor binding proteins (IGFBP) with special reference to human IGFBP-1 was evaluated in five endometrial adenocarcinoma cell lines (HEC 1A, HEC 1B, KLE, RL952 and AN3CA) in continuous culture. Two of the cell lines (HEC 1B and KLE) produced immunoreactive IGFBP-1. The production was inhibited by clomiphene and progesterone, whereas estrogen, cortisol and insulin had no effect on IGFBP-1 secretion. The two cell lines which secreted immunoreactive IGFBP-1 also had IGF-I receptors, whereas the cell lines RL952 and AN3CA, not producing IGFBP-1, had no saturable IGF membrane binding sites. IGF-I receptor binding to HEC 1B and KLE cells was inhibited in the presence of purified IGFBP-1. In addition to IGFBP-1, the endometrial cancer cells secreted several other forms of IGFBPs as determined by cross-linking. Immunoprecipitation of IGF-BP complexes with a polyclonal antiserum against IGFBP-3 indicated that all cell lines secreted binding proteins antigenically related to IGFBP-3 with molecular weights ranging from 20 to 39 kDa.

Differential expression of mRNAs for endothelin-related proteins in human endometrium, myometrium and leiomyoma.

The expression of mRNAs encoding endothelin-1 (ET-1) and its receptors (ETA-R and ETB-R) as well as the ET degrading enzyme, neutral endopeptidase 24.11 (NEP), was determined in tissue samples of endometrium, myometrium and leiomyoma by using a reverse transcriptase polymerase chain reaction (RT-PCR) technique. ET-1 mRNA was detected in all samples studied. The level of ET-1 mRNA was higher in endometrium than in myometrium (p < 0.01) and leiomyoma (p < 0.001). The ETA-R mRNA was more abundant in endometrium than in myometrium (p < 0.001). For ETB-R mRNA there was no difference between these tissues. In contrast to ETA-R mRNA, which was more abundant in leiomyoma than in myometrium (p < 0.01), the ETB-R mRNA was less abundant in leiomyoma (p < 0.01). The NEP mRNA was detected in all endometrial samples but not in myometrium and leiomyoma. Our results show that the expression and relative levels of mRNAs encoding ET-1, ETA-R, ETB-R, and NEP vary in different tissue compartments of the human uterus. Since the net biological action of ET-1 in a particular cell type presumably depends on the balance between the peptide itself, its receptors and degrading enzymes, these results suggest different roles for ET-1 action in uterine endometrium, myometrium and leiomyoma. The difference in relative abundance of ETA-R and ETB-R mRNAs between myometrium and leiomyoma suggests that an altered ET-R gene expression may be a contributing factor in myomal growth.

Human endometrial adenocarcinoma cells express endothelin-1.

The expression of endothelin-1 (ET-1) in five human endometrial adenocarcinoma cell lines was studied. Using specific radioimmunoassay, immunoreactive ET-1 was detected in conditioned medium from two of the cell lines (RL 952 and HEC 1A). In reverse-phase high-performance liquid chromatography (HPLC), synthetic ET-1 and immunoreactive ET-1 from conditioned media revealed the same elution profile. By amplification of cDNA using the polymerase chain reaction, normal human endometrium as well as cell lines RL 952 and HEC 1A were shown to express ET-1 mRNA. In addition, cell line HEC 1B and KLE, which did not produce measurable amounts of immunoreactive ET-1, contained ET-1 specific mRNA whereas cell line AN3CA had no detectable ET-1 mRNA and did not secrete immunoreactive ET-1.

Differential expression of keratinocyte growth factor and its receptor in the human uterus.

The expression of mRNAs for keratinocyte growth factor (KGF) (also called FGF-7) and its receptor was evaluated in normal human endometrium and myometrium as well as in myoma and in endometrial adenocarcinoma cell lines using reverse transcriptase polymerase chain reaction. Both KGF and its receptor mRNA are expressed in the human endometrium throughout the menstrual cycle, whereas fibroblast growth factor receptor 2 (FGFR-2) mRNA expression is low in this tissue. In endometrial stromal cell enriched preparations KGF mRNA dominates with little expression of KGF receptor (KGFR) and FGFR-2, whereas in the epithelial cell-enriched fraction the KGFR mRNA dominates. Human myometrium and myoma express mRNA for KGF, but not for KGFR. FGFR-2 is expressed in both myometrial and myoma tissues. None of the five endometrial adenocarcinoma cell lines studied expressed KGF mRNA, whereas all cell lines expressed mRNA for either KGFR or FGFR-2 or for both receptors. The results show a selective expression of KGFR and the closely related FGFR-2 in the human uterus with the former being expressed in the endometrium and the latter predominantly in the adjacent myometrium. In the endometrial tissue, selective expression of KGF in stromal cells and KGFR in epithelial cells supports the paracrine function of KGF in epithelial tissue.

The role of mass spectrometry in proteome studies.

Mass spectrometry (MS) is an important tool in modern protein chemistry. In proteome analyses the expression of hundreds or thousands of proteins can be monitored at the same time. First, complex protein mixtures are separated by two-dimensional gel electrophoresis (2-DE) and then individual proteins are identified by using MS followed by database searches. Recent developments in this field have made it possible to do automated, high-throughput protein identification that is needed in proteome analyses. MS can also be used to characterize post-translational modifications in proteins and to study protein complexes. This review will introduce the current MS methods used in proteome studies, and discuss their advantages and disadvantages. New instrumental MS developments are also presented that are useful in these analyses.

Effect of intrauterine contraceptive devices on cytokine messenger ribonucleic acid expression in the human endometrium.

OBJECTIVE: To compare cytokine messenger RNA (mRNA) expression in the endometrium exposed to an intrauterine contraceptive device (IUD) and in normal cycling endometrium. DESIGN: Quantitative reverse-transcribed polymerase chain reaction was used to assess interleukin (IL) 1 beta, IL-6, tumor necrosis factor alpha (TNF-alpha), and colony-stimulating factor 1 (CSF-1) mRNA expression in endometrial tissue samples obtained by curettage or at hysterectomy from 11 women using a copper-releasing IUD, 10 women having a levonorgestrel-releasing IUD, and 13 fertile women during different phases of the menstrual cycle. RESULTS: The mRNAs encoding the studied cytokines were detected in all endometrial samples. The mean IL-1 beta, IL-6, and TNF-alpha mRNA levels were higher in the late secretory menstrual phase compared with the proliferative early secretory phase of the menstrual cycle both in endometria exposed to the copper IUD and in the control samples. The IL-1 beta and TNF-alpha mRNA levels were significantly higher in endometria exposed to the copper IUD compared with the control endometria in the late secretory menstrual phase, whereas no difference was found in the expression of these cytokine mRNAs during the proliferative early secretory phase. In contrast, the mean IL-6 mRNA level was higher in the copper IUD-exposed endometria than in the control endometria in the proliferative early secretory but not in the late secretory menstrual phase. In the levonorgestrel IUD-exposed endometria, the mean levels of IL-1 beta, TNF-alpha, and CSF-1 mRNA were similar to those in normal endometrium in the late secretory menstrual phase. The mean IL-6 mRNA level in levonorgestrel IUD-exposed endometria was lower than that in late secretory menstrual phase of controls but, because of a great individual variation in the control samples, the difference did not reach significance. No significant cyclic change nor any difference in relative CSF-1 mRNA levels between the three study groups was detected. CONCLUSIONS: These data provide the first evidence that the use of IUDs is associated with alterations in endometrial cytokine expression and that the alterations differ depending on the cytokine and the type of IUD. We speculate that cytokines are involved in intrauterine contraceptive effects of IUDs.

Glutamine: fructose-6-phosphate amidotransferase activity and gene expression are regulated in a tissue-specific fashion in pregnant rats.

We examined whether regulation of glutamine: fructose-6-phosphate amidotransferase (GFA), the rate-limiting enzyme of the hexosamine pathway, is tissue specific and if so whether such regulation occurs at the level of gene expression. We compared GFA activity and expression and levels of UDP-hexosamines and UDP-hexoses between insulin-sensitive (liver and muscle) tissues and a glucose-sensitive (placenta) tissue from 19 day pregnant streptozotocin diabetic and non-diabetic rats. In pregnant non-diabetic rats GFA activities averaged (1521+/-75 pmol/mg protein x min) in the placenta, 895+/-74 in the liver and 81+/-11 in muscle (p<0.001 between each tissue). In the diabetic rats, GFA activities were approximately 50% decreased both in the liver (340+/-42 pmol/mg protein x min, p<0.05 vs control rats) and in skeletal muscle (46+/-3, p<0.05) compared to control rats. In the placenta, GFA activities were identical between diabetic (1519+/-112 pmol/mg protein x min) and non-diabetic (1521+/-75) animals. In the liver, the reduction in GFA activity could be attributed to a significant decrease in GFA mRNA concentrations, while GFA mRNA concentrations were similar in the placenta between diabetic and non-diabetic animals. UDP-N-acetylglucosamine (UDP-GlcNAc), the end product of the hexosamine pathway, was significantly reduced in the liver and in skeletal muscle but similar in the placenta between diabetic and non-diabetic rats. In summary, GFA activity and expression and the concentration of UDP-GlcNAc are decreased in the liver but unaltered in the placenta, although GFA activity is almost 2-fold higher in this tissue than in the liver. These data provide the first evidence for tissue specific regulation of GFA and for its regulation at the level of gene expression.

Determination of nine beta-blockers in serum by micellar electrokinetic capillary chromatography.

beta-Adrenergic blocking agents are used for the treatment of angina pectoris, cardiac arrhythmia, hypertension, anxiety attacks, thyrotoxicosis, migraine and glaucoma. Owing to their sedative effect, they are also used as doping agents in sport. All beta-blockers have an alkanol amine side chain terminating in a secondary amino group in their structure. The pKa values vary from 9.2 to 9.8. Because some beta-blockers are hydrophilic and some lipophilic, simultaneous determination is difficult. In this work, a method based on micellar electrokinetic capillary chromatography (MECC) was developed for the separation and determination of beta-blockers in serum. The phosphate buffer 0.08 M (pH 6.7) solution contained 15 mM N-cetyl-N,N,N-trimethylammonium bromide. Nine parent beta-blockers could be separated in a single run and the concentrations determined by internal standard (ephedrine) method. The simple clean-up procedure consisted of enzyme hydrolysis (Helix pomatia), protein precipitation, and filtration through 0.5-microns PTFE membranes. The MECC method exhibited good repeatability and a linear range of 75-300 micrograms/ml. The method was successfully applied after concentration to the determination of propranolol in real samples.

Anticancer compound ABT-263 accelerates apoptosis in virus-infected cells and imbalances cytokine production and lowers survival rates of infected mice.

ABT-263 and its structural analogues ABT-199 and ABT-737 inhibit B-cell lymphoma 2 (Bcl-2), BCL2L1 long isoform (Bcl-xL) and BCL2L2 (Bcl-w) proteins and promote cancer cell death. Here, we show that at non-cytotoxic concentrations, these small molecules accelerate the deaths of non-cancerous cells infected with influenza A virus (IAV) or other viruses. In particular, we demonstrate that ABT-263 altered Bcl-xL interactions with Bcl-2 antagonist of cell death (Bad), Bcl-2-associated X protein (Bax), uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA). ABT-263 thereby activated the caspase-9-mediated mitochondria-initiated apoptosis pathway, which, together with the IAV-initiated caspase-8-mediated apoptosis pathway, triggered the deaths of IAV-infected cells. Our results also indicate that Bcl-xL, Bcl-2 and Bcl-w interact with pattern recognition receptors (PRRs) that sense virus constituents to regulate cellular apoptosis. Importantly, premature killing of IAV-infected cells by ABT-263 attenuated the production of key pro-inflammatory and antiviral cytokines. The imbalance in cytokine production was also observed in ABT-263-treated IAV-infected mice, which resulted in an inability of the immune system to clear the virus and eventually lowered the survival rates of infected animals. Thus, the results suggest that the chemical inhibition of Bcl-xL, Bcl-2 and Bcl-w could potentially be hazardous for cancer patients with viral infections.

Acute in vivo effects of insulin on gene expression in adipose tissue in insulin-resistant and insulin-sensitive subjects.

AIMS/HYPOTHESIS: We determined the response of selected genes to in vivo insulin in adipose tissue in 21 non-diabetic women. MATERIALS AND METHODS: The women were divided into insulin-sensitive and -resistant groups based on their median whole-body insulin sensitivity (8.7+/-0.4 vs 4.2+/-0.3 mg kg(-1) min(-1) for insulin-sensitive vs -resistant group). Subcutaneous adipose tissue biopsies were obtained before and after 3 and 6 h of i.v. maintained euglycaemic hyperinsulinaemia. Adipose tissue mRNA concentrations of facilitated glucose transporter, member 1 (SLC2A1, previously known as GLUT1), facilitated glucose transporter, member 4 (SLC2A4, previously known as GLUT4), peroxisome proliferator-activated receptor gamma ( PPARG), peroxisome proliferator-activated receptor gamma co-activator 1alpha (PPARGC1A), 11beta-hydroxysteroid dehydrogenase-1 (HSD11B1), TNF, adiponectin (ADIPOQ), IL6 and the macrophage marker CD68 were measured using real-time PCR. RESULTS: Basal expression of 'insulin-sensitivity genes' SLC2A4 and ADIPOQ was lower while that of 'insulin-resistance genes', HSD11B1 and IL6 was significantly higher in the insulin-resistant than in the insulin-sensitive group. Insulin significantly increased expression of 'insulin-sensitivity genes' SLC2A4, PPARG, PPARGC1A and ADIPOQ in the insulin-sensitive group, while only expression of PPARG and PPARGC1A was increased in the insulin-resistant group. The expression of 'insulin-resistance genes' HSD11B1 and IL6 was increased by insulin in the insulin-resistant group, but insulin failed to increase HSD11B1 expression in the insulin-sensitive group. At 6 h, expression of HSD11B1, TNF and IL6 was significantly higher in the insulin-resistant than in the insulin-sensitive group. IL6 expression increased significantly more in response to insulin in the insulin-resistant than in the insulin-sensitive group. CD68 was overexpressed in the insulin-resistant as compared with the insulin-sensitive group at both 0 and 6 h. CONCLUSIONS/INTERPRETATION: These data suggest that genes adversely affecting insulin sensitivity hyperrespond to insulin, while genes enhancing insulin sensitivity hyporespond to insulin in insulin-resistant human adipose tissue in vivo.