2100 MilliporeProtein-Concentrate Kit (Micro)

Exposures to two commercial nanofilm spray products (NFPs), a floor sealant (NFP 1) and a coating product for tiles (NFP 2), were investigated for airway irritation, airway inflammation and lung damage in a mouse inhalation model. The particle-exposure was characterized by particle number, particle size-distribution and gravimetric analysis. BALB/cJ mice were exposed for 60 min to the aerosolized products at 3.3-60 mg/m(3) (10(5) - 10(6) fine particles/cm(3)) measured in the breathing zone of the mice. Lung inflammation and lung damage were assessed by study of bronchoalveolar lavage fluid (BALF) cytology, protein in BALF and histology. Mass spectral analysis showed that NFP 1 and NFP 2 contained hydrolysates and condensates of a perfluorosilane and alkylsilane, respectively. NFP 1 induced a concentration-dependent decrease of the tidal volume lasting for at least one day. Exposure concentrations above 16.1 mg/m(3) (2.5x10(6) fine particles/cm(3)) gave rise to significant increases of protein level in BALF, reduced body weight, and histological examination showed atelectasis, emphysema and hemorrhages. A narrow interval between the no-effect level (16.1 mg/m(3)) and lethal concentrations (18.4 mg/m(3)) was observed. The alkylsilane based product (NFP 2) had no effect at the concentrations studied. Experiments with different types of perfluorinated silanes and siloxanes showed that the toxic effects did not arise solely from the perfluorination. The number of free hydroxyl groups in the silanes/siloxanes was also critical for the toxicity.

Intravenous administration of apolipoprotein (apo) A-I complexed with phospholipid has been shown to rapidly reduce plaque size in both animal models and humans. Short synthetic amphipathic peptides can mimic the antiatherogenic properties of apoA-I and have been proposed as alternative therapeutic agents. In this study, we investigated the atheroprotective effect of the 5A peptide, a bihelical amphipathic peptide that specifically effluxes cholesterol from cells by ATP-binding cassette transporter 1 (ABCA1). 5A stimulated a 3.5-fold increase in ABCA1-mediated efflux from cells and an additional 2.5-fold increase after complexing it with phospholipid (1:7 mol/mol). 5A-palmitoyl oleoyl phosphatidyl choline (POPC), but not free 5A, was also found to promote cholesterol efflux by ABCG1. When incubated with human serum, 5A-POPC bound primarily to high-density lipoprotein (HDL) but also to low-density lipoprotein (LDL) and promoted the transfer of cholesterol from LDL to HDL. Twenty-four hours after intravenous injection of 5A-POPC (30 mg/kg) into apoE-knockout (KO) mice, both the cholesterol (181%) and phospholipid (219%) content of HDL significantly increased. By an in vivo cholesterol isotope dilution study and monitoring of the flux of cholesterol from radiolabeled macrophages to stool, 5A-POPC treatment was observed to increase reverse cholesterol transport. In three separate studies, 5A when complexed with various phospholipids reduced aortic plaque surface area by 29 to 53% (n = 8 per group; p < 0.02) in apoE-KO mice. No signs of toxicity from the treatment were observed during these studies. In summary, 5A promotes cholesterol efflux both in vitro and in vivo and reduces atherosclerosis in apoE-KO mice, indicating that it may be a useful alternative to apoA-I for HDL therapy.

We investigated the ability of AMP-activated protein kinase (AMPK) to activate PPARgamma coactivator-1alpha (PGC-1alpha) in the brain, liver and brown adipose tissue (BAT) of the NLS-N171-82Q transgenic mouse model of Huntington's Disease (HD). In the striatum of the HD mice, the baseline levels of PGC-1alpha, NRF1, NRF2, Tfam, COX-II, PPARdelta, CREB and ERRalpha mRNA, and mitochondrial DNA (mtDNA), were significantly reduced. Administration of the creatine analog beta guanidinopropionic acid (GPA), reduced ATP and PCr levels, and increased AMPK mRNA in both the cerebral cortex and striatum. Treatment with GPA significantly increased expression of PGC-1alpha, NRF1, Tfam, and downstream genes in the striatum and cerebral cortex of wildtype (WT) mice, but there was no effect on these genes in the HD mice. The striatum of the untreated HD mice showed microvacuolation in the neuropil, as well as gliosis and huntingtin aggregates, which were exacerbated by treatment with GPA. GPA treatment produced a significant increase in mtDNA in the cerebral cortex and striatum of WT mice, but not in HD mice. The HD mice treated with GPA had impaired activation of liver PGC-1alpha, and developed hepatic steatosis with accumulation of lipids, degeneration of hepatocytes, and impaired activation of gluconeogenesis. The BAT in the HD mice showed vacuolation due to accumulation of neutral lipids, and age-dependent impairment of UCP-1 activation and temperature regulation. Impaired activation of PGC-1alpha, therefore plays an important role in the behavioral phenotype, metabolic disturbances, and pathology of HD, which suggests the possibility that agents which enhance PGC-1alpha function, will exert therapeutic benefits in HD patients.

Mutations in glucose transporter 10 (GLUT10) alter angiogenesis and cause arterial tortuosity syndrome (ATS); however, the mechanisms by which these mutations cause disease remain unclear. It has been reported that in most cells, mitochondria are the major source of reactive oxygen species (ROS). Moreover, mitochondria are known to incorporate as well as recycle vitamin C, which plays a critical role in redox homeostasis, although the molecular mechanism(s) underlying mitochondrial vitamin C uptake are poorly understood. We report here that GLUT10 localizes predominantly to the mitochondria of smooth muscle cells and insulin-stimulated adipocytes, where GLUT10 is highly expressed. We further demonstrate that GLUT10 facilitates transport of l-dehydroascorbic acid (DHA), the oxidized form of vitamin C, into mitochondria, and also increases cellular uptake of DHA, which in turn protects cells against oxidative stress. This protection is compromised when GLUT10 expression in mitochondria is inhibited. In addition, we found that aortic smooth muscle cells from GLUT10-mutant mice have higher ROS levels than those from wild-type mice. Our results identify the physiological role of GLUT10 as the mitochondrial DHA transporter, and demonstrate that GLUT10 protects cells from oxidative injury. Furthermore, our findings provide a mechanism to explain the ascorbate in mitochondria and show how loss-of-function GLUT10 mutations may lead to arterial abnormalities in ATS. These results also reinforce the importance of vitamin C and ROS in degenerative diseases.

Mitochondrial respiratory chain (RC) deficiency is among the most common causes of inherited metabolic disease, but its physiological consequences are poorly characterized. We studied the skeletal muscle gene expression profiles of mice with late-onset mitochondrial myopathy. These animals express a dominant patient mutation in the mitochondrial replicative helicase Twinkle, leading to accumulation of multiple mtDNA deletions and progressive subtle RC deficiency in the skeletal muscle. The global gene expression pattern of the mouse skeletal muscle showed induction of pathways involved in amino acid starvation response and activation of Akt signaling. Furthermore, the muscle showed induction of a fasting-related hormone, fibroblast growth factor 21 (Fgf21). This secreted regulator of lipid metabolism was also elevated in the mouse serum, and the animals showed widespread changes in their lipid metabolism: small adipocyte size, low fat content in the liver and resistance to high-fat diet. We propose that RC deficiency induces a mitochondrial stress response, with local and global changes mimicking starvation, in a normal nutritional state. These results may have important implications for understanding the metabolic consequences of mitochondrial myopathies.

Ligation of two oligonucleotide probes hybridized adjacently to a DNA template has been widely used for detection of genome alterations. The multiplex ligation-dependent probe amplification (MLPA) technique allows simultaneous screening of multiple target sequences in a single reaction by using pairs of probes that carry tails for binding of common amplification primers. Resolution of the various targets is achieved by electrophoresis on the basis of predefined differences in amplicon length. In the conventional MLPA approach, one of the two target probes is generated by cloning in a single-stranded bacteriophage vector to introduce a sequence of defined length between the primer binding site and the specific target sequence. Here we demonstrate that differences in amplicon length can be achieved by using multiple short synthetic probes for each target sequence. When joined by a DNA ligase, these probes will form a single amplifiable template whose length is defined by the number and lengths of the individual probes. We have used this principle to establish a methylation-specific MLPA (MS-MLPA) assay that simultaneously determines the methylation status of five promoter CpG islands, and we have used this assay to analyze DNA from tumor tissue and corresponding urine samples from patients with bladder cancer. Our data show that the use of multiple short synthetic probes provides a simple means for custom-designed MS-MLPA analysis.

Areas and layers of the cerebral cortex are specified by genetic programs that are initiated in progenitor cells and then, implemented in postmitotic neurons. Here, we report that Tbr1, a transcription factor expressed in postmitotic projection neurons, exerts positive and negative control over both regional (areal) and laminar identity. Tbr1 null mice exhibited profound defects of frontal cortex and layer 6 differentiation, as indicated by down-regulation of gene-expression markers such as Bcl6 and Cdh9. Conversely, genes that implement caudal cortex and layer 5 identity, such as Bhlhb5 and Fezf2, were up-regulated in Tbr1 mutants. Tbr1 implements frontal identity in part by direct promoter binding and activation of Auts2, a frontal cortex gene implicated in autism. Tbr1 regulates laminar identity in part by downstream activation or maintenance of Sox5, an important transcription factor controlling neuronal migration and corticofugal axon projections. Similar to Sox5 mutants, Tbr1 mutants exhibit ectopic axon projections to the hypothalamus and cerebral peduncle. Together, our findings show that Tbr1 coordinately regulates regional and laminar identity of postmitotic cortical neurons.

We demonstrate that single interneurons can toggle the output neurons of the cerebellar cortex (the Purkinje cells) between their two states. The firing of Purkinje cells has previously been shown to alternate between an "up" state in which the cell fires spontaneous action potentials and a silent "down" state. We show here that small hyperpolarizing currents in Purkinje cells can bidirectionally toggle Purkinje cells between down and up states and that blockade of the hyperpolarization-activated cation channels (H channels) with the specific antagonist ZD7288 (10 microM) blocks the transitions from down to up states. Likewise, hyperpolarizing inhibitory postsnyaptic potentials (IPSPs) produced by small bursts of action potentials (10 action potentials at 50 Hz) in molecular-layer interneurons induce these bidirectional transitions in Purkinje cells. Furthermore, single interneurons in paired interneuron --> Purkinje cell recordings, produce bidirectional switches between the two states of Purkinje cells. The ability of molecular-layer interneurons to toggle Purkinje cells occurs when Purkinje cells are recorded under whole-cell patch-clamp conditions as well as when action potentials are recorded in an extracellular loose cell-attached configuration. The mode switch demonstrated here indicates that a single presynaptic interneuron can have opposite effects on the output of a given Purkinje cell, which introduces a unique type of synaptic interaction that may play an important role in cerebellar signaling.

The DM domain proteins Doublesex- and MAB-3-related transcription factors (DMRTs) are widely conserved in metazoan sex determination and sexual differentiation. One of these proteins, DMRT1, plays diverse and essential roles in development of the vertebrate testis. In mammals DMRT1 is expressed and required in both germ cells and their supporting Sertoli cells. Despite its critical role in testicular development, little is known about how DMRT1 functions as a transcription factor or what genes it binds and regulates. We combined ChIP methods with conditional gene targeting and mRNA expression analysis and identified almost 1,400 promoter-proximal regions bound by DMRT1 in the juvenile mouse testis and determined how expression of the associated mRNAs is affected when Dmrt1 is selectively mutated in germ cells or Sertoli cells. These analyses revealed that DMRT1 is a bifunctional transcriptional regulator, activating some genes and repressing others. ChIP analysis using conditional mutant testes showed that DNA binding and transcriptional regulation of individual target genes can differ between germ cells and Sertoli cells. Genes bound by DMRT1 in vivo were enriched for a motif closely resembling the sequence DMRT1 prefers in vitro. Differential response of genes to loss of DMRT1 corresponded to differences in the enriched motif, suggesting that other transacting factors may modulate DMRT1 activity. DMRT1 bound its own promoter and those of six other Dmrt genes, indicating auto- and cross-regulation of these genes. Many of the DMRT1 target genes identified here are known to be important for a variety of functions in testicular development; the others are candidates for further investigation.

In working environments, especially in confined spaces like greenhouses, elevated concentrations of airborne microorganisms may become a problem for workers' health. Additionally, the use of microbial pest control agents may increase exposure to microorganisms. The aim of this study was to investigate tomato growers' exposure to naturally occurring bioaerosol components (dust, bacteria, fungi, actinomycetes, (1-->3)-beta-D-glucans and endotoxin) and microbial pest control agents applied by drip irrigation. Airborne dust was collected with filter samplers and analyzed for microorganisms by plate counts and total counts in microscope. Analysis of (1-->3)-beta-D-glucan and endotoxin content were performed by kinetic, chromatic Limulus Amoebocyte Lysate tests. The fungal strain (Trichoderma harzianum) from the biocontrol product Supresivit(R) was identified by PCR analysis. Measurements were performed on the day of drip irrigation and one week, one month and three months after the irrigation. T. harzianum from Supresivit(R) could only be detected on the day of treatment. Streptomyces griseoviridis, an applied microbial pest control agent, was not detected in the air during this investigation. We found that bioaerosol exposure increases during the growth season and that exposure to fungi, bacteria, and endotoxin can reach levels during the harvest period, that may cause respiratory symptoms in growers. The collected data indicates that MPCAs applied by drip irrigation do not become airborne later in the season.