Immunohistochemical localization of cytochrome P450 3A in human pulmonary carcinomas and normal bronchial tissue

The cytochrome P450 (CYP) enzymes metabolize drugs and other xenobiotics in liver and also in some extrahepatic tissues. We have studied the expression and localization of CYP3A in primary lung tumours and normal lung tissue from the same patients. Thirtytwo patients undergoing partial or total lung resection for therapy of primary pulmonary carcinoma were included in this study. Immunohistochemical staining for CYP3A was performed with a modification of the ABC technique. Eight of the 32 cases of primary pulmonary carcinoma showed expression of CYP3A. In 12 of the 32 cases of normal tissue, the seromucous glands were positive for CYP3A. The bronchial epithelium was positive for CYP3A in 11 cases. We observed no correlation between CYP3A expression in tumour tissue and that in seromucous glands or bronchial epithelium. We conclude that CYP3A is present in both normal and cancerous lung tissue. Our findings suggest, however, no co-expression of CYP3A in lung cancer.     

Effects of γ-aminobutyric acid on 33Pi incorporation into rat brain phospholipids in vivo

The effects of γ-aminobutyric acid (GABA), bicuculline and strychnine on the incorporation $\text{in vivo}$ of ³³Pi into phospholipids of rat brain were studied at 10 and 30 minutes after intracisternal injection of the radionuclide. GABA inhibited labeling of phospholipids in the three brain regions studied at both times. Bicuculline by itself had no significant effect on ³³Pi incorporation, but totally blocked the inhibitory effect of GABA in all three brain regions. Strychnine by itself inhibited phospholipid labeling in the brain stem and forebrain, had no significant effect on GABA inhibition of ³³Pi incorporation in the cerebellum and forebrain, and partially blocked the GABA effect in the brain stem. GABA inhibited ³³Pi incorporation into phosphatidic acid, phosphatidylinositol, phosphatidyl choline and phosphatidyl ethanolamine but had no effect on phosphatidyl serine. The data suggest that the inhibitory effects of GABA on CNS phospholipid labeling are mediated specifically through GABA receptor sites.     

Subtypes of Depression—Diagnosis and Medical Management

Depression is both a common and a greatly undertreated illness in the United States today. The focus of this review is a definition of the characteristics of four subtypes of depression which appear to be differentially sensitive to four different classes of medications. The tricyclic antidepressants should be used for patients with unipolar depression and vegetative symptoms. Lithium appears to be most effective for bipolar depressives. The monoamine oxidase (MAO) inhibitors are best used for patients with atypical depression. Antipsychotic medications appear to be useful for depressed patients with psychotic symptoms or agitation. Recent pharmacokinetic and biochemical data, including serum lithium levels, plasma tricyclic levels, and the predictive ability of pretreatment urinary 3-methoxy-4-hydroxyphenylglycol (MHPG) levels are also reviewed.     

Wind-sensitive interneurones in the spider CNS (Cupiennius salei ): directional information processing of sensory inputs from trichobothria on the walking legs

Spiders can use air particle movements to localize moving prey. We studied the responses of 32 wind-sensitive interneurones in the hunting spider Cupiennius salei to prey stimuli. Stimulation with a tethered flying fly or with artificial air pulses activated plurisegmental interneurones that responded to changes in air movement velocity and were thus well suited to represent the highly fluctuating air stream typical of prey stimuli. In most interneurones (n = 18) the responses to the stimulation of different legs were not significantly different from each other. Different interneurones had different response characteristics and their latencies largely overlapped suggesting that there is parallel processing of the signals by populations of interneurones with different response characteristics. In two interneurones the number of spikes and the spiking pattern elicited by stimulation of each of the eight legs markedly differed depending on the leg stimulated. These neurones may play an important role in directional information processing. Stimulation of the adjacent legs from front to back or from back to front revealed two interneurones sensitive to the direction of successive stimulation of the legs. These neurones may be able to detect the motion of an air movement source in a preferred direction and thus act as nearfield motion detectors to localize a moving prey item. Accepted: 28 September 1996     

Real-time monitoring of cell viability and cell density on the basis of a three dimensional optical reflectance method (3D-ORM): investigation of the effect of sub-lethal and lethal injuries

Cell density and cell viability have been followed on-line by using a three-dimensional optical reflectance method (3D-ORM) probe. This method has allowed to highlight the differences between a well-mixed and a scale-down bioreactor configured in order to reproduce mixing deficiencies during a fed-batch culture of Escherichia coli. These differences have been observed both for the obscuration factor (OBF) and the coincidence probability delivered by the probe. These parameters are correlated to flow cytometry measurement based on the PI-uptake test and cell density based on optical density measurement. This first set of results has pointed out the fact that the 3D-ORM probe is sensitive to sub-lethal injuries encountered by microbial cells in process-related conditions. The effect of lethal injuries has been further investigated on the basis of additional experiments involving heat stress and a sharp increase of the OBF has been observed indicating that cells are effectively injured by the increase of temperature. However, further improvement of the probe are needed in order to give access to single-cell measurements.     

Clonal Expansion Analysis of Transposon Insertions by High-Throughput Sequencing Identifies Candidate Cancer Genes in a PiggyBac Mutagenesis Screen

Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10⁶ reads corresponding to >10⁴ insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors.     

Simultaneous Surface Display and Secretion of Proteins from Mammalian Cells Facilitate Efficient in Vitro Selection and Maturation of Antibodies

A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation.     

Genome wide association (GWA) study for early onset extreme obesity supports the role of fat mass and obesity associated gene (FTO) variants

Background

Obesity is a major health problem. Although heritability is substantial, genetic mechanisms predisposing to obesity are not very well understood. We have performed a genome wide association study (GWA) for early onset (extreme) obesity.

Methodology/Principal Findings

a) GWA (Genome-Wide Human SNP Array 5.0 comprising 440,794 single nucleotide polymorphisms) for early onset extreme obesity based on 487 extremely obese young German individuals and 442 healthy lean German controls; b) confirmatory analyses on 644 independent families with at least one obese offspring and both parents. We aimed to identify and subsequently confirm the 15 SNPs (minor allele frequency ≥10%) with the lowest p-values of the GWA by four genetic models: additive, recessive, dominant and allelic. Six single nucleotide polymorphisms (SNPs) in FTO (fat mass and obesity associated gene) within one linkage disequilibrium (LD) block including the GWA SNP rendering the lowest p-value (rs1121980; log-additive model: nominal p = 1.13×10⁻⁷, corrected p = 0.0494; odds ratio (OR)_CT 1.67, 95% confidence interval (CI) 1.22–2.27; OR_TT 2.76, 95% CI 1.88–4.03) belonged to the 15 SNPs showing the strongest evidence for association with obesity. For confirmation we genotyped 11 of these in the 644 independent families (of the six FTO SNPs we chose only two representing the LD bock). For both FTO SNPs the initial association was confirmed (both Bonferroni corrected p<0.01). However, none of the nine non-FTO SNPs revealed significant transmission disequilibrium.

Conclusions/Significance

Our GWA for extreme early onset obesity substantiates that variation in FTO strongly contributes to early onset obesity. This is a further proof of concept for GWA to detect genes relevant for highly complex phenotypes. We concurrently show that nine additional SNPs with initially low p-values in the GWA were not confirmed in our family study, thus suggesting that of the best 15 SNPs in the GWA only the FTO SNPs represent true positive findings.