Pom1 regulates the assembly of Cdr2-Mid1 cortical nodes for robust spatial control of cytokinesis.

Proper division plane positioning is essential to achieve faithful DNA segregation and to control daughter cell size, positioning, or fate within tissues. In Schizosaccharomyces pombe, division plane positioning is controlled positively by export of the division plane positioning factor Mid1/anillin from the nucleus and negatively by the Pom1/DYRK (dual-specificity tyrosine-regulated kinase) gradients emanating from cell tips. Pom1 restricts to the cell middle cortical cytokinetic ring precursor nodes organized by the SAD-like kinase Cdr2 and Mid1/anillin through an unknown mechanism. In this study, we show that Pom1 modulates Cdr2 association with membranes by phosphorylation of a basic region cooperating with the lipid-binding KA-1 domain. Pom1 also inhibits Cdr2 interaction with Mid1, reducing its clustering ability, possibly by down-regulation of Cdr2 kinase activity. We propose that the dual regulation exerted by Pom1 on Cdr2 prevents Cdr2 assembly into stable nodes in the cell tip region where Pom1 concentration is high, which ensures proper positioning of cytokinetic ring precursors at the cell geometrical center and robust and accurate division plane positioning.

Human epidermal Langerhans cells express the tight junction protein claudin-1 and are present in human genetic claudin-1 deficiency (NISCH syndrome).

Claudin-1 (CLDN1) is a structural tight junction (TJ) protein and is expressed in differentiating keratinocytes and Langerhans cells in the epidermis. Our objective was to identify immunoreactive CLDN1 in human epidermal Langerhans cells and to examine the pattern of epidermal Langerhans cells in genetic human CLDN1 deficiency [neonatal ichthyosis, sclerosing cholangitis (NISCH) syndrome]. Epidermal cells from healthy human skin labelled with CLDN1-specific antibodies were analysed by confocal laser immunofluorescence microscopy and flow cytometry. Skin biopsy sections of two patients with NISCH syndrome were stained with an antibody to CD1a expressed on epidermal Langerhans cells. Epidermal Langerhans cells and a subpopulation of keratinocytes from healthy skin were positive for CLDN1. The gross number and distribution of epidermal Langerhans cells of two patients with molecularly confirmed NISCH syndrome, however, was not grossly altered. Therefore, CLDN1 is unlikely to play a critical role in migration of Langerhans cells (or their precursors) to the epidermis or their positioning within the epidermis. Our findings do not exclude a role of this TJ molecule once Langerhans cells have left the epidermis for draining lymph nodes.

Physiology of GLP-1--lessons from glucoincretin receptor knockout mice.

GLP-1 has both peripheral and central actions, as this hormone is secreted by gut endocrine cells and brainstem neurons projecting into the hypothalamus and other brain regions. GLP-1 has multiple regulatory functions participating in the control of glucose homeostasis, beta-cell proliferation and differentiation, food intake, heart rate and even learning. GLP-1 action depends on binding to a specific G-coupled receptor linked to activation of the adenylyl cyclase pathway. Analysis of mice with inactivation of the GLP-1 receptor gene has provided evidence that absence of GLP-1 action in the mouse, despite this hormone potent physiological effects when administered in vivo, only leads to mild abnormalities in glucose homeostasis without any change in body weight. However, a critical role for this hormone and its receptor was demonstrated in the function of the hepatoportal vein glucose sensor, in contrast to that of the pancreatic beta-cells, although absence of both GLP-1 and GIP receptors leads to a more severe phenotype characterized by a beta-cell-autonomous defect in glucose-stimulated insulin secretion. Together, the studies of these glucoincretin receptor knockout mice provide evidence that these hormones are part of complex regulatory systems where multiple redundant signals are involved.

Neurotoxicity of dibutyltin in aggregating brain cell cultures.

Dibutyltin (DBT) compounds are used primarily as stabilizers for polyvinyl chloride (PVC) plastics. Small quantities can be released from PVC containers into stored liquids. The neurotoxicological potential of DBT was tested in aggregating brain cell cultures after a 10-day treatment with concentrations ranging from 10(-10) to 10(-6)m, either during an early developmental period, or during a phase of advanced maturation. Changes in protein content, DNA labelling and cell type-specific enzyme activities were measured as end points. DBT caused general cytotoxicity at 10(-6)m in both immature and differentiated cultures. At 10(-7)m, it affected the myelin content and the cholinergic neurons in both states of maturation, while GABAergic neurons remained unchanged. Astrocyte and oligodendrocyte markers were diminished at 10(-7)m of DBT exclusively in immature cultures. DBT uptake by undifferentiated and differentiated cells was similar at this concentration. Whereas trimethyltin (TMT) is known to induce gliosis and triethyltin (TET) to cause demyelination and affect GABAergic neurons, DBT appeared to be more toxic than TMT, and to present a distinct toxicological pattern.

Long-lasting antidiabetic effect of a dipeptidyl peptidase IV-resistant analog of glucagon-like peptide-1.

Glucagon-like peptide-1(7-37) (GLP-1) is the most potent insulinotropic hormone characterized thus far. Because its activity is preserved in non-insulin-dependent diabetes mellitus (NIDDM) patients, it is considered a potential new drug for the treatment of this disease. One limitation in its therapeutic use is a short half-life in vivo (5 minutes), due in part to a fast degradation by the endoprotease dipeptidylpeptidase IV (DPPIV). Recently, it was reported that GLP-1 became resistant to DPPIV when the alanine residue at position 8 was replaced by a glycine (GLP-1-Gly8). We report here that this change slightly decreased the affinity of the peptide for its receptor (IC50, 0.41 +/- 0.14 and 1.39 +/- 0.61 nmol/L for GLP-1 and GLP-1-Gly8, respectively) but did not change the efficiency to stimulate accumulation of intracellular cyclic adenosine monophosphate (cAMP) (EC50, 0.25 +/- 0.05 and 0.36 +/- 0.06 nmol/L for GLP-1 and GLP-1-Gly8, respectively). Second, we demonstrate for the first time that this mutant has an improved insulinotropic activity compared with the wild-type peptide when tested in vivo in an animal model of diabetes. A single injection of 0.1 nmol GLP-1-Gly8 in diabetic mice fed a high-fat diet can correct fasting hyperglycemia and glucose intolerance for several hours, whereas the activity of 1 nmol GLP-1 vanishes a few minutes after injection. These actions were correlated with increased insulin and decreased glucagon levels. Interestingly, normoglycemia was maintained over a period that was longer than the predicted peptide half-life, suggesting a yet undescribed long-term effect of GLP-1-Gly8. GLP-1-Gly8 thus has a markedly improved therapeutic potential compared with GLP-1, since it can be used at much lower doses and with a more flexible schedule of administration.

Stable carbon isotope Composition of c9,t11-conjugated linoleic acid in cow's milk as related to dietary fatty acids

This study explores the potential use of stable carbon isotope ratios (delta C-13) of single fatty acids (FA) as tracers for the transformation of FA from diet to milk, with focus on the metabolic origin of c9,t11-18:2. For this purpose, dairy cows were fed diets based exclusively on C-3 and C-4 plants. The FA in milk and feed were fractionated by silver-ion thin-layer chromatography and analyzed for their delta C-13 values. Mean delta C-13 values of FA from C-3 milk were lower compared to those from C-4 milk (-30.1aEuro degrees vs. -24.9aEuro degrees, respectively). In both groups the most negative delta C-13 values of all FA analyzed were measured for c9,t11-18:2 (C-3 milk = -37.0 +/- A 2.7aEuro degrees; C-4 milk -31.4 +/- A 1.4aEuro degrees). Compared to the dietary precursors 18:2n-6 and 18:3n-3, no significant C-13-depletion was measured in t11-18:1. This suggests that the delta C-13-change in c9,t11-18:2 did not originate from the microbial biohydrogenation in the rumen, but most probably from endogenous desaturation of t11-18:1. It appears that the natural delta C-13 differences in some dietary FA are at least partly preserved in milk FA. Therefore, carbon isotope analyses of individual FA could be useful for studying metabolic transformation processes in ruminants.

Pituitary blastoma: a pathognomonic feature of germ-line DICER1 mutations.

Individuals harboring germ-line DICER1 mutations are predisposed to a rare cancer syndrome, the DICER1 Syndrome or pleuropulmonary blastoma-familial tumor and dysplasia syndrome [online Mendelian inheritance in man (OMIM) #601200]. In addition, specific somatic mutations in the DICER1 RNase III catalytic domain have been identified in several DICER1-associated tumor types. Pituitary blastoma (PitB) was identified as a distinct entity in 2008, and is a very rare, potentially lethal early childhood tumor of the pituitary gland. Since the discovery by our team of an inherited mutation in DICER1 in a child with PitB in 2011, we have identified 12 additional PitB cases. We aimed to determine the contribution of germ-line and somatic DICER1 mutations to PitB. We hypothesized that PitB is a pathognomonic feature of a germ-line DICER1 mutation and that each PitB will harbor a second somatic mutation in DICER1. Lymphocyte or saliva DNA samples ascertained from ten infants with PitB were screened and nine were found to harbor a heterozygous germ-line DICER1 mutation. We identified additional DICER1 mutations in nine of ten tested PitB tumor samples, eight of which were confirmed to be somatic in origin. Seven of these mutations occurred within the RNase IIIb catalytic domain, a domain essential to the generation of 5p miRNAs from the 5' arm of miRNA-precursors. Germ-line DICER1 mutations are a major contributor to PitB. Second somatic DICER1 "hits" occurring within the RNase IIIb domain also appear to be critical in PitB pathogenesis.

Lack of original antigenic sin in recall CD8(+) T cell responses.

In the real world, mice and men are not immunologically naive, having been exposed to numerous antigenic challenges. Prior infections sometimes negatively impact the response to a subsequent infection. This can occur in serial infections with pathogens sharing cross-reactive Ags. At the T cell level it has been proposed that preformed memory T cells, which cross-react with low avidity to epitopes presented in subsequent infections, dampen the response of high-avidity T cells. We investigated this with a series of related MHC class-I restricted Ags expressed by bacterial and viral pathogens. In all cases, we find that high-avidity CD8(+) T cell precursors, either naive or memory, massively expand in secondary cross-reactive infections to dominate the response over low-avidity memory T cells. This holds true even when >10% of the CD8(+) T cell compartment consists of memory T cells that cross-react weakly with the rechallenge ligand. Occasionally, memory cells generated by low-avidity stimulation in a primary infection recognize a cross-reactive epitope with high avidity and contribute positively to the response to a second infection. Taken together, our data show that the phenomenon of original antigenic sin does not occur in all heterologous infections.

The evolution of lncRNA repertoires and expression patterns in tetrapods.

Only a very small fraction of long noncoding RNAs (lncRNAs) are well characterized. The evolutionary history of lncRNAs can provide insights into their functionality, but the absence of lncRNA annotations in non-model organisms has precluded comparative analyses. Here we present a large-scale evolutionary study of lncRNA repertoires and expression patterns, in 11 tetrapod species. We identify approximately 11,000 primate-specific lncRNAs and 2,500 highly conserved lncRNAs, including approximately 400 genes that are likely to have originated more than 300 million years ago. We find that lncRNAs, in particular ancient ones, are in general actively regulated and may function predominantly in embryonic development. Most lncRNAs evolve rapidly in terms of sequence and expression levels, but tissue specificities are often conserved. We compared expression patterns of homologous lncRNA and protein-coding families across tetrapods to reconstruct an evolutionarily conserved co-expression network. This network suggests potential functions for lncRNAs in fundamental processes such as spermatogenesis and synaptic transmission, but also in more specific mechanisms such as placenta development through microRNA production.

Encapsulated, genetically engineered cells, secreting glucagon-like peptide-1 for the treatment of non-insulin-dependent diabetes mellitus.

Non-insulin-dependent, or type II, diabetes mellitus is characterized by a progressive impairment of glucose-induced insulin secretion by pancreatic beta cells and by a relative decreased sensitivity of target tissues to the action of this hormone. About one third of type II diabetic patients are treated with oral hypoglycemic agents to stimulate insulin secretion. These drugs however risk inducing hypoglycemia and, over time, lose their efficacy. An alternative treatment is the use of glucagon-like peptide-1 (GLP-1), a gut peptidic hormone with a strong insulinotropic activity. Its activity depends of the presence of normal blood glucose concentrations and therefore does not risk inducing hypoglycemia. GLP-1 can correct hyperglycemia in diabetic patients, even in those no longer responding to hypoglycemic agents. Because it is a peptide, GLP-1 must be administered by injection; this may prevent its wide therapeutic use. Here we propose to use cell lines genetically engineered to secrete a mutant form of GLP-1 which has a longer half-life in vivo but which is as potent as the wild-type peptide. The genetically engineered cells are then encapsulated in semi-permeable hollow fibers for implantation in diabetic hosts for constant, long-term, in situ delivery of the peptide. This approach may be a novel therapy for type II diabetes.

Influence of environmental factors on the expression of phenotypic characters by chicken dorsal root ganglion cells in culture.

Primary sensory neurons were grown under four conditions of culture. The influence of nonneuronal cells, horse serum or both was studied on the phenotypic expression of certain neuronal subpopulations. The number of neurons expressing acetylcholinesterase, alpha-bungarotoxin-binding sites or a high uptake capacity for glutamine was enhanced by nonneuronal cells. The horse serum increases the neuronal subpopulation exhibiting a carbonic anhydrase activity. Certain phenotypic changes fit conditions consistent with an epigenetic induction rather than a cell selection.

A genetic basis for functional hypothalamic amenorrhea.

Background: Functional hypothalamic amenorrhea is a reversible form of gonadotropin-releasing hormone (GnRH) deficiency commonly triggered by stressors such as excessive exercise, nutritional deficits, or psychological distress. Women vary in their susceptibility to inhibition of the reproductive axis by such stressors, but it is unknown whether this variability reflects a genetic predisposition to hypothalamic amenorrhea. We hypothesized that mutations in genes involved in idiopathic hypogonadotropic hypogonadism, a congenital form of GnRH deficiency, are associated with hypothalamic amenorrhea. Methods: We analyzed the coding sequence of genes associated with idiopathic hypogonadotropic hypogonadism in 55 women with hypothalamic amenorrhea and performed in vitro studies of the identified mutations. Results: Six heterozygous mutations were identified in 7 of the 55 patients with hypothalamic amenorrhea: two variants in the fibroblast growth factor receptor 1 gene FGFR1 (G260E and R756H), two in the prokineticin receptor 2 gene PROKR2 (R85H and L173R), one in the GnRH receptor gene GNRHR (R262Q), and one in the Kallmann syndrome 1 sequence gene KAL1 (V371I). No mutations were found in a cohort of 422 controls with normal menstrual cycles. In vitro studies showed that FGFR1 G260E, FGFR1 R756H, and PROKR2 R85H are loss-of-function mutations, as has been previously shown for PROKR2 L173R and GNRHR R262Q. Conclusions: Rare variants in genes associated with idiopathic hypogonadotropic hypogonadism are found in women with hypothalamic amenorrhea, suggesting that these mutations may contribute to the variable susceptibility of women to the functional changes in GnRH secretion that characterize hypothalamic amenorrhea. Our observations provide evidence for the role of rare variants in common multifactorial disease. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT00494169.)

Optimization of HS-SPME/GC-MS analysis and its use in the profiling of illicit ecstasy tablets (Part 1)

A headspace solid-phase microextraction procedure (HS-SPME) was developed for the profiling of traces present in 3,4-methylenedioxymethylampethamine (MDMA). Traces were first extracted using HS-SPME and then analyzed by gas chromatography-mass spectroscopy (GC-MS). The HS-SPME conditions were optimized using varying conditions. Optimal results were obtained when 40 mg of crushed MDMA sample was heated at 80 °C for 15 min, followed by extraction at 80 °C for 15 min with a polydimethylsiloxane/divinylbenzene coated fibre. A total of 31 compounds were identified as traces related to MDMA synthesis, namely precursors, intermediates or by-products. In addition some fatty acids used as tabletting materials and caffeine used as adulterant, were also detected. The use of a restricted set of 10 target compounds was also proposed for developing a screening tool for clustering samples having close profile. 114 seizures were analyzed using an SPME auto-sampler (MultiPurpose Samples MPS2), purchased from Gerstel GMBH & Co. (Germany), and coupled to GC-MS. The data was handled using various pre-treatment methods, followed by the study of similarities between sample pairs based on the Pearson correlation. The results show that HS-SPME, coupled with the suitable statistical method is a powerful tool for distinguishing specimens coming from the same seizure and specimens coming from different seizures. This information can be used by law enforcement personnel to visualize the ecstasy distribution network as well as the clandestine tablet manufacturing.