Axonal targeting of the 5-HT1B serotonin receptor relies on structure-specific constitutive activation

By analogy to other axonal proteins, transcytotic delivery following spontaneous endocytosis from the somatodendritic membrane is expected to be essential for polarized distribution of axonal G-protein coupled receptors (GPCRs). However, possible contribution from constitutive activation, which may also result in constitutive GPCR endocytosis, is poorly known. Using two closely related but differentially distributed serotonin receptors, here we demonstrate higher constitutive activation and spontaneous endocytosis for the axonal 5-HT(1B) R, as compared to the somatodendritic 5-HT(1A) R, both in non-neuronal cells and neurons. Activation-dependent constitutive endocytosis is crucial for axonal targeting, because inverse-agonist treatment, which prevents constitutive activation, leads to atypical accumulation of newly synthesized 5-HT(1B) Rs on the somatodendritic plasma membrane. Using receptor chimeras composed of different domains from 5-HT(1A) R and 5-HT(1B) R, we show that the complete third intracellular loop of 5-HT(1B) R is necessary and sufficient for constitutive activation and efficient axonal targeting, both sensitive to inverse-agonist treatment. These results suggest that activation and targeting of 5-HT(1B) Rs are intimately interconnected in neurons.     

DNA methylation changes associated with risk factors in tumors of the upper aerodigestive tract

Cancers of the upper aerodigestive tract (UADT) are common forms of malignancy associated with tobacco and alcohol exposures, although human papillomavirus and nutritional deficiency are also important risk factors. While somatically acquired DNA methylation changes have been associated with UADT cancers, what triggers these events and precise epigenetic targets are poorly understood. In this study, we applied quantitative profiling of DNA methylation states in a panel of cancer-associated genes to a case-control study of UADT cancers. Our analyses revealed a high frequency of aberrant hypermethylation of several genes, including MYOD1, CHRNA3 and MTHFR in UADT tumors, whereas CDKN2A was moderately hypermethylated. Among differentially methylated genes, we identified a new gene (the nicotinic acetycholine receptor gene) as target of aberrant hypermethylation in UADT cancers, suggesting that epigenetic deregulation of nicotinic acetycholine receptors in non-neuronal tissues may promote the development of UADT cancers. Importantly, we found that sex and age is strongly associated with the methylation states, whereas tobacco smoking and alcohol intake may also influence the methylation levels in specific genes. This study identifies aberrant DNA methylation patterns in UADT cancers and suggests a potential mechanism by which environmental factors may deregulate key cellular genes involved in tumor suppression and contribute to UADT cancers.Key words: DNA methylation, upper aerodigestive tract, cancer, risk factors, biomarkers     

Effects of intravenous infusion of prostacyclin (PGI2) in man

Prostacyclin infused intravenously in human volunteers induces ex vivo inhibition of platelet aggregation, tachycardia and hypotension. The inhibition of platelet aggregation is obtained with slightly lower doses than those which exhibit cardiovascular effects.The cardiovascular effects disappeared within a few minutes after discontinuing the infusion of prostacyclin but the platelet effects were longer lasting.Prostacyclin did not have any effect on platelet count, platelet factor 3, accelerated partial thromboplastin time, prothrombin time, euglobulin clot lysis time, fibrinogen degradation products, blood glucose concentration or urine sodium potassium ratio.     

Evaluation of the Inheritance of the Complex Vertebral Malformation Syndrome by Breeding Studies

To investigate the congenital complex vertebral malformation syndrome (CVM) in Holstein calves, two breeding studies were performed including 262 and 363 cows, respectively. Cows were selected from the Danish Cattle Database based on pedigree and insemination records. Selected cows were progeny of sires with an established heterozygous CVM genotype and pregnant after insemination with semen from another sire with heterozygous CVM genotype. Following calving the breeders should state, if the calf was normal and was requested to submit dead calves for necropsy. In both studies, significantly fewer CVM affected calves than expected were obtained; a finding probably reflecting extensive intrauterine mortality in CVM affected foetuses. The findings illustrate increased intrauterine mortality as a major potential bias in observational studies of inherited disorders.     

A gamma 2(R43Q) mutation, linked to epilepsy in humans, alters GABAA receptor assembly and modifies subunit composition on the cell surface

Genetic defects leading to epilepsy have been identified in gamma2 GABA(A) receptor subunit. A gamma2(R43Q) substitution is linked to childhood absence epilepsy and febrile seizure, and a gamma2(K289M) mutation is associated with generalized epilepsy with febrile seizures plus. To understand the effect of these mutations, surface targeting of GABA(A) receptors was analyzed by subunit-specific immunofluorescent labeling of living cells. We first transfected hippocampal neurons in culture with recombinant gamma2 constructs and showed that the gamma 2(R43Q) mutation prevented surface expression of the subunit, unlike gamma2(K289M) substitution. Several gamma2-subunit constructs, bearing point mutations within the Arg-43 domain, were expressed in COS-7 cells with alpha3- and beta3-subunits. R43Q and R43A substitutions dramatically reduced surface expression of the gamma2-subunit, whereas R43K, P44A, and D39A substitutions had a lesser, but still significant, impact and K289M substitution had no effect. Whereas the mutant gamma2(R43Q) was retained within intracellular compartments, alphabeta complexes were still targeted at the cell membrane. Coimmunoprecipitation experiments showed that gamma2(R43Q) was able to associate with alpha3- or beta3-subunits, although the stoichiometry of the complex with alpha3 was altered. Our data show that gamma2(R43Q) is not a dominant negative and that the mutation leads to a modification of GABA(A) receptor subunit composition on the cell surface that impairs the synaptic targeting in neurons. This study reveals an involvement of the gamma2-Arg-43 domain in the control of receptor assembly that may be relevant to the effect of the heterozygous gamma2(R43Q) mutation leading to childhood absence epilepsy and febrile seizure.     

Incorporation of 15N from ammonium into the N-linked oligosaccharides of an immunoadhesin glycoprotein expressed in Chinese hamster ovary cells

Elevated ammonium concentrations in the medium of cultivated cells have been shown to increase the intracellular levels of uridine-5'-diphospho- N-acetylglucosamine (UDP-GlcNAc) and uridine-5'-diphospho-N- acetylgalactosamine (UDP-GalNAc; Ryll et al., 1994). These sugar nucleotides are substrates for glycosyltransferases in the glycosylation pathway. In our experiments, recombinant Chinese hamster ovary cells producing an immunoadhesin glycoprotein (GP1-IgG) have been cultivated under controlled cell culture conditions in the presence of different ammonium concentrations.15N-Labeled ammonium chloride (15NH4Cl) was added exogenously to the cell culture media to determine if ammonium was incorporated into UDP-GlcNAc and cytidine-5'- monophospho-N-acetylneuraminic acid (CMP-NANA) pools, and subsequently incorporated into GP1-IgG as N-linked glycans. The intracellular pools of UDP-activated hexosamines (UDP-GNAc) were followed during the time course of the experiment. To assess the extent of15NH4+incorporation into the glycans of GP1-IgG, the glycoprotein was first purified to homogeneity by protein A chromatography. Enzymatically released N- glycans were then analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. N-Glycans synthesized in the presence of15NH4Cl revealed an N-glycan-dependent increase in mass-to-charge of 2.5-4.8 Da. These results indicate that 60-70% of the total nitrogen containing monosaccharides had incorporated15N. Presumably,15NH4+was incorporated into GlcNAc and N- acetylneuraminic acid as proposed earlier (Ryll et al., 1994). This might be a universal and previously not described reaction in mammalian cells when exposed to nonphysiological but in cell culture commonly found concentrations of ammonium. The data presented here are of significance for glycoprotein production in mammalian cell culture, since it has been shown previously that elevated levels of UDP- activated hexosamines affect N-glycan characteristics such as branching and degree of amino sugar incorporation. In addition, our results demonstrate that isotope labeling in combination with MALDI-TOF-MS can be used as an alternate tool to radioactive labeling of sugar substrates in metabolic studies.      

In vivo and in vitro characterization of novel microparticulates based on hyaluronan and chitosan hydroglutamate

This study examined the application of previously characterized microparticles composed of hyaluronan (HA) and chitosan hydroglutamate (CH) as well as novel microparticles consisting of both polymers (HA/CH) to improve the nasal delivery of a model drug. The rabbit bioavailabilities of gentamicin incorporated in HA, CH, and HA/CH microparticles were increased 23-, 31-, and 42-fold, respectively, compared with the control intranasal solution of gentamicin, indicating that all test microparticles were retained for longer periods on the nasal mucosa of the rabbits as supported by previous in vitro dissolution as well as frog palate mucoadhesion studies, thereby improving drug absorption. The higher bioavailabilities of CH-based formulations (CH and HA/CH) suggest the penetration-enhancing effects of CH may also be partially responsible for the improvement. A model was developed, based on a glass impinger device, to deliver dry powder formulations reproducibly onto the surface of cultured cell monolayers. In vitro permeability and fluorescence microscopy studies on the tight junctions of the 16HBE14o- cell lines further confirmed the ability of CH-based formulations to enhance penetration. Furthermore, the in vitro absorption profile from cell culture studies was consistent with those determined from in vivo studies. The complementary effect from the mucoadhesive nature of HA coupled with the penetration-enhancing effects of CH makes the novel HA/CH formulation a promising nasal delivery system.     

Geographic distribution of methyltransferases of Helicobacter pylori: evidence of human host population isolation and migration

Background  

Helicobacter pylori colonizes the human stomach and is associated with gastritis, peptic ulcer, and gastric cancer. This ubiquitous association between H. pylori and humans is thought to be present since the origin of modern humans. The H. pylori genome encodes for an exceptional number of restriction and modifications (R-M) systems. To evaluate if R-M systems are an adequate tool to determine the geographic distribution of H. pylori strains, we typed 221 strains from Africa, America, Asia, and Europe, and evaluated the expression of different 29 methyltransferases.