Cloning, genomic structure, and expression profiles of TULIP1 (GARNL1), a brain-expressed candidate gene for 14q13-linked neurological phenotypes, and its murine homologue

Previously, we have described the clinical and molecular characterization of a de novo 14q13.1-q21.1 microdeletion, less than 3.5 Mb in size, in a patient with severe microcephaly, psychomotor retardation, and other clinical anomalies. Here we report the characterization of the genomic structure of the human tuberin-like protein gene 1 (TULIP1; approved gene symbol GARNL1), a CpGisland-associated, brain-expressed candidate gene for the neurological findings in our patient, and its murine homologue. The human TULIP1 gene was mapped to chromosome band 14q13.2 by fluorescence in situ hybridization of BAC clone RP11-355C3 (GenBank Accession No. AL160231), containing the 3' region of the gene. TULIP1 spans about 271 kb of human genomic DNA and is divided into 41 exons. An untranscribed, processed pseudogene of TULIP1 was found on human chromosome band 9q31.1. The active locus TULIP1, encoding a predicted protein of 2036 amino acids, is expressed ubiquitously in pre- and postnatal human tissues. The murine homologue Tulip1 spans about 220 kb of mouse genomic DNA and is also divided into 41 exons, encoding a predicted protein of 2035 amino acids. No pseudogene could be found in the available mouse sequence data. Several splicing variants were found. Considering the location, expression profile, and predicted function, TULIP1 is a strong candidate for several neurological features seen in 14q deletion patients. Additionally we searched for mutations in the coding region of TULIP1 in subjects from a family with idiopathic basal ganglia calcification (IBGC; Fahr disease), previously linked to chromosome 14q. We identified two novel SNPs in the intron-exon boundaries; however, they did not segregate only with affected subjects in the predicted model of an autosomal dominant disease such as IBGC.     

Antifouling potential of lubricious, micro-engineered, PDMS elastomers against zoospores of the green fouling alga Ulva (Enteromorpha)

The settlement and release of Ulva spores from chemically modified, micro-engineered surface topographies have been investigated using poly(dimethyl siloxane) elastomers (PDMSe) with varying additions of non-network forming poly(dimethyl siloxane) based oils. The topographic features were based on 5 microns wide pillars or ridges separated by 5, 10, or 20 microns wide channels. Pattern depths were 5 or 1.5 microns. Swimming spores showed no marked difference in settlement on smooth surfaces covered with excess PDMS oils. However, incorporation of oils significantly reduced settlement density on many of the surfaces with topographic features, in particular, the 5 microns wide and deep channels. Previous results, confirmed here, demonstrate preferences by the spores to settle in channels and against pillars with spatial dimensions of 5 microns, 10 microns and 20 microns. The combination of lubricity and pillars significantly reduced the number of attached spores compared to the control, smooth, unmodified PDMSe surfaces when exposed to turbulent flow in a flow channel. The results are discussed in relation to the energy needs for spores to adhere to various surface features and the concepts of ultrahydrophobic surfaces. A factorial, multi-level experimental design was analyzed and a 2nd order polynomial model was regressed for statistically significant effects and interactions to determine the magnitude and direction of influence on the spore density measurements between factor levels.     

Aminomethylpyrimidines as novel DPP-IV inhibitors: a 10(5)-fold activity increase by optimization of aromatic substituents

The influence of aromatic substitution on a newly discovered class of inhibitors of dipeptidyl peptidase IV was investigated. A 10(5)-fold increase in potency was achieved by the optimization of aromatic substituents in a parallel chemistry program. The observed SAR could be explained by an X-ray structure of the protein-ligand complex.     

APOBEC3F properties and hypermutation preferences indicate activity against HIV-1 in vivo

APOBEC3G (CEM15 ) deaminates cytosine to uracil in nascent retroviral cDNA. The potency of this cellular defense is evidenced by a dramatic reduction in viral infectivity and the occurrence of high frequencies of retroviral genomic-strand G --> A transition mutations. The overwhelming dinucleotide hypermutation preference of APOBEC3G acting upon a variety of model retroviral substrates is 5'-GG --> -AG. However, a distinct 5'-GA --> -AA bias, which is difficult to attribute to APOBEC3G alone, prevails in HIV-1 sequences derived from infected individuals (e.g., ). Here, we show that APOBEC3F is also a potent retroviral restrictor but that its activity, unlike that of APOBEC3G, is partially resistant to HIV-1 Vif and results in a clear 5'-GA --> -AA retroviral hypermutation preference. This bias is also apparent in a bacterial mutation assay, suggesting that it is an intrinsic APOBEC3F property. Moreover, APOBEC3F and APOBEC3G appear to be coordinately expressed in a wide range of human tissues and are independently able to inhibit retroviral infection. Thus, APOBEC3F and APOBEC3G are likely to function alongside one another in the provision of an innate immune defense, with APOBEC3F functioning as the major contributor to HIV-1 hypermutation in vivo.     

No evidence for DUP25 in patients with panic disorder using a quantitative real-time PCR approach

A duplication of chromosome 15q24-q26 (DUP25) has been reported to be associated with anxiety disorders. We tested for the presence of DUP25 in a sample of 50 patients with panic disorder and 50 controls using a quantitative real-time PCR approach. Contrary to the original finding, our results were compatible with the absence of DUP25, and no significant difference could be detected between patients and controls (P=1.0). Thus, our study does not support the hypothesis of an involvement of DUP25 in panic disorder.     

2-Phenylimidazo[2,1-i]purin-5-ones: structure-activity relationships and characterization of potent and selective inverse agonists at Human A3 adenosine receptors

Structure-activity relationships of 2-phenyl-imidazo[2,1-i]purin-5-ones as ligands for human A(3) adenosine receptors (ARs) were investigated. An ethyl group in the 8-position of the imidazoline ring of 4-methyl-2-phenyl-imidazopurinone leading to chiral compounds was found to increase affinity for human A(3) ARs by several thousand-fold. Propyl substitution instead of methyl at N4 decreased A(3) affinity but increased A(1) affinity leading to potent A(1)-selective AR antagonists. The most potent A(1) antagonist of the present series was (S)-8-ethyl-2-phenyl-4-propyl-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one (S-3) exhibiting a K(i) value of 7.4 nM at rat A(1) ARs and greater than 100-fold selectivity versus rat A(2A) and human A(3) ARs. At human A(1) ARs 2-phenylimidazo[2,1-i]purin-5-ones were generally less potent and therefore less A(1)-selective (S-3: K(i)=98 nM). 2-, 3-, or 4-Mono-chlorination of the 2-phenyl ring reduced A(3) affinity but led to an increase in affinity for A(1) ARs, whereas di- (3,4-dichloro) or polychlorination (2,3,5-trichloro) increased A(3) affinity. The most potent and selective A(3) antagonist of the present series was the trichlorophenyl derivative (R)-8-ethyl-4-methyl-2-(2,3,5-trichlorophenyl)-4,5,7,8-tetrahydro-1H-imidazo[2,1-i]purin-5-one (R-8) exhibiting a subnanomolar K(i) value at human A(3) ARs and greater than 800-fold selectivity versus the other AR subtypes. Methylation of 4-alkyl-2-phenyl-substituted imidazo[2,1-i]purin-5-ones led exclusively to the N9-methyl derivatives, which exhibited largely reduced AR affinities as compared to the unmethylated compounds. [35S]GTP gamma S binding studies of the most potent 2-phenyl-imidazo[2,1-i]purin-5-ones at membranes of Chinese hamster ovary cells expressing the human A(3) AR revealed that the compounds were inverse agonists at A(3) receptors under standard test conditions. Due to their high A(3) affinity, selectivity, and relatively high water-solubility, 2-phenyl-imidazo[2,1-i]purin-5-ones may become useful research tools.     

Effects of injury and progesterone treatment on progesterone receptor and progesterone binding protein 25-Dx expression in the rat spinal cord

Progesterone provides neuroprotection after spinal cord injury, but the molecular mechanisms involved in this effect are not completely understood. In this work, expression of two binding proteins for progesterone was studied in intact and injured rat spinal cord: the classical intracellular progesterone receptor (PR) and 25-Dx, a recently discovered progesterone membrane binding site. RT-PCR was employed to determine their relative mRNA levels, whereas cellular localization and relative protein levels were investigated by immunocytochemistry. We observed that spinal cord PR mRNA was not up-regulated by estrogen in contrast to what is observed in many brain areas and in the uterus, but was abundant as it amounted to a third of that measured in the estradiol-stimulated uterus. In male rats with complete spinal cord transection, levels of PR mRNA were significantly decreased, while those of 25-Dx mRNA remained unchanged with respect to control animals. When spinal cord-injured animals received progesterone treatment during 72 h, PR mRNA levels were not affected and remained low, whereas 25-Dx mRNA levels were significantly increased. Immunostaining of PR showed its intracellular localization in both neurons and glial cells, whereas 25-Dx immunoreactivity was localized to cell membranes of dorsal horn and central canal neurons. As the two binding proteins for progesterone differ with respect to their response to lesion, their regulation by progesterone, their cellular and subcellular localizations, their functions may differ under normal and pathological conditions. These observations point to a novel and potentially important role of the progesterone binding protein 25-Dx after injury of the nervous system and suggest that the neuroprotective effects of progesterone may not necessarily be mediated by the classical progesterone receptor but may involve distinct membrane binding sites.     

Regional and local population structure of the pioneer wood-decay fungus Trichaptum abietinum

The population structure of 11 Fennoscandian geographic populations of the pioneer wood-decay basidiomycete Trichaptum abietinum was assessed with PCR-RFLPs, intersequence simple repeats (ISSRs) and mating studies. The three codominant PCR-RFLP markers (1) internal transcribed spacer 2 (nrDNA), (2) glyceraldehyde-3-phosphate dehydrogenase and (3) translation elongation factor 1α showed that genotype distributions in most cases (94%) agreed with Hardy-Weinberg expectations and that random association of alleles occurred across loci. The molecular data suggest that T. abietinum is a highly outcrossing fungus that regularly proliferates and spreads by sexual spores. Interstock mating reactions suggest a high number of mating factors among individuals and that biological barriers to gene flow are nonexistent in the region. The three PCR-RFLP loci gave an overall F(ST) = 0.03, indicating a low level of genetic differentiation and presumably high gene flow among the geographic populations. The ISSR markers revealed no systematic substructuring and the among-population variance component was low (6.1%) in AMOVA. However, all PCR-RFLP and most ISSR markers (7/12) showed significant deviation from the null hypothesis of an even distribution of allele frequencies across the 11 geographic populations. Allele frequencies varied in an apparently random manner, suggesting that genetic drift might be an important structuring factor in T. abietinum. The spatial small-scale distribution of heterokaryons on three selected substrate units (logs) showed that most isolates represented discrete individuals and that a number of genets (19) may occupy a single log. The small-scale genotype distributions (within logs) were in agreement with panmictic Hardy-Weinberg expectations.