Identification of novel "pathologs" (human disease-related gene candidates) from the RIKEN full-length mouse cDNA data set

The majority of common diseases such as cancer, allergy, diabetes, or heart disease are characterized by complex genetic traits, in which genetic and environmental components contribute to disease susceptibility. Our knowledge of the genetic factors underlying most of such diseases is limited. A major goal in the post-genomic era is to identify and characterize disease susceptibility genes and to use this knowledge for disease treatment and prevention. More than 500 genes are conserved across the invertebrate and vertebrate genomes. Because of gene conservation, various organisms including yeast, fruitfly, zebrafish, rat, and mouse have been used as genetic models.

Identification of a minimal promoter sequence for the human N-acetyltransferase Type I gene that binds AP-1 (activator protein 1) and YY-1 (Yin and Yang 1)

Human N-acetyltransferase Type I (NAT1) catalyses the acetylation of many aromatic amine and hydrazine compounds and it has been implicated in the catabolism of folic acid. The enzyme is widely expressed in the body, although there are considerable differences in the level of activity between tissues. A search of the mRNA databases revealed the presence of several NAT1 transcripts in human tissue that appear to be derived from different promoters. Because little is known about NAT1 gene regulation, the present study was undertaken to characterize one of the putative promoter sequences of the NAT1 gene located just upstream of the coding region. We show with reverse-transcriptase PCR that mRNA transcribed from this promoter (Promoter 1) is present in a variety of human cell-lines, but not in quiescent peripheral blood mononuclear cells. Using deletion mutant constructs, we identified a 20 bp sequence located 245 bases upstream of the translation start site which was sufficient for basal NAT1 expression. It comprised an AP-1 (activator protein 1)-binding site, flanked on either side by a TCATT motif. Mutational analysis showed that the AP-1 site and the 3' TCATT sequence were necessary for gene expression, whereas the 5' TCATT appeared to attenuate promoter activity. Electromobility shift assays revealed two specific bands made up by complexes of c-Fos/Fra, c-Jun, YY-1 (Yin and Yang 1) and possibly Oct-1. PMA treatment enhanced expression from the NAT1 promoter via the AP-1-binding site. Furthermore, in peripheral blood mononuclear cells, PMA increased endogenous NAT1 activity and induced mRNA expression from Promoter I, suggesting that it is functional in vivo.

Expression analysis of putative vitellogenin and lipophorin receptors in honey bee (Apis mellifera L.) queens and workers

Two members of the low density lipoprotein receptor (LDLR) family were identified as putative orthologs for a vitellogenin receptor (Amvgr) and a lipophorin receptor (Amlpr) in the Apis mellifera genome. Both receptor sequences have the structural motifs characteristic of LDLR family members and show a high degree of similarity with sequences of other insects. RT-PCR analysis of Amvgr and Amlpr expression detected the presence of both transcripts in different tissues of adult female (ovary, fat body, midgut, head and specifically hypopharyngeal gland), as well as in embryos. In the head RNA samples we found two variant forms of AmLpR: a full length one and a shorter one lacking 29 amino acids in the O-linked sugar domain. In ovaries the expression levels of the two honey bee LDLR members showed opposing trends: whereas Amvgr expression was upregulated as the ovaries became activated, Amlpr transcript levels gradually declined. In situ hybridization analysis performed on ovaries detected Amvgr mRNA exclusively in germ line cells and corroborated the qPCR results showing an increase in Amvgr gene expression concomitant with follicle growth. (C) 2008 Elsevier Ltd. All rights reserved.

Sequence analysis, chromosomal distribution and long-range organization show that rapid turnover of new and old pBuM satellite DNA repeats leads to different patterns of variation in seven species of the Drosophila buzzatii cluster

We aimed to study patterns of variation and factors influencing the evolutionary dynamics of a satellite DNA, pBuM, in all seven Drosophila species from the buzzatii cluster (repleta group). We analyzed 117 alpha pBuM-1 (monomer length 190 bp) and 119 composite alpha/beta (370 bp) pBuM-2 repeats and determined the chromosome location and long-range organization on DNA fibers of major sequence variants. Such combined methodologies in the study of satDNAs have been used in very few organisms. In most species, concerted evolution is linked to high copy number of pBuM repeats. Species presenting low-abundance and scattered distributed pBuM repeats did not undergo concerted evolution and maintained part of the ancestral inter-repeat variability. The alpha and alpha/beta repeats colocalized in heterochromatic regions and were distributed on multiple chromosomes, with notable differences between species. High-resolution FISH revealed array sizes of a few kilobases to over 0.7 Mb and mutual arrangements of alpha and alpha/beta repeats along the same DNA fibers, but with considerable changes in the amount of each variant across species. From sequence, chromosomal and phylogenetic data, we could infer that homogenization and amplification events involved both new and ancestral pBuM variants. Altogether, the data on the structure and organization of the pBuM satDNA give insights into genome evolution including mechanisms that contribute to concerted evolution and diversification.

Colonization sequence of non-pollinating fig wasps associated with Ficus citrifolia in Brazil

Mutualisms, such as the fig-agaonid wasp association, are susceptible to colonization by parasitic species, which exploit the resources involved therein. In most cases, they oviposit into the figs from outside without providing any pollination service. In this study, we used several different methods (adhesive traps and direct standardized field observations) to assess the colonization sequence of a diverse fig wasp fauna associated with Ficus citrifolia, section Americana, in Brazil. They consistently showed a temporal partitioning in colonization among non-pollinating fig wasp species. Idarnes species belonging to the flavicollis and incerta groups colonized figs just before or during the fig receptive phase. In contrast, Idarnes females belonging to the carme group oviposited one to three weeks later, mainly in the middle of the inter-floral phase. Eurytoma, Heterandrium, Physothorax and Torymus were later colonizers, and laid eggs either in the middle or during the late inter-floral phase. The results suggest that these Neotropical fig wasps have different strategies of resource exploitation, even among species belonging to the same genus.

cDNA microarray analysis of differentially expressed genes in penile tissue after treatment with tadalafil

To evaluate differential gene expression in penile tissue after treatment with the phosphodiesterase 5 (PDE5) inhibitor tadalafil, as of the three clinically available PDE5 inhibitors (sildenafil, tadalafil, and vardenafil) used for the treatment of erectile dysfunction (ED), tadalafil has a long half-life and low incidence of side-effects. In all, 32 adult rats were divided into two groups. The control group received 0.5 mL of drinking water alone, while the tadalafil group was treated with tadalafil at a dose of 0.27 mg/kg. At 4 h after treatment with water or tadalafil the rats were killed and the penile tissue was removed. The total RNA was isolated from the penile tissue from both groups and differentially expressed genes were identified by cDNA microarray analysis. To validate the expression data from the microarray analysis, quantitative real-time polymerase chain reaction (PCR) and immunohistochemistry were used. In all, 153 genes were differentially expressed between the control group and the tadalafil group. We validated the microarray results by quantitative PCR for the insulin-like growth factor binding protein 6 (IGFBP-6) gene and the neuronal calcium sensor 1 (NCS-1) gene, both of which were up-regulated in the tadalafil group, and for the natriuretic peptide receptor 1 (NPR-1) gene that was down-regulated in this group. Immunohistochemistry showed localization of the NCS-1 protein in sinusoid trabeculae of the corpus cavernosum in control and tadalafil-treated rats. There was differential expression in 153 genes after tadalafil treatment. Some of these genes such as IGFBP-6, NPR-1 and NCS-1, might result in new targets in the treatment of ED.

The p.A2215D Thyroglobulin Gene Mutation Leads to Deficient Synthesis and Secretion of the Mutated Protein and Congenital Hypothyroidism with Wide Phenotype Variation

Context: Thyroglobulin (TG) is a large glycoprotein and functions as a matrix for thyroid hormone synthesis. TG gene mutations give rise to goitrous congenital hypothyroidism (CH) with considerable phenotype variation. Objectives: The aim of the study was to report the genetic screening of 15 patients with CH due to TG gene mutations and to perform functional analysis of the p. A2215D mutation. Design: Clinical evaluation and DNA sequencing of the TG gene were performed in all patients. TG expression was analyzed in the goitrous tissue of one patient. Human cells were transfected with expression vectors containing mutated and wild-type human TG cDNA. Results: All patients had an absent rise of serum TG after stimulation with recombinant human TSH. Sequence analysis revealed three previously described mutations (p. A2215D, p. R277X, and g. IVS30 + 1G > T), and two novel mutations (p. Q2142X and g. IVS46-1G > A). Two known (g. IVS30 + 1G/p. A2215D and p. A2215D/p. R277X) and one novel (p. R277X/g. IVS46-1G > A) compound heterozygous constellations were also identified. Functional analysis indicated deficiency in TG synthesis, reduction of TG secretion, and retention of the mutant TG within the cell, leading to an endoplasmic reticulum storage disease, whereas small amounts of mutant TG were still secreted within the cell system. Conclusion: All studied patients were either homozygous or heterozygous for TG gene mutations. Two novel mutations have been detected, and we show that TG mutation p. A2215D promotes the retention of TG within the endoplasmic reticulum and reduces TG synthesis and secretion, causing mild hypothyroidism. In the presence of sufficient iodine supply, some patients with TG mutations are able to compensate the impaired hormonogenesis and generate thyroid hormone. (J Clin Endocrinol Metab 94: 2938-2944, 2009)

Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium - Serum-free growth of IGF-I producing CHO cells

Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/10(6) cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.

Transient expression of a novel serine protease in the ectoderm of the ascidian Herdmania momus during development

We have studied gene expression during ascidian embryonic development using the technique of differential display and isolated partial cDNA sequences of 12 genes. Developmental regulation of these genes has been confirmed by northern hybridization analysis. Further cDNA cloning and sequence analysis of an mRNA that is present during gastrulation, neurulation and tailbud formation reveals that it encodes a novel serine protease containing a single kringle motif and catalytic domain. The spatial expression of this gene, designated Hmserp1, is restricted to precursor cells of the epidermis. The structure and expression of Hmsery1 is discussed in relation to possible functions during development.

A Mox homeobox gene in the gastropod mollusc Haliotis rufescens is differentially expressed during larval morphogenesis and metamorphosis

We have isolated a homeobox-containing cDNA from the gastropod mollusc Haliotis rufescens that is most similar to members of the Mox homeobox gene class, The derived Haliotis homeodomain sequence is 85% identical to mouse and frog Mox-2 homeodomains and 88.9% identical to the partial cnidarian cnox5-Hm homeodomain. Quantitative reverse transcription-polymerase chain reaction analysis of mRNA accumulation reveals that this gene, called HruMox, is expressed in the larva, but not in the early embryo, Transcripts are most prevalent during larval morphogenesis from trochophore to veliger. There are also transient increases in transcript prevalence 1 and 3 days after the intitiation of metamorphosis from veliger to juvenile. The identification of a molluscan Mox homeobox gene that is more closely related to vertebrate genes than other protostome (e.g. Drosophila) genes suggests the Mox class of homeobox genes may consist of several different families that have been conserved through evolution, (C) 1997 Federation of European Biochemical Societies.

Differential expression of mitochondrial NADH dehydrogenase in ethanol-treated rat brain: Revealed by differential display

The technique of polymerase chain reaction (PCR) differential display was used to detect alterations in gene expression after chronic alcohol administration. Male Wistar rats were treated with ethanol vapor for 14 days. The cDNA generated from mRNA isolated from the hippocampi of ethanol-treated and control animals was compared by PCR differential display. A differentially expressed cDNA fragment was used to screen mRNA samples by Northern analysis. The level of a mRNA was significantly elevated (x 2.5) in the hippocampus, but not the cortex of alcohol-treated rats up to 48 hr after withdrawal. Sequence analysis of the cDNA fragment revealed an almost perfect homology to rat mitochondrial NADH dehydrogenase subunit 4 mRNA. The selective induction of this mRNA in alcohol-treated rat brain areas suggests altered metabolic processes and possible dysfunction of the mitochondria. The technique of PCR differential display may prove useful in further analysis of gene expression during alcohol dependence and withdrawal.

Application of inter simple sequence repeat (ISSR) markers to plant genetics

Microsatellites or simple sequence repeats (SSRs) are ubiquitous in eukaryotic genomes. Single-locus SSR markers have been developed for a number of species, although there is a major bottleneck in developing SSR markers whereby flanking sequences must be known to design 5'-anchors for polymerase chain reaction (PCR) primers. Inter SSR (ISSR) fingerprinting was developed such that no sequence knowledge was required. Primers based on a repeat sequence, such as (CA)(n), can be made with a degenerate 3'-anchor, such as (CA)(8)RG or (AGC)(6)TY. The resultant PCR reaction amplifies the sequence between two SSRs, yielding a multilocus marker system useful for fingerprinting, diversity analysis and genome mapping. PCR products are radiolabelled with P-32 or P-33 via end-labelling or PCR incorporation, and separated on a polyacrylamide sequencing gel prior to autoradiographic visualisation. A typical reaction yields 20-100 bands per lane depending on the species and primer. We have used ISSR fingerprinting in a number of plant species, and report here some results on two important tropical species, sorghum and banana. Previous investigators have demonstrated that ISSR analysis usually detects a higher level of polymorphism than that detected with restriction fragment length polymorphism (RFLP) or random amplified polymorphic DNA (RAPD) analyses. Our data indicate that this is not a result of greater polymorphism genetically, but rather technical reasons related to the detection methodology used for ISSR analysis.

Relative Bioavailability of Two Oral Formulations of Risperidone 2 mg: A Single-Dose, Randomized-Sequence, Open-Label, Two-Period Crossover Comparison in Healthy Brazilian Volunteers

Background: Risperidone (RSP) is a benzisoxazole antipsychotic agent used to treat schizophrenia and other psychiatric illnesses in adults and children (including those with autism). After oral administration, RSP is completely absorbed from the gastrointestinal tract and undergoes hydroxylation to yield 9-hydroxyrisperidone (9-OH-RSP), an active metabolite that has a pharmacologic profile and potency similar to RSP. Objectives: The aims of this study were to compare the relative bioavailability of a pharmaceutical-equivalent (test) formulation with a reference formulation of oral RSP 2 mg, both available commercially on the Brazilian pharmaceutical market, and to generate data regarding the oral bioavailability of the tested drug in healthy Brazilian volunteers. Methods: This single-dose, randomized-sequence, open-label, 2-period crossover study was conducted in healthy Brazilian volunteers from August to December 2008. Subjects were randomly assigned to receive the test formulation followed by the reference formulation or vice versa, with a 30-day washout period between doses. Study drugs were administered after a 12-hour overnight fast. For pharmacokinetic analysis, blood samples were drawn at 0 (baseline), 0.25, 0.5, 1, 1.5, 3, 5, 8, 12, 24, 48, 72, 96, and 120 hours after administration. Plasma concentrations of RSP and 9-OH-RSP were determined using LC-MS/MS. The test and reference formulations were to be considered bioequivalent if the 90% CIs for the geometric mean test/reference ratios were within a predetermined range of 80% to 125%, in accordance with the policies of the Brazilian Sanitary Surveillance Agency and the US Food and Drug Administration. Tolerability was determined using clinical assessments, monitoring of vital signs, analysis of laboratory test results, and subject interviews regarding adverse events. Results: A total of 22 subjects were enrolled (11 men, 11 women; mean [SD] age, 32 [12] years [range, 18-58 years]; weight, 70.4 [11.9] kg [range, 50-103 kg]; height, 1.67 [0.08] m [range, 1.56-1.80 m]; and body mass index, 25 [4] kg/m(2) [range, 18-29 kg/m(2)]). For RSP, mean (SD) C(max) values were 12.6 (2.7) and 16.0 (2.3) ng/mL for the test and reference formulations, respectively. For 9-OH-RSP, mean C(max) values were 17.8 (1.3) and 21.0 (1.7) ng/mL for the test and reference formulations. The 90% CIs for the mean test/reference ratios for RSP C(max), AUC(0-120), and AUC(0-infinity) were 74% to 82%, 75% to 85%, and 76% to 85%, respectively, and 83% to 87%, 75% to 79%, and 75% to 78% for 9-OH-RSP. The related adverse events (headache, low back pain, drowsiness, standing hypotension, local postvenipuncture ecchymoses, insomnia, nausea, and vomiting) were transient and mild. Conclusions: This single-dose study found that the test and reference formulations of oral RSP 2 mg did not meet the Brazilian and US regulatory criteria for bioequivalence in these fasting, healthy volunteers. The study formulations appeared to be well tolerated. (Clin Ther 2010;32:2106-2115) (C) 2010 Elsevier HS Journals, Inc.