Molecular cloning and characterization of a novel mRNA present in the squid giant axon

Previously, we reported the presence of a heterogeneous population of mRNAs in the squid giant axon. The construction of a cDNA library to this mRNA population has facilitated the identification of several of the constituent mRNAs which encode several cytoskeletal and motor proteins as well as enolase, a glycolytic enzyme. In this communication, we report the isolation of a novel mRNA species (pA6) from the axonal cDNA library. The pA6 mRNA is relatively small (550 nucleotides in length) and is expressed in both nervous tissue and skeletal muscle. The axonal localization of pA6 mRNA was unequivocally established by in situ hybridization histochemistry. The results of quantitative RT-PCR analysis indicate that there are 1.8 x 10(6) molecules of pA6 mRNA (approximately 0.45 pg) in the analyzed segment of the giant axon and suggest that the level of pA6 mRNA in the axonal domain of the giant fiber system might be equal to or greater than the level present in the parental cell soma. Sequence analysis of pA6 suggests that the mRNA encodes an integral membrane protein comprising 84 amino acids. The putative protein contains a single transmembrane domain located in the middle of the molecule and a phosphate-binding loop situated near the N terminus. The C-terminal region of the protein contains two potential phosphorylation sites. These four structural motifs manifest striking similarity to domains present in the ryanodine receptor, raising the possibility that pA6 represents a cephalopod intracellular calcium release channel protein.     

Enteropathogenic E. coli (EPEC) transfers its receptor for intimate adherence into mammalian cells

Enteropathogenic E. coli (EPEC) belongs to a group of bacterial pathogens that induce epithelial cell actin rearrangements resulting in pedestal formation beneath adherent bacteria. This requires the secretion of specific virulence proteins needed for signal transduction and intimate adherence. EPEC interaction induces tyrosine phosphorylation of a protein in the host membrane, Hp90, which is the receptor for the EPEC outer membrane protein, intimin. Hp90-intimin interaction is essential for intimate attachment and pedestal formation. Here, we demonstrate that Hp90 is actually a bacterial protein (Tir). Thus, this bacterial pathogen inserts its own receptor into mammalian cell surfaces, to which it then adheres to trigger additional host signaling events and actin nucleation. It is also tyrosine-phosphorylated upon transfer into the host cell.     

Hepatic lacI and cII mutation in transgenic (lambdaLIZ) rats treated with dimethylnitrosamine

The recent introduction of a transgenic rat in vivo mutation assay is a much needed supplement to the transgenic mouse models and offers the tools necessary for collecting target tissue specific genotoxicity data in this species. The utility of the Big Blue(R) rat for the detection of in vivo mutations was investigated by studying spontaneous and dimethylnitrosamine (DMN)-induced hepatic mutations. High molecular weight DNA isolated from Big Blue(R) rat livers typically yielded good transgene rescue efficiency of up to 5x105 plaque forming units per packaging reaction. DMN, when administered by oral gavage at dose levels of 0.2, 0.6, 2.0, and 6.0 mg kg-1 day-1, induced up to a 4.5-fold increase in mutations at the highest dose level. There was no apparent difference between the lacI vs. cII target genes of the shuttle vector in either the background or DMN-induced mutant frequencies. These results suggest that the transgenic rat model is a useful tool for studying potential genotoxicity in target organs and, with further validation, the selectable cII target could be an attractive alternative to the conventional lacI color screening method for the detection of mutations in the lambdaLIZ shuttle vector.     

Association between uncoupling protein polymorphisms (UCP2-UCP3) and energy metabolism/obesity in Pima indians

The UCP2-UCP3 gene cluster maps to chromosome 11q13 in humans, and polymorphisms in these genes may contribute to obesity through effects on energy metabolism. DNA sequencing of UCP2 and UCP3 revealed three polymorphisms informative for association studies: an Ala-->Val substitution in exon 4 of UCP2, a 45 bp insertion/deletion in the 3'-untranslated region of exon 8 of UCP2 and a C-->T silent polymorphism in exon 3 of UCP3. Initially, 82 young (mean age = 30 +/- 7 years), unrelated, full-blooded, non-diabetic Pima Indians were typed for these polymorphisms by direct sequencing. The three sites were in linkage disequilibrium ( P < 0.00001). The UCP2 variants were associated with metabolic rate during sleep (exon 4, P = 0.007; exon 8, P = 0.016) and over 24 h (exon 8, P = 0.038). Heterozygotes for UCP2 variants had higher metabolic rates than homozygotes. The UCP3 variant was not significantly associated with metabolic rate or obesity. In a further 790 full-blooded Pima Indians, there was no significant association between the insertion/deletion polymorphism and body mass index (BMI). However, when only individuals >45 years of age were considered, heterozygotes (subjects with the highest sleeping metabolic rate) had the lowest BMI (P = 0.04). The location of the insertion/deletion polymorphism suggested a role in mRNA stability; however, it appeared to have no effect on skeletal muscle UCP2 mRNA levels in a subset of 23 randomly chosen Pima Indians. In conclusion, these results suggest a contribution from UCP2 (or UCP3) to variation in metabolic rate in young Pima Indians which may contribute to overall body fat content later in life.     

Increased expression of NGFI-A mRNA in the rat striatum following burst stimulation of the medial forebrain bundle

Using in situ hybridization, we examined the mRNA expression for several immediate early genes in dopamine-innervated brain areas following electrical burst vs. regular stimulation of the medial forebrain bundle in anaesthetized rats. Two hours after 5 Hz burst stimulation, the expression of the nerve growth factor-inducible clone A (NGFI-A) mRNA was increased in the medial part of the striatum. This increase was prevented by pretreatment with the dopamine-D1 receptor antagonist, SCH23390 (0.1 mg/kg i.p.). After 8 Hz burst stimulation, NGFI-A mRNA expression was increased in the medial, central and lateral parts of the striatum. Induction occurred predominantly in cells expressing mRNAs for the dopamine-D1 receptor, substance P and dopamine and cAMP-regulated phosphoprotein (DARP-32). Regular stimulation had no effect on NGFI-A mRNA expression. The induction of NGFI-A was related to the levels of dopamine released by burst or regular stimulation as demonstrated with in vivo amperometry. Two hours after stimulation, the expression of none of the other genes studied was altered. One hour after 8 Hz burst stimulation, the expression of NGFI-A, NGFI-B and jun-B mRNAs was increased in the striatum and that of NGFI-A, NGFI-B, c-fos, fos-B and jun-B mRNAs was variably increased in the nucleus accumbens and lateral septum. These results provide additional support for the physiological importance of burst firing activity in midbrain dopamine neurons for the activation of their target cells. They demonstrate a spatial and temporal specificity as regards the brain region, the gene activated, the receptor involved and the phenotype of the cells affected.     

Maternal smoking during pregnancy and the risk of conduct disorder in boys

(BACKGROUND): We studied the clinical efficacy of transurethral microwave thermotherapy (TUMT) using Endotherm UMW system (OLYMPUS). (METHODS): TUMT was performed in 28 patients with benign prostatic hyperplasia (BPH). Three patients of them were catheterized because of urinary retention. The treatment was performed in a single session for an hour. The urethral surface temperature was set at 39 degrees C, and the coolant flow of the urethral applicator (21 Fr balloon catheter) was set at 30 ml/min, to heat up the broad area of the prostate up to 45 degrees C. The clinical efficacy was evaluated by analyzing subjective responses, using the International Prostate Symptom Score (I-PSS) scale (S) and QOL score (L), and objective responses, using peak urinary flow rate (Qmax), average flow rate (Qave), residual urine volume and prostate volume following the treatment. (RESULTS): At 24 weeks after the treatment, significant improvement were observed in S score (41%), L score (37%), Qmax (53%) and Qave (62%). Although there was no significant decrease in residual urine and prostate volume. The three patients, with a catheter indwelled because of urinary retention, were all free of the catheter within 4 weeks after the treatment. During and after the treatment, no severe adverse effects, including transient urinary retention needed for indwelling a catheter, was detected. (CONCLUSION): A single session of TUMT by Endotherm UMW considered to be safe and useful for symptomatic BPH patients, even who are not indicated for transurethral resection of the prostate (TUR-P) because of underlying disorders.