Clinical Pharmacology & Therapeutics (2007) 81, S10–S12. doi:10.1038/sj.clpt.6100173

The oxytocin receptor gene (OXTR) has been studied in autism because of the role of oxytocin (OT) in social cognition. Linkage has also been demonstrated to the region of OXTR in a large sample. Two single nucleotide polymorphisms (SNPs) and a haplotype constructed from them in OXTR have been associated with autism in the Chinese Han population. We tested whether these associations replicated in a Caucasian sample with strictly defined autistic disorder. We genotyped the two previously associated SNPs (rs2254298, rs53576) in 57 Caucasian autism trios. Probands met clinical, ADI-R, and ADOS criteria for autistic disorder. Significant association was detected at rs2254298 (p = 0.03) but not rs53576. For rs2254298, overtransmission of the G allele to probands with autistic disorder was found which contrasts with the overtransmission of A previously reported in the Chinese Han sample. In both samples, G was more frequent than A. However, in our Caucasian autism trios and the CEU Caucasian HapMap samples the frequency of A was less than that reported in the Chinese Han and Chinese in Bejing HapMap samples. The haplotype test of association did not reveal excess transmission from parents to affected offspring. These findings provide support for association of OXTR with autism in a Caucasian population. Overtransmission of different alleles in different populations may be due to a different pattern of linkage disequilibrium between the marker rs2254298 and an as yet undetermined susceptibility variant in OXTR.

To assess the molecular changes associated with pancreatic beta-cell dysfunction occurring during the onset of type 2 diabetes, we profiled pancreatic islet mRNAs from diabetic male and high-fat-fed female Zucker diabetic fatty (ZDF) rats and their nondiabetic lean counterparts on custom islet-specific oligonucleotide arrays. The most prominent changes in both the male and female models of type 2 diabetes were increases in the mRNAs encoding proteases and extracellular matrix components that are associated with tissue remodeling and fibrosis. The mRNAs for metalloproteinase (MMP)-2, -12, and -14 were sharply increased with the onset of islet dysfunction and diabetes. Zymography of islet extracts revealed a concurrent, >10-fold increase in MMP-2 protease activity in islets from 9-week-old male ZDF rats. Treatment of female ZDF rats receiving a diabetogenic diet with PD166793, a broad-spectrum MMP inhibitor, substantially prevented diabetes. The effect of this compound was due in part to marked beta-cell expansion. These studies indicate that MMPs contribute to islet fibrosis and insulin insufficiency in ZDF rats. Class-targeted protease inhibitors should be explored for their potential therapeutic utility in preservation of beta-cell mass in type 2 diabetes.

The hyperlipidemia and hyperglycemia of the diabetic state accelerate beta-cell dysfunction, yet the mechanisms are not fully defined. We used rat islet-specific oligonucleotide arrays (Metabolex Rat Islet Genechips) to identify genes that are coordinately regulated by high glucose and free fatty acids (FFA). Exposure of rat islets to FFA (125 muM for 2 days) or glucose (27 mM for 4 days) reduced glucose-stimulated insulin secretion by 70 +/- 5 and 40 +/- 4%, respectively, relative to control-cultured islets. These treatments also substantially reduced the insulin content of the islets. Islet Genechips analysis revealed that the mRNA levels of cAMP response element modulator (CREM)-17X and inducible cAMP early repressor were significantly increased in both 27 mM glucose- and FFA-treated islets. Removing FFA or high glucose from the culture medium restored glucose- stimulated insulin secretion and the mRNA levels of the two CREM repressors to normal. Northern blot analysis revealed a 5-fold increase in the abundance of CREM-17X mRNA and a concomitant 50% reduction in the insulin mRNA in FFA-treated islets. Transient transfection of the insulin-secreting betaHC9 cells with CREM-17X suppressed rat insulin promoter activity by nearly 50%. Overexpression of CREM-17X in intact islets via adenovirus infection decreased islet insulin mRNA levels and insulin content and resulted in a significant decrease in glucose- or KCl-induced insulin secretion. Taken together, these data suggest that up-regulation of CREM repressors by either FFA or high glucose exacerbates beta-cell failure in type 2 diabetes by suppressing insulin gene transcription.

Two members of a family of spore germination-specific cDNAs, celA and celB, are expressed coordinately, exclusively during spore germination. In the present study the regulatory sequence elements responsible for celA germination-specific expression have been identified. The very AT-rich 81 bp sequence between -664 and -584 upstream of the translation initiation site was required for proper temporal transcription of the celA gene. This sequence is comprised of two cis elements, each of which was active by itself in allowing celA expression. Electrophoretic mobility shift assays showed that a factor(s) in an extract prepared from germinating spores bound to the celA regulatory region. One of the three complexes formed was specific for the germinating spore extract. The results are consistent with the notion that the factor(s) that binds to this regulatory region is involved in expression of celA.

During Dictyostelium discoideum spore germination, degradation of the cellulose-containing spore wall is required to allow the amoeba to emerge. The CelA gene, which is transcribed and expressed exclusively during spore germination, codes for a 705-amino-acid protein that has cellulase activity [endo-(1,4)-beta-D-glucanase]. Amoebae transformed by a vector containing the CelA coding sequence or portions of it transcribed from a heterologous promoter expressed and secreted full-length or suitably truncated proteins during vegetative growth when, under normal conditions, these proteins are not made. The gene constructs divided the CelA protein into three domains: a 461-amino-acid N-terminal region that has significant similarity to those of other cellulases and that has been shown to be the catalytic domain; a contiguous 91-residue repeat containing the motif threonine-glutamic acid-threonine-proline, which is glycosylated; and, joined to the repeat, a C-terminal 153-amino-acid sequence that most probably defines a cellulose-binding domain.

Two different cDNA clones denoted pTO270-6 and pTO270-11 represent two mRNAs that are developmentally regulated during spore germination in Dictyostelium discoideum. The respective mRNAs are found only during early germination and are not present in other stages of growth or multicellular development. Four different genomic clones that hybridize to sequences that are common to both of the 270 cDNA clones were isolated from Dictyostelium libraries and sequenced. Two are the genes for the two cDNAs, and the other two represent genes that do not seem to be transcribed. All four genomic sequences possess a very unusual internal feature in the deduced protein sequences composed of a monotonous repeat of the tetrapeptide threonine-glutamic acid-threonine-proline. The other portions of the proteins have no homology among themselves. The deduced protein corresponding to the 270-6 gene is very similar to avocado (Persea americana) cellulase. Since cellulose in the spore wall has to be digested during spore germination this suggests that this protein may function as an endo-(1,4)-beta-D-glucanase during germination.

A member of the 270 spore germination-specific gene family in Dictyostelium discoideum is shown to encode a cellulase (endo-(1,4)-beta-D-glucanase, EC 3.2.1.4) activity. The 270-6 deduced protein shows 38% identity and 58% similarity to an avocado (Persea americana) cellulase. During spore germination in Dictyostelium extracellular cellulase activity starts to accumulate coincident with the appearance of the 270-6 gene transcript. Amoebae transformed by a vector containing the 270-6 mRNA sequence express an extracellular cellulase during vegetative growth when there would otherwise be no cellulase activity. In addition, the expression of a truncated 270-6 polypeptide lacking the 270 gene family-defining tetrapeptide repeat and the C-terminal region, in suitably transformed amoebae, also produces an extracellular cellulase activity. Several differently sized cellulase activities are shown to accumulate during spore germination, and it is possible that the 270 gene family represents a coordinately expressed family of cellulases.

We have examined the effect of protein synthesis and of ribosome loading on the estrogen-mediated stabilization of hepatic Xenopus laevis vitellogenin mRNA. Removal of estradiol-17 beta from the culture medium, which destabilizes vitellogenin mRNA, does not alter the density of ribosomes on polysomal vitellogenin mRNA, or change the proportion of vitellogenin mRNA associated with the endoplasmic reticulum. Cycloheximide, which inhibits elongation, without changing the density of ribosomes on vitellogenin mRNA, does not block estrogen-mediated stabilization. In contrast, 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamide, (MDMP), which inhibits initiation, greatly reduces the density of ribosomes on vitellogenin mRNA, and completely blocks estrogen-mediated stabilization. Vitellogenin mRNA in MDMP treated cells is degraded at a rate similar to that seen when untreated cells are transferred from medium containing estrogen to estrogen-free medium. This suggests that a ribosome-associated degradative system may not be responsible for vitellogenin mRNA degradation. The failure of estrogen to stabilize vitellogenin mRNA in MDMP-treated cells is not due to the release of vitellogenin mRNA from the endoplasmic reticulum. Vitellogenin mRNA in MDMP-treated cells remains associated with the endoplasmic reticulum in small polysomes containing 3-5 ribosomes. These data demonstrate that maintaining a high density of ribosomes on vitellogenin mRNA, but not continuing protein synthesis, is necessary for estrogen-mediated stabilization of vitellogenin mRNA.