Pax proteins and eye development

Male and female rats were used to investigate the effects of type of dietary carbohydrate (CHO), copper, and ethanol consumption on lung antioxidant enzyme activities and levels of phosphorylated compounds in whole blood. Copper-deficient female rats exhibited a greater degree of copper deficiency than males, as assessed by hepatic copper concentration and hepatic copper superoxide dismutase (CuSOD) activity. However, copper-deficient male rats fed fructose-containing diets exhibited greater growth retardation, anemia, and heart hypertrophy than females consuming the same diets and males fed starch. In addition, one of 10 copper-deficient male rats that ate a fructose-based diet and drank water and one of 10 copper-deficient male rats that ate a starch-based diet and drank ethanol died. Copper-deficient, starch-fed males exhibited the highest activities of glutathione peroxidase (GSH-Px) and catalase as compared with fructose-fed rats. Ethanol consumption elevated the activities of GSH-Px and catalase. Copper-deficient female rats exhibited higher catalase but lower GSH-Px activities than males. It is suggested that in copper deficiency, the ability to increase antioxidant enzyme activities in rats consuming starch is greater than in rats consuming fructose. Rats fed starch are provided with a greater degree of protection against oxidative damage than rats fed fructose. In addition, polyphosphorylated compounds in blood were reduced in copper-deficient male rats that consumed fructose-based diets. This may impair supply of oxygen to tissues.     

Genetic background effects on dental and other craniofacial abnormalities in homozygous small eye (Pax6Sey/Pax6Sey) mice

Blockade of the N-methyl D-aspartate (NMDA) receptor by the ion-channel-blocking drug aptiganel hydrochloride (CNS 1102, Cerestat) is neuroprotective in focal cerebral ischemia. Short intravenous infusions of up to 30 microg/kg have been well tolerated by healthy male volunteers. We undertook a randomized, double-blind, placebo-controlled study in 20 male volunteers to examine the safety, tolerability, and cardiovascular and psychomotor effects of a dosing paradigm similar to that envisaged for therapeutic use. Aptiganel HCl was infused over 4 h in total doses of 15, 32, 50, or 73 microg kg. Mean arterial pressure increased significantly with dose group (p < 0.01, analysis of covariance). Motor reaction time was related to maximal plasma concentration (r2 = 0.21, p < 0.001). Transient symptoms and signs of peripheral paresthesiae, light-headedness, and euphoria were seen at total doses of 32 microg/ kg. Higher doses were associated with motor retardation, perceptual disturbances, and hallucinations (one case). Clearance was 125 +/- 55 L/h, and volume of distribution was 537 +/- 1,261. Total doses of up to 32 microg/kg of aptiganel HCl infused over 4 h are well tolerated by healthy males. Aptiganel HCl causes elevation of blood pressure and is associated with central nervous system symptoms and signs similar to other noncompetitive NMDA antagonists.     

Alternative splicing of LTBP-3

LTBPs bind the 100-kDa latent TGF-beta complex and thereby regulate TGF-beta assembly, tissue localization, and function. However, the 100-kDa complex is not always associated with LTBP, and, conversely, evidence suggests that LTBP has a distinct role in the extracellular matrix. As yet, there are no data to explain how the binding interaction between LTBP and the 100-kDa complex is regulated. This report provides the first direct evidence of alternative splicing of an LTBP gene. Two alternative splice sites in the mouse LTBP-3 gene have been identified based on in vivo and in vitro studies. Alternative splicing at one site in particular was found to disrupt a structural motif involved in the binding interaction with the 100-kDa latent TGF-beta complex. Therefore, alternative splicing may represent a molecular mechanism by which the uncomplexed form of LTBP-3 is produced, and, as a corollary, by which the 100-kDa latent TGF-beta 1 complex is produced.     

Alternative splicing and programmed cell death

Programmed cell death (PCD) is critical for development and homeostasis of multicellular organisms. Genetic and biochemical studies have revealed that PCD is under complex and delicate regulation. An important level of such regulation may be pre-mRNA splicing as suggested by the observation that a number of PCD regulatory genes are expressed as functionally distinct or even antagonistic isoforms as a result of alternative splicing. Studies on alternative splicing of these genes are reviewed here. Expression and function of a large number of genes involved in PCD are regulated by alternative splicing, including death receptors and intracellular components of the death machinery. Alternative splicing affects not only intracellular distribution but also functional activity of these death regulators, providing a fine-tuning mechanism in modulating a presumably tightly controlled process of cell death.     

Group II intron splicing in vivo by first-step hydrolysis

Group I, group II and spliceosomal introns splice by two sequential transesterification reactions. For both spliceosomal and group II introns, the first-step reaction occurs by nucleophilic attack on the 5' splice junction by the 2' hydroxyl of an internal adenosine, forming a 2'-5' phosphodiester branch in the intron. The second reaction joins the two exons with a 3'-5' phosphodiester bond and releases intron lariat. In vitro, group II introns can self-splice by an efficient alternative pathway in which the first-step reaction occurs by hydrolysis. The resulting linear splicing intermediate participates in normal second-step reactions, forming spliced exon and linear intron RNAs. Here we show that the group II intron first-step hydrolysis reaction occurs in vivo in place of transesterification in the mitochondria of yeast strains containing branch-site mutations. As expected, the mutations block branching, but surprisingly still allow accurate splicing. This hydrolysis pathway may have been a step in the evolution of splicing mechanisms.     

Alternative splicing of GTBP in normal human tissues

The relationship between serum hepatitis Be antigen (HBeAg) and serum hepatitis B virus (HBV) DNA determined by two commercially available assays was examined in 345 Chinese patients with chronic HBV infection. HBV DNA was detected by these commercial assays in 85% of the HBeAg-positive patients. Discrepancies between test results were found to occur when serum HBV DNA levels were low (< 5 pg ml-1 for the Abbott Genostics and < 100 MEq ml-1 for Chiron Quantiplex assays). An equation for the conversion between results generated by these two assays was derived, which was found to be very similar to the equation recently described by Kapke et al.     

Detection and analysis of Tetrahymena pyriformis 26S ribosomal DNA domain sequences, differing in degree of evolutionary conservation

Domains of different evolutionary conservatism were defined in the 26S rDNA sequence of T. pyriformis. The fragment of studied DNA (1212 bp) showing high evolutionary conservatism was cloned. It was shown this fragment of DNA may be used to a probe for blot-hybridization analysis of the structure of rDNA from various taxa, protists to mammals. Superconservative and hypervariable domains were defined. The first are good for the primers for PCR analysis of rDNA from various taxa, the second--for species specific primers.     

Alternative splicing of human NrCAM in neural and nonneural tissues

HEC1A endometrial cancer cells express the wild-type form of the estrogen receptor (ER) and 17beta-estradiol (E2) induces proliferation of these cells. In contrast, tamoxifen only causes a minimal increase (<20%) in cell proliferation. In HEC1A cells transiently transfected with the C3-Luc plasmid derived from the complement C3 gene, both E2 and tamoxifen exhibited ER agonist activity and tamoxifen was also a partial antagonist for this response. The relative ER agonist/antagonist activities of E2, tamoxifen and ICI 182,780 were also investigated in HEC1A1 cells transiently transfected with two E2-responsive plasmids, pCATHD-CAT and pCKB-CAT which contain 5'-promoter inserts from the cathepsin D and creatine kinase B genes, respectively. The results showed that E2 and tamoxifen induced reporter gene activity in cells transiently transfected with both constructs. ICI 182,780 exhibited partial ER agonist activity only in cells transiently transfected with pCKB-CAT and antagonized E2-induced reporter gene activity using both the CKB- and CATHD-derived constructs. These results demonstrate that HEC1A endometrial cancer cells are E2-responsive and represent a useful cell culture model for understanding hormone/antihormone-induced endometrial cell responses.     

Synteny conservation between parts of human chromosome 4q and bovine and ovine chromosomes 6

Booroola Merino sheep are characterized by a high ovulation rate attributable to the presence of the FecB allele of the FEC gene. This gene, which has been assigned to sheep chromosome 6, is linked to the gene for EGF, which in man is located on the long arm of chromosome 4 (HSA 4q). To increase the number of known markers on sheep chromosome 6, we used comparative mapping data from sheep, man, and cattle. In our study, we show a synteny between EGF and the genes PDHA2 and FGF5 (from HSA 4q) and microsatellites ILSTS018, ILSTS090, and ILSTS093 (from bovine chromosome 6) in sheep. We also show that the conservation between HSA 4q and sheep chromosome 6 is disrupted between EGF and FGF2.     

Alternative splicing of CYP2D mRNA in human breast tissue

Cerebrovascular arteriovenous malformations (AVMs) display abnormal vascular development and dysautoregulation of blood flow. Genetic mechanisms that contribute to the pathogenesis and phenotype of cerebral AVMs are unknown. As a first step in understanding the pathophysiology of AVMs, the authors investigated the hypothesis that endothelial dysfunction-specifically, deregulation of endothelin-1 (ET-1) secretion-contributes to the abnormal vascular phenotype and the lack of hemodynamic autoregulation elaborated by these lesions. Endothelin-1 peptide and preproendothelin-1 (ppET1) messenger RNA were not detected in the intranidal vasculature of all 17 patients with AVMs studied, but were prominently expressed in human control subjects with normal cerebrovasculature (p < 0.01). Although AVM vasculature lacked ET-1, its expression was prominent in vasculature distant from these lesions, suggesting local repression of the ppET-1 gene. Local repression of ET-1 was specific to AVMs; ET-1 in vascular malformations of patients with Sturge-Weber disease was actually elevated compared to normal controls (p < 0.01). Repression of the ppET-1 gene was an intrinsic phenotype of AVM endothelial cells and was not due to factors in the AVM microenvironment. The authors also showed that ETA receptor expression was low in AVM vasculature compared to normal controls. Together, these results demonstrate that the ppET-1 gene is locally repressed in AVM lesions and suggest a role for abnormal ppET-1 gene regulation in the pathogenesis and clinical sequelae of cerebral AVMs.     

Contrasting population structure from nuclear intron sequences and mtDNA of humpback whales

Powerful analyses of population structure require information from multiple genetic loci. To help develop a molecular toolbox for obtaining this information, we have designed universal oligonucleotide primers that span conserved intron-exon junctions in a wide variety of animal phyla. We test the utility of exon-primed, intron-crossing amplifications by analyzing the variability of actin intron sequences from humpback, blue, and bowhead whales and comparing the results with mitochondrial DNA (mtDNA) haplotype data. Humpback actin introns fall into two major clades that exist in different frequencies in different oceanic populations. It is surprising that Hawaii and California populations, which are very distinct in mtDNAs, are similar in actin intron alleles. This discrepancy between mtDNA and nuclear DNA results may be due either to differences in genetic drift in mitochondrial and nuclear genes or to preferential movement of males, which do not transmit mtDNA to offspring, between separate breeding grounds. Opposing mtDNA and nuclear DNA results can help clarify otherwise hidden patterns of structure in natural populations.