Faculty Bibliography

Glycogen storage disease type Ib is caused by mutations in the glucose 6-phosphate transporter (G6PT) in the endoplasmic reticulum membrane in liver and kidney. Twenty-eight missense and two deletion mutations that cause the disease were previously shown to reduce or abolish the transporter's activity. However, the mechanisms by which these mutations impair transport remain unknown. On the basis of the recently determined crystal structure of its Escherichia coli homologue, the glycerol 3-phosphate transporter, we built a three-dimensional structural model of human G6PT by homology modeling. G6PT is proposed to consist of 12 transmembrane alpha-helices that are divided into N- and C-terminal domains, with the substrate-translocation pore located between the two domains and the substrate-binding site formed by R28 and K240 at the domain interface. The disease-causing mutations were found to occur at four types of positions: (I) in the substrate-translocation pore, (II) at the N-/C-terminal domain interface, (III) in the interior of the N- and C-terminal domains, and (IV) on the protein surface. Whereas class I mutations affect substrate binding directly, class II mutations, mostly involving changes in side chain size, charge, or both, hinder the conformational change required for substrate translocation. On the other hand, class III and class IV mutations, often introducing a charged residue into a helix bundle or at the protein-lipid interface, probably destabilize the protein. These results also suggest that G6PT operates by a similar antiport mechanism as its E. coli homologue, namely, the substrate binds at the N- and C-terminal domain interface and is then transported across the membrane via a rocker-switch type of movement of the two domains

The passage of an individual's genome to future generations is essential for the maintenance of species and is mediated by highly specialized cells, the germ cells. Genetic studies in a number of model organisms have provided insight into the molecular mechanisms that control specification, migration and survival of early germ cells. Focusing on Drosophila, we will discuss the mechanisms by which germ cells initially form and remain transcriptionally silent while somatic cells are transcriptionally active. We will further discuss three separate attractive and repellent guidance pathways, mediated by a G-protein coupled receptor, two lipid phosphate phosphohydrolases, and isoprenylation. We will compare and contrast these findings with those obtained in other organisms, in particular zebrafish and mice. While aspects of germ cell specification are strikingly different between these species, germ cell specific gene functions have been conserved. In particular, mechanisms that sense directional cues during germ cell migration seem to be shared between invertebrates and vertebrates

Subcellular compartmentalization is an emerging paradigm in signaling pathways including the Ras/MAPK pathway. In a recent issue of Developmental Cell, Torii et al. (2004) characterize a new MAPK scaffold, Sef, that resides on the Golgi apparatus, binds active MEK/ERK complexes, and permits signaling to cytosolic substrates but not nuclear targets

The effect of different dose, mode and duration of estradiol administration was examined in the different brain catecholaminergic areas in ovariectomized (OVX) female rats. We determined changes in mRNA levels of tyrosine hydroxylase (TH), rate-limiting enzyme in catecholamine (CA) biosynthesis of GTP cyclohydrolase I (GTPCH), rate-limiting enzyme in biosynthesis as well as of tetrahydrobiopterin (BH4), and concentration of BH4, which is an essential cofactor for TH, tryptophan hydroxylase and nitric oxide synthase. Short-term administration of estradiol benzoate (EB) by five injections of 15 or 40 microg/kg 12 h apart led to increase in TH and GTPCH mRNA levels in dopaminergic and noradrenergic cell bodies of the ventral tegmental area (VTA), substantia nigra (SN), locus coeruleus (LC) and the nucleus of solitary tract (NTS) depending on dose of administration. Estrogen-elicited alterations in BH4 concentrations were mostly correlated with changes in GTPCH mRNA levels, except in SN. Long-term administration of estradiol by injections (EB: 25 microg/kg, 16 injections 26 h apart; 50 microg/kg, 16 injections 48 h apart) or pellets (0.1 mg 17 beta-estradiol, 14 days) were not very effective in modulating mRNA levels for both genes in most locations except the NTS. Long-term injections of EB elevated GTPCH mRNA levels throughout the NTS and in microvessels. Administration of estradiol by pellets led to decline of TH mRNA in rostral-medial and elevation in caudal parts of the NTS. Thus, estradiol has a complex and differential effect on TH and GTPCH gene expression in a tissue specific manner and depends on the mode of administration.

The ability of nicotine infusion to modulate plasma aldosterone levels in response to different stressors was investigated. Sprague-Dawley rats given nicotine (5 mg/kg/day) or saline for 14 days were subjected to stress. Baseline plasma aldosterone (86+/-17 pmol/l) was unaffected by nicotine. Aldosterone was significantly elevated by restraint (450+/-72 pmol/l) and especially with cold (1249+/-172 pmol/l) or immobilization (1779+/-247 pmol/l) stress. Nicotine infusion attenuated the rise in aldosterone with restraint and cold stress, but not immobilization. These results reveal that nicotine infusion can attenuate the aldosterone response, depending on the type of stress.

Mammalian genomes are densely populated with long duplicated sequences. In this paper, we demonstrate the existence of doublets, short duplications between 25 and 100 bp, distinct from previously described repeats. Each doublet is a pair of exact matches, separated by some distance. The distribution of these intermatch distances is strikingly nonrandom. An unexpectedly high number of doublets have matches either within 100 bp (adjacent) or at distances tightly concentrated approximately 1,000 bp apart (nearby). We focus our study on these proximate doublets. First, they tend to have both matches on the same strand. By comparing nearby doublets shared in human and chimpanzee, we can also see that these doublets seem to arise by an insertion event that produces a copy without markedly affecting the surrounding sequence. Most doublets in humans are shared with chimpanzee, but many new pairs arose after the divergence of the species. Doublets found in human but not chimpanzee are most often composed of almost tandem matches, whereas older doublets (found in both species) are more likely to have matches spaced by approximately 1 kb, indicating that the nearly tandem doublets may be more dynamic. The spacing of doublets is highly conserved. So far, we have found clearly recognizable doublets in the following genomes: Homo sapiens, Mus musculus, Arabidopsis thaliana, and Caenorhabditis elegans, indicating that the mechanism generating these doublets is widespread. A mechanism that generates short local duplications while conserving polarity could have a profound impact on the evolution of regulatory and protein-coding sequences