An<Emphasis Type="Italic"> Alu</Emphasis>-mediated rearrangement as cause of exon skipping in Hunter disease

Hunter syndrome (Mucopolysaccharidosis type II), a rare X-linked lysosomal storage disorder, results from deleterious mutations in the iduronate-2-sulfatase ( IDS) gene located on Xq27.3-q28. Partial or complete deletions and large rearrangements have been extensively reported in the IDS gene as the basis of Hunter disease. The present report, however, is the first report on a Hunter patient in which Alu-mediated recombinations are implicated. Our patient showed the skipping of exon 8 at the cDNA level, without any splice-junction defects at the genomic level, where a new large rearrangement was identified instead. This new mutant allele consisted of an extensive deletion of IDS sequence of about 3 kb, as well as an additional inserted sequence of 157 bp. Two different computer programs were necessary to elucidate the nature of the insert. NCBI-BLAST query detected a single match for 126 bp out of 157 of the fragment that aligned exactly with a specific chromosomal region, Xq25-27.1, where an AluSg sequence is adjacent to an L1. Instead, the Repeat Masker program identified only 83 bp out of 157 of the insert, which was confirmed as an AluS. The observed homology between the AluSc sequence in the IDS intron 8 and the inserted AluS element, as well as the closeness of 26 bp Alu core sequence, considered to be a recombination hotspot, made us hypothesise upon the fact that both an Alu retrotransposition and an Alu-mediated deletion underlie the disease-producing rearrangement. We, therefore, now propose a mechanism that led to the large genomic deletion causing the production of the aberrant mRNA splicing.     

AIP1/ALIX is a binding partner for HIV-1 p6 and EIAV p9 functioning in virus budding

HIV-1 and other retroviruses exit infected cells by budding from the plasma membrane, a process requiring membrane fission. The primary late assembly (L) domain in the p6 region of HIV-1 Gag mediates the detachment of the virion by recruiting host Tsg101, a component of the class E vacuolar protein sorting (Vps) machinery. We now show that HIV Gag p6 contains a second region involved in L domain function that binds AIP1, a homolog of the yeast class E Vps protein Bro1. Further, AIP1 interacts with Tsg101 and homologs of a subunit of the yeast class E Vps protein complex ESCRT-III. AIP1 also binds to the L domain in EIAV p9, and this binding correlates perfectly with L domain function. These observations identify AIP1 as a component of the viral budding machinery, which serves to link a distinct region in the L domain of HIV-1 p6 and EIAV p9 to ESCRT-III.     

Sudden exposure to solar UV-B radiation reduces net CO(2) uptake and photosystem I efficiency in shade-acclimated tropical tree seedlings

Tree seedlings developing in the understory of the tropical forest have to endure short periods of high-light stress when tree-fall gaps are formed, and direct solar radiation, including substantial UV light, reaches the leaves. In experiments simulating the opening of a tree-fall gap, the response of photosynthesis in leaves of shade-acclimated seedlings (Anacardium excelsum, Virola surinamensis, and Calophyllum longifolium) to exposure to direct sunlight (for 20-50 min) was investigated in Panama (9 degrees N). To assess the effects of solar UV-B radiation (280-320 nm), the sunlight was filtered through plastic films that selectively absorbed UV-B or transmitted the complete spectrum. The results document a strong inhibition of CO(2) assimilation by sun exposure. Light-limited and light-saturated rates of photosynthetic CO(2) uptake by the leaves were affected, which apparently occurred independently of a simultaneous inhibition of potential photosystem (PS) II efficiency. The ambient UV-B light substantially contributed to these effects. The photochemical capacity of PSI, measured as absorbance change at 810 nm in saturating far-red light, was not significantly affected by sun exposure of the seedlings. However, a decrease in the efficiency of P700 photooxidation by far-red light was observed, which was strongly promoted by solar UV-B radiation. The decrease in PSI efficiency may result from enhanced charge recombination in the reaction center, which might represent an incipient inactivation of PSI, but contributes to thermal dissipation of excessive light energy and thereby to photoprotection.     

Circadian dynamics of cytosolic and nuclear Ca2+ in single suprachiasmatic nucleus neurons

Intracellular free Ca(2+) regulates diverse cellular processes, including membrane potential, neurotransmitter release, and gene expression. To examine the cellular mechanisms underlying the generation of circadian rhythms, nucleus-targeted and untargeted cDNAs encoding a Ca(2+)-sensitive fluorescent protein (cameleon) were transfected into organotypic cultures of mouse suprachiasmatic nucleus (SCN), the primary circadian pacemaker. Circadian rhythms in cytosolic but not nuclear Ca(2+) concentration were observed in SCN neurons. The cytosolic Ca(2+) rhythm period matched the circadian multiple-unit-activity (MUA)-rhythm period monitored using a multiple-electrode array, with a mean advance in phase of 4 hr. Tetrodotoxin blocked MUA, but not Ca(2+) rhythms, while ryanodine damped both Ca(2+) and MUA rhythms. These results demonstrate cytosolic Ca(2+) rhythms regulated by the release of Ca(2+) from ryanodine-sensitive stores in SCN neurons.     

Rescue of molybdenum cofactor biosynthesis in gephyrin-deficient mice by a Cnx1 transgene

Gephyrin is a bifunctional protein which is essential for both synaptic clustering of inhibitory neurotransmitter receptors in the central nervous system and the biosynthesis of the molybdenum cofactor (MoCo) in peripheral tissues. Mice deficient in gephyrin die early postnatally and display a loss of glycine receptors (GlyRs) and many GABA(A) receptor (GABA(A)R) subtypes from postsynaptic sites. In addition, the activities of the MoCo-dependent enzymes xanthine dehydrogenase and sulfite oxidase are reduced to background levels in the liver and intestine of these animals. To genetically separate the different consequences of gephyrin deficiency, we expressed a transgene of the plant homolog Cnx1, known to rescue mammalian MoCo deficiency, on the background of gephyrin knockout mice. Cnx1 partially restored sulfite oxidase activity in the liver of the transgenic animals, whereas early lethality and the loss of GlyR clustering were unaltered. Our data suggest that the loss of neurotransmitter receptor clustering at inhibitory synapses causes the early lethality of gephyrin deficient mice.     

Rac2 regulation of phospholipase C-beta 2 activity and mode of membrane interactions in intact cells

Phospholipase C-beta (PLCbeta) isozymes play important roles in transmembrane signaling. Their activity is regulated by heterotrimeric G proteins. The PLCbeta(2) isozyme is unique in being stimulated also by Rho GTPases (Rac and Cdc42). However, the mechanism(s) of this stimulation are still unclear. Here, we employed fluorescence recovery after photobleaching to investigate the interaction of green fluorescent protein (GFP)-PLCbeta(2) with the plasma membrane. For either GFP-PLCbeta(2) or GFP-PLCbeta(2)Delta, a C-terminal deletion mutant lacking the region required for stimulation by Galpha(q), these interactions were characterized by a mixture of exchange with a cytoplasmic pool and lateral diffusion. Constitutively active Rac2(12V) stimulated the activity of both GFP-PLCbeta(2) and GFP-PLCbeta(2)Delta in live cells, and enhanced their membrane association as evidenced by the marked reduction in their fluorescence recovery rates. Both effects required the putative N-terminal pleckstrin homology (PH) domain of PLCbeta(2). Importantly, Rac2(12V) dramatically increased the contribution of exchange to the fluorescence recovery of GFP-PLCbeta(2), but had the opposite effect on GFP-PLCbeta(2)Delta, where lateral diffusion became dominant. Our results demonstrate for the first time the regulation of membrane association of a PLCbeta isozyme by a GTP-binding protein and assign a novel function to the PLCbeta(2) C-terminal region, regulating its exchange between membrane-bound and cytosolic states.     

The autoxidation of tetrahydrobiopterin revisited. Proof of superoxide formation from reaction of tetrahydrobiopterin with molecular oxygen

It has been known for quite some time that tetrahydrobiopterin (H4B) is prone to autoxidation in the presence of molecular oxygen. Evidence has been presented that in this process superoxide radicals may be released, although their intermediacy never has been directly proven. In the present study, the autoxidation of H4B was reinvestigated with the aim to find direct evidence for superoxide formation. By means of two specific assays, namely elicitation of luminescence from lucigenin and ESR-spectrometric detection of the DEPMPO-OOH radical adduct, the release of free superoxide radicals was unequivocally demonstrated. The production of superoxide radicals was further corroborated by interaction with nitric oxide. The kinetics of the autoxidation process was established. Our data fully confirm earlier conclusions that the direct reaction between H4B and oxygen serves as an initiation reaction for the further, rapid reaction of the thus formed superoxide with H4B, thereby very likely establishing a chain reaction process involving reduction of molecular oxygen by the intermediary tetrahydrobiopterin radical. Conclusively, because H4B can per se induce oxidative stress, an in vivo overproduction of this pterin, as is evident in various diseases, may be responsible for the observed acceleration of pathophysiological pathways.     

The urbilaterian brain: developmental insights into the evolutionary origin of the brain in insects and vertebrates

Classical phylogenetic, neuroanatomical and neuroembryological studies propose an independent evolutionary origin of the brains of insects and vertebrates. Contrasting with this, data from three sets of molecular and genetic analyses indicate that the developmental program of brains of insects and vertebrates might be highly conserved and suggest a monophyletic origin of the brain of protostomes and deuterostomes. First, recent results of molecular phylogeny imply that none of the currently living animals correspond to evolutionary intermediates between protostomes and deuterostomes, thus making it impossible to infer the morphological organization of an ancestral bilaterian brain from living specimens. Second, recent molecular genetic evidence provides support for the body axis inversion hypothesis, which implies that a dorsoventral inversion of the body axis occurred in protostomes versus deuterostomes, leading to the inverted location of neurogenic regions in these animal groups. Third, recent developmental genetic analyses are uncovering the existence of structurally and functionally homologous genes that have comparable and interchangeable functions in early brain development in insect and vertebrate model systems. Thus, development of the anteriormost part of the embryonic brain in both insects and vertebrates depends upon the otd/Otx and ems/Emx genes; development of the posterior part of the embryonic brain in both insects and vertebrates involves homologous control genes of the Hox cluster. These findings, which demonstrate the conserved expression and function of key patterning genes involved in embryonic brain development in insects and vertebrates support the hypothesis that the brains of protostomes and deuterostomes are of monophyletic, urbilaterian origin.