The Qo site of cytochrome b6f complexes controls the activation of the LHCII kinase.

We created a Qo pocket mutant by site-directed mutagenesis of the chloroplast petD gene in Chlamydomonas reinhardtii. We mutated the conserved PEWY sequence in the EF loop of subunit IV into PWYE. The pwye mutant did not grow in phototrophic conditions although it assembled wild-type levels of cytochrome b6f complexes. We demonstrated a complete block in electron transfer through the cytochrome b6f complex and a loss of plastoquinol binding at Qo. The accumulation of cytochrome b6f complexes lacking affinity for plastoquinol enabled us to investigate the role of plastoquinol binding at Qo in the activation of the light-harvesting complex II (LHCII) kinase during state transitions. We detected no fluorescence quenching at room temperature in state II conditions relative to that in state I. The quantum yield spectrum of photosystem I charge separation in the two state conditions displayed a trough in the absorption region of the major chlorophyll a/b proteins, demonstrating that the cells remained locked in state I. 33Pi labeling of the phosphoproteins in vivo demonstrated that the antenna proteins remained poorly phosphorylated in both state conditions. Thus, the absence of state transitions in the pwye mutant demonstrates directly that plastoquinol binding in the Qo pocket is required for LHCII kinase activation.     

Intestinal SGLT1-mediated absorption and metabolism of benzyl beta-glucoside contained in Prunus mume: carrier-mediated transport increases intestinal availability

The intestinal absorption of benzyl beta-glucoside (BNZ beta glc) contained in the fruit of Prunus mume SIEB. et ZUCC. (Rosaceae), which is traditionally used as a medicinal food in Japan, was studied in rat intestines. BNZ beta glc was absorbed from the mucosal to serosal sides. Its metabolite, benzyl alcohol (BAL), was also detected on both the mucosal and serosal sides. In the presence of phloridzin (Na(+)/glucose cotransporter (SGLT1) inhibitor) or in the absence of Na+ (driving force), BNZ beta glc absorption was significantly decreased. Transport clearance of BNZ beta glc across the brush border membrane decreased as its concentration increased. These results indicate that BNZ beta glc is transported by SGLT1. Metabolic clearance of BNZ beta glc also decreased as its concentration increased. The amount ratio of BNZ beta glc to BAL on the serosal side increased with the increase of BNZ beta glc concentration. The intestinal availability of BNZ beta glc was lower in the absence of Na+ than in the presence of Na+, indicating that the SGLT1-mediated transport of BNZ beta glc increases intestinal availability by decreasing the intestinal extraction ratio. This neutraceutical study concluded that intestinal carrier-mediated transport across the brush border membrane improves the intestinal availability of nutritionally, pharmacologically or physiologically active compounds that undergo intestinal metabolism (first-pass effect).     

Molecular evolution of human T-cell leukemia virus

Summary In light of previous data, which suggested thatAc-like sequences might have undergone a significant radiation in the recent past, I examined the copy number ofAc-like sequences in representatives of all theZea taxa, both maize and teosinte. The maize and teosinte samples contained approximately equal numbers ofAc-like sequences. FewAc-like sequences were in unmethylated regions of DNA. Unmethylated elements were distributed randomly among both maize and teosinte lines. The appearance in a line of a discrete band resulting from digestion with one methylation-sensitive restriction enzyme was correlated with the appearance of discrete bands with other methylation-sensitive bands. This suggests that individualAc-like elements are occasionally demethylated in many sites. No unmethylated element having restriction fragments of the lengths predicted from the publishedAc sequence was seen in the approximately 326 elements examined.     

Genetic mapping of glutaric aciduria, type 3, to chromosome 7 and identification of mutations in c7orf10

While screening Old Order Amish children for glutaric aciduria type 1 (GA1) between 1989 and 1993, we found three healthy children who excreted abnormal quantities of glutaric acid but low 3-hydroxyglutaric acid, a pattern consistent with glutaric aciduria type 3 (GA3). None of these children had the GCDH c.1262C→T mutation that causes GA1 among the Amish. Using single-nucleotide polymorphism (SNP) genotypes, we identified a shared homozygous 4.7 Mb region on chromosome 7. This region contained 25 genes including C7orf10, an open reading frame with a putative mitochondrial targeting sequence and coenzyme-A transferase domain. Direct sequencing of C7orf10 revealed that the three Amish individuals were homozygous for a nonsynonymous sequence variant (c.895C→T, Arg299Trp). We then sequenced three non-Amish children with GA3 and discovered two nonsense mutations (c.322C→T, Arg108Ter, and c.424C→T, Arg142Ter) in addition to the Amish mutation. Two pathogenic alleles were identified in each of the six patients. There was no consistent clinical phenotype associated with GA3. In affected individuals, urine molar ratios of glutarate to its derivatives (3-hydroxyglutarate, glutarylcarnitine, and glutarylglycine) were elevated, suggesting impaired formation of glutaryl-CoA. These observations refine our understanding of the lysine-tryptophan degradation pathway and have important implications for the pathophysiology of GA1.     

Increased sleep fragmentation leads to impaired off-line consolidation of motor memories in humans

A growing literature supports a role for sleep after training in long-term memory consolidation and enhancement. Consequently, interrupted sleep should result in cognitive deficits. Recent evidence from an animal study indeed showed that optimal memory consolidation during sleep requires a certain amount of uninterrupted sleep.Sleep continuity is disrupted in various medical disorders. We compared performance on a motor sequence learning task (MST) in relatively young subjects with obstructive sleep apnea (n = 16; apnea-hypopnea index 17.1±2.6/h [SEM]) to a carefully matched control group (n = 15, apnea-hypopnea index 3.7±0.4/h, p<0.001. Apart from AHI, oxygen nadir and arousal index, there were no significant differences between groups in total sleep time, sleep efficiency and sleep architecture as well as subjective measures of sleepiness based on standard questionnaires. In addition performance on the psychomotor vigilance task (reaction time and lapses), which is highly sensitive to sleep deprivation showed no differences as well as initial learning performance during the training phase. However there was a significant difference in the primary outcome of immediate overnight improvement on the MST between the two groups (controls = 14.7±4%, patients = 1.1±3.6%; P = 0.023) as well as plateau performance (controls = 24.0±5.3%, patients = 10.1±2.0%; P = 0.017) and this difference was predicted by the arousal index (p = 0.02) rather than oxygen saturation (nadir and time below 90% saturation. Taken together, this outcome provides evidence that there is a clear minimum requirement of sleep continuity in humans to ensure optimal sleep dependent memory processes. It also provides important new information about the cognitive impact of obstructive sleep apnea and challenges its current definitions.     

IGF-I and insulin receptor signal transduction in trout muscle cells

In this study, primary cultures of trout skeletal muscle cells were used to investigate the main signal transduction pathways of insulin and IGF-I receptors in rainbow trout muscle. At different stages of in vitro development (myoblasts on day 1, myocytes on day 4, and fully developed myotubes on day 11), we detected in these cells the presence of immunoreactivity against ERK 1/2 MAPK and Akt/PKB proteins, components of the MAPK and the phosphatidylinositol 3-kinase-Akt pathways, respectively, two of the main intracellular transduction pathways for insulin and IGF-I receptors. Both insulin and IGF-I activated both pathways, although the latter provoked higher immunoreactivity of phosphorylated MAPKs and Akt proteins. At every stage, increases in total MAPK immunoreactivity levels were observed when cells were stimulated with IGF-I or insulin, while total Akt immunoreactivity levels changed little under stimulation of peptides. Total Akt and total MAPK levels increased as skeletal muscle cells differentiated in culture. Moreover, when cells were incubated with IGF-I or insulin, MAPK-P immunoreactivity levels showed greater increases over the basal levels on days 1 and 4, with no effect observed on day 11. Although Akt-P immunoreactivity displayed improved responses on days 1 and 4 as well, a stimulatory effect was still observed on day 11. In addition, the present study demonstrates that purified trout insulin receptors possess higher phosphorylative activity per unit of receptor than IGF-I receptors. In conclusion, these results indicate that trout skeletal muscle culture is a suitable model to study the insulin and IGF-I signal transduction molecules and that there is a different regulation of MAPK and Akt pathways depending on the developmental stage of the muscle cells.     

Role of HiPIP as electron donor to the RC-bound cytochrome in photosynthetic purple bacteria

High-Potential Iron-Sulfur Proteins (HiPIP) are small electron carriers, present only in species of photosynthetic purple bacteria having a RC-bound cytochrome. Their participation in the photo-induced cyclic electron transfer was recently established for Rubrivivax gelatinosus, Rhodocyclus tenuis and Rhodoferax fermentans (Schoepp et al. 1995; Hochkoeppler et al. 1996a, Menin et al. 1997b). To better understand the physiological role of HiPIP, we extended our study to other selected photosynthetic bacteria. The nature of the electron carrier in the photosynthetic pathway was investigated by recording light-induced absorption changes in intact cells. In addition, EPR measurements were made in whole cells and in membrane fragments in solution or dried immobilized, then illuminated at room temperature. Our results show that HiPIP plays an important role in the reduction of the photo-oxidized RC-bound cytochrome in the following species: Ectothiorhodospira vacuolata, Chromatium vinosum, Chromatium purpuratum and Rhodopila globiformis. In Rhodopseudomonas marina, the HiPIP is not photo-oxidizible in whole cells and in dried membranes, suggesting that this electron carrier is not involved in the photosynthetic pathway. In Ectothiorhodospira halophila, the photo-oxidized RC-bound cytochrome is reduced by a high midpoint potential cytochrome c, in agreement with midpoint potential values of the two iso-HiPIPs (+ 50 mV and + 120 mV) which are too low to be consistent with their participation in the photosynthetic cyclic electron transfer.     

Tumour class prediction and discovery by microarray-based DNA methylation analysis

Aberrant DNA methylation of CpG sites is among the earliest and most frequent alterations in cancer. Several studies suggest that aberrant methylation occurs in a tumour type-specific manner. However, large-scale analysis of candidate genes has so far been hampered by the lack of high throughput assays for methylation detection. We have developed the first microarray-based technique which allows genome-wide assessment of selected CpG dinucleotides as well as quantification of methylation at each site. Several hundred CpG sites were screened in 76 samples from four different human tumour types and corresponding healthy controls. Discriminative CpG dinucleotides were identified for different tissue type distinctions and used to predict the tumour class of as yet unknown samples with high accuracy using machine learning techniques. Some CpG dinucleotides correlate with progression to malignancy, whereas others are methylated in a tissue-specific manner independent of malignancy. Our results demonstrate that genome-wide analysis of methylation patterns combined with supervised and unsupervised machine learning techniques constitute a powerful novel tool to classify human cancers.