Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector

Pre-clinical studies in mice and haemophilic dogs have shown that introduction of an adeno-associated viral (AAV) vector encoding blood coagulation factor IX (FIX) into skeletal muscle results in sustained expression of F.IX at levels sufficient to correct the haemophilic phenotype. On the basis of these data and additional pre-clinical studies demonstrating an absence of vector-related toxicity, we initiated a clinical study of intramuscular injection of an AAV vector expressing human F.IX in adults with severe haemophilia B. The study has a dose-escalation design, and all patients have now been enrolled in the initial dose cohort (2 x 10(11) vg/kg). Assessment in the first three patients of safety and gene transfer and expression show no evidence of germline transmission of vector sequences or formation of inhibitory antibodies against F.IX. We found that the vector sequences are present in muscle by PCR and Southern-blot analyses of muscle biopsies and we demonstrated expression of F.IX by immunohistochemistry. We observed modest changes in clinical endpoints including circulating levels of F.IX and frequency of FIX protein infusion. The evidence of gene expression at low doses of vector suggests that dose calculations based on animal data may have overestimated the amount of vector required to achieve therapeutic levels in humans, and that the approach offers the possibility of converting severe haemophilia B to a milder form of the disease.     

Respiration as the main determinant of carbon balance in European forests

Carbon exchange between the terrestrial biosphere and the atmosphere is one of the key processes that need to be assessed in the context of the Kyoto Protocol. Several studies suggest that the terrestrial biosphere is gaining carbon, but these estimates are obtained primarily by indirect methods, and the factors that control terrestrial carbon exchange, its magnitude and primary locations, are under debate. Here we present data of net ecosystem carbon exchange, collected between 1996 and 1998 from 15 European forests, which confirm that many European forest ecosystems act as carbon sinks. The annual carbon balances range from an uptake of 6.6 tonnes of carbon per hectare per year to a release of nearly 1 t C ha(-1) yr(-1), with a large variability between forests. The data show a significant increase of carbon uptake with decreasing latitude, whereas the gross primary production seems to be largely independent of latitude. Our observations indicate that, in general, ecosystem respiration determines net ecosystem carbon exchange. Also, for an accurate assessment of the carbon balance in a particular forest ecosystem, remote sensing of the normalized difference vegetation index or estimates based on forest inventories may not be sufficient.     

Intraprotein radical transfer during photoactivation of DNA photolyase

Amino-acid radicals play key roles in many enzymatic reactions. Catalysis often involves transfer of a radical character within the protein, as in class I ribonucleotide reductase where radical transfer occurs over 35 A, from a tyrosyl radical to a cysteine. It is currently debated whether this kind of long-range transfer occurs by electron transfer, followed by proton release to create a neutral radical, or by H-atom transfer, that is, simultaneous transfer of electrons and protons. The latter mechanism avoids the energetic cost of charge formation in the low dielectric protein, but it is less robust to structural changes than is electron transfer. Available experimental data do not clearly discriminate between these proposals. We have studied the mechanism of photoactivation (light-induced reduction of the flavin adenine dinucleotide cofactor) of Escherichia coli DNA photolyase using time-resolved absorption spectroscopy. Here we show that the excited flavin adenine dinucleotide radical abstracts an electron from a nearby tryptophan in 30 ps. After subsequent electron transfer along a chain of three tryptophans, the most remote tryptophan (as a cation radical) releases a proton to the solvent in about 300 ns, showing that electron transfer occurs before proton dissociation. A similar process may take place in photolyase-like blue-light receptors.     

Rewarding effects of opiates are absent in mice lacking the receptor for substance P

Modulation of substance P activity offers a radical new approach to the management of depression, anxiety and stress. The substance P receptor is highly expressed in areas of the brain that are implicated in these behaviours, but also in other areas such as the nucleus accumbens which mediate the motivational properties of both natural rewards such as food and of drugs of abuse such as opiates. Here we show a loss of the rewarding properties of morphine in mice with a genetic disruption of the substance P receptor. The loss was specific to morphine, as both groups of mice responded when cocaine or food were used as rewards. The physical response to opiate withdrawal was also reduced in substance P receptor knockout mice. We conclude that substance P has an important and specific role in mediating the motivational aspects of opiates and may represent a new pharmacological route for the control of drug abuse.     

CpG methylation is maintained in human cancer cells lacking DNMT1

Hypermethylation is associated with the silencing of tumour susceptibility genes in several forms of cancer; however, the mechanisms responsible for this aberrant methylation are poorly understood. The prototypic DNA methyltransferase, DNMT1, has been widely assumed to be responsible for most of the methylation of the human genome, including the abnormal methylation found in cancers. To test this hypothesis, we disrupted the DNMT1 gene through homologous recombination in human colorectal carcinoma cells. Here we show that cells lacking DNMT1 exhibited markedly decreased cellular DNA methyltransferase activity, but there was only a 20% decrease in overall genomic methylation. Although juxtacentromeric satellites became significantly demethylated, most of the loci that we analysed, including the tumour suppressor gene p16INK4a, remained fully methylated and silenced. These results indicate that DNMT1 has an unsuspected degree of regional specificity in human cells and that methylating activities other than DNMT1 can maintain the methylation of most of the genome.     

Superconductivity in molecular crystals induced by charge injection

Progress in the field of superconductivity is often linked to the discovery of new classes of materials, with the layered copper oxides being a particularly impressive example. The superconductors known today include a wide spectrum of materials, ranging in complexity from simple elemental metals, to alloys and binary compounds of metals, to multi-component compounds of metals and chalcogens or metalloids, doped fullerenes and organic charge-transfer salts. Here we present a new class of superconductors: insulating organic molecular crystals that are made metallic through charge injection. The first examples are pentacene, tetracene and anthracene, the last having the highest transition temperature, at 4 K. We anticipate that many other organic molecular crystals can also be made superconducting by this method, which will lead to surprising findings in the vast composition space of molecular crystals.