Response of aged parkinsonian monkeys to in vivo gene transfer of GDNF.

This study assessed the potential for functional and anatomical recovery of the diseased aged primate nigrostriatal system, in response to trophic factor gene transfer. Aged rhesus monkeys received a single intracarotid infusion of MPTP, followed one week later by MRI-guided stereotaxic intrastriatal and intranigral injections of lentiviral vectors encoding for glial derived neurotrophic factor (lenti-GDNF) or beta-galactosidase (lenti-LacZ). Functional analysis revealed that the lenti-GDNF, but not lenti-LacZ treated monkeys displayed behavioral improvements that were associated with increased fluorodopa uptake in the striatum ipsilateral to lenti-GDNF treatment. GDNF ELISA of striatal brain samples confirmed increased GDNF expression in lenti-GDNF treated aged animals that correlated with functional improvements and preserved nigrostriatal dopaminergic markers. Our results indicate that the aged primate brain challenged by MPTP administration has the potential to respond to trophic factor delivery and that the degree of neuroprotection depends on GDNF levels.

Consecutive aquatic jump-gliding with water-reactive fuel.

Robotic vehicles that are capable of autonomously transitioning between various terrains and fluids have received notable attention in the past decade due to their potential to navigate previously unexplored and/or unpredictable environments. Specifically, aerial-aquatic mobility will enable robots to operate in cluttered aquatic environments and carry out a variety of sensing tasks. One of the principal challenges in the development of such vehicles is that the transition from water to flight is a power-intensive process. At a small scale, this is made more difficult by the limitations of electromechanical actuation and the unfavorable scaling of the physics involved. This paper investigates the use of solid reactants as a combustion gas source for consecutive aquatic jump-gliding sequences. We present an untethered robot that is capable of multiple launches from the water surface and of transitioning from jetting to a glide. The power required for aquatic jump-gliding is obtained by reacting calcium carbide powder with the available environmental water to produce combustible acetylene gas, allowing the robot to rapidly reach flight speed from water. The 160-gram robot could achieve a flight distance of 26 meters using 0.2 gram of calcium carbide. Here, the combustion process, jetting phase, and glide were modeled numerically and compared with experimental results. Combustion pressure and inertial measurements were collected on board during flight, and the vehicle trajectory and speed were analyzed using external tracking data. The proposed propulsion approach offers a promising solution for future high-power density aerial-aquatic propulsion in robotics.

Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo.

Retroviral vectors derived from lentiviruses such as HIV-1 are promising tools for human gene therapy because they mediate the in vivo delivery and long-term expression of transgenes in nondividing tissues. We describe an HIV vector system in which the virulence genes env, vif, vpr, vpu, and nef have been deleted. This multiply attenuated vector conserved the ability to transduce growth-arrested cells and monocyte-derived macrophages in culture, and could efficiently deliver genes in vivo into adult neurons. These data demonstrate the potential of lentiviral vectors in human gene therapy.

Transgene expression in the guinea pig cochlea mediated by a lentivirus-derived gene transfer vector.

The utility of lentivirus as a gene delivery vector in the cochlea was evaluated in vitro and in vivo. Lentivirus transduction was assessed through expression analysis of a reporter gene, green fluorescent protein (GFP), integrated within the viral genome. In vitro characterization of lentivirus-GFP was assessed by infection of explants from cochleas of neonatal rat. The lentiviral vector transduced both spiral ganglion neurons (SGNs) and glial cells. In vivo characterization of lentivirus-GFP was assessed by directly infusing the vector into the guinea pig cochlea via an osmotic minipump. Sections of lentivirus-infused cochlea revealed a highly restricted fluorescence pattern limited to the periphery of the perilymphatic space. Transduction of SGNs and glial cells by lentivirus in vitro but not in vivo suggests limited dissemination of the viral vector from the perilymphatic space. The cellular and tissue architecture of the lentivirus-infused cochlea was intact and free of inflammation. Restricted transduction of cell types confined to the periphery of the perilymphatic space by the lentivirus is ideal for stable production of gene products secreted into the perilymph.

Self-inactivating lentiviral vectors with enhanced transgene expression as potential gene transfer system in Parkinson's disease.

Glial cell line-derived neurotrophic factor (GDNF) is able to protect dopaminergic neurons against various insults and constitutes therefore a promising candidate for the treatment of Parkinson's disease. Lentiviral vectors that infect quiescent neuronal cells may allow the localized delivery of GDNF, thus avoiding potential side effects related to the activation of other brain structures. To test this hypothesis in a setting ensuring both maximal biosafety and optimal transgene expression, a self-inactivating (SIN) lentiviral vector was modified by insertion of the posttranscriptional regulatory element of the woodchuck hepatitis virus, and particles were produced with a multiply attenuated packaging system. After a single injection of 2 microl of a lacZ-expressing vector (SIN-W-LacZ) in the substantia nigra of adult rats, an average of 40.1 +/- 6.0% of the tyrosine hydroxylase (TH)-positive neurons were transduced as compared with 5.0 +/- 2.1% with the first-generation lentiviral vector. Moreover, the SIN-W vector expressing GDNF under the control of the mouse phosphoglycerate kinase 1 (PGK) promoter was able to protect nigral dopaminergic neurons after medial forebrain bundle axotomy. Expression of hGDNF in the nanogram range was detected in extracts of mesencephalon of animals injected with an SIN-W-PGK-GDNF vector, whereas it was undetectable in animals injected with a control vector. Lentiviral vectors with enhanced expression and safety features further establish the potential use of these vectors for the local delivery of bioactive molecules into defined structures of the central nervous system.

Heme C incorporation into the c-type cytochromes FixO and FixP is essential for assembly of the Bradyrhizobium japonicum cbb3-type oxidase.

The monoheme and diheme c-type cytochromes FixO and FixP are two of the subunits of the respiratory cbb3-type oxidase of Bradyrhizobium japonicum. The cysteines of the respective heme C binding motifs CXXCH were changed to serines by site-directed mutagenesis, which led to inactive oxidases in all mutants. Western blot analyses showed that an intact heme binding site in the FixO polypeptide is a prerequisite not only for the synthesis of holo-FixO protein but also for the formation of the entire cbb3-type oxidase complex. Both heme binding sites of FixP were essential for maturation and assembly of this subunit. It was not possible to create stable FixP variants that contained only one heme C.

Histidine 131, not histidine 43, of the Bradyrhizobium japonicum FixN protein is exposed towards the periplasm and essential for the function of the cbb3-type cytochrome oxidase.

In subunit I (FixN) of the Bradyrhizobium japonicum cbb3-type oxidase, only five instead of the normal six strictly conserved histidines (H) could be unambiguously assigned as the putative heme or copper ligands. The ambiguity concerned H43 or H131 as the presumptive N-terminal ligands of the low-spin heme B. We report here that a H43A replacement had a wild-type phenotype, whereas the H131A mutant was defective in oxidase function and subunit assembly or stability, suggesting that H131 serves as the N-terminal low-spin heme ligand. Topological studies revealed that H131 resides on the periplasmic side of helix 2, where one of the low-spin heme ligands is normally found in conventional heme-copper oxidases.

The Bradyrhizobium japonicum fixGHIS genes are required for the formation of the high-affinity cbb3-type cytochrome oxidase.

We report structural and functional analyses of the Bradyrhizobium japonicum fixGHIS genes, which map immediately downstream of the fixNOQP operon for the symbiotically essential cbb3-type heme-copper oxidase complex. Expression of fixGHIS, like that of fixNOQP, is strongly induced in cells grown microaerobically or anaerobically. A fixGHI deletion led to the same prominent phenotypes as those known from a fixNOQP deletion: defective symbiotic nitrogen fixation (Fix-) and decreased cytochrome oxidase activity in cells grown under oxygen deprivation. Only traces, if any, of cytochrome cbb3 subunits were present in membranes isolated from the delta fixGHI strain, as revealed by Western blot analysis with subunit-specific antibodies. This effect was not due to lack of fixNOQP transcription. The results suggested a critical involvement of the fixGHIS gene products in the assembly and/or stability of the cbb3-type heme-copper oxidase. On the basis of sequence similarities between the FixI protein and a Cu-transporting P-type ATPase (CopA) of Enterococcus hirae, and between FixG and a membrane-bound oxidoreductase (RdxA) of Rhodobacter sphaeroides, we postulate that a membrane-bound FixGHIS complex might play a role in uptake and metabolism of copper required for the cbb3-type heme-copper oxidase.

A stable system for the high-titer production of multiply attenuated lentiviral vectors.

Lentiviral vectors open exciting perspectives for the genetic treatment of a wide array of inherited and acquired diseases, owing to their ability to govern the efficient delivery, integration, and long-term expression of transgenes into nondividing cells both in vitro and in vivo. The genomic complexity of HIV, where a whole set of genes encode virulence factors essential for pathogenesis but not required for gene transfer, allowed a major step toward clinical acceptability through the creation of multiply attenuated packaging systems. Until now, however, vector particles could only be produced by transient transfection because no high-output, stable packaging cell line was available that produced the latest generation of HIV-based vectors. Here we describe such a line, based on the doxycycline-repressible expression of HIV-1 Rev/Gag/Pol and of the vesicular stomatitis virus G envelope (VSV G) in 293 human embryonic kidney cells. Upon induction, the LVG clones can produce 1 to 20 HeLa-transducing units per cell per day for about a week, a yield that compares favorably with that of transiently transfected 293T cells. These virions exhibit functional properties similar to those of viruses produced transiently, in particular the ability to transduce nonmitotic targets. This system will facilitate the further development of lentiviral vectors for gene therapy.

The A985G mutation in the medium-chain acyl-CoA dehydrogenase gene: high prevalence in the Swiss population resident in Geneva.

We have determined the frequency of the A985G mutation in the medium-chain acyl-CoA dehydrogenase (MCAD) gene in a cohort of 1142 healthy babies born in two Geneva hospitals. Among babies with at least one Swiss parent, heterozygotes were detected at a frequency of 1/52, with a 95% confidence range from 1/82 to 1/38. The high frequency of the carrier state for this mutation suggests that MCAD-deficient babies are born with a frequency of 1/10,000 in the Swiss population. This number is in sharp contrast with the low number of symptomatic MCAD-deficient patients diagnosed in this country. Thus, the fraction of homozygotes who remain asymptomatic is likely to be very high in the Swiss population, and possibly higher than in other countries of northern Europe.

Assembly and function of the cytochrome cbb3 oxidase subunits in Bradyrhizobium japonicum.

The Bradyrhizobium japonicum cbb3-type cytochrome oxidase, which supports microaerobic respiration, is a multisubunit enzyme encoded by the genes of the fixNOQP operon. We investigated the contribution of the individual subunits to function and assembly of the membrane-bound complex. In-frame deletion mutants of fixN, fixO, and fixQ, and an insertion mutant of fixP were constructed. All mutants, except the fixQ mutant, showed clearly altered absorption difference spectra of their membranes and decreased oxidase activities, and they were unable to fix nitrogen symbiotically. The presence of the individual subunits was assayed by Western blot analysis, using subunit-specific antibodies, and by heme staining of the c-type cytochromes FixO and FixP. These analyses led to the following conclusions: (i) FixN and FixO are necessary for assembly of the multimeric oxidase, (ii) FixN and FixO assemble independently of FixP, and (iii) FixQ is not required for complex formation and, therefore, does not seem to be an essential subunit. The possible oxidase biogenesis pathway involves the formation of a primary core complex consisting of FixN and FixO, which allows the subsequent association with FixP to form the complete enzyme.

[Silicosis in Switzerland. A half century of observation. The physician's viewpoint]. / La silicose en Suisse. Un demi-siècle d'observation. Le point de vue du médecin.

In Switzerland, the first cases of silicosis were recorded by Zangger in 1900. Since 1930, patients with silicosis have been provided with certain services by the "Caisse Nationale Suisse d'Assurances" (i.e. Swiss National Insurance Fund), an organization enforcing the Occupational Accidents and Diseases Bill. However, it took another two years before an effectively organized struggle against silicosis was started. Eventually, by 1938, this specific pneumoconiosis was acknowledged as an occupational disease under Swiss Law. Thus, the CNSA has been concerned with this disease for half a century, and it seems relevant now to take stock of the situation. From 1930 to late 1980, 9690 cases of silicosis were accepted by the CNSA. Nearly one half (46%) of these silicotic patients were still alive on December 31, 1980; another third had died of silicosis and the rest of other affections not related The origin of cases has remained remarkably constant over the course of time. Underground working and the stone-working industry account for the majority of cases (70%), followed by smelting works (16%) and the ceramic industry (5%). The remaining 9% are due to various causes. Silicosis hazards have declined but still remain real. In late 1980, 1287 companies in Switzerland were being monitored from this standpoint. More than two-thirds (67%) belong to the stone-working industry (even though only 30% of hazard-exposed workers are employed in this sector), 10% are involved in underground work (10% of hazard-exposed workers), 10% are smelting industries (36% exposed) and 6% belong to the ceramic industry (17% exposed). Since 1950, the number of hazard-exposed people has fluctuated between relatively narrow limits (i.e. 15,000 and 20,000). Corresponding figures for previous periods are not known. Some facts indicate that silicosis is becoming less problematic: a) The annual incidence rate of silicosis in Switzerland has evolved in three distinct phases. From 1930 to 1940 the number of new cases recorded each year rose regularly. From 1940 to the late 1960s, the incidence levelled off (200-300 cases yearly); then, from 1974, it dropped rather sharply and less than 100 new cases have been recorded yearly since 1978 (97 in 1978, 69 in 1979 and 68 in 1980). b) The average age at diagnosis of silicosis has regularly increased. Until 1940, the average age of recorded silicotic patients was about 40. The threshold of 50 years was reached between 1953 and 1957. More recently (between 1978 and 1980) the corresponding figure was 68.2.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of tGLP-1, a Golgi and lysosome-associated, transmembrane glycoprotein of African trypanosomes.

Purification of endosomal/lysosomal vesicles of Trypanosoma brucei brucei bloodstream forms and generation of monoclonal antibodies led to the isolation of antibodies directed against an 85 kDa, Golgi and endocytic traffic-associated protein termed tGLP-1, Trypanosoma Golgi/lysosome protein-1. Preliminary immunoelectron microscopical analysis revealed that the protein is present in, but not restricted to, the limiting membrane of multivesicular lysosomes and is more abundant in bloodstream forms compared to the procyclic stage. The corresponding gene was cloned and is present as a single copy. Blast searches did not reveal any homologies to other proteins and genes published. The nucleotide sequence of the gene (1848 base pairs) predicted a type 1 membrane topology with an N-terminal signal sequence (20 aa), a luminal domain with 2 N-glycosylation sites (524 aa), a transmembrane domain (23 aa), and a long cytosolic tail domain (49 aa). Polyclonal antibodies raised against the cytosolic tail confirmed the localization of the gene product to multivesicular lysosomes but revealed that the majority of the protein was in the Golgi apparatus. Colabelling with an antibody against p67, a lysosomal glycoprotein of trypanosomes, revealed extensive overlap between the proteins with opposing relative abundance. Expression of the tGLP-1 open reading frame in Leishmania resulted in Golgi localization, and in Toxoplasma, in localization to both the Golgi and endoplasmic reticulum. These data indicate conservation in the functionality of the Golgi-targeting sequence of tGLP-1.

How replacements of the 12 conserved histidines of subunit I affect assembly, cofactor binding, and enzymatic activity of the Bradyrhizobium japonicum cbb3-type oxidase.

Alignments of the amino acid sequences of subunit I (FixN or CcoN) of the cbb3-type oxidases show 12 conserved histidines. Six of them are diagnostic of heme-copper oxidases and are thought to bind the following cofactors: the low spin heme B and the binuclear high spin heme B-CuB center. The other six are FixN(CcoN)-specific and their function is unknown. To analyze the contribution of these 12 invariant histidines of FixN in cofactor binding and function of the Bradyrhizobium japonicum cbb3-type oxidase, they were substituted by valine or alanine by site-directed mutagenesis. The H131A mutant enzyme had already been reported previously to be defective in oxidase assembly and function (Zufferey, R., Th¿ny-Meyer, L., and Hennecke, H. (1996) FEBS Lett. 394, 349-352). Four of the remaining histidines were not essential for activity or assembly (positions 226, 246, 333, and 457); by contrast, histidines 331, 410, and 418 were required both for activity and stability of the enzyme. The last group of mutant enzymes, H420A, H280A, H330A, and H316V, were assembled but not functional. To purify the latter mutant proteins and the wild-type enzyme, a six-histidine tag was added to the C terminus of subunit I. The His6-tagged cbb3-oxidase complexes were purified 20-fold by a three-step purification protocol. With the exception of the H420A mutant oxidase, the mutant enzymes H280A, H316V, and H331A contained normal amounts of copper and heme B, and they displayed similar visible light spectroscopic characteristics like the wild-type His6-tagged enzyme. The His6-tagged H420A mutant oxidase differed from the His6-tagged wild-type protein by showing altered visible light spectroscopic characteristics. No stable mutant oxidase lacking copper or heme B was obtained. This strongly suggests that copper and heme B incorporations in subunit I are prerequisites for assembly of the enzyme.

A specific screen for oligosaccharyltransferase mutations identifies the 9 kDa OST5 protein required for optimal activity in vivo and in vitro.

The central reaction in the process of N-linked protein glycosylation in eukaryotic cells, the transfer of the oligosaccharide Glc(3)Man(9)GlcNAc(2) from the lipid dolicholpyrophosphate to selected asparagine residues, is catalyzed by the oligosaccharyltransferase (OTase). This enzyme consists of multiple subunits; however, purification of the complex has revealed different results with respect to its protein composition. To determine how many different loci are required for OTase activity in vivo, we performed a novel, specific screen for mutants with altered OTase activity. Based on the synthetic lethal phenotype of OTase mutants in combination with a deficiency of dolicholphosphoglucose biosynthesis which results in non-glucosylated lipid-linked oligosaccharide, we identified seven complementation groups with decreased OTase activity. Beside the known OTase loci, STT3, OST1, WBP1, OST3, SWP1 and OST2, a novel locus, OST5, was identified. OST5 is an intron-containing gene encoding a putative membrane protein of 9.5 kDa present in highly purified OTase preparations. OST5 protein is not essential for growth but its depletion results in a reduced OTase activity. Suppression of an ost1 mutation by overexpression of OST5 indicates that this small membrane protein directly interacts with other OTase components, most likely with Ost1p. A strong genetic interaction with a stt3 mutation implies a role in complex assembly.

A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum.

It has been a long-standing hypothesis that the endosymbiotic rhizobia (bacteroids) cope with a concentration of 10 to 20 nM free O2 in legume root nodules by the use of a specialized respiratory electron transport chain terminating with an oxidase that ought to have a high affinity for O2. Previously, we suggested that the microaerobically and anaerobically induced fixNOQP operon of Bradyrhizobium japonicum might code for such a special oxidase. Here we report the biochemical characteristics of this terminal oxidase after a 27-fold enrichment from membranes of anaerobically grown B. japonicum wild-type cells. The purified oxidase has TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine) oxidase activity as well as cytochrome c oxidase activity. N-terminal amino acid sequencing of its major constituent subunits confirmed that presence of the fixN,fixO, and fixP gene products. FixN is a highly hydrophobic, heme B-binding protein. FixO and FixP are membrane-anchored c-type cytochromes (apparent Mrs of 29,000 and 31,000, respectively), as shown by their peroxidase activities in sodium dodecyl sulfate-polyacrylamide gels. All oxidase properties are diagnostic for it to be a member of the cbb3-type subfamily of heme-copper oxidases. The FixP protein was immunologically detectable in membranes isolated from root nodule bacteroids, and 85% of the total cytochrome c oxidase activity in bacteroid membranes was contributed by the cbb3-type oxidase. The Km values for O2 of the purified enzyme and of membranes from different B. japonicum wild-type and mutant strains were determined by a spectrophotometric method with oxygenated soybean leghemoglobin as the sole O2 delivery system. The derived Km value for O2 of the cbb3-type oxidase in membranes was 7 nM, which is six- to eightfold lower than that determined for the aerobic aa3-type cytochrome c oxidase. We conclude that the cbb3-type oxidase supports microaerobic respiration in endosymbiotic bacteroids.

Overproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichia coli.

We report on a system to improve expression of mature c-type cytochromes in Escherichia coli. It is based on the use of plasmid pEC86 that expresses the E. coli cytochrome c maturation genes ccmABCDEFGH constitutively, whereby the production of both endogenous and foreign c-type cytochromes was increased substantially. The periplasmic soluble domains of the c-type cytochrome subunits FixO and FixP of the Bradyrhizobium japonicum cbb3 oxidase could be expressed in E. coli only when pEC86 was provided in a degP-deficient strain. This shows that a stimulation of heme attachment by the Ccm maturase system combined with the diminished proteolytic activity in the periplasm can increase c-type cytochrome yields.

Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors.

The expression of genes delivered by retroviral vectors is often inefficient, a potential obstacle for their widespread use in human gene therapy. Here, we explored the possibility that the posttranscriptional regulatory element of woodchuck hepatitis virus (WPRE) might help resolve this problem. Insertion of the WPRE in the 3' untranslated region of coding sequences carried by either oncoretroviral or lentiviral vectors substantially increased their levels of expression in a transgene-, promoter- and vector-independent manner. The WPRE thus increased either luciferase or green fluorescent protein production five- to eightfold, and effects of a comparable magnitude were observed with either the immediate-early cytomegalovirus or the herpesvirus thymidine kinase promoter and with both human immunodeficiency virus- and murine leukemia virus-based vectors. The WPRE exerted this influence only when placed in the sense orientation, consistent with its predicted posttranscriptional mechanism of action. These results demonstrate that the WPRE significantly improves the performance of retroviral vectors and emphasize that posttranscriptional regulation of gene expression should be taken into account in the design of gene delivery systems.

Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery.

In vivo transduction of nondividing cells by human immunodeficiency virus type 1 (HIV-1)-based vectors results in transgene expression that is stable over several months. However, the use of HIV-1 vectors raises concerns about their safety. Here we describe a self-inactivating HIV-1 vector with a 400-nucleotide deletion in the 3' long terminal repeat (LTR). The deletion, which includes the TATA box, abolished the LTR promoter activity but did not affect vector titers or transgene expression in vitro. The self-inactivating vector transduced neurons in vivo as efficiently as a vector with full-length LTRs. The inactivation design achieved in this work improves significantly the biosafety of HIV-derived vectors, as it reduces the likelihood that replication-competent retroviruses will originate in the vector producer and target cells, and hampers recombination with wild-type HIV in an infected host. Moreover, it improves the potential performance of the vector by removing LTR sequences previously associated with transcriptional interference and suppression in vivo and by allowing the construction of more-stringent tissue-specific or regulatable vectors.