Biotherapies of neuromuscular disorders.

This review focuses on the most recent data on biotherapeutic approaches, using DNA, RNA, recombinant proteins, or cells as therapeutic tools or targets for the treatment of neuromuscular diseases. Many of these novel technologies have now reached the clinical stage and have or are about to move to the market. Others, like genome editing are still in an early stage but hold great promise.

Variations in the microcystin production of Planktothrix rubescens (cyanobacteria) assessed from a four-year survey of Lac du Bourget (France) and from laboratory experiments.

Between 1999 and 2002, a routine survey of water quality in the Lac du Bourget was performed to study the dynamics and microcystin (MC) production of Planktothrix rubescens. Using liquid chromatography coupled to diode array detection and mass spectrometry, we found that two main variants ([D-Asp3] and [D-Asp3, Dhb7] microcystin-RR) were produced. The proportion of these two variants was not influenced by the depth or season of sampling. Expressed in microcystin-LR equivalents, high microcystin concentrations were recorded from August to December each year, reaching values of up to 6.7 microg L-1. A significant correlation was found between the microcystin cell content and the cell densities of P. rubescens. Cellular quotas of microcystins ranged from 0.1 to 0.3 pg cell-1. Simultaneously, laboratory experiments were performed on a strain of P. rubescens isolated from the lake to assess the potential impact of various P-PO4 (3-) concentrations on intra- and extracellular microcystin production. Unlike natural populations, this strain only produced [D-Asp3] MC-RR. The intracellular microcystin content was similarly correlated to the cell density, but the cellular quota was slightly higher (0.3-0.7 pg cell-1) than in the natural population. Again, as in the natural population, a linear relationship was found between growth rate and microcystin production rate. These findings support the hypothesis that environmental factors, such as phosphate concentrations, have no direct impact on microcystin production by P. rubescens, but act indirectly by affecting growth rate.

Toxicological comparison of diverse Cylindrospermopsis raciborskii strains: evidence of liver damage caused by a French C raciborskii strain.

The freshwater cyanobacterium Cylindrospermopsis raciborskii is known to produce toxic effects in several countries. Acute and chronic exposures to C. raciborskii in Australia have been linked to liver damage (hepatotoxicity) with concomitant effects on the kidneys, adrenal glands, small intestine, lungs, thymus, and heart. The alkaloid cylindrospermopsin, which produces these toxic effects, is thought to be a potent inhibitor of protein synthesis. C. raciborskii strains producing cylindrospermopsin or analogue alkaloids have also been reported in Florida, USA, and Thailand. Brazilian isolates of C. raciborskii are also toxic but act by a different mechanism, causing acute death in mice with neurotoxic symptoms similar to those induced by the saxitoxins. In this article we compare the toxicity in the mouse of a C. raciborskii French strain with C. raciborskii strains from various other sources (Australia, Brazil, Mexico, and Hungary). We tested the toxicity of cell extracts by a mouse bioassay. Acute, fatal neurotoxicity was produced by the Brazilian strain, which was confirmed by liquid chromatography with fluorescence detection of the cell extracts, which revealed the presence of saxitoxin, neosaxitoxin, and decarbamoylsaxitoxin, along with two unidentified compounds. Acute hepatotoxicity with severe liver, kidney, and thymus damage was observed with the Australian cylindrospermopsin-producing strain. The Mexican and Hungarian strains were not found to be toxic to mice in our experimental conditions. No animals died after exposure to the extracts of the French C. raciborskii strain. Histological examination of the liver revealed moderate, multifocal necrosis characterized by small areas of hepatocellular necrosis, combined with disorganization of the parenchyma and congestion of the inner sinusoid. These symptoms and lesions resembled those induced by cylindrospermopsin, but the chemical analysis performed by liquid chromatography coupled with either a diode array detector or a mass spectrometer demonstrated that this toxin was not present in our culture extract.

Environmental context of Cylindrospermopsis raciborskii (Cyanobacteria) blooms in a shallow pond in France.

Cylindrospermopsis raciborskii, a potentially toxic blooming cyanobacterium (blue-green alga), responsible for public health problems in Australia, was identified in France in 1994 in a shallow pond south of Paris. A program monitoring the occurrence of C. raciborskii in this pond was conducted from July 1998 to October 1999. The phytoplankton assemblages were studied, and limnological parameters (water temperature, dissolved oxygen, pH, conductivity, and dissolved inorganic nutrients) were measured. By multivariate analysis (principal component analysis), we showed that sufficiently high temperatures to allow the germination of akinetes, relatively low nutrient concentrations (soluble reactive phosphorus with a mean concentration of 1 microM and nitrate between 0 and 5 microM, except in February 1999 (21 microM)) and a characteristic high and constant sulfate concentration (8981+/-471 microM) seemed to be the main factors involved in the proliferation of C. raciborskii in the "Francs-Pêcheurs" (FP) pond. In the light of these findings and of bibliographic data, C. raciborskii would seem to be characterized by good adaptability, but also by low competitiveness with other phytoplanktonic species in the temperate study area.

Partners of Rpb8p, a small subunit shared by yeast RNA polymerases I, II and III.

Rpb8p, a subunit common to the three yeast RNA polymerases, is conserved among eukaryotes and absent from noneukaryotes. Defective mutants were found at an invariant GGLLM motif and at two other highly conserved amino acids. With one exception, they are clustered on the Rpb8p structure. They all impair a two-hybrid interaction with a fragment conserved in the largest subunits of RNA polymerases I (Rpa190p), II (Rpb1p), and III (Rpc160p). This fragment corresponds to the pore 1 module of the RNA polymerase II crystal structure and bears a highly conserved motif (P.I.KP.LW.GKQ) facing the GGLLM motif of Rpb8p. An RNA polymerase I mutant (rpa190-G728D) at the invariant glycyl of P.I.KP.LW.GKQ provokes a temperature-sensitive defect. Increasing the gene dosage of another common subunit, Rpb6p, suppresses this phenotype. It also suppresses a conditional growth defect observed when replacing Rpb8p by its human counterpart. Hence, Rpb6p and Rpb8p functionally interact in vivo. These two subunits are spatially separated by the pore 1 module and may also be possibly connected by the disorganized N half of Rpb6p, not included in the present structure data. Human Rpb6p is phosphorylated at its N-terminal Ser2, but an alanyl replacement at this position still complements an rpb6-Delta null allele. A two-hybrid interaction also occurs between Rpb8p and the product of orphan gene YGR089w. A ygr089-Delta null mutant has no detectable growth defect but aggravates the conditional growth defect of rpb8 mutants, suggesting that the interaction with Rpb8p may be physiologically relevant.

Cross talk between tRNA and rRNA synthesis in Saccharomyces cerevisiae.

Temperature-sensitive RNA polymerase III (rpc160-112 and rpc160-270) mutants were analyzed for the synthesis of tRNAs and rRNAs in vivo, using a double-isotopic-labeling technique in which cells are pulse-labeled with [(33)P]orthophosphate and coextracted with [(3)H]uracil-labeled wild-type cells. Individual RNA species were monitored by Northern blot hybridization or amplified by reverse transcription. These mutants impaired the synthesis of RNA polymerase III transcripts with little or no influence on mRNA synthesis but also largely turned off the formation of the 25S, 18S, and 5.8S mature rRNA species derived from the common 35S transcript produced by RNA polymerase I. In the rpc160-270 mutant, this parallel inhibition of tRNA and rRNA synthesis also occurred at the permissive temperature (25 degrees C) and correlated with an accumulation of 20S pre-rRNA. In the rpc160-112 mutant, inhibition of rRNA synthesis and the accumulation of 20S pre-rRNA were found only at 37 degrees C. The steady-state rRNA/tRNA ratio of these mutants reflected their tRNA and rRNA synthesis pattern: the rpc160-112 mutant had the threefold shortage in tRNA expected from its preferential defect in tRNA synthesis at 25 degrees C, whereas rpc160-270 cells completely adjusted their rRNA/tRNA ratio down to a wild-type level, consistent with the tight coupling of tRNA and rRNA synthesis in vivo. Finally, an RNA polymerase I (rpa190-2) mutant grown at the permissive temperature had an enhanced level of pre-tRNA, suggesting the existence of a physiological coupling between rRNA synthesis and pre-tRNA processing.

A protein-protein interaction map of yeast RNA polymerase III.

The structure of the yeast RNA polymerase (pol) III was investigated by exhaustive two-hybrid screening using a library of random genomic fragments fused to the Gal4 activation domain. This procedure allowed us to identify contacts between individual polypeptides, localize the contact domains, and deduce a protein-protein interaction map of the multisubunit enzyme. In all but one case, pol III subunits were able to interact in vivo with one or sometimes two partner subunits of the enzyme or with subunits of TFIIIC. Four subunits that are common to pol I, II, and III (ABC27, ABC14.5, ABC10alpha, and ABC10beta), two that are common to pol I and III (AC40 and AC19), and one pol III-specific subunit (C11) can associate with defined regions of the two large subunits. These regions overlapped with highly conserved domains. C53, a pol III-specific subunit, interacted with a 37-kDa polypeptide that copurifies with the enzyme and therefore appears to be a unique pol III subunit (C37). Together with parallel interaction studies based on dosage-dependent suppression of conditional mutants, our data suggest a model of the pol III preinitiation complex.

Intramolecular cross-overs generate deleted mitochondrial DNA molecules in Podospora anserina.

The unavoidable senescence process that limits the vegetative growth of Podospora anserina is always associated with an accumulation of various classes of circular, tandemly arranged, defective mitochondrial DNA molecules (senDNAs). The monomers of the senDNAs belonging to the so-called beta class share a common core, but differ in both their length and termini. To understand the mechanism leading to their formation, we have determined the junction sequence of 36 senDNA beta monomers present in various senescent cultures. In most cases, we observe that: (1) short direct repeats precisely bound the senDNA beta termini and (2) one copy of the repeats is retained in the senDNA sequence. Moreover, PCR analysis of the mitochondrial DNA of some of the senescent cultures, has allowed us to detect another genome which is exactly lacking the sequence of the senDNA beta found in the culture. These results demonstrate that an intramolecular unequal cross-over occurring between short direct repeats can generate deleted mtDNA molecules in P. anserina. In addition, the polymorphism displayed by one pair of repeats allows us to establish that this cross-over may be associated with a short conversion tract spanning a few (about 15) nucleotides.