Aquaporins are multifunctional water and solute transporters highly divergent in living organisms.

Aquaporins (AQPs) are ubiquitous membrane proteins whose identification, pioneered by Peter Agre's team in the early nineties, provided a molecular basis for transmembrane water transport, which was previously thought to occur only by free diffusion. AQPs are members of the Major Intrinsic Protein (MIP) family and often referred to as water channels. In mammals and plants they are present in almost all organs and tissues and their function is mostly associated to water molecule movement. However, recent studies have pointed out a wider range of substrates for these proteins as well as complex regulation levels and pathways. Although their relative abundance in plants and mammals makes it difficult to investigate the role of a particular AQP, the use of knock-out and mutagenesis techniques is now bringing important clues regarding the direct implication of specific AQPs in animal pathologies or plant deficiencies. The present paper gives an overview about AQP structure, function and regulation in a broad range of living organisms. Emphasis will be given on plant AQPs where the high number and diversity of these transport proteins, together with some emerging aspects of their functionalities, make them behave more like multifunctional, highly adapted channels rather than simple water pores.

Isolation and characterization of cDNA clones encoding the skeletal and smooth muscle Xenopus laevis beta tropomyosin isoforms.

cDNAs clones corresponding to the skeletal and smooth muscle beta tropomyosins isoforms were isolated from a Xenopus laevis embryo cDNA library. Sequence analysis indicated that the two isoforms are coded by a single gene that uses two couples of alternative exons. The expression of the X. laevis beta tropomyosin gene closely resembles that of the mammalian gene but differs from the avian gene.

Developmental program expression of myosin alkali light chain and skeletal actin genes in the rainbow trout Oncorhynchus mykiss.

We have isolated MLC1(F) (tMLC1(F)), MLC3(F) (tMLC3(F)) and skeletal actin cDNAs from the teleost Oncorhynchus mykiss. Sequence analysis indicates that tMLC1(F) and tMLC3(F) are not produced from differentially spliced mRNAs as reported in avians and rodents but are encoded by different genes. Results from RNase protection analysis showed that the corresponding transcripts are expressed in fast skeletal muscles. Whole-mount in situ hybridisation revealed distinct expression patterns of the myosin alkali light chains and skeletal actin genes during skeletal muscle development in the embryo.

Two skeletal alpha-tropomyosin transcripts with distinct 3'UTR have different temporal and spatial patterns of expression in the striated muscle lineages of Xenopus laevis.

The Xenopus laevis alpha-tropomyosin (TM) gene, like its vertebrates counterparts, encodes muscle and non-muscle isoforms through two promoters and alternatively spliced exons. In the present study we describe a cDNA clone (XTMalpha7) encoding a skeletal muscle isoform of the gene that differs from the previously described skeletal TM transcript (XTMalpha2) by its 3'UTR sequence. The two skeletal alpha-TM encoding mRNAs are generated through distinct 3'end processing using different polyA signals and distinct patterns of exon splicing. Using RNAse protection and RNA in situ hybridization, we have analysed the developmental and spatial expression of the two transcripts. Both are expressed in the embryo, but XTMalpha7 is by far the most prevalent of the two. In contrast, only XTMalpha2 is expressed in adult striated muscle tissues. In the embryo, the spatial expression of XTMalpha7 is restricted to the somites whereas XTMalpha2 is expressed in both somites and embryonic heart.

Differential expression of two skeletal muscle beta-tropomyosin mRNAs during Xenopus laevis development.

A cDNA clone for a Xenopus laevis skeletal muscle beta-tropomyosin (beta-TMad) isoform was isolated from an adult skeletal muscle cDNA library. Sequence analysis revealed that this clone corresponded to a second beta-tropomyosin mRNA distinct from the one that was previously characterized (beta-TMemb). The two skeletal beta-TM mRNAs originate from distinct genes and are differentially expressed during development. Beta-TMemb mRNA is expressed only in the somites of the early embryo while beta-TMad mRNA is expressed in pre-metamorphic tadpoles and adult skeletal muscles. We have isolated the promoter region of the beta-TMemb gene and shown that a DNA construct containing 2.9 kb of promoter region is properly expressed after injection in the embryo.

The Xenopus laevis TM-4 gene encodes non-muscle and cardiac tropomyosin isoforms through alternative splicing.

A full-length cDNA clone coding for a 248-amino-acid tropomyosin (TM) was isolated from a Xenopus laevis (Xl) oocyte cDNA library. This TM is very similar to the members of the non-muscle (nm) TM family that includes rat TM-4, human TM30pl and chicken cardiac fibroblast FT-C. An RNase protection assay showed that the corresponding transcript is expressed ubiquitously and revealed the presence of an alternative transcript in adult heart RNA. A full-length cDNA clone was isolated from an adult heart cDNA library using a nm TM-encoding cDNA probe. It encodes a muscle TM homologous to chicken FT-C and the corresponding mRNA is expressed in adult RNA and to a very low level in adult skeletal muscle. The two cDNAs correspond to two alternatively spliced isoforms of TM generated from the Xl TM-4 gene. These data, together with published observations, demonstrated that, in this amphibian, the cardiac muscle TM are synthesized from the TM alpha and TM-4 genes.

The cloning and characterization of a cDNA encoding Xenopus laevis DNA ligase I.

A cDNA clone coding for DNA ligase I (LigI) was isolated from a Xenopus laevis oocyte cDNA library. The 3766-bp sequence showed a putative ORF capable of encoding a 1070-amino-acid protein whose overall identity with two mammalian sequences is 63%. This identity, however, rises to 72.5% in the C-terminal portion of the protein that contains the active site. Expression of the cDNA in a prokaryotic system produces a protein that is immunologically identical to LigI and can be adenylated. The 180-kDa size of the recombinant protein is similar to the LigI detected in oocyte. Northern blot analysis of ovary and embryo RNAs revealed the expression of two (4.1 and 6 kb) LigI transcripts.

A novel tropomyosin isoform encoded by the Xenopus laevis alpha-TM gene is expressed in the brain.

A full-length cDNA clone encoding a novel non-muscle tropomyosin (nmTM) termed X alpha TMB5, as yet unidentified in vertebrates, was isolated from a Xenopus laevis (Xl) brain cDNA library. X alpha TMB5 is derived from the XL alpha-tropomyosin gene (X alpha TM), which was previously found to express striated muscle and nmTM isoforms via an alternative splicing mechanism. The deduced amino-acid sequence reveals that X alpha TMB5 contains 248 amino acids. The protein differs from the skeletal muscle alpha-TM isoform only at its NH2-terminal region. The mRNA encoding X alpha TMB5 is expressed mainly in brain and striated muscle. Genomic clone analysis reveals that, unlike mammals and avian, the X alpha TM gene is devoid of the brain-specific exon 9c. The amphibian alpha-TM gene is a complex transcription unit containing 14 exons, including two alternative promoters, two internal mutually exclusive exons, and two alternatively spliced 3' exons that encode two different COOH-terminal coding regions. Therefore, a total of at least five distinct mRNAs are expressed from the X alpha TM gene in a cell-type-specific manner.

Towards amperometric immunosensor devices.

In contrast to optical immunosensors, the electrochemical detection of an immunanalytical reaction does require a labeling, but allows an easier discrimination of specific and non-specific binding. We present a concept and first results for a multivalent amperometric immunosensor system which is based on silicon technology. The capture molecule streptavidin, covalently immobilized on silica, allows the immobilization of biotinylated antigens at a defined density. A nanostructured gold electrode serving as a stable network of nanowires is expected to be beneficial for the electrochemical detection of bound ferrocene-labeled antibody molecules. The results presented focus on site-specific immobilization of streptavidin on silica and reduction of non-specific binding of proteins.

An array of Pt-tip microelectrodes for extracellular monitoring of activity of brain slices.

A microelectrode array (MEA) consisting of 34 silicon nitride passivated Pt-tip microelectrodes embedded on a perforated silicon substrate (porosity 35%) has been realized. The electrodes are 47 microns high, of which only the top 15 microns are exposed Pt-tips having a curvature of 0.5 micron. The MEA is intended for extracellular recordings of brain slices in vitro. Here we report the fabrication, characterization and initial electrophysiological evaluation of the first generation of Pt-tip MEAs.

Biocompatibility of silicon-based arrays of electrodes coupled to organotypic hippocampal brain slice cultures.

In this study we examined the passive biocompatibility of a three-dimensional microelectrode array (MEA), designed to be coupled to organotypic brain slice cultures for multisite recording of electrophysiological signals. Hippocampal (and corticostriatal) brain slices from 1-week-old (and newborn) rats were grown for 4-8 weeks on the perforated silicon chips with silicon nitride surfaces and 40 microm sized holes and compared with corresponding tissue slices grown on conventional semiporous membranes. In terms of preservation of the basic cellular and connective organization, as visualized by Nissl staining, Timm sulphide silver-staining, microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP) immunostaining, the slice cultures grown on chips did not differ from conventionally grown slice cultures. Neither were there any signs of astrogliosis or neurodegeneration around the upper recording part of the 47-microm-high platinum-tip electrodes. Slice cultures grown on a separate set of chips with platinum instead of silicon nitride surfaces also displayed normal MAP2 and GFAP immunostaining. The width of the GFAP-rich zone (glia limitans) at the bottom surface of the slice cultures was the same ( approximately 20 microm) in cultures grown on chips with silicon nitride and platinum surfaces and on conventional insert membranes. The slice cultures grown on chips maintained a normal, subfield differentiated susceptibility to the glutamate receptor agonist N-methyl-D-aspartate (NMDA) and the neurotoxin trimethyltin (TMT), as demonstrated by the cellular uptake of propidium iodide (PI), which was used as a reproducible and quantifiable marker for neuronal degeneration. We conclude that organotypic brain slice cultures can grow on silicon-based three-dimensional microelectrode arrays and develop normally with display of normal subfield differentiated susceptibilities to known excito- and neurotoxins. From this it is anticipated that the set-up, designed for recording of electrophysiological parameters, can be used for long-term studies of defined neuronal networks and provide valuable information on both normal, neurotoxicological and neuropathological conditions.

Microelectrode arrays for electrophysiological monitoring of hippocampal organotypic slice cultures.

A three-dimensional platinum (Pt) microelectrode array embedded on a micromachined silicon (Si) substrate (porosity of 13%, via hole diameter of 40 microns) has been developed. Electrodes are 35-micron wide and 20-microns high, spaced 200 microns apart and arranged in an elliptic geometry. Integrated within a microperfusion chamber, the devices were used for stimulation and recording experiments of hippocampal slice cultures over a period of several days.

Characterization of a mammalian smooth muscle cell line that has retained transcriptional and posttranscriptional potencies.

Unlike skeletal and cardiac muscle cells that differentiate irreversibly, smooth muscle cells (SMCs) retain a high degree of plasticity. During the so-called phenotypic modulation, SMCs can undergo transition between a contractile phenotype and a highly proliferative synthetic phenotype, as apparent from the extinction of numerous smooth muscle (SM) markers when they are passaged in culture. It would be very useful to have an SMC line that can be indefinitely propagated for the cellular and molecular analysis of the mechanisms that underlie the control of SM differentiation. This report describes an immortalized rabbit aorta SMC-derived cell line (U8A4) that has conserved differentiated properties through multiple subcultures. U8A4 cells can grow in the absence of serum and express the SMC markers studied, including SM alpha-actin, SM calponin, SM22alpha, SM alpha-tropomyosin (alpha-TM), SM myosin heavy chain (SM-MHC), and myocardin. U8A4 cells can activate SMC-restricted promoters like those of SM22alpha, SM calponin, and SM-MHC genes as efficiently as described previously for rat SMC lines (PAC1, A7r5, and A10). These cells can also process exogenous alpha-TM transcripts according to an SM-specific pattern. These results demonstrate that the U8A4 cell line constitutes a good alternative model to existing SMC lines that could facilitate the study of the transcriptional and posttranscriptional regulatory mechanisms underlying SMC differentiation.

Potential of freezing in wastewater treatment: soluble pollutant applications.

When wastewater containing only soluble pollutants is frozen gradually, ice crystals grow from the pure water only, while pollutants are rejected into the liquid phase thus increasing their concentration. In this way, pure water can be removed from various wastewaters. Two kinds of wastewater were studied: synthetic wastewater containing water and one or more soluble pollutants, and industrial wastewaters (urban wastewater and cutting oil wastewater). In most cases, separation efficiency close to 100% was achieved. A large range of pollutant concentrations was studied in order to determine the limits of freezing separation.

DNA ligase I from Xenopus laevis eggs.

We have purified the major DNA ligase from Xenopus laevis eggs and raised antibodies against it. Estimates from SDS PAGE indicate that this DNA ligase is a 180 kDa protein. This enzyme is similar to the mammalian type I DNA ligase which is presumed to be involved in DNA replication. We have also analysed DNA ligase activity during X. laevis early development. Unfertilized eggs contain the highest level of activity reflecting the requirement for a large amount of DNA replicative enzymes for the period of intense replication following fertilization. In contrast with previous studies on the amphibians axolotl and Pleurodeles, the major DNA ligase activity detected during X. laevis early development is catalysed by a single enzyme: DNA ligase I. And the presence of this DNA ligase I in Xenopus egg before fertilization clearly demonstrates that the exclusion process of two forms of DNA ligase does not occur during X. laevis early development.

Isolation of the messenger RNA for 8S DNA ligase in early developing axolotl egg and its cell free translation.

A new DNA ligase activity is expressed when the Axolotl eggs enter cleavage. The messenger RNA can be labelled by [3H] uridine thereby indicating its de novo synthesis. This new genetic expression is occurring just before cleavage and is the earliest found during Amphibian development. The newly synthesized [3H] mRNA can be translated in vitro in the rabbit reticulocyte lysate system. The resulting product is a 160 K protein specifically immunoprecipitated with the antiserum directed against 8S DNA ligase. This in vitro translated polypeptide exhibits 8S DNA ligase activity specific of activated or fertilized eggs but does not display 6S DNA ligase activity of non activated eggs.

Evidence for a change in molecular for of DNA ligase in early development of the sea urchin Psammechinus miliaris.

A change in the molecular form of DNA ligase appears when the sea urchin egg enters cleavage. Sucrose gradient analysis and DNA cellulose chromatography show that a slower migrating form (7 S) of enzyme exists in unfertilized eggs and in sperm. A faster migrating form of DNA ligase (7.8 S) is present in developing embryos as well as in artificially activated eggs. The timing of this early biochemical event has been determined, following fertilization or activation. The change in molecular form of DNA ligase has been shown to be sensitive to drugs inhibiting protein synthesis, gene transcription, or DNA replication. Consequently the appearance of the faster migrating form of enzyme is assumed to result from expression of the corresponding gene, transcription, and translation. RNA extracted from testes and from cleaving stages, assayed in vitro and in vivo, have been shown to carry the information for, respectively, 7 S and 7.8 S DNA ligase according to the origin of the RNA.

Glider and Vision: two new families of miniature inverted-repeat transposable elements in Xenopus laevis genome.

We have characterised from Xenopus laevis two new short interspersed repetitive elements, we have named Glider and Vision, that belong to the family of miniature inverted-repeat transposable elements (MITEs). Glider was first characterised in an intronic region of the alpha-tropomyosin (alpha-TM) gene and database search has revealed the presence of this element in 10 other Xenopus laevis genes. Glider elements are about 150 bp long and for some of them, their terminal inverted repeats are flanked by potential target-site duplications. Evidence for the mobility of Glider element has been provided by the presence/absence of one element at corresponding location in duplicated alpha-TM genes. Vision element has been identified in the promoter region of the cyclin dependant kinase 2 gene (cdk2) where it is boxed in a Glider element. Vision is 284bp long and is framed by 14-bp terminal inverted repeats that are flanked by 7-bp direct repeats. We have estimated that there are about 20,000 and 300 copies of Glider and Vision respectively scattered throughout the Xenopus laevis genome. Every MITEs elements but two described in our study are found either in 5' or in 3' regulatory regions of genes suggesting a potential role in gene regulation.

Reinvestigation of DNA ligase I in axolotl and Pleurodeles development.

We have recently shown that the exclusion process causing the replacement of DNA ligases II by DNA ligase I in amphibian eggs after fertilization does not occur in the case of Xenopus laevis [Hardy, S., Aoufouchi, S., Thiebaud, P., and Prigent, C., (1991) Nucleic Acids Res. 19, 701-705]. Since this result is in contradiction with the situation reported in axolotl and Pleurodeles we decided to reinvestigate such results in both species. Three different approaches have been used: (1) the substrate specificity of DNA ligase I; (2) the DNA ligase-AMP adduct reaction and (3) the immunological detection using antibodies raised against the X.laevis DNA ligase I. Our results clearly demonstrate that DNA ligase I activity is associated with a single polypeptide which is present in oocyte, unfertilized egg and embryo of both amphibians. Therefore, the hypothesis of a change in DNA ligase forms, resulting from an expression of the DNA ligase I gene in axolotl and Pleurodeles early development must be rejected. We also show that, in contradiction with published data, the unfertilized sea urchin egg contains a DNA ligase activity able to join blunt ended DNA molecules.