Micro-Watt building blocks for biomedical RF tranceivers.

The development of Wireless Body Area Network (WBAN) is a key point enabling the mobility health. Among the most critical constrains in WBAN implementation is the power consumption of wireless featuring nodes. This work focuses on the development of ultra low power radio building blocks dedicated to 2.4 GHz ISM band. A novel design approach based on device optimization is first presented. It is then applied to the implementation of a Low Noise Amplifier (LNA) and a mixer in a 0.13 µm CMOS technology. The LNA provides a 13.1 dB gain and a 5.3 dB NF for a 60 µW/0.4 V power consumption. The mixer achieves a conversion gain of 17.5 dB and a NF of 12 dB at 0 dBm LO power. It consumes 350 µW for a 0.8 V supply.

Detection of circulating cells expressing chromogranin A gene transcripts in patients with lung neuroendocrine carcinoma.

High grade lung neuroendocrine carcinomas, like small and large cell neuroendocrine carcinomas, pose therapeutic problems. Most initially respond to chemotherapeutic agents, but early relapses are frequent and are resistant to the presently available treatments. Our study reports for the first time the development and evaluation of a test for detecting the presence of circulating tumour cells by measuring chromogranin A gene transcripts with reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blotting. The test is specific and sensitive (detection of 10 cancer cells/ml blood), and only minimally invasive. Positivity is statistically correlated to high grade neuroendocrine carcinomas and to a poor prognosis with a 3-fold higher lethal risk. The test now needs to be assessed for its usefulness as a tool in the initial staging procedures and follow-up by comparison with the recent immunoradiometric assay (RIA) for detection of chromogranin A in the serum.

Identification and characterization of a gene encoding a subtilisin-like serine protease induced during the vegetative incompatibility reaction in Podospora anserina.

In the filamentous fungi, cell fusion between unlike individuals generally triggers a cell-death reaction known as vegetative incompatibility. In Podospora anserina, it was shown that, during this cell-death reaction, there is a strong increase in proteolytic activity. Here, we report the purification of a 36-kDa protease that is induced during the incompatibility reaction. An internal peptide of this protein displayed a strong similarity with the PEPC subtilisin-like serine protease from Aspergillus niger. This led us to use the pepC gene as a probe to clone the homologous gene from P. anserina, which we called pspA. The expression of pspA is upregulated at the transcript level during the progress of the incompatibility reaction. This induction is diminished in strains bearing suppressors of the vegetative incompatibility reaction. The fact that pspA is homologous to PrB, a vacuolar protease involved in autophagy in yeast, suggests that the incompatibility cell-death reaction and autophagy might be related processes.

In vivo aggregation of the HET-s prion protein of the fungus Podospora anserina.

We have proposed that the [Het-s] infectious cytoplasmic element of the filamentous fungus Podospora anserina is the prion form of the HET-s protein. The HET-s protein is involved in a cellular recognition phenomenon characteristic of filamentous fungi and known as heterokaryon incompatibility. Under the prion form, the HET-s protein causes a cell death reaction when co-expressed with the HET-S protein, from which it differs by only 13 amino acid residues. We show here that the HET-s protein can exist as two alternative states, a soluble and an aggregated form in vivo. As shown for the yeast prions, transition to the infectious prion form leads to aggregation of a HET-s--green fluorescent protein (GFP) fusion protein. The HET-s protein is aggregated in vivo when highly expressed. However, we could not demonstrate HET-s aggregation at wild-type expression levels, which could indicate that only a small fraction of the HET-s protein is in its aggregated form in vivo in wild-type [Het-s] strains. The antagonistic HET-S form is soluble even at high expression level. A double amino acid substitution in HET-s (D23A P33H), which abolishes prion infectivity, suppresses in vivo aggregation of the GFP fusion. Together, these results further support the model that the [Het-s] element corresponds to an abnormal self-perpetuating aggregated form of the HET-s protein.

Vegetative incompatibility in filamentous fungi: Podospora and Neurospora provide some clues.

In filamentous fungi, vegetative cell fusion between genotypically distinct individuals leads to a cell-death reaction known as vegetative or heterokaryon incompatibility. Genes involved in this reaction have been characterised molecularly. We can now begin to get a better understanding of the mechanism and the biological significance of this intriguing phenomenon.

Characterization of hch, the Podospora anserina homolog of the het-c heterokaryon incompatibility gene of Neurospora crassa.

The het-c locus controls heterokaryon formation in Neurospora crassa. It is subject to balancing selection operating to maintain polymorphism at that locus in natural populations. We have isolated hch, the het-c homolog from the related species Podospora anserina (hch for het-c homolog), in order to determine if this gene also functions as a het gene in that species. The het-c and hch sequences are highly similar but differ in the region defining allele specificity in N. crassa het-c. Analysis of hch variability in 11 natural P. anserina isolates with different het genotypes revealed no polymorphism. This suggested that hch does not function as a het gene. However, heterologous expression of the N. crassa het-cPA allele in P. anserina triggers a growth defect reminiscent of the het-c incompatibility reaction.

Concept, design and fabrication of smart orthopedic implants.

Extensive clinical experience has been built up using orthopaedic implants instrumented with strain gauges connected to a Wheatstone bridge by means of percutaneous leads. This research showed the medical relevance of the monitoring of the deformation of implants as a powerful tool to evaluate nursing and rehabilitation exercises, for tracing dangerous overloads and anticipating implant failure and also to observe the healing process. The IMPACT 3500 project focuses on the instrumentation of femoral implants with on board sensors: regular Benoist-Girard implants have been modified, to contain a 'sensing cell', and thoroughly tested in vitro and in vivo. The implant deformations are measured with resistive strain gauges, and the signal is transferred to a personal computer for processing and display, via a hard wired connection, or via a telemetry system. Two fully implantable wireless designs, called Linkstrain and Sealstrain, are powered from the outside by magnetic induction. As Sealstrain contains the whole telemetric system in its cavity, the highest miniaturization was required; this seriously deteriorates the efficiency of the inductive power link.

Mutational analysis of the [Het-s] prion analog of Podospora anserina. A short N-terminal peptide allows prion propagation.

The het-s locus is one of nine known het (heterokaryon incompatibility) loci of the fungus Podospora anserina. This locus exists as two wild-type alleles, het-s and het-S, which encode 289 amino acid proteins differing at 13 amino acid positions. The het-s and het-S alleles are incompatible as their coexpression in the same cytoplasm causes a characteristic cell death reaction. We have proposed that the HET-s protein is a prion analog. Strains of the het-s genotype exist in two phenotypic states, the neutral [Het-s*] and the active [Het-s] phenotype. The [Het-s] phenotype is infectious and is transmitted to [Het-s*] strains through cytoplasmic contact. het-s and het-S were associated in a single haploid nucleus to generate a self-incompatible strain that displays a restricted and abnormal growth. In the present article we report the molecular characterization of a collection of mutants that restore the ability of this self-incompatible strain to grow. We also describe the functional analysis of a series of deletion constructs and site-directed mutants. Together, these analyses define positions critical for reactivity and allele specificity. We show that a 112-amino-acid-long N-terminal peptide of HET-s retains [Het-s] activity. Moreover, expression of a mutant het-s allele truncated at position 26 is sufficient to allow propagation of the [Het-s] prion analog.

MOD-D, a Galpha subunit of the fungus Podospora anserina, is involved in both regulation of development and vegetative incompatibility.

Cell death via vegetative incompatibility is widespread in fungi but molecular mechanism and biological function of the process are poorly understood. One way to investigate this phenomenon was to study genes named mod that modified incompatibility reaction. In this study, we cloned the mod-D gene that encodes a Galpha protein. The mod-D mutant strains present developmental defects. Previously, we showed that the mod-E gene encodes an HSP90. The mod-E1 mutation suppresses both vegetative incompatibility and developmental defects due to the mod-D mutation. Moreover, we isolated the PaAC gene, which encodes an adenylate cyclase, as a partial suppressor of the mod-D1 mutation. Our previous results showed that the molecular mechanisms involved in vegetative incompatibility and developmental pathways are connected, suggesting that vegetative incompatibility may result from disorders in some developmental steps. Our new result corroborates the involvement of mod genes in signal transduction pathways. As expected, we showed that an increase in the cAMP level is able to suppress the defects in vegetative growth due to the mod-D1 mutation. However, cAMP increase has no influence on the suppressor effect of the mod-D1 mutation on vegetative incompatibility, suggesting that this suppressor effect is independent of the cAMP pathway.

Regulation of gene expression during the vegetative incompatibility reaction in Podospora anserina. Characterization of three induced genes.

Vegetative incompatibility in fungi limits the formation of viable heterokaryons. It results from the coexpression of incompatible genes in the heterokaryotic cells and leads to a cell death reaction. In Podospora anserina, a modification of gene expression takes place during this reaction, including a strong decrease of total RNA synthesis and the appearance of a new set of proteins. Using in vitro translation of mRNA and separation of protein products by two-dimensional gel electrophoresis, we have shown that the mRNA content of cells is qualitatively modified during the progress of the incompatibility reaction. Thus, gene expression during vegetative incompatibility is regulated, at least in part, by variation of the mRNA content of specific genes. A subtractive cDNA library enriched in sequences preferentially expressed during incompatibility was constructed. This library was used to identify genomic loci corresponding to genes whose mRNA is induced during incompatibility. Three such genes were characterized and named idi genes for genes induced during incompatibility. Their expression profiles suggest that they may be involved in different steps of the incompatibility reaction. The putative IDI proteins encoded by these genes are small proteins with signal peptides. IDI-2 protein is a cysteine-rich protein. IDI-2 and IDI-3 proteins display some similarity in a tryptophan-rich region.

The mod-A suppressor of nonallelic heterokaryon incompatibility in Podospora anserina encodes a proline-rich polypeptide involved in female organ formation.

Vegetative incompatibility in fungi results from the control of heterokaryon formation by the genes present at het loci. Coexpression of antagonistic het genes in the same hyphae leads to a lethal process. In Podospora anserina, self-incompatible strains containing nonallelic incompatible genes in the same nucleus are inviable as the result of a growth arrest and a lytic process. Mutations in suppressor genes (mod genes) can restore the viability. These mod mutations also interfere with developmental processes, which suggests common steps between the incompatibility reaction and cellular differentiation. The mod-A locus, responsible for growth arrest in the self-incompatible strains, is also involved in the control of the development of female organs. The mod-A gene was isolated. An open reading frame 687 amino acids long was identified. The MOD-A-encoded polypeptide is rich in proline residues, which are clustered in a domain containing a motif that displays similarity to SH3-binding motifs, which are known to be involved in protein-protein interactions. Construction of a strain deleted for mod-A confirmed that the product of this gene involved in differentiation is a key regulator of growth arrest associated with vegetative incompatibility.

Characterization of a gene from the filamentous fungus Podospora anserina encoding an aspartyl protease induced upon carbon starvation.

In an attempt to characterize proteases associated with vegetative incompatibility, a Podospora anserina gene (papA) encoding an aspartyl protease (podosporapepsin) was cloned using a heterologous probe. The deduced papA coding region was 1278 nucleotides long, interrupted by a single 71bp intron. The corresponding amino acid sequence presented a high degree of similarity to other aspartyl proteases. Sequence analysis and proteolytic activity measurement suggested that the podosporapepsin could be intracellular rather than secreted. The papA gene was expressed under carbon starvation, but not under nitrogen starvation conditions. Its disruption led to a slight decrease in the growth rate of the mutant strain when bovine serum albumin was the sole carbon source in the medium. Disruption or overexpression of papA gene had no obvious consequence on vegetative incompatibility. Transcription of papA induced by carbon starvation was strongly reduced in the presence of a suppressor of vegetative incompatibility. This result suggests a relationship between adaptation for starvation and vegetative incompatibility.

A mutation in an HSP90 gene affects the sexual cycle and suppresses vegetative incompatibility in the fungus Podospora anserina.

Vegetative incompatibility is widespread in fungi but its molecular mechanism and biological function are still poorly understood. A way to study vegetative incompatibility is to investigate the function of genes whose mutations suppress this phenomenon. In Podospora anserina, these genes are known as mod genes. In addition to suppressing vegetative incompatibility, mod mutations cause some developmental defects. This suggests that the molecular mechanisms of vegetative incompatibility and development pathway are interconnected. The mod-E1 mutation was isolated as a suppressor of the developmental defects of the mod-D2 strain. We show here that mod-E1 also partially suppresses vegetative incompatibility, strengthening the link between development and vegetative incompatibility. mod-E1 is the first suppressor of vegetative incompatibility characterized at the molecular level. It encodes a member of the Hsp90 family, suggesting that development and vegetative incompatibility use common steps of a signal transduction pathway. The involvement of mod-E in the sexual cycle has also been further investigated.

The protein product of the het-s heterokaryon incompatibility gene of the fungus Podospora anserina behaves as a prion analog.

The het-s locus of Podospora anserina is a heterokaryon incompatibility locus. The coexpression of the antagonistic het-s and het-S alleles triggers a lethal reaction that prevents the formation of viable heterokaryons. Strains that contain the het-s allele can display two different phenotypes, [Het-s] or [Het-s*], according to their reactivity in incompatibility. The detection in these phenotypically distinct strains of a protein expressed from the het-s gene indicates that the difference in reactivity depends on a posttranslational difference between two forms of the polypeptide encoded by the het-s gene. This posttranslational modification does not affect the electrophoretic mobility of the protein in SDS/PAGE. Several results suggest a similarity of behavior between the protein encoded by the het-s gene and prions. The [Het-s] character can propagate in [Het-s*] strains as an infectious agent, producing a [Het-s*] --> [Het-s] transition, independently of protein synthesis. Expression of the [Het-s] character requires a functional het-s gene. The protein present in [Het-s] strains is more resistant to proteinase K than that present in [Het-s*] mycelium. Furthermore, overexpression of the het-s gene increases the frequency of the transition from [Het-s*] to [Het-s]. We propose that this transition is the consequence of a self-propagating conformational modification of the protein mediated by the formation of complexes between the two different forms of the polypeptide.

Reactivity in vegetative incompatibility of the HET-E protein of the fungus Podospora anserina is dependent on GTP-binding activity and a WD40 repeated domain.

The het-e gene of the filamentous fungus Podospora anserina is involved in vegetative incompatibility. Co-expression of antagonistic alleles of the unlinked loci het-e and het-c triggers a cell death reaction that prevents the formation of viable heterokaryons between strains that contain incompatible combinations of het-c and het-e alleles. The het-elA gene encodes a polypeptide that contains a putative GTP-binding site and WD40 repeats. The role of these two domains in the reactivity of the HET-E protein in incompatibility was analyzed. An in vitro assay confirmed that the first domain is functional and can bind GTP and not ATP, suggesting that GTP-binding is essential for triggering the incompatibility reaction. The relationship between the number of WD40 repeats and the reactivity of the protein in incompatibility was investigated by estimating this number in different wild-type and mutant het-e alleles. It was deduced that reactive alleles contain a minimal number of ten WD40 repeats. These results demonstrate that the reactivity of the HET-E protein depends on two functional elements, a GTP-binding domain and several WD40 repeats. These motifs are present in separate polypeptides in trimeric G proteins, suggesting that HET-E polypeptides are also involved in signal transduction. Disruption of the het-e locus does not impair the phenotype of strains but DNA hybridization analyses revealed that het-e may belong to a multigenic family.

An additional copy of the adenylate cyclase-encoding gene relieves developmental defects produced by a mutation in a vegetative incompatibility-controlling gene in Podospora anserina.

To identify cellular functions involved in vegetative incompatibility in filamentous fungi, we have initiated the cloning of Podospora anserina (Pa) mod genes. These genes interfere with the lethal reaction triggered by interaction between incompatible het genes. A gene (Pa AC) has been cloned by complementation of developmental defects caused by a mutation in the mod-D gene. This gene encodes a protein of 2145 amino acids (aa)that exhibits strong similarities with many adenylate cyclases (AC). About 65% aa identity has been found between the sequence of the polypeptide encoded by this Pa AC gene and the AC of Neurospora crassa. The organization of peptidic domains in the polypeptide encoded by Pa AC is closely related to that of Saccharomyces cerevisiae CYR1. Restriction-fragment-length polymorphism (RFLP) and genetic analysis have shown that Pa AC and mod-D are distinct genes.

A gene responsible for vegetative incompatibility in the fungus Podospora anserina encodes a protein with a GTP-binding motif and G beta homologous domain.

The het-e-1 gene of the fungus Podospora anserina is responsible for vegetative incompatibility through specific interactions with different alleles of the unlinked gene, het-c. Coexpression of two incompatible genes triggers a cell death reaction that prevents heterokaryon formation. The het-e1 allele has been cloned to get information on the function of the locus. It encodes a putative 1356-amino-acid polypeptide that displays two sequence motifs that have not yet been reported to be present on a single polypeptide. They are a GTP-binding domain and a repeated region that shares similarity with that of the beta-transducin. Contrary to other members of the beta-transducin family, sequence conservation between the repeated units is very strong and the number of repeats is different in wild-type het-e alleles.

Self-cloning in filamentous fungi: application to the construction of endothiapepsin overproducers in Cryphonectria parasitica.

The filamentous ascomycete fungus Cryphonectria parasitica naturally secretes endothiapepsin, an aspartic proteinase. It is cultured on a commercial scale as a source of the milk-clotting enzyme for cheese making. Our objective was to increase enzyme production of an industrial C. parasitica strain by a new technique of self-cloning; it consisted in the screening for transformants producing higher levels of endothiapepsin and having integrated only the DNA fragment of interest. Such genetically improved strains that are devoid of any foreign genes should be more readily acceptable for the production of food-grade enzymes.

Ontogeny of the D1 dopamine receptor in the rat striatonigral system: an immunohistochemical study.

Antibodies were raised against a recombinant protein to analyse the pre- and postnatal ontogeny of the neurons expressing the D1 dopamine receptor in the striatum by immunohistochemistry. We report that D1 immunoreactivity is detectable from gestational day (G) 15 and is distributed homogeneously throughout the striatum from G15 to G18. From G19-20 to postnatal day (P) 3, D1 immunoreactivity becomes heterogeneous and predominates in cell bodies of the patch compartment while very limited immunoreactivity is detectable in the matricial compartment. The differential intensity between patches and matrix reaches its peak around P0. From P2, the pattern of D1 immunoreactivity progressively assumes the homogeneous distribution characteristic of the adult striatum. The expression of D1 mRNA in striatal neurons, as investigated by in situ hybridization, displays a similar pattern during this period. Substance P mRNA is also preferentially expressed in the patch compartment during the same period. D1 immunoreactivity appears at G17 in the substantia nigra as clusters of fibres and increases subsequently until reaching its adult form during the first postnatal week. These results demonstrate that the two compartments of the developing striatum display differential transcriptional and translational activity for the D1 gene and consequently two different and successive patterns of expression of D1 protein: patch neurons first express D1 receptor intensely while matrix neurons express it later and in smaller amounts so that D1 receptor appears transiently during the perinatal period as a marker of the patch compartment.

Sequence diversity and unusual variability at the het-c locus involved in vegetative incompatibility in the fungus Podospora anserina.

The het-c locus of the filamentous fungus Podospora anserina controls heterokaryon formation through genetic interaction with alleles of the unlinked loci het-e and het-d. We have isolated four wild-type and two mutant alleles of the het-c locus. A comparison of the predicted proteins encoded by the different wild-type alleles revealed an unusual high level of amino-acid replacements compared to silent polymorphisms but only one amino-acid difference is sufficient to modify the specificity of het-c alleles. Chimeric genes constructed in vitro may exhibit a new specificity different from that of any known wild-type allele.