Patterns of calcium-binding proteins in human inferior colliculus: identification of subdivisions and evidence for putative parallel systems.

The subdivisions of human inferior colliculus are currently based on Golgi and Nissl-stained preparations. We have investigated the distribution of calcium-binding protein immunoreactivity in the human inferior colliculus and found complementary or mutually exclusive localisations of parvalbumin versus calbindin D-28k and calretinin staining. The central nucleus of the inferior colliculus but not the surrounding regions contained parvalbumin-positive neuronal somata and fibres. Calbindin-positive neurons and fibres were concentrated in the dorsal aspect of the central nucleus and in structures surrounding it: the dorsal cortex, the lateral lemniscus, the ventrolateral nucleus, and the intercollicular region. In the dorsal cortex, labelling of calbindin and calretinin revealed four distinct layers.Thus, calcium-binding protein reactivity reveals in the human inferior colliculus distinct neuronal populations that are anatomically segregated. The different calcium-binding protein-defined subdivisions may belong to parallel auditory pathways that were previously demonstrated in non-human primates, and they may constitute a first indication of parallel processing in human subcortical auditory structures.

Identifying knots in proteins.

Polypeptide chains form open knots in many proteins. How these knotted proteins fold and finding the evolutionary advantage provided by these knots are among some of the key questions currently being studied in the protein folding field. The detection and identification of protein knots are substantial challenges. Different methods and many variations of them have been employed, but they can give different results for the same protein. In the present article, we review the various knot identification algorithms and compare their relative strengths when applied to the study of knots in proteins. We show that the statistical approach based on the uniform closure method is advantageous in comparison with other methods used to characterize protein knots.

Controlled expression of recombinant proteins in Physcomitrella patens by a conditional heat-shock promoter: a tool for plant research and biotechnology.

The ability to express tightly controlled amounts of endogenous and recombinant proteins in plant cells is an essential tool for research and biotechnology. Here, the inducibility of the soybean heat-shock Gmhsp17.3B promoter was addressed in the moss Physcomitrella patens, using beta-glucuronidase (GUS) and an F-actin marker (GFP-talin) as reporter proteins. In stably transformed moss lines, Gmhsp17.3B-driven GUS expression was extremely low at 25 degrees C. In contrast, a short non-damaging heat-treatment at 38 degrees C rapidly induced reporter expression over three orders of magnitude, enabling GUS accumulation and the labelling of F-actin cytoskeleton in all cell types and tissues. Induction levels were tightly proportional to the temperature and duration of the heat treatment, allowing fine-tuning of protein expression. Repeated heating/cooling cycles led to the massive GUS accumulation, up to 2.3% of the total soluble proteins. The anti-inflammatory drug acetyl salicylic acid (ASA) and the membrane-fluidiser benzyl alcohol (BA) also induced GUS expression at 25 degrees C, allowing the production of recombinant proteins without heat-treatment. The Gmhsp17.3B promoter thus provides a reliable versatile conditional promoter for the controlled expression of recombinant proteins in the moss P. patens.

Ultra-High Field (7 Tesla) MRI for Gamma Knife Surgery Targeting of the Ventro-Intermediate Nucleus: A Pilot in Vivo Study

Aim: Gamma Knife surgery (GKS) is a non-invasive neurosurgical stereotactic procedure, increasingly used as an alternative to open functional procedures. This includes the targeting of the ventro-intermediate (Vim) nucleus of the thalamus for tremor. We currently perform an indirect targeting, using the "quadrilatere of Guyot," as the Vim nucleus is not visible on current 3 Tesla (T) MRI acquisitions. The primary objective of the current study was to enhance anatomic imaging for Vim GKS using high-field (7 T) MRI, with the aim of refining the visualization and precision of anatomical targeting. Method: Five young healthy subjects (mean age 23 years) were scanned both on 3 and 7 T MRI in Lausanne University Hospital (CHUV) and Center for Biomedical Imaging (CIBM). Classical T1-weighted MPRAGE, T2 CISS sequences (replacing former ventriculography) and diffusion tensor imaging were acquired at 3T. We obtained high-resolution susceptibility weighted images (SWI) at 7T for the visualization of thalamic subparts. SWI was further integrated for the first time into Leksell Gamma Plan® (LGP) software and co-registered with the 3T images. A simulation of targeting of the Vim was done using the "quadrilatere of Guyot" methodology on the 3T images. Furthermore, a correlation with the position of the found target on SWI was performed. The atlas of Morel et al. was used to confirm the findings on a detailed computer analysis outside LGP. Also, 3T and 7T MRI of one patient undergoing GKS Vim thalamotomy, were obtained before and 2 years after the procedure, and studied similarly. Results: The use of SWI provided a superior resolution and improved image contrast within the central gray matter. This allowed visualization and direct delineation of groups of thalamic nuclei in vivo, including the Vim. The position of the target, as assessed with the "quadrilatere of Guyot" method on 3 T, perfectly matched with the supposed one of the Vim on the SWI. Furthermore, a 3-dimensional model of the Vim target area was created on the basis of 3T and 7T images. Conclusion: This is the first report of the integration of SWI high-field MRI into the LGP in healthy subjects and in one patient treated GKS Vim thalamotomy. This approach aims at the improvement of targeting validation and further direct targeting of the Vim in tremor. The anatomical correlation between the direct visualization on 7T and the current targeting methods on 3T seems to show a very good anatomical matching.

The emerging importance of the SPX domain-containing proteins in phosphate homeostasis

Plant growth and development are strongly influenced by the availability of nutrients in the soil solution. Among them, phosphorus (P) is one of the most essential and most limiting macro-elements for plants. In the environment, plants are often confronted with P starvation as a result of extremely low concentrations of soluble inorganic phosphate (Pi) in the soil. To cope with these conditions, plants have developed a wide spectrum of mechanisms aimed at increasing P use efficiency. At the molecular level, recent studies have shown that several proteins carrying the SPX domain are essential for maintaining Pi homeostasis in plants. The SPX domain is found in numerous eukaryotic proteins, including several proteins from the yeast PHO regulon, involved in maintaining Pi homeostasis. In plants, proteins harboring the SPX domain are classified into four families based on the presence of additional domains in their structure, namely the SPX, SPX-EXS, SPX-MFS and SPX-RING families. In this review, we highlight the recent findings regarding the key roles of the proteins containing the SPX domain in phosphate signaling, as well as providing further research directions in order to improve our knowledge on P nutrition in plants, thus enabling the generation of plants with better P use efficiency.

Clinical and molecular characterization of Diastrophic Dysplasia in the Portuguese population.

SLC26A2-related dysplasias encompass a spectrum of diseases: from lethal achondrogenesis type 1B (ACG1B; MIM #600972) and atelosteogenesis type 2 (AO2; MIM #256050) to classical diastrophic dysplasia (cDTD; MIM #222600) and recessive multiple epiphyseal dysplasia (rMED; MIM #226900). This study aimed at characterizing clinically, radiologically and molecularly 14 patients affected by non-lethal SLC26A2-related dysplasias and at evaluating genotype-phenotype correlation. Phenotypically, eight patients were classified as cDTD, four patients as rMED and two patients had an intermediate phenotype (mild DTD - mDTD, previously 'DTD variant'). The Arg279Trp mutation was present in all patients, either in homozygosity (resulting in rMED) or in compound heterozygosity with the known severe alleles Arg178Ter or Asn425Asp (resulting in DTD) or with the mutation c.727-1G>C (causing mDTD). The 'Finnish mutation', c.-26+2T>C, and the p.Cys653Ser, both frequent mutations in non-Portuguese populations, were not identified in any of the patients of our cohort and are probably very rare in the Portuguese population. A targeted mutation analysis for p.Arg279Trp and p.Arg178Ter in the Portuguese population allows the identification of approximately 90% of the pathogenic alleles.

Simultaneous quantification of selective serotonin reuptake inhibitors and metabolites in human plasma by liquid chromatography-electrospray mass spectrometry for therapeutic drug monitoring.

A simple and sensitive liquid chromatography-electrospray ionization mass spectrometry method was developed for the simultaneous quantification in human plasma of all selective serotonin reuptake inhibitors (citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline) and their main active metabolites (desmethyl-citalopram and norfluoxetine). A stable isotope-labeled internal standard was used for each analyte to compensate for the global method variability, including extraction and ionization variations. After sample (250μl) pre-treatment with acetonitrile (500μl) to precipitate proteins, a fast solid-phase extraction procedure was performed using mixed mode Oasis MCX 96-well plate. Chromatographic separation was achieved in less than 9.0min on a XBridge C18 column (2.1×100mm; 3.5μm) using a gradient of ammonium acetate (pH 8.1; 50mM) and acetonitrile as mobile phase at a flow rate of 0.3ml/min. The method was fully validated according to Société Française des Sciences et Techniques Pharmaceutiques protocols and the latest Food and Drug Administration guidelines. Six point calibration curves were used to cover a large concentration range of 1-500ng/ml for citalopram, desmethyl-citalopram, paroxetine and sertraline, 1-1000ng/ml for fluoxetine and fluvoxamine, and 2-1000ng/ml for norfluoxetine. Good quantitative performances were achieved in terms of trueness (84.2-109.6%), repeatability (0.9-14.6%) and intermediate precision (1.8-18.0%) in the entire assay range including the lower limit of quantification. Internal standard-normalized matrix effects were lower than 13%. The accuracy profiles (total error) were mainly included in the acceptance limits of ±30% for biological samples. The method was successfully applied for routine therapeutic drug monitoring of more than 1600 patient plasma samples over 9 months. The β-expectation tolerance intervals determined during the validation phase were coherent with the results of quality control samples analyzed during routine use. This method is therefore precise and suitable both for therapeutic drug monitoring and pharmacokinetic studies in most clinical laboratories.

Evolution of proteins after whole-genome duplication

Evolution of proteins after whole-genome duplicationGene and genome duplication are considered major mechanisms in the creation of newfunctions in genomes, or in the refinement of networks by the division of function amongmore genes. In animals, the best demonstrated whole genome duplication occurred at theorigin of Teleost fishes. This makes fishes an ideal model to study the consequences ofgenome duplication, particularly since we have a good sampling of genome sequences,abundant functional information, and a very well studied outgroup: the tetrapodes (includinghuman). More specifically, I studied the consequences of duplication on proteins usingevolutionary models to infer adaptive events. I analysed the influence of positive selection invertebrate genes, by contrasting singleton genes and duplicated genes. The conclusion of theanalyses was threefold: (i) positive selection affects diverse phylogenetic branches anddiverse gene categories during vertebrate evolution; (ii) it concerns only a small proportion ofsites (1%-5%); and (iii) whole genome duplication had no detectable impact on theprevalence of this positive selection.I also studied evolution at the amino acid level with different methods to detect functionalshifts (covarion process and constant-but-different process). As in my previous research, Ifound similar numbers of functional shifts between duplicates and between orthologs.The accepted framework for studies of molecular evolution is that orthologs share the samefunction, whereas the function of paralogs diverges. This framework gives a special place togene duplication in evolution, as the main mechanism for generating novelty. With myprevious results showing that duplication and speciation are not so different, we investigatedthe literature to question the evidence for similar or divergent evolution of gene function afterduplication relative to speciation genes. This led us to propose a more rigorous design offuture studies of gene duplication.Finally, based on my automated protocol, we built a database of positive selection invertebrates' genes, Selectome. This database is freely available on the web and will helpfuture evolutionary as well as biochemical studies.

Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones.

When massively expressed in bacteria, recombinant proteins often tend to misfold and accumulate as soluble and insoluble nonfunctional aggregates. A general strategy to improve the native folding of recombinant proteins is to increase the cellular concentration of viscous organic compounds, termed osmolytes, or of molecular chaperones that can prevent aggregation and can actively scavenge and convert aggregates into natively refoldable species. In this study, metal affinity purification (immobilized metal ion affinity chromatography [IMAC]), confirmed by resistance to trypsin digestion, was used to distinguish soluble aggregates from soluble nativelike proteins. Salt-induced accumulation of osmolytes during induced protein synthesis significantly improved IMAC yields of folding-recalcitrant proteins. Yet, the highest yields were obtained with cells coexpressing plasmid-encoded molecular chaperones DnaK-DnaJ-GrpE, ClpB, GroEL-GroES, and IbpA/B. Addition of the membrane fluidizer heat shock-inducer benzyl alcohol (BA) to the bacterial medium resulted in similar high yields as with plasmid-mediated chaperone coexpression. Our results suggest that simple BA-mediated induction of endogenous chaperones can substitute for the more demanding approach of chaperone coexpression. Combined strategies of osmolyte-induced native folding with heat-, BA-, or plasmid-induced chaperone coexpression can be thought to optimize yields of natively folded recombinant proteins in bacteria, for research and biotechnological purposes.

Different behaviour of mouse-human chimeric antibody F(ab')2 fragments of IgG1, IgG2 and IgG4 sub-class in vivo.

Mouse-human chimeric monoclonal antibodies (MAbs) of 3 different human IgG sub-classes directed against carcinoembryonic antigen (CEA) have been produced in SP-0 cells transfected with genomic chimeric DNA. F(ab')2 fragments were obtained by pepsin digestion of the purified chimeric MAbs of human IgG1, IgG2 and IgG4 sub-class and of parental mouse MAb IgG1. The 4 F(ab')2 fragments exhibit similar molecular weight by SDS-PAGE. They were labelled with 125I or 131I and high binding (80 to 87%) to purified unsolubilized CEA was observed. In vivo, double labelling experiments indicate that the longest biological half-life and the highest tumour-localization capacity is obtained with F(ab')2 from chimeric MAb of human IgG2 sub-class, whereas F(ab')2 from chimeric MAb IgG4 give very low values for these 2 parameters. F(ab')2 from chimeric MAb IgG1 and from parental mouse MAb yield intermediate results in vivo. Our findings should help to select the appropriate human IgG sub-class to produce chimeric or reshaped MAb F(ab')2 to be used for tumour detection by immunoscintigraphy and for radioimmunotherapy.

Epigenetic regulation of histone H3 serine 10 phosphorylation status by HCF-1 proteins in C. elegans and mammalian cells.

BACKGROUND: The human herpes simplex virus (HSV) host cell factor HCF-1 is a transcriptional coregulator that associates with both histone methyl- and acetyltransferases, and a histone deacetylase and regulates cell proliferation and division. In HSV-infected cells, HCF-1 associates with the viral protein VP16 to promote formation of a multiprotein-DNA transcriptional activator complex. The ability of HCF proteins to stabilize this VP16-induced complex has been conserved in diverse animal species including Drosophila melanogaster and Caenorhabditis elegans suggesting that VP16 targets a conserved cellular function of HCF-1. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the role of HCF proteins in animal development, we have characterized the effects of loss of the HCF-1 homolog in C. elegans, called Ce HCF-1. Two large hcf-1 deletion mutants (pk924 and ok559) are viable but display reduced fertility. Loss of Ce HCF-1 protein at reduced temperatures (e.g., 12 degrees C), however, leads to a high incidence of embryonic lethality and early embryonic mitotic and cytokinetic defects reminiscent of mammalian cell-division defects upon loss of HCF-1 function. Even when viable, however, at normal temperature, mutant embryos display reduced levels of phospho-histone H3 serine 10 (H3S10P), a modification implicated in both transcriptional and mitotic regulation. Mammalian cells with defective HCF-1 also display defects in mitotic H3S10P status. CONCLUSIONS/SIGNIFICANCE: These results suggest that HCF-1 proteins possess conserved roles in the regulation of cell division and mitotic histone phosphorylation.

Differential phosphorylation of tau proteins during kitten brain development and Alzheimer's disease.

Differential distribution and phosphorylation of tau proteins were studied in developing kitten brain by using several antibodies, and was compared to phosphorylation in Alzheimer's disease. Several antibodies demonstrated the presence of phosphorylated tau proteins during kitten brain development and identified pathological structures in human brain tissue. Antibody AD2, recognized tau in kittens and adult cats, but reacted in Alzheimer's tissue only with a pathological tau form. Antibody AT8 was prominent in developing kitten neurons and was found in axons and dendrites. After the first postnatal month this phosphorylation type disappeared from axons. Furthermore, dephosphorylation of kitten tau with alkaline phosphatase abolished immunoreactivity of AT8, but not that of AD2, pointing to a protection of the AD2 epitope in cats. Tau proteins during early cat brain development are phosphorylated at several sites that are also phosphorylated in paired helical filaments during Alzheimer's disease. In either event, phosphorylation of tau may play a crucial role to modulate microtubule dynamics, contributing to increased microtubule instability and promoting growth of processes during neuronal development or changing dynamic properties of the cytoskeleton and contributing to the formation of pathological structures in neurodegenerative diseases.

Human immunoglobulins and Fc fragments promote microtubule assembly via tau proteins and induce conformational changes of neuronal microtubules in vitro.

The influence of human immunoglobulins (Ig) in neuronal cytoskeleton stability was studied in vitro. Here we show that human Ig and Fc fragments stimulate animal and human microtubule assembly by binding to microtubules via tau isoforms. In presence of Ig, microtubules show increased aggregation, twisting and rigidity. Non-immune Ig and Fc fragments promote microtubule assembly in temperature-dependent manner and stabilize microtubules at a molecular ratio of 1 Ig per 4 tubulin dimers. These in vitro data provide an experimental support for an immuno-mediated modulation of the cytoskeleton. In conjunction with previous neuropathological data, they suggest that Ig could participate in early stages of neurodegeneration by affecting the microtubule stability in vivo.