Balanced reciprocal translocation t(5;13)(q33;q12) and 9q31.1 microduplication in a man suffering from infertility and pollinosis

We describe the first case of two chromosomal abnormalities, balanced reciprocal translocation t(5;13)(q33;q12.1) and a microduplication in the region 9q31.1, in a man suffering from infertility and pollinosis. In the region 13q12.1 is located the TUBA3C (tubulin, alpha 3c) gene, which plays an important dynamic role in the motility of flagella. This case might support the opinion that haploinsufficiency of the TUBA3C gene could be the cause of sperm immotility and abnormal sperm morphology, resulting in infertility in the patient. Single-nucleotide polymorphism (SNP) array analysis revealed a novel 9q31.1 microduplication inherited from both parents, which contributes to the genomic instability.     

A Chimeric HS4-SAR Insulator (IS2) That Prevents Silencing and Enhances Expression of Lentiviral Vectors in Pluripotent Stem Cells

Chromatin insulators, such as the chicken β-globin locus control region hypersensitive site 4 (HS4), and scaffold/matrix attachment regions (SARs/MARs) have been incorporated separately or in combination into retroviral vectors (RVs) in order to increase transgene expression levels, avoid silencing and reduce expression variability. However, their incorporation into RVs either produces a reduction on titer and/or expression levels or do not have sufficient effect on stem cells. In order to develop an improved insulator we decided to combine SAR elements with HS4 insulators. We designed several synthetic shorter SAR elements containing 4 or 5 MAR/SARs recognition signatures (MRS) and studied their effects on a lentiviral vector (LV) expressing eGFP through the SFFV promoter (SE). A 388 bp SAR element containing 5 MRS, named SAR2, was as efficient or superior to the other SARs analyzed. SAR2 enhanced transgene expression and reduced silencing and variability on human embryonic stem cells (hESCs). We next compared the effect of different HS4-based insulators, the HS4-Core (250 bp), the HS4-Ext (400 bp) and the HS4-650 (650 bp). All HS4 elements reduced silencing and expression variability but they also had a negative effect on transgene expression levels and titer. In general, the HS4-650 element had a better overall effect. Based on these data we developed a chimeric insulator, IS2, combining the SAR2 and the HS4-650. When incorporated into the 3′ LTR of the SE LV, the IS2 element was able to enhance expression, avoid silencing and reduce variability of expression on hESCs. Importantly, these effects were maintained after differentiation of the transduced hESCs toward the hematopoietic linage. Neither the HS4-650 nor the SAR2 elements had these effects. The IS2 element is therefore a novel insulator that confers expression stability and enhances expression of LVs on stem cells.     

Tissue-specific autophagy alterations and increased tumorigenesis in mice deficient in Atg4C/autophagin-3

Atg4C/autophagin-3 is a member of a family of cysteine proteinases proposed to be involved in the processing and delipidation of the mammalian orthologues of yeast Atg8, an essential component of an ubiquitin-like modification system required for execution of autophagy. To date, the in vivo role of the different members of this family of proteinases remains unclear. To gain further insights into the functional relevance of Atg4 orthologues, we have generated mutant mice deficient in Atg4C/autophagin-3. These mice are viable and fertile and do not display any obvious abnormalities, indicating that they are able to develop the autophagic response required during the early neonatal period. However, Atg4C-/--starved mice show a decreased autophagic activity in the diaphragm as assessed by immunoblotting studies and by fluorescence microscopic analysis of samples from Atg4C-/- GFP-LC3 transgenic mice. In addition, animals deficient in Atg4C show an increased susceptibility to develop fibrosarcomas induced by chemical carcinogens. Based on these results, we propose that Atg4C is not essential for autophagy development under normal conditions but is required for a proper autophagic response under stressful conditions such as prolonged starvation. We also propose that this enzyme could play an in vivo role in events associated with tumor progression.     

Variation in life history responses of Daphnia to toxic Microcystis aeruginosa

Three clones of Daphnia pulex and two clones of Daphnia longispinawere exposed to toxic Microcystis aeruginosa for 21 days ina lifetable experiment. The growth and reproduction of individualdaphnids were followed daily to study the long-term effectsof toxic Microcystis. Exposure to Microcystis increased mortality,decreased growth, delayed maturation and decreased offspringproduction, indicating nutritional deficiency and toxic effects.We found variation in life history responses between speciesand among clones. Our results suggest that toxic cyanobacteriamay act as a modifying agent in zooplankton communities at boththe species and clonal level.     

Characterization of 69- and 100-kDa forms of 2-5A-synthetase from interferon-treated human cells

The existence of distinct 69- and 100-kDa forms of 2-5A-synthetase in addition to the smaller (40 and 46 kDa) forms has recently been established. Using specific monoclonal antibodies we investigated the induction, synthesis, and activity of 69- and 100-kDa 2',5'-oligoadenylate (2-5A) synthetases in interferon-treated human Daudi cells. Although induction of these synthetases is detectable in cells treated with as little as 1-5 units/ml of human alpha-interferon, higher concentrations are required for maximum synthesis of the 100 kDa than the 69-kDa protein. At 5 units/ml of interferon, enhanced synthesis of both proteins is detectable at 4 h with maximum synthesis occurring between 8 to 12 and 12 to 16 h for 69- and 100-kDa 2-5A-synthetases, respectively. At 24 h after addition of interferon, synthesis of these synthetases declines due to a decrease of active interferon in the culture medium. The synthesis of both synthetases is blocked by actinomycin D, and the half-life of these proteins is estimated to be 8 h. The activities of immunoaffinity purified 69- and 100-kDa synthetases are dependent on double-stranded (ds)RNA but show different requirements for optimum concentration of dsRNA and pH of the reaction. The apparent Km of 69- and 100-kDa synthetases for ATP is 1.7 X 10(-3) M and 3.6 X 10(-3) M, respectively. At optimum conditions for the activity of these enzymes, the pattern of 2',5'-linked oligoadenylates synthesized are different, the 69-kDa protein synthesizing higher oligomers than the 100-kDa species. Taken together, these results indicate that the 69- and 100-kDa 2-5A-synthetases are distinct proteins each with specific characteristics of induction and enzymatic activity.     

Amino acid sequence and function of rebredoxin from Desulfovibrio vulgaris Miyazaki

Rubredoxin was purified from Desulfovibrio vulgaris Miyazaki. It was sequenced and some of its properties determined. Rubredoxin is composed of 52 amino acids. It is highly homologous to that from D. vulgaris Hildenborough. Its N-methionyl residue is partially formalated. The millimolar absorption coefficients of the rubredoxin at 489 nm and 280 are 8.1 and 18.5, respectively, and the standard redox potential is +5 mB, which is slightly higher than those of other rubredoxins. Rubredoxin, as well as cytochrome c-553, was reduced with lactate by the action of lactate dehydrogenase of this organism, and the rection was stimulated with 2-methyl-1, 4-naphthoquinone. It is suggested that rubredoxin, in collaboration with membraous quinone, functions as natural electron carrier for cytoplasmic lactate dehydrogenase of this organism, whereas cytochrome c-553 plays the same role for periplasmic lactate dehydrogenase.     

A family of Ca2+-dependent activator proteins for secretion: comparative analysis of structure, expression, localization, and function

Ca2+-dependent activator protein for secretion (CAPS) 1 is an essential cytosolic component of the protein machinery involved in large dense-core vesicle (LDCV) exocytosis and in the secretion of a subset of neurotransmitters. In the present study, we report the identification, cloning, and comparative characterization of a second mammalian CAPS isoform, CAPS2. The structure of CAPS2 and its function in LDCV exocytosis from PC12 cells are very similar to those of CAPS1. Both isoforms are strongly expressed in neuroendocrine cells and in the brain. In subcellular fractions of the brain, both CAPS isoforms are enriched in synaptic cytosol fractions and also present on vesicular fractions. In contrast to CAPS1, which is expressed almost exclusively in brain and neuroendocrine tissues, CAPS2 is also expressed in lung, liver, and testis. Within the brain, CAPS2 expression seems to be restricted to certain brain regions and cell populations, whereas CAPS1 expression is strong in all neurons. During development, CAPS2 expression is constant between embryonic day 10 and postnatal day 60, whereas CAPS1 expression is very low before birth and increases after postnatal day 0 to reach a plateau at postnatal day 21. Light microscopic data indicate that both CAPS isoforms are specifically enriched in synaptic terminals. Ultrastructural analyses show that CAPS1 is specifically localized to glutamatergic nerve terminals. We conclude that at the functional level, CAPS2 is largely redundant with CAPS1. Differences in the spatial and temporal expression patterns of the two CAPS isoforms most likely reflect as yet unidentified subtle functional differences required in particular cell types or during a particular developmental period. The abundance of CAPS proteins in synaptic terminals indicates that they may also be important for neuronal functions that are not exclusively related to LDCV exocytosis.     

Auditory Short-Term Memory Activation during Score Reading

Performing music on the basis of reading a score requires reading ahead of what is being played in order to anticipate the necessary actions to produce the notes. Score reading thus not only involves the decoding of a visual score and the comparison to the auditory feedback, but also short-term storage of the musical information due to the delay of the auditory feedback during reading ahead. This study investigates the mechanisms of encoding of musical information in short-term memory during such a complicated procedure. There were three parts in this study. First, professional musicians participated in an electroencephalographic (EEG) experiment to study the slow wave potentials during a time interval of short-term memory storage in a situation that requires cross-modal translation and short-term storage of visual material to be compared with delayed auditory material, as it is the case in music score reading. This delayed visual-to-auditory matching task was compared with delayed visual-visual and auditory-auditory matching tasks in terms of EEG topography and voltage amplitudes. Second, an additional behavioural experiment was performed to determine which type of distractor would be the most interfering with the score reading-like task. Third, the self-reported strategies of the participants were also analyzed. All three parts of this study point towards the same conclusion according to which during music score reading, the musician most likely first translates the visual score into an auditory cue, probably starting around 700 or 1300 ms, ready for storage and delayed comparison with the auditory feedback.     

Typing of Verotoxins by DNA Colony Hybridization with Poly- and Oligonucleotide Probes,a Bead-Enzyme-Linked Immunosorbent Assay,and Polymerase Chain Reaction

To identify the type of Verotoxins (VT) produced by Verocytotoxin-producing Escherichia coli (VTEC), a sensitive bead-enzyme-linked immunosorbent assay and polymerase chain reaction with common and specific primers to various VTs (VT1, VT2, VT2vha, VT2vhb, and VT2vp1) were developed. Together with colony hybridization tests with oligo- and polynucleotide probes, these methods were applied to VTEC isolates to type the VT produced. The toxin types of 26 of 37 strains were identified, but the reaction profiles in assays of the remaining 11 strains suggested the existence of new VT2 variants. The application of these identification procedures may be useful as a tool for clinical and epidemiological studies of VTEC infection.