Zebrafish serotonin-N-acetyltransferase-2 gene regulation: pineal-restrictive downstream module contains a functional E-box and three photoreceptor conserved elements

Pineal function is defined by a set of very narrowly expressed genes that encode proteins required for photoperiodic transduction and rhythmic melatonin secretion. One of these proteins is serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT), which controls the daily rhythm in melatonin production. Here, pineal-specific expression of the zebrafish aanat-2 (zfaanat-2) was studied using in vivo transient expression analyses of promoter-reporter constructs; this revealed that specificity is determined by two regions located 12 kb away from each other. One is the 5'-flanking region, and the other is a 257-bp sequence, located 6 kb downstream of the transcribed region. This 3'-sequence, designated pineal-restrictive downstream module (PRDM), has a dual function: enhancement of pineal expression and inhibition of extrapineal expression. The former is an autonomic property of PRDM whereas the later function requires interaction with the upstream regulatory region of zfaanat-2. Functional analyses of the PRDM sequence revealed that three photoreceptor conserved elements (TAATC) and a single perfect E-box (CACGTG) are crucial for the dual function of PRDM. These results indicate that pineal specificity of zfaanat-2 is determined by the dual functionality of the PRDM and the interaction between upstream regulatory region and downstream photoreceptor conserved elements and E-box element.     

The lanceolate hair rat phenotype results from a missense mutation in a calcium coordinating site of the desmoglein 4 gene

Desmosomal cadherins are essential cell adhesion molecules present throughout the epidermis and other organs, whose major function is to provide mechanical integrity and stability to epithelial cells in a wide variety of tissues. We recently identified a novel desmoglein family member, Desmoglein 4 (Dsg4), using a positional cloning approach in two families with localized autosomal recessive hypotrichosis (LAH) and in the lanceolate hair (lah) mouse. In this study, we report cloning and identification of the rat Dsg4 gene, in which we discovered a missense mutation in a naturally occurring lanceolate hair (lah) rat mutant. Phenotypic analysis of lah/lah mutant rats revealed a striking hair shaft defect with the appearance of a lance head within defective hair shafts. The mutation disrupts a critical calcium binding site bridging the second and third extracellular domains of Dsg4, likely disrupting extracellular interactions of the protein.     

Depletion of deoxyribonucleoside triphosphate pools in tumor cells by nitric oxide

Nitric oxide displays pro- and anti-tumor activities, prompting further studies to better understand its precise role. Nitric oxide inhibits ribonucleotide reductase (RnR), the limiting enzyme for de novo dNTP synthesis. We report here the first detailed analysis of dNTP variations induced in tumor cells by NO. NO prodrugs induced a depletion in dNTP pools and an activation of the pyrimidine salvage pathway, as did hydroxyurea, the prototypic RnR inhibitor. In the presence of dipyridamole, which blocked salvaged dNTP synthesis, depletion of dNTP pools was also observed in tumor cells cocultured with macrophages expressing the high-output iNOS activity. This effect was rapid, reversible, blocked by NO scavengers, and cGMP independent. It was quantitatively correlated to iNOS activity. In the absence of dipyridamole, NO still induced a decrease in dATP concentration in tumor cells cocultured with macrophages, whereas surprisingly, concentrations of dCTP and dTTP expanded considerably, resulting in a strong imbalance in dNTP pools. NO prodrugs did not cause such an increase in pyrimidine dNTP, suggesting that pyrimidine nucleosides were released by NO-injured macrophages. Altered dNTP levels have been reported to promote mutagenesis and apoptosis. It is suggested that abnormal changes in dNTP pools in tumors might contribute to NO-dependent toxicity.     

Basal defenses induced in pepper by lipopolysaccharides are suppressed by Xanthomonas campestris pv. vesicatoria

The nonpathogenic hrcC mutant of Xanthomonas campestris pv. vesicatoria 85-10::hrpA22 multiplied in pepper leaves if it was mixed with pathogenic strains of X. campestris pv. vesicatoria. Reactions to the mutant alone included localized deposition of phenolics and callose in papillae, and alterations to the plant cell wall leading to increased electron density. Electron microscopy showed that the localized responses were suppressed in the presence of wild-type bacteria but other wall changes occurred at some sites, involving cellulose-rich ingrowth of the wall. Multiplication of the hrp mutant in mixed inocula was confirmed by tagging 85-10::hrpA22 using immunocytochemical location of AvrBs3 expressed from the plasmid pD36. Elicitors of callose deposition and other wall changes were isolated from the hrcC mutant. Activity in extracts of bacteria was attributed to the presence of high molecular weight lipopolysaccharides (LPS). Wild-type X. campestris pv. vesicatoria suppressed induction of structural changes caused by purified LPS. Results obtained suggest that effector proteins produced by phytopathogenic bacteria and delivered by the type III secretion system may have a key role in suppressing the basal defense responses activated by bacterial LPS, which lead to restricted multiplication of nonpathogens such as hrp mutants.     

Microglia promote the death of developing Purkinje cells

The loss of neuronal cells, a prominent event in the development of the nervous system, involves regulated triggering of programmed cell death, followed by efficient removal of cell corpses. Professional phagocytes, such as microglia, contribute to the elimination of dead cells. Here we provide evidence that, in addition to their phagocytic activity, microglia promote the death of developing neurons engaged in synaptogenesis. In the developing mouse cerebellum, Purkinje cells die, and 60% of these neurons that already expressed activated caspase-3 were engulfed or contacted by spreading processes emitted by microglial cells. Apoptosis of Purkinje cells in cerebellar slices was strongly reduced by selective elimination of microglia. Superoxide ions produced by microglial respiratory bursts played a major role in this Purkinje cell death. Our study illustrates a mammalian form of engulfment-promoted cell death that links the execution of neuron death to the scavenging of dead cells.     

Recent contributions of electronic circular dichroism to the investigation of oligopeptide conformations

Recent applications in our laboratories of electronic circular dichroism to the study of peptide secondary structures and their changes under external stimuli are briefly reviewed. More specifically, this article deals with: 1). characterization of a novel peptide conformation; 2). origin of amino acid homo-chirality on Earth; 3). bend and helical peptides as spacers; and 4). transfer and propagation of chirality in peptides.     

Elongated mouse chromosomes suitable for enhanced molecular cytogenetics

Characterization of genetic disorders in humans and animal models requires identification of chromosomal aberrations. However, identifying fine deletions or insertion in metaphase chromosomes has been always a challenge due to limitations of resolution. In this study we developed a rapid method for chromosome elongation using two different intercalating agents: ethidium bromide and 5-bromo-2′-deoxyuridine (BrdU), together with a short-term mitotic block using colcemid. About 70% of the chromosomes from cells that underwent this elongation procedure reached three times longer than those prepared from control cells. FISH experiments using elongated chromosomes revealed a duplicated region of chromosome 11 that was not visible in cells prepared with conventional methods.     

A lysR-type regulator is involved in the negative regulation of genes encoding selenium-free hydrogenases in the archaeon Methanococcus voltae

The archaeon Methanococcus voltae encodes two pairs of NiFe-hydrogenase isoenzymes. One hydrogenase of each pair contains selenium in the active site, whereas the other one is selenium-free. The gene groups for the selenium-free hydrogenases, called vhc and frc, are linked by a common intergenic region. They are only transcribed under selenium limitation. A protein binding to a negative regulatory element involved in the regulation of the two operons was purified by DNA-affinity chromatography. Through the identification of the corresponding gene the protein was found to be a LysR-type regulator. It was named HrsM (hydrogenase gene regulator, selenium dependent in M. voltae). hrsM knockout mutants constitutively transcribed the vhc and frc operons in the presence of selenium. A putative HrsM binding site was also detected in the intergenic region in front of the hrsM gene. Northern blot analysis indicated that the hrsM gene might be autoregulated.     

Noise, delays, robustness, canalization and all that

A biological system such as a developing embryo can withstand many perturbations. What is the basis of this robustness both against noise and mutation? Recent advances in modeling may throw new light on this old problem. First, recent theoretical and experimental work clearly demonstrates the importance of noise and time delays for the proper functioning of genetic networks: noise and delays are simply part of the normal operating constraints. By contrast, sweeping statements have been made recently about a so-called 'robustness' of biological processes, based on work that neglects noise and delays completely. I submit that studying the stability of complex biological systems with such omissions is an unnecessary, inadequate and potentially disastrous simplification. I review the existing alternatives and propose using them to construct a modeling framework that overcomes all serious limitations.