Development of the digestive system of Argentine hake,Merluccius hubbsi,larvae

The Argentine hake Merluccius hubbsi is an important fishery resource of the Southwestern Atlantic Ocean and it is also a potential species for cultivation. In this work, the digestive system development in field-caught hake larvae was studied using histological and histochemical approaches. The digestive tract of larvae was divided into: oropharyngeal cavity (OPC), esophagus, stomach (that develops in the preflexion stage), and intestine. The annexed digestive glands consisted of the liver and the exocrine pancreas. At the beginning of the preflexion stage, teeth were developed in the OPC. There were mucous cells in the esophagus secreting different glycoconjugates from hatching. The enterocytes in the posterior intestine exhibited supranuclear vesicles associated with protein absorption. Mucous cells were observed in the posterior intestine in the preflexion stage and, in the anterior region, ending the flexion stage. Each type of glycoconjugates has a specific role. Acidic mucins lubricate and protect from mechanical damage, sialomucines protect from bacterial infections and neutral mucins regulate the acidity of mucus secretion, protect against abrasion and participate in the formation of the chyme, indicating a pregastric digestion. The liver was present since hatching with pancreatic tissue inside and increased in size acquiring the typical structure with hepatocyte cords, sinusoids, vacuoles, and hepatic duct. The hepatocytes vacuolization increased with larval development. The pancreas became extra-hepatic, with basophilic acinar cells and acidophilic zymogen granules. Throughout the ontogeny, the increased structural and functional complexity of the digestive system reflected the transition to exogenous feeding and nutritional increasing needs.     

Dimer–monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer

An ad hoc bioconjugation/fluorescence resonance energy transfer (FRET) assay has been designed to spectroscopically monitor the quaternary state of human thymidylate synthase dimeric protein. The approach enables the chemoselective engineering of allosteric residues while preserving the native protein functions through reversible masking of residues within the catalytic site, and is therefore suitable for activity/oligomerization dual assay screenings. It is applied to tag the two subunits of human thymidylate synthase at cysteines 43 and 43′ with an excitation energy donor/acceptor pair. The dimer–monomer equilibrium of the enzyme is then characterized through steady‐state fluorescence determination of the intersubunit resonance energy transfer efficiency.     

Synthesis of novel nitric oxide (NO)-releasing esters of timolol

A novel class of timolol derivatives with nitric oxide (NO)-donating moieties achieved chemical stability yet under physiologically relevant conditions released timolol and NO. Hindered esters A were designed and synthesized, whose ‘triggered’ release relied on enzymatic hydrolysis of the nitrate ester in A to B, that in turn cyclized to liberate timolol.     

The immunoglobulin heavy chain switch: structural features of gamma 1 recombinant switch regions

The immunoglobulin heavy chain isotype switch is mediated by a DNA rearrangement involving specific genomic segments referred to as switch regions. Switch regions are composed of tandemly repeated simple sequences. The role of the tandemly repeated structure of switch regions in the switch recombination process is not understood. We mapped eight recombination sites--six in the gamma 1 and two in the gamma 3 tandem arrays. In addition, we obtained molecular clones representing three of the six gamma 1 rearrangements, and determined the nucleotide sequences of the recombination sites in each. In general, the rearrangements are confined to the tandem repeat units, and are not clustered in a particular portion of either the gamma 3 or gamma 1 switch region. Nucleotide sequence analysis of one of the recombinant clones, gamma M35, reveals evidence for a successive switch event wherein a recombination between S mu and S gamma 3 was followed by recombination 57 bp downstream with S gamma 1. gamma 1 sequence data from the molecular clones we obtained, together with similar data from other investigators regarding the gamma 1, gamma 2b, and gamma 2a switch regions, reveals that recombinations tend to occur at homologous positions of the respective gamma-unit repeats, adjacent to the elements AGCT and GGGG found in each. This finding suggests that the cutting and religation step of the recombination process is mediated by a recombinase common to the four gamma-isotypes.     

Altered Intracellular Localization of SOD1 in Leukocytes from Patients with Sporadic Amyotrophic Lateral Sclerosis

Several lines of evidence support the hypothesis of a toxic role played by wild type SOD1 (WT-SOD1) in the pathogenesis of sporadic amyotrophic lateral sclerosis (SALS). In this study we investigated both distribution and expression profile of WT-SOD1 in leukocytes from 19 SALS patients and 17 healthy individuals. Immunofluorescence experiments by confocal microscopy showed that SOD1 accumulates in the nuclear compartment in a group of SALS subjects. These results were also confirmed by western blot carried out on soluble nuclear and cytoplasmic fractions, with increased nuclear SOD1 level (p<0.05). In addition, we observed the presence of cytoplasmic SOD1 aggregates in agreement with an increased amount of the protein recovered by the insoluble fraction. A further confirmation of the overall increased level of SOD1 has been obtained from single cells analysis using flow cytometry as cells from SALS patients showed an higher SOD1 protein content (p<0.05). These findings add further evidence to the hypothesis of an altered WT-SOD1 expression profile in peripheral blood mononuclear cells (PBMCs) from patients with ALS suggesting that WT-SOD1 species with different degrees of solubility could be involved in the pathogenesis of the disease.     

The Dark Recovery Rate in the Photocycle of the Bacterial Photoreceptor YtvA Is Affected by the Cellular Environment and by Hydration

We report thermal recovery kinetics of the lit state into the parental dark state, measured for the blue light-sensing photoreceptor YtvA inside overexpressing E. coli and B. subtilis bacterial cells, performed for the wild type and several mutated proteins. Recovery was followed as a recovery of the fluorescence, as this property is only found for the parental but not for the photochemically generated lit state. When cells were deposited onto a microscope glass plate, the observed thermal recovery rate in the photocycle was found ca. ten times faster in comparison to purified YtvA in solution. When the E. coli or B. subtilis colonies were soaked in an isotonic buffer, the dark relaxation became again much slower and was very similar to that observed for YtvA in solution. The observed effects show that rate constants can be tuned by the cellular environment through factors such as hydration.     

The application of oligonucleotide probes and microarrays for the identification of freshwater diatoms

Diatoms are major contributors to global carbon fixation and constitute a significant portion of biofilms found in lotic ecosystems. Despite their widespread abundance and the fact that extensive studies have been performed on morphological features of frustules, molecular tools for the identification of diatoms are not commonly available. This study focuses on the development of oligonucleotide probes for the detection of diatom species relevant to water quality assessment. The selected panel of diatoms covers all the species found in water of varying quality from the rivers of central-East Apennine (Italy). Small subunit rRNA-targeted probes were applied to a microarray platform as well as to a new technique termed Primer–Probe, with the aim of obtaining a molecular tool suitable for accurate identification of both single and mixed species diatom populations. The Primer–Probe technique together with dot-blot assays proved to be ideal for the preliminary screening of a large set of DNA oligonucleotides designed by ARB software. It was shown that microarrays, as a promising technology for rapid and simultaneous detection of a wide range of species-specific genetic markers, can be adapted to monitor changes within a diatom community. It is suggested that microarrays will provide a molecular basis for microbial identification to support standard microscopy techniques used by ecologists and environmental scientists for monitoring water quality.     

Identification of an Interstitial 18p11.32-p11.31 Duplication Including the EMILIN2 Gene in a Family with Porokeratosis of Mibelli

Porokeratosis is a rare disease of epidermal keratinization characterized by the histopathological feature of the cornoid lamella, a column of tightly fitted parakeratocytic cells, whose etiology is still unclear. Porokeratosis of Mibelli is a subtype of porokeratosis presenting a single plaque or a small number of plaques of variable size located unilaterally on limbs. It frequently appears in childhood and occurs with a higher incidence in males. Cytogenetic analyses were performed in all members of the family on lesioned and uninvolved skin. An array-CGH analysis was also performed utilizing the Human Genome CGH Microarray Kit G3 400 with 5.3 KB overall median probe spacing. Gene expression was performed on skin fibroblasts. In this study, we describe a Caucasian healthy 4-year-old child and his father showing features of porokeratosis of Mibelli. Array-CGH analysis revealed an interstitial 429.5 Kb duplication of chromosome 18p11.32-p11.3 containing four genes, namely: SMCHD1, EMILIN2, LPIN2, and MYOM1 both in patient and his father. EMILIN2 resulted overexpressed on skin fibroblasts. Also other members of this family, without evident signs of porokeratosis, carried the same duplication. Among these genes, we focused our attention on elastin microfibril interfacer 2 (EMILIN2) gene. Apoptosis plays a fundamental role in maintaining epidermal homeostasis, balancing keratinocytes proliferation, and forming the stratum corneum. EMILIN2 is known to trigger the apoptosis of different cell lines negatively affecting cell survival. It is expressed in the skin. We could speculate that the duplication and overexpression of EMILIN2 cause an abnormal apoptosis of epidermal keratinocytes and alter the process of keratinization, even if other epigenetic and genetic factors could also be involved. Our results could contribute to a better understanding of the pathogenesis of porokeratosis of Mibelli.