Mapping of neuropeptide Y expression in Octopus brains

Neuropeptide Y (NPY) is an evolutionarily conserved neurosecretory molecule implicated in a diverse complement of functions across taxa and in regulating feeding behavior and reproductive maturation in Octopus. However, little is known about the precise molecular circuitry of NPY-mediated behaviors and physiological processes, which likely involve a complex interaction of multiple signal molecules in specific brain regions. Here, we examined the expression of NPY throughout the Octopus central nervous system. The sequence analysis of Octopus NPY precursor confirmed the presence of both, signal peptide and putative active peptides, which are highly conserved across bilaterians. In situ hybridization revealed distinct expression of NPY in specialized compartments, including potential “integration centers,” where visual, tactile, and other behavioral circuitries converge. These centers integrating separate circuits may maintain and modulate learning and memory or other behaviors not yet attributed to NPY-dependent modulation in Octopus. Extrasomatic localization of NPY mRNA in the neurites of specific neuron populations in the brain suggests a potential demand for immediate translation at synapses and a crucial temporal role for NPY in these cell populations. We also documented the presence of NPY mRNA in a small cell population in the olfactory lobe, which is a component of the Octopus feeding and reproductive control centers. However, the molecular mapping of NPY expression only partially overlapped with that produced by immunohistochemistry in previous studies. Our study provides a precise molecular map of NPY mRNA expression that can be used to design and test future hypotheses about molecular signaling in various Octopus behaviors.     

Balancing crosstalk between 20-hydroxyecdysone-induced autophagy and caspase activity in the fat body during Drosophila larval-prepupal transition

In the fruitfly, Drosophila melanogaster, autophagy and caspase activity function in parallel in the salivary gland during metamorphosis and in a common regulatory hierarchy during oogenesis. Both autophagy and caspase activity progressively increase in the remodeling fat body, and they are induced by a pulse of the molting hormone (20-hydroxyecdysone, 20E) during the larval-prepupal transition. Inhibition of autophagy and/or caspase activity in the remodeling fat body results in 25–40% pupal lethality, depending on the genotypes. Interestingly, a balancing crosstalk occurs between autophagy and caspase activity in this tissue: the inhibition of autophagy induces caspase activity and the inhibition of caspases induces autophagy. The Drosophila remodeling fat body provides an in vivo model for understanding the molecular mechanism of the balancing crosstalk between autophagy and caspase activity, which oppose with each other and are induced by the common stimulus 20E, and blockage of either path reinforces the other path.     

A new biallelic DNA polymorphism of the human COL5A1 gene

A cDNA probe of the human COL5A1 gene detects a frequent biallelic PstI polymorphism. Allele A has a frequency of 54% whereas that of allele B is 46%. This restriction fragment length polymorphism provides a useful marker for linkage analysis in 9q34.3.     

Progressive changes in the structure and dynamics of the phytoplankton community along a pollution gradient in a lowland river — a multivariate approach

The phytoplankton of the River Lujan (Buenos Aires, Argentina) was studied for a period of 18 months, together with physical and chemical variables, in relation to a pollution gradient. 167 taxa were recorded within a seasonal succession characterized by dominance of diatoms with a brief summer green algae facies. A combination of several biotic indices and multivariate analysis was employed to assess the impact of pollution on the phytoplankton community. The biotic indices used were species diversity and richness, algal quotients (green algae/diatom ratio, Centrales/Pennales ratio) and the SD succession rate index. Multivariate procedures included cluster analysis and ordination by PCA of both species and samples, stepwise discriminant analysis and multiple discriminant analysis of variance (MANOVA). Results indicate that community dynamism is attenuated at the more polluted sites, concomitant with an increased predominance of a broad-tolerance algal assemblage, co-dominated by Cyclotella meneghiniana and Nitzschia stagnorum. The changes in the community structure and dynamics described herein involved alterations in the distribution and relative proportions of the algae, rather than modifications in the basic species composition. These changes may not be readily detectable by methods which over-simplify the ecological information, such as systems of indicator species and biotic indices, designed to assess the degree of pollution. The suitability of multivariate analysis and biotic indices in river phytoplankton studies is further discussed.     

Periphyton on artificial substrata from three lakes of different trophic status at Hope Bay (Antarctica)

The nutrient characteristics of aquatic environments at Hope Bay (Antarctica) depend on different factors, penguin enrichment being one of the most important. In this study we surveyed three water bodies of different morphometry and trophic status: Esperanza Lake (oligotrophic), Boeckella Lake (meso-eutrophic), and Pingüi Pond (hypereutrophic). This research provides a preliminary characterization of the periphyton colonization on artificial substrata (glass slides) of these water bodies from the end of January to mid-March 2000. Qualitative and quantitative analyses of the algae, chlorophyll a, dry weight, ash-free dry weight, and physical and chemical variables of the lakes were assessed. The Autotrophic Index and the Lakatos system of classification were also applied. All three lakes differed clearly in all the variables considered. The periphyton from Esperanza Lake was first dominated by Bacillariophyceae and subsequently by the chrysophyce Phaeogloea mucosa. P. mucosa was the dominant species at Boeckella Lake and Chlamydomonas subcaudata was dominant at Pingüi Pond. Highest mean maximum density values were reached at Pingüi Pond, followed by Boeckella and Esperanza Lakes. Highest biomass figures were registered with increasing trophic status. According to the Lakatos index, the periphyton of Esperanza Lake was the least developed and it was mainly organic and heterotrophic; Boeckella Lake showed intermediate development in periphyton mass, which was autotrophic and mainly inorganic whereas Pingüi Pond showed the highest development in periphytic mass, and it was mainly autotrophic.     

CD8+ CD28- T regulatory lymphocytes inhibiting T cell proliferative and cytotoxic functions infiltrate human cancers

Tumor growth is allowed by its ability to escape immune system surveillance. An important role in determining tumor evasion from immune control might be played by tumor-infiltrating regulatory lymphocytes. This study was aimed at characterizing phenotype and function of CD8+ CD28- T regulatory cells infiltrating human cancer. Lymphocytes infiltrating primitive tumor lesion and/or satellite lymph node from a series of 42 human cancers were phenotypically studied and functionally analyzed by suppressor assays. The unprecedented observation was made that CD8+ CD28- T regulatory lymphocytes are almost constantly present and functional in human tumors, being able to inhibit both T cell proliferation and cytotoxicity. CD4+ CD25+ T regulatory lymphocytes associate with CD8+ CD28- T regulatory cells so that the immunosuppressive activity of tumor-infiltrating regulatory T cell subsets, altogether considered, may become predominant. The infiltration of regulatory T cells seems tumor related, being present in metastatic but not in metastasis-free satellite lymph nodes; it likely depends on both in situ generation (via cytokine production) and recruitment from the periphery (via chemokine secretion). Collectively, these results have pathogenic relevance and implication for immunotherapy of cancer.     

Investigating the role of active site residues of Rhodotorula gracilis D-amino acid oxidase on its substrate specificity

D-amino acid oxidase (DAAO) is a flavoprotein that catalyzes stereospecifically the oxidative deamination of D-amino acids. The wild-type DAAO is mainly active on neutral D-amino acids, while basic D-amino acids are poor substrates and the acidic ones are virtually not oxidized. To present a comprehensive picture of how the active site residues can modulate the substrate specificity a number of mutants at position M213, Y223, Y238, R285, S335, and Q339 were prepared in the enzyme from the yeast Rhodotorula gracilis. All DAAO mutants have spectral properties similar to those of the wild-type enzyme and are catalytically active, thus excluding an essential role in catalysis; a lower activity on neutral and basic amino acids was observed. Interestingly, an increase in activity and (k(cat)/K(m))(app) ratio on D-aspartate was observed for all the mutants containing an additional charged residue in the active site. The active site of yeast DAAO appears to be a highly evolved scaffold built up through evolution to optimize the oxidative deamination of neutral D-amino acids without limiting its substrate specificity. It is noteworthy, that introduction of a sole, additional, positively charged residue in the active site is sufficient to optimize the reactivity on acidic D-amino acids, giving rise to kinetic properties similar to those of D-aspartate oxidase.     

New biotech applications from evolved D-amino acid oxidases

D-Amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxygen-dependent oxidative deamination of amino acid D-isomers with absolute stereospecificity, which results in α-keto acids, ammonia and hydrogen peroxide. Recently, the extraordinary functional plasticity of DAAO has become evident; in turn, boosting research on this flavoprotein. Protein engineering has allowed for a redesign of DAAO substrate specificity, oxygen affinity, cofactor binding, stability, and oligomeric state. We review recent developments in utilizing DAAO, including as a biocatalyst for resolving racemic amino acid mixtures, as a tool for biosensing, and as a new mechanism of herbicide resistance. Perspectives for future biotechnological applications of this oxidative biocatalyst are also outlined.