Neuronal and Glial Properties Coexist in a Novel Mouse CNS Immortalized Cell Line

A mes-c-myc A1 (A1) cell line was generated by retroviral infection of cultured embryonic mesencephalic cells and selected by neomycin resistance. A1 cells cease to divide and undergo morphological differentiation after serum withdrawal or addition of c-AMP. Proliferating or morphologically differentiated A1 cells are all positive for vimentin and nestin, a marker of neural precursor, and show neuronal markers such as microtubule-associated protein 1, neuron-specific enolase and peripherin, and the glial marker glial fibrillary acidic protein. Neuronal and glial markers coexist in single cells. Furthermore, A1 cells show presence of glutamic acid decarboxylase 67 mRNA and its embryonic form EP10 and accumulate the neurotransmitter GABA. Electrophysiological studies demonstrate that morphologically differentiated A1 cells display voltage-gated sodium and potassium channels in response to depolarizing stimuli. A1 cells thus represent a novel, bipotent neural cell line useful for studying CNS differentiation and plasticity, as well as the molecular mechanisms underlying development of GABAergic neurotransmission.     

A new phosphotriesterase from Sulfolobus acidocaldarius and its comparison with the homologue from Sulfolobus solfataricus

The phosphotriesterase PTE, identified in the soil bacterium Pseudomonas diminuta, is thought to have evolved in the last several decades to degrade the pesticide paraoxon with proficiency approaching the limit of substrate diffusion (k(cat)/K(M) of 4 x 10(7)M(-1)s(-1)). It belongs to the amidohydrolase superfamily, but its evolutionary origin remains obscure. The enzyme has important potentiality in the field of the organophosphate decontamination. Recently we reported on the characterization of an archaeal member of the amidohydrolase superfamily, namely Sulfolobus solfataricus, showing low but significant and extremely thermostable paraoxonase activity (k(cat)/K(M) of 4 x 10(3)M(-1)s(-1)). Looking for other thermostable phosphotriesterases we assayed, among others, crude extracts of Sulfolobus acidocaldarius and detected activity. Since the genome of S. acidocaldarius has been recently reported, we identified there an open reading frame highly related to the S. solfataricus enzyme. The gene was cloned, the protein overexpressed in Escherichia coli, purified, and proven to have paraoxonase activity. A comparative analysis detected some significant differences between the two archaeal enzymes.     

Changes in growth and maturation parameters of Pacific sardine Sardinops sagax collected off California during a period of stock recovery from 1994 to 2010

Whether fluctuation in density influenced the growth and maturation variables of three aggregated cohorts (fish born during the 1986–1993, 1996–2003 and 2004–2008 periods) of Pacific sardine Sardinops sagax caeruleus collected off the Californian coast from 2004 to 2010 was investigated. Using a von Bertalanffy mixed‐effects model with aggregated cohorts as covariates, estimated growth rate significantly covaried with aggregated cohorts. Growth rate (K) was modelled as a fixed effect and estimated to be 0·264 ± 0·015 (±s.e ). Statistical contrasts among aggregated cohorts showed that the 1996–2003 cohorts had a significantly lower growth rate than the other two aggregated cohorts. The theoretical age at length zero (t₀) and the standard length at infinity (L_S∞) were modelled as random effects, and were estimated to be ?2·885 ± 0·259 (±s.e ) and 273·13 ± 6·533 mm (±s.e ). The relation of ovary‐free mass at length was significantly different among the three aggregated cohorts, with the allometric coefficient estimated to be 2·850 ± 0·013 (±s.e ) for the S. sagax population. The age‐at‐length trajectory of S. sagax born between 1986 and 2008 showed strong density dependence effects on somatic growth rates. In contrast to the density‐dependent nature of growth, the probability to be mature at‐size or at‐age was not significantly affected by aggregated cohort density. The size and the age‐at‐50% maturity were estimated to be 150·92 mm and 0·56 years, respectively. Stock migration, natural fluctuations in biomass and removal of older and larger S. sagax by fishing might have been interplaying factors controlling growth parameters during 1986–2010.     

Immune imbalance in the synovial fluid of rheumatoid arthritis patients: effects of intra-articular injection of thymopentin.

T cell subsets in the synovial fluid (SF) and peripheral blood (PB) of RA patients and controls suffering from different forms of chronic synovitis have been investigated. The immunological evaluation showed a reduction of CD4 subsets in RA-SF compared to RA-PB (p less than 0.001), and an almost complete absence of the suppressor-inducer/naive T cells in RA-SF compared to RA-PB and SF from patients with other forms of chronic synovitis. The CD8 subpopulation showed an increased proportion of cytotoxic cells only in RA-SF. On the basis of these results, an intra-articular immunomodulating treatment with thymopentin has been performed: its effects were characterized by an increase of CD8+CD11b+ T cells in the CD8 subset parallel to the enhancement of the suppressor-inducer/naive T cells in the CD4 subset with a statistically significant correlation. The enhanced levels of soluble CD8 decreased after treatment in RA-SF, whereas the soluble IL-2R levels were not significantly modified. Clinical evaluation showed a significative amelioration in all considered parameters.     

Enzyme promiscuity in the hormone-sensitive lipase family of proteins

The number of enzymes endowed with the capacity to catalyse other reactions than the main, physiological one, a feature that has been called promiscuity, is increasing at a fast pace. Promiscuity is a highly pervasive phenomenon that is present at each level of life complexity. For enzymes, promiscuity encompasses interesting aspects related to their physiological role, evolution and biotechnological applications. Herein, at first we will describe some general aspects of enzyme promiscuity and then we will report some examples from the α/β hydrolase superfamily of proteins, with particular emphasis to the hormone-sensitive lipase family.     

Expression of GFRalpha1 receptor splicing variants with different biochemical properties is modulated during kidney development

The glial cell line-derived neurotrophic factor (GDNF) family coreceptor alpha1 (GFRalpha1) is a critical component of the RET receptor kinase signal-transducing complex. The activity of this multicomponent receptor is stimulated by the glial cell line-derived neurotrophic factor (GDNF) and is involved in neuronal cells survival and kidney development. GFRalpha1 pre-mRNA is alternatively spliced and produces two isoforms: GFRalpha1a, which includes the exon 5; and GFRalpha1b, which excludes it. Here we show that the Gfralpha1a isoform is predominantly expressed in neuronal tissues and in PC12 cells differentiated toward a neuronal phenotype. GFRalpha1 splicing is also regulated during kidney development, GFRalpha1a is the minor isoform before birth and then rapidly becomes the major form after birth. We established cell lines expressing either GFRalpha1 isoforms and demonstrated that the GFRalpha1b isoform binds GDNF more efficiently than GFRalpha1a. Consistently, GFRalpha1b promotes a stronger RET phosphorylation than GFRalpha1a. These results indicate that specific inclusion of the GFRalpha1 exon 5 in neuronal tissues or during kidney development may alter the binding properties of GDNF to GFRalpha1, and thus could constitute an additional regulatory mechanism of the RET signaling pathway.