Clonal Human Fetal Ventral Mesencephalic Dopaminergic Neuron Precursors for Cell Therapy Research

A major challenge for further development of drug screening procedures, cell replacement therapies and developmental studies is the identification of expandable human stem cells able to generate the cell types needed. We have previously reported the generation of an immortalized polyclonal neural stem cell (NSC) line derived from the human fetal ventral mesencephalon (hVM1). This line has been biochemically, genetically, immunocytochemically and electrophysiologically characterized to document its usefulness as a model system for the generation of A9 dopaminergic neurons (DAn). Long-term in vivo transplantation studies in parkinsonian rats showed that the grafts do not mature evenly. We reasoned that diverse clones in the hVM1 line might have different abilities to differentiate. In the present study, we have analyzed 9 hVM1 clones selected on the basis of their TH generation potential and, based on the number of v-myc copies, v-myc down-regulation after in vitro differentiation, in vivo cell cycle exit, TH⁺ neuron generation and expression of a neuronal mature marker (hNSE), we selected two clones for further in vivo PD cell replacement studies. The conclusion is that homogeneity and clonality of characterized NSCs allow transplantation of cells with controlled properties, which should help in the design of long-term in vivo experiments.     

Phosphate control over nitrogen metabolism in Streptomyces coelicolor: direct and indirect negative control of glnR, glnA, glnII and amtB expression by the response regulator PhoP

Bacterial growth requires equilibrated concentration of C, N and P sources. This work shows a phosphate control over the nitrogen metabolism in the model actinomycete Streptomyces coelicolor. Phosphate control of metabolism in Streptomyces is exerted by the two component system PhoR-PhoP. The response regulator PhoP binds to well-known PHO boxes composed of direct repeat units (DRus). PhoP binds to the glnR promoter, encoding the major nitrogen regulator as shown by EMSA studies, but not to the glnRII promoter under identical experimental conditions. PhoP also binds to the promoters of glnA and glnII encoding two glutamine synthetases, and to the promoter of the amtB-glnK-glnD operon, encoding an ammonium transporter and two putative nitrogen sensing/regulatory proteins. Footprinting analyses revealed that the PhoP-binding sequence overlaps the GlnR boxes in both glnA and glnII. ‘Information theory’ quantitative analyses of base conservation allowed us to establish the structure of the PhoP-binding regions in the glnR, glnA, glnII and amtB genes. Expression studies using luxAB as reporter showed that PhoP represses the above mentioned nitrogen metabolism genes. A mutant deleted in PhoP showed increased expression of the nitrogen metabolism genes. The possible conservation of phosphate control over nitrogen metabolism in other microorganisms is discussed.     

New Carboniferous brachiopods from the eastern Great Basin, Nevada, USA: implications for loop ontogeny and evolution in Late Palaeozoic terebratuloids

Terebratuloid brachiopods from two localities in the eastern Great Basin, Nevada, USA, include the new taxa Cryptacanthia savagei sp. nov., Fletcherithyris infrequens sp. nov., Cryptonella simplex sp. nov., and Albelenina alvarezi gen. et sp. nov. The faunas are considered to be mid Desmoinesian (late Moscovian) in age. The brachiopods were found associated in clusters, and many of the specimens are well preserved. Records of Pennsylvanian terebratuloid brachiopods are uncommon, and these faunas have enabled an understanding of internal features previously unknown or poorly understood. Systematic analyses of the faunas have generated some new concepts on the ontogeny and evolution of the loop in Late Palaeozoic terebratuloids. From ontogenetic analyses, it is inferred that the stage of loop development may be the most important aspect in taxonomic classification. Analyses of loop evolution suggest the possibility of a transition from a teloform stage to long-flanged deltiform and deltiform stages. These transitions may be possible through reduction in length of the descending lamellae of the crura by resorption of calcium carbonate or posteroventral folding of flanges. Subsequently, these changes in loop morphology are retained by paedomorphosis within long-term evolutionary processes.     

The Total Quasi-Steady-State Approximation for Fully Competitive Enzyme Reactions

The validity of the Michaelis–Menten–Briggs–Haldane approximation for single enzyme reactions has recently been improved by the formalism of the total quasi-steady-state approximation. This approach is here extended to fully competitive systems, and a criterion for its validity is provided. We show that it extends the Michaelis–Menten–Briggs–Haldane approximation for such systems for a wide range of parameters very convincingly, and investigate special cases. It is demonstrated that our method is at least roughly valid in the case of identical affinities. The results presented should be useful for numerical simulations of many in vivo reactions.     

Fibroblast growth factor-2 in hyperplastic pituitaries of D2R knockout female mice

Dopamine D2 receptor (D2R) knockout (KO) female mice develop chronic hyperprolactinemia and pituitary hyperplasia. Our objective was to study the expression of the mitogen fibroblast growth factor (FGF2) and its receptor, FGFR1, comparatively in pituitaries from KO and wild-type (WT) female mice. We also evaluated FGF2 subcellular localization and FGF2 effects on pituitary function. FGF2-induced prolactin release showed a similar response pattern in both genotypes, even though basal and FGF2-stimulated release was higher in KO. FGF2 stimulated pituitary cellular proliferation (MTS assay and [(3)H]thymidine incorporation), with no differences between genotypes. FGF2 concentration (measured by ELISA) in whole pituitaries or cultured cells was lower in KO (P < 0.00001 and 0.00014). Immunofluorescence histochemistry showed less FGF2 in pituitaries from KO females and revealed a distinct FGF2 localization pattern between genotypes, being predominantly nuclear in KO and cytosolic in WT pituitaries. Finally, FGF2 could not be detected in the conditioned media from pituitary cultures of both genotypes. FGFR1 levels (Western blot and immunohistochemistry) were higher in pituitaries of KO. Basal concentration of phosphorylated ERKs was lower in KO cells (P = 0.018). However, when stimulated with FGF2, a significantly higher increment of ERK phosphorylation was evidenced in KO cells (P < or = 0.02). We conclude that disruption of the D2R caused an overall decrease in pituitary FGF2 levels, with an increased distribution in the nucleus, and increased FGFR1 levels. These results are important in the search for reliable prognostic indicators for patients with pituitary dopamine-resistant prolactinomas, which will make tumor-specific therapy possible.     

Complement dependent amplification of the innate response to a cognate microbial ligand by the long pentraxin PTX3

The long pentraxin PTX3 is a fluid-phase pattern recognition receptor, which plays a nonredundant role in resistance against selected pathogens. PTX3 has properties similar to Abs; its production is induced by pathogen recognition, it recognizes microbial moieties, activates complement, and facilitates cellular recognition by phagocytes. The mechanisms responsible for the effector function of PTX3 in vivo have not been elucidated. OmpA, a major outer membrane protein of Gram-negative Enterobacteriaceae, is a microbial moiety recognized by PTX3. In the air pouch model, KpOmpA induces an inflammatory response, which is amplified by coadministration of PTX3 in terms of leukocyte recruitment and proinflammatory cytokine production. PTX3 did not affect the inflammatory response to LPS, a microbial moiety not recognized by PTX3. As PTX3 binds to C1q and modulates the activation of the complement cascade, we assessed the involvement of complement in the amplification of the response elicited by KpOmpA and PTX3. Experiments performed using cobra venom factor, C1-esterase inhibitor, and soluble complement receptor 1 indicate that PTX3 amplifies the inflammatory response to KpOmpA through complement activation. The results reported here demonstrate that PTX3 activates a complement-dependent humoral amplification loop of the innate response to a microbial ligand.     

Chemical composition of flavonoids and styrylpyrones and the genetic variability of isozymes in natural populations of Cryptocarya mandioccana Meisner (Lauraceae)

The chemical composition of leaves of 57 trees of Cryptocarya mandioccana from three populations of southeastern Brazil was investigated through HPLC, assaying six flavonoids, seven styrylpyrones, and seven unidentified compounds. From 51 of the former trees, genotypes were obtained from 40 polymorphic loci of 19 isozymes. Cluster analyses of the phytochemical and genetical variation revealed that trees exhibited four chemotypes and five clusters from isozymes, respectively. Discriminant analyses from selected variables of the isozymic and chemical data sets were performed, respectively, in relation to the four chemotypes and the five isozyme clusters. The classification of individuals presented respective error estimates of 9.16% and 13.57%, indicating that the genetic data could explain the clusters from chemotypes and vice versa at acceptable error levels. Linear regressions with Dummy variable showed significant association of locus Skdh-2 with quercetin-3-O-β-d-glucopyranoside and cryptofolione, indicating that its alleles would be responsible for the chemotype variation between individuals.     

Male reproductive senescence: the price of immune-induced oxidative damage on sexual attractiveness in the blue-footed booby

In animals, male reproduction is commonly a function of sexual attractiveness, based on the expression of sexually dimorphic traits that advertise genuinely the male's quality. Male performance may decline with age because physiological functions underlying sexual attractiveness may be affected by senescence. Here we show that a sexual signal (foot colour) declines with age, due probably to the deleterious effects of oxidative damage. We found that in the blue-footed booby Sula nebouxii foot colour during courtship was less attractive in senescent than in middle-aged males. In addition, we increased reactive oxygen species experimentally by immunizing males with lipopolysaccharide, a bacterial cell wall component that induces marked oxidative stress in animals. The immune system activation induced greater lipid peroxidation and invoked changes on colour expression (less attractive), particularly in senescent males. These results support the idea that oxidative stress affects reproductive senescence, and suggest that oxidative damage might be a proximal mechanism underlying age-reproductive patterns in long-lived animals.     

Neuroendocrine immunity in patients with Alzheimer's disease: toward translational epigenetics

The emerging domain of epigenetics in molecular medicine finds application for a variety of patient populations. Here, we present fundamental neuroendocrine immune evidence obtained in patients with senile dementia of the Alzheimer's type (sDAT), and discuss the implications of these data from the viewpoint of translational epigenetics of Alzheimer's disease. We followed 18 subjects with mild sDAT treated with acetylcholinesterase inhibitors, and 10 control subjects matched for age in a repeated measure design every six months for 18 months. We monitored psychosocial profile (Mini-Mental State Examination, Functional Assessment Staging, Independence in Activities of Daily Living, Depression, Profile of Moods States) in parallel to immunophenotypic parameters of T cell subpopulations by flow cytometry. Based on change in the mini-mental state score at entry and at 18 months, patients with sDAT were assigned to a "fast progression" (delta greater than 2 points) or to a "slow progression" group (delta less than or equal to 2 points). The change in circulating activated T cells (CD3+Dr+) with time in patients with sDAT was significantly inversely correlated with the change in time in natural killer (NK) cytotoxic activity to cortisol modulation in these patients, which was greater in patients with fast progression, compared to slow progression sDAT. These data indicate underlying neuroendocrine immune processes during progression of sDAT. Our observations suggest that psychoimmune measures such as those we have monitored in this study provide relevant information about the evolving physiological modulation in patients with sDAT during progression of Alzheimer's disease, and point to new or improved translational epigenetic treatment interventions.