BDNF increases the early expression of TRH mRNA in fetal TrkB+ hypothalamic neurons in primary culture.

Known effects of neurotrophins in the developing central nervous system include induction or regulation of peptide expression. Hypothalamic postmitotic thyrotropin-releasing hormone (TRH)-producing neurons may require neurotrophins for survival and/or differentiation. This issue was investigated using primary cell cultures derived from 17-day-old fetal rat hypothalamus seeded in serum-free medium and analysed up to 4 days in vitro culture. Neurotrophin receptor (TrkB and TrkC) mRNA expression was detected by RT-PCR in fetal hypothalamus and throughout the culture period. Western blots confirmed the expression of the full-length proteins in vitro. Semi-quantitative RT-PCR showed that the addition of brain-derived neurotrophic factor (BDNF) increases TRH mRNA levels while the addition of neurotrophin-3 does not. TRH cell content was not modified. Studies on the effect of cell density or homologous conditioned medium demonstrated that endogenous factors probably contribute to determine TRH mRNA levels. One of these factors was BDNF because basal TRH mRNA levels were reduced by the addition of a Trk inhibitor or anti-BDNF. TrkB mRNA was expressed in 27% of cells and TRH mRNA in 2% of cells. The number of TRH+ cells was not affected by BDNF treatment. Forty-eight per cent of TRH neurons contained TrkB mRNA; these neurons had higher amounts of TRH mRNA than TrkB- neurons. Only TrkB+ cells responded to BDNF by increasing their TRH mRNA levels suggesting that BDNF may directly affect TRH biosynthesis. In conclusion, fetal hypothalamic TRH neurons are probably heterogeneous in regard to the neurotrophic factors enhancing peptide and mRNA levels. BDNF enhances TRH mRNA levels in a population of TrkB+ fetal hypothalamic TRHergic neurons in primary culture. However, additional influences may be necessary for the establishment of peptide phenotype in the TrkB+ neurons.

Membranes from pituitary intermediate lobe cells enhance differentiation of fetal hypothalamic dopaminergic neurons in primary culture.

Coculture of adult pituitary intermediate lobe (IL) cells, a target for hypothalamic dopaminergic neurons, with fetal rat hypothalamic cells accelerate differentiation of dopaminergic neurons. This involves long range diffusible as well as additional factors which may be membrane-bound. To determine whether IL membrane-bound factors contribute to the differentiating effect of IL cells, IL membranes were added to dispersed fetal hypothalamic neurons. This stimulated the outgrowth of dopaminergic neurites and elevated TH levels. Limited trypsin proteolysis of IL cell surface abolished the effect on TH levels. Addition of adenohypophyseal membranes was ineffective. Joint treatment with IL membranes, and medium conditioned (CM) over IL cells, produced the same effect on TH levels as did coculture with the same number of IL cells. The results demonstrate that IL cells express on their surface a membrane-bound factor promoting differentiation of fetal dopaminergic neurons in vitro; this factor acts in addition to diffusible activities.

Effect of the selection marker on the viability and plasmid stability of two human proteins with neurotrophic action expressed in Escherichia coli.

Most developed expression systems rely on the production of fusion proteins or the change of selection marker increasing genetic stability to avoid toxicity of heterologous proteins to Escherichia coli host cells. According to this, we analyzed the effect of the selection marker on the viability and plasmid stability of vectors pYMK5 and pYMK7 that codify neurotrophic factors brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF). We also analyzed the influence of two different lac promoter inducers on these parameters. We found that the addition of IPTG to culture medium produced a significant decrease of viability and plasmid stability for both expression vectors compared with values reached with lactose. There was no increase of both parameters when we changed the selection marker, so we can conclude that, in our case, a change of antibiotic does not solve the problem of low plasmid stability values.

[Genetic expression of nerve growth factor in the central nervous system. Evaluation of an experimental model for Alzheimer's dementia]. / Expresión genética del factor de crecimiento neural en el sistema nervioso central. Evaluación en un modelo experimental de demencia de Alzheimer.

INTRODUCTION AND OBJECTIVE: Several authors have suggested that loss of neuronal trophic support may be an important element in the physiopathology of degenerative conditions of the central nervous system such as Alzheimer's dementia, Parkinson's disease or amyotrophic lateral sclerosis amongst others. In the light of present knowledge, the survival of cholinergic populations of the anterior basal cerebrum, closely involved with cognitive processes of memory and learning, is associated with adequate function of the neural growth factor (NGF). These populations are markedly damaged in Alzheimer's disease, and this has been correlated with the progressive loss of memory and intellectual involvement seen in this disorder. The model used in this study was based on section of the septohippocampal connecting pathways, so that transport of regulatory impulses from the hippocampus to the medial septum was interrupted. This has lethal results for the cholinergic neurons of the latter. We have developed a study designed to characterize the expression of the gene of NGF in different regions of the brain, involved in cholinergic neurotransmission in healthy and in damaged tissue. MATERIAL AND METHODS: We used a molecular hybridization technique with a cDNA catheter complementary to the radio-isotope marked NGF human gene. RESULTS AND CONCLUSIONS: The highest levels of expression were found in the healthy cortex and hippocampus. The reduction in the levels of mRNA of NGF in the damaged hippocampus supports the current thesis which considers synaptic activity to be a major regulator of the synthesis of this molecule in the brain.

Expresión genética del factor de crecimiento neural en el sistema nervioso central: evaluación en un modelo experimental de demencia de Alzheimer / Genetic expression of the neural growth factor in the central nervous system: evaluation of an experimental model of Alzheimerïs dementia

Introducción y objetivo. Varios autores han sugerido que la pérdida de un soporte trófico neuronal puede ser un elemento importante en la fisiopatología de enfermedades degenerativas del sistema nervioso como la demencia de Alzheimer, el Parkinson o la Esclerosis Lateral Amiotrófica, entre otros. A la luz de los conocimientos actuales, la supervivencia de poblaciones colinérgicas del cerebro basal anterior, íntimamamente relacionados con procesos cognitivos de memoria y aprendizaje, está asociada a una función adecuada del factor de crecimiento neural (FCN). Estas poblaciones sufren un deterioro importante en la enfermedad de Alzheimer, que ha sido correlacionado con la progresiva pérdida de memoria y afectación intelectual presente en esta enfermedad. El modelo utilizado en este trabajo se basa en la selección de las vías de conexión septohipocampales, con lo cual se interrumpe el transporte de señales regulatorias del hipocampo al septum medial; esto trae consecuencias letales sobre las neuronas colinérgicas de este último. Hemos desarrollado un estudio dirigido a caracterizar la expresión del gen de FCN en diferentes regiones cerebrales implicadas en la neurotransmisión colinérgica en el tejido sano y en el lesionado. Material y métodos. Fue utilizada una técnica de hibridación molecular con sonda de ADNc complementaria al gen del FCN humano marcada radioisotópicamente. Resultados y conclusiones. Se encontraron los mayores niveles de expresión en el hipocampo y la corteza sana. La disminución en los niveles de ARNm de FCN en el hipocampo lesionado apoya la tesis actual de considerar la actividad sináptica como una importante reguladora de la síntesis de esta molécula en el cerebro(AU)

Expresión gen tica del factor de crecimiento neural en el sistema nervioso central: evaluaci›n en un modelo experimental de demencia de Alzheimer / Genetic expression of the neural growth factor in the central nervous system: evaluation of an experimental model of Alzheimer's disease

Introducci›n y objetivo. Varios autores han sugerido que la p rdida de un soporte tr›fico neuronal puede ser un elemento importante en la fisiopatolog­a de enfermedades degeneerativas del sistema nervioso como la demencia de Alzheimer, el Parkinson o la Esclerosis Lateral Amiotr›fica, entre otras. A la luz de los conocimientos actuales, la supervivencia de poblaciones colin rgicas del cerebro basal anterior, ­ntimamente relacionados con procesos cognitivos de memoria y aprendizaje, estÿ asociada a una funci›n adecuada del factor de crecimiento neural (FCN). Estas poblaciones sufren un deterioro importante en la enfermedad de Alzheimer, que ha sido correlacionado con la progresiva p rdida de memoria y afectaci›n intelectual presente en esta enfermedad. El modelo utilizado en este trabajo se basa en la secci›n de las v­as de conexi›n septohipocampales con lo cual se interrumpe el transporte de se ales regulatorias del hipocampo al septum medial; esto trae consecuencias letales sobre las neuronas colin rgicas de este œltimo. Hemos desarrollado un estudio dirigido a caracterizar la expresi›n del gen de FCN en diferentes regiones cerebrales implicadas en la neurotransmisi›n colin rgica en el tejido sano y en el lesionado. Material y m todos. Fue utilizada una t cnica de hibridaci›n molecular con sonda de ADNc complementaria al gen de FCN humano marcada radioisot›picamente. Resultados y conclusiones. Se encontraron los mayores niveles de expresi›n en el hipocampo y la corteza sana. La disminuci›n en los niveles de ARNm de FCN en el hipocampo lesionado apoya la tesis actual de considerar la actividad sinÿptica como una importante reguladora de la s­ntesis de esta mol cula en el cerebro(AU)

Expression and folding of an interleukin-2-proinsulin fusion protein and its conversion into insulin by a single step enzymatic removal of the C-peptide and the N-terminal fused sequence.

We report the expression in E. coli of a proinsulin fusion protein carrying a modified interleukin-2 N-terminal peptide linked to the N-terminus of proinsulin by a lysine residue. The key aspects investigated were: (a) the expression of the fused IL2-PI gene, (b) the folding efficiency of the insulin precursor when still carrying the N-fused peptide and (c) the selectivity of the enzymatic cleavage reaction with trypsin in order to remove simultaneously the C-peptide and the N-terminal extension. It was found that this construction expresses the chimeric proinsulin at high level (20%) as inclusion bodies; the fused protein was refolded at 100-200 micrograms/ml to yield about 80% of correctly folded proinsulin and then it was converted into insulin by prolonged reaction (5 h) with trypsin and carboxypeptidase B at a low enzyme/substrate rate (1:600). This approach is based on a single enzymatic reaction for the removal of both the N-terminal fused peptide and the C-peptide and avoids the use of toxic cyanogen bromide.

Protein flexibility and aggregation state of human epidermal growth factor. A time-resolved fluorescence study of the native protein and engineered single-tryptophan mutants.

A time-resolved fluorescence spectroscopic study of the recombinant human epidermal growth factor (hEGF), a bis(tryptophan)-containing protein (Trp49-Trp50), and of the two single-tryptophan-containing engineered mutants with Trp49 or Trp50 replaced by Phe ([W49F]hEGF, [W50F]hEGF), was undertaken in order to gain insight into the conformational dynamics of the C-terminal region. Quite different position-dependent microenvironments for the two Trp residues are shown by comparing the fluorescence intensity decay of both mutants. Trp50 in the single-tryptophan mutant [W49F]EGF probably undergoes a dominant interaction with the solvent. A more heterogeneous environment of Trp49 in the [W50F]hEGF mutant is found. Moreover, the fluorescence decay of the native hEGF is not simply the additive result of the decays of both mutants: the Trp2 sequence confers a conformation of the C-terminal sequence which is more in contact with the rest of the protein molecule. By contrast, the fluorescence anisotropy decay of the native protein is quite similar to that of the single-tryptophan mutants. A high degree of rotational freedom in the C-terminal region of the protein is demonstrated. The resonance energy transfer, which could contribute to the anisotropy decay, appears therefore not to be highly efficient with respect to the depolarization motions. In addition to these local conformational and dynamic aspects of the hEGF C-terminal sequence, the fluorescence anisotropy decay data demonstrate the existence of a dimerization process of the native protein which is dependent on pH and protein concentration. This phenomenon influences the excited-state lifetime profiles and, therefore, the local conformational equilibrium of the C-terminal region.

Expresión de interferones humanos en E. coli / Expression of human interferons in E. coli

Los genes codificantes para los interferones alfa-2 y gamma humanos fueron clonados y expresados eficientemente bajo el control del promotor triptófano de E. coli. La adición de una señal de terminación de la transcripción en ambos ARNn dió como resultado un aumento de la expresión de las respectivas proteinas (AU)

Expresión de interferones humanos en E. coli / Expression of human interferons in E. coli

Los genes codificantes para los interferones alfa-2 y gamma humanos fueron clonados y expresados eficientemente bajo el control del promotor triptófano de E. coli. La adición de una señal de terminación de la transcripción en ambos ARNn dió como resultado un aumento de la expresión de las respectivas proteinas

Síntesis químico-enzimática del gen de la proinsulina humana / Chemical-enzymatic synthesis of the human proinsulin gene

En el presente trabajo se reporta la síntesis químoco-enzimática del gen que codifica para la proinsulina humana, hormona proteica de 86 aminoácidos. Para realizar la síntesis del gen de 270/266 pares de bases, fueron preparados 24 oligodesoxinucleótidos en fase sólida por el método del B-cianoetilfosforamidito. El gen sintético, que contiene los codones más frecuentes de E. coli y levadura, fue insertado en el plasmidio pUC18 y secuenciado por el método de Sanger (AU)

Síntesis químico-enzimática del gen de la proinsulina humana / Chemical-enzymatic synthesis of the human proinsulin gene

En el presente trabajo se reporta la síntesis químoco-enzimática del gen que codifica para la proinsulina humana, hormona proteica de 86 aminoácidos. Para realizar la síntesis del gen de 270/266 pares de bases, fueron preparados 24 oligodesoxinucleótidos en fase sólida por el método del B-cianoetilfosforamidito. El gen sintético, que contiene los codones más frecuentes de E. coli y levadura, fue insertado en el plasmidio pUC18 y secuenciado por el método de Sanger

Production and characterization of human gamma interferon from Escherichia coli.

The production of human gamma interferon as intracellular inclusion bodies in Escherichia coli, which simplified the purification process, is described. An expression plasmid carrying lipoprotein and the tryptophane promoters in tandem was used. Preparation of highly pure interferon was achieved using high resolution chromatography after denaturation and renaturation steps. Structural characteristics of this protein were verified by mass spectrometric analysis. Additional control tests have shown the suitability of the final product for clinical purposes.

[Site-specific endonuclease CauB31 from Chloroflexus aurantiacus B3]. / Sait-spetsificheskaia éndonukleaza CauB31 iz Chloroflexus aurantiacus B3.

A sequence-specific endonuclease CauB3I has been isolated from cell extracts of Chloroflexus aurantiacus and partially purified by chromatography on heparin-sepharose; the yield was 3000 units per 1 g of cells. The final preparation is free of non-specific nucleases. It is shown that endonuclease CauB3I recognizes 5' T decreases CCGGA 3' sequence in double-stranded DNA and cleaves it as shown by an arrow. Methylation of adenine in the recognition sequence makes it resistant to CauB3I.