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Novel therapeutic peptides are increasingly making their way into clinical application. The cationic and amphipathic properties of certain peptides allow them to cross biological membranes in a non-disruptive way without apparent toxicity increasing drug bioavailability. By modifying the primary structure of the Limulus-derived LALF32–51 peptide we designed a novel peptide, L-2, with antineoplastic effect and cell-penetrating capacity. Interestingly, L-2 induced cellular cytotoxicity in a variety of tumor cell lines and systemic injection into immunocompetent and nude mice bearing established solid tumor, resulted in substantial regression of the tumor mass and apoptosis. To isolate the gene transcripts specifically regulated by L-2 in tumor cells, we conducted suppressive subtractive hybridization (SSH) analysis and identified a set of genes involved in biological processes relevant to cancer biology. Our findings describe a novel peptide that modifies the gene expression of the tumor cells and exhibits antitumor effect in vivo, indicating that peptide L-2 is a potential candidate for anticancer therapy. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.

Brain derived neurotrophic factor (BDNF) increases the levels of pre-pro-thyrotropin releasing hormone (TRH) mRNA in fetal rodent hypothalamic neurons that express TrkB receptors. The present studies aimed at better understanding the role of BDNF in establishing and maintaining the TRH phenotype in hypothalamic neurons during early development. To determine where BDNF regulates the expression of pre-pro-TRH mRNA in vivo, we compared the hypothalamic distribution of pre-pro-TRH mRNA to that of TrkB mRNA. Full-length TrkB (FL-TrkB) mRNA was detected earlier in development than pre-pro-TRH mRNA in the region that gives rise to the paraventricular nucleus of the hypothalamus (PVN). We also evaluated the effects of BDNF on the expression of pre-pro-TRH mRNA in vitro. BDNF up-regulated the levels of pre-pro-TRH mRNA in primary cell cultures obtained from the hypothalamus or the PVN of 17 days old fetuses or newborn rats. This effect was abolished by PD98059, an inhibitor of the mitogen-activated protein kinase kinase (MEK) 1/2 or 5. The effect of BDNF on pre-pro-TRH mRNA levels was reversible. The continuous application of BDNF led to a desensitization of the response at day 10 in vitro, an effect that correlated with a drop in the levels of FL-TrkB protein. In conclusion, BDNF enhances the expression of pre-pro-TRH mRNA in PVN neurons. This effect is reversible, decreases with time, and requires an active MEK. BDNF may contribute to the enhancement of pre-pro-TRH mRNA expression in the hypothalamic PVN during development.

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.

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.

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.

A combination of antiviral drugs known as antiretroviral therapy (ART) has shown effectiveness against the human immunodeficiency virus (HIV). ART has markedly decreased mortality and morbidity among HIV-infected patients, having even reduced HIV transmission. However, an important current disadvantage, resistance development, remains to be solved. Hope is focused on developing drugs against cellular targets. This strategy is expected to prevent the emergence of viral resistance. In this study, using a comparative proteomic approach in MT4 cells treated with an anti-HIV leukocyte extract, we identified vimentin, a molecule forming intermediate filaments in the cell, as a possible target against HIV infection. We demonstrated a strong reduction of an HIV-1 based lentivirus expressing the enhanced green fluorescent protein (eGFP) in vimentin knockdown cells, and a noteworthy decrease of HIV-1 capsid protein antigen (CAp24) in those cells using a multiround infectivity assay. Electron micrographs showed changes in the structure of intermediate filaments when MT4 cells were treated with an anti-HIV leukocyte extract. Changes in the structure of intermediate filaments were also observed in vimentin knockdown MT4 cells. A synthetic peptide derived from a cytoskeleton protein showed potent inhibitory activity on HIV-1 infection, and low cytotoxicity. Our data suggest that vimentin can be a suitable target to inhibit HIV-1.

Novel therapeutic peptides are increasingly making their way into clinical application. The cationic and amphipathic properties of certain peptides allow them to cross biological membranes in a non-disruptive way without apparent toxicity increasing drug bioavailability. By modifying the primary structure of the Limulus-derived LALF(32-51) peptide we designed a novel peptide, L-2, with antineoplastic effect and cell-penetrating capacity. Interestingly, L-2 induced cellular cytotoxicity in a variety of tumor cell lines and systemic injection into immunocompetent and nude mice bearing established solid tumor, resulted in substantial regression of the tumor mass and apoptosis. To isolate the gene transcripts specifically regulated by L-2 in tumor cells, we conducted suppressive subtractive hybridization (SSH) analysis and identified a set of genes involved in biological processes relevant to cancer biology. Our findings describe a novel peptide that modifies the gene expression of the tumor cells and exhibits antitumor effect in vivo, indicating that peptide L-2 is a potential candidate for anticancer therapy.

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.

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.

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.