Glycosylated cell-penetrating peptides and their conjugates to a proapoptotic peptide: preparation by click chemistry and cell viability studies

Cell-penetrating peptides (CPPs), which are usually short basic peptides, are able to cross cell membranes and convey bioactive cargoes inside cells. CPPs have been widely used to deliver inside cells peptides, proteins, and oligonucleotides; however, their entry mechanisms still remain controversial. A major problem concerning CPPs remains their lack of selectivity to target a specific type of cell and/or an intracellular component. We have previously shown that myristoylation of one of these CPPs affected the intracellular distribution of the cargo. We report here on the synthesis of glycosylated analogs of the cell-penetrating peptide (R6/W3): Ac-RRWWRRWRR-NH₂. One, two, or three galactose(s), with or without a spacer, were introduced into the sequence of this nonapeptide via a triazole link, the Huisgen reaction being achieved on a solid support. Four of these glycosylated CPPs were coupled via a disulfide bridge to the proapoptotic KLAK peptide, (KLAKLAKKLAKLAK), which alone does not enter into cells. The effect on cell viability and the uptake efficiency of different glycosylated conjugates were studied on CHO cells and were compared to those of the nonglycosylated conjugates: (R6/W3)S-S-KLAK and penetratinS-S-KLAK. We show that glycosylation significantly increases the cell viability of CHO cells compared to the nonglycosylated conjugates and concomitantly decreases the internalization of the KLAK cargo. These results suggest that glycosylation of CPP may be a key point in targeting specific cells.     

Taenia solium metacestode immunodominant peptides recognized by IgG antibodies in cerebrospinal fluid and serum paired samples from patients with active and inactive neurocysticercosis

The aim of this study was to test if serological distinction between patients with active and inactive neurocysticercosis (NCC), could be accomplished by the recognition of immunodominant peptides in total saline antigenic extract of Taenia solium metacestodes by IgG antibody in cerebrospinal fluid (CSF) and serum paired samples. CSF and serum samples of 10 each, active NCC patients, inactive NCC, and individuals with other neurological disorders, were used to recognize the antigenic peptides by western blot (WB). In the active NCC the 28-32 and 39-42 kDa peptides were more frequently detected in CSF than in sera (p < 0.05). The 47-52, 64-68, and 70 kDa antigens showed high frequencies in both samples from patients with active NCC. All the CSF samples of inactive NCC and other neurological disorder (control) patients tested negative, while serum samples from these last two groups recognized mainly the 80, 86, 95, and 98 kDa bands. This finding eliminates the use of the high molecular weigh bands (>or= 80 kDa) for diagnosis of NCC. The final conclusions were that the difference between active and inactive NCC may be done with the detection of peptides only in the CSF samples and that the 47-52, 64-68, and 70 kDa bands may be included as specific markers for active NCC when detected in CSF samples by WB using total saline extract of T. solium metacestode.     

Antioxidant properties of Krebs cycle intermediates against malonate pro-oxidant activity in vitro: a comparative study using the colorimetric method and HPLC analysis to determine malondialdehyde in rat brain homogenates

A variety of Krebs cycle intermediaries has been shown to possess antioxidant properties in different in vivo and in vitro systems. Here we examined whether citrate, succinate, malate, oxaloacetate, fumarate and alpha-ketoglutarate could modulate malonate-induced thiobarbituric acid-reactive species (TBARS) production in rat brain homogenate. The mechanisms involved in their antioxidant activity were also determined using two analytical methods: 1) a popular spectrophotometric method (Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Analytical Biochemistry 95, 351-358.) and a high performance liquid chromatographic (HPLC) procedure (Grotto, D., Santa Maria, L. D., Boeira, S., Valentini, J., Char?o, M. F., Moro, A. M., Nascimento, P. C., Pomblum, V. J., Garcia, S. C., 2006. Rapid quantification of malondialdehyde in plasma by high performance liquid chromatography-visible detection. Journal of Pharmaceutical and Biomedical Analysis 43, 619-624.). Citrate, malate, and oxaloacetate reduced both basal and malonate-induced TBARS production. Their effects were not changed by pre-treatment of rat brain homogenates at 100 degrees C for 10 min. alpha-Ketoglutarate increased basal TBARS without changing malonate-induced TBARS production in fresh and heat-treated homogenates. Succinate reduced basal--without altering malonate-induced TBARS production. Its antioxidant activity was abolished by KCN or heat treatment. Fumarate reduced malonate-induced TBARS production in fresh homogenates; however, its effect was completely abolished by heat treatment. There were minimal differences among the studied methods. Citrate, oxaloacetate, malate, alpha-ketoglutarate and malonate showed iron-chelating activity. We suggest that antioxidant properties of citrate, malate and oxaloacetate were due to their ability to cancel iron redox activity by forming inactive complexes, whereas alpha-ketoglutarate and malonate pro-oxidant activity can be due to formation of active complexes with iron. In contrast, succinate and fumarate antioxidant activity was probably due to some enzymatic system.     

High affinity Grb2-SH3 domain ligand incorporating Cbeta-substituted prolines in a Sos-derived decapeptide

Peptide ligands that disrupt MAPK pathways are of great interest for a better understanding of these signalling cascades and represent therefore an attractive target to control cell degenerative processes. In that context, selective disruption of the upstream Grb2/Sos complex in the Ras/MAPK cascade has focused extensive work. The Sos PPII decapeptide, which interacts with the Grb2-SH3 domains, has been modified in various positions and the best inhibitors designed so far are either dimeric ligands or peptoid analogues of the VPPPVPPRRR sequence. We report the synthesis of new Grb2 ligands in which the key Val5 residue has been replaced by a cis C(beta)-substituted proline. Both fluorescence and ITC assays have been employed to measure the affinity of these substituted peptides for a recombinant Grb2 protein. Whereas proline in position 5 completely abolished the binding potency, a cis C(beta)-methyl-L-proline restored the affinity. Other cis C(beta)-proline substituents led to a complete loss of binding potency. Combining the best modifications: a cis C(beta)-methylproline 5, N-acetylation, C-carboxamide and dimerization yielded a 560-fold affinity enhancement compared to the wild-type VPPPVPPRRR sequence. This study shows that C(beta)-substituted prolines may constitute a new alternative for PPII ligands, combining entropy and enthalpy beneficial effects.     

Demographic study of two population outbreaks of Elasmoderus wagenknechti (Liebermann) (Orthoptera: Tristiridae) in the transitional desert of Chile

Under certain environmental conditions, several species of grasshoppers inhabiting rangeland areas (e.g., inter-valleys) of the Chilean transitional desert can irrupt demographically. One of them is Elasmoderus wagenknechti (Liebermann), an endemic species. We studied two outbreaks occurred in 1996 and 1999. The objectives of the research were to (i) estimate some demographic parameters associated with the aforementioned events, and (ii) compare between them the population parameters. The parameters we studied were density (ind/m(2)), sexual proportion, female fertility (i.e., eggs/female), and the relationship eggs/female versus female size. Density (ind/m(2)) ranged from 0.2 to 0.4 in 1996 and from 0.5 to 0.6 in 1999. ANOVA-tests registered differences in density among study sites and between years. In some of the study sites, females were more abundant than males (e.g., Lomas del Sauce). Females fertility ranged from 27 to 39 eggs per female, with significant differences among sites in 1996 but not in 1999. For females of 30-50 mm body size, a linear relationship was detected between egg number/female and body size. It is concluded that the E. wagenknechti outbreaks are site-specific in the study area and that the demographic parameters examined show a high variability among sites and between events.     

Cell therapy of burns

Severe burns remain a life-threatening local and general inflammatory condition often with serious sequelae, despite remarkable progress in their treatment over the past three decades. Cultured epidermal autografts, the first and still most up-to-date cell therapy for burns, plays a key role in that progress, but drawbacks to this need to be reduced by using cultured dermal-epidermal substitutes. This review focuses on what could be, in our view, the next major breakthrough in cell therapy of burns - use of mesenchymal stromal cells (MSCs). After summarizing current knowledge, including our own clinical experience with MSCs in the pioneering field of cell therapy of radiation-induced burns, we discuss the strong rationale supporting potential interest in MSCs in treatment of thermal burns, including limited but promising pre-clinical and clinical data in wound healing and acute inflammatory conditions other than burns. Practical options for future therapeutic applications of MSCs for burns treatment, are finally considered.     

A DNA methylation microarray-based study identifies ERG as a gene commonly methylated in prostate cancer

DNA methylation of promoter regions is a common event in prostate cancer, one of the most common cancers in men worldwide. Because prior reports demonstrating that DNA methylation is important in prostate cancer studied a limited number of genes, we systematically quantified the DNA methylation status of 1505 CpG dinucleotides for 807 genes in 78 paraffin-embedded prostate cancer samples and three normal prostate samples. The ERG gene, commonly repressed in prostate cells in the absence of an oncogenic fusion to the TMPRSS2 gene, was one of the most commonly methylated genes, occurring in 74% of prostate cancer specimens. In an independent group of patient samples, we confirmed that ERG DNA methylation was common, occurring in 57% of specimens, and cancer-specific. The ERG promoter is marked by repressive chromatin marks mediated by polycomb proteins in both normal prostate cells and prostate cancer cells, which may explain ERG's predisposition to DNA methylation and the fact that tumors with ERG DNA methylation were more methylated, in general. These results demonstrate that bead arrays offer a high-throughput method to discover novel genes with promoter DNA methylation such as ERG, whose measurement may improve our ability to more accurately detect prostate cancer.     

ABCB1 identifies a subpopulation of uveal melanoma cells with high metastatic propensity

Metastasis of tumor cells to distant organs is the leading cause of death in melanoma. Yet, the mechanisms of metastasis remain poorly understood. One key question is whether all cells in a primary tumor are equally likely to metastasize or whether subpopulations of cells preferentially give rise to metastases. Here, we identified a subpopulation of uveal melanoma cells expressing the multidrug resistance transporter ABCB1 that are highly metastatic compared to ABCB1(-) bulk tumor cells. ABCB1(+) cells also exhibited enhanced clonogenicity, anchorage-independent growth, tumorigenicity and mitochondrial activity compared to ABCB1(-) cells. A375 cutaneous melanoma cells contained a similar subpopulation of highly metastatic ABCB1(+) cells. These findings suggest that some uveal melanoma cells have greater potential for metastasis than others and that a better understanding of such cells may be necessary for more successful therapies for metastatic melanoma.