Protein microarray for complex apoptosis monitoring of dysplastic oral keratinocytes in experimental photodynamic therapy

Background

Photodynamic therapy is an alternative treatment of muco-cutaneous tumors that uses a light source able to photoactivate a chemical compound that acts as a photosensitizer. The phthalocyanines append to a wide chemical class that encompasses a large range of compounds; out of them aluminium-substituted disulphonated phthalocyanine possesses a good photosensitizing potential.

Results

The destructive effects of PDT with aluminium-substituted disulphonated phthalocyanine are achieved by induction of apoptosis in tumoral cells as assessed by flow cytometry analysis. Using protein microarray we evaluate the possible molecular pathways by which photodynamic therapy activates apoptosis in dysplastic oral keratinocytes cells, leading to the tumoral cells destruction. Among assessed analytes, Bcl-2, P70S6K kinase, Raf-1 and Bad proteins represent the apoptosis related biomolecules that showed expression variations with the greatest amplitude.

Conclusions

Up to date, the intimate molecular apoptotic mechanisms activated by photodynamic therapy with this type of phthalocyanine in dysplastic human oral keratinocytes are not completely elucidated. With protein microarray as high-throughput proteomic approach a better understanding of the manner in which photodynamic therapy leads to tumoral cell destruction can be obtained, by depicting apoptotic molecules that can be potentially triggered in future anti-tumoral therapies.     

PrimerHunter: a primer design tool for PCR-based virus subtype identification

Rapid and reliable virus subtype identification is critical for accurate diagnosis of human infections, effective response to epidemic outbreaks and global-scale surveillance of highly pathogenic viral subtypes such as avian influenza H5N1. The polymerase chain reaction (PCR) has become the method of choice for virus subtype identification. However, designing subtype-specific PCR primer pairs is a very challenging task: on one hand, selected primer pairs must result in robust amplification in the presence of a significant degree of sequence heterogeneity within subtypes, on the other, they must discriminate between the subtype of interest and closely related subtypes. In this article, we present a new tool, called PrimerHunter, that can be used to select highly sensitive and specific primers for virus subtyping. Our tool takes as input sets of both target and nontarget sequences. Primers are selected such that they efficiently amplify any one of the target sequences, and none of the nontarget sequences. PrimerHunter ensures the desired amplification properties by using accurate estimates of melting temperature with mismatches, computed based on the nearest neighbor model via an efficient fractional programming algorithm. Validation experiments with three avian influenza HA subtypes confirm that primers selected by PrimerHunter have high sensitivity and specificity for target sequences.     

In Vitro Effects of Calcium Fructoborate upon Production of Inflammatory Mediators by LPS-stimulated RAW 264.7 Macrophages

The present study is supported by our previous findings suggesting that calcium fructoborate (CF) has anti-inflammatory and antioxidant properties. Thus, we investigated the effects of CF on a model for studying inflammatory disorders in vitro represented by lipopolysaccharide (LPS)-stimulated murine macrophage RAW 264.7 cells. This investigation was performed by analyzing the levels of some mediators released during the inflammatory process: cytokines such as tumor necrosis factor-α (TNF-α), interleukins IL-1β and IL-6 as well as cyclooxygenase-2 (COX-2), the main enzyme responsible for endotoxin/LPS-induced prostaglandin synthesis by macrophages. We also measured production of nitric oxide (NO) that plays an important role in the cytotoxicity activity of macrophages towards microbial pathogens. After CF treatment of LPS-stimulated macrophages we found an up-regulation of TNF-α protein level in culture medium, no significant changes in the level of COX-2 protein expression and a decrease in NO production as well as in IL-1β and IL-6 release. Collectively, this series of experiments indicate that CF affect macrophage production of inflammatory mediators. However, further research is required in order to establish whether CF treatment can be beneficial in suppression of pro-inflammatory cytokine production and against progression of endotoxin-related diseases.     

Acid sphingomyelinase mediated release of ceramide is essential to trigger the mitochondrial pathway of apoptosis by galectin-1

The mechanism of apoptosis induced by human galectin-1, a mammalian beta-galactoside-binding protein with a remarkable cytotoxic effect on activated peripheral T cells and tumor T cell lines has been extensively investigated in this study. Here we first show that galectin-1 initiate the acid sphingomyelinase mediated release of ceramide and this event is critical in the further steps. Elevation of ceramide level coincides with exposure of phosphatidylserine on the outer cell membrane. The downstream events, decrease of Bcl-2 protein amount, depolarization of the mitochondria and activation of the caspase 9 and caspase 3 depend on production of ceramide. All downstream steps, including production of ceramide, require the generation of membrane rafts and the presence of two tyrosine kinases, p56(lck) and ZAP70. Based on our findings we suggest a model of the mechanism of galectin-1 triggered cell death.     

Type I IFNs stimulate nitric oxide production and resistance to Trypanosoma cruzi infection

The participation of type I IFNs (IFN-I) in NO production and resistance to Trypanosoma cruzi infection was investigated. Adherent cells obtained from the peritoneal cavity of mice infected by the i.p. route produced NO and IFN-I. Synthesis of NO by these cells was partially inhibited by treatment with anti-IFN-alphabeta or anti-TNF-alpha Abs. Compared with susceptible BALB/c mice, peritoneal cells from parasite-infected resistant C57BL/6 mice produced more NO (2-fold), IFN-I (10-fold), and TNF-alpha (3.5-fold). Later in the infection, IFN-I levels measured in spleen cell (SC) cultures from 8-day infected mice were greater in C57BL/6 than in infected BALB/c mice, and treatment of the cultures with anti-IFN-alphabeta Ab reduced NO production. IFN-gamma or IL-10 production by SCs was not different between the two mouse strains; IL-4 was not detectable. Treatment of C57BL/6 mice with IFN-I reduced parasitemia levels in the acute phase of infection. Mice deprived of the IFN-alphabetaR gene developed 3-fold higher parasitemia levels in the acute phase in comparison with control 129Sv mice. Production of NO by peritoneal macrophages and SCs was reduced in mice that lacked signaling by IFN-alphabeta, whereas parasitism of macrophages was heavier than in control wild-type mice. We conclude that IFN-I costimulate NO synthesis early in T. cruzi infection, which contributes to a better control of the parasitemia in resistant mice.     

Enantiodifferent Proton Exchange in Alanine and Asparagine in the Presence of H 2 17 O

Using Time Domain ¹H Nuclear Magnetic Resonance with H₂¹⁷O (H₂¹⁷O-TD-¹HNMR), we found [H₂¹⁷O]- and pH-controlled chiral differences in proton exchange properties in alanine (Ala) and asparagine (Asn). To minimize and equalize chemical impurities, Asn enantiomers were purified by crystallization from racemic solution. At <0.1 M H₂¹⁷O, a shift in isoelectric pH (pI) occurred, ~1.14 kJ mol⁻¹ l-d-Asn ΔΔG ^o′ in the 5.91–6.42 pH range. One potential source for this asymmetry is the enantio-different magnetic moments (lμ↑ ≠ dμ↓) produced by neutral ring currents in the chiral center, leading to enantio-different nuclear spin organization and charge distribution in the amino group. At ≥pI, dissimilar interactions may occur in the hydration of the amino group with H₂¹⁷O (NH₂/H₂¹⁷O ≠ NH₂/H₂¹⁶O; NH₃ ⁺/H₂¹⁷O ≠ NH₂/H₂¹⁷O; l-*C-NH₂/H₂¹⁷O ≠ d-*C-NH₂/H₂¹⁷O). As lμ↑ ≠ dμ↓, the l-*C-amino and the d-*C-amino groups are diastereo spin-isomers. The nuclear spin of ¹⁷O may be parallel or antiparallel with the ortho-¹H¹H pair; hence two ortho-H₂¹⁷O molecules exist, also diastereo spin-isomers. As the pK of H₂¹⁷O is different from H₂¹⁶O, dissimilarities between l-*C- and d-*C-amino groups are converted into proton exchange differences. During H₂¹⁷O-TD-¹HNMR, the H₂¹⁷O molecule is a “probe” of the state of the amino group. Regarding prebiotic evolution: prebiotic chirality may not require stochastic symmetry breaking or preexisting chiral conditions; chemical chiral effects due to lμ↑ ≠ dμ↓ are small and need chiral amplification to generate an enantiomeric excess significant for prebiotic evolution; and prebiotic symmetry breaking was homochiral because the effect of lμ↑ and dμ↓ on the amino group should be similar in all alpha amino acids.     

Abstracts: Albany 2005, The 14th Conversation

Abstract

We report a cost efficient approach for amino-acid-type selective isotope labeling of proteins expressed in Leishmania tarentolae. The method provides an economically advantageous alternative to recently established protocol for isotopic labeling using expensive synthetic media. The method is based on cultivation of the L. tarentolae expression strain in a cheap complex medium supplemented with labeled amino acid(s). In this protocol, a labeled amino acid is deliberately diluted in the medium of undefined composition, which leads to a low-level isotope enrichment upon protein over-expression. The economic advantage of the protocol is achieved by avoiding large volumes of expensive synthetic medium. Decreased sensitivity of a NMR experiment due to low-level isotope enrichment is compensated by a five- to seven-fold increase of the yield of the recombinant protein in complex medium as compared to that in the synthetic medium. In addition, the decreased sensitivity can be compensated by using a higher magnetic field, cryo-detection system or higher number of transients during the NMR data acquisition. We show that enrichment as low as 5% does not compromise a NMR experiment and makes preparation of the recombinant proteins over- expressed in L. tarentolae economically viable. The method is demonstrated by selective labeling of the ～27 kDa enhanced green fluorescent protein (EGFP) with ¹⁵N-labeled valine.     

Ethanol exposure modulates hepatic S-adenosylmethionine and S-adenosylhomocysteine levels in the isolated perfused rat liver through changes in the redox state of the NADH/NAD(+) system

Methionine metabolism is disrupted in patients with alcoholic liver disease, resulting in altered hepatic concentrations of S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), and other metabolites. The present study tested the hypothesis that reductive stress mediates the effects of ethanol on liver methionine metabolism. Isolated rat livers were perfused with ethanol or propanol to induce a reductive stress by increasing the NADH/NAD(+) ratio, and the concentrations of SAM and SAH in the liver tissue were determined by high-performance liquid chromatography. The increase in the NADH/NAD(+) ratio induced by ethanol or propanol was associated with a marked decrease in SAM and an increase in SAH liver content. 4-Methylpyrazole, an inhibitor the NAD(+)-dependent enzyme alcohol dehydrogenase, blocked the increase in the NADH/NAD(+) ratio and prevented the alterations in SAM and SAH. Similarly, co-infusion of pyruvate, which is metabolized by the NADH-dependent enzyme lactate dehydrogenase, restored the NADH/NAD(+) ratio and normalized SAM and SAH levels. The data establish an initial link between the effects of ethanol on the NADH/NAD(+) redox couple and the effects of ethanol on methionine metabolism in the liver.     

Calcium Fructoborate—Potential Anti-inflammatory Agent

Calcium fructoborate is a boron-based nutritional supplement. Its chemical structure is similar to one of the natural forms of boron such as bis-manitol, bis-sorbitol, bis-fructose, and bis-sucrose borate complexes found in edible plants. In vitro studies revealed that calcium fructoborate is a superoxide ion scavenger and anti-inflammatory agent. It may influence macrophage production of inflammatory mediators, can be beneficial for the suppression of cytokine production, and inhibits progression of endotoxin-associated diseases, as well as the boric acid and other boron sources. The mechanisms by which calcium fructoborate exerts its beneficial anti-inflammatory effects are not entirely clear, but some of its molecular biological in vitro activities are understood: inhibition of the superoxide within the cell; inhibition of the interleukin-1β, interleukin-6, and nitric oxide release in the culture media; and increase of the tumor necrosis factor-α production. Also, calcium fructoborate has no effects on lipopolysaccharide-induced cyclooxygenase-2 protein express. The studies on animals and humans with a dose range of 1–7 mg calcium fructoborate (0.025–0.175 mg elemental boron)/kg body weight/day exhibited a good anti-inflammatory activity, and it also seemed to have negligible adverse effect on humans.