Photodynamic Diagnosis Using 5‐Aminolevulinic Acid in 41 Biopsies for Primary Central Nervous System Lymphoma

We evaluated the feasibility of 5‐aminolevulinic acid (5‐ALA)‐mediated photodynamic diagnosis (PDD) in the biopsy for primary central nervous system lymphoma (PCNSL). 5‐ALA (20 mg kg^?1) was administered orally 4 hours preoperatively. Forty‐one biopsies obtained under PDD in 47 consecutive biopsies (46 patients) that were finally pathologically diagnosed as PCNSL were evaluated. Positive fluorescence was observed in 34 of those 41 biopsies (82.9%). An intraoperative pathological diagnosis (IOD) of suspected PCNSL was made in 21 of the biopsies with positive fluorescence (61.8%). However, the eight IODs in the remaining 13 biopsies (23.5%) were not correct (atypical cell, 4; high‐grade glioma, 1; gliosis, 1; unremarkable, 2). In those 8 biopsies, PCNSL was confirmed by the final pathological diagnosis. There was no difference in the mean Mib‐1 labeling index between the biopsies with positive fluorescence (86.5%) and those without positive fluorescence (90.0%). IOD was not performed in 6 biopsies; however, 5 of those biopsies (83.3%) showed positive fluorescence and were finally pathologically diagnosed as PCNSL. Use of PDD in biopsies for patients with suspected PCNSL is a reliable way of obtaining specimens of adequate quality for the final pathological diagnosis and may lead to improved diagnostic yield in the biopsy of PCNSL.     

The p53‐Dependent Expression of Frataxin Controls 5‐Aminolevulinic Acid‐Induced Accumulation of Protoporphyrin IX and Photo‐Damage in Cancerous Cells

Mitochondrial frataxin is involved in various functions such as iron homeostasis, iron–sulfur cluster biogenesis, the protection from oxidative stress and apoptosis and acts as a tumor suppressor protein. We now show that the expression of frataxin is stimulated in a p53‐dependent manner and prove that frataxin is a direct p53 target gene by showing that the p53‐responsive element in the promoter of the mouse frataxin gene is bound by p53. The bacterial expression of human frataxin stimulated maturation of human ferrochelatase, which catalyzes the insertion of iron into protoporphyrin at the last step of heme biosynthesis. Overexpression of frataxin in human cancer A431 and HeLa cells lowered 5‐aminolevulinic acid(ALA)‐induced accumulation of protoporphyrin and induced resistance to ALA‐induced photo‐damage, whereas p53 silencing with siRNA in non tumor HEK293T cells down‐regulated the expression of frataxin and increased the accumulation of protoporphyrin. Thus, the decrease of the expression of frataxin unregulated by p53 in tumor cells enhances ALA‐induced photo‐damage, by down‐regulation of mitochondrial functions.     

Interaction of dimethyltin(IV) dichloride with 5′‐IMP and 5′‐UMP

The formation constants of the species formed in the systems H⁺ + dimethyltin(IV) + 5′‐IMP and 5′‐UMP, H⁺ + 5′‐IMP and H⁺ + 5′‐UMP have been determined in aqueous solution in the pH range 1.5–9.5 at constant temperature (25 °C) and constant ionic strength (0.1 mol dm⁻³ NaClO₄), using spectrophotometric and potentiometric techniques. ¹H and ³¹P NMR investigations in aqueous solution confirmed the species formation. The precipitated complexes of IMP and UMP by Me₂Sn(IV)²⁺ at low pH values were characterized by elemental analysis and FTIR spectroscopy methods, the bonding sites of the ligands were determined and ruled out purine and pyrimidine moieties (N‐7 and N‐1 in IMP and N‐3 in UMP, respectively) while a bidentated coordination of the phosphate group is concluded in both cases. Finally, the experiments revealed the existence of complexes with trigonal bipyramidal structures that is in agreement with similar systems resulted previously. Copyright © 2008 John Wiley & Sons, Ltd.     

Alkyne‐Mediated Approach to the Synthesis of (4R,5R)‐5‐Hydroxy‐4‐decanolide and (−)‐Muricatacin

The asymmetric synthesis of two naturally occurring 5‐hydroxy‐γ‐butyrolactones, (4R,5R)‐5‐hydroxy‐4‐decanolide ( 1a ) and (?)‐muricatacin ( 2 ), is described using a general alkyne‐mediated strategy. The key steps involved are Sonogashira coupling for the desired carbon‐chain extension followed by Sharpless asymmetric dihydroxylation to construct the hydroxy‐lactone framework.     

Neurotransmitter Transporter Family Including SLC6A6 and SLC6A13 Contributes to the 5‐Aminolevulinic Acid (ALA)‐Induced Accumulation of Protoporphyrin IX and Photodamage,through Uptake of ALA by Cancerous Cells

δ‐Aminolevulinic acid (ALA)‐induced protoporphyrin accumulation is widely used in the treatment of cancer, as photodynamic therapy (PDT). To clarify the mechanisms of ALA uptake by tumor cells, we have examined the ALA‐induced accumulation of protoporphyrin by the treatment of colon cancer DLD‐1 and epithelial cancer HeLa cells with γ‐aminobutyric acid (GABA)‐related compounds. When the cells were treated with GABA, taurine and β‐alanine, the level of protoporphyrin was decreased, suggesting that plasma membrane transporters involved in the transport of neurotransmitters contribute to the uptake of ALA. By transfection with neurotransmitter transporters SLC6A6, SLC6A8 and SLC6A13 cDNA, the ALA‐ and ALA methylester‐dependent accumulation of protoporphyrin markedly increased in HEK293T cells, dependent on an increase in the uptake of ALA. When ALA‐treated cells were exposed to white light, the extent of photodamage increased in SLC6A6‐ and SLC6A13‐expressing cells. Conversely, knockdown of SLC6A6 or SLC6A13 with siRNAs in DLD‐1 and HeLa cells decreased the ALA‐induced accumulation. The expression of SLC6A6 and SLC6A13 was found in some cancer cell lines. Immunohistochemical studies revealed that the presence of these transporters was elevated in colon cancerous cells. These results indicated that neurotransmitter transporters including SLC6A6 and SLC6A13 mediate the uptake of ALA and can play roles in the enhancement of ALA‐induced accumulation of protoporphyrin in cancerous cells.     

1,3‐Dipolar cycloadditions of (4R*,5R*)‐1‐alkylidene‐4‐(benzoylamino)‐5‐phenyl‐3‐pyrazolidinon‐1‐azomethine imines

1,3‐Dipolar cycloadditions of azomethine imines 3a and 3b , available by acid‐catalyzed treatment of 3‐pyrazolidinone 1 with acetone ( 2a ) and butyraldehyde ( 2b ), respectively, were studied. Reactions of 3a with DMAD ( 4 ) afforded a mixture of products 9 and 10a , whilst treatment of 3b with DMAD ( 4 ) gave a mixture of compound 9 and epimeric cycloadducts 10 / 10′b . On the other hand, cycloadducts 13a,b‐16a,b were isolated as single diastereomers in 9–37% yields upon reactions of 3a,b with olefinic dipolarophiles 5–8 . The structures of cycloadducts 9, 10a, 10/10′b , and 13a,b‐16a,b were determined by ¹H nmr and NOESY spectroscopy. The structure of compound 13a was confirmed by X‐ray diffraction.     

A “Cell‐Friendly” Window for the Interaction of Cells with Hyaluronic Acid/Poly‐l‐Lysine Multilayers

Polyelectrolyte multilayers assembled from hyaluronic acid (HA) and poly‐l ‐lysine (PLL) are most widely studied showing excellent reservoir characteristics to host molecules of diverse nature; however, thick (HA/PLL)_n films are often found cell repellent. By a systematic study of the adhesion and proliferation of various cells as a function of bilayer number “n” a correlation with the mechanical and chemical properties of films is developed. The following cell lines have been studied: mouse 3T3 and L929 fibroblasts, human foreskin primary fibroblasts VH‐Fib, human embryonic kidney HEK‐293, human bone cell line U‐2‐OS, Chinese hamster ovary CHO‐K and mouse embryonic stem cells. All cells adhere and spread well in a narrow “cell‐friendly” window identify in the range of n = 12–15. At n < 12, the film is inhomogeneous and at n > 15, the film is cell repellent for all cell lines. Cellular adhesion correlates with the mechanical properties of the films showing that softer films at higher “n” number exhibiting a significant decrease of the Young's modulus below 100 kPa are weakly adherent to cells. This trend cannot be reversed even by coating a strong cell‐adhesive protein fibronectin onto the film. This indicates that mechanical cues plays a major role for cell behavior, also in respect to biochemical ones.     

Physicochemical Interaction of ZnO Fine Particles with 5‐Mono‐(4‐carboxyphenyl)‐10,15,20‐Triphenylporphyrin

This study deals with an investigation on the preparation and physicochemical interactions of ZnO nanoparticles with acid functionalized porphyrin [5‐mono‐(4‐carboxyphenyl)‐10,15,20‐triphenylporphyrin (CPTPP)] for photovoltaic applications in a detailed manner. Zinc acetate and sodium hydroxide were used as the starting materials for the synthesis of ZnO nanoparticles at 60 °C in an alcoholic medium. The freshly prepared fine particles were then functionalized with CPTPP. Both the virgin and pregnant ZnO particles were characterized by using UV‐Visible spectrophotometry (UV), fluorescence emission (PL), Fourier transform infrared spectroscopy (FTIR), X‐ray diffraction (XRD) and scanning electron microscopy (SEM). The band gap energy obtained for ZnO particles, having a value of 3.47 eV, shows significant quantum confinement effect and enhanced photophysical activity. FTIR analysis of the doped ZnO nanostructures showed the presences of some chemical species. SEM analysis revealed a clear change in the surface morphologies of undoped ZnO. The average crystallite size of nanoparticles, calculated from XRD peaks, was found in the nano regime. The lattice parameters calculated for ZnO nanocrystals were also found in good agreement with those given in the literature. From the enhancement in the red shift of the UV‐Vis spectra, it is concluded that hybridization of acid functionalized porphyrin can cause a significant expansion in the total absorption region of ZnO semiconductor for photovoltaic applications.     

Determination of 5‐Aminosalicylic Acid by Catalase‐Peroxidase Based Biosensor

5‐Aminosalicylic acid is an antiinflammatory drug used to treat inflammation of the digestive tract (Crohn's disease) and mild to moderate ulcerative‐colitis. 5‐Aminosalicylic acid is a bowel‐specific aminosalicylate drug. It was developed an amperometric biosensor for determination of 5‐aminosalicylic acid concentration and measurement technique is based on substrate‐competition. The biosensor is more suitable especially for routine 5‐aminosalicylic acid analysis because it is simple to construct and sensitive, specific and does not require any expensive apparatus. This enzyme based biosensor was made with a couple of enzymes which uses the same substrate. The electrode was developed to determine measurement conditions and also characterized.     

Interaction of 3‐(2‐Aminophenyl)‐6‐R1‐1,2,4‐triazin‐5‐ones with Acylating Reagents: An Efficient Method for Preparation of 6‐Substituted 3‐R1‐2H‐[1,2,4]triazino[2,3‐c]quinazolin‐2‐ones

The series of 6‐substituted 3‐R¹‐2H‐[1,2,4]triazino[2,3‐c]quinazolin‐2‐one was prepared via condensation of 3‐(2‐aminophenyl)‐6‐R¹‐1,2,4‐triazin‐5‐ones with acylating reagents. Particularities of ¹H NMR spectra have been also discussed based on the comparison of experimental and theoretical results for 3‐methyl‐6‐phenyl‐2H‐[1,2,4]triazino[2,3‐c]quinazolin‐2‐one and its 4,3‐isomer.     

The First Synthesis of (4S,5R,6R)‐5,6‐Dihydroxy‐4‐(prop‐1‐en‐2‐yl)cyclohex‐1‐ene‐1‐carboxylic Acid

A putative acid metabolite of a novel highly effective antiparkinsonian agent, (4S,5R,6R)‐5,6‐dihydroxy‐4‐(prop‐1‐en‐2‐yl)cyclohex‐1‐ene‐1‐carboxylic acid ( 5 ), was synthesized for the first time. Several synthetic approaches based on different transformations of O‐bearing monoterpenoids of the pinane and p‐menthane series were developed and tested in the course of the study. Acid 5 was synthesized starting from a commercially available monoterpenoid, (?)‐verbenone, in a total yield of 4.4% over eight steps.     

On the Structure of PHB (=Poly[(R)‐3‐hydroxybutanoic Acid]) in Phospholipid Bilayers: Preparation of Trifluoromethyl‐Labeled Oligo[(R)‐3‐hydroxybutanoic Acid] Derivatives

Oligomers of (R)‐3‐hydroxybutanoate (OHB) have previously been shown to transport cations through lipid bilayers. The ion‐transport activity has been attributed to the formation of hydrophobic aggregates or pores, which have been identified by fluorescence‐microscopy measurements of membrane‐incorporated fluorescence‐labeled OHBs. To obtain more information about these aggregates, we describe here the synthesis of the specifically F‐labeled HB oligomers II – IV for structural investigation by means of solid‐state ¹⁹F‐NMR spectroscopic techniques.     

Chemical Proteomic Profiling of Protein 4′‐Phosphopantetheinylation in Mammalian Cells

Protein 4′‐phosphopantetheinylation is an essential post‐translational modification (PTM) in prokaryotes and eukaryotes. So far, only five protein substrates of this specific PTM have been discovered in mammalian cells. These proteins are known to perform important functions, including fatty acid biosynthesis and folate metabolism, as well as β‐alanine activation. To explore existing and new substrates of 4′‐phosphopantetheinylation in mammalian proteomes, we designed and synthesized a series of new pantetheine analogue probes, enabling effective metabolic labelling of 4′‐phosphopantetheinylated proteins in HepG2 cells. In combination with a quantitative chemical proteomic platform, we enriched and identified all the currently known 4′‐phosphopantetheinylated proteins with high confidence, and unambiguously determined their exact sites of modification. More encouragingly, we discovered, using targeted chemical proteomics, a potential 4′‐phosphopantetheinylation site in the protein of mitochondrial dehydrogenase/reductase SDR family member 2 (DHRS2).     

(+)‐(R,Z)‐5‐Muscenone and (−)‐(R)‐Muscone by Enantioselective Aldol Reaction and Grob Fragmentation

(+)‐(R,Z)‐5‐Muscenone ((R)‐ 1 ) was synthesized by an enantioselective aldol reaction, catalyzed by new ephedrine‐type Ti reagents (up to 70 % enantiomeric excess). Substrate‐directed diastereoselective reduction of the aldol product and Grob fragmentation of the tosylate of the resultant 1,3‐diol afforded (+)‐ 1 . This approach also gave access to (?)‐(R,E)‐5‐muscenone and (?)‐(R)‐muscone.     

Light‐Induced Protein Dimerization by One‐ and Two‐Photon Activation of Gibberellic Acid Derivatives in Living Cells

We developed a highly efficient system for light‐induced protein dimerization in live cells using photo‐caged derivatives of the phytohormone gibberellic acid (GA₃). We demonstrate the application of the photo‐activatable chemical inducer of dimerization (CID) for the control of protein translocation with high spatiotemporal precision using light as an external trigger. Furthermore, we present a new two‐photon (2P)‐sensitive caging group, whose exceptionally high two‐photon cross section allows the use of infrared light to efficiently unleash the active GA₃ for inducing protein dimerization in living cells.     

Effects of Silencing Heme Biosynthesis Enzymes on 5‐Aminolevulinic Acid‐mediated Protoporphyrin IX Fluorescence and Photodynamic Therapy

Aminolevulinic acid (ALA)‐mediated protoporphyrin IX (PpIX) production is being explored for tumor fluorescence imaging and photodynamic therapy (PDT). As a prodrug, ALA is converted in heme biosynthesis pathway to PpIX with fluorescent and photosensitizing properties. To better understand the role of heme biosynthesis enzymes in ALA‐mediated PpIX fluorescence and PDT efficacy, we used lentiviral shRNA to silence the expression of porphobilinogen synthase (PBGS), porphobilinogen deaminase (PBGD) and ferrochelatase (FECH) in SkBr3 human breast cancer cells. PBGS and PBGD are the first two cytosolic enzymes involved in PpIX biosynthesis, and FECH is the enzyme responsible for converting PpIX to heme. PpIX fluorescence was examined by flow cytometry and confocal fluorescence microscopy. Cytotoxicity was assessed after ALA‐mediated PDT. Silencing PBGS or PBGD significantly reduced ALA‐stimulated PpIX fluorescence, whereas silencing FECH elevated basal and ALA‐stimulated PpIX fluorescence. However, compared with vector control cells, the ratio of ALA‐stimulated fluorescence to basal fluorescence without ALA was significantly reduced in all knockdown cell lines. PBGS or PBGD knockdown cells exhibited significant resistance to ALA‐PDT, while increased sensitivity to ALA‐PDT was found in FECH knockdown cells. These results demonstrate the importance of PBGS, PBGD and FECH in ALA‐mediated PpIX fluorescence and PDT efficacy.