Experimental and Computational Analysis of Synthesis Conditions of Hybrid Nanoflowers for Lipase Immobilization.

This work presents a framework for evaluating hybrid nanoflowers using Burkholderia cepacia lipase. It was expanded on previous findings by testing lipase hybrid nanoflowers (hNF-lipase) formation over a wide range of pH values (5-9) and buffer concentrations (10-100 mM). The free enzyme activity was compared with that of hNF-lipase. The analysis, performed by molecular docking, described the effect of lipase interaction with copper ions. The morphological characterization of hNF-lipase was performed using scanning electron microscopy. Fourier Transform Infrared Spectroscopy performed the physical-chemical characterization. The results show that all hNF-lipase activity presented values higher than that of the free enzyme. Activity is higher at pH 7.4 and has the highest buffer concentration of 100 mM. Molecular docking analysis has been used to understand the effect of enzyme protonation on hNF-lipase formation and identify the main the main binding sites of the enzyme with copper ions. The hNF-lipase nanostructures show the shape of flowers in their micrographs from pH 6 to 8. The spectra of the nanoflowers present peaks typical of the amide regions I and II, current in lipase, and areas with P-O vibrations, confirming the presence of the phosphate group. Therefore, hNF-lipase is an efficient biocatalyst with increased catalytic activity, good nanostructure formation, and improved stability.

Invasive candidiasis in dogs: A case report and review of the literature.

Invasive candidiasis is characterized by the systemic dissemination of Candida spp. and colonization of multiple organs. We are reporting a case of invasive candidiasis in a 3.5-year-old female mixed-breed dog with a history of limb injury. After clinical evaluation and complementary examinations a sepsis diagnose was established. The patient remained hospitalized under antibiotic therapy, dying three days later. Necropsy revealed white, nodular (pyogranulomas), and multifocal areas on the liver, button ulcers in the stomach and intestines, and a random lung consolidation. Impression smears were made from the liver and lung surface lesions during necropsy showing yeast and pseudohyphae structures. Fragments of these organs were sent for fungal culture and subsequent molecular etiologic characterization, identifying it as Candida albicans. Histological examination of different organs showed pyogranulomatous inflammation surrounding the necrosis areas, which were full of yeast and pseudohyphae, as evidenced by periodic acid Schiff and immunohistochemistry. Neutropenia, as a consequence of sepsis, associated with the use of antibiotics may have allowed yeast invasion and proliferation in the mucosa of the gastrointestinal tract, reaching the liver and lungs through hematogenous route. Invasive candidiasis is a rare canine disease, and no other cases of neutropenia associated with antibiotic therapy, as a predisposing factors, have been reported.

Preparation of gentamicin sulfate eluting fiber mats by emulsion and by suspension electrospinning.

This work investigates the immobilization of the antibiotic gentamicin sulfate (GS) in electrospun fiber mats composed of poly(lactic acid) (PLA), poly(ε-caprolactone) (PCL) and the copolymer poly(lactic-co-glycolic acid) (PLGA). Since GS is highly water soluble but weakly soluble in the organic solvents commonly used in the electrospinning process, two methods of immobilization were investigated: by suspension electrospinning, in which GS particles were directly dispersed in the polymeric organic solutions, and by emulsion electrospinning, in which GS was solubilized in an aqueous phase that was then dispersed in the organic polymeric solution containing the surfactant SPAN80. Fibers with distinct diameters and morphologies were obtained for the different methods and compositions. Contrary to the fibers prepared by suspension electrospinning, emulsion electrospinning based fibers exhibited an excellent wettability, allegedly due to the effect of the surfactant SPAN80. Despite the differences between both methods the produced mats presented similar GS release profiles, with a considerable burst release in the first 8 h followed by a gradual release of the remaining drug during the next 4-6 days. Finally, all GS loaded fiber mats proved to have an antibacterial effect against the bacterial strain Staphylococcus aureus.

Photodynamic therapy: Dermatology and ophthalmology as main fields of current applications in clinic.

Photodynamic therapy (PDT) of skin tumors or pre-cancerous lesions and of age-related macular degeneration combines the administration of porphyrins or porphyrin precursors and illumination with red light at the diseased sites. Photosensitizers absorbing light beyond 630 nm where tissues have the highest transmittance produce singlet oxygen, a highly reactive activated oxygen species and a major cytotoxin. The PDT of age-related macular degeneration is performed with red laser light after i.v. injection of verteporfin (Visudyne) a hydrophobic porphyrin carried by serum lipoproteins whose endocytosis leads to accumulation of the porphyrin in endothelial cells of choroidal neo-vessels. In the PDT of skin cancers, local synthesis of the photosensitizer occurs after topical application of the natural protoporphyrin IX precursor delta-aminolevulinic acid (or its ester forms) on the lesions. In all the cases, the photosensitizers should be rapidly excreted to avoid a long lasting skin photosensitivity.

Photodynamic therapies: principles and present medical applications.

Photodynamic therapy (PDT) by porphyrins and related tetrapyrrole derivatives is an emerging new treatment modality of tumors of lung, eosophagus and skin and of age-related macular degeneration. Phase III clinical trials for other applications such as re-stenosis after angioplasty are also underway. Under systemic conditions, the transport of porphyrin photosensitizers by serum low density lipoproteins and their specific delivery to tumor cells and vasculature is a determinant of treatment effectiveness. However, this effectiveness can be improved by increasing the selectivity of the photosensitizer uptake by tumors and by using photosensitizers absorbing light in the 660-800 nm range where tissues have the highest transmittance. Another treatment showing great promise is the PDT of skin cancers after topical application of the protoporphyrin IX precursor delta-aminolevulinic acid (or its ester forms). In all the cases, the photosensitizers should be rapidly excreted to avoid a long lasting skin photosensitivity.

Mechanisms of flavonoid repair reactions with amino acid radicals in models of biological systems: a pulse radiolysis study in micelles and human serum albumin.

Neutral tryptophan (*Trp) and tyrosine (TyrO(*)) radicals are repaired by certain flavonoids in buffer, in micelles and in human serum albumin (HSA) with corresponding formation of semioxidized flavonoid radicals. In deaerated buffer, *Trp but not TyrO(*) radicals react with catechin. In micelles, quercetin and rutin repair both *Trp and TyrO(*) radicals. In addition to amino acid reactivity, microenvironmental factors and nature of the flavonoids govern this repair. Electron transfer efficiencies from quercetin to negatively charged *Trp radicals are 100% in the micellar pseudophases of positively charged cetyltrimethylammonium bromide, (CTAB), and neutral Triton X100 (TX100), but 55% in negatively charged sodium dodecyl sulfate (SDS). In oxygen-saturated CTAB micelles, quercetin also reacts with the superoxide radical anion. When bound to domain IIA of HSA, quercetin repairs, by intra- or intermolecular encounter, less than 20% of oxidative damage to HSA. Quercetin can also repair freely circulating oxidized molecules with repair efficiencies falling to 7% for oxidized Trp, Tyr and alpha-MSH and to less than 2% for urate radical. This limited effectiveness is attributed both to the inaccessibility of bound quercetin and rutin toward radicals of circulating molecules and to the diffusion-controlled recombination of these radicals.

Mechanisms of flavonoid repair reactions with amino acid radicals in models of biological systems: a pulse radiolysis study in micelles and human serum albumin.

Neutral tryptophan (*Trp) and tyrosine (TyrO(*)) radicals are repaired by certain flavonoids in buffer, in micelles and in human serum albumin (HSA) with corresponding formation of semioxidized flavonoid radicals. In deaerated buffer, *Trp but not TyrO(*) radicals react with catechin. In micelles, quercetin and rutin repair both *Trp and TyrO(*) radicals. In addition to amino acid reactivity, microenvironmental factors and nature of the flavonoids govern this repair. Electron transfer efficiencies from quercetin to negatively charged *Trp radicals are 100% in the micellar pseudophases of positively charged cetyltrimethylammonium bromide, (CTAB), and neutral Triton X100 (TX100), but 55% in negatively charged sodium dodecyl sulfate (SDS). In oxygen-saturated CTAB micelles, quercetin also reacts with the superoxide radical anion. When bound to domain IIA of HSA, quercetin repairs, by intra- or intermolecular encounter, less than 20% of oxidative damage to HSA. Quercetin can also repair freely circulating oxidized molecules with repair efficiencies falling to 7% for oxidized *Trp, Tyr and alpha-MSH and to less than 2% for urate radical. This limited effectiveness is attributed both to the inaccessibility of bound quercetin and rutin toward radicals of circulating molecules and to the diffusion-controlled recombination of these radicals.

Polyhydroxylated 2-styrylchromones as potent antioxidants.

Four polyhydroxylated 2-styrylchromones, structurally related to flavones and cinnamic acid, have been studied. An SC derivative with OH groups only at positions 3' and 4' on the styryl moiety and another SC bearing an additional OH group at position 5 on the benzopyrone ring were more potent inhibitors of the Cu2+-induced peroxidation of LDL than the flavonoid quercetin. Fluorescence and absorption spectroscopies suggested that one LDL particle may bind 40 SC molecules. A pulse radiolysis study in pH 7 buffered micellar solutions of neutral TX100 and positively charged CTAB demonstrated that one-electron oxidation by *Br2-, *O2- and tryptophan radicals (8Trp) depends strongly on the micellar microenvironment. All SCs were readily oxidized by *O2- in CTAB micelles (rate constants: 6-18 x 10(6) M(-1) s(-1)). In TX100 micelles only the SC derivative with OH groups in position 3' and 4' reacted with *O2- (rate constant: 1.1 x 10(6) M(-1)s(-1)). In CTAB, electron transfer to *Trp radicals was observed for all SCs with rate constants > or =3.2 x 10(7) M(-1) s(-1). In TX100 micelles, this reaction occurred solely with the derivative bearing OH groups only at positions 3' and 4'.

Oxygen-copper (II) interplay in the repair of semi-oxidized urate by quercetin bound to human serum albumin.

The 1:1 complex of copper (II) and human serum albumin (HSA) slowly reacts with radiolytically generated *O2- radical-anion at a rate constant of 6.1 x 10(6) M(-1) s(-1). Absorbance and fluorescence spectroscopies demonstrate that addition of an equimolar portion of quercetin (QH2) to the solution of the copper (II)-HSA complex induces a relocalization of the copper resulting in a ternary copper (II)-QH2-HSA complex. This form of quercetin slowly oxidizes in air-saturated solutions. A 10-fold excess urate, a plasma antioxidant, cannot displace copper (II) bound to HSA. In N2O-saturated solutions the ternary complex form of QH2 can repair the urate radical with a rate constant of 2.7 x 10(6) M(-1) s(-1) by an electron transfer reaction similar to that observed in the absence of copper (II). In O2-saturated solutions and in the absence of copper, HSA-bound QH2 fails to repair the urate radical because of the fast competitive reaction of *O2- with urate radicals. However, addition of equimolar copper (II) restores the electron transfer from QH2 to the urate radical. These contrasting results are tentatively explained either by an enhanced reactivity of copper (II) with *O2- in the ternary complex or by direct production of quercetin radicals via a copper-catalyzed reduction of the *O2- radicals by QH2.

Plasma lipoproteins as mediators of the oxidative stress induced by UV light in human skin: a review of biochemical and biophysical studies on mechanisms of apolipoprotein alteration, lipid peroxidation, and associated skin cell responses.

There are numerous studies concerning the effect of UVB light on skin cells but fewer on other skin components such as the interstitial fluid. This review highlights high-density lipoprotein (HDL) and low-density lipoprotein (LDL) as important targets of UVB in interstitial fluid. Tryptophan residues are the sole apolipoprotein residues absorbing solar UVB. The UVB-induced one-electron oxidation of Trp produces (•)Trp and (•)O2 (-) radicals which trigger lipid peroxidation. Immunoblots from buffered solutions or suction blister fluid reveal that propagation of photooxidative damage to other residues such as Tyr or disulfide bonds produces intra- and intermolecular bonds in apolipoproteins A-I, A-II, and B100. Partial repair of phenoxyl tyrosyl radicals (TyrO(•)) by α -tocopherol is observed with LDL and HDL on millisecond or second time scales, whereas limited repair of α -tocopherol by carotenoids occurs in only HDL. More effective repair of Tyr and α -tocopherol is observed with the flavonoid, quercetin, bound to serum albumin, but quercetin is less potent than new synthetic polyphenols in inhibiting LDL lipid peroxidation or restoring α -tocopherol. The systemic consequences of HDL and LDL oxidation and the activation and/or inhibition of signalling pathways by oxidized LDL and their ability to enhance transcription factor DNA binding activity are also reviewed.

The alkyl chain length of 3-alkyl-3',4',5,7-tetrahydroxyflavones modulates effective inhibition of oxidative damage in biological systems: illustration with LDL, red blood cells and human skin keratinocytes.

It is shown that the relationship between the alkyl chain length of 3-alkyl-3',4',5,7 tetrahydroxyflavones (FnH) bearing alkyl chains of n=1, 4, 6, 10 carbons and their capacity to counter oxidative damage varies markedly with the nature of the biological system. In Cu(2+)-induced lipid peroxidation of low-density lipoprotein (LDL), the less hydrophobic short-chain F1H and F4H are probably located in the outer layer of LDL and parallel the reference flavonoid antioxidant, quercetin (Q) as effective inhibitors of lipid peroxidation. A marked inhibition of haemolysis induced in red blood cells (RBC) suspensions by the membrane-permeant oxidant, tert-butylhydroperoxide (t-BuOOH), is observed with F4H and F6H present at concentration in the micromolar range. However, F10H the most hydrophobic FnH is even more effective than Q against both haemolysis and lipid peroxidation as measured by malondialdehyde (MDA) equivalents. In oxidation of RBC by H(2)O(2,) at least 50 times more F6H and F10H than by t-BuOOH are required to only partly inhibit haemolysis and MDA production. The F1H, F4H and Q are found rather inactive under these conditions. At concentrations in the micromolar range, a marked protection against the cytotoxic effects of the t-BuOOH-induced oxidative stress in human skin NCTC 2544 keratinocytes is also exhibited by the four FnH antioxidants and is comparable to that of Q. Thus, the four FnH species under study may be considered as potent antioxidants which manifest complementary anti-oxidative actions in biological systems of markedly different complexity.

Albumin-bound quercetin repairs vitamin E oxidized by apolipoprotein radicals in native HDL3 and LDL.

In the minor fraction of HDL3 containing alpha-tocopherol (alphaTocOH), selective one-electron oxidation of Trp and Tyr residues of apolipoproteins A-I and A-II by *Br2- radical-anions produces the corresponding semioxidized species, TyrO* and *Trp. Repair of TyrO* by endogenous alphaTocOH generates the alpha-tocopheroxyl radical (alphaTocO*). Fast spectroscopic studies show that two populations representing 80% of alphaTocO* initially formed are repaired over several seconds with rate constants of 3.0 x 10(6) and 1.5 x 10(5) M-1 s-1 by quercetin bound to human serum albumin (HSA) at physiologically relevant concentration. Formation of HSA-bound quercetin radicals (*Qb) is observed. In the major fraction of HDL3 particles lacking alphaTocOH, TyrO* and *Trp are repaired by free and HSA-bound quercetin. In LDL particles which all contain alphaTocOH, alphaTocO* radicals are formed in the millisecond time scale by repair of TyrO* radicals produced in apolipoprotein B. Then, 75% of initial alphaTocO* are repaired over seconds by HSA-bound quercetin (rate constant: 2.0 x 10(6) M-1 s-1). HSA-bound quercetin can also repair *Trp radicals. In O2-saturated solutions, the fraction of alphaTocO* radicals (more than 50%) not repaired by superoxide radical-anions can be repaired by HSA-bound quercetin with formation of *Qb but to a much lesser extent in LDL than in HDL.

Interplay of oxygen, vitamin E, and carotenoids in radical reactions following oxidation of Trp and Tyr residues in native HDL3 apolipoproteins. Comparison with LDL. A time-resolved spectroscopic analysis.

It has been recently shown that the inhibition of apolipoprotein A-I (apoAI) reverse cholesterol transport activity during oxidation of HDL by myeloperoxidase may involve myeloperoxidase electron transfer pathways other than those leading to tyrosine chlorination. To better understand how such mechanisms might be initiated, the role of semioxidized Tyr and Trp residues in loss of apoAI and apolipoprotein A-II (apoAII) integrity has been assessed using selective Trp and Tyr one-electron oxidation by *Br2(-) radical-anions in HDL3 as well as in unbound apoAI and apoAII. Behavior of these radicals in apolipoprotein B of LDL has also been assessed. Formation of semioxidized Tyr in HDL3 is followed by partial repair during several milliseconds via reaction with endogenous alpha-tocopherol to form the alpha-tocopheroxyl radical. Subsequently, 2% of alpha-tocopheroxyl radical is repaired by HDL3 carotenoids. With LDL, a faster repair of semioxidized Tyr by alpha-tocopherol is observed, but carotenoid repair of alpha-tocopheroxyl radical is not. Only a small fraction of HDL3 particles contains alpha-tocopherol and carotenoids, which explains limited repair of semioxidized Tyr by alpha-tocopherol. All LDL particles normally contain multiple alpha-tocopherol and carotenoid molecules, and the lack of repair of alpha-tocopheroxyl radical by carotenoids probably results from hindered mobility of carotenoids in the lipid core. Western blots of gamma-irradiated HDL3 comparable to those reported for apoAI myeloperoxidase oxidation show that the incomplete repair of semioxidized Tyr and Trp induces apoAI and apoAII permanent damage including formation of a heterodimer of one apoAI with a monomeric apoAII at about 36 kDa.

Contrasting action of flavonoids on phototoxic effects induced in human skin fibroblasts by UVA alone or UVA plus cyamemazine, a phototoxic neuroleptic.

The potential protective effects of the flavanol catechin, the flavonol quercetin, the flavones, luteolin and rutin, and the isoflavones, genistein and daidzein, against the photo-oxidative stress induced by ultraviolet A radiation (UVA) and by phototoxic reactions resulting from the interaction of UVA with drugs and chemicals, has been assessed with cultured human skin fibroblasts. Lipid peroxidation and cell death have been chosen as model photobiological damage induced by UVA alone or photosensitized by cyamemazine (CMZ) and its photoproduct possessing phototoxic properties. Contrasting effects of flavonoids are observed. The flavanol, the flavonol and the flavones may protect against lipid peroxidation and cell death induced by 30 J cm(-2) of UVA alone or CMZ plus 10 J cm(-2) UVA. On the other hand, an amplification of the photodamage may be observed with isoflavones. A concentration-dependence study demonstrates that among the protective flavonoids, quercetin is the most efficient. The very effective protection brought by quercetin may result from its ability to scavenge reactive oxygen species produced by the photo-oxidative stress. However, the modification of membrane properties and the alteration of the lysosomal function by quercetin may not be neglected in these protective effects. The amplification of the photodamage by isoflavones is in sharp contrast with previous literature data demonstrating photoprotection by genistein. As a consequence, it may be concluded that an eventual antioxidant action of genistein may strongly depend on cells and photosensitizers. Furthermore such contrasting pro-versus anti-oxidant effects have to be taken into account when using flavonoid mixtures of plant extracts.

Repair of amino acid radicals of apolipoprotein B100 of low-density lipoproteins by flavonoids. A pulse radiolysis study with quercetin and rutin.

Selective oxidative damage to apolipoprotein B in LDL can be effected radiolytically by (*)Br(2)(-) radicals. Twenty-seven Trp residues constitute major primary sites of oxidation, but two-thirds of oxidized Trps ((*)Trp) that are formed are repaired by intramolecular electron transfer from Tyr residues with formation of phenoxyl radicals (TyrO(*)). Analysis of (*)Trp and TyrO(*) transient absorbance changes suggests that other apolipoprotein B residues, probably Cys, are oxidized. LDL-bound quercetin can efficiently repair this damage. Absorption studies show that a LDL particle has the capacity to bind approximately 10 quercetin molecules through interaction with apolipoprotein B. The repair occurs by intramolecular electron transfer characterized by a rate constant of 2 x 10(3) s(-)(1). In contrast, rutin, a related flavonoid which does not bind to LDL, cannot repair oxidized apolipoprotein B. Urate is a hydrophilic plasma antioxidant which displays synergistic antioxidant properties with flavonoids. Urate radicals produced by (*)Br(2)(-) can also be repaired by LDL-bound quercetin. This repair occurs with a reaction rate constant of 6.8 x 10(7) M(-)(1) s(-)(1). Comparison with previous studies conducted with human serum albumin-bound quercetin suggests that quercetin analogues tailored to be carried preferentially by lipoproteins might be more powerful plasma antioxidants than natural quercetin carried by serum albumin.