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Approximately 70% of the initial ferriprotoporphyrin IX polymerizing activity in cell-free preparations of erythrocytes infected with Plasmodium berghei was recovered in a chloroform extract. No polymerizing activity remained in the residue. In studies to identify substances that promote FP polymerization, arachidonic, linoleic, oleic, and palmitoleic acids, 1-mono- and di-oleoylglycerol, and the detergents, SDS, Tween 80, and n-octyl-glucopyranoside, were active. Tri-oleoylglycerol, cholesterol, di-oleoylphosphatidylethanolamine, and stearic and palmitic acids were inactive. The model lipid, mono-oleoylglycerol (250 nmol), co-precipitated with FP from a 0.09 M acetate medium at pH 5 and promoted the polymerization of 215 nmol (61%) of the ferriprotoporphyrin IX in the precipitate during a 24-h incubation at 37 degrees C. Polymerization was maximal at pH 5, it was approximately linear for 2 h, and it continued at a decreasing rate for 24 h. The polymer contained exclusively ferriprotoporphyrin IX (97+/-1.3%, mean+/-S.E., n=4) and exhibited the solubility and the electronic absorption and infrared spectral characteristics of the sequestered ferriprotoporphyrin IX of hemozoin. Detergents presumably promote polymerization in an acid medium by helping to dissolve monomeric FP. We suggest that unsaturated lipids co-precipitate with FP in the parasite's acidic food vacuole and also dissolve sufficient monomeric FP to allow polymerization.

Bloodstains are common forensic samples, which can often be problematic for genotyping. Besides hematin naturally occurring in bloodstain extracts, components of the substrate on which forensic evidence is found can affect DNA amplification as powerful polymerase chain reaction (PCR) inhibitors. The aim of this study was to overcome the PCR inhibition by using powdered activated carbon (PAC) purification. We evaluated the effectiveness of PAC-purification at 37˚C with 60 minutes contact time vs. ultrafiltration using Amicon®Ultra-4 unit (100K) in removing hematin and soluble humic substances. Genomic DNA from bloodstains on peat soil substrate and from blood without substrate was extracted using Chelex®100 and purified by PAC or Amicon®Ultra-4. In addition, for some samples purification was not performed. Real-time PCR was used for DNA quantity estimation and AmpFlSTR®SGM Plus® kit for amplification. Moreover, concentrations of residual inhibitors were determined by UV/VIS spectrometry and compared to unpurified samples. PAC more effectively removed hematin, without DNA loss and provided high DNA quality. Our results show that purification significantly decreased humic substances content. Conversely, both purification methods significantly increased average DNA yield and provided equivalently good DNA quality. In conclusion, this study provides the first insight into a new application of PAC as a well-known adsorbent, based solely on temperature and contact time without introducing any reagents that may interfere with subsequent downstream analysis and may provide the basis for its further application in forensic casework.

The goal of this manuscript was to study the immobilization of horseradish peroxidase (HRP) and hematin on different supports at selected conditions to be used in the removal of two synthetic dyes: Alizarin Red (AR) and Eriochrome Blue Black R (EBBR). Reuse in the target reactions was tested for the best biocatalysts. Three supports were selected: magnetite-activated carbon Fe 3O 4/C, activated carbon C and chitosan CS. Several reasons led to discard the use of Fe 3O 4/C and C as supports for hematin and HRP. CS-glutaraldehyde (GA)-HRP not only has low ability to remove EBBR, but also the enzymatic activity of HRP could not be separated from the dye adsorption on CS. However, some adsorptive functions of CS-GA were lost when HRP was supported on it. In the case of CS-GA-hematin, the most important factors for EBBR removal are dye concentration and mass of catalyst, whereas the temperature has almost no effect on the conversion of dye, considering in all cases the H 2O 2 concentration (0.021M) constant. There was not a marked decrease in the removal of AR even after the ninth reuse in the case of CS-GA-hematin. For EBBR removal, with CS-GA-HRP, in the second reuse the catalyst was completely deactivated, whereas with CS-GA-hematin this was found only in the fourth reuse. Fil: Pirillo, Silvina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Química del Sur. Universidad Nacional del Sur. Departamento de Química. Instituto de Química del Sur; Argentina Fil: Rueda, Elsa Haydee. Universidad Nacional del Sur. Departamento de Química; Argentina Fil: Ferreira, María Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Planta Piloto de Ingeniería Química. Universidad Nacional del Sur. Planta Piloto de Ingeniería Química; Argentina. Universidad Nacional del Sur. Departamento de Química; Argentina

Tip-enhanced Raman scattering (TERS) spectroscopy is a promising technique for nanoscale chemical analysis. However, there are several challenges preventing widespread application of this technology, including reproducible fabrication of efficient TERS probes. These problems reflect a lack of clear understanding of the origins of, and the parameters influencing TERS. It is believed that the coating characteristics at the apex of the tip have a major effect on the near-field optical enhancement and thus the TERS activity of a metalized probe. Here we show that the aspect ratio of the tip can play a significant role in the efficiency of TERS probes. We argue that the electrostatic field arising from the lightning-rod effect has a substantial role in the observed TERS effect. This argument is supported by 'edge-enhanced Raman scattering' which is shown for a noble metal film. Furthermore, it is reported that an associated tip-surface-enhanced Raman scattering effect can be achieved by using a TERS-inactive metalized probe on a surface-enhanced Raman spectroscopy-inactive roughened surface. This observation can be explained by an interparticle enhancement of the electromagnetic field. Refereed/Peer-reviewed

Forenzični biološki uzorci često sadržavaju male količine DNA uz prisustvo smjese inhibitora umnažanja DNA. Tijekom izolacije DNA s podloge dijelovi podloge kao potencijalni inhibitori mogu se izolirati zajedno s genomskom DNA, čime se smanjuje ili u potpuno onemogućuje umnažanje mikrosatelitnih lokusa. Cilj ovog istraživanja jest ukloniti inhibitore umnažanja DNA pomoću aktivnog ugljena uz što manji gubitak humane DNA. Kvaliteta izolirane DNA testirana je umnažanjem deset mikrosatelitnih lokusa. Uspoređena je efikasnost neafinitetnih metoda izolacije genomske DNA (Chelex i organska izolacija), kao i metoda pročišćavanja ultrafiltracijom (Amicon komercijalna kolona) i adsorpcijom (aktivni ugljen). Općenito, Chelex se pokazao kao učinkovitija metoda izolacije DNA, a usporedbom metoda pročišćavanja nije bilo statistički značajne razlike u kvaliteti umnožene DNA. Primjena aktivnog ugljena pokazala se jednostavnom, brzom, ekonomičnom i reproducibilnom metodom pročišćavanja. Aktivni ugljen uspješno je adsorbirao hematin, prirodni inhibitor iz krvi, bez značajnih gubitaka DNA. Nadalje, smanjena je koncentracija inhibitora iz podloge kao što su humusne tvari iz zemlje i tanini iz drva. Amicon se pokazao kao efikasnija metoda za uklanjanje indiga iz trapera kao podloge. Pročišćavanje bioloških uzoraka temeljeno na fizikalnim i kemijskim svojstvima aktivnog ugljena dosad nije provedeno, stoga spoznaje utvrđene u ovom istraživanju daju prvi uvid u novu primjenu tog dobro poznatog adsorbensa. Forensic biological samples often contain small quantities of DNA in presence of mixture of DNA amplification inhibitors. During the genomic DNA extraction, parts of the substrate, as potential inhibitors, could be co-extracted with genomic DNA and reduce or completely disable amplification of microsatellite loci. Aim of this study is to eliminate inhibitors of DNA amplification using activated carbon, without significant loss of human DNA. Quality of extracted DNA was tested by amplification of ten microsatellite loci. In this doctoral thesis, efficiency of non-affinity DNA extraction methods (Chelex and organic extraction) as well as purification methods based on ultrafiltration (Amicon commercial column) and adsorption (activated carbon) were compared. Generally, Chelex extraction method was more efficient, while no statistically significant difference was obtained if purification methods were compared. Application of activated carbon was shown as simple, fast, economic and reproducible purification method. Activated carbon effectively adsorbed hematin, as inhibitor naturally originated from blood, without significant loss of DNA. In addition, concentration of inhibitors present in the substrate, such as humic substances in soil substrate, as well as tannins in wood, was reduced. Amicon was more efficient method for elimination of indigo dye as inhibitor present in denim substrate. Since purification of biological samples based solely on chemical and physical characteristics of activated carbon has not yet been performed, results of this research provide the first insight into a new application of this well known adsobent.

We describe herein the design, synthesis, biological evaluation, and structure-activity relationship (SAR) studies of an innovative class of antimalarial agents based on a polyaromatic pharmacophore structurally related to clotrimazole and easy to synthesize by low-cost synthetic procedures. SAR studies delineated a number of structural features able to modulate the in vitro and in vivo antimalarial activity. A selected set of antimalarials was further biologically investigated and displayed low in vitro toxicity on a panel of human and murine cell lines. In vitro, the novel compounds proved to be selective for free heme, as demonstrated in the beta-hematin inhibitory activity assay, and did not show inhibitory activity against 14-alpha-lanosterol demethylase (a fungal P450 cytochrome). Compounds 2, 4e, and 4n exhibited in vivo activity against P. chabaudi after oral administration and thus represent promising antimalarial agents for further preclinical development.

This manuscript presents the UV-vis study of the aniline elimination by polymerization using peroxidases from different sources and hematin, free and supported. The use of magnetite-supported hematin has potential in the implementation of aniline elimination by polymerization in acidic media at pH 4. The temperature must be selected between 45 and 65°C. Crude preparations of Pleurotus sajor caju (PSC) are an alternative to purified Horseradish peroxidase (HRP) as the catalyst of the reaction. Pernigraniline is the main species found in the reaction media after aniline elimination procedure. Supported oxidoreductases are active biocatalysts and conversion of aniline in the 30-90% range can be obtained, depending on the catalyst. The UV-vis spectra show bands corresponding to base and protoned (blue and purple-deep red) pernigraniline, a contribution of quinoid species at 480 nm and a minor contribution of base blue emeraldine and another oxidation intermediate states between pernigraniline and emeraldine. At selected conditions, the recovered solid showed greenish/reddish coloration. The goal is the precipitation of the aniline as polyaniline. Supported biocatalysts were less active than free enzymes. Fil: Saidman, Silvana Beatriz. Universidad Nacional del Sur; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Rueda, Elsa Haydee. Universidad Nacional del Sur; Argentina Fil: Ferreira, María Luján. Universidad Nacional del Sur; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Planta Piloto de Ingeniería Química. Universidad Nacional del Sur. Planta Piloto de Ingeniería Química; Argentina

Porphyrins related to the naturally occurring pigment heme were found to effectively interfere with the aggregation of beta-amyloid peptides as determined by an immunoassay configured for the detection of beta-amyloid oligomers. Oligomerisation of beta-amyloid is believed to be a key event in the progression of Alzheimer's disease. Inhibition of this aggregation is thus an important strategy in combating this commonest form of senile dementia. Evidence was also generated for hemin and hematin mediated protection of cultured cells against the neurotoxic effects of beta-amyloid. These data are discussed with reference to the known pathology of Alzheimer's disease and the chemistry of porphyrins.

Histidyl-tRNA synthetase, an enzyme against which antibodies are directed in some patients with polymyositis, has been purified 5000-fold from HeLa cells, but was extremely labile to dilution or on storage at -80 degrees C. In order to facilitate study of the biochemical and immunological properties of the enzyme, a stabilizer was sought. Hemoglobin at 2 mg/ml was found to stimulate the enzyme and also partially preserved the activity of the enzyme stored at a low concentration (less than 10 micrograms/ml). Hematin, but not the globin protein, could substitute for hemoglobin in stimulating the enzyme.

X-ray absorption spectroscopy is used to determine the local atomic structure around the iron atom from a soluble synthetic analogue of malaria pigment (hemozoin), cf. ferrimesoporphyrin IX of mesohematin anhydride, in the absence or presence of chloroquine (CQ) in dimethyl sulfoxide (DMSO). Of particular note are the CQ:induced changes in the structure of mesohematin anhydride, which might confirm the formation of CQ-ferrimesoporphyrin IX complex. Examination of solutions of mesohematin anhydride dissolved in DMSO reveals preservation of the dimerlike structure with the central iron atoms of the ferric porphyrin IX reciprocally linked by propionate side chains, which is typical for hematin anhydride (beta-hematin). In the presence of CO, additional light atoms, such as nitrogen, carbon, and oxygen, were detected surrounding the iron in a distance ranging from 2.48 to 3.77 angstrom. The changes introduced by CQ in DMSO are different from that observed in the acetic acid solution.

International audience; Malaria is an infectious disease caused by Plasmodium type parasites transmitted by the bites of infected female anopheles mosquitoes. The malaria parasite multiplies in red blood cells where it degrades hemoglobin. This degradation of hemoglobin proteins releases hematin, an iron-containing porphyrin, which provokes membrane disruption and lysis. The malaria parasite blocks hematin-induced lysis by biocrystallization, a process that converts hematin into insoluble and chemically inert crystals. Hematin molecules are especially prone to self-assembly as dimers, oligomers and aggregates depending on environmental conditions (pH, solvent, temperature, concentration, ionic strength). Considering the different forms of hematin-based assemblies, it is still unclear which are the ones able to interact with membranes. We have prepared hematin under different conditions to form hematin-based assemblies and to measure their ability to interact and to disorganize membranes. Our results show that different forms of hematin molecules are able to penetrate lipid membranes. Interestingly, this membrane activity is spontaneously inhibited at acidic pH and it can be restored under neutral pH. By contrast, the oligomers of β-hematin were found to be completely harmless toward lipid membranes. Finally, the AFM visualization of hematin interaction with supported lipid bilayers showed for the first time its preferential interaction with defaults in membranes, at the boundaries between two distinct lipid phases. The superficial adsorption of aggregates on membranes and the absence of effect due to oligomers were also confirmed with AFM.