Cheese Whey Milk Adulteration Determination Using Casein Glycomacropeptide as an Indicator by HPLC.

Raw milk adulteration with cheese whey is a major problem that affects the dairy industry. The objective of this work was to evaluate the adulteration of raw milk with the cheese whey obtained from the coagulation process, with chymosin enzyme using casein glycomacropeptide (cGMP) as an HPLC marker. Milk proteins were precipitated with 24% TCA; with the supernatant obtained, a calibration curve was established by mixing raw milk and whey in different percentages, which were passed through a KW-802.5 Shodex molecular exclusion column. A reference signal, with a retention time of 10.8 min, was obtained for each of the different percentages of cheese whey; the higher the concentration, the higher the peak. Data analysis was adjusted to a linear regression model, with an R2 of 0.9984 and equation to predict dependent variable (cheese whey percentage in milk) values. The chromatography sample was collected and analyzed by three tests: a cGMP standard HPLC analysis, MALDI-TOF spectrometry, and immunochromatography assay. The results of these three tests confirmed the presence of the cGMP monomer in adulterated samples with whey, which was obtained from chymosin enzymatic coagulation. As a contribution to food safety, the molecular exclusion chromatography technique presented is reliable, easy to implement in a laboratory, and inexpensive, compared with other methodologies, such as electrophoresis, immunochromatography, and HPLC-MS, thus allowing for the routine quality control of milk, an important product in human nutrition.

Response Surface Methodology (RSM) for analysing culture conditions of Acidocella facilis strain USBA-GBX-505 and Partial Purification and Biochemical Characterization of Lipase 505 LIP / Metodología de Superficie de Respuesta (RSM) para el análisis de las condiciones de cultivo de Acidocella facilis cepa USBA-GBX-505 y la purificación parcial y caracterización bioquímica de la lipasa 505 LIP / Metodologia de Superfície de Resposta (MSR) para a análise das condições de cultivo de Acidocella facilis cepa USBA-GBX-505 e purificação parcial e caracterização bioquímica da lip ase 505 LIP

Abstract Using Response Surface Methodology (RSM) we evaluated the culture conditions (nitrogen source, carbon source, pH and agitation rate) that increase the biomass of Acidocellafaalis strain USBA-GBX-505 and therefore enhance the production of its lipolytic enzyme, 505 LIP. RSM results revealed that yeast extract and agitation were key culture factors that increased the growth-associated lipolytic activity by 4.5-fold (from 0.13 U.mg-1 to 0.6 U.mg-1). The 505 LIP lipase was partially purified using size-exclusion chromatography and ion-exchange chromatography. Its molecular weight was >77 kDa. The enzyme shows its optimum catalytic activity at 55 °C and pH 7.5. EDTA, PMSF, 1-butanol and DMSO inhibited enzymatic activity, whereas Tween 20, acetone, glycerol and methanol increased it. Metallic ions are not required for the activity of 505 LIP, and even have an inhibitory effect on the enzyme. This study shows the potential use of A. facilis strain USBA- GBX-505 for the production of a newly identified lipolytic enzyme, 505 LIP, which is stable at moderate temperatures and in the presence of organic solvents. These are important characteristics for the synthesis of many useful products.

Resumen Por medio de la Metodología de Respuesta de Superficie (RSM) evaluamos las condiciones de cultivo (fuente de N, fuente de C, pH y tasa de agitación) que incrementan la biomasa de Acidocella facilis cepa USBA-GBX-505 y, como consecuencia, la producción de su enzima lipolítica, llamada 505 LIP. Los resultados de la RSM revelaron que el extracto de levadura y la agitación fueron factores de cultivo claves, que incrementaron de 4 a 5 veces la actividad lipolítica asociada al crecimiento (de 0.13 U.mg-1 a 0.6 U.mg-1). La lipasa 505 LIP se purificó parcialmente usando cromatografía de exclusión por tamaño y cromatografía de intercambio iónico. Su peso molecular fue > 77 kDa. La enzima muestra su actividad catalítica óptima a 55 °C y pH 7.5. El EDTA, el PMSF, el 1-butanol y el DMSO inhibieron la actividad enzimática, mientras que el Tween 20, la acetona, el glicerol y el metanol la incrementaron. La enzima 505 LIP no requiere iones metálicos para su actividad, e incluso se inhibe en presencia de ellos. Este estudio muestra el uso potencial de A. facilis cepa USBA-GBX-505 para la producción de una nueva enzima lipolítica, 505 LIP, que es estable a temperaturas moderadas y en la presencia de solventes orgánicos. Estas son características importantes en la síntesis de muchos productos útiles.

Resumo Utilizando a Metodologia de Superfície de Resposta (MSR) avaliamos as condições de cultivo (fontes de nitrogénio e carbono, pH e taxa de agitação) que aumentam a biomassa de Acidocella facilis cepa USBA-GBX-505, e, portanto, elevam a produção de sua enzima lipolítica 505 LIP. Os resultados da MSR revelaram que o extrato de levedura e a agitação foram fatores de cultivo chave que permitiram aumentar 4 a 5 vezes a atividade lipolítica associada ao crescimento (de 0,13 U.mg-1 a 0,6 U.mg-1). A lipase 505 LIP foi parcialmente purificada utilizando cromatografia por exclusão de tamanho e cromatografia de intercambio iónico. Seu peso molecular foi > 77 kDa. A enzima mostra sua atividade catalítica ótima a 55 °C e pH 7,5. EDTA, PMSF, 1-butanol e DMSO inibiram a atividade enzimática, enquanto que Tween 20, acetona, glicerol e metanol aumentaram esta atividade. Íons metálicos não são necessários para a atividade da 505 LIP, apresentando inclusive efeito inibitório da enzima. Este estudo demonstra o potencial uso de A. facilis cepa USBA-GBX-505 para a produção de uma nova enzima lipolítica, 505 LIP, a qual é estável a moderadas temperaturas e na presença de solventes orgánicos. Estas características são importantes para a síntese de diversos produtos úteis.

De novo DESIGN AND SYNTHESIS OF AN ICE-BINDING, DENDRIMERIC, POLYPEPTIDE BASED ON INSECT ANTIFREEZE PROTEINS / SÍNTESIS Y DISEÑO De novo DE UN POLIPÉPTIDO DENDRÍMERO CON UNIÓN AL HIELO BASADO EN LAS PROTEINAS ANTICONGELANTES DE INSECTOS / Síntes e desenhoo de novo de um polipetídeos dendrímero com uniâo ao gelo baseado nas proteínas anticongelantes de insetos

A new strategy is presented for the design and synthesis of peptides that exhibit ice-binding and antifreeze activity. A pennant-type dendrimer polypeptide scaffoíd combining an a-helical backbone with four short (β-strand branches was synthesized in solid phase using Fmoc chemistry in a divergent approach. The 51-residue dendrimer was characterized by reverse phase high performance liquid chromatography, mass spectrometry and circular dichroism. Each (β-strand branch contained three overlapping TXT amino acid repeats, an ice-binding motif found in the ice-binding face of the spruce budworm (Choristoneura fumiferana) and beetle (Tenebrio molitor) antifreeze proteins. Ice crystals in the presence of the polypeptide monomer displayed fiat, hexagonal plate morphology, similar to that produced by weakly active antifreeze proteins. An oxidized dimeric form of the dendrimer polypeptide also produced fiat hexagonal ice crystals and was capable of inhibiting ice crystal growth upon temperature reduction, a phenomenon termed thermal hysteresis, a defining property of antifreeze proteins. Linkage of the pennant-type dendrimer to a tri-functional cascade-type polypeptide produced a trimeric macromolecule that gave flat hexagonal ice crystals with higher thermal hysteresis activity than the dimer or monomer and an ice crystal burst pattern similar to that produced by samples containing insect antifreeze proteins. This macromolecule was also capable of inhibiting ice recrystallization.

Una nueva estrategia se presenta para el diseño y síntesis de péptidos que se unen al hielo y evidencian actividad anticongelante. Un polipéptido dendrímero del tipo banderín, que combina en su estructura un núcleo a-hélice con cuatro ramificaciones cortas de hojas β, se sintetizó en fase sólida utilizando la química Fmoc con una estrategia divergente. El dendrímero de 51 residuos se caracterizó por cromatografía líquida de alta resolución, espectrometría de masas y dicroís-mo circular. Cada ramifcación de hoja β contiene tres repeticiones de los motivos de aminoácidos TxT sobrelapados, un motivo de unión al hielo presente en la cara de unión de las proteínas anticongelantes del gusano de brotes de abeto (Choristoneura fumiferana) y en el escarabajo (Tenebrio molitor). Los cristales de hielo en presencia del polipéptido monomérico presentan una morfología hexagonal plana, similar a la producida por las proteínas anticongelantes con una débil actividad. Un dímero oxidado del polipéptido también produce cristales de hielo hexagonales planos que fueron capaces de inhibir el crecimiento de los cristales de hielo a medida que se disminuía la temperatura, un fenómeno conocido como histéresis térmica, esto es, una propiedad que define las proteínas anticongelantes. La vinculación del dendrímero tipo banderín a un polipéptido tipo cascada trifuncional produjo una macro-molécula trimérica que generó cristales de hielo hexagonales planos con una mayor actividad de histéresis térmica que los dímeros y los monómeros y un patrón de estallido del cristal de hielo muy similar al producido por las muestras que contienen proteínas anticongelantes de insectos. Estas moléculas además fueron capaces de inhibir la recristianización del hielo.

Uma nova estratégia é apresentada para o desenho e síntese de peptídeos que se unem ao gelo e apresentam atividade anticongelante. Um polipeptídeo dendrímero do tipo pennant que combina em sua estrutura um núcleo a-hélice com quatro ramifcacoes curtas de folhas β foi sintetizado em fase sólida utilizando a química Fmoc com uma estratégia divergente. O dendrímero de 51 resíduos foi caracterizado por cromatografa líquida de alta resolução, espectrometria de massas e dicroísmo circular. Cada ramifcacao de folha (β contém três repeticoes dos motivos de aminoácidos TxT sobrepostos, um motivo de união ao gelo presente na cara de união das proteínas anticongelantes do verme de Choristoneura fumiferana e no escaravelho (Tenebrio molitor). Os cristais de gelo, em presença do polipeptídeo monomérico, apresentam urna morfologia hexagonal plana, similar à produzida pelas proteínas anticongelantes com uma atividade fraca. Um dímero oxidado do polipeptídeo também produz cristais de gelo hexagonais planos e fo-ram capazes de inibir o crescimento dos cristais de gelo à medida que a temperatura diminuia, um fenômeno conhecido como histerese térmica una propriedade que def ne as proteínas anticongelantes. A vinculação do dendrímero tipo pen-nant a um polipeptídeo tipo cascata tri-funcional produziu uma macromolécula trimérica que gerou cristais de gelo hexa-gonais planos com uma maior atividade de histerese térmica que os dímeros e os monómeros e um padrão de estouro do cristal de gelo muito similar ao produzi-do pelas amostras que contêm proteínas anticongelantes de insetos. Estas moléculas, aliás, foram capazes de inibir a recristalização do gelo.

Characterising Mycobacterium tuberculosis Rv1510c protein and determining its sequences that specifically bind to two target cell lines.

The process of Mycobacterium tuberculosis infection of the macrophage implies a very little-known initial recognition and adherence step, important for mycobacterial survival; many proteins even remain like hypothetical. The Rv1510c gene, encoding a putatively conserved membrane protein, was investigated by analysing the M. tuberculosis genome sequence data reported by Cole et al. and a previous report that used PCR assays to show that the Rv1510 gene was only present in M. tuberculosis. This article confirmed all the above and identified the transcribed gene in M. tuberculosis, Mycobacterium africanum, and in M. tuberculosis clinical isolates. Antibodies raised against peptides from this protein recognised a 44 kDa band, corresponding to Rv1510c theoretical mass (44,294 Da). Assays involving synthetic peptides covering the whole protein binding to U937 and A549 cell lines led to recognising five high activity binding peptides in the Rv1510 protein: 11094, 11095, 11105, 11108, and 11111. Their affinity constants and Hill coefficients were determined by using U937 cells. Cross-linking assays performed with some of these HABPs showed that they specifically bound to a U937 cell line 51 kDa protein, but not to Hep G2 or red blood cell proteins, showing this interaction's specificity.

Amino terminal peptides from the Plasmodium falciparum EBA-181/JESEBL protein bind specifically to erythrocytes and inhibit in vitro merozoite invasion.

Several EBA-175 paralogues (EBA-140, EBA-165, EBA-175, EBA-181, and EBL-1) have been described among the Plasmodium falciparum malaria parasite proteins, which are important in the red blood cell (RBC) invasion process. EBA-181/JESEBL is a 181 kDa protein expressed in the late schizont stage and located in the micronemes; it belongs to the Plasmodium Duffy binding-like family and is able to interact with the erythrocyte surface. Here, we describe the synthesis of 78, 20-mer synthetic peptides derived from the reported EBA-181/JESEBL sequence and their ability to bind RBCs in receptor-ligand assays. Five peptides (numbered 30030, 30031, 30045, 30051, and 30060) displayed high specific binding to erythrocytes; their equilibrium binding parameters were then determined. These peptides interacted with 53 and 33 kDa receptor proteins on the erythrocyte surface, this binding being altered when RBCs were pretreated with enzymes. They were able to inhibit P. falciparum merozoite invasion of RBCs when tested in in vitro assays. According to these results, these five EBA-181/JESEBL high specific erythrocyte binding peptides, as well as the entire protein, were seen to be involved in the molecular machinery used by the parasite for invading RBCs. They are thus suggested as potential candidates in designing a multi-sub-unit vaccine able to combat the P. falciparum malaria parasite.

Human papillomavirus type 16 and 18 L1 protein peptide binding to VERO and HeLa cells inhibits their VLPs binding.

Human papillomaviruses (HPVs) are the cause of epithelial lesions, HPV type 16 and type 18 being associated with the development of anogenital cancer. The L1 Major Capsid Protein (L1) represents about 90% of total HPV protein and is involved in virus-host cell interaction, but little is known about this binding process. L1 sequences from HPV types 16 and 18 were synthesized in 56 20-mer peptides, covering the entire protein, HPLC-purified, (125)I-radiolabeled and tested in VERO and HeLa cell-binding assays to identify those peptides with high specific binding activity. Peptides 18283 (residues 54-77) and 18294 (274-308) from HPV16 L1, as well as 18312 (59-78) and 18322 (259-278) from HPV18 L1, presented high specific target cell binding activity. Peptide 18283 and 18294 affinity constants were 300 and 600 nM, respectively. Enzyme cell treatment before binding assay indicated that VERO and HeLa cell peptide receptor is a surface-exposed protein. There was a 60% reduction in peptide 18283 binding to heparin lyase-treated cells. Cross-linking assays showed that these proteins molecular weights were around 69 and 54 kDa. Peptides 18283 and 18294 specifically inhibited HPV-16 VLP binding to HeLa cells. According to the L1- and VLP-reported structure, both peptides are close on the VLP-surface, belonging to the outer surface broad pockets suggested as being potential receptor sites. Furthermore, it has been reported that a conserved motif from peptide 18294 is the target for neutralizing antibodies. These results suggest that such binding sequences are used by the virus as cell-binding regions.