RESUMEN
Among the lipids in bovine milk, minor components such as conjugated linoleic acids and phospholipids are more attractive than triacylglycerols from the standpoint of biological activity. To explore novel functions of bovine milk polar lipids (MPL), topical application to murine dorsal skin was introduced as an assay system. The acetone-insoluble lipid fraction derived from bovine milk was dispersed in ethanol and applied to 9-wk-old C57BL/6N female mice for 3 wk. In combination with visual assessment of the dorsal pigmentation, the progression of the hair cycle was estimated by calculating the ratio of subcutis to dermis thickness. The administration of MPL led to earlier progression of the hair cycle compared with administration of the vehicle. In some cases, the extent of MPL-induced hair cycle progression was comparable to that in animals treated with minoxidil, the most well-known reagent that initiates anagen. These results indicate that the MPL preparation contains a dermal penetrative component that can regulate the hair cycle and, thus, this preparation possesses potential for cosmetic use.
Asunto(s)
Cabello/efectos de los fármacos , Lípidos/farmacología , Leche/metabolismo , Administración Tópica , Animales , Bovinos , Femenino , Cabello/crecimiento & desarrollo , Lípidos/administración & dosificación , Lípidos/aislamiento & purificación , Ratones , Ratones Endogámicos C57BLRESUMEN
Several attempts have been made to incorporate whey proteins into curd to increase cheese yield. For some types of cheese, degradation of whey proteins that have been incorporated into the curd would be required to obtain acceptable flavor and texture. On the basis of the high potential for protease synthesis in Aspergillus oryzae, sodium nitrate as a nitrogen source in a minimal medium for fungi, known as Czapek-Dox medium, was replaced with whey protein isolate to induce the protease to hydrolyze whey protein using A. oryzae AHU7146. A solid-phase medium adjusted to pH 6 was suitable for this purpose when incubation was carried out at 25°C for 2 wk. The application of column chromatography enabled the resolution of 3 proteolytic components (1, 2, and 3). With respect to optimal temperature and zymographic analysis, component 1 was similar to component 3. In contrast, component 2 was less abundant than the other components and exhibited activity in the alkaline pH region. The degradation of ß-lactoglobulin and α-lactalbumin in whey protein isolate solution by the crude enzyme was primarily attributed to the action of components 1 and 3, based on HPLC analysis and the N-terminal amino acid sequences; however, zymography demonstrated evident proteolysis due to component 2. Because heat-denatured whey protein aggregates were digestible by the crude enzyme, the proteolytic system from A. oryzae has the potential as an additive to stimulate the ripening of cheese enriched with whey protein.
Asunto(s)
Aspergillus oryzae/metabolismo , Proteínas de la Leche/metabolismo , Péptido Hidrolasas/biosíntesis , Animales , Aspergillus oryzae/crecimiento & desarrollo , Queso/microbiología , Medios de Cultivo/química , Manipulación de Alimentos/métodos , Microbiología de Alimentos , Péptido Hidrolasas/química , Proteína de Suero de LecheRESUMEN
In the present study, lactoferrin binding to bifidobacteria and detection of lactoferrin-binding protein in membrane fractions of several bifidobacteria have been demonstrated. This is the first report showing the binding of bovine lactoferrin to four Bifidobacterium spp. (B. infantis, B. breve, B. bifidum, B. longum) incubated with biotinylated lactoferrin and fluorescein conjugated-avidin and observed under an inverted confocal laser scanning microscope. Fluorescence staining showed lactoferrin binding at the pole of the bacterial cells. A lactoferrin-binding protein with a molecular weight of approximately 67 kDa was also detected in the membrane fraction of Bifidobacterium spp. by far western blotting technique using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin. Based on the results of this and previously reported studies, we suggest that binding of lactoferrin to Bifidobacterium longum is strain-dependent.
Asunto(s)
Proteínas Bacterianas/metabolismo , Bifidobacterium/metabolismo , Pared Celular/metabolismo , Lactoferrina/metabolismo , Animales , Bifidobacterium/citología , Bovinos , Lactoferrina/farmacología , Microscopía Confocal , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Especificidad de la EspecieRESUMEN
To evaluate the potential of yeasts of dairy origin as probiotics, we tested 8 species including Candida humilis, Debaryomyces hansenii, Debaryomyces occidentalis, Kluyveromyces lactis, Kluyveromyces lodderae, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Yarrowia lipolytica, isolated from commercial blue cheese and kefir. Strains were randomly selected from each species and tested for their ability to adhere to human enterocyte-like Caco-2 cells in culture. Among the 8 species, K. lactis showed higher adhesive ability than K. marxianus, K. lodderae, and D. hansenii. The other 4 species were poorly adhesive. All species other than K. marxianus and C. humilis were resistant to acidic conditions. In the presence of bile acid, growth inhibition was undetectable when incubation was carried out at 27 degrees C; however, it was evident for C. humilis and a strain of D. occidentalis when incubated at 37 degrees C. Moreover, the influence of proteinase treatment of living cells of K. lactis and K. lodderae on their adhesion to Caco-2 cells was evaluated. Although a slight reduction was recognized when K. lactis was treated with proteinase K, the influence of intestinal protease treatments of pepsin followed by trypsin was negligible. These results indicated that a proteinaceous factor was unlikely to be involved in adhesion of K. lactis and K. lodderae to Caco-2 cells. No stimulation of IL-8 synthesis by Caco-2 cells was recognized in the presence of K. lactis. In conclusion, K. lactis was the most attractive to continue study for use as probiotic microorganisms.
Asunto(s)
Adhesión Celular/fisiología , Productos Lácteos/microbiología , Alimentos Orgánicos/microbiología , Probióticos/aislamiento & purificación , Levaduras/aislamiento & purificación , Levaduras/fisiología , Animales , Células CACO-2/microbiología , Queso/microbiología , Microbiología de Alimentos , Humanos , Concentración de Iones de Hidrógeno , Kluyveromyces/aislamiento & purificación , Kluyveromyces/fisiología , Saccharomyces/aislamiento & purificación , Saccharomyces/fisiología , Saccharomycetales/aislamiento & purificación , Saccharomycetales/fisiología , Temperatura , Yarrowia/aislamiento & purificación , Yarrowia/fisiologíaRESUMEN
Degradation of dextran beads was observed when the water-soluble fraction of a blue cheese extract was applied to the top of a Sephadex G-150 or G-200 column. This phenomenon suggests the presence of a specific enzyme that can hydrolyze dextran. After removal of casein components from the blue cheese fraction, ammonium sulfate treatment and gel filtration chromatography were performed to isolate the enzyme fraction. The enzymatic products were analyzed by thin-layer chromatography and gel filtration chromatography and identified as isomaltooligosaccharides. The isoelectric point of this enzyme fraction was approximately 4.9, as determined by isoelectric focusing using Rotofor, and the molecular weight of the fraction was 65 kDa, as estimated by sodium dodecyl sulfate (SDS)-PAGE. Optimum pH for enzymatic activity was 5.0 to 5.3. A partial N-terminal amino acid sequence of 20 residues was determined to be ATPDEWRSRSIYFMLTDRGA from an enzyme fraction further purified by ion-exchange chromatography and native PAGE. This sequence showed a maximum homology of 80% with alpha-amylase or Taka amylase that originated from various microorganisms.
Asunto(s)
Queso , Dextranos/metabolismo , Oligosacáridos/química , Penicillium/enzimología , Secuencia de Aminoácidos , Amilasas , Cromatografía en Gel/veterinaria , Cromatografía en Capa Delgada/veterinaria , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica , Punto Isoeléctrico , Datos de Secuencia Molecular , Peso Molecular , Oligosacáridos/aislamiento & purificación , Homología de Secuencia de AminoácidoRESUMEN
Porcine mammary epithelial cells were isolated to culture on collagen gel followed by gel floating treatment to evaluate differentiation under the culture conditions of serum-free medium, supplemented with combinations of insulin, hydrocortisone, and prolactin. After the culture period, the mammary cells attached to the collagen gels were recovered to observe expression of beta-casein, beta-lactoglobulin, and lactoferrin by reverse transcriptase polymeric chain reaction method. Expression of beta-casein was observed in the presence of insulin, hydrocortisone, and prolactin whereas transcription of beta-lactoglobulin and lactoferrin occured irrespective of hydrocortisone and prolactin. Immunoblot analysis demonstrated synthesis and secretion of lactoferrin in the fraction of recovered cells and the culture medium.
Asunto(s)
Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/fisiología , Proteínas de la Leche/metabolismo , Animales , Técnicas de Cultivo de Célula/métodos , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Medio de Cultivo Libre de Suero/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/fisiología , Femenino , Hidrocortisona/farmacología , Insulina/farmacología , Glándulas Mamarias Animales/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Proteínas de la Leche/efectos de los fármacos , Proteínas de la Leche/genética , Prolactina/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , PorcinosRESUMEN
Lactoferrin is a multifunctional, iron-binding glycoprotein found in physiological fluids of mammals. In the present study, a gene encoding the N-terminal half (N-lobe) of bovine lactoferrin was cloned and expressed in cultured insect cells using a baculovirus expression system. One mutation was found in the lactoferrin N-lobe gene, but it resulted in no amino acid substitution. The recombinant lactoferrin N-lobe was secreted into the culture medium and partially purified by means of an immobilized heparin column. The recombinant lactoferrin N-lobe secreted was not glycosylated, but it possessed antimicrobial activity toward Escherichia coli O111. The recombinant product synthesized and accumulated in the host cells exhibited greater electrophoretic mobility on SDS-PAGE than the secreted product and showed no potency to inhibit the growth of bacteria. It is thought that the product accumulated intracellularly lacks antimicrobial ability due to its degradation in the host cells or due to disruption of the active conformation.
Asunto(s)
Antiinfecciosos/metabolismo , Lactoferrina/metabolismo , Spodoptera , Animales , Antibacterianos , Antiinfecciosos/química , Baculoviridae/genética , Western Blotting , Bovinos , Línea Celular , Clonación Molecular , Medios de Cultivo Condicionados/metabolismo , Electroforesis en Gel de Poliacrilamida , Escherichia coli/inmunología , Glicosilación , Heparina/metabolismo , Lactoferrina/biosíntesis , Lactoferrina/química , Lactoferrina/genética , Mutación/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADNRESUMEN
Biochemical properties of bovine lactoperoxidase isolated from milk and recombinant bovine lactoperoxidase expressed by Chinese hamster ovary cells were compared. The natural and recombinant lactoperoxidases showed the same conformational features as determined by circular dichroism (CD) measurements. The alpha-helix, beta-structure, and unordered structure contents were found to be 17. 8, 54.2, and 28.0% for the natural lactoperoxidase and 18.6, 50.1, and 31.3% for the recombinant lactoperoxidase, respectively. The microenvironments of aromatic amino acid residues in both lactoperoxidases seemed to be the same, although the CD spectral band due to the Soret band differed slightly. A difference in the pH-dependent spectral changes of absorbance at 413 nm was observed. From a pepsin hydrolysate of lactoperoxidase, a heme-binding peptide was isolated by reverse-phase HPLC and its amino acid sequence was examined.
Asunto(s)
Lactoperoxidasa , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Cricetinae , Concentración de Iones de Hidrógeno , Lactoperoxidasa/química , Lactoperoxidasa/genética , Lactoperoxidasa/metabolismo , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
The antimicrobial activity of lactoferrin isolated from Korean native goat (KN goat) milk was studied and its antimicrobial domain was identified using synthetic peptides. Antimicrobial activity was assayed by a micro-method using 96-well microplates and a microplate reader. The amino acid sequence of the antimicrobial domain was suggested to be YQWQRRMRKLGAPSIT and this sequence corresponds to amino acid residues 20 to 35 of KN goat lactoferrin. Five peptides with certain amino acid residues deleted were synthesized in an effort to identify the residues essential for antimicrobial activity and it was found that the part with the sequence RRMRK (24-28) is the region most important for this activity. On the other hand, the conformation of the peptides did not influence the antimicrobial activity.
Asunto(s)
Lactoferrina/farmacología , Secuencia de Aminoácidos , Animales , Cabras , Corea (Geográfico) , Lactoferrina/química , Leche/química , Datos de Secuencia Molecular , Fragmentos de Péptidos/síntesis química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
The gene encoding the proteinase from Pseudomonas fluorescens was cloned and sequenced in an effort to identify the cleavage sites involved in its autolysis at 50 degrees C. A single open reading frame consisting of 1449 nucleotides, encoding a protein of 482 amino acids, was found. Analysis of the N-terminal amino acid sequence of the purified proteinase indicated the presence of a prosequence consisting of 13 amino acid residues. The molecular weight of the mature protein was calculated as 48,900 based on the deduced amino acid sequence, which was consistent with that of the purified proteinase as determined by sodium dodecylsulfate-PAGE. Greater than 90% loss of proteolytic activity was observed upon heating at 50 degrees C for 2 min compared with the unheated enzyme. Incubation of the proteinase at 50 degrees C led to disappearance of the intact enzyme, as shown by sodium dodecyl sulfate-PAGE, whereas it was stable in the presence of the protease inhibitor o-phenanthroline. Autolytic fragments were fractionated by reverse-phase HPLC and subjected to N-terminal amino acid sequence analysis in an effort to determine the cleavage sites. The cleavage profile was not definitive; however, amino acid residues with small side chain groups, such as glycine or alanine, were frequently found adjacent to the cleavage sites.
Asunto(s)
Proteínas Bacterianas , Endopeptidasas/metabolismo , Pseudomonas fluorescens/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Clonación Molecular , ADN Bacteriano/química , Electroforesis en Gel de Poliacrilamida , Endopeptidasas/química , Endopeptidasas/genética , Calor , Datos de Secuencia Molecular , Pseudomonas fluorescens/genética , Mapeo Restrictivo , Alineación de SecuenciaRESUMEN
In Pseudomonas fluorescens no. 33, the lipase gene is clustered with the genes for alkaline protease, AprDEF exporter, and two homologue proteins of Serratia serine proteases (pspA and pspB). Secretion of the lipase and alkaline protease through AprDEF was shown in the Escherichia coli cells. Interestingly, the E. coli cells carrying the pspA gene secreted PspA to the media AprDEF-independently.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Lipasa/genética , Familia de Multigenes , Pseudomonas fluorescens/genética , Serina Endopeptidasas/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Escherichia coli/genética , Proteínas de Choque Térmico/genética , Lipasa/metabolismo , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Plásmidos , Pseudomonas fluorescens/enzimología , Homología de Secuencia de AminoácidoRESUMEN
We purified lactoferrin from the colostrum of the Korean native goat (Capra hircus) by ion-exchange chromatography using CM-Toyopearl 650M followed by affinity chromatography on AF-Heparin Toyopearl 650M. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis suggested the molecular mass of Korean native goat lactoferrin is 82 kDa with an iron saturation of 30% as estimated by spectroscopic analysis. Circular dichroism analysis shows goat lactoferrin molecule contains 24.5%, alpha-helix; 36.0%, beta-structure; 13.5%, beta-turn and 26.0%, unordered structure. Heparin binding affinity is the same as that of bovine lactoferrin, but lower than that of human lactoferrin. An analysis using synthetic peptides shows that the peptide from residue 22 to 31--WQRRMRKLGA--exerts a positive heparin-binding ability.
Asunto(s)
Lactoferrina/química , Animales , Dicroismo Circular , Calostro/química , Cabras , Heparina/metabolismo , Hierro/análisis , Corea (Geográfico) , Lactoferrina/aislamiento & purificación , Lactoferrina/metabolismo , Peso Molecular , Fragmentos de Péptidos/farmacología , Unión Proteica , Estructura Secundaria de Proteína , Alineación de Secuencia , Análisis EspectralRESUMEN
A heparin-binding peptide was isolated from a proteolytic hydrolysate of bovine lactoferrin by affinity chromatography using an immobilized heparin column. Analysis of amino acid sequences at the N-terminus showed that this heparin-binding peptide is derived from the region beginning at the 17th amino acid residue of the bovine lactoferrin sequence. The molecular mass of this peptide was 3195.5 as measured by matrix-assisted laser desorption-time of flight mass spectrometry. This peptide is the same as the bactericidal peptide lactoferricin B. In an aqueous environment, this peptide displays mainly a beta-sheet structure and an unordered structure as assessed by measurements of circular dichroism spectra. When this peptide was mixed with heparin, a distinct spectral change was induced because of conformational alteration of the peptide. This spectral change was reversible. Analysis of data from peptide synthesis indicated that binding by the sequence Arg28-Met29-Lys30-Lys31 of bovine lactoferrin is significant and that there is a synergistic contribution from Lys18-Cys19-Arg20-Arg21, and Arg38-Arg39.
Asunto(s)
Heparina/metabolismo , Lactoferrina/análisis , Fragmentos de Péptidos/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Bovinos , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Concentración de Iones de Hidrógeno , Lactoferrina/química , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transferrina/metabolismoRESUMEN
We sequenced the pepP gene of Lactococcus lactis, which encodes an aminopeptidase P (PepP), and demonstrated that the X-prolyl dipeptidyl aminopeptidase PepX plays a more important role than PepP in nitrogen nutrition. PepP shares homology with methionine aminopeptidases and could play a role in the maturation of nascent proteins.
Asunto(s)
Aminopeptidasas/genética , Genes Bacterianos , Lactococcus lactis/enzimología , Lactococcus lactis/genética , Leche/microbiología , Secuencia de Aminoácidos , Aminopeptidasas/biosíntesis , Aminopeptidasas/química , Aminopeptidasas/metabolismo , Animales , Mapeo Cromosómico , Cromosomas Bacterianos , Secuencia Conservada , Lactococcus lactis/crecimiento & desarrollo , Metionil Aminopeptidasas , Datos de Secuencia Molecular , Familia de Multigenes , Fenotipo , Alineación de Secuencia , Homología de Secuencia de AminoácidoAsunto(s)
Células Epiteliales/fisiología , Lactoferrina/biosíntesis , Lactoferrina/genética , Glándulas Mamarias Animales/fisiología , Animales , Células Cultivadas , Femenino , Expresión Génica , Técnicas de Transferencia de Gen , Humanos , Glándulas Mamarias Animales/citología , Ratas , Ratas WistarRESUMEN
The gene encoding an extracellular lipase from Pseudomonas fluorescens No. 33 was cloned and sequenced. A single open reading frame consisting of 1,428 nucleotides that encoded a mature protein of 476 amino acids was recognized. Sequence analysis showed that the deduced molecular weight of 50,209 agreed with the molecular weight of the purified lipase as measured by SDS-PAGE and the lipase lacked a signal peptide. The presence of a repeating motif, GXXGXDXXX, suggested that the lipase might be exported and secreted via a system that involves the ATP-binding cassette protein.
Asunto(s)
Genes Bacterianos , Lipasa/genética , Pseudomonas fluorescens/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Código Genético , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Pseudomonas fluorescens/enzimologíaRESUMEN
Bovine lactoferricin (LFcin B) is a strong antimicrobial peptide derived from N-lobe of lactoferrin. To study the immunochemical and structural properties of LFcin B, monoclonal antibody (mAb) was prepared and the amino acid sequence concerning with the binding to mAb has been identified. Mice injected with LFcin B showed no production of antibody specific to this peptide, whereas those with LFcin B-KLH conjugate produced anti-LFcin B antibodies. None of the mAb reacted with bovine lactoferrin C-lobe, human lactoferrin or LFcin H. By the reactivity of the mAb against the peptides synthesized on cellulose membranes using SPOTs and against chemically modified derivatives of LFcin B, the antigenic determinant of LFcin B was identified to be the sequence of "QWR".
Asunto(s)
Antibacterianos/química , Epítopos/análisis , Lactoferrina/análogos & derivados , Lactoferrina/química , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales , Bovinos , Femenino , Humanos , Inmunohistoquímica , Lactoferrina/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
The effects of some milk proteins on the thermostability of the lipase from Pseudomonas fluorescens 33 were investigated. All purified milk protein fractions except kappa-casein that dissolved in phosphate buffer were effective for thermostabilization of the lipase. Thermal behavior of the lipase containing beta-lactoglobulin was so specific that, after heating at 80 to 90 degrees C, activity remained high and was comparable with that of unheated treatment. The thermostability of the lipase containing whey proteins in synthetic salts solution was extensively lowered, but that containing casein micelles retained 50% of original activity after heat treatment at 80 degrees C for 10 min. Low temperature inactivation of the lipase was influenced by concomitant milk proteins.
Asunto(s)
Calor , Lipasa/metabolismo , Proteínas de la Leche/farmacología , Pseudomonas fluorescens/enzimología , Caseínas/farmacología , Estabilidad de Enzimas , Lactoglobulinas/farmacología , Proteína de Suero de LecheRESUMEN
The extracellular proteinase from Pseudomonas fluorescens No. 33 was purified to electrophoretic homogeneity by a procedure including precipitation with HCl and (NH4)2SO4, and column chromatography. The enzyme was purified 170-fold giving a yield of 7% of the original activity. The molecular mass of the purified enzyme was 48,000 by SDS-PAGE. The optimum pH and temperature for the hydrolysis of casein were 8.0-9.8 and 30-35 degrees C respectively. The enzyme was more thermostable in synthetic milk salts solution than in 0.1 M-sodium phosphate buffer, but was heat-labile at 50 degrees C in both buffer systems. The activity was inhibited by o-phenanthroline, Hg2+, Cu2+, Fe2+ and, to a lesser extent, Ni2+. Caseins were susceptible to the proteinase, but degradation patterns were dependent on the form of the casein.