RESUMEN
The theory behind laser hair removal and pulsed flashlamps is briefly reviewed, as are the devices currently available for this indication. Today, the treatment procedure has been firmly established so that clear and precise information can be provided to patient on the results obtained with these techniques, both for medical and esthetic indications. The potential complications are discussed with respect to the treatment procedures and a thorough understanding of the theoretical foundations.
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Remoción del Cabello/métodos , Rayos Láser , Humanos , Rayos Láser/efectos adversosRESUMEN
The theory behind laser hair removal and pulsed flashlamps is briefly reviewed, as are the devices currently available for this indication. Today, the treatment procedure has been firmly established so that clear and precise information can be provided to patient on the results obtained with these techniques, both for medical and esthetic indications. The potential complications are discussed with respect to the treatment procedures and a thorough understanding of the theoretical foundations.
Asunto(s)
Remoción del Cabello/métodos , Rayos Láser , Humanos , Rayos Láser/efectos adversosRESUMEN
INTRODUCTION: Cathelicidins are antimicrobial peptides found in epithelial and mucosal tissues as well as the secondary granules of neutrophils. SMAP29, a sheep cathelicidin, has differential antimicrobial properties against various pathogens, including periodontal organisms. The purpose of this study was to evaluate the antimicrobial properties and cytotoxicity of SMAP29, SMAP28, and three congeners (SMAP18A, SMAP18D, and SMAP14A). METHODS: The peptides at concentrations ranging from 0.25 to 250 microg/ml were tested for their activity against multiple strains of Streptococcus mutans, Streptococcus sanguis, Actinomyces israelii, Actinomyces naeslundii, Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Peptostreptococcus micros, and Porphyromonas gingivalis using a radial diffusion assay. Cytotoxicity of keratinocytes was evaluated by measuring lactate dehydrogenase release after incubation with the individual peptides. RESULTS: SMAP28, thought to be the biologically active peptide, was the most potent antimicrobial (range of minimum inhibitory concentrations 0.06-7.03 microg/ml, P < 0.05); however, the activity of SMAP28 and SMAP29 was strongly associated (r = 0.933). The congeners also demonstrated antimicrobial activity against the bacteria tested (range of minimum inhibitory concnetrations 0.21-79 microg/ml). Overall, F. nucleatum was the most susceptible organism, while P. gingivalis was the least susceptible. Keratinocyte cytotoxicity was dependent on peptide length and dose. SMAP28 was the most cytotoxic, while SMAP14A was the least cytotoxic. CONCLUSION: The antimicrobial activities against oral microorganisms and the minimal toxicity seen in this study suggest that the congeners of SMAP29 may serve as an alternative to traditional antibiotics in the prevention and treatment of periodontal and other oral diseases.
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Péptidos Catiónicos Antimicrobianos/farmacología , Bacterias Anaerobias/efectos de los fármacos , Proteínas Sanguíneas/farmacología , Catelicidinas/farmacología , Encía/efectos de los fármacos , Actinomyces/efectos de los fármacos , Aggregatibacter actinomycetemcomitans/efectos de los fármacos , Animales , Péptidos Catiónicos Antimicrobianos/toxicidad , Proteínas Sanguíneas/toxicidad , Catelicidinas/toxicidad , Fusobacterium nucleatum/efectos de los fármacos , Encía/citología , Humanos , Queratinocitos/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Peptostreptococcus/efectos de los fármacos , Porphyromonas gingivalis/efectos de los fármacos , Oveja Doméstica , Streptococcus/efectos de los fármacosRESUMEN
INTRODUCTION: Verrucous epidermal nevus are benign epidermal hyperplasias. Their treatments are disappointing because of recurrences and anaesthetic scars. The aim of this study was to evaluate the efficacy of continuous carbon dioxide laser in the treatment of verrucous epidermal nevus. MATERIAL AND METHODS: It was a retrospective study concerning all patients treated for epidermal verrucous nevus by carbon dioxide laser from 1991 to 2003. Several criteria were evaluated by the patients, a dermatologist and a staff (external observers). Aesthetic result, recurrences, pain due to treatment and global result were evaluated by the patients when they came back. RESULTS: Twenty-one patients (12 F and 9 M), medium age 20.4, were evaluated with a 40.4 months follow-up (7 to 165 months). The epidermal verrucous nevus was situated on the neck or on the head for 62% of them (n = 13). Among these 21 patients, 86% (n = 19) estimated their skin as "cured" or "nearly normal" or "much improved". The rate was the same for the dermatologist. As for the staff, thanks to photos, only 53% of results were satisfying. The recurrences were never complete, but moderate in 38% of patients. Five patients, medium age 12,5, had hypertrophic scars. Among them, three were only partially hypertrophic. DISCUSSION: A literature review has been made. Our satisfaction rates (nearly 90%) were slightly higher than other studies rates using carbon dioxide laser (74%) or other types of lasers (87%). The medium follow-up was longer than the one of other laser CO2 studies (26.7 months) or other lasers studies' (15.8 months). This study shows that continuous wave carbon dioxide laser is an easy and effective treatment of verrucous epidermal nevus. Aesthetic results are satisfying but moderate recurrences often occur. To prevent hypertrophic scars, we suggest to exclude teenagers.
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Hamartoma/cirugía , Terapia por Láser , Enfermedades de la Piel/cirugía , Adolescente , Adulto , Niño , Cicatriz Hipertrófica/prevención & control , Epidermis , Estética , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Satisfacción del Paciente , Recurrencia , Estudios Retrospectivos , Factores de Tiempo , Resultado del TratamientoRESUMEN
The severity of periodontal disease is dependent on a combination of host, microbial agent and environmental factors. One strong correlate related to periodontal disease pathogenesis is the immune status of the host. Here we show that human neutrophil peptide (HNP) defensins or human beta-defensins (HBD), co-administered intranasally with the antigen ovalbumin (OVA), induce unique immune responses that if used with microbial antigens may have the potential to hinder the pathogenesis of periodontal disease. C57BL/6 mice were immunized intranasally with phosphate buffered saline (PBS) containing 1 micro g HNP-1, HNP-2, HBD1 or HBD2 with and without 50 microg OVA. At 21 days, isotypes and subclasses of OVA-specific antibodies were determined in saliva, serum, nasal wash, bronchoalveolar lavage fluid, and fecal extracts. OVA-stimulated splenic lymphoid cell cultures from immunized mice were assessed for interferon (IFN)-gamma, Interleukin (IL)-4 and IL-10. In comparison with mice immunized with only OVA, HNP-1 and HBD2 induced significantly higher (P < 0.05) OVA-specific serum IgG, lower, but not significant, serum IgM and significantly lower (P < 0.05) IFN-gamma. In contrast, HNP-2 induced low OVA-specific serum IgG and higher, but not significant, serum IgM. HBD1 induced significantly higher (P < 0.05) OVA-specific serum IgG, higher, but not significant, serum IgM, and significantly higher (P < 0.05) IL-10. The elevated serum IgG subclasses contained IgG1 and IgG2b.
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Adyuvantes Inmunológicos/farmacología , Defensinas/inmunología , Enfermedades Periodontales/prevención & control , Análisis de Varianza , Animales , Anticuerpos/análisis , Anticuerpos/sangre , Líquido del Lavado Bronquioalveolar/inmunología , Defensinas/farmacología , Heces/química , Femenino , Humanos , Inmunidad Activa/efectos de los fármacos , Inmunoglobulina G/análisis , Inmunoglobulina G/sangre , Inmunoglobulina M/análisis , Inmunoglobulina M/sangre , Interferón gamma/análisis , Interleucinas/análisis , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ovalbúmina/inmunología , Enfermedades Periodontales/inmunología , Saliva/inmunología , alfa-Defensinas/inmunología , beta-Defensinas/inmunologíaRESUMEN
The effects of cathelicidins against oral bacteria and clinically important oral yeasts are not known. We tested the susceptibilities of Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus sanguis, Candida krusei, Candida tropicalis and Candida albicans to the following cathelicidins: FALL39, SMAP29, and CAP18. SMAP29 and CAP18 were antimicrobial, whereas FALL39 did not exhibit antimicrobial activity. Future studies are needed to determine the potential use of these antimicrobial peptides in prevention and treatment of oral infections.
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Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Mamíferos/metabolismo , Boca/microbiología , Levaduras/efectos de los fármacos , Animales , Péptidos Catiónicos Antimicrobianos/farmacología , Catelicidinas , Recuento de Colonia Microbiana , Pruebas de Sensibilidad MicrobianaRESUMEN
SMAP29, an ovine cathelicidin, was systematically altered to create a family of 23 related peptides for MIC and minimum bactericidal concentration determinations. SMAP28, SMAP29, and a derivative of SMAP29 called ovispirin were all antimicrobial. However, many congeners of SMAP29 and ovispirin were not as active as the parent molecules. With immunoelectron microscopy, SMAP29 was seen on membranes and within the cytoplasm of Pseudomonas aeruginosa PAO1.
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Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Bacterias/ultraestructura , Proteínas Sanguíneas/farmacología , Enfermedades de las Ovejas/microbiología , Ovinos/microbiología , Secuencia de Aminoácidos , Animales , Catelicidinas , Pruebas de Sensibilidad Microbiana , Microscopía Inmunoelectrónica , Datos de Secuencia MolecularRESUMEN
The orientation and dynamics of an 18-residue antimicrobial peptide, ovispirin, has been investigated using solid-state NMR spectroscopy. Ovispirin is a cathelicidin-like model peptide (NH(2)-KNLRRIIRKIIHIIKKYG-COOH) with potent, broad-spectrum bactericidal activity. (15)N NMR spectra of oriented ovispirin reconstituted into synthetic phospholipids show that the helical peptide is predominantly oriented in the plane of the lipid bilayer, except for a small portion of the helix, possibly at the C-terminus, which deviates from the surface orientation. This suggests differential insertion of the peptide backbone into the lipid bilayer. (15)N spectra of both oriented and unoriented peptides show a reduced (15)N chemical shift anisotropy at room temperature compared with that of rigid proteins, indicating that the peptide undergoes uniaxial rotational diffusion around the bilayer normal with correlation times shorter than 10(-4) s. This motion is frozen below the gel-to-liquid crystalline transition temperature of the lipids. Ovispirin interacts strongly with the lipid bilayer, as manifested by the significantly reduced (2)H quadrupolar splittings of perdeuterated palmitoyloleoylphosphatidylcholine acyl chains upon peptide binding. Therefore, ovispirin is a curved helix residing in the membrane-water interface that executes rapid uniaxial rotation. These structural and dynamic features are important for understanding the antimicrobial function of this peptide.
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Péptidos Catiónicos Antimicrobianos/química , Membrana Dobles de Lípidos/química , Modelos Químicos , Fosfolípidos/química , Sitios de Unión , Difusión , Espectroscopía de Resonancia Magnética , Membranas Artificiales , Rotación , Propiedades de Superficie , Termodinámica , Agua/químicaRESUMEN
Endogenous peptide antibiotics are under investigation as inhaled therapeutic agents for cystic fibrosis (CF) lung disease. The bactericidal activities of five cathelicidin peptides (LL37 [human], CAP18 [rabbit], mCRAMP [mouse], rCRAMP [rat], and SMAP29 [sheep]), three novel alpha-helical peptides derived from SMAP29 and termed ovispirins (OV-1, OV-2, and OV-3), and two derivatives of CAP18 were tested by broth microdilution assays. Their MICs were determined for multiply antibiotic-resistant Pseudomonas aeruginosa (n = 24), Burkholderia cepacia (n = 5), Achromobacter xylosoxidans (n = 5), and Stenotrophomonas maltophilia (n = 5) strains isolated from CF patients. SMAP29 was most active and inhibited mucoid and nonmucoid P. aeruginosa strains (MIC, 0.06 to 8 microg/ml). OV-1, OV-2, and OV-3 were nearly as active (MIC, 0.03 to 16 microg/ml), but CAP18 (MIC, 1.0 to 32 microg/ml), CAP18-18 (MIC, 1.0 to >32 microg/ml), and CAP18-22 (MIC, 0.5 to 32 microg/ml) had variable activities. LL37, mCRAMP, and rCRAMP were least active against the clinical isolates studied (MIC, 1.0 to >32 microg/ml). Peptides had modest activities against S. maltophilia and A. xylosoxidans (MIC range, 1.0 to > 32 microg/ml), but none inhibited B. cepacia. However, CF sputum inhibited the activity of SMAP29 substantially. The effects of peptides on bacterial cell membranes and eukaryotic cells were examined by scanning electron microscopy and by measuring transepithelial cell resistance, respectively. SMAP29 caused the appearance of bacterial membrane blebs within 1 min, killed P. aeruginosa within 1 h, and caused a dose-dependent, reversible decrease in transepithelial resistance within 5 h. The tested cathelicidin-derived peptides represent a novel class of antimicrobial agents and warrant further development as prophylactic or therapeutic agents for CF lung disease.
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Péptidos Catiónicos Antimicrobianos/farmacología , Fibrosis Quística/microbiología , Resistencia a Múltiples Medicamentos/fisiología , Alcaligenes/efectos de los fármacos , Secuencia de Aminoácidos , Proteínas Sanguíneas/farmacología , Burkholderia cepacia/efectos de los fármacos , Catelicidinas , Sinergismo Farmacológico , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Péptidos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Homología de Secuencia de Aminoácido , Esputo/química , Stenotrophomonas maltophilia/efectos de los fármacos , Factores de TiempoRESUMEN
beta-Defensins are endogenous cationic peptides with broad-spectrum antimicrobial activity that are thought to play a role in the innate immune response. Two human beta-defensins, beta-defensin-1 (HBD-1) and beta-defensin-2 (HBD-2), have been identified. These peptides have recently been characterized in several human tissues. The presence of these peptides in the paranasal sinuses has not been investigated. We examined maxillaary sinus secretions from six patients with sinusitis and 10 patients without signs, symptoms, or radiologic evidence of sinus disease for the presence of beta-defensins. Cationic peptides were extracted from antral lavage specimens and examined for the presence of HBD-1 and HBD-2 by Western blot. Normal maxillary sinus epithelium was obtained from two patients and analyzed by RT-PCR for the presence of HBD-1 and HBD-2 mRNA. Tissue immunostaining for the two peptides was also used. Western blot analysis identified HBD-1 in two of 10 patients in the control group and in three of six patients in the sinusitis group. HBD-2 was identified in one of 10 patients in the control group and in four of six patients in the sinusitis group. RT-PCR revealed HBD-1 mRNA in one of two normal controls tested. Immunostaining localized HBD-1 and HBD-2 to the epithelial cell cytoplasm. This is the first demonstration of HBD-1 and HBD-2 production in the paranasal sinuses. In the present study, HBD-1 and HBD-2 were detected more frequently in the maxillary sinus fluid of patients with inflamed sinuses than in normal controls.
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Seno Maxilar/química , beta-Defensinas/análisis , Adulto , Anciano , Femenino , Humanos , Inmunohistoquímica , Masculino , Sinusitis Maxilar/metabolismo , Persona de Mediana Edad , Membrana Mucosa/químicaRESUMEN
Human beta-defensin-2 (HBD-2) is a member of the defensin family of antimicrobial peptides. HBD-2 was first isolated from inflamed skin where it is posited to participate in the killing of invasive bacteria and in the recruitment of cells of the adaptive immune response. Static light scattering and two-dimensional proton nuclear magnetic resonance spectroscopy have been used to assess the physical state and structure of HBD-2 in solution. At concentrations of < or = 2.4 mM, HBD-2 is monomeric. The structure is amphiphilic with a nonuniform surface distribution of positive charge and contains several key structural elements, including a triple-stranded, antiparallel beta-sheet with strands 2 and 3 in a beta-hairpin conformation. A beta-bulge in the second strand occurs at Gly28, a position conserved in the entire defensin family. In solution, HBD-2 exhibits an alpha-helical segment near the N-terminus that has not been previously ascribed to solution structures of alpha-defensins or to the beta-defensin BNBD-12. This novel structural element may be a factor contributing to the specific microbicidal or chemokine-like properties of HBD-2.
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Fragmentos de Péptidos/química , beta-Defensinas/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Enlace de Hidrógeno , Luz , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular/métodos , Conformación Proteica , Estructura Secundaria de Proteína , Dispersión de Radiación , SolucionesRESUMEN
Epithelial beta-defensins are broad-spectrum cationic antimicrobial peptides that also act as chemokines for adaptive immune cells. In the human genome, all known defensin genes cluster to a <1 Mb region of chromosome 8p22-p23. To identify new defensin genes, the DNA sequence from a contig of large-insert genomic clones from the region containing human beta-defensin-2 (HBD-2) was analyzed for the presence of defensin genes. This sequence survey identified a novel beta-defensin, termed HBD-3. The HBD-3 gene contains two exons, is located 13 kb upstream from the HBD-2 gene, and it is transcribed in the same direction. A partial HBD-3 cDNA clone was amplified from cDNA derived from IL-1beta induced fetal lung tissue. The cDNA sequence encodes for a 67 amino acid peptide that is approximately 43% identical to HBD-2 and shares the beta-defensin six cysteine motif. By PCR analysis of two commercial cDNA panels, HBD-3 expression was detected in adult heart, skeletal muscle, placenta and in fetal thymus. From RT-PCR experiments, HBD-3 expression was observed in skin, esophagus, gingival keratinocytes, placenta and trachea. Furthermore, in fetal lung explants and gingival keratinocytes, HBD-3 mRNA expression was induced by IL-1beta. Additional sequence analysis identified the HE2 (human epididymis secretory protein) gene 17 kb upstream from the HBD-3 gene. One splice variant of this gene (HE2beta1) encodes a beta-defensin consensus cysteine motif, suggesting it represents a defensin gene product. HE2beta1 mRNA expression was detected in gingival keratinocytes and bronchial epithelia using RT-PCR analysis. The discovery of these novel beta-defensin genes may allow further understanding of the role of defensins in host immunity at mucosal surfaces.
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Proteínas Portadoras , Genómica , Proteínas Recombinantes , beta-Defensinas/genética , Adulto , Secuencia de Aminoácidos , Antígenos de Superficie/genética , Secuencia de Bases , Mapeo Contig , ADN/química , ADN/genética , Exones , Femenino , Feto/metabolismo , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genes/genética , Biblioteca Genómica , Glicopéptidos/genética , Glicoproteínas , Humanos , Intrones , Masculino , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Distribución Tisular , Proteínas de Transporte VesicularRESUMEN
The presence of the complement-derived anaphylatoxin peptides, C3a and C5a, in the lung can induce respiratory distress characterized by contraction of the smooth muscle walls in bronchioles and pulmonary arteries and aggregation of platelets and leukocytes in pulmonary vessels. C3a and C5a mediate these effects by binding to their specific receptors, C3aR and C5aR, respectively. The cells that express these receptors in the lung have not been thoroughly investigated, nor has their expression been examined during inflammation. Accordingly, C3aR and C5aR expression in normal human and murine lung was determined in this study by immunohistochemistry and in situ hybridization. In addition, the expression of these receptors was delineated in mice subjected to LPS- and OVA-induced models of inflammation. Under noninflamed conditions, C3aR and C5aR protein and mRNA were expressed by bronchial epithelial and smooth muscle cells of both human and mouse lung. C3aR expression increased significantly on both bronchial epithelial and smooth muscle cells in mice treated with LPS; however, in the OVA-challenged animals only the bronchial smooth muscle cells showed increased C3aR expression. C5aR expression also increased significantly on bronchial epithelial cells in mice treated with LPS, but was not elevated in either cell type in the OVA-challenged mice. These results demonstrate the expression of C3aR and C5aR by cells endogenous to the lung, and, given the participation of bronchial epithelial and smooth muscle cells in the pathology of diseases such as sepsis and asthma, the data suggest a role for these receptors during lung inflammation.
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Antígenos CD/biosíntesis , Asma/inmunología , Bronquios/metabolismo , Complemento C3a/metabolismo , Complemento C5a/metabolismo , Endotoxemia/inmunología , Proteínas de la Membrana , Músculo Liso/metabolismo , Receptores de Complemento/biosíntesis , Aerosoles , Secuencia de Aminoácidos , Animales , Antígenos CD/genética , Asma/metabolismo , Asma/patología , Bronquios/irrigación sanguínea , Bronquios/inmunología , Bronquios/patología , Células Cultivadas , Modelos Animales de Enfermedad , Endotoxemia/metabolismo , Endotoxemia/patología , Humanos , Inyecciones Intraperitoneales , Lipopolisacáridos/administración & dosificación , Pulmón/citología , Pulmón/inmunología , Pulmón/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Músculo Liso/inmunología , Músculo Liso/patología , Músculo Liso Vascular/inmunología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Ovalbúmina/administración & dosificación , Ovalbúmina/inmunología , Receptor de Anafilatoxina C5a , Receptores de Complemento/genética , Mucosa Respiratoria/irrigación sanguínea , Mucosa Respiratoria/inmunología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patologíaRESUMEN
Human beta-defensin-1 (HBD-1) was detected in breast milk in concentrations of approximately 1 to 10 microg/mL. Breast tissue during lactation showed HBD-1 expression in mammary gland epithelia and within luminal secretions. The peptide demonstrated antimicrobial activity against Escherichia coli. HBD-1 may augment neonatal host defenses through antimicrobial effects or prime the adaptive immune system at mucosal surfaces.
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Mama/citología , Leche Humana/química , beta-Defensinas/análisis , beta-Defensinas/inmunología , Epitelio/química , Humanos , Inmunidad Materno-Adquirida/inmunología , Inmunidad Mucosa/inmunología , Lactancia/fisiología , Pruebas de Sensibilidad MicrobianaRESUMEN
Cathelicidins are antimicrobial peptides from sheep (SMAP29 and SMAP34), rabbits (CAP11 and CAP18), rodents (CRAMP), and humans (FALL39, LL37, and h/CAP18). In a broth microdilution assay against nine ovine pathogens, SMAP29, SMAP34, mouse CRAMP, CAP18, CAP18(31), CAP18(28), CAP18(22), and CAP18(21a) were the most active, with MICs as low as 0.6 microg/ml. Other cathelicidins were less active. In lambs with pneumonia, 0.5 mg of SMAP29 reduced the concentration of bacteria in both bronchoalveolar lavage fluid and consolidated pulmonary tissues. Hence, the antimicrobial activity of SMAP29 suggests that it has applications in the treatment of respiratory tract infections.
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Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas Sanguíneas , Proteínas/farmacología , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/veterinaria , Enfermedades de las Ovejas/microbiología , Animales , Catelicidinas , Pulmón/microbiología , Pseudomonas aeruginosa/efectos de los fármacos , OvinosRESUMEN
Airway surface liquid contains multiple factors thought to provide a first line of defense against bacteria deposited in the airways. Although the antimicrobial action of individual factors has been studied, less is known about how they work in combination. We examined the combined action of six antimicrobial peptides found in airway surface liquid. The paired combinations of lysozyme-lactoferrin, lysozyme-secretory leukocyte protease inhibitor (SLPI), and lactoferrin-SLPI were synergistic. The triple combination of lysozyme, lactoferrin, and SLPI showed even greater synergy. Other combinations involving the human beta-defensins, LL-37, and tobramycin (often administered to cystic fibrosis patients by inhalation) were additive. Because the airway surface liquid salt concentration may be elevated in cystic fibrosis patients, we examined the effect of salt on the synergistic combinations. As the ionic strength increased, synergistic interactions were lost. Our data suggest that the antibacterial potency of airway surface liquid may be significantly increased by synergistic and additive interactions between antimicrobial factors. These results also suggest that increased salt concentrations that may exist in cystic fibrosis could inhibit airway defenses by diminishing these synergistic interactions.
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Antibacterianos/farmacología , Escherichia coli/efectos de los fármacos , Lactoferrina/farmacología , Muramidasa/farmacología , Proteínas/farmacología , Antibacterianos/aislamiento & purificación , Defensinas/farmacología , Interacciones Farmacológicas , Sinergismo Farmacológico , Espacio Extracelular/química , Espacio Extracelular/fisiología , Humanos , Cinética , Leche Humana/química , Leche Humana/fisiología , Proteínas Inhibidoras de Proteinasas Secretoras , Proteínas Recombinantes/farmacología , Mucosa Respiratoria , Inhibidor Secretorio de Peptidasas Leucocitarias , Tobramicina/farmacologíaRESUMEN
Terminase is the enzyme that mediates lambda DNA packaging into the viral prohead. The large subunit of terminase, gpA (641 amino acid residues), has a high-affinity ATPase activity (K(m)=5 microM). To directly identify gpA's ATP-interacting amino acids, holoterminase bearing a His(6)-tag at the C terminus of gpA was UV-crosslinked with 8-N(3)-[alpha-(32)P]ATP. Tryptic peptides from the photolabeled terminase were purified by affinity chromatography and reverse-phase HPLC. Two labeled peptides of gpA were identified. Amino acid sequencing failed to show the tyrosine residue of the first peptide, E(43)SAY(46)QEGR(50), or the lysine of the second peptide, V(80)GYSK(84)MLL(87), indicating that Y(46) and K(84) were the 8-N(3)-ATP-modified amino acids. To investigate their roles in lambda DNA packaging, Y(46) was changed to E, A, and F, and K(84) was changed to E and A. Purified His(6)-tagged terminases with changes at residues 46 and 84 lacked the gpA high-affinity ATPase activity, though the cos cleavage and cohesive end separation activities were near to those of the wild-type enzyme. In virion assembly reactions using virion DNA as a packaging substrate, the mutant terminases showed severe defects. In summary, the results indicate that Y(46) and K(84) are part of the high-affinity ATPase center of gpA, and show that this ATPase activity is involved in the post-cos cleavage stages of lambda DNA packaging.
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Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Bacteriófago lambda/enzimología , ADN Viral/metabolismo , Endodesoxirribonucleasas/química , Endodesoxirribonucleasas/metabolismo , Ensamble de Virus , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos/genética , Sitios de Ligazón Microbiológica/genética , Bacteriófago lambda/genética , Bacteriófago lambda/fisiología , Secuencia de Bases , Sitios de Unión , Cromatografía Líquida de Alta Presión , ADN Viral/genética , Endodesoxirribonucleasas/genética , Holoenzimas/química , Holoenzimas/genética , Holoenzimas/metabolismo , Hidrólisis , Cinética , Mutación/genética , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Etiquetas de Fotoafinidad , Huella de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/genética , Tripsina/metabolismo , Rayos Ultravioleta , Virión/enzimología , Virión/genética , Virión/fisiologíaRESUMEN
beta-Defensins are broad spectrum antimicrobial peptides expressed at epithelial surfaces. Two human beta-defensins, HBD-1 and HBD-2, have been identified. In the lung, HBD-2 is an inducible product of airway epithelia and may play a role in innate mucosal defenses. We recently characterized rat homologs (RBD-1, RBD-2) of the human genes and used these sequences to identify novel mouse genes. Mouse beta-defensin-4 (MBD-4) was amplified from lung cDNA using polymerase chain reaction primers designed from conserved sequences of RBD-2 and HBD-2. A full-length cDNA was cloned which encodes a putative peptide with the sequence MRIHYLLFTFLLVLLSPLAAFTQIINNPITCMTNGAICWGPCPTAFRQIGNCGHFKVRCCKIR. The peptide shares approximately 40% identity with HBD-2. MBD-4 mRNA was expressed in the esophagus, tongue, and trachea but not in any of 20 other tissues surveyed. Cloning of the genomic sequence of MBD-4 revealed two nearly (>99%) identical sequences encoding MBD-4 and the presence of numerous additional highly similar genomic sequences. Radiation hybrid mapping localized this gene to a region of chromosome 8 near several other defensins, MBD-2, MBD-3, and alpha-defensins (cryptdins)-3 and -17, consistent with a gene cluster. Our genomic cloning and mapping data suggest that there is a large beta-defensin gene family in mice. Identification of murine beta-defensins provides an opportunity to understand further the role of these peptides in host defense through animal model studies and the generation of beta-defensin-deficient animals by gene targeting.