Structure and function of human-derived lysozyme: a review / 生物工程学报

Human-derived lysozyme is a general term for a group of naturally occurring alkaline proteins in the human body that are capable of lysing bacterial cell walls. Its action is characterized by its ability to cleave the β-(1,4)-glycosidic bond between N-acetylglucosamine and N-acetylmuramic acid in peptidoglycan. Human-derived lysozyme has a variety of properties such as antibacterial, anti-inflammatory, antiviral and immune enhancing, and is therefore widely used in the domestic and international pharmaceutical markets. This review summarizes the structural features, expression sites, biological functions of human-derived lysozymes and its market applications.

Preparação e caracterização de nanocarreadores de beta-lactose à base de pectina e lisozima / Preparation and characterization of beta-lactose nanocarriers based on pectin and lysozyme

O objetivo deste trabalho foi preparar e caracterizar nanocarreadores via auto-organização a partir da pectina de citros e lisozima para o encapsulamento da ß-lactose. Foram estudadas três condições de interação entre os biopolímeros variando a razão molar pectina/lisozima (3:1, 2:1, 1:1, 1:2 e 1:3), o pH e o tempo de aquecimento. A confirmação da interação foi determinada por espectroscopia no infravermelho por transformada de Fourier (FTIR) e por calorimetria de varredura diferencial (DSC). Os espectros de infravermelho evidenciaram que ligações de hidrogênio foram as principais forças envolvidas na formação dos nanocarreadores e sugeriram a ausência de ß-lactose livre na superfície das nanopartículas. Os termogramas evidenciaram que as nanopartículas formadas na presença de ß-lactose têm maior estabilidade térmica do que as nanopartículas sem ß-lactose. Para ambas as formulações estudadas, na presença e na ausência de ß-lactose, a formação das nanopartículas ocorreu entre os valores de pKa e ponto isoelétrico (pI) da pectina e lisozima, respectivamente, sendo a melhor razão de interação pectina/lisozima 1:2, em pH 10, a 80 ºC por 30 min. As nanopartículas foram formadas via auto-organização e todos as partículas apresentaram distribuição de tamanho homogênea, formato esférico, diâmetro inferior a 100 nm e carga superficial negativa. A morfologia e o tamanho das partículas pouco alteraram com a incorporação da -lactose. A eficiência de encapsulação (EE) da ß-lactose foi superior a 96% para as concentrações estudadas. Ensaios preliminares in vitro, em células epiteliais de câncer de cólon (HCT-116), evidenciaram que as nanopartículas formadas são capazes de adentrar no meio intracelular, possivelmente, por via endocitose

This work aimed to prepare and characterize nanocarriers via self-assembly using citrus pectin and lysozyme for ß-lactose encapsulation. Three interaction conditions between the biopolymers were studied, varying the pectin/lysozyme molar ratio (3:1, 2:1, 1:1, 1:2 and 1:3), pH and heating time. Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) determined the interaction's confirmation. The infrared spectra showed that hydrogen bonds were the main forces involved in the formation of nanocarriers and suggested the absence of free ß-lactose on the surface of the nanoparticles. The thermograms showed that nanoparticles formed in the presence of ß-lactose have greater thermal stability than nanoparticles without ß-lactose. For both formulations studied, in the presence and absence of lactose, the formation of nanoparticles occurred between the pKa and isoelectric point (pI) values of pectin and lysozyme, respectively, with the best pectin/lysozyme interaction molar ratio 1:2, at pH 10, at 80 °C for 30 min. Nanoparticles were formed via self-assembly, and all particles presented homogeneous size distribution, spherical shape, diameter less than 100 nm, and negative surface charge. The morphology and size of the particles changed little with the incorporation of ß-lactose. The encapsulation efficiency (EE) of ß-lactose was higher than 96% for the concentrations studied. Preliminary in vitro assays in colon cancer epithelial cells (HCT-116) showed that the nanoparticles formed are capable of entering the intracellular medium, possibly via endocytosis

Immunomodulation of Juvenile Pacu, Piaractus mesopotamicus, by Different ß(1-3)(1-6)-D glucan Products

Abstract Stress in intensive fish farming hamper immune function of fish and cause losses by disease outbreaks, a situation that can be minimized, but cannot be completely circumvented, by the use of immunomodulators. Addition of immunomodulators to aquafeeds has thus become a common practice. β-glucan (BG) is one of most studied and effective immunomodulators, aquaculture purposes included. Extracted from cell walls of bacteria, fungi and selected cereals, BG activity depends on the source and extraction methods. This study evaluated effects of two BG products (BG1 and BG2), extracted from Saccharomyces cerevisiae under varying extraction methods and with different immune activity, on the feeding of pacu Piaractus mesopotamicus juveniles. BG1 provided higher leukocytes respiratory activity when fed at 0.5% inclusion for 10 days and 0.1% inclusion for 15 days. Both products seems to cause negative effect on lysozyme concentration and monocytes profile when fed to pacu for 15 days at 0.5% inclusion. Although the results for BG2 did not differ from control (diet devoid of BG), the proximity with the BG1 behavior is a indicative that a commercial product with smaller BG concentration can be effective when more refined technology is used in extraction process.

Impact of environmental pollution on the ocular surface of Sjögren's syndrome patients / Impacto da poluição ambiental na superfície ocular de pacientes com síndrome de Sjögren

ABSTRACT Purpose: To evaluate the effect of air pollution on the ocular surface of patients with Sjögren's syndrome. Methods: We investigated the ocular surfaces of thirty patients with Sjögren's syndrome and thirty healthy volunteers (control group) living in the Metropolitan Area of Buenos Aires. We used nitrogen dioxide as an indicator of exposure to air pollution. An ocular symptoms questionnaire was answered by all subjects, who also underwent a complete ocular surface ophthalmic examination-including an Ocular Surface Disease Index questionnaire, biomicroscopy, tear breakup time, Schirmer 1 test, corneal and conjunctival vital staining with fluorescein and lissamine green, tear lysozyme concentration, and impression cytology. Results: In almost all ocular surface test findings, we found a positive and significant correlation between higher levels of exposure to air pollution and higher levels of ocular surface damage in both the control group and Sjögren's syndrome patients. In Sjögren's syndrome patients, the Ocular Surface Disease Index questionnaire, tear breakup time, vital staining and impression cytology showed a significant correlation between high levels of air pollution and ocular surface disease. In the control group, the Ocular Surface Disease Index questionnaire, tear breakup time, and impression cytology showed a significant correlation between high levels of air pollution and ocular surface disease. Conclusions: Here we demonstrated that in patients with dry eye syndrome associated with Sjögren, abnormalities of the ocular surface and eye irritation related to air pollution are more severe than those in the control group. We believe that measuring air quality should be not only an integral part of the evaluation of ocular surface disease but also a therapeutic consideration.

RESUMO Objetivo: Avaliar o efeito da poluição do ar na superfície ocular de pacientes com síndrome de Sjögren. Métodos: Foram investigadas as superfícies oculares de trinta pacientes com síndrome de Sjögren e trinta voluntários saudáveis (grupo controle) residentes na Região Metropolitana de Buenos Aires. Usamos o dióxido de nitrogênio como um indicador de exposição à poluição do ar. Um questionário de sintomas oculares foi respondido por todos os indivíduos, que também foram submetidos a um exame oftalmológico completo da superfície ocular - incluindo um questionário do Índice da Doença da Superfície Ocular, biomicroscopia, tempo de ruptura do filme lacrimal, teste de Schirmer 1, coloração da córnea e conjuntiva com fluoresceína e lissamina verde, concentração de lisozima lacrimal e citologia de impressão. Resultados: Em quase todos os achados do teste de superfície ocular, encontramos uma correlação positiva e significativa entre níveis mais altos de exposição à poluição do ar e níveis mais elevados de danos na superfície ocular tanto no grupo controle quanto nos pacientes com síndrome de Sjögren. Em pacientes com síndrome de Sjögren, o questionário do Índice da Doença da Superfície Ocular, tempo de ruptura do filme lacrimal, coloração vital e citologia de impressão mostraram uma correlação significativa entre altos níveis de poluição do ar e doença da superfície ocular. No grupo controle, o questionário do Índice de Doenças da Superfície Ocular, tempo de ruptura do filme lacrimal e citologia de impressão mostraram uma correlação significativa entre altos níveis de poluição do ar e doença da superfície ocular. Conclusões: Aqui demonstramos que, pacientes com síndrome de olho seco associada a Sjögren, as anormalidades da superfície ocular e a irritação ocular relacionadas à poluição do ar são mais graves do que aquelas no grupo controle. Acreditamos que a medição da qualidade do ar não deve ser apenas uma parte integral da avaliação da doença da superfície ocular, mas também uma consideração terapêutica.

Anti-Bacterial Effect of Lactobacillus rhamnosus Cell-Free Supernatant Possessing Lysozyme Activity Against Pathogenic Bacteria

Recently, there has been a growing demand for natural preservatives because of increased consumer interest in health. In this study, we produced Lactobacillus rhamnosus cell-free supernatant (LCFS) and evaluated and compared its antimicrobial activity with existing natural preservatives against pathogenic microorganisms and in chicken breast meat contaminated with Escherichia coli and Staphylococcus aureus. Lactobacillus rhamnosus cell-free supernatant possessed 30 units of lysozyme activity and contained 18,835 mg/L of lactic acid, 2,051 mg/L of citric acid and 5,060 mg/L of acetic acid. Additionally, LCFS inhibited the growth of fourteen pathogenic bacteria, S. aureus, Bacillus cereus, Listeria monocytogenes, Vibrio parahaemolyticus, Listeria innocua, S. epidermidis, L. ivanovii, E. coli, Pseudomonas aeruginosa, Shigella sonnei, Shi. flexneri, Proteus vulgaris, Pseudomonas fluorescens, and Klebsiella pneumoniae. The antibacterial activity of LCFS was stronger than that of egg white lysozyme (EWL), Durafresh (DF) and grapefruit seed extract (GSE). Additionally, LCFS maintained its antimicrobial activity after heat treatment at 50℃~95℃ and at pH values of 3~9. Moreover, LCFS inhibited the growth of E. coli and S. aureus in chicken breast meat. In conclusion, it is expected that LCFS, which contains both lysozyme and three organic acids, will be useful as a good natural preservative in the food industry.

The role of human lysozyme-like protein 4 in fertilization and its enzymatic properties / 中华男科学杂志

Objective@#To elucidate the possible role of human lysozyme-like protein 4 (LYZL4) in fertilization and characterize its enzymatic properties.@*METHODS@#The localization of LYZL4 in human spermatozoa was investigated by immunofluorescence staining, the sources of LYZL4 on the sperm surface examined by RT-PCR, and the role of LYZL4 in fertilization assessed by the zona-free hamster egg penetration test. The recombinant plasmid pPIC9K-LYZL4 was constructed and its expression induced with methanol after transformed into competent Pichia pastoris GS115. The recombinant LYZL4 protein (rLYZL4) was purified from the fermentation supernatant and subsequently identified by Western blot. The hyaluronan binding ability of rLYZL4 was determined by ELISA and the muramidase activity, hyaluronidase activity, and free radical scavenging ability examined by spectrophotometric methods.@*RESULTS@#Immunodetection with a specific antiserum localized LYZL4 on the acrosomal membrane of mature spermatozoa, which was exclusively secreted from the testis and epididymis as shown by RT-PCR. Immunoneutralization of LYZL4 significantly decreased the number of human spermatozoa bound to zona-free hamster eggs in a dose-dependent manner in vitro. The recombinant protein was expressed successfully by the P. pastoris strain GS115. Purified rLYZL4 exhibited a potent hyaluronan binding ability and a strong free radical scavenging ability but no muramidase or hyaluronidase activity.@*CONCLUSIONS@#LYZL4 secreted from the testis and epididymis is localized on the acrosomal membrane of mature spermatozoa and plays a role in sperm-egg binding as well as in binding hyaluronan and scavenging free radicals, which suggests that it might be a multi-functional molecule contributive to sperm protection and sperm-egg binding.

Effect of Various Agents on Oral Bacterial Phagocytosis in THP-1 Cells

Phagocytosis is a fundamental process in which phagocytes capture and ingest foreign particles including pathogenic bacteria. Several oral pathogens have anti-phagocytic strategies, which allow them to escape from and survive in phagocytes. Impaired bacteria phagocytosis increases inflammation and contributes to inflammatory diseases. The purpose of this study is to investigate the influences of various agents on oral pathogenic phagocytosis. To determine phagocytosis, Streptococcus mutans, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were stained with 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), and was measured using flowcytometery and confocal microscopy. The influencing factors on phagocytosis were evaluated through the pretreatment of ROS inhibitor (N-acetyl-L-cysteine (NAC)), lysozyme, potassium chloride (KCI) and adenosine triphosphate (ATP) in THP-1 cells. Expression of pro-inflammatory cytokines was determined by enzyme-linked immunosorbent assay (ELISA). The phagocytosis of various bacteria increased in a MOI-dependent manner. Among the tested bacteria, phagocytosis of P. gingivalis showed the highest fluorescent intensity at same infection time. Among the tested inhibitors, the NAC treatment significantly inhibited phagocytosis in all tested bacteria. In addition, NAC treatment indicated a similar pattern under the confocal microscopy. Moreover, NAC treatment significantly increased the bacteria-induced secretion of IL-1β among the tested inhibitors. Taken together, we conclude that the phagocytosis occurs differently depending on each bacterium. Down-regulation by ROS production inhibited phagocytosis and lead increased of oral pathogens-associated inflammation.

Lysozyme gene treatment in testosterone induced benign prostate hyperplasia rat model and comparasion of its' effectiveness with botulinum toxin injection

ABSTRACT Objectives: To compare the effects and histopathological changes of botulinum neurotoxin type A and lysozyme gene injections into prostate tissue within a testosterone induced benign prostate hyperplasia rat model. Materials and Methods: 40 male Wistar rats were randomized into four Groups. Group-1: Control, Group-2: Testosterone replacement, Group-3: Testosterone+botulinum neurotoxin type A, Group-4: Testosterone+plazmid DNA/liposome complex. Results: Estimated prostate volume of the testosterone injected Groups were higher than the control (p <0.05). Actual prostate weight of the testosterone injected Groups was higher than the control Group (p <0.05). Testosterone undecanoate increased the prostate weight by 39%. Botulinum neurotoxin type A treatment led to an estimated prostate volume and actual prostate weights decreased up to 32.5% in rats leading to prostate apoptosis. Lysozyme gene treatment led to an estimated prostate volume and actual prostate weights decrease up to 38.7%. Conclusion: Lysozyme gene and botulinum neurotoxin type A treatments for prostate volume decreasing effect have been verified in the present study that could be anew modality of treatment in prostatic benign hyperplasia that needs to be verified in large randomized human experimental studies.

Proteínas Totales, Fosfatasa Alcalina, Prostaglandinas E2 y Lisozima como Biomarcadores Salivales en Pacientes Adultos con Periodontitis Crónica / Total Protein, Alkaline Phosphatase, Prostaglandin E2 and Lysozyme as Salivary Biomarkers in Patients with Chronic Periodontitis

RESUMEN: La periodontitis crónica es una inflamación de los tejidos que rodean y dan soporte a los dientes. Diversos biomarcadores químicos y pro inflamatorios están aumentados durante el transcurso de la enfermedad. El objetivo del estudio fue determinar los distintos niveles salivales de proteínas totales, fosfatasa alcalina, prostaglandina E2 (PGE2) y lisozima en pacientes con periodontitis crónica. Se obtuvieron muestras de saliva de 31 pacientes con periodontitis crónica y se realizó un estudio de serie de casos para la determinación cuantitativa de los biomarcadores. La concentración de proteínas totales se encontró por sobre los rangos de referencia en 22 pacientes, la actividad de la fosfatasa alcalina se vio aumentada en 9 pacientes y la concentración de PGE2 se vio por sobre los rangos de referencia en 30 pacientes. Las proteínas totales y PGE2 son biomarcadores salivales en estos pacientes con periodontitis, no así la fosfatasa alcalina y la lisozima.

ABSTRACT: Chronic periodontitis is an inflammation of tissue that surrounds and supports the teeth; during the course of the disease there is an increase of different chemical and pro-inflammatory biomarkers. The objective of the study was to determine different levels in saliva of total protein, alkaline phosphatase, lysozyme and prostaglandin E2 (PGE2) in patients with chronic periodontitis. We used saliva samples from 31 patients who had chronic periodontitis and the study was a case of series. Our results showed 22 patients with increased concentrations of protein concentration, nine patients with increased alkaline phosphatase and PGE2 concentration was above the reference range in 30 patients: The total protein and PGE2 are good salivary biomarkers in patients with periodontitis, but not the alkaline phosphatase and lysozyme.

Preparation of a polyclonal antibody against human LYZL4 and its expression in the testis / 中华男科学杂志

Objective@#To prepare a polyclonal antibody against human lysozyme-like protein 4 (LYZL4) expressed in the prokaryotic system and identify the distribution of LYZL4 in the testis.@*METHODS@#The full-length cDNA of LYZL4 was cloned into the pET32a plasmid and the expression of the recombinant LYZL4 (rLYZL4) was induced by IPTG. The rLYZL4 was purified by Ni-NTA and chitin affinity chromatography respectively and its bactericidal activity was observed by bilayer agar plate diffusion assay. The purified rLYZL4 was used as an immunogen to generate the polyclonal antibody, followed by examination of the antibody titer by ELISA and its specificity by Western blot. The distribution of LYZL4 in human tissue, sperm and seminal plasma was identified and its subcellular localization in the testis was determined by immunohistochemistry.@*RESULTS@#rLYZL4 was expressed efficiently in the prokaryotic system and exhibited no bacteriolytic activity against M. lysodeikticus and E. coli. The anti-rLYZL4 polyclonal antibody could bind the recombinant protein with a high sensitivity and specificity. LYZL4 was identified in the testis, epididymis and sperm protein extracts and localized in the acrosomal region of round and elongating spermatids.@*CONCLUSIONS@#An anti-rLYZL4 polyclonal antibody was successfully prepared using the prokaryotic expression system. LYZL4 was detected in the acrosomal region of round and elongating spermatids, suggesting an association with the structure and function of the acrosome.

Identification of microbiome with 16S rRNA gene pyrosequencing and antimicrobial effect of egg white in bovine mastitis

Bovine mastitis is an important microbial disease in the dairy industry. We investigated the frequencies of bacterial pathogens in 62 farms and pathogen antibiotic resistance from mastitis samples (n = 748). We tested the antimicrobial activity of chicken and duck egg white and lysozyme purified from chicken egg white. Moreover, we compared the microbiomes of normal and mastitic raw milk obtained by 16S rRNA gene pyrosequencing and culture methods. The results showed that the frequencies of Gram-positive pathogens (Enterococcus faecalis 37% and Staphylococcus aureus 36%) were higher than that of a Gram-negative pathogen (Escherichia coli 15%). Resistance frequencies to ampicillin and norfloxacin were lowest in Staphylococcus aureus (21%), Enterococcus faecalis (23%), and Escherichia coli (33%), and the antimicrobial activity of chicken egg white was higher than those of lysozyme and duck egg white. Pyrosequencing results revealed clear differences between the microbiomes of mastitic and normal raw milk samples and revealed a slightly similar, but clearly different, composition of pathogens compared to that from the culture method. Thus, pyrosequencing may be useful for elucidating changes in microbiomes during mastitis progression and treatment. A chicken egg white and antibiotic combination may help with mastitis treatment; however, further studies are needed.

Identification of CEA-interacting proteins in colon cancer cells and their changes in expression after irradiation

PURPOSE: The serum carcinoembryonic antigen (CEA) level has been recognized as a prognostic factor in colorectal cancer, and associated with response of rectal cancer to radiotherapy. This study aimed to identify CEA-interacting proteins in colon cancer cells and observe post-irradiation changes in their expression. MATERIALS AND METHODS: CEA expression in colon cancer cells was examined by Western blot analysis. Using an anti-CEA antibody or IgG as a negative control, immunoprecipitation was performed in colon cancer cell lysates. CEA and IgG immunoprecipitates were used for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. Proteins identified in the CEA immunoprecipitates but not in the IgG immunoprecipitates were selected as CEA-interacting proteins. After radiation treatment, changes in expression of CEA-interacting proteins were monitored by Western blot analysis. RESULTS: CEA expression was higher in SNU-81 cells compared with LoVo cells. The membrane localization of CEA limited the immunoprecipitation results and thus the number of CEA-interacting proteins identified. Only the Ras-related protein Rab-6B and lysozyme C were identified as CEA-interacting proteins in LoVo and SNU-81 cells, respectively. Lysozyme C was detected only in SNU-81, and CEA expression was differently regulated in two cell lines; it was down-regulated in LoVo but up-regulated in SNU-81 in radiation dosage-dependent manner. CONCLUSION: CEA-mediated radiation response appears to vary, depending on the characteristics of individual cancer cells. The lysozyme C and Rab subfamily proteins may play a role in the link between CEA and tumor response to radiation, although further studies are needed to clarify functional roles of the identified proteins.

Identification of CEA-interacting proteins in colon cancer cells and their changes in expression after irradiation

PURPOSE: The serum carcinoembryonic antigen (CEA) level has been recognized as a prognostic factor in colorectal cancer, and associated with response of rectal cancer to radiotherapy. This study aimed to identify CEA-interacting proteins in colon cancer cells and observe post-irradiation changes in their expression. MATERIALS AND METHODS: CEA expression in colon cancer cells was examined by Western blot analysis. Using an anti-CEA antibody or IgG as a negative control, immunoprecipitation was performed in colon cancer cell lysates. CEA and IgG immunoprecipitates were used for liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis. Proteins identified in the CEA immunoprecipitates but not in the IgG immunoprecipitates were selected as CEA-interacting proteins. After radiation treatment, changes in expression of CEA-interacting proteins were monitored by Western blot analysis. RESULTS: CEA expression was higher in SNU-81 cells compared with LoVo cells. The membrane localization of CEA limited the immunoprecipitation results and thus the number of CEA-interacting proteins identified. Only the Ras-related protein Rab-6B and lysozyme C were identified as CEA-interacting proteins in LoVo and SNU-81 cells, respectively. Lysozyme C was detected only in SNU-81, and CEA expression was differently regulated in two cell lines; it was down-regulated in LoVo but up-regulated in SNU-81 in radiation dosage-dependent manner. CONCLUSION: CEA-mediated radiation response appears to vary, depending on the characteristics of individual cancer cells. The lysozyme C and Rab subfamily proteins may play a role in the link between CEA and tumor response to radiation, although further studies are needed to clarify functional roles of the identified proteins.

Identification of 10 Candidate Biomarkers Distinguishing Tuberculous and Malignant Pleural Fluid by Proteomic Methods

PURPOSE: Pleural effusion, an accumulation of fluid in the pleural space, usually occurs in patients when the rate of fluid formation exceeds the rate of fluid removal. The differential diagnosis of tuberculous pleurisy and malignant pleural effusion is a difficult task in high tuberculous prevalence areas. The aim of the present study was to identify novel biomarkers for the diagnosis of pleural fluid using proteomics technology. MATERIALS AND METHODS: We used samples from five patients with transudative pleural effusions for internal standard, five patients with tuberculous pleurisy, and the same numbers of patients having malignant effusions were enrolled in the study. We analyzed the proteins in pleural fluid from patients using a technique that combined two-dimensional liquid-phase electrophoresis and matrix assisted laser desorption/ionization-time of flight-mass spectrometry. RESULTS: We identified a total of 10 proteins with statistical significance. Among 10 proteins, trasthyretin, haptoglobin, metastasis-associated protein 1, t-complex protein 1, and fibroblast growth factor-binding protein 1 were related with malignant pleural effusions and human ceruloplasmin, lysozyme precursor, gelsolin, clusterin C complement lysis inhibitor, and peroxirexdoxin 3 were expressed several times or more in tuberculous pleural effusions. CONCLUSION: Highly expressed proteins in malignant pleural effusion were associated with carcinogenesis and cell growth, and proteins associated with tuberculous pleural effusion played a role in the response to inflammation and fibrosis. These findings will aid in the development of novel diagnostic tools for tuberculous pleurisy and malignant pleural effusion of lung cancer.

Immunocytochemical characterization of primary cell culture in canine transmissible venereal tumor / Caracterização imunocitoquímica de culturas primárias de tumor venéreo transmissível canino

Immunochemistry with anti-vimentin, anti-lysozyme, anti-alpha 1 antitrypsin, anti-CD3 and anti-CD79α antibodies has been used for characterization of primary cell culture in the transmissible venereal tumor (TVT). Samples for primary cell culture and immunohistochemistry assays were taken from eight dogs with cytological and clinical diagnosis of TVT. To validate the immunochemical results in the primary cell culture of TVT, a chromosome count was performed. For the statistical analysis, the Mann-Whitney test with p<0.05 was used. TVT tissues and culture cells showed intense anti-vimentin immunoreactivity, lightly to moderate immunoreactivity for anti-lysozyme, and mild for anti-alpha-antitrypsin. No marking was achieved for CD3 and CD79α. All culture cells showed chromosomes variable number of 56 to 68. This is the first report on the use of immunocytochemical characterization in cell culture of TVT. Significant statistic difference between immunochemistry in tissue and culture cell was not established, what suggests that the use of this technique may provide greater certainty for the confirmation of tumors in the primary culture. This fact is particularly important because in vitro culture of tumor tissues has been increasingly used to provide quick access to drug efficacy and presents relevant information to identify potential response to anticancer medicine; so it is possible to understand the behavior of the tumor.(AU)

Os anticorpos anti-vimentina, anti-lisozima, anti-alfa 1 antitripsina, anti-CD3 e anti-CD79α foram empregados para a caracterização de culturas primárias de tumor venéreo transmissível canino (TVT). Amostras para cultura primária e imuno-histoquímica foram coletadas de oito cães com diagnóstico clínico e citológico de TVT. Para validar o resultado inmunocitoquímico nas culturas de TVT foi realizada a contagem de cromossomos. Para a análise estatística o teste de Mann-Whitney foi empregado a um nível de significância de p<0.05. As culturas e os tecidos de TVT apresentaram intensa reatividade para vimentina, moderada a leve para Lisozima, moderada para alfa-antitripsina e não houve marcação para CD3 e CD79α. Finalmente, todas as culturas apresentaram números de cromossomos que variaram de 56 a 68. Este é o primeiro relato que apresenta o uso da immunocitoquímica para a caracterização de culturas de TVT. Assim, e devido ao fato de se observar semelhança entre a imunomarcação em células e tecidos, sugere-se que o uso desta técnica possa auxiliar na confirmação de culturas primárias do tumor, fato muito importante porque a utilização da cultura do tumor pode permitir o acesso a informação relevante sobre resposta potencial a um tratamento e conhecimento do comportamento biológico do tumor.(AU)

Tratamientos farmacológicos en pacientes con síndrome de boca urente: revisión sistemática / Pharmacologic treatment of burning mouth syndrome: a systematic review

El objetivo fue determinar mediante una revisión sistemática, cuáles tratamientos farmacológicos para el Síndrome de Boca Urente (SBU) logran una reducción de síntomas, según Escala Visual Análoga (EVA). Se realizó una búsqueda bibliográfica en la bases de datos PubMed y SciELO, Trip Database, Scopus Database, EBSCO host y LILACS entre el 2005 y 2015. De 72 artículos, se seleccionaron un total de 11. Los tratamientos sistémicos usados fueron, Hipericum perforatum, Catuama, Clonazepam, Ácido alfa lipoico y Lafutidina. Entre los tratamientos tópicos, Aceite de oliva virgen enriquecido con licopeno, Lisozima lactoperoxidasa, Clonazepam y Capsaicina. Los fármacos que obtuvieron mejores resultados para el tratamiento del SBU fueron Lafutidina, Catuama, Clonazepam tópico y sistémico, y en menor grado Capsaicina.

The aim of this study was to determine through a systematic review, which is the best drug treatment for burning mouth syndrome (SBU), measured on a Visual Analogue Scale. A scientific literature search was conducted in PubMed and SciELO, Trip Database, Database Scopus, EBSCO host and LILACS data between 2005 and 2015. Of a total of 72 articles, 11 were included for analysis. Systemic treatments were Lycopene-enriched virgin olive oil, Hypericum perforatum, Catuama, Clonazepam, Alpha lipoic acid; topical treatments were Lysozyme lactoperoxidase, Clonazepam, Capsaicin and Lafutidine. The best results obtained were with Lafutidine, Catuama, topical and systemic Clonazepam, and to a lesser degree Capsaicin.

Supplementation with humic substances affects the innate immunity in layer hens in posfasting phase / El suplemento de sustancias húmicas afecta la inmunidad innata en gallinas ponedoras en fase posmuda

Objective. Asses the effect of supplementation with Humic substances (HS) over some innate immunity parameters (serum bactericidal activity, phagocytosis, bacterial agglutination, respiratory burst and lisozyme activity) in phase after fasting of layer hens. Materials and methods. 120 posfasting phase Hy Line Brown layer hens were taken which were distributed into four groups: The first and the second were supplemented with 0.1 and 0.2% of HS, respectively. The third group was supplemented with 0.25 mg/kg on levamisole hydrochloride and fourth group have no supplementation; during sixty days period. Blood samples were collected on 8th, 30th and 60th of experiment day. Results. The phagocytic index and respiratory burst increased significantly at day 30th in HS supplemented groups. Alike, serum bactericidal activity and lisozyme activity improved on 8 th day, nevertheless, changes were no evident latter. The bacterial agglutination was high in supplemented groups evaluated at everyone times. Conclusions. Results showed that HS behave as immunostimulant in the early phase after fasting layer hens.

Objetivos. Evaluar el efecto de las sustancias húmicas (SH) sobre algunos parámetros de la inmunidad innata (actividad bactericida del suero, fagocitosis, aglutinación bacteriana, explosión respiratoria y actividad de la lisozima) en la fase posmuda de gallinas ponedoras. Materiales y métodos. Se utilizaron 120 gallinas ponedoras Hy Line Brown en la fase de posmuda, las cuales fueron divididas en cuatro grupos: Los dos primeros fueron suplementados con 0.1 y 0.2% de SH respectivamente, el tercer grupo fue suplementado con 0.25 mg/kg de Clorhidrato de levamisol y el cuarto grupo control sin suplemento; durante un período de 60 días. Las muestras sanguíneas se tomaron los días 8, 30 y 60 del experimento. Resultados. El Índice fagocítico y la explosión respiratoria se incrementaron significativamente a partir del día 30 de suplementación con SH. De la misma manera, la actividad bactericida del suero y la actividad de la lisozima aumentaron al día 8; no obstante no se evidenciaron cambios posteriores. La aglutinación bacteriana fue significativamente mayor en los grupos suplementados en todos los tiempos evaluados. Conclusiones. Los resultados demuestran que las SH se comportan como agentes inmunoestimulantes en la fase temprana de la posmuda en gallinas ponedoras Hy Line Brown.

Oxidação de resíduos de triptofano em proteínas: formação de ligação cruzada ditriptofano e implicações patofisiológicas / Oxidation of tryptophan residues in proteins: formation of the ditryptophan cross-link and pathophysiological implications

Apesar de extensa investigação das modificações oxidativas irreversíveis sofridas pelas proteínas in vitro e in vivo, os produtos formados pela oxidação de resíduos de triptofano ainda permanecem apenas parcialmente conhecidos. Recentemente, nosso grupo caracterizou uma ligação cruzada de ditriptofano produzida pela recombinação de radicais hSOD1-triptofanila gerados pelo ataque do radical carbonato produzido durante a atividade peroxidásica da enzima superóxido dismutase humana (hSOD1). Neste trabalho, examinamos se a ligação ditriptofano pode ser formada em outras proteínas, além da hSOD1 e por outros oxidantes, além do radical carbonato. A lisozima da clara do ovo e a beta cristalino bovina foram utilizadas como alvos de oxidação. A lisozima foi utilizada por ser uma enzima pequena (129 aminoácidos) e de estrutura bem conhecida, contendo seis resíduos de Trp. Os resultados mostraram que o radical carbonato, gerado enzimatica ou fotoliticamente, promove a oxidação, dimerização e inativação da lisozima. Os principais produtos de oxidação caracterizados por análise de nano-ESI-Q-TOF-MS/MS foram hidroxi-triptofano e N-formilquinurenina juntamente com um dímero de lisozima (lisozima-Trp28-Trp28-lisozima) e um hetero dímero lisozima-hSOD1 (lisozima-Trp28-Trp32-hSOD1), ambos ligados por uma ligação ditriptofano. Também demonstramos que a irradiação da lisozima com luz UVC leva à formação do dímero lisozima-Trp28-Trp28-lisozima. Em consequência, resolvemos tratar a beta cristalino bovina com radical carbonato gerado fotoliticamente ou com luz UVC, e a proteína também sofreu oxidação, dimerização e agregação. Os principais produtos de oxidação caracterizados por nano-ESI-Q-TOF-MS/MS foram hidroxi-triptofano, N-formilquinurenina, DOPA e um dímero de beta cristalino (ßB2-Trp151-Trp151-ßB2). A irradiação com luz UVC também levou à formação de um dímero intra-cadeia, caracterizado como ßA2-Trp78-Trp81. Quando a beta cristalino foi irradiada com um simulador de luz solar (UVA e UVB) também foi possível observar um dímero, caracterizado como ßA2-Trp150-Trp150-ßA2. A presença de produtos de oxidação de resíduos de Trp, dentre eles a ligação cruzada ditritpofano, também foi avaliada in vivo, utilizando o cristalino de pacientes que foram submetidos a cirurgia para remoção de catarata. Beta, alfa e gama cristalino foram as principais proteínas identificadas nas frações solúvel e insolúvel do cristalino. A principal modificação pós-traducionais identificada foi deamidação. Um alto conteúdo de resíduos de metionina e triptofano oxidados foram identificados nas proteínas presentes na fração insolúvel. Os principais produtos de oxidação de Trp identificados por nano-ESI-Q-TOF-MS/MS foram quinurenina e N-formilquinurenina. A presença de dímeros covalentes no cristalino com catarata foi confirmada por análises de massas. A completa caracterização desses dímeros (ßB1-Trp127-Trp127-ßB1 e ßB1-Trp193-Trp193-ßB1) confirmou que as cadeias polipeptídicas foram ligadas por uma ligação ditriptofano. Em síntese, nossos dados demonstraram que o radical carbonato e a luz UV podem produzir dímeros de ditriptofano em diferentes proteínas. Também, a presença da ligação cruzada de ditriptofano in vivo (catarata humana) foi pela primeira vez detectada

Despite extensive investigation of irreversible oxidative modifications suffered by proteins in vitro and in vivo, the products formed by oxidation of tryptophan residues remain partially characterized. Our group recently described a ditryptophan cross-link produced by recombination of hSOD1-tryptophanyl radicals generated by attack of the carbonate radical produced during the peroxidase activity of the human superoxide dismutase (hSOD1) enzyme. Here, we examine whether the ditryptophan cross-link can be produced in others proteins besides the hSOD1 and by other oxidants, in addition to the carbonate radical. The egg white lysozyme and bovine beta crystalline were used as targets. Lysozyme was used because it is a small enzyme (129 amino acids) with a well-known structure, containing six Trp residues. The results showed that the carbonate radical, generated enzymatically or photolytically, promotes lysozyme oxidation, inactivation and dimerization. The major oxidation products characterized by nano-ESI-Q-TOF-MS/MS analysis were hydroxy-tryptophan and N-formylkynurenine together with a dimer of lysozyme (lysozyme-Trp28-Trp28-lysozyme) and a hetero dimer hSOD1-lysozyme (lysozyme-Trp28-Trp32-hSOD1), both bound by a ditryptophan cross-link. Also, it was demonstrated that lysozyme irradiation with UVC light leads to the formation of the dimer lysozyme-Trp28-Trp28-lysozyme. In view of these results, we decided to treat beta crystalline bovine with photolytically generated carbonate radical and UVC. Beta crystalline also suffered oxidation, dimerization and aggregation. The major oxidation products characterized were hydroxy-tryptophan, N-formylkynurenine, DOPA and a beta crystalline dimer (ßB2-Trp151-Trp151-ßB2) by nano-ESI-Q-TOF-MS/MS. Irradiation with UVC light also led to the formation of an intra-chain dimer, which was characterized as ßA2-Trp78-Trp81. When beta crystalline was irradiated with a solar simulator (UVA and UVB), it was also possible to observe a dimer which was characterized as ßA2-Trp150-Trp150-ßA2. The presence of oxidized tryptophan products, including the ditryptophan cross-link, was also evaluated in vivo in the lenses of patients submitted to cataract removal. Beta, alpha and gamma crystalline were the main proteins identified in soluble and insoluble fractions of the lenses. The main post translational modification identified was deamidation. A high content of oxidized methionine and tryptophan residues were identified in proteins present in the insoluble fraction. The main tryptophan oxidation products identified by nano-ESI-Q-TOF-MS/MS were kynurenine and N-formylkynurenine. The presence of covalent dimers in the lenses with cataract was demonstrated by mass analysis. Full MS/MS characterization of the dimers ßB1-Trp127-Trp127-ßB1 and ßB1-Trp193-Trp193-ßB1 confirmed that they were linked by a ditryptophan bond. In summary, our data demonstrate that the carbonate radical and UV light can produce ditryptophan dimers in different proteins. Also, the presence of the ditryptophan cross-link was first detected in vivo (human cataract)

Staphylococcus aureus biofilm influences the expression of lysozyme, SLPI and gp340 in a human sinonasal explant model / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To investigate the influences of staphylococcus aureus in planktonic and biofilm forms on the expression of lysozyme, SLPI and gp340 in the human sinonasal explant model.@*METHOD@#Mucosa samples from ethmoid sinus were collected from ten patients of cerebrospinal fluid leak and were cultured with and without S. aureus biofilms and planktonic cells. After the infection, the explant model was confirmed by CLSM, and the secretion of lysozyme, SLPI and gp340 was detected by enzyme-linked immunosorbent assay (ELISA) at 8, 16, and 24 h after S. aureus challenge. Expressions of lysozyme, SLPI and gp340 in mRNA and protein levels after 24 h S. aureus challenge were detected using RT-PCR, immunohistochemistry and Western bolt assay respectively.@*RESULT@#The secretion of lysozyme, SLPI and gp340 in the explant model was observed with a trend to increase in a time-dependent manner. At 8 and 16 h after S. aureus challenge, the secretion of lysozyme, SLPI and gp340 in biofilms group was significantly higher than these in planktonic cells group and control group (P 0.05), the biofilms enhanced the expressions of lysozyme, SLPI and gp340 significantly compared with planktonic cells and controls (P < 0.05).@*CONCLUSION@#S. aureus biofilm induced the expressions of lysozyme, SLPI and gp340 to a higher level than planktonic cells, indicating that S. aureus biofilm was an influencing factor on the innate immune system.

Expression of human lysozyme-like protein 6 in the male reproductive system / 中华男科学杂志

<p><b>Objective</b>To study the expression of human lysozyme-like protein 6 (LYZL6) in the male reproductive system and its physiological role.</p><p><b>METHODS</b>The recombinant P. pastoris strain was cultured and induced with methanol to express LYZL6, followed by purification using chitin affinity chromatography. The bactericidal activity of the recombinant LYZL6 was observed by bilayer agar plate diffusion assay, and then the recombinant protein was used as an immunogen to generate polyclonal antibodies, whose specificity was examined by ELISA. The distribution of LYZL6 in the human tissue and semen was identified by Western blotting and the subcellular localization in the testis was investigated by immunohistochemistry.</p><p><b>RESULTS</b>At pH 5.6, recombinant LYZL6 exhibited a high bacteriolytic activity against M. lysodeikticus. ELISA analysis showed that the anti-LYZL6 polyclonal antibodies could bind the recombinant protein with a high specificity. Western blot manifested the expression of LYZL6 in the testis and epididymis, higher in the former than in the latter. LYZL6 was also detected in the sperm protein extract, while protein bands were not observed in the seminal plasma. Immunodetection with a specific antiserum localized the LYZL6 protein in the late spermatocytes and round spermatids.</p><p><b>CONCLUSIONS</b>LYZL6 has a higher bacteriolytic activity under low pH condition and is bound to spermatozoa after secreted in the testicular epithelia, suggesting that LYZL6 could act as a potential hydrolase for carbohydrates in zona pellucida penetration.</p>