Bilayer hydrogel dressing with lysozyme-enhanced photothermal therapy for biofilm eradication and accelerated chronic wound repair

Biofilms are closely associated with the tough healing and dysfunctional inflammation of chronic wounds. Photothermal therapy (PTT) emerged as a suitable alternative which could destroy the structure of biofilms with local physical heat. However, the efficacy of PTT is limited because the excessive hyperthermia could damage surrounding tissues. Besides, the difficult reserve and delivery of photothermal agents makes PTT hard to eradicate biofilms as expectation. Herein, we present a GelMA-EGF/Gelatin-MPDA-LZM bilayer hydrogel dressing to perform lysozyme-enhanced PTT for biofilms eradication and a further acceleration to the repair of chronic wounds. Gelatin was used as inner layer hydrogel to reserve lysozyme (LZM) loaded mesoporous polydopamine (MPDA) (MPDA-LZM) nanoparticles, which could rapidly liquefy while temperature rising so as to achieve a bulk release of nanoparticles. MPDA-LZM nanoparticles serve as photothermal agents with antibacterial capability, could deeply penetrate and destroy biofilms. In addition, the outer layer hydrogel consisted of gelatin methacryloyl (GelMA) and epidermal growth factor (EGF) promoted wound healing and tissue regeneration. It displayed remarkable efficacy on alleviating infection and accelerating wound healing in vivo. Overall, the innovative therapeutic strategy we came up with has significant effect on biofilms eradication and shows promising application in promoting the repair of clinical chronic wounds.

Biocompatible silver nanoparticles:An investigation into their protein binding efficacies,anti-bacterial effects and cell cytotoxicity studies / 药物分析学报

Green synthesis of silver nanoparticles (AgNPs) has garnered tremendous interest as conventional methods include the use and production of toxic chemicals,products,by-products and reagents.In this regard,the synthesis of AgNPs using green tea (GT) extract and two of its components,(-)-epi-gallocatechin gallate (EGCG) and (+)-catechin (Ct) as capping/stabilizing agents,is reported.The syn-thesized AgNPs showed antibacterial activity against the bacterial strains Staphylococcus aureus and Escherichia coli,along with anticancer activity against HeLa cells.After administering nanoparticles to the body,they come in contact with proteins and results in the formation of a protein corona;hence we studied the interactions of these biocompatible AgNPs with hen egg white lysozyme (HEWL) as a carrier protein.Static quenching mechanism was accountable for the quenching of HEWL fluorescence by the AgNPs.The binding constant (Kb) was found to be higher for EGCG-AgNPs ((2.309 ± 0.018) × 104 M-1)than for GT-AgNPs and Ct-AgNPs towards HEWL.EGCG-AgNPs increased the polarity near the binding site while Ct-AgNPs caused the opposite effect,but GT-AgNPs had no such observable effects.Circular dichroism studies indicated that the AgNPs had no such appreciable impact on the secondary structure of HEWL.The key findings of this research included the synthesis of AgNPs using GT extract and its con-stituent polyphenols,and showed significant antibacterial,anticancer and protein-binding properties.The-OH groups of the polyphenols drive the in situ capping/stabilization of the AgNPs during synthesis,which might offer new opportunities having implications for nanomedicine and nanodiagnostics.

Stability of lactoferrin and lysozyme in human milk at various temperatures and duration of storage

@#Introduction: Exclusive breastfeeding, especially in the first six months, is essential for infants as it provides nutrition and protection against various diseases. Colostrum, which is found in the first breast milk produced, contains various protective factors, such as lactoferrin and lysozyme. Human milk can be stored at room temperature, refrigerated, or kept frozen. Several factors affect the stability of the bioactive content in human milk, such as temperature and storage time. The aim of this study was to measure the stability of lactoferrin and lysozyme levels in human milk during the first six hours (h) at different temperatures and compare it with that of frozen human milk. Methods: Human milk samples were obtained from 11 breastfeeding mothers using certain criteria. The human milk was stored at room temperature and 4°C for 1, 3, and 6 h and classified as never frozen, while frozen human milk was stored at -20°C for 1, 3, and 6 days. Measurement of the lactoferrin and lysozyme levels was performed using enzyme-linked immunosorbent assay. Results: The results showed that storage at room temperature significantly reduced lactoferrin and lysozyme levels. Lactoferrin levels in frozen human milk did not significantly decrease during the first six days. Meanwhile, the lysozyme levels in frozen human milk decreased significantly. Conclusion: The levels of lactoferrin and lysozyme in frozen human milk stored for the first six days were more stable than those stored at room temperature and 4°C in the first 6 h.

Immunological and Morphological substantiation of the use of dental stick with biopharmaceuticals in the complex therapy of inflammatory periodontal diseases

Aim of the study: clinical, immunological and morphological substantiation of the use of a dental stick with biopharmaceuticals in the complex therapy of inflammatory periodontal diseases. Research methods: leukocyte migration test, cytograms, the study of quantitative and qualitative content of spontaneously released mixed saliva, measurement of the total content of protein, the content of secretory immunoglobulin A (sIgA) and lysozyme in it. Clinical improvement of the periodontal tissues condition after treatment by 77% was registered. OHI-S (hygienic index), PI (periodontal disease index), PMA (papillary-marginal-alveolar index) were 7.5, 3.4, and 8.7 times lower, respectively, compared to the pre-treatment group. The depth of periodontal pockets decreased 2.6-3.4 times, the number of sessions per treatment course was reduced to 3-8 visits to the doctor. The amount of mixed saliva (in comparison with the initial data) increased 2.7 times, normal levels were restored and were 1.2 ml higher than control indicators. Protein levels, lysozyme and sIgA concentrations increased and exceeded the pre-treatment level 1.8 times and by 44.5%, respectively. Cytograms data revealed that the number of red blood cells (in one field of view) in the gingiva specimens in inflammatory periodontal diseases (IPD) patients was 2.3 times lower, count of leukocytes with signs of destruction was 13.4 lower and intact leukocyte count was 3.8 times lower. Lymphocyte count was 2 times lower, indicating that the inflammatory process in periodontal tissues was reduced as a re sult of the reduction of the microbial burden: staphylococci and actinomycetes were detected 3 times less often, diplococci ­ 3.5 times less often, filamentous bacteria and streptococci ­ 4 times less often, protists ­ 5 times less often, respectively. The number of cocci microcolonies was 2.7 times higher. The number of fibroblasts (in one field of view) increased threefold, the content of collagen filaments of the normally oriented structure increased 1.4 times, compared to the original data, which indicated the formation of favorable conditions for healing and reparative regeneration processes. The results of the study showed that the use of a dental stick during local IPD therapy is an effective way of correcting changes in clinical and laboratory indicators of local immunity and that dental stick is a promising new dosage form in practical periodontology.

Influence of some environmental variables and addition of r-lysozyme on efficacy of Vibrio harveyi phage for therapy

Bacteriophage therapy is a viable proposition for controlling luminous vibriosis caused by Vibrio harveyi in shrimpaquaculture. However, environmental factors influence the growth and activity of phage and affect its efficiency incontrolling bacterial diseases. An essential problem in the use of vibrio phage as a therapeutic agent was the development ofresistance to phage attachment, rendering them resistant to the lytic action of phage. This problem could be overcome byapplying a cocktail of phages. This study aimed to evaluate the effect of salinity and pH on the phage activity and also tostudy the role of recombinant shrimp lysozyme on the performance of the V. harveyi phage. Out of three different levels ofsalinity (20, 25 and 30 ppt) and pH (6, 7 and 8) tested, optimum phage activity was observed at a salinity of 25 ppt and atneutral pH. Application of recombinant shrimp lysozyme in combination with V. harveyi phage significantly improved theactivity of phage in in vitro assay as well as in microcosm study using seawater. The application of phage along withlysozyme can be a useful approach to overcome the inability of phage to enter the bacteria and thus eliminate or reduce fish/shrimp pathogenic bacteria in aquaculture.

Differential Expression of Serum Lysozyme Allelic Variants in Muzaffarnagri Sheep

Lysozyme degrades the bacterial cell walls and gives rise to degradation product that stimulates and activates the immune system. Several gram positive and gram negative bacteria were found to be susceptible to different degree of purified lysozyme. Variation in promoter region may regulate the expression of a particular gene. Hence, considering lysozyme gene a potential marker for general immune response, expression pattern of various genotypes on the basis of variations in promoter region is investigated in Muzaffarnagri sheep. A 268 bp fragment spanning partial promoter, exon 1 and partial intron 1 of serum lysozyme gene were amplified and sequenced. Sequencing revealed five genotypes AA, AB, AC, BB and CC and consequently three alleles A, B and C in Muzaffarnagri sheep. Differential expression study of various genotypes by real time pcr revealed significant difference (P≤0.05) in the serum lysozyme expression in animals having different genotypes. Animals having AA genotype showed higher expression of serum lysozyme than the animals having AB, AC and BB genotype.

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.

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.

Affinity of Hydrogel Nanoparticles to Lysozyme / 分析化学

Hydrogel nanoparticles (NPs) were synthesized by precipitation polymerization method with N-isopropyl acrylamide (NIPAm), acrylic acid (Aac), N-tert butyl acrylamide (tBAM) and N,N'-methylene bisacrylamide(Bis) as thermosensitive monomer,negative monomer,hydrophobic monomer and crosslinker, respectively. The morphology of the resulting NPs was characterized by scanning electron microscopy(SEM),and the size and the particle size distribution were investigated by dynamic light scattering (DLS). The dynamics test was also carried out to investigate adsorption property of NPs. The results showed that NPs was spherical with uniform particle size and narrow distribution. NPs had the best adsorption performance to lysozyme when the monomer molar ratio was optimized to Aac 20%,tBAM 40%,NIPAm 38% and Bis 2%. Meanwhile, when the particle size of NPs decreased from 386. 20 nm to 77. 25 nm, the adsorption capacity increased gradually. The adsorption rate could reach up to 67.8% within 5 minutes. The thermosensitive of NPs provided a new candidate for the adsorption and separation of lysozyme with good reusability.

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.

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.

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.

Purification, identification and characterization of an anti-microbial hexapeptide from Sus scrofa lysozyme / 生物工程学报

Sus scrofa lysozyme (SSL) was digested by different proteases to find peptides with enhanced antibacterial activity against gram-negative bacteria. Hydrolysate with the highest anti-bacterial activity was loaded onto a gel filtration chromatography column followed by a reversed-phase one. The obtained substance was identified by liquid chromatography-mass spectrometry, synthesized to test its antibacterial spectrum and analyzed for bioinformatics. The hydrolysate of trypsin showed the highest antibacterial activity. By purification and identification, the functional peptide with sequence of A-W-V-A-W-K was obtained. The peptide was synthesized and proved to retain partial function of SSL and had activity against gram-negative bacteria. By bioinformatics analysis, the peptide was found to locate in a helix-loop-helix structure, suggesting that the peptide may kill cells by penetrating cell membrane and cause the outflow of cell contents. The discovery of the peptide could lay the foundation for improving the antibacterial activity of SSL.

Influence of isoniazid on immune function of rat / 药物评价研究

Objective To study the influence of isoniazid on lymphocyte factor expression and macrophage function of rats.Methods Healthy male SD rats were randomly divided into three groups,which was treated for one month,three months and withdrawal for one month after treated for three months,and each group was randomly divided into isoniazid group and control group.The isoniazid groups were ig with isoniazid at dose of 120 mg/kg every other day and control groups were fed on normal saline.At the corresponding time points,the level of interleukin-12 (IL-12) and interferon-γ (IFN-γ) was detected with ELISA method,detected serum lysozyme content by agar plate method,and Comori method was used for the detection of acid phosphatase levels in peritoneal fluid.Results At all the time points,levels of IL-12,IFN-γ and lysozyme in isoniazid group were not significantly different compared with control group.There were statistically significant differences in acid phosphatase between isoniazid group and control group after treated for one month (P ＜ 0.05),but the significant differences disappeared at the next two time points.Conclusion Isoniazid of 120 mg/kg may have no obvious influence on the immune function of rat.We don't detect the immune injury.

Influence of isoniazid on immune function of rat / 药物评价研究

Objective To study the influence of isoniazid on lymphocyte factor expression and macrophage function of rats.Methods Healthy male SD rats were randomly divided into three groups,which was treated for one month,three months and withdrawal for one month after treated for three months,and each group was randomly divided into isoniazid group and control group.The isoniazid groups were ig with isoniazid at dose of 120 mg/kg every other day and control groups were fed on normal saline.At the corresponding time points,the level of interleukin-12 (IL-12) and interferon-γ (IFN-γ) was detected with ELISA method,detected serum lysozyme content by agar plate method,and Comori method was used for the detection of acid phosphatase levels in peritoneal fluid.Results At all the time points,levels of IL-12,IFN-γ and lysozyme in isoniazid group were not significantly different compared with control group.There were statistically significant differences in acid phosphatase between isoniazid group and control group after treated for one month (P ＜ 0.05),but the significant differences disappeared at the next two time points.Conclusion Isoniazid of 120 mg/kg may have no obvious influence on the immune function of rat.We don't detect the immune injury.

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)

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>

A study on the effect of indomethacin on SiO2 induced lysozyme over-expression in rat alveolar macrophages / 预防医学

Objective To learn the effect of indomethacin (IND)of different concentrations on the expression of lysozyme protein induced by SiO2 in rat alveolar macrophages (AM).Methods Pure AM was prepared with the method of bronchoalveolar lavage in rats.In the model group,the silica dust poisoning model was replicated by adding SiO2 dust suspension (50 μg /ml).In the intervention group,following the adding of SiO2 dust suspension,the IND of 1 0 -5 ,1 0 -4 , and 1 0 -3 g/ml were added respectively.In the control group,the same volume of PBS was given.After 8 h,1 6 h,the cell morphology was observed.Results Compared with the model group,the AM cells in the intervention group were relatively complete,and that there was a concentration dependent trend.The expression of LSZ protein in AMof the model group was significantly higher than that of the control group (P<0. 05 ),while the expression of LSZ protein in the intervention group decreased compared with that of the model group.After incubation with IND and SiO2 ,the expression of LSZ protein in the intervention group decreased compared with that of the model group.Conclusion IND can inhibit the increased expression of LSZ protein in AMcaused by the stimulation of SiO2dust,and can reduce the damage of SiO2on the membrane of alveolar macrophage and thus has the protective effect on the AM cell membrane.