Turbidimetric determination of lysozyme with Micrococcus lysodeikticus cells: Reexamination of reaction conditions

Mörsky, Pertti

doi:10.1016/0003-2697(83)90347-0

Cited by 94 publications

(35 citation statements)

References 24 publications

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“…Lysozyme was measured as described previously [14], but on microtitration trays and with a Multiskan MS photometer (Labsystems, Helsinki, Finland). This method is a modification of the turbidimetric method described by Mörsky [20]. The analysis of NAGase was carried out by a method developed for microtitration plate fluorometry [21].…”

Section: Laboratory Analysesmentioning

confidence: 99%

Effect of frozen semen on the uterus of mares with pathological uterine changes

2004

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-Pregnancy rates after frozen semen inseminations (AI), particularly in older and problem mares, are lower than after fresh semen AI. Uterine contractility and the inflammatory reaction after frozen semen insemination were studied in two groups of mares: the abnormal group comprised of 6 old barren mares categorized in biopsy category IIB or III, and the control group including 6 reproductively normal young maiden mares in biopsy category I or IIA. All 12 mares were inseminated in the first cycle with 2 mL of phosphate-buffered saline (PBS) and in their second cycle with 2 mL of frozen semen containing 800 × 10 6 spermatozoa. Before and 1, 2, 4, 8, and 20 to 24 h after this treatment, all mares were examined by ultrasonography for intrauterine fluid accumulations (IUFA). The examinations were videotaped to count the number of uterine contractions later. Uterine fluid was obtained by tampon before treatment, and by the tampon method followed by uterine lavage after the last examination. Fluids were cultured bacteriologically, and polymorphonuclear leukocytes (PMN) were counted. Trypsin-inhibitor capacity (TIC), lysozyme concentration, and β-glucuronidase (BGase) and N-acetyl-β-D-glucosaminidase (NAGase) activities were determined in frozen-thawed tampon and lavage fluids. Both treatments induced significant neutrophilia in the uterine lumen. Although PMN concentrations were numerically higher after frozen semen AI than after PBStreatment, the difference was not significant. There was not any difference between the mare groups either. The amount of IUFA differed only in the normal group between frozen semen AI and PBS treatment, and between 0-and 24-h samples for frozen semen AI. Although abnormal mares showed consistently more fluid than normal mares, this difference was not significant. Uterine contractions and enzyme concentrations between groups did not differ. None of the variables showed significant differences between the normal and abnormal mares in their reaction to frozen semen AI.horse / insemination / frozen semen / uterine fluid / inflammation / leukocyte

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Section: Laboratory Analysesmentioning

confidence: 99%

Effect of frozen semen on the uterus of mares with pathological uterine changes

2004

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“…The intra-and interassay CV was 2% for NAGase and 5% for B-Gase. A modification of the turbidimetric method described by Mörsky [24] was used to determine lysozyme concentrations [27]. The detection limit of the assay was…”

Section: Biochemical Analysis Of Uterine Lavage Fluidsmentioning

confidence: 99%

Relationship between embryo recovery rate and uterine lavage fluid composition in postpartum mares

Reilas¹,

Huhtinen²,

Oksanen

et al. 2000

Reprod. Nutr. Dev.

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-The aim of the study was to determine whether neutrophil numbers (PMN), trypsininhibitor capacity (TIC), lysozyme, N-acetyl-β-D-glucosaminidase (NAGase), β-glucuronidase (B-Gase), total protein, and plasmin in uterine lavage fluid of postpartum (p.p.) mares, either at the time of foal heat insemination or around the time of arrival of the embryo in the uterus, could be used in predicting conception. Fifteen mares were inseminated within 13 h after the first p.p. ovulation. Uterine lavage fluids were successfully collected from 9 out of 12 mares before insemination and from all 15 mares before embryo recovery 7 to 8 days after insemination. The embryo recovery rate was 53% (8/15). Prior to insemination, PMN, TIC and lysozyme levels were elevated in 3/4 mares not producing embryos. However, only 1/5, 1/5 and 0/5 mares producing embryos had elevated levels of PMN, TIC, and lysozyme, respectively. None of the parameters was significantly different in mares with or without embryos, but lysozyme was the closest to significance (p = 0.07). In both groups of mares, activities of NAGase (p < 0.01) and B-Gase (p < 0.05) were significantly higher in dioestrus than immediately after ovulation. At embryo recovery, NAGase was higher in mares not producing embryos (p < 0.05). The results suggest that a long-lasting inflammation is the best explanation for low pregnancy rates during the first p.p. oestrus. Further research is needed to establish whether lysozyme, or possibly TIC, could be used in predicting conception at foal heat. mare / foal heat / uterine fluid / inflammation / embryo recovery Résumé -Relations entre le taux de récupération des embryons et la composition du liquide de lavage de l'utérus chez la jument lors du post-partum. Le taux de récupération des embryons a été relié à la composition du fluide utérin chez 15 juments qui ont été inséminées dans les 13 h suivant la première ovulation post-partum. Les liquides de lavage ont été récupérés avant l'insémination chez 9 juments sur 12 et chez 15 sur 15 avant la collecte des embryons 7 à 8 jours après. Les mesures ont porté sur le nombre des neutrophiles, les teneurs en plasmine et en protéines totales, la concentration en lysozyme, l'activité de l'inhibiteur de la trypsine (TIC), les activités de la N-acétyl-D-glucosaminidase (N-Gase), et de la β-glucoronidase (B-Gase). Le taux de récupération des Reprod. Nutr. Dev. 40 (2000) 383-391 383

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“…The r-hLY assay was carried out using a method adapted from Morsky (13). An enzyme solution of 300 µl was mixed with 2.7 ml Micrococcus lysodeikticus WX-2008 cell suspension (OD 450 approximately 0.7) in 50 mM phosphate buffer, pH 6.2.…”

Section: Construction Of the Recombinant Expression Plasmidmentioning

confidence: 99%

Cloning and functional expression of a human lysozyme gene (hly) from human leukocytes in Pichia pastoris

Wei

Tang

et al. 2012

Mol Med Report

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Abstract. hly is a cDNA gene derived from human leukocytes that encodes a mature human lysozyme (abbreviated to hLY). The aim of the present study was to determine the effect of cloned hly on recombinant hLY (r-hLY) activity under optimized conditions. hly was amplified by RT-PCR and ligated into the pPIC9K plasmid. The cloned cDNA (hly) was 393 bp in length, encoding a 130 amino acid hLY with a calculated molecular mass of 14,698 Da. The recombinant expression plasmid, designated as pPIC9K-hly, was linearized with SacⅠ and transformed into Pichia pastoris GS115 (his4, Mut + ) by electroporation. The integration of hly into the P. pastoris genome was confirmed by PCR analysis using 5'-AOX1 and 3'-AOX1 primers. Yeast extract peptone dextrose (YPD) plates containing different concentrations of geneticin (G418) were used for the screening of P. pastoris transformants (His + , Mut + ) with multiple hly copies. One transformant resistant to 4.0 mg/ ml of G418, designated as P. pastoris GShLY4-6, expressing the highest r-hLY activity was selected by the shake-flask test, and used for the optimization of expression conditions. When the P. pastoris GShLY4-6 was induced under optimized conditions, the expressed r-hLY activity was up to 533 U/ml, which was 1.52 times as high as that (351 U/ml) expressed using the standard protocol. SDS-PAGE assay demonstrated that the r-hLY with an apparent molecular mass of approximately 14.7 kDa was extracellularly expressed in P. pastoris. In conclusion, r-hLY increased following the cloning of hly and the optimized conditions as compared to standard protocol. IntroductionHuman lysozyme (EC 3.2.1.17) is a bacteriolytic enzyme that is widely distributed in a variety of tissues and body fluids (1). It hydrolyzes preferentially the β-1,4 glycosidic linkages between the N-acetylmuramic acid and N-acetylglucosamine residues that occur in the peptidoglycan cell wall structures of certain microorganisms, particularly those of Gram-positive bacteria, and therefore appears to have a role in host defense (2). The hLY comprises 130 amino acid residues and belongs to the group of lysozymes called c-type lysozymes (3). Due to its bacteriolytic activity, it is considered to be useful for medical and food uses. There is a reemergence of uses of lysozyme in various clinical trials. The addition of lysozyme to baby formulas was proposed (4), and lysozyme had also been shown to inhibit the growth of HIV-1 in vitro. Of note, the anti-HIV activity of lysozyme is independent of its muramidase activity (5). In addition, findings of previous studies have shown that it is effective in inactivating spores of Bacillus cereus in cheese by causing high hydrostatic pressure following the addition of lysozyme (6).Currently, hLY is mainly extracted from human milk and placenta, which is restricted by many factors, such as the lack of raw materials and high cost of purification. Therefore, the hen egg-white lysozyme is the most readily available form of commercial lysozyme, since it is relatively inexpensive compared...

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Turbidimetric determination of lysozyme with Micrococcus lysodeikticus cells: Reexamination of reaction conditions

Cited by 94 publications

References 24 publications

Effect of frozen semen on the uterus of mares with pathological uterine changes

Effect of frozen semen on the uterus of mares with pathological uterine changes

Relationship between embryo recovery rate and uterine lavage fluid composition in postpartum mares

Cloning and functional expression of a human lysozyme gene (hly) from human leukocytes in Pichia pastoris

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