Heparin-dependent aggregation of hen egg white lysozyme reveals two distinct mechanisms of amyloid fibrillation.

Heparin, a biopolymer possessing high negative charge density, is known to accelerate amyloid fibrillation by various proteins. Using hen egg white lysozyme, we studied the effects of heparin on protein aggregation at low pH, raised temperature, and applied ultrasonic irradiation, conditions under which amyloid fibrillation was promoted. Heparin exhibited complex bimodal concentration-dependent effects, either accelerating or inhibiting fibrillation at pH 2.0 and 60 °C. At concentrations lower than 20 µg/ml, heparin accelerated fibrillation through transient formation of hetero-oligomeric aggregates. Between 0.1 and 10 mg/ml, heparin rapidly induced amorphous heteroaggregation with little to no accompanying fibril formation. Above 10 mg/ml, heparin again induced fibrillation after a long lag time preceded by oligomeric aggregate formation. Compared with studies performed using monovalent and divalent anions, the results suggest two distinct mechanisms of heparin-induced fibrillation. At low heparin concentrations, initial hen egg white lysozyme cluster formation and subsequent fibrillation is promoted by counter ion binding and screening of repulsive charges. At high heparin concentrations, fibrillation is caused by a combination of salting out and macromolecular crowding effects probably independent of protein net charge. Both fibrillation mechanisms compete against amorphous aggregation, producing a complex heparin concentration-dependent phase diagram. Moreover, the results suggest an active role for amorphous oligomeric aggregates in triggering fibrillation, whereby breakdown of supersaturation takes place through heterogeneous nucleation of amyloid on amorphous aggregates.

Effect of Technically Relevant X-Ray Doses on the Structure and Function of Alcohol Dehydrogenase and Hen Egg-White Lysozyme.

PURPOSE: The effect of different irradiation doses on the structure and activity of lyophilized powders of Hen Egg-White Lysozyme (HEWL) and alcohol dehydrogenase (ADH) was investigated using these substances as models for robust and sensitive proteins, respectively. Three doses were selected to cover the ranges of radio-sterilization (25kGy), treatment of blood products (25Gy) and annual background radiation dose (approximately 2mGy). The results offer an initial screening of different irradiation doses and support the development of X-ray imaging methods as non-destructive process analytical technology (PAT) tools for detecting the visible particulate matters in such products. METHODS: HEWL and ADH were exposed to X-rays in the solid state. The effect of irradiation was determined directly after irradiation and after storage. Structural changes and degradation were investigated using SAXS, SDS-PAGE and HPLC-MS. Protein functionality was assessed via activity assays. RESULTS: Lower irradiation doses of 25Gy and 2mGy had no significant impact on the structure and enzyme activity. The dose of 25kGy caused a significant decrease in the enzyme activity and structural changes immediately after irradiation of ADH and after storage of irradiated HEWL at -20°C. CONCLUSION: The results emphasize the importance of careful selection of radiation doses for development of X-ray imaging methods as PAT tools inspection of solid biopharmaceutical products.

[The role of human lysozyme-like protein 4 in fertilization and its enzymatic properties].

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.

[The influence of trans-cranial electrical stimulation on the cells of the macrophagal system of the women presenting with the climacteric syndrome]. / Vliianie transkranial'noi élektrostimuliatsii na kletki makrofagal'noi sistemy u zhenshchin s klimaktericheskim sindromom.

BACKGROUND: The search for the harmless and effective methods for the drug-free correction of various vegetovascular manifestations associated with the climacteric syndrome (CS) is currently a serious challenge facing modern medicine. Dysfunction of the ovaries during the perimenopause is characterized by the impaired production of sex steroids. The estrogens and progesterone, in their turn, are able to interact with macrophages via the specific receptors of steroid hormones localized on the surface of these cells. In this context, the trans-cranial electrical stimulation (TES) technique is of great interest due to its strong influence on the functional state of the central nervous system, subcortical brain structures, the vegetative and hormonal balance. The immunotropic effects of TES therapy described in the literature provide a basis for anticipating its beneficial action on the functional activity of monocytes/macrophages in the women presenting with the climacteric syndrome. AIM: The objective of the present study was to evaluate the influence of trans-cranial electrical stimulation on the state and stability of lysosomal membranes of blood monocytes and peritoneal macrophages, the secretory and synthetic activity of these cells in the women exhibiting the vegetovascular manifestations of the climacteric syndrome. MATERIAL AND METHODS: The influence of the transcranial electrical stimulation (TES) technique on the stability of lysosomal membranes of blood monocytes and peritoneal macrophages, the secretory and synthetic activity of these cells was studied in 23 women at the age from 45 to 52 years presenting with vegetovascular manifestations of the climacteric syndrome. The control group was comprised of 16 healthy women of the same age. The state of blood monocytes and peritoneal macrophages was evaluated before and after the course of TES therapy. To determine stability of lysosomal membranes of blood monocytes and peritoneal macrophages and calculate the stability index (PSI), the isolated cells were cultured in a medium 199 supplemented by sterile 0.5% L-glutamine and 2.5% mixed human serum previously heated at 560 °C for 30 min and at 37 °C for 12-15 hours. The micro-method was employed to determine secreted lysozyme (Lsec) as well as total lysozyme (L total=L secreted plus L intracellular) following 4-6-fold freeze-thawing of the cultured cells. The data obtained on the levels of secreted and total lysozymes were used to calculate PSI by the formula: PSI=Lsec/Ltotal´100%. The increase in PSI above the optimal value (53- 58%) was regarded as giving evidence of labilization of the lysosomal membranes and the decrease of this parameter as the indicator of membrane stabilization. Based on the difference between the total lysozyme levels before and after cultivation, the amount of newly synthesized lysozyme (L int) was determined. RESULTS: The results of the present study give evidence that the women with climacteric syndrome experience labilization of the blood monocytes and peritoneal macrophages; their lysozyme secretion increases while its synthesis decreases. In other words, the therapeutic application of the transcranial electrical stimulation technique contributed to the disappearance or reduction of 'hot flashes' and the change of these features to the level characteristic of the healthy women. CONCLUSIONS: The data obtained extend our knowledge about the mechanisms underlying the action of TES therapy on the macrophagic cells; moreover, they allow to draw the conclusion that the course of medication with the application this technique not only decreases the intensity of manifestations of the climacteric syndrome but also corrects the function of the immune system during the perimenopause.

[Lysozyme--occurrence in nature, biological properties and possible applications]. / Lizozym--wystepowanie w przyrodzie, wlasciwosci biologiczne i mozliwosci zastosowan.

Lysozyme (LZ, muramidase, N-acetylmuramylhydrolase) is a protein occuring in animals, plants, bacteria and viruses. It can be found e.g. in granules of neutrophils, macrophages and in serum, saliva, milk, honey and hen egg white. The enzyme hydrolyzes the ß-1,4 glycosidic bonds between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) of cell wall peptidoglycan (PG) in Gram-positive and Gram-negative bacteria. In the animal kingdom, three muramidase types have been identified: the c-type (chicken type), the g-type (goose-type) and the i-type (invertebrates). The c-type LZ from hen egg white is a model for the study of protein structure and function. Muramidase shows bactericidal activity mainly against Gram-positive bacteria. Cytolytic activity against cells of Gram-negative bacteria has not been proved. Bacterial cells have developed defense mechanisms that allow them to avoid the action of LZ. They are based e.g. on the production of enzyme inhibitors or modification of the PG. LZ is one of the most studied enzymes and yet not all aspects characterizing this protein are fully understood. One of the most important unresolved issues concerning the biological function of LZ is the role of muramidase in the bactericidal action of serum against Gram-negative bacteria. In order to clarify the function of LZ, the enzyme is e.g. removed from the serum by adsorption onto bentonite (montmorillonite, MMT). By using X-ray diffraction techniques it has been shown that MMT after contact with the serum is delaminated. The problems associated with folding of muramidase and LZ participation in the development of amyloidoses also await explanation.

Constraint Network Analysis (CNA): a Python software package for efficiently linking biomacromolecular structure, flexibility, (thermo-)stability, and function.

For deriving maximal advantage from information on biomacromolecular flexibility and rigidity, results from rigidity analyses must be linked to biologically relevant characteristics of a structure. Here, we describe the Python-based software package Constraint Network Analysis (CNA) developed for this task. CNA functions as a front- and backend to the graph-based rigidity analysis software FIRST. CNA goes beyond the mere identification of flexible and rigid regions in a biomacromolecule in that it (I) provides a refined modeling of thermal unfolding simulations that also considers the temperature-dependence of hydrophobic tethers, (II) allows performing rigidity analyses on ensembles of network topologies, either generated from structural ensembles or by using the concept of fuzzy noncovalent constraints, and (III) computes a set of global and local indices for quantifying biomacromolecular stability. This leads to more robust results from rigidity analyses and extends the application domain of rigidity analyses in that phase transition points ("melting points") and unfolding nuclei ("structural weak spots") are determined automatically. Furthermore, CNA robustly handles small-molecule ligands in general. Such advancements are important for applying rigidity analysis to data-driven protein engineering and for estimating the influence of ligand molecules on biomacromolecular stability. CNA maintains the efficiency of FIRST such that the analysis of a single protein structure takes a few seconds for systems of several hundred residues on a single core. These features make CNA an interesting tool for linking biomacromolecular structure, flexibility, (thermo-)stability, and function. CNA is available from http://cpclab.uni-duesseldorf.de/software for nonprofit organizations.

Parity-dependent association between TNF-α and LTF gene polymorphisms and clinical mastitis in dairy cattle.

BACKGROUND: One major problem in dairy cattle husbandry is the prevalence of udder infections. In today's breeding programmes, top priority is being given to making animal evaluation more cost-effective and reliable and less time-consuming. We proposed tumor necrosis factor α (TNF-α), lactoferrin (LTF) and macrophage-expressed lysozyme (mLYZ) genes as potential DNA markers in the improvement of immunity to mastitis.This study included 588 Polish Holstein-Friesian cows kept on one farm located in the north-western region of Poland. All clinical cases of mastitis in the herd under study were recorded by a qualified veterinarian employed by the farm. The following indicators were applied to determine udder immunity to mastitis in the cows under study: morbidity rate (MR), duration of mastitis (DM) and extent of mastitis (EM). TNF-α, mLYZ and LTF genotypes were identified by real-time PCR method, using SimpleProbe technology. Due to the very low frequency of mLYZ allele T, the gene was excluded from further analysis.A statistical analysis of associations between TNF-α and LTF genes and immunity to mastitis were performed using three models: 1) a parity-averaged model including only additive effects of the genes; 2) a parity-averaged model including both additive and epistatic effects of the genes; and 3) a parity-specific model including only additive effects of the genes. RESULTS: With the first and second models it was revealed that the genes effects on the applied indicators of immunity to mastitis were non-significant whereas with the third one the effects were found to be statistically significant. Particularly noteworthy was the finding that the effects of TNF-α and LTF varied depending on age (parity). The alleles which were linked to high immunity to mastitis in lower parities appeared to be less favourable in higher parities. CONCLUSIONS: These interactions might be related to inflamm-ageing, that is an increased susceptibility to infection due to immune system deregulation that progresses with age. Such pattern of interactions makes it impossible to use the genes in question in marker-assisted selection aimed at reducing heritable susceptibility to mastitis. This is because the immune mechanisms behind resistance to infections proved to be too complex.

Correlation between the activity of digestive enzymes and nonself recognition in the gut of Eisenia andrei earthworms.

Earthworms Eisenia andrei, similarly to other invertebrates, rely on innate defense mechanisms based on the capability to recognize and respond to nonself. Here, we show a correlation between the expression of CCF, a crucial pattern-recognition receptor, and lysozyme, with enzyme activities in the gut of E. andrei earthworms following a microbial challenge. These data suggest that enzyme activities important for the release and recognition of molecular patterns by pattern-recognition molecules, as well as enzymes involved in effector pathways, are modulated during the microbial challenge. In particular, protease, laminarinase, and glucosaminidase activities were increased in parallel to up-regulated CCF and lysozyme expression.

Bacillus subtilis CwlP of the SP-{beta} prophage has two novel peptidoglycan hydrolase domains, muramidase and cross-linkage digesting DD-endopeptidase.

For bacteria and bacteriophages, cell wall digestion by hydrolases is a very important event. We investigated one of the proteins involved in cell wall digestion, the yomI gene product (renamed CwlP). The gene is located in the SP-ß prophage region of the Bacillus subtilis chromosome. Inspection of the Pfam database indicates that CwlP contains soluble lytic transglycosylase (SLT) and peptidase M23 domains, which are similar to Escherichia coli lytic transglycosylase Slt70, and the Staphylococcus aureus Gly-Gly endopeptidase LytM, respectively. The SLT domain of CwlP exhibits hydrolytic activity toward the B. subtilis cell wall; however, reverse phase (RP)-HPLC and mass spectrometry revealed that the CwlP-SLT domain has only muramidase activity. In addition, the peptidase M23 domain of CwlP exhibited hydrolytic activity and could cleave d-Ala-diaminopimelic acid cross-linkage, a property associated with dd-endopeptidases. Remarkably, the M23 domain of CwlP possessed a unique Zn(2+)-independent endopeptidase activity; this contrasts with all other characterized M23 peptidases (and enzymes similar to CwlP), which are Zn(2+) dependent. Both domains of CwlP could hydrolyze the peptidoglycan and cell wall of B. subtilis. However, the M23 domain digested neither the peptidoglycans nor the cell walls of S. aureus or Streptococcus thermophilus. The effect of defined point mutations in conserved amino acid residues of CwlP is also determined.

Antibacterial properties of chicken intestinal phospholipase A2.

BACKGROUND: The presence of chicken group-IIA PLA2 (ChPLA2-IIA) in the intestinal secretion suggests that this enzyme plays an important role in systemic bactericidal defence. We have analyzed the bactericidal activity of purified ChPLA2-IIA, on several gram-positive and gram-negative bacteria by using the diffusion well and dilution methods. RESULTS: ChPLA2-IIA displays potent bactericidal activity against gram-positive bacteria but lacks bactericidal activity against gram negative ones. We have also demonstrated a synergic action of ChPLA2-IIA with lysozyme when added to the bacteria culture prior to ChPLA2-IIA. The bactericidal efficiency of ChPLA2-IIA was shown to be dependent upon the presence of calcium ions and then a correlation could be made to its hydrolytic activity of membrane phospholipids. Interestingly ChPLA2-IIA displays a higher dependence to Ca²+ ions than to Mg²+ ions. CONCLUSION: We conclude that the main physiological role of ChPLA2-IIA could be the defence of the intestine against bacterial invasions.

Lyzl4, a novel mouse sperm-related protein, is involved in fertilization.

The role of Chicken-type (c-type) lysozyme, a prototype lysozyme, in immunity has been characterized in many organisms. In this study, we cloned a novel c-type lysozyme-like gene, Lyzl4, which was located on mouse chromosome 9F4 and encoded 145 amino acids with a putative signal peptide and a protease cleavage site. The mature recombinant Lyzl4 protein expressed in yeast did not show the bacteriolytic activity. Sequence alignment analysis demonstrated that 3 of the 20 invariant residues in c-type lysozymes were changed in Lyzl4. One of the 'changed' amino acids (D52G) is located in the catalytic domain. Lyzl4 mRNA was selectively expressed in testis and epididymis in adult mice, with varying expression level across different developmental stages. High level of Lyzl4 protein was found on the spermatozoa of acrosomal region and principal piece of tail. Immuno-neutralization of Lyzl4 protein in spermatozoa with its specific antibody significantly decreased in vitro fertilization percentage in a dose-dependent manner, suggesting that Lyzl4 might be important for fertilization.

Rheological properties of hyaluronic acid and its effects on salivary enzymes and candida.

OBJECTIVE: To investigate the viscosity and wettability of hyaluronic acid (HA), its effects on lysozyme and peroxidase activities, and its candidacidal activity. MATERIALS AND METHODS: Human whole saliva, HA, hen egg-white lysozyme (HEWL), and bovine lactoperoxidase (bLPO) were used. Viscosity was measured with a cone-and-plate digital viscometer, while wettability was determined by measuring the contact angle. Lysozyme activity was determined by the turbidimetric method. Peroxidase activity was determined with NbsSCN assay. Candidacidal activity was determined by comparing colony forming units. RESULTS: The viscosity of HA solutions was proportional to its concentration, with 0.05 mg ml(-1) of HA in distilled water or 0.5 mg ml(-1) in simulated salivary buffer displaying similar viscosity values to stimulated whole saliva. The contact angle of HA solutions showed no significant differences according to the tested materials and tested HA concentrations. Contact angles of HA solutions on acrylic resin were higher than those of human saliva. HA did not affect lysozyme or peroxidase activities of whole saliva as well as HEWL or bLPO activities. HA also showed no candidacidal activity. CONCLUSIONS: The viscoelastic properties of HA compared with human saliva were objectively confirmed, indicating a vital role for HA in the development of effective salivary substitutes.

Yak (Bos grunniens) stomach lysozyme: molecular cloning, expression and its antibacterial activities.

The cDNA coding for stomach lysozyme in yak was cloned. The cloned cDNA contains a 432 bp open reading frame and encodes 143 amino acids (16.24 KDa) with a signal peptide of 18 amino acids. Further analysis revealed that its amino acid sequence shares many common properties with cow milk lysozyme. Expression of this gene was also detected in mammary gland tissue by RT-PCR. Phylogenetic relationships among yak stomach lysozyme and 8 cow lysozymes indicated that the yak enzyme is more closely related to both cow milk lysozyme and the pseudogene PsiNS4 than cow stomach lysozyme. Recombinant yak lysozyme purified by Ni(2+)-column showed a molecular weight of 33.78 kDa and exhibited lytic activity against Staphylococcus aureus, providing evidence of its antibacterial activities.

Cellular membrane disruption by amyloid fibrils involved intermolecular disulfide cross-linking.

Accumulating evidence has strongly suggested that amyloid fibrils of protein or peptide are cytotoxic. Fibrillar species appear to lead to disruption of cell membrane structures and thereby cause cell death. In this study, human erythrocytes were used as an in vitro model to examine the disruptive effect of lysozyme fibrils on the plasma membrane. Both the protofibrils and mature fibrils induced hemolysis and aggregation of erythrocytes. Treating ghost membranes with the fibrils resulted in aggregation of membrane proteins through intermolecular disulfide cross-linking. LC-ESI-MS/MS and Western blotting analysis showed that lysozyme fragments were incorporated into the aggregates of ghost membrane proteins, which suggested that thio-disulfide exchange among lysozyme and membrane proteins was triggered when the fibrils interacted with erythrocyte membranes. Metal-ion chelators, radical scavengers, and antioxidants had no effect on the amyloid-induced disulfide cross-linking. The exposure of interior hydrophobic residues and the increased level of solvent-accessible disulfides in the lysozyme fibrils are thought to be involved in membrane disruption. These results may unveil a novel pathway for the cytotoxicity of amyloid fibrils.

Control of enzyme activity by an engineered disulfide bond.

A novel approach to the control of enzyme catalysis is presented in which a disulfide bond engineered into the active-site cleft of bacteriophage T4 lysozyme is capable of switching the activity on and off. Two cysteines (Thr21----Cys and Thr142----Cys) were introduced by oligonucleotide-directed mutagenesis into the active-site cleft. These cysteines spontaneously formed a disulfide bond under oxidative conditions in vitro, and the catalytic activity of the oxidized (cross-linked) T4 lysozyme was completely lost. On exposure to reducing agent, however, the disulfide bond was rapidly broken, and the reduced (non-cross-linked) lysozyme was restored to full activity. Thus an enzyme has been engineered such that redox potential can be used to control catalytic activity.

The defensive role of lysozyme in human gingiva in inflammatory periodontal disease.

BACKGROUND AND OBJECTIVE: The presence of lysozyme in human gingiva has not previously been demonstrated. In this study, we looked for evidence for the potential role of lysozyme as a protector of gingival elastic fibres. The objective of this study was also to determine the ex vivo susceptibility to hydrolysis of gingival elastic fibres from patients with or without periodontal disease by human leukocyte elastase and by human cathepsin G. MATERIALS AND METHODS: Using gingival tissue sections from eight control, 10 gingivitis and 10 periodontitis patients, we evaluated the area fraction occupied by gingival elastic fibres (after selective staining) by the use of automated image analysis. In the ex vivo experiments, serial tissue sections from four control, four gingivitis, four young periodontitis and four aged periodontitis patients were submitted to the action of human leukocyte elastase and cathepsin G, after which enzymatic activities were determined by image analysis. Indirect immunodetection of lysozyme was also done on tissue sections for all patients included in this study. RESULTS: Large variations of the area fraction occupied by elastic fibres were observed in human gingiva from young and aged patients with and without periodontal disease. In control and gingivitis patients, leukocyte elastase and cathepsin G had high comparable elastin solubilizing activities. With young and aged periodontitis patients, the two serine proteinases had weak elastin solubilizing activities. Lysozyme appeared to be present at the periphery of gingival elastic fibres in periodontitis patients. CONCLUSION: Lysozyme can be considered an important natural protector of elastic fibres in pathological gingiva.

Crystallization and preliminary X-ray analysis of a C-terminal fragment of FlgJ, a putative flagellar rod cap protein from Salmonella.

The formation of the bacterial flagellar axial structure, including the filament, the hook and the rod, requires the attachment of a cap complex to the distal end of the growing structure. Because the rod penetrates the peptidoglycan (PG) layer, the rod cap complex is thought to have PG-hydrolyzing activity. FlgJ is a putative rod cap protein whose C-terminal region shows sequence similarity to known muramidases. In this study, FlgJ(120-316), a C-terminal fragment of FlgJ which contains the muramidase region, was overproduced, purified and crystallized. Crystals were obtained by the sitting-drop vapour-diffusion technique using PEG 3350 as a crystallizing agent and belonged to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 38.8, b = 43.9, c = 108.5 A. Anomalous difference Patterson maps calculated from the diffraction data set of a selenomethionine-labelled crystal showed significant peaks in the Harker sections, indicating that the data were suitable for structure determination.

The up-regulation of two identified wound healing specific proteins-HSP70 and lysozyme in regenerated Eisenia fetida through transcriptome analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Disposed earthworm has been used to treat various common ailments including burns, arthritis, itching, and inflammation for thousands of years in China. As their remarkable ability to fully regenerate in a scar-free manner, regenerated tissue homogenate of amputated Eisenia fetida (E. fetida) have been considered as an excellent wound repair therapy in our previous study. We have demonstrated that regenerated earthworm (G-90') can perform higher wound repair ability to non-regeneration tissue (G-90) through significant promotion of cutaneous wound repair in mice after their administration into wound beds. OBJECTIVE: In the present study, we aimed to reveal the mechanism of G-90' and to explore a potential wound healing accelerated strategy. METHODS AND RESULTS: Two functional proteins- HSP70 and lysozyme in G-90' were confirmed by cross-identification of LC-MS/MS and transcriptome analyses. Followed with semi-quantitative PCR and western blot, their expression were validated to up-regulate in 3-day regenerated tissues (G-90'). CONCLUSION: This study implies the therapeutic potency of G-90' for wound recovery and provides a new strategy to assess other natural materials targeting wound healing with the tail-amputated E .fetida as a model organism.

Lysozyme transgenic goats' milk influences gastrointestinal morphology in young pigs.

Transgenesis provides a method of expressing novel proteins in milk to increase the functional benefits of milk consumption. Transgenic goats expressing human lysozyme (hLZ) at 67% of the concentration in human breast milk were produced, thereby enhancing the antimicrobial properties of goats' milk. The objective of this study was to investigate the impact of pasteurized milk containing hLZ on growth, the intestinal epithelium, and an enteropathogenic Escherichia coli (EPEC) infection in young weaned pigs. Pigs were placed into 4 groups and fed a diet of solid food and either control (nontransgenic) goats' milk or milk from hLZ-transgenic goats. Growth was assessed by weight gain. Nonchallenged pigs were necropsied after 6 wk, whereas the remaining pigs were necropsied at 7 wk following bacterial challenge. We determined the numbers of total coliforms and E. coli and examined small intestinal histology for all pigs. Complete blood counts were also determined pre- and postchallenge. Challenged pigs receiving hLZ milk had fewer total coliforms (P = 0.029) and E. coli (P = 0.030) in the ileum than controls. hLZ-fed pigs also had a greater duodenal villi width (P = 0.029) than controls. Additionally, nonchallenged hLZ-fed pigs had fewer intraepithelial lymphocytes per micron of villi height (P = 0.020) than nonchallenged controls. These results indicate that the consumption of pasteurized hLZ goats' milk has the potential to improve gastrointestinal health and is protective against an EPEC in young weaned pigs. These same benefits may occur in young children if they were to consume milk from hLZ-transgenic goats.

Molecular cloning of an invertebrate goose-type lysozyme gene from Chlamys farreri, and lytic activity of the recombinant protein.

Lysozyme is a widely distributed hydrolase possessing lytic activity against bacterial peptidoglycan, which enables it to protect the host against pathogenic infection. In the present study, the cDNA of an invertebrate goose-type lysozyme (designated CFLysG) was cloned from Zhikong scallop Chlamys farreri by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of CFLysG consisted of 829 nucleotides with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame (ORF) of 603 bp encoding a polypeptide of 200 amino acid residues with a predicted molecular weight of 21.92 kDa and theoretical isoelectric point of 7.76. The high similarity of CFLysG with goose-type (g-type) lysozymes in vertebrate indicated that CFLysG should be an invertebrate counterpart of g-type lysozyme family, which suggested that the origin of g-type lysozyme preceded the emergence of urochordates and even preceded the emergence of deuterostomes. Similar to most g-type lysozymes, CFLysG possessed all conserved features critical for the fundamental structure and function of g-type lysozymes, such as three catalytic residues (Glu 82, Asp 97, Asp 108). By Northern blot analysis, mRNA transcript of CFLysG was found to be most abundantly expressed in the tissues of gills, hepatopancreas and gonad, weakly expressed in the tissues of haemocytes and mantle, while undetectable in the adductor muscle. These results suggested that CFLysG could possess combined features of both the immune and digestive adaptive lysozymes. To gain insight into the in vitro lytic activities of CFLysG, the mature peptide coding region was cloned into Pichia pastoris for heterogeneous expression. Recombinant CFLysG showed inhibitive effect on the growth of both Gram-positive and Gram-negative bacteria with more potent activities against Gram-positive bacteria, which indicated the involvement of CFLysG in the innate immunity of C. farreri.