Lysozyme Gene Research Articles

Molecular Characterization of a Lysozyme Gene and Its Altered Expression Profile in Crowded Beet Webworm (Loxostege sticticalis).

There is growing evidence that insects living in high-density populations exhibit an increase in immune function to counter a higher risk of disease. This phenomenon, known as density-dependent prophylaxis, has been experimentally tested in a number of insect species. Although density-dependent prophylaxis is especially prevalent in insects exhibiting density-dependent phase polyphenism, the molecular mechanism remains unclear. Our previous study demonstrated that the antibacterial activity of lysozyme is important for this process in the beet webworm Loxostege sticticalis. In this study, a lysozyme cDNA from L. sticticalis was cloned and characterized. The full-length cDNA is 1078 bp long and contains an open reading frame of 426 bp that encodes 142 amino acids. The deduced protein possesses structural characteristics of a typical c-type lysozyme and clusters with c-type lysozymes from other Lepidoptera. LsLysozyme was found to be expressed throughout all developmental stages, showing the highest level in pupae. LsLysozyme was also highly expressed in the midgut and fat body. Elevated LsLysozyme expression was observed in L. sticticalis larvae infected by Beauveria bassiana and in larvae reared under crowding conditions. In addition, the expression level of LsLysozyme in infected larvae reared at a density of 10 larvae per jar was significantly higher compared to those reared at a density of l or 30 larvae per jar. These results suggest that larval crowding affects the gene expression profile of this lysozyme. This study provides additional insight into the expression of an immune-associated lysozyme gene and helps us to better understand the immune response of L. sticticalis under crowding conditions.

The Invertebrate Lysozyme Effector ILYS-3 Is Systemically Activated in Response to Danger Signals and Confers Antimicrobial Protection in C. elegans.

Little is known about the relative contributions and importance of antibacterial effectors in the nematode C. elegans, despite extensive work on the innate immune responses in this organism. We report an investigation of the expression, function and regulation of the six ilys (invertebrate-type lysozyme) genes of C. elegans. These genes exhibited a surprising variety of tissue-specific expression patterns and responses to starvation or bacterial infection. The most strongly expressed, ilys-3, was investigated in detail. ILYS-3 protein was expressed constitutively in the pharynx and coelomocytes, and dynamically in the intestine. Analysis of mutants showed that ILYS-3 was required for pharyngeal grinding (disruption of bacterial cells) during normal growth and consequently it contributes to longevity, as well as being protective against bacterial pathogens. Both starvation and challenge with Gram-positive pathogens resulted in ERK-MAPK-dependent up-regulation of ilys-3 in the intestine. The intestinal induction by pathogens, but not starvation, was found to be dependent on MPK-1 activity in the pharynx rather than in the intestine, demonstrating unexpected communication between these two tissues. The coelomocyte expression appeared to contribute little to normal growth or immunity. Recombinant ILYS-3 protein was found to exhibit appropriate lytic activity against Gram-positive cell wall material.

C-type lysozyme gene haplotypes in Rhode Island Red layer chickens are associated with hatchability and survival.

C-type lysozyme gene haplotypes in Rhode Island Red layer chickens are associated with hatchability and survival.

Gene duplications and the evolution of c-type lysozyme during adaptive radiation of East African cichlid fish

The adaptive radiation of African cichlids is a prime model system for studying vertebrate speciation. Cichlid species are distributed in lakes and rivers with various water conditions such as various pH values. The innate immune system in fish is particularly important because water contains a wide range of pathogenic microorganisms. To investigate the evolution of the host defense system in cichlids, we isolated the c-type lysozyme gene, which functions in the innate immune system of fish. Southern blot and sequence analyses showed that the lysozyme gene underwent several gene duplication events and evolved with amino acid replacements during the adaptive radiation of cichlids. The inferred 3D structure revealed that the amino acid substitutions were localized on the lysozyme surface. Moreover, more than half of the surface substitutions changed the charge of amino acid residues, suggesting changes in the optimum pH for enzymatic activity. In African cichlids, the lysozyme genes may have played and still play an important role in defense against pathogens.

Immune related transcriptional responses and performance of Litopenaeus vannamei post-larvae fed on dietary probiotic PrimaLac(®).

The present study investigated the effects of various levels of multi-strain probiotic on the immune related gene expression, digestive enzyme activity, growth performance, body chemical composition and survival of Litopenaeus vannamei post-larvae. After transferring post-larvae of L. vannamei to indoor conditions and subsequent acclimation to laboratory condition for 14 days, the shrimp were fed multi-strain probiotic at four different doses of 0, 0.25, 0.5 and 1.0gkg(-1) for eight weeks. Shrimp fed 0.5 and 1.0gkg(-1) probiotic PrimaLac(®) exhibited significantly (p<0.05) higher weight gain, specific growth rate, body crude protein as well as lower FCR compared to other groups. Feeding on 0.5 and 1gkg(-1) dietary multi-strain probiotic significantly (p<0.05) increased the level of body crude protein. Oral administration of 0.5 and 1.0gkg(-1) multi-strain probiotic significantly (p<0.05) decreased body crude lipid and body moisture respectively. 30 days after feeding, protease, amylase and lipase activity increased in groups fed 0.5 and 1.0gkg(-1) probiotic PrimaLac(®). However, on the 60th day, specific protease and amylase activity in all treatment groups were significantly higher than control group (p<0.05) but lipase activity was higher (p<0.05) in groups fed 0.5 and 1.0gkg(-1) multi-strain probiotic. Oral administration of 1.0gkg(-1) probiotic increased (p<0.05) the level of prophenoloxidase and g-type lysozyme gene on day 30th and 60th after treatment. On day 30th and 60th, penaeidin gene expression was significantly higher in all treatment groups compared to the control group (p<0.05). In general, findings of this study demonstrated that oral administration of 0.5 and 1.0gkg(-1) multi-strain probiotic improved the performance of the fish and increased the expression of immune related genes.

Campylobacter jejuni in Musca domestica: An examination of survival and transmission potential in light of the innate immune responses of the house flies.

The house fly, Musca domestica, has been implicated as a vector of Campylobacter spp., a major cause of human disease. Little is known whether house flies serve as biological amplifying hosts or mechanical vectors for Campylobacter jejuni. We investigated the period after C. jejuni had been ingested by house flies in which viable C. jejuni colonies could be isolated from whole bodies, the vomitus and the excreta of adult M. domestica and evaluated the activation of innate immune responses of house flies to ingested C. jejuni over time. C. jejuni could be cultured from infected houseflies soon after ingestion but no countable C. jejuni colonies were observed > 24 h postingestion. We detected viable C. jejuni in house fly vomitus and excreta up to 4 h after ingestion, but no viable bacteria were detected ≥ 8 h. Suppression subtractive hybridization identified pathogen-induced gene expression in the intestinal tracts of adult house flies 4-24 h after ingesting C. jejuni. We measured the expression of immune regulatory (thor, JNK, and spheroide) and effector (cecropin, diptericin, attacin, defensing, and lysozyme) genes in C. jejuni-infected and -uninfected house flies using quantitative real time PCR. Some house fly factor, or combination of factors, eliminates C. jejuni within 24 h postingestion. Because C. jejuni is not amplified within the body of the housefly, this insect likely serves as a mechanical vector rather than as a true biological, amplifying vector for C. jejuni, and adds to our understanding of insect-pathogen interactions.

Multiple specialised goose-type lysozymes potentially compensate for an exceptional lack of chicken-type lysozymes in Atlantic cod.

Previous analyses of the Atlantic cod genome showed unique combinations of lacking and expanded number of genes for the immune system. The present study examined lysozyme activity, lysozyme gene distribution and expression in cod. Enzymatic assays employing specific bacterial lysozyme inhibitors provided evidence for presence of g-type, but unexpectedly not for c-type lysozyme activity. Database homology searches failed to identify any c-type lysozyme gene in the cod genome or in expressed sequence tags from cod. In contrast, we identified four g-type lysozyme genes (LygF1a-d) constitutively expressed, although differentially, in all cod organs examined. The active site glutamate residue is replaced by alanine in LygF1a, thus making it enzymatic inactive, while LygF1d was found in two active site variants carrying alanine or glutamate, respectively. In vitro and in vivo infection by the intracellular bacterium Francisella noatunensis gave a significantly reduced LygF1a and b expression but increased expression of the LygF1c and d genes as did also the interferon gamma (IFNγ) cytokine. These results demonstrate a lack of c-type lysozyme that is unprecedented among vertebrates. Our results further indicate that serial gene duplications have produced multiple differentially regulated cod g-type lysozymes with specialised functions potentially compensating for the lack of c-type lysozymes.

The mucosal expression signatures of g-type lysozyme in turbot (Scophthalmus maximus) following bacterial challenge.

The mucosal surfaces constitute the first line of host defense against infection, and also serve as the dynamic interfaces that simultaneously mediate a diverse array of critical physiological processes, while in constantly contact with a wide range of pathogens. The lysozymes are considered as key components for innate immune response to pathogen infection with their strong antibacterial activities. But their activities in mucosal immune responses were always overlooked, especially for g-type lysozymes, whose expression patterns in mucosal tissues following bacterial challenge are still limited. Towards to this end, here, we characterized the g-type lysozymes, Lyg1 and Lyg2 in turbot, and determined their expression patterns in mucosal barriers following different bacterial infection. The phylogenetic analysis revealed the turbot g-type lysozyme genes showed the closest relationship to Cynoglossus semilaevis. The two lysozyme genes showed different expression patterns following challenge. Lyg2 was significantly up-regulated in mucosal tissues following Vibrio anguillarum and Streptococcus iniae challenge, while Lyg1 showed a general trend of down-regulation. The significant mucosal expression signatures of g-type lysozyme genes indicated their key roles to prevent pathogen attachment and entry in the first line of host defense system. Further functional studies should be carried out to better characterize the availability of utilization of g-type lysozyme to increase the disease resistance in the mucosal surfaces and facilitate the disease resistant breeding selection.

Cloning and expression analysis of c-type lysozyme gene in golden pompano, Trachinotus ovatus.

It is well known that lysozymes are key proteins to teleosts in the innate immune system and possess high bactericidal properties. In the present study, a c-type lysozyme gene (To-lysC) was cloned from golden pompano, Trachinotus ovatus. The To-lysC cDNA is composed of 743bp with a 36bp of 5'-UTR, 432bp open reading frame (ORF) and 275bp 3'-UTR, encoding a polypeptide of 144 amino acids (GenBank accession no: KT935522). Phylogenetic analysis revealed that To-lysC showed highest similarity to Perca flavescens lysC. Quantitative real-time PCR (qRT-PCR) analysis showed that To-lysC had relatively high expression level in the head kidney, gill and brain. After Vibrio harveyi infection, transcripts of To-lysC increased and reached its peak at 12h p.i. These results indicated that To-lysC may play an important role in innate immune response to bacteria.

Cloning and expression analysis of c-type and g-type lysozymes in yellow catfish (Pelteobagrus fulvidraco)

Lysozymes have important roles in innate immune system. Here, a c-type and a g-type lysozyme were identified from yellow catfish (Pelteobagrus fulvidraco). The deduced amino acid sequences of both lysozymes were conserved in catalytic sites and structural features as compared to their counterparts from other species. It was interesting that the g-type lysozyme possessed a signal peptide. The c-type and g-type lysozymes had the highest identity 89.4 and 76.2 % with that from channel catfish respectively. Phylogenetic analysis showed that the two lysozymes had a closely relationship with that from channel catfish and Astyanax mexicanus. Lysozymes from one order could form more than one clade in the phylogenetic tree, which indicated the gene duplications in evolution. Expression analysis with real time quantitative PCR revealed that the two lysozyme genes were constitutively expressed in all the tested tissues. The highest expression of c-type lysozyme was observed in liver, followed by spleen, head kidney, and trunk kidney, while the g-type lysozyme had highest expression in intestine, followed by spleen, head kidney, and trunk kidney. The mRNA levels of both genes were all up-regulated after challenging with Aeromonas hydrophila. However, there were differences in tissues and time points when the mRNA levels reached its peak between the two lysozymes. It indicated the diversity in regulation mechanisms and detailed functions among lysozymes. Taking together, these results will benefit the understanding of yellow catfish lysozymes.

Molecular cloning and expression of i-type lysozyme and phage-type like lysozyme gene from freshwater shellfish

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, A i-type lysozyme, designated as CpLYZ3, from Cristaria plicata and a phage-type lysozyme, designated as HcPLYZ, from Hyriopsis cumingii were identified by rapid amplification of cDNA ends and nested PCR. The complete CpLYZ3 cDNA sequence comprises of 968 bp, and 432 bp of open reading frame (ORF) coded for a 143 amino acids. The multiple sequence alignment analysis indicated that this gene had the two highly conserved i-type lysozyme activity sites (Glu61 and Ser72). The sequence homology analysis found that this gene had 27~87% the high homology with the other i-type lysozyme gene. The full-length cDNA sequence of HcPLYZ was 829 bp with the open reading frame of 468 bp, and encoded a polypeptide of 154 amino acids. The deduced amino sequence of HcPLYZ had no signal peptide and the mature peptide harbored three cysteine residues, and contained two highly conserved phage-type lysozyme activity sites (Glu20 and Asp29). Realtime quantitative-PCR analysis showed that the mRNA expression of CpLYZ3 was significant high in hepatopancreas, followed by the mantle and gill. The transcripts of HcPLYZ was constitutive expression in all detected tissues with a high level in gills. The expression levels of CpLYZ3 and HcPLYZ in hemocytes, hepatopancreas and gills of C. plicata or H. cumingii were increased significantly after the Aeromonas hydrophila and PGN challenged. These results indicated the involvement of CpLYZ3 and HcPLYZ in the innate immunity of freshwater shellfish. In order to further inquire into the functions of HcPLYZ, PET30-PLYZ prokaryotic expression system was constructed by double digestion, and prokaryotic  Corresponding author: Tel.: +86-0791-3969530; fax: +86-0791-3969530. E-mail address: cgwen@ncu.edu.cn (CG, Wen) expression plasmid was transformed into Escherichia coli (DE3). The recombinant protein was successfully expressed after IPTG induction, and was purified by using the native Ni affinity chromatography. Micrococcus luteus as substrate, the optimum pH of the recombinant HcPLYZ was 5.5, and the optimum temperature was 50 °C .

Keratinocyte growth factor supports pulmonary innate immune defense through maintenance of alveolar antimicrobial protein levels and macrophage function.

Keratinocyte growth factor (KGF) is an epithelial mitogen that has been reported to protect the lungs from a variety of toxic and infectious insults. In prior studies we found that recombinant human KGF accelerates clearance of bacteria from the murine lung by augmenting the function of alveolar macrophages (AM). In this study we tested the hypothesis that endogenous KGF plays a role in the maintenance of innate pulmonary defense against gram-negative bacterial infections. KGF-deficient mice exhibited delayed clearance of Escherichia coli from the lungs, attenuated phagocytosis by AM, and decreased antimicrobial activity in bronchoalveolar lavage (BAL) fluid, due in part to reductions in levels of surfactant protein A, surfactant protein D, and lysozyme. These immune deficits were accompanied by lower alveolar type II epithelial cell counts and reduced alveolar type II epithelial cell expression of collectin and lysozyme genes on a per cell basis. No significant between-group differences were detected in selected inflammatory cytokines or BAL inflammatory cell populations at baseline or after bacterial challenge in the wild-type and KGF-deficient mice. A single intranasal dose of recombinant human KGF reversed defects in bacterial clearance, AM function, and BAL fluid antimicrobial activity. We conclude that KGF supports alveolar innate immune defense through maintenance of alveolar antimicrobial protein levels and functions of AM. Together these data demonstrate a role for endogenous KGF in maintenance of normal pulmonary innate immune function.

Venom of Parasitoid Pteromalus puparum Impairs Host Humoral Antimicrobial Activity by Decreasing Host Cecropin and Lysozyme Gene Expression.

Insect host/parasitoid interactions are co-evolved systems in which host defenses are balanced by parasitoid mechanisms to disable or hide from host immune effectors. Here, we report that Pteromalus puparum venom impairs the antimicrobial activity of its host Pieris rapae. Inhibition zone results showed that bead injection induced the antimicrobial activity of the host hemolymph but that venom inhibited it. The cDNAs encoding cecropin and lysozyme were screened. Relative quantitative PCR results indicated that all of the microorganisms and bead injections up-regulated the transcript levels of the two genes but that venom down-regulated them. At 8 h post bead challenge, there was a peak in the transcript level of the cecropin gene, whereas the peak of lysozyme gene occurred at 24 h. The transcripts levels of the two genes were higher in the granulocytes and fat body than in other tissues. RNA interference decreased the transcript levels of the two genes and the antimicrobial activity of the pupal hemolymph. Venom injections similarly silenced the expression of the two genes during the first 8 h post-treatment in time- and dose-dependent manners, after which the silence effects abated. Additionally, recombinant cecropin and lysozyme had no significant effect on the emergence rate of pupae that were parasitized by P. puparum females. These findings suggest one mechanism of impairing host antimicrobial activity by parasitoid venom.

The activity parameters, cDNA cloning and mRNA expression of lysozyme in Cyclina sinensis (Bivalvia, Veneridae) responding to the pathogenic bacterium Vibrio anguillarum

ABSTRACTLysozyme is an important component of the innate immune response against pathogen infection. The functional forms have been well-studied in the c-type lysozymes of vertebrates but less so in i-type lysozymes prevalent in most invertebrate animals. Cyclina sinensis is a commercially important marine venerid bivalve that is abundant and widely distributed around the maritime coasts of Asia. We obtained an i-type lysozyme (i-lyz) gene in C. sinensis by large scale EST sequencing of a SMART-cDNA library. In C. sinensis, the i-lyz gene encodes 181 amino acids. The lysozyme activities and the mRNA levels of the i-lyz gene in haemocytes were upregulated during the infection by a bacterium, Vibrio anguillarum. The lysozyme activity in haemocytes was increased after 12 h infection by V. anguillarum, and reached the highest level at 24 h post-infection, which was significantly higher than that of the control group (P < 0.01). The expression level of the i-lyz gene in haemocytes was increased after 3 h infection of Vibrio, and reached the highest level at 6 h post-infection. A recombinant i-lyz was obtained through prokaryotic expression which, when isolated and purified, had a molecular weight of approximately 19 kDa. Western blotting was used to validate the expression of the fusion protein. Furthermore, we demonstrate that the recombinant i-lyz protein has obvious bacteriostatic activity upon Escherichia coli whereby the K value of the curve was significantly lower than control and blank groups. Our study shows that the C. sinensis i-type lysozyme plays an important role in the immune defence against V . anguillarum and provides a theoretical reference for clarifying the mechanism of immune response against pathogen invasion in this bivalve.

Escherichia coli Challenging Modulate the Humoral Immune Response of Lucilia cuprina(Diptera: Calliphoridae)

Antimicrobial compounds are recently emerging as anti-infectious agents that can be applied topically or systemically to speed up wound healing process. Maggot debridement therapy has become more prevalent in the treatment of chronic wound. This study focuses on studying the activity of commonly used natural compounds, which can be extracted from the whole and fat bodies of Lucilia cuprina. The current report was designed to investigate the effect of injecting bacterial suspension of Escherichia coli during the development of Lucilia cuprina at different time points. Measurements involved the assay of (a) total protease activities, (b) semi-quantitative RT-PCR for selected antimicrobial peptides genes expressions and (c) antioxidant capacity measurement as part of normal responding elements to humoral innate immunity (represented by lipid peroxide (Malondialdehyde) and glutathione reduced levels). Total lipid content in immature stages was also assessed. The results revealed an increase in total proteases activities and antioxidant capacities (low Malondialdehyde and high reduced glutathione levels) during the 3rd instar larvae and pupal phases after bacterial challenge. Detection of an increased mRNA expression levels in the fat body for lysozyme, cecropin, and attacin genes after bacterial-infection indicate high antibacterial activity in early pupae at 1h after infection. In conclusion, our findings may draw attention proposing that naturally extracted compounds from the whole and fat body of Lucilia cuprina, which are rich in proteolytic enzymes, as novel larval-based therapy in parallel to the traditional known ones. Further studies are required to illustrate the exact components of the extracts.

Dietary Effect of Lonicera japonica on Immune Expression in Olive Flounder Paralichthys olivaceus, Challenged with Vibrio anguillarum

Respiratory burst, lysozyme, phagocytic activities, and immune gene expression levels were assessed in olive flounder (Paralichthys olivaceus, 10.52±2.5 g) fed with 0%, 0.025%, 0.05%, 0.1%, 0.2%, and 0.4% Lonicera japonica leaf powder (LjLP) for four weeks. All flounder fish fed LjLP showed significantly increased respiratory burst, lysozyme and phagocytic activity. Total immunoglobulin levels in LjLP diet fed fish groups were higher than in fish fed the basal diet (BD). In flounder fed LjLP, immune gene expression and antioxidant activity were significantly enhanced after 4 weeks of culture. One week following a challenge with Vibrio anguillarum, fish fed with different concentrations of LjLP showed decreased cumulative mortality and enhanced immunity all compared to the BD diet group. Our findings have shown that Lonicera japonica leaf powder enhanced immune response and resistance to V. anguillarum infection in olive flounder.

Molecular characterization and in-silico analysis of complete coding sequence of bubaline mLYS

The role of immune-relevant genes involved in disease biology as well as innate and adaptive immunity is at the centre stage while exploring the genetic basis of disease occurrence. The macrophage expressed lysozyme gene product contributes to innate immunity by cleaving the ß- 1,4 linkage of bacterial peptidoglycan in phagocytosis. Higher specific activity observed in bubaline milk/ serum lysozymes over their bovine counterparts is expected to contribute higher resistances to diseases in general and mastitis in particular. To explore the reasons for higher activity in terms of sequence variations, macrophage expressed lysozyme cDNA was synthesized, cloned and sequenced. The 593 bp mRNA sequence revealed a 444 bp (27nt to 470nt) ORF bearing usual start codon ATG and end codon TAA with GC content of 46 % coding for precursor polypeptide of 147 amino acids. The comparative sequence analysis of cattle and buffalo revealed the difference at 10 places leading to 3 non-synonymous (5th, 116th and 142th) substitutions which did not affect the predicted 3D structure. The mLYS polypeptide had signal sequence (1st to 18th residue) and the mature peptide had two lysozyme catalytic sites, three Ca++ binding sites as well as eleven catalytic clefts prominent among the conserved domains. Secondly, bubaline mLYS was phylogenetically closer to the abomasum type than mammary gland type. The high similarity in the coding sequences and predicted structure suggested that bubaline lysozyme gene if hyper-expressed either in native or recombinant form in bovine udder may result in better udder health.

Expression profiles of lysozyme- and prophenoloxidase-encoding genes in Spodoptera species challenged with entomopathogenic fungus, Metarhizium anisopliae (Metchnikoff) Sorokin using qRT-PCR

Lysozyme and prophenoloxidase are two immunity-related enzymes in insect. In our attempt to understand their roles in defence, we profiled expressions of the encoding genes in Spodoptera exigua Hübner and Spodoptera litura Fabricius, during the infection process by an entomopathogenic fungus, Metarhizium anisopliae var. anisopliae. Gene expressions were analysed using quantitative real-time reverse transcription-PCR. Sampling was done at 0, 2, 12 and 24 h after infection, and when the infected larvae reached the moribund stage at 36 and 60 h after infection. The expression profiles of the two genes were similar in both larvae. At 24 h after infection, the lysozyme gene was expressed at a higher level in the infected larvae (fivefold) when compared to the healthy larvae, and reached the highest level (13-fold in S. exigua and 7-fold in S. litura) at the moribund stage. The expression levels of the prophenoloxidase gene in the infected larvae were similar to that of the control, except for a slight increase in expression at 2 and 24 h (~1.5-fold) after infection. These findings provide new information on the changes in transcriptional levels of LYZ and PPO, and indicate the importance of these genes in insect defence response against fungal infection.

Molecular Cloning and Characterization of a New C-type Lysozyme Gene from Yak Mammary Tissue.

Milk lysozyme is the ubiquitous enzyme in milk of mammals. In this study, the cDNA sequence of a new chicken-type (c-type) milk lysozyme gene (YML), was cloned from yak mammary gland tissue. A 444 bp open reading frames, which encodes 148 amino acids (16.54 kDa) with a signal peptide of 18 amino acids, was sequenced. Further analysis indicated that the nucleic acid and amino acid sequences identities between yak and cow milk lysozyme were 89.04% and 80.41%, respectively. Recombinant yak milk lysozyme (rYML) was produced by Escherichia coli BL21 and Pichia pastoris X33. The highest lysozyme activity was detected for heterologous protein rYML5 (M = 1,864.24 U/mg, SD = 25.75) which was expressed in P. pastoris with expression vector pPICZαA and it clearly inhibited growth of Staphylococcus aureus. Result of the YML gene expression using quantitative polymerase chain reaction showed that the YML gene was up-regulated to maximum at 30 day postpartum, that is, comparatively high YML can be found in initial milk production. The phylogenetic tree indicated that the amino acid sequence was similar to cow kidney lysozyme, which implied that the YML may have diverged from a different ancestor gene such as cow mammary glands. In our study, we suggest that YML be a new c-type lysozyme expressed in yak mammary glands that plays a role as host immunity.

Roles of receptor for activated protein kinase C1 for modulating immune responses in white shrimp Litopenaeus vannamei.

Complementary (c)DNA encoding a receptor for activated protein kinase C1 (RACK1) messenger (m)RNA of the white shrimp Litopenaeus vannamei, designated LvRACK1, consisted a 1136-bp cDNA containing an open reading frame (ORF) of 954 bp, a 111-bp 5'-untranslated region (UTR), and a 71-bp 3'-UTR, which is a 36 kDa cytosolic protein, belonging to the Trp-Asp40 (WD40) family of proteins, characterized by containing seven highly conserved Trp-Asp40 (WD40) internal repeats, and a poly A tail. The WD repeat of LvRACK1 can be predicted to form a seven-bladed propeller structure with each WD repeat composed of four antiparallel β-sheets. The WD40 domains have been implicated in protein-protein interactions. A comparison of amino acid sequences showed that LvRACK1 was closely related to arthropods RACK1. LvRACK1 cDNA was synthesized in all tested tissues detected with real-time PCR including haemocytes, hepatopancreas, gills, muscles, subcuticular epithelium, intestines, abdominal nervous ganglia, thoracic nervous ganglia, lymphoid organ, stomach, heart, and antennal gland, especially in subcuticular epithelium and gill. LvRACK1 mRNA transcription in haemocytes of L. vannamei injected with Vibrio alginolyticus decreased. The depletion of LvRACK1 of haemocytes in L. vannamei received its dsRNA revealed the increased respiratory bursts per haemocyte, superoxide dismutase (SOD), activity, glutathione peroxidase (GPx) activity, and clotting time, but showed the decreased total haemocyte count (THC), hyaline cells (HCs), phagocytic activity, and transglutaminase (TG) activity. LvRACK1 silenced shrimp showed the upregulated gene expressions of cyMnSOD, mtMnSOD, peroxinectin (PE), and TGI, and showed the downregulated α2-macroglobulin (α2-M), clottable protein (CP), lysozyme, and crustin gene expressions. It is therefore concluded that LvRACK1 is involved in immune defense and signaling transduction in haemocytes of L. vannamei infected with V. alginolyticus.