Effect of amino acid substitutions in the epitope regions of pyolysin from Arcanobacterium pyogenes

Pyolysin (PLO), secreted by Arcanobacterium pyogenes, is a novel member of the thiol-activated cytolysin (TACY, cholesterol-dependent cytolysin) family of bacterial toxins. Recently, we demonstrated that the epitopes of monoclonal antibodies (mAbs) S, H, C, and G lie in the regions of amino acids regions 55-73, 123-166, 482-506, and 482-506 of PLO, respectively, by the reaction of mAbs with truncated PLOs. In this study, we substituted the amino acids in these epitope regions of PLO by site-directed mutagenesis and examined the effect of these amino acid substitutions. Mutants I70S/R71A/L73S, Y131S/P132S, and L163S/P164S for mAbs H or S completely lost the hemolytic activity of the proteins, but these mutants still bound to erythrocyte membranes. Mutants L495S/W497S and W500S/W501S for mAbs C and G also completely lost their hemolytic activity, but still bound to erythrocyte membranes. In the undecapeptide region of PLO, the cysteine residue required for thiol activation is replaced with alanine. Therefore, we substituted Ala-492 of the undecapeptide region for Cys. The hemolytic activity of this mutant A492C decreased by adding hydrogen peroxide or storing at 4 degrees C, and the decreased hemolytic activity was restored by adding L-cysteine.     

Structural gene and strain specificity of a novel cysteine protease produced by Staphylococcus aureus isolated from a diseased chicken

Recently whole genome sequencing of Staphylococcus aureus has revealed the genes encoding cysteine proteases such as staphopain and SspB. In this study, we cloned and sequenced the structural gene (ScpA) encoding a cysteine (thiol) protease of S. aureus strain CH-91 from a chicken with dermatitis using polymerase chain reaction (PCR) and inverse PCR methods. The sequence information revealed a coding sequence (CDS) of 1200 nucleotides encoding the ScpA preproenzyme of 399 amino acids with a molecular mass of 45,071 Da. The deduced amino acid sequence of the ScpA differed at many positions from those of staphopain and SspB with identities of 64 and 42%, respectively. In the Southern blot analysis with a total DNA of S. aureus strain CH-91, the ScpA probe hybridized with a single 7.7 kb XbaI fragment or 2.8 and 0.8 kb EcoRI fragments, whereas the staphopain and SspB probes did not hybridize with these DNA fragments. These results suggest that this ScpA gene is a single-copy gene and is a novel gene, which is not found in the published whole genome sequences of S. aureus. In immunoblot, PCR, and Southern blot assays, the ScpA or its gene was detected in high protease-producing strains from chickens, but was not recognized in bovine and porcine strains or low protease-producing avian strains. These results indicate that the ScpA of CH-91 type may be specific to the high protease-producing strains of S. aureus from chickens, namely, there is a strain specificity of the ScpA.     

Congenital cutaneous fibropapillomatosis without evidences of papillomavirus infection in a Holstein-Friesian calf

A male Holstein-Friesian calf was born with multiple, cauliflower-like, pale pink cutaneous masses on the head and limbs. On histopathological examination, the cutaneous masses were diagnosed as congenital cutaneous fibropapillomatosis. Those lesions involved focal proliferation of sebaceous gland in the dermis. There were no histological findings to suggest bovine papillomavirus infection, such as the presence of intranuclear inclusion bodies, large keratohyalin granules, and koilocytosis. Furthermore, papillomaviral antigens and DNA were not detected by immunohistochemistry and polymerase chain reaction, respectively. These results suggested that there was no association between these cutaneous lesions and bovine papillomavirus infection, and the lesions were considered as harmartomatous changes.     

Effects of Estradiol on Expression of Estrogen Receptor and Collagen mRNAs in Chick Skin

Skin thickness and strength differ between male and female chickens. This study aimed to clarify the effects of estradiol on the expression of estrogen receptors and collagen mRNA in chicken skin. Estradiol was administered to male chicks for 3 weeks, then cryosections of skin collected from the cervical, thoracic, dorsal, and pelvic limb regions were stained with hematoxylin and eosin, and dermal thickness was measured. Estrogen receptor and collagen mRNA expression was assessed using real-time RT-PCR, and collagen contents were determined. Estradiol did not alter dermal thickness or the collagen content of the skin from any tested region. Among the estrogen receptors, significantly more ESR1 mRNA was expressed in the thoracic skin of chicks administered with estradiol compared with vehicle (control), and in the thoracic skin compared with skin from other regions within each group. Estradiol did not affect ESR2, GPER, and COL1A1 mRNA expression. These results suggested that estradiol stimulates ESR1 expression in thoracic skin, but does not affect collagen synthesis in skin from any other region of male chicks.     

Changes of histochemical profiles of myofibers in pectoralis and supracoracoideus fasciculi induced by breeding for large or small body size in Japanese quails

The histochemical profiles of myofibers in Musculus pectoralis (PT) and M. supracoracoideus (SC) fasciculi were compared among Japanese quail strains with large, normal and small body sizes. In male and female adults, both the PT and SC muscles had attained a 2.5–2.7-fold weight gain in the large strain and conversely a 0.43–0.50-fold change in the small strain relative to those of the normal size. The muscles were composed of fasciculi with a central cluster of type IIA fibers surrounded by a peripheral layer of type IIB fibers. In the large strain, the cross sectional area (CSA) of the fasciculus and CSA of the fibers in each type were significantly enlarged compared with those in the normal size, with the exception of the fasciculus in the deep region of the male PT muscle. The hypertrophied type IIA fibers in the large strain showed considerable variation in nicotinamide adenine dinucleotide dehydrogenase activity, some of which might represent a transitional form into type IIB fibers. In the small strain, the fasciculus CSA did not significantly differ from that of the normal size except for the PT surface region of the male. However, fiber atrophy was observed in type IIB fibers of the PT surface region in both sexes, and type IIA fibers of the PT deep region and SC muscle in the small strain male quails. The relative fiber type composition of a fasciculus in each region showed only a slight change across the strains. These results indicate that breast muscle hypertrophy in the large strain could be based mainly on fasciculus and fiber hypertrophy, but muscle atrophy in the small strain is not induced by fasciculus and fiber atrophy.     

Comparative observations on the growth changes of the histochemical property and collagen architecture of the Musculus pectoralis from Silky, layer-type and meat-type cockerels

Growth‐related changes in the histochemical properties and collagen architecture of the Musculus pectoralis were compared among Silky, layer‐type and meat‐type cockerels. Histochemical and immunohistochemical methods were used and collagen architecture was studied using scanning electron microscopy. The total amount of collagen present was also measured. The diameter of type IIB myofibers was similar or rather larger in the layer‐type birds compared with the meat‐type. The collagen content was generally low for 5–10 weeks across the breeds and then increased in the other breeds except for Silky. In the perimysium, the collagen bundles gradually increased in size and the density of the fibrils also increased during growth. At 30 weeks of age, the layer‐type birds showed compact collagen bundles while the meat‐type had loose bundles. The endomysial collagen network appeared relatively denser in the meat‐type chicks compared to the others at week 1. At 30 weeks of age, compact and felt‐like structure of endomysium was shown by Silky and layer‐type chickens, while the meat‐type showed a relatively loose arrangement of tissue in the endomysial collagen. From these results, it appears that the meat‐type chicken can produce a large M. pectoralis with many, relatively thinner myofibers and a relatively undeveloped form of intramuscular collagen structure.