Analysis of the functional domains of Arcanobacterium pyogenes pyolysin using monoclonal antibodies.

Pyolysin (PLO), secreted by Arcanobacterium pyogenes, is a novel member of the thiol-activated cytolysin (TACY) family of bacterial toxins. Four monoclonal antibodies (mAbs) to PLO were prepared for the analysis of functional domains of this toxin. Two (mAbs S and H) of these markedly inhibited the hemolytic activity of PLO, but the inhibiting activity of the other two antibodies (mAbs C and G) was weaker. Subsequently, nine truncated PLOs were derived from recombinant Escherichia coli by various deletions from the N-terminus. Strong hemolytic activity was recognized in truncates of PLO following the deletion of 30 or 55 amino acids, but not in the truncate with deletion of 74 residues. Truncated PLOs were used in immunoblotting experiments to locate the epitopes for the mAbs. The epitope for mAbs C and G lies within the undecapeptide region (amino acids 487-505) of the C-terminus of PLO, which seems to be the binding site to erythrocytes. In contrast, the epitopes for mAbs S and H, which showed strong neutralizing activity, were found to lie in the N-terminal regions of the PLO ranging from 55 to 73 and 123 to 166 amino acids, respectively. From these results, it seems that the N-terminal region of PLO, in particular, the region of amino acids 55-74 is important for hemolytic activity.

Variation of the agr locus in Staphylococcus aureus isolates from cows with mastitis.

Staphylococcus aureus isolates from mastitic cow's milk were examined for production of alpha-hemolysin and protein A and their accessory gene regulator (agr locus) was analyzed. An inverse relationship between alpha-hemolysin and protein A production was found in most of the 76 isolates, suggesting that the isolates tested may be classified into group I (high alpha-hemolysin/low protein A), II (low alpha-hemolysin/high protein A), or III (low alpha-hemolysin/low protein A). The agr locus, which consists of hld, agrB, agrD, agrC, and agrA, was detected in most of the 78 isolates including two reference strains (Wood 46 and Cowan I) by polymerase chain reaction (PCR). When the PCR products for agr locus of 22 isolates from groups I and II were digested with restriction enzyme MboI, seven bands of the expected lengths were recognized in strain Wood 46, but not in the other isolates tested. Nucleotide sequence analysis of PCR products from six isolates revealed that the agr locus sequence of strain Wood 46 corresponded to that of the published sequence data, but the other five isolates from groups I and II diverged at agrB and agrD sequences and thus the deduced amino acid sequences. These variations of agr locus in S. aureus bovine isolates differed from those reported by Ji et al. [Science 276 (1997) 2027].

Genetic analysis of exfoliative toxin A-producing Staphylococcus aureus isolated from mastitic cow's milk.

Exfoliative toxin A (ETA), produced by Staphylococcus aureus, is the causative agent of staphylococcal scalded-skin syndrome (SSSS) in children. Recently, we reported that ETA was detected by reverse passive latex agglutination in three isolates of S. aureus from cow's milk, but that these ETA-positive isolates did not cause the so-called Nikolsky sign in neonatal mice. In this study, therefore, the eta gene encoding ETA and regulatory genes of these bovine isolates were analyzed by the polymerase chain reaction (PCR) and sequencing. The eta gene was amplified from three bovine isolates by PCR and their resulting nucleotide sequences found to correspond to the eta gene from the human isolate, except for three nucleotides in the upstream region of the eta open reading frame (ORF). An accessory gene regulator (agr), which is a global regulatory locus, was detected in these bovine isolates by PCR amplification. In addition, the ORF (J-4), located 120 bp upstream from the eta ORF of the human isolate, was also amplified from these bovine isolates, with their nucleotide sequences differing at 32 positions from the human isolate. Bovine and human ORF J-4 equally enhanced production of ETA in the recombinants of the eta gene, suggesting that the variation in bovine ORF J-4 may be not be the cause of the difference in amount of ETA produced by bovine and human isolates.

Intercellular junctions between palisade nerve endings and outer root sheath cells of rat vellus hairs.

Hair follicles have a longitudinal set of sensory nerve endings called palisade nerve endings (PN). We examined the junctional structures between the PN and outer root sheath (ORS) cells of hair follicles in the rat external ear. Transmission electron microscopy of serial thin sections showed that the processes of the ORS cells penetrated the basal lamina of the hair follicle, forming intercellular junctions with the PN (PN-ORS junctions). Two types of junctions were found: junctions between nerve endings and ORS cells (N-ORS junctions) and those between Schwann cell processes and ORS cells (S-ORS junctions). The N-ORS junctions had two subtypes: 1) a short process or small eminence of the ORS cell was attached to the nerve ending (type I); or 2) a process of the ORS cell was invaginated into the nerve ending (type II). The S-ORS junctions also had two subtypes: 1) a short process or small eminence of the ORS cell was abutted on the Schwann cell process (type I); or 2) a process of the ORS cell was invaginated into the Schwann cell process (type II). Vesicles, coated pits, coated vesicles, and endosomes were sometimes seen in nerve endings, Schwann cells, and ORS cells near the junctions. Computer-aided reconstruction of the serial thin sections displayed the three-dimensional structure of these junctions. These results suggested that the PN-ORS junctions provided direct relationships between the PN and ORS in at least four different patterns. The discovery of these junctions shows the PN-ORS relationship to be closer than previously realized. We speculate that these junctions may have roles in attachment of the PN to the ORS, contributing to increases in the sensitivity of the PN, and in chemical signaling between the PN and ORS.

Antibody response to toxic shock syndrome toxin-1 of Staphylococcus aureus in dairy cows.

Antibody response to toxic shock syndrome toxin-1 (TSST-1) of Staphylococcus aureus in dairy cows was examined by enzyme linked immunosorbent assay (ELISA). Serum antibody to TSST-1 was not detected in 39 (76.5%) of 51 calves, which were 1-6 months of age. In contrast, TSST-1 antibody was demonstrated in 1728 (72.6%) of 2380 lactating cows housed on 36 dairy farms. The ELISA values of antibody ranged from 0.2 to 3.0 OD and presented a distribution with the peak at 1.6 OD. The mean ELISA value differed between farms, and it increased slightly along with parturient history. Somatic cell counts of milk from 174 lactating cows was compared with TSST-1 antibody and tst1,000,000 cells per ml. The mean ELISA values in milk were lower than those of sera, but they rose as somatic cells increased. The tst gene of S. aureus detected in 76.0-86.2% of the milk samples containing somatic cells > 500,000 cells per ml, a level which indicates mastitis. The data suggests that many lactating cows may be infected by TSST-1- producing S. aureus.

Genetic and biochemical properties of a hemolysin (pyolysin) produced by a swine isolate of Arcanobacterium (Actinomyces) pyogenes.

Arcanobacterium (Actinomyces) pyogenes, a causative agent of various pyogenic diseases in domestic animals, produces a hemolysin which is thought to be an important virulence factor. This hemolysin was purified from the culture supernatant of A. pyogenes swine isolate. The purified hemolysin showed a single band with a molecular mass of 56 kDa on SDS-polyacrylamide gel electrophoresis, and its isoelectric point was 9.2. The activity of this hemolysin was not enhanced by the addition of L-cysteine or sodium thioglycolate, but it was inhibited by cholesterol. The gene encoding the hemolysin was cloned, sequenced and expressed in Escherichia coli by means of ZAP Express vector. Analysis by SDS-polyacrylamide gel electrophoresis with immunoblotting showed that the molecular weight of the hemolysin expressed in E. coli is the same as that of the hemolysin purified from A. pyogenes. Nucleotide sequence analysis revealed an open reading frame of 1,605 bp encoding a 534 amino acid protein of 57,989 Da. The nucleotide sequence of the hemolysin gene from A. pyogenes swine isolate differed only slightly (97.6% identity) from the sequence of plo gene from A. pyogenes strain BBR1 reported by Billington et al (J. Bacteriol. 179: 6100-6106, 1997). The cysteine residue existed in the undecapeptide region of the hemolysin, which is highly conserved in thiol-activated cytolysins (cholesterol-binding cytolysins), and is replaced with alanine. Therefore, the hemolysin of A. pyogenes seems to be a novel member of the thiol-activated cytolysin family.

A metalloprotease is common to swine, avian and bovine isolates of Staphylococcus hyicus.

Staphylococcus hyicus is considered to be an etiological agent of exudative epidermitis in young pigs, but is frequently isolated from chickens and cows. In the present study, the proteases of 58 S. hyicus isolates from pigs, chickens and cows were examined by skim milk agar plate culture, gelatinolytic zymogram and polymerase chain reaction (PCR). These isolates showed proteolytic activity on skim milk agar plate, but activity differed amongst the isolates. In the gelatinolytic zymogram, one main band was observed in all the porcine, avian and bovine isolates, while one to two other bands were recognized in some isolates. The formation of the main band was inhibited by EDTA, suggesting that this protease is a metalloprotease. When the Shp1 gene, which codes for one the metalloproteases of S. hyicus as reported previously, was examined by PCR, one band arising from an open reading frame (ORF) was detected in all of 58 isolates tested. In addition, upstream nucleotides containing the promoter region of Shp1 gene were amplified and sequenced. From these results, it seems likely that the metalloprotease is common to porcine, avian and bovine isolates of S. hyicus.

Three-dimensional architecture of the keratin filaments in epithelial cells surrounding taste buds in the rat circumvallate papilla.

The three-dimensional architectures of the perigemmal cells and their keratin bundles in the rat circumvallate papillae were studied by transmission and scanning electron microscopy. The perigemmal cells were classified into three layers: basal, middle and upper. The basal layer consisted of polygonal cells located close to the basal lamina, the middle layer comprised longitudinally elongated cells fitting the lateral convexity of the taste bud, and the upper layer was imbricating flat cells along the upper portion of the taste bud. When fresh specimens were jointly treated with Triton X-100 and sonication, the taste buds were often detached and the cytoplasmic matrices of the perigemmal cells were effectively removed. Consequently, we were able to demonstrate an extensive network of the subplasmalemmal keratin bundles of the perigemmal cells. The framework appeared either as a thin lacework, a thick fence-like structure, or a lattice work in the basal, middle, and upper layers, respectively. The thin lacework in the basal layer was considered to be a developing process of the framework. The thick fence-like structure in the middle layer probably plays a primary role in supporting the taste bud. The latticework in the upper layer is believed to reflect a remodeling in reducing the keratin framework.

Scanning electron microscopic observation on the equatorial region of the rat intrafusal muscle fibres, especially on the subsarcolemmal sarcoplasmic reticulum.

We examined intracellular structures in the equatorial region of the muscle spindles of rat soleus muscles by scanning electron microscopy, paying particular attention to the ultrastructure of the sarcoplasmic reticulum (SR) beneath the sarcolemma. The subsarcolemmal SR was more developed in nuclear chain fibres than in nuclear bag fibres as was reported in the sleeve and extracapsular regions. In addition, the subsarcolemmal SR of the chain fibre formed a fenestrated sheet, whereas that of the bag fibre organized a layer of fenestrated bands beneath the sarcolemma where the sensory nerve endings are associated. No T-tubules were discerned in the subsarcolemmal SR of both fibres, which may be concerned with the little contraction of the equatorial region.

Three-dimensional structure of the synaptic contact of the neuromuscular junction in the rat lumbrical muscle.

This study examined the three-dimensional structures of the synaptic contact in rat lumbrical muscles by scanning electron microscopy using three different methods: the aldehyde prefix-osmium-dimethyl sulfoxide-osmium method (A-O-D-O method), the cell-extraction method, and the NaOH-digestion method. These three methods visualized the motor nerve endings, subneural basal lamina and postsynaptic sarcolemma, respectively. The motor nerve endings were composed of a cluster of spherical and cylindrical terminals. Pores on the presynaptic membrane were considered openings of exocytotic vesicles. The postsynaptic side of the subneural basal lamina showed numerous ridges, corresponding to junctional folds. Most of the ridges rose vertically from their base. The ridges showed widening, narrowing, and branching. The subneural basal lamina appeared to be composed of small granular substances. The basal lamina of the primary synaptic clefts had pores 25-30 nm in diameter, which may facilitate the transport of acetylcholine (ACh) without being hydrolyzed by ACh esterase in the lamina. On the outer surface of the postsynaptic sarcolemma in a sole plate, the primary synaptic clefts were composed of a mixture of depressions and gutters; so far as we know, this represents the only example of such a phenomenon. These depressions and gutters seem to fit respectively into the spherical and cylindrical terminals of the motor nerve endings. The openings of the junctional folds consisted of a mixture of many slits and a few pits in the primary synaptic clefts.

Purification and characterization of protease produced by Staphylococcus aureus isolated from a diseased chicken.

A protease produced by Staphylococcus aureus, isolated from a chicken suffering from dermatitis, was purified by successive precipitation with ammonium sulfate, ion-exchange chromatography on Q-Sepharose FF, Sp-Sepharose FF and Mono-Q columns. By Mono-Q column chromatography, two proteases (protease 1 and 2) were obtained. The molecular weights of protease 1 and 2 were estimated at 23.1 and 22.7 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. Their isoelectric points were 5.85 and 5.55, respectively, and they possessed antigenic similarity when examined by the immunoblotting. The N-terminal amino acid sequences of both the proteases were identical (RAQYVNQLKNFKIRETQ). The activities of both the proteases were strongly increased by reducing agents such as L-cysteine and sodium thioglycolate. Their activity was inhibited by thiol protease inhibitors, but was not inhibited by metalloprotease or serine protease inhibitors. From the results, it seems likely that these proteases, produced by S. aureus from diseased chickens, might belong to the thiol protease group.

Changes in VEGF expression and in the vasculature during the growth of early-stage ethylnitrosourea-induced malignant astrocytomas in rats.

Vascular endothelial growth factor (VEGF), a potent angiogenic and vascular permeability factor, may be important as a mediator of brain tumour progression. However, it is still not clear whether VEGF plays a causative role in the early stage of glioma development. We investigated the relationship between VEGF protein expression (as assayed by immunohistochemistry) and different morphological parameters reflecting tumour progression (tumour diameter, vascular density and vascular diameter) in tumours at various stages. As a tumour model, ethylnitrosourea (ENU)-induced rat malignant astrocytoma was used. Tumours were classified by size and level of vascularity estimated by the von Willebrand factor (vWF) staining. Tumours less than 10 mm in diameter were designated early stage neoplastic lesions. All 34 early astroglial tumours were found to be VEGF positive. Increase in the VEGF immunopositive rate of tumour cells correlated significantly with increase in vascular density and vascular diameter. We suggest that VEGF induces angiogenesis and growth of microvessels, promoting growth of the early stage malignant astrocytoma.

Ultrastructural changes of the myoepithelium of the dilator pupillae during miosis and mydriasis in the rat iris.

Changes in the three-dimensional structures of the myoepithelium of the dilator pupillae (MDP) during mydriasis and miosis were investigated in the rat by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Following fixation, SEM specimens were treated with sodium hydroxide to expose the muscle surface. Significant morphological differences were noted in the anterior surface of the MDP between mydriasis and miosis. In the mydriatic eye, a highly rugged structure with numerous linear folds was oriented circularly or obliquely together with spherical bulges. These structures, presumably corresponding to the highly contractile portion of the myoepithelial cells, were more prominent near the pupillary margin than near the ciliary margin, indicating that the MDP may contract much more strongly in the pupillary margin. In the miotic eye, the anterior surface of the MDP showed less conspicuous linear folds in the pupillary area, and was almost flat in the ciliary area. Radially oriented ridges were observed only in the pupillary area. These findings suggest that the contraction of the sphincter pupillae in miosis induces a stretching of the MDP toward the pupil and a circular shrinkage of the MDP. Ultrastructural changes of the MDP particularly near the pupillary margin may play an important role in regulation of the pupil diameter as a diaphragm of the eye because morphological changes such as the linear folds, spherical bulges, and ridges were more prominent near the pupillary margin than those near the ciliary margin.

Production of toxic shock syndrome toxin by Staphylococcus aureus isolated from mastitic cow's milk and farm bulk milk.

The production of toxic shock syndrome toxin (TSST) by Staphylococcus aureus isolated from mastitic cow's milk and farm bulk milk was examined by a reverse passive latex agglutination method (RPLA). TSST was detected in 25 (58.1%) of 43 isolates from clinical mastitic cow's milk, in 79 (76.7%) of 103 isolates from subclinical mastitic cow's milk, and in 95 (75.4%) of 126 isolates from farm bulk milk, respectively. When the quantity of TSST in the isolates was determined by RPLA, the titers ranged from 5 to 2560. TSST with RPLA titers of 640 to 2,560 was produced by 83 (30.5%) of 272 isolates tested. Almost all of the isolates showing RPLA titers of 640 and over produced enterotoxin C, whereas isolates showing titers of 5 to 320 produced enterotoxin C or both enterotoxin A and C. SDS-polyacrylamide gel electrophoresis and isoelectric focusing with immunoblotting showed that the TSST from bovine isolates had same molecular size (22 kDa) and isoelectric point (7.2) as TSST-1 from human isolates. These findings are consistent with previous reports.

Detection of Clostridium septicum hemolysin gene by polymerase chain reaction.

A polymerase chain reaction (PCR) was developed for the detection of the hemolysin (alpha toxin) gene of Clostridium septicum. The PCR primers were designed from the sequence of the hemolysin gene and synthesized. A DNA fragment of 270 bp was amplified from 10 strains of C. septicum, but was not from strains of C. chauvoei, C. perfringens, C. novyi, or C. haemolyticum. When the PCR product was digested with Sau3AI, two DNA fragments of the expected 148 bp and 122 bp were recognized. The lowest detectable threshold of PCR for the hemolysin gene was 3.8 x 10(3) cells/ml. The PCR technique may be useful for rapid detection or identification of C. septicum associated with malignant edema.

Characterization of the putative alpha subunit of a heterotrimeric G protein in rice.

A recombinant protein with a cDNA that encodes the putative alpha subunit of a rice heterotrimeric G protein was synthesized in Escherichia coli and purified. The recombinant protein (rGrice alpha) with an apparent molecular mass of 45 kDa was bound with guanosine 5'-(3-O-thio)triphosphate with an apparent association constant (kapp) of 0.36. The protein also hydrolyzed GTP and its kcat was 0.44. rGrice alpha was ADP-ribosylated by activated cholera toxin. Monoclonal antibodies raised against rGrice alpha reacted with a 45 kDa polypeptide localized in the plasma membrane of rice seedlings. The peptide map of this polypeptide after digestion with V8 protease was identical to that of rGrice alpha. A 45 kDa polypeptide in the plasma membrane, as well as rGrice alpha, was ADP-ribosylated by activated cholera toxin. The GTPase activity of the plasma membrane was stimulated 2.5-fold by mastoparan 7 but not mastoparan 17. These properties were similar to those of the alpha subunits of heterotrimeric G proteins in animals, suggesting that the putative alpha subunit is truly the alpha subunit itself.

Detection of toxic shock syndrome toxin-1 gene in Staphylococcus aureus bovine isolates and bulk milk by the polymerase chain reaction.

Staphylococcus aureus isolates from mastitic cow's milk and farm bulk milk were examined for toxic shock syndrome toxin-1 (TSST-1) gene (tst gene) by the polymerase chain reaction (PCR). The 179 bp band of tst gene was observed in almost all the bovine isolates which showed TSST positive in a latex agglutination, as well as in human strain FRI 1169, but was not observed in bovine isolates of TSST negative. The lowest detectable threshold of the PCR for tst gene was 1.2 x 10(3) cells/ml. When 125 bovine milk samples were cultured selectively for staphylococci and examined by PCR, the tst gene was detected in 10 of the 35 culture fluids, in which staphylococci were recognized by Gram's staining.

Plasmodium gallinaceum: mosquito peritrophic matrix and the parasite-vector compatibility.

Transmission of malaria parasites occurs by relatively few species of mosquitoes. One proposed mechanism of refractoriness is an inability of certain Plasmodium spp. to cross the peritrophic matrix (PM) in the midgut of an incompatible mosquito. We have tested this hypothesis by studying sporogonic development of Plasmodium gallinaceum in susceptible (Aedes aegypti and Anopheles gambiae G3) and refractory (Anopheles stephensi) mosquito species in the presence and absence of the PM. In the presence of the PM the number of oocytes that developed in A. gambiae G3 was about 20% of that in A. aegypti, whereas no oocysts developed in A. stephensi. To disrupt PM formation we added, to an infectious bloodmeal, either exogenous fungal chitinase or polyoxin D, the latter being a potent inhibitor of chitin synthase. The absence of the PM did not increase the susceptibility of A. aegypti and A. gambiae nor did it make A. stephensi susceptible to P. gallinaceum infection. The data indicate that the PM is not the primary determinant of P. gallinaceum compatibility in these mosquitoes and suggest that determinant(s) of refractoriness occurs after the parasite crosses the mosquito PM.

Assay of proteases from Actinomyces pyogenes isolated from pigs and cows by zymography.

Extracellular proteases of Actinomyces pyogenes were assayed by zymography on SDS-polyacrylamide gel with the concentrated culture supernatant of 7 isolates from pigs and cows. When gelatin was used as a substrate, three proteases with molecular weights of 69, 59 and 55 kilodalton (kDA) were detected in all the isolates from both the pigs and cows. In addition to these proteases, 108 and 102 kDa proteases were detected in the swine and bovine isolates respectively. No protease bands appeared, however, when the calcium ion was absent from the buffer solution of the zymography. When casein was used as a substrate, the bands of 69, 59 and 55 kDa proteases were present, but 108 and 102kDa proteases were not detected in any of the isolates. The activity of all proteases was completely inhibited by phenylmethyl-sulfonyl fluoride and diisopropyl fluorophosphate but not by other protease inhibitors. From this result, it seems likely that the five proteases produced by A, pyogenes, originating from pigs and cows, are serine proteases.

Effect and localization of trifluralin in Plasmodium falciparum gametocytes: an electron microscopic study.

Trifluralin, a herbicide which is known to bind to plant and algal tubulin, induced ultrastructural changes in the microtubules of the mature Plasmodium falciparum gametocytes in vitro. Trifluralin treatment led to disassembly of the well ordered subpellicular microtubules, whereas it had no effect on microtubules of human platelets or of rat neuronal cells in vitro. The disassembled microtubules showed fragmented large tubular structures, which frequently were associated with the pellicular membranes. Electron microscopic autoradiography showed radioactive trifluralin associated with the microtubule fragments. These results provide evidence that trifluralin selectively binds to microtubules in malaria parasites and causes disruption of their structure.