Tail-associated structural protein gp61 of Staphylococcus aureus phage φMR11 has bifunctional lytic activity

A tailed bacteriophage, φMR11 (siphovirus), was selected as a candidate therapeutic phage against Staphylococcus aureus infections. Gene 61, one of the 67 ORFs identified, is located in the morphogenic module. The gene product (gp61) has lytic domains homologous to CHAP (corresponding to an amidase function) at its N-terminus and lysozyme subfamily 2 (LYZ2) at its C-terminus. Each domain of gp61 was purified as a recombinant protein. Both the amidase [amino acids (aa) 1–150] and the lysozyme (aa 401–624) domains but not the linker domain (aa 151–400) caused efficient lysis of S . aureus . Immunoelectron microscopy localized gp61 to the tail tip of the φMR11 phage. These data strongly suggest that gp61 is a tail-associated lytic factor involved in local cell-wall degradation, allowing the subsequent injection of φMR11 DNA into the host cytoplasm. Staphylococcus aureus lysogenized with φMR11 was also lysed by both proteins. Staphylococcus aureus strains on which φMR11 phage can only produce spots but not plaques were also lysed by each protein, indicating that gp61 may be involved in 'lysis from without'. This is the first report of the presence of a tail-associated virion protein that acts as a lysin, in an S. aureus phage.     

Purification and Properties of Prostaglandin H-E Isomerase from the Cytosol of Human Brain: Identification as Anionic Forms of Glutathione S-Transferase

Abstract: Prostaglandin H-E isomerase (EC 5.3.99.3) was purified from human brain cytosol. Purification was by ammonium sulfate fractionation, diethylaminoethyl-Sephar-ose chromatography, gel filtration on a BioGel P-100 column, GSH-agarose chromatography, and MonoQ chromatography. The activity was eluted in two peaks from the MonoQ column, which were designated peaks 1 and 2. The molecular weights of peaks 1 and 2, determined by gel filtration, were 42,000 and 44,000, respectively. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peak 1 showed two bands at the molecular weights of 24,500 and 25,000, and peak 2 showed a single band at the molecular weight of 25,000, results suggesting that both were dimeric proteins. The pI values of both enzymes were ∼5.4. The enzymes catalyzed selective conversion of prostaglandin H₂ to prostaglandin E₂. The K _m values for prostaglandin H₂ of peaks 1 and 2 were 147 and 308 μ M , respectively, and the V _max values were 380 and 720 nmol/min/mg of protein, respectively. GSH was required for the catalysis of both enzymes, and no other sulfhydryl compounds could support the reaction. A part of glutathione S -transferase (EC 2.5.1.18) was copurified with peaks 1 and 2 of prostaglandin H-E isomerase. Prostaglandin H-E isomerase activity of peak 2 enzyme was competitively inhibited by 1-chloro-2,4-dinitrobenzene, a substrate of glutathione S -transferase. These results suggested that prostaglandin H-E isomerases in human brain cytosol were identical with anionic forms of glutathione S -transferase.     

Ontogeny of absence-like and tonic seizures in the spontaneously epileptic rat

The ontogeny of epileptic seizures in spontaneously epileptic rats (SER; zi/zi, tm/tm) was studied by examining behaviour and electroencephalogram (EEG) simultaneously. Weight gain and survival time were also studied. Compared with the control Kyo:Wistar rats, SER showed a much smaller increase in body weight. All male and female SER died before 20 and 18 weeks of age, respectively. Body tremor was observed at 2 weeks of age but disappeared after 11 weeks. Staggering gait appeared after 7 weeks of age, and intensified with age. Absence-like seizures characterized by paroxysmal appearance of 5-7 Hz spike-wave-like complexes were observed in the cortical or hippocampal EEG after 5 weeks of age, and tonic seizures with low voltage fast waves were observed after 6 weeks of age. All SER exhibited both absence-like and tonic seizures with high frequencies from 12 weeks of age. Differences with other spontaneous rat models of epilepsy and application methods for estimating seizure-inhibitory effects of anti-epileptic drugs are discussed.     

Clinical significance of adrenal computed tomography in Addison's disease.

Adrenal computed tomographic (CT) scanning was conducted in twelve patients with Addison's disease during the clinical course. In tuberculous Addison's disease (n = 8), three of four patients examined during the first two years after disease onset had bilaterally enlarged adrenals, while one of four had a unilaterally enlarged one. At least one adrenal gland was enlarged after onset in all six patients examined during the first four years. Thereafter, the adrenal glands may atrophy bilaterally, in contrast to adrenal glands in idiopathic Addison's disease, which atrophy bilaterally from disease onset (n = 2). Adrenal calcification was a less sensitive clue in tracing pathogenesis, i.e., adrenal calcification was observed in five of eight patients with tuberculous Addison's disease, but not in idiopathic patients. Thus, adrenal CT scanning could show the etiology of Addison's disease (infection or autoimmunity) and the phase of Addison's disease secondary to tuberculosis, which may be clinically important for initiating antituberculous treatment.     

Mast cells contain spleen-type prostaglandin D synthetase

Prostaglandin D synthetase activity in the cytosol (100,000 x g, 1-h supernatant) fraction of peritoneal mast cells of adult rats (105.0 nmol/min/mg protein) was the highest among such activities in various rat tissues and cells. As judged by the absolute requirement for glutathione for the reaction (Km = 300 microM), the Km value for prostaglandin H2 (200 microM), and insensitivity of the activity to 1 mM 1-chloro-2,4-dinitrobenzene, the enzyme in mast cells was similar to rat spleen prostaglandin D synthetase and differed from rat brain prostaglandin D synthetase or glutathione S-transferase, all of which catalyze the isomerase reaction from prostaglandin H2 to prostaglandin D2. In immunotitration analyses, the activity in mast cells showed a titration curve exactly identical with that of the purified spleen-type enzyme and almost completely absorbed by an excess amount of antibody against this enzyme, but it remained unchanged after incubation with antibodies against the brain-type enzyme and glutathione S-transferase isozymes thus far purified. In Western blot after two-dimensional electrophoresis of crude extracts of mast cells, a single immunoreactive spot was observed with antibody against the spleen-type enzyme at the same position as that of the purified enzyme (Mr = 26,000, pI = 5.2). Furthermore, the immunoreactive protein obtained from mast cells showed the same peptide fingerprints as those of the purified spleen-type enzyme, after partial digestion with Staphylococcus aureus V8 protease or trypsin. In immunoperoxidase staining, the immunoreactivity of the spleen-type enzyme was found in the cytosol of tissue mast cells in various organs such as thymus, intestine, stomach, and skin of adult rats. These findings indicate that prostaglandin D2 is produced by the spleen-type synthetase in mast cells of various tissues.     

A novel site-specific recombination system derived from bacteriophage phiMR11

We report identification of a novel site-specific DNA recombination system that functions in both in vivo and in vitro, derived from lysogenic Staphylococcus aureus phage phiMR11. In silico analysis of the phiMR11 genome indicated orf1 as a putative integrase gene. Phage and bacterial attachment sites (attP and attB, respectively) and attachment junctions were determined and their nucleotide sequences decoded. Sequences of attP and attB were mostly different to each other except for a two bp common core that was the crossover point. We found several inverted repeats adjacent to the core sequence of attP as potential protein binding sites. The precise and efficient integration properties of phiMR11 integrase were shown on attP and attB in Escherichia coli and the minimum size of attP was found to be 34bp. In in vitro assays using crude or purified integrase, only buffer and substrate DNAs were required for the recombination reaction, indicating that other bacterially encoded factors are not essential for activity.     

Genomic and phylogenetic traits of Staphylococcus phages S25-3 and S25-4 (family Myoviridae,genus Twort-like viruses)

The genomes of Staphylococcus phages S25-3 and S25-4 (family Myoviridae, genus Twort-like viruses) were sequenced and analyzed from an evolutionary perspective. The genome-based phylogeny and genome analyses of phages S25-3 and S25-4 showed that they had diverged evolutionarily from the majority of this viral genus based on the presence of mobile genetic elements, i.e., a putative transposase and the homing endonuclease I-MsaI. These results suggest that genetic elements such as transposases and homing endonucleases are likely to be involved with the evolution of some Twort-like phages, including phages S25-3 and S25-4.     

Inhibition by enkephalin of medial vestibular nucleus neurons responding to horizontal pendular rotation

Electrophysiological studies were performed to determine whether or not enkephalin modulates the activities of medial vestibular nucleus (MVN) neurons responding to horizontal pendular rotation using alpha-chloralose anesthetized cats. The effects of microiontophoretically applied drugs were examined in type I and type II neurons identified according to responses to horizontal, sinusoidal rotation; type I and type II neurons showed an increase and decrease in firing with rotation ipsilateral to the recording site and vice versa with contralateral rotation, respectively. Iontophoretic application of enkephalin suppressed spike firing induced by rotation of the animals in type I neuron, but not in type II neuron. The spike firing induced by iontophoretically applied glutamate was also inhibited during the application of enkephalin. The inhibition by enkephalin of both rotation- and glutamate-induced firing was antagonized by naloxone which was given simultaneously. These results suggest that enkephalin acts on MVN type I neuron to inhibit transmission from the vestibule, thereby controlling vestibulo-ocular reflex.     

Prostaglandin D2 formation and characterization of its synthetases in various tissues of adult rats

When the amounts of primary prostaglandins formed from endogenous arachidonic acid were determined in homogenates of various tissues of adult rats, prostaglandin D2 was the major prostaglandin found in most tissues. It was formed actively in the spleen (3100 ng/g tissue/5 min at 25 degrees C), intestine (2600), bone marrow (2400), lung (1100), and stomach (630); moderately in the epididymis, skin, thymus, and brain (140-340); and weakly in other tissues (less than 100). Addition of exogenous arachidonic acid (1 mM) accelerated the formation of prostaglandin D2 in all tissues as follows: spleen (15,000); bone marrow, intestine, thymus, liver, and lung (1600-5200); stomach, adrenal gland, epididymis, brain, salivary gland, skin, spinal cord, and seminal vesicle (380-1000); and other tissues (80-310). The activity of prostaglandin D synthetase (prostaglandin-H2 D-isomerase) was detected in 100,000g supernatants of almost all tissues. As judged by glutathione requirement for the reaction, inhibition of the activity by 1-chloro-2,4-dinitrobenzene, and immunotitration or immunoabsorption analyses with specific antibodies, the enzyme in the epididymis, brain, and spinal cord (1.8-9.2 nmol/min/mg protein) was glutathione-independent prostaglandin D synthetase (Y. Urade, N. Fujimoto, and O. Hayaishi (1985) J. Biol. Chem. 260, 12410-12415). The enzyme in the spleen, thymus, bone marrow, intestine, skin, and stomach (2.0-57.1) was glutathione-requiring prostaglandin D synthetase (Y. Urade, N. Fujimoto, M. Ujihara, and O. Hayaishi (1987) J. Biol. Chem. 262, 3820-3825). The activity in the kidney and testis (3.7-4.5) was catalyzed by glutathione S-transferase. The activity in the liver, lung, adrenal gland, salivary gland, heart, pancreas, and muscle (0.6-5.1) was due to both the glutathione-requiring synthetase and the transferase.