Affinity purification of Helicobacter pylori urease. Relevance to gastric mucin adherence by urease protein.

A simple, reproducible and high yield method of Helicobacter pylori urease enzyme purification was developed using a heparinoid (Cellufine sulfate) affinity gel. The purification method involved two sequential steps using the same gel that takes advantage of the differential affinity of urease to the heparinoid at two levels of hydrogen ion concentration. SDS-polyacrylamide gel electrophoresis analysis of affinity-purified urease revealed two major protein bands with about 62- and 30-kDa molecular mass. When whole cell lysates of clinical and laboratory strains of H. pylori were probed by Western blot, anti-urease hyperimmune serum produced by affinity-purified urease in rabbit recognized only the two bands corresponding to the urease A and B subunits. To probe the molecular relevance of affinity gel adherence to mucin adherence, the purified urease was derivatized with N-hydroxysuccinimidobiotin and used in adherence assays. Competitive inhibition tests revealed commonality of urease receptors among gastric mucin, heparin, and heparinoid. Composite data on adherence kinetics modulated by pH, salt, incubation time, and concentration of urease or mucin were indicative of conformation-dependent ligand-receptor interaction.

Oral passive immunization against experimental salmonellosis in mice using chicken egg yolk antibodies specific for Salmonella enteritidis and S. typhimurium.

The efficacy of chicken egg yolk homotypic antibodies specific for outer membrane proteins (OMP), lipopolysaccharide (LPS) or flagella (Fla) in controlling experimental salmonellosis in mice was investigated. Mice challenged orally with 2 x 10(9) c.f.u. of Salmonella enteritidis or 2 x 10(7) c.f.u. of S. typhimurium were orally treated with 0.2 ml anti-OMP, -LPS or -Fla yolk antibody three times a day for three consecutive days. In mice challenged with S. enteritidis, antibody treatment resulted in a survival rate of 80%, 47% and 60% using OMP, LPS or Fla specific antibodies respectively, in contrast to only 20% in control mice. In the S. typhimurium trial, survival rate was 40%, 30% and 20% using OMP, LPS or Fla specific antibodies respectively in contrast to 0% in control mice. In vitro adhesion of S. enteritidis and S. typhimurium to HeLa cells was significantly reduced by anti-OMP, -LPS, and -Fla homotypic antibodies. Results suggest that egg yolk antibodies specific for Salmonella OMP, LPS, and Fla may protect mice from experimental salmonellosis when passively administered orally. Of these antibodies, anti-OMP exhibited the highest level of protection in vivo and in vitro.

Prevention of fatal salmonellosis in neonatal calves, using orally administered chicken egg yolk Salmonella-specific antibodies.

OBJECTIVE: To protect neonatal calves against fatal salmonellosis within the first 2 weeks after birth, using chicken egg yolk antibodies specific against Salmonella typhimurium or S dublin. ANIMALS: 38 neonatal Holstein calves from Salmonella-free farms. PROCEDURE: After removal of the lipid components with hydroxypropylmethylcellulose phthalate, egg yolk antibodies were spray dried. At 4 days of age, calves were challenge exposed by oral inoculation with 10(11) virulent S typhimurium (experiment 1) or S dublin (experiment 2). Starting from the challenge-exposure day, egg yolk antibody preparations were administered orally 3 times a day for 7 to 10 days. RESULTS: In passive immunization trials, the orally administered antibodies conferred dose-dependent protection against infection with each of the homologous strains of Salmonella. Within 7 to 10 days after challenge exposure, all control calves died, whereas low-titer antibody-treated calves had 60 to 100% mortality. Only fever and diarrhea, but no deaths (P < 0.01), were observed in calves given the highest titer of antibody. CONCLUSIONS AND CLINICAL RELEVANCE: Compared with that in control calves, survival was significantly higher among calves given antibodies with titers of 500 (P < 0.05) and 1,000 (P < 0.01) homotypic for S typhimurium and with titer of 5,000 (P < 0.01) for S dublin. Egg yolk antibodies specific for whole cell S typhimurium or S dublin are protective against fatal salmonellosis when given in sufficiently high concentration, and may be clinically useful during a salmonellosis outbreak.

Effect of oral egg antibody in experimental F18+ Escherichia coli infection in weaned pigs.

The protection conferred by egg antibody specific for F18-fimbriae against infection with F18+ Escherichia coli was studied in controlled passive immunization trials involving weaned pigs. Parameters of protection consisted of body weight gain, frequency and severity of diarrhea and recovery of the challenge strain of F18+ E. coli. Weaned pigs at four weeks of age were challenge exposed once daily for three days by oral inoculation with 10(11) cfu of virulent F18+ E. coli followed by daily administration of antibody supplemented feed for 9 days starting from the first challenge day 0. Results showed a dose-dependent response to antibody treatment. The group of pigs given 1:50 titer of antibody in feed had less frequency of diarrhea (P < 0.01-0.05), higher rate of gain (P < 0.01) and lower isolation rate of challenge strain in rectal and intestinal swabs (P < 0.01) compared to non-treated control. In the same manner, the anti-F18 antibody significantly reduced adherence of F18+ E. coli to pig intestinal epithelial cells in vitro (P < 0.01). Results suggest that egg antibodies specific for the F18 fimbriae is a suitable immunotherapeutic agent for pigs infected with F18+ E. coli and that pigs can be protected from overt clinical disease and the subsequent reduced performance arising from infection with this pathogen.

Chronological sequences and blood-brain barrier permeability changes in local injury as assessed by nuclear magnetic resonance (NMR) images from sliced rat brain.

Two experiments were done with a prototype mini-NMR imager to evaluate the potential application of nuclear magnetic resonance (NMR) imaging in neuropathology. Cryo-injury-induced brain edema in brain slices from 22 adult male rats was imaged for observing the chronological sequences. Blood-brain barrier permeability changes were evaluated in 12 other brain slice images. EDTA-2Na-Mn solution was intravenously injected as an indicator of blood-brain barrier permeability. Contrast enhancement was achieved by changing the NMR imaging parameters. High resolution imaging permitted visualization of the corpus callosum, the thickness of which was only 0.2-0.4 mm. The extent of edema in gray matter was clearly shown with a striking contrast; no consistent findings were seen with slight differences in water content between edema and the surrounding normal cortex. As a result, the chronological sequences of brain edema were clearly observed. Mn-EDTA leaking from the circulating blood through the damaged capillary wall had a "paradoxical enhancement" effect on the NMR images; this effect might be suitable for evaluating blood-brain barrier permeability changes in NMR images.

High-resolution synchronized-quantum-beat spectroscopy using modulation sidebands: measurement of pressure shifts of the hyperfine splitting in sodium 3(2)S((1/2)).

Pressure shifts of the hyperfine splitting in Na 3(2)S((1/2)) that are due to buffer gases have been measured for the first time to our knowledge by all-optical means using the method of synchronized-quantum-beat spectroscopy reported previously [Y. Fukuda et al., Opt. Commun. 38, 357 (1981)]. The substate coherence is excited and detected with amplitude-modulated mode-locked laser pulses tuned to the D(1) line (pulse-repetition frequency, f(p); modulation frequency, f(m)), and the resonances observed at f(0) = nf(p) + nf(m), (n and n' are integers) are used for the measurement. The pressure-shift coefficients obtained at 210 degrees C are 82, 61, -1, and -47 Hz/Torr for He, N(2), Ar, and Kr, respectively, within an accuracy of +/-3 Hz/Torr.

Sublevel echoes selectively excited by light-pulse trains: synchronized-quantum-beat echoes.

We report on a new type of sublevel echoes, synchronized-quantum-beat (SQB) echoes, generated by a successive application of two resonant light-pulse trains. The echoes are selectively generated for a sublevel pair whose frequency splitting is equal to an integral multiple of the repetition frequency of the light pulses. By using mode locked laser pulses tuned to the D(1) transition, we observed SQB echoes (and free induction decay) for all Zeeman and hyperfine transitions in the ground state of sodium atoms in low magnetic fields.

[Nuclear magnetic resonance (NMR) imaging of brain edema from sliced rat brain: preliminary report].

A series of the sliced rat brain were imaged by proton nuclear magnetic resonance (1H-NMR) using a Carr-Purcell-Meiboom-Gill sequence. The slices were obtained from the adult male Wistar rats, normal or suffering from vasogenic brain edema at 2, 6, 24Hr, 2Wk and 2Mo period after the application of cryo-injury. The imaging time was arranged 17 to 22 minutes dependently upon signal to noise ratio. The slice thickness was 1.2 mm, and pixel dimensions were 0.2 X 0.2 mm. Whereas a voxel size in our images is mere 1/1,200 compared to that reported about the prototype human NMR imaging devices, high resolution has been realized. The spatial resolution is very fine, as evidenced by the appearance of major internal structures of rat brain, and the object contrast is so high that cerebral white-gray contrast is excellent, although the difference in water concentration between them is only 7%. It has been impossible to measure serially in vivo change in water concentration of small organs as rat brain, NMR has the capability to generate images in response to tissue state of hydration, therefore we can easily recognize the extent and intensity of the brain edema, which has been defined as an expansion of the brain volume resulting from an increase in its fluid content. And in this study, using the prototype mini-NMR-CT of excellent spatial and contrast resolution, we have been able to show this advantage of NMR as a new tool for research in the field of neurological surgery.

[Intravenous chronic toxicity of lentinan in rats: 6-month treatment and 3-month recovery (author's transl)].

Chronic toxicity of lentinan was studied in male and female JCL : SD rats. Lentinan was given intravenously into tail vein. Dosage levels employed were 0 (5% mannitol), 0.01, 0.1, 1 (with or without dextran), and 10 mg/kg/day for 6 months in a volume of 1 ml/100 g body weight. After 6 months, the treatment was discontinued and a recovery study was performed for 3 months. Rats receiving 10 mg/kg had redness and necrosis of the tail, the treatment was stopped at week 5, and the rats were sacrificed. Rats receiving 1 mg/kg showed redness of the ear, tail, and scrotum, which was remarkable in the 2nd and 3rd months. Body weight gains were not adversely affected. Laboratory examinations revealed an increase in leukocyte count, decreases in differential eosinophil count and platelet count, and an increase in serum beta-globulin level in drug-treated rats. At autopsy after 6 months, rats from the drug-treated groups had pulmonary hemorrhage and enlargements of the spleen and mesenteric lymph nodes. Histologic changes attributable to treatment included (1) activation of reticulo-endothelial system such as small epithelioid cell nodule in the liver, spleen, and mesenteric lymph nodes, and mobilization of Kupffer cells; (2) arteritis in various organs, especially notable in the spleen, testis, and epididymis ; (3) hemorrhage in the lung; and (4) hypospermatogenesis. All these changes described above had a propensity to recover. The maximum no effect level was estimated to be less than 0.01 mg/kg in the present study in male and female rats.

[Chronic toxicity test of 4-ethoxy-2-methyl-5-morpholino-3(2H)-pyridazinone (M73101) in beagle dogs (author's transl)].

Male and female dogs, aged 17--21 months, were administered orall M 73101 (0, 60, 120 and 240 mg/kg/day), a new analgesic and antiinflammatory drug, for 27 weeks, and following recovery test was carried out for 5 weeks. Dead animals were not found throughout the experimental period. Body weight gain, and food and water consumption were not affected due to M 73101 administration. Except for a slight increase of vomitting in the highest dose, there were no abnormal symptoms. Biochemical examination showed the slight increase in serum alkaline phosphatase activity and free cholesterol level. Pathological examination revealed a dose-dependent increase of liver weight and hypertrophy of hepatocytes due to proliferation of smooth endoplasmic reticulum. In addition, mitochondria became irregularly large in the highest dose. There were no abnormal findings in the gastro-intestinal tracts except for an erosion of gastric mucosa, which was noted in a female dog treated 240 mg/kg/day of M 73101. From these results, it was suggested that the maximum non-toxic dose was 60 mg/kg/day or less, and the greatest safety dose was 120 mg/kg/day in beagle dogs.

[Subacute toxicity test of AMI-U-II, a new amino acid solution for renal failure (author's transl)].

Subacute toxicity test of AMI-U-II was carried out using male and female JCL:SD rats. The animals were given intravenously AMI-U-II (80, 40 and 20ml/kg) or reference solution (80 and 40ml/kg) for five weeks. Tachypnea, depression of spontaneous activity, blepharoptosis and edema of face were observed in rats treated with AMI-U-II or reference solution at highest dose. Food consumptions and gaining body weight were slightly reduced in male of these animals, but water intakes were increased in both sexes. Autopsies of the animals which died during five weeks showed pulmonary congestion and/or edema, ascites and pleural effusion. Microscopically, hydropic degeneration of liver cells and dilation of renal tubules and Bowman's capsules were shown. It seems likely that most of these findings were caused by hypervolumic administration of amino acid solution.