Effect of Bifidobacterium bifidum fermented milk on Helicobacter pylori and serum pepsinogen levels in humans.

Helicobacter pylori infection is an important risk factor for gastric diseases. Some probiotics are useful for suppressing H. pylori infection. Bifidobacterium bifidum YIT 4007 can improve the experimental gastric injury in rats and the disease stages on the gastric mucosa in peptic ulcer patients. We evaluated the fermented milk using a clone (BF-1) having the stronger ability to survive in the product than this parent strain to clarify the in vitro suppressive effect of BF-1 on H. pylori and the in vivo efficacy of BF-1 fermented milk on H. pylori and gastric health. In the mixed culture assay of BF-1 and H. pylori, the number of pathogens was decreased such that it was not detected after 48 h in the Brucella broth with a decrease in pH values. In the cell culture experiment with human gastric cells, the H. pylori infection-induced IL-8 secretion was suppressed by the preincubation of BF-1. In a human study of 12-wk ingestion (BF-1 group, n = 40; placebo group, n = 39) with a randomized double-blind placebo-control design, the H. pylori urease activity and gastric situation were evaluated using a urea breath test (UBT) and the serum pepsinogen (PG) levels as biomarkers for inflammation or atrophy, respectively. In the H. pylori-positive subjects, the difference (DeltaUBT) of the UBT value from the baseline value in the BF-1 group (n = 34) was lower than that in the placebo group (n = 35) at 8 wk. The baseline UBT values showed a negative correlation with DeltaUBT values at 8 and 12 wk in the BF-1 group but not in the placebo. In the PG-positive subjects classified by the PG test method, the BF-1 group was lower in DeltaUBT values than the placebo group at 8 and 12 wk. In the active gastritis class by PG levels, the BF-1 group was lower in their DeltaUBT values than the placebo at 8 and 12 wk. The PG I levels in the BF-1 group were lower than the placebo at 12 wk. The PG II levels in the BF-1 group did not change during the ingestion period, but the placebo was increased. The PG I/II ratios slightly decreased from baseline at 12 and 20 wk in the BF-1 and placebo groups. These patterns were also observed in the H. pylori-positive subjects. The improving rates of upper gastrointestinal symptomatic subjects and total symptom numbers in the BF-1 group were higher than those in the placebo. These results indicate that BF-1 fermented milk may affect H. pylori infection or its activity, gastric mucosal situation, and the emergence of upper gastrointestinal symptoms.

Fasudil (HA1077), an intracellular calcium antagonist, improves neurological deficits and tissue potassium loss in focal cerebral ischemia in gerbils.

The effects of an intracellular calcium antagonist fasudil (HA 1077) on protein synthesis, ion derangement, brain edema, and the neurological signs associated with focal cerebral ischemia, were investigated via a nine-hour occlusion of the left common carotid artery (CCA) in Mongolian gerbils. Protein synthesis was evaluated by the amount of incorporation of [14C]methionine into the tissue. Intravenous infusion of fasudil hydrochloride (1 mg kg-1) just after CCA ligation was effective in reducing neurological deficits six and nine hours after CCA ligation, as well as loss of tissue potassium. However, fasudil did not ameliorate the brain edema or the accumulation of sodium in the tissue. On the other hand, fasudil improved the uptake of [14C]methionine, but not its incorporation into the tissue. The improved uptake of tracer into the tissue may indicate the facilitation of collateral blood flow by fasudil. These results suggest that fasudil may mitigate ischemic damage in the penumbral zone, possibly by ameliorating collateral blood flow and preventing calcium-related cell damage.

Does the anabolic metabolism of L-[2-18F]fluorophenylalanine and L-[2,6-3H]phenylalanine differ in the cerebrum and the cerebellum?

The anabolism of isotopically labeled amino acids was compared between the cerebrum and the cerebellum in conscious rat at three feeding conditions. After L-[2-18F]fluorophenylalanine and L-[2,6-3H]phenylalanine injections, the incorporation rate of both radioactivity into protein fraction showed no difference between the cerebrum and the cerebellum at normal condition, but the lipid fraction in the cerebellum was higher than that in the cerebrum in any conditions. These results show the usefulness of L-[2-18F]fluorophenylalanine as a positron emission tomography tracer and different anabolic rate of the amino acids to lipid between the cerebrum and cerebellum.

Quantitative distribution of rat brain monoamine oxidase A by [14C]clorgyline autoradiography.

The distribution of functionally active monoamine oxidase type A (MAO-A) was investigated by in vivo quantitative autoradiography using [14C]clorgyline in normal, conscious rat brain. [14C]clorgyline was synthesized by the methylation reaction of N-desmethylclorgyline using [14C]methyliodide. Sixty minutes after [14C]clorgyline administration (1.58 MBq/animal i.v.), the brains were removed and prepared for autoradiography by washing the brain sections with 5% trichloroacetic acid solution to remove the nonbinding free tracer. The amount of MAO-A was calculated from the regional acid-insoluble tissue radioactivity and the specific activity of the tracer. The highest amount of MAO-A (5.84 nmol/g tissue) was found in the locus coeruleus. The interpeduncular nucleus, habenular nucleus, fasciculus retroflexus, and solitary tract nucleus possessed over 1.6 nmol/g tissue of MAO-A. Among 23 regions of interest, the lowest amount of MAO-A (0.37 nmol/g tissue) was found in the globus pallidus. The findings of this study suggest that the pattern of MAO-A parallels both in neuroanatomical distribution and in density that of norepinephrine and serotonin innervation. The MAO-A concentration was, however, relatively low in the dopamine-related areas. This corresponded to the previous results obtained by histochemical analysis. In addition, among the white matter structures, a high amount of MAO-A was found specifically in the fasciculus retroflexus.

L-[2-18F]fluorophenylalanine and L-[U-14C]phenylalanine: a comparative study of their transport to rat brain.

The transport rates of L-[2-18F]fluorophenylalanine ([18F]Phe) and L-[U-14C]phenylalanine ([14C]Phe) across the blood-brain barrier were compared in saline, large neutral amino acids (NAA) and large basic amino acids (BAA) loaded rats. The fraction of transported tracer (FTT) was expressed as the ratio of brain radioactivity to integrated plasma radioactivity at 5 min after injection of the tracers. Cerebral FTTs of [18F]Phe and [14C]Phe in the control group were 0.042 and 0.045, respectively, and decreased to 0.016 and 0.019 in the NAA loaded group. The ratios of FTT of [18F]Phe and that of [14C]Phe were approx. 0.90, and did not differ among the three groups. These results indicate that the transport of [18F]Phe from the blood to the brain can be considered to be mediated by the NAA transport system and that its transport rate is almost the same as that of [14C]Phe.

A convenient method for regional monoamine oxidase-A determination by [14C]clorgyline autoradiography.

The availability of clorgyline for regional monoamine oxidase-A (MAO-A) determination was examined using [14C]clorgyline in rat. [14C]Clorgyline was synthesized by the methylation reaction of N-desmethyl-clorgyline and [14C]methyliodide in dimethylformamide with high radiochemical yield. The MAO-A distribution map by autoradiography correlated with that by histochemical technique and its quantity was consistent with the calculated MAO-A amount based on previous reports. The combination of labeled clorgyline and autoradiographic technique will promise the quantitative measurement of regional MAO-A distribution.

Molecular characterization of monoclonal anti-steroid antibodies: primary structures of the variable regions of seven antibodies specific for 17 alpha-hydroxyprogesterone or 11-deoxycortisol and their pH-reactivity profiles.

The variable region nucleotide sequences of seven monoclonal anti-steroid antibodies that are specific for the closely related progesterone derivative, 11-deoxycortisol or 17 alpha-hydroxyprogesterone (17-OHP), have been determined by genomic cloning and DNA-sequencing or by direct mRNA-sequencing. As for their heavy chain variable regions, the nucleotide sequences of the SCET.M8.1 (SCET) and OHP 4B2.2.1 (4B2) antibodies were classified into the VH-9 family, while OHP 7D7.2.3 (7D7), 1E9.3.1 (1E9), 57.G6.1 (57) and 138.H8.1 (138) used VH-3 family genes. OHP 101.B11.1 (101) used a gene of the VH-1 family. For their light chain variable regions, SCET and 57 used VK-28 group genes, while 4B2, 7D7, 1E9, 101 and 138 antibodies used genes of the VK-21 subgroups (21A, 21B or 21C). All of the antibodies used different combinations of genes in the VH families and VK groups or subgroups. This indicates that the antibody response against the steroid hapten, 17-OHP, is fairly polyclonal, and several VH/VL combinations show high affinity for progesterone-related steroids. Although the primary structures of hypervariable loop regions of the mAbs were relatively diverse, generally, hydrophobic and aromatic amino acids were rich in these regions. Moreover, the length of heavy chain CDR3 was constant in all the antibodies investigated in this paper as well as the previously reported anti-progesterone monoclonal antibodies (mAbs). This suggests that the length of VH CDR3 in these mAbs has a considerable influence on the formation of antigen-combining pockets. The pH-reactivity profiles for the anti-17-OHP mAbs indicated that the the steroid-mAb binding was independent of pH between pH 4 and 11 in most of the mAbs. The results suggest that the steroid-mAb interactions are not largely affected by the electrostatic environments near the combining sites of these mAbs. Taken together, these data imply that the shape of hydrophobic depressions in the combining sites is important for the binding of relatively large, hydrophobic and rigid haptens like steroids.

Effects of HA1077, an intracellular calcium antagonist, on neurotransmitter metabolism in rat brain in vivo.

The effect of HA1077, an intracellular calcium antagonist, on neurotransmitter metabolism in rat brain was investigated in vivo. After administration of HA1077, at doses of 0.1, 0.3, and 3 mg/kg, 5-hydroxyindoleacetic acid (5-HIAA) levels increased in most regions except midbrain. In the striatum, parallel increases of both serotonin (5-HT) and 5-HIAA levels were observed at 0.3 mg/kg, but only the 5-HT level increased at 0.1 mg/kg. These results suggest that HA1077 may activate the turnover or synthesis of 5-HT. After administration of HA1077 at 0.3, 1, and 3 mg/kg, the dopamine (DA) level was increased in the striatum, but 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid levels were unchanged. After HA1077 administration at 1 mg/kg, both DA and DOPAC levels increased in the hypothalamus and only DA level increased in the cerebral cortex. By contrast, DOPAC level decreased in the midbrain after HA1077 treatment at 0.1 and 0.3 mg/kg, and in the brainstem at 0.1 and 10 mg/kg. The ratio of [3H]-N-methylspiperone accumulation relative to that in the cerebellum did not change after HA1077 treatment at any of the doses employed. Thus, the effects of HA1077 on neurotransmitter metabolism are complex and vary depending on the dosage and sites of the brain. Although the dose-dependent effects of HA1077 on neurotransmitter metabolism are similar to those of calcium entry blockers, HA1077 can facilitate DA synthesis in the hypothalamus and striatum, unlike the calcium entry blockers.

Effect of thyrotropin-releasing hormone on cerebral blood flow in conscious rat.

The effect of thyrotropin-releasing hormone (TRH) was studied on local CBF (LCBF) in normal conscious rats. LCBF was measured by the autoradiographic [14C]iodoantipyrine method 5 min after TRH (5 mg/kg, i.v.) administration. TRH significantly increased LCBF in 22 of 33 brain regions. This increase of LCBF exceeded 100% of the control values in the cerebral cortices, whereas there was no significant increase in white matter or in some gray matter structures. The increase of CBF following TRH administration was abolished by pretreatment with indomethacin (5 mg/kg, i.v.). The mechanisms underlying the increase of CBF following TRH administration are discussed in relation to prostaglandin metabolism.

Effect of methotrexate on local cerebral blood flow in conscious rats.

The effect of methotrexate (MTX) on local cerebral blood flow (I-CBF) was studied with autoradiographic [14C]-iodoantipyrine methods in normal conscious rats. I-CBF was reduced by 30-57% in the rats given MTX (100 mg or 200 mg/body) as compared to that in the saline injected control group, but no dose-dependent effect was observed. The mechanism of I-CBF reduction by MTX was discussed.

[Effects of guanfacine on the levels of cyclic nucleotides in anesthetized rat brain regions].

Effects of an antihypertensive drug, guanfacine, on brain regional cyclic AMP and cyclic GMP levels were studied in anesthetized rats. Cyclic nucleotides were analyzed in seven brain regions. Guanfacine decreased blood pressure and heart rate 20 min after administration. Yohimbine inhibited these hemodynamic effects of guanfacine. Guanfacine reduced cyclic AMP levels in the hypothalamus. The reducing effect of guanfacine on cyclic AMP was antagonized by yohimbine in the hypothalamus. Guanfacine lowered cyclic GMP in the cerebellum, medulla oblongata and hypothalamus. Yohimbine inhibited the effect of guanfacine on cyclic GMP in the cerebellum, medulla oblongata and hypothalamus. Prazosin showed no effect on guanfacine induced change of cyclic nucleotides in any brain region. From these results, it is concluded that guanfacine decreased cyclic AMP and cyclic GMP in the hypothalamus. In addition, it is suggested that alpha-2 adrenoceptors mainly modulate these changes of cyclic nucleotides.

[Effects of clonidine on the levels of cyclic nucleotides in rat brain regions: the change in cyclic nucleotides after clonidine alone or in combination with alpha-antagonists].

The following 4 Wistar rat groups were sacrificed by microwave irradiation after measuring blood pressure and heart rate: 1) saline control (1 ml/kg, i.v.), 2) clonidine alone (50 micrograms/kg, i.v.). 3) prazosin pretreated (0.1 mg/kg, i.v.), 4) yohimbine pretreated (1 mg/kg, i.v.). Cyclic nucleotides were analyzed in seven brain regions. Clonidine decreased blood pressure and heart rate 20 min after administration. These effects of clonidine were inhibited by yohimbine. Clonidine increased the levels of cyclic AMP in the medulla oblongata (including pons) and hypothalamus. Prazosin attenuated the cyclic AMP-increasing action of clonidine in the cerebellum. Yohimbine inhibited this action of clonidine in the cerebellum, striatum and hippocampus. Clonidine reduced cyclic GMP levels in the hypothalamus and striatum. Prazosin potentiated the cyclic GMP-reducing action of clonidine in the medulla oblongata. Yohimbine attenuated this action of clonidine in the medulla oblongata and hypothalamus. From these results, it is concluded that clonidine changes the levels of cyclic nucleotides in brain regions, especially in the part of the autonomic nervous center. In addition, it is indicated that alpha 1- or alpha 2-adrenoceptor plays a different role in the regulation of brain cyclic nucleotides, region by region.

Conditionally lethal nusAts mutation of Escherichia coli reduces transcription termination but does not affect antitermination of bacteriophage lambda.

Termination of transcription at bacteriophage lambda terminators as well as at the Escherichia coli trp a attenuator was examined in the conditionally lethal mutant (nusAts11) defective in the NusA protein of E. coli. Experiments using terminator-assay lambda vectors revealed that the efficiency of termination at both rho-dependent (lambda tL1) and rho-independent (lambda tL2 and trp a) terminators decreases in the mutant. The mutation does not block lambda phage growth at either permissive or nonpermissive temperatures, nor does it affect the lambda Q protein antitermination activity at the t6s terminator. These results indicate that NusA is required for transcription termination, and that lambda N and Q-mediated antitermination may not require the NusA protein function in the nusAts11 mutant.

Evaluation of N-isopropyl-[125I]p-iodoamphetamine affinity sites in rat brain by autoradiography.

Affinity sites of N-isopropyl-[125I]p-iodoamphetamine (IMP) were studied in rat brain employing in vitro technique of receptor autoradiography. High degrees of IMP accumulation were observed in almost regions of white matter and some of gray matter. Phenethylamine, glutamic acid and serotonin suppressed IMP accumulation in all brain regions, in cerebral cortex and in hypothalamus, respectively. These results indicated that IMP was accumulated mainly due to its physicochemical property of lipophilicity and might have some interactions with neurotransmitter receptors.

Nucleotide sequence of the coding region of the mouse N-myc gene.

A genomic clone for the mouse N-myc gene was isolated and the total nucleotide sequence (4807 bp) of the two coding exons and an intron located between them was determined. The amino acid sequence of the N-myc protein was deduced from the DNA sequence. This protein is composed of 462 amino acids, slightly larger than human and mouse c-myc proteins, and is rich in proline like the c-myc protein. Comparison of the amino acid sequences of the mouse N-myc and c-myc proteins showed that conserved sequences are located in eight regions: four regions are in the N-terminal half of the N-myc protein and are separated from each other by regions poorly homologous to those of the c-myc protein, and the four others are located in the C-terminal half, throughout which certain homology exists. A remarkable sequence containing 13 successive acidic amino acids is present in one of the conserved regions located in the middle of the N-myc protein.

Regulatory defects of a conditionally lethal nusAts mutant of Escherichia coli. Positive and negative modulator roles of NusA protein in vivo.

Previous studies have attributed two activities to the NusA protein of Escherichia coli; namely, termination and antitermination of transcription. To examine these activities, we isolated a temperature-sensitive mutant of the nusA gene (nusAts11). The mutant cells produce a thermolabile NusA protein and grow at 32 degrees C, but not at 42 degrees C. At 42 degrees C, nusAts11 is recessive to nusA+ and nusA1, indicating the absence of its active gene product at that temperature. In the mutant, the efficiency of termination at the lambda tR1 terminator decreases, resulting in an increased expression of distal gene(s). On the other hand, the synthesis of the beta-galactosidase and beta beta' subunits of RNA polymerase is reduced in the mutant. This mimics effects seen in vitro when NusA protein is removed from a coupled transcription-translation system. These results suggest that the NusA protein plays both negative and positive modulator roles in vivo. The mutation nusAts11, unlike nusA1, does not block lambda phage growth at non-permissive temperatures, suggesting that NusA protein is not required for N antitermination in the mutant. Besides, the nusAts11 allows lambda Nam7Nam53byp phage growth under sup0 conditions, indicating that the N antitermination function is dispensable (at least partly) in this mutant.

Improvement of the dideoxy chain termination method of DNA sequencing by use of deoxy-7-deazaguanosine triphosphate in place of dGTP.

The dideoxy chain termination method using deoxy-7-deazaguanosine triphosphate (dc7GTP) in place of dGTP was found to be very useful. Sequencing of a part of the human N-myc gene having 85% GC content is impossible by the original method using dGTP, because of compression of bands. However, the nucleotide sequence of this part was unambiguously determined by analysis of both strands by the modified method. Use of dc7GTP is concluded to improve the dideoxy chain termination method for DNA sequencing.

In vivo evidence that the nusA and infB genes of E. coli are part of the same multi-gene operon which encodes at least four proteins.

Previous work has shown that the Escherichia coli nusA gene codes for a protein which regulates transcription termination. The 16.0-kb EcoRI DNA fragment that includes the nusA gene, codes for at least eight bacterial proteins of mol. wts. 48 000 (argG), 21 000 (p21), 64 000 (nusA), 120 000 (IF2 alpha)-(infB), 91 000 (IF2 beta)(infB), 15 000 (p15), 10 000 (rpsO) and 85 000 (pnp). We have constructed several deletion and fusion derivatives from this cloned DNA and examined in vivo the structure and expression of these genes. First, the promoter functional in vivo for the nusA gene was mapped at approximately 800 bp upstream of the nusA structural gene. Second, the synthesis of five proteins, p21, NusA, IF2 alpha, IF2 beta (and p15) proteins, was affected by the deletion of the nusA promoter. Third, these same five proteins were hyperproduced after fusion of the DNA fragment to the lambda pL promoter. In addition, subcloning experiments revealed that the p15 gene is expressed by the read-through transcription from the infB gene. These results lead us to conclude that the genes coding for the p21, NusA, InfB (IF2 alpha and IF2 beta), and p15 proteins form a single-transcriptional unit ('nusA-infB operon') in vivo and that rpsO and pnp genes do not belong to the same operon. The in vivo attenuation site of this operon is described.