The binding and utilization of hemoglobin by Prevotella intermedia.

Prevotella intermedia, a putative periodontopathic microorganism, requires iron for growth. Hemoglobin can be a major source of iron for bacterial growth in vivo since it is present in the crevicular fluid collected from periodontitis sites. Experiments studying the growth of P. intermedia in iron-depleted Todd-Hewitt broth supplemented with human hemoglobin showed that the bacteria were able to utilize human hemoglobin as a source of iron. The uptake of iron from hemoglobin by P. intermedia appears to be initiated by the binding of hemoglobin to the bacteria as shown by direct binding studies using 125I-labeled human hemoglobin. Scatchard analysis of saturation binding data revealed that 125I-labeled human hemoglobin had a dissociation constant (Kd) of 2.53 x 10(-8) M for the receptor on P. intermedia. Binding of labeled hemoglobin to P. intermedia was competitively inhibited by unlabeled human hemoglobin showing that the binding was specific. The ability of bovine hemoglobin, but not hemin or non-hemoglobin heme-containing compounds, to inhibit binding competitively suggested that the globin moiety of the hemoglobin molecule is recognized by the hemoglobin binding receptors.

Human galactocerebrosidase gene: promoter analysis of the 5'-flanking region and structural organization.

Galactocerebrosidase (GALC; EC 3.2.1.46) is a lysosomal enzyme which hydrolyzes several galactolipids and the deficiency of GALC is responsible for Krabbe disease. Recently, we cloned cDNAs for human and murine GALC. In this study we characterized the genomic organization and the promoter of the human gene. The gene was about 60 kb in length and consisted of 17 exons as reported by Luzi et al. DNA sequence analysis showed that the 5'-flanking region of the first exon was GC-rich and had not typical TATA-box but ten GC-box-like sequences within a 200 bp sequence upstream from the initiation codon. Another inframe ATG, which has better Kozak consensus sequence, was found at 48 bp upstream to the first ATG reported]. Promoter analysis using a luciferase assay in COS 7 cells showed that the -149 to -112 nucleotide (from the initiation codon A) region has dominant promoter activity. In this region three GC-box-like sequence and one YY1 binding site were detected. Primer extension revealed several transcription start sites within the region of -146 to -103 nucleotide. In this study we firstly demonstrated that the YY1 binding site and subsequent GC-box-like sequences could be a promoter in a housekeeping gene.

Differential effect of codeine on coughs caused by mechanical stimulation of two different sites in the airway of guinea pigs.

We studied the difference in the effects of codeine on coughs caused by mechanical stimulation to the larynx and to the bifurcation of the trachea in lightly anaesthetized guinea pigs. Mechanical stimulation to the larynx or the bifurcation of trachea caused a stable cough response. The response was reproducible over 60 min, when stimulation was repeatedly applied at 20-min intervals. No significant difference was found between the amplitudes of the responses to mechanical stimulation of the larynx and of the tracheal bifurcation. Codeine, 10, 20 and 50 mg/kg, dose dependently depressed the coughs caused by larynx stimulation. The antitussive, however, failed to depress the cough caused by stimulation to the tracheal bifurcation, although a large dose, 50 mg/kg, significantly depressed the cough. In capsaicin-treated guinea pigs, codeine at 20 mg/kg significantly depressed the cough caused by stimulation to the tracheal bifurcation. The present results suggest that cough caused by mechanical stimulation to the larynx might be more sensitive to codeine treatment than cough caused by stimulation to the bifurcation of trachea. Furthermore, it is suggested that coughs caused by mechanical stimulation to both sites might consist of at least two components as regards their pharmacological nature.

The RY sequence is necessary but not sufficient for the transcription activation of a winged bean chymotrypsin inhibitor gene in developing seeds.

A winged bean Kunitz-type chymotrypsin inhibitor (WCI) is expressed in seeds and tuberous roots. In seeds, the expression of WCI is restricted to the period between the mid- and late-maturation stage. To understand the mechanisms that regulate the expression of WCI genes, we analyzed the promoter activity of the upstream region of the WCI-3b gene, which encodes a major WCI protein, in transgenic tobacco plants. By using a series of constructs with 5' deletions in the upstream sequences, the region between -882 and -623, relative to the transcription start site, was shown to contain multiple sequences which are responsible for high level expression in mid-maturation stage seeds. However, when this region was fused to the cauliflower mosaic virus 35S core promoter in both orientations, the chimeric promoters showed only a weak transcription activity in transgenic tobacco plants. Further analyses using internal deletion constructs revealed that the region between -882 and -174 is required for the transcription activation. Disruption of the RY sequence at -517, which is conserved in many seed protein genes, resulted in a drastic reduction of the transcription activity in seeds. These results suggest that sequences necessary for high level induction of the WCI-3b gene transcription in developing seeds are dispersed in the region between -882 and -174, and that the RY sequence is one of these sequences.

A gene encoding a major Kunitz proteinase inhibitor of storage organs of winged bean is also expressed in the phloem of stems.

Winged bean Kunitz chymotrypsin inhibitor (WCI) accumulates abundantly in seeds and tuberous roots, and small amounts of the WCI protein and mRNA can also be detected in stems. In this study, we analyzed the localization of the WCI protein in stems of winged bean. The results demonstrated that the WCI protein was localized in sieve tubes. Furthermore, we showed that the 5' region of the WCI-3b gene, which exhibited strong transcriptional activity in developing seeds, also promoted transcription of a reporter gene in the phloem of stems of transgenic tobacco.

Structures and contribution to the antigenicity of oligosaccharides of Japanese cedar (Cryptomeria japonica) pollen allergen Cry j I: relationship between the structures and antigenic epitopes of plant N-linked complex-type glycans.

The oligosaccharide structures of Cry j I, a major allergenic glycoprotein of Cryptomeria japonica (Japanese cedar, sugi), were analysed by 400 MHz 1H-NMR and two-dimensional sugar mapping analyses. The four major fractions comprised a series of biantennary complex type N-linked oligosaccharides that share a fucose/xylose-containing core and glucosamine branches including a novel structure with a nongalactosylated fucosylglucosamine branch. Rabbit polyclonal anti-Cry j I IgG antibodies cross-reacted with three different plant glycoproteins having the same or shorter N-linked oligosaccharides as Cry j I. ELISA and ELISA inhibition studies with intact glycoproteins, glycopeptides and peptides indicated that both anti-Cry j I IgGs and anti-Sophora japonica bark lectin II (B-SJA-II) IgGs included oligosaccharide-specific antibodies with different specificities, and that the epitopic structures against anti-Cry j I IgGs include a branch containing alpha 1-6 linked fucose and a core containing fucose/xylose, while those against anti-B-SJA-II IgGs include nonreducing terminal mannose residues. The cross-reactivities of human allergic sera to miraculin and Clerodendron Trichotomum lectin (CTA) were low, and inhibition studies suggested that the oligosaccharides on Cry j I contribute little or only conformationally to the reactivity of specific IgE antibodies.

Ginsenoside-Ia: a novel minor saponin from the leaves of Panax ginseng.

A new saponin, named ginsenoside-I (a), was isolated from leaves of Panax Ginseng, together with nine known saponins, and its structure was elucidated as 3beta,6alpha,12beta,20(S)-tetrahydroxyldammar-24(25)-ene (20- O-beta-D-glucopyranosyl)-3-O-beta-D-glucopyranoside on the basis of chemical and 2D-NMR methods.

Transcriptional activities of a winged bean Kunitz chymotrypsin inhibitor gene promoter in stable and transient expression systems.

Sequential deletions of the promoter region of the WCI-3b gene, which encodes the major chymotrypsin inhibitor of winged bean, were constructed and their expression was analyzed in transgenic tobacco plants and in bombarded winged bean seeds. In transgenic tobacco plants, a critical promoter region which is important for high levels of expression in seeds was identified, but deletion of this region had essentially no effect when bombarded into winged bean seeds.

Effect of KRN2391, a novel vasodilator, on various experimental anginal models in rats.

The antianginal effect of KRN2391, N-cyano-N'-(2-nitroxyethyl)-3-pyridinecarboximidamide monomethanesulfonate, on various anginal models in rats was compared with those of nifedipine and nicorandil. Angina pectoris was induced by methacholine or isoproterenol, and the change in the ST-segments in the electrocardiogram (ECG) was used as the parameter to indicate angina pectoris. The intracoronary administration of methacholine (3 micrograms) produced an elevation in the ST-segment of the ECG. This ST-elevation was inhibited by the intravenous administration of KRN2391 (30 and 100 micrograms/kg), nifedipine (100 and 300 micrograms/kg) and nicorandil (1000 and 3000 micrograms/kg). The administration of isoproterenol (10 micrograms/kg/min, i.v.) produced a depression of the ST-segment of the ECG. The intravenous administration of KRN2391 (100 micrograms/kg), nifedipine (100 micrograms/kg) and nicorandil (3000 micrograms/kg) inhibited the ECG changes induced by isoproterenol. These results suggest that KRN2391 exerts a potent protective effect on angina pectoris models compared with nifedipine and nicorandil. KRN2391 appears to be useful as an antianginal drug.

Abnormal glucagon response to arginine and its normalization in obese hyperinsulinaemic patients with glucose intolerance: importance of insulin action on pancreatic alpha cells.

An excessive glucagon secretion to intravenous arginine infusion was found in obese hyperinsulinaemic patients with glucose intolerance. This study was designed to determine whether the glucagon hyperresponsiveness to arginine in these patients would improve by insulin infused at a high enough dose to overcome insulin resistance. By infusing high dose insulin during arginine infusion, the previously exaggerated glucagon response to arginine could be normalized. To normalize the abnormal glucagon response, insulin doses of 4.2 +/- 0.7 and 3.8 +/- 0.5 IU were required during arginine infusion in obese hyperinsulinaemic patients with impaired glucose tolerance and Type 2 (non-insulin-dependent) diabetes mellitus, respectively. This achieved plasma peak insulin levels 3 to 4 times higher than those observed in non-obese healthy subjects. Furthermore, we clarified whether or not the effect of normalizing insulin action and/or glycaemic excursions contributed to normalizing the exaggerated glucagon response to arginine in these patients. Blood glucose was clamped while high dose insulin was infused at the same levels as observed during the arginine infusion test with no insulin infusion. As a result, normalization of the exaggerated plasma glucagon response was achieved, whether hyperglycaemia existed or not. These results clearly demonstrate that, similar to non-obese hypoinsulinaemic Type 1 (insulin-dependent) and Type 2 (non-insulin-dependent) diabetic patients, the exaggerated Alpha-cell response to arginine infusion in obese hyperinsulinaemic patients with glucose intolerance is secondary to the reduction of insulin action on the pancreatic Alpha cell, and that the expression of insulin action plays an important part in normalizing these abnormalities.

[Hepatocellular carcinoma with a complete remission of bone metastasis achieved by arterial chemotherapy].

Reported is the case of a hepatocellular carcinoma with a complete remission of the bone metastasis by arterial chemotherapy. The patient was 60 year old male, with the chief complaint being a tumor at the right side of the chest. The diagnosis was a hepatocellular carcinoma with a bone metastasis at the right 10th rib. The bone tumor showed no decrease in size after an intra-arterial injection of adriamycin, and radiation and or hyperthermia. Therefore, intra-arterial injections of mitomycin C mixed with lipiodol were instituted through the intercostal artery, and no bone tumor was noted after start of this therapy. Although the patient subsequently died, microscopic examination of a specimen obtained at autopsy revealed no malignant cells at the right 10th rib. Intra-arterial injections of anticancer agents mixed with lipiodol therefore are thought to be useful for the treatment of a bone metastasis.

Antimicrobial susceptibilities of Eubacterium, Peptostreptococcus, and Bacteroides isolated from root canals of teeth with periapical pathosis.

Eubacterium, Peptostreptococcus, and Bacteroides were isolated in high frequency from root canals with acute periapical inflammation. The antimicrobial susceptibilities of these strains were studied by determining minimum inhibitory concentrations of different agents. Although all three kinds of isolates were susceptible to penicillins, the isolates other than black-pigmented Bacteroides were less susceptible to cephems, tetracyclines, and macrorides with several resistant strains. All strains were uniformly resistant to aminoglycosides. Some differences in susceptibilities were observed among species of Eubacterium and Peptostreptococcus, while penicillins were effective for both species. Black-pigmented Bacteroides showed good susceptibilities to all agents except for aminoglycosides. The susceptibility of Bacteroides gingivalis was superior to that of Bacteroides intermedius. There were many resistant strains in non-black-pigmented but not in black-pigmented Bacteroides isolates. Penicillins were the most effective for Eubacterium, Peptostreptococcus, and Bacteroides, indicating that penicillins are suitable for treatment of root canals with acute apical periodontitis.

A novel method of conjugation of daunomycin with antibody with a poly-L-glutamic acid derivative as intermediate drug carrier. An anti-alpha-fetoprotein antibody-daunomycin conjugate.

In studies on antitumor antibody-cytotoxic drug conjugates as potential antitumor agents with improved tumor specificity, daunomycin (DM) was first linked to a poly-L-glutamic acid (PLGA) derivative having a single masked thiol group. At the thiol group, DM-linked PLGA was bound to horse anti-rat alpha-fetoprotein (AFP) antibody. The anti-AFP antibody-PLGA-DM conjugate (anti-AFP conjugate, DM/PLGA/Ig molar binding ratio, 7.5/1.2/1.0) retained most of the antigen-binding activity of the parent antibody and was more potent than either unconjugated DM, a conjugate similarity prepared with normal horse immunoglobulin (normal conjugate), or an unconjugated mixture of anti-AFP antibody and DM in an in vitro cytotoxicity assay against the AFP-producing rat ascites hepatoma cell line AH66. Anti-AFP conjugate tended to be less cytotoxic than DM against the AFP-nonproducing rat ascites hepatoma AH272 cells, and in this case there was no difference between the cytotoxicities of anti-AFP conjugate and of normal conjugate.

[Depressive effects of benzodiazepines on penicillin-induced epileptic discharges].

Effects of clonazepam, nitrazepam and diazepam on penicillin induced primary, spread and reactive epileptoform discharges were investigated in gallamine immobilized cats. Different strengths of seizure foci were induced by penicillin G 1000, 3000 and 6000 U injected into the cortex, amygdala and intralaminal thalamus, and the spread of epileptic discharges in the subcortex or surrounding area and to the contralateral area was followed. Benzodiazepines 5 mg/kg i.v. shortened the duration of primary epileptoform discharges and prolonged the interictal interval in the cortical, amygdaloid and intralaminal thalamic epileptogenesis induced by a high concentration of penicillin G. When a low concentration of penicillin G was injected into the cortex, amygdala and intralaminal thalamus, benzodiazepines abolished the spread of primary epileptoform discharges and the reactive discharges, but did not suppress completely the primary epileptogenic discharges and the contralateral reflective activity. Suppression of the discharges necessitated administration of a high dose. The greatest degree of suppression was seen with clonazepam. It is concluded that the anticonvulsive effect of benzodiazepines may be due to the blackades of neuronal pathways which spread the seizure discharges from the site of origin (focus) to the effector organ, and the elevation of convulsive thresholds.

Studies on Tetrahymena membranes. In vivo manipulating of membrane lipids by 1-O-hexadecyl glycerol-feeding in Tetrahymena pyriformis.

1. Tetrahymena pyriformis NT-I cells were grown in the medium supplemented with 1-O-hexadecyl glycerol which is the precursor for alkyl ether-containing phospholipids; choline phosphoglyceride and 2-aminoethylphosphonolipid, and alterations in the plasma membrane and microsome lipid composition were examined. No incorporation of supplemented 1-O-hexadecyl glycerol was seen in ethanolamine phosphoglyceride. 2. The hexadecyl glycerol fed membranes contain more polyunsaturated fatty acids than do the native membranes. However, the level of oleic acid (C 18:1) drops strikingly in the phospholipids of plasma and microsome membranes. 3. The hexadecyl glycerol-feeding induced a remarkable alteration in the polar headgroup composition of plasma membrane, especially a large increase in 2-amino-ethylphosphonolipid with a compensatory decrease in ethanolamine phosphoglyceride of plasma membranes. 4. The fatty acyl chain composition of phospholipids, especially ethanolamine phosphoglyceride, of the hexadecyl glycerol-fed plasma membranes and microsomes was found to be significantly different from that of the native membranes. 5. These results may indicate that marked alterations in polar headgroup as well as fatty acyl chain composition of membranes induced by glyceryl ether-feeding would be required for maintaining proper membrane fluidity.

Studies on tetrahymena membranes. Modification of surface membrane lipids by replacement of tetrahymanol by exogenous ergosterol in Tetrahymena pyriformis.

Tetrahymena pyriformis WH-14 cells were grown in the medium supplemented with ergosterol (1 mg/100 ml) and the effects of replacement of tetrahymanol by ergosterol upon the lipid composition in the surface membranes (cilia and pellicles) were examined. 1. By scanning and freeze-etch electron microscopy it was suggested that exogenous ergosterol would be inserted into the lipid regions in the surface membranes. Although freeze-etched faces of filipin-treated membranes containing the native tetrahymanol showed a random distribution of 85-a protein particles, the ergosterol-replaced membranes after the same polyene treatment revealed the marked ultrastructural alterations on the fracture faces. 2. The replacement of tetrahymanol in membranes by ergosterol induced a profound alteration in the phospholipid class composition and a marked increase in phosphatidylethanolamine with a compensatory decrease in phosphatidylcholine and 2-aminoethylphosphonolipid. 3. There are significant and quantitative but not qualitative changes in the fatty acid composition of total lipids from the ergosterol-replaced membranes. There are also increases in saturated and decreases in unsaturated fatty acids. Phosphatidylethanolamine acyl chains particularly become more saturated, as compared with two other phospholipids, in ergosterol-replaced pellicles. This increase in saturation is due to an appreciable increase in C14:0, C16:0 and iso-C17:0, and a decrease in C18:1(delta9), C18:2(delta9,12) and C18:3(delta6,9,12). 4. These results suggest that profound alterations in phospholipids as well as in their fatty acyl chains are required to modify the overall membrane lipid composition for the maintenance of proper membrane fluidity. Our data would also support the thesis that polat head groups are involved in the membrane lipid organization and that sterols interact selectively with phospholipid molecules containing the appropriate fatty acyl chain composition in biological membranes.