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H Araki

Publications and source records attributed to H Araki.

At least 37 records · Page 2Linked to original sources
PubMed2001

Monochloramine impairs mucosal blood flow response and healing of gastric lesions in rats: relation to capsaicin-sensitive sensory neurons.

AIMS: We examined the effects of monochloramine (NH2Cl) on the gastric mucosal blood flow (GMBF) response and the healing of ethanol-induced gastric lesions in rats. METHODS: Rats fasted for 18 h were given the 99% ethanol p.o. for induction of gastric lesions, and were fed normally from 1 h later onwards. Monochloramine, at non-ulcerogenic doses (5 to approximately 20 mmol/L), was given p.o. twice daily for 7 days, starting 2 h after ethanol treatment. RESULTS: Gastric lesions caused by ethanol healed almost completely within 7 days with re-epithelialization. The repeated administration of NH2Cl significantly delayed the healing of ethanol-induced gastric lesions in a dose-dependent manner. The damaged mucosa showed a marked rise in H+ permeability, resulting in luminal acid loss, but this process was accompanied by an increase of mucosal blood flow. Monochloramine did not affect the increased mucosal H+ permeability observed in the stomach after damage by ethanol, but significantly inhibited the mucosal hyperemic response associated with luminal acid loss. Prior exposure of the mucosa to NH2Cl (20 mmol/L) did not affect the gastric hyperemic response caused by mucosal application of misoprostol (a prostaglandin E1 derivative) or NOR-3 (a nitric oxide donor), but totally attenuated the increase of GMBF in response to intragastric capsaicin. Impaired healing and GMBF responses were also observed in rats following chemical ablation of capsaicin-sensitive sensory neurons. CONCLUSIONS: These results suggest that NH2Cl impaired the healing of acute gastric mucosal lesions at low concentrations, and this action may be attributable, at least partly, to the impairment of gastric hyperemic response caused by the dysfunction of capsaicin-sensitive sensory neurons.

Afferent Pathways↗
PubMed2001

Molecular evolution of duplicated amylase gene regions in Drosophila melanogaster: evidence of positive selection in the coding regions and selective constraints in the cis-regulatory regions.

In this study, we randomly sampled Drosophila melanogaster from Japanese and Kenyan natural populations. We sequenced duplicated (proximal and distal) Amy gene regions to test whether the patterns of polymorphism were consistent with neutral molecular evolution. F(st) between the two geographically distant populations, estimated from Amy gene regions, was 0.084, smaller than reported values for other loci, comparing African and Asian populations. Furthermore, little genetic differentiation was found at a microsatellite locus (DROYANETSB) in these samples (G'st = -0.018). The results of several tests (Tajima's, Fu and Li's, and Wall's tests) were not significantly different from neutrality. However, a significantly higher level of fixed replacement substitutions was detected by a modified McDonald and Kreitman test for both populations. This indicates that positive selection occurred during or immediately after the speciation of D. melanogaster. Sliding-window analysis showed that the proximal region 1, a part of the proximal 5' flanking region, was conserved between D. melanogaster and its sibling species, D. simulans. An HKA test was significant when the proximal region 1 was compared with the 5' flanking region of Alcohol dehydrogenase (Adh), indicating a severe selective constraint on the Amy proximal region 1. These results suggest that natural selection has played an important role in the molecular evolution of Amy gene regions in D. melanogaster.

Alleles↗
PubMed2001

Purification, molecular cloning, and immunohistochemical localization of dipeptidyl peptidase II from the rat kidney and its identity with quiescent cell proline dipeptidase.

We purified dipeptidyl peptidase II (DPP II) to homogeneity from rat kidney and determined its physicochemical properties, including its molecular weight, substrate specificity, and partial amino acid sequence. Furthermore, we screened a rat kidney cDNA library, isolated the DPP II cDNA and determined its structure. The cDNA was composed of 1,720 base pairs of nucleotides, and 500 amino acid residues were predicted from the coding region of cDNA. Human quiescent cell proline dipeptidase (QPP) cloned from T-cells is a 58-kDa glycoprotein existing as a homodimer formed with a leucine zipper motif. The levels of amino acid homology were 92.8% (rat DPP II vs. mouse QPP) and 78.9% (rat DPP II vs. human QPP), while those of nucleotide homology were 93.5% (rat DPP II vs. mouse QPP) and 79.4% (rat DPP II vs. human QPP). The predicted amino acid sequences of rat DPP II and human and mouse QPP possess eight cysteine residues and a leucine zipper motif at the same positions. The purified DPP II showed similar substrate specificity and optimal pH to those of QPP. Consequently, it was thought that DPP II is identical to QPP. Northern blot analysis with rat DPP II cDNA revealed prominent expression of DPP II mRNA in the kidney, and the order for expression was kidney >> testis > or = heart > brain > or = lung > spleen > skeletal muscle > or = liver. In parallel with Northern blot analysis, the DPP II antigen was detected by immunohistochemical staining in the cytosol of epithelial cells in the kidney, testis, uterus, and cerebrum.

Amino Acid Sequence↗
PubMed2001

Prostaglandin E inhibits indomethacin-induced gastric lesions through EP-1 receptors.

BACKGROUNDS AND AIMS: We examined the effect of various prostaglandin E (PGE) analogs specific to EP receptor subtypes on indomethacin-induced gastric lesions in rats and investigated which EP receptor subtype is involved in the protective action of PGE(2) using EP-receptor knockout mice. METHODS: Gastric lesions were induced by subcutaneous administration of indomethacin (35 mg/kg). Gastric motility was measured using a balloon method, while neutrophil chemotaxis determined using a Boyden chamber. RESULTS: Indomethacin-induced gastric lesions were significantly prevented by PGE(2) as well as atropine, and the former effect was mimicked by sulprostone (EP(1)/EP(3)) and 17-phenyl PGE(2) (EP(1)) and antagonized by an EP(1) antagonist, ONO-AE-829. Neither butaprost (EP(2)), ONO-NT-012 (EP(3)) nor 11-deoxy PGE(1) (EP(3)/EP(4)) showed any protection on the lesions. Indomethacin caused a marked increase in gastric motility; the response preceded the onset of lesions and was inhibited by atropine as well as PGE derivatives acting as EP(1) receptors. Neutrophil chemotaxis was inhibited by PGE(2), butaprost and slightly by 11-deoxy PGE(1), but not by either 17-phenyl PGE(2), ONO-NT-012 or atropine. In addition, indomethacin caused damage similarly in both wild-type and knockout mice lacking EP(1) or EP(3) receptors, yet the protective action of PGE(2) was observed in wild-type and EP(3) receptor knockout mice but totally disappeared in mice lacking EP(1) receptors. CONCLUSION: PGE(2) inhibits indomethacin-induced gastric lesions, through EP(1) receptors, and this effect may be functionally associated with inhibition of gastric motility but not of neutrophil activation/migration.

Animals↗
PubMed2001

The protease-activated receptor-2 agonist induces gastric mucus secretion and mucosal cytoprotection.

Protease-activated receptor-2 (PAR-2), a receptor activated by trypsin/tryptase, modulates smooth muscle tone and exocrine secretion in the salivary glands and pancreas. Given that PAR-2 is expressed throughout the gastrointestinal tract, we investigated effects of PAR-2 agonists on mucus secretion and gastric mucosal injury in the rat. PAR-2-activating peptides triggered secretion of mucus in the stomach, but not in the duodenum. This mucus secretion was abolished by pretreatment with capsaicin, which stimulates and ablates specific sensory neurons, but it was resistant to cyclo-oxygenase inhibition. In contrast, capsaicin treatment failed to block PAR-2-mediated secretion from the salivary glands. Intravenous calcitonin gene-related peptide (CGRP) and neurokinin A markedly elicited gastric mucus secretion, as did substance P to a lesser extent. Specific antagonists of the CGRP1 and NK2, but not the NK1, receptors inhibited PAR-2-mediated mucus secretion. Pretreatment with the PAR-2 agonist strongly prevented gastric injury caused by HCl-ethanol or indomethacin. Thus, PAR-2 activation triggers the cytoprotective secretion of gastric mucus by stimulating the release of CGRP and tachykinins from sensory neurons. In contrast, the PAR-2-mediated salivary exocrine secretion appears to be independent of capsaicin-sensitive sensory neurons.

Animals↗
PubMed2001

[Assessment of anti-tremorogenic drugs using nicotine-induced tail-tremor model and elucidation of the mechanism].

Repeated administration of nicotine causes a tremor only in the tail (tail-tremor) of rats. The tremor is accompanied with locomotor hyperactivity without rigidity and immobility of the whole body, suggesting the involvement of the mechanism associated with the movement. The tail-tremor induced by nicotine was suppressed by nicotinic acethylcholine (nACh) receptor antagonists, but not by muscarinic acethylcholine (mACh) receptor antagonists. Moreover, the tail-tremor was suppressed by beta-adrenoceptor antagonists and benzodizepines. The tremor at rest is observed only in Parkinson's disease, which is improved by the use of mACh receptor antagonists. An essential tremor is one of the typical tremor connected with the movement (postural tremor) and improved with beta-adrenoceptor antagonists. These findings and results suggest that the nicotine-induced tail-tremor is useful for the study of the essential tremor as an animal model. On the other hand, daily administration of nicotine resulted in an augmentation of the tail-tremor. The development of the tail-tremor was suppressed by nACh receptor antagonists, N-methyl-D-aspartate (NMDA) receptor antagonists and nitric oxide (NO) synthase inhibitors. These results suggest that central nACh receptors are essential for the onset and further development of the tail-tremor induced by repeated administration of nicotine, and that NO formation mediated by NMDA receptors is involved in the developmental mechanisms.

Adrenergic beta-Antagonists↗
PubMed2001

[The development of new drugs for osteoporosis in Japan].

In a rapidly aging society, the number of patients becoming bed-ridden due to osteoporosis-related fracture has become a socially important health issue that includes the problem of adequate nursing. Recently, the introduction of a rapidly acting bone absorption suppressant with a clear mechanism accelerated the development of osteoporosis treatment. A selective estrogen receptor modulator in a estrogen preparation is expected, because it has antagonism for the acceptor of the reproductive organ. It is noted that a new steroid with a weak androgen action and estrogen- and progestogen-like actions both prevents bone quantity decrease in women and increases bone quantity in osteoporotic patients after menopause. The second and third generation of bisphosphanate is more powerful than etidronate, the first generation of bisphosphanate, allows continual medication, and is now in clinical trial. The introduction of new drugs, which have clear efficacy and fewer side effects may be expected, along with the combined use of the drugs in which action mechanisms differ. The introduction of pharmaceutical new approaches is also expected as the expression mechanism of osteoporosis is further clarified by basic studies.

Anabolic Agents↗
PubMed2001

Nitrooxy alkyl apovincaminate activates K+ currents in rat neocortical neurons.

The effects of nitrooxy alkyl apovincaminate VA-045 ((+)-eburunamenine-14-carboxylic acid(2-nitroxy-ethyl ester), VA) were investigated in acutely dissociated rat neocortical neurons by using a nystatin-perforated patch recording configuration. VA activated a steady-state outward current in a concentration-dependent manner, with an EC50 of 0.65 microM. The reversal potential for the current shifted 56.5 mV with tenfold changes in the extracellular K+ concentration, suggesting that the current was carried by K+. The VA-induced current was not suppressed by apamin (1 microM), charybdotoxin (1 microM), Cs+ (3 mM), Ba2+ (3 mM), 4-aminopyridine (10 mM) or glibenclamide (10 microM), whereas tetraethylammonium suppressed the current with an IC50 of 1.4 mM. These pharmacological properties of the VA-induced current were compatible with a slowly inactivating delayed rectifier current (I(K)). It was suggested that the current activated by VA was I(K). The VA-induced current was not affected by Ca2+ depletion or by staurosporine (0.1 microM), quinacrine (10 microM), wortmanin (1 microM) or genistein (1 microM). The intracellular perfusion of GDPbetaS (0.4 mM) also had no significant effect. Thus, VA may directly activate the K+ channels.

Animals↗
PubMed2001

Deep rooting in winter wheat: rooting nodes of deep roots in two cultivars with deep and shallow root systems.

Deep rooting of wheat has been suggested that it influences the tolerance to various environmental stresses. In this study, the nodes from which the deepest penetrated roots had emerged were examined in winter wheat. The wheat was grown in long tubes with or without mechanical stress and in large root boxes. The length and growth angle of each axile root were examined to analyze the difference in the vertical distribution of the roots between the two wheat cultivars, one with a deep and one with a shallow root system. In Shiroganekomugi, a Japanese winter wheat cultivar with a shallow root system, the rooting depths of the seminal and nodal roots decreased as the rooting nodes advanced acropetally. Six out of nine deepest roots were seminal root in the non-mechanical stress conditions. In Mutsubenkei, a Japanese winter wheat cultivar with a deep root system, grown in root boxes, not only the seminal roots but also the coleoptilar and the first nodal roots penetrated to a depth of more than 1.3 m in the root box, and became the deepest roots. In both cultivars, the seminal roots became the deepest roots under the mechanical stress conditions. There were no clear tendencies in the root growth angles among the rooting nodes in the wheat root system. This indicates that the length of the axile roots can explain the differences in the rooting depths among axile roots in a wheat root system. On the other hand, the axile roots of Mutsubenkei elongated significantly more vertically than those of Shiroganekomugi. This suggests that not only seminal but also nodal roots exhibit strong positive gravitropism and penetrate deeply in a cultivar with a deep root system. In wheat cultivars, it is likely that the extent of its Root Depth Index results partly from the gravitropic responses of both seminal and nodal roots.

Acclimatization↗
PubMed2001

Severe fenitrothion poisoning complicated by rhabdomyolysis in psychiatric patient.

Non-traumatic rhabdomyolysis associated with organophosphate intoxication has not been generally reported. We report here in a severe case of fenitrothion poisoning complicated by rhabdomyolysis. A 43-year-old woman ingested approximately 100 ml of fenitrothion emulsion (50%) in an attempt to commit suicide. On day 3 after admission, her creatine phosphokinase (CPK) peaked at 47,762 IU/L. She received supportive treatment included sodium bicarbonate and fluid resuscitation. However, muscarinic symptoms including excessive miosis and salivation developed on day 5 when her CPK levels decreased. The delay in cholinergic symptoms might have been due to the trihexyphenidyl she took with the antipsychotic drugs. Fortunately, the present patient recovered from the acute cholinergic crisis, and acute renal failure was prevented by early diagnosis. This is a case of organophosphate poisoning complicated by rhabdomyolysis in a psychiatric patient. The masking of acute cholinergic symptoms should be taken into consideration in such patients.

Adult↗
PubMed2001

Dopaminergic neurotransmission triggers ischemia-induced hyperactivity in Mongolian gerbils.

It is recognized that sustained ischemia-induced hyperactivity is related to abnormalities in dopamine function. However, it is unclear that dopaminergic neurotransmission triggers such ischemia-induced hyperactivity. Therefore, the relationship between dopaminergic neurotransmission and ischemia-induced hyperactivity was investigated in an animal model using Mongolian gerbils. When haloperidol 2 mg/kg was administered i.p. 30 min after ischemia, the ischemia-induced hyperactivity at 24 h after ischemia was blocked. General behavior was similar to that of sham-operated animals. Haloperidol at doses of 0.1 and 0.2 mg/kg had no effect on locomotor activity in sham-operated animals and decreased ischemia-induced hyperactivity when the drug was administered 24 h after ischemia; these doses did not have any effect on ischemia-induced hyperactivity when the drug was administered 30 min after ischemia. On the other hand, when the animal was confined to a small, restrictive cage for the 24 h period immediately following ischemic injury, locomotor activity at 24 h after ischemia increased. Such behavior also increased in animals when they were returned to their original more permissive cages immediately after ischemia. It is conceivable that the decrease in the level of activity was not related to ischemia-induced hyperactivity. These data suggested that the inhibition of ischemia-induced hyperactivity can be induced by complete blockage of dopaminergic receptors immediately after ischemia.

Animals↗
PubMed2001

Adaptive gastric cytoprotection is mediated by prostaglandin EP1 receptors: a study using rats and knockout mice.

Endogenous prostaglandins (PGs) play a central role in adaptive cytoprotection induced in the stomach by mild irritants. In the present study, we used taurocholate (TC) as a mild irritant in both rats and EP-receptor knockout mice, and examined which EP receptor is responsible for the adaptive gastric cytoprotection. Gastric lesions were induced by p.o. administration of HCl/ethanol (60% ethanol in 150 mM HCl). TC (5-20 mM) or PGE2 was administered p.o. 30 min before HCl/ethanol. HCl/ethanol-induced gastric lesions were dose dependently prevented by TC, and the effect at 20 mM was equivalent to that induced by PGE2 at 0.3 mg/kg. The protective effect of TC was significantly attenuated by indomethacin as well as ONO-AE-829, the EP1 antagonist, but not by either NS-398, the selective cyclooxygenase (COX)-2 inhibitor, or chemical ablation of capsaicin-sensitive sensory neurons. Likewise, the protective action of PGE2 was also antagonized by ONO-AE-829 but not chemical deafferentation. TC significantly increased PGE2 contents in the stomach, with or without chemical deafferentation, and this effect was blocked in the presence of indomethacin but not NS-398 or ONO-AE-829. TC increased the mucosal PGE2 contents similarly in both wild-type and knockout mice lacking EP1 or EP3 receptors, yet the protective action of TC against HCl/ethanol was observed in both wild-type and EP3 receptor knockout mice, but not in mice lacking EP1 receptors. The present findings confirmed a role for endogenous PGE2 produced by COX-1 in adaptive gastric cytoprotection and suggested that this action is mediated by activation of EP1-receptors but not associated with capsaicin-sensitive afferent neurons.

Adaptation, Physiological↗
PubMed2001

[Relationship between prothrombin time international normalized ratio and thrombo test (%)].

OBJECTIVES: The optimal therapeutic range for laboratory evaluation of oral anticoagulant therapy is now defined by the prothrombin time international normalized ratio (PT-INR). However, the thrombo test (TT), an alternative method to measure intensity of anticoagulation, is also currently used throughout Japan. The relationship between PT-INR and TT (%) has yet to be clarified. This study investigated the relationship between PT-INR and TT (%). METHODS: The PT-INR and TT (%) were simultaneously measured of 505 consecutive samples from patients treated with warfarin in our hospital. Fourteen functions were used for regression analyses: a fractional function (Y = a/X + b), a square root function (Y = aX0.5 + b), a natural logarithmic function (Y = a.lnX + b), a power series function (Y = aXb), a quotient function (Y = abX), and polynomial functions [Y = anXn + an - 1Xn - 1 +......+ a1X1 + b, (1 < or = n < or = 9)]. The results were confirmed by the same methods in 383 samples and 296 samples from another two laboratories. RESULTS: The power series function showed the most significant (p < 0.0001) and highest adjusted R2 (0.858) correlation, with a regression formula of TT (%) = e4.48 (PT-INR)-2.09 in our laboratory. Using the same analyses, the power series function also showed the most significant and highest adjusted R2 in samples from the other two laboratories. CONCLUSIONS: This study showed that a power series function is the most appropriate for expressing the relationship between PT-INR and TT (%) among the 14 functions. The function between PT-INR and TT (%) is mainly derived from the relationship between TT (%) and TT (sec). Both internal validity and external validity confirmed the relationship between PT-INR and TT (%).

Aged↗
PubMed2000

Effect of consecutive lower-dose cisplatin in enhancement of 5-fluorouracil cytotoxicity in experimental tumor cells in vivo.

It is known that cisplatin (CDDP) potentiates the cytotoxicity of 5-fluorouracil (5-FU), and that the biochemical mechanism is an increase in the intracellular reduced folate levels in the tumor cells. We investigated the effect of consecutive administration with lower-dose CDDP on intracellular accumulation of reduced folate and the activity of methionine synthase, a key enzyme in intracellular methionine synthesis. When CDDP (1 mg/kg) was administered i.p. to ascitic Yoshida sarcoma-bearing rats for 4 consecutive days, both the reduced folate levels and methionine synthase activity in the cells significantly increased, as the same as a single 5 mg/kg dose of CDDP. Furthermore, when Yoshida sarcoma-bearing rats were pre-treated with 1 mg/kg CDDP for 5 consecutive days, [14C]L-methionine incorporation into the isolated ascitic cells was significantly inhibited as compared to that in non-treated cells, suggesting that consecutive administration of lower-dose CDDP is capable of inducing the intracellular modulation of reduced folate levels and methionine synthase activity via inhibition of cellular uptake of methionine. In addition, 5-day administration of lower-dose (1 mg/kg) CDDP potentiated the antitumor effect of 5 mg/kg S-1, a new oral preparation of tegafur, given for 7 consecutive days, and this combined effect was almost similar to the antitumor effect of a combination of S-1 and a single conventional dose (5 mg/kg) of CDDP. Consecutive lower-dose CDDP also may be concluded to act as an important modulator of the enhancement of 5-FU cytotoxicity in experimental tumors.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed2000

Involvement of 5-hydroxytryptamine(1A) receptors in nicotine-induced tail tremor in rats.

Involvement of the serotonergic system in tail tremor induced by repeated administration of nicotine was investigated in rats. Tail tremor induced by nicotine (0.5 mg/kg, s.c.) was suppressed by a 5-HT(1A) receptor antagonist, N-¿2-[4-(2-methoxyphenyl)-1-piperazinyl-]ethyl¿-N-(2-pyridinyl)cycloh exanecarboxamide trihydrochloride (WAY-100635; 0.3-3 mg/kg, i.p.), but not by a 5-HT(2) receptor antagonist, ketanserin (0.1-0.3 mg/kg, i.p). The 5-HT(1A) receptor agonists, buspirone (1-20 mg/kg, i.p.), gepirone (1-10 mg/kg, i.p.), tandospirone (1-10 mg/kg, i.p.) and (+/-)-8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT; 0.01-0.1 mg/kg, s.c.), enhanced the tail tremor. The enhancement of tail tremor by buspirone (10 mg/kg, i.p.) was blocked by WAY-100635 (0.3-3 mg/kg, i.p.). These findings suggest that nicotine-induced tail tremor is mediated by 5-HT(1A) receptors and that 5-HT(1A) receptor antagonists are effective in the treatment of tremor.

Animals↗
PubMed2000

Characterization of the protease-activated receptor-1-mediated contraction and relaxation in the rat duodenal smooth muscle.

Activation of protease-activated receptor-1 (PAR-1) produces a dual action, apamin-sensitive relaxation followed by contraction, in the rat duodenal smooth muscle, which is partially dependent on activation of L-type Ca2+ channels, protein kinase C (PKC) or tyrosine kinase (TK), and resistant to tetrodotoxin. The present study further characterized the PAR-1-mediated duodenal responses. Removal of extracellular Ca2+ as well as SK&F96365 reduced the contraction due to the PAR-1 agonist TFLLR-NH2 (TFp-NH2) by 60-80% that was similar to the extent of the inhibition by nifedipine. Lowering of the extracellular Na+ concentration, but not IAA-94, a Cl- channel inhibitor, reduced both the PAR-1-mediated contraction and relaxation by about 50%. U73122, a phospholipase C (PLC) inhibitor, or wortmannin, a phosphatidyl inositol 3'-kinase (PI3K) inhibitor, significantly reduced the PAR-1-mediated contraction, but not the relaxation, by itself, as the PKC inhibitor GF109203X and the TK inhibitor genistein did. U73122 or wortmannin, like GF109203X, when applied in combination with genistein, significantly reduced the PAR-1-mediated relaxation. The relaxation was resistant to antagonists of PACAP receptors, VIP receptors and P2 purinoceptors. Thus, the PAR-1-mediated contraction is considered to be dependent on intracellular and extracellular Ca2+, the influx of the latter being induced through activation of L-type Ca2+ channels triggered by the enhanced Na+ permeability, and that PLC and PI3K, in addition to PKC and TK, are involved in the PAR-1-mediated dual responses. Furthermore, non-adrenergic, non-cholinergic nerve neurotransmitter candidates that may modulate K+ channels do not appear to contribute to the relaxation by PAR-1 activation.

Androstadienes↗
PubMed2000

An advantage for use of isotope labeling and NMR chemical shifts to analyze the structure of four homologous IgG-binding domains of staphylococcal protein A.

Because of the complexity arising from the large molecular size and the amino acid sequence homologies of IgG-binding domains of Staphylococcal Protein A (SpA), we have introduced, a combination of stable isotope labeling and both qualitative and quantitative investigations of the structural dependence of the NMR chemical shifts for its structure analysis. In order to enable selective isotope labeling with high efficiency, a mutated low molecular weight Protein A (LPA; MWt = 27 kDa) which consists of E, D, A, B and 13 residues of the C-domain was used in this study. Amide proton chemical shifts, measured using uniformly 15N-labeled LPA and LPA labeled selectively with 15N-alanine, show that the turn between helices 1 and 2, and its tertiary interactions with helix 3, are very similar in all domains. This contradicts previous results obtained using independent structure calculations on isolated domains. The close similarity in NH and 15N chemical shifts of alanine residues in the interdomain linker suggests that the linker maintains a similar structure both in isolated domains and in the intact protein. We show that the high-field shifted methyl signal of Ala 48 is affected by the ring-current effect arising from Phe 30, and has a very similar helical environment in all four domains. Thus, helix 3 is present in all domains, as we previously reported [Kikuchi et al., J Biochem Biophys Method, 1999:38:203-208], even though it is not observed in the crystal structure [Deisenhofer J. Biochemistry 1981;20:2361-2370].

Alanine↗