A region of the muscarinic-gated atrial K+ channel critical for activation by G protein beta gamma subunits.

Complementary DNAs encoding two types of inwardly rectifying K+ channels, GIRK1 and IRK1, have been cloned from rat atrium and mouse macrophage, respectively. GIRK1 expressed in Xenopus oocytes was activated by acetylcholine when m2 muscarinic acetylcholine receptor was coexpressed. The acetylcholine-induced activation of GIRK1 was enhanced by coexpression with the G protein beta 1 gamma 2 subunit but not the beta 1 gamma 1 or alpha subunits. Deletion of the C-terminus of GIRK1 impaired the channel activation associated with the beta 1 gamma 2 subunit. Moreover, replacement of the C-terminus of IRK1 with that of GIRK1 produced a chimera channel that was activated by the beta 1 gamma 2 subunit, whereas intact IRK1 was not activated by the beta 1 gamma 2 subunit. These findings define the C-terminus of GIRK1 as a regulatory region for the G protein beta gamma subunit.

Application of sonoelastography for evaluating the stiffness of equine superficial digital flexor tendon during healing.

Sonoelastography can assess the inner stiffness of tissues. Sonoelastographic evaluation of injured equine superficial digital flexor tendons (SDFTs) is considered to be useful for assessing the stiffness of a lesion even during late-stage rehabilitation. The purpose of this study was to investigate and compare the sonoelastographic appearance of injured SDFTs over time from the onset of the injury. Eighteen horses were classified into three groups according to the length of time from injury onset: group A, within two weeks after injury; group B, approximately five months after injury; and group C, approximately nine months after injury. Longitudinal and transverse images of all injured SDFTs were obtained using grey-scale ultrasonography and sonoelastography. Grey-scale and sonoelastographic images were evaluated by two observers using echogenicity-grading and colour-grading systems, respectively. The authors evaluated the interobserver agreement and compared the grades among the three groups. The results indicated almost perfect interobserver agreement. Significant differences were found in the sonoelastography among the three groups, whereas no significant difference was found in the grey-scale ultrasonography between groups B and C. Sonoelastography is a feasible and useful modality to evaluate the equine injured SDFTs in vivo and to distinguish between them among the different phases even during the chronic phase.

The use of sonoelastography to assess the recovery of stiffness after equine superficial digital flexor tendon injuries: A preliminary prospective longitudinal study of the healing process.

BACKGROUND: The objective assessment of the mechanical properties of the superficial digital flexor tendon (SDFT) could provide useful information for the rehabilitation of horses with SDFT injuries. Assessment of strain ratio (the strain of a standard reference divided by that of lesions) is a quantitative method in sonoelastography for evaluating tissue stiffness in vivo. As yet, no longitudinal studies have used strain ratio to evaluate the progression of stiffness in SDFT injuries. OBJECTIVES: To test the hypothesis that strain ratio can evaluate the recovery of stiffness during the healing of SDFT injuries. STUDY DESIGN: Prospective and longitudinal study with observer-blinded evaluation. METHODS: Ultrasonography, including sonoelastography, was performed in seven Thoroughbred horses with naturally occurring SDFT injuries at five time points: within 20 days of the injury, and at 2, 3, 6 and 9 months after the injury. Blinded sonoelastographic images were independently evaluated by two veterinarians to assess interobserver agreement. The recovery of stiffness and echogenicity in lesions were evaluated using the strain ratio and grey-scale ratio (echogenicity of lesions divided by that of the surrounding area), respectively. RESULTS: Interobserver agreement was assessed as 'almost perfect'. Strain ratios were significantly higher at 9 months after injury than at the other time points (all P<0.05). Strain ratios at 6 months after injury were significantly higher than those at earlier time points (P<0.05). Grey-scale ratios within 20 days of injury were significantly lower than those at the other time points (all P<0.05). MAIN LIMITATIONS: Validations of SDFT status were evaluated only by recovery of the echogenicity in lesions and not by histopathological examination. CONCLUSIONS: Although further studies are needed to validate the relationships between injured SDFT status and sonoelastographic findings, this preliminary study shows that strain ratio may provide a means to monitor the recovery of stiffness in lesions during rehabilitation, even when the grey-scale ratio remains unchanged from a few months after SDFT injury. The Summary is available in Chinese - see Supporting Information.

Functional expression of cloned cDNA encoding the alpha-subunit of adenylate cyclase-stimulating G-protein.

Cloned cDNA encoding the alpha-subunit of the adenylate cyclase-stimulating G-protein (Gs), carried by a simian virus 40 vector, has been introduced into the cyc- variant of S49 lymphoma cells by electroporation. In contrast to untransfected cys- cells, clones transformed with the cDNA exhibit an increase in intracellular cyclic AMP concentration in response to a beta-adrenergic agonist.

Primary structure of the alpha-subunit of bovine adenylate cyclase-stimulating G-protein deduced from the cDNA sequence.

The primary structure of the alpha-subunit of the adenylate cyclase-stimulating G-protein (Gs) has been deduced from the nucleotide sequence of cloned DNA complementary to the bovine cerebral mRNA encoding the polypeptide. Comparison of the amino acid sequences of the alpha-subunits of Gs and transducin reveals that some of the highly conserved regions show sequence homology with elongation factor-Tu and ras p21 proteins and correspond to functional regions of guanine nucleotide-binding proteins.

Amino acid residues in the transmembrane domain of the type 1 sigma receptor critical for ligand binding.

The type 1 sigma receptor expressed in Xenopus oocytes showed binding abilities for the sigma-1 ligands, [3H](+)pentazocine and [3H]NE-100, with similar kinetic properties as observed in native tissue membranes. Amino acid substitutions (Ser99Ala, Tyr103Phe and di-Leu105,106di-Ala) in the transmembrane domain did not alter the expression levels of the type 1 sigma receptor as determined by immunoblot analysis using an anti-type 1 sigma receptor antiserum. By contrast, ligand binding was significantly suppressed by the substitutions. These findings provide evidence that the transmembrane domain of the type 1 sigma receptor plays a critical role in ligand binding of this receptor.

Primary structure of the alpha-subunit of bovine adenylate cyclase-inhibiting G-protein deduced from the cDNA sequence.

The primary structure of the alpha-subunit of the adenylate cyclase-inhibiting G-protein (Gi) has been deduced from the nucleotide sequence of cloned DNA complementary to the bovine cerebral mRNA encoding the polypeptide. A much higher degree of amino acid sequence homology is observed between the alpha-subunits of Gi and transducin (68%) than between those of Gi and the adenylate cyclase-stimulating G-protein (Gs) (43%) or between those of transducin and Gs (42%).

Primary structure of the beta-subunit of bovine transducin deduced from the cDNA sequence.

DNA complementary to the bovine retinal mRNA coding for the beta-subunit of transducin has been cloned by screening a cDNA library with oligodeoxyribonucleotide probes. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 340 amino acid residues (including the initiating methionine). Furthermore, cDNA hybridizable with a transducin beta-subunit cDNA probe has been cloned from a library derived from bovine brain poly(A)+ RNA. Comparison of the cloned cDNAs, in conjunction with blot hybridization analysis and S1 nuclease mapping of poly(A)+ RNA from bovine retina, brain and liver, suggests that the mRNAs coding for the beta-subunits of transducin and other guanine nucleotide binding proteins have the same protein-coding sequence but partly different 5'-noncoding sequences.

Myoclonus in a case of suspected progressive rubella panencephalitis.

Predominantly unilateral myoclonus induced by movement of the affected parts in a 21-year-old Japanese man with suspected progressive rubella panencephalitis appeared in the course of treatment for epilepsy with anticonvulsant drugs after five years. Antirubella antibody levels in the serum and CSF were elevated and IgM antibody against rubella was found in the serum. An EEG recorded while the patient was awake showed diffuse 3- to 4-Hz theta activity with high voltage. The neurological symptoms are progressing slowly.

Internal carotid occlusion: assessment, by Tc-99m-tagged microspheres, of bypass from superficial temporal to middle cerebral artery.

The assessment of regional blood perfusion through the STA-MCA anastomosis was performed by the method of intracarotid injection of Tc-99m-labeled human albumin microspheres (HAM scintigraphy). This study included 11 anastomoses in 10 cases with occlusion of the internal carotid artery, who were treated by bypass surgery. The area of intracranial perfusion through the bypass was well defined in all cases by HAM scintigraphy. This method is excellent for imaging the regional blood perfusion through the anastomotic vessel, and the scintigraphic findings agree well with those of postoperative angiography. HAM scintigraphy will partially replace postoperative angiography because of its simplicity and low invasiveness.

Thromboxane generation in patients with essential hypertension or cerebrovascular disease and effect of oral aspirin.

The ability of platelets to synthesize thromboxane B2 (TxB2) from arachidonic acid (AA) or prostaglandin H2 (PGH2) was studied in 26 control subjects, 40 patients with essential hypertension, 20 patients with cerebrovascular disease (CVD) not taking aspirin and 11 patients with CVD taking aspirin. The activity of platelets to form TxB2 from AA or PGH2 was measured using 1-14C arachidonic acid or 1-14C PGH2 as a substrate. There was no significant difference in TxB2 generation from AA or PGH2 among the platelets collected from the control subjects, hypertensive patients and CVD patients not taking aspirin. In CVD patients taking aspirin, marked suppression was observed in TxB2 synthesis from AA, but no suppression in TxB2 synthesis from PGH2. At least 750 mg aspirin per day were required for nearly complete suppression of TxB2 generation from AA.

Voltage and pH dependent block of cloned N-type Ca2+ channels by amlodipine.

1. Two types of Ca2+ channel alpha1-subunits were co-expressed in Xenopus oocytes with the Ca2+ channel alpha2- and beta1-subunits. The Ba2+ current through the alpha1C alpha2beta and the alpha1B alpha2beta channels had electrophysiological and pharmacological properties of L- and N-type Ca2+ channels, respectively. 2. Amlodipine had a strong blocking action on both the L-type and N-type Ca2+ channels expressed in the oocyte. The potency of the amlodipine block on the N-type Ca2+ channel was comparable to that on the L-type Ca2+ channel. At -100 mV holding potential, the IC50 values for amlodipine block on the L-type and N-type Ca2+ channel were 2.4 and 5.8 microM, respectively. 3. The blocking action of amlodipine on the N-type Ca2+ channel was dependent on holding potential and extracellular pH, as has been observed with amlodipine block on the L-type Ca2+ channel. A depolarized holding potential and high pH enhanced the blocking action of amlodipine. 4. The time course of block development by amlodipine was similar for L-type and N-type Ca2+ channels. However, it was slower than the time course of block development by nifedipine for the L-type Ca2+ channel.

Importance of N-terminal regions of G protein alpha subunits for the activation of phospholipase C in Xenopus oocytes.

The alpha subunits of Gq family G proteins, GL1 alpha and GL2 alpha, are bovine homologues of mouse G14 alpha and G11 alpha, respectively, and are closely related to each other. When expressed in Xenopus oocytes together with metabotropic glutamate receptors, GL2 alpha activates endogenous phospholipase C (PLCx) in response to glutamate stimulation, whereas GL1 alpha inhibits the activation of PLCx. By examining the properties of 10 chimeras between GL1 alpha and GL2 alpha and their mutants, we tried to identify the regions on the G alpha proteins that are important for the activation of PLCx. The results indicated that a necessary (but not sufficient) condition for a chimeric G alpha protein to be able to clearly activate PLCx was that its N-terminal quarter portion should be derived from GL2 alpha. No correlation was found between the origin (GL1 alpha or GL2 alpha) of C-terminal regions of the chimeras and the ability of chimeras to activate PLCx. One of the chimeras is different from GL2 alpha at only four amino acid residues in the N-terminal region, and yet it could not activate PLCx. When one of the four residues, Ser-59, in the chimera was mutated back to Ala as in the original GL2 alpha, the resulting mutant became capable of activating PLCx. This residue is localized in the midst of the N-terminal linker connecting the two major domains in the G alpha proteins. These results indicate that Ala-59 is critical for the activation of PLCx, and that the linker may play important roles in determining functions of G alpha proteins.

Behcet's disease with sinus thrombosis and arteriovenous malformation in brain.

A 55-year-old man suffered from Behcet's disease with bilateral sigmoid sinus thrombosis and dural arteriovenous malformation has been studied clinically and pathologically. A possible causal relationship between Behcet's disease and sigmoid sinus thrombosis is discussed.

Multiple pathways of sigma(1) receptor ligand uptakes into primary cultured neuronal cells.

Although many antipsychotics have affinities for sigma receptors, the transportation pathway of exogenous sigma(1) receptor ligands to intracellular type-1 sigma receptors are not fully understood. In this study, sigma(1) receptor ligand uptakes were studied using primary cultured neuronal cells. [(3)H](+)-pentazocine and [(3)H](R)-(+)-1-(4-chlorophenyl)-3-[4-(2-methoxyethyl)piperazin-1-yl]methyl-2-pyrrolidinone L-tartrate (MS-377), used as a selective sigma(1) receptor ligands, were taken up in a time-, energy- and temperature-dependent manner, suggesting that active transport mechanisms were involved in their uptakes. sigma(1) receptor ligands taken up into primary cultured neuronal cells were not restricted to agonists, but also concerned antagonists. The uptakes of these ligands were mainly Na(+)-independent. Kinetic analysis of [(3)H](+)-pentazocine and [(3)H]MS-377 uptake showed K(m) values (microM) of 0.27 and 0.32, and V(max) values (pmol/mg protein/min) of 17.4 and 9.4, respectively. Although both ligands were incorporated, the pharmacological properties of these two ligands were different. Uptake of [(3)H](+)-pentazocine was inhibited in the range 0.4-7.1 microM by all the sigma(1) receptor ligands used, including N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine monohydrochloride (NE-100), a selective sigma(1) receptor ligand. In contrast, the inhibition of [(3)H]MS-377 uptake was potently inhibited by haloperidol, characterized by supersensitivity (IC(50), approximately 2 nM) and was inhibited by NE-100 with low sensitivity (IC(50), 4.5 microM). Moreover, kinetic analysis revealed that NE-100 inhibited [(3)H]MS-377 uptake in a noncompetitive manner, suggesting that NE-100 acted at a site different from the uptake sites of [(3)H]MS-377. These findings suggest that there are at least two uptake pathways for sigma(1) receptor ligands in primary cultured neuronal cells (i.e. a haloperidol-sensitive pathway and another, unclear, pathway). In addition, pretreatment of cells with a calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (W-7), a myosin light chain kinase inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)homopiperazine (ML-9), or microsomal Ca(2+)-ATPase inhibitors resulted in a reduction of the amount of sigma receptor ligand uptake. These findings suggest that the Ca(2+) pump on the endoplasmic reticulum and/or calmodulin-related events might be involved in the regulation of the uptake of sigma receptor ligands into primary neuronal cells.

The reproducibility of color Doppler duplex sonography in the measurement of renal arterial blood velocity.

In this study, the reproducibility of color Doppler duplex sonography for repeated measurements of renal blood flow was evaluated in 14 healthy subjects. We examined the reproducibility for different examiners and different time intervals between the examinations. Doppler frequency sonograms were analyzed with several parameters, and statistical evaluation was performed by calculating both the correlation coefficient (r) and coefficient of variation (CV). Peak systolic velocity (S), early diastolic velocity (D1) and mean velocity (MV) showed good reproducibility (r = 0.902-0.992, CV = 2.15-8.16%). On the other hand, end-diastolic velocity (D2), acceleration time (AT) and acceleration index (AI) showed poor reproducibility. We conclude that the reproducibility of this method is acceptable for repeated measurements of renal blood velocity, using suitable parameters S, D1 and MV.

A validation study on the reproducibility of transcranial Doppler velocimetry.

The transcranial Doppler method for the measurement of intracranial arterial blood flow velocity is a useful noninvasive technique with a number of applications. The present study validated the reproducibility of this method for repeated measurements of flow velocity in the middle cerebral and basilar arteries. Fifteen healthy volunteers were studied. Measurements were made twice by one examiner and once by another in a single day and again by the first examiner on another day. The reproducibility was evaluated by calculating the correlation coefficient (r) and the coefficient of variation (CV) of the difference between the values obtained from each pair of measurements. Although depending on the examiner, the time interval between the examinations and the vessels studied some differences were noted in the reproducibility, both the r (0.69-0.95) and CV (6.7%-19.5%) values in the whole study were good enough to warrant the applicability of this method for the repeated measurements of the intracranial arterial blood flow velocity in future studies.

Effect of transcranial Doppler intensity on successful recording in Japanese patients.

The major limitation of transcranial Doppler sonography (TCD) is the failure to obtain data for all patients. The purpose of this study was to determine in detail the effect of increasing ultrasonic acoustic intensity on the rate of successful recording of intracranial blood velocity signals. The study was performed in 239 Japanese patients using a 2-MHz range-gated, pulsed-wave TCD. The middle cerebral artery flow signals were recorded at 76, 152, 228, 304, 380, 456 and 532 mW/cm2 and the results analyzed by age, gender and intensity. The rate of successful recording showed significant increase with the ultrasonic intensity in both genders (45.7% at 76 mW/cm2 vs. 81.1% at 532 mW/cm2 in males and 29.5% vs. 60.7% in females). However, recording was only successful in 54% of aged (50-89 gamma) female patients at the highest ultrasonic intensity used. It should be possible to significantly increase TCD usefulness in an aging Japanese population by further increasing TCD acoustic intensity within safety limitation.

Synthesis of an octasaccharide fragment of high-mannose-type glycans of glycoproteins.

O-(alpha-D-Mannopyranosyl)-(1----2)-O-(alpha-D-mannopyranosyl)-(1----3)- O- [(alpha-D-mannopyranosyl)-(1----2)-O-(alpha-D-mannopyranosyl)-(1----6)]- O- (alpha-D-mannopyranosyl)-(1----6)-O-(beta-D-mannopyranosyl)-(1----4)-O-( 2- acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-2-deoxy- glucopyranose, an octasaccharide fragment of high-mannose type glycan of glycoproteins, was synthesized. Crucial glycosylation of trisaccharide intermediate, benzyl O-(2,4-di-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-(2-acetamido-3,6-di -O- benzyl-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-3,6-di-O-benz yl-2- deoxy-beta-D-glucopyranoside, was successful only with a di-O-acetyltetradeca-O-benzyl-D-mannopentaosyl chloride. The use of the corresponding hexadeca-O-acetyl-D-mannopentaosyl bromide did not give the desired product.

Synthesis of an appropriately protected core glycotetraoside, a key intermediate for the synthesis of "bisected" complex-type glycans of a glycoprotein.

A stereocontrolled synthetic route to a glycotetraoside, allyl O-(3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1--- -4)-O- (3,6-di-O-allyl-2-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-3, 6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1----4)-3-O- benzyl- 2-deoxy-6-O-p-methoxy-phenyl-2-phthalimido-beta-D-glucopyranoside, an important intermediate for the synthesis of "bisected" complex type glycans of glycoproteins has been established by employing two glycosyl donors, 3,4,6-tri-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl trichloroacetimidate and 4-O-acetyl-3,6-di-O-allyl-2-O-benzyl-alpha-D-mannopyranosyl bromide, and a glycosyl acceptor, allyl O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl)-(1----4) -3-O- benzyl-2-deoxy-6-O-p-methoxyphenyl-2-phthalimido-beta-D-glucopyranoside.