Disruption of the splicing enhancer sequence within exon 27 of the dystrophin gene by a nonsense mutation induces partial skipping of the exon and is responsible for Becker muscular dystrophy.

The mechanism of exon skipping induced by nonsense mutations has not been well elucidated. We now report results of in vitro splicing studies which disclosed that a particular example of exon skipping is due to disruption of a splicing enhancer sequence located within the exon. A nonsense mutation (E1211X) due to a G to T transversion at the 28th nucleotide of exon 27 (G3839T) was identified in the dystrophin gene of a Japanese Becker muscular dystrophy case. Partial skipping of the exon resulted in the production of truncated dystrophin mRNA, although the consensus sequences for splicing at both ends of exon 27 were unaltered. To determine how E1211X induced exon 27 skipping, the splicing enhancer activity of purine-rich region within exon 27 was examined in an in vitro splicing system using chimeric doublesex gene pre-mRNA. The mutant sequence containing G3839T abolished splicing enhancer activity of the wild-type purine-rich sequence for the upstream intron in this chimeric pre-mRNA. An artificial polypurine oligonucleotide mimicking the purine-rich sequence of exon 27 also showed enhancer activity that was suppressed by the introduction of a T nucleotide. Furthermore, the splicing enhancer activity was more markedly inhibited when a nonsense codon was created by the inserted T residue. This is the first evidence that partial skipping of an exon harboring a nonsense mutation is due to disruption of a splicing enhancer sequence.

Ion selectivity of volume regulatory mechanisms present during a hypoosmotic challenge in vestibular dark cells.

Volume regulation during a hypoosmotic challenge (RVD) in vestibular dark cells from the gerbilline inner ear has previously been shown to depend on the presence of cytosolic K+ and Cl-, suggesting that it involves KCl efflux. The aim of the present study was to characterize hypoosmotically-induced KCl transport under conditions where a hypoosmotic challenge causes KCl influx via the pathways normally used for efflux. Net osmolyte movements were monitored as relative changes in cell volume measured as epithelial cell height (CH). A hypoosmotic challenge (298 to 154 mosM) in the presence of 3.6 or 25 mM K+ and loop-diuretics (piretanide or bumetanide) caused an increase in CH by about a factor of 1.2 presumably due to the net effect of primary swelling defined as osmotic dilution of the cytosol and RVD involving KCl efflux. A hypoosmotic challenge in the presence of 79 mM K+ and loop-diuretics, however, caused CH to increase by a factor of over 2.4. Presumably, this large increase in CH was due to the sum of primary and secondary swelling. Secondary swelling depended on the presence of extracellular K+ and Cl- suggesting that it involved KCl influx followed by water. The ion selectivity of secondary swelling was K+ = Rb+ > Cs+ >> Na+ = NMDG+ and Cl- = NO3- = SCN- >> gluconate-. Secondary swelling was not inhibited by Ba2+, tetraethylammonium, quinidine, lidocaine, amiloride, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, 4-acetamido-4'-diisothiocyanatostilbene-2,2'-disulfonic acid, 4,4'-dinitrostilbene-2,2'-disulfonic acid, 5-nitro-2(3-phenylpropylamino)benzoic acid, acetazolamide, or ethoxyzolamide. These data define a profile of the hypoosmotically-induced KCl transport pathways. The ion selectivity and the blocker insensitivity are consistent with the involvement of the apical slowly activating K+ channel (IsK or minK channel) and the basolateral 360 pS Cl- channel. The involvement of these channels, however, remains to be demonstrated.

Systemic endothelial function is preserved in men with both active and inactive variant angina pectoris.

To test the hypothesis that coronary spasm could be a coronary manifestation of systemic endothelial dysfunction and that the activity of coronary spasm could influence systemic endothelial function, we examined brachial flow-mediated, endothelium-dependent vasodilation and nitroglycerin-induced endothelium-independent vasodilation with high-resolution ultrasound in 11 men with variant angina pectoris (6 active and 5 inactive) without established coronary atherosclerosis. Endothelium-dependent vasodilation in peripheral circulation was preserved in men with active and inactive variant angina pectoris, suggesting that systemic endothelial dysfunction is not involved in either the pathogenesis or the activity of coronary spasm.

Mutational analysis of the cardiac actin gene in familial and sporadic dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) results in part from genetic disorders. Recently, missense mutations of the cardiac actin gene have been reported to cause DCM. We studied 136 Japanese DCM cases to elucidate how frequently the gene mutations are involved in its pathogenesis. Genomic DNA samples were obtained from 136 DCM cases (107 males, 29 females), containing 30 familial DCM (5 confirmed and 25 suspected). All six exons of the cardiac actin gene were analyzed by polymerase chain reaction, single-strand conformation polymorphism, and sequencing. We detected no mutations of the disease causation previously reported (G867A or A1014G) but two silent mutations (G979C and C1018T) in exon 6 and one point mutation (T1080A) in the 3'-untranslated region. As a result of screening 128 healthy subjects, these novel silent mutations were found to be mere genetic polymorphisms, not responsible for the disease. Although some genetic polymorphisms exist in the cardiac actin gene, mutations of the gene are rarely responsible for DCM, at least in the Japanese patients.

Effects of ethanol on meal-stimulated secretion of pancreatic polypeptide and cholecystokinin: comparison of healthy volunteers, heavy drinkers, and patients with chronic pancreatitis.

Tiscornia and Dreiling (Physiopathogenic Hypothesis of Alcoholic Pancreatitis: Supranormal Ecbolic Stimulation of the "Pancreon" Units Secondary to the Loss of the Negative Component of Pancreas Innervation. Pancreas 1987;2:604-612.) proposed that hypertonicity of intrapancreatic cholinergic neurons provoked by chronic alcoholism may contribute to the pathogenesis of chronic pancreatitis (CP). In the present study, the validity of this hypothesis was investigated in humans by studying the effects of atropine, cisapride, and ethanol on the meal-stimulated secretion of pancreatic polypeptide (PP) and cholecystokinin (CCK) in healthy volunteers, heavy drinkers, and CP patients. In healthy volunteers, the early phase PP response (0-40 min) to a test meal was completely blocked by atropine, whereas it was augmented by cisapride, an enhancer of acetylcholine release from cholinergic nerves. The early phase PP response to a test meal was inhibited by ethanol in healthy volunteers, whereas, in heavy drinkers, the response was augmented and the inhibition by ethanol was abrogated. In CP patients, ethanol tended to enhance the early phase PP response. Ethanol did not affect the early phase CCK response to a test meal in any group, but it significantly enhanced the late phase CCK response (40-120 min) in CP patients. These results suggest that: (i) oral ethanol may inhibit the postprandial activation of the cholinergic neural pathway to the pancreas in healthy subjects, (ii) in heavy drinkers, postprandial cholinergic tone may be augmented and become resistant to the inhibition by ethanol, and (iii) the ethanol-induced increase in the postprandial CCK response in CP patients may play some role in the pathophysiology of this disease.

Vestibular dark cells contain the Na+/H+ exchanger NHE-1 in the basolateral membrane.

Vestibular dark cells and strial marginal cells transport K+ by similar mechanisms. We have shown that K+ transport in vestibular dark cells is sensitive to the cytosolic pH (pHi) (Wangemann et al. (1995a): J. Membrane Biol. 147: 255-262). The present study addresses pharmacologically the questions whether vestibular dark cells and strial marginal cells from the gerbil contain a Na+/H+ exchanger (NHE) and in which membrane, apical or basolateral, NHE is located. Further, the study addresses the question which NHE subtype is present in vestibular dark cells. pHi was measured micro-fluorometrically with the pH-sensitive dye 2',7'-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF), cell volume which is a measure of the net balance between ion influx and efflux was monitored as cell height (CH) and the equivalent short circuit current (Isc) which is a measure of transepithelial K+ secretion was calculated from measurements of the transepithelial voltage (Vt) and the transepithelial resistance (Rt). Changes in pHi were induced by 20 or 40 mM propionate. In the presence of propionate a transient acidification of pHi was observed in vestibular dark cells as well as a subsequent alkalinization to pHi values exceeding those under control conditions. The alkalinization of pHi in the presence of propionate was inhibited by the NHE blockers amiloride and EIPA. Propionate-induced swelling of vestibular dark cells was inhibited by amiloride. The NHE blocker amiloride caused in vestibular dark cells an acidification of pHi and a decrease in CH. Amiloride caused in both vestibular dark cells and strial marginal cells a transient stimulation of Isc when added to the basolateral side but not added to the apical side. Similar effects and pHi were observed in vestibular dark cells with the amiloride analog ethyl-isopropyl-amiloride (EIPA) and similar effects on Isc were observed with EIPA and the NHE blocker HOE694 when applied to the basolateral side of vestibular dark cell epithelium. The IC50 for these basolateral effects of EIPA, HOE694 and amiloride on Isc in vestibular dark cells were 2 x 10(-7) M, 8 x 10(-7) M and 4 x 10(-5) M. These observation suggest that vestibular dark cells and strial marginal cells contain NHE in their basolateral membrane, that K+ transport in strial marginal cells in pHi sensitive similar to K+ transport in vestibular dark cells and that NHE in vestibular dark cells consists of the subtype NHE-1.

The Na+/H+ exchanger in transitional cells of the inner ear.

The cytosolic pH (pHi) of transitional cells from the ampulla of the gerbil was measured micro-fluorometrically with the pH-sensitive dye 2',7'-bicarboxyethyl-5(6)-carboxyfluorescein (BCECF) to assess the possible contribution of a Na+/H+ exchanger to the regulation of pHi. All experiments were conducted in virtually HCO3(-)-free solutions. Under control conditions, pHi was 7.19 and addition of 10(-5) M ethylisopropylamiloride (EIPA), a blocker of Na+/H+ exchange, caused a small but significant acidification of pHi. A transient exposure to 21.4 mM NH4Cl caused a rapid cytosolic alkalinization followed by a brisk acidification and prompt recovery of pHi to its control value. The cytosolic buffer capacity (Bi) was determined in the absence of Na+ from changes in pHi which were elicited by [NH4+] steps. Bi was 4.7 mM/pH at pHi 7.19 and varied with pHi. The initial net proton flux JH, representative of Na+/H+ exchange activity, was calculated from the product of the initial rate of alkalinization after an NH4(+)-prepulse and Bi at the corresponding pHi. JH was dependent on the extracellular Na+ with an apparent Km of 64 mM, sensitive to the cytosolic [H+] with an apparent Km of 2.7 * 10(-7) M (i.e. pH 6.6), and inhibited by EIPA with an IC50 of 5 * 10(-7) M. These data suggest that transitional cells contain in the basolateral membrane a Na+/H+ exchanger of the amiloride-sensitive subtype.

Effect on cochlear potentials of lateral semicircular canal destruction.

Recording of the cochlear potentials was successfully performed during experimental labyrinthectomy in the guinea pig and in three patients with acoustic neuromas during translabyrinthine removal of the tumors. In the guinea pig, complete interruption of the duct of the lateral semicircular canal including the endolymphatic canal caused little change in the endocochlear DC potential of the first cochlear turn and input-output function curve of the N1 component of the compound action potential elicited by 8-kHz tone bursts. Further drilling of the vestibular labyrinth in the guinea pig caused decline of these potentials when the vestibular was opened. In patients with acoustic neuromas, the interruption of the duct of the lateral semicircular canal hardly altered the N1 input-output function curve and N1 input-latency function curve during the 1-hour observation period. Consistent preservation of cochlear function even after interruption of lateral semicircular canals suggests the possibility of partial surgical labryrinthectomy with preservation of hearing for lesions involving semicircular canals.

A rare case of severe acute pancreatitis complicated with pancreatic pseudocysts, obstructive jaundice and intraperitoneal hemorrhage.

A 58-year-old man visited our hospital because of back pain. Blood examinations revealed the presence of acute inflammation and an increase of pancreatic enzymes. Abdominal computed tomography indicated pseudocysts in the pancreas. The patient was diagnosed as having acute pancreatitis with pseudocysts formation. During the course of the disease, a newly formed pseudocyst in the pancreatic head compressed the common bile duct, leading to the obstructive jaundice. In addition, the rupture of a pseudocyst in the pancreatic tail caused intraperitoneal hemorrhage. This is an interesting case of acute pancreatitis with pseudocysts in which two rare complications developed.

Effect of perilymphatic air perfusion on cochlear potentials.

Perilymphatic fistula is now widely recognized to cause acute profound hearing loss. It is still controversial, however, which mechanism it is that causes the reversible hearing loss. Recently, it has been suggested by two groups of researchers that the intrusion of air bubbles into the perilymphatic space (a condition called pneumolabyrinth or aerolabyrinth) through the ruptured labyrinthine window(s) may be one of the causes. In order to examine the mechanism underlying the hearing loss associated with pneumolabyrinth, the perilymphatic space of the guinea pig cochlea was perfused with air and cochlear potentials were recorded. Although perfusion of the scala tympani with air at a rate as high as 200 microliter/min caused an immediate and drastic decrease of the cochlear microphonics (CM) and the compound action potential (AP), it had little effect on the endocochlear dc potential (EP) during perfusion for 20 min. A decline in EP was seen in half the ears, but only when the duration of perfusion exceeded 30 min. These results show that the EP has an amazing resistance to air trapped in the scala tympani of the cochlea and that the initial decrease of hearing acuity after the elimination of perilymph from the scala tympani (or introduction of air into the scala tympani) is probably due to interference in CM and AP generation mechanisms rather than to strial dysfunction.

A case of tick bite.

A 30-year-old women suffered from right superior eyelid infestation of Ixodes ovatus Neumann, which was mechanically removed with forceps after application of benzine. No mouth parts were retained in the eyelid. Removed mouth parts were examined by a scanning electron microscope. There were no complications except focal trichotylosis in the involved area. The mode of attachment of the tick to the host and the method of removal were discussed.

[Nationwide study of the antimicrobial susceptibility of clinical isolates of Proteus group in Japan].

We discussed the antimicrobial susceptibilities of Proteus group isolated in 1983 and 1984 and also the annual changes of the susceptibilities from 1980 to 1984. The tested strains were isolated in 103 hospitals in Japan. Antibiotics tested for this study were ampicillin (ABPC), cefazolin (CEZ), cefmetazole (CMZ), and gentamicin (GM). The MIC's were determined by the standard method of the Japan Society of Chemotherapy. Susceptibilities of the bacterial strains to the 4 antibiotics were described below: 1. Proteus mirabilis had good susceptibilities to all the antibiotics tested. 2. Susceptibilities of Proteus vulgaris were low to ABPC and CEZ, but high to CMZ and GM. 3. Proteus morganii showed low susceptibilities to ABPC and CEZ, and moderate to CMZ and GM. 4. Susceptibilities of Proteus rettgeri were low to ABPC and CEZ, and 25-40% of the strains were resistant to CMZ and GM. 5. Proteus inconstance had low susceptibilities to ABPC and CEZ, but fairly good to CMZ. About 55% of the strains showed resistance to GM. 6. There were no significant annual changes in susceptibilities of P. mirabilis, P. vulgaris and P. morganii to ABPC, CEZ and CMZ during the period from 1980 to 1984, but decreased susceptibilities to GM were noted in 1982. 7. There was no evidence of changes in susceptibilities of strains of P. rettgeri to ABPC and CMZ, but a tendency of decreasing susceptibilities to CEZ was shown from 1981. 8. P. inconstance showed no major changes in susceptibilities to ABPC, CMZ and GM. 9. Frequencies of resistant strains with MIC of 25 micrograms per ml or more in P. mirabilis, P. morganii and P. rettgeri were higher in 1982 and/or 1983 than the other years.

[A nationwide study of the antimicrobial susceptibility of clinical isolates of Escherichia coli in Japan].

We studied the antimicrobial susceptibility of E. coli isolated in 1983 and 1984 and also the yearly changes of the susceptibility of E. coli from 1980 to 1984 isolated in 103 hospitals in Japan. Antibiotics used for MIC determination were ampicillin (ABPC) as a penicillin, cefazolin (CEZ) as a cephalosporin, cefmetazole (CMZ) as a cephamycin and gentamicin (GM) as an aminoglycoside. Numbers of isolates tested were 2,321 strains in 1983 and 1,965 strains in 1984. CMZ was the most effective agent among the 4 antibiotics tested, followed by GM and CEZ in this order. A large number of the isolates were inhibited by 1.56 micrograms/ml on CMZ and GM and by 6.25 micrograms/ml of CEZ. About two-thirds of the isolates were inhibited at a concentration of 12.5 micrograms/ml or less of ABPC. The susceptibility of E. coli against the 4 antibiotics changed little year by year and the tendency of the appearance of resistant strains did not increase in the last 5 years.

[A nationwide study of the antimicrobial susceptibility of clinical isolates of Klebsiella sp. in Japan].

We have reported the antimicrobial susceptibility of clinical isolates of Escherichia coli in 1983 and 1984 to 4 antibiotics, ampicillin (ABPC), cefazolin (CEZ), cefmetazole (CMZ) and gentamicin (GM), in the previous report. In this paper, we described the antimicrobial susceptibility of Klebsiella sp. to the same antibiotics during the same period reported for E. coli before. Numbers of isolates tested were 1,671 strains in 1983 and 1,374 strains in 1984. We determined MICs of the 4 antibiotics against isolates by the standard method of the Japan Society of Chemotherapy. The most effective agent was GM among the 4 antibiotics. CMZ had a similar antimicrobial activity to GM. Resistance rates were low for both agents. CEZ had a fairly good antimicrobial activity, but it was less active than GM and CMZ. ABPC was not effective. There was neither significant annual change of antimicrobial activity of each agent against isolates nor an increasing tendency of resistant strains in the last 5 years. Depending on isolates from clinical materials, differences in the susceptibility were noted and there was a high ratio of resistant organisms among isolates from urine.

[Evaluation of angiographic delayed vasospasm due to ruptured aneurysm in comparison with cerebral circulation time measured by IA-DSA].

Delayed vasospasm due to ruptured aneurysm has been basically evaluated by angiographic changes in contrast to clinical features such as delayed ischemic neurological deficits (DIND). However, the discrepancies between angiographic and clinical findings have been pointed out. In this study, angiographic changes and cerebral circulation time in ruptured aneurysms were simultaneously investigated with IA-DSA. Thirty-two patients, who had ruptured aneurysms at the anterior circle of Willis and neck clippings at the acute stage, were investigated. Carotid angiogram was performed with IA-DSA on the 7-13th day after the attack. Angiographic changes were evaluated by Fischer's classification and circulation time was calculated in the following way. A time-density curve was obtained at the two ROI's; the C3-C4 portion and the rolandic vein. Circulation time was defined by the difference between the time showing peak optical density at the carotid and the venous portion. The control value of this circulation time obtained from 20 cases with non-rupture aneurysm and epilepsy was 3.4 sec (53 year old) on the average. X-ray CT scan examination was performed at the same time and clinical features were observed everyday. Angiographically, 3 cases were free from vasospasm, 18 cases were found to present slight to moderate vasospasm, and 11 cases showed severe vasospasm. Circulation time in patients with no spasm was 3.6 seconds, in patients with slight to moderate vasospasm it was 4.3 seconds and in patients with severe vasospasm it was 6.8 seconds. Ten patients showing cerebral infarction on CT scans demonstrated significantly long circulation time, 7.0 second on the average.(ABSTRACT TRUNCATED AT 250 WORDS)

[Ruptured distal anterior cerebral artery aneurysms presenting with acute subdural hematoma: report of two cases].

Two cases of ruptured distal anterior cerebral-artery aneurysms presenting with acute subdural hematoma are reported. Case 1 was a 55-year-old male, who showed abrupt disturbance of consciousness. An emergency CT revealed acute subdural hematoma at the right parietal convexity and interhemispheric fissure with moderate midline shift. There was no evidence of subarachnoid hemorrhage. Right carotid angiography showed an aneurysm at the right distal anterior cerebral artery. An emergency external decompression was performed and the aneurysm was clipped successfully through the interhemispheric fissure. In the operative field, subarachnoid hemorrhage could not been seen, and the patient had uneventful recovery. Case 2 was a 66-year-old female, who complained of severe headache. She deteriorated rapidly and become comatous with development of anisocoria. An emergency CT revealed acute subdural hematoma on the bilateral parietal convexities and interhemispheric fissure with severe midline shift. There was no evidence of subarachnoid hemorrhage. Carotid angiography showed right distal anterior cerebral artery aneurysm. An emergency external decompression was performed, then the aneurysm was clipped successfully. She recovered with disorientation and hemiparesis. Ruptured distal anterior cerebral artery aneurysms presenting with acute subdural hematoma without subarachnoid hemorrhage are rare. It is suggested that CT scans and history of patients are most important but an emergency angiography was prerequisite for correct diagnosis. Surgical treatment should be the best management in such cases.

[Mechanism of volume regulation of vestibular dark cells of the gerbil].

The volume response of vestibular dark cells of the gerbil to a hyposmotic challenge was investigated. Tissues including dark cells were perfused in preparations in which the perfusate had access to both sides of the epithelium and the height of the dark cell layer was measured as an indicator of its volume. We found that dark cells showed a fast and strong regulatory volume decrease (RVD) and prevented cell swelling in hypotonic media. This mechanism was dependent upon extracellular [K+] and [Cl-]. Ion selectivity of this mechanism was K+ = Rb+ > Cs+ > Na+ = NMDG+ (N-methyl-D-glucamine) for cations and Cl- = SCN- = NO3- > > gluconate- for anions. RVD of dark cells was inhibited by K(+)- channel blockers barium, quinidine and lidocaine, by Cl(-)-channel blockers 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, by an Na(+)-K+ ATPase inhibitor ouabain and by low temperature, but was not inhibited by a loop diuretic bumetanide, by carbonic anhydrase inhibitors acetazolamide and ethoxyzolamide, by a K(+)-channel blocker tetraethylammonium, by a Cl(-)-channel blocker 5-nitro-2 (3-phenylpropylamino)-benzoic acid or by an inhibitor of the Na(+)-H+ exchanger amiloride. These data suggest that the RVD of dark cells occurs via separate K+ and Cl- channels which are different from those active under isosmotic condition, and is presumably activated by a hyposmotic stimulus.

[Bilateral simultaneous sudden deafness--a report of five cases].

Five cases of bilateral simultaneous sudden deafness were reported. Two patients were male and three patients were female. The age of the patients ranged from five to 54 years. All the five cases were profoundly deaf on both sides. The elevation of the mumps virus antibody titer was found in the three cases out of four in those examined. The recovery pattern of hearing was seen symmetrically on both sides in four cases. Although complete recovery of hearing was obtained in one case and remarkable improvement was observed in two cases bilaterally, no improvement was seen in one case on either side. The last case showed remarkable improvement of hearing on one side with only a slight recovery on the other. Although the prognosis of the five cases of bilateral simultaneous sudden deafness was found comparable to that of the unilateral case by other investigators, the recovery was slower and the prognosis was less favorable in cases with the elevation of mumps antibody titer.

[Mutations of the dystrophin gene in dilated cardiomyopathy].

X-linked dilated cardiomyopathy(XLDCM) is caused by mutations of the dystrophin gene, which was originally cloned as the responsible gene for Duchenne muscular dystrophy and Becker muscular dystrophy. Mutations due to XLDCM are centered on 5' end of the gene, especially M-promoter and the adjacent region. However, other mutations are dispersed and cannot be characterized. Three mechanisms have been proposed by which the involvement of cardiac muscle is so severe in spite of the lack of skeletal muscle symptoms; 1) up-regulation of B- and P-dystrophin in merely skeletal muscle compensating for the defect of M-dystrophin, 2) dysfunction of some parts of dystrophin specifically essential to cardiac muscle, 3) different expression patterns of mutant mRNA between cardiac and skeletal muscle.