Substeps within the 8-nm step of the ATPase cycle of single kinesin molecules.

Kinesin is a molecular motor that moves processively by regular 8-nm steps along microtubules. The processivity of this movement is explained by a hand-over-hand model in which the two heads of kinesin work in a coordinated manner. One head remains bound to the microtubule while the other steps from the alphabeta-tubulin dimer behind the attached head to the dimer in front. The overall movement is 8 nm per ATPase cycle. To investigate elementary processes within the 8-nm step, we have developed a new assay that resolves nanometre displacements of single kinesin molecules with microsecond accuracy. Our data show that the 8-nm step can be resolved into fast and slow substeps, each corresponding to a displacement of approximately 4 nm. The substeps are most probably generated by structural changes in one head of kinesin, leading to rectified forward thermal motions of the partner head. It is also possible that the kinesin steps along the 4-nm repeat of tubulin monomers.

Two types of Chlamydomonas flagellar mutants missing different components of inner-arm dynein.

Two types of Chlamydomonas reinhardtii flagellar mutants (idaA and idaB) lacking partial components of the inner-arm dynein were isolated by screening mutations that produce paralyzed phenotypes when present in a mutant missing outer-arm dynein. Of the currently identified three inner-arm subspecies I1, I2, and I3, each containing two heterologous heavy chains (Piperno, G., Z. Ramanis, E. F. Smith, and W. S. Sale. 1990. J. Cell Biol. 110:379-389), idaA and idaB lacked I1 and I2, respectively. The 13 idA isolates comprised three genetically different groups (ida1, ida2, ida3) and the two idaB isolates comprised a single group (ida4). In averaged cross-section electron micrographs, inner dynein arms in wild-type axonemes appeared to have two projections pointing to discrete directions. In ida1-3 and ida4 axonemes, on the other hand, either one of them was missing or greatly diminished. Both projections were weak in the double mutant ida1-3 x ida4. These observations suggest that the inner dynein arms in Chlamydomonas axonemes are aligned not in a single straight row, but in a staggered row or two discrete rows. Both ida1-3 and ida4 swam at reduced speed. Thus, the inner-arm subspecies missing in these mutants are not necessary for flagellar motility. However, the double mutants ida1-3 x ida4 were nonmotile, suggesting that axonemes with significant defects in inner arms cannot function. The inner-arm dynein should be important for the generation of axonemal beating.

Immunological detection of actin in the 14S ciliary dynein of Tetrahymena.

The association of actin with Tetrahymena ciliary dyneins was examined using a polyclonal antibody against Tetrahymena actin. Western blotting shows that actin is present in the 14S dynein fraction, but not in the 22S dynein fraction, which comprises the outer arm. By anion-exchange chromatography, 14S dynein can be further separated into three major fractions that contain four distinct heavy chains in total. When each fraction was tested by anti-actin immunoblotting, all three fractions contained actin in nearly stoichiometric amounts with the heavy chain. Since Tetrahymena actin differs significantly from actins of other species, the association with inner-arm dynein may be a conserved property of actin.

Expression of inducible nitric oxide synthase in T lymphocytes and macrophages of cholesterol-fed rabbits.

While endothelial nitric oxide synthase has been reported to be expressed in the endothelial cells of normal and atherosclerotic vessels, there are few reports about inducible nitric oxide synthase (iNOS). We investigated the expression of iNOS and its relation to inflammatory cells in atheroma. New Zealand White rabbits were fed 1 of 4 diets: (i) normal diet for 9 weeks; (ii) normal plus 1% cholesterol diet (atherogenic diet) for 9 weeks; (iii) atherogenic diet for 9 weeks, then normal diet for 9 weeks; (iv) atherogenic diet for 9 weeks, then the normal diet for 36 weeks. The aortas were examined by immunohistochemical staining for anti-iNOS antibody, as well as antibodies for macrophages, T lymphocytes, and muscle actin. No iNOS was detected in normal aortas, intimal thickenings, or fatty streaks. Although iNOS was detected in necrotic cores of advanced plaque, it was not seen in smooth muscle-derived cells or endothelial cells but was found in some macrophage-derived cells and in T lymphocytes. In regressive atherosclerotic aortas, iNOS was detected only in necrotic cores, not in macrophage-derived cells but in T lymphocytes. These findings suggest that T lymphocytes and some macrophages induce iNOS through cytokine production in atheroma. This is the first report of iNOS expression in atheromatous plaque.

Rupture of congenital peripheral pulmonary aneurysm.

A 58-year-old man presenting with solitary aneurysm of a peripheral pulmonary artery was treated by left lower lobectomy. Histologically, the aneurysmal wall showed medial hypertrophy with the loss of smooth muscle fibers, without evidence of a mycotic process or inflammatory exudate. The aneurysm appeared to be congenital in origin.

The beta adrenoreceptor antagonist, nipradilol, preserves the endothelial nitric oxide response in atherosclerotic vessels of rabbit.

We evaluated the effects of nipradilol, a beta-adrenoreceptor antagonist which contains a nitroxy residue, for vascular response in atherosclerosis of rabbits. Four groups of rabbits received different diets (standard diet; standard diet plus 10 mg/kg/day nipradilol; atherogenic diet [standard diet plus 1% cholesterol]; atherogenic diet plus 10 mg/kg/day nipradilol) for 9 weeks. Plasma lipids, blood pressure, vascular function, nitric oxide (NO), activity of NO synthase, cGMP, and histological atherosclerotic changes were evaluated. Neither the atherogenic diet nor nipradilol treatment affected significantly the animals' body weight, blood pressure, or heart rate. The atherogenic diet increased total cholesterol and triglycerides, which were not altered by nipradilol. The atherogenic diet diminished the acetylcholine-induced NO mediated relaxation. Nipradilol treatment restored this relaxation. Analyses using a NO-sensitive selective electrode showed that nipradilol released NO in the presence of cells and that NO release was greater in atherosclerotic aorta with than without nipradilol treatment. Nipradilol treatment increased the basal NO release as evaluated by the aortic tissue cyclic GMP (cGMP) levels in atherosclerotic vessel, and reduced the esterified cholesterol levels in atherosclerotic vessel. Conclusively, NO released by nipradilol may protect endothelium derived relaxation in atherosclerotic vessels, and may partially inhibit the accumulation of cholesterol in the atherosclerotic lesions.

Endothelial-constitutive nitric oxide synthase exists in airways and diesel exhaust particles inhibit the effect of nitric oxide.

Diesel exhaust particles (DEP) are an important cause of air pollution and are thought to be responsible for some respiratory ailments, but the exact mechanism is not known. We evaluated whether DEP inhibit nitric oxide (NO) synthesis in bronchi as NO is present in the exhaled air and has a physiological role in the respiratory tract. Aortic rings were also examined for comparison. We observed that acetylcholine (ACh) induced contraction of the bronchi was partially attenuated by the simultaneous release of NO. When bronchial rings were incubated either with NG-methyl-L-arginine (L-NMA): an inhibitor of NO synthase (NOS) or with DEP, the contraction to ACh was abolished. The source of the NOS was the bronchial epithelium and this endothelial-constitutive NOS was demonstrated by immunohistochemistry. DEP like L-NMA inhibited the ACh induced endothelium dependent relaxation in the aortic rings. The inhibition of NO release by DEP and L-NMA from bronchial and aortic rings was also confirmed by a selective NO electrode. We conclude that inhibition of NO availability by DEP may in part be responsible for the adverse respiratory effects seen by inhalation of these particles in polluted air.

A case of renal sarcoidosis showing central necrosis and abnormal expression of angiotensin converting enzyme in the granuloma.

We describe a 66-year-old man who developed renal failure related to granulomatous renal sarcoidosis without systemic manifestations. Renal failure was severe enough to require hemodialysis transiently. Renal biopsy of this patient revealed the central necrosis of the granuloma which is usually absent in sarcoid granuloma. Serum level of angiotensin converting enzyme (ACE) was not helpful for diagnosis in this patient because serum ACE level is often elevated in the condition of chronic renal failure. Immunohistochemical detection of ACE was of diagnostic value in this patient. Subsequent course in which glucocorticoid was used for therapy was consistent with the diagnosis. This is the first report of identification of ACE in renal sarcoid granuloma.

Food resource utilization of the skates Rioraja agassizii (Müller & Henle, 1841) and Psammobatis extenta (Garman, 1913) on the continental shelf off Ubatuba, South-eastern Brazil.

The feeding habits of Rioraja agassizii (syn. Raja agassizii) and Psammobatis extenta (syn. Psammobatis glansdissimilis) of the South-eastern Brazilian coast were studied by means of stomach content analysis. The samples were obtained on eight seasonal oceanographic cruises, carried out between October 1985 and July 1987. The importance of each food item was evaluated on the basis of the Index of Relative Importance and the feeding similarity by Percentage of Similarity. The results indicated that both species are benthic feeders, preying mainly on Crustacea, especially Amphipoda, Caridea and Brachyura. Teleostei were also important for R. agassizii. Seasonal variation of the diet seems to be associated with the availability of the prey, whose distribution and abundance are related to the dynamics of the water masses of the region. Juveniles and adults of P. extenta exploited the same resources while juveniles and adults of R. agassizii presented low diet similarity during most of the year. Caridea were an important food for all length classes of R. agassizii, while Amphipoda were for smaller specimens, and Teleostei for larger ones. The feeding overlap between the two species was higher during autumn 1986, winter 1986 and winter 1987.

[10 cases of congenital cystic dilatation of the common bile duct].

Ten cases of congenital cystic dilatation of the common bile duct (including insufficiency form) were examined for mode of pancreatico-biliary anastomosis and cholangiographic findings. The results were as follows. Male to female ratio was 1:9 in this series. Regarding the mode of pancreatico-biliary anastomosis, type I (pancreatic duct flowing into common bile duct) tended to occur frequently in patients 30 years old or more, while type II (common bile duct flowing into pancreatic duct) in patients under 30 years old. With respect to clinical symptoms, no cases had triad (jaundice, abdominal pain and palpation of mass), but abdominal pain occurred in all the cases. Jaundice and palpation of mass were seen in 30% and 10% of the cases, respectively. Regarding the age at the onset and the duration of the disease, type I occurred in elderly cases with a longer duration of disease, while type II occurred in young cases with a shorter duration of disease. The degree of distention increased in proportion to the severity of the peripheral stenosis of the biliary duct. Complication with biliary tract carcinoma was seen in 2 type I cases. Among the type I cases, those with mild distention were considered to be insufficient form.

HRCT in detection of pulmonary infections from nontuberculous mycobacteria: personal experience.

PURPOSE: The authors sought to assess the role of high-resolution computed tomography (HRCT) in the detection and follow-up of nontuberculous mycobacteria (NTM) pulmonary infection in immunocompetent patients and to identify the most common radiological patterns for diagnosis. MATERIALS AND METHODS: Plain chest radiographs and HRCT scans of 42 consecutive patients with NTM pulmonary infection (M/F 26/16; mean age 57, range 41-83) were retrospectively reviewed. Ten of these patients were followed up for 18 months after diagnosis. Small nodules (<10 mm), nodules 10- to 30-mm in diameter, lobar/segmental consolidation, cavitations, bronchiectasis and tree-in-bud pattern were analysed. RESULTS: Small nodules were more frequent than nodules 10- to 30-mm in diameter, and segmental consolidation was more frequent than lobar. Cavitations, tree-in-bud and bronchiectasis were more frequently located in the upper lobes. Four of the followed-up patients had cavitation of preexisting nodules, and five had progression of bronchiectasis. CONCLUSIONS: HRCT allows accurate detection and followup of the most frequent presentation patterns: diffuse small nodules, bronchiectasis, upper lobe segmental consolidation and cavitations. The appearance of new bronchiectasis and progression of old disease are due to pulmonary infection.

Kinetics of force generation by single kinesin molecules activated by laser photolysis of caged ATP.

To relate transients of force by single kinesin molecules with the elementary steps of the ATPase cycle, we measured the time to force generation by kinesin after photorelease of ATP from caged ATP. Kinesin-coated beads were trapped by an infrared laser and brought onto microtubules fixed to a coverslip. Tension was applied to a kinesin-microtubule rigor complex using the optical trap, and ATP was released by flash photolysis of caged ATP with a UV laser. Kinesin started to generate force and move stepwise with a step size of 8 nm at average times of 31, 45, and 79 ms after photorelease of 450, 90, and 18 microM ATP, respectively. The kinetics of force generation were consistent with a two-step reaction: ATP binding, with an apparent second-order rate constant of 0.7 microM-1.s-1, followed by force generation at 45 s-1 per kinesin molecule. The transient rate of force generation was close to the rate of the ATPase cycle in solution, suggesting that the rate-limiting step of ATPase cycle is involved with the force generation.

Mechanics of single kinesin molecules measured by optical trapping nanometry.

We have analyzed the mechanics of individual kinesin molecules by optical trapping nanometry. A kinesin molecule was adsorbed onto a latex bead, which was captured by an optical trap and brought into contact with an axoneme that was bound to a glass surface. The displacement of kinesin during force generation was determined by measuring the position of the beads with nanometer accuracy. As the displacement of kinesin was attenuated because of the compliance of the kinesin-to-bead and kinesin-to-microtubule linkages, the compliance was monitored during force generation and was used to correct the displacement of kinesin. Thus the velocity and the unitary steps could be obtained accurately over a wide force range. The force-velocity curves were linear from 0 to a maximum force at 10 microM and 1 mM ATP, and the maximum force was approximately 7 pN, which is larger by approximately 30% than values previously reported. Kinesin exhibited forward and occasionally backward stepwise displacements with a size of approximately 8 nm. The histograms of step dwell time show a monotonic decrease with time. Model calculations indicate that each kinesin head steps by 16-nm, whereas kinesin molecule steps by 8-nm.

In vivo ESR measurement of free radicals in whole mice.

The in vivo measurement of nitroxide radicals in whole mouse was carried out by L-band ESR spectroscopy. Spectra were successively observed in hepatic and bladder domains of female mice after intravenous administration of spin-labeled compounds (CPROXYL or TEMPOL). The signal intensities from both domains decreased gradually. The kinetic constants of clearance in the hepatic domain were 0.09/min for CPROXYL and 0.71/min for TEMPOL. The clearance constants in the bladder domain coincided with those in the hepatic domain within experimental error, whereas the constants in collected blood were 1/7-1/10 of those in the hepatic or bladder domains. The mechanism of clearance of nitroxide radicals in whole mice is discussed.

Outcomes of written living wills in Japan--a survey of the deceased ones' families.

A study was recently done to investigate the outcomes of written living wills in Japan, which, unlike the United States, does not legally recognize advance directives. This study collected demographic information and predictors of living will use among families and guardians who used the living will form provided by the Japan Society for Dying with Dignity. According to the results of the study, affliction with cancer and death in a hospital was the best predictor of written living will use. Research on physicians' treatment limitation and withdrawal behaviors in the clinical setting is needed to better understand the actual impact of living wills. Such research can provide better understanding of where boundaries of self-determination are drawn and how to respect autonomy in Japan.

Motility of single one-headed kinesin molecules along microtubules.

The motility of single one-headed kinesin molecules (K351 and K340), which were truncated fragments of Drosophila two-headed kinesin, has been tested using total internal reflection fluorescence microscopy. One-headed kinesin fragments moved continuously along the microtubules. The maximum distance traveled until the fragments dissociated from the microtubules for both K351 and K340 was approximately 600 nm. This value is considerably larger than the space resolution of the measurement system (SD approximately 30 nm). Although the movements of the fragments fluctuated in forward and backward directions, statistical analysis showed that the average movements for both K340 and K351 were toward the plus end of the microtubules, i.e., forward direction. When BDTC (a 1.3-S subunit of Propionibacterium shermanii transcarboxylase, which binds weakly to a microtubule), was fused to the tail (C-terminus) of K351, its movement was enhanced, smooth, and unidirectional, similar to that of the two-headed kinesin fragment, K411. However, the travel distance and velocity of K351BDTC molecules were approximately 3-fold smaller than that of K411. These observations suggest that a single kinesin head has basal motility, but coordination between the two heads is necessary for stabilizing the basal motility for the normal level of kinesin processivity.

Physiological concentrations of 17beta-estradiol inhibit the synthesis of nitric oxide synthase in macrophages via a receptor-mediated system.

We investigated the effect of estrogen on inducible nitric oxide synthase (iNOS), which is not well understood, in contrast to the known effect of estrogen on endothelial nitric oxide synthase (eNOS). When J774 cells, a murine macrophage cell line, were incubated with interferon-gamma and lipopolysaccharide, iNOS was induced, and a large amount of NO was released. Pre- or coincubation with 17beta-estradiol inhibited this induction of iNOS protein and NO release; however, 17beta-estradiol did not have a direct effect on enzyme activity of iNOS. The analog, 17alpha-estradiol, did not have such an effect. Tamoxifen, an antiestrogen, and ICI182780, an estrogen-receptor antagonist, inhibited the influence of 17beta-estradiol on iNOS. Thus 17beta-estradiol inhibited the induction of iNOS by a classic receptor-mediated pathway. The inhibition of the NO release from iNOS by 17beta-estradiol is in contrast to the reported augmentation of continuous NO release from eNOS. These harmonious effects of estrogen on iNOS and eNOS may have some role in the antiatherosclerotic effects of 17beta-estradiol.

Direct inhibition of microtubule-based kinesin motility by local anesthetics.

Local anesthetics are known to inhibit neuronal fast anterograde axoplasmic transport (FAAT) in a reversible and dose-dependent manner, but the precise mechanism has not been determined. FAAT is powered by kinesin superfamily proteins, which transport membranous organelles, vesicles, or protein complexes along microtubules. We investigated the direct effect of local anesthetics on kinesin, using both in vitro motility and single-molecule motility assays. In the modified in vitro motility assay, local anesthetics immediately and reversibly stopped the kinesin-based microtubule movement in an all-or-none fashion without lowering kinesin ATPase activity. QX-314, a permanently charged derivative of lidocaine, exerted an effect similar to that of lidocaine, suggesting that the effect of anesthetics is due to the charged form of the anesthetics. In the single-molecule motility assay, the local anesthetic tetracaine inhibited the motility of individual kinesin molecules in a dose-dependent manner. The concentrations of the anesthetics that inhibited the motility of kinesin correlated well with those blocking FAAT. We conclude that the charged form of local anesthetics directly and reversibly inhibits kinesin motility in a dose-dependent manner, and it is the major cause of the inhibition of FAAT by local anesthetics.

A case of coexisting malignant carcinoid tumor and adenocarcinoma in the papilla of Vater.

A 47-year-old Japanese woman in whom obstructive jaundice had already been diagnosed, was found to have a dome-shaped elevated tumor approximately 3 cm in diameter located in the area very close to the papilla of Vater on endoscopical and radiographical investigations. Histopathologically, the resected tumor was composed mainly of solid nests of atypical argyrophilic cells, and partially of an area of well differentiated tubular adenocarcinoma, showing mutual transition in the mucosal layer. Both immunohistochemical and ultrastructural analyses confirmed the difference in character of tumor cells between these two areas: neuroendocrine cell carcinoma and tubular adenocarcinoma of common type in the intestine. To the best of our knowledge, this is only the third case reported to be a coexisting malignant carcinoid tumor and adenocarcinoma arising in the periampullary region.