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Modulation of spindle oscillations by acetylcholine, cholecystokinin and 1S,3R-ACPD in the ferret lateral geniculate and perigeniculate nuclei in vitro.

The transition from sleep to waking is associated with the abolition of spindle waves and the appearance of tonic activity in thalamocortical neurons and thalamic reticular/perigeniculate GABAergic cells. We tested the possibility that changes such as these may arise through modulation of the leak potassium current, IKL, by examining the effects of neurotransmitters known to modulate this current on spindle wave generation in the ferret geniculate slice maintained in vitro. Local application of agents that reduce IKL in thalamocortical neurons, including acetylcholine, DL-muscarine chloride and the glutamate metabotropic receptor agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), to spontaneously spindling thalamocortical neurons resulted in a 5-10 mV membrane depolarization and the abolition of spindle waves. Local application of 1S,3R-ACPD and cholecystokinin-8-sulfate, both of which reduce IKL, to GABAergic neurons of the perigeniculate nucleus resulted in a 10-20 mV membrane depolarization, appearance of tonic discharge and the abolition of spindle wave generation. Local application of 1S,3R-ACPD and cholecystokinin to the perigeniculate nucleus while recording from thalamocortical neurons resulted in the abolition of spindle wave-associated inhibitory postsynaptic potentials and the occurrence of a continuous barrage of smaller amplitude inhibitory postsynaptic potentials, presumably in response to depolarization and tonic discharge of perigeniculate neurons. These results indicate that modulation of IKL in thalamocortical neurons and perigeniculate neurons is capable of abolishing the generation of spindle waves in thalamic networks. Through the modulation of IKL, ascending and descending activating systems may control the state of the thalamus such that the transition from slow wave sleep to waking is associated with the abolition of slow, synchronized rhythms and the facilitation of a state that is conducive to sensory receptor field analysis, arousal and perception.

Acetylcholine↗

Applications of a new device (spindle) for improved characterization of drug release (dissolution) of pharmaceutical products.

A crescent spindle (patent pending) is described which may be used in place of the USP paddle component in USP dissolution apparatus 2. The new spindle is curve shaped, corresponding to the bottom of a dissolution vessel, with attached bristles to fill in the gap between the spindle and the surface of the vessel. The geometry of the new spindle provides more efficient mixing than the USP paddle and prevents accumulation of disintegrated material (no cone formation). Using the new spindle, in comparison with the USP paddle, dissolution characteristics of three drug products: 250 mg amoxicillin capsules, 15.6 g acetylsalicylic acid (ASA) boluses and 200 mg carbamzepine tablets were evaluated. The experimental conditions for dissolution testing with the two stirring devices included; 900 ml of 0.05 M phosphate buffer, pH 6.8 with 50 rpm, 900 ml of 0.05 M acetate buffer, pH 4.5-ethanol (7:3) with 50 rpm, and water containing 1% sodium lauryl sulphate with 75 rpm for amoxicillin capsules, ASA boluses and carbamazepine tablets, respectively. Uncharacteristic of the test products, which are fast release, the USP paddle provides significantly slower drug release. For example, 90 min for <80% drug release vs. 10 min for >90% for amoxicillin capsules and 6 h for 80% vs. 30 min for >90% for ASA boluses with USP paddle vs. the new spindle. In case of the carbamazepine tablets, three products which are bioequivalent and prescribed interchangeably, the USP paddle method shows significantly different dissolution characteristics. However, with the new device, all these products show similar drug release characteristics, a better reflection of product release characteristics and in vivo drug release behaviour. Compared with the USP paddle, the suggested device (spindle) provides improved stirring and mixing which appears to provide more appropriate (biorelevant) characterization of pharmaceutical products.

Amoxicillin↗

Spindle-cell carcinoma of the oral cavity and larynx. Immunohistochemical aspects.

Five cases of monophasic and 7 cases of biphasic spindle-cell carcinomas were analyzed immunohistochemically for the presence of vimentin and keratin type intermediate filaments in the pleomorphic spindle cells. Vimentin reactivity proved to be a consistent feature but keratin reactivity was more variable, this latter filament being lost in two cases initially presenting as pure squamous cell carcinomas showing dedifferentiation towards a pure monophasic spindle-cell tumour when recurring. The converse was also noted: acquisition of keratin in a monophasic spindle-cell tumour that recurred as squamous cell carcinoma. These results were considered to support the concept that spindle-cell tumours of the upper aerodigestive tract are a peculiar type of carcinoma and not a product of a pluripotent stem cell exhibiting bidirectional differentiation. Diagnostic implications are as follows: keratin positivity in a spindle-cell tumour substantiates its carcinomatous nature but its absence does not rule out a diagnosis of spindle-cell carcinoma.

Aged↗

Benign spindle cell tumor of the breast with prominent adipocytic component.

We report a rare case of "benign spindle cell tumor of the breast with prominent adipocytic component" and present a review of the literature on the topic. The close morphologic resemblance with myofibroblastoma and spindle cell lipoma of the breast is emphasized and used as a starting point for diagnostic and histogenetic considerations. Although immunocytochemistry and electron microscopy showed features favoring a diagnosis of spindle cell lipoma, they were not conclusive. The tumor described here contributes to widen the spectrum of the benign spindle cell tumors of the breast variously named "benign spindle cell tumor," myofibroblastoma, spindle cell lipoma, and fibroma. It is important to distinguish this lesion from spindle cell metaplastic carcinoma and aggressive fibromatosis.

Adipocytes↗

The effects of normal aging on sleep spindle and K-complex production.

OBJECTIVES: Despite a relatively large body of literature describing the characteristics of sleep spindles and K-complexes in young adults, relatively little research has been conducted in older individuals. The general consensus from the few studies that have addressed this issue is that there is a progressive decrease in the number of spindles and K-complexes with age, although there is large intra-individual variation. Whether or not these changes are an inevitable consequence of the aging process can be addressed by studying healthy older adults who provide an example of the effects of age independently from those of disease. METHODS: Fourteen young adults (mean age=21.4+/-2.5 years) and 20 older adults (mean age=75.5+/-6.3 years) participated in the study. All subjects were neurologically and medically healthy and were not taking any medications with a known effect on the central nervous system or sleep. For each subject, a number of characteristics were determined including the number, density (SS/min), amplitude and frequency of all spindles as well as the number and density of K-complexes (KC/min). RESULTS: Spindle number, density and duration as well as K-complex number and density were all significantly lower in the elderly compared to the young adults. The EEG frequency within the spindles was significantly higher in the elderly, although the absolute difference was less than 0.5 Hz. Multiple regression analysis indicated that spindle duration and K-complex density were able to predict over 90% of the variance in age. CONCLUSIONS: The age-related decrease in sleep spindle and K-complex density is consistent with previous reports and may be interpreted as an age-related alteration of thalamocortical regulatory mechanisms.

Adult↗

Spindle cell squamous carcinoma of the oesophagus: an analysis of 17 cases, with new immunohistochemical evidence for a clonal origin.

AIMS: To study the morphological and immunohistochemical characteristics of spindle cell squamous carcinoma of the oesophagus, in order better to understand the histogenesis of this tumour. METHODS AND RESULTS: In this study we analysed the morphological and immunohistochemical characteristics of 17 cases of spindle cell squamous carcinoma of the oesophagus. Most tumours were polypoid, but tumours with an ulcerated and infiltrative pattern were also observed. Histologically, most tumours were of superficial type, with a characteristic morphological aspect consisting of two types of tumour cells, i.e. differentiated squamous cells, and spindle cells with transition zones between the two components. On immunohistochemistry, the squamous cells were positive for cytokeratin and the spindle cells showed variable expression of cytokeratin, vimentin and smooth muscle actin. p53 protein was over-expressed in 10 cases, both tumour cell types showing strong nuclear positivity. In most tumours, E-cadherin was expressed in the squamous cells and absent in the spindle cells. CONCLUSIONS: The similar pattern of p53 protein expression in the two tumour cell types of spindle cell squamous carcinoma of the oesophagus suggests their common origin. The change in adhesion molecule expression with loss of E-cadherin expression may be associated with the acquisition of spindle cell morphology by the squamous tumour cells.

Actins↗

Differential Ki67 and bcl-2 immunoexpression in solid-glandular and spindle cell components of biphasic synovial sarcoma: a double immunostaining assessment with cytokeratin and vimentin.

AIMS: Synovial sarcoma is a malignant soft tissue of uncertain histogenesis that may show a biphasic (spindle and solid/glandular components) or a monophasic histological appearance. In previous studies, we demonstrated that the solid/glandular component possesses higher proliferation rates than the spindle cell component of biphasic synovial sarcomas and that the spindle cell component may exhibit a progressive transition from or to the solid-glandular component in biphasic synovial sarcoma. To evaluate this hypothesis further, we designed a novel approach to correlate immunoexpression of Ki67, bcl-2 and bax in the spindle cell and in the solid-glandular component of biphasic synovial sarcomas. We also performed a double-immunohistochemical assessment of the Ki67 proliferative indices and the immunoexpression of anti-apoptotic protein bcl-2 in neoplastic cells expressing either vimentin or cytokeratin. METHODS AND RESULTS: Immunohistochemistry for vimentin (10 cases), bcl-2 (10 cases), Ki67(10 cases), cytokeratin (10 cases), and bax (eight cases), and double-immunostaining for vimentin/Ki67 (10 cases), vimentin/bcl-2 (nine cases), cytokeratin/Ki67 (10 cases), and cytokeratin/bcl-2 (10 cases) assays were performed in 10 cases of primary biphasic synovial sarcoma. Semiquantitative assessment was adopted for each case in both components. Statistical analysis was performed using Fisher's exact test or chi2 test. On conventional immunohistochemistry, the solid/glandular component revealed more expression of Ki67, bax and cytokeratin than the spindle cell component (P=0.0004, P=0.082, and P < 0.0001, respectively); on the other hand, the latter showed higher expression of bcl-2 and vimentin than the former (P=0.0281 and P=0.059, respectively). Double immunohistochemistry analysis revealed higher co-expression levels of cytokeratin/Ki67 and cytokeratin/bcl-2 than the spindle cell component (P=0.015 and P < 0.0001, respectively); conversely, the latter presented higher co-expression of vimentin/bcl-2 than the former (P=0.0007). All cases showed no more than 10% of cells coexpressing cytokeratin/bcl-2, cytokeratin/Ki67, and no case revealed cells coexpressing vimentin/Ki67. CONCLUSION: Our findings indicate that in biphasic synovial sarcoma the acquisition of epithelial phenotype (solid/glandular component) is associated with a high expression of pro-apoptotic proteins and a high proliferative differentiation status, and conversely, mesenchymal phenotype (spindle cell component) is associated with a high expression of apoptosis-inhibitor bcl-2 and a low proliferative terminal-type differentiation status.

Adult↗

Optimization of sigma amplitude threshold in sleep spindle detection.

Sleep spindles are transient EEG waveforms of non-rapid eye movement sleep. There is considerable intersubject variability in spindle amplitudes. The problem in automatic spindle detection has been that, despite this fact, a fixed amplitude threshold has been used. Selection of the spindle detection threshold value is critical with respect to the sensitivity of spindle detection. In this study a method was developed to estimate the optimal recording-specific threshold value for each all-night recording without any visual scorings. The performance of the proposed method was validated using four test recordings each having a very different number of visually scored spindles. The optimal threshold values for the test recordings could be estimated well. The presented method seems very promising in providing information about sleep spindle amplitudes of individual all-night recordings.

Electroencephalography↗

Regional differences in the circadian modulation of human sleep spindle characteristics.

Electroencephalographic oscillations in the sleep spindle frequency range (11-16 Hz) are a key element of human nonrapid eye movement sleep. In the present study, sleep spindle characteristics along the anterior-posterior axis were analysed during and outside the circadian phase of melatonin secretion. Sleep electroencephalograms were recorded during naps distributed over the entire circadian cycle and analysed with two different methodological approaches, the classical fast Fourier transform in the frequency-domain and a new method for instantaneous spectral analysis, the fast time frequency transform that yields high-resolution parameters in the combined time-frequency-domain. During the phase of melatonin secretion, spindle density was generally increased and intraspindle frequency variation reduced. Furthermore, lower spindle frequencies were promoted: peak frequencies shifted towards the lower end of the spindle frequency range, and spindle amplitude was enhanced in the low-frequency range (11-14.25 Hz) and reduced in the high-frequency range (approximately 14.5-16 Hz). The circadian variation showed a clear dependence on brain topography such that it was maximal in the parietal and minimal in the frontal derivation. Our data provide evidence that the circadian pacemaker actively promotes low-frequency sleep spindles during the biological night with a parietal predominance.

Adult↗

Multiple spindle cell lipomas and dermatofibrosarcoma protuberans within a single patient: evidence for a common neoplastic process of interstitial dendritic cells?

This case report describes a 48-year-old man with multiple spindle cell lipomas of the neck and a dermatofibrosarcoma protuberans (DFSP) with fibrosarcomatous transformation of the chest. The presence of familial and nonfamilial multiple spindle cell lipomas within a single patient is a rare event, with only two reports in the current literature. This case represents the first report of multiple spindle cell lipomas occurring in association with a DFSP. It is of particular interest in that both spindle cell lipoma and DFSP represent, at least in part, neoplastic proliferations of CD34(+) spindled cells. The exact nature and differentiation of these spindled cells remains controversial, but prior studies have suggested that they could represent neoplastic interstitial dendritic cells. The association of DFSP and spindle cell lipoma within this single patient suggests that these two tumors (and their histologic variants) may well be linked, conceptually, as neoplastic proliferations of CD34(+) interstitial dendritic cells.

Dendritic Cells↗

Prolonged hyperpolarizing potentials precede spindle oscillations in the thalamic reticular nucleus.

The thalamic reticular (RE) nucleus is a key structure in the generation of spindles, a hallmark bioelectrical oscillation during early stages of sleep. Intracellular recordings of RE neurons in vivo revealed the presence of prolonged hyperpolarizing potentials preceding spindles in a subgroup (30%) of neurons. These hyperpolarizations (6-10 mV) lasted for 200-300 ms and were present just before the onset of spontaneously occurring spindle waves. Corticothalamic volleys also were effective in generating such hyperpolarizations followed by spindles in RE neurons. A drop of up to 40% in the apparent input resistance (R(in)) was associated with these hyperpolarizing potentials, suggesting an active process rather than disfacilitation. Accordingly, the reversal potential was approximately -100 mV for both spontaneous and cortically elicited hyperpolarizations, consistent with the activation of slow K(+) conductances. QX-314 in the recording pipettes decreased both the amplitude and incidence of prolonged hyperpolarizations, suggesting the participation of G protein-dependent K(+) currents in the generation of hyperpolarizations. Simultaneous extracellular and intracellular recordings in the RE nucleus demonstrated that some RE neurons discharged during the hyperpolarizations and, thus, may be implicated in their generation. The prolonged hyperpolarizations preceding spindles may play a role in the transition from tonic to bursting firing of RE neurons within a range of membrane potential (-60 to -65 mV) at which they set favorable conditions for the generation of low-threshold spike bursts that initiate spindle sequences. These data are further arguments for the generation of spindles within the thalamic RE nucleus.

Action Potentials↗

Two microtubule-associated proteins required for anaphase spindle movement in Saccharomyces cerevisiae.

In many eucaryotic cells, the midzone of the mitotic spindle forms a distinct structure containing a specific set of proteins. We have isolated ASE1, a gene encoding a component of the Saccharomyces cerevisiae spindle midzone. Strains lacking both ASE1 and BIK1, which encodes an S. cerevisiae microtubule-associated protein, are inviable. The analysis of the phenotype of a bik1 ase1 conditional double mutant suggests that BIK1 and ASE1 are not required for the assembly of a bipolar spindle, but are essential for anaphase spindle elongation. The steady-state levels of Ase1p are regulated in a manner that is consistent with a function during anaphase: they are low in G1, accumulate to maximal levels after S phase and then drop as cells exit mitosis. Components of the spindle midzone may therefore be required in vivo for anaphase spindle movement. Additionally, anaphase spindle movement may depend on a dedicated set of genes whose expression is induced at G2/M.

Anaphase↗

On the mechanism of anaphase spindle elongation in Diatoma vulgare.

Central spindles from five dividing cells (one metaphase, three anaphase, and one telophase) of Diatoma vulgare were reconstructed from serial sections. Each spindle is made up of two half-spindles that are composed almost entirely of polar microtubules. A small percentage of continuous microtubules and free microtubules were present in every stage except telophase. The half-spindles interdigitate at the midregion of the central spindle, forming a zone of overlap where the microtubules from one pole intermingle with those of the other. At metaphase the overlap zone is fairly extensive, but as elongation proceeds, the spindle poles move apart and the length of the overlap decreases because fewer microtubules are sufficiently long to reach from the pole to the zone of interdigitation. At telophase, only a few tubules are long enough to overlap at the midregion. Concurrent with the decrease in the length of the overlap zone is an increase in the staining density of the intermicrotubule matrix at the same region. These changes in morphology can most easily be explained by assuming zone mechanochemical interaction between microtubules in the overlap zone which results in a sliding apart of the two half-spindles.

Eukaryota↗

Role of spindle microtubules in the control of cell cycle timing.

Sea urchin eggs are used to investigate the involvement of spindle microtubules in the mechanisms that control the timing of cell cycle events. Eggs are treated for 4 min with Colcemid at prophase of the first mitosis. No microtubules are assembled for at least 3 h, and the eggs do not divide. These eggs show repeated cycles of nuclear envelope breakdown (NEB) and nuclear envelope reformation (NER). Mitosis (NEB to NER) is twice as long in Colcemid-treated eggs as in the untreated controls. Interphase (NER to NEB) is the same in both. Thus, each cycle is prolonged entirely in mitosis. The chromosomes of treated eggs condense and eventually split into separate chromatids which do not move apart. This "canaphase" splitting is substantially delayed relative to anaphase onset in the control eggs. Treated eggs are irradiated after NEB with 366-nm light to inactivate the Colcemid. This allows the eggs to assemble normal spindles and divide. Up to 14 min after NEB, delays in the start of microtubule assembly give equal delays in anaphase onset, cleavage, and the events of the following cell cycle. Regardless of the delay, anaphase follows irradiation by the normal prometaphase duration. The quantity of spindle microtubules also influences the timing of mitotic events. Short Colcemid treatments administered in prophase of second division cause eggs to assemble small spindles. One blastomere is irradiated after NEB to provide a control cell with a normal-sized spindle. Cells with diminished spindles always initiate anaphase later than their controls. Telophase events are correspondingly delayed. This work demonstrates that spindle microtubules are involved in the mechanisms that control the time when the cell will initiate anaphase, finish mitosis, and start the next cell cycle.

Animals↗

Micromanipulation studies of chromosome movement. II. Birefringent chromosomal fibers and the mechanical attachment of chromosomes to the spindle.

The degree of mechanical coupling of chromosomes to the spindles of Nephrotoma and Trimeratropis primary spermatocytes varies with the stage of meiosis and the birefringent retardation of the chromosomal fibers. In early prometaphase, before birefringent chromosomal fibers have formed, a bivalent can be displaced toward a spindle pole by a single, continuous pull with a microneedle. Resistance to poleward displacement increases with increased development of the chromosomal fibers, reaching a maximum at metaphase. At this stage kinetochores cannot be displaced greater than 1 micrometer toward either spindle pole, even by a force which is sufficient to displace the entire spindle within the cell. The abolition of birefringence with either colcemid or vinblastine results in the loss of chromosome-spindle attachment. In the absence of birefringent fibers a chromosome can be displaced anywhere within the cell. The photochemical inactivation of colcemid by irradiation with 366-nm light results in the reformation of birefringent chromosomal fibers and the concomitant re-establishment of chromosome attachment to the spindle. These results support the hypothesis that the birefringent chromosomal fibers anchor the chromosomes to the spindle and transmit the force for anaphase chromosome movement.

Animals↗

Functional autonomy of monopolar spindle and evidence for oscillatory movement in mitosis.

The oscillations of chromosomes associated with a single spindle pole in monocentric and bipolar spindles were analysed by time-lapse cinematography in mitosis of primary cultures of lung epithelium from the newt Taricha granulosa. Chromosomes oscillate toward and away from the pole in all stages of mitosis including anaphase. The duration, velocity, and amplitude of such oscillations are the same in all stages of mitosis. The movement away from the pole in monocentric spindle is rapid enough to suggest the existence of a previously unrecognized active component in chromosome movement, presumably resulting from a pushing action of the kinetochore fiber. During prometaphase oscillations, chromosomes may approach the pole even more closely than at the end of anaphase. Together, these observations demonstrate that a monopolar spindle is sufficient to generate the forces for chromosome transport, both toward and away from the pole. The coordination of the aster/centrosome migration in prophase with the development of the kinetochore fibers determines the course of mitosis. After the breaking of the nuclear envelope in normal mitosis, aster/centrosome separation is normally followed by the rapid formation of bipolar chromosomal fibers. There are two aberrant extremes that may result from a failure in coordination between these processes: (a) A monocentric spindle will arise when aster separation does not occur, and (b) an anaphaselike prometaphase will result if the aster/centrosomal complexes are already well-separated and bipolar chromosomal fibers do not form. In the latter case, the two monopolar prometaphase half-spindles migrate apart, each containing a random number of two chromatid (metaphase) monopolar-oriented chromosomes. This random segregation of prometaphase chromosome displays many features of a standard anaphase and may be followed by a false cleavage. The process of polar separation during prometaphase occurs without any visible interzonal structures. Aster/centrosomes and monopolar spindles migrate autonomously by an unknown mechanism. There are, however, firm but transitory connections between the aster center and the kinetochores as demonstrated by the occasional synchrony of centrosome-kinetochore movement. The data suggest that aster motility is important in the progress of both prometaphase and anaphase in normal mitosis.

Animals↗

Taxol-requiring mutant of Chinese hamster ovary cells with impaired mitotic spindle assembly.

In the accompanying paper (Cabral, F., 1982, J. Cell. Biol., 97:22-29) we described the isolation and properties of taxol-requiring mutants of Chinese hamster ovary cells. We now show that at least one of these mutants, Tax-18, has an impaired ability to form a spindle apparatus. Immunofluorescence studies using antibodies to tubulin demonstrate that, when incubated in the absence of taxol, Tax-18 forms only a rudimentary spindle with few and shortened microtubules associated with the spindle poles. Furthermore, midbodies were not observed, consistent with an absence of cytokinesis. Essentially normal spindles and midbodies are seen in the presence of taxol. Electron microscopic examination indicates that centrioles and kinetochores are morphologically normal in the mutant strain. Pole-to-kinetochore microtubules were seen but interpolar microtubules were not. Taxol-deprived mutant cells stained with anti-centrosome serum show an elevated centriole content, indicating that the defect in Tax-18 does not affect centriole replication or prevent progression through the cell cycle. Although Tax-18 cells do not form a complete spindle in the absence of taxol, cytoplasmic microtubule assembly occurs in association with microtubule-organizing centers, and microtubules with apparently normal morphology exist throughout the cytoplasm. Observation of chromosome movement indicates that the defect in these cells occurs after prometaphase. These studies demonstrate that the formation of spindle microtubules requires cellular conditions that are different from those required for cytoplasmic microtubule formation. They further show that a normal spindle may be necessary for cytokinesis but not for progress of the cells through the cell cycle.

Alkaloids↗

Control mechanisms of the cell cycle: role of the spatial arrangement of spindle components in the timing of mitotic events.

To characterize the control mechanisms for mitosis, we studied the relationship between the spatial organization of microtubules in the mitotic spindle and the timing of mitotic events. Spindles of altered geometry were produced in sea urchin eggs by two methods: (a) early prometaphase spindles were cut into half spindles by micromanipulation or (b) mercaptoethanol was used to indirectly induce the formation of spindles with only one pole. Cells with monopolar spindles produced by either method required an average of 3 X longer than control cells to traverse mitosis. By the time the control cells started their next mitosis, the experimental cells were usually just finishing the original mitosis. In all cases, only the time from nuclear envelope breakdown to the start of telophase was prolonged. Once the cells entered telophase, events leading to the next mitosis proceeded with normal timing. Once prolonged, the cell cycle never resynchronized with the controls. Several types of control experiments showed that were not an artifact of the experimental techniques. These results show that the spatial arrangement of spindle components plays an important role in the mechanisms that control the timing of mitotic events and the timing of the cell cycle as a whole.

Animals↗